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Volume 41

Advances in Genetics

This Page Intentionally Left Blank

Volume 41

Advances in Genetics Edited by Jeffrey C. Hall

Jay C. Dunlap

Department of Biology Brandeis University Waltham, Massachusetts

Department of Biochemistry Dartmouth Medical School Hanover, New Hampshire

Theodore Friedmann

Francesco Giannelli

Center for Molecular Genetics University of California at San Diego School of Medicine La Jolla, California

Division of Medical and Molecular Genetics United Medical and Dental Schools of Guy’s and St Thomas’ Hospitals London Bridge, London United Kingdom

Academic Press San Diego

London

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This book is printed on acid-free paper

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Copyright 1999 by ACADEMIC PRESS All Rights Reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U S Copyright Law This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale Copy fees for pre-1999 chapters are as shown on the title pages If no fee code appears on the title page, the copy fee is the same as for current chapters 0065-2660/99 $30 00 Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given

Academic Press A arcourt Science and Technology Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www apnet com

Academic Press 24-28 Oval Road, London NW1 7DX, UK http://www hbuk co uk/ap/ International Standard Book Number: 0-12-017641-6 PRINTED IN THE UNITED STATES OF AMERICA 99 00 01 02 03 04 EB 9 8 7 6 5

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Contents Contributors

vii

1 Mosquito Genomes: Structure, Organization, and Evolution

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Karamjit S Rai and William C Black IV I II III IV V VI VII VIII IX

Overview 1 Mosquito Taxonomy, Evolution, and the Fossil Record Cladistic Analysis of Culicidae 3 Chromosome Number Is Conserved in Culicidae 5 Sex Chromosome Evolution in Culicidae 7 Genome Size and General Genome Organization 7 Heterochromatin: Localization, Variation, and Expression 17 Saturated Linkage Maps Generated through Multipoint Mapping 23 Summary 26 References 27

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2 Seeing the Light: News in Neurospora Blue Light Signal Transduction

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H Linden, P Ballario, G Arpaia, and G Macino I II III IV V VI

Introduction 36 The Perception of Light in Neurospora 37 The Interplay of Blue Light and Other Regulatory Pathways in Neurospora 41 Mutational Analysis of Blue Light Signal Transduction in Neurospora 42 The Neurospora Blue Light Regulatory Proteins WC-1 and WC-2 44 Concluding Remarks 50 References 51 v

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Contents

3 X-Linked Mental Retardation

55

Giovanni Neri and Pietro Chiurazzi I II III IV

Introduction 56 Syndromal XLMR 57 Nonsyndromal XLMR (MRX) Conclusion 83 References 83

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4 Pharmaceutical Perspectives of Nonviral Gene Therapy

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Ram I Mahato, Louis C Smith, and Alain Rolland I II III IV V VI VII VIII IX

Why a Gene-Based Approach for Protein Therapy? 97 Commercialization of Gene Therapy Products 100 Basic Components of Gene Expression Plasmids 104 Gene Delivery Systems 110 Formulation Factors Influencing Gene Transfer 123 Biodistribution and Pharmacokinetics of Plasmids 124 Intracellular Trafficking of Gene Medicines 130 Biological Opportunities for Gene Therapy 134 Concluding Remarks 143 References 144

5 Mutational Analysis of 23S Ribosomal RNA Structure and Function In Escherichia coli

157

Kathleen L Triman I II III IV

Index

Introduction 157 Methods of Detection of rRNA Mutants in Escherichia coli 158 Mutational Analysis of 23S rRNA Structure and Function 162 Conclusions 169 References 187

197

Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

G. Arpaia ( 5) Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari, Sezione di Genetica Molecolare, Universita` di Roma “La Sapienza,” 00161 Roma, Italy P. Ballario ( 5) Dipartimento di Genetica e Biologia Molecolare, Centro di Studio per gli Acidi Nucleici, Universita` di Roma “La Sapienza,” 00185 Roma, Italy William C. Black IV (1) Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556 Pietro Chiurazzi (55) Centro Ricerche per la Disabilita` Mentale e Motoria, Associazione Anni Verdi, 00168 Roma, Italy H. Linden ( 5) Lehrstuhl fur Physiologic and Biochemie der Pflanzen, Universitat Konstanz, D-784 4 Konstanz, Germany G. Macino ( 5) Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari, Sezione di Genetica Molecolare, Universita` di Roma “La Sapienza,” 00161 Roma, Italy Ram I. Mahato (95) Copernicus Therapeutics, Inc., Cleveland, Ohio 44106 Giovanni Neri (55) Istituto di Genetica Medica, Facolta` di Medicina e Chirurgia “A. Gemelli,” Universita` Cattolica del Sacro Cuore, 00168 Roma, Italy Karamjit S. Rai (1) Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556 Alain Rolland (95) Valentis, Inc., The Woodlands, Texas 77 81 Louis C. Smith (95) Valentis, Inc., The Woodlands, Texas 77 81 Kathleen L. Triman (157) Department of Biology, Franklin and Marshall College, Lancaster, Pennsylvania 17604

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Mosquito Genomes: Structure, Organization, and Evolution Karamjit S. Rai* and William C. Black IV† Depar men of Biological Sciences Universi y of No re Dame No re Dame, Indiana 46556

I. II. III. IV. V. VI.

Overview Mosqui o Taxonomy, Evolu ion, and he Fossil Record Cladis ic Analysis of Culicidae Chromosome Number Is Conserved in Culicidae Sex Chromosome Evolu ion in Culicidae Genome Size and General Genome Organiza ion A. In erspecific Varia ion and Genome Organiza ion B. In raspecific Genome Size Varia ion VII. He erochroma in: Localiza ion, Varia ion, and Expression VIII. Sa ura ed Linkage Maps Genera ed hrough Mul ipoin Mapping IX. Summary Acknowledgmen s References

I. OVERVIEW The family Culicidae is composed of more han 3,400 mosqui o species, many of which are major vec ors of arboviruses, malaria, and filariasis. In view of heir * Address for correspondence: E-mail: Karamji [email protected]. Fax (219) 631-7413. Telephone: (219) 631-6584. † Presen address: College of Ve erinary Medicine and Biomedical Sciences, Depar men of Microbiology, Colorado S a e Universi y, For Collins, Colorado 80523. E-mail: wcb4@lamar. colos a e.edu. Advances in Genetics, Vol. 41 Copyrigh 1999 by Academic Press All righ s of reproduc ion in any form reserved. 0065-2660/99 $30.00

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K. S. Rai and W. C. Black IV

impor ance as vec ors, many mosqui o genera and species have been he subjec of ex ensive cy ological and gene ic inves iga ions over he las 40 years (Ki zmiller 1953, 1976; Whi e, 1980; Rai et al., 1982; Rai, 1991). As a resul , here is a voluminous li era ure on mosqui o genomes sca ered in various en omological and gene ics journals. The purpose of his review is o highligh he salien fea ures of mosqui o genomes and heir evolu ion. I is indeed surprising ha , excep for a couple of minireviews (Besansky and Collins, 1992; Kumar and Rai, 1993), he various face s of his work have no been reviewed earlier. We begin wi h a general review of mosqui o sys ema ics, highligh ing and summarizing recen s udies ha employed modern cladis ic analysis of morphological and molecular charac ers o es ima e phylogene ic rela ionships among sis er families o Culicidae and among Culicidae subfamilies, ribes, genera, subgenera, and species. We nex review he ex ensive li era ure on karyo ypes, emphasizing ha he number of chromosomes has remained a a cons an 2n ⫽ 6 despi e a rela ively ancien origin for Culicidae, he evolu ion of bo h homomorphic and he eromorphic sex chromosomes, and evidence of ex ensive ransloca ions and inversions. The li era ure on he evolu ion of genome size and organiza ion in Culicidae is summarized and considered in ligh of curren phylogene ic rela ionships. Genome evolu ion is also reviewed in he con ex of he now-ex ensive s udies on he erochroma in dis ribu ion and in erms of he linkage maps ha are beginning o arise hrough various recen in ensive genome mapping projec s in Culicidae.

II. MOSQUITO TAXONOMY, EVOLUTION, AND THE FOSSIL RECORD The family Culicidae, which includes all mosqui oes, is divided in o hree subfamilies, Anophelinae, Toxorhynchi inae, and Culicinae (Knigh and S one, 1977; Knigh , 1978; Ward, 1984, 1992; Service, 1993). Anophelinae includes hree genera, he neo ropical Chagasia (4 species), he Aus ralasian Bir nella (9 species in 3 subgenera), and he nearly cosmopoli an An pheles wi h some 422 species grouped in 6 subgenera. Toxorhynchi inae includes a single genus, T x rhynchites wi h 76 species. Culicinae is by far he larges subfamily: i is subdivided in o 10 ribes, 33 genera, and 117 subgenera and includes abou 2,925 described species. Al hough mosqui o sys ema ics is in a s a e of flux (Muns ermann, 1995), he o al numbers of genera, subgenera, and species in Culicidae curren ly s and a 37, 129, and 3,436, respec ively (Service, 1993). The genus Aedes, which includes some 962 species grouped in 43 subgenera, is one of he bes s udied cy ogene ically (Rai et al., 1982). Based on he fossil record, scan y hrough i is, and zoogeographic evidence involving pas in ercon inen al connec ions and faunis ic composiion, i has been sugges ed ha mosqui oes had evolved by he Jurassic, approx-

1. Mosquito Genomes: Structure, Organization, and Evolution

3

ima ely 210 million years ago (MYA) (Edwards, 1932). This is abou he ime con inen al drif began (Wilson, 1963). The con inen al breakup led o fragmen a ion and geographical isola ion of popula ions. This may have been accompanied by grea ecological flux ha promo ed rapid specia ion (McClelland, 1967). Ross (1951) proposed ha a burs of Culicinae lineages arose approxima ely 120 MYA. By he end of he Cre aceous, some 65 MYA, he generic composi ion of family Culicidae was well es ablished (Belkin, 1968; Rohdendorf, 1974). New Zealand has been in i s presen posi ion of isola ion for approxima ely he las 50 million years (Rick, 1970). Wi h he excep ion of hree species, Aedes n t scriptus, Aedes australicus, and Culex quinquefasciatus, he presen -day mosqui o fauna of New Zealand is relic and endemic. This provides circums an ial evidence ha he genus Aedes exis ed prior o he island’s separa ion from Aus ralia and ha i was probably widely dispersed during he Cre aceous, which began 145 MYA (Belkin, 1968). Fossils of family Culicidae (Culex, Aedes) and i s sis er family Chaoboridae are well known from he Eocene (Ter iary) and Oligocene, which began 60 and 55 MYA, respec ively (Rohdendorf, 1974).

III. CLADISTIC ANALYSIS OF CULICIDAE The phylogene ic rela ionship of Culicidae rela ive o o her nema ocerous diperan families has been evalua ed using modern cladis ic analysis. Muns ermann and Conn (1997) have reviewed he impac of molecular biology and cladis ic analysis on sys ema ics of selec ed axa of Culicidae wi h par icular emphasis on he Aedes and An pheles species. Phylogenies have been es ima ed wi h sui es of morphological charac ers (Oos erbroek and Cour ney, 1995) and nucleo ide sequences from he 18S and 5.8S nuclear ribosomal DNA (rDNA) (Miller et al., 1997) and 28S rDNA (Pawlowski et al., 1996). The morphological and 18S da ase s are congruen in iden ifica ion of Chaoboridae (phan om midges) as a sis er group o Culicidae and in placemen of Core hrellidae as a basal clade o Chaoboridae – Culicidae. The 28S da ase suppor ed monophyly of hese hree families bu consis en ly indica ed Chaoboridae – Core hrellidae as sis er axa. Phylogenies of high-order rela ionships among hese hree families and Chironomidae, Cera opogonidae, Dixidae, Psychodidae, and Simulidae are incongruen in all hree s udies. Each s udy ci es several independen lines of suppor for he higher-order rela ionships derived from heir respec ive phylogenies bu all s udies also indica e ha hese rela ionships were suppor ed by few charac ers or lack s rong boo s rap suppor . The rDNA papers use differen species in each family, obvia ing a combined analysis as a means o resolve his conflic . The rDNA s udies also suffer from sampling of single species in mos families,

4


preven ing iden ifica ion of synapomorphies for each family. These rela ionships should be explored fur her wi h more comple e axon sampling and an examina ion of single-copy nuclear genes. The rela ionship of Culicidae subfamilies has been examined wi h nucleo ide da ase s using rDNA genes (Pawlowski et al., 1996; Miller et al., 1997) and he single-copy nuclear gene white (Besansky and Fahey, 1997): All hree s udies were congruen in placemen of Anophelinae as he basal clade in Culicidae. Fur hermore, he 18S and white genes were consis en in placing Toxorhynchi inae as basal o he Culicinae. Rela ionships among ribes, genera, and species in Culicinae have also been evalua ed using modern cladis ic analysis. Judd (1996) examined 59 morphological charac ers in 37 axa wi hin he ribe Sabe hini. Cladis ic analysis, using Eretmap dites quinquevittatus and Haemag gus spegazzinii as ou groups, suppor ed Sabe hini as a monophyle ic group bu s rongly sugges ed paraphyle ic rela ionships among species in a leas hree genera (Runch myia, Tripter ides, and Wye myia). Wesson et al. (1992) sequenced he 5.8S – 28S half of he in ernal ranscribed spacer of he rDNA cis ron (ITS2) o examine phylogene ic rela ionships among seven species in hree genera (Aedes, Haemag gus, and Ps r ph ra) of Aedini. Their analyses sugges ed paraphyle ic rela ionships among species in he Aedes subgenus Steg myia and sugges ed ha Haemag gus and Ps r ph ra arose wi hin Aedes. The resolved phylogeny also provided evidence for biogeographical rela ionships among Aedini species: one clade conained Old World species (Ae. aegypti, Ae. simps ni, Ae. vexans, and Ae. alb pictus); a second clade con ained he New World axa Ae. triseriatus, Haemag gus mes dentatus, and Ps r ph ra ver x. Besansky and Fahey (1997) performed a horough axon sampling of varia ion in he white gene among axa in ribes Culicini, Sabe hini, and Aedini in he Culicinae. Their analysis suppor ed placemen of Sabe hini as basal o Culicini and Aedini. Like he analysis of Wesson et al. (1992), his analysis of he white gene placed old World Aedini (Ae. aegypti and Ae. alb pictus) in a separa e clade from he New World species (Ae. triseriatus, Haemag gus equinus) wi h high boo s rap suppor . The rDNA genes (Pawlowski et al., 1996; Miller et al., 1997) and white gene all suppor a monophyle ic rela ionship be ween Culicini and Aedini. Miller et al. (1996) examined sequence divergence in he en ire in ernal ranscribed spacer (ITS) among 14 species in four subgenera of he genus Culex. Species in he subgenera Culex, Lutzia, and Ne Culex were monophyle ic. There was low boo s rap suppor for monophyly of species in he subgenus Culex bu only single species were examined in he subgenera Lutzia and Ne Culex. Some rela ionships among species and species complexes were also examined. Kumar et al. (1998) cons ruc ed res ric ion maps of he rDNA cis ron of 12 species of mosqui oes in six genera of he subfamily Culicinae using eigh


5

6-bp recogni ion res ric ion enzymes. An pheles albimanus was used as an ou group. Clades wi hin he RFLP (res ric ion fragmen leng h morphism) phylogeny were no well suppor ed and were incongruen wi h he morphology charac er based and molecular phylogenies previously discussed. The lack of resolu ion in he RFLP da ase was probably due o homoplasy arising from frequen independen loss or possibly, hough less likely, from gain of res ric ion si es among unrela ed axa. S udies by Kumar et al. (1998) showed ha only rela ionships among closely rela ed axa were well suppor ed. As in Besansky and Fahey (1997), Ae. triseriatus and Ha. equinus were monophyle ic. The sis er species, Ae. epactius and Ae. atr palpus, were also monophyle ic. Species in he Aedes alb pictus and he Aedes scutellaris subgroups of he Aedes scutellaris group were monophyle ic in he (RFLP) phylogeny. Based on a correla ion of he allozyme differen ia ion among cer ain species and heir geological his ories and calibra ion of a well-es ablished geologic even in he Sou h Pacific, Pashley et al. (1985) concluded ha he Ae. alb pictus and he Ae. scutellaris subgroups diverged rela ively recen ly. In summary, modern cladis ic analyses of morphological and molecular charac ers consis en ly suppor Chaoboridae – Core hrellidae as sis er axa o Culicidae. All analyses suppor Anophelinae as he basal clade in Culicidae and are consis en in placing Toxorhynchi inae as basal o he Culicinae. Wi hin Culicinae, he ribe Sabe hini is basal o Culicini and Aedini. All da ase s suppor a monophyle ic rela ionship be ween Culicini and Aedini. Many subgeneric rela ionships wi hin Sabe hini, Culicini, and Aedini may be paraphyle ic and warran axonomic revision. These s udies do no address he key ques ion of whe her Toxorhynchi inae arose wi hin Anophelinae or as a separa e lineage from a common ances or wi h Anophelinae. This becomes a pivo al issue in discussing he origins of some major gene ic differences be ween anopheline and culicine mosqui oes la er in his chap er. This issue may become resolved in he fu ure hrough examina ion of addi ional gene sequences and in ensive sampling of primi ive and derived members of bo h Toxorhynchi inae and Anophelinae. However, i is also qui e possible ha ances ral axa are ex inc in ei her or bo h subfamilies and ha he issue will never be adequa ely resolved.

IV. CHROMOSOME NUMBER IS CONSERVED IN CULICIDAE Chromosomal karyo ypes have been es ablished for “no less han” 19 genera, 35 subgenera, and 200 species in family Culicidae (Whi e, 1980). Over he las several years, addi ional species have been cy ologically examined (Rao and Rai, 1987a, 1990). One of he mos remarkable findings of his karyo ypic survey is ha , despi e he ancien origin of he group and despi e ex ensive repa ern-

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ing of he genome involving ransloca ions and inversions (Ma hews and Muns ermann, 1994; Mori et al., 1998), he basic chromosome number (2n ⫽ 6) has remained unchanged. The only excep ion, Chagasia bathana (2n ⫽ 8) of he subfamily Anophelinae, possesses hree au osome pairs and a he eromorphic pair of sex chromosomes (Kreu zer, 1978). All o her anophelines possess wo pairs of generally me acen ric chromosomes of unequal size and one pair of he eromorphic sex chromosomes ha of en show ex ensive polymorphism in overall leng h and of he quan i y and quali y of he erochroma in differen ia ion among various species (Whi e, 1980). The posi ion of he cen romeres in he he eromorphic X and Y chromosomes in Anophelinae varies from sub elocen ric or acrocen ric o subme acen ric and me acen ric (Baimai et al., 1993a, b, 1995). In con ras , species of he subfamilies Toxorhynchi inae and Culicinae all possess hree pairs of homomorphic me acen ric and/or sligh ly subme acenric chromosomes: a pair of small chromosomes, a pair of large chromosomes, and a pair of in ermedia e-sized chromosomes (Rai, 1963; McDonald and Rai, 1970; Rai et al., 1982, Rao and Rai, 1987a). In culicine mosqui oes, sex is de ermined by a gene a a single locus. Females are homozygous recessive a his locus, and males are he erozygous for a dominan allele (Gilchris and Haldane, 1947; McClelland, 1962). In species in which linkage group – chromosome correla ions have been made, he shor es chromosome con ains he sex locus and is herefore sex de ermining (McDonald and Rai, 1970; Baker et al., 1971; Dennho¨fer, 1972). Differences clearly exis in overall leng hs and arm ra ios of individual chromosomes, bo h wi hin and be ween species, bu can be easily overlooked if careful measuremen s of each arm of a chromosome are no made (Rai, 1980; Rai et al., 1982). To al chromosomal leng h varies almos fivefold, from 8.2 ␮m in An pheles quadrimaculatus o 39.3 ␮m in Aedes alcasidi. Wi hin he genus Aedes, here is a hreefold varia ion in chromosome leng h (Table 1.1) Conserva ion of chromosome number in Culicidae does no indica e syn eny. Ma hews and Muns ermann (1994) and Severson et al. (1995) clearly documen ha groups of allozyme loci have remained linked and colinear in a varie y of culicine axa bu ha hese linkage groups have ransloca ed and are inver ed ex ensively across he hree culicine chromosomes. The ex ensive varia ion in chromosome number in mos dip era axa s udied does no predic he ex reme conserva ion found in Culicidae. For example, he chromosome number ranges from n ⫽ 3 o 7 in he genus Dr s phila (see Whi e, 1973) and from n ⫽ 3 o 8 in he genus Gl ssina (Mauldin 1970). In family Muscidae, mos species possess six pairs of chromosomes; however, six species have only five pairs each (Boyes, 1967). Never heless, cer ain o her dip eran families such as Simulidae (Ro hfels, 1979) and Sarcophagidae also show ex ensive conserva ion of chromosome number, al hough some excep ions do occur (Whi e,


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1973). No logical explana ion exis s for he ex raordinary conserva ion of he haploid chromosome number in Culicidae. The chromosomal karyo ype da a from Culicidae in general suppor Whi e’s (1973) sugges ion ha here may be some kind of barrier ha main ains chromosome number in he Dip era. Never heless, we know no hing abou he ac ual na ure of such a barrier.

V. SEX CHROMOSOME EVOLUTION IN CULICIDAE Curren dogma sugges s ha he eromorphic sex chromosomes evolved from vir ually iden ical homologues. Bo h heore ical considera ions (Charleswor h, 1978) and considerable experimen al evidence sugges ha i is he gradual accumula ion of repe i ive sequences on he Y chromosome followed by loss of recombina ion be ween he he eromorphic pair ha leads o he differen ia ion of X and Y chromosomes. Theory predic s even ual loss of func ion and evenual ex inc ion of he Y chromosome (S einemann et al., 1993; Morell, 1994; Rice, 1994, 1996). This direc ionali y is generally referred o as he “rise and fall of he Y chromosome” (Morell, 1994). Evolu ion of a he eromorphic Y chromosome may have occurred only once or possibly may have been reversed in he evolu ion of sex chromosomes in Culicidae. The primi ive Nema ocera families Tipulidae and Dixidae possess homomorphic sex chromosomes. However, he sis er families Chaoboridae – Core hrellidae con ain genera wi h homomorphic (Euc rethra, C rethrella, Cha b rus) and he eromorphic (M chl nyx) sex chromosomes (Rao and Rai, 1987a). If homomorphy was ances ral in Culicidae, hen i was re ained in he lineages leading o Toxorhynchi inae and Culicinae, while he eromorphy probably evolved early in he evolu ion of Anophelinae and was re ained in all axa. This scenario is suppor ed by he curren dogma concerning he evolu ion of sex chromosomes (Rice, 1996). Al ernaively, if, as proposed by Rao and Rai (1987a), Culicidae arose from a Mochlonyx-like ances or, hen Anophelinae re ained he eromorphic sex chromosomes, while homomorphic sex chromosomes evolved hrough euchroma iniza ion or loss of he Y in Toxorhynchi inae and Culicinae.

VI. GENOME SIZE AND GENERAL GENOME ORGANIZATION A. Interspecific variation and genome organization Considerable effor has been expended in recen years o de ermine haploid nuclear DNA amoun s in he superfamily Culicoidea (Jos and Mameli, 1972; Rao and Rai, 1987b, 1990; Black and Rai, 1988; Kumar and Rai, 1990). This has been done hrough quan i a ive cy opho ome ry of Feulgen-s ained primary

8 Table 1.1. Mean Chromosomal Leng hs in 30 Represen a ive Species Belonging o 8 Genera of Mosqui oes and Rela ed Taxa in Superfamily Culicoidae Mean chromosome leng h (␮m) Family

Genus/species

I

II

III

TCLa (I ⫹ II ⫹ III)

References

Chaoboridae

M chl nyx velutinus Cha b rus americanus

2.2(X); 1.3(Y) 2.3

4.6 3.1

5.4 3.3

12.2 8.7

Rao and Rai, 1987a Rao and Rai, 1987a

Culicidae

An pheles quadrimaculatus Culex pipiens Culex territans Culex restuans T x rhynchites splendens Wye myia smithii Haem ag gus Equinus Aedes t g i Ae. metallicus Ae. hebrideus Ae. aegypti Ae. heischii Ae. kesseli Ae. atr palpus Ae. pseud scutellaris Ae. unilineatus

1.4(x); 0.9(y) 2.4 2.6 3.0 3.4 4.6 6.6 3.0 5.2 5.1 5.4 6.3 6.3 6.2 6.9 6.4

3.0 4.2 4.1 5.4 4.7 5.8 9.6 4.6 6.3 6.3 6.9 7.4 7.8 8.4 9.6 9.1

3.8 5.0 5.4 6.2 5.0 6.2 10.7 5.4 7.8 7.9 7.6 8.0 9.4 9.2 9.9 10.0

8.2 11.6 12.1 14.6 13.1 13.0 26.9 13.0 19.3 19.3 19.9 21.6 23.5 23.8 24.6 25.5

Rai, 1963 Rai, 1963 Rai, 1963 Rai, 1963 Rao and Rai, 1987a Rai, 1963 Rao and Rai, 1987a Rai, 1963 Rao and Rai, 1987a Rao and Rai, 1987a Rai, 1963 Rao and Rai, 1987a Dev and Rai, 1984 Rai, 1963 Dev and Rai, 1984 Rao and Rai, 1987a

Ae. c ki Ae. seat i Ae. p lynesiensis Ae. katherinensis Ae. stimulans Ae. pseud alb pictus Ae. malayensis Ae. flav pictus Ae. triseriatus Ae. z s phus Ae. alcasidi Ae. alb pictus Oahu, Hawaii Calcu a, India Kolar, India Mauri ius Tananareve, Madagascar Pune, India Delhi, India TCL: To al chromosomal leng h in microme ers.

a

6.9 7.3 7.4 7.5 7.6 7.9 7.9 8.3 9.9 9.1 9.9

8.8 9.3 9.4 9.6 10.7 10.3 10.3 11.5 10.7 14.4 13.6

9.4 10.5 11.1 12.6 11.5 11.8 12.1 13.5 15.2 14.8 15.8

25.6 27.1 27.9 29.7 29.8 30.0 30.4 33.3 35.8 38.3 39.3

Dev and Rai, 1984 Rao and Rai, 1987a Dev and Rai, 1984 Rao and Rai, 1987a Rai, 1963 Rao and Rai, 1987a Dev and Rai, 1984 Rao and Rai, 1987a Rao and Rai, 1987a Rao and Rai, 1987a Dev and Rai, 1984

6.0 6.5 6.6 7.2 8.3 8.0 9.2

6.3 8.1 7.9 9.3 11.0 11.2 11.4

8.9 9.3 9.5 10.4 11.8 12.8 12.2

21.2 23.9 24.0 26.9 31.1 32.0 32.8

Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b

9

10


sperma ocy es and in a few cases hrough analyses of rena ura ion kine ics of nuclear DNA (Black and Rai, 1988; Warren and Cramp on, 1991; Besansky and Powell, 1992). As a resul , haploid genome sizes have been es ablished for 44 species belonging o 13 genera of mosqui oes and rela ed Culicoidea families (Table 1.2). Genome size is generally small in Anophelinae (0.23 – 0.29 pg/haploid genome) (Jos and Mameli, 1972; Black and Rai 1988; Besansky and Powell, 1992). The single species T x rhynchites splendens, examined in subfamily Toxorynchi inae possesses an in ermedia e-size genome of 0.62 pg as do Sabethes cyaneus and Wye myia smithii (Sabe hini).The haploid genomes of Culex species examined ranged from 0.54 o 1.02 pg and hose of Culiseta species (Culicini) from 0.92 o 1.25 pg. Armigeres subalbatus and Haemag gus equinus (Aedini) con ained 1.24 and 1.12 pg, respec ively. A he generic level, he cosmopoli an genus Aedes showed more han hreefold varia ion in nuclear DNA amoun s, wi h he Polynesian species Ae. pseud scutellaris and Ae. c ki (belonging o he Ae. scutellaris subgroup in he subgenus Steg myia) possessing he lowes genome size of 0.59 pg and Ae. z s phus(subgenus Pr t macleaya) possesing he highes genome size of 1.9 pg among he 23 species examined (Rao and Rai, 1987b, 1990). Placed in he con ex of phylogene ic rela ionships discussed earlier, hese figures sugges a general increase in genome size during he evolu ion of Culicidae. Black and Rai (1988) demons ra ed ha all classes of repe i ive DNA sequences increased linearly in amoun wi h o al genome size. Fur hermore, linear regression analysis of a fairly large da ase involving 28 species belonging o 11 genera of he superfamily Culicoidea showed a highly significan posi ive correla ion (r ⫽ 0.87; p ⫽ 0.0001) be ween o al chromosomal leng h and haploid genome size (Rao and Rai, 1987b). Never heless, eigh fold varia ion in haploid genome size was accompanied by only an approxima e fivefold varia ion in he o al chromosomal leng h, indica ing ha DNA amoun s have increased almos wice as much as he increase in chromosomal size. S udies using reassocia ion kine ics have provided informa ion on genome organiza ion in Anophelinae and Culicinae (Black and Rai, 1988; Warren and Cramp on, 1991; Besansky and Powell, 1992). Genome organiza ion refers o he amoun s, complexi y, and dispersion of repe i ive elemen s in a genome. Two basic forms of genome organiza ion have been described in eukaryo es (Davidson et al., 1975). The firs ype is ermed “shor period in erspersion” and describes a pa ern in which single-copy sequences, 1000 – 2000 bp in leng h, al erna e regularly wi h shor (200 – 600 bp) and modera ely long (1000 – 4000 bp) repe i ive sequences. This charac erizes genome organiza ion in he majori y of animal species and was found in he culicine species Culex pipiens, Ae. aegypti, Ae. alb pictus, and Ae. triseriatus (Black and Rai, 1988). The second ype of genome organiza ion is ermed “long-period in erspersion”


11

and describes a pa ern of long (⬎ 5600 bp) repea s al erna ing wi h very long (⬎ 13,000 bp) unin errup ed s re ches of unique sequences. Repea s in An. quadrimaculatus (Black and Rai, 1988) and An. gambiae (Besansky and Powell, 1992) follow a long-period in erspersion pa ern. Genome organiza ion is of he long-in erspersion ype in Chir n mus tentans (Wells et al., 1976) bu has no been de ermined in sis er families Chaoboridae – Core hrellidae. However, haploid DNA amoun s of 0.47, 0.55, and 0.40 pg were observed in he hree principal genera C rethrella, M chl nyx, and Cha b rus, respec ively (Table 1.2; Rao and Rai, 1990). In insec s, long period in erspersion is charac eris ic of mos species wi h small genome sizes (0.1 – 0.5 pg/haploid genome), while shor -period in erspersion ends o be associa ed wi h larger genomes wi h larger amoun s of repe i ive DNA (Palmer and Black, 1997). I is difficul o predic genome organiza ion in Chaoboridae – Core hrellidae based on genome size, because hey fall in o he upper limi for long-in erspersed species. Thus here remain wo compe ing hypo heses for ances ral genome evolu ion in Culicidae. I is possible ha longperiod in erspersion was ances ral in Culicidae and was re ained in he lineage leading o Anophelinae, while larger genomes developed hrough accumula ion of shor -period in erspersed repe i ive elemen s in Culicinae. The al erna ive hypo hesis is ha Culicidae arose from a shor -period in erspersed species and, while ha organiza ion was re ained in he Culicinae, repe i ive elemen s were shed and became organized in o a long-period in erspersion pa ern in he Anophelinae. This is he scenario considered by Rao and Rai (1990), who proposed a phylogeny of he superfamily Culicoidea based on genome sizes (Figure 1.1). They sugges ed ha he line ha possibly gave rise o Anophelinae from a Mochlonyx-like ances or underwen many dele ions of highly repe i ive DNA. However, his scenario lacks any empirical evidence from evolu ion of genome size in o her sys ems. Cullis (1983) sugges ed ha nuclear DNA is organized in o cons an and fluid domains. The fluid domain, which is composed mainly of repe i ive DNA sequences (Cavallini et al., 1986), shif s in response o changing environmen s and developmen al and physical s imuli (Walbo and Cullis, 1985). Genome size shif s dynamically as a resul of DNA amplifica ion, burs s of ransposi ions, unequal crossing-over ha can simul aneously cause elimina ion and gain of cer ain DNA sequences (Bassi et al., 1984; Na ali et al., 1986; Al amura et al., 1987), and in ragenomic drif (Cavalier-Smi h, 1985a). However, hese mechanisms generally cause genomes o accumula e repe i ive elemen s, and very few mechanisms have been proposed for genomewide “shedding” of repe i ive elemen s. Considering hese argumen s, i is mos parsimonious o sugges ha long-period in erspersion was ances ral in Culicidae. However, i is cri ical o de ermine genome organiza ion in Chaoboridae or Core hrellidae o es his hypo hesis. Fur hermore, analysis of genome orga-

12 Table 1.2. Haploid Genome Size (Picogram DNA) in 44 Species Belonging o 13 Genera of Mosqui oes and Rela ed Taxa

Family Dixidae Chaoboridae

Culcidae

Subfamily

Tribe

Genus

Sabe hini

Dixa C rethrella M chl nyx Cha b rus An pheles An. An. An. An. An. T x rhynchites Sabethes Wye myia Culex Cx. Cx. Cx. Culiseta Cu. Cu. Haemag gus Armigeres Aedes

Core hrellinae Chaoborinae Chaoborinae Anophelinae

Toxorhynchi inae Culicinae

Culicini

Culise ini

Aedini

Subgenus

Chaoborus Anopheles

Cellia Toxohynchi es Sabe hes Wyeomyia Culex

Culicella Climacura Haemagogus Armigeres S egomyia

Species

pg DNA/haploid genome ⫾ SE

bscura brakeleyi velutinus americanus labranchiae atr parvus quadrimaculatus freeb rni stephensi gambiae splendens cyaneus smithii pipiens pipiens quinquefasciatus restuans lit rea m rsitans melanura equinus subalbanus pseud scutellaris

0.156 0.47 ⫾ 0.02 0.55 ⫾ 0.02 0.40 ⫾ 0.02 0.234 0.242 0.245 ⫾ 0.01 0.294 0.242 0.27 0.618 ⫾ 0.019 0.786 ⫾ 0.02 0.855 ⫾ 0.011 1.02 ⫾ 0.19 0.540 ⫾ 0.012 0.54 ⫾ 0.01 1.02 ⫾ 0.04 0.92 1.21 ⫾ 0.04 1.25 ⫾ 0.005 1.120 ⫾ 0.023 1.124 ⫾ 0.027 0.591 ⫾ 0.012

References Jos and Mameli, 1972 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 1990 Jos and Mameli, 1972 Jos and Mameli, 1972 Rao and Rai, 1990 Jos and Mameli, 1972 Jos and Mameli, 1972 Besansky and Powell, 1992 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 1990 Jos and Mameli, 1972 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 1990 Jos and Mameli, 1972 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 1987b

Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae. Ae.

Aedes Howardina Ochlero abus

Pro omaclyeaya

c ki p lynesiensis aegypti aegypti malayensis hebrideus seat i alcasidi unilineatus metallicus heischii katherinensis pseud alb pictus flav pictus cinereus bahamensis canadensis c mmunis caspius stimulans excrucianus triseriatus z s phus

0.594 ⫾ 0.027 0.725 ⫾ 0.018 0.812 ⫾ 0.031 0.83 0.943 ⫾ 0.025 0.965 ⫾ 0.031 0.971 ⫾ 0.023 0.974 ⫾ 0.016 1.064 ⫾ 0.04 1.093 ⫾ 0.033 1.121 ⫾ 0.039 1.277 ⫾ 0.02 1.29 ⫾ 0.028 1.33 ⫾ 0.024 1.210 ⫾ 0.03 1.375 ⫾ 0.03 0.904 ⫾ 0.02 1.013 ⫾ 0.05 0.988 1.439 ⫾ 0.039 1.500 ⫾ 0.03 1.520 ⫾ 0.062 1.902 ⫾ 0.062

Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Warren and Cramp on, 1991 Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Jos and Mameli, 1972 Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b

13

14


Figure 1.1. Genome sizes in some members of Culicoidea and he proposed phylogeny. (Dis ances be ween members are arbi rary.) Af er Rao and Rai (1990).

niza ion in Sabe hini and Toxorhynchi inae is impera ive o de ermine when shor -period in erspersion arose in culicine evolu ion.

B. Intraspecific genome size variation Genome s udies in Rai’s labora ory have also focused on in raspecific varia ion in genome size and have indica ed unequivocally ha DNA amoun s are no fixed wi hin species (Ferrari and Rai, 1989; Rao and Rai, 1987b; Kumar and Rai, 1990). An analysis of 47 geographic popula ions of Ae. alb pictus from 18 coun ries showed a 2.5-fold varia ion in DNA amoun s, ranging from 0.62 pg in he Koh Samui popula ion from Thailand o 1.66 pg in a popula ion from Hous on, Texas recen ly in roduced o he con inen al Uni ed S a es (Table 1.3). Fur hermore, ex ensive varia ion exis ed among and wi hin popula ions from con iguous geographic loca ions. For example, he haploid DNA amoun s of wo popula ions each of Ae. alb pictus from Singapore (Ken Ridge and Amoy) and Brazil (San a Tereza and Cariacica) were significan ly differen from each o her. Six Duncan’s groupings of genome sizes were observed among he 37 popula ions of Ae. alb pictus s udied by Kumar and Rai (1990). Genome size was independen of geographic origin in he various popula ions examined. For example, 12 popula ions from he Uni ed S a es belonged o four groupings ha also con ained popula ions from o her geographic areas (Kumar and Rai, 1990). Using DNA-reassocia ion kine ics, Black and Rai (1988) showed ha

15

1. Mosquito Genomes: Structure, Organization, and Evolution Table 1.3. Haploid Genome Size (Picogram DNA) in 47 Geographic Popula ions of Aedes alb pictus from 18 Coun ries

Genus

Species

Aedes

alb pictus Ge graphic p pulati ns Koh Samui, Thailand Korea Tananareve, Madagascar Sri Lanka Pon ianak, Indonesia Ndo Ndo Creek, Solomon Island Tananareve, Madagascar Hong Kong Mauri ius Saigon, Vie nam Taipei, Taiwan Malaysia Ger ak Sanguul Malaysia Perak Road Sabah Singap re Ken Ridge Amoy India Calcu a Kolar Hardwar Delhi Pune Shalimar Bagh Hawaii Makiki Oahu Manoa Japan Nagasaki Saga Kabeshima Ebina Seburi Zama Tokyo Brazil Cariacica San a Tereza


References

0.62 0.69 0.78 0.92 1.07 1.12 1.15 1.26 1.32 1.36 1.48

⫾ 0.02 ⫾ 0.03 ⫾ 0.03 ⫾ 0.05 ⫾ 0.044 ⫾ 0.06 ⫾ 0.026 ⫾ 0.026 ⫾ 0.035 ⫾ 0.04 ⫾ 0.05

Kumar and Rai, 1990 Kumar and Rai, 1990 Rao and Rai, 1987b Kumar and Rai, 1990 Rao and Rai, 1987b Kumar and Rai, 1990 Kumar and Rai, 1990 Rao and Rai, 1987b Rao and Rai, 1987b Kumar and Rai, 1990 Kumar and Rai, 1990

0.64 0.81 0.83 0.85

⫾ 0.02 ⫾ 0.03 ⫾ 0.03 ⫾ 0.02

Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990

0.75 ⫾ 0.02 1.29 ⫾ 0.06

Kumar and Rai, 1990 Kumar and Rai, 1990

0.86 0.94 0.96 1.02 1.07 1.42

⫾ 0.03 ⫾ 0.025 ⫾ 0.02 ⫾ 0.008 ⫾ 0.62 ⫾ 0.05

Rao and Rai, 1987b Rao and Rai, 1987b Kumar and Rai, 1990 Rao and Rai, 1987b Rao and Rai, 1987b Kumar and Rai, 1990

0.75 ⫾ 0.03 1.24 ⫾ 0.032 1.47 ⫾ 0.06

Kumar and Rai, 1990 Rao and Rai, 1987b Kumar and Rai, 1990

0.76 0.80 0.82 0.85 1.11 1.16 1.29

Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Rao and Rai, 1987b

⫾ 0.03 ⫾ 0.02 ⫾ 0.03 ⫾ 0.03 ⫾ 0.04 ⫾ 0.05 ⫾ 0.032

0.98 ⫾ 0.04 1.18 ⫾ 0.02

Kumar and Rai, 1990 Kumar and Rai, 1990 c ntinues

16


Table 1.3. (c ntinued)

Genus Aedes

Species alb pictus United States Chambers Coun y, TX Chicago, IL Jacksonville, FL Memphis, IN Hous on, TX Indianapolis, IN Milford, DE New Orleans, LA Brazoria Coun y, TX Evansville, IN Savannah, GA Hous on 61, TX


1.03 1.11 1.13 1.23 1.33 1.34 1.46 1.48 1.50 1.59 1.65 1.66

⫾ 0.03 ⫾ 0.09 ⫾ 0.10 ⫾ 0.13 ⫾ 0.08 ⫾ 0.09 ⫾ 0.05 ⫾ 0.26 ⫾ 0.05 ⫾ 0.11 ⫾ 0.07 ⫾ 0.08

References

Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990 Kumar and Rai, 1990

he in raspecific varia ion in DNA con en in wo s rains of Ae. alb pictus was due mainly o highly repe i ive DNA sequences. Fur her, MacLain et al. (1987) showed ha popula ions of Ae. alb pictus ha were significan ly differen in DNA con en also varied in he frequency of differen classes of highly repe iive DNA. Thus, he varia ion in DNA con en among popula ions of Ae. alb pictus appears o be due mainly o repe i ive DNA sequences ha are under rapid change. This sugges s ha he amoun of repe i ive DNA is dynamic in Ae. alb pictus and probably o her mosqui o species. Significan varia ion in haploid DNA con en has been observed among inver ebra es (Papeschi, 1991; Palmer and Pe i pierre, 1996), ver ebra es (Walker et al., 1991), and plan s (Flavell et al., 1974; Jasienski and Bazzaz, 1995). In addi ion, several s udies have shown a direc correla ion of genome size wi h nuclear and cellular surfaces and volumes (Walker et al., 1991). Genome size also varies wi h he dura ion of he cell cycle (Benne , 1987); he life his ory, phenology, and dis ribu ion of species (Macgillivray and Grime, 1995); he dura ion of genera ion ime (Ferrari and Rai, 1989; Flavell et al., 1974; Jasienski and Bazzaz, 1995); and he body size (Palmer and Pe i pierre, 1996). Cavalier-Smi h (1985b) proposed ha varia ion in DNA amoun is subjec o na ural selec ion and plays an adap ive role. Al hough exac func ion(s) of highly repe i ive DNA have long been deba ed, biologically significan roles in various species have been ascribed. For example, i has been sugges ed ha he propor ion of repe i ive DNA in Plasm dium berghei maybe direc ly correla ed wi h mosqui o infec ivi y (Birago et al., 1982). A s rain con aining 18% repe i ive DNA produced viable game o-


17

cy es in mice, while ano her s rain wi h 3% repe i ive DNA did no . Also, repe i ive elemen s of he bac erium Myc plasma genitalium con ribu e o he an igenic varia ion in pro eins of he MgPa cellular adhesion operon (Pe erson et al., 1995). Wi hin species, Benne and Benne (1992) sugges ed ha smaller genomes are associa ed wi h popula ions ha occur in s ressful environmen s where rapid developmen , a shor lifespan, and a high reproduc ive ra e are favored (“r-selec ed”), while larger genomes are found mos of en in popula ions in environmen s ha favor slower developmen imes, increased longevi y, delayed reproduc ion, and of en a lower fecundi y (“k-selec ed”).

VII. HETEROCHROMATIN: LOCALIZATION, VARIATION, AND EXPRESSION The applica ion of Giemsa C-banding and o her banding procedures o soma ic and meio ic chromosomes has provided impor an insigh s in o linear differenia ion and evolu ion of chromosomes in Culicidae. S udies have been comple ed in 36 species belonging o seven-genera of Culicinae (Aedes, Mans nia, Culiseta, Armigeres, Sabethes, Wye myia, and T x rhynchites) including 28 Aedes species (Mo ara and Rai, 1977, 1978; Rao and Rai, 1987a), hree species of Culex (Mo ara 1982), and several An pheles species (Ga i et al., 1977; Baimai, 1988; Baimai et al., 1993a, b, 1995, 1996; Marchi and Mezzano e, 1990). Cbanding pa erns were also s udied in represen a ive species of Tipulidae, Dixidae, and Cha b ridae in order o examine how chromosomes have evolved in hese families (Rao and Rai, 1987a). These s udies es ablished ha he dis ribu ion of he erochroma in is markedly differen in anopheline and culicine mosqui oes, par icularly in he he eromorphic sex chromosomes. All species showed he presence of he erochroma in around he cen romeres of he au osomes, al hough here are of en large in er-and in raspecific differences in amoun s of he same. Using differen banding echniques, hree ypes of he erochroma in were iden ified on he basis of s aining charac eris ics in he pericen romeric regions in he Culicini species Culiseta l ngiare lata (Mezzano e et al., 1979; Marchi and Mezzano e, 1988). In addi ion o cen rome ric bands, he au osomes in species such as Ae. bahamensis (Rao and Rai, 1987a) and he long arms of he sex chromosomes in An. atr parvus (Fraccaro et al., 1976) possess elomeric C-bands also. The organiza ion of he erochroma in is markedly differen in he wo homologues of he sex chromosome pair in mos Aedes species as well as be ween anopheline and culicine mosqui oes. Mo ara and Rai (1977, 1978) repor ed wo dis inc ypes, cons i u ive and facul a ive he erochroma in, in Aedes mosqui oes. The former is presen around he cen romere region of all hree chromosome pairs and he la er in an in ers i ial posi ion on one of he arms of he female-de ermining (m) chromosome in mos Aedes species (Figure

18


1.2). The in ercalary band is loca ed proximal o he cen romere in Ae. annandalei and in elomeric posi ion on bo h he male- and female-de ermining chromosomes in Ae. vittatus. Ae. mascarensis (Figure 1.2), Ae. katherinesis, Ae. excrucians, Ae. stimulans, Ae. cinereus, and Ae. triseriatus lack he in ercalary band (Figure 1.3). The fac ha hese species belong o hree differen subgen-

Figure 1.2. Schema ic represen a ion of C-banding karyo ypes in Steg myia mosqui oes. Af er Mo ara and Rai (1978).

19 Figure 1.3. Chromosome number, morphology, and C-banding pa erns in some genera of Nema ocerous (Dip era: Nema ocera) families. Af er Rao and Rai (1987a).

20


era sugges s ha he erochroma iniza ion of par icular segmen s is species-specific. The male-de ermining chromosome (M) in Ae. aegypti lacks even he cen romeric he erochroma in (Figure 1.2). The cons i u ive and facul a ive he erochroma in replica e a differen imes in he cell cycle (Marchi and Rai, 1986). Unlike in Aedes, he in ercalary he erochroma in is no presen on he female-de ermining chromosome in Armigeres subalbatus or T x rhynchites splendens bu on an arm of one of he au osomes (chromosome II in he former and chromosome III in he la er) (Figure 1.3 and Rao and Rai, 1987a). Rai et al. (1982) sugges ed a possible evolu ionary deriva ion of he various he erochroma in pa erns observed in Aedes species. The overall pa erns observed among various genera (Figure 1.3) are also sugges ive of he role chromosome repa erning played in genome evolu ion. The expression of he in ercalary C-band on he sex chromosome in a par icular species varies as a func ion of he gene ic background in which i is placed. This was revealed by Giemsa C-banding of he F1 hybrids and progeny of cer ain backcrosses be ween wo closely rela ed species, Ae. aegypti and Ae. mascarensis (Mo ara and Rai, 1977). Crosses involving Ae. aegypti females and Ae. mascarensis males produced F1 progeny in which he expression of he dis al in ercalary C-band on he female-de ermining (M) chromosome of Ae. aegypti was suppressed in bo h he males and he females (Figure 1.4a). This indica ed ha he dis al region of he female-de ermining (M) chromosome represen ed by he he erochroma ic C-band was derepressed and ha i became euchroma ic. When F1 males from his cross were backcrossed o Ae. aegypti females, a propor ion of he sons developed in o in ersexes and differed from normal males in heir C-banding pa ern (Figure 1.4c). Thus, i was possible o rela e abnormal sexual developmen of adul males in he backcross progeny o a selec ive ac iva ion of a discre e chromosomal locus on he male-de ermining chromosome of heir fa hers (Mo ara and Rai, 1977). Reciprocal crosses (Ae. mascarensis females ⫻ Ae. aegypti males) gave expec ed resul s (Figure 1.4b,d). The reversible gene ic regula ion of he facul a ive C-band apparen ly represen s selec ive con rol of a chromosomal segmen of one species (e.g., Ae. aegypti) hrough gene ic in erac ion wi h ano her, Ae. mascarensis (Mo ara and Rai, 1977). Such gene ic regula ion, which was also observed in progeny of crosses involving Ae. katherinensis and Ae. hebrideus (Rao and Rai, 1987a), may be widespread among aedine mosqui oes and may help pro ec species in egri y. In anopheline species, he he eromorphic chromosomes of en show ex ensive differences in he amoun , dis ribu ion, and ypes of he erochroma in. The Y chromosome may be en irely he erochroma ic in mos An pheles species while he X chromosomes may be he erochroma ic from less han one-half o grea er han hree-four hs of heir leng h, even among closely rela ed species. Fur hermore, several of hese species — for example, he Hyrcanus group (sub-


21

Figure 1.4 a– d. Diagramma ic represen a ions of Ae aegypti and A. mascarensis C-banding pa erns: (a and b) Summary of he expec ed and observed resul s in he F1 in reciprocal crosses and (c and d) among he backcross progeny. Because chromosomes II and III show expec ed banding pa erns in all cases, hey are excluded from he F1 and BC1 drawings ( o enhance readabili y). Af er Mo ara and Rai (1977).

genus An pheles), he macula us group (subgenus Cellia), and o hers — are polymorphic for he size of he X chromosome and for he amoun of he erochroma in (Baimai et al., 1993a,b, 1995, 1996). Such differences are diagnos ic and allow unambiguous iden ifica ion of species whose poly ene chromosome-banding pa ern is vir ually homosequen ial (Green et al., 1985). Presumably, differen densi ies of he Giemsa bands on he X and he Y chromosomes in hese species reflec differen ypes of cons i u ive he erochroma in (Baimai, 1988). Four sa elli e DNAs defined on Hoechs 3325S – CsCl densi y gradien s are similarly reflec ive of he presence of differen ypes of he erochroma in in he An. stephensi genome (Redfern, 1981). In conclusion, here seems li le doub ha changes in amoun s, ypes, and loca ions of he erochroma in are associa ed wi h mosqui o specia ion, par icularly in he subfamily Anophelinae and Culicinae. In situ chromosomal localiza ion of four cloned repe i ive DNA fragmen s (H-76, 61, H-19, and H-85) indica ed ha hey are dispersed hroughou he leng hs of he hree pairs of chromosomes in all Aedes species examined

22


(Kumar and Rai, 1991a,b). Al hough he sequences homologous o hese cloned repe i ive DNA fragmen s are presen in o her culicid genera, Haemag gus equinus, Tripter ideres bambusa, and An pheles quadrimaculatus, significan differences in heir abundance and dis ribu ion were observed (Kumar and Rai, 1991a,b). Unlike such dispersed pa ern in Aedes, Sa elli e 1 was localized o he he erochroma ic arms of he X and he Y chromosomes and he cen romere regions of chromosome 3 in An. stephensi (Redfern, 1981). Similarly, a highly repe i ive DNA clone isola ed from Ae. alb pictus (H115) was shown o be loca ed a an in ercalary posi ion on chromosome 1 in all Aedes species examined (Kumar and Rai, 1992). Sou hern hybridiza ion of his DNA fragmen wi h genomic DNA of An. quadrimaculatus, on he o her hand, showed a dispersed pa ern. An impor an difference in chromosome organiza ion wi h regard o he erochroma in dis ribu ion be ween anophelines and mos culicines may be cri ical in de ermining whe her poly ene chromosomes can be easily mapped. There is generally a good resolu ion of individual bands on each of he euchroma ic chromosome arms in he anophelines, while culicines are largely refracory o his ype of analysis. In anophelines, apparen ly much of he he erochroma in is clus ered around he cen romeres of each of he hree pairs of chromosomes, resul ing in he forma ion of a chromocen er in poly ene chromosome prepara ions. Of he eigh mosqui o genera in which poly ene chromosome morphology has been s udied, An pheles alone possesses a chromocener. All o her genera (Aedes, Culex, Mans nia, T x rhynchites, Orth p d myia, Wye myia, and Sabethes) lack a dis inc chromocen er (Sharma et al., 1979; Dennho¨fer, 1968; Tewfik and Barr, 1974; Verma et al., 1987; Chaudhry, 1972; Whi e, 1980; Muns ermann et al., 1985; Moeur and Is ock, 1982; Muns ermann and Marchi, 1986). Never heless, Orth p d myia pulcripalpas (Muns ermann et al., 1985) and Sabathes cyaneus (Muns ermann and Marchi, 1986) have yielded well-resolved poly ene chromosomes. This sugges s ha hese axa have longperiod in erspersion and may be more basal in culicid evolu ion. Fur hermore, as indica ed earlier, repe i ive DNA cons i u es a large propor ion of he genome in culicine mosqui oes. Since his DNA undergoes la e replica ion during he S period (Marchi and Rai, 1986), such dispersed sequences may conceivably ac like microchromocen ers, hereby preven ing effec ive separa ion of individual chromosomes. In examining karyo ypes and C-banding pa erns in species of Tipulidae, Dixidae, Cha b ridae, and Culicidae, Rao and Rai (1987a) concluded ha Culicidae arose from a chaoborid Mochlonyx-like ances or and ha he Anophelinae and Culicinae evolved along separa e lineages from a common ancesral s ock (Figure 1.5). The Chagasia karyo ype was considered o be primi ive for Anophelinae, while he Toxorhynchi es karyo ype was considered primi ive for Culicinae. The cladis ic analyses discussed earlier, suppor his proposal.


23

Figure 1.5. Proposed chromosomal evolu ion in some nema ocerous axa. Arabic numerals represen chromosomes; chromosomes no drawn o scale. Afer Rao and Rai (1987a).

VIII. SATURATED LINKAGE MAPS GENERATED THROUGH MULTIPOINT MAPPING Over he las decade, a new paradigm has emerged in gene ic linkage mapping, where hundreds or housands of markers are mapped simul aneously in one or a few crosses. Molecular gene ic me hods ha allow for amplifica ion of many loci from small amoun s of genomic DNA have been ins rumen al in he applica ion

24


of his echnology o small ar hropods. Recen echniques make i possible o analyze many regions of a genome simul aneously. All of hese incorpora e he polymerase chain reac ion (PCR) for he amplifica ion of markers from small amoun s of genomic empla e DNA. Random amplified polymorphic DNA (RAPD) markers and arbi rarily primed (AP) markers are amplified wi h PCR using shor oligonucleo ide primers wi h arbi rary sequence (Williams et al., 1990; Welsh and McClelland, 1990). PCR is also used for amplifica ion of genomic regions for analysis by res ric ion enzymes (Severson et al., 1993) or single-s rand conforma ion polymorphism (SSCP) analysis (Ori a et al., 1989). The discovery of abundan microsa elli es in eukaryo ic genomes. (Weber, 1990; Beckmann and Soller, 1989, 1990) has provided a ple hora of markers for mapping many eukaryo ic genomes. In addi ion o hese molecular gene ic echniques, he developmen of sof ware for maximum-likelihood es ima ion of linkage rela ionships among mul iple cosegrega ing markers (Lander et al., 1987; S am, 1993) has been ins rumen al in allowing mul ipoin mapping wi h a varie y of molecular markers. This echnology has also allowed a number of differen labora ories o cons ruc mul ipoin linkage maps of en ire mosqui o genomes. This was firs accomplished by Severson et al. (1993), who cons ruc ed a linkage map of Ae. aegypti using 50 RFLP markers from 42 random cDNA clones, 3 random genomic clones, and 5 cDNAs of known origin. The leng hs of chromosomes I, II, and III were 49, 60, and 56 cM, respec ively (165 cM o al). An olin et al. (1996) cons ruc ed a linkage map of Ae. aegypti using SSCP analysis of 94 RAPD markers. The leng hs of linkage groups I, II, and III were 52, 58, and 57 cM, respec ively (168 cM o al), remarkably similar o he cDNA map. Mu ebi et al. (1997) cons ruc ed a linkage map of Ae. alb pictus using SSCP analysis of 68 RAPD markers. The leng hs of chromosomes I, II, and III were 54, 67, and 104 cM, respec ively (225 cM o al). Severson et al. (1995) showed ha cDNA markers are colinear in Ae. aegypti and Ae. alb pictus. These s udies using molecular markers sugges a large (57 cM) increase in he recombina ional size of he Ae. alb pictus genome. Fur hermore, mos of his appears o be due o increased recombina ion on chromosome III. I is uncer ain whe her hese differences are due o varia ions in DNA amoun or o differences in he dis ribu ion and frequency of chiasma a on chromosome III of he wo species. Aedes species are known o vary widely in chiasma a dis ribu ion and frequency (Dev and Rai, 1984; Sherron and Rai, 1984). The leng hs of he hree linkage maps involving morphological and enzyme loci calcula ed from observed chiasma a frequencies were 62, 86, and 80 cM, respec ively ( o al 228 cM in Ae. aegypti) (Muns ermann and Craig, 1979). However, large s re ches of all hree linkage maps were devoid of any markers, par icularly on linkage group III on which he 17 observed markers were clus ered in a 44-uni map, while he chiasma a-based model predic s an 80-uni map.


25

More recen ly, a linkage map of Armigeres subalbatus has been cons ruc ed using 26 RFLP markers involving cDNA clones from Ae. aegyti. The overall leng hs of linkage groups I, II, and III were 51, 72, 58 cM, respec ively (181 cM o al), and, excep for one marker, he linear order was he same as in Ae. aegypti (Ferdig et al., 1998). A similar RFLP linkage map has been cons ruc ed for Culex pipiens using 21 cDNA clones from Ae. aegypti (Mori et al., 1998). The o al map spans 165.8 cM. The linkage maps for chromosomes I, II, and III of Cx. pipiens were 7.1, 80.4, and 78.3 cM, respec ively. However, based on he rela ively small number of markers used, hese es ima es do no accura ely coincide wi h leng hs of corresponding linkage maps of o her culicine species, par icularly for linkage group I. This necessi a es work wi h addi ional molecular markers. The compara ive linkage maps for chromosomes II and III in Cx. pipiens and Ae. aegypti reflec whole-arm ransloca ions (Mori et al., 1998). Zheng et al. (1996) mapped 131 microsa elli e markers in An pheles gambiae. Chromosomes I, II, and II were, respec ively, 49, 72, and 94 cM in leng h (215 cM o al). In egra ion of RAPD markers in o his map increased he overall densi y of markers wi hou affec ing he overall leng h (Dimopoulos et al., 1996). I is ins ruc ive o consider linkage map size, an indica ion of he amoun of recombina ion on individual chromosomes, rela ive o he genome sizes discussed earlier in his chap er. The observed linkage map sizes are 165 cM in Ae. aegypti, 225 cM in Ae. alb pictus, 166 cM in Cx. pipiens, 181 cM in Ar. subalbatus, and 215 cM in An. gambiae. These do no correspond in any way o he genome sizes of 0.83, 0.86 – 1.32, 0.54 – 1.02, 1.12 and 0.27 pg/haploid genome, respec ively, in hese species. The rela ionship of physical o recombina ion dis ance is approxima ely 3 – 6 Mb DNA/cM in Ae. aegypti, Ae. alb pictus, and he wo o her culicine species s udied, and 1.2 Mb DNA/cM in An. gambiae (Table 1.4). Thus here appears o be li le rela ionship be ween genome size and recombina ion frequency. The frequency of recombina ions remains high in An. gambiae despi e i s having a genome size one- hird o onefif h he size of he Aedes and o her culicine species genomes. DNA reassociaion kine ic analysis has shown ha he amoun of repe i ive DNA sequences in culicine species is generally much higher han ha in An pheles species (Table 1.4; Black and Rai, 1988; Warren and Cramp on, 1991; Besansky and Powell, 1992). Since recombina ion is considerably res ric ed in chromosomal regions rich in repea ed DNA sequences (Charleswor h et al., 1986), overall An pheles would be expec ed o show higher recombina ion ra es. Also, his predic s a closer rela ionship be ween physical and linkage maps in An pheles and a higher likelihood of success in mapped-based posi ional cloning of genes ha con rol he pheno ype of a charac er under s udy. Fur hermore, he fac ha he sizes of he linkage maps do no vary by more han 60 cM in hese

26


Table 1.4. Comparison of Linkage Maps (To al cM), DNA Amoun s (pg), Propor ions of Unique/Repe i ive DNA Sequences, and Ra io of Haploid DNA Amoun s o Linkage Map Size (cM) DNA Species Culicinae Ae. aegypti Ae. alb pictus Calcutta Mauritius Ar. subalbatus Cx. pipiens Anophelinae An. gambiae An. quadrimaculatus

Linkage map ( o al cM)

% Repe i ive

Haploid DNA/cM (Mb)

To al (pg)

bp

% Unique

0.83a

8.0 ⫻ 108

60d

32d

4.85

0.86a 1.32a 1.12a 1.02a 0.54a

8.3 1.3 1.1 1.0 5.2

⫻ 108 ⫻ 109 ⫻ 109 ⫻ 109 ⫻ 109

36c 33c

54c 57c

22c

67c

3.7 5.8 6.1 6.0 3.1

0.27a 0.24a

2.6 ⫻ 108 2.3 ⫻ 108

61b 80c

33b 16c

1.2 1.1

165 225

181 166

215

Da a from Tables 1.2 and 1.3. Da a from Besansky and Powell, 1992. cDa a from Black and Rai, 1988. dDa a from Warren and Cramp on, 1991. a b

hree species sugges s ha he number of chiasma a have remained rela ively cons an despi e increases in genome size and chromosome leng h in he evolu ion of Culicidae.

IX. SUMMARY A grea deal of informa ion has been accumula ed on chromosome numbers and he erochroma in dis ribu ion as well as on genome size and organiza ion in he mosqui o family Culicidae. A number of rends in genome evolu ion emerge when hese da a are reviewed in ligh of recen cladis ic phylogenies of Culicidae and i s sis er families. Anophelinae have he eromorphic sex chromosomes and a small genome size, and repe i ive elemen s are dis ribu ed in a long-period in erspersion pa ern. In con ras , Culicinae have homomorphic sex chromosomes, and repe i ive DNA is organized in a shor -period in erspersion pa ern. There has been a general increase in genome size during he evolu ion of culicine ribes. The organiza ion of he ances ral culicid genome remains uncerain awai ing s udies on genome organiza ion in Chaoboridae – Core hrellidae axa.


27

The mos parsimonious hypo hesis for he evolu ion of sex chromosomes and genome organiza ion in Culicidae would be ha homomorphic sex chromosomes and a long-period in erspersion pa ern was ances ral in lineages leading o Toxorhynchi inae and Culcinae. Larger genomes developed in subsequen culicine lineages hrough accumula ion of shor -period in erspersed repe i ive elemen s. He eromorphic sex chromosomes evolved early in he evolu ion of Anophelinae, and a long-period in erspersion pa ern was re ained. The al erna ive scenario proposed by Rao and Rai (1987a) is ha Culicidae arose from a chaoborid M chl nyx-like ances or wi h he eromorphic sex chromosomes and possibly shor -period in erspersion. This scenario would require he loss of he eromorphic sex chromosomes in he lineage leading o Toxorhynchi inae and Culicinae and he “shedding” of repe i ive elemen s in he lineage leading o Anophelinae. Several in eres ing pa erns have emerged from s udies of C-banding, and he dis ribu ion of he erochroma in in Culicidae and phylogenies derived from hese s udies are suppor ed by he modern cladis ic analyses. Recen in ensive mul ipoin linkage map s udies sugges ha recombina ion frequencies per genome have remained rela ively cons an over he course of culicid evolu ion such ha Anophelinae, wi h a rela ively small genome size, has a linkage map of similar size o Aedini. As a consequence, axa in Anophelinae have higher amoun s of recombina ion per haploid genome size han Culicinae. Al hough several key ques ions have ye o be addressed, he Culicidae remain one of he bes -s udied sys ems of genome evolu ion in animals.

Acknowledgments The original work in he senior au hor’s labora ory included in his chap er was suppor ed by NIH Research Gran 5R01 AI 21443, by NIH Training Gran 5T30 AI 07030, and by he Universi y of No re Dame. We hank Doc ors Nora Besansky and David Severson for a cri ical review and for making several sugges ions for he improvemen of he manuscrip . We also express our sincere hanks o Ka hleen Merz for her invaluable help in keyboarding and for several revisions of he manuscrip .

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Seeing the Light: News in Neurospora Blue Light Signal Transduction H. Linden Lehrs uhl fur Physiologie und Biochemie der Pflanzen Universi a Kons anz D-78434 Kons anz, Germany

P. Ballario Dipar imen o di Gene ica e Biologia Molecolare, Cen ro di S udio per gli Acidi Nucleici Universi a` di Roma “La Sapienza” 00185 Roma, I aly

G. Arpaia, and G. Macino* Is i u o Pas eur Fondazione Cenci Bologne i Dipar imen o di Bio ecnologie Cellulari, Sezione di Gene ica Molecolare Universi a` di Roma “La Sapienza” 00161 Roma, I aly

I. In roduc ion II. The Percep ion of Ligh in eurospora A. eurospora Perceives Ligh Only in he Ul raviole /Blue Ligh Range B. Blue Ligh Ac iva es Gene Expression C. eurospora Is Capable of Adap ing o Differen Ligh In ensiies D. Pro ein Kinase C Is Involved in he Pho oadap a ion Process of . crassa * To whom correspondence should be addressed. Advances in Genetics, Vol. 41 Copyrigh 1999 by Academic Press All righ s of reproduc ion in any form reserved. 0065-2660/99 $30.00

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III. The In erplay of Blue Ligh and O her Regula ory Pa hways in eurospora IV. Mu a ional Analysis of Blue Ligh Signal Transduc ion in eurospora V. The eurospora Blue Ligh Regula ory Pro eins WC-1 and WC-2 A. The WC-1 and WC-2 Pro eins Are Pu a ive Transcrip ion Fac ors Involved in Blue-Ligh -Induced Transcrip ional Conrol B. WC-1 and WC-2 Domains for Dimeriza ion and Signal Transduc ion C. How Do WC-1 and WC-2 Func ion in eurospora Blue Ligh Signaling? VI. Concluding Remarks Acknowledgmen s References

I. INTRODUCTION Ligh is one of he mos impor an environmen al fac ors for plan s, algae, bac eria, and fungi and regula es developmen al and physiological processes. Plan s are able o perceive ligh over he whole sunligh spec rum, and percepion of ligh is carried ou by a leas hree differen families of pho orecep ors: he phy ochromes (red and far-red ligh absorp ion), ul raviole recep or(s), and blue ligh pho orecep or(s) (Deng, 1994). In our a emp o unravel he mys erious process of blue ligh percep ion and he ransduc ion of he ligh signal, we are using he ascomyce e eurospora crassa, which has been proven o be an ideal organism for pho obiological, biochemical, and gene ic s udies. In addi ion o he more general advan ageous fea ures of eurospora, such as a small eukaryo ic genome (es ima ed as 47 megabases; Orbach et al., 1988), fas grow h, s raigh forward gene ics, and fas ransforma ion wi h foreign DNA, here are more specific reasons o use . crassa as a model organism o s udy ligh regula ed processes. In con ras o higher plan s, . crassa is capable of sensing ligh only in he blue ligh range, and blue ligh is he s imulus for several differen processes. During he asexual life cycle, mycelial caro enoid biosyn hesis (Harding and Turner, 1981), forma ion of vege a ive spores (macroconidia)(Klemm and Ninnemann, 1978; Lau er, 1996), and circadian rhy hmici y (Sargen and Briggs, 1967) are regula ed by blue ligh . In addi ion, blue ligh responses such as forma ion of pro operi hecia (Degli-Innocen i et al., 1983) and he pho o ropism of peri hecial beaks (Harding and Melles, 1983), have been observed during he eurospora sexual life cycle. Regula ion of hese processes seems o occur mainly a he level of gene expression, and o da e

2. Seeing the Light: News in Neurospora Blue Light Signal Transduction

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several blue-ligh -regula ed genes have been cloned. Fur hermore, many eurospora mu an s ha seem o be impaired in ligh percep ion and/or ransduc ion of he ligh signal have been isola ed and charac erized. During he pas decades, a weal h of da a have been published regarding he eurospora blue ligh responses, blue-ligh -regula ed genes, and he pu a ive na ure of he blue ligh pho orecep or and componen s of he signal ransduc ion chain ha have only recen ly been reviewed in de ail (Lau er, 1996; Linden et al., 1997a; Ballario and Macino, 1997). The purpose of he presen review is o discuss he recen progress ha has been made in he cloning and charac eriza ion of wo coopera ing par ners of he eurospora blue ligh signal ransduc ion chain. Fur hermore, we ou line some new and lessknown aspec s of blue ligh regula ion in . crassa.

II. THE PERCEPTION OF LIGHT IN Neurospora A. Neurospora perceives light only in the ultraviolet/blue light range Several ac ion spec ra for differen eurospora blue ligh responses have been published. An ac ion spec rum reflec s he waveleng h dependency of he sensi ivi y for a specific response. Da a from DeFabo et al. (1976) for ligh -regula ed biosyn hesis of caro enoids in eurospora and from Sargen and Briggs (1967) for he pho osuppression of conidial banding clearly demons ra ed he sensi ivi y of he eurospora pho orecep or(s) no only for blue ligh bu also for UV ligh . Their resul s also revealed ha eurospora is “blind” oward ligh beyond 520 nm. Schro (1980, 1981) repor ed fluence response curves for ligh -induced caro enoid biosyn hesis in . crassa. A sa ura ion of ligh -induced caroenogenesis was observed when he mycelia were exposed o fluence ra es beyond 0.3 W m⫺2 for up o 16 min. Fur hermore, he fluence response was shown o be biphasic; an ex ension of he illumina ion ime beyond 16 min resul ed in a second increase in he amoun of caro enoids syn hesized during he subsequen dark period. A emporary insensi ivi y oward ligh be ween he firs and he second phase of he biphasic fluence response curve was described (Schro , 1981). A period of 2 h af er a firs illumina ion was found o be necessary for res oring maximum compe ence for a second ligh induc ion. Schro sugges ed ha he pho orecep or and/or elemen s of he signal ransducion chain become deple ed during he firs phase. Consequen ly, such a period of res ora ion may be necessary before he sensi ivi y oward ligh is recovered. Corrochano et al. (1995) also repor ed a wo-phase s imulus – response curve. They prepared a ransla ional fusion of he ligh inducible con-10 promo er and he Escherichia coli lacZ gene. Af er ransforma ion of eurospora and pho oinduc ion of he mycelia, he ␤-galac osidase ac ivi y was de ermined. Following

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a ligh induc ion of 1 o 15 min, a firs induc ion of ␤-galac osidase ac ivi y reached a pla eau 1 min af er onse of ligh . Upon fur her illumina ion for 30 min, a second response ha doubled he ␤-galac osidase ac ivi y was observed. In eres ingly, his biphasic response has never been observed on he level of ranscrip ion. All he blue-ligh -regula ed genes isola ed oday show a one-phase response curve only. Therefore, a pos ranscrip ional even may be responsible for he observed biphasic response on he level of enzyme ac ivi y.

B. Blue light activates gene expression Many ligh -regula ed genes have been cloned in eurospora o da e. When dark-grown mycelia are illumina ed wi h cons an ligh , mos of he ligh regula ed genes show a ransien expression pa ern (Figures 2.1A and 2.1B). The only excep ion o his rule is he eurospora gene frequency (frq), which encodes a cen ral componen of he circadian clock (Loros, 1995). The mRNA of frequency shows a fas increase in response o ligh and remains eleva ed in comparison o he levels observed in cons an darkness (Figure 2.1C; Cros hwai e et al., 1995). I is impor an o no e ha Figure 2.1 cons i u es a schema ic represen a ion only and does no ake in o accoun he quan i a ive differences in rela ive mRNA s eady-s a e levels. For example, a 90-fold increase of some blue-ligh -inducible mRNAs has been described o occur af er ligh induc ion, whereas o her genes show a much lower induc ion (3-fold) wi h respec o heir dark levels (Sommer et al., 1989). Due o heir expression pa ern, he blue-ligh -regula ed genes can be divided in o early ligh -regula ed genes, wi h a mRNA peak a abou 20 – 30 min af er onse of ligh , and la e ligh -regula ed genes, wi h a mRNA peak a 45 – 120 min. The caro enoid biosyn hesis genes al-1, al-2, and al-3 (Baima et al., 1991; Li and Schmidhauser, 1995), he cen ral regula or of blue ligh responses wc-1 (Ballario et al., 1996), he blue-ligh -induced genes bli-3 and bli-4 (Sommer et al., 1989), he conidiaion genes con-5 and con-10 (Lau er and Russo, 1991), and he clock-con rolled genes ccg-4 and ccg-6 (Bell-Pedersen et al., 1996b) are fas ligh -regula ed genes (Figure 2.1A), while during conidia ion he clock-con rolled genes ccg-1, ccg-2 (eas), and ccg-9 (Arpaia et al., 1993, 1995a; Bell-Pedersen et al., 1996b) and al1, al-2, and al-3 reveal a delayed induc ion af er exposure o ligh (Figure 2.1B).

C. Neurospora is capable of adapting to different light intensities A desensi iza ion phenomenon in which a con inuous s imula ion resul s in a decreased sensi ivi y for he s imulus has been described in animal cells and higher plan s (Bowler et al., 1994). Kine ic examina ion of he al-3 mRNA induc ion using differen ligh and dark incuba ion periods, as well as differen ligh in ensi ies, indica ed he presence also of a pho osensory adap a ion mech-


39

Figure 2.1. Schema ic represen a ion of he expression of early ligh -regula ed (A) and la e ligh regula ed genes (B) as well as he expression pa ern of frequency (C) in cons an ligh . The rela ive mRNA levels are given in arbi rary uni s.

anism in eurospora (Macino et al., 1993). No al-3 mRNA was de ec ed af er a con inuous ligh induc ion of 100 min, whereas he al-3 mRNA was found o be inducible by a second ligh pulse af er a firs ligh pulse and a subsequen dark period of 60 min. This dark period of a leas 60 min seemed o be necessary o recover he sensi ivi y of he pho osensory sys em. These resul s comply wi h he emporary insensi ivi y oward ligh for he biosyn hesis of caro enoids af er a firs ligh pulse described by Schro (1981). If he observed ransien expression of he al-3 gene and he emporary insensi ivi y oward ligh are due o an ac ive process of desensi iza ion, irradia on of he mycelia

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wi h a higher ligh in ensi y should overcome he insensi ivi y af er a firs pulse of lower ligh in ensi y. In fac , a second, albei lower, peak of al-3 expression was observed when a higher ligh in ensi y was used for a second ligh pulse, and he expression pa ern was again shown o be ransien (G. Arpaia e al., 1999). These resul s indica e he capaci y of eurospora o adap o differen ligh in ensi ies.

D. Protein kinase C is involved in the photoadaptation process of N. crassa A biochemical approach has been used o inves iga e he phy ochrome signal ransduc ion pa hway in higher plan s (Neuhaus et al., 1993; Bowler et al., 1994). The au hors used specific inhibi ors and agonis s o iden ify signal ransduc ion componen s of he phy ochrome signal ransduc ion chain. A similar approach has been carried ou in our labora ory o inves iga e blue ligh signaling in eurospora (Arpaia et al., 1999). Moni oring of he expression of he al3 gene revealed wo differen ligh -inducible ranscrip s during mycelial grow h and conidia ion, as discussed in de ail la er (Arpaia et al., 1995b). Al hough many differen inhibi ors and agonis s were used in his inves iga ion, only pro ein kinase C-direc ed compounds showed a reproducible effec on he blueligh -regula ed expression of he al-3 gene. During conidia ion, pro ein kinase C inhibi ors comple ely blocked he ligh induc ion of he conidia ion-specific al-3 ranscrip . Normally, he al-3 mycelial mRNA shows a ransien expression pa ern even under cons an ligh condi ions and, af er a ligh induc ion of 2 h, no eleva ed mRNA levels can be observed (Figure 2.1A). Applica ion of proein kinase C inhibi ors during mycelial grow h resul ed in a normal increase in mycelial mRNA up o he ime of maximal expression; however, he mRNA levels remained high for a leas 90 min, indica ing ha pro ein kinase C is a leas in par responsible for he inhibi ion of he ligh signaling cascade leading o desensi iza ion. I would herefore appear ha pro ein kinase C has a dual role in eurospora blue ligh signal ransduc ion. On he one hand, during conidia ion pro ein kinase C media es ligh induc ion of he conidia ion-specific al-3 ranscrip , while on he o her hand, during mycelial grow h pro ein kinase C is responsible for he nega ive con rol of ligh signaling. Consequen ly, he repor ed emporary insensi ivi y ou lined above seems o be due o an ac ive adap a ion mechanism and desensi iza ion of he pho orecep or and/or he signal ransduc ion machinery o a given ligh in ensi y ra her han o a depleion of signal ransduc ion elemen s as was sugges ed previously. Pro ein kinase C represen s he firs componen of his adap a ion machinery in . crassa.


41

III. THE INTERPLAY OF BLUE LIGHT AND OTHER REGULATORY PATHWAYS IN Neurospora As ou lined earlier, here are many differen morphological, developmen al, and physiological processes regula ed by blue ligh in . crassa. Some of hese processes are regula ed by more han one environmen al s imulus a a ime bu seem o be under a complex con rol mechanism. For example, he forma ion of conidia is influenced by glucose limi a ion, carbon dioxide levels, desicca ion, and blue ligh (Springer, 1993; Sokolovsky et al., 1992). The forma ion of pro operi hecia during he sexual cycle of eurospora is influenced by grow h empera ure, ni rogen, oxygen, carbon dioxide, suspensions of heir own conidia, and blue ligh (Degli-Innocen i et al., 1983, 1984a, and references quo ed herein). Consequen ly, gene expression was shown o be under mul iple conrol of numerous ex racellular and in racellular s imuli. This became eviden when iden ical genes were isola ed in differen screening approaches. The bli-7 gene was iden ified in a search for blue-ligh -inducible genes, while ccg-2 was cloned in a screening for clock-con rolled genes (Sommer et al., 1989; Loros et al., 1989). Bo h genes were subsequen ly proven o be allelic and under he con rol of blue ligh and he circadian clock (Bell-Pedersen et al., 1992; Lau er et al., 1992). Similarly, he al-1, al-2, bli-4, ccg-2/bli-7, con-5, and con-10 genes are influenced by blue ligh and he amoun of ni rogen supplemen ed o he grow h media (Sokolovsky et al., 1992). The developmen al process of conidiaion, blue ligh , and he circadian clock all regula e he expression of he conidia ion-specific genes con-6 and con-10 (Lau er and Yanofsky, 1993). Ano her example of a complexly regula ed gene in eurospora is he circadian clock gene frequency (frq). The FRQ pro ein was shown o be par of an au oregula ory nega ive feedback loop in which he FRQ pro ein nega ively regula es i s own expression (Aronson et al., 1994). I was sugges ed ha his nega ive feedback loop represen ed a cen ral componen of he eurospora circadian oscilla or. Fur hermore, i was found ha he frq gene is rapidly induced by ligh and his ligh induc ion was correla ed wi h he rese ing and en rainmen of he circadian clock (Cros hwai e et al., 1995). Using eurospora mu an s ha lack a func ional circadian clock, Arpaia et al. (1993, 1995a) were able o show ha he ligh induc ion of he clock-con rolled genes ccg-1 and ccg-2 is direc and does no depend on he circadian clock. Bell-Pedersen et al. (1996a), in s udying he ccg-2 promo er, also iden ified separa e regula ory cis elemen s for ligh and he circadian clock in accord wi h he findings of Arpaia et al. (1993). On inves iga ion of he regula ion of he al-3 gene by ligh and by developmen al s imuli, wo overlapping ranscrip s of 2.2 and 1.6 kb were iden ified (Arpaia et al., 1995b). The 2.2-kb ranscrip revealed a long, un rans-

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la ed leader sequence and occurred only in conidia ing cul ures. Fur hermore, he 2.2-kb ranscrip was no observed in he wo mu an s, acon-2 and fl, ha were blocked in differen s ages of conidia ion and herefore seemed o represen a conidia ion-specific ranscrip wi h a specific iming of expression. The al-3 conidia ion-specific ranscrip is also ligh inducible and under circadian clock con rol bu only during conidia ion. The expression of he o her ligh -regula ed caro enoid biosyn hesis genes, al-1 and al-2, was also repor ed o be influenced by ligh and conidia ion, al hough no differen ranscrip s were iden ified (Li and Schmidhauser, 1995). Gene expression and promo er s udies sugges ha differen s imuli address dis inc regula ory cis elemen s in promo ers. A leas he blue ligh signal ransduc ion chain seems o be separa ed from o her signal ransduc ion chains. This is indica ed by he fac ha al hough almos every ligh induc ion of eurospora genes is dependen on he wo blue-ligh -regula ory whi e collar pro eins (WC-1 and WC-2), hese pro eins do no seem o in erfere wi h o her signal ransduc ion pa hways excep for a peculiar role in circadian clock con rol proposed by Cros hwai e et al. (1997) and ou lined la er. In view of he recen resul s sugges ing ha bo h wc-1 and wc-2 gene produc s are involved in ranscrip ional ac iva ion, a common mechanism for all ligh -regula ed genes can be presumed. Promo er-specific differences, such as he sequence of he ligh regula ory cis elemen s, heir si ua ion in he promo er, and addi ional ac ion of repressors and/or ranscrip ional ac iva ors, may accoun for he observed differences in gene expression in response o ligh . This gives rise o a complex pa ern of ranscrip ional con rol ha enables . crassa o respond o ex raand in racellular s imuli and o adap o environmen al condi ions.

IV. MUTATIONAL ANALYSIS OF BLUE LIGHT SIGNAL TRANSDUCTION IN Neurospora During he pas decades, a considerable effor has been made in he gene ic dissec ion of he eurospora blue ligh ransduc ion pa hway. Numerous muan s ha seem o affec or par icipa e in blue ligh signaling have been isola ed (for review, see Linden et al., 1997a). The mos impor an and bes examined eurospora mu an s in blue ligh signal ransduc ion isola ed o da e are he white collar mu an s (Perkins et al., 1982; Harding and Shropshire, 1980). The white collar mu an s have pigmen ed conidia, whereas he mycelia are whi e due o a specific deficiency in ligh -induced caro enoid biosyn hesis. This is in con ras o he albino mu an s, which reveal whi e mycelia and whi e conidia due o mu a ions in s ruc ural genes of caro ene biosyn hesis. The wc-1 and wc2 mu an s have been shown o be comple ely “blind” for almos all eurospora blue ligh responses, and mos of he blue-ligh -regula ed genes cloned were


43

repor ed o be no inducible by blue ligh in ei her a wc-1 or wc-2 mu an background. Mos of he mu an s repor ed previously, including several wc mu an alleles, were isola ed ei her by chance or by visual screening wi hou he applica ion of a selec ion sys em (Degli-Innocen i and Russo, 1984b). In order o isola e new regula ory mu an s ha affec blue ligh percepion in . crassa and o carry ou a sa ura ing gene ic dissec ion of “blind” mu an s, a selec ion sys em has been developed (Cara oli et al., 1995). Taking advan age of he fac ha blindness does no seem o be le hal in eurospora, all nonredundan blue ligh signal ransduc ion componen s could be iden ified wi h his selec ion sys em. The ligh -induced al-3 promo er was fused o he coding region of he mtr gene, he produc of which is responsible for he up ake of neu ral alipha ic and aroma ic amino acids in eurospora (S uar et al., 1988). Af er ransforma ion of a mtr⫺/trp⫺ s rain wi h his cons ruc , he resul ing s rain (13-1) became ligh dependen for he up ake of ryp ophan and of a oxic analogue of phenylalanine, p-fluorophenylalanine (Linden et al., 1997c). S rain 13-1 was able o grow on a medium supplemen ed wi h p-fluorophenylalanine in darkness only, as he al-3::mtr gene cons ruc is no expressed under hese condi ions. In con ras , in he ligh he al-3::mtr promo er is induced, causing mtr expression and he up ake of he drug, which inhibi s cell grow h. Therefore, only mu an s impaired in blue ligh percep ion or signal ransduc ion will grow in he ligh in he presence of p-fluorophenylalanine. This selec ion sys em was successfully applied o he isola ion of mu an s ha showed a decreased sensi ivi y for blue-ligh -regula ed processes (Cara oli et al., 1995). The blue-ligh -regula or mu an s blr-1 and blr-2 revealed a pale-orange pheno ype indica ing decreased ligh induc ion of mycelial caro enoid biosyn hesis. Furhermore, he mu an s had decreased s eady-s a e levels of mRNA for all ligh regula ed genes examined. In sexual crossing experimen s, he mu a ions blr-1 and blr-2 fell in o differen segrega ion groups from wc-1 and wc-2. Consequen ly, hey do no represen leaky alleles of he wc loci. In addi ion, he selec ion sys em was used for he isola ion of wc mu an s af er ul raviole mu agenesis (Linden et al., 1997c). In spi e of an exhaus ive screening, no addi onal wc loci o her han wc-1 and wc-2 were isola ed. Therefore, he wc-1 and wc-2 genes seem o be he only nonredundan loci presen in eurospora ha lead o a comple e “blindness” oward ligh . The selec ion sys em jus described has a fur her applica ion: The selec ion s rain 13-1 is unable o ake up aroma ic amino acids in he dark. Af er ul raviole mu agenesis, grow h of 13-1 on ryp ophan in darkness resul ed in he isola ion of mu an s ccb-1 and ccb-2 (for cons i u ive caro enoid biosynhesis), which showed a ligh -grown pheno ype even in he dark (Linden et al., 1997c). In spi e of cons i u ive mycelial caro enoid biosyn hesis in darkness, he mu an s did no show increased mRNA levels of ligh -regula ed genes in he dark. However, an increased expression of some ligh -regula ed genes in

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comparison o he wild ype occurred af er ligh induc ion, indica ing a func ion in blue ligh signaling a leas for ccb-1. I s recessive na ure oge her wi h he specific effec s on ligh induc ion of caro enoid biosyn hesis sugges ed a role for he ccb-1 gene produc as ranscrip ional repressor of some ligh -regula ed genes. The iden ifica ion of dark repression si es in promo ers of ligh -regula ed genes poin ed o he presence of such repressors in eurospora (Kaldenhoff and Russo, 1993). On he o her hand, he ccb-2 gene produc was proposed o ac during he developmen al process of conidia ion.

V. THE Neurospora BLUE LIGHT REGULATORY PROTEINS WC-1 AND WC-2 A. The WC-1 and WC-2 proteins are putative transcription factors involved in blue-light-induced transcriptional control The wc-1 gene was cloned by chromosome walking and complemen a ion of he wc-1 mu an pheno ype (Ballario et al., 1996); inser ional mu agenesis oge her wi h he applica ion of he selec ion sys em for blue ligh regula ory mu an s resul ed in cloning of he wc-2 gene (Linden and Macino, 1997b). The wc-1 gene encodes a 125-kDa pro ein consis ing of 1154 amino acids, whereas he WC-2 pro ein is a smaller polypep ide (57 kDa) wi h 530 amine acids. In a search of pro ein da abases, no overall homology wi h o her pro eins was found for he WC-1 pro ein. In con ras , an overall homology wi h WC-2 was deec ed for ano her fungal pro ein, he so-called palindrome-binding pro ein PBP isola ed from Fusarium solani (EMBL Da a Bank Accession No. U23722), which seems o play a role in he induc ion of he cu inase gene in Fusarium (Li and Kola ukudy, 1995). Al hough he PBP pro ein has no been discussed in conex wi h blue ligh signal ransduc ion, i is in eres ing o no e ha Fusarium shows blue ligh responses and ac ion spec ra similar o hose of eurospora (Rau, 1967). Due o he high overall homology wi h WC-2 (61.3%), we believe ha PBP is he WC-2 homologue from Fusarium. Therefore, he blue ligh regula ory pro ein WC-2 does no seem o be res ric ed o . crassa. I would be in eres ing o know if he same is rue for WC-1. Al hough no overall homology exis s be ween WC-1 and WC-2, he pro eins share several common fea ures (Figure 2.2A). Bo h pro eins con ain a single pu a ive zinc-finger DNA-binding domain ha shows similari y o he DNA-binding domain of GATA fac ors. In con ras o he o her GATA fac ors from ver ebra es ha con ain wo zinc-finger domains wi h 17-amino-acid loops, WC-1 and WC-2 reveal only one pu a ive zinc finger wi h an 18-amino-acid loop. In addi ion, pu a ive ranscrip ional ac iva ion domains have been charac erized in bo h WC-1 and WC-2 pro eins. The amino- erminal region of


45

Figure 2.2. Domain s ruc ure of WC-1 and WC-2 (A) compared wi h he AH-recep or (AHR) and ARNT (B) (according o Burbach et al., 1992). The posi ion of pu a ive PAS domains in WC-1 and WC-2 as well as PAS A and PAS B in he dioxin recep or componen s are indica ed by ha ched boxes. O her regions indica ed include he proline-rich (P-rich) and acidic domains and he region of homology wi h he pho oac ive yellow pro ein (PYP) in WC-2, he pu a ive zinc-finger domain in WC-1 and WC-2, he glu ama e-rich (Q-rich) regions, and pu a ive helix– loop– helix domains in he AH recep or and ARNT as well as he ligand-binding region of he AH recep or.

WC-1 con ains a s re ch of 28 glu amine residues, whereas proline-rich and acidic regions have been found in WC-2. These domains have been described for many o her ranscrip ion fac ors and have been implica ed in ranscrip ional ac iva ion. Pu a ive nuclear arge ing signals may indica e he localiza ion of WC-1 and WC-2 in he nucleus. Bandshif experimen s using ei her WC-1 or WC-2 fusion pro eins have shown ha WC-1 and WC-2 are capable of binding a DNA fragmen of he ligh -regula ed promo er of he caro enoid biosyn hesis gene al-3. I was concluded ha bo h WC-1 and WC-2 accomplish heir func ion in blue ligh signal ransduc ion by binding o promo ers of ligh regula ed genes. This idea was suppor ed by he finding ha several wc-2 mu an alleles show mu a ion or disrup ion of he pu a ive zinc-finger binding domain. The exis ence of a eurospora ligh -responsive elemen (LRE) has been hypo hesized by several au hors; however, a comparison of he 5⬘ ups ream regions of all he ligh -regula ed genes in eurospora has failed o uncover universally conserved cis elemen s (see Lau er, 1996, and Linden et al., 1997a, for a lis of

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known eurospora ligh -regula ed promo ers). A presen , wo sequences, GATA and APE, are he bes candi a es for LREs. Bo h mo ifs are presen in he al-3 promo er fragmen recognized by WC-1 and WC-2 binding domains. On he basis of he compe i ion experimen s repor ed by Ballario et al. (1996) and Linden and Macino (1997b), GATA mo ifs cer ainly form par of he recogni ion si e of he WC pro eins under he experimen al condi ions used; however, he absence of GATA mo ifs in some of he known ligh -regula ed eurospora promo ers weakens i s general func ion. The APE sequence has been shown o be involved in al-3 ligh regula ion by dele ion analysis (Cara oli et al., 1994) and o be able o confer ligh inducibili y o a repor er gene (Cara oli et al., 1995); however, i has been iden ified only in a subse of he ligh regula ed genes, including he caro enoid biosyn hesis gene al-3, he clock con rol gene 2 (ccg-2 or eas or bli-7) (Bell-Pedersen et al., 1996a), and he conidia ion gene 10 (con-10) (Corrocchano et al., 1995). In con-10, he APE sequence does no seem o par icipa e in he ligh regula ion of ranscrip ion (Corrocchano et al., 1995).

B. WC-1 and WC-2 domains for dimerization and signal transduction Addi ional domains were iden ified in bo h WC pro eins ha showed a similari y o a dimeriza ion domain called PAS (for PER-ARNT-SIM). A PAS domain is a region of homology of approxima ely 300 amino acids con aining wo degenera e direc repea s of 50 amino acids, called PAS A and PAS B. The WC-2 PAS domain, however, differs from o her PAS domains repor ed so far, including WC-1, since i does no comprise he usual PAS A and PAS B repea s bu seems o consis of only one PAS repea . This PAS domain is presen in he Drosophila pro ein Period (PER) and o her regula ory pro eins, e.g., in bo h subuni s of he mammalian dioxin recep or AHR (aryl hydrocarbon recep or) and ARNT (aryl hydrocarbon recepor nuclear ransloca or) (Huang et al., 1993; Figure 2.2B). Huang et al. (1993) found ha he PER PAS domains func ion as pro ein dimeriza ion mo ifs in vitro no only wi h PER PAS i self bu also wi h he PAS mo ifs of SIM ( he Drosophila single-minded gene produc ) and ARNT. A recen finding is ha PER can in erac wi h TIM ( he produc of Drosophila timeless gene), a circadian clock elemen lacking a PAS domain (Zeng et al., 1996). The presence of he PAS dimeriza ion mo ifs in bo h eurospora blue ligh regula ory pro eins led o he hypo hesis ha WC-1 and WC-2 in erac in order o carry ou heir func ion in blue ligh signaling. In fac , we were able o show in pro ein – pro ein in erac ion experimen s in vitro ha no only homodimeriza ion bu also he erodimeriza ion occurred be ween WC-1 and WC-2 and ha dimeriza ion was dependen on he presence of WC-1 and WC-2 PAS domains. Moreover, an associa ion of he WC-1 PAS domain wi h o her PAS


47

pro eins, such as ARNT and AHR, in vitro suppor ed our idea ha WC-1 and WC-2 are also members of he PAS pro ein family (Ballario et al., 1998). In WC-1, in addi ion o a canonical PAS domain wi h wo repea s, a second region weakly reminiscen of a PAS domain (amino acids 399 – 504) has been iden ified. The same region shows remarkable homology (35% iden i y) wi h Ba (Gropp and Be lach, 1994), a ranscrip ion fac or required for he oxygen-media ed expression of he Halobacterium halobium bac eriopsin and wi h NIFL (Blanco et al., 1993) (29% iden i y), a pro ein ha regula es nif gene ranscrip ion in response o environmen al oxygen concen ra ions in Klebsiella pneumoniae and Azotobacter vinelandii. All hese pro eins seem o be involved in oxygen binding and sensing. In par icular, NIFL, a flavopro ein ha uses FAD (flavin adenine dinucleo ide) as a pros he ic group, does no sense molecular oxygen direc ly bu is responsive o he oxida ion s a e of he chromophore, hus represen ing an example of redox-sensi ive pro ein (Hill et al., 1996). I is useful o repor in his con ex ha experimen s wi h a s rong reducing agen , such as di hioni e, have demons ra ed ha he oxygen is essenial for ligh induc ion in fungi (Harding and Shropshire, 1980; Arpaia and Macino, unpublished resul s). O her in eres ing similari ies were iden ified in all he PAS pro ein sec ions of WC-1 and WC-2 (Figure 2.3). The WC-2 PAS domain showed a similari y of 48% over 62 amino acids wi h he pho oac ive yellow pro ein PYP (Figure 2.3A), and a more limi ed similari y has also been iden ified wi h WC1 PAS domains. PYP is a small pro ein consis ing of 125 amino acids ha seem o encode a blue ligh pho orecep or involved in nega ive pho o axis of he halophilic purple pho o rophic bac erium Ectothiorhodospira (Baca et al., 1984). In addi ion, a more limi ed similari y was iden ified be ween he PAS domain of he WC polypep ides and phy ochromes, he red ligh pho orecep ors of plan s (Figure 2.3B). The WC-2 pro ein revealed a similari y of 38% over 56 amino acids wi h Arabidopsis PHYC, whereas a similari y of 43% over 46 amino acids was found be ween WC-1 and Arabidopsis PHYE. Al hough hese similari ies were comparably low, i was in eres ing o find in bo h he regions of similari y overlap cases of a conserved direc repea domain of phy ochromes (Figure 2.3B). Phy ochrome is a homodimeric pro ein wi h each subuni having wo major func ional domains. The amino- erminal domain is involved in ligh percep ion and con ains he chromophore-binding si e, whereas he carboxyerminal domain is involved in signal ransduc ion and in dimeriza ion of he wo subuni s (Quail et al., 1995). The conserved direc repea s are loca ed in he phy ochrome carboxyerminal domain and were sugges ed o media e a leas in par he subuni con ac of he phy ochrome dimer (Jones and Edger on, 1994). However, more recen da a implica ed hese phy ochrome repea s and adjacen pro ein regions as being involved in he ac iva ion of downs ream

48 Figure 2.3. Alignmen of he WC-1 and WC-2 pu a ive PAS domains wi h o her polypep ides from he SwissPro pro ein sequence da a base (A) Comparison of he WC-2 PAS domain wi h he amino acid sequence of he pho oac ive yellow pro ein (PYP). Similar residues are boxed. A hyphen indica es a gap in roduced o maximize alignmen . (B) Comparison of he WC-2 and WC-1 pu a ive PAS domains wi h he Arabidopsis phy ochrome C and E, respec ively. The phy ochrome repea I and repea II consensus regions according o Jones and Edger on (1994) are prin ed in bold. The regions of WC-1 and WC-2 ha show similari ies are underlined. The number of he firs amino acid of each sequence is given in paren hesis on he lef .


49

signaling componen s. Wagner and Quail (1995) described four PHYB mu an s ha were isola ed in a screening for regula ory mu an s. Al hough hese mu an s were normal wi h respec o pho opercep ion and dimeriza ion, a loss of biological ac ivi y was observed. All four mu a ions fell wi hin a small carboxyerminal region, which overlaps one of he direc repea s. Fur hermore, dele ion of he firs of he wo repea s led o he reduc ion of maximal biological ac ivi y of PHYB wi hou a decrease in he efficiency of ligh percep ion (Wagner et al., 1996). In addi ion, mos of he mu a ions iden ified in PHYA and PHYB were clus ered in his direc repea pro ein region (Quail et al., 1995). As ou lined by Lagarias et al. (1995), he direc phy ochrome repea s also show similari ies o o her regula ory pro eins, such as he bac erial wo-componen pro ein kinases, he ni rogen-fixa ion regula ory pro ein NIFL, and he opsin-ac iva or pro ein Ba . A general consequence of he observa ions repor ed earlier is ha he PAS domains of WC-1 and WC-2 seem o be widespread in animals, plan s, fungi, and bac eria. They have been iden ified in many regula ory pro eins wi h func ions in signal ransduc ion and he recep ion of differen s imuli, such as ligh , chemical compounds, and oxygen. This domain may herefore serve as a general pro ein in erface for he in erac ion be ween recep ors and signal ransduc ion componen s.

C. How do WC-1 and WC-2 function in Neurospora blue light signaling? As ou lined earlier, bo h WC pro eins are pu a ive ranscrip ion fac ors ha con rol all blue-ligh -regula ed phenomena. The indis inguishable pheno ypes of wc-1 and wc-2 mu an s and he similari y of heir func ional domains seem o sugges an iden ical role for he white collar genes in he biology of eurospora. Never heless, WC-1 and WC-2 seem o play differen roles in a leas some blue-ligh -regula ed phenomena. Cros hwai e et al. (1997) have recen ly proposed a differen ia ed role for WC-1 and WC-2 in sus aining circadian rhy hm in eurospora. WC-1 appears o be essen ial for he rese ing of he circadian cycle by ligh and for he induc ion of frequency (frq) ranscrip ion upon a pulse of blue ligh . In con ras , WC-2 is no required for ligh -induced ranscrip ion of frq bu is proposed o be a new componen of he circadian clock, ac ing as a posi ive ranscrip ion fac or necessary for main aining circadian cycling (Cros hwai e et al., 1997). Al hough he exac role of WC-1 and WC-2 in he clock is s ill unknown, i is clear ha bo h pro eins mus be presen for sus ained rhy hmici y in he dark. Fur hermore, he previously observed ligh inducibili y of wc-2 (Linden and Macino, 1997b) and ccg-1 (Arpaia et al., 1995a) in wc-2 gene ic backgrounds again sugges s dis inc roles for he wo WC pro eins.

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WC-1 and WC-2 represen he firs wo ranscrip ion fac ors characerized in any organism ha seem o be dedica ed o ligh -ac iva ed gene regula ion. Fur hermore, in vitro experimen s indica e ha WC-1 and WC-2 are capable of forming a complex via heir pu a ive PAS dimeriza ion domains presen in bo h pro eins. Na urally, numerous ques ions arise regarding heir mode of ac ion in vivo: Do WC-1 and WC-2 form he ero- and homodimeric complexes also in vivo? Are o her pro eins implica ed in he forma ion of he erodimers (i.e., wi h FRQ)? Wha is he ranscrip ionally ac ive complex and how does ligh influence hese complexes? Wha are he o her signal ransduc ion componen s, and, mos impor an , wha is he na ure of he eurospora blue ligh pho orecep or? Are he whi e collar pro eins hemselves involved in ligh percep ion and ransduc ion? A conceivable model would be a ligh -induced he erodimeriza ion of WC-1 and WC-2 ha resul s in binding and ranscrip ional ac iva ion of ligh -regula ed genes. This would be analogous o he basic helix – loop – helix PAS pro eins AHR and ARNT (Figure 2.2). In he absence of he ligand, he AH recep or was found in a complex wi h he hea shock pro ein hsp90 in he cy oplasm (An onsson et al., 1995). Upon addi ion of he ligand, he complex dissolves and he AH recep or he erodimerizes wi h i s par ner ARNT (Burbach et al., 1992). This AH recep or – ARNT complex is hen ranspor ed in o he nucleus, where i leads o ranscrip ional ac iva ion. Analogous o he AHR – ARNT model, WC-1 and WC-2 may no only func ion in ranscrip ional ac iva ion bu also par icipa e in blue ligh signal ransduc ion. A role of WC-1 and WC-2 beyond ranscrip ional regulaion would, for example, explain he fac ha WC-1 and WC-2 also seem o be necessary for blue ligh processes ha are independen of ranscrip ional gene regula ion, such as pro ein phosphoryla ion and changes in elec rophysiological parame ers of he cell membrane (Levina et al., 1988; Oda and Hasunume, 1994). Fur hermore, i would explain he finding ha , in spi e of ex ensive mu an searches, only wc-1 and wc-2 mu an s were isola ed as reliable candida es for blue ligh signal ransduc ion pro eins in eurospora.

VI. CONCLUDING REMARKS The finding of a pho osensory adap a ion mechanism in eurospora, oge her wi h he iden ifica ion of he firs pu a ive componen of he adap a ion machinery, uncovered an unan icipa ed complexi y of eurospora blue ligh signal ransduc ion. The increasing number of mu an s ha seem o in erfere wi h blue ligh signaling and ligh -regula ed ranscrip ion also suppor s his idea. In con ras , he presence of only wo regula ory mu an s ha comple ely inhibi blue ligh signal ransduc ion and ha seem o be ubiqui ously involved in all ligh responses indica es a very shor signaling cascade consis ing of only a few


51

componen s, as proposed by Ballario et al. (1996). Fur hermore, he similari ies of WC-1 and WC-2 o pro eins involved in ligh percep ion and signal ransduc ion, as well as he involvemen of he wo polypep ides in he eurospora circadian clock, may be corrobora ive of a complex regula ory func ion of WC1 and WC-2 beyond ranscrip ional con rol. A horough inves iga ion of he func ion of he WC pro eins in vivo as well as he iden ifica ion of he missing componen s of blue ligh regula ion will be necessary before we begin o unders and he way eurospora sees he ligh .

Acknowledgments G.M. and P.B. hank people in heir labs for s imula ing discussion. This work was suppor ed in par by gran s from Is i u o Pas eur Fondazione Cenci Bologne i and from Minis ero delle Risorse Agricole, Alimen ari e Fores ali, Piano Nazionale Bio ecnologie Vege ali.

References An onsson, C., Whi elaw, M. L., McGuire, J., Gus afsson, J. A., and Poellinger, L. (1995). Dis inc roles of he molecular chaperone hsp90 in modula ing dioxin recep or func ion via he basic helix– loop– helix and PAS domains. Mol. Cell. Biol. 15, 756– 765. Aronson, B. D., Johnson, K. A., Loros, J. J., and Dunlap, J. C. (1994). Nega ive feedback defining a circadian clock: Au oregula ion of he clock gene frequency. Science 263, 1578– 1584. Arpaia, G., Loros, J. J., Dunlap, J. C., Morelli, G., and Macino, G. (1993). The in erplay of ligh and circadian clock. Independen dual regula ion of clock-con rolled gene ccg-2 (eas). Plant Physiol. 102, 1299– 1305. Arpaia, G., Loros, J. J., Dunlap, J. C., Morelli, G., and Macino, G. (1995a). Ligh induc ion of he clock-con rolled gene ccg-1 is no ransduced hrough he circadian clock in eurospora crassa. Mol. Gen. Genet. 247, 157– 163. Arpaia, G., Cara oli, A., and Macino, G. (1995b). Ligh and developmen regula e he expression of he albino-3 gene in eurospora crassa. Dev. Biol. 170, 626– 635. Arpaia, G., Cerri, F., Baima, S., and Macino, G. (1999). Pro ein kinase C may be a novel componen of he blue ligh ransduc ion pa hway in eurospora crassa. Mol. Gen. Genet. in press. Baca, M., Borgs ahl, G. E. O., Boissino , M., Burke, P. M., Williams, D. R., Sla er, K. A., and Ge zoff, E. D. (1994). Comple e chemical s ruc ure of pho oac ive yellow pro ein: Novel hioeser-linked 4-hydroxycinnamyl chromophore and pho ocycle chemis ry. Biochemistry 33, 14,369– 14,377. Baima, S., Macino, G., and Morelli, G. (1991). Pho oregula ion of he albino-3 gene in eurospora crassa. J. Photochem. Photobiol. 11, 107– 115. Ballario, P., Vi orioso, P., Magrelli, A., Talora, C., Cabibbo, A., and Macino, G. (1996). Whi e collar-1, a cen ral regula or of blue ligh responses in eurospora, is a zinc finger pro ein. EMBO J. 15, 1650– 1657. Ballario, P., and Macino G. (1997). Whi e collar pro eins. PASsing he ligh signal in eurospora crassa. Trends Microbiol. 458– 462. Ballario, P., Talora, C., Galli, D., Linden, H., and Macino, G. (1998). Roles in dimeriza ion and blue ligh pho oresponse of he PAS and LOV domains of eurospora crassa Whi e Collar pro eins. Mol. Microbiology 29, 719– 731.

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Bell-Pedersen, D., Dunlap, J. C., and Loros, J. J. (1992). The eurospora circadian clock-con rolled gene, ccg-2, is allelic o eas and encodes a fungal hydrophobin required for forma ion of he conidial rodle layer. Genes Dev. 6, 2382– 2394. Bell-Pedersen, D., Dunlap, J. C., and Loros, J. J. (1996a). Dis inc cis-ac ing elemen s media e clock, ligh , and developmen al regula ion of he eurospora crassa eas (ccg-2) gene. Mol. Cell. Biol. 16, 513– 521. Bell-Pedersen, D., Shinohara, M. L., Loros, J. J., and Dunlap, J. C. (1996b). Circadian clockcon rolled genes isola ed from eurospora crassa are la e-nigh o early morning-specific. Proc. atl. Acad. Sci. USA 3, 13,096– 13,101. Blanco, G., Drummond, M., Woodley, P., and Kennedy, C. (1993). Sequence and molecular analysis of he nifL gene of Azotobacter vinelandii. Mol. Microbiol. , 869– 879. Bowler, C., Yamaga a, H., Neuhaus, G., and Chua, N. H. (1994). Phy ochrome signal ransduc ion pa hways are regula ed by reciprocal con rol mechanisms. Genes Dev. 8, 2188– 2202. Burbach, K. M., Poland, A., and Bradfield, C. A. (1992). Cloning of he AH-recep or cDNA reveals a dis inc ive ligand-ac iva ed ranscrip ion fac or. Proc. atl. Acad. Sci. USA 8 , 8185– 8189. Cara oli, A., Cogoni, C., Morelli, G., and Macino, G. (1994). Molecular charac eriza ion of ups ream regula ory sequences con rolling he pho oinduced expression of he albino-3 gene of eurospora crassa. Mol. Microbiol. 13, 787– 795. Cara oli, A., Ka o, E., Rodriguez-Franco, M., S uar , W. D., and Macino, G. (1995). A chimeric ligh -regula ed amino acid ranspor sys em allows he isola ion of blue ligh regula or (blr) mu an s of eurospora crassa. Proc. atl. Acad. Sci. USA 2, 6612– 6616. Corrocchano, L. M., Lau er, F. R., Ebbole, D. J., and Yanofsky, C. (1995). Ligh and developmen al regula ion of he gene con-10 of eurospora crassa. Dev. Biol. 167, 190– 200. Cros hwai e, S. K., Loros, J. J., and Dunlap, J. C. (1995). Ligh -induced rese ing of a circadian clock is media ed by a rapid increase in frequency ranscrip . Cell 81, 1003– 1012. Cros hwai e, S. K., Dunlap, J. C., and Loros, J. J. (1997). eurospora wc-1 and wc-2: Transcrip ion, pho oresponses, and he origin of he circadian rhy hmici y. Science 276, 763– 769. DeFabo, E. C., Harding, R. W., and Shropshire, W. (1976). Ac ion spec rum be ween 260 and 800 nanome ers for he pho oinduc ion of caro enoid biosyn hesis in eurospora crassa. Plant Physiol. 57, 440– 445. Degli-Innocen i, F., Pohl, U., and Russo, V. E. A. (1983). Pho oinduc ion of pro operi hecia in eurospora crassa by blue ligh . Photochem. Photobiol. 37, 49– 51. Degli-Innocen i, F., Chambers, J. A. A., and Russo, V. E. A. (1984a). Conidia induce he forma ion of pro operi hecia in eurospora crassa: Fur her charac eriza ion of whi e collar mu an s. J. Bacteriol. 15 , 808– 810. Degli-Innocen i, F., and Russo, V. E. A. (1984b). Isola ion of new whi e collar mu an s of eurospora crassa and s udies on heir behavior in he blue ligh -induced forma ion of pro operi hecia. J. Bacteriol. 15 , 757– 761. Deng, X. W. (1994). Fresh view of ligh signal ransduc ion in plan s. Cell 76, 423– 426. Gropp, F., and Be lach, M. C. (1994) The bat gene of Halobacterium halobium encodes a ransac ing oxygen inducibili y fac or Proc. atl. Acad. Sci. USA 1, 5475– 5479. Harding, R. W., and Shropshire, W. (1980). Pho ocon rol of caro enoid biosyn hesis. Annu. Rev. Plant Physiol. 31, 217– 238. Harding, R. W., and Turner, R. V. (1981). Pho oregula ion of he caro enoid biosyn he ic pa hway in albino and whi e collar mu an s of eurospora crassa. Plant Physiol. 68, 745– 749. Harding, R. W., and Melles, S. (1983). Gene ic analysis of he pho o rophism of eurospora crassa peri hecial beaks using whi e collar and albino mu an s. Plant Physiol. 72, 996– 1000. Hill, S., Aus in, S., Eydmann, T., Jones, T., and Dixon, R. (1996). Azotobater vinelandii NIFL is a flavopro ein ha modula es ranscrip ional ac iva ion of in ron ni rogen-fixa ion genes via a redox sensi ive swi ch Proc. atl. Acad. Sci. USA 3, 2143– 2148.


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Huang, Z. J., Edery, I., and Rosbach, M. (1993). PAS is a dimeriza ion domain common o Drosophila period and several ranscrip ion fac ors. ature 364, 259– 262. Jones, A. M., and Edger on, M. D. (1994). The ana omy of phy ochrome, a unique pho orecep or in plan s. Sem. Cell Biol. 5, 295– 302. Kaldenhoff, R., and Russo, V. E. A. (1993). Promo er analysis of he bli-7/eas gene. Curr. Genet. 24, 394– 399. Klemm, E., and Ninnemann, H. (1978). Correla ion be ween absorbance changes and a physiological response induced by blue ligh in eurospora crassa. Photochem. Photobiol. 28, 227– 230. Lagarias, D. M. Wu, S. H., and Lagarias, J. C. (1995). A ypical phy ochrome gene s ruc ure in he green algae Mesotaenium caldariorum. Plant Mol. Biol. 2 , 1127– 1142. Lau er, F. R. (1996). Molecular gene ics of fungal pho obiology. J. Genet. 75, 375– 386. Lau er, F. R., and Russo, V. E. A. (1991). Blue ligh induc ion of conidia ion specific genes in eurospora crassa. ucleic Acids Res. 1 , 6883– 6886. Lau er, F. R., Russo, V. E. A., and Yanofsky, C. (1992). Developmen al and ligh regula ion of eas, he s ruc ural gene for he rodle pro ein of eurospora. Genes Dev. 6, 2373– 2381. Lau er, F. R., and Yanofsky, C. (1993). Day/nigh and circadian rhy hm con rol of con gene expression in eurospora. Proc. atl. Acad. Sci. USA 0, 8249– 8253. Lau er, F. R., Yamashiro, C. T., and Yanofsky, C. (1997). Ligh s imula ion of conidia ion in eurospora crassa: S udies wi h wild ype and mu an s wc-1, wc-2, and acon-2. J. Photochem. Photobiol. B. 37, 203– 211. Levina, N. N., Belozerskaya, T. A., Kri sky, M. S., and Po apova, T. V. (1988). Pho oelec rical responses of eurospora crassa mu an whi e collar 1. Exp. Mycol. 12, 77– 79. Li, C., and Schmidhauser, T. J. (1995). Developmen al and pho oregula ion of al-1 and al-2, s ruc ural genes for wo enzymes essen ial for caro enoid biosyn hesis in eurospora. Dev. Biol. 16 , 90– 95. Li, D., and Kola ukudy, P. E. (1995). Cloning and expression of cDNA encoding a pro ein ha binds a palindromic promo er elemen essen ial for induc ion of fungal cu inase by plan cu in. J. Biol. Chem. 270, 11,753– 11,756. Linden, H., Ballario, P., and Macino, G. (1997a). Blue ligh regula ion in eurospora crassa. Fungal Genet. Biol. (in press). Linden, H., and Macino, G. (1997b). Whi e collar 2, a par ner in blue ligh signal ransduc ion, con rolling expression of ligh -regula ed genes in eurospora crassa. EMBO J. 16, 98– 109. Linden, H., Rodriguez-Franco, M., and Macino, G. (1997c). Regula ory mu an s of eurospora crassa in blue ligh percep ion. Mol. Gen. Genet. 254, 111– 118. Loros, J. (1995). The molecular basis of he eurospora clock. eurosciences 7, 3– 13. Loros, J. J., Denome, S. A., and Dunlap, J. C. (1989). Molecular cloning of genes under con rol of he circadian clock in eurospora. Science 243, 385– 388. Macino, G., Baima, S., Cara oli, A., Morelli, G., and Valle, E. M. (1993). Blue ligh -regula ed expression of geranylgeranyl pyrophospha e syn he ase (albino-3) gene in eurospora crassa. In “Molecular Biology and I s Applica ion o Medical Mycology” (B. Meresca, G. S. Kobayashi, and H. Yamaguchi, eds.), pp. 117– 124. Na o Asi Series. Vol. H 69, Springer-Verlag, Berlin Heidelberg. Neuhaus, G., Bowler, C., Kern, R., and Chua, N. H. (1993). Calcium/calmodulin-dependen and independen phy ochrome signal ransduc ion pa hways. Cell 73, 937– 952. Oda, K., and Hasunume, K. (1994). Ligh signals are ransduced o he phosphoryla ion of 15 kDa pro eins in eurospora crassa. FEBS Lett. 345, 162– 166. Orbach, M. J., Vollra h, D., Davis, R. W., and Yanofsky, C. (1988). An elec rophore ic karyo ype of eurospora crassa. Mol. Cell. Biol. 8, 1469– 1473. Perkins, D. D., Radford, A., Newmeyer, D., and Bjorkmann, M. (1982) Chromosomal loci of eurospora crassa. Microbiol. Rev. 46, 426– 570.

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3

X-Linked Mental Retardation Giovanni Neri* Is i u o di Gene ica Medica Facol a` di Medicina e Chirurgia “A. Gemelli” Universi a` Ca olica del Sacro Cuore 00168 Roma, I aly

Pietro Chiurazzi Cen ro Ricerche per la Disabili a` Men ale e Mo oria Associazione Anni Verdi 00168 Roma, I aly

I. In roduc ion II. Syndromal XLMR A. Fragile X Syndrome B. Simpson – Golabi – Behmel Syndrome C. ATR-X Syndrome D. Opi z/G-BBB Syndrome E. The Aarskog – Sco Syndrome F. The Coffin – Lowry Syndrome III. Nonsyndromal XLMR (MRX) A. FMR2 B. GDI1 C. OPH 1 D. PAK3 IV. Conclusion References

* To whom correspondence should be addressed. E-mail: [email protected] .i . Telephone: ⫹39063054449. Fax: ⫹39-063050031. Advances in Genetics, Vol. 41 Copyrigh 1999 by Academic Press All righ s of reproduc ion in any form reserved. 0065-2660/99 $30.00

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I. INTRODUCTION I has been known for a long ime ha here is an excess of males among he men ally re arded, especially if one considers men al re arda ion (MR) of mildo-modera e degree. In his famous “Colches er Survey,” conduc ed in an ins iu ion for he men ally re arded, Penrose (1938) es ima ed his excess o be as high as 25%, a ribu ing i mainly o ascer ainmen bias. In preparing his doc oral hesis approxima ely 30 years la er, Lehrke analyzed a sample of menally re arded individuals ha included a number of familial cases wi h X-linked inheri ance. This analysis led him o formula e he hypo hesis ha he excess of MR among males is due o he exis ence of a number of condi ions caused by X-linked mu an genes, and herefore MR is bound o be more, if no exclusively, expressed in hemizygous males. This concep of X-linked MR (XLMR) was formally defined by Lehrke in a la er publica ion (Lehrke, 1974) and is now largely accep ed. However, he idea ha ex rinsic fac ors may also con ribu e o he excess of MR among males, should no be o ally dismissed. Al hough accura e epidemiologic da a prospec ively collec ed from sufficien ly large popula ions are vir ually nonexis en , one can s ill calcula e, based on available da a, ha XLMR represen s approxima ely 5% of all MR, corresponding o a prevalence in he general popula ion of abou 1.8 per 1000 (Herbs and Miller, 1980). In 1991, Neri et al. published he firs of a series of XLMR gene upda es, hus es ablishing a ca alog of he corresponding clinical condi ions, which con ained, in he firs edi ion, 39 en ries. The mos recen edi ion, published in 1999, con ains 179 en ries (Lubs et al., 1999), he large increase being due in par o he discovery of new condi ions in he in ervening years and in par o he adop ion of more inclusive cri eria. All lis ed disorders are subdivided in o wo major groups, one for he syndromal forms of XLMR and he o her for he nonsyndromal ones. The former group is composed of hose condi ions ha are clinically recognizable because of a specific pa ern of physical, neurological, or me abolic abnormali ies. The la er includes all hose disorders whose only consis en clinical manifes a ion is MR. These disorders can be dis inguished from each o her only on he basis of he differen regional assignmen of he corresponding locus on he X chromosome. Table 3.1 provides a summary of he curren s a us of XLMR genes – disorders and also indica es he number of genes cloned and/or regionally mapped.

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3. X-Linked Mental Retardation Table 3.1. Coun s of XLMR Condi ions

Syndromal XLMR Malforma ion syndromes Me abolic disorders Neuromuscular diseases Dominan condi ions (le hal in males) To al Nonsyndromal XLMR (MRX) To al En ries

To al coun

Mapped

Cloned

68 13 32 7 120 59 179

30 2 15 5 52 55 107

6 10 6 1 23 4 27

II. SYNDROMAL XLMR Owing o he prac ical impossibili y of giving a de ailed descrip ion of all known XLMR syndromes, only some will be repor ed here based on heir rela ively higher frequency and be er charac eriza ion. Those ha are also fairly common and well known, such as Duchenne muscular dys rophy and Hun er syndrome, are ex ensively rea ed in specialized books (e.g., Scriver et al., 1995). O hers ha are very rare and some imes repor ed in a single family are summarized in he recen review by Lubs et al. (1999). Cloned and mapped genes are graphically displayed in Figure 3.1 which makes i apparen ha many regional assignmen s are qui e ex ended and largely overlapping. However, given he pheno ypic differences among he various clinical condi ions, i can be safely assumed ha even overlapping loci correspond o dis inc condi ions un il proven o herwise. The fragile X syndrome will be rea ed more ex ensively han o her syndromes because of i s impor ance as an arche ypal model of XLMR.

A. Fragile X syndrome The fragile X syndrome is he pro o ype of a growing lis of disorders known o be caused by he so-called dynamic mu a ions resul ing from he ins abili y and expansion of riple repea s (Djian, 1998). The mu an gene, FMR1, is loca ed in Xq27.3 and harbors a repea ed CGG riple in i s 5⬘ un ransla ed region (Verkerk et al., 1991). The syndrome derives i s name from he fragile si e FRAXA, which is colocalized wi h he CGG repea in Xq27.3 and was firs observed by Lubs (1969) in four men ally re arded males and hree obliga e carrier females of he same family. The expression of he fragile X si e is bes induced when cells are cul ured wi h low fola e concen ra ion and when ei her

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Figure 3.1. X chromosome ideogram wi h he known localiza ions of genes responsible for syndromal XLMR. The bars on he righ indica e he locus assignmen for hose pu a ive genes ha have been regionally mapped. The arrows on he lef indica e he posi ion of he cloned genes.

3. X-Linked Mental Retardation

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fluorodeoxyuridine or an excess of hymidine is added (Jacky et al., 1991). The fragile si e is usually expressed in 30 o 50% of he cells examined. However, a lower expression is no unusual, and, in fac , i can be some imes as low as 4 or 5%, especially in carrier females. I appears as a decondensed chroma in gap be ween Xq28 and he res of he X chromosome. I has been shown ha DNA replica ion is delayed well af er he S phase in he region con aining he expanded CGG repea and could be incomple e a mi osis, hus de ermining he chromosomal “fragili y” (Hansen et al., 1993). The firs large family wi h men al re arda ion and macroorchidism in males ransmi ed in an X-linked fashion and la er confirmed o have fragile X syndrome was described over 50 years ago by Mar in and Bell (1943), and heir names have been of en used as an eponym for he syndrome.

1. Clinical pheno ype The clinical pheno ype of he fragile X syndrome can be qui e variable. In ypical cases here is all s a ure and rela ive macrocephaly, a long and narrow face wi h prominen forehead and mandible, and midface hypoplasia wi h hypo eloric, sunken eyes. The ears are large and he pala e is highly arched. Tes es are generally large, wi h volumes up o 100 ml. Generalized muscular hypo onia is a vir ually cons an finding and is usually accompanied by join laxi y. These la er findings migh be caused by an underlying connec ive issue dysplasia, which could also be responsible for he frequen ly observed mi ral valve prolapse. MR is usually of modera e degree and behavior ends o be in rover ed, wi h poor eye con ac and avoidance of new and unexpec ed si ua ions. In ex reme cases his behavior can be described as au is ic. The pheno ype is usually more sub le in newborns and children, in whom facial rai s end o be less pronounced and macroorchidism is less obvious. Increased bir hweigh and generalized congeni al hypo onia may be he only significan findings. Hyperac ivi y and a en ion defici disorder have been described in children. Seizures may also occur during infancy and a charac eris ic EEG pa ern of rains of medium-high vol age spikes discharging from he emporal regions during sleep has been repor ed (Musumeci et al., 1991). Epilep ic seizures, if presen , generally disappear before puber y and end o respond well o rea men . Brain MRI shows volume conserva ion of brain issue wi h a diminished whi e- o-gray ma er ra io and a rela ively enlarged cauda e nucleus and hippocampus, while cerebrospinal fluid is increased, especially in he la eral ven ricles (Reiss et al., 1995). The four h ven ricle is also enlarged in correspondence o a smaller pos erior cerebellar vermis (Reiss et al., 1991). Among non ypical cases of he syndrome a subgroup ha , because of obesi y and shor s a ure bore some resemblance o he Prader – Willi syndrome,

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was iden ified (de Vries et al., 1993). Al hough his is no hing more han a superficial similari y, i is a good reminder of he pi falls of a purely clinical diagnosis and jus ifies he view ha every men ally re arded person should be es ed for fragile X syndrome in he absence of ano her reasonable diagnosis. The affec ed females, who represen abou one- hird of all females carrying a full mu a ion, usually do no demons ra e a charac eris ic physical pheno ype. They are mildly re arded or may presen only a learning disabili y and have a shy and in rover ed personali y.

2. Diagnosis and prevalence Molecular diagnosis of he CGG amplifica ion, which cons i u es grea er han 95% of he fragile X mu a ions, has been available since he cloning of he FMR1 gene in 1991 and fundamen ally relies on Sou hern blo ing and hybridiza ion of probes specific for he promo er region, whereas PCR is employed o measure he leng h of he CGG repea rac in he normal and premu a ion range. Screening for full and premu a ions should hus combine bo h PCR and Sou hern blo ing, possibly using a pooling – reanalysis s ra egy as in Rousseau et al. (1995). Cy ogene ic es ing can s ill be considered in looking for full mu a ions in males, al hough posi ive cases should be checked wi h DNA analysis and may lead o he iden ifica ion of a few FRAXE individuals (Knigh et. al., 1993). A rapid me hod based on an ibody de ec ion of he FMR1 pro ein in cells of blood smears has been described and valida ed by Willemsen et al. (1995) and is useful in screening affec ed males. In our opinion, prena al diagnosis s ill depends on he availabili y of sufficien DNA o perform a Sou hern blo ing af er double diges ion ha includes a me hyla ion-sensi ive enzyme (usually EagI or BssHII). The sex of he fe us can be de ermined wi h a s andard karyo ype or by Y-specific PCR analysis. Bo h false posi ives, due o subop imal amplifica ion, and false negaives, due o he possible presence of rever ed alleles in he wildype range, can occur when PCR alone is performed on a sample from a male fe us. Fur hermore, only direc DNA analysis af er diges ion wi h a me hyla ion-sensi ive enzyme can demons ra e he ac ual me hyla ion s a us of he FMR1 CpG island, especially in he presence of a full mu a ion. Given he occurrence of unme hyla ed full mu a ions in unaffec ed ransmi ing males (Rousseau et al., 1994; Smee s et al., 1995) and he evidence ha in ex raembryonic issues, such as chorionic villi, a full mu a ion may remain largely underme hyla ed un il 10 – 11 weeks of ges a ion (Su cliffe et al., 1992; Luo et al., 1993; Cas ellvi-Bel et al., 1995), CVS migh no display he hyperme hyla ion already presen in he embryonic issues and may need confirma ion wi h amniocen esis. De ec ion of he FMR1 pro ein is also possible in amniocy es (Willemsen et al., 1997) and chorionic villi (Willemsen et al., 1996a), bu given he semiquan i a ive na ure


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of he assay, he role of he pro ein es can only be viewed as confirma ory of he DNA analysis. Al hough he fragile X syndrome is s ill believed o accoun for he majori y of XLMR cases, i seems ha i s prevalence is no as high as ini ially es ima ed. A recen reevalua ion of he he same popula ion ha yielded he much-quo ed figure of 1:1300 males (Webb et al., 1986) led o he conclusion ha 1:4000 males is probably a more realis ic figure (Turner et al., 1996). Mos likely, his apparen discrepancy can be explained by he use in he res udy of he molecular es , which is more accura e and specific han he cy ogene ic es previously available. No general popula ion screening has been done on unselec ed popula ions, such as consecu ive newborns. Surveys have concenra ed on children wi h MR or learning disabili ies or ins i u ionalized pa ien s, where he prevalence of he fragile X syndrome is approxima ely 5% (van den Ouweland et al., 1994). Some da a on he prevalence of heal hy female carriers have been provided by a French-Canadian s udy ha screened 10,624 unselec ed women and found 41 (1:259) carriers of FMR1 premu a ed alleles wi h 55 – 101 CGG repea s (Rousseau et al., 1995). Addi ional similar s udies are needed o es ablish whe her his unexpec edly high prevalence of premu a ion carriers is unique o he specific popula ion s udied or applies o o her popula ions as well, as seems more likely (Sherman et al., 1995). Evidence ha expansion o full mu a ion upon ransmission from a premu a ed mo her is more likely o occur in male han in female fe uses has been provided by Loesch and co-workers (1995) and may explain a rela ive lack of premu a ed males in he general popula ion (Rousseau et al., 1996). Large popula ion s udies on unselec ed series of newborns would be useful o se le he ques ion of he rue prevalence of affec ed (fully mu a ed) and normal ransmi ing (premu a ed) males and of full-mu a ion and premu a ion carrier females. Al hough very few fragile X cases have been repor ed wi hou amplifica ion of he CGG repea and wi h ei her poin mu a ions or dele ions in o her par s of he FMR1 gene (Gronskov et al., 1998), i is wor h considering ha he prevalence of hese “nondynamic” mu a ions migh be underes ima ed because mos molecular diagnos ic s ra egies es only he s a us of he CGG repea and i s flanking sequences.

3. Gene ics a. Gene structure and protein isoforms The FMR1 gene s ruc ure has been de ermined in de ail. The 17 exons of he gene are embedded in 38 kb of genomic sequence in Xq27.3 (Eichler et al., 1993). The polymorphic CGG repea is loca ed in he 5⬘ un ransla ed region of exon 1 and is included in all FMR1 ranscrip s (Verkerk et al., 1993). FMR1

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was shown o be ubiqui ously ranscribed during murine and human embryogenesis (Hinds et al., 1993) wi h he highes level of expression in differen ia ed neurons of he hippocampus and basal ganglia (Abi bol et al., 1993). In adul mice, FMR1 is expressed only in neurons and in sperma ogonia. FMR1 pro ein has been de ec ed in synapses, dendri ic spines, and he soma of ra neurons bu no in he nucleus or axon, and ac ive FMR1 pro ein produc ion has been demons ra ed near synapses in response o neuro ransmi er ac iva ion (Weiler et al., 1997). FMR1 ac ion is probably required for normal ma ura ion of synap ic connec ions, which appear imma ure and reduced in number in fragile X brains (Hin on et al., 1991). The 4.4-kb full-leng h mRNA can code for a pro ein wi h a maximum leng h of 632 amino acids and an apparen molecular weigh of 70 – 80 kDa (Verheij et al., 1993; Devys et al., 1993), and al hough 20 differen ranscrip s migh be produced by al erna ive splicing (Verkerk et al., 1993; Ashley et al., 1993), only 4 o 5 of hem and heir corresponding pro ein produc s are ac ually de ec ed in various issues. Isoform 7 (ISO7), which lacks only he 21 amino acids of exon 12, makes up almos all he FMR1 pro ein wi h an approxima e molecular weigh of 80 kDa, (Si ler et al., 1996). Two KH domains (KH1 and KH2) and one RGG box, common o several RNA-binding pro eins, have been iden ified in exons 8, 10, and 15, respec ively (Siomi et al., 1993). I was shown ha FMR1 could bind syn he ic RNAs in vitro, and he impor ance of KH domains was underscored by he descrip ion of a severely re arded fragile X pa ien wi h a poin mu a ion changing a highly conserved isoleucine of KH2 in o asparagine (Ile304Asn) (De Boulle et al., 1993), which impaired he RNAbinding ac ivi y of FMR1 (Siomi et al., 1994). I is s ill no clear whe her FMR1 binds mRNAs and par icipa es in mRNP (ribonucleopro ein) par icle forma ion (Corbin et al., 1997; Feng et al., 1997) or if i binds o ribosomal RNA (Tamanini et al., 1996; Siomi et al., 1996). The major isoform (ISO7) is localized in he cy oplasm (Verheij et al., 1993; Devys et al., 1993), whereas he minor isoforms, lacking exon 14 and wi h a differen C- erminus (ISO6 or ISO12), are confined o he nucleus (Si ler et al., 1996). The N- erminus of he pro ein (exons 1 – 5) seems o con ain a pu a ive nuclear localiza ion signal (NLS) (Si ler et al., 1996), whereas exon 14 con ains sequences capable of ac ing like he nuclear expor signal (NES) of he HIV-1 Rev regula ory pro ein (Fridell et al., 1996). A shu ling be ween cy oplasm and nucleus was herefore envisaged and, in fac , FMR1 has been de ec ed by elec ron microscopy free and associa ed wi h ribosomes in he cy oplasm, bu also in he nucleoli (Willemsen et al., 1996b). The serendipi ous discovery of a fragile-X-rela ed pro ein, FXR1 (Siomi et al., 1995), led o he search for o her FXR pro eins possibly in erac ing wi h FMR1 or complemen ing i s func ions. FXR2 was iden ified by using he yeas wo-hybrid sys em (Zhang et al., 1995) and is also able o bind FXR1. The


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FXR1 and FXR2 genes have been mapped o 3q28 and 17p13.1, respec ively, and an in ronless form of FXR1 has been localized o 12q13 (Coy et al., 1991). Bo h FXRs and FMR1 are highly homologous in he N- erminal por ion, including he KH domains, he RGG box, and he firs half of exon 14 ( he ribosome-binding si e coinciding wi h he NES) (Zhang et al., 1995), and heir genes possibly evolved from a common ances or. All hree pro eins can in erac wi h each o her and form he ero- as well as homodimers in vitro (Zhang et al., 1995). One isoform of FXR1 is highly expressed in skele al muscle and pos meio ic sperma ids and absen in differen ia ed neurons and in sperma ogonia; o her isoforms of FXR1 and FXR2 are ranscribed in neurons in bo h he cerebellum and he cor ex (Coy et al., 1995).

b. Amplification mechanisms I is now known ha in more han 95% of cases, he fragile X syndrome is caused by a single ype of mu a ion (“full mu a ion”), i.e., he expansion and hyperme hyla ion of a po en ially uns able CGG rinucleo ide repea in he 5⬘ UTR of he FMR1 gene. Depending on he leng h of he CGG repea , hree general classes of alleles are observed in he FMR1 gene: wildype alleles (6 – 50 repea s), premu a ions (50 – 200 repea s), and full mu a ions (200 – 1000 repea s and more). However, he boundaries be ween hese classes are no absolu e, and he ini ial ins abili y depends no only on he o al leng h bu also on he repea configura ion (Hirs et al., 1995). De ailed analysis of over 400 wildype alleles showed ha he CGG repea s re ch is commonly in errup ed by AGG riple s, usually wo, occurring every 9 o 10 CGGs (Eichler et al., 1994; Hirs et al., 1994; Kuns et al., 1996), which apparen ly have a s abilizing effec by preven ing replica ion slippage (Heale and Pe es, 1995). In vitro replica ion s udies of expanded CTG and CGG repea s demons ra ed ha DNA polymerase pauses af er copying 29 o 31 pure repea uni s (Kang et al., 1995). This is likely o allow he forma ion of secondary s ruc ures on he nascen s rand, including unimolecular hairpins (reviewed by Darlow and Leach, 1998), which resul s in a more subs an ial increase in repea leng h (Wells, 1996). Subsequen ly, when he so-called expansion hreshold (abou 70 pure CGG repea s) has been reached (Eichler et al., 1994), mul iple hairpins and/or s em-and-loop s ruc ures can form. This can also happen because Okazaki fragmen s exclusively composed of CGG repea s may slip a bo h ends (Richards and Su herland, 1994). Such s ruc ures are ex remely uns able and, af er inappropria e repair, may resul in a varie y of expanded full mu a ions ha are frequen ly accompanied by smaller or even dele ed alleles. Ac ually, fragile X pa ien s are of en mosaics for full mu a ions and premu a ions (Chiurazzi et al., 1994a), alleles of normal size (van den Ouweland et al., 1994), or dele ions of he en ire CGG s re ch and par of i s flanking sequences (de Graaff et al., 1995; Mila et al., 1996).

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c. FMR1 instability and founder effects FMR1 full mu a ions appear o be genera ed by a mul is ep process requiring he sequen ial ac ion of differen mechanisms (Mor on and Macpherson, 1992; Richards and Su herland, 1994; Chiurazzi et al., 1996). Thus far, no direc conversion of a wildype o a full-mu a ion allele has been observed in fragile X families; all mo hers of affec ed individuals were found o be carriers of an already expanded CGG riple . The ini ial even s leading o he ins abili y of a wildype allele are apparen ly much more rare han hose de ermining he final ransi ion from premu a ion o full mu a ion (Chiurazzi et al., 1996). Replica ion slippage is known o cause varia ion of a few repea uni s in microsa elli es due o a local misalignmen of he empla e and nascen s rands during a brief de achmen of he DNA polymerase (Levinson and Gu man, 1987; Schloe erer and Tau z, 1992). As a consequence, bo h small reduc ions and amplifica ions of allele leng h have been de ec ed in single sperm cells of wo males carrying, respec ively, a 29 and a 55 CGG repea allele (Morne et al., 1996). More rarely, slippage of a whole AGG(CGG)9 rac can de ermine a 10-uni increase of a normal allele (Macpherson et al., 1995). I is wor h no ing ha he majori y of al era ions in repea leng h occur a he 3⬘ end of he CGG repea (Kuns and Warren, 1994). This polari y may derive from he differen mu abili y of he leading and lagging s rand and was shown in vitro o depend on he local direc ion of replica ion (Wells, 1996; Hirs and Whi e, 1998). The loss of he dis al AGG, mos probably due o a poin mu a ion (A- o-C ransversion), observed in many premu a ed alleles is implica ed in a fas er rou e o ins abili y, as i crea es a longer pure CGG rac (Kuns and Warren, 1994; Eichler et al., 1994, 1996). However, i is likely ha only a few alleles can reach he ins abili y hreshold of approxima ely 30 unin errup ed CGG repea s. These few alleles, some imes referred o as protomutations, mus be linked o a limi ed number of ances ral haplo ypes. Some of hese founder chromosomes, which accoun for he linkage disequilibrium de ec ed in differen popula ions (reviewed in Chiurazzi et al., 1996), have apparen ly increased heir frequency in he general popula ion by gene ic drif and cons i u e large pools of a -risk alleles (Mandel, 1994). These in ermedia e pools, which may be difficul o dis inguish from wildype alleles, mos likely explain he rela ively high frequency of he fragile X syndrome in spi e of he low fi ness of affec ed individuals and he limi ed number of founder chromosomes observed.

d. Full mutations As he (CGG)n repea in he firs exon of he FMR1 gene exceeds he illdefined hreshold of 200 repea s, mos cy osine residues in he repea i self and in he ups ream CpG island become comple ely me hyla ed, as if hey were on an inac ive X chromosome (S oeger et al., 1997; Luo et al., 1993; Hansen et


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al., 1992). The FMR1 gene is herefore ranscrip ionally silenced (Piere i et al., 1991), and no pro ein is presen in affec ed males (Verheij et al., 1993). I seems ha he ex reme expansion of he CGG repea allows he forma ion of abnormal s ruc ures, like hairpins and e raplex DNA, on he lagging s rand during replica ion (reviewed by Darlow and Leach, 1998; Mi as, 1997), which in urn a rac DNA me hyl ransferases (Bes or and Tycko, 1996; Kho et al., 1998). The hyperme hyla ion of he CGG repea hen spreads o he surrounding CpG island, possibly af er in erac ion wi h me hylcy osinebinding pro eins (MeCP2 and/or MBDs) (Boyes and Bird, 1992; Lewis et al., 1992) or o her rinucleo ide repea -binding pro eins (Deissler et al., 1996). Hyperme hyla ion is mos likely responsible no only for he ranscrip ional silencing (Piere i et al., 1991; Su cliffe et al., 1992; Hwu et al., 1993) bu also for he delayed replica ion of he FMR1 gene (Hansen et al., 1993, 1996; Samadashwily et al., 1997), which supposedly causes he cy ogene ic fragili y (Laird et al., 1987). The iming of pre- o full-mu a ion expansion and of i s me hyla ion is s ill being inves iga ed. I has been observed ha only premu a ions are presen in he sperm of fragile X pa ien s (Reyniers et al., 1993), and i has been proposed ha pre- o full ransi ion would occur only pos zygo ically during embryogenesis. This hypo hesis would require he ac ion of some “imprin ing” signal ha dis inguishes ma ernally and pa ernally derived premu aions, because a premu a ion never becomes full when ransmi ed from he fa her (Rousseau et al., 1991). On he con rary, Mal er et al. (1997) have presen ed evidence ha full-mu a ion alleles can be de ec ed in oocy es and in fe al sperma ogonia, al hough premu a ions seem o be selec ed in fe al es es. In his scenario, pre- o full-mu a ion ransi ion is limi ed o meiosis, while pos zygo ic (mi o ic) ins abili y genera es mosaicism wi hin he range of full mu a ions as well as reduced alleles in he premu a ion range or even dele ions (Chiurazzi et al., 1994a). In any case, premu a ions would be selec ed in fe al es es, hus explaining why all daugh ers of premu a ed males always re ain a premu a ion and he sperm of fragile X pa ien s only harbors premu a ions. As for me hyla ion, Mal er et al. (1997) showed ha full mu a ions are unme hyla ed in oocy es, al hough hey were comple ely me hyla ed in all soma ic issues of a 13-week-old fe us. Me hyla ion is herefore likely o ake place af er fer iliza ion and during early embryogenesis.

e. Mutations other than CGG expansion The iden ifica ion of mu a ions o her han he expansion of he CGG repea , even hough in a minori y of pa ien s, was impor an in confirming ha he fragile X syndrome is a single-gene disorder. Thus, poin mu a ions (De Boulle et al., 1993; Lugenbeel et al., 1995) or small in ragenic dele ions (Meijer et al., 1994) ruled ou he possibili y ha he abnormal hyperme hyla ion associa ed

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wi h he full mu a ion migh no be res ric ed o he FMR1 promo er bu could affec he expression of o her genes in ha chromosomal region. Several larger dele ions, even encompassing he en ire FMR1 gene (Tarle on et al., 1993), have also been repor ed. A review of he dele ion cases has been published by Hammond et al. (1997). Careful analysis of small dele ions limi ed o he promo er region can help define he essen ial regula ory sequences governing FMR1 ranscrip ion (Gronskov et al., 1997). Finally, i is wor h no ing ha some rare pa ien s wi h all he pheno ypic manifes a ions of he fragile X syndrome show no de ec able al era ion of he FMR1 gene, which is apparen ly no involved in he pa hogenesis of heir condi ion (Chiurazzi et al., 1994b). Considering he in erac ions be ween he FMR1 and he FXR pro eins, all presen in neurons, i may be possible ha hese pa ien s have a mu a ion in ei her he FXR1 or he FXR2 gene.

f. Animal models Fmr1 knockou mice have been genera ed by homologous recombina ion of a arge ing vec or in errup ing exon 5 in embryonic s em (ES) cells (Bakker et al., 1994). I is impor an o no e ha no reduced fer ili y of mu an s of ei her sex has been observed, and he erozygous females had a normal li er size wi h he expec ed dis ribu ion of offspring wi h he mu an allele. Thus, Fmr1 is no necessary for sperma ogenesis or oogenesis in mice, nor for normal embryonic developmen or pos na al viabili y. Fmr1 knockou mice show no over ana omical or his ological abnormali ies bu do have macroorchidism and exhibi hyperac ivi y and learning defici s (Bakker et al., 1994). Apparen ly, an increased Ser oli cell prolifera ion during es icular developmen is responsible for he macroorchidism, al hough his increase does no appear o be he resul of major changes in FSH signal ransduc ion in knockou mice (Sleg enhors Eegdeman et al., 1998). Experimen al designs can now be made o in roduce ransgenic copies of FMR1 in o various issues (brain, gonads) of he knockou mice in order o dissec he pa hogene ic componen s of he fragile X pheno ype. Transgenic mouse lines wi h a fusion gene consis ing of an Escherichia coli ␤-galac osidase repor er gene (lacZ) linked o he FMR1 promo er region have already been es ablished (Hergersberg et al., 1995) and showed an expression pa ern closely resembling he endogenous one, indica ing ha he 2.8-kb fragmen 5⬘ of he CGG repea con ains mos cis-ac ing elemen s regula ing i s ranscrip ion.

4. Trea men Useful guidelines for heal h supervision of fragile X children have been published by he American Academy of Pedia rics (1996) and include advice for bo h physical and behavioral aspec s of he syndrome. Af er confirma ion of he


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diagnosis wi h he molecular es and appropria e gene ic counseling of he paren s for subsequen pregnancies, a series of medical examina ions can be envisaged, depending on he age of he child. Developmen during he firs year of life may be normal, al hough hypo onia and irri abili y may be apparen . In early childhood i is impor an o give an oph halmologic examina ion (s rabismus, myopia), o perform an echocardiogram if a murmur or click is presen (mi ral valve prolapse), and o check for or hopedic problems (fla fee , scoliosis, and loose join s). An inguinal hernia should also be excluded. A his ory of seizures or s aring episodes should be reviewed and an EEG migh be appropria e, hough an iepilep ic medica ion af er a single seizure is no advisable given he self-limi ing course of epilep ic manifes a ions in adolescence (Musumeci et al., 1991). Hyperac ive behavior (head banging, hand bi ing, e c.) and severe a en ion defici , which are major concerns in he school-age period, can be rea ed pharmacologically (Hagerman, 1997). However, socializa ion and school in egra ion, possibly wi hin a mains ream program wi h individual suppor , are ex remely impor an in helping o overcome hese problems. Spor s and regular physical ac ivi y (e.g., swimming) are impor an for coun erac ing he hypo onic pos ure and improving mo or coordina ion. Speech, language, and occupa ional herapy should be goal orien ed and help adolescen s and young adul s o a ain as much au onomy as possible. Suppor from family organiza ions is ex remely impor an , especially for he paren s and sibs, because i eases he sense of isola ion and helplessness ha of en follows he diagnosis. Trea men s specifically aimed a recovering he func ion of he FMR1 gene have been a emp ed wi h folic acid because of i s ac ion on he cy ogene ic expression of he fragile si e, and al hough a few repor s indica ed some effec on he behavior (Hagerman et al., 1986), o hers did no confirm hese observa ions (Fros er-Iskenius et al., 1986; Webb et al., 1990). I can be safely concluded ha fola e supplemen a ion has no efficacy for he rea men of fragile X syndrome pa ien s. Recen observa ions of in ellec ually normal (Rousseau et al., 1994; Smee s et al., 1995) or minimally affec ed (McConkie-Rosell et al., 1993; Hagerman et al., 1994) males wi h an unme hyla ed full mu a ion confirmed ha he abnormally amplified CGG rac per se can s ill be ranscribed and ransla ed. Even if ransla ion may no be comple ely efficien (Feng et al., 1995), lymphoblas oid cell lines con aining only unme hyla ed full mua ions of wo such males have clearly shown he presence of FMR1 pro ein in every cell, al hough a a reduced level (Smee s et al., 1995). Given he observa ion of hese excep ional individuals and knowing ha he coding sequence of he mu a ed FMR1 gene was in ac , we es ed he possibili y of res oring i s ac ivi y in vitro employing a DNA deme hyla ion pro ocol. We ob ained in vitro reac iva ion of FMR1 expression af er inducing DNA deme hyla ion wi h 5azadeoxycy idine in he pa ien s’ lymphoblas oid cells. Specific mRNA was de ec ed by RT-PCR, he presence of he pro ein produc was verified by

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immunocy ochemis ry, and he ac ual promo er deme hyla ion was confirmed by me hylase-sensi ive res ric ion analysis. These resul s clarify he clinical inerpre a ion of he rare cases of male individuals wi h unme hyla ed full mu aions and normal IQ and pave he way o fu ure a emp s a pharmacologically res oring FMR1 gene ac ivi y in vivo (Chiurazzi et al., 1998). However, only less oxic drugs can be envisaged for in vivo applica ions and much informa ion is s ill needed abou he main enance of he deme hyla ion – reac iva ion effec af er a ime-limi ed rea men .

B. Simpson – Golabi – Behmel syndrome The firs repor of wha became la er known as he Simpson – Golabi – Behmel syndrome (SGBS) was published in 1975 by Simpson et al., who described wo cousins, ma ernally rela ed, wi h macrocephaly, “coarse” face, broad hands wi h dysplas ic fingernails, and apparen ly normal in elligence. In 1984, Behmel et al. described a similar condi ion in several males of a large family, calling a en ion o a number of addi ional findings, such as hear defec s, polydac yly, and a high ra e of infan mor ali y. They confirmed he X-linked inheri ance of he rai and also no ed a mild expression in carrier females. A approxima ely he same ime, Golabi and Rosen (1984) repor ed ye ano her family in which several affec ed males had addi ional malforma ions of in ernal organs and early dea h. Opi z et al. (1984) also described severely affec ed males in a family from Michigan, al hough here is some ques ion whe her his ins ance may represen a differen condi ion. In 1988, Neri et al., repor ing on an I alian family, explici ly no ed ha he hree affec ed males in his family had he same clinical condi ion previously repor ed by Simpson et al. (1975), Behmel et al. (1984), and Golabi and Rosen (1984) and coined he eponym “Simpson – Golabi – Behmel syndrome.” The clinico-gene ic findings in SGBS have been recen ly reviewed (Neri et al., 1998a).

1. Clinical pheno ype SGBS is a syndrome charac erized by overgrow h, mul iple congeni al anomalies, and dysplasia and caused by an X-linked mu an gene. The spec rum of i s clinical manifes a ions is very broad, varying from very mild forms in carrier females o infan ile le hal forms in affec ed males. I has been calcula ed ha as many as 50% of affec ed males die neona ally (Neri et al., 1988), al hough he causes of his high mor ali y remain unknown. Overgrow h is of prena al onse and con inues pos na ally. Bir h measuremen s (heigh , leng h, head circumference) of affec ed males are usually well above he 97 h cen ile, and final adul heigh can exceed 2.0 m, al hough wi h ample varia ion depending on background fac ors, such as average family


69

heigh . In mos pa ien s, he facial rai s are “coarse,” ypically wi h hyper elorism, downslan ing palpebral fissures wi h epican hic folds, shor nose, macrosomia wi h macroglossia, severe den al malocclusion, and cen ral groove of he lower lip. Clef lip and pala e have been occasionally repor ed. Hands and fee are rela ively shor and broad and may display a variable combina ion of deformi ies (me a arsus varus, clubfoo ), dysplasias (fingernail hypoplasia, especially of he index finger, various degrees of in erdigi al webbing or cu aneous syndacyly), and malforma ions (pos axial polydac yly). A comple e ransverse palmar crease is a common finding, oge her wi h s riking derma oglyphic changes, including an excess of iradii and in erdigi al loops and an irregular mix ure of arches, loops, and whorls on finger ips. Consis en ly presen on he ches are supernumerary nipples. Thickened and/or darkened skin and skin ags may also be presen . Geni alia are usually normal, al hough hypospadias and cryp orchidism have been repor ed in a number of pa ien s. The in ernal organs may be involved in many differen ways. Organomegaly is common, affec ing especially he liver, spleen, and kidneys. Kidneys may be mul icys ic wi h dysplas ic changes. Lung segmen a ion defec s have been no ed. A diaphragma ic defec has been repor ed in several pa ien s. The hear may be affec ed in more han one- hird of cases, wi h ei her s ruc ural defec s, such as ven ricular sep al defec , pa en duc us ar eriosus, pulmonic s enosis, or func ional defec s, especially arry hmias (Lin et al., 1999). In one pa ien , he developmen of a dila ed cardiomyopa hy was no ed, al hough i was impossible o ell whe her his was primary or secondary o a preexis ing congeni al hear defec (Gurrieri et al., 1992). In any case, he hear func ion should be wa ched closely in SGBS pa ien s because i can be a cause of early dea h (Ko¨nig et al., 1991). An X-ray survey of he skele on will demons ra e, in a ypical case, advanced bone age, ver ebral segmen a ion defec s such as fusion of C2/C3, cervical ribs, usually wi h 13 pairs of ribs, 6 lumbar ver ebrae, sacral and coccygeal defec s, and scoliosis. The mos consis en neurological finding in SGBS is congeni al muscular hypo onia, which may appear in s riking con ras o he big, s ocky build of he pa ien s. Several minor anomalies can be considered a direc consequence of he congeni al hypo onia: he mou h-brea hing face wi h highly arched pala e and den al malocclusion, pec us excava um, downsloping shoulders, dias asis rec i, umbilical and inguinal hernias, and cryp orchidism. The ques ion of men al re arda ion in SGBS is much deba ed. I is possible, and even likely, ha severely affec ed pa ien s are men ally re arded, al hough in mos of hese cases early dea h preven s a formal psychome ric evalua ion. However, i is clearly es ablished ha he majori y of pa ien s are no men ally re arded. This is no o say ha hese pa ien s do no have psychological problems; in fac , qui e he opposi e. The coarse appearance and

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he speech difficul ies, due o macroglossia and mou h malocclusion, give he impression ha hese pa ien s are men ally re arded, an impression of which hey become acu ely and painfully aware. There is an increased risk of neoplasia in SGBS ha mus be carefully considered, especially in young pa ien s. A Wilms umor of he kidney was diagnosed in several members of affec ed families in Canada (Hughes-Benzie et al., 1992; Xuan et al., 1994), and a hepa ocellular carcinoma was repor ed in a young child (Lapunzina et al., 1998). Because o her infan ile umors can be expec ed, every pa ien should be considered a increased risk of neoplasia and consequen ly wa ched for a leas he firs 5 years of life.

2. Diagnosis SGBS belongs o he family of he overgrow h syndromes. Therefore, a ques ion of differen ial diagnosis may easily arise wi h one or more of he clinical en i ies included in his family of syndromes. Conversely, a diagnosis of SGBS should be considered for any pa ien , especially if male, presen ing wi h excessive grow h. However, he ruly cri ical nosologic issue is wi h he Beckwi h – Wiedemann syndrome (BWS). Several pa ien s who were reassessed and rediagnosed as SGBS af er an ini ial diagnosis of BWS are on record (Neri et al., 1988; Punne , 1994). Overgrow h a bir h, coarse face wi h macroglossia, hernias, visceromegaly, congeni al hypo onia, and increased incidence of umors, especially Wilms umor, are fea ures common o bo h SGBS and BWS. Midline capillary hemangiomas, body asymme ries wi h hemihyper rophy, and a endency o decelera ed grow h can be considered more ypical of BWS. Persis ing overgrow h, congeni al hear defec s, diaphragma ic defec s, polydacyly, ex ra nipples, and familial occurrence wi h evidence of X-linkage are more ypical of SGBS. However, many cases in which he clinical diagnosis will remain suspended and in which only he molecular diagnosis will be decisive are o be expec ed. Recen ly, Verloes et al. (1995) poin ed ou he clinical overlap beween SGBS and he Perlman syndrome, an au osomal recessive overgrow h syndrome charac erized by enlarged, dysplas ic kidneys and a high risk of developing a Wilms umor (Neri et al., 1984). However, he facial rai s, he clinical course, and he mode of inheri ance are sufficien ly differen in SGBS and in he Perlman syndrome o make he wo condi ions easily dis inguishable. I should also be men ioned ha a pa ien ini ially diagnosed as having Weaver syndrome (Tsukahara et al., 1984) was subsequen ly recognized as having SGBS (Kajii and Tsukahara, 1984). The las , and s ill unresolved, nosological issue concerns he possibili y ha SGBS is a he erogeneous condi ion, clinically as well as gene ically. A family repor ed by Opi z (1984) was of en ques ioned as being a bona fide case


71

of SGBS because of he severi y of he clinical presen a ion. Ano her severely affec ed family was recen ly described by Terespolsky et al. (1995). Given ha X-linked inheri ance was apparen in bo h families, fu ure molecular s udies will de ermine whe her severe forms of SGBS are caused by allelic mu a ions a he same locus or by ano her X-linked gene. The la er hypo hesis is suppor ed by a very recen observa ion (Brzus owicz et al., 1998).

3. Gene ics SGBS is an X-linked dominan rai wi h mild expression in he erozygous females and full expression in affec ed males. The mu an gene was ini ially mapped o he Xq25 – q27 region by linkage analysis in a Du ch-Canadian family (Xuan et al., 1994). Close linkage o he HPRT locus in Xq26 was demons ra ed by Or h et al. (1994) hrough he s udy of wo European families. This loca ion coincides wi h he cy ogene ic breakpoin of an X;1 ransloca ion in he previously men ioned pa ien who was originally diagnosed as having BWS bu who was subsequen ly recognized as having SGBS (Punne , 1994). Ac ually, his pa ien became very cri ical for he cloning of he SGBS gene recen ly repor ed by Pilia et al. (1996). The gene, encoding an ex racellular pro eoglycan, designa ed glypican 3 (GPC3), spans more han 500 kb and con ains eigh exons. The cDNA measures 2.2 kb. The X;1 ransloca ion in errup s he gene in he second in ron, and ano her ransloca ion, X;16, from a pa ien described as having he Klippel – Feil anomaly, in errup s he gene be ween exons 7 and 8. In he hree families also analyzed by Pilia et al. (1996), hree differen dele ions were found: one involving exon 2, one involving he las hree exons, and he hird one also involving he las hree exons bu ex ending fur her in he 3⬘ direc ion. Addi ional dele ions were repor ed subsequen ly, al hough here are several bona fide pa ien s in whom nei her a dele ion nor a poin mu a ion can be found (Lindsay et al., 1997). According o Hughes-Benzie et al. (1996), lack of correla ion be ween he ex en of he dele ions and he pheno ypic expression of he disease sugges s ha “classical” cases of SGBS are likely due o he loss of func ion of GPC3. The gene is expressed in a number of mesoderm-derived issues, including lung, liver, and kidney issues, and he level of expression is higher in issues from mouse embryos han in murine and human adul issues. There is no expression in whi e blood cells. The GPC3 pro ein belongs o he glypican family of heparan sulfa e pro eoglycans (David, 1993) and can func ion on he cell surface as a recep or or par of a recep or complex. Mos in eres ingly, i is capable of in erac ing wi h IGF2, he insulinlike grow h fac or, which has been sugges ed as a causal fac or in BWS (Pilia et al., 1996; Weksberg et al., 1996).

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4. Trea men There is no specific rea men for SGBS. However, symp oms should be addressed according o needs. Surgery may be indica ed for congeni al hear defec s, diaphragma ic defec s, and gas roin es inal and geni ourinary malforma ions. Or hogna hic rea men should be considered of he grea es imporance. The correc ion of den al malocclusion and he reduc ion of macroglossia, if indica ed, should lead o speech improvemen , an essen ial s ep oward he es ablishmen of normal social rela ions. This should be accompanied, when needed, by appropria e psychological suppor aimed a improving he self-image of he pa ien s. I is impera ive ha every effor be made o elimina e he impression ha SGBS pa ien s are men ally re arded or, even worse, aggressive. Carrier females should be properly iden ified and adequa ely counseled wi h respec o recurrence risks and prena al diagnosis.

C. ATR-X syndrome Af er he seminal paper by Wea herall et al. was published in 1981, several o her repor s appeared describing pa ien s in whom MR is associa ed wi h a mild form of ␣- halassemia. This combina ion has since been known by he acronym ATR. Analysis of he clinical pheno ype and he pa ern of inheriance in familial cases and molecular s udies led o he iden ifica ion of wo dis inc syndromes. A group of pa ien s had large dele ions a he ip of he shor arm of chromosome 16 wi hin band 16p13.3, including he ␣-globin gene complex. This explained he presence of mild (hemizygous) ␣- halassemia in addi ion o MR and a pa ern of physical anomalies whose variabili y likely depended on he size of he dele ion (Wilkie et al., 1990a; Lamb et al., 1993). These condi ions can be in erpre ed as ypical of a con iguous gene syndrome. The o her group of pa ien s, all males, was charac erized by a more specific physical pheno ype, in ac ␣-globin gene complex and familial inheriance consis en wi h X-linkage (Wilkie et al., 1990b; Cole et al., 1991; Donnai et al., 1991). A new condi ion whose main charac eris ics were ␣- halassemia (no from dele ion), MR, and X-linkage was herefore recognized and designa ed ATR-X.

1. The clinical pheno ype The main clinical findings of he ATR-X syndrome are a charac eris ic face, geni al anomalies, and severe men al re arda ion (Gibbons et al., 1991, 1995a; Wilkie et al., 1991). The face can be described as coarse, wi h hyper elorism, epican hic folds, a fla nasal bridge, midface hypoplasia, a shor nose of riangular shape wi h an ever ed nares and fla phil rum, an inver ed V shape of he


73

upper lip and ever ed lower lip, macroglossia, and widely spaced incisors. The ears may be small, simple, low-se , and pos eriorly angula ed. Geni alia are usually abnormal, wi h small, undescended or dysgene ic es es, a shawl-like or hypoplas ic scro um, and a small penis wi h hypospadias. O her fairly common physical findings are microcephaly, shor s a ure, alipes equinovarus, and gasroin es inal problems, including gas roesophageal reflux and cons ipa ion. Xray inves iga ions have shown delayed bone age, minor digi al abnormali ies, and kyphoscoliosis. Psychomo or developmen appears o be delayed from early on and is accompanied by generalized muscular hypo onia. Men al re arda ion is usually severe, wi h vir ually absen speech and minimal comprehension. Seizures have been repor ed in some pa ien s. Brain imaging occasionally shows cerebral a rophy. Carrier females are subs an ially normal, bo h physically and men ally, al hough mild midfacial anomalies have been no ed in some (Donnai et al., 1991).

2. Diagnosis The pheno ypic diagnosis of he ATR-X syndrome can be confirmed in he labora ory by a rela ively simple blood es . The mild form of ␣- halassemia in hese pa ien s is reflec ed in he presence of HbH inclusions in a propor ion of red cells, varying from 1 o 40%. The amoun of HbH de ec ed elec rophore ically is also variable, ranging from 0 o abou 7% (Gibbons et al., 1991). Occasionally, a very few ery hrocy es wi h inclusions have been no ed in carrier females. Al hough hese HbH findings can be aken as diagnos ic evidence for bo h carriers and affec ed individuals, he opposi e is no rue. Unequivocal diagnosis is now available, based on direc mu a ional analysis of he responsible gene (vide infra).

3. Gene ics Linkage analysis of several pedigrees segrega ing he ATR-X syndrome localized he corresponding locus o an in erval of 11 cM in Xq12 – q21.31 (Gibbons et al., 1992). This observa ion was followed 3 years la er by he cloning of he gene. Gibbons et al. (1995b) showed ha he ATR-X syndrome is caused by mu a ions of XH2, a gene belonging o he helicase superfamily, whose pro ein produc s carry ou a number of regula ory func ions ranging from DNA recombina ion and repair o con rol of ranscrip ion. More specifically, he pro ein belongs o he SNF2 subgroup, probably ac ing as a regula or of gene expression (Picke s et al., 1996). Analysis of several independen pa ien s showed he exis ence of a varie y of diverse mu a ions, including prema ure s op mu a ions, missense mu a ions, and dele ions (Gibbons et al., 1995b). In a subsequen repor , Gibbons et al. (1997) showed he exis ence of a mu a ional ho spo in

74


a segmen of he gene encoding a cys eine-rich zinc-finger domain ha accoun s for more han 60% of known mu a ions. I is of he grea es in eres ha fur her recen mu a ional analyses of XH2 led o he discovery ha his gene can be involved in he causa ion of condi ions ha were originally described as independen en i ies. For example, he so-called Juberg – Marsidi syndrome, also mapped o he Xq12 – q21 region and whose pheno ypic manifes a ions include deafness in addi ion o men al re arda ion and mul iple physical anomalies, was shown o be due o a mu a ion of he X P gene, a differen designa ion for he same XH2 gene (Villard et al., 1996a). Likewise, a frameshif mu a ion of XH2 ha genera ed a prema ure s op codon was repor ed o segrega e in a family in which he affec ed males had a pheno ype resembling ha of he ATR-X syndrome bu wi hou ␣- halassemia and wi h male- o-female sex reversal (Ion et al., 1996b). The absence of ␣halassemia was also no ed in a pa ien wi h an XH2 mu a ion causing a proline- o-serine ransi ion in he helicase II domain (Villard et al., 1996). Taken oge her, hese observa ions suppor he no ion ha XH2 mu a ions downregula e he expression of several genes, including he ␣-globin genes. This would explain he complexi y of he ATR-X pheno ype.

4. Trea men There is no specific rea men for he ATR-X syndrome. Female carriers have a 50% risk of heir male offspring being affec ed. I is herefore of he grea es impor ance ha women a risk of being carriers be properly iden ified by molecular es s, adequa ely counseled, and offered prena al diagnosis when indica ed.

D. Opitz/G-BBB syndrome The G and BBB syndromes were originally repor ed as wo separa e condi ions even hough bo h involved defec s of he midline developmen al field (Opi z et al., 1969a,b). The G syndrome appeared o have an au osomal dominan mode of inheri ance, whereas in he case of he BBB syndrome, X-linkage seemed o be more likely, al hough no clearly proven. Subsequen ly, he s riking phenoypic similari ies led o he provisional conclusion ha he wo condi ions should be considered one and he same under he commom designa ion of Opi z syndrome un il proven o herwise (Cappa et al., 1987). More recen ly, Robin et al. (1995) performed linkage s udies on several Opi z syndrome families, including he original G family, and found gene ic he erogenei y: one locus was iden ified on he X chromosome and ano her one on chromosome 22. The Xlinked gene has now been cloned (Quaderi et al., 1997).


75

1. Clinical pheno ype Opi z syndrome can s ill be described pheno ypically as a single en i y, and i is charac erized by a series of defec s of he midline. The face is ypical, wi h widely spaced eyes, broad or hypoplas ic nasal sella, a large nose, and a hypoplas ic phil rum or clef ing of he upper lip and pala e. Tracheoesophageal defec s range from simple swallowing difficul ies o a racheo esophageal fis ula. Pec us excava um, umbilical hernia, and hypospadias in males are also common findings. The hear frequen ly has a varie y of abnormali ies, including sep al and cono runcal defec s. Brain imaging has demons ra ed agenesis of he corpus callosum in some pa ien s (Neri et al., 1987). In ellec ual developmen may range from normal o mildly re arded. In familial cases one paren occasionally will show mild physical signs, sugges ing variable expressivi y of he mu an gene(s). This is par icularly rue in mo hers of affec ed boys, suppor ing he no ion ha a par ially dominan , X-linked mu a ion segrega es in some families.

2. Gene ics As already men ioned, Opi z syndrome is gene ically he erogeneous, wi h possibly several differen genes involved in differen families. One of hese genes is X-linked, and he corresponding locus was found o map wi hin an 18-cM in erval on band Xp22 (Robin et al., 1995). The gene has now been cloned from a pericen ric inversion wi h breakpoin s in Xp22 and Xq26 found in affec ed members of an Opi z syndrome family, and has been designa ed MID1 (Midline 1). MID1 is ubiqui ously expressed as a 7-kb ranscrip in fe al and adul human issues. I encodes a member of he B-box family of pro eins con aining a RING finger mo if, which is involved in pro ein in erac ion. Mu a ions of MID1 were found in unrela ed Opi z syndrome pa ien s, confirming i s pa hogenic role in his condi ion. The second locus involved in Opi z syndrome, iden ified hrough linkage analysis of o her families, maps o chromosome 22q in a 32-cM in erval wi hin band 22q11.2, which coincides wi h he velo-cardio-facial – DiGeorge syndrome region (Robin et al., 1995). A comparison of clinical findings in Xlinked cases and in 22-linked cases did no show any significan pheno ypic differences (Robin et al., 1996).

3. Trea men Opi z syndrome pa ien s should be rea ed for heir anomalies or complica ions hereof. Surgical in erven ion may be indica ed for he correc ion of a clef lip,

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he laryngo-esophageal defec s, he umbilical hernia, and he hypospadias. Swallowing difficul ies may require a fundoplica ion of he s omach.

E. Aarskog – Scott syndrome Aarskog – Sco syndrome, also known as faciogeni al dysplasia (FGDY), owes i s eponym o he au hors who described i independen ly a abou he same ime (Aarskog, 1970; Sco , 1971). The more descrip ive acronym FGDY nicely summarizes he major componen s of he clinical pheno ype, s ressing he mulisys emic involvemen . Familial cases sugges ed ha he syndrome is gene ic in origin, wi h an X-linked mode of ransmission (Gorlin et al., 1990). X-linkage was fur her proven by he observa ion of pa ien s carrying an X – au osome ransloca ion involving he p arm of he X chromosome (Bawle et al., 1984; Glover et al., 1993).

1. Clinical pheno ype Grow h re arda ion is a cons an fea ure of Aarskog – Sco syndrome, wi h mos pa ien s reaching an adul heigh below he hird cen ile. The hands are dispropor iona ely shor , wi h some degree of webbing be ween fingers. Mos ypical is he hyperex ensibili y of he proximal in erphalangeal join s and flexion of he dis al join s. A single ransverse palmar crease and fif h-finger clinodac yly are of en presen . Similarly, fee are shor and broad wi h splayed oes. The face is ypically rounded, wi h a broad forehead and small chin. There are hyperelorism, epican hic folds, downslan ing of he eyes, p osis of he upper eyelids, a shor nose wi h an ever ed nares, long, fla phil rum, and a cupid’s bow shape o he upper lip. The ears are usually low-se and pos eriorly angula ed and have a hick lobe. The ee h may show delayed erup ion and enamel hypoplasia. Pec us excava um and umbilical and inguinal hernias are rela ively common findings. Urogeni al anomalies include shawl scro um, cryp orchidism, hypospadias, and kidney hypoplasia. X-ray s udies of he skele on have shown delayed bone age and a number of anomalies affec ing mos ly he hands and spine. There are hypoplasia of he erminal phalanges of he fingers in a majori y of pa ien s and cervical spina bifida occul a or o her ver ebral defec s, such as hypoplasia of he firs cervical ver ebra and segmen a ion defec s. Men al developmen is usually normal, wi h only a few cases showing mild delay. Carrier mo hers may show some a enua ed manifes a ions of he syndrome, including shor ness of s a ure and of hands, round face, and hyperelorism. An excellen clinical descrip ion of he syndrome and a horough


review of he li era ure can be found in Syndromes of the Head and et al., 1990).

77 eck (Gorlin

2. Gene ics The mu an gene responsible for Aarskog – Sco syndrome was mapped o he pericen romeric region of he X chromosome bo h by linkage analysis in informa ive families (Por eous et al., 1992; S evenson et al., 1994) and by he observa ion of an X;8 reciprocal ransloca ion in a mo her and son showing clinical manifes a ions of he syndrome (Bawle et al., 1984). The X chromosome breakpoin of his ransloca ion was subsequen ly localized o a region in band Xp11.21 flanked by markers ALAS2 and DXS323 (Glover et al., 1993). This finding paved he way o he cloning of FGD1, a candida e gene for he syndrome (Pas eris et al., 1994). FGD1 encodes a pro ein of 961 amino acids ha shows s rong homologies o he guanine nucleo ide exchange fac ors Rho/ Rac and con ains a zinc-fingerlike region as well as wo SH3-binding regions. Pro eins of his family are known o be involved in grow h regula ion and signal ransduc ion. In fac , FGD1 was found expressed in a varie y of fe al issues, including hear , brain, lung, and kidney issues. FGD1 is runca ed by he previously men ioned X;8 ransloca ion, and a produc ive mu a ion was found o segrega e in affec ed members of a family by he inser ion of a guanine residue a nucleo ide 2122. The resul ing frameshif mu a ion was predic ed o cause a ransla ional runca ion a residue 469 (Pas eris et al., 1994). All hese findings are s rong evidence ha FGD1 is indeed he gene responsible for Aarskog – Sco syndrome, a no ion suppor ed by he recen observa ion of a missense mu a ion segrega ing wi h he disease in ano her affec ed family (Neri et al., 1998b).

3. Trea men Once again, here is no specific rea men for Aaskorg – Sco syndrome. In erven ions should be direc ed a hose anomalies ha may become clinically relevan , for example, severe palpebral p osis and hypospadias.

F. Coffin – Lowry syndrome Coffin – Lowry syndrome (CLS) owes i s name o he au hors who independen ly described i 5 years apar . The firs descrip ion was by Coffin et al. in 1966 and he second by Lowry et al. in 1971. However, i was Tem amy et al. (1975) who, in repor ing eigh pa ien s from hree differen families, recognized ha he pa ien s had he same condi ion previously described by hese au hors

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and ha i was indeed one and he same disorder, probably inheri ed as a sexlinked rai . Since hen, several o her pa ien s have been described, sugges ing ha he syndrome may no be very rare. Thanks o hese repor s, especially hose of Hun er et al. (1982), Gilgenkran z et al. (1988), and Young (1988), Coffin – Lowry syndrome is pheno ypically well delinea ed. Several family s udies suppor inheri ance as an X-linked dominan rai wi h reduced expression in he he erozygous females. The gene locus was ini ially mapped o he Xp22 region (Hanauer et al., 1988; Par ing on et al., 1988; Bird et al., 1995) and subsequen ly cloned (Trivier et al., 1996).

1. Clinical pheno ype According o one of he original descrip ions (Lowry et al., 1971), CLS consis s of “men al re arda ion, small s a ure, re arded bone age, hypo onia, apering fingers, a charac eris ic facies which includes hyper elorism, up urned nares, and prominen fron al region, and possibly arres ed hydrocephalus.” Grow h seems o be normal prena ally bu is defini ely delayed pos na ally. Shor s a ure is eviden from early childhood, wi h adul heigh below he hird cen ile in vir ually all affec ed males and in a large propor ion of carrier females. Re arded bone age was observed in nearly all repor ed cases. Microcephaly has been no ed in only a few cases, possibly hose ha do no have hydrocephaly. Ven ricular dila a ion was repor ed in several pa ien s, al hough i is no clear whe her his is due o increased in racranial pressure or ra her o cerebral a rophy (hydrocephalus ex vacuo). The face is qui e dis inc ive and i can be described as coarse. There is a prominen forehead and hick supraorbi al ridges, hyper elorism, narrow and downslan ing palpebral fissures, and a broad nose wi h a hick sep um and an ever ed nares. The phil rum is high and narrow, and here is den al malocclusion wi h large and widely spaced upper incisors. The hands have a qui e ypical appearance. They are broad, sof , and puffy, wi h broad erminal phalanges and join hyperlaxi y. Similar findings can be observed in he fee . A charac eris ic horizon al crease in he hypo henar region has been no ed in many pa ien s. Geni alia are normal and puber al developmen seems o occur normally. A skele al survey in many pa ien s has consis en ly shown skull hyperos osis, a drums ick aspec of he erminal phalanges of he fingers, and an involvemen of he spine, including an erior webbing of he ver ebral bodies and decreased in erver ebral spaces resul ing in severe kyphoscoliosis. Several of he clinical findings repor ed so far have sugges ed ha in CLS here may be an involvemen of he connec ive issue. Reduced elas in and abnormal vacuolaion were observed by Tem amy et al. (1975) in skin biopsies, and an abnormali y of chondrocy es had already been men ioned by Coffin et al. (1966) in


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heir original repor . Au opsy findings of panacinar emphysema, nodular ransforma ion of he liver, renal microcys s, and pleural calcific plaques suppor he concep of a generalized connec ive issue disorder as par of he syndrome. A visceral neuropa hy, which could have been he cause of in es inal pseudoobs ruc ions or diver icular disease, was also observed pos mor em (Machin et al., 1987). Severe MR is one of he hallmarks of he syndrome; IQ values are of en below 20 and here is a vir ual absence of speech in he majori y of affec ed males. Generalized epilep iform seizures have been repor ed (Fryns et al., 1977), as well as sensorineural hearing loss and prema ure ca arac (Har sfield et al., 1993). In affec ed females he pheno ype is much milder, including mild men al delay, shor ness of s a ure, and facial changes such as a broad and prominen forehead, broad nose, and fleshy, ever ed lips.

2. Diagnosis The diagnosis of CLS is based on he ypical facial and hand changes and can be confirmed by mu a ional analysis. The differen ial diagnosis is as wi h o her syndromes also charac erized by MR, coarse face, and shor s a ure. Borjeson – Forssman – Lehmann syndrome, also X-linked, can be dis inguished on he basis of obesi y and hypogeni alism. Pa ien s wi h A kin – Flai z syndrome (1985), as well as hose wi h a similar condi ion repor ed by Clark and Barai ser (1987), have macrocephaly and macro-orchidism. In sporadic cases i is probably wor h ruling ou Williams syndrome, for which a simple labora ory es now exis s. Differen ial diagnosis wi h he ATRX syndrome should also be considered. The wo condi ions can now be dis inguished on he basis of molecular es s.

3. Gene ics Early linkage s udies assigned he CLS locus o band Xp22 in a 13-cM in erval be ween markers DXS43 and DXS41 (Hanauer et al., 1988; Par ing on et al., 1988). This localiza ion was progressively narrowed, firs o a 7-cM in erval be ween DXS207 and DXS274 (Biancalana et al., 1992) and hen o a 5-cM in erval in band Xp22.1. (Biancalana et al., 1994). More recen ly, he observaion of a recombina ion in a carrier female from a Bri ish family has fur her reduced he cri ical region o 3.4 cM be ween markers AFM291wf5 and DXS365 (Bird et al., 1995). This was an impor an s ep oward he cloning of a candida e gene, which was accomplished shor ly hereaf er. The CLS gene encodes he 740-amino-acid pro ein RSK-2, a ribosomal S6 kinase belonging o a family of grow h-fac or-regula ed serine – hreonine kinases. This pro ein has a role in he regula ion of cell prolifera ion and differen a ion. Dele ions, nonsense, missense, and splice-si e mu a ions were found in a number of pa ien s

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(Trivier et al., 1996). All families s udied so far are linked o he Xp22 locus, consis en wi h he no ion ha he CLS is gene ically homogeneous.

4. Trea men There is no specific rea men for CLS. In erven ions will be symp oma ic for any problems ha migh arise. A favorable social milieu is probably helpful in minimizing he progressive de eriora ion, in erms of men al re arda ion, ha has been repor ed in some pa ien s. Wi hin families, carrier females mus be properly counseled, and prena al diagnosis can be offered in informa ive cases.

III. NONSYNDROMAL XLMR (MRX) Nonsyndromal XLMR includes, by defini ion, hose condi ions in which MR is no accompanied by dis inc ive clinical signs. These condi ions can be recognized only if hey presen as familial cases wi h X-linked inheri ance, and can be dis inguished from each o her based only on linkage o differen polymorphic markers of he X chromosome. Each individual en i y is designa ed by he acronym MRX, followed by a progressive number (MRX1, MRX2, e c.). A presen , he MRX coun o als 59 en ries (Table 3.1), bu his number changes rapidly. A comple e lis of published MRXs can be found in he review of Lubs et al. (1999); an ideogram of he X chromosome wi h he localiza ions of each MRX is depic ed in Figure 3.2. I is immedia ely apparen ha here are large regions of overlap, sugges ing ha ul ima ely some MRXs ha are now separa ed will be lumped, jus as happened o MRX41 and MRX48 (vide infra). In fac , based on heir regional assignmen , no more han 10 of he curren ly mapped, pu a ive loci could accoun for all MRXs. Only he cloning of individual genes from he affec ed families will allow he emergence of a clear pic ure and con ribu e o he exac coun of hose genes in he X chromosome ha can cause MR. So far, only four genes have been cloned, FMR2, GDI1, OPH 1, and PAK3.

A. FMR2 The FMR2 gene coincides wi h he fola e-sensi ive fragile si e FRAXE, approxima ely 600 kb dis al o he FRAXA si e. Ac ually, he firs families carrying a mu a ion of FMR2 were ascer ained as fragile X syndrome families by es ing posi ive o he cy ogene ic fragili y assay. However, subsequen molecular analysis failed o show a mu a ion of he FMR1 gene. In 1993, Knigh et al. cloned FMR2 from he fragile si e FRAXE and found ha he mu a ional mechanism is essen ially iden ical o ha of FMR1. Even hough in he promo er region of


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Figure 3.2. X chromosome ideogram wi h he known localiza ions of genes responsible for nonsyndromal XLMR (MRX). The bars indica e indica e he locus assignmen for hose pu a ive genes ha have been regionally mapped. The arrows indica e he posi ion of he cloned genes.

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he wildype gene here is a sequence of GCC repea s ranging in number from 6 o 25 and affec ed individuals have more han 200 copies, wi h hyperme hyla ion of he CpG island, heir physical pheno ype is no dis inc ive and MR usually varies from mild o borderline.

B. GDI1 The gene GDI1 encodes ␣-GDI, a pro ein highly expressed in he brain and whose func ion is o con rol he recycling of he Rab-GTPases across cell membranes (Bione et al., 1993; Wu et al., 1996), wi h special emphasis on i s role as regula or of neuro ransmi er release (Gepper et al., 1994). D’Adamo et al. (1998) found mu a ions in GDI1 in affec ed individuals from families MRX41 and MRX48 ha map in Xq28. In one family (MRX41) he mu a ion was a T : C ransi ion a posi ion 433 of he cDNA, resul ing in subs i u ion of a leucine wi h a proline in posi ion 92, which was responsible for reduced binding and recycling of RAB3A. The mu a ion in he o her family (MRX48) was a C : T ransi ion a posi ion 366 of he cDNA, causing he inser ion of a prema ure s op codon. Lymphoblas s of affec ed individuals did no express ␣GDI, as expec ed.

C. OPHN1 OPH 1 is a newly charac erized gene cloned from a men ally re arded female pa ien carrying an X;12 ransloca ion wi h a breakpoin in Xq12 (Billuar et al., 1998). The gene, which was found highly expressed in fe al brain, encodes a 91-kDa pro ein of 802 amino acids (oligophrenin-1) charac erized by he presence of a domain ypical of a Rho-GTPase-ac iva ing pro ein involved in signaling pa hways ha affec differen ia ion and migra ion of neurons. The pa hogenic role of a loss of func ion of his pro ein was confirmed in an independen family (MRX60), in which affec ed individuals were shown o have a one-base-pair dele ion corresponding o nucleo ide 1578.

D. PAK3 The PAK3 gene, originally cloned in he mouse (Manser et al., 1995), is a member of he family of p21-ac iva ing kinase genes. I encodes PAK3, a serine – hreonine kinase wi h a cri ical role in linking Rho-GTPases o he ac in cy oskele on. Allen et al. (1998) cloned he human gene and showed ha i is mu a ed in affec ed individuals from family MRX30 and maps o Xq22. The mu a ion consis s of a C : T ransi ion ha inser s a s op codon (TGA) in place of an arginine codon (CGA), corresponding o amino acid 419. This resul s in a runca ed pro ein ha lacks a region essen ial for normal kinase


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func ion. The pa hogenic role of PAK3 is fur her suppor ed by he observa ion ha i is highly expressed in fe al brain bu no in o her fe al organs.

IV. CONCLUSION Cloning and charac erizing XLMR genes will have a number of consequences. I will improve our insigh in o he nosology of MR; i will genera e a useful model for he searching ou of au osomal MR genes; i will shed ligh on he pa hophysiology of complex clinical condi ions; and i will provide new ools for prena al diagnosis, carrier de ec ion, and gene ic counseling. Ul ima ely, i may lead o he developmen of gene herapy, a leas in some cases. The impor ance of discovering genes whose mu a ions cause “pure” MR canno be overemphasized. Two aspec s appear o be par icularly significan . One is ha a common pa hway seems o emerge hrough which he produc s of differen genes opera e in he cen ral nervous sys em. Rho and Rab GTPases are cri ical fac ors in an in rica e ne work of in ercellular in erac ions and play a cen ral role in he con rol of neural cell differen ia ion, migra ion, and signaling (An onarakis and Van Aels , 1998). Ano her impor an aspec is he obvious implica ion ha hrough malfunc ion we may learn more abou normal func ion, ha is, ha unders anding men al re arda ion will ul ima ely lead us o unders and normal brain func ioning and he molecular bases of in elligence.

Acknowledgments The personal work quo ed in his review was par ially suppor ed by a gran from Tele hon, I aly (No. E-245). P.C. is he recipien of a Tele hon in erna ional fellowship. The au hors are indeb ed o Mrs. Luciana Ama o for skilled secre arial assis ance and for yping he manuscrip .

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4

Pharmaceutical Perspectives of Nonviral Gene Therapy Ram I. Mahato* Copernicus Therapeu ics, Inc. Cleveland, Ohio 44106

Louis C. Smith and Alain Rolland Valen is, Inc. The Woodlands, Texas 77381

I. Why a Gene-Based Approach for Pro ein Therapy? A. Why Soma ic Gene Therapy? B. Gene Therapy Approaches C. Plasmid-Based Gene Medicines D. Advan ages of Gene Medicines II. Commercializa ion of Gene Therapy Produc s A. Commercial Challenges B. Regula ory Issues C. Clinical Trials III. Basic Componen s of Gene Expression Plasmids A. Bac erial Elemen s B. Mammalian Transcrip ion Uni C. Promo er/Enhancer D. Un ransla ed Regions (UTR) E. In ron F. Poly(A) Signal G. Gene Swi ches

* Corresponding au hor: Telephone: (216) 231-0227. Fax: (216) 231-9477. E-mail: maha [email protected]. Advances in Genetics, Vol. 41 Copyrigh 1999 by Academic Press All righ s of reproduc ion in any form reserved. 0065-2660/99 $30.00

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R. I. Mahato, L. C. Smith, and A. Rolland

IV. Gene Delivery Sys ems A. Lipid-Based Gene Delivery B. Pep ide-Based Gene Delivery C. Polymer-Based Gene Delivery V. Formula ion Fac ors Influencing Gene Transfer A. DNA Topology B. DNA Condensa ion C. DNA Condensing Agen s D. DNA Aggrega ion VI. Biodis ribu ion and Pharmacokine ics of Plasmids A. Ana omical and Physiological Considera ions B. Influence of (Pa ho)physiology on Biodis ribu ion C. Biodis ribu ion and Pharmacokine ics of Plasmid DNA VII. In racellular Trafficking of Gene Medicines A. Cellular Up ake Mechanisms B. In racellular Trafficking C. Nuclear Envelope and Nuclear Pore Complex D. Nuclear Localiza ion Signal (NLS) Sequence VIII. Biological Oppor uni ies for Gene Therapy A. Sys emic Gene Therapy B. Cancer Gene Therapy C. Pulmonary Gene Therapy D. Gene ic Vaccines IX. Concluding Remarks

ABSTRACT The use of nonviral plasmid-based gene medicines represen s an a rac ive in vivo gene ransfer s ra egy ha is simple and lacks many risks ha are inheren o viral sys ems. Commercializa ion of gene medicines requires a horough analysis of business oppor uni ies, unme clinical needs, compe i ive produc s under developmen , and issues rela ed o in ellec ual proper y. Syn he ic gene delivery sys ems are designed o con rol he loca ion of a gene wi hin he body by affec ing dis ribu ion and access of a gene expression sys em o he arge cell, and/or recogni ion by a cell surface recep or and up ake followed by in racellular and nuclear ransloca ion. Plasmid-based gene expression sys ems are designed o con rol he level, fideli y, and dura ion of in vivo produc ion of a herapeu ic gene produc . This review will provide insigh s in o he poen ials of plasmid-based gene herapy and cri ical evalua ion of gene delivery sciences and clinical applica ions of gene medicines. 1999 Academic Press.

4. Pharmaceutical Perspectives of Nonviral Gene Therapy

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I. WHY A GENE-BASED APPROACH FOR PROTEIN THERAPY? Each cell in he body has he abili y o produce housands of differen pro eins ha are essen ial for cellular s ruc ure, func ion, and grow h. Genes are segmen s of deoxyribonucleic acid (DNA) and provide informa ion needed by he cells for pro ein produc ion (Berg and Singer, 1992; Drlika, 1996). The pro ein expressed in a par icular cell may be limi ed o he cell i self (autocrine or cis func ion), i could be secre ed and ac on o her cells (paracrine or trans funcion), or i could be secre ed in o he blood or lymph nodes (endocrine func ion) (Vega, 1995). Plasmid expression sys ems are being cons ruc ed ha lead o he secre ion of a herapeu ic gene produc in o he sys emic circula ion for an endocrine effec . Expression plasmids are also being cons ruc ed o express genes locally a he si e of adminis ra ion for autocrine or paracrine effec s. The disease arge s range widely, including gene ic diseases (cys ic fibrosis, hemophilia, Duchenne muscular dys rophy), me abolic disorders (e.g., diabe es and hyper choles erolemia), and differen forms of cancer (Rolland and Felgner, 1998).

A. Why somatic gene therapy? The body con ains a ple hora of pro eins (including enzymes, hormones, and recep ors) ha regula e biological func ions. The absence or overproduc ion of a specific pro ein can lead o a varie y of clinical manifes a ions, depending on he s ruc ural or func ional role ha he pro ein normally plays in he body. Many severe and debili a ing diseases (e.g., diabe es, hemophilia, cys ic fibrosis) and several chronic diseases (i.e., hyper ension, ischaemic hear disease, as hma, Parkinson’s disease, mo or neuron disease, mul iple sclerosis) remain inadequa ely rea ed by conven ional pharmaceu ical approaches (Dalgleish, 1997). Recombinan DNA echnology has allowed he large-scale produc ion and biological charac eriza ion of several herapeu ic pro eins, including granulocy e-macrophage colony s imula ing fac or (GM-CSF), ery hropoie in (EPO), in erleukins, insulin-like grow h fac or-I (IGF-I), human fac or VIII and IX, and issue plasminogen ac iva or ( -PA). However, he clinical use of many pro ein drugs is limi ed by heir inappropria e concen ra ion in blood, poor oral bioavailabili y, manufac uring cos , chemical and biological ins abili y, and/or rapid hepa ic me abolism and renal excre ion (Tomhnson, 1992). In addi ion, few pro ein drugs can efficien ly en er arge cells unless adminis ered a very high doses, which can lead o oxic side effec s. These limi a ions lead o heir frequen adminis ra ion wi h an increased rea men cos and reduced pa ien compliance (Woodley, 1994). Gene herapy is a me hod for he rea men or preven ion of disease ha uses genes o provide he pa ien ’s soma ic cells wi h he gene ic informaion necessary o produce specific herapeu ic pro eins needed o correc or o

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modula e a disease. The promise of soma ic gene herapy is o overcome limi aions associa ed wi h he adminis ra ion of herapeu ic pro eins, including low bioavailabili y, inadequa e pharmacokine ic profiles, and high cos of manufacure. Providing a herapeu ic gene as a “predrug” o a pa ien o allow ei her he produc ion of herapeu ic pro eins ha may be difficul o adminis er exogenously or he inhibi ion of abnormal pro ein produc ion may circumven some limi aions associa ed wi h he use of recombinan herapeu ic pro eins (Ledley, 1996).

B. Gene therapy approaches Gene herapy approaches curren ly in developmen may be dis inguished by he me hods used o ransfer or deliver herapeu ic genes o he pa ien . The me hods include he use of (i) cells ha have been al ered ex vivo (ou side he body) wi h viruses (such as re rovirus, adenovirus, adenoassocia ed virus, herpes simplex virus, and vaccinia virus) or o her gene ransfer me hods (e.g., elec ropora ion) and (ii) in vivo (inside he body) wi h viruses, which have been gene ically modified so ha hey canno mul iply and infec o her cells or wi h syn he ic formula ions of plasmids (Eck and Wilson, 1996). Ex vivo approaches have significan clinical and commercial limi aions. These approaches involve complex procedures whereby he arge cells mus be removed from he pa ien , modified wi h he herapeu ic gene, expanded in number, cleansed of con aminan s, and hen rein roduced in o he pa ien . In addi ion, mos ex vivo gene herapy procedures produce a permanen gene ic al era ion of he cell, which generally precludes he abili y o modula e rea men in response o herapeu ic needs. Al hough a number of viral gene herapies are curren ly used for direc in vivo adminis ra ion, safe y issues may limi heir fur her developmen . These include inflamma ion as well as cellular and humoral immune responses. There are also concerns abou he possibili y of in egra ion of viral vec ors in o he hos genome (e.g., re roviral vec ors) (Miller and Vile, 1995). Nonviral me hods involve he direc adminis ra ion of plasmid-based gene expression sys ems. The plasmids con ain a herapeu ic gene, as well as gene ic sequences, ha direc he cell o ranscribe and ransla e his gene accura ely and efficien ly in o a herapeu ic pro ein. In he majori y of cases, plasmid-based gene herapy requires he use of a syn he ic gene delivery sys em o con rol he delivery of he gene expression sys em from he si e of adminisera ion in he body o he nucleus of specific arge cells. Nonviral gene delivery sys ems can be adminis ered o pa ien s by conven ional rou es, such as direc injec ion, inhala ion, or in ravenous injec ion, hus providing increased safe y over viral gene herapy approaches. Moreover, he nonviral gene delivery sys ems can be degraded by he body using na ural processes, allowing he gene medicine o be adminis ered repea edly (Maha o et al., 1997a).


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C. Plasmid-based gene medicines A gene medicine con ains hree componen s: a herapeu ic gene ha encodes a specific herapeu ic pro ein, a plasmid-based gene expression sys em ha con rols he func ioning of a gene wi hin a arge cell; and a gene delivery sys em ha con rols he delivery of he plasmid expression sys em o specific loca ions wi hin he body. The gene and he gene expression sys em are he componen s of he plasmid (Maha o et al., 1997b). The gene delivery sys em dis ribu es he plasmid o he desired arge cell, af er which he plasmid is in ernalized in o he cell by a number of mechanisms (e.g., phagocy osis, macropinocy osis, recep or-media ed endocy osis, and caveolae-media ed endocy osis) (Wolff et al., 1992; Friend et al., 1996; Laba -Moleur et al., 1996. Li and Huang, 1996). Once inside he cy oplasm, he plasmid can hen ransloca e o he nucleus, where gene expression begins, leading o he produc ion of a herapeuic pro ein hrough he s eps of ranscrip ion and ransla ion. The gene expression sys em can be engineered o con rol whe her he resul ing pro ein will remain wi hin he cell for an in racellular effec or will be secre ed ou of he cell for ei her a local or sys emic ac ion. The gene expression sys em can also be adjus ed o con rol he level of pro ein produc ion as well as he fideli y and dura ion of gene expression (Figure 4.1).

Gene Delivery Systems

Gene Expression Systems

Distribution

Amount Promoters RNA processing Synthetic intron 5’and 3’UTR

Stability Dispersion

Access

Regulation

Passive uptake Opsonization

Recognition

Proteins mRNA

Receptor-mediated

Timing

Trafficking

Persistence Drug-controlled

Endosomal release Decomplexation Nuclear entry

A

Cell-specificity GeneSwitch Fidelity Post-translation

Gene Expression

B

Figure 4.1. Spa ial and emporal modula ion of gene expression. (A) Gene delivery sys ems are designed o con rol he loca ion of a gene wi hin he body by affec ing dis ribu ion and access of a gene expression sys em o he arge cell recep or followed by in racellular and nuclear ransloca ion. (B) Plasmid-based gene expression sys ems are designed o con rol he level and dura ion of in vivo produc ion of a herapeu ic gene produc .

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D. Advantages of gene medicines Small molecular-weigh drugs usually func ion by in erac ing wi h pro eins hroughou he body. Pro ein drugs are large molecules ha generally ac as replacemen s for he body’s own pro eins. Bo h small molecular-weigh drugs and pro ein drugs are designed o ac on chemical recep ors on a cell’s surface. Shor , normally single-s rand, an isense olgonucleo ides are designed o inhibi he produc ion of aberran pro eins by hybridizing wi h he coding (sense) RNA. However, here is li le con rol of he pharmacokine ic profiles of small molecular-weigh drugs, pro ein drugs, and oligonucleo ides. These molecules are widely dis ribu ed hroughou he body and rapidly cleared hrough he kidney. The use of plasmid-based gene medicines is in ended o be analogous o conven ional medicines in erms of con rolled dosing, convenien sys emic or local adminis ra ion, and well-charac erized pharmacokine ics. Plasmid expression sys ems can persis for a defined ime in he nucleus as nonin egra ed episomes before hey are degraded. I should herefore be possible o use gene medicines like conven ional medicines. Gene medicines could be adminis ered repe i ively o a pa ien according o a dosing schedule ha ma ches he ex en and severi y of he disease, rea ing ei her acu e or chronic diseases. They are in ended o have low oxici y due o he use of syn he ic carriers and nonin egra ing plasmids. Al hough a single dose of curren gene medicines generally has a low herapeu ic effec , heir repea ed injec ions may be effec ive for several clinical arge s. Compared o viral vec ors, gene medicines presen several po en ial advan ages, including (i) low cos s, (ii) noninfec ivi y, (iii) absence of immunogenici y, (iv) good compliance, (v) well-defined charac eris ics and (vi) possibili y of repea ed clinical adminis ra ion (Maha o et al., 1999).

II. COMMERCIALIZATION OF GENE THERAPY PRODUCTS Gene delivery sys ems need o be developed o increase and main ain an adequa e level of in vivo gene expression over a defined period of ime. The even ual goal is o achieve cell- or issue-specific expression and o regula e gene expression wi hin he cells. A basic unders anding of disease pa hogenesis is required o define he mechanisms by which gene defec s lead o disease. Fur hermore, knowledge of disease (pa ho)physiology is crucial for be er unders anding of appropria e arge cells for effec ive herapy, levels of gene expression required for clinical efficacy, and regula ion of gene expression. Animal models also need o be developed o es experimen al hypo heses and specific herapies prior o rials in human (Ross et al., 1996). The produc ion of gene herapy produc s as well as heir research and developmen ac ivi ies are subjec o regula ion for safe y, efficacy, and quali y by governmen al au hori ies in he Uni ed S a es and o her coun ries. Safe y and regula ory aspec s of gene herapy can be addressed along hree lines: (i)


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experimen al and preclinical research, (ii) manufac uring of gene medicines, (iii) clinical rials and developmen . Gene herapy represen s a field of daun ing complexi y for he regula ory au hori ies (Cohen-Haguenaur, 1996, 1997).

A. Commercial challenges The fundamen al commercial challenges facing gene herapy produc s as hey proceed o he marke will be o provide herapeu ic benefi wi hin he confines of an accep able safe y profile. Gene herapy is a new and rapidly evolving field. Major advances in gene ics and he abili y o con rol gene delivery and expression will bring revolu ionary novel herapeu ic me hods in he upcoming millennium. Many pharmaceu ical and bio echnology companies as well as academic ins i u ions are exploring he field of soma ic gene herapy. Rapid echnological developmen may produce po en ial produc s or echnologies ha could become obsole e before a company recovers i s research, developmen , and capi al expendi ures. Basic informa ion and echnological advances ha would normally be published in scien ific journals are of en delayed for incorpora ion in o pa en applica ions. Fur hermore, numerous pa en s are being issued ha cover he broad concep s of echnology (Figure 4.2), which can inhibi he developmen of new echnologies and produc s ha are direc ly applicable o a produc (Bossar and Pearson, 1995).

Figure 4.2. Rapid grow h of gene herapy in ellec ual proper y.

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There are curren ly no marke ed gene herapy produc s. The exis ing clinical da a on he safe y and efficacy of po en ial gene herapy produc s are s ill limi ed. Fur hermore, he resul s of preclinical s udies do no necessarily predic safe y or efficacy in humans. All of he po en ial produc s under developmen are in research, preclinical, or clinical developmen . These po en ial produc s will con inue o require significan addi ional research and developmen , as well as clinical inves iga ion effor s, prior o commercial use (Persidis and Tomczyk, 1997). Residual RNA, pro eins, and bac erial DNA are considered con aminan s and hus heir presence should be reduced or elimina ed in he produc according o defined specifica ions. Toxic chemicals such as e hidium bromide and cesium chloride should ei her be avoided in plasmid produc ion or heir amoun in he final produc should be quan ified. Gene expression is influenced by he plasmid forms and hus he percen age of supercoiled and linear DNA in he prepara ion should be quan ified (Hermann, 1996). Aberran expression of some pro eins in non arge organs may lead o an inappropria e ac iva ion of he immune sys em, resul ing in acu e or chronic inflamma ory and immune responses and po en ial damage of normal issues. Therefore, s udies should be conduc ed over reasonably long periods of ime o allow de ec ion of po en ial immune reac ions (Ledley, 1991).

B. Regulatory issues The marke ing of a new pharmaceu ical produc in he Uni ed S a es requires • Preclinical labora ory es s and in vivo preclinical s udies • Submission of an Inves iga ional New Drug (IND) applica ion o he FDA for human clinical es ing • Human clinical rials for es ablishing produc safe y and efficacy • Submission of a New Drug Applica ion (NDA) o he FDA for a Biologics License Applica ion (BLA) • FDA approval of he NDA or BLA prior o any commercial sale The Uni ed S a es is a leader in he developmen of safeguards for he clinical applica ion of human soma ic gene herapy, which is subjec o rigorous regula ion by he Food and Drug Adminis ra ion (FDA) (Kessler et al., 1993; Marcel and Grausz, 1997; Ledley, 1991; Cohen-Haguenauer, 1995). The Naional Ins i u es of Heal h Recombinan DNA Advisory Commi ee (RAC) serves in an advisory func ion and as a public forum for many gene herapy issues ra her han as a body involved in case-by-case approval. The clinical performance of gene ransfer experimen s is s ill in an early phase of developmen . As of June 1996, 161 clinical pro ocols have been approved in he Uni ed S a es and 46 rials in Europe (Mar in and Thomas, 1998). In he Uni ed


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S a es, such produc s are regula ed under he Federal Food, Drug, and Cosme ic Ac . As biological produc s, in addi ion, hey are subjec o cer ain provisions of his ac and are regula ed under he Public Heal h Service Ac . These laws and he regula ions promulga ed hereunder govern, among o her hings, es ing, manufac uring, safe y, efficacy, labeling, s orage, record keeping, adver ising, and promo ional prac ices involving drugs and biological produc s. A he FDA, he Cen er for Biologics Evalua ion and Research is responsible for he regulaion of biological produc s and has regula ed all gene herapy produc s o da e. Each herapeu ic produc con aining a par icular gene will likely be regula ed as a separa e biological produc , depending on i s in ended use and he FDA policies in effec a he ime. To commercialize any produc s, he company mus sponsor and file an IND applica ion for each proposed produc and will be responsible for ini ia ing and overseeing he clinical s udies o demons ra e he safe y and efficacy ha are necessary o ob ain FDA approval of any such produc s. Gene herapy is a novel me hod of rea men and hus regula ory requiremen s are cons an ly evolving and changing. Even if regulaory approvals are ob ained, hey may include limi a ions on he indica ed uses for which a produc may be marke ed. In addi ion, a marke ed produc is subjec o con inual FDA review. La er discovery of previously unknown problems may resul in res ric ions on he marke ing of a produc or wi hdrawal of he produc from he marke . Preclinical es s include labora ory evalua ion of he produc as well as animal s udies o assess he po en ial safe y and efficacy of he produc . Compounds mus be produced according o applicable curren Good Manufac uring Prac ices (GMP), and preclinical safe y es s mus be conduc ed by labora ories ha comply wi h FDA regula ions regarding Good Labora ory Prac ices (GLP). The resul s of he preclinical es s, oge her wi h manufac uring informa ion and analy ical da a, are submi ed o he FDA as par of an IND, which mus become effec ive before human clinical rials commence.

C. Clinical trials The gene herapy clinical rials aim a answering he crucial ques ions rela ed o he safe y and efficacy of a gene herapy produc (Ledley, 1991). Clinical rials involve he adminis ra ion of he inves iga ional produc o heal hy volun eers or o pa ien s under he supervision of a qualified principal inves iga or. Clinical rials are conduc ed in accordance wi h Good Clinical Prac ices (GCP) under pro ocols ha de ail he objec ives of he s udy, he parame ers o be used o moni or safe y, and he efficacy cri eria o be evalua ed. Each pro ocol mus be submi ed o he FDA as par of an IND. Fur her, each clinical s udy mus be reviewed and approved by an independen ins i u ional review board a he ins i u ion a which he s udy will be conduc ed. The ins i u ional review

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board will consider, among o her hings, e hical fac ors and he safe y of human subjec s. Clinical rials ypically are conduc ed in hree sequen ial phases, bu he phases may overlap. Phase I s udies involve he very firs es ing of a po en ial gene herapy produc in humans, wi h he aim of evalua ing safe y and olerabili y. Phase II s udies are modera e-scale dose-escala ion s udies designed o inves iga e efficacy in pa ien s while con inuing o accumula e safe y da a. Once a rea men has been shown o have a herapeu ic effec in a number of pa ien s, large-scale Phase III pivo al rials need o be under aken o provide adequa e s a is ical proof of efficacy and safe y of he effec observed in Phase II s udies and also o compare he new rea men wi h s andard herapies, if such herapies exis . The resul s of he pharmaceu ical developmen , preclinical s udies, and clinical s udies are submi ed o he FDA in he form of an NDA or BLA for approval of he manufac ure, marke ing, and commercial shipmen of he drug or biologic. The FDA may deny an NDA or BLA if applicable regula ory cri eria are no sa isfied, require addi ional es ing or informa ion, or require pos marke ing es ing and surveillance o moni or he safe y or efficacy of a produc . Among he condi ions for NDA or BLA approval is he requiremen ha he prospec ive manufac urer’s quali y con rol and manufac uring procedures conform o cGMP, which mus be followed a all imes. Foreign regula ory requiremen s governing human clinical rials and marke ing approval for drugs may vary from hose of he Uni ed S a es. In Europe, he approval process for he commencemen of clinical rials varies from coun ry o coun ry (Mar in and Thomas, 1998). Since he beginning of human gene herapy in 1990, a large percen age of pro ocols are s ill in Phase I. Indeed, of he 48 gene herapy rials ini ia ed since January 1996, 77% are in Phase I and 15% in Phase I/II. Several gene herapy rials did no produce expec ed resul s, and hus he FDA direc ed hem o under ake fur her preclinical evalua ion. This “back- o- he-bench” rend is apparen among he 23 exis ing cys ic fibrosis rials. Al hough 75% of hese rials were ini ia ed before Sep ember 1995, hey s ill remain in Phase I or I/II and none has ye reached phase III.

III. BASIC COMPONENTS OF GENE EXPRESSION PLASMIDS Plasmids are circular double-s randed DNA molecules, which can be manufacured a high yields in a cos -effec ive manner. Plasmids are chemically s able under appropria e condi ions for prolonged periods. Plasmid-based gene expression sys ems con ain a cDNA sequence coding for ei her a full gene or a minigene and several o her gene ic elemen s, including in rons, polyadenyla ion


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sequences, and ranscrip s abilizers o con rol ranscrip ion, ransla ion, and pro ein s abili y, and secre ion from he hos cell (Brown, 1990). Op ional componen s can be added o an expression plasmid, such as “gene swi ch,” which enables expression of he herapeu ic pro ein o be urned on or off a he ranscrip ional level by oral adminis ra ion of a specific low molecularweigh drug (Wang et al., 1994).

A. Bacterial elements Plasmids encode wo fea ures ha are impor an for heir propaga ion in bac eria. One is he bac erial origin of replica ion, usually derived from a high-copy plasmid, such as pUC plasmid (Vieira and Messing, 1982). The second required elemen is a selec able marker, usually a gene ha confers resis ance o an an ibio ic, such as kanamycin or neomycin. These “prokaryo ic” plasmid segmen s permi he produc ion of large quan i ies of a given plasmid in bac eria. The prokaryo ic origin of replica ion is a specific DNA sequence ha binds o fac ors ha regula e replica ion of plasmid and, in urn, con rol he number of copies of plasmid per bac erium.

B. Mammalian transcription unit The minimal ranscrip ion uni ha is required for he expression of a herapeuic pro ein consis s of 5⬘ enhancer/promo er ups ream of he gene encoding for he herapeu ic pro ein and a poly(A) signal downs ream of he gene. A he erologous in ron is of en inser ed in o he 5⬘ or 3⬘ un ransla ed region (UTR) of he ranscrip ion uni . This kind of “inser ion” leads o eleva ion in mRNA levels. A single in ron inser ed in o he 5⬘ UTR of he ranscrip ion uni is he mos common arrangemen .

C. Promoter/Enhancer A promo er is defined as a DNA region, usually a he 5⬘ end of a gene, ha binds o ranscrip ion fac ors and RNA polymerase during he ini ia ion of ranscrip ion of a gene a he correc nucleo ide si e. To da e, a ple hora of promo ers origina ing from eukaryo ic viruses, such as cy omegalovirus (CMV), simian virus 40 (SV40), Moloney murine leukemia virus (MoMLV), and Rous Sarcoma virus (RSV), are widely used because hey are known o be s rong promo ers (Qin et al., 1997). However, hese promo ers appear o show a decrease in in vivo ac ivi y when differen gene delivery sys ems are used. Cy okines, such as in erferon-␥ (IFN-␥) and umor necrosis fac or-␣ (TNF-␣), have been shown o inhibi ransgene expression from hese promo er-based gene expression sys ems (Gribaudo et al., 1995; Tzen and Sco , 1993; S ein et

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al., 1993). The combina ion of bo h IFN-␥ and TNF-␣ was shown o have s ronger inhibi ory effec s han ei her cy okine individually. However, hese cy okines were shown no o affec he ranscrip ion of he ac in promo er, which is a cellular promo er (Qin et al., 1997). Tissue-specific promo ers are designed o in erac wi h he ranscripion fac ors or o her nuclear pro eins ha are presen in he desired arge cells. The chicken skele al ␣-ac in promo er is an a rac ive candida e for a musclespecific plasmid-based expression sys em. The ␣-ac in promo er con ains posiive cis-ac ing elemen s ha are required for efficien ranscrip ional ac ivi y in myogenic cells. Skele al ␣-ac in accoun s for approxima ely 8% of he poly(A) RNA in adul chicken skele al muscle (Pe ropoulos et al., 1989, Hayward and Schwar z, 1986). Therefore, an ␣-ac in promo er could direc high expression of recombinan pro ein in skele al muscle. Muscle-specific expression of insulinlike grow h fac or-I (IGF-I), human grow h hormone (hGH), and human facor-IX (hFIX) has been demons ra ed af er in ramuscular adminis ra ion of plasmids ha encode hese genes and con ain skele al ␣-ac in (SK) promo er/ enhancer (Coleman et al., 1995; Alila et al., 1997; Anwer et al., 1998). To genera e higher levels of a gene produc , several sys ems have been developed ha can ranscribe ransgenes in he cy oplasm of ransfec ed cells. One of hese sys ems con ains a repor er gene driven by he bac eriophage T7 promo er and he purified T7 RNA polymerase (Elroy-S ein and Moss, 1990). T7 RNA polymerase does no en er he nucleus. The ranscrip ional ac ivi y has been shown o be grea er han ha of he eukaryo ic RNA polymerase. The level of expression increased wi h an increase in he amoun of T7 RNA polymerase from bac eriophage T7 specifically recognizes and s ar s ranscrip ion a a 19-bp DNA sequence: he T7 promo er. Expression casse es consis ing of a repor er gene under ranscrip ional con rol of a T7 promo er sequence can be used o genera e he repor er pro ein in cells ha express T7 polymerase.

D. Untranslated regions (UTR) The 5⬘ un ransla ed region (5⬘ UTR) is he region of he mRNA ranscrip ha is loca ed be ween he cap si e and he ini ia ion codon. The linkage be ween me hyla ed G residue and a 5⬘ o 5⬘ riphospha e bridge is known as he cap structure, which is essen ial for efficien ini ia ion of pro ein syn hesis. The 5⬘ UTR is known o influence mRNA ransla ion efficiency. In eukaryo ic cells, ini ia ion fac ors firs in erac wi h he 5⬘ cap s ruc ure and prepare he mRNA by unwinding i s secondary s ruc ure. An efficien 5⬘ UTR is usually modera e in leng h, devoid of s rong secondary s ruc ure, devoid of ups ream ini ia ion codons, and has AUG wi hin an op imal con ex . Any of he following fea ures


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ha influence he accessibili y of he 5⬘ cap s ruc ure o ini ia ion fac ors will influence mRNA ransla abili y (Kozak, 1991, 1992): • Ini ia ion codon (AUG) appears o be bes recognized when i is in he con ex of he sequence CCRCCAUGG wi h purine (R) a ⫺3 and/or guanidine (G) a ⫹4 (A of he AUG is numbered ⫹1). • If an AUG occurs alone, or an AUG in conjuc ion wi h a shor open reading frame, is loca ed be ween he cap si e and he genuine AUG, ransla ion will be inhibi ed. • Secondary s ruc ures of he UTRs inhibi ransla ion. • 5⬘ UTR leng hs ha are grea er han 32, bu less han 100, nucleo ides permi efficien recogni ion of he firs AUG. Mos na urally occuring 5⬘ UTRs are 50 o 100 nucleo ides in leng h. The 3⬘ UTR is defined as he mRNA sequences following he ermina ion codon. The 3⬘ UTR is hough o play a po en ial role in mRNA s abili y. AU-rich mo ifs are commonly found in he 3⬘ UTR of mRNA of cy okines, grow h fac ors, and oncogenes. These mo ifs are mRNA ins abili y elemen s and should be elimina ed for maximal levels of expression. This is usually accomplished by using s andard 3⬘ UTR sequences in place of he one found in he cDNA. The mos commonly u ilized 3⬘ UTR sequences are from he bovine grow h hormone and rabbi ␤-globin genes. Ano her approach is o minimize he leng h of he 3⬘ UTR by placing he hexanucleo ide of he poly(A) signal immedia ely downs ream of he s op codon (Har ikka et al., 1996). Inclusion of 5⬘ and 3⬘ UTR in rons may provide issue specifici y and long- erm gene expression. The 3⬘ UTR from he chicken skele al muscle ␣ac in gene con ains a s abiliza ion elemen ha improves mRNA s abili y and con rols grow h and differen ia ion of myoblas s. The pSK-hGH-SK expression plasmid was shown o produce ⬃3 – 5 imes more hGH han pSK-hGH-GH expression plasmid in he muscle (Figure 4.3) (Alila et al., 1997). Replacemen of 3⬘ UTR from hGH gene by SK of a muscle-specific hIGF-I expression sys em has also been shown o produce higher accumula ion and perinuclear localizaion of hIGF-I in he muscle af er in ramuscular injec ion (Alila et al., 1997).

E. Intron The pro ein-coding region in he eukaryo ic gene is of en in errup ed by s re ches of noncoding DNA called introns. Transcrip s from he in ronless genes are degraded rapidly in he nuclear compar men , leading o reduc ion in gene expression (Ryu and Mer z, 1989). Therefore, for maximal gene expression in eukaryo ic cells, a leas one in ron should be included wi hin he ranscrip-

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Figure 4.3. Muscle-specific gene expression sys em. (A) Cons ruc ion maps of human grow h hormone (hGH) gene expression sys ems pSK-hGH-GH and pSK-hGH-SK (driven by chicken skele al ␣-ac in promo er elemen s), ␣-SKP, chicken skele al ␣-ac in promo er, hGH, human grow h hormone genomic DNA, ␣-SKI, chicken skele al ␣-ac in in ron. (B) Levels of hGH in ibialis cranalis and gas rocnemius muscle ex rac 21 days af er he in ramuclular injec ion of pSK-hGH-SK or pSK-hGH-GH in 5% polyvinylpyrrolidone (PVP) in o hyposec omized ra s. Values are mean ⫾ S.E.M. (n ⫽ 5) (modified from Alila et al., 1997, wi h permission).

ion uni . Incorpora ion of in rons in o cDNA expression sys ems has been shown o enhance gene expression in cell cul ure up o 100-fold (Huang and Gorman, 1990).

F. Poly(A) signal The poly(A) ail is a homopolymeric s re ch of A residues added o he primary ranscrip by a nuclear mechanism known as polyadenylation. A poly(A) signal is required for he forma ion of he 3⬘ end of mos eukaryo ic mRNA. The signal direc s wo RNA processing reac ions: si e-specific endonucleoly ic cleav-


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age of he RNA ranscrip , and s epwise addi ion of adenyla es o he newly genera ed 3⬘ end o form he poly(A) ail. The efficiency of polyadenyla ion is impor an for gene expression, as ranscrip s ha fail o be cleaved and polyadenyla ed are rapidly degraded in he nuclear compar men . The poly(A) signals u ilized in gene expression plasmids are chosen from a se of mammalian poly(A) signals, such as bovine grow h hormone, rabbi ␤-globin, and SV40. These mammalian poly(A) RNAs have been ex ensively s udied and charac erized as s rong (Goodwin and Ro man, 1992). The bovine grow h hormone and rabbi ␤-globin poly(A) signals are essen ially equivalen in heir abili y o enhance gene expression and are more effec ive han he SV40 la e poly(A) signal (Yew et al., 1997). A modified version of he rabbi ␤-globin poly(A) signal yielded an approxima ely wofold increase in expression compared o he bovine grow h hormone poly(A) signal (Har ikka et al., 1996).

G. Gene switches Many endogenous pro eins are produced according o circadian rhy hms. Therefore, in vivo pulsa ile produc ion of cer ain herapeu ic pro eins may be beneficial for heir clinical applica ions. This can be achieved by including gene swi ches in a gene expression sys em o urn on or off he ranscrip ion of an adminis ered gene. In addi ion, a gene swi ch adds ano her safe y level in ha excessive gene expression can be con rolled. A gene swi ch is designed o be par of a gene expression sys em ha con ains bo h he gene swi ch and a herapeu ic gene. In he posi ive sys em, he arge gene will be inac ive un il he adminis ra ion of an exogenous compound or ligand. Such inducing agen s or drugs include proges erone an agonis s (Wang et al., 1997a), e racycline (Gossen et al., 1995), ecdysone (No et al., 1996), and rapamycin (Wang et al., 1997b). A common approach is ha a chimeric ranscrip ion ac iva or reversely binds o a arge gene cons ruc in response o he adminis ered drug or ligand. Several differen ypes of gene swi ches have been proposed, including one based on a modified proges erone recep or. This modified recep or has a dele ion of 42 amino acid residues a i s carboxy erminus and is linked o bo h he yeas Ga14 DNA-binding domain and he herpes simplex virus pro ein VP16 ranscrip ional ac iva ion domain (Figure 4.4). The mu a ed proges erone recep or does no bind o endogenous s eroids, bu selec ively binds o an iproges in drugs, such as mifepris one, which ac a very low concen ra ions (1 nM) as an agonis (Wang et al., 1997b). An iproges ins dis ribu e o mos cells in he body af er oral adminis ra ion and can bind he expressed gene swi ch pro ein, causing i s dimeriza ion in he cy oplasm. The ac iva ed gene swi ch ransloca es o he nucleus and hen binds o he Ga14-binding sequence ha is buil in o he gene expression sys em and con rols he expression of he herapeu ic gene. The herapeu ic gene produc would only be expressed when

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Figure 4.4. Mode of ac ion of an an iproges in gene swi ch. Gene expression plasmid con aining ransac iva or GLVP linked wi h liver-specific rans hrei in (TTR) promo er/enhancer was used o genera e ransac iva or mice. These mice were hen crossed wi h human grow h hormone (hGH) arge gene mice o genera e bigenic mice harboring bo h ransgenes (TTR-GLVP-hGH). Serum hGH was measured bo h prior o and 12 hr pos adminis ra ion of mifepris one (250 ␮g/kg, in raperi oneally). The hGH ransgene expression declined following me abolism of mifepris one (3 weeks la er) and could be reac iva ed following a fur her mifepris one injec ion (adap ed from Wang et al., 1997b, wi h permission).

he pa ien akes an an iproges in drug — for ins ance, orally — and gene expression is urned off when he an iproges in is elimina ed from he arge cells (Wang et al., 1997b). The expression of arge gene hGH has been shown o be dependen on he presence of mifepris one and correla ed wi h he rela ive issue-specific expression pa ern of he ransac iva or GLVP (Figure 4.4). O her gene swi ches have been cons ruc ed based on e racycline and rifamycin. Alhough very small doses of hese low molecular-weigh drugs are being used, heir chronic adminis ra ion is cer ainly a concern. For example, he slow clearance ra e of e racycline and he ac iva ion of ecdysone recep ors by muris erone may be harmful.

IV. GENE DELIVERY SYSTEMS Gene delivery sys ems are designed o con rol he loca ion of a gene wi hin he body by affec ing he dis ribu ion and access of a gene expression sys em o he


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arge cell, and/or recogni ion by a cell-surface recep or followed by in racellular rafficking and nuclear ransloca ion (Rolland, 1996). Gene delivery sys ems should serve bo h o pro ec a gene expression sys em from prema ure degradaion in he ex racellular milieu and o affec he nonspecific or cell-specific delivery o a arge cell. O her elemen s in a gene delivery sys em may facili a e he in racellular rafficking of a gene expression sys em. This sec ion describes he developmen of several lipid-, pep ide-, and polymer-based gene delivery sys ems.

A. Lipid-based gene delivery Liposomes are microscopic vesicles composed of uni- or mul ilamellar lipid bilayers surrounding aqueous compar men s. Plasmids may be incorpora ed in o anionic or neu ral liposomes o ensure pro ec ion agains degrada ion by nucleases in biological fluids, o con rol disposi ion profiles, and o enhance in racellular delivery (Ellens et al., 1984). However, he encapsula ion efficiency of plasmids is very low. The uncondensed plasmids are large compared o he in ernal diame er of he vesicles. pH-sensi ive liposomes are fusogenic a acidic pH and hus can be used o facili a e he endosomal disrup ion and subsequen release of plasmids in he cy oplasm. pH-sensi ive liposomes usually consis of dioleoylphospha idyle hanolamine (DOPE) and a lipophilic anionic componen con aining a i ra able head group. Examples are oleic acid, palmi oylhomocys eine, choles erol hemisuccina e morpholine sal (CHEMS), and dioleoylsuccinylglycerol (DOSG) (Wang and Huang, 1987a,b, Legendre and Szoka, 1992). The in vitro ransfecion efficiency of pH-sensi ive liposomes, composed of CHEMS:DOPE, has been compared o hose of non-pH-sensi ive liposomes, composed of CHEMS:dioleoylphospha idylcholine (DOPC) and phospha idylserine (PS):choles erol Non-pH-sensi ive liposomes were unable o ransfec expression plasmids in o monkey fibroblas CV-1 cells, whereas pH-sensi ive liposomes efficien ly ransfec ed plasmids in o hese cells (Legendre and Szoka et al., 1992). The pHsensi ive immunoliposomes have been shown o media e ⬃6 – 8 imes higher levels of hymidine kinase (TK) gene expression in o mouse lymphoma cells compared o non-pH-sensi ive immunoliposomes. Pro eoliposomes, also known as virosomes or chimerasomes, have been used for plasmid delivery o cells bo h in vitro and in vivo (Tikchonenko et al., 1988, Gould-Fogeri e et al., 1989). Pro eoliposomes incorpora e viral pro eins, fusogenic pep ides, nuclear pro eins, or nuclear localiza ion pep ides, which induce fusion of liposomes wi h he cell membranes and facili a e DNA release and ranspor hrough he cy oplasm. Cochlea es can also be used for plasmid delivery. A nega ively charged phospholipid such as phospha idylserine, phospha idic acid, or phospha idyl

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glycerol, in he absence or presence of choles erol, are u ilized o produce a suspension of mul ilamellar vesicles con aining plasmids, which are hen conver ed o small unilamellar vesicles by sonica ion. These vesicles are dialyzed agains buffered divalen ca ions (e. g., calcium chloride) o produce an insoluble precipi a e referred o as cochlea es. Cochlea es have been shown o encapsula e plasmid and enhance plasmid s abili y and ransfec ion efficiency (Mannino and Gould-Fogeri e, 1996). Since he in roduc ion of he ransfec ion reagen Lipofec in, a ca ionic liposome composed of 1:1 (w/w) mix ure of he ca ionic lipid N[1-(2,3dioleyloxy)propyl]-N,N,N- rime hylammonium chloride) (DOTMA) and he colipid DOPE (Felgner et al., 1987), many ca ionic lipid formula ions have been es ed for in vitro and in vivo ransfec ion of plasmids. The flexibili y in he design of ca ionic lipid s ruc ure has suppor ed he view ha ca ionic lipids can be used for gene ranfer in vivo (Felgner et al., 1987; Lasic and Temple on, 1996). Ca ionic lipids in erac elec ros a ically wi h he nega ively charged phospha e backbone of DNA, neu ralizing he charges and promo ing he condensa ion of DNA in o a more compac s ruc ure. Usually, ca ionic lipids are mixed wi h a zwi erionic or neu ral colipid such as DOPE (Farhood et al., 1995; Hui et al., 1996) or choles erol (Benne et al., 1995), respec ively, o form liposomes or micelles. The lipid mix ures are mixed in chloroform, which is hen evapora ed o dryness, followed by vacuum drying. Wa er is added o he dried lipid film and he hydra ed films hen ei her ex ruded or sonica ed o form ca ionic liposomes. Ca ionic liposomes have also been prepared by an e hanol injec ion echnique (Campbell, 1995). Inclusion of a colipid is no always essen ial. For ins ance, he ca ionic lipid DOTAP is ac ive in he absence of a colipid in a varie y of cells in vitro (McLachlan et al., 1994).

1. Ca ionic lipid s ruc ures The general s ruc ure of a ca ionic lipid has hree par s: (i) a hydrophobic lipid anchor group, which helps in forming liposomes (or micellar s ruc ures) and can in erac wi h cell membranes; (ii) a linker group; and (iii) a positively charged headgroup, which in erac s wi h plasmid, leading o i s condensa ion. The hydrophobic lipid anchors can be ei her fa y chains (e.g., derived from oleic or myris ic acid) or a choles erol group. Lipid anchors de ermine he physical proper ies of a lipid bilayer, such as membrane rigidi y and ra e of lipid exchange be ween lipid membranes. The linker group is an impor an componen and de ermines he chemical s abili y and biodegradabili y of a ca ionic lipid. The head groups of ca ionic lipid appear o be cri ical for ransfec ion and cy o oxici y of corresponding liposome formula ions. The ca ionic amphiphiles differ


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markedly in s ruc ure and may be single- or mul iple-charged as primary, secondary, er iary, and/or qua ernary amines. Examples are lipospermine, ca ionic choles erol, ca ionic de ergen , or lipopolysine. The physicochemical proper ies of plasmid/lipid complexes are s rongly influenced by he rela ive propor ions of each componen and he s ruc ure of he headgroup. Many effec ive ca ionic lipids con ain pro ona able polyamines linked o dialkyl or choles erol anchors. In he case of DOTMA, he hydrophobic domain is an oleoyl alcohol group ha is connec ed o a glycerol-like, hreecarbon backbone via an e her bond. A rime hyl qua ernary amine is linked direc ly o he hree-carbon backbone. 1, 2-dimyris yloxypropyl-3-dime hylhydroxye hyl ammonium bromide (DMRIE) is a deriva ive of DOTMA ha con ains a hydroxye hyl group a ached o he qua ernary amine. To increase he biodegradabili y of ca ionic lipids, a series of carbonic lipids have been syn hesized in which he e her bonds were replaced wi h es er bonds (Felgner et al., 1994). The s ruc ure of 1,2-bis(oleoyloxy)-3-( rime hylammonio)propane (DOTAP) is similar o DOTMA excep ha DOTAP con ains es er bonds (McLachlan et al., 1994). 3␤(N⬘, N⬘-dime hylaminoe hane)-carbamoyl] choleserol (DC-Chol) con ains a choles erol-linked via carbamoyl bond and e hyl group o a rime hyl, qua ernary amine (Gao and Huang, 1991). Several ca ionic lipids, including 2,3-dioleyloxy-N-[2(sperminecarboxyamido)e hyl]-N,Ndime hyl-1-propanaminium rifuoroace a e (DOSPA), con ain a spermine group for binding o DNA (Hawley-Nelson et al., 1993). Al hough ca ionic lipid-based gene delivery sys ems are being ex ensively inves iga ed and novel ca ionic lipid molecules are syn hesized rou inely, a defini e s ruc ure-ac ivi y rela ionship has no clearly emerged. Lee et al. (1996) recen ly a emp ed o es ablish a s ruc ure-ac ivi y rela ionship by sysema ically analyzing a large number of differen ca ionic lipid s ruc ures bo h in vitro and in vivo. Ca ionic lipids con aining 3-␤-(N4-spermine carbamoyl) choles erol (lipid #67) and 3-␤-(N4-spermidine carbamoyl) choles erol (lipid #53) in a “T-shape” configura ion ra her han a linear configura ion were found o be more effec ive han s ruc ures con aining only a single pro ona able amine (e.g., DC-Chol). However, here was a poor correla ion be ween in vitro and in vivo resul s wi h various lipids used in ha s udy (Lee et al., 1996). Al hough he ca ionic lipids dioc adecylamidoglyl spermine (DOGS) (Behr et al., 1989) and DOSPA also con ain spermine headgroups, hey were less ac ive han he car ionic lipid #67, possibly due o he following differences in heir s ruc ures: (i) he headgroup of lipid #67 is a ached o he linker via a ni rogen a om, whereas hose of DOGS and DOSPA are a ached hrough a carbon a om; (ii) bo h DOGS and DOSPA con ain a dialkyl chain as heir lipid anchor groups, whereas lipid #67 con ains a choles erol anchor; and (iii) lipid #67 is in a free-base form, whereas DOGS and DOSPA are in sal forms.

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2. Role of colipids Al hough subs an ial a en ion has been paid o he func ioning of ca ionic lipids, he role of colipids in gene ransfer is less well defined. DOPE has been shown o be more effec ive han several o her neu ral co-lipids a facili a ing ca ionic lipid-media ed ransfec ion. The neu ral colipid may facili a e escape of DNA from he endosome in o he cy oplasm and increase he abili y of he DNA o dissocia e from he plasmid/lipid complex. The effec of he colipid on gene ransfer depends on he ype of ca ionic lipid, molar ra io of ca ionic lipid o colipid, and he arge cell. DOPE is a phospholipid, which exhibi s a high endency o form inver ed hexagonal phase a acidic pH. Dioleoylphospha idylcholine (DOPC), a s ruc ural analog of DOPE, has no such ac ivi y (Farhood et al., 1995, Felgner et al., 1994). DOPE has been proposed o promo e fusion wi h he endosome membrane allowing release of DNA in o he cy oplasm (Fasbender et al., 1997). DOPE may be more effec ive in disrup ing membranes because i ends o assume a nonbilayer s ruc ure, whereas DOPC ends o form a s able bilayer (Wimley and Thompson, 1991).

3. Polyca ion/Lipid hybrid sys ems Since a plasmid has a hydrodynamic diame er of ⬃100 – 200 nm, depending on he number of i s base pairs and opology, i is difficul o produce compac par icles wi hou efficien DNA condensa ion (S ernberg et al., 1994). A hybrid DNA sys em consis ing of a polyca ion-condensed plasmid core and a lipid coa ing are being developed o allow efficien condensa ion of plasmid DNA. Ei her ca ionic or anionic lipids can be used in heir cons ruc ion. Moreover, anionic lipids may be conjuga ed wi h a arge ing ligand for issue-specific gene delivery. Gao and Huang (1996) prepared plasmid/lipid complexes by adding DNA o he mix ure of a polyca ion (such as poly-L-lysine or pro amine) and DC-Chol:DOPE liposomes. The resul ing suspension was hen subjec ed o sucrose densi y gradien ul racen rifuga ion o separa e he complex from free ca ionic liposomes.

4. In erac ion wi h biomolecules In vitro ransfec ion wi h ca ionic lipids is generally bes ob ained when plasmid/ lipid complexes bear a s rong posi ive charge. However, posi ively charged complexes may in erac wi h serum pro eins, lipopro eins, heparin, and glycosaminoglycans in he ex racellular ma rix, leading o he aggrega ion or release of DNA from he complexes even before reaching he arge cells. The poor correla ion be ween in vitro and in vivo ransfec ion ac ivi ies of plasmid/lipid complexes may be in par due o a differen biological environmen encompass-


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ing he cells (Remy et al., 1994). Ca ionic liposomes alone or complexed wi h plasmid have been demons ra ed o in erac wi h plasma complemen in vitro (Plank et al., 1996). Complemen ac iva ion may, herefore, lead o he coa ing of complex wi h complemen pro eins, hereby arge ing i o complemen recep ors presen on pulmonary endo helium. Al hough posi ively charged plasmid/lipid complexes ac iva e he complemen sys em o a considerable degree, no significan difference was seen in biodis ribu ion and gene expression beween he complemen -in ac and complemen -deple ed mice (Barron et al., 1998). This implies ha he in erac ion be ween plasmid/lipid complexes and complemen pro eins does no al er he proper ies of he injec ed complexes o he ex en ha gene delivery is al ered. The prepara ion of nega ively charged plasmid/lipid complexes or surface modifica ion of hese complexes wi h a s eric s abilizer such as polye hylelene glycol (PEG) are likely o fur her minimize and possibly avoid ac iva ion of he complemen sys em.

5. Targe specifici y Ca ionic lipid-based gene delivery sys ems lack arge specifici y, which resul s in low ransfec ion efficiency in cer ain issues due o he in erference from ca ionic lipid-binding macromolecules ei her in he circula ion or in he ex racellular ma rix. The elec ros a ic in erac ion be ween he posi ively charged plasmid/lipid complexes and he cell membrane usually does no provide cell specifici y. To circumven his problem, neu ral plasmid/lipospermine complexes con aining a rigalac olipid have been prepared and shown o efficien ly ransfec hepa oma HepG2 cells bearing asialoglycopro ein recep or. Addi ion of 25% (mol/mol) of he rian ennary galac olipid increased he ransfec ion efficiency by a housandfold, compared o he lipid-based sys em wi h no arge ing ligand (Remy et al., 1995). An efficien ransfec ion of ␤-galac osidase in o HeLa cells has been accomplished wi h he combina ion of ransferrin and ca ionic liposome Lipofec in, whereas Lipofec in alone had low ransfec ion efficiency (Cheng, 1995). Asialofe uin is an asialoglycopro ein con aining erminal galac osyl residues ha have been used o arge liposomes o he liver. (Hara et al., 1995) Temple on et al. (1997) demons ra ed sevenfold enhancemen in CAT expression in he liver when succinyla ed asialofe uin was added o preformed plasmid/DOTAP:Chol complexes o provide a ligand for hepa ic asialoglycopro ein recep or.

6. Toxici y Ca ionic lipids may no be readily me abolized or secre ed and, herefore, may accumula e in he body following adminis ra ion, po en ially producing undesirable side effec s. Lipids con aining es er or amide linkages are more likely o be

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rapidly me abolized han hose wi h e her linkages because of he presence of high concen ra ions of es erases and pep idases in he body. The degree of oxici y induced by plasmid/lipid complexes has been shown o be dose dependen , which was diminished wi h ime (San et al., 1993). A rela ively low concen ra ions of plasmid/lipid complexes, li le or no oxic effec has been repor ed in mice, rabbi s, and pigs af er sys emic or local injec ion of he complex (Canonico et al., 1994; S ewar et al., 1992; Nabel et al., 1992). There was no evidence of au oimmuni y, biochemical abnormali ies, or issue pa hology in hese animal models, and he gonadal issue did no con ain plasmids af er in ravenous and in ra-ar erial adminis ra ion (Nabel et al., 1992). Safe y s udies have also been performed in nonhuman prima es by once-a-week in ravenous injec ion of plasmid/DMRIE:DOPE complexes for hree weeks (San et al., 1993). The plasmid/lipid complexes did no produce au oimmuni y or oxici y, and here were no or mild changes in clinical chemis ries, hema ology, and issue his opa hology. A high doses, acu e inflamma ion was observed, primarily from he ca ionic lipid componen of he plasmid/lipid complex.

B. Peptide-based gene delivery For si e-specific delivery of plasmids, posi ively charged macromolecules such as poly(L-lysine) (PLL), his ones, pro amine, or poly(L-orni hine) may be linked o a cell-specific ligand and hen bound o plasmids via elec ros a ic in erac ion. The resul ing complexes re ain heir abili y o in erac specifically wi h arge cell recep ors, leading o recep or-media ed in ernaliza ion of he complex in o he cells. Recep or ligands curren ly being inves iga ed include glycopro eins (Wu and Wu, 1988; Findeis et al., 1994), ransferrin (Wagner et al., 1990), polymeric immunoglobulin (Ferkol et al., 1993), insulin (Hucke et al., 1990), epidermal grow h fac or (EGF) (Chen et al., 1994a), lec ins (Cheng and Yin, 1994), fola e (Go schalk et al., 1994), malaria circumsporozoi e pro ein (Ding et al., 1995), ␣2-macroglobulin (Schneider et al., 1996), CD3-T cell (Buschle et al., 1995), sugars (Chen et al., 1994b; Erbacher et al., 1996), in egrins (Har et al., 1995), hrombomodulin (Trube skoy et al., 1992), surfac an pro ein A and B (Ross et al., 1995; Baa z et al., 1995), mucin (Thurnher et al., 1994), and he c-ki recep or (Schwarzenberger et al., 1996). Si e-specific gene delivery and expression are influenced by he ex en of DNA condensa ion, he me hod of complexa ion, he molecular weigh s of bo h polyca ions and plasmid, and he number of ligand residues bound per polyca ion molecule (Erbacher et al., 1995).

1. Poly(L-lysine)-based sys ems Recep or ligands usually have been conjuga ed o poly(L-lysine) for si e-specific gene delivery. Galac osyla ed poly(L-lysine) (Gal-PLL) was, for ins ance, synhesized by reac ing PLL (⬃2000 kDa) wi h ␣-D-galac opyranosyl phenyliso-


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hiocyana e for delivery and expression of genes in o he ra hepa ocy es (Perales et al., 1994). Similarly, mannosyla ed poly(L-lysine) (Man-PLL), syn hesized using PLL (⬃2000 kDa) and ␣-D-mannopyranosyl phenyliso hiocyana e, has been shown o express genes in murine macrophages isola ed from peri oneal exuda es in vitro and macrophages residing in he liver and spleen of adul animals (Ferkol et al., 1996). Poly(L-lysine) is commercially available in molecular weigh s ranging from approxima ely 1 kDa o 300 kDa. However, he prepara ions are he erogenous, complica ing formula ion and charac eriza ion of DNA condensa es. Due o he high polydispersi y of poly(L-lysine), he individual molecular species of he polyca ion in erac wi h DNA wi h individually dis inc kine ics, for bo h elec ros a ic and hydrophobic in erac ions. The ex reme he erogenei y grea ly confounds bo h he kine ics of DNA/poly(L-lysine) in erac ion and he hermodynamic s abili y of he final DNA complexes. In addi ion o i s molecular he erogenei y, poly(L-lysine) is oxic o living cells in nM concen ra ions, which limi s i s general applicabili y (Smi h et al., 1998).

2. Syn he ic pep ide-based sys ems To avoid high cy o oxici y, molecular he erogenei y, and possible immunogenici y of poly(L-lysine), molecularly homogenous lysine-rich syn he ic pep ides have been used for gene ransfer. The ac ive si es of enzymes, recep or ligands, and an ibodies involve abou 5 o 20 amino acids. Thus, i should be possible o use small syn he ic pep ides o emula e he ac ive si es of viral pro eins and fomula e syn he ic DNA complexes ha are as efficien as viruses, wi h few limi a ions (Tomlinson and Rolland, 1996; Duguid et al., 1998). A syn he ic pep ide-based gene delivery sys ems has he po en ial abili y o ake advan age of specific pep ide sequences o overcome ex ra- and in racellular barriers o gene delivery. Specific sequences of in eres for gene delivery include DNA binding and pro ec ing pep ides, pep ide ligands for recep or-media ed up ake, pep ides wi h endosomoly ic proper ies o release DNA from he endosomes, and pep ides ha facili a e nuclear ranspor of DNA. Syn he ic pep ide-based gene delivery sys ems consis ing of a lysinerich DNA binding mo if and a pH-sensi ive endosome-ly ic mo if have been developed for in vivo gene delivery and expression (Tomlinson and Rolland, 1996; Wadhwa et al., 1997). Molecular modeling of condensing and endosomoly ic pep ides is shown in Figure 4.5. One example of such a gene delivery sys em is composed of (i) a galac osyla ed pep ide ha bo h condenses he plasmid in o monodisperse nanopar icles of abou 100 nm in diame er and enables specific recogni ion and binding o asialoglycopro ein recep ors, and (ii) an amphipa hic, pH-selec ive pep ide ha enables he plasmid o leave he endosomes prior o heir fusion wi h lysosomes and en ry in o he cy oplasm (Plank et al., 1994; Go schalk et al., 1996).

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Figure 4.5. Molecular configura ion of pep ides. Condensing and endosomoly ic pep ides.

3. Lipopep ides The improved DNA binding and condensa ion provided by amino acids such as ryp ophan sugges ha he inclusion of hydrophobic in erac ions wi hin DNA complexes may be beneficial. Pep ides wi h moi ies ha provide coopera ive hydrophobic behavior of he alkyl chains of ca ionic lipids would improve he s abili y of he pep ide-based DNA delivery sys ems. Smi h and associa es (1998) have cons ruc ed wo general classes of lipopep ide analogs of he TyrLys-Ala-Lysn-Trp-Lys pep ides by including a hydrophobic anchor. The general s ruc ures are N, N-dialkyl-Gly-Tyr-Lys-Ala-Lysn-Trp-Lys and N␣,N⑀-diacyl-LysLysn-Trp-Lys. These pep ides differ from he paren s ruc ures in ha hey selfassocia e o form micelles in aqueous solu ions. The inclusion of dialkyl or diacyl chains in he ca ionic pep ides improves he pep ide abili y o bind DNA and reduces aggrega ion of he complexes in ionic media.


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4. Endosomoly ic pep ides Shor syn he ic pep ides con aining he firs 23 amino acids of he HA2 subuni of influenza hemagglu inin pro ein (HA) are a rac ive because of heir pHdependen ly ic proper ies, wi h li le ac ivi y a pH 7 bu grea er han or equal o a 100-fold increase in ransfec ion efficiency a pH 5. The ly ic charac eris ics of he pep ides are revealed as he carboxyl groups of he aspar yl and glu amyl side chains are pro ona ed, which allows he pep ides o assume an ␣-helical conforma ion ha can be inser ed in o he membrane bilayer (Rafalski et al., 1991; Lear and De Grado, 1987). Plank et al. (1994) have used a series of hese pep ides derived from influenza HA o achieve endosomal rup ure and hereby enhanced gene expression in vitro. Go schalk et al. (1996) developed an amphipa hic membrane-associa ing pep ide, JTS-1, Gly-Ileu-Phe-Glu-Ala-Leu-Leu-Glu-Ser-Leu-Trp-Glu-LeuLeu-Leu-Glu-Ala. The hydrophobic face con ains only s rongly apolar amino acids, while nega ively charged glu amic acid residues domina e he hydrophilic face a physiological pH. The hydrophobic face of JTS-1 causes self-associa ion and forms pores in one side of he endosomal membrane, hereby des abilizing he membrane, which leads o i s rup ure. The ca ionic DNA complex formed wi h he condensing pep ide Tyr-Lys-Ala-Lys8-Trp-Lys is rapidly mixed wi h nega ively charged JTS-1, which spon aneously incorpora es hrough elec ros a ic in erac ions o form he er iary complex. A a given charge ra io of condensing pep ide o plasmid, he ransfec ion efficiency has been shown o be propor ional o he concen ra ion of he endosomoly ic pep ide added o he complex. The pH-selec ive pep ides form ␣-helices a acidic pH bu no a pH 7 (Figure 4.6). This s ruc ural conforma ion favors par i ioning of he amphipa hic pep ides in o he endosomal membrane and promo es DNA release from he endosomal compar men in o he cy oplasm. In vitro ransfec ion efficiency was up o 10,000-fold higher han ha of DNA/Tyr-Lys-Ala-Lys8-Trp-Lys complex alone (Go schalk et al., 1996).

C. Polymer-based gene delivery

1. Noncondensing polymer-based sys ems Pro ec ive, in erac ive, noncondensing (PINC) sys ems, such as polyvinyl polymers, have been pos ula ed o form hydrogen bonds wi h DNA base pairs, resul ing in a hydrophobic coa ing of he plasmid by he vinyl backbone (Mumper et al., 1996). Polyvinyl pyrrolidone (PVP)-based formula ions are hyperosmo ic and resul in an improved dispersion of plasmids hrough he ex racellular ma rix of solid issues (e.g., muscle), possibly by (i) pro ec ing plasmids from nuclease degrada ion, (ii) dispersing plasmids in he muscle, and (iii) facili a ing heir up ake by muscle cells. By increasing he hydrophobici y of plasmids and

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Figure 4.6. Effec of pH on he molecular configura ion of ly ic pep ide Gly-Leu-Glu-Ala-Leu-GluGlu-Leu-Trp-Glu-Ala-Lys.

reducing heir ne nega ive surface charge, he PINC polymers may facili a e he up ake of plasmids by muscle cells. In ramuscular injec ion of PVP-based plasmid formula ions in ra s significan ly increased he number and dis ribu ion of expressing cells, as compared o unformula ed plasmid (Mumper et al., 1998). Up o a 10-fold enhancemen of gene expression over unformula ed plasmid has been observed in mouse and ra muscle. N-me hyl-2-pyrrolidone (NM2P), which is a me hyla ed monomer of PVP, also enhances gene expression in ra skele al muscle. Five percen NM2P in saline con aining 100 ␮g CMV-driven ␤-galac osidase (␤-gal) expression plasmids has been shown o express levels of ␤-gal, which are approxima ely wo-fold higher han ha observed using a PVPbased formula ion (Mumper et al., 1996). Kabanov and associa es (1991 and 1995) have proposed he forma ion of condensed in erpolyelec roly e complexes be ween polyvinyl pyridinium, and DNA has been proposed o bo h pro ec DNA from nuclease degrada ion and facili a e i s cellular up ake by hydrophobic in erac ions wi h cell membranes (Kabanov et al., 1991 and 1995). The increased hydrophobici y of he complex may enhance in erac ion wi h cell membranes and facili a e cell up ake.


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2. Ca ionic polymer-based sys ems Ca ionic polymers such as polybrene and die hylaminoe hyldex ran (DEAEdex ran) have been used for ransfec ion of genes in o cul ured cells (Hol er et al., 1989). However, hese polymers canno be used for in vivo applica ion due o heir poor ransfec ion efficiency and high cy o oxici y. S arburs polyamidoamine (PAMAM) dendrimers are a class of highly branched spherical polymers whose surface charge and diame er are de ermined by he number of syn he ic s eps (Tomalia et al., 1990). For example, five polymeriza ion cycles produce he 5 h-genera ion dendrimers. The major s ruc ural differences in PAMAM dendrimers rela e o he core molecules, ei her ammonia or e hylenediamine, wi h which he s epwise polymeriza ion process begins and which dic a es he overall shape, densi y, and surface charge of he molecule. Dendrimers can condense plasmids hrough elec ros a ic in erac ions of heir erminal primary amines wi h he DNA phospha e groups. The effec of colloidal and surface charac eris ics of plasmid/dendrimer complexes on gene ransfer has been examined (Mumper et al., 1995). These complexes were monodisperse wi h a mean hydrodynamic diame er of abou 200 nm . The par icle size, surface charge, and gene ransfer efficiency of plasmid/dendrimer complexes prepared wi h he 5 h-genera ion of dendrimers has been shown o be influenced by dendrimer concen ra ion in he complexes. Fur hermore, covalen a achmen of fusogenic pep ide GALA o he dendrimer has been shown o significan ly enhance gene ransfer efficiency (Haensler and Szoka, 1993). Kukowska-La alla et al. (1996) have recen ly shown ha DEAE-dex ran facili a es he forma ion of small par icles from he large dendrimer/plasmid aggrega es and significan ly improves ransfec ion in vitro. Polye hyleneimine (PEI) is a branched ca ionic polymer and has been shown o condense plasmids in o colloidal par icles ha effec ively ransfec genes in o a varie y of cells in vitro (Boussif et al., 1995). In addi ion o enhancing cellular up ake of plasmids by nonspecific adsorp ive mechanisms, PEI may also enhance he in racellular rafficking of plasmids by buffering he endosomal compar men s, hus pro ec ing plasmids agains degrada ion and enabling endosomal release of plasmid via lysosomal osmo ic swelling and disrup ion (Abdallah et al., 1996; Dunlap et al., 1997). Conjuga ion of arge ing ligands, such as ransferrin or an i-CD3 an ibody, o PEI has recen ly been shown o enhance ransfec ion efficiency by ⬃30 – 1000-fold compared o ligand-free PEI in various umor cell lines. This ac ivi y depends on he ligand/ recep or in erac ion and has also been observed a low PEI/DNA charge ra ios where ligand-free PEI lacks efficiency (Kircheis et al., 1997). Chi osan is a biodegradable polysaccharide composed of wo subuni s, D-glucosamine and N-ace yl-D-glucosamine, linked oge her by ␤(1,4) glycosidic bond (Tang and Szoka, 1997a; Richardson et al., 1997). Chi osan has been

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shown o in erac wi h he phospha e groups of DNA, condensing plasmids in o spherical and oroidal par icles. The colloidal and surface proper ies of plasmid/ chi osan complexes have been shown o depend on he molecular weigh of chi osan, he ra io of plasmid o chi osan, and he prepara ion medium. Smaller nanopar icles have been observed wi h low molecular weigh chi osan (2 kDa) as compared o high molecular weigh chi osan (540 kDa). A number of cell lines have been ransfec ed wi h plasmid/chi osan complexes (Mumper et al., 1995). Poly(2-dime hylamino)e hyl me hacryla e (PDMAEMA) has also been evalua ed for ransfec ing plasmids encoding he ␤-galac osidase gene in COS7 cell lines in vitro (Cherng et al., 1996). The op imal ransfec ion efficiency was found a a PDMAEMA/plasmid ra io of 3:1 (w/w), he ra io a which homogeneous complexes of abou 150 nm in diame er could be formed. In eres ingly, he ransfec ion efficiency of he complexes was no affec ed by he presence of serum pro eins, even hough he presence of serum is known o adversely affec he ransfec ion efficiency (Zelpha i et al., 1998). Poly(e hylene glycol)-poly(L-lysine) block co-polymers have been shown o form complexes wi h DNA ha can ransfec human embryonal kidney cells in vitro (Wolfer et al., 1996).

3. S ruc ures of ca ionic polymers Poly(L-lysine) is a linear polymer, whereas dendrimers and polye hyleneimine are branched polymers. The s ruc ures of branched polymers can be fur her dis inguished by heir symme ry of branching. Dendrimers are radially branched, whereas polye hyleneimine lacks a defined cen er of symme ry. Dendrimers can be ei her in ac or frac ured. In ac dendrimer has wo arms ex ending from every branch poin , whereas frac ured dendrimer has zero, one, or wo arms ex ending from each branch poin (Tang and Szoka, 1997a). The major differences be ween he ca ionic polymers wi h respec o chemical s ruc ure are he ype and rela ive number of pro ona able amines. All he polymers possess primary amines, which are predominan ly pro ona ed a neu ral pH. The acid/base i ra ion curves of dendrimers and polye hylenimine exhibi considerable buffer capaci y over almos he en ire pH range. In conras , poly(L-lysine) shows li le buffer capaci y below pH 8, as shown by he nearly ver ical slope of he i ra ion curve below his poin . The complex par icle size in solu ion of poly(L-lysine) or in ac dendrimer is much larger han ha of he frac ured dendrimer and polye hylenimine. All he polymers demons ra e heir maximum ransfec ion ac ivi ies a charge ra ios wi h an excess of primary amines o DNA phospha es. Despi e he vas differences in s ruc ure of hese ca ionic polymers, he plasmid/ca ionic polymer complexes have similar oroid morphology. The diame ers of oroids vary sligh ly wi h he ca ionic polymer s ruc ure, al hough he differences do no appear o correla e


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wi h he physical size of he ca ionic polymer. For example, in ac dendrimers yield oroids ha have a significan ly smaller diame er han oroids formed from he frac ured dendrimer, al hough he in ac dendrimer has nearly wice he molecular weigh of he frac ured dendrimer (Tang and Szoka, 1997a).

V. FORMULATION FACTORS INFLUENCING GENE TRANSFER A. DNA topology Plasmids may exis in hree er iary s ruc ures: supercoiled, open circular, and linear. An open circular molecule is formed by nicking one s rand of he DNA, which relaxes he orsional s ress on he supercoiled plasmid. A linear molecule is formed by breaking he double-s randed DNA sequences (Ledley, 1996). DNA opology influences bo h he colloidal behavior and condensa ion of DNA. Topologically cons rained circular DNA may con ribu e bending energy o he condensing sys em hrough orsional elas ici y. Thus, supercoiled plasmids should yield smaller oroids. For ins ance, Wilson and Bloomfield (1979) observed for hexamidine cobal (III) condensa es ha closed circular plasmid yielded mul imolecular oroids 25 – 30% smaller in diame er han hose made up of linearized plasmid.

B. DNA condensation The ex en of DNA condensa ion has grea implica ions for gene delivery and expression. Recen progress in our unders anding of DNA condensa ion includes he observa ion of DNA collapse, grea er insigh s in o he in ramolecular forces driving condensa ion, he recogni ion of helical s ruc ure per urba ion in condensed DNA, and he increasing recogni ion of he likely biological consequences of condensa ion (Bloomfield, 1991). Unfavorable free energies associa ed wi h DNA bending, en ropy of mixing, and elec ros a ic repulsion forces mus be overcome o condense plasmid hrough he use of mul ivalen organic or inorganic ca ions. Al hough small mul ivalen ca ions bind and condense DNA, hey are highly mobile. Therefore, hey can easily be displaced by compounds wi h higher charge, and heir complexes have a endency o aggrega e (Bloomfield, 1996).

C. DNA condensing agents Ca ions of hree or more charges condense DNA in aqueous suspension primarily o oroids, al hough o her condensa ion s a es have been observed. Divalen ca ions, such as Mg2⫹, can also condense DNA in he presence of sufficien mole frac ion of alcohol (Sharp and Honig, 1995). Toroids of similar shape and size can also be formed by spermine, spermidine, hexamine cobal (III), and

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conjuga ed polylysines as well as various branched ca ionic polymers (Tang and Szoka, 1997b). Flexibili y in he spa ial arrangemen of posi ive charges provides many op ions in he design of ca ionic agen s ha can effec ively condense DNA. In addi ion o sal -dependen elec ros a ic in erac ion, he ion a mosphere and dielec ric cons an s are major fac ors in de ermining he s abili y, s ruc ure, reacivi y, and binding behavior of nucleic acids (Sharp and Honig, 1995). The DNA condensa e size is dependen on he ype of condensing agen s used. For ins ance, calf hymus DNA condensed by hexamine cobal (III) has been shown o yield oroids ha were subs an ially smaller in diame er han hose of spermidine or me hyla ed spermidine analog (Sharp and Honig, 1995). Nonelec ros a ic fac ors, such as bridging be ween helices, hydra ion forces, or degree of hydrogen bonding may influence he con ribu ion of ca ionspecific in erac ion o oroid size. The ex en of DNA condensa ion depends on a number of variables, including he me hod of complexa ion, ypes of ca ionic carriers, buffers, coun er-ions, and he size, sequence, and opology of plasmid.

D. DNA aggregation Ca ionic carriers form complexes wi h plasmid via elec ros a ic in erac ions. The large popula ion of hese complexes has wide par icle-size dis ribu ion due o he he erogenei y of some condensing carriers (Perales et al., 1994). Nearneu ral (“isoelec ric”) plasmid/ca ionic carrier complexes usually have a s rong endency o form large aggrega es over ime, whereas complexes carrying a ne nega ive or posi ive charge are rela ively s able. Aggrega ion is probably a resul of charge and/or hydrophobic in erac ions be ween he plasmid/ca ionic carrier complexes. Complexes prepared a very high ionic s reng h or formed a a high DNA concen ra ion generally have a grea er endency o form aggrega es over ime. Insufficien or rapid vor exing of plasmid/ca ionic carrier complexes can also lead o aggrega ion. The echnical difficul y in forming s able plasmid/ ca ionic carrier complexes a high DNA concen ra ions may be par ially overcome by formula ion using a large excess of ca ionic carriers. The excess in posi ive charge preven s rapid aggrega ion of he complexes during mixing. The resul ing uncomplexed carriers can hen be separa ed from he complex formula ions by sucrose densi y gradien cen rifuga ion (Lee and Huang, 1997). However, aggrega ion of purified complexes can occur following in erac ion wi h blood componen s.

VI. BIODISTRIBUTION AND PHARMACOKINETICS OF PLASMIDS Since plasmids and he carrier molecules have very differen physicochemical proper ies, a horough unders anding of he ana omy and (pa ho)physiology of


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arge organs as well as he physicochemical charac eris ics of bo h ac ive and carrier molecules is necessary. Biodis ribu ion of plasmid DNA o ei her ex racellular or in racellular arge s is dependen on he s ruc ure of capillary walls, (pa ho)physiological condi ions, he ra e of blood and lymph supply, and he physicochemical proper ies of plasmid and i s carrier molecules. These properies include molecular size, elec rical charge, and physical forms and arge ing group (if presen ), and an in erac ion wi h blood pro eins (Tomlinson, 1987). The fa e of plasmid af er in vivo adminis ra ion is illus ra ed in Figure 4.7.

A. Anatomical and physiological considerations The blood capillary walls are generally comprised of four layers, namely plasmaendo helial in erface, endo helium, basal lamina, and adven ia. The endo helium is a monolayer of me abolically ac ive cells, which media e and moni or he bidirec ional exchange of fluid be ween he plasma and he in ers i ial fluid. There are several differen pa hways by which macromolecules can cross he endo helial barrier (Simionescu, 1983; Taylor and Granger, 1984): (i) hrough he cy oplasm of endo helial cells hemselves; (ii) across he endo helial cell membrane vesicles; (iii) hrough in erendo helial cell junc ions; and (iv) hrough endo helial cell fenes rae. Based on he morphology and con inui y of he endo helial layer and he basemen membrane, capillary endo helium can be divided in o hree ca egories con inuous, fenes ra ed, and discon inuous endo helium.

Figure 4.7. Fa e of plasmid DNA af er in vivo adminis ra ion.

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The con inuous capillaries are found in skele al, cardiac, and smoo h muscles, as well as in lung, skin, and subcu aneous and mucous membranes. The endo helial layer of he brain microvascula ure is he igh es endo helium, wi h no fenes ra ions. This endo helial barrier forms a con inuous cellular layer be ween he blood and brain in ers i ium, which is impermeable o plasmids. Capillaries wi h fenes ra ed endo helia and a con inuous basemen membrane are generally found in he kidney, small in es ine, and salivary glands. Mos of hese capillaries have diaphragmed fenes rae, which are circular openings of 40 – 60 nm in diame er. The discon inuous capillaries, also known as sinusoidal capillaries, are common in he liver, spleen, bone marrow, and o her organs of he re iculoendo helial sys em. These capillaries show large in erendo helial junc ion (fenes ra ions up o 150 nm). Depending on he issue or organ, he basal membrane in sinusoidal capillaries is ei her absen (e.g., in liver) or presen as a discon inuous membrane (e.g., in spleen and bone marrow) (Venka achalam and Rennke, 1978). The sinusoids of he liver are lined by highly phagocy ic Kupffer cells, and hose of he bone marrow by fla ened, phagocy ic re iculoendo helial cells. In he spleen, he endo helial cells are grea ly elonga ed and con ain a large number of pinocy ic vesicles (up o 100 nm in diame er). Due o heir large molecular weigh (grea er han 1000 kDa) and hydrodynamic diame er in aqeuous suspension of 100 nm (Ledley, 1996), plasmids ex ravasa e poorly via con inuous capillaries because of igh junc ions be ween he cells. However, plasmids can easily ex ravasa e o sinusoidal capillaries of liver and spleen. Formula ing plasmids in o unimeric par icles of 20 – 40 nm in diame er may enhance ex ravasa ion of plasmids across con inuous and fenes ra ed capillaries.

B. Influence of (patho)physiology on biodistribution Inflamma ion is associa ed wi h regional changes in he s ruc ure, chemical composi ion, and increased permeabili y of he endo helium. Increase in ranspor of macromolecules a inflamma ion si es is due o openings in he endo helium a he level of pos capillary venules. Molecules grea er han 50 kDa usually do no ex ravasa e in normal issues; in inflamed and umor issues his limi is significan ly increased (Arfors et al., 1979). The (pa ho)physiology and microana omy of umors are significan ly differen from normal issues. A umor con ains vessels recrui ed from he preexis ing ne work and vessels resul ing from angiogenic response reduced by cancer cells. There is a considerable varia ion in he cellular composi ion and basemen membranes and in he size of he in erendo helial cell fenes ra ions (Jain, 1989). Tumor in ers i ium is charac erized by large in ers i ial volume and high diffusion ra e (Takakura and Hashida, 1995). The high in ers i ial pressure of he umor re ards he ex ravasa ion of macromolecules, whereas large


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vascular permeabili y and high in ers i ial diffusivi y of macromolecules facilia e heir migra ion o umor issues. Tumor accumula ion of plasmid could resul from he enhanced permeabili y of he umor vascula ure, combined wi h reduced clearance from he umor due o he absence of he lympha ic sys em.

C. Biodistribution and pharmacokinetics of plasmid DNA The biodis ribu ion of plasmid DNA can be de ermined by measuring he ra e of disappearance of radio-labeled DNA from he bloods ream and i s accumulaion in issues or by he use of fluorescence microscopy o race he leakage of dye-labeled plasmids from he vascula ure. Pharmacokine ic analysis of in vivo disposi ion profiles of radio-labeled plasmid DNA provides useful informa ion on he overall dis ribu ion charac eris ics of sys emically adminis ered plasmids, wi h one cri ical limi a ion. The radio label represen s bo h in ac plasmid and i s me aboli es. The plasma half-life of plasmid is less han 10 min (Kawaba a et al., 1995), and hence issue dis ribu ion and pharmacokine ic parame ers of plasmid DNA calcula ed on he basis of o al radioac ivi y are no valid a longer ime poin s. Thus, polymerase chain reac ion (PCR) and Sou hern-blo analysis are required o es ablish he ime a which he radio lable is no longer an index of plasmid dis ribu ion. Even af er local adminis ra ion, i is impor an o unders and pharmacokine ics a bo h he organ and sys emic levels because a par of injec ed plasmid will en er he blood circula ion. Sys emic disposi ion processes involve in erac ion wi h blood componen s and/or vascular endo helial cells, organ dis ribu ion, and up ake by re iculoendo helial sys ems (RES) before reaching he arge si e. In case of paren eral adminis ra ion, movemen in he issues and absorp ion via capillary and lympha ic rou es should be considered. In he early phase of dis ribu ion, he movemen of plasmid DNA from he circula ion o organs is roughly a unidirec ional process in many organs. Thus, he disposi ion charac eris ics of plasmid DNA can be charac erized using organ up ake clearance (Clorg) as an essen ial index of dis ribu ion o each organ. To al body clearance (Cl o al) is equal o he sum of individual organ clearance values (Figure 4.8). The deposi ion of plasmids af er sys emic adminis ra ion is res ric ed o he in ravascular space due o i s low microvascular permeabili y in mos organs wi h con inuous capillary bed. Some organs wi h fenes ra ed capillaries, such as liver, spleen, and bone marrow, provide some oppor uni ies for ex ravasa ion of plasmid DNA. In ravenously injec ed plasmids ini ially perfuse he pulmonary vascular beds, maximizing he po en ial up ake of plasmid DNA in he lung endo helial cells soon af er adminis ra ion. Based on he clearance concep , Kawaba a et al., (1995) and Maha o et al. (1995a,b) de ermined he pharmacokine ic parame ers of plasmid DNA af er ail vein rejec ion of [32P]pCMV-CAT in mice. The radioac ivi y was rapidly

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Figure 4.8. Physiological pharmacokine ic model for evalua ing biodis ribu ion of plasmid (adap ed from Maha o et al., 1997a, wi h permission).

elimina ed from he circula ion due o he ex ensive up ake by he lung and liver, while i was no suscep ible o glomerular fil ra ion because of he presence of he basemen membrane. Pharmacokine ic analysis under condi ions wi h minimal enzyma ic degrada ion, derived from [32P]pCMV-CAT up o 1 min af er injec ion, has demons ra ed ha he hepa ic up ake clearance of pCMVCAT is almos iden ical o he plasma flow ra e in he liver (Figure 4.9) which indica es ha plasma DNA is cleared subs an ially on firs -pass of he liver. A he la er phase following in ravenous injec ion of [32P]pCMV-CAT, he proporion of he radioac ivi y accumula ed in he liver decreased wi h ime, probably due o he release of degrada ion produc s in o he plasma pool and accumulaion of radioac ivi y in he kidney. In addi ion, pCMV-CAT was prefen ially aken up by he liver nonparenchymal cells (NPC). Scavenger recep or-media ed processes are involved in he up ake of large anionic molecules (Kawaba a et al., 1995; Yoshida et al., 1996). Au oradiography of mouse whole body af er in ravenous injec ion of [32P]plasmid/lipid complexes has shown DNA localiza ion predominan ly in he lung, wi h no able up ake in he liver and o her issues con aining RES cells. In cons ras , he au oradiograph of mouse whole body af er in ravenous injecion of free [33P]plasmid DNA showed he highes levels of radioac ivi y in he


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Figure 4. . Rela ionship be ween hepa ic and urinary clearances of plasmid and macromolecules af er in ravenous injec ion in mice. BSA, bovine serum albumin; Gal-BSA, galac osyla ed BSA; Man-BSA, mannosyla ed BSA, pCAT, plasmid encoding chloramphenicol ace yl ransferase; 3⬘-M5⬘B-T10, 3⬘-me hoxye hylamine 5⬘-bio in-deca hymidylic acid; T10-CMD, carboxyme hyldex ran-deca hymidylic acid conjuga e; PO, phosphodies er oligonucleo ides; PS, phosphoro hioa e oligonucleo ides (adap ed from Maha o et al., 1997, wi h permission).

liver, followed by o her issues con aining RES cells (Osaka et al., 1996). Sou hern-blo analysis of blood showed he rapid degrada ion of plasmid DNA, wi h a half-life of less han 5 min for in ac plasmid, and ha i was no longer de ec able a 1 hr pos injec ion. By Sou hern-blo analysis, here was no de ec able plasmid in he brain, large in es ine, small in es ine, or gonads a he 1-hr ime poin . Sou hern analysis also demons ra ed ha plasmid DNA remained in he liver, spleen, lung, marrow, and muscle, al hough a diminished levels, up o 24 hr pos injec ion. Af er 7 days, no in ac plasmid DNA was de ec able by Sou hern-blo analysis. However, he plasmid was de ec able by PCR analysis in all issues examined a 7 and 28 days pos injec ion PCR analysis a he sixmon h ime poin revealed ha only muscle had any significan levels of plasmid above background (Lew et al., 1995).

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Biodis ribu ion charac eris ics of plasmid DNA in umors has been assessed by using issue-isola ed umor prepara ions of Walker 256 carcinoma af er in ra-ar erial or direc injec ion of radio-labeled plasmids in o he umor (Nomura et al., 1997). This sys em, which is composed of a solid umor wi h a supplying ar ery and a draining vein, permi s he measuremen of he amoun of he drug ha flows in and ou of a umor. From such informa ion, he amoun of drug ha is re ained can be calcula ed. The venous ou flow pa erns can be analyzed using s a is ical momen heory as described by Kaku ani et al. (1985). Two hours af er he in ra umoral injec ion of [32P]-labeled plasmid DNA, only 40% of he radioac ivi y was elimina ed from he umor issue and in ac plasmid was found in he venous ou flow (Nomura et al., 1997).

VII. INTRACELLULAR TRAFFICKING OF GENE MEDICINES The degree o which ex racellular and in racellular barriers limi plasmid movemen following up ake of formula ed plasmid by cells is dependen upon he gene delivery sys em and he arge issue. In erac ion of he formula ed plasmid wi h biofluids and pene ra ion of he ex racellular ma rix, if presen , are he major ex racellular barriers. The plasma membrane is he nex obs acle o be overcome in delivering genes in o a cell. Gene delivery sys ems rely on binding o cell surface molecules, ei her specific, nonspecific, or bo h, prior o cellular in ernaliza ion. The surface-bound ma erial usually gains en ry in o he cell ei her by endocy osis or membrane fusion. Successful in racellular rafficking of plasmids mus surmoun several barriers, including release from endosomes, DNA uncoa ing, movemen hrough he cy oplasm, associa ion wi h he nuclear membrane, and ranspor o he nucleus, probably hrough he nuclear pore, before ranscrip ion fac ors become limi ing (Meyer et al., 1997).

A. Cellular uptake mechanisms The in racellular fa e of plasmids, wi h or wi hou a delivery sys em, depends on he ype of endocy ic process involved in heir cellular in ernaliza ion (Scheule and Cheng, 1996). There is a consensus ha formula ed plasmids en er cells via an endocy o ic pa hway (Friend et al., 1996). In his pa hway, DNA complexes firs bind o he cell surface hen migra e o cla hrin-coa ed pi s abou 150 nm in diame er and are in ernalized from he plasma membrane o form coa ed vesicles. The ransi ion from coa ed vesicle o early endosome is accompanied by acidifica ion of he vesicular lumen ha con inues in o he la e endosomal and lysosomal compar men s, reaching a final pH in he perinuclear lysosome of approxima ely 4.5. Such acidifica ion associa ed wi h endosome


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ma ura ion provides he means by which cer ain viruses gain access o he cy osol. Acid-induced conforma ional changes in he viral pro eins rigger ransloca ion across he endosomal membrane via he fusion process (Whi e et al., 1992). By aking advan age of he endosomal acidifica ion, pH-sensi ive liposomes (Wang and Huang, 1987), adenovirus (Curiel et al., 1991), and fusogenic or ly ic pep ides (Wagner et al., 1992) have been used o facili a e he release of plasmids in o he cy oplasm prior o lysosomal degrada ion. Noncla hrin-coa ed pi in ernaliza ion can occur hrough smoo h invagina ion of 150 – 300 nm vesicles or via po ocy osis. Po ocy osis involves invagina ion of a caveolae-rich 50 – 100-nm-diame er vesicles from he cell surface. This pa hway has been shown o be involved in he ranspor of fola e and o her small molecules in o he cy oplasm (Ro hberg et al., 1990; Anderson et al., 1992). Plasmids are aken up by muscles hrough he T- ubules sys em and caveolae via po ocy osis (Wolff et al., 1992). Apar from coa ed or uncoa ed pi pa hways, plasmid/ca ionic carrier complexes may also be aken up by cells via plasma membrane des abiliza lon (Laba -Moleur et al., 1996). Par icles grea er han 200 nm in diame er are no efficien ly aken up by endocy osis, bu some larger plasmid/ca ionic carrier complexes may also be aken up by cells via phagocy osis (Li and Huang, 1996; Zhou and Huang, 1994). Li le is known abou he escape of plasmid from he endosomal or po osomal compar men and i s movemen wi hin he cells and rafficking o he nucleus. I has been proposed ha plasmid/lipid complexes in ernalize in o he endosome and ini ia e he des abiliza ion of endosomal membrane (Zhou and Huang, 1994). This des abiliza ion would induce diffusion of anionic lipids from he ex ernal layer of he endosomal membrane in o he complexes and form charge-neu ralized ion pairs wi h he ca ionic lipids. This phenomenon may displace he plasmid from he complex and permi DNA en ry in o he cy oplasm (Figure 4.10). This hypo hesis is par ly based on he evidence ha free plasmids injec ed in o he nucleus will express, whereas plasmid/lipid complexes injec ed in o he nucleus will no (Capecchi, 1980; Zabner et al., 1995). This s rongly sugges s ha plasmids need o be released from he plasmid/lipid complexes prior o en ering he nucleus for expression o occur. The mechanism illus ra ed in Figure 4.10, however, does no explain why endocy osis is required. Des abiliza ion and/or fusion of he complex wi h he plasma membrane would permi he same anionic lipids o diffuse o he surface as would fusion wi h he endosomal membrane. Release of he condensed DNA from he ca ionic lipid in he endosome is likely o genera e a mechanical or osmo ic s ress ha rup ures he endosomal bilayer and releases DNA in o he cy oplasm. In conras , DNA release from complexes on he cell surface migh be unable o s ress he membrane o a degree sufficien o rup ure i .

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Plasmid/Lipid Complex

Endosomal membrane Cationic lipid

Anionic lipid

Zwitterionic lipid

Figure 4.10. Mechanism of plasmid release from ca ionic liposomes. Following endocy osis of plasmid/lipid complexes, he endosomal membrane is des abilized, allowing diffusion of anionic phospholipid from he complex. The anionic cy oplasmic lipids diffuse in o he complex and form a charge-neu ral ion pair wi h ca ionic lipids. The DNA dissocia es from he complex and is released in o he cy oplasm (adap ed from Xu and Szoka, 1996, wi h permission).

B. Intracellular trafficking Even af er being released from he complex in o he cy oplasm, he plasmid is s ill oo large o en er he nucleus by simple diffusion, as he aqueous channel of he nuclear pore allows free diffusion of only small par icles (less han approxima ely 70 kDa). Ye some plasmids reach he nucleus, because gene expression is de ec ed. Nucleoplasmic ranspor is affec ed by he mi o ic and cellular s a e. Cy oplasmic injec ion of plasmid has been shown o produce rela ively high levels of gene expression in myo ubes. This provides irrefu able evidence ha plasmid can en er he karyoplasm of a pos mi o ic nucleus wi h in ac membranes. When plasmids were injec ed far from he nuclei (approxima ely 60 – 90 ␮m) pro ein expression significan ly decreased compared o injec ions near he nuclei. This sugges s ha he in racellular rafficking of plasmid is cons rained by cy oplasmic elemen s (Dow y et al., 1995). A fundamen al limi a ion o gene expression using a lipid-based sys em is he inabili y of plasmid in he cy oplasm o migra e in o he nucleus. To


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design s ra egies o enhance in racellular and nuclear rafficking of plasmid, a horough unders anding of cy oskele al componen s, nuclear envelope, nuclear pore complex, and nuclear localiza ion of plasmids as well as nuclear localiza ion signal sequences (NLSs) will be needed (Cole and Lippinco -Schwar z, 1995). The mos likely candida es for ranspor mechanisms involve par icipa ion of cy oskele al componen s, such as micro ubules and ac in filamen s. These cy oskele al componen s are believed o main ain in racellular dis ribuion of organelles and o facilila e rafficking be ween organelles. I may be possible o u ilize hese sys ems o ranspor plasmid o he nucleus o facili a e gene delivery. Mo or pro eins, mo or pro ein recep ors, or he relevan pep ide sequences may be conjuga ed o or complexed wi h plasmid. This may resul in associa ion of plasmids wi h myo ubules or ac in filamen s for more efficien ranspor hrough he cy oplasm o regions bordering he nucleus (GarciaBus os et al., 1991).

C. Nuclear envelope and nuclear pore complex The nucleus is bound by he nuclear envelope, which encloses chroma in and he machinery necessary for gene ranscrip ion. The nucleus is a dynamic s ruc ure, which disassembles a he onse of mi osis and reassembles during elophase. The major barrier be ween he cy osolic and nucleoplasmic compar men s is he hydrophobic double-bilayered barrier of he nuclear envelope. Access of plasmids and o her large molecules in o he nucleus is res ric ed and regula ed by he nuclear envelope and he nuclear pore complex (NPC). The NPC accommoda es bo h passive diffusion and ac ive ranspor . Small molecules or pro eins of less han approxima ely 70 kDa passively diffuse hrough he NPC in and ou of he nucleus, al hough passive diffusion becomes ra e limi ing a approxima ely 20 kDa. Larger macromolecules require ac ive ranspor for nuclear en ry. The up ake of endogenous nucleopro eins (e.g., his ones, ranscrip ion fac ors) in o he nucleus is achieved by ac ive ranspor hrough he NPC. The exac mechanism by which plasmid is ranspor ed hrough he NPC has no ye been de ermined. S udies examining rafficking of plasmid hrough he NPC have no ye been done adequa ely, and hus i is no clear how plasmid, exceeding he size of NPC, is able o pass hrough he nuclear pore. Plasmids may gain access in o he nucleus hrough he NPC during elophase, when he nuclear envelope reassembles af er cell division (Nakanishi et al., 1996).

D. Nuclear localization signal (NLS) sequences NLS sequences are ypically shor pep ide sequences responsible for direc impor of pro eins in o he nucleus. In general, hese sequences con ain a high

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propor ion of he basic amino acids lysine and arginine (Lanford and Bu el, 1984). Frequen ly, amino acids such as proline, which disrup helical domains, are also presen . NLS sequences are frequen ly presen wi hin viral pro eins, or, as in he case of adenovirus, he nuclear localizing pro eins are covalen ly linked o he 5⬘ end of he DNA a he erminal dCMP. A varie y of viral nucleic acids (HIV-2, influenza virus, SV40, and adenovirus) are guided hrough he nuclear pore complex wi h assis ance from a leas one NLS-con aining viral pro ein. The bes charac erized NLS is ha presen in 92-kDa SV40 large Tan igen. Cy oplasmic microinjec ion of a plasmid complexed wi h Cys-Gly-GlyPro-Lys-Lys-Lys-Arg-Lys-Val-Gly-amide has been shown o give enhanced gene expression (Fraley et al., 1980). However, mu a ions of any lysine residue of SV40 large T-an igen are repor ed o abolish nuclear accumula ion. The minimal sequence ha direc ed a pyruva e kinase fusion pro ein o he nucleus was Pro-Lys-Lys-Lys-Arg-Lys-Val. Syn he ic pep ides con aining his sequence also arge ed cross-linked carrier pro eins o he nucleus. Unlike SV40 large Tan igen, he NLS sequence of influenza virus nucleopro ein, Ala-Ala-Phe-GluAsp-Leu-Arg-Val-Leu-Ser, has only one basic residue. NLS sequences have been iden ified for many pro eins of viral and cellular origins and generally resemble ei her he single basic-domain SV40 large T-an igen NLS (e.g., Pro-Lys-Lys-Lys-Arg-Lys-Val) or he double basicdomain nucleoplasmin NLS (e.g., Lys-Arg-Pro-Ala-Ala-Thr-Lys-Lys-Arg-GlyQln-Arg-Lys-Lys-Lys-Lys). The presence of addi ional copies of a nuclear arge ing sequence in a molecule increases he ini ial ra e and final s eady-s a e level of nuclear accumula ion such ha a par ially defec ive sequence can bring abou comple e nuclear accumula ion when a number of copies of he sequence are presen (Chelsky et al., 1989), Targe ing can also be modula ed by cy oplasmic anchoring pro eins or phosphoryla ion of flanking sequences.

VIII. BIOLOGICAL OPPORTUNITIES FOR GENE THERAPY Gene herapy is being inves iga ed for monogenic diseases such as adenosine deaminase, cys ic fibrosis, familial hypercholes erolemia, Gaucher’s disease, and Duchenne muscular dys rophy as well as for more complex disease processes such as cancer and infec ious diseases like AIDS. This sec ion discusses biological oppor uni ies for sys emic, cancer, and pulmonary gene herapy as well as for nucleic acid-based vaccines.

A. Systemic gene therapy In vivo produc ion and secre ion of herapeu ic pro eins may be con rolled by an appropria e mode of adminis ra ion. Bo h sys emic and local adminis ra ion of gene medicines offer several biological oppor uni ies for gene herapy. The


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sys emic rou e allows noninvasive access o many arge cells and issues ha are no accessible o herwise by direc adminis ra ion. Sys emic gene delivery can be broadly ca egorized as passive and ac ive arge ing. Passive arge ing refers o he exploi a ion of na ural disposi ion profiles of gene medicines, which depend on he physicochemical proper ies of formula ed plasmid DNA and he ana omical and physiological charac eris ics of he body. On he o her hand, ac ive arge ing refers o an al era ion in he na ural disposi ion pa ern of plasmids by means of arge -specific ligands, which can bind specifically o recep ors on he surface of arge cells (Tomlinson, 1990).

1. Passive arge ing Passive arge ing is an a rac ive approach for delivery and expression of herapeu ic genes o normal endo helia (e.g., lung, liver), various phagocy ic cells, and po en ially dissemina ed umors and me as ases. Following in ravenous injec ion of plasmid/lipid complexes, gene expression was de ec ed in various organs, wi h high expression in he lung (Brigham et al., 1989; Zhu et al., 1993; Liu et al., 1995), possibly due o expression by lung endo helia (McLean et al., 1997; Maha o et al., 1998). This sugges s ha he lung endo helium could be used as a bioreac or o produce pro eins for sys emic dis ribu ion. Numerous genes, including human grow h hormone, ␣-an i rypsin, pros aglandin G/H syn hase, and cys ic fibrosis ransmembrane conduc ance regula or (CFTR) genes have been shown o be expressed in he lungs af er in ravenous adminisra ion (Brigham et al., 1993; Canonico et al., 1994; Hyde et al., 1993; Caplen et al., 1994). In ravenous injec ion of pros aglandin G/H syn hase expression plasmid complexed wi h DOTMA:DOPE liposomes has also been shown o pro ec rabbi s and pigs agains endo oxin-induced pulmonary hyper ension (Conary et al., 1994). The liver is he si e of many essen ial me abolic and secre ory func ions and hus also cons i u es an impor an arge for gene herapy. Po en ial herapies include he rea men of inheri ed hepa ic me abolic and infec ious disorders, such as hyperlipidemia, phenylke onuria, familial hypercholes erolemia, organic acidemia, urea cycle disorders, hepa i is, cirrhosis, and hemophilia. The liver may also be used as a bioreac or for he sus ained produc ion and secre ion of herapeu ic pro eins, such as blood-clo ing fac ors (fac or VIII or fac or IX), ery hropoie in, grow h fac ors, and ␣-1 an i rypsin. Gene expression in he liver af er in ravenous injec ion of plasmid/lipid complexes of en remains low (Maha o et al., 1995a), as hese complexes are largely aken up by Kupffer cells via phagocy osis, which presumably leads o he degrada ion of he DNA and inefficien gene expression (Maha o et al., 1995b). However, he preproinsulin I gene was shown o be expressed in hepa ocy es and endo helial cells af er in ravenous injec ion of he preproinsulin I expression plasmid encapsula ed in o anionic liposomes (Nicolau et al., 1983). Inclusion of lac osylceramide in

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he liposomes subs an ially increased he amoun of exogenous DNA in bo h hepa ocy es and liver endo helial cells (Soriano et al., 1983). Sys emic adminis ra ion of mul ilamellar vesicles (MLV) encapsula ing human fac or IX (hFIX) expression plasmids has also been repor ed o resul in de ec able levels of hFIX in mouse plasma, liver, and spleen (Baru et al., 1995). Similarly, gene expression in he mouse liver has also been shown o be significan ly increased af er in ravenous injec ion of plasmid/lipid complexes prepared using 1-[2-(9(Z)oc adecenoyloxy e hyl]-2(8-(Z)hep adecenyl-3-hydroxye hylimidazolium chloride (DOTIM)/Chol (1:1 mol/mol) MLV, as compared o DOTIM/Chol (1:1 mol/mol) SUV (Liu et al., 1997). Prolonged re en ion of gene medicines in he blood circula ion migh be beneficial for passive dis ribu ion of genes o bo h he in ravascular spaces and o he highly vascularized issues, such as umors (Jain, 1994). Ca ionic liposomes con aining amino-polye hylene glycol (PEG)-phospha idylcholine may be used o minimize he nonspecific in erac ion of ca ionic lipid-based gene medicines wi h he blood componen s and heir up ake by he re iculoendo helial cells (Zalipsky et al., 1994). Even wi hou he use of s erically s abilized liposomes, passive arge ing may s ill be possible for gene delivery o cer ain umors. For example, repea ed ail-vein injec ion of he umor suppressor gene p53 complexed wi h DOTMA:DOPE liposomes in o breas umor-bearing nude mice has been shown o significan ly decrease he umor size as well as he number of me as a ic cells in he lung.

2. Ac ive- arge ing Endo helial cells, hepa ocy es, umors, and blood cells may be able o process bo h soluble macromolecules and par icula e ma erials via recep or-media ed endocy osis. Hepa ocy es represen an a rac ive arge for he following reasons: a large and well-perfused cell popula ion accessible by ex ravasa ion; he presence of a unique cell-surface in ernalizing recep or; he po en ial of rea ing many hepa ic disorders; and he po en ial of u ilizing normal hepa ocy es for he secre ion of herapeu ic pro eins. Effec ive hepa ocy e gene herapy requires par icula e sys ems wi h he appropria e size (less han 100 nm in diame er) and colloidal proper ies for ex ravasa ion hrough he sinusoidal hepa ic endo helium and access o he Space of Disse, while avoiding nonspecific up ake in o numerous non arge si es. The recep or-binding ligand on he surface of he formula ed plasmid mus also compe e wi h endogenous ligands for cell binding and in ernaliza ion and mus avoid masking by adsorbed serum pro eins. Hepaocy es are quiescen cells ha normally do no undergo mi osis. Incorpora ion of hepa ocy e-specific promo er elemen s ( ha con ain binding si es for hepaocy e ranscrip ion fac ors) wi hin plasmid cons ruc s may allow long dura ion and high levels of issue-specific gene expression. Asialo-glycopro ein recep or- arge ed polypep ide-based sys ems have


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been employed o deliver genes o he hepa ocy es in vivo. Plasmid/asialoorosomucoid-poly(L-lysine) complexes have been used o ob ain expression of genes in hepa ocy es of normal animals (Chowdhury et al., 1992), as well as expression of low-densi y lipopro ein (LDL) recep ors in LDL-deficien rabbi s (Wilson et al., 1992), albumin in analbuminemic ra s (Wu et al., 1991), and me hyla ed CoA mu ase in mice (S ankovics et al., 1994). Evidence of hepa ocy e cell-specific gene expression in vivo has been ob ained wi h he use of hepa ocy e-specific promo ers (Wu et al., 1989). However, prolonged gene expression required par ial (66%) hepa ec omy 15 min before in ravenous injecion of he complex in o ra s, probably due o s imula ion of liver cell regenera ion. Tomlinson and Rolland (1996) have described a hepa ocy e-specific gene delivery sys em comprised of a condensing glycopep ide, pH-sensi ive pep ide, and a hepa ocy e-specific gene expression sys em. This promising sys em is undergoing fur her op imiza ion.

B. Cancer gene therapy Gene herapy provides a significan oppor uni y o devise novel s ra egies for he con rol or cure of cancer. Cancer gene herapy accoun s for almos 65% of he gene herapy clinical rials (Sikora, 1996). Several approaches o cancer gene herapy are curren ly being inves iga ed: (i) enhancing cellular and humoral immune responses o umors, (ii) inser ing genes in o umor cells o evoke “cell suicide,” and (iii) modifying umor suppressor genes or an ioncogenes. Such herapeu ic genes include he ones ha conver prodrugs in o oxic me aboli es, such as he herpes simplex hymidine kinase (HSV k) gene driven by T7 promo er followed by ganciclovir rea men (Chen et al., 1998); cy okine genes, which s imula e he immune sys em o elimina e cancer cells (e.g., IL-2) (Plau z et al., 1993; Parmiani et al., 1997; Tepper and Mule, 1994); cos imulaory molecules (e.g., gene B7-1) ha augmen an igen presen a ion of umorspecific an igens by he umor o he T cells (Townsend and Allinson, 1993); foreign his ocompa ibili y genes ha s imula e a polyclonal alloreac ive immune response (Nabel et al., 1994); gene ic vaccines ha genera e umor specific immuni y (Conry et al., 1994); replacemen of wildype umor suppressor genes, such as p53 (Shaw et al., 1992), and an isense genes arge ed a oncogenes (e.g., ras oncogenes) (Bos, 1989).

1. Prodrug-conver ing enzyme genes Prodrug-conver ing enzyme genes, also known as suicide genes, have been used o subsequen ly ac iva e cy o oxic drugs selec ively in ransfec ed umor cells. A gene encoding an enzyme ha is capable of ac iva ing a prodrug cloned in o an expression plasmid permi s specific expression of he gene only in he issue affec ed by he umor. This specifici y was achieved by coupling a promo er for

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a umor-associa ed specific an igen ups ream of he drug-ac iva ing gene. The mos widely used is ha of he HSV k, which conver s he rela ively non oxic prodrug ganciclovir (GCV) in o riphospha es ha in erfere wi h DNA syn hesis and give preferen ial dea h of he ransduced umor cells (Chen et al., 1998). The plasmid used in his s udy con ains a T7 RNA polymerase gene driven by a T7 promo er and a human HSV k gene driven by a second T7 promo er. Even hough only a small frac ion of umor cells ac ually expressed HSV k, his rea men produced ex ensive umor cell dea h in an animal model. The mechanism is believed o involve diffusion of he oxic me aboli es of ganciclovir from ransduced cells via gap junc ions o he surrounding non ransduced cells.

2. Cy okine gene herapy Several cy okine genes have been found o reduce umors by s imula ing localized inflamma ory and/or immune responses. These include in erleukin-1 (IL1), IL-2, IL-4, IL-6, IL-7, IL-12, in erferon gamma (IFN-␥), umor necrosis fac or-␣ (TNF-␣), and granulocy e-macrophage colony-s imula ing fac or (GMCSF) (Plau z et al., 1993; Parmiani et al., 1997; Tepper and Mule, 1994; Whar enby et al., 1995). Ac iva ion and differen ia ion of cy o oxic T lymphocy es (CD8⫹ T cells) (CTLs) require in erplay of various cy okines and cells. During he presen a ion of umor-specific an igens by an igen-presen ing cells (APCs) o helper T cells (CD4⫹ T cells), cy okines presen in he microenvironmen con rol he helper immune response o develop in o ei her a cellular or a humoral response. CD4⫹ T cells have been classified in o Th1 and Th2 subse s according o he pa ern of cy okines hey produce. Th1 clones secre e IL-12 and IFN-␥, whereas Th2 clones secre e IL-4, IL-5, IL-6, and IL-10. Th1 immune response is beneficial for he developmen of he cellular cy o oxic (CD8) immune response, whereas Th2 immune response is inhibi ory o cy ooxic response (Whar enby et al., 1995). IFN-␣ is a ype 1 in erferon ha also promo es Th1 ype an i umor immuni y, reduces umor cell grow h, and inhibi s angiogenesis (Gajewski et al., 1995). GM-CSF may enhance a specific immune response by inducing differen ia ion of hemopoie ic progeni or cells in o APCs. In addi ion o macrophages, GM-CSF s imula es he prolifera ion and differen ia ion of dendri ic cells, which are bone-marrow-derived cells involved in an igen presen a ion and which play a key role in he ini ia ion of T-cell-media ed immune responses by presen ing immunogenic epi opes o CD4⫹ T cells. An igen presen a ion ac ivi y is downregula ed by IL-10.

3. Cos imula ory molecules CD28 and i s s ruc ural homologue CTLA-4 are expressed on he surface of bo h CD4⫹ and CD8⫹ peripheral T lymphocy es. B7.1 (CD80) and B7.2


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(CD86) are curren ly he bes -charac erized cos imula ory molecules expressed by dendri ic cells and by o her APCs. These molecules are recognized by CD28, which is expressed by naive CD4⫹ and CD8⫹ T cells. An igen presen a ion in he absence of cos imula ion no only does no s imula e he an igen-specific T cells bu also changes hem. Thus, lack of B7 expression by many umors has been hough o be a fac or con ribu ing o he lack of heir immunogenici y. The presence of B7 on he umors was found o be cri ical for T-cell induc ion bu no for effec or-cell func ion (Ta sumi et al., 1997). The combined gene ransfer of IL-2 cy okine and B7.1, however, demons ra ed synergis ic effec s in genera ing efficacious an i umor immuni y in animal umor models (Lanier et al., 1995).

4. Foreign his ocompa ibili y genes The immune sys em has he abili y o reac very s rongly o foreign his ocompa abili y an igens, even ones ha have no been seen before. This proper y of he immune sys em has been u ilized o genera e immune responses agains umors. A Phase I/II clinical rial is underway using in ra umoral injec ion of plasmid/lipid complex ha resul s in he expression of HLA-B7, a class I major his ocompa ibili y an igen (MHC class-I), on he umor cell surfaces. The plasmid used in his s udy encodes a biscis ronic mRNA ha produces HLA-B7 (heavy chain) and ␤-microglobulin (ligh chain) in equimolar amoun s. The expression of he HLA-B7 pro ein by cancer cells is expec ed o s imula e he pa ien ’s immune sys em o recognize hese ransfec ed cells as “foreign” and o selec ively des roy he umor. This may also facili a e he presen a ion of umorspecific an igens o he immune sys em and help he developmen of umorspecific immuni y. Preclinical resul s ob ained af er in ra umoral injec ion of plasmid encoding a murine allogeneic MHC an igen and complexed wi h ca ionic liposomes also sugges ha he immune response genera ed agains he primary umor may be effec ive in elimina ing secondary umors or me as ases (Plau z et al., 1994).

5. Tumor suppressor genes Tumor suppressor genes ac ively repress cell grow h and heir loss leads o umor developmen . The p53 umor-suppressor gene deficiency is observed in mos cancers. The p53 gene has been shown o be involved in he con rol of he cell cycle, ranscrip ional regula ion, DNA replica ion, and induc ion of apop osis. The p53 gene can suppress cell ransforma ion and malignan cell grow h. In roduc ion of he wildype p53 gene in a colon cancer xenograf model has been shown o reduce umor regression due o apop osis (Ro h et al., 1996). Lung cancer cells are frequen ly deficien in p53 and are suscep ible o he induc ion of apop osis by overexpressed p53, making his umor par icularly

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sui able for gene herapy by p53. Sys emic adminis ra ion of he umor-suppressor gene p53 complexed wi h ca ionic liposomes significan ly reduced umor grow h and me as ases of nude mice injec ed wi h cancer cells (Lesoon-Wood et al., 1995; Xu et al., 1997).

C. Pulmonary gene therapy Pulmonary gene herapy is a rac ive for he rea men of chronic bronchi is, cys ic fibrosis, ␣-1 an i rypsin deficiency, familial emphysema, as hma, pulmonary infec ions, surfac an deficiency, pulmonary hyper ension, lung cancer, and malignan meso helioma (Curiel et al., 1996; Caplen et al., 1993; Schwarz et al., 1996; S ribling et al., 1992). The pulmonary endo helium may ac as a bioreac or for he produc ion and secre ion of herapeu ic pro eins, such as blood-clo ing fac ors and ery hropoie in in o he blood circula ion. There is a po en ial benefi for acquired lung diseases, as well as cancers, o be con rolled and possibly rea ed by expression of cy okines, surfac an , an ioxidan enzymes, or mucopro eins wi hin lung cells. There are wo obvious ways o deliver plasmid o he lung: (i) in ra racheally by inhala ion or ins illa ion of a formula ed plasmid, and (ii) in ravenous delivery o he respira ory endo helium. Human airways have complex macroscopic branching s ruc ures and con ain a varie y of cells whose s ruc ure and func ion vary from rachea o dis al bronchioli. The ranspor from he airway lumen o he vascular endo helium represen s ano her significan barrier. Gene delivery o he submucosal glands of he upper airway is of par icular in eres , hough challenging for rea men of cys ic fibrosis, where correc ion of he gene ic defec s in he glands may improve he al era ions in he pa ien s’ secre ions (Al on et al., 1993). The aqueous layers ha lie on op of heal hy pulmonary epi helium are composed of mucins, sal s, and pro eins. In a damaged lung, here may be pro eases and pro eins ha can bind o plasmid/ca ionic carrier complexes and ha may lower gene ransfer efficiency. In diseases associa ed wi h chronic infec ion and inflamma ion, such as cys ic fibrosis, he pa ien frequen ly has a massive infil ra ion of neu rophils in o he lung. The neu rophils even ually lyse and release heir DNA in o he racheobronchial secre ions. The large amoun of par ially degraded neu rophil-derived DNA could compe e for he ca ionic carrier and disrup he plasmid/carrier complexes. Aerosoliza ion requires monodisperse par icles, because he deposi ion of inhaled par icles in he airways depends on par icle size. Larger par icles (grea er han 5 ␮m mass median diame er) end o deposi mainly in he larynx and upper airways. Wi h drople s less han 5 ␮m, here is an increase in airway and alveoli deposi ion, bu alveolar deposi ion is far grea er (Eas man et al., 1997). The inhaled dose is dependen on he minu e volume and en rainmen efficiency of he subjec , and herefore has considerable in ersubjec variabili y.


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Such variabili y will only be accen ua ed in many diseases in which he airways are obs ruc ed by mucus. S ill, aerosoliza ion of plasmid/lipid complexes has been successfully used for gene delivery in o he airways of mice and rabbi s wi h a pump spray device (Middle on et al., 1994; Logan et al., 1995) and is under clinical rials for gene delivery o he nasal epi helium (Sorscher et al., 1994; Gill et al., 1997; Por eous et al., 1997). In ra racheal ins illa ion bypasses he barrier of he endo helial cell layer ha is associa ed wi h sys emic gene delivery. Almos 80 – 90% of he s ar ing ma erial is was ed in aerosol gene delivery irrespec ive of he inhala ion device employed (Aldjei and Gup a, 1997). Therefore, in ra racheal ins illa ion of plasmid/lipid complexes is being inves iga ed as an al erna ive o deliver a varie y of repor er and po en ially herapeu ic genes o he lung (Yoshimura et al., 1992; Meyer et al., 1995; Tsan et al., 1995). Plasmid/lipid complexes, no plasmid alone, are effec ive in aerosoliza ion, whereas plasmids alone can efficien ly be ransfec ed o ra and mouse airway epi helial issues when given by he in ra racheal rou e (Meyer et al., 1995; Tsan et al., 1995). The dis ribu ion of plasmid o he bronchial ree can be varied by al ering he physicochemical charac eris ics of he formula ed plasmids. Immunohis ochemical s udies of lung issues af er in ra racheal adminis ra ion of plasmid/lipid complexes have shown gene expression mainly wi hin he epi helial cell layers lining he bronchus (Canonico et al., 1994).

D. Genetic vaccines Gene ic vaccina ion can be carried ou by injec ing plasmids encoding an igens direc ly in o muscle or skin, resul ing in hos immuni y agains his an igen (Johns on and Tang, 1994; Rabinovich et al., 1994; Fynan et al., 1993; Donnelly et al., 1997). Depending on he si e of expression and he na ure of he an igen, in vivo expression of plasmids encoding an igen can provide superior cellular, humoral, and mucosal immuni y. The efficacy of gene ic vaccines could be enhanced or modula ed hrough he use of formula ions ha increase nucleic-acid s abili y or dis ribu ion in he issue, he coexpression of immune molecules ha affec he processing of an igens, or hrough he use of adjuvan s ha affec he immune response. Gene ic vaccina ion has been applied o several sys ems, including immune responses agains cancer an igens (Spooner et al., 1995), mycoplasma (Lai et al., 1995), uberculosis (Lowrie et al., 1994), malaria (Doolan et al., 1996), parasi es (Yang et al., 1995), and viral infec ions (Yokoyama et al., 1995).

1. Mechanism of immuniza ion Two ypes of immuni y may be reduced in response o an an igen — namely, humoral immuni y media ed by an igen-specific an ibodies produced by B lym-

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phocy es, and cell-media ed immuni y produced by ac iva ed macrophages and cy o oxic T lymphocy es. An ibodies may neu ralize pa hogens, whereas cy ooxic T lymphocy es can des roy infec ed cells or con rol infec ion by noncy oly ic means. Ac iva ed macrophages can kill bac eria ha are seques ered inside hem. An ibody-media ed immuni y effec ively preven s infec ion by binding o he infec ious organisms and hen elimina ing ei her direc ly or via phagocy ic inges ion by neu rophils and/or monocy es. An ibodies also bind o he surface of infec ed cells expressing he specific an igen. Cell-media ed immuni y involves T cells, which recognize an igen presen ed by APCs via molecules encoded by he major his ocompa ibili y complex (MHC) genes. MHC class I molecules presen pep ides derived from an igens ha are syn hesized endogenously by he cells. CD8⫹ T cells differen ia e in o cy o oxic lymphocy es (CTLs) upon ac iva ion by such pep ideMHC class I complex-expressing an igen-presen ing cells. CD4⫹ T cells differen ia e upon recogni ion of pep ide-MHC class II complexes, which are genera ed from he processing of exogenous an igens, developing in o T helper cells (Davis, 1997). The CD4⫹ T helper cells can broadly be divided in o wo major sub ypes: Th1 CD4⫹ cells are implica ed in delayed- ype hypersensi ivi y reac ions and he genera ion and main enance of CTL responses, while Th2 cells are necessary for he genera ion and main enance of adequa e an ibody responses.

2. Delivery of gene ic vaccines Several rou es have been inves iga ed for he adminis ra ion of nucleic-acidbased vaccines. These include in ramuscular, subcu aneous, in ravenous, in radermal, nasal, and oral adminis ra ion. Of hese rou es, in ramuscular injec ion of gene ic vaccines genera ed he bes response (Wolff et al., 1990; Tang et al., 1997). Ma ure myo ube has been shown o be he arge for he up ake of plasmid af er in ramuscular adminis ra ion. Plasmid can en er he bloods ream and lympha ic sys em af er in ramuscular adminis ra ion and raffic o he spleen, liver, kidney, lymph nodes, and bone marrow. I is no clear whe her he produc ion of an igens in muscle has unique proper ies wi h respec o he elici a ion of a prolonged immune response or whe her expression in any issue in he periphery is sufficien for he induc ion of an an igen-specific immune response. Subcu aneous injec ion leads o DNA up ake and expression in kerainocy es, macrophages, and Langerhans cells. Single injec ion provides for a full humoral and T cell response for 60 – 70 weeks, wi h he an ibody i er being higher han ha achieved by in ramuscular injec ion. The an igen-producing epidermal kera inocy es and myocy es canno properly presen o he immune sys em wi hou special APCs, dendri ic cells and macrophages. The la er are


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more abundan in epidermis han in muscle. DNA can be in roduced in he epidermal cells by he ballis ic me hod, i.e., hrough bombardmen wi h gold par icles carrying DNA (“gene gun” echnique) or even by simple applica ion of a DNA suspension on skin (Tang et al., 1997). Skin is rich in dendri ic cells, which are po en ini ia ors of immune responses and possess he cos imula ory and adhesion molecules required for Tcell ac iva ion. In addi ion, dendri ic cells possess a unique abili y o process and presen ex racellular an igens in he con ex of bo h class I and class II molecules. Thus, ransfec ion of plasmids in o hese cells is likely o elici bo h cellular and humoral responses. Specific arge ing of dendri ic cells residing in he lymph nodes will likely represen an a rac ive s ra egy for providing a robus immune response wi h nucleic-acid vaccines.

IX. CONCLUDING REMARKS Nonviral gene herapy holds grea promise for improving he delivery and herapeu ic use of pro eins ha have poor pharmacokine ic profiles. Al hough almos 85% of curren gene herapy clinical rials are employing viral vec ors, hese gene herapies, in general, have no ye me expec a ions in erms of safe y and clinical efficacy. Therefore, here is a growing in eres in developing efficien nonviral gene delivery sys ems o con rol he loca ion and func ion of genes af er heir in vivo adminis ra ion o pa ien s. Several nonviral approaches are already in clinical rials and offer he po en ial of safe and effec ive gene herapy. To enhance he herapeu ic efficacy of pro eins using plasmid-based expression sys ems, many fundamen al ques ions rela ed o heir pharmaceu ical formula ion, biodis ribu ion, and in racellular rafficking s ill need o be addressed. Gene medicines are designed o provide a safe and cos -effec ive rea men for a varie y of severe and debili a ing diseases as well as o enhance pa ien compliance as compared o conven ional biopharmaceu ical produc s. They offer unique oppor uni ies in he developmen of novel produc s ha produce in racellular pro eins. Improvemen s will be needed for he exis ing nonviral delivery sys ems o fur her enhance si e specifici y, cellular en ry, and in racellular dis ribu ion.

Acknowledgments We would like o acknowledge our colleagues a Valen is, Inc. for heir useful commen s and cons ruc ive discussion. In par icular, we wish o hank Drs. Ross Durland, Jack Schaumberg, and Norman Hardman for reviewing he manuscrip and Oscar Monera for drawing Figures 4.5 and 4.6.

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5

Mutational Analysis of 23S Ribosomal RNA Structure and Function in Escherichia coli Kathleen L. Triman Depar men of Biology Franklin and Marshall College Lancas er, Pennsylvania 17604

I. In roduc ion II. Me hods of De ec ion of rRNA Mu an s in Escherichia coli A. Plasmid Expression of rRNA Mu a ions B. In roduc ion of Mu a ions III. Mu a ional Analysis of 23S rRNA S ruc ure and Func ion A. Secondary S ruc ure of 23S rRNA B. Mu a ions in domain I of 23S rRNA C. Mu a ions in domain II of 23S rRNA D. Mu a ions in domain III of 23S rRNA E. Mu a ions in domain IV of 23S rRNA F. Mu a ions in domain V of 23S rRNA G. Mu a ions in domain VI of 23S rRNA IV. Conclusions

I. INTRODUCTION The ribosome is responsible for he ransla ion of he gene ic code in all living organisms. The s ruc ural complexi y of he ribosome presen s an obs acle o defini ion of he molecular mechanism of ribosome ac ion. The Escherichia coli ribosome is he bes -charac erized sys em in which ransla ion has been s udied a he molecular level. The E. coli ribosome is a (70S) complex of RNA and pro ein composed of wo subuni s, he small (30S) subuni and he large (50S) subuni . The 30S subuni is composed of one species of RNA, 16S ribosomal Advances in Genetics, Vol. 41 Copyrigh 1999 by Academic Press All righ s of reproduc ion in any form reserved. 0065-2660/99 $30.00

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RNA, and 21 ribosomal pro eins. The 50S subuni is composed o wo RNA species, 23S ribosomal RNA and 5S ribosomal RNA, and 31 ribosomal pro eins (L1, L2, e c.; see Riley, 1993, and references herein). Evidence from bo h biochemical and gene ic approaches sugges s ha ribosomal RNA plays a func ional role in he process of ransla ion (reviewed in Dahlberg, 1989; Green and Noller, 1997; Noller, 1991; Noller, 1993a, 1993b; see also relevan chap ers in Ma heson et al., 1995, and in Zimmermann and Dahlberg, 1996). An excellen his orical perspec ive on research in he ribosome field was published recen ly by San er and Dahlberg (1996). Gene ic approaches have proved useful for he iden ifica ion of new aspec s of ribosomal RNA s ruc ure and func ion ha are no accessible o s udy by biochemical me hods alone. A review of he effec s of mu a ions in roduced in o 16S rRNA in E. coli was published previously (Triman, 1995). This review ou lines gene ic s ra egies designed o improve our unders anding of he s rucure and func ion of 23S ribosomal RNA in E. coli.

II. METHODS OF DETECTION OF rRNA MUTANTS IN Escherichia coli Gene ic analysis of he s ruc ure and func ion of ribosomal RNA has proved difficul because (1) expression of rRNA genes is essen ial and (2) here are seven copies of he rRNA genes in he E. coli genome (Riley, 1993). Bo h of hese challenges have been me by he use of plasmids con aining a single copy of one of he seven operons (rrnA, rrn , rrnC, rrnD, rrnE, rrnG, and rrnH) found in he genome. This subjec has been reviewed elsewhere (Triman, 1995; O’Connor et al., 1995), so he gene ic approaches are summarized briefly in his sec ion.

A. Plasmid expression of rRNA mutations Plasmid pKK3535, a deriva ive of pBR322, is a high copy number plasmid con aining he in ac rrn operon (Brosius et al., 1981a, 1981b, 1981c). Plasmid pLC7-21 is a recombinan plasmid ha con ains rrnH on a ColE1 vehicle (Sigmund and Morgan, 1982). O her plasmids con ain a copy of an rRNA operon under he con rol of an inducible promo er – opera or such as bac eriophage lambda pL (Gourse et al., 1985) permi ing condi ional rRNA expression in s rains con aining he empera ure-sensi ive cI857 repressor (Jacob et al., 1987). Appropria e bac erial hos s rains can be used o main ain plasmids con aining dele erious mu a ions a low copy number (O’Connor et al., 1992). Plasmids provide he oppor uni y o manipula e rRNA genes direc ly and, in some cases, o con rol expression of manipula ed rRNA genes. The general

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gene ic approach o he s udy of ribosomal RNA s ruc ure and func ion in E. coli has involved mu agenesis of plasmid rRNA genes (reviewed in DeS asio et al., 1988; Tapprich et al., 1990b; O’Connor et al., 1995; Triman, 1995).

1. In vivo expression Transformed cells can be grown under condi ions in which bo h chromosomally encoded rRNA and plasmid-encoded rRNA are expressed. Mu a ions in roduced by si e-direc ed mu agenesis in o plasmid rRNA genes may confer an al ered grow h pheno ype demons ra ing a dominan effec in ransformed cells. Plasmid-derived rRNA con aining a dominan mu a ion may in erfere wi h he normal func ion of chromosomally encoded rRNA. In ex reme cases, he defec may be a dominan le hal mu a ion, he expression of which causes cell dea h. A special class of dominan mu a ions is represen ed by he condi ional dominan , which confers mu an grow h proper ies, for example, a low empera ure bu no a higher empera ures. Sigmund et al. (reviewed in 1988) selec ed a number of an ibio ic resis ance mu a ions in rrnH by chemical mu agenesis of plasmid pLC7-21 and he use of media con aining an ibio ics. The de ec ion of recessive rRNA mu a ions was made possible by he in roduc ion of wo of hese selec able an ibio ic resis ance markers in o plasmid rRNA. Plasmid pSTL102 (Triman et al., 1989) was cons ruc ed from pKK3535 by in roduc ion of a spec inomycinresis ance allele (C o U change a posi ion 1192) in o he 16S rRNA gene and an ery hromycin-resis ance allele (A o G change a posi ion 2058) in o he 23S rRNA gene (Sigmund et al., 1984; Morgan et al., 1988). Mu a ions leading o loss of func ion of he cloned 16S rRNA gene cause loss of spec inomycinresis ance (Spcr), and mu a ions in he cloned 23S rRNA gene may affec ery hromycin-resis ance (Eryr). Spec inomycin-resis ance can be used o con rol agains ranscrip ional defec s when 23S rRNA mu an s are being sough . Recessive 23S rRNA mu an grow h pheno ypes can be de ec ed only under condi ions ha selec for ery hromycin-resis ance, whereas dominan mu an grow h pheno ypes can be de ec ed in he absence of ei her spec inomycin or ery hromycin. S ark et al. (1982) developed a maxicell procedure for expression of plasmid-coded rRNA in he comple e absence of hos -coded rRNA syn hesis. Maxicells are derived from s rains of E. coli unable o repair UV-ligh -damaged DNA; ribosomes isola ed from maxicells con aining mu agenized plasmids can be analyzed for he effec s of specific mu a ions on rRNA processing, pro ein – rRNA in erac ion, and subuni assembly (Dahlberg, 1986; Jemiolo et al., 1988). Hui and DeBoer (1987) developed a unique sys em involving he use of specialized ribosomes. This in vivo sys em involves expression of mu an rRNA ha also con ains an al ered an i-Shine-Delgarno sequence (e.g., 5⬘

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GGAGG). When rRNA con aining his al ered sequence is ranscribed and assembled in o ribosomes, i can ransla e only specifically engineered mRNAs con aining he complemen ary Shine-Delgarno sequence (e.g., 5⬘ CCUCC). Thus he ransla ion of a repor er mRNA can be followed, allowing s udy of he effec s of ribosomal mu a ions on ransla ion.

2. In vitro expression In vitro expression of mu an rRNA has been facili a ed by he cons ruc ion of plasmids in which he bac erial promo ers normally used for ranscrip ion of he rrn operon are replaced wi h a promo er for bac eriophage T7 RNA polymerase no normally found in E. coli (S een et al., 1986; Lewicki et al., 1993; Adamski et al., 1996). These plasmids were designed o provide (a) a “silen ” copy of he 23S rRNA gene, o avoid po en ial dele erious physiological effec s of mu an rRNA genes, and (b) he oppor uni y for expression of mu an 23S rRNA in vitro using T7 RNA polymerase. However, 23S rRNA ranscribed from he T7 promo er fails o assemble in o ca aly ically ac ive 50S subuni s in an in vitro recons i u ion reac ion (Lewicki et al., 1993; Green and Noller, 1996). Plasmid cons ruc s con aining DNA fragmen s corresponding o specific regions of he 23S rRNA sequence under he con rol of he promo er of bac eriophage T7 polymerase have also been genera ed. In vitro ranscrip ion from hese cons ruc s produces syn he ic RNA fragmen s sui able for use in fil er binding assays o s udy RNA – pro ein in erac ions or in conforma ional s udies (reviewed in Draper, 1996).

3. Allele-specific s ruc ural probing of plasmid-derived 23S rRNA Rapid chemical and enzyma ic probing me hods have permi ed he iden ificaion of residues in rRNA ha in erac wi h ribosomal pro eins, RNA, elongaion fac ors, and an ibio ics (reviewed in Noller, 1991). Biochemical charac eriza ion of mu an ribosomes in vitro has been hindered, however, by he fac ha ribosomes isola ed from cells are he erogeneous, con aining bo h mu an plasmid-derived rRNA and wildype chromosomally derived rRNA. Aagaard et al., (1991) have addressed his problem by cons ruc ing deriva ives of plasmid pKK3535, each carrying one of four specific mu a ions, priming si es, ha allow for selec ive probing of mu an ribosomes using reverse ranscrip ion from he engineered si es (see also Powers and Noller, 1993). Each of he mu a ions, in roduced in o a phylogene ically variable region of 23S rRNA, is pheno ypically silen . These specific priming-si e mu a ions will be presen ed in he appropria e sec ion of his chap er according o heir localiza ion in 23S rRNA.


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B. Introduction of mutations

1. Random mu agenesis Sigmund and Morgan (1982) rea ed cells con aining plasmid pLC7-21 in vivo wi h me hanesulfonic acid e hyl es er, pla ed hem on media con aining an ibio ics, and succeeded in isola ing a number of an ibio ic-resis ance mu a ions in rrnH (reviewed in Sigmund et al., 1988). These mu a ions included (1) he G o A change a posi ion 2057 of 23S rRNA ha confers resis ance o chloramphenicol and 14-a om lac one ring macrolides (MLS) (E ayebi et al., 1985) and (2) he A o U change a posi ion 2058 of 23S rRNA ha confers ery hromycin resis ance (Sigmund et al., 1984). Trea men of plasmid pSTL 102 wi h hydroxylamine yielded a number of mu an s con aining G o A or C o U al era ions in 23S rRNA (Dou hwai e et al., 1985). In each case, mu an s were iden ified among ransforman s conaining mu agenized DNA by he par icular grow h pheno ype associa ed wi h in roduc ion of a specific al era ion. The mos convenien me hods of de ec ion of randomly in roduced mu a ions involve (1) selec ion or screen for grow h on pla es con aining an ibio ics ha specifically arge rRNA (e.g., spec inomycin, ery hromycin, or s rep omycin; [Cundliffe, 1987; 1990]) or (2) selec ion or screen for grow h a ex reme empera ures ou side he op imum range for E. coli (e.g., 26 C or 42 C; [Ingraham, 1987]). Iden ifica ion of randomly in roduced mu a ions requires a mapping echnique, such as res ric ion fragmen exchanges be ween mu an and wildype plasmids, in order o (1) limi he region of DNA o be subjec ed o sequence analysis and (2) rule ou he presence of one or more secondary mu a ions.

2. Si e-direc ed mu agenesis Me hodologies for cons ruc ion of dele ion mu a ions, ransi ion mu a ions, or oligonucleo ide-direc ed mu a ions in rRNA have been described in a review by Tapprich et al. (1990a).

a. Mutagenesis targeted to regions of 23S rR A Gourse et al. (1982) and Skinner et al. (1985) isola ed si e-direc ed rRNA mu an s by limi ed exonuclease Bal-31 diges ion from selec ed res ric ion si es in a plasmid. These mu an s included some con aining dele ions be ween bases 318 and 365 in 23S rRNA. Some of he dele ions affec ed 70S binding o 30S subuni s. S ark et al. (1985) also charac erized a single-base dele ion a residue 1985 in 23S rRNA ha affec ed processing of he pre30S-rRNA ranscrip by RNAse III. Dou hwai e et al. (1989) used exonuclease diges ion from a res ric-

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ion si e o genera e a series of dele ion mu a ions in he 1220 – 1250 region. A remarkable s udy by Liiv et al. (1996) repor s he resul s of analysis of a series of 32 overlapping dele ions ha cover posi ions 40 – 2773 in 23S rRNA of E. coli. Plasmid cons ruc s con aining specific DNA fragmen s corresponding o regions of 23S rRNA sequence under he con rol of he promo er of phage T7 polymerase have been u ilized o ob ain syn he ic RNA fragmen s sui able for mu a ional analysis of RNA – pro ein complex forma ion and fil er binding assays. Examples of in erac ions defined by his s ra egy include he 23S rRNA binding si es for ribosomal pro eins L1 (e.g., Said et al., 1988), L11 (e.g., Ryan et al., 1991), and L23 (e.g., Kooi et al., 1993).

b. Mutagenesis targeted to a specific 23S rR A base The use of M13 cons ruc s (Ves er and Garre , 1987) or phagemids derived from Bluescrip (S ra agene) vec ors (e.g., Samaha et al., 1995) permi s prepara ion of 23S rDNA in single-s randed form for oligonucleo ide-direc ed mu agenesis. The 23S rRNA gene can be manipula ed in he mu agenesis plasmid and hen ransplan ed in o an expression vec or.

III. MUTATIONAL ANALYSIS OF 23S rRNA STRUCTURE AND FUNCTION Mu a ional analysis of he promo er region of he E. coli rrn operon has been carried ou by a number of groups. These s udies of he con rol of expression of rRNA are beyond he scope of his review, bu he de ails can be found in an excellen review by Gourse et al. (1996) and he references herein. Wha follows is a brief summary of represen a ive classes of mu a ions found in 23S rRNA. Appropria e references are provided so ha he reader can pursue he de ails of experimen al work ci ed here.

A. Secondary structure of 23S rRNA Figures 5.1a and 5.1b illus ra e he higher-order s ruc ure diagram for E. coli 23S rRNA (from Gu ell, 1996, wi h permission). The 23S rRNA molecule is subdivided in o six major s ruc ural domains by long-range base-paired in eracions. These are referred o as domains I (residues 16 – 524), II (residues 579 – 1261, a region ha includes he EG-F binding si e and GTP hydrolysis domain), III (residues 1295 – 1645), IV (residues 1648 – 2009), V (residues 2043 – 2625, a region ha includes he ErmE me hyla ion si e and he pep idyl ransferase


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Figure 5.1a. Higher-order s ruc ure diagram for Escherichia coli 23S rRNA, 5⬘ half; reproduced by permission of R. R. Gu ell and CRC Press (Fig. 1B, p. 115 in “Ribosomal RNA S ruc ure, Evolu ion, Processing and Func ion in Pro ein Biosyn hesis,” 1996).

cen er), and VI (residues 2630 – 2882, a region ha includes he EF-G binding si e and he ␣-sarcin and ricin loops) (Noller, 1984). Examples of he resul s of mu a ional analysis of 23S rRNA s ruc ure and func ion presen ed in his review are organized according o he s ruc ural domain in which par icular mu a ions are loca ed.

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Figure 5.1b. Higher-order s ruc ure diagram for Escherichia coli 23S rRNA, 3⬘ half; reproduced by permission of R. R. Gu ell and CRC Press (Fig. 1C, p. 116 in “Ribosomal RNA S ruc ure, Evolu ion, Processing and Func ion in Pro ein Biosyn hesis,” 1996).

B. Mutations in domain I of 23S rRNA

1. Mu a ions in ribosomal pro ein binding si es The localiza ion of he binding domain of ribosomal pro ein L24 has been defined by a varie y of biochemical me hods (reviewed in Draper, 1996) o include he posi ions 9 – 252 and 1276 – 386. In ernal dele ions from posi ions 318 – 392 block ribosome assembly and diminish binding of L24 (Skinner et al., 1985). Nishi and Schnier repor ed (1986) he isola ion of C33U, a mu a ion ha suppresses he empera ure-sensi ive pheno ype produced by an L24 pro ein


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mu a ion. Liiv et al. (1996) repor he associa ion of pro eins L4, L20, L22, and L24 wi h mu an rRNA con aining dele ions ha leave domain I of 23S rRNA in ac .

2. Mu a ion affec ing 4.5S RNA requiremen O’Connor et al. (1995) repor ed he isola ion of G424A, a mu a ion ha suppresses he requiremen for 4.5S RNA in ransla ion of na ural mRNAs by cell ex rac s. 4.5S RNA (114 nucleo ides) is he signal recogni ion par icle (SRP) RNA homolog in E. coli.

C. Mutations in domain II of 23S rRNA

1. Ery hromycin-resis an mu an s Dou hwai e et al. (1985) repor ed he isola ion of a dele ion in domain II of 23S rRNA ha included posi ions 1219 – 1230 and confers resis ance o ery hromycin. The dele ion was presumed o in erfere wi h he usual in erac ion be ween ery hromycin and a si e defined by nucleo ides in domain V ha are involved in con ac s wi h domain II (reviewed in Dou hwai e et al., 1989, 1993). Recen ly, however, mu a ions in domain II have been demons ra ed o facili a e ransla ion of a 23S rRNA-encoded pen apep ide conferring er hromycin resis ance (Dam et al., 1996).

2. Mu a ions in he L11 ribosomal pro ein binding si e The binding si e for pro ein L11 has been defined by a varie y of gene ic and biochemical echniques o include posi ions wi hin 1052 – 1112 in 23S rRNA (reviewed in Lu and Draper, 1995; Draper, 1996; Huang et al., 1996; Foun ain et al., 1996; Wang et al., 1996). A recen repor revealed coopera ive in eracions of his region of 23S rRNA and hios rep on an ibio ic wi h wo domains of ribosomal pro ein L11 (Xing and Draper, 1996). Thios rep on resis ance is conferred by al era ions a posi ions 1067 (Thompson et al., 1988) and 1095 (Rosendahl and Dou hwai e, 1994).

3. He erologous cons ruc s The ex reme conserva ion of he secondary s uc ure of he L11 binding domain of 23S rRNA is demons ra ed by he resul s of experimen s involving he erologous cons ruc s. Replacemen of he S. cerevesiae GTPase cen er (helix 39 – 40 region of domain II) wi h i s coun erpar from E. coli did no affec assembly of he large subuni RNA in o func ional subuni s (Mus ers et al., 1991). Likewise,

166

K. L. Triman

he erologous cons ruc s ha replace he E. coli L11 binding region wi h i s homolog from yeas have been shown o produce hybrid ribosomes ha in erac wi h hios rep on and func ion compe en ly in pro ein syn hesis (Thompson et al., 1993). The in ervening sequence from he 23S rRNA of he rrnD operon of Salmonella typhimurium was inser ed by Gregory et al. (1996) in o domain II of 23S rRNA of E. coli, where i was found o be pheno ypically silen . Ribosomes con aining he hybrid 23S rRNA appeared o func ion normally, wi h or wi hou processing of he IVS by RNAse III.

4. Priming si e mu a ion for s ruc ural probing of he GTPase region Aagaard et al. (1991) in roduced a pheno ypically silen mu a ion consis ing of five single-base al era ions in he 1170 region (A1169G, C1170U, A1175G, G1179A, U1180C) o crea e a unique sequence ha permi s allele-specific priming. The 1170 priming si e allows s ruc ural probing of he GTPase region in 23S rRNA.

D. Mutations in domain III of 23S rRNA

1. L23 ribosomal pro ein binding si e The L23 ribosomal pro ein binding si e has been defined by a varie y of biochemical me hods (reviewed in Draper, 1996) o include he posi ions 1304 – 1416 and 1588 – 1613. Mus ers et al., (1991) demons ra ed ha subs i u ion of he E. coli region 1371 – 1373 for he yeas sequences pro ec ed by L23 (V9) was olera ed by yeas 60S subuni s. Liiv et al. (1996) repor ed he associa ion of pro eins L10, L11, L13, and L23 wi h E. coli dele ion mu an s in which domain II was in ac .

2. Mu a ion affec ing 4.5S RNA requiremen O’Connor et al. (1995) repor ed he isola ion of G1423A, a mu a ion ha suppresses he requiremen for 4.5S RNA in ransla ion of na ural mRNAs by cell ex rac s.

3. Priming si e mu a ion for s ruc ural probing of he 1200– 1250 region Aagaard et al. (1991) in roduced a pheno ypically silen mu a ion consis ing of hree single-base al era ions in he 1360 region (C1362A, A1366G, G1367U)


167

o crea e a unique sequence ha permi s allele-specific priming. The priming si e allows s ruc ural probing of he 1200 – 1250 region in 23S rRNA.

E. Mutations in domain IV of 23S rRNA Leviev et al. (1995) described he resul s of random mu agenesis experimen s o sa ura e he highly conserved region of domain IV of 23S rRNA (posi ions 1900 – 1981). Of nine recessive le hal mu a ions ob ained (a posi ions be ween 1926 and 1984), wo yielded 50S subuni s defec ive in subuni – subuni associa ion bu ac ive in pep idyl ransferase ac ivi y, and five were defec ive in bo h subuni – subuni associa ion and pep idyl ransferase ac ivi y. The au hors propose ha he primary role of he region is he main enance of subuni – subuni in erac ions, while pep idyl ransferase ac ivi y migh be a secondary role. There is specula ion ha he region may be involved in he alignmen of pro ein L2, which binds in domain IV and is implica ed in pep idyl ransferase ac ivi y recons i u ed in vitro (Joseph and Noller, 1996). Domain IV also con ains a pos ranscrip ionally modified 3-me hylpseudouridine residue a posi ion 1915 and wo psuedouridines a posi ions 1911 and 1917 (Bakin and Ofengand, 1993; Kowalak et al., 1996). There is speculaion ha hese modified nucleo ides may play a role in pep idyl ransferase ac ivi y. O’Connor and Dahlberg (1995) repor ed he isola ion of a group of mu a ions in he 1916 loop of domain IV ha promo e read hrough of s op codons and increase frameshif ing. The 1916 loop has been localized o he subuni in erface of he ribosome and is implica ed in RNA – ribosome in erac ions.

F. Mutations in domain V of 23S rRNA

1. L1 ribosomal pro ein binding si e The L1 ribosomal pro ein binding si e has been ideni ified by a varie y of biochemical me hods (reviewed in Draper, 1996) o include he posi ions 2108 – 2129 and 2159 – 2181. Liiv et al. (1996) repor ed he associa ion of pro eins L1, L5, L6, L18, and L25 in dele ion mu an s in which domain V was in ac .

2. Pep idyl ransferase The essen ial fea ures of ribosome-ca alyzed pep ide bond forma ion and he involvemen of rRNA in his process have been reviewed elsewhere (e.g., Zimmermann et al., 1990; Lieberman and Dahlberg, 1995; Zimmermann, 1996). The pep idyl ransfer si e has been localized o he cen er of domain V of 23S rRNA. Relevan o considera ion of he role of domain V are he con ac s

168

K. L. Triman

be ween 5S rRNA and 23S rRNA and heir rela ive orien a ion o he pep idyl ransferase cen er, as explored in he work of Don sova et al. (1994) and Dokudovskaya et al. (1996). Also relevan are repor s of he involvemen of he “DEAD box” pro ein DbpA, an RNA helicase, in binding o 23S rRNA a he region of he pep idyl ransferase cen er (Nicol and Fuller-Pace, 1995) as well as o o her regions of 23S rRNA (Boddeker et al., 1997).

a. Antibiotic resistance mutations An ibio ic binding si es have proved useful in he de ermina ion of he s ruc ure of he pep idyl ransferase cen er by a varie y of biochemical me hods (reviewed in Rodriguez-Fonseca et al., 1995 and he references herein). An ibio ic resis ance mu a ions are clus ered o he cen ral par of domain V, an uns ruc ured circle in he secondary s ruc ure model. Mu a ions a conserved nucleo ides (e.g., 2058) in his region confer an ibio ic resis ances o pep idyl ransferase inhibi ors (chloramphenicol and anisomycin) as well as drugs ha in erfere wi h chloramphenicol binding (ery hromycin, MLS- ype an ibio ics; [Skinner et al., 1983; Ves er and Garre , 1988]). The me hyla ion of A2058 confers MLS resis ance as well (Egebjerg and Garre , 1991; Ves er et al., 1995). Mu a ions a 2438 confer amice in resis ance in halobac eria (Leviev et al., 1994) bu no in E. coli. The al era ions a 2438 are le hal a high empera ure in E. coli, however (Mankin, personal communica ion).

3. Priming si e mu a ion for s ruc ural probing of he pep idyl ransferase region Aagaard et al. (1991) in roduced a pheno ypically silen mu a ion consis ing of four single-base al era ions in he 2140 region (G2141U, A2142G, U2149C, C2150A) o crea e a unique sequence ha permi s allele-specific priming. The priming si e allows s ruc ural probing of he pep idyl ransferase region in 23S rRNA.

G. Mutations in domain VI of 23S rRNA

1. L3 pro ein binding si e Liiv et al. (1996) repor ed ha L3 ribosomal pro ein is associa ed wi h mu an par icles ha con ain dele ions of 23S rRNA in which domain VI is in ac . This is consis en wi h he biochemical analysis of L3 binding.

2. ␣-sarcin and ricin loops ␣-sarcin and ricin are ribo oxins ha in erac a nucleo ides 2660, 2661, and 2655 in E. coli rRNA and a analogous posi ions in eukaryo ic 23S-like rRNA (Gluck and Wool, 1996). Cleavage a he ␣-sarcin si e in E. coli 23S rRNA


169

(2661) in erferes wi h he binding of elonga ion fac ors EF-Tu and EF-G bu no wi h pep ide bond forma ion or subuni associa ion (Gluck et al. 1994 and he references herein). Ricin ca alyzes depurina ion a posi ion 2660. Marchan and Har ley (1995) repor ed he effec s of mu a ions a posi ion 2661 and 2663 ha resul in he loss of depurina ion by ricin bu have no effec on he ac ion of ano her ribosomal inac iva ing pro ein, pokeweed an iviral pro ein. O’Connor et al. (1995) have reviewed he effec s of mu a ions in his region on ransla ional accuracy.

3. Priming si e mu a ion for s ruc ural probing of he region Aagaard et al. (1991) in roduced a pheno ypically silen mu a ion consis ing of four single-base al era ions in he 2800 region (C2795A, U2796C, A2800G, G2801U) o crea e a unique sequence ha permi s allele-specific priming. The priming si e allows s ruc ural probing of he ␣-sarcin region in 23S rRNA.

IV. CONCLUSIONS One objec ive of his review was o ou line gene ic s ra egies designed o improve our unders anding of he s ruc ure and func ion of 23S ribosomal RNA in E. coli. A second objec ive has been o a emp o abula e he effec s of mu a ions in roduced in o 23S rRNA. The abula ion of 16S rRNA mu a ions for “Mu a ional Analysis of 16S Ribosomal RNA S ruc ure and Func ion in Escherichia coli” (see Table 1.2 in Triman, 1995) led o he crea ion of da abases of ribosomal RNA mu a ions. The Ribosomal RNA Mu a ion Da abases (16SMDB and 23SMDB) provide lis s of mu a ed posi ions in 16S and 23S ribosomal RNA from E. coli and he iden i y of each al era ion. The 16S Ribosomal RNA Mu a ion Da abase (16SMDB) consis s of an anno a ed lis of 233 al era ions dis ribu ed over 134 posi ions in 16S ribosomal RNA from E. coli (Triman, 1994, 1996a; Triman and Adams, 1997). The 23S Ribosomal RNA Mu a ion Da abase (23SMDB) consis s of an anno a ed lis of 235 al era ions dis ribu ed over 129 posi ions in 23S ribosomal RNA from E. coli (Triman, 1996b; Triman and Adams, 1997). Expanded versions of each da abase are also available (Triman et al., 1996; Triman and Adams, 1997) and include da a from E. coli and from o her organisms; hese files are en i led 16SMDBexp and 23SMDBexp. Table 5.1 provides examples of mu a ions a posi ions included in he 23SMDBexp file and illus ra es he forma of he da abase files. Mu a ed posi ions are arranged in order beginning wi h he 5⬘ end of 23S rRNA and ending wi h he 3⬘ end. For da a from organisms o her han E. coli, nucleo ide posi ions are iden ified by he corresponding pos ion in he E. coli s ruc ure. Pheno ypes associa ed wi h each al era ion are briefly described and designa ed as o whe her he

170

K. L. Triman

Table 5.1. 23SMDBexp: Single & Double Mu a ions in 23S and 23S-like Ribosomal RNA Posi ion

Al era ion

33

C oU

424

G oA

1005

C oG

C1005G/C1006U C1005G/G1138C

1006

C oU

C1006U/C1005G C1006U/G1137A

1056

G oA

G oA G oC 1062

G oA

1064

C oU

C1064U/C1075U

1067

A o U, C or G

Pheno ypea, b

Reference(s)

Suppressor of empera ure-sensi ive pro ein L24 mu a ion.a Suppressed requiremen for 4.5S RNA in ransla ion of na ural mRNAs by cell ex rac s.b Slow grow h under na ural promo er; (wi h 2058G and ery hromycin) severe grow h re arda ion.a Slow grow h under pL promo er; (wi h 2058G and ery hromycin) Erys.a Res ores normal grow h under pL promo er; (wi h 2058G and ery hromycin) Eryr.a Le hal under na ural promo er; under pL promo er (wi h 2058G and ery hromycin) Erys.a Slow grow h under pL promo er; (wi h 2058G and ery hromycin) Erys.a Res ores normal grow h under pL promo er; (wi h 2058G and ery hromycin) Eryr.a S oichiome ric L11 binding.b (Wi h 2058G and ery hromycin) reduced grow h ra e.a Binding of bo h L11 and hios rep on is weakened in RNA fragmen s.b Binding of hios rep on is weakened in RNA fragmen s.b Much reduced L11 binding.b (wi h 2058G and ery hromycin) reduced grow h ra e.a S oichiome ric L11 binding.b (Wi h 2058G and ery hromycin) reduced grow h ra e.a Normal in vivo assembly of L11 in o ribosomes.a

Nishi and Schnier, 1986.

A o C or U confers high-level resis ance o hios rep on, whereas A o G confers in ermedia e-level resis ance; drug binding affini y is reduced similarly.a, b Expression by hos RNA polymerase resul s in forma ion of ac ive ribosomal subuni s in vivo.a Reduced binding of micrococcin.b

O’Connor et al., 1995.

Rosendahl et al., 1995.

Rosendahl et al., 1995. Rosendahl et al., 1995.


Rosendahl et al., 1995. Rosendahl et al., 1995.

Dou hwai e et al., 1993; Rosendahl and Dou hwai e, 1995. Ryan and Draper, 1991. Ryan and Draper, 1991. Dou hwai e et al., 1993; Rosendahl and Dou hwai e, 1995. Dou hwai e et al., 1993; Rosendahl and Dou hwai e, 1995. Dou hwai e et al., 1993; Rosendahl and Dou hwai e, 1995. Thompson et al., 1988; Thompson and Cundliffe, 1991; Lewicki et al., 1993; Rosendahl and Dou hwai e, 1994.

171

5. Mutational Analysis of 23S rRNA Structure and Function in Escherichia coli Table 5.1. continued Posi ion 1067

Al era ion A oG

A o G or U A oU

1068

G oA

G1068A/G1099A 1071

G oA G1071A/G1106A

1072

C oU

1075

C oU C1075U/C1064U

Pheno ypea, b Suppressed requiremen for 4.5S RNA in ransla ion of na ural mRNAs by cell ex rac s.b Thios rep on resis ance in Halobacterium sp. Increased read hrough a UAG. Suppressed by combina ion wi h G2583A, C, or U. Reduced L11 binding.b (Wi h 2058G) le hal when expressed from rrnB or pL promo or in presence of ery hromycin.a Suppression of 1068A; le hali y only in absence of ery hromycin.a (Wi h 2058G) empera ure sensi ivi y.a Suppression of empera ure sensi ivi y of 1071A.a Le hal when expressed from rrnB or pL promo or in presence of ery hromycin.a No effec on L11 binding.b

1076

C oU

Normal in vivo assembly of L11 in o ribosomes.a No effec on L11 binding.b

1079

C oU

No effec on L11 binding.b

1082

U oC

Binding of bo h L11 and hios rep on is weakened in RNA fragmen s.b Bo h L11 and hios rep on bind RNA fragmen s wi h abou wildype affini y.b Bo h L11 and hios rep on bind RNA fragmen s wi h abou wildype affini y.b Reduced L11 binding.b Binding of bo h L11 and hios rep on is weakened in RNA fragmen s.b Bo h L11 and hios rep on bind fragmen s wi h abou wildype affini y.b Bo h L11 and hios rep on bind RNA fragmen s wi h abou wildype affini y.b Reduced L11 binding.b

U1082C/A1086G

U1082A/A1086U

1085 1086

A o G, C or U A oG A1086G/U1082C A1086U/U1082A

1087

G oA

Reference(s) Brown, 1989.

Hummel and Bo¨ck, 1987a. Saarma et al., 1993.

Dou hwai e et al., 1993; Rosendahl and Dou hwai e, 1995. Rosendahl and Dou wai e, 1995. Rosendahl and Dou wai e, 1995. Rosendahl and Dou wai e, 1995. Rosendahl and Dou wai e, 1995.

hhhh-

Rosendahl and Dou hwai e, 1995. Rosendahl and Dou hwai e, 1995. Rosendahl and Dou hwai e, 1995. Rosendahl and Dou hwai e, 1995. Ryan and Draper, 1991. Ryan and Draper, 1991.

Ryan and Draper, 1991.

Dou hwai e et al., 1993. Ryan and Draper, 1991. Ryan and Draper, 1991. Ryan and Draper, 1991.

Dou hwai e et al., 1993. continues

172

K. L. Triman

Table 5.1. continued Posi ion 1091 1092

Al era ion G oA C oU C1092U/G1099A C1092U/C1109U

1093

G oA

Pheno ypea, b

Reference(s)

Reduced L11 binding. (Wi h 2058G) empera ure sensi ivi y.a Suppression of 1092U empera ure sensi ivi y.a Par ial suppression of 1092U emperaure sensi ivi y.a Reduced L11 binding; trpA UGA suppressor; empera ure sensi ive.

Dou hwai e et al., 1993. Rosendahl and Dou hwai e, 1995. Rosendahl and Dou hwai e, 1995. Rosendahl and Dou hwai e, 1995. Dou hwai e et al., 1993; Jemiolo et al., 1995; Murgola et al., 1995. Dou hwai e et al., 1993; Jemiolo et al., 1995; Murgola et al., 1995. Xu and Murgola, 1996.

b

⌬G

trpA UGA suppressor

G oU

trpA UGA suppressor; empera ure sensi ive. trpA UGA suppressor; empera ure sensi ive. trpA UGA suppressor; empera ure sensi ive. trpA UGA suppressor; empera ure sensi ive. Reduced L11 binding; trpA UGA suppressor; empera ure sensi ive.

G oC G oA G1093A/A1098G 1094

U oA

1095

A o U, C or G

empera ure

Xu and Murgola, 1996.

1096

⌬A

trpA UGA suppressor

1097

⌬U

trpA UGA suppressor

1098

A oU A oG A oC

Normal grow h pheno Normal grow h pheno trpA UGA suppressor; sensi ive. trpA UGA suppressor; sensi ive. Reduced L11 binding.

G oA

G1099A/G1068A G1099A/C1092U


ype. ype. empera ure

⌬A

1099


Dou hwai e et al., 1993; Jemiolo et al., 1995; Murgola et al., 1995. Rosendahl and Dou hwai e, 1994. Jemiolo et al., 1995; Murgola et al., 1995. Jemiolo et al., 1995; Murgola et al., 1995. Jemiolo et al., 1995; Murgola et al., 1995. Xu and Murgola, 1996. Xu and Murgola, 1996. Xu and Murgola, 1996.

Reduced hios rep on and micrococcin binding. trpA UGA suppressor

A1098G/G1093A


Suppression of 1068A le hali y, bu only in absence of ery hromycin. Suppression of 1092U empera ure sensi ivi y.a

Dou hwai e et al., 1993; Rosendahl and Dou hwai e, 1995. Rosendahl et al., 1995. Rosendahl and Dou hwai e, 1995.

173

5. Mutational Analysis of 23S rRNA Structure and Function in Escherichia coli Table 5.1. continued Posi ion

Al era ion

Pheno ypea, b

1100

C oU

Reduced L11 binding.

1102

C oU

Reduced L11 binding.

1104

C oU

Reduced L11 biding.

1106

G1106A/G1071A

Reduced L11 bindingb; suppression of G1071A empera ure sensi ivi y.

1107 1109

G oA C oU C1109U/C1092U

1115

G oA

1137

G oA

G1137A/C1006U

G1137A/G1138C

1138

G oC

G1138C/C1005G

G1138C/G1137A

1206 1207 1208

1211

G oA G1206A/G1228A C oU C1207U/C1243U C oU C1208U/C1211U C1208U/C1243U C1211U/C1208U

No effec on L11 binding. Par ial suppression of empera ure sensi ivi y. No effec on L11 binding. Wi h 2058G and ery hromycin, le hal when expressed from rrnB promo er. Res ores normal grow h under pL promo or; (wi h 2058G and ery hromycin) Eryr. Wi h 2058G and ery hromycin, le hal when expressed from rrnB promo er. Wi h 2058G and ery hromycin, le hal when expressed from rrnB promo er. Res ores normal grow h under pL promo or; (wi h 2058G and ery hromycin) Eryr. Wi h 2058G and ery hromycin, le hal when expressed from rrnB promo er. Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin resis an .a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin resis an .a Ery hromycin sensi ive.a

Reference(s) Dou hwai e et al., 1993; Rosendahl and Dou hwai e, 1995. Dou hwai e et al., 1993; Rosendahl and Dou hwai e, 1995. Dou hwai e et al., 1993; Rosendahl and Dou hwai e, 1995. Dou hwai e et al., 1993; Rosendahl and Dou hwai e, 1995. Rosendahl and Dou hwai e, 1995. Rosendahl and Dou hwai e, 1995. Rosendahl and Dou hwai e, 1995. Rosendahl et al., 1995.






Dam et Dam et Dam et Dam et Dam et Dam et Dam et Dam et

al., 1996. al., 1996. al., 1996. al., 1996. al., 1996. al., 1996. al., 1996. al., 1996. continues

174

K. L. Triman

Table 5.1. continued Posi ion

Al era ion

1215 1218 1220 1221

1245 1248 1262

G oA G1218A/G1245A G1220A/G1239A C1221U/C1229U C1221U/C1233U C1221U/C1243U G oA ⌬1225/⌬1226 ⌬1226/⌬1225 G1227A/G1236A G1228A/G1206A C oU ⌬1230/⌬1231 ⌬1231/⌬1230 G1232A/G1238A C oU U oC U1234C/⌬1235 G oA ⌬1235/U1234C G oA G1236A/G1227A G oA G1238A/G1232A G1239A/G1220A C1243U/C1207U C1243U/C1208U C1243U/C1221U G1245A/G1218A G oA A o G or C

Ery hromycin sensi ive. Ery hromycin sensi ive.a Ery hromycin resis an .a Ery hromycin resis an .a Ery hromycin resis an .a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Ery hromycin resis an .a Ery hromycin resis an .a Ery hromycin resis an .a Ery hromycin resis an .a Ery hromycin sensi ive.a Ery hromycin sensi ive.a Wi h ery hromycin, le hal.

1262

A oU

Wi h ery hromycin, reduced grow h ra e. Wi h ery hromycin, reduced grow h ra e. Wi h ery hromycin, reduced grow h ra e. Suppression of grow h effec s; wildype grow h on ery hromycin. Suppression of grow h effec s; wildype grow h on ery hromycin. Suppressed requiremen for 4.5S RNA in ransla ion of na ural mRNAs by cell ex rac s.b

1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1238 1239 1243

A1262U/U2017G A1262C/U2017G A1262G/U2017C A1262U/U2017A 1423

G oA

Pheno ypea, b a

Reference(s) Dam et al., 1996. Dam et al., 1996. Dam et al., 1996. Dam et al., 1996. Dam et al., 1996. Dam et al., 1996. Dam et al., 1996. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dam et al., 1996. Dam et al., 1996. Dam et al., 1996. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dam et al., 1996. Dam et al., 1996. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dam et al., 1996. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Dou hwai e et al., 1989. Aagaard and Dou hwai e, 1994. Aagaard and Dou hwai e, 1994. Aagaard and Dou hwai e, 1994. Aagaard and Dou hwai e, 1994. Aagaard and Dou hwai e, 1994. Aagaard and Dou hwai e, 1994. O’Connor et al., 1995.

175

5. Mutational Analysis of 23S rRNA Structure and Function in Escherichia coli Table 5.1. continued Posi ion

Al era ion

Pheno ypea, b

1698

A oG

Suppresses 2555 mu a ions.

1819

A oG

Suppresses 2555 mu a ions.

1914

C oU

Promo es misreading; trpE91 frameshif suppressor. Li le or no effec on ransla ional fideli y. Promo es misreading; trpE91 frameshif suppressor. Li le or no effec on ransla ional fideli y. Reduced 70S ribosome forma ion in vivo; reduced pep idyl ransferase ac ivi y in 50S subuni s. Sligh ly reduced 70S ribosome formaion in vivo; sligh ly reduced pep idyl ransferase ac ivi y in 50S subuni s. Low-level 70S ribosome forma ion in vivo; pep idyl ransferase ac ivi y in 50S subuni s no de ec ed. Normal 70S ribosome forma ion in vivo; pep idyl ransferase ac ivi y in 50S subuni s normal. Low-level 70S ribosome forma ion in vivo; pep idyl ransferase ac ivi y in 50S subuni s normal. Low-level 70S ribosome forma ion in vivo; pep idyl ransferase ac ivi y in 50S subuni s normal. Sligh ly reduced 70S ribosome formaion in vivo; pep idyl ransferase acivi y in 50S subuni s no de ec ed. 70S ribosome forma ion in vivo no de ec ed; pep idyl ransferase ac ivi y in 50S subuni s no de ec ed.

C oA 1916

⌬1916

1921

G oC

1926

U oC

1940

U oA

1946

U oC

1951

U oC

1955

U oG

1956

U oA

1979

U oC

1982

U oA

1984

⌬1984 G oA

2017

U o G, C or A

Low 70S ribosome forma ion in vivo; pep idyl ransferase ac ivi y in 50S subuni s no de ec ed. Reduced grow h ra e on ery hromycin.

U2017G/A1262U

Reduced grow h ra e on ery hromycin.

Reference(s) O’Connor and Dahlberg, unpublished. O’Connor and Dahlberg, unpublished. O’Connor et al., 1995. O’Connor and Dahlberg, unpublished. O’Connor et al., 1995. O’Connor and Dahlberg, unpublished. Leviev et al., 1995.

Leviev et al., 1995.







Gourse et al., 1982. Leviev et al., 1995.

Aagaard and Dou hwai e, 1994. Aagaard and Dou hwai e, 1994. continues

176

K. L. Triman


Al era ion

Pheno ypea, b

Reference(s)

2017

U2017G/A1262C


U2017C/A1262G


U2017A/A1262U

Suppression of grow h effec s; wildype grow h on ery hromycin. Suppresses 2555 mu a ions.

Aagaard and Dou hwai e, 1994. Aagaard and Dou hwai e, 1994. Aagaard and Dou hwai e, 1994. O’Connor and Dahlberg, unpublished. Dou hwai e, 1992; Dou hwai e and Aagaard, 1993; Ves er et al., 1995. Dou hwai e, 1992; Dou hwai e and Aagaard, 1993; Ves er et al., 1995. Dou hwai e, 1992; Dou hwai e and Aagaard, 1993; Ves er et al., 1995. Dou hwai e, 1992; Dou hwai e and Aagaard, 1993; Ves er et al., 1995. Cseplo¨ et al., 1988. E ayebi et al., 1985; Dou hwai e and Aagaard, 1993; Ves er et al., 1995. Harris et al., 1989. Dou hwai e, 1992; Dou hwai e and Aagaard, 1993. Ves er et al., 1995.

2026

U oC

2032

G oA

Eryhs, Cdr, Cmr; no effec on me hylaion.

G2032A/G2057A

Eryr, Cdr, Cmr

G2032A/A2058G

Erys, Cds, Cms

G2032A/A2058U G oA

Eryhs, Cds, Cms; lincomycin resis ance in obacco chloroplas s.

G oA

Eryr, clinidamycin (Cd)s, chloramphemicol (Cm)r; reduces me hylaion of 23S rRNA by ErmE.

G oA

Eryr in Chlamydomonas chloroplas s.

G2057A/G2032A G2057A/C2611U A oU

Eryr, Cdr, Cmr Sligh ly Eryr; reduced me hyla ion. Eryr, Cdr, Cms; abolishes me hyla ion of 23S rRNA by ErmE.

A oG

Eryr, Cdr, Cms; abolishes me hyla ion of 23S rRNA by ErmE.

A oG

Ery hromycin resis ance in yeas mi ochondria.

2057

2058

Sigmund et al., 1984; Vannuffel et al., 1992b; Dou hwai e and Aagaard, 1993; Ves er et al., 1995. Ves er and Garre , 1987; Dou hwai e and Aagaard, 1993; Ves er et al., 1995. Sor and Fukuhara, 1982.

177


Al era ion A oG

A2058G/G2032A

Lincomycin resis ance in obacco chloroplas s Eryr, Lincomycin, and clindamycin resis ance in Chlamydomonas chloroplas s. Lincomycin resis ance in Solanum nigrum. chloroplasts. Clari hromycin resis ance in Helicobacter pylori. EryS, Cds, Cms.

A2058U/G2032A

Eryhs, Cds, Cms.

A oG

Lincomycin resis ance in obacco chloroplas s. Clari hromycin resis ance in Helicobacter pylori. Le hal.

A oG

A oG A oG

2059

A oG 2060

A oC

2061

G oA

2062

A oC

2123

G2123C/G2124C

2124

G2124C/G2123C

2125

G o A or C G oU G2125C/A2126C

2126

Pheno ypea, b

A o G or C A2126C/G2125U

2174

C2174C/C2175G

2175

C2175G/C2174G

2251

G o A, C or U

Cloramphenicol resis ance in ra mi ochondria Chloramphenicol resis ance in Halobacterium halobium. Reduced L1 binding in RNA fragmen s.a, b Reduced L1 binding in RNA fragmen s.a, b Reduced L1 binding in RNA fragmen s.a, b Sligh ly reduced L1 binding in RNA fragmen s.a, b Reduced L1 binding in RNA fragmen s.a, b Reduced L1 binding in RNA fragmen s.a, b Reduced L1 binding in RNA fragmen s.a, b Reduced L1 binding in RNA fragmen s.a, b Reduced L1 binding in RNA fragmen s.a, b Dominan le hal subuni associa ion defec .

Reference(s) Cseplo¨ et al., 1988. Harris et al., 1989.

Kavanagh et al., 1994. Versalovic et al., 1996. Dou hwai e, 1992; Dou hwai e and Aagaard, 1993. Dou hwai e, 1992; Dou hwai e and Aagaard, 1993. Cseplo¨ et al., 1988. Versalovic et al., 1996. Ves er and Garre , 1988; Koike et al. ci ed in Veser and Garre , 1988. Mankin and Garre , 1991. Said et al., 1988. Said et al., 1988. Said et al., 1988. Said et al., 1988. Said et al., 1988. Said et al., 1988. Said et al., 1988. Said et al., 1988. Said et al., 1988. Gregory and Dahlberg, unpublished. continues

178

K. L. Triman

Table 5.1. continued Posi ion 2252

Al era ion G o A, C or U

G2252C/G2253C

2253

Severely de rimen al o cell grow h; promo ed frameshif ing and readhrough of nonsense codons. Reduced pep idyl ransferase ac ivi y; severely de rimen al o cell grow h.

Gregory et al., 1994; Lieberman and Dahlberg, 1994. Lieberman and Dahlberg, 1994; Samaha et al., 1995; O’Connor et al. 1995. Lieberman and Dahlberg, 1994; Samaha et al., 1995; O’Connor et al. 1995. Porse et al., 1996.

No effec on grow h ra e.

G o A, C or U

Less han 5% of con rol level pep idyl ransferase ac ivi y. Promo ed frameshif ing and readhrough of nonsense codons.

G o C or U

G oC G2253C/G2252C

Slow grow h ra e. Severely de rimen al o cell grow h; reduced ra e of pep ide bond formaion in vitro.

G o U or A G2253A

No effec on cell grow h. 19% of con rol level pep idyl ransferase ac ivi y. 42% con rol level pep idyl ransferase ac ivi y. Less han 5% con rol level pep idyl ransferase ac ivi y. Amice in resis ance in Halobacterium halobium. Amice in resis ance and reduced grow h ra e in Halobacterium halobium. Uns able in presence or absence of amice in in Halobacterium halobium. Chloramphenicol resis ance in yeas mi ochondria. Anisomycin resis ance in Halobacterium sp. Le hal.

G2253U U oC U oA

U oG 2447

Reference(s)

G o U or A

G2253C

2438

Pheno ypea, b

G oA G oC

2450

A oC

2451

A oU

Chloramphenicol resis ance in mouse mi ochondria.

Lieberman and Dahlberg, 1994; Samaha et al., 1995; O’Connor et al. 1995. Gregory et al., 1994. Lieberman and Dahlberg, 1994; Samaha et al., 1995; O’Connor et al. 1995. Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Leviev et al., 1994. Leviev et al., 1994.

Leviev et al., 1994. Dujon, 1980. Hummel and Bo¨ck, 1987b. Ves er and Garre , 1988. Kearsey and Craig, 1981.

179


Al era ion C oA

G oC

Chloramphenicol resis ance in human mi ochondria. Chloramphenicol resis ance in mouse mi ochondria. Anisomycin resis ance in Halobacterium sp. Anisomycin resis ance in Tetrahymena thermophila. Chloramphenicol resis ance in Halobacterium halobium. Low-level sparsomycin resis ance in Halobacterium halobium. Anisomycin resis ance in Halobacterium sp. Promo es misreading; lack of mu an ribosomes in ransla ing polysome pool. Increased misreading.

G2458A/U2493C

Increased misreading.

G2458C/U2493C

Increased misreading.

U o G, C or A

Promo es misreading; trpE91 frameshif suppressor. Promo es misreading; trpE91 frameshif suppressor. Promo es misreading; trpE91 frameshif suppressor. Promo es misreading; trpE91 frameshif suppressor. No effec on grow h ra e or ranslaional fideli y. Promo es misreading; trpE91 frameshif suppressor. Promo es misreading; trpE91 frameshif suppressor. Promo es misreading; trpE91 frameshif suppressor. Promo es misreading; trpE91 frameshif suppressor. Promo es misreading; trpE91 frameshif suppressor.

C oU C oU C oU C oU C oU 2453

A oC

2458

G oA

2460

Pheno ypea, b

U2460G/G2490A U2460C/G2490C

2477

U2460G/G2490U U2460G/G2490C U o C or A

2490

G o A, C or U G2490C/U2460G G2490C/U2460C G2490U/U2460G G2490C/U2460G

Reference(s) Blanc et al., 1981 Slo et al., 1983. Hummel and Bo¨ck, 1987b. Sweeney et al., 1991. Mankin and Garre , 1991. Tan et al., 1996. Hummel and Bo¨ck, 1987b. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. continues

180

K. L. Triman


Al era ion

Pheno ypea, b

2492

U o C, G or A

Frameshif suppressors.

2493

U o A or C

(Wi h A2058G and ery hromycin) lehal grow h effec s. Frameshif suppressors. (Wi h A2058G and ery hromycin) lehal grow h effec s. Frameshif suppressors. (Wi h A2058G and ery hromycin) lehal grow h effec s. Frameshif suppressors. Dele erious effec s on ribosome funcion. Dele erious effec s on ribosome funcion. (Wi h A2058G and ery hromycin) reduced grow h ra e. Sparsomycin resis ance in Halobacterium halobium. Low-level sparsomycin resis ance in Halobacterium halobium. Decreased grow h ra e.

U o C, G or A

⌬U

U2493C/G2458A U2493C/G2458C 2497

A oG

2499

C oU

2500

U oC

2502

G oA

2503

A oC A oG A oC

2504

U o A or C U oC

2505

G oC

2505

G oA G oC

2506

G oU U oA

Decreased grow h ra e; CAMr. (Wi h A2058G and ery hromycin) slow grow h ra e. CAMr Chloramphenicol resis ance in yeas mi ochondria. Increased read hrough of s op codons and frameshif ing; le hal. Chloramphenicol resis ance in human mi ochondria. (Wi h A1067U and Thios rep on) empera ure-sensi ive grow h.a Hypersensi ivi y o Cam; increased sensi ivi y of in vitro ransla ion. Sligh increase in sensi ivi y o licomycin.b No effec on ransla ional accuracy. 14% ac ivi y of 70S ribosomes. Excluded from 70S ribosomes; 17% ac ivi y of 70S ribosomes. ⬍5% ac ivi y of 70S ribosomes. Dominan le hal; 5% ac ivi y of 70S ribosomes.

Reference(s) O’Connor and Dahlberg, 1995. Porse and Garre , 1995; O’Connor et al., 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. O’Connor and Dahlberg, 1995. Porse and Garre , 1995. Tan et al., 1996. Tan et al., 1996. Ves er and Garre , 1988. Porse and Garre , 1995. Porse and Garre , 1995. Dujon, 1980. O’Connor et al., 1995. Kearsey and Craig, 1981; Blanc et al., 1981. Saarma and Remme, 1992; Saarma et al., 1993.

Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Porse et al., 1996.

181


Al era ion

2508

U oC U oG G2508U

2514

U oC

2516

A oU

2528

U oA

U oC 2530

A oG

2546

U oC

2550

G oA

2552

U oA U oC

2555

U oC

U o A or G

U oC 2557

G oA

2561

U oC

2565

A oU

2580

U oC

Pheno ypea, b

Reference(s)

20% ac ivi y of 70S ribosomes. ⬍5% ac ivi y of 70S ribosomes. Con rol-level pep idyl ransferase acivi y. Con rol-level pep idyl ransferase acivi y. Con rol-level pep idyl ransferase acivi y. (Wi h A2058G and ery hromycin) slow grow h ra e. Con rol-level pepidyl ransferase ac ivi y. Con rol-level pep idyl ransferase acivi y. (Wi h A2058G and ery hromycin) slow grow h ra e. Con rol-level pep idyl ransferase acivi y. (Wi h A2058G and ery hromycin) slow grow h ra e. (Wi h A2058G and ery hromycin) slow grow h ra e. (Wi h A2058G and ery hromycin) slow grow h ra e. (Wi h A2058G and ery hromycin) slow grow h ra e. Con rol-level pepidyl ransferase ac ivi y. S imula es read hrough of s op codons and frameshif ing; U o A is trpE91 frameshif suppressor; viable in low copy number plasmids bu le hal when expressed cons i u ively from lambda pL promo er. No effec .

Porse et al., 1996. Porse et al., 1996. Porse and Garre , 1995; Porse et al., 1996. Porse and Garre , 1995.

(Wi h A2058G and ery hromycin) slow grow h ra e. In ermedia e decrease in pep idyl ransferase ac ivi y. Small decrease in pep idyl ransferase ac ivi y. (Wi h A2058G and ery hromycin) slow grow h ra e. Very low pep idyl ransferase ac ivi y. (Wi h A2058G and ery hromycin) lehal grow h effec s. No pep idyl ransferase ac ivi y.

Porse and Garre , 1995. Porse and Garre , 1995.

Porse and Garre , 1995. Porse and Garre , 1995. Porse and Garre , 1995. Porse and Garre , 1995. Porse and Garre , 1995. Porse and Garre , 1995. Porse and Garre , 1995.

O’Connor and Dahlberg, 1993; O’Connor et al., 1995.

O’Connor and Dahlberg, 1993. Porse and Garre , 1995.

Porse and Garre , 1995. Porse and Garre , 1995.

Porse and Garre , 1995.

continues

182

K. L. Triman

Table 5.1. continued Posi ion 2580

Al era ion U2580A U2580C U2580G

2581

G2581A G2581C G2581U

2582

G2582A G2582C G2582U

2583

G o A, U or C

G oA G oC G oU 2584

U oG

U oA U oC

2585

U oG U oA

Pheno ypea, b Dele erious; ⬍5% ac ivi y of 70S ribosomes. Dominan le hal; 12% ac ivi y of 70S ribosomes. Dele erious; 6% ac ivi y of 70S ribosomes. Dele erious; 22% ac ivi y of 70S ribosomes. Dele erious; 13% ac ivi y of 70S ribosomes. Dele erious; 18% ac ivi y of 70S ribosomes. Less han 5% of con rol-level pep idyl ransferase ac ivi y. Less han 5% of con rol-level pep idyl ransferase ac ivi y. Less han 5% of con rol-level pep idyl ransferase ac ivi y. Decreased misreading in vitro; increased ribosome sensi ivi y o chloramphenicol; (wi h A1067U and hios rep on, empera ure-sensiive grow ha and suppression of 1067U mis ransla ion effec s). Hypersensi ivi y o Cam increased sensi ivi y of in vitro ransla ion. Sligh increase in sensi ivi y o lincomycin.b Increased ransla ional accuracy C ⬎ U ⬎ A ⬎ G. Less han 5% of con rol-level pep idyl ransferase ac ivi y. Less han 5% of con rol-level pep idyl ransferase ac ivi y. Dominan le hal; less han 5% of conrol-level pep idyl ransferase ac ivi y. (Wi h A2058G and ery hromycin) lehal grow h effec s. No pep idyl ransferase ac ivi y. Dele erious; 20% ac ivi y of 70S ribosomes. Dele erious; 20% ac ivi y of 70S ribosomes. Dominan le hal Dominan le hal; less han 6% of conrol-level pep idyl ransferase ac ivi y.

Reference(s) Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Saarma and Remme, 1992; Saarma et al., 1993.

Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Porse and Garre , 1995.

Porse et al., 1996. Porse et al., 1996. Porse et al., 1996. Porse et al., 1996.

183


Al era ion U oC

U oG 2589

A oG

2600

A oU

2607 2608 2611

G oC G o A, U or C C oU C oU C oG C oG

C2611U/G2057A C o U or G

2654

A oG A o G or C A oC A oU A oG A2654G/C2666U A2654G/C2666G A2654C/C2666U A2654C/C2666G A2654U/C2666A

Pheno ypea, b Dominan le hal; less han 6% of conrol-level pep idyl ransferase ac ivi y. Dominan le hal; 36% of con rol-level pep idyl ransferase ac ivi y. (Wi h A2058G and ery hromycin) slow grow h ra e. S rong reduc ion in pep idyl ransferase ac ivi y. Small decrease in pep idyl ransferase ac ivi y. (Wi h A1067U) (Wi h A1067U) Eryr; reduces me hyla ion of 23SrRNA by ErmE. Spiramycin resis ance in yeas mi ochondria. Ery hromycin and spiramycin resis ance in yeas mi ochondria. Ery hromycin and spiramycin resis ance in Chlamydomonas chloroplas s. Eryr; reduces me hyla ion of 23SrRNA by ErmE. Eryr and low-level lincomycin and clindamycin resis ance in Chlamydomonas chloroplas s. S imula es read hrough of s op codons and frameshif ing. No effec . Mildly res ric ive effec on fideli y.a Minor increase in s op codon readhrough and frameshif ing.a Significan increase in s op codon read hrough and frameshif ing.a Significan increase in s op codon read hrough and frameshif ing.a Increased s op codon read hrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a

Reference(s) Porse et al., 1996.

Porse et al., 1996. Porse et al., 1996.

Porse et al., 1996. Saarma et al., 1993. Saarma et al., 1993. Vannuffel et al., 1992a; Ves er et al., 1995. Sor and Fukuhara, 1984. Sor and Fukuhara, 1984. Gau hier et al., 1988.

Ves er et al., 1995. Harris et al., 1989.

O’Connor et al., 1995. O’Connor et al., 1995. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. continues

184

K. L. Triman


Al era ion

Pheno ypea, b

Reference(s)

2654

A2654U/C2666G

Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Unde ec able levels of mu an 23S rRNA in 50S, 70S, or polysome frac ions. Reduced levels of mu an 23S rRNA in 50S, 70S, or polysome frac ions. Unde ec able levels of mu an 23S rRNA in 50S, 70S, or polysome frac ions. Decreased misreading; s rep omycin dependen when expressed wi h Smr, hyperaccura e S12 mu a ion.

O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. Marchan and Har ley, 1994.

A2654U/C2666U A2654C/C2666A A2654G/C2666A 2658

C oG

C2658G/G2663C 2660

A oG

2661

G oC

G oU G oA 2663

G oC G2663C/C2658G

2664

G oC

2666

C oU C o A or G C oU C oG C oA C2666U/A2654G

Decreased misreading; no effec on s rep binding. Grow h unaffec ed; incorpora ed in o ribosomes a wildype levels. Reduced levels of mu an 23S rRNA in 50S, 70S, or polysome frac ions. Reduced levels of mu an 23S rRNA in 50S, 70S, or polysome frac ions. Decreased grow h ra e, reduced viabili y and incorpora ion in o polysomes. Promo es misreading; trpE91 frameshif suppressor. No effec . Increased s op codon read hrough and frameshif ing.a No effec on s op codon read hrough or frameshif ing.a No effec on s op codon read hrough or frameshif ing.a Significan increase in s op codon read hrough and frameshif ing.a

Marchan and Har ley, 1994. Marchan and Har ley, 1994. Tapprich and Dahlberg, 1990; Tapio and Isaksson, 1991; Melancon et al., 1992; Bilgin and Ehrenberg, 1994; O’Connor et al., 1995. Melancon et al., 1992. Marchan 1994. Marchan 1994. Marchan 1994. Marchan 1994.

and Har ley, and Har ley, and Har ley, and Har ley,

O’Connor et al., 1995. O’Connor et al., 1995. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996.


185


Al era ion

Pheno ypea, b

Reference(s)

2666

C2666G/A2654G

Increased s op codon read hrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a Minor increase in s op codon readhrough and frameshif ing.a

O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996. O’Connor and Dahlberg, 1996.

C2666U/A2654C C2666G/A2654C C2666A/A2654U C2666G/A2654U C2666U/A2654U C2666A/A2654C C2666A/A2654G

a

in vivo in vitro

b

pheno ypes were de ec ed in vivo or in vitro. Appropria e references are provided for each al era ion. Files con aining only he da a from organisms o her han E. coli are en i led 16S-likeMDB and 23S-likeMDB (Triman et al., 1996; Triman and Adams, 1997). Individuals wi h access o he In erne elecommunica ions ne work may ob ain ex files of he da abases by anonymous file ransfer pro ocol. The f p si e is Acad.FandM.edu. The direc ory is /NAR. The da abases are also available a he following URL: http://www.fandm.edu/Departments/ iology/Databases/RNA.html on he World Wide Web (Su¨hnel, 1997). Ul ima ely, he goal of his work is o provide da abases ha can be queried for specific kinds of informa ion. The plan is o organize he da a so ha one can access, for example, (1) all he da a from one specific organism, (2) all he da a for one specific nucleo ide posi ion (e.g., see Felciano et al., 1997), or (3) all he da a for one specific pheno ype. Ideally, informa ion abou new mu a ions will also be submi ed elec ronically o he da abase! Mu a ional analysis of 23S ribosomal RNA s ruc ure and func ion has proven o be a powerful approach o he s udy of he role of his RNA in he process of ransla ion. There is also grea promise in he following novel gene ic approaches o he s udy of 23S rRNA: (1) inac iva ion of as many as four chromosomal rrn operons in E. coli by inser ion – dele ion mu agenesis using

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an ibio ic resis ance casse es (reviewed in Condon et al., 1995); (2) he in roduc ion of an ibio ic resis ance mu a ions in o he single chromosomal rRNA operon of archaebac eria (e.g., Mankin et al., 1992; Mankin, 1994; Mankin et al., 1994; Aagaard et al., 1994; Tan et al., 1996); and (3) s udies in eukaryo ic sys ems amenable o gene ic analysis of he ribosome (e.g., Mus ers et al., 1991; Newman et al., 1991; Sweeney et al., 1991; Liu and Liebman, 1996). We are for una e o have he guidance of hose who produce s ruc ural models based on compara ive sequence analysis (e.g., Gu ell, 1992, 1993, 1996; Gau here et al., 1994, 1995; Gu ell et al., 1994; Schnare et al., 1996) and he insigh provided by s udies ha reveal he significance of pos ranscrip ional modifica ion for 23S rRNA s ruc ure and func ion (e.g., Smi h et al., 1992; Bakin and Ofengand, 1993; Green and Noller, 1996; Lazaro et al., 1996; Ofengand and Bakin, 1997). The con ex for our gene ic s udies may be found in he hree-dimensional maps of he ribosome (reviewed by Moore, 1995; see also Moore, 1997) genera ed by cryo-elec ron microscopy in combina ion wi h angular recons i u ion (e.g., S ark et al., 1995) and hree-dimensional recons rucion (Agrawal et al. 1996), as well as in he resul s of cross-linking experimen s ha map RNA crosslinks o rRNA (e.g., Rinke-Appel et al., 1995) and he pa h of pep ides biosyn hesized in situ on E. coli ribosomes (e.g., S ade et al., 1995). Our knowledge of he process of ribosome syn hesis and ac ivi y is also being advanced rapidly as a resul of analyses of ribosomes con aining an isense DNA hybridized o various sequences of 23S rRNA (e.g., Meyer et al., 1996) and s udies of he effec s of mu an release fac or on ransla ion ermina ion (e.g., Zhang et al., 1996). Wha lies ahead also includes a grea er unders anding of (1) he role of he 50S ribosomal subuni and i s 23S rRNA on pro ein folding in E. coli, as implied by he resul s from in vitro s udies by Das et al. (1996); (2) he in erac ion be ween he ribosome and he signal recogni ion par icle (e.g., Powers and Wal er, 1996); (3) he po en ial role of pep ides in regula ion of he ac ivi y of ransla ing ribosomes (e.g., Harrod and Love , 1995; Love , 1996); and (4) s udies on ribosome-inac iva ing pro eins (e.g., Holmberg and Nygard, 1996). Fur hermore, here are in riguing repor s abou he effec s of he bacerial pro ein Fis (fac or for inversion s imula ion) on ini ia ion of chromosome replica ion and on ac iva ion of he P1 promo ers of rRNA and RNA genes (e.g., Zhang and Bremer, 1996), as well as abou he effec s of differen grow h condi ions on ribosomal RNA promo er ac ivi y (Liebig and Wagner, 1995) and on frameshif ing (Barak et al., 1996). The common heme of all hese kinds of s udies is he remarkable power of molecular ools for gene ic and biochemical manipula ion of RNA and pro ein o uncover fascina ing and unan icipa ed fea ures of s ruc ure and funcion in he ribosome.


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Acknowledgments This work was suppor ed by he NSF (MCB-9315443) and gran s from he Dean of Franklin and Marshall College. I am par icularly gra eful o S avros Vavoulis for his help wi h he prepara ion of his manuscrip and o Alexander Mankin for his insigh ful commen s.

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Index Aarskog-Scott syndrome, 76– 77 ␣-Actin promoter, 106 Aedes, 2 chromosome length, 6, 8– 9t cladistic analysis, 4, 5 evolutionary history, 3 genome size, 10 variation within, 14– 17 heterochromatin, 17– 20 multipoint linkage mapping, 24 A aegypti, 4, 10, 20, 21f, 24, 25 A albopictus, 4, 5, 10, 14– 17, 22, 24, 25 A alcasidi, 6 A annandalei, 18 A atropalpus, 5 A bahamensis, 17 A cinereus, 18 A cooki, 10 A epactius, 5 A excrucians, 18 A hebrideus, 20 A katherinesis, 18, 20 A mascarensis, 18, 20, 21f A pseudoscutellaris, 10 A scutellaris, 5 A simpsoni, 4 A stimulans, 18 A triseriatus, 4, 10, 18 A vexans, 4 A vittatus, 18 A zoosophus, 10 Aedes aegypti, 4 comparing linkage maps size with genome size, 25 genome organization, 10 heterochromatin, 20, 21f linkage mapping, 24 Aedes albopictus, 4, 5 comparing linkage maps size with genome size, 25 genome organization, 10 genome size variation within, 14– 17

linkage mapping, 24 repetitive DNA, 22 Aedini, 5 cladistic analysis, 4 genome size, 10 Aerosolization, in pulmonary gene therapy, 140– 141 AHR, see Aryl hydrocarbon receptor al genes (Neurospora), 38– 40, 41– 42 light-regulated promoter, 45, 46 Anchor groups, 112 Anopheles, 2 comparing linkage map size to genome size, 25– 26 heterochromatin on sex chromosomes, 20– 21 A albimanus, 5 A gambiae, 11, 25 A quadrimaculatus, 6, 11, 22 A stephensi, 21, 22 Anophelinae chromosome number, 6 cladistic analysis, 4, 5 comparing linkage map size to genome size, 25– 26 evolution of, 22, 26– 27 genome organization, 10, 11, 12t genome size, 10, 12t, 14f heterochromatin, 17, 20– 21, 22, 23f polytene chromosomes, 22 sex chromosomes, 6, 7 taxonomy, 2 Antibiotic resistance mutations, in E coli 23S rRNA, 159, 165, 168 Antigen-presenting cells, 138, 139, 142 Antiprogestin gene switches, 109– 110 Anti-Shine-Delgarno sequences, 159– 160 Armigeres subalbatus, 10, 20, 25 ARNT, see Aryl hydrocarbon receptor nuclear translocator Aryl hydrocarbon receptor (AHR), 45f, 46, 47, 50

1 7

198

Index

Aryl hydrocarbon receptor nuclear translocator (ARNT), 45f, 46, 47, 50 Asialofetuin, 115 Atkin-Fleitz syndrome, 79 ATR- syndrome, 72– 74 Autocrine proteins, 97

Ballistic particle delivery, nucleic acid-based vaccines, 143 Bat transcription factor, 47 BBB syndrome, 74; see also Opitz syndrome Beckwith-Wiedemann syndrome, 70, 71 Biologics License Application, 102, 104 Bironella, 2 Blood tests, for ATR- syndrome, 73 Blood vessels, interactions with gene expression plasmids, 125– 126 blr mutants (Neurospora), 43 Blue light responses, see also Neurospora blue light responses in Fusarium, 44 Borjeson-Forssman-Lehmann syndrome, 79

Cancer gene therapy, see also Tumors costimulatory molecules, 138– 139 cytokine genes, 138, 139 foreign histocompatibility genes, 139 prodrug-converting enzyme genes, 137– 138 tumor suppressor genes, 139– 140 Capillaries interactions with gene expression plasmids, 125– 126 sinusoidal, 126 Cap structure, 106 Cationic lipids, gene delivery systems with, see Lipid-based gene delivery systems Cationic polymers gene delivery systems with, 121– 122 toroid morphology, 122– 123 structure of, 122 C-banding, see Heterochromatin ccb mutants (Neurospora), 43– 44 ccg genes (Neurospora), 41, 46 Cell-mediated immune response cancer gene therapy, 138– 139 nucleic acid-based vaccines and, 142 Ceratopogonidae, 3

Cesium chloride, 102 Chagasia, 2 C bathana, 6, 23f Chaoboridae, 3 chromosome length, 8t cladistic analysis, 5 genome organization, 11 genome size, 12t, 14f sex chromosomes, 7 Chaoborus, 11, 23f CHEMS, 111 Cherasomes, 111 Chironomidae, 3 Chironomus tentans, 11 Chitosan, 121– 122 Cholesterol hemisuccinate morpholine salt, see CHEMS Chromocenter, 22 Chromosome length, in mosquitoes, 6, 8– 9t Chromosome number, in Culicidae, 5– 7 Circadian clock, in Neurospora, 41– 42, 50 Clinical trials, in gene therapy development, 103– 104 Cochleates, 111– 112 Coffin-Lowry syndrome, 77– 80 Colipids, 114 Colon cancer, 139 Complement system, interaction with gene expression plasmids, 115 con-10 gene (Neurospora), 46 Condensing peptides, 117, 118f Conidia, development, factors influencing, 41 Corethrella, 11, 23f Corethrellidae, 3 cladistic analysis, 5 genome organization, 11 sex chromosomes, 7 Culex, 4, 10 C pipens, 10, 25 Culicidae chromosome length, 6, 8– 9t chromosome number, 5– 7 cladistic analysis, 3– 5 evolution in, 2– 3, 22, 26– 27 genome organization, 10– 11, 14 genome size, 7, 10, 12– 13t intrapsecifc variation, 14– 17 heterochromatin, 17– 23 multipoint linkage mapping, 23– 25 comparing map size to genome size, 25– 26

Index overview of, 1– 2 polytene chromosomes, 22 r- and k-selection in, 17 repetitive DNA, 16– 17, 21– 22 sex chromosomes, 6, 7 evolution of, 7, 27 heterochromatin organization, 17– 18, 20 taxonomy, 2 Culicinae chromosome number, 6 cladistic analysis, 4– 5 comparing linkage map size to genome size, 25, 26f evolution in, 22, 26, 27 genome organization, 10, 12– 13t genome size, 12– 13t, 14f heterochromatin, 17– 20, 21– 22, 23f polytene chromosomes, 22 sex chromosomes, 7 taxonomy, 2 Culicini, 5, 10 Culicoidea, 7, 10 Culiseta, 10 C longiareolata, 17 Cystic fibrosis, nonviral gene therapy, 140 Cytokine gene therapy, 138, 139 Cytokines, inhibition of transgene expression, 105– 106 Cytoplasm, trafficking of gene expression plasmids, 132– 133 Cytoskeleton, trafficking of gene expression plasmids, 133 Cytotoxic lymphocytes, 142

DC-Chol, 113, 114 DEAE-dextran, 121 Demethylation, FMR1 gene and, 67– 68 Dendritic cells cancer gene therapy, 138, 139 skin, nucleic acid-based vaccines, 143 Depurination, 169 Diethylaminoethyldextran, see DEAE-dextran Dioctadecylamidoglyl spermine, see DOGS Dioleoylphophatidylcholine, see DOPC Dioleoylphophatidylethanoamine, see DOPE Dioleoylsuccinylglycerol, see DOSG Diptera, chromosome number, 6 Dixidae, 3, 7, 12t

199

DMRIE, 113 DNA in nonviral gene therapy aggregation, 124 condensation, 114, 123 condensing agents, 123– 124 topology, 123 repetitive, in mosquitoes, 16– 17, 21– 22 DOGS, 113 DOPC, 111, 114 DOPE, 111, 112, 114 DOSG, 111 DOSPA, 113 DOTAP, 112, 113 DOTMA, 112, 113

Endocrine proteins, 97 Endocytosis, uptake of gene expression plasmids, 130– 131, 136 Endosomal release, 131, 132f Endosomolytic peptides, 117, 118f, 119 Endothelial cells, interactions with gene expression plasmids, 125– 126 Enhancers, in gene expression plasmids, 105– 106 Eretmapodites quinquevittatus, 4 Erythromycin resistance, in E coli, 159, 165 Escherichia coli G424A mutation, 165 ribosomal RNA mutations affecting 4.5S RNA requirement, 165, 166 databases, 169– 185 introduction of, 161– 162 methods of detection, 158– 162 plasmid expression, 158– 160 ribosome subunits, 157– 158 23S rRNA future studies, 186 mutational analysis of, 162, 164– 169 mutation database, 169– 185 novel research approaches, 185– 186 plasmid-derived, allele-specific structural probing of, 160 secondary structure, 162– 164 site-directed mutagenesis of, 161– 162 in vitro expression, 160 Ethidium bromide, 102

200

Index

Faciogenital dysplasia, 76; see also AarskogScott syndrome FDA, see Food and Drug Administration Federal Food, Drug, and Cosmetic Act, 102 Fenestrated capillaries, interactions with gene expression plasmids, 126 FGD1 gene, 77 Fis bacterial protein, 186 FMR1 gene amplification of triplet repeats, 63 animal models for, 66 demethylation treatment, 67– 68 founder effects, 64 fragile site, 57 full mutation state, 63, 64– 65 instability in, 64 localization, 62 “nondynamic” mutations, 61 premutation state, 63, 65 protomutations, 64 recovering with folic acid treatment, 67 structure of, 61– 62 FMR1 protein fragile- -related proteins and, 62– 63 isoforms, 62 localization, 62 FMR2 gene, 80, 82 Folic acid, FMR1 gene and, 67 Food and Drug Administration (FDA), regulation of commercial gene therapy development, 102, 103, 104 Foreign histocompatibility genes, cancer gene therapy, 139 4.5S RNA, in E coli, mutations affecting, 165, 166 Fragile- -related (F R1) proteins, 62– 63 Fragile syndrome, see also FMR1 gene clinical phenotype, 59– 60 diagnosis, 60– 61 fragile site, 57, 59 genetics, 61– 66 prevalence, 61 treatment, 66– 68 FRA A fragile site, 57, 59 FRA E fragile site, FMR2 gene and, 80 frequency gene (Neurospora), 38, 39f, 41, 49 FRQ protein, 41 Fusarium, blue light response, 44 F R1 proteins, see Fragile- -related proteins

G424A E coli mutation, 165 Ganciclovir, 138 GDI1 gene, 82 Gene delivery systems, see also Nonviral gene therapy lipid-based, 111– 116 overview of, 110– 111 peptide-based, 116– 119 polymer-based, 119– 123 Gene expression plasmids, 98– 99, see also Nonviral gene therapy biodistribution and pharmacokinetics analysis of, 127– 130 anatomical and physiological factors, 125– 126 effects of pathophysiology on, 126– 127 overview of, 124– 125 components of, 104– 105 bacterial elements, 105 gene switches, 109– 110 introns, 107– 108 mammalian transcription unit, 105 poly(A) signals, 108– 109 promoters/enhancers, 105– 106 untranslated regions, 106– 107 DNA aggregation, 124 DNA condensation, 114, 123 DNA condensing agents, 123– 124 DNA topology, 123 Gene gun, nucleic acid-based vaccines, 143 Gene medicines, see also Gene therapy; Nonviral gene therapy advantages of, 99– 100 biodistribution and pharmacokinetics analysis of, 127– 130 anatomical and physiological factors, 125– 126 effects of pathophysiology on, 126– 127 overview of, 124– 125 components of, 98– 99 future of, 143 intracellular trafficking, 130– 134 Gene regulation, in Neurospora, 38, 41– 42 Gene switches, in gene expression plasmids, 109– 110 Gene therapy, see also Nonviral gene therapy aberrant gene expression, 102 commercialization clinical trials, 103– 104 commercial challenges, 101– 102

Index regulation of, 100, 102– 103 safety issues, 102 technical challenges in, 100, 102 ex vivo approaches, 98 rationales for, 96– 98 in vivo administration, 98 Genome organization long period interspersions, 10– 11 in mosquitoes, 10– 11, 14 short period interspersions, 10, 11 Genome size factors affecting, 11 in mosquitoes, 7, 10, 12– 13t comparing to linkage map size, 25– 26 Giemsa C-banding, see Heterochromatin GM-CSF, see Granulocyte-macrophage colonystimulating factor Good Clinical Practices, 103 Good Laboratory Practices, 103 Good Manufacturing Practices, 103, 104 GPC3 proteoglycan, 71 Granulocyte-macrophage colony-stimulating factor (GM-CSF), 138 G syndrome, 74; see also Opitz syndrome

Haemagogus cladistic analysis, 4 H equinus, 4, 10, 22 H mesodentatus, 4 H spegazzinii, 4 HbH inclusions, ATR- syndrome and, 73 Headgroups, 112, 113 Heart problems, in Simpson-Golabi-Behmel syndrome, 69 Hemaglutinin protein, 119 Hepatocyte gene therapy, 136– 137 Heterochromatin, in mosquitoes, 17– 23 Heterologous constructs, 165– 166 Hexamine cobalt (III), 124 Highly repetitive DNA, see also Repetitive DNA in mosquitoes, 16– 17 Hydroxylamine, 161

Immune response cancer gene therapy, 138– 139

201

nucleic acid-based vaccines, 141– 142 Inflammation, effects on gene expression plasmids, 126 Influenza hemaglutinin protein, 119 Instillation, intratracheal, in pulmonary gene therapy, 140, 141 Insulin-like growth factor (IGF2), 71 Interferon, 138 inhibition of transgene expression, 105– 106 Intramuscular administration, nucleic acidbased vaccines, 142 Intratracheal instillation, pulmonary gene therapy, 140, 141 Introns, in gene therapy transgenes, 107– 108 Investigational New Drug application, 102, 103

JTS-1 peptide, 119 Juberg-Marsidi syndrome, 74

Knockout studies, in fragile Kupffer cells, 126, 135

FMR1 gene, 66

L1 E coli ribosomal protein, 167 L3 E coli ribosomal protein, 168 L11 E coli ribosomal protein, 165 L24 E coli ribosomal protein, 164– 165 Light, Neurospora perception of, 37– 38 Light induction, see also Neurospora blue light responses in fungi, oxygen and, 47 Light-regulated genes, in Neurospora, 38, 39f, 41– 42 white collar proteins and, 49– 50 Light-responsive elements, Neurospora and, 45– 46 Linkage mapping, in mosquitoes, 23– 25 comparing map size with genome size, 25– 26 Linker groups, 112 Lipid-based gene delivery systems cationic lipids structure, 112– 113 structure-activity relationship, 113 colipids, 114 interactions with biomolecules, 114– 115

202

Index

intracellular trafficking, 132– 133 overview of, 111– 112 pH sensitive, 111 plasmid release from, 131, 132f polycation/lipid hybrids, 114 target specificity, 115 toxicity, 115– 116 Lipofectin, 112, 115 Lipopeptides, in peptide-based gene delivery systems, 118 Liposomes, in lipid-based gene delivery systems, 111 Liver gene expression plasmid uptake, 128, 129 systemic gene therapy, 135– 137 Long period interspersions, in mosquitoes, 10– 11 Lung cancer, 139– 140 Lungs gene expression plasmid uptake, 127, 128 systemic gene therapy, 135 Lutzia, 4

Major histocompatibility molecules cancer gene therapy, 139 nucleic acid-based vaccines, 142 Maxicells, 159 M chromosomes, in mosquitoes, heterochromatin organization, 17– 18, 20 Mental retardation, -linked catalog of genes and disorders, 56, 57t nonsyndromal, 57t, 80 FMR2 gene, 80, 82 GDI1 gene, 82 genes responsible for, 81f OPHN1 gene, 82 PAK3 gene, 82– 83 origin of concept, 56 prevalence, 56 research in, benefits of, 83 syndromal, 57 Aarskog-Scott syndrome, 76– 77 ATR- syndrome, 72– 74 Coffin-Lowry syndrome, 77– 80 fragile syndrome, 57, 59– 68 genes involved in, 58f Opitz syndrome, 74– 76 Simpson-Golabi-Behmel syndrome, 68– 72

Methanesulfonic acid ethyl ester, 161 N-methyl-2-pyrrolione, 120 Methylation, in fragile syndrome, 64– 65 MIDI gene, 75 Mifepristone, gene switches and, 109, 110 Mochlonyx, 11, 23f Mosquitoes, see Culicidae Multipoint mapping in mosquitoes, 23– 25 comparing map size with genome size, 25– 26 overview of, 22– 23 Mutagenesis, in E coli ribosomal RNA, 161– 162 Mycoplasma genitalium, 17

Nematocera, 7 NeoCulex, 4 Neoplasia, Simpson-Golabi-Behmel syndrome and, 70 Neurospora blue light responses action spectra, 37 adaptation to varying light intensity, 38– 40 biphasic, 37– 38 in conjunction with other regulatory pathways, 41– 42 light-regulated genes, 38, 39f mutational analysis, 42– 44 overview of, 36– 37, 50– 51 protein kinase C, 40 white collar proteins, 44– 50 Neutrophils, pulmonary gene therapy, 140 New Drug Application, 102, 104 New Zealand, mosquito fauna, 3 NIFL protein, 47 NM2P, 120 Noncondensing polymer-based gene delivery systems, 119– 120 Nonviral gene therapy, see also Gene medicines biological opportunities cancer therapy, 137– 140 nucleic acid-based vaccines, 141– 143 pulmonary therapy, 140– 141 systemic administration, 134– 137 cellular uptake mechanisms, 130– 132 commercialization clinical trials, 103– 104 commercial challenges, 101– 102

Index regulation of, 100, 102– 103 safety issues, 102 technical challenges in, 100, 102 DNA condensation, 114, 123 DNA factors influencing gene transfer, 123– 124 future of, 143 gene delivery systems lipid-based, 111– 116 overview of, 99, 100, 110– 111 peptide-based, 116– 119 polymer-based, 119– 123 gene expression in, 99 aberrant, 102 gene expression plasmids, 98– 99, 104– 105 bacterial elements, 105 biodistribution and pharamcokinetics, 124– 130 gene switches, 109– 110 introns, 107– 108 mammalian transcription unit, 105 poly(A) signal, 108– 109 promoters/enhancers, 105– 106 untranslated regions, 106– 107 intracellular trafficking, 130, 132– 133 cellular uptake, 130– 132 endosomal release, 131, 132f nuclear envelope, 133 nuclear localization signals, 133– 134 nuclear pore complex, 133 overview of, 98 Nuclear envelope, trafficking of gene expression plasmids, 133 Nuclear localization signals, 133– 134 Nuclear pore complex, trafficking of gene expression plasmids, 133 Nuclear transport, of gene expression plasmids, 132, 133– 134 Nucleic acid-based vaccines delivery, 142– 143 mechanism of immunization, 141– 142 overview of, 141

OPHN1 gene, 82 Opitz syndrome, 74– 76 Organomegaly, in Simpson-Golabi-Behmel syndrome, 69 Organ uptake clearance, 127, 128f

203

Origin of replication, bacterial, in gene expression plasmids, 105 Orthopodomyia pulcripalpas, 22 Overgrowth, see Simpson-Golabi-Behmel syndrome Oxygen, in fungi light induction, 47

p53 gene, 139– 140 PAK3 gene, 82– 83 Palindrome-binding protein, 44 PAMAM, 121 Paracrine proteins, 97 PAS domains, Neurospora white collar proteins and, 45f, 46– 47, 48f, 49, 50 PCR, see Polymerase chain reaction PDMAEMA, 122 PEI, see Polyethyleneimine Peptide-based gene delivery systems endosomolytic peptides, 119 lipopeptides, 118 overview of, 116 polylysine-based, 116– 117 synthetic peptides, 117, 118f Peptidyl transferase, E coli 23S rRNA and, 167– 168 Period protein, 46 Perlman syndrome, 70 Phagocytosis, 131 Pharmacokinetics, of gene medicines, 124– 130 Phosphatidylserine (PS), 111 Photoactive yellow protein, 45f, 47, 48f Phytochromes, Neurospora white collar proteins and, 47– 49 PINC systems, 119, 120 pKK3535 plasmid, 158, 159, 160 Plasmids expression of E coli rRNA mutations, 158– 160, 161 in nonviral gene therapy, see Gene expression plasmids Plasmodium berghei, 16– 17 pLC7-21 plasmid, 158, 159, 161 Polyadenylation, 108 Polyamidoamine, 121 Polybrene, 121 Polycation/lipid gene delivery systems, 114 Polyethylene glycol, cationic liposome complexes and, 115

204

Index

Polyethyleneimine, 121, 122 Poly(2-dimethylamino)ethyl methacrylate, 122 Polylysine gene delivery systems with, 114, 116– 117 structure, 122 Polymerase chain reaction (PCR), in analysis of gene expression plasmid biodistribution, 127, 129 Polymer-based gene delivery systems cationic, 121– 122 toroid morphology, 122– 123 noncondensing, 119– 120 Polytene chromosomes, in mosquitoes, 22 Polyvinyl pyrrolidone, 119– 120 Potocytosis, 131 Priming site mutations, in E coli 23S rRNA, 160, 166– 167, 168, 169 Prodrug-converting enzyme genes, 137– 138 Promoters, in gene expression plasmids, 105– 106 Protamine, in polycation/lipid complexes, 114 Protein kinase C, in Neurospora blue light signal transduction, 40 Proteins diseases and disorders caused by, 97 functional modes, 97 somatic gene therapy, 97– 98 Protein therapy, see Gene therapy; Nonviral gene therapy Proteoliposomes, 111 Protomutations, in fragile syndrome, 64 Protoperithecia, 41 PS, see Phosphatidylserine Psorophora verox, 4 pSTL102 plasmid, 159, 161 Psychodidae, 3 Public Health Service Act, 102 Pulmonary gene therapy, 140– 141

Rab-GTPases, 83 Recombinant DNA Advisory Committee, 102 Regulatory processes, in gene therapy development, 102– 103 Repetitive DNA, in mosquitoes, 16– 17, 21– 22 Rho-GTPases, 83 Ribosomal proteins, binding site mutations in E coli 23S rRNA, 164– 165, 166, 167, 168

Ribosomal RNA Mutation Database, 169– 185 Ribosomal RNA mutations, in E coli affecting 4.5S RNA requirement, 165, 166 analysis of 23S rRNA with, 162, 164– 169 databases, 169– 185 future studies, 186 methods of detection, 158– 160 methods of induction, 161– 162 novel study approaches, 185– 186 plasmid expression, 158– 160 Ribosomes, in E coli, subunits, 157– 158 Ribotoxins, 168– 169 Ricin, 168, 169 Rifamycin, gene switches and, 110 Runchomyia, 4

Sabethes cyaneus, 10, 22 Sabethini, 4, 5, 10 ␣-Sarcin, 168– 169 Selection, in mosquitoes, genome size and, 17 Sex chromosomes, in mosquitoes, 6 evolution of, 7 heterochromatin organization, 17– 18, 20 Sex determination, in mosquitoes, 6 Shine-Delgarno sequences, 159– 160 Short period interspersions, in mosquitoes, 10, 11 SIM, see Single-minded gene product (SIM) Simpson-Golabi-Behmel syndrome clinical phenotype, 68– 70 diagnosis, 70– 71 first reports of, 68 genetics, 71 mental retardation in, 69– 70 treatment, 72 Simulidae, 3 Single-minded gene product (SIM), 46 Sinusoidal capillaries, 126 Site-directed mutagenesis, of E coli 23S rRNA, 161– 162 16S Ribosomal RNA Mutation Database, 169 Skeletal problems in Coffin-Lowry syndrome, 78 in Simpson-Golabi-Behmel syndrome, 69 Southern blot analysis, in analysis of gene expression plasmid biodistribution, 127, 129 Spectinomycin resistance, in E coli, 159 Stegomyia, 4

Index Subcutaneous administration, nucleic acidbased vaccines, 142– 143 Suicide genes, 137– 138 SV40 large T-antigen, 134 Systemic gene therapy, 134– 135 active targeting, 136– 137 passive targeting, 135– 136

T7 promoter, 138 T7 RNA polymerase, 106 in vitro expression of E coli rRNA mutations, 160 Targeting in polymer-based gene delivery systems, 121 in systemic gene therapy active, 136– 137 passive, 135– 136 T cells cancer gene therapy, 138– 139 nucleic acid-based vaccines, 142 Tetracycline, gene switches and, 110 ␣-Thalassemia, 72; see also ATR- syndrome T helper cells, 142 Thiostrepton resistance, in E coli, 165 Timeless gene product, 46 Tipulidae, 7 Toroids DNA condensing agents and, 124 of plasmid/cationic polymer complexes, 122– 123 Total body clearance, 127, 128f Toxicity, of cationic liposome complexes, 115– 116 Toxorhynchites, 2 T splendens, 10, 20, 23f Toxorhynchitinae, 27 chromosome number, 6 cladistic analysis, 4, 5 genome size, 10, 12t, 14f sex chromosomes, 7 taxonomy, 2 Transferrin, in targeting gene expression plasmids, 115 Trigalactolipids, 115 Triplet repeats, in fragile syndrome, 57 amplification of, 63 diagnosis, 60

205

in FMR1 gene and transcript, 61– 62 methylation of, 64– 65 Tripteroideres, 4 T bambusa, 22 Tumor necrosis factor, inhibition of transgene expression, 105– 106 Tumors, see also Cancer gene therapy biodistribution of gene expression plasmids, 126– 127, 130 cancer gene therapy, 139– 140 passive targeting, 135– 136 Tumor suppressor genes, 139– 140 23S Ribosomal RNA Mutation Database, 169– 185 23S rRNA (E coli) mutational analysis, 162 database, 169– 185 domain I, 164– 165 domain II, 165– 166 domain III, 166– 167 domain IV, 167 domain V, 167– 168 domain VI, 168– 169 future studies, 186 novel approaches, 185– 186 plasmid-derived, allele-specific structural probing of, 160 secondary structure, 162– 164 site-directed mutagenesis of, 161– 162 in vitro expression, 160 in vivo expression, 159

Untranslated regions, in gene therapy transgenes, 106– 107

Vaccinations, nucleic acid-based, 141– 143 Viral vectors, compared to plasmid-based gene medicines, 100 Virosomes, 111

WC-1, see White collar proteins WC-2, see White collar proteins wc genes (Neurospora), 44, 49 wc mutants (Neurospora), 42– 43, 49

206 Weaver syndrome, 70 white collar mutants (Neurospora), 42– 43 White collar proteins (Neurospora) in blue light signal transduction, 42, 49– 50, 51 common features of WC-1 and WC-2, 44– 45 domains for dimerization, 46– 47, 48f domains for signal transduction, 47– 49 homology with other proteins, 44 light-responsive elements and, 45– 46 localization, 45 sizes of, 44 white gene (Culicidae), 4 Williams syndrome, 79

Index Wyeomyia, 4 W smithii, 10

chromosomes, in mosquitoes, 20– 21 XH2 gene, 73– 74 -linked mental retardation, see Mental retardation, -linked XNP gene, 74; see also XH2 gene

Y chromosomes, in mosquitoes, 7, 20, 21

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