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Volume 41 Advances in Genetics Edited by Jeffrey C. Hall Jay C. Dunlap Department of Biology Brandeis University W al...

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Volume 41

Advances in Genetics Edited by Jeffrey C. Hall

Jay C. Dunlap

Department of Biology Brandeis University W altham , 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

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Copyright 0 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 US.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. 0065-2660199 $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

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

G. Arpaia ( 3 5 ) Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari, Sezione di Genetica Molecolare, Universiti di Roma “La Sapienza,” 00161 Roma, Italy P. Ballario ( 3 5 ) Dipartimento di Genetica e Biologia Molecolare, Centro di Studio per gli Acidi Nucleici, Universiti 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 DisabilitP Mentale e Motoria, Associazione Anni Verdi, 00168 Roma, Italy H. Linden (35) Lehrstuhl fur Physiologic and Biochemie der Pflanzen, Universitat Konstanz, D-78434 Konstanz, Germany G. Macino ( 3 5 ) Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari, Sezione di Genetica Molecolare, Universiti di Roma “La Sapienza,” 00161 Roma, Italy Ram I. Mahato (95) Copernicus Therapeutics, Inc., Cleveland, Ohio 44106 Giowanni Neri (55) Istituto di Genetica Medica, FacoltP 6 Medicina e Chirurgia “A. Gemelli,” Universiti 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 77381 Louis C. Smith (95) Valentis, Inc., The Woodlands, Texas 77381 Kathleen L. Triman (157) Department of Biology, Franklin and Marshall College, Lancaster, Pennsylvania 17604

vii

I

Mosquito Genomes: Structure, Organization, and Evolution

Karamlit S. Rai* and William C. Black IVP

Department of Biological Sciences University of Notre Dame Notre Dame, Indiana 46556

I. Overview

11. Mosquito Taxonomy, Evolution, and the Fossil Record 111. Cladistic Analysis of Culicidae IV. Chromosome Number Is Conserved in Culicidae

V. Sex Chromosome Evolution in Culicidae VI. Genome Size and General Genome Organization A. Interspecific Variation and Genome Organization B. Intraspecific Genome Size Variation VII. Heterochromatin: Localization, Variation, and Expression VIII. Saturated Linkage Maps Generated through Multipoint Mapping

IX. Summary Acknowledgments References

1. OVERVIEW The family Culicidae is composed of more than 3,400 mosquito species, many of which are major vectors of arboviruses, malaria, and filariasis. In visw of their *Address for correspondence: E-mail: [email protected] (219) 631-7413. Telephone: (219) 631-6584. Present address: College of Veterinary Medicine and Biomedical Sciences, Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523. E-mail: wcb40lamar. colostate.edu. Advances in Genetics, Vol. 41 Copyright 0 1999 by Academic Press All rights of reproduction in any form reserved. 0065-2660/99 $30.00

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

importance as vectors, many mosquito genera and species have been the subject of extensive cytological and genetic investigations over the last 40 years (Kitzmiller 1953, 1976; White, 1980; Rai et al., 1982; Rai, 1991). As a result, there is a voluminous literature on mosquito genomes scattered in various entomological and genetics journals. The purpose of this review is to highlight the salient features of mosquito genomes and their evolution. It is indeed surprising that, except for a couple of minireviews (Besansky and Collins, 1992; Kumar and Rai, 1993), the various facets of this work have not been reviewed earlier. We begin with a general review of mosquito systematics, highlighting and summarizing recent studies that employed modem cladistic analysis of morphological and molecular characters to estimate phylogenetic relationships among sister families to Culicidae and among Culicidae subfamilies, tribes, genera, subgenera, and species. We next review the extensive literature on karyotypes, emphasizing that the number of chromosomes has remained at a constant 2n = 6 despite a relatively ancient origin for Culicidae, the evolution of both homomorphic and heteromorphic sex chromosomes, and evidence of extensive translocations and inversions. The literature on the evolution of genome size and organization in Culicidae is summarized and considered in light of current phylogenetic relationships. Genome evolution is also reviewed in the context of the now-extensive studies on heterochromatin distribution and in terms of the linkage maps that are beginning to arise through various recent intensive genome mapping projects in Culicidae.

II. MOSQUITO TAXONOMY, EVOLUTION, AND THE FOSSIL RECORD The family Culicidae, which includes all mosquitoes, is divided into three subfamilies, Anophelinae, Toxorhynchitinae, and Culicinae (Knight and Stone, 1977; Knight, 1978; Ward, 1984, 1992; Service, 1993). Anophelinae includes three genera, the neotropical Chagusia (4 species), the Australasian Bironella (9 species in 3 subgenera), and the nearly cosmopolitan Anopheles with some 422 species grouped in 6 subgenera. Toxorhynchitinae includes a single genus, Toxorhynchites with 76 species. Culicinae is by far the largest subfamily: it is subdivided into 10 tribes, 33 genera, and 117 subgenera and includes about 2,925 described species. Although mosquito systematics is in a state of flux (Munstermann, 1995), the total numbers of genera, subgenera, and species in Culicidae currently stand at 37, 129, and 3,436, respectively (Service, 1993). The genus Aed.es, which includes some 962 species grouped in 43 subgenera, is one of the best studied cytogenetically (Rai et al., 1982). Based on the fossil record, scanty through it is, and zoogeographic evidence involving past intercontinental connections and faunistic composition, it has been suggested that mosquitoes had evolved by the Jurassic, approx-

1. Mosquito Genomes: Structure, Organization, and Evolution

3

imately 210 million years ago (MYA) (Edwards, 1932). This is about the time continental drift began (Wilson, 1963). The continental breakup led to fragmentation and geographical isolation of populations. This may have been accompanied by great ecological flux that promoted rapid speciation (McClelland, 1967). Ross (1951) proposed that a burst of Culicinae lineages arose approximately 120 MYA. By the end of the Cretaceous, some 65 MYA, the generic composition of family Culicidae was well established (Belkin, 1968; Rohdendorf, 1974). New Zealand has been in its present position of isolation for approximately the last 50 million years (Rick, 1970). With the exception of three species, Aedes notoscriptus, Aedes australicus, and Culex quinquefmciatus, the present-day mosquito fauna of New Zealand is relict and endemic. This provides circumstantial evidence that the genus Aedes existed prior to the island’s separation from Australia and that it was probably widely dispersed during the Cretaceous, which began 145 MYA (Belkin, 1968). Fossils of family Culicidae (Culex, Aedes) and its sister family Chaoboridae are well known from the Eocene (Tertiary) and Oligocene, which began 60 and 55 MYA, respectively (Rohdendorf, 1974).

The phylogenetic relationship of Culicidae relative to other nematocerous dipteran families has been evaluated using modern cladistic analysis. Munstermann and Conn (1997) have reviewed the impact of molecular biology and cladistic analysis on systematics of selected taxa of Culicidae with particular emphasis on the Aedes and Anopheles species. Phylogenies have been estimated with suites of morphological characters (Oosterbroek and Courtney, 1995) and nucleotide sequences from the 18s and 5.8s nuclear ribosomal DNA (rDNA) (Miller et al., 1997) and 28s rDNA (Pawlowski et al., 1996). The morphological and 18s datasets are congruent in identification of Chaoboridae (phantom midges) as a sister group to Culicidae and in placement of Corethrellidae as a basal clade to Chaoboridae -Culicidae. The 28s dataset supported monophyly of these three families but consistently indicated Chaoboridae-Corethrellidae as sister taxa. Phylogenies of high-order relationships among these three families and Chironomidae, Ceratopogonidae, Dixidae, Psychodidae, and Simulidae are incongruent in all three studies. Each study cites several independent lines of support for the higher-order relationships derived from their respective phylogenies but all studies also indicate that these relationships were supported by few characters or lack strong bootstrap support. The rDNA papers use different species in each family, obviating a combined analysis as a means to resolve this conflict. The rDNA studies also suffer from sampling of single species in most families,

4


preventing identification of synapomorphies for each family. These relationships should be explored further with more complete taxon sampling and an examination of single-copy nuclear genes. The relationship of Culicidae subfamilies has been examined with nucleotide datasets using rDNA genes (Pawlowski et al., 1996; Miller et al., 1997) and the single-copy nuclear gene white (Besansky and Fahey, 1997): All three studies were congruent in placement of Anophelinae as the basal clade in Culicidae. Furthermore, the 18s and white genes were consistent in placing Toxorhynchitinae as basal to the Culicinae. Relationships among tribes, genera, and species in Culicinae have also been evaluated using modem cladistic analysis. Judd (1996) examined 59 morphological characters in 37 taxa within the tribe Sabethini. Cladistic analysis, using Eretmapodites quinquevittatus and Haemagogus spegazzinii as outgroups, supported Sabethini as a monophyletic group but strongly suggested paraphyletic relationships among species in at least three genera (Runchomyia, Tripteroides, and Wyeomyia). Wesson et al. (1992) sequenced the 5.8s-28s half of the internal transcribed spacer of the rDNA cistron (ITS2) to examine phylogenetic relationships among seven species in three genera (Aedes, Haemagogus, and Psorophora) of Aedini. Their analyses suggested paraphyletic relationships among species in the Aedes subgenus Stegomyia and suggested that Haemagogus and Psorophora arose within Aedes. The resolved phylogeny also provided evidence for biogeographical relationships among Aedini species: one clade contained Old World species (Ae. aegypti, Ae. simpsoni, Ae. vexans, and Ae. albopictus); a second clade contained the New World taxa Ae. triseriatus, Haemagogus mesodentatus, and Psorophora verox. Besansky and Fahey (1997) performed a thorough taxon sampling of variation in the white gene among taxa in tribes Culicini, Sabethini, and Aedini in the Culicinae. Their analysis supported placement of Sabethini as basal to Culicini and Aedini. Like the analysis of Wesson et al. (1992), this analysis of the white gene placed old World Aedini (Ae. aegypti and Ae. albopictus) in a separate clade from the New World species (Ae. niseriutus, Haemagogus equinus) with high bootstrap support. The rDNA genes (Pawlowski et al., 1996; Miller et al., 1997) and white gene all support a monophyletic relationship between Culicini and Aedini. Miller et al. (1996) examined sequence divergence in the entire internal transcribed spacer (ITS) among 14 species in four subgenera of the genus Cukx. Species in the subgenera Cukx, Lutzja, and NeoCukx were monophyletic. There was low bootstrap support for monophyly of species in the subgenus Cukx but only single species were examined in the subgenera Lutda and NeoCUtex. Some relationships among species and species complexes were also examined. Kumar et al. (1998) constructed restriction maps of the rDNA cistron of 12 species of mosquitoes in six genera of the subfamily Culicinae using eight

1. Mosquito Genornes: Structure, Organization, and Evolution

5

6-bp recognition restriction enzymes. Anopheles albimanus was used as an outgroup. Clades within the RFLP (restriction fragment length morphism) phylogeny were not well supported and were incongruent with the morphology character based and molecular phylogenies previously discussed. The lack of resolution in the RFLP dataset was probably due to homoplasy arising from frequent independent loss or possibly, though less likely, from gain of restriction sites among unrelated taxa. Studies by Kumar et al. (1998) showed that only relationships among closely related taxa were well supported. As in Besansky and Fahey (1997), Ae. criseriatus and Ha. equinus were monophyletic. The sister species, Ae. epuctius and Ae. atropulptus, were also monophyletic. Species in the Aedes albopictus and the Aedes scutelhris subgroups of the Aedes scutellaris group were monophyletic in the (RFLP) phylogeny. Based on a correlation of the allozyme differentiation among certain species and their geological histories and calibration of a well-established geologic event in the South Pacific, Pashley et al. (1985) concluded that the Ae. albopictus and the Ae. scutellaris subgroups diverged relatively recently. In summary, modern cladistic analyses of morphological and molecular characters consistently support Chaoboridae-Corethrellidae as sister taxa to Culicidae. All analyses support Anophelinae as the basal clade in Culicidae and are consistent in placing Toxorhynchitinae as basal to the Culicinae. Within Culicinae, the tribe Sabethini is basal to Culicini and Aedini. All datasets support a monophyletic relationship between Culicini and Aedini. Many subgeneric relationships within Sabethini, Culicini, and Aedini may be paraphyletic and warrant taxonomic revision. These studies do not address the key question of whether Toxorhynchitinae arose within Anophelinae or as a separate lineage from a common ancestor with Anophelinae. This becomes a pivotal issue in discussing the origins of some major genetic differences between anopheline and culicine mosquitoes later in this chapter. This issue may become resolved in the future through examination of additional gene sequences and intensive sampling of primitive and derived members of both Toxorhynchitinae and Anophelinae. However, it is also quite possible that ancestral taxa are extinct in either or both subfamilies and that the issue will never be adequately resolved.

IV. CHROMOSOME NUMBER IS CONSERVED IN CULlClDAE Chromosomal karyotypes have been established for “no less than” 19 genera, 35 subgenera, and 200 species in family Culicidae (White, 1980). Over the last several years, additional species have been cytologically examined (Rao and Rai, 1987a, 1990). One of the most remarkable findings of this karyotypic survey is that, despite the ancient origin of the group and despite extensive repattern-

6


ing of the genome involving translocations and inversions (Matthews and Munstermann, 1994; Mori et al., 1998), the basic chromosome number (2n = 6) has remained unchanged. The only exception, Chugasia bathana (2n = 8) of the subfamily Anophelinae, possesses three autosome pairs and a heteromorphic pair of sex chromosomes (Kreutzer, 1978). All other anophelines possess two pairs of generally metacentric chromosomes of unequal size and one pair of heteromorphic sex chromosomes that often show extensive polymorphism in overall length and of the quantity and quality of heterochromatin differentiation among various species (White, 1980). The position of the centromeres in the heteromorphic X and Y chromosomes in Anophelinae varies from subtelocentric or acrocentric to submetacentric and metacentric (Baimai e t al., 1993a, b, 1995). In contrast, species of the subfamilies Toxorhynchitinae and Culicinae all possess three pairs of homomorphic metacentric and/or slightly submetacentric chromosomes: a pair of small chromosomes, a pair of large chromosomes, and a pair of intermediate-sized chromosomes (Rai, 1963; McDonald and Rai, 1970; Rai et al., 1982, Rao and Rai, 1987a). In culicine mosquitoes, sex is determined by a gene at a single locus. Females are homozygous recessive at this locus, and males are heterozygous for a dominant allele (Gilchrist and Haldane, 1947; McClelland, 1962). In species in which linkage group-chromosome correlations have been made, the shortest chromosome contains the sex locus and is therefore sex determining (McDonald and Rai, 1970; Baker et al., 1971; Dennhofer, 1972). Differences clearly exist in overall lengths and arm ratios of individual chromosomes, both within and between species, but can be easily overlooked if careful measurements of each arm of a chromosome are not made (Rai, 1980; Rai e t al., 1982). Total chromosomal length varies almost fivefold, from 8.2 pm in Anopheles quadrimaculatus to 39.3 pm in Aedes alcasidi. Within the genus Aedes, there is a threefold variation in chromosome length (Table

1.1)

Conservation of chromosome number in Culicidae does not indicate synteny. Matthews and Munstermann (1994) and Severson et al. (1995) clearly document that groups of allozyme Ioci have remained linked and colinear in a variety of culicine taxa but that these linkage groups have translocated and are inverted extensively across the three culicine chromosomes. The extensive variation in chromosome number in most diptera taxa studied does not predict the extreme conservation found in Culicidae. For example, the chromosome number ranges from n = 3 to 7 in the genus Drosophila (see White, 1973) and from n = 3 to 8 in the genus Glossina (Mauldin 1970). In family Muscidae, most species possess six pairs of chromosomes; however, six species have only five pairs each (Boyes, 1967). Nevertheless, certain other dipteran families such as Simulidae (Rothfels, 1979) and Sarcophagidae also show extensive conservation of chromosome number, although some exceptions do occur (White,


7

1973). No logical explanation exists for the extraordinary conservation of the haploid chromosome number in Culicidae. The chromosomal karyotype data from Culicidae in general support White’s (1973) suggestion that there may be some kind of barrier that maintains chromosome number in the Diptera. Nevertheless, we know nothing about the actual nature of such a barrier.

v.

SEX CHROMOSOMEEVOLUT~ONNCULICIDAE

Current dogma suggests that heteromorphic sex chromosomes evolved from virtually identical homologues. Both theoretical considerations (Charlesworth, 1978) and considerable experimental evidence suggest that it is the gradual accumulation of repetitive sequences on the Y chromosome followed by loss of recombination between the heteromorphic pair that leads to the differentiation of X and Y chromosomes. Theory predicts eventual loss of function and eventual extinction of the Y chromosome (Steinemann et ul., 1993; Morell, 1994; Rice, 1994, 1996). This directionality is generally referred to as the “rise and fall of the Y chromosome” (Morell, 1994). Evolution of a heteromorphic Y chromosome may have occurred only once or possibly may have been reversed in the evolution of sex chromosomes in Culicidae. The primitive Nematocera families Tipulidae and Dixidae possess homomorphic sex chromosomes. However, the sister families Chaoboridae-Corethrellidae contain genera with homomorphic (Eucorethru, Corethrella, Chaoborus) and heteromorphic (Mochlonyx) sex chromosomes (Rao and Rai, 1987a). If homomorphy was ancestral in Culicidae, then it was retained in the lineages leading to Toxorhynchitinae and Culicinae, while heteromorphy probably evolved early in the evolution of Anophelinae and was retained in all taxa. This scenario is supported by the current dogma concerning the evolution of sex chromosomes (Rice, 1996). Altematively, if, as proposed by Rao and Rai (1987a), Culicidae arose from a Mochlonyx-like ancestor, then Anophelinae retained heteromorphic sex chromosomes, while homomorphic sex chromosomes evolved through euchromatinization or loss of the Y in Toxorhynchitinae and Culicinae.

VI. GENOME SIZE AND GENERAL GENOME ORGANIZATION A. lnterspecific variation and genome organization Considerable effort has been expended in recent years to determine haploid nuclear DNA amounts in the superfamily Culicoidea (Jost and Mameli, 1972; Rao and Rai, 198713, 1990; Black and Rai, 1988; Kumar and Rai, 1990). This has been done through quantitative cytophotometry of Feulgen-stained primary

m

Table 1.1. Mean Chromosomal Lengths in 30 Representative Species Belonging to 8 Genera of Mosquitoes and Related Taxa in SuperfamilyCulicoidae Mean chromosome length (pm) Family

Genus/species

1

I1

111

(I

TCLQ

+ I1 + 111)

References

Chaoboridae

Mochlonyx welutinus C h a o h amencanus

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

Anopheks qdnmaculatus Cukx pipiem culex temtans

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.O 4-2 4.1 5.4

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

Cukx restuans Toxorhynchites spkndens Wyeomyiu smihii Haemoagogus Equinus Aedes togoi Ae. metallicw Ae. hebrideus Ae. aegypti Ae. heischii Ae. kesseli Ae. anopalpus Ae. pseudoscutekris Ae. unilinentus

4.7

5.8 9.6 4.6 6.3 6.3 6.9 7.4 7.8 8.4 9.6 9.1

Ae. cook Ae. seatoi Ae. polynesiensis Ae. katheTinensis

Ae. stirnulam

Ae . pseudoalbopicrus Ae. malayensis Ae. fivopictus Ae. mseriatus Ae . zoosophw

Ae. alcasidi Ae. albopictw Oahu, Hawaii Calcutta, India Kolar, India Mauritius Tananareve, Madagascar Pune, India Delhi, India

oTCL:Total chromosomal length in micrometers.

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, Rao and Rai, Dev and Rai, Rao and Rai, Rai, 1963 Rao and Rai, Dev and Rai, Rao and Rai, Rao and Rai, Rao and Rai, Dev and Rai,

1987a 1984 1987a 1987a 1987a 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, Rao and Rai, Rao and Rai, Rao and Rai, Rao and Rai, Rao and Rai, Rao and Rai,

1987b 1987b 1987b 1987b 198713 198713 1987b

1984 1987a 1984 1987a

10


spermatocytes and in a few cases through analyses of renaturation kinetics of nuclear DNA (Black and Rai, 1988; Warren and Crampton, 1991; Besansky and Powell, 1992). As a result, haploid genome sizes have been established for 44 species belonging to 13 genera of mosquitoes and related Culicoidea families (Table 1.2). Genome size is generally small in Anophelinae (0.23 -0.29 pghaploid genome) (Jost and Mameli, 1972; Black and Rai 1988; Besansky and Powell, 1992). The single species Toxorhynchites spkndens, examined in subfamily Toxorynchitinae possesses an intermediate-size genome of 0.62 pg as do Sabethes cyanern and Wyeomyia smithii (Sabethini).The haploid genomes of Cukx species examined ranged from 0.54 to 1.02 pg and those of Culiseta species (Culicini) from 0.92 to 1.25 pg. Armigeres subalbarus and Haemagogus equinw (Aedini) contained 1.24 and 1.12 pg, respectively. At the generic level, the cosmopolitan genus Aedes showed more than threefold variation in nuclear DNA amounts, with the Polynesian species Ae. psewEoscutelIaris and Ae. cooki (belonging to the Ae. scutellaris subgroup in the subgenus Stegomyia) possessing the lowest genome size of 0.59 pg and Ae. roosophw(subgenus Protomackaya)possesing the highest genome size of 1.9 pg among the 23 species examined (Rao and Rai, 1987b, 1990). Placed in the context of phylogenetic relationships discussed earlier, these figures suggest a general increase in genome size during the evolution of Culicidae. Black and Rai (1988) demonstrated that all classes of repetitive DNA sequences increased linearly in amount with total genome size. Furthermore, linear regression analysis of a fairly large dataset involving 28 species belonging to 11 genera of the superfamily Culicoidea showed a highly significant positive correlation ( r = 0.87; p = 0.0001) between total chromosomal length and haploid genome size (Rao and Rai, 1987b). Nevertheless, eightfold variation in haploid genome size was accompanied by only an approximate fivefold variation in the total chromosomal length, indicating that DNA amounts have increased almost twice as much as the increase in chromosomal size. Studies using reassociation kinetics have provided information on genome organization in Anophelinae and Culicinae (Black and Rai, 1988; Warren and Crampton, 1991; Besansky and Powell, 1992). Genome organization refers to the amounts, complexity, and dispersion of repetitive elements in a genome. TWObasic forms of genome organization have been described in eukaryotes (Davidson et al., 1975). The first type is termed “short period interspersion” and describes a pattern in which single-copy sequences, 1000-2000 bp in length, alternate regularly with short (200-600 bp) and moderately long ( 1000-4000 bp) repetitive sequences. This characterizes genome organization in the majority of animal species and was found in the culicine species Cukx pipiens, Ae. aegypti, Ae. albopictus, and Ae. triseriatus (Black and Rai, 1988). The second type of genome organization is termed “long-period interspersion”


11

and describes a pattern of long (> 5600 bp) repeats alternating with very long (> 13,000 bp) uninterrupted stretches of unique sequences. Repeats in An. ~uadrimacuhtus(Black and Rai, 1988) and An. gambiae (Besansky and Powell, 1992) follow a long-period interspersion pattern. Genome organization is of the long-interspersion type in Chironomus tentans (Wells et al., 1976) but has not been determined in sister families Chaoboridae-Corethrellidae. However, haploid DNA amounts of 0.47, 0.55, and 0.40 pg were observed in the three principal genera Corethrelh, Mochlonyx, and Chaoborus, respectively (Table 1.2; Rao and Rai, 1990). In insects, long period interspersion is characteristic of most species with small genome sizes (0.1-0.5 pgkaploid genome), while short-period interspersion tends to be associated with larger genomes with larger amounts of repetitive DNA (Palmer and Black, 1997). It is difficult to predict genome organization in Chaoboridae-Corethrellidae based on genome size, because they fall into the upper limit for long-interspersed species. Thus there remain two competing hypotheses for ancestral genome evolution in Culicidae. It is possible that longperiod interspersion was ancestral in Culicidae and was retained in the lineage leading to Anophelinae, while larger genomes developed through accumulation of short-period interspersed repetitive elements in Culicinae. The alternative hypothesis is that Culicidae arose from a short-period interspersed species and, while that organization was retained in the Culicinae, repetitive elements were shed and became organized into a long-period interspersion pattern in the Anophelinae. This is the scenario considered by Rao and Rai (1990), who proposed a phylogeny of the superfamily Culicoidea based on genome sizes (Figure 1.1). They suggested that the line that possibly gave rise to Anophelinae from a Mochlonyx-like ancestor underwent many deletions of highly repetitive DNA. However, this scenario lacks any empirical evidence from evolution of genome size in other systems. Cullis (1983) suggested that nuclear DNA is organized into constant and fluid domains. The fluid domain, which is composed mainly of repetitive DNA sequences (Cavallini et al.,1986), shifts in response to changing environments and developmental and physical stimuli (Walbot and Cullis, 1985). Genome size shifts dynamically as a result of DNA amplification, bursts of transpositions, unequal crossing-over that can simultaneously cause elimination and gain of certain DNA sequences (Bassi et al., 1984; Natali et al., 1986; Altamura et al., 1987), and intragenomic drift (Cavalier-Smith, 1985a). However, these mechanisms generally cause genomes to accumulate repetitive elements, and very few mechanisms have been proposed for genomewide “shedding” of repetitive elements. Considering these arguments, it is most parsimonious to suggest that long-period interspersion was ancestral in Culicidae. However, it is critical to determine genome organization in Chaoboridae or Corethrellidae to test this hypothesis. Furthermore, analysis of genome orga-

c

N

Table 1.2. Haploid Genome Size (Picogram DNA) in 44 Species Belonging to 13 Genera of Mosquitoes and Related Taxa Family Dixidae Chaoboridae

Culcidae

Subfamily

Tribe

Subgenus

Dlxa Corethrella Mochkmyx

Corethrellinae Chaoborinae Chaoborinae Anophelinae

Toxorhynchitinae Culicinae

Genus

ChaObOW

Sabethini Culicini

Culisetini

Aedini

Anopheles An. An. An. An. An. Toxorhynchites Sabethes Wyeomyia Culex cx. cx. cx. Cufisera c u. Cu. Hapmagogus Annigeres Aedes

chaoborus Anopheles

Species obscura brkkyi oelutinus americanw lnbranchiae atropaw

Cellia

qwdrimnculatus fieeborni stephensi gambim

Toxohynchites Sabethes Wyeomyia Culex

Culicella Climacura Haemagogus Armigeres Stegomyia

SP&

cyaneus smithii pipiens pipiens quinquefaciatw resmns litwea mwsitans melanura equinus subahanw pseudoscutellaris

pg DNA/haploid genome ? SE 0.156 0.47 2 0.02 0.55 2 0.02 0.40 2 0.02 0.234 0.242 0.245 2 0.01 0.294 0.242 0.27 0.618 2 0.019 0.786 2 0.02 0.855 2 0.011 1.02 ? 0.19 0.540 2 0.012 0.54 ? 0.01 1.02 2 0.04 0.92 1.21 ? 0.04 1.25 2 0.005 1.120 ? 0.023 1.124 ? 0.027 0.591 2 0.012

References Jost and Mameli, 1972 Rao and Rai, 1Y90 Rao and Rai, 1990 Rao and Rai, 1990 lost and Mameli, 1972 Jost and Mameli, 1972 Rao and Rai, 1990 lost and Mameli, 1972 Jost and Mameli, 1972 Besansky and Powell, 1992 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 1990 lost and Mameli, 1972 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 1990 lost and Mameli, 1972 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 1990 Rao and Rai, 198713

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

cooki polynesiensis WYPh %YPh

malayensis hebrideus seatoi afcasidi

unilineatus metallicus

Aedes Howardina Ochlerotahus

heischii kutherinensis pseudoalbopictus j7avopictus cinereus bahamensis

canadensis communis

caspius stimuhns

Protomaclyeaya

exmicianus Riseriutus zoosophus

0.594 t 0.027 0.725 t 0.018 0.812 -C 0.031 0.83 0.943 t 0.025 0.965 -t 0.031 0.971 -C 0.023 0.974 -C 0.016 1.064 -C 0.04 1.093 -t 0.033’ 1.121 -t 0.039 1.277 ? 0.02 1.29 -C 0.028 1.33 -t 0.024 1.210 t 0.03 1.375 -C 0.03 0.904 -t 0.02 1.013 ? 0.05 0.988 1.439 -t 0.039 1.500 -C 0.03 1.520 -C 0.062 1.902 2 0.062

Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Warren and Crampton, 1991 Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rat, 198710 Rao and Rai, 19871, Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987h Rao and Rai, 1987h Rao and Rai, 1987h Jost and Mameli, 1972 Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b Rao and Rai, 1987b

14


Culicinae gn=6; 1.18pg)

Anophelinae Qn=6,80.245Pg)

Qn=6,8; 0.1spg) Figure 1.1. Genome sizes in some members of Culicoidea and the proposed phylogeny. (Distances becween members are arbitrary.) After Rao and Rai (1990).

nization in Sabethini and Toxorhynchitinae is imperative to determine when short-period interspersion arose in culicine evolution.

B. lntraspecific genome size variation Genome studies in Rai’s laboratory have also focused on intraspecific variation in genome size and have indicated unequivocally that DNA amounts are not fixed within species (Ferrari and Rai, 1989; Rao and Rai, 1987b; Kumar and Rai, 1990). An analysis of 47 geographic populations of Ae. albopictus from 18 countries showed a 2.5-fold variation in DNA amounts, ranging from 0.62 pg in the Koh Samui population from Thailand to 1.66 pg in a population from Houston, Texas recently introduced to the continental United States (Table 1.3). Furthermore, extensive variation existed among and within populations from contiguous geographic locations. For example, the haploid DNA amounts of two populations each of Ae. albopictus from Singapore (Kent Ridge and Amoy) and Brazil (Santa Tereza and Cariacica) were significantly different from each other. Six Duncan’s groupings of genome sizes were observed among the 37 popuIations of Ae. albopictus studied by Kumar and Rai (1990). Genome size was independent of geographic origin in the various populations examined. For example, 12 populations from the United States belonged to four groupings that also contained populations from other geographic areas (Kumar and Rai, 1990). Using DNA-reassociation kinetics, Black and Rai (1988) showed that

15

1. Mosquito Genomes: Structure, Organization, and Evolution Table 1.3. Haploid Genome Size (Picogram DNA) in 47 Geographic Populations of Aedes albopictus horn 18 Countries Species

Genus

Aedes

pg DNA/haploid genome f SE

References

0.62 f 0.02 0.69 f 0.03 0.78 f 0.03 0.92 2 0.05 1.07 f 0.044 1.12 f 0.06 1.15 f 0.026 1.26 f 0.026 1.32 f 0.035 1.36 f 0.04 1.48 f 0.05

Kumar and Rai, 1990 Kumar and Rai, 1990 Rao and Rai, 198713 Kumar and Rai, 1990 Rao and Rai, 198713 Kumar and Rai, 1990 Kumar and Rai, 1990 Rao and Rai, 1987b Rao and Rai, 198713 Kumar and Rai, 1990 Kumar and Rai, 1990

0.64 -t 0.02 0.81 f 0.03 0.83 f 0.03 0.85 2 0.02

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

0.75 f 0.02 1.29 f 0.06

Kumar and Rai, 1990 Kumar and Rai, 1990

0.86 f 0.03 0.94 f 0.025 0.96 f 0.02 1.02 f 0.008 1.07 f 0.62 1.42 f 0.05

Rao and Rai, 1987b Rao and Rai, 1987h Kumar and Rai, 1990 Rao and Rai, 198713 Rao and Rai, 1987b Kumar and Rai, 1990

0.75 f 0.03 1.24 f 0.032 1.47 f 0.06

Kumar and Rai, 1990 Rao and Rai, 1987h Kumar and Rai, 1990

0.76 t 0.03 0.80 f 0.02 0.82 t 0.03 0.85 t 0.03 1.11 f 0.04 1.16 f 0.05 1.29 f 0.032

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, 1987h

0.98 f 0.04 1.18 f 0.02

Kumar and Rai, 1990 Kumar and Rai, 1990

ahopictus

Geographic populations Koh Samui, Thailand Korea Tananareve, Madagascar Sri Lanka Pontianak, Indonesia Ndo Ndo Creek, Solomon Island Tananareve, Madagascar Hong Kong Mauritius Saigon, Vietnam Taipei, Taiwan Malaysia Gertak Sanguul Malaysia Perak Road Sahah Singapore Kent Ridge Amoy India Calcutta Kolar Hardwar Delhi Pune Shalimar Bagh Hawaii Makiki Oahu Manoa Japan Nagasaki Saga Kaheshima Ebina Sehuri Zama Tokyo Brazil Cariacica Santa Tereza

1990 1990 1990 1990

continues


16 Table 1.3. (continued)

Genus Aedes

Species nlbopictus United States Chambers County, TX Chicago, IL Jacksonville, FL Memphis, IN Houston, TX Indianapolis, IN Milford, DE New Orleans, LA Brazoria County, TX Evansville, IN Savannah, GA Houston 61, TX

pg DNAhaploid genome 2 SE

1.03 2 0.03 1.11 2 0.09 1.13 2 0.10 1.23 0.13 1.33 2 0.08 1.34 0.09 1.46 % 0.05 1.48 t 0.26 1.50 2 0.05 1.59 2 0.11 1.65 2 0.07 1.66 2 0.08

* *

References

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

1990 1990 1990 1990 1990 1990 1990 1990 1990 1990 1990 1990

the intraspecific variation in DNA content in two strains of Ae. albopictus was due mainly to highly repetitive DNA sequences. Further, MacLain et al. (1987) showed that populations of Ae. albopictus that were significantly different in DNA content also varied in the frequency of different classes of highly repetitive DNA. Thus, the variation in DNA content among populations of Ae. albopictus appears to be due mainly to repetitive DNA sequences that are under rapid change. This suggests that the amount of repetitive DNA is dynamic in Ae. albopictus and probably other mosquito species. Significant variation in haploid DNA content has been observed among invertebrates (Papeschi, 1991; Palmer and Petitpierre, 1996), vertebrates (Walker et al., 1991), and plants (Flavell et al., 1974; Jasienski and Bazzaz, 1995). In addition, several studies have shown a direct correlation of genome size with nuclear and cellular surfaces and volumes (Walker et al., 1991). Genome size also varies with the duration of the cell cycle (Bennett, 1987);the life history, phenology, and distribution of species (Macgillivray and Grime, 1995); the duration of generation time (Ferrari and Rai, 1989; Flavell et at., 1974; Jasienski and Bazzaz, 1995); and the body size (Palmer and Petitpierre, 1996). Cavalier-Smith (1985b) proposed that variation in DNA amount is subject to natural selection and plays an adaptive role. Although exact function(s) of highly repetitive DNA have long been debated, biologically significant roles in various species have been ascribed. For example, it has been suggested that the proportion of repetitive DNA in P h rnodium berghei maybe directly correlated with mosquito infectivity (Birago et al., 1982). A strain containing 18% repetitive DNA produced viable gameto-


17

cytes in mice, while another strain with 3% repetitive DNA did not. Also, repetitive elements of the bacterium Mycoplasm genitalium contribute to the antigenic variation in proteins of the MgPa cellular adhesion operon (Peterson et al., 1995). Within species, Bennett and Bennett (1992) suggested that smaller genomes are associated with populations that occur in stressful environments where rapid development, a short lifespan, and a high reproductive rate are favored (“r-selected”), while larger genomes are found most often in populations in environments that favor slower development times, increased longevity, delayed reproduction, and often a lower fecundity (“k-selected”).

VII. HETEROCHROMATIN: LOCALIZATION, VARIATION, AND EXPRESSION The application of Giemsa C-banding and other banding procedures to somatic and meiotic chromosomes has provided important insights into linear differentiation and evolution of chromosomes in Culicidae. Studies have been completed in 36 species belonging to seven-genera of Culicinae (Aedes, Mansonia, Culiseta, Armigeres, Sabethes, Wyeomyia, and Toxorhynchites) including 28 Aedes species (Motara and Rai, 1977, 1978; Rao and Rai, 1987a), three species of Cukx (Motara 1982), and several Anopheles species (Gatti et al., 1977; Baimai, 1988; Baimai et al., 1993a, b, 1995, 1996; Marchi and Mezzanotte, 1990). Cbanding patterns were also studied in representative species of Tipulidae, Dixidae,and Chaoboridae in order to examine how chromosomes have evolved in these families (Rao and Rai, 1987a). These studies established that the distribution of heterochromatin is markedly different in anopheline and culicine mosquitoes, particularly in the heteromorphic sex chromosomes. All species showed the presence of heterochromatin around the centromeres of the autosomes, although there are often large inter-and intraspecific differences in amounts of the same. Using different banding techniques, three types of heterochromatin were identified on the basis of staining characteristics in the pericentromeric regions in the Culicini species Culisetu longiareolatu (Mezzanotte et al., 1979; Marchi and Mezzanotte, 1988). In addition to centrometric bands, the autosomes in species such as Ae. bahmensis (Rao and Rai, 1987a) and the long arms of the sex chromosomes in An. atroparvus (Fraccaro et al., 1976) possess telomeric C-bands also. The organization of heterochromatin is markedly different in the two homologues of the sex chromosome pair in most Aedes species as well as between anopheline and culicine mosquitoes. Motara and Rai (1977, 1978) reported two distinct types, constitutive and facultative heterochromatin, in Aedes mosquitoes. The former is present around the centromere region of all three chromosome pairs and the latter in an interstitial position on one of the arms of the female-determining (m) chromosome in most Aedes species (Figure

18

K.

S. Rai and W. C. Black IV

1.2). The intercalary band is located proximal to the centromere in Ae. unnun&lei and in telomeric position on both the male- and female-determining chromosomes in Ae. vittatus. Ae. mascurensis (Figure 1.2), Ae. katherinesis, Ae. excrucians, Ae . stimulans, Ae . cinereus, and Ae . triseriatus lack the intercalary band (Figure 1.3). The fact that these species belong to three different subgen~

Females

Species

A. olbopictus

Males

I

A. polynesiensis

A. sculellaris

A. seafoi

I

A. pseudofbopiclus A. metalticus

Figure 1.2. Schematic representation of C-banding karyotypes in Stegomyia mosquitoes. After Motara and Rai (1978).

~~

CATEGORY CHROMOSOME I

--

CHROMOSOME I1

CHROMOSOME Ill CHROMOSOME IV

I

I

I

-

0

VII IV

IV

W

SPECIES'

1

I-

0

Qicmrn C-band

' 7

#

n n n

I

Tipula sp

I I

I I I

C h a o b o ~ americanur s C h a o b m flavicanr Chaoborus puncriprnnis Tororliynchilrr splsndrnr Wpomyia mrilhii Saberher cynneus

Culiscls malanura C. morsilanr Monsonia oerlurbans

A. kalh erinendr, ucnrcinns, slimulanr, cineitus, triserintut A. bahamcnris

Silver nitrate pwttivc

* Arranged Phylogeneticaliy

Figure 1.3. Chromosome number, morphology, and C-banding patterns in some genera of Nematocerous (Diptera: Nematocera) families. After Rao and Rai (1987a).

20


era suggests that heterochromatinization of particular segments is species-specific. The male-determining chromosome (M) in Ae. aegypti lacks even the centromeric heterochromatin (Figure 1.2). The constitutive and facultative heterochromatin replicate at different times in the cell cycle (Marchi and Rai, 1986). Unlike in Aedes, the intercalary heterochromatin is not present on the female-determining chromosome in Amigeres subulbutus or Toxorhynchites splendens but on an arm of one of the autosomes (chromosome I1 in the former and chromosome I11 in the latter) (Figure 1.3 and Rao and Rai, 1987a). Rai et ul. ( 1982) suggested a possible evolutionary derivation of the various heterochromatin patterns observed in Aedes species. The overall patterns observed among various genera (Figure 1.3) are also suggestive of the role chromosome repatteming played in genome evolution. The expression of the intercalary C-band on the sex chromosome in a particular species varies as a function of the genetic background in which it is placed. This was revealed by Giemsa C-banding of the F, hybrids and progeny of certain backcrosses between two closely related species, Ae. mgypti and Ae. mascarensis (Motara and Rai, 1977). Crosses involving Ae. uegypti females and Ae. mascarensis males produced F1 progeny in which the expression of the distal intercalary C-band on the female-determining (M) chromosome of Ae. aegypti was suppressed in both the males and the females (Figure 1.4a). This indicated that the distal region of the female-determining (M) chromosome represented by the heterochromatic C-band was derepressed and that it became euchromatic. When F, males from this cross were backcrossed to Ae. uegypti females, a proportion of the sons developed into intersexes and differed from normal males in their C-banding pattern (Figure 1 . 4 ~ )Thus, . it was possible to relate abnormal sexual development of adult males in the backcross progeny to a selective activation of a discrete chromosomal locus on the male-determining chromosome of their fathers (Motara and Rai, 1977). Reciprocal crosses (Ae. mascarensis females X Ae. uegypti males) gave expected results (Figure 1.4b,d). The reversible genetic regulation of the facultative C-band apparently represents selective control of a chromosomal segment of one species (e.g., Ae. aegypti) through genetic interaction with another, Ae. mascarensis (Motara and Rai, 1977). Such genetic regulation, which was also observed in progeny of crosses involving Ae. katherinensis and Ae. hebrideus (Rao and Rai, 1987a), may be widespread among aedine mosquitoes and may help protect species integrity. In anopheline species, the heteromorphic chromosomes often show extensive differences in the amount, distribution, and types of heterochromatin. The Y chromosome may be entirely heterochromatic in most Anopheles species while the X chromosomes may be heterochromatic from less than one-half to greater than three-fourths of their length, even among closely related species. Furthermore, several of these species-for example, the Hyrcanus group (sub-

21


A aegypti

9

Expected

8's

Amascarensis d

Eapected

Observed

a

9

FI,

81 11

9 Expected

Observed

Figure

18 11

A.aegypti

a"l

d

b

A.aegypti

C

9

9

(1

11

ObrarvHI

Amascarensis

d

Amascarensis

d

d

d

normal

9

intersex

d

1.4 a-d. Diagrammatic representations of Ae wgypti and A . mnscarensis C-banding patterns: (a and b) Summary of the expected and observed results in the F, in reciprocal crosses and (c and d ) among the backcross progeny. Because chromosomes I1 and 111 show expected banding patterns in all cases, they are excluded from the F, and BC, drawings (to enhance readability). After Motara and Rai (1977).

genus Anopheles), the maculatus group (subgenus Cetliu),and others-are polymorphic for the size of the X chromosome and for the amount of heterochromatin (Baimai et ul., 1993a,b, 1995, 1996). Such differences are diagnostic and allow unambiguous identification of species whose polytene chromosome-handing pattern is virtually homosequential (Green et al., 1985). Presumably, different densities of the Giemsa hands on the X and the Y chromosomes in these species reflect different types of constitutive heterochromatin (Baimai, 1988). Four satellite DNAs defined on Hoechst 3325s-CsC1 density gradients are similarly reflective of the presence of different types of heterochromatin in the An. stephemi genome (Redfem, 1981). In conclusion, there seems little doubt that changes in amounts, types, and locations of heterochromatin are associated with mosquito speciation, particularly in the subfamily Anophelinae and Culicinae. In situ chromosomal localization of four cloned repetitive DNA fragments (H-76,61, H-19, and H-85) indicated that they are dispersed throughout the lengths of the three pairs of chromosomes in all Aedes species examined

22

K.


(Kumar and Rai, 1991a,b). Although the sequences homologous to these cloned repetitive DNA fragments are present in other culicid genera, Humagogus equinus , Tripteroideres bambusa, and Anopheks quadrimaculatus, significant differences in their abundance and distribution were observed (Kumar and Rai, 1991a,b). Unlike such dispersed pattern in Aedes, Satellite 1 was localized to the heterochromatic arms of the X and the Y chromosomes and the centromere regions of chromosome 3 in An. stephensi (Redfern, 1981). Similarly, a highly repetitive DNA clone isolated from Ae. ulbopictw (H115) was shown to be located at an intercalary position on chromosome 1 in all Aedes species examined (Kumar and Rai, 1992). Southern hybridization of this DNA fragment with genomic DNA of An. quadrimaculatus, on the other hand, showed a dispersed pattern. An important difference in chromosome organization with regard to heterochromatin distribution between anophelines and most culicines may be critical in determining whether polytene chromosomes can be easily mapped. There is generally a good resolution of individual bands on each of the euchromatic chromosome arms in the anophelines, while culicines are largely refractory to this type of analysis. In anophelines, apparently much of the heterochromatin is clustered around the centromeres of each of the three pairs of chromosomes, resulting in the formation of a chromocenter in polytene chromosome preparations. Of the eight mosquito genera in which polytene chromosome morphology has been studied, Anopheks alone possesses a chromocenter. All other genera (Aedes, Cukx, Mansonia, Toxorhynchites, Orthopodomyia, Wyeomyia, and Sabethes) lack a distinct chromocenter (Sharma et al., 1979; Dennhofer, 1968; Tewfik and Barr, 1974; Verma et al., 1987; Chaudhry, 1972; White, 1980; Munstermann et al., 1985; Moeur and Istock, 1982; Munstermann and Marchi, 1986). Nevertheless, Orthopodomyia pulnipatpus (Munstermann et al., 1985) and Subathes cyuneus (Munstermann and Marchi, 1986) have yielded well-resolved polytene chromosomes. This suggests that these taxa have longperiod interspersion and may be more basal in culicid evolution. Furthermore, as indicated earlier, repetitive DNA constitutes a large proportion of the genome in culicine mosquitoes. Since this DNA undergoes late replication during the S period (Marchi and Rai, 1986), such dispersed sequences may conceivably act like microchromocenters, thereby preventing effective separation of individual chromosomes. In examining karyotypes and C-banding patterns in species of Tipulidae,Dixidae, Chaoboridae, and Culicidae, Rao and Rai (1987a) concluded that Culicidae arose from a chaoborid Mochlonyx-like ancestor and that the Anophelinae and Culicinae evolved along separate lineages from a common ancestral stock (Figure 1.5). The Chagmia karyotype was considered to be primitive for Anophelinae, while the Toxorhynchites karyotype was considered primitive for Culicinae. The cladistic analyses discussed earlier, support this proposal.

23


1 2 3 CULlClNAE

'/

1 2 3 Anopheles sp.

1 2 3 4 Mochlonvx

1 2 3 4 Chegasia bafhans

1 2 3 4 Dixa recene

1 2 3 )Tipula sp.

Ancestral Tlpulldaa

Figure 1.5. Proposed chromosomal evolution in some nematocerous taxa. Arabic numerals represent chromosomes; chromosomes not drawn to scale. Afer Rao and Rai (1987a).

VIII. SATURATED LINKAGE MAPS GENERATED THROUGH MULTIPOINT MAPPING Over the last decade, a new paradigm has emerged in genetic linkage mapping, where hundreds or thousands of markers are mapped simultaneously in one or a few crosses. Molecular genetic methods that allow for amplification of many loci from small amounts of genomic DNA have been instrumental in the application

24


of this technology to small arthropods. Recent techniques make it possible to analyze many regions of a genome simultaneously. All of these incorporate the polymerase chain reaction (PCR) for the amplification of markers from small amounts of genomic template DNA. Random amplified polymorphic DNA (RAPD) markers and arbitrarily primed (AP) markers are amplified with PCR using short oligonucleotide primers with arbitrary sequence (Williams et al., 1990; Welsh and McClelland, 1990). PCR is also used for amplification of genomic regions for analysis by restriction enzymes (Severson et al., 1993) or single-strand conformation polymorphism (SSCP) analysis (Orita et al., 1989). The discovery of abundant microsatellites in eukaryotic genomes. (Weber, 1990; Beckmann and Soller, 1989, 1990) has provided a plethora of markers for mapping many eukaryotic genomes. In addition to these molecular genetic techniques, the development of software for maximum-likelihood estimation of linkage relationships among multiple cosegregating markers (Lander et al., 1987; Stam, 1993) has been instrumental in allowing multipoint mapping with a variety of molecular markers. This technology has also allowed a number of different laboratories to construct multipoint linkage maps of entire mosquito genomes. This was first accomplished by Severson et al. (1993), who constructed a linkage map of Ae. mgypti using 50 RFLP markers from 42 random cDNA clones, 3 random genomic clones, and 5 cDNAs of known origin. The lengths of chromosomes I, 11, and 111 were 49, 60, and 56 cM, respectively (165 cM total). Antolin et al. (1996) constructed a linkage map of Ae. mgypti using SSCP analysis of 94 RAPD markers. The lengths of linkage groups I, 11, and 111 were 52, 58, and 57 cM, respectively (168 cM total), remarkably similar to the cDNA map. Mutebi et al. (1997) constructed a linkage map of Ae. albopictus using SSCP analysis of 68 RAPD markers. The lengths of chromosomes I, 11, and 111 were 54, 67, and 104 cM, respectively (225 cM total). Severson et al. (1995) showed that cDNA markers are colinear in Ae. mgypti and Ae. albopictus. These studies using molecular markers suggest a large (57 cM) increase in the recombinational size of the Ae. albopictus genome. Furthermore, most of this appears to be due to increased recombination on chromosome 111. It is uncertain whether these differences are due to variations in DNA amount or to differences in the distribution and frequency of chiasmata on chromosome 111 of the two species. Aedes species are known to vary widely in chiasmata distribution and frequency (Dev and Rai, 1984; Sherron and Rai, 1984). The lengths of the three linkage maps involving morphological and enzyme loci calculated from observed chiasmata frequencies were 62, 86, and 80 cM, respectively (total 228 cM in Ae. mgyptl) (Munstermann and Craig, 1979). However, large stretches of all three linkage maps were devoid of any markers, particularly on linkage group 111 on which the 17 observed markers were clustered in a 44-unit map, while the chiasmata-based model predicts an 80-unit map.


25

More recently, a linkage map of Armigeres subalbatus has been constructed using 26 RFLP markers involving cDNA clones from Ae. aegyti. The overall lengths of linkage groups I, 11, and 111 were 51, 72, 58 cM, respectively (181 cM total), and, except for one marker, the linear order was the same as in Ae. uegypti (Ferdig et al., 1998). A similar RFLP linkage map has been constructed for Culex pipiens using 21 cDNA clones from Ae. aegypti (Mori et al., 1998). The total map spans 165.8 cM. The linkage maps for chromosomes I, 11, and 111 of Cx. pipiens were 7.1, 80.4, and 78.3 cM, respectively. However, based on the relatively small number of markers used, these estimates do not accurately coincide with lengths of corresponding linkage maps of other culicine species, particularly for linkage group I. This necessitates work with additional molecular markers. The comparative linkage maps for chromosomes I1 and I11 in Cx. pipiens and Ae. uegypti reflect whole-arm translocations (Mori et al., 1998). Zheng et al. (1996) mapped 131 microsatellite markers in Anopheles gambiue. Chromosomes I, 11, and I1 were, respectively, 49, 72, and 94 cM in length (215 cM total). Integration of RAPD markers into this map increased the overall density of markers without affecting the overall length (Dimopoulos et al., 1996). It is instructive to consider linkage map size, an indication of the amount of recombination on individual chromosomes, relative to the genome sizes discussed earlier in this chapter. The observed linkage map sizes are 165 cM in Ae. aegypti, 225 cM in Ae. albopictus, 166 cM in Cx. pipiens, 181 cM in Ar. subalbatus, and 215 cM in An. gambiue. These do not correspond in any way to the genome sizes of 0.83, 0.86-1.32, 0.54-1.02, 1.12 and 0.27 pg/haploid genome, respectively, in these species. The relationship of physical to recombination distance is approximately 3-6 Mb DNA/cM in Ae. mgypti, Ae. albopictus, and the two other culicine species studied, and 1.2 Mb DNA/cM in An. gambiae (Table 1.4). Thus there appears to be little relationship between genome size and recombination frequency. The frequency of recombinations remains high in An. gambiae despite its having a genome size one-third to onefifth the size of the Aedes and other culicine species genomes. DNA reassociation kinetic analysis has shown that the amount of repetitive DNA sequences in culicine species is generally much higher than that in Anopheles species (Table 1.4; Black and Rai, 1988; Warren and Crampton, 1991; Besansky and Powell, 1992). Since recombination is considerably restricted in chromosomal regions rich in repeated DNA sequences (Charlesworth et al., 1986), overall Anopheles would be expected to show higher recombination rates. Also, this predicts a closer relationship between physical and linkage maps in Anopheks and a higher likelihood of success in mapped-based positional cloning of genes that control the phenotype of a character under study. Furthermore, the fact that the sizes of the linkage maps do not vary by more than 60 cM in these

26

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

Table 1.4. Comparison of Linkage Maps (Total cM), DNA Amounts (pg), Proportions of Uniquemepetitive DNA Sequences, and Ratio of Haploid DNA Amounts to Linkage Map Size (cM)

DNA Species Culicinae Ae. aegypti

Ae. albopictw Calcutta Mauritius Ar. subalbatus Cx. pipiens Anophelinae An. gambiae An. quadrimaculattu

Linkage map (total cM)

% Repetitive

Haploid DNA/cM (Mb)

Total (pg)

bp

% Unique

0.8P

8.0 X 108

6od

32*

4.85

0.8@ 8.3 X lo8 1.32a 1.3 X lo9 1 . 1 ~ 1.1 x 109 1 . o ~ 1.0 x 109 0 . s ~ 5.2 x 109

36c 33c

54' 5?

22

67'

3.1 5.8 6.1 6.0 3.1

0.27" 0.24"

61b 8&

33b 1&

1.2 1.1

165 225 181 166 215

2.6 X los 2.3 X lo8

Q a t a from Tables 1.2 and 1.3. bData from Besansky and Powell, 1992. 'Data from Black and Rai, 1988. dData from Warren and Crampton, 1991.

three species suggests that the number of chiasmata have remained relatively constant despite increases in genome size and chromosome length in the evolution of Culicidae.

IX. SUMMARY A great deal of information has been accumulated on chromosome numbers and heterochromatin distribution as well as on genome size and organization in the mosquito family Culicidae. A number of trends in genome evolution emerge when these data are reviewed in light of recent cladistic phylogenies of Culicidae and its sister families. Anophelinae have heteromorphic sex chromosomes and a small genome size, and repetitive elements are distributed in a longperiod interspersion pattern. In contrast, Culicinae have homomorphic sex chromosomes, and repetitive DNA is organized in a short-period interspersion pattern. There has been a general increase in genome size during the evolution of culicine tribes. The organization of the ancestral culicid genome remains uncertain awaiting studies on genome organization in Chaoboridae-Corethrellidae taxa.


27

The most parsimonious hypothesis for the evolution of sex chromosomes and genome organization in Culicidae would be that homomorphic sex chromosomes and a longperiod interspersion pattern was ancestral in lineages leading to Toxorhynchitinae and Culcinae. Larger genomes developed in subsequent culicine lineages through accumulation of short-period interspersed repetitive elements. Heteromorphic sex chromosomes evolved early in the evolution of Anophelinae, and a long-period interspersion pattern was retained. The alternative scenario proposed by Rao and Rai (1987a) is that Culicidae arose from a chaoborid Mochlonyx-like ancestor with heteromorphic sex chromosomes and possibly short-period interspersion. This scenario would require the loss of heteromorphic sex chromosomes in the lineage leading to Toxorhynchitinae and Culicinae and the “shedding” of repetitive elements in the lineage leading to Anophelinae. Several interesting patterns have emerged from studies of C-banding, and the distribution of heterochromatin in Culicidae and phylog enies derived from these studies are supported by the modem cladistic analyses. Recent intensive multipoint linkage map studies suggest that recombination frequencies per genome have remained relatively constant over the course of culicid evolution such that Anophelinae, with a relatively small genome size, has a linkage map of similar size to Aedini. As a consequence, taxa in Anophelinae have higher amounts of recombination per haploid genome size than Culicinae. Although several key questions have yet to be addressed, the Culicidae remain one of the best-studied systems of genome evolution in animals.

Acknowledgments The original work in the senior author’s laboratory included in this chapter was supported by NIH Research Grant 5R01 A1 21443, by NIH Training Grant 5T30 A1 07030, and by the University of Notre Dame. We thank Doctors Nora Besansky and David Severson for a critical review and for making several suggestions for the improvement of the manuscript. We also express our sincere thanks to Kathleen Merz for her invaluable help in keyboarding and for several revisions of the manuscript.

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H. linden Lehrstuhl fur Physiologie und Biochemie der Pflanzen Universitat Konstanz D-78434 Konstanz, Germany

P. Ballario Dipartimento di Genetica e Biologia Molecolare, Centro di Studio per gli Acidi Nucleici Universitk di Roma “La Sapienza” 00185 Roma, Italy

6. Arpaia, and 6. Macino* lsrituto Pasteur Fondazione Cenci Bolognetti Dipartimento di Biotecnologie Cellulari, Sezione di Genetica Molecolare Universita di Roma “La Sapienza” 00161 Roma, Italy

I. Introduction 11. The Perception of Light in Neurospora A. Neurosporu Perceives Light Only in the Ultraviolet/Blue Light Range

B. Blue Light Activates Gene Expression

C. Neurosporu Is Capable of Adapting to Different Light Intensities

D. Protein Kinase C Is Involved in the Photoadaptation Process of N . crassa *To whom correspondence should be addressed. Advances in Genetics, Voi. 41 Copyright 0 1999 by Academic Press All rights of reproduction in a n y form reserved. 0065-2660/99$30.00

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H. Linden eta/.

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111. The Interplay of Blue Light and Other Regulatory Pathways in Neurosporu IV. Mutational Analysis of Blue Light Signal Transduction in Neurosporu V. The Neurosporu 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 B. WC-1 and WC-2 Domains for Dimerization and Signal Transduct ion C. How Do WC-1 and WC-2 Function in Neurospmu Blue Light Signaling? VI. Concluding Remarks Acknowledgments References

1. INTRODUCTION Light is one of the most important environmental factors for plants, algae, bacteria, and fungi and regulates developmental and physiological processes. Plants are able to perceive light over the whole sunlight spectrum, and perception of light is carried out by at least three different families of photoreceptors: the phytochromes (red and far-red light absorption), ultraviolet receptor( s), and blue light photoreceptor(s) (Deng, 1994). In our attempt to unravel the mysterious process of blue light perception and the transduction of the light signal, we are using the ascomycete Neurospora crussu, which has been proven to be an ideal organism for photobiological, biochemical, and genetic studies. In addition to the more general advantageous features of Neurospora, such as a small eukaryotic genome (estimated as 47 megabases; Orbach et ul., 1988),fast growth, straightforward genetics, and fast transformation with foreign DNA, there are more specific reasons to use N. c r a w as a model organism to study lightregulated processes. In contrast to higher plants, N. crussu is capable of sensing light only in the blue light range, and blue light is the stimulus for several different processes. During the asexual life cycle, mycelial carotenoid biosynthesis (Harding and Turner, 1981), formation of vegetative spores (macroconidia)(Klemm and Ninnemann, 1978; Lauter, 1996), and circadian rhythmicity (Sargent and Briggs, 1967) are regulated by blue light. In addition, blue light responses such as formation of protoperithecia (Degli-Innocenti et al., 1983) and the phototropism of perithecial beaks (Harding and Melles, 1983), have been observed during the Neurosporu sexual life cycle. Regulation of these processes seems to occur mainly at the level of gene expression, and to date

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

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several blue-light-regulated genes have been cloned. Furthermore, many Neurospora mutants that seem to be impaired in light perception and/or transduction of the light signal have been isolated and characterized. During the past decades, a wealth of data have been published regarding the Neurospora blue light responses, blue-light-regulated genes, and the putative nature of the blue light photoreceptor and components of the signal transduction chain that have only recently been reviewed in detail (Lauter, 1996; Linden et al., 1997a; Ballario and Macino, 1997). The purpose of the present review is to discuss the recent progress that has been made in the cloning and characterization of two cooperating partners of the Neurospora blue light signal transduction chain. Furthermore, we outline some new and lessknown aspects of blue light regulation in N.crmsa.

II. THE PERCEPTION OF LIGHT IN Nemspora A. Neurospora perceives light only in the ultraviolet/blue light range Several action spectra for different Neurospora blue light responses have been published. An action spectrum reflects the wavelength dependency of the sensitivity for a specific response. Data from DeFabo et al. (1976) for light-regulated biosynthesis of carotenoids in Neurospora and from Sargent and Briggs (1967) for the photosuppression of conidial banding clearly demonstrated the sensitivity of the Neurospma photoreceptor(s) not only for blue light but also for UV light. Their results also revealed that Neurospora is “blind” toward light beyond 520 nm. Schrott (1980, 1981) reported fluence response curves for light-induced carotenoid biosynthesis in N.crassa. A saturation of light-induced carotenogenesis was observed when the mycelia were exposed to fluence rates beyond 0.3 W m-2 for up to 16 min. Furthermore, the fluence response was shown to be biphasic; an extension of the illumination time beyond 16 min resulted in a second increase in the amount of carotenoids synthesized during the subsequent dark period. A temporary insensitivity toward light between the first and the second phase of the biphasic fluence response curve was described (Schrott, 1981). A period of 2 h after a first illumination was found to be necessary for restoring maximum competence for a second light induction. Schrott suggested that the photoreceptor and/or elements of the signal transduction chain become depleted during the first phase. Consequently, such a period of restoration may be necessary before the sensitivity toward light is recovered. Corrochano et al. (1995) also reported a two-phase stimulus-response curve. They prepared a translational fusion of the light inducible con-10 promoter and the Escherichia coli lac2 gene. After transformation of Neurospora and photoinduction of the mycelia, the P-galactosidase activity was determined. Following

38

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a light induction of 1 to 15 min, a first induction of 0-galactosidase activity reached a plateau 1 min after onset of light. Upon further illumination for 30 min, a second response that doubled the P-galactosidase activity was observed. Interestingly, this biphasic response has never been observed on the level of transcription. All the blue-light-regulated genes isolated today show a one-phase response curve only. Therefore, a posttranscriptional event may be responsible for the observed biphasic response on the level of enzyme activity.

B. Blue light activates gene expression Many light-regulated genes have been cloned in Neurospora to date. When dark-grown mycelia are illuminated with constant light, most of the lightregulated genes show a transient expression pattern (Figures 2.1A and 2.1B). The only exception to this rule is the Neurospora gene frequency (frq), which encodes a central component of the circadian clock (Loros, 1995). The mRNA of frequency shows a fast increase in response to light and remains elevated in comparison to the levels observed in constant darkness (Figure 2.1C; Crosthwaite et al., 1995). It is important to note that Figure 2.1 constitutes a schematic representation only and does not take into account the quantitative differences in relative mRNA steady-state levels. For example, a 90-fold increase of some blue-light-inducible mRNAs has been described to occur after light induction, whereas other genes show a much lower induction (3-fold) with respect to their dark levels (Sommer et al., 1989). Due to their expression pattern, the blue-light-regulated genes can be divided into early light-regulated genes, with a mRNA peak at about 20-30 min after onset of light, and late light-regulated genes, with a mRNA peak at 45-120 min. The carotenoid biosynthesis genes al-I , al-2, and al-3 (Baima et al., 1991; Li and Schmidhauser, 19951, the central regulator of blue light responses wc-I (Ballario et al., 1996), the blue-light-induced genes bli-3 and bli-4 (Sommer et al., 1989), the conidiation genes c o n 5 and con-10 (Lauter and RUSSO, 1991), and the clock-controlled genes ccg-4 and ccg-6 (Bell-Pedersen et al., 1996b) are fast light-regulated genes (Figure 2.1A), while during conidiation the clock-controlled genes ccg-l , ccg-2 (em), and ccg-9 (Arpaia et al., 1993, 1995a; Bell-Pedersen et al., 199613) and al1 , aC2, and al-3 reveal a delayed induction after exposure to light (Figure 2. l B).

C. Neurospora is capable of adapting to different light intensities A desensitization phenomenon in which a continuous stimulation results in a decreased sensitivity for the stimulus has been described in animal cells and higher plants (Bowler et al., 1994). Kinetic examination of the al-3 mRNA induction using different light and dark incubation periods, as well as different light intensities, indicated the presence also of a photosensory adaptation mech-

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

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~

early lightregulated genes

A

1

1

1

,

1

,

,

-

,

,

,

,

-

frequency

C

I

1

1

1

1

1

1

1

l

1

1

Incubation in constant light (min) Figure 2.1. Schematic representation of the expression of early light-regulated (A) and late lightregulated genes (B) as well as the expression pattern offreqwncy ( C ) in constant light. The relative mRNA levels are given in arbitrary units.

anism in Neurospma (Macino et al., 1993). No al-3 mRNA was detected after a continuous light induction of 100 min, whereas the ale3 mRNA was found to be inducible by a second light pulse after a first light pulse and a subsequent dark period of 60 min. This dark period of at least 60 min seemed to be necessary to recover the sensitivity of the photosensory system. These results comply with the temporary insensitivity toward light for the biosynthesis of carotenoids after a first light pulse described by Schrott (1981). If the observed transient expression of the al-3 gene and the temporary insensitivity toward light are due to an active process of desensitization, irradiaton of the mycelia

40

H. Linden 81 a/.

with a higher light intensity should overcome the insensitivity after a first pulse of lower light intensity. In fact, a second, albeit lower, peak of al-3 expression was observed when a higher light intensity was used for a second light pulse, and the expression pattern was again shown to be transient (G. Arpaia et al., 1999). These results indicate the capacity of Neurospora to adapt to different light intensities.

D. Protein kinase C is involved in the photoadaptation process of Iv. crassa A biochemical approach has been used to investigate the phytochrome signal transduction pathway in higher plants (Neuhaus et al., 1993; Bowler et al., 1994). The authors used specific inhibitors and agonists to identify signal transduction components of the phytochrome signal transduction chain. A similar approach has been carried out in our laboratory to investigate blue light signaling in Neurospora (Arpaia et al., 1999). Monitoring of the expression of the al3 gene revealed two different light-inducible transcripts during mycelial growth and conidiation, as discussed in detail later (Arpaia et al., 1995b). Although many different inhibitors and agonists were used in this investigation, only protein kinase C-directed compounds showed a reproducible effect on the bluelight-regulated expression of the al-3 gene. During conidiation, protein kinase C inhibitors completely blocked the light induction of the conidiation-specific al-3 transcript. Normally, the al-3 mycelial mRNA shows a transient expression pattern even under constant light conditions and, after a light induction of 2 h, no elevated mRNA levels can be observed (Figure 2.1A). Application of protein kinase C inhibitors during mycelial growth resulted in a normal increase in mycelial mRNA up to the time of maximal expression; however, the mRNA levels remained high for at least 90 min, indicating that protein kinase C is at least in part responsible for the inhibition of the light signaling cascade leading to desensitization. It would therefore appear that protein kinase C has a dual role in Neurospora blue light signal transduction. O n the one hand, during conidiation protein kinase C mediates light induction of the conidiation-specific al-3 transcript, while on the other hand, during mycelial growth protein kinase C is responsible for the negative control of light signaling. Consequently, the reported temporary insensitivity outlined above seems to be due to an active adaptation mechanism and desensitization of the photoreceptor and/or the signal transduction machinery to a given light intensity rather than to a depletion of signal transduction elements as was suggested previously. Protein kinase C represents the first component of this adaptation machinery in N. crassa.

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

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111. THE INTERPLAY OF BLUE LIGHT AND OTHER REGULATORY PATHWAYS IN Neuruspura As outlined earlier, there are many different morphological, developmental, and physiological processes regulated by blue light in N. crassa. Some of these processes are regulated by more than one environmental stimulus at a time but seem to be under a complex control mechanism. For example, the formation of conidia is influenced by glucose limitation, carbon dioxide levels, desiccation, and blue light (Springer, 1993; Sokolovsky et al., 1992). The formation of protoperithecia during the sexual cycle of Neurospora is influenced by growth temperature, nitrogen, oxygen, carbon dioxide, suspensions of their own conidia, and blue light (Degli-Innocenti et al., 1983, 1984a, and references quoted therein). Consequently, gene expression was shown to be under multiple control of numerous extracellular and intracellular stimuli. This became evident when identical genes were isolated in different screening approaches. The bli-7 gene was identified in a search for blue-light-inducible genes, while ccg-2 was cloned in a screening for clock-controlled genes (Sommer et al., 1989; Loros et al., 1989). Both genes were subsequently proven to be allelic and under the control of blue light and the circadian clock (Bell-Pedersen et al., 1992; Lauter et al., 1992). Similarly, the al-1, al-2, bli-4, ccg-2/bli-7, con-5, and con-10 genes are influenced by blue light and the amount of nitrogen supplemented to the growth media (Sokolovsky et al., 1992). The developmental process of conidiation, blue light, and the circadian clock all regulate the expression of the conidiation-specific genes con-6 and cowl0 (Lauter and Yanofsky, 1993). Another example of a complexly regulated gene in Neurospora is the circadian clock gene frequency (frq). The FRQ protein was shown to be part of an autoregulatory negative feedback loop in which the FRQ protein negatively regulates its own expression (Aronson et al., 1994). It was suggested that this negative feedback loop represented a central component of the Neurospora circadian oscillator. Furthermore, it was found that the fiq gene is rapidly induced by light and this light induction was correlated with the resetting and entrainment of the circadian clock (Crosthwaite et al., 1995). Using Neurospora mutants that lack a functional circadian clock, Arpaia et al. (1993, 1995a) were able to show that the light induction of the clock-controlled genes ccg-l and ccg-2 is direct and does not depend on the circadian clock. Bell-Pedersen et al. (1996a), in studying the ccg-2 promoter, also identified separate regulatory cis elements for light and the circadian clock in accord with the findings of Arpaia et al. (1993). O n investigation of the regulation of the at-3 gene by light and by developmental stimuli, two overlapping transcripts of 2.2 and 1.6 kb were identified (Arpaia et al., 199%). The 2.2-kb transcript revealed a long, untrans-

42

H. Linden eta/.

lated leader sequence and occurred only in conidiating cultures. Furthermore, the 2.2-kb transcript was not observed in the two mutants, acon-2 and j2, that were blocked in different stages of conidiation and therefore seemed to represent a conidiation-specific transcript with a specific timing of expression. The al-3 conidiation-specific transcript is also light inducible and under circadian clock control but only during conidiation. The expression of the other light-regulated carotenoid biosynthesis genes, d-1 and d-2, was also reported to be influenced by light and conidiation, although no different transcripts were identified (Li and Schmidhauser, 1995). Gene expression and promoter studies suggest that different stimuli address distinct regulatory cis elements in promoters. At least the blue light signal transduction chain seems to be separated from other signal transduction chains. This is indicated by the fact that although almost every light induction of Neurospora genes is dependent on the two blue-light-regulatory white collar proteins (WC-1 and WC-2), these proteins do not seem to interfere with other signal transduction pathways except for a peculiar role in circadian clock control proposed by Crosthwaite et al. (1997) and outlined later. In view of the recent results suggesting that both wc-I and wc-2 gene products are involved in transcriptional activation, a common mechanism for all light-regulated genes can be presumed. Promoter-specific differences, such as the sequence of the lightregulatory cis elements, their situation in the promoter, and additional action of repressors and/or transcriptional activators, may account for the observed differences in gene expression in response to light. This gives rise to a complex pattern of transcriptional control that enables N. crussa to respond to extraand intracellular stimuli and to adapt to environmental conditions. ~

IV. MUTATIONAL ANALYSIS OF BLUE LIGHT SIGNAL TRANSDUCTION IN Neurospora During the past decades, a considerable effort has been made in the genetic dissection of the Neurospora blue light transduction pathway. Numerous mutants that seem to affect or participate in blue light signaling have been isolated (for review, see Linden et al., 1997a). The most important and best examined Neurospora mutants in blue light signal transduction isolated to date are the white collar mutants (Perkins et al., 1982; Harding and Shropshire, 1980). The white collar mutants have pigmented conidia, whereas the mycelia are white due to a specific deficiency in light-induced carotenoid biosynthesis. This is in contrast to the albino mutants, which reveal white mycelia and white conidia due to mutations in structural genes of carotene biosynthesis. The wc-f and wc2 mutants have been shown to be completely “blind” for almost all Neurospora blue light responses, and most of the blue-light-regulated genes cloned were


43

reported to be not inducible by blue light in either a wc-1 or wc-2 mutant background. Most of the mutants reported previously, including several wc mutant alleles, were isolated either by chance or by visual screening without the application of a selection system (Degli-Innocenti and Russo, 1984b). In order to isolate new regulatory mutants that affect blue light perception in N. crasa and to carry out a saturating genetic dissection of “blind” mutants, a selection system has been developed (Carattoli et al., 1995). Taking advantage of the fact that blindness does not seem to be lethal in Neurospora, all nonredundant blue light signal transduction components could be identified with this selection system. The light-induced al-3 promoter was fused to the coding region of the mtr gene, the product of which is responsible for the uptake of neutral aliphatic and aromatic amino acids in Neurospora (Stuart et al., 1988). After transformation of a mtr-/trp- strain with this construct, the resulting strain (13-1) became light dependent for the uptake of tryptophan and of a toxic analogue of phenylalanine, p-fluorophenylalanine (Linden et al., 1 9 9 7 ~ ) . Strain 13-1 was able to grow on a medium supplemented with p-fluorophenylalanine in darkness only, as the aC3::mtr gene construct is not expressed under these conditions. In contrast, in the light the al-3::mtr promoter is induced, causing mtr expression and the uptake of the drug, which inhibits cell growth. Therefore, only mutants impaired in blue light perception or signal transduction will grow in the light in the presence of p-fluorophenylalanine. This selection system was successfully applied to the isolation of mutants that showed a decreased sensitivity for blue-light-regulated processes (Carattoli et al., 1995). The blue-light-regulator mutants blr-1 and blr-2 revealed a pale-orange phenotype indicating decreased light induction of mycelial carotenoid biosynthesis. Furthermore, the mutants had decreased steady-state levels of mRNA for all lightregulated genes examined. In sexual crossing experiments, the mutations blr-1 and blr-2 fell into different segregation groups from wc-l and wc-2. Consequently, they do not represent leaky alleles of the wc loci. In addition, the selection system was used for the isolation of wc mutants after ultraviolet mutagenesis (Linden et at., 1 9 9 7 ~ ) In . spite of an exhaustive screening, no additonal wc loci other than wc-1 and wc-2 were isolated. Therefore, the wc-1 and wc-2 genes seem to be the only nonredundant loci present in Neurospora that lead to a complete “blindness” toward light. The selection system just described has a further application: The selection strain 13-1 is unable to take up aromatic amino acids in the dark. After ultraviolet mutagenesis, growth of 13-1 on tryptophan in darkness resulted in the isolation of mutants ccb-1 and ccb-2 (for constitutive carotenoid biosynthesis), which showed a light-grown phenotype even in the dark (Linden et al., 1 9 9 7 ~ )In . spite of constitutive mycelial carotenoid biosynthesis in darkness, the mutants did not show increased mRNA levels of light-regulated genes in the dark. However, an increased expression of some light-regulated genes in

44

H. Linden eta/.

comparison to the wild type occurred after light induction, indicating a function in blue light signaling at least for ccb-I. Its recessive nature together with the specific effects on light induction of carotenoid biosynthesis suggested a role for the ccb- I gene product as transcriptional repressor of some light-regulated genes. The identification of dark repression sites in promoters of light-regulated genes pointed to the presence of such repressors in Neurospora (Kaldenhoff and RUSSO, 1993). O n the other hand, the ccb-2 gene product was proposed to act during the developmental process of conidiation.

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 complementation of the wc-I mutant phenotype (Ballario et al., 1996); insertional mutagenesis together with the application of the selection system for blue light regulatory mutants resulted in cloning of the wc-2 gene (Linden and Macino, 199713). The wc-I gene encodes a 125-kDa protein consisting of 1154 amino acids, whereas the WC-2 protein is a smaller polypeptide (57 kDa) with 530 amine acids. In a search of protein databases, no overall homology with other proteins was found for the WC-1 protein. In contrast, an overall homology with WC-2 was detected for another fungal protein, the so-called palindrome-binding protein PBP isolated from Fusarium solani (EMBL Data Bank Accession No. U23722), which seems to play a role in the induction of the cutinase gene in Fusarium (Li and Kolattukudy, 1995). Although the PBP protein has not been discussed in context with blue light signal transduction, it is interesting to note that Fusarium shows blue light responses and action spectra similar to those of Neurospora (Rau, 1967). Due to the high overall homology with WC-2 (61.3%), we believe that PBP is the WC-2 homologue from Fusarium. Therefore, the blue light regulatory protein WC-2 does not seem to be restricted to N.crassa. It would be interesting to know if the same is true for WC-1. Although no overall homology exists between WC-1 and WC-2, the proteins share several common features (Figure 2.2A). Both proteins contain a single putative zinc-finger DNA-binding domain that shows similarity to the DNA-binding domain of GATA factors. In contrast to the other GATA factors from vertebrates that contain two zinc-finger domains with 17-amino-acid loops, WC-1 and WC-2 reveal only one putative zinc finger with an 18-amino-acid loop. In addition, putative transcriptional activation domains have been characterized in both WC.1 and WC-2 proteins. The amino-terminal region of


A wc2 TG

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45

1

N. crassa blue light signal transduction partners

]

1I

Human dioxin receptor components

I

Figure 2.2. Domain structure of WC-1 and WC-2 (A) compared with the AH-receptor (AHR) and ARNT (B) (according to Burbach et d., 1992). The position of putative PAS domains in WC-1 and WC-2 as well as PAS A and PAS B in the dioxin receptor components are indicated by hatched boxes. Other regions indicated include the proline-rich (P-rich) and acidic domains and the region of homology with the photoactive yellow protein (PYP) in WC-2, the putative zinc-finger domain in WC-1 and WC-2, the glutamate-rich (Q-rich) regions, and putative helix-loop-helix domains in the. A H receptor and ARNT as well as the ligand-binding region of the A H receptor.

WC-1 contains a stretch of 28 glutamine residues, whereas proline-rich and acidic regions have been found in WC-2. These domains have been described for many other transcription factors and have been implicated in transcriptional activation. Putative nuclear targeting signals may indicate the localization of WC-1 and WC-2 in the nucleus. Bandshift experiments using either WC-1 or WC-2 fusion proteins have shown that WC-1 and WC.2 are capable of binding a DNA fragment of the light-regulated promoter of the carotenoid biosynthesis gene al-3. It was concluded that both WC-1 and WC-2 accomplish their function in blue light signal transduction by binding to promoters of lightregulated genes. This idea was supported by the finding that several wc-2 mutant alleles show mutation or disruption of the putative zinc-finger binding domain. The existence of a Neurospora light-responsive element (LRE) has been hypothesized by several authors; however, a comparison of the 5' upstream regions of all the light-regulated genes in Neurospora has failed to uncover universally conserved cis elements (see Lauter, 1996, and Linden et al., 1997a, for a list of

46

H. linden at a/.

known Neurospora light-regulated promoters). A t present, two sequences, GATA and APE, are the best canditates for LREs. Both motifs are present in the al-3 promoter fragment recognized by WC-1 and WC-2 binding domains. O n the basis of the competition experiments reported by Ballario et al. (1996) and Linden and Macino (1997b), GATA motifs certainly form part of the recognition site of the WC proteins under the experimental conditions used; however, the absence of GATA motifs in some of the known light-regulated Neurospora promoters weakens its general function. The APE sequence has been shown to be involved in al-3 light regulation by deletion analysis (Carattoli et al., 1994) and to be able to confer light inducibility to a reporter gene (Carattoli et al., 1995); however, it has been identified only in a subset of the lightregulated genes, including the carotenoid biosynthesis gene al-3, the clock control gene 2 (ccg-2 or e m or bli-7) (Bell-Pedersen et al., 1996a), and the conidiation gene 10 (con-10)(Corrocchano et al., 1995). In con-10, the APE sequence does not seem to participate in the light regulation of transcription (Corrocchano et al., 1995).

B. WC-1 and WC-2 domains for dimerization and signal transduction Additional domains were identified in both WC proteins that showed a similarity to a dimerization domain called PAS (for PER-ARNT-SIM). A PAS domain is a region of homology of approximately 300 amino acids containing two degenerate direct repeats of 50 amino acids, called PAS A and PAS B. The WC-2 PAS domain, however, differs from other PAS domains reported so far, including WC-I, since it does not comprise the usual PAS A and PAS B repeats but seems to consist of only one PAS repeat. This PAS domain is present in the Dosophila protein Period (PER) and other regulatory proteins, e.g., in both subunits of the mammalian dioxin receptor AHR (aryl hydrocarbon receptor) and ARNT (aryl hydrocarbon receptor nuclear translocator) (Huang et al., 1993; Figure 2.2B). Huang et al. (1993) found that the PER PAS domains function as protein dimerization motifs in eritro not only with PER PAS itself but also with the PAS motifs of SIM (the Drosofihila single-minded gene product) and ARNT. A recent finding is that PER can interact with TIM (the product of Dosophila timekss gene), a circadian clock element lacking a PAS domain (Zeng et al., 1996). The presence of the PAS dimerization motifs in both Neurospora blue light regulatory proteins led to the hypothesis that WC-1 and WC-2 interact in order to carry out their function in blue light signaling. In fact, we were able to show in protein-protein interaction experiments in vitro that not only homodimerization but also heterodimerization occurred between WC-1 and WC-2 and that dimerization was dependent on the presence of WC-1 and WC-2 PAS domains. Moreover, an association of the WC-1 PAS domain with other PAS


47

proteins, such as ARNT and AHR, in vitro supported our idea that WC-1 and WC-2 are also members of the PAS protein family (Ballario et ul.,

1998).

In WC-1, in addition to a canonical PAS domain with two repeats, a second region weakly reminiscent of a PAS domain (amino acids 399-504) has been identified, The same region shows remarkable homology (35% identity) with Bat (Gropp and Betlach, 1994), a transcription factor required for the oxygen-mediated expression of the Hubbacterium halobium bacteriopsin and with NIFL (Blanco et al., 1993) (29% identity), a protein that regulates nif gene transcription in response to environmental oxygen concentrations in Kkbsielh pneumoniue and Azotobacter vinehndii. All these proteins seem to be involved in oxygen binding and sensing. In particular, NIFL, a flavoprotein that uses FAD (flavin adenine dinucleotide) as a prosthetic group, does not sense molecular oxygen directly but is responsive to the oxidation state of the chromophore, thus representing an example of redox-sensitive protein (Hill et ul., 1996). It is useful to report in this context that experiments with a strong reducing agent, such as dithionite, have demonstrated that the oxygen is essential for light induction in fungi (Harding and Shropshire, 1980; Arpaia and Macino, unpublished results). Other interesting similarities were identified in all the PAS protein sections of WC-1 and WC-2 (Figure 2.3). The WC-2 PAS domain showed a similarity of 48% over 62 amino acids with the photoactive yellow protein PYP (Figure 2.3A), and a more limited similarity has also been identified with WC1 PAS domains. PYP is a small protein consisting of 125 amino acids that seem to encode a blue light photoreceptor involved in negative phototaxis of the halophilic purple phototrophic bacterium Ectothiorhodospira (Baca et al., 1984). In addition, a more limited similarity was identified between the PAS domain of the WC polypeptides and phytochromes, the red light photoreceptors of plants (Figure 2.3B). The WC-2 protein revealed a similarity of 38% over 56 amino acids with Arubidopsis PHYC, whereas a similarity of 43% over 46 amino acids was found between WC-1 and Arubidopis PHYE. Although these similarities were comparably low, it was interesting to find in both the regions of similarity overlap cases of a conserved direct repeat domain of phytochromes (Figure 2.3B). Phytochrome is a homodimeric protein with each subunit having two major functional domains. The amino-terminal domain is involved in light perception and contains the chromophore-binding site, whereas the carboxyterminal domain is involved in signal transduction and in dimerization of the two subunits (Quail et al., 1995). The conserved direct repeats are located in the phytochrome carboxy-terminal domain and were suggested to mediate at least in part the subunit contact of the phytochrome dimer (Jones and Edgerton, 1994). However, more recent data implicated these phytochrome repeats and adjacent protein regions as being involved in the activation of downstream

P

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I I I I I I1 I1 VCDVTLNDCPIIYVSDNFQN L T G Y S R H E I V G R N C R F J , O A P D G NV E A G T U E FVE NNAVY TL K KT I4 I11 1111111

(396)

V

Figure 2.3. Alignment of the WC-1 and WC-2 putative PAS domains with other polypeptides from the SwissProt protein sequence data base (A) Comparison of the WC-2 PAS domain with the amino acid sequence of the photoactive yellow protein (PYP). Similar residues are boxed. A hyphen indicates a gap introduced to maximize alignment. (B) Comparison of the WC-2 and WC-1 putative PAS domains with the Arabldopsis phytochrome C and E, respectively. The phytochrome repeat I and repeat I1 consensus regions according to Jones and Edgerton (1994) are printed in bold. The regions of WC-1 and WC-2 that show similarities are underlined. The number of the first amino acid of each sequence is given in parenthesis on the left.

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

49

signaling components. Wagner and Quail ( 1995) described four PHYB mutants that were isolated in a screening for regulatory mutants. Although these mutants were normal with respect to photoperception and dimerization, a loss of biological activity was observed. All four mutations fell within a small carboxyterminal region, which overlaps one of the direct repeats. Furthermore, deletion of the first of the two repeats led to the reduction of maximal biological activity of PHYB without a decrease in the efficiency of light perception (Wagner et al., 1996). In addition, most of the mutations identified in PHYA and PHYB were clustered in this direct repeat protein region (Quail et ul., 1995). As outlined by Lagarias et al. (1995), the direct phytochrome repeats also show similarities to other regulatory proteins, such as the bacterial two-component protein kinases, the nitrogen-fixation regulatory protein NIFL, and the opsin-activator protein Bat. A general consequence of the observations reported earlier is that the PAS domains of WC-1 and WC-2 seem to be widespread in animals, plants, fungi, and bacteria. They have been identified in many regulatory proteins with functions in signal transduction and the reception of different stimuli, such as light, chemical compounds, and oxygen. This domain may therefore serve as a general protein interface for the interaction between receptors and signal transduction components.

C. How do WC-1 and WC-2 function in Neurospora blue light signaling? As outlined earlier, both WC proteins are putative transcription factors that control all blue-light-regulated phenomena. The indistinguishable phenotypes of wc-1 and wc-2 mutants and the similarity of their functional domains seem to suggest an identical role for the white collar genes in the biology of Neurosporu. Nevertheless, WC-1 and WC-2 seem to play different roles in at least some blue-light-regulated phenomena. Crosthwaite et al. (1997) have recently proposed a differentiated role for WC-1 and WC-2 in sustaining circadian rhythm in Neurospora. WC-1 appears to be essential for the resetting of the circadian cycle by light and for the induction of frequency (frq) transcription upon a pulse of blue light. In contrast, WC-2 is not required for light-induced transcription of frq but is proposed to be a new component of the circadian clock, acting as a positive transcription factor necessary for maintaining circadian cycling (Crosthwaite et al., 1997). Although the exact role of WC-1 and WC-2 in the clock is still unknown, it is clear that both proteins must be present for sustained rhythmicity in the dark. Furthermore, the previously observed light inducibility of wc-2 (Linden and Macino, 1997b) and ccg-l (Arpaia et al., 1995a) in wc-2 genetic backgrounds again suggests distinct roles for the two WC proteins.

50

H. Linden ef a/.

WC-1 and WC-2 represent the first two transcription factors characterized in any organism that seem to be dedicated to light-activated gene regulation. Furthermore, in v i m experiments indicate that WC-1 and WC-2 are capable of forming a complex via their putative PAS dimerization domains present in both proteins. Naturally, numerous questions arise regarding their mode of action in vivo: Do WC-1 and WC-2 form hetero- and homodimeric complexes also in vivo? Are other proteins implicated in the formation of heterodimers (i.e., with FRQ)? What is the transcriptionally active complex and how does light influence these complexes? What are the other signal transduction components, and, most important, what is the nature of the Neurospora blue light photoreceptor? Are the white collar proteins themselves involved in light perception and transduction? A conceivable model would be a light-induced heterodimerization of WC-1 and WC-2 that results in binding and transcriptional activation of light-regulated genes. This would be analogous to the basic helix-loop-helix PAS proteins AHR and ARNT (Figure 2.2). In the absence of the ligand, the AH receptor was found in a complex with the heat shock protein hsp90 in the cytoplasm (Antonsson et al., 1995). Upon addition of the ligand, the complex dissolves and the AH receptor heterodimerizes with its partner ARNT (Burbach et al., 1992). This AH receptor-ARNT complex is then transported into the nucleus, where it leads to transcriptional activation. Analogous to the AHR-ARNT model, WC-1 and WC-2 may not only function in transcriptional activation but also participate in blue light signal transduction. A role of WC-1 and WC-2 beyond transcriptional regulation would, for example, explain the fact that WC-1 and WC-2 also seem to be necessary for blue light processes that are independent of transcriptional gene regulation, such as protein phosphorylation and changes in electrophysiological parameters of the cell membrane (Levina et al., 1988; Oda and Hasunume, 1994). Furthermore, it would explain the finding that, in spite of extensive mutant searches, only wc-l and wc-2 mutants were isolated as reliable candidates for blue light signal transduction proteins in Neurospora.

VI. CONCLUDING REMARKS The finding of a photosensory adaptation mechanism in Neurospora, together with the identification of the first putative component of the adaptation machinery, uncovered an unanticipated complexity of Neurospora blue light signal transduction. The increasing number of mutants that seem to interfere with blue light signaling and light-regulated transcription also supports this idea. In contrast, the presence of only two regulatory mutants that completely inhibit blue light signal transduction and that seem to be ubiquitously involved in all light responses indicates a very short signaling cascade consisting of only a few


51

components, as proposed by Ballario et al. (1996). Furthermore, the similarities of WC-1 and WC-2 to proteins involved in light perception and signal transduction, as well as the involvement of the two polypeptides in the Neurospora circadian clock, may be corroborative of a complex regulatory function of WC1 and WC-2 beyond transcriptional control. A thorough investigation of the function of the WC proteins in vivo as well as the identification of the missing components of blue light regulation will be necessary before we begin to understand the way Neurospora sees the light.

Acknowledgments G.M. and P.B. thank people in their labs for stimulating discussion. This work was supported in part by grants from Istituto Pasteur Fondazione Cenci Bolognetti and from Minister0 delle Risorse Agricole, Alimentari e Forestali, Piano Nazionale Biotecnologie Vegetali.

References Antonsson, C., Whitelaw, M. L., McGuire, J., Gustafsson, J. A,, and Poellinger, L. (1995). Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains. Mol. Cell. Bid. 15, 756-765. Aronson, B. D., Johnson, K. A., Loros, J. J., and Dunlap, J. C. (1994). Negative feedback defining a circadian clock: Autoregulation of the clock gene frequency. Science 263, 1578-1584. Arpaia, G., Loros, J. J., Dunlap, 1. C., Morelli, G., and Macino, G. (1993). The interplay of light and circadian clock. Independent dual regulation of clock-controlled gene ccg-2 (eas) . Plant Physiol. 102, 1299-1305. Arpaia, G., Loros, J. J., Dunlap, 1. C., Morelli, G., and Macino, G. (1995a). Light induction of the clock-controlled gene ccg- 1 is not transduced through the circadian clock in Neurosporu crassa. Mol. Gen. Genet. 247, 157-163. Arpaia, C., Carattoli, A., and Macino, G. (1995b). Light and development regulate the expression of the albino-3 gene in Neurosporu crassu. Dev. Biol. 170,626-635. Arpaia, G., Cerri, F., Baima, S., and Macino, G. (1999). Protein kinase C may be a novel component of the blue light transduction pathway in Neurospu nassu. Mol. Gen. Genet. in press. Baca, M., Borgstahl, G. E. O., Boissinot, M., Burke, P. M., Williams, D. R., Slater, K. A., and Getzoff, E. D. (1994). Complete chemical structure of photoactive yellow protein: Novel thioester-linked 4-hydroxycinnamyl chromophore and photocycle chemistry. Biochemistry 33, 14,36914,377. Baima, S., Macino, G., and Morelli, G. (1991). Photoregulation of the uhno-3 gene in Neurospura crassa. J. Photochem. Photobiol. 11, 107-115. Ballario, P., Vittorioso, P., Magrelli, A., Talora, C., Cabibbo, A., and Macino, G. (1996). White collar-1, a central regulator .of blue light responses in Neurospora, is a zinc finger protein. EMBO J. 15, 1650-1657. Ballario, P., and Macino G. (1997). White collar proteins. PASsing the light signal in Neurospora crassa. Trends Microbiol. 458-462. Ballario, P., Talora, C., Galli, D., Linden, H., and Macino, G. (1998). Roles in dimerization and blue light photoresponse of the PAS and LOV domains of Neurospoom crassu White Collar proteins. Mol. Microbiology 29, 719-731.

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~~

Bell-Pedersen, D., Dunlap, J. C., and Loros, J. J. (1992). The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer. Genes Dew. 6, 2382-2394. Bell-Pedersen, D., Dunlap, J. C., and Loros, J. J. (1996a). Distinct cis-acting elements mediate clock, light, and developmenral regulation of the Neurospora crassa eas (ccg-2) gene. Mol. Cell. Biol. 16, 513-521. Bell-Pedersen, D., Shinohara, M. L., Loros, J. J., and Dunlap, J. C. (199613). Circadian clockcontrolled genes isolated from Neurospora crassa are late-night to early morning-specific. Proc. Natl. Acad. Sci. USA 93, 13,096-13,101. Blanco, G., Drummond, M., Woodley, P., and Kennedy, C. (1993). Sequence and molecular analysis of the nifi gene of Azotobacter winelandii. Mol. Microbiol. 9, 869-879. Bowler, C., Yamagata, H., Neuhaus, G., and Chua, N. H. (1994). Phytochrome signal transduction pathways are regulated by reciprocal control mechanisms. Genes Dew. 8, 2188-2202. Burbach, K. M., Poland, A., and Bradfield, C. A. (1992). Cloning of the AH-receptor cDNA reveals a distinctive ligand-activated transcription factor. Proc. Nutl. Acad. Sci. USA 89, 81858189. Carattoli, A., Cogoni, C., Morelli, G., and Macino, G. (1994). Molecular characterization of upstream regulatory sequences controlling the photoinduced expression of the albino-3 gene of Neurospora crassa. Mol. Microbiol. 13, 787-795. Carattoli, A., Kato, E., Rodriguez-Franco, M., Stuart, W. D., and Macino, G. (1995). A chimeric light-regulated amino acid transport system allows the isolation of blue light regulator (blr) mutants of Neurospora crassa. Proc. Natl. Acad. Sci. USA 92, 6612-6616. Corrocchano, L. M., Lauter, F. R., Ebbole, D. J., and Yanofsky, C. (1995). Light and developmental regulation of the gene con-J0 of Neurospora crassa. Deo. Biol. 167, 190-200. Crosthwaite, S. K., Loros, J. J., and Dunlap, J. C. (1995). Light-induced resetting of a circadian clock is mediated by a rapid increase infrequency transcript. Cell 81, 1003-1012. Crosthwaite, S. K., Dunlap, J. C., and Loros, J. J. (1997). Neurospora wc-I and wc-2: Transcription, photoresponses, and the origin of the circadian rhythmicity. Science 276, 763-769. DeFabo, E. C., Harding, R. W., and Shropshire, W. (1976). Action spectrum between 260 and 800 nanometers for the photoinduction of carotenoid biosynthesis in Neurospora crassa. Plant Physiol. 57,440-445. Degli-Innocenti, F., Pohl, U., and Russo, V. E. A. (1983). Photoinduction of protoperithecia in Neurospora crassa by blue light. Photochem. Photobiol. 37, 49-51. Degli-lnnocenti, F., Chambers, J. A. A., and Russo, V. E. A. (1984a). Conidia induce the formation of protoperithecia in Neurospora crassa: Further characterization of white collar mutants. 1. Bacterial. 159,808-810. Degli-Innocenti, F., and Russo, V. E. A. (1984b). Isolation of new white collar mutants of Neurospora crassa and studies on their behavior in the blue light-induced formation of protoperithecia. I. Bacteriol. 159,757-761. Deng, X. W. (1994). Fresh view of light signal transduction in plants. Cell 76, 423-426. Gropp, F., and Betlach, M. C. (1994) The bat gene of Halobacterium halobium encodes a transacting oxygen inducibility factor Proc. Natl. Acad. Sci. USA 91, 5475-5479. Harding, R. W., and Shropshire, W. (1980). Photocontrol of carotenoid biosynthesis. Annu. Rew. Plant Physiol. 31, 217-238. Harding, R. W., and Turner, R. V. (1981). Photoregulation of the carotenoid biosynthetic pathway in albino and white collar mutants of Neurospora CTCISSLI. Plant Physiol. 68, 745-749. Harding, R. W., and Melles, S. (1983). Genetic analysis of the phototrophism of Neurospora crassa perithecial beaks using white collar and albino mutants. Plant Physiol. 72, 996-1000. Hill, S., Austin, S., Eydmann, T., Jones, T., and Dixon, R. (1996). Arotobater vinelandii NIFL is a flavoprotein that modulates transcriptional activation of intron nitrogen-fixation genes via a redox sensitive switch Proc. Natl. Acad. Sci. USA 93, 2143-2148.

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

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Huang, Z. J., Edery, I., and Rosbach, M. (1993). PAS is a dimerization domain common to Drosophila period and several transcription factors. Nature 364, 259-262. Jones, A. M., and Edgerton, M. D. (1994). The anatomy of phytochrome, a unique photoreceptor in plants. Sem. Cell Bid. 5 , 295-302. Kaldenhoff, R., and Russo, V. E. A. (1993). Promoter analysis of the bli-7/eas gene. Cum. Genet. 24,394-399. Klemm, E., and Ninnemann, H. (1978). Correlation between absorbance changes and a physiological response induced by blue light in Neurospora crassa. Photochem. Photobiol. 28, 227-230. Lagarias, D. M. Wu, S. H., and Lagarias, J. C. (1995). Atypical phytochrome gene structure in the green algae Mesotaenium caldariurum. Plant Mol. Biol. 29, 1127-1142. Lauter, F. R. (1996). Molecular genetice of fungal photohiology. 1. Genet. 75, 375-386. Lauter, F. R., and Russo, V. E. A. (1991). Blue light induction of conidiation specific genes in Neurospora crassa. Ntackic Acids Res. 19, 6883-6886. Lauter, F. R., Russo, V. E. A,, and Yanofsky, C. (1992). Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora. Genes Dev. 6, 2373-2381. Lauter, F. R., and Yanofsky, C. (1993). Day/night and circadian rhythm control of con gene expression in Neurospora. Proc. Natl. Acad. Sci. USA 90, 8249-8253. Lauter, F. R., Yamashiro, C. T., and Yanofsky, C. (1997). Light stimulation of conidiation in Neurospora crassa: Studies with wild type and mutants wc-1, wc-2, and acon-2. 1. Photochem. Photobiol. B. 37, 203-211. Levina, N. N., Belozerskaya, T. A,, Kriwky, M. S., and Potapova, T. V. (1988). Photoelectrical responses of Neurospura crassa mutant white collar 1. Exp. Mycol. 12, 77-79. Li, C., and Schmidhauser, T. J. (1995). Developmental and photoregulation of al-I and al-2, structural genes for two enzymes essential for carotenoid hiosynthesis in Neurospora. Deu. Biol. 169,90-95. Li, D., and Kolattukudy, P. E. (1995). Cloning and expression of cDNA encoding a protein that binds a palindromic promoter element essential for induction of fungal curinase by plant cutin. 1. Bid. Chem. 270, 11,753-11,756. Linden, H., Ballario, P., and Macino, G. (1997a). Blue light regulation in Neurospora crassa. Fungal Genet. Biol. (in press). Linden, H., and Macino, G. (199713). White collar 2, a partner in blue light signal transduction, controlling expression of light-regulated genes in Neurospma nassa. EMBO 1. 16, 98- 109. . mutants of Neurospora Linden, H., Rodriguez-Franco, M., and Macino, G. ( 1 9 9 7 ~ ) Regulatory massa in blue light perception. Mol. Gen. Genet. 254, 111-118. Loros, J. (1995). The molecular basis of the Neurospora clock. Neurosciences 7, 3-13. Loros, J. J., Denome, S. A., and Dunlap, J. C. (1989). Molecular cloning of genes under control of the circadian clock in Neurospora. Science 243, 385-388. Macino, G., Baima, S., Carattoli, A., Morelli, G., and Valle, E. M. (1993). Blue light-regulated expression of geranylgeranyl pyrophosphate synthetase (albino-3) gene in Neurospora crassa. In “Molecular Biology and Its Application to Medical Mycology” (B. Meresca, G. S.Kohayashi, and H. Yamaguchi, eds.), pp. 117-124. Nato Asi Series. Vol. H 69, Springer-Verlag, Berlin Heidelberg. Neuhaus, G., Bowler, C., Kern, R., and Chua, N. H. (1993). Calcium/calmodulin-dependent and independent phytochrome signal transduction pathways. Cell 73, 937-952. Oda, K., and Hasunume, K. (1994). Light signals are transduced to the phosphorylation of 15 kDa proteins in Neurospora crassa. FEBS Lett. 345, 162-166. Orbach, M. J., Vollrath, D., Davis, R. W., and Yanofsky, C. (1988). An electrophoretic karyotype of Neurospora crassa. Mol. Cell. Biol. 8, 1469-1473. Perkins, D. D., Radford, A., Newmeyer, D., and Bjorkmann, M. (1982) Chromosomal loci of Neurospora crassa. Microbiol. Rev. 46, 426-570.

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Quail, P. H., Boylan, M. T., Parks, B. M., Short, T. W., Xu, Y.,and Wagner, D. (1995). Phytochromes: Photosensory perception and signal transduction. Science 268,675-680. Rau, W. (1967). Untersuchungen ueber die lichtabhkgige Carotinoidsynthese.Plantu 72, 14-28. Sargent, M. L., and Briggs, W. R. (1967). The effects of light on a circadian rhythm of conidiaton in Neurospora Plant Physiol. 42,1504-1510. Schrott, E. L. (1980). Fluence response relationship of carotenogenesis in Neurospora crassa. Plantu 150,174-179. Schrott, E. L. (1981). The biphasic fluence response of carotenogenesis in Neurospora crassa: Temporary insensitivity of the photoreceptor system. Pkmtu 151, 371-374. Sokolovsky, V. Y., Lauter, F. R., Mueller-Roeber, B., Ricci, M., Schmidhauser, T. J., and Russo, V. E. A. (1992). Nitrogen regulation of blue light-inducible genes in Neurospora crassa. J. Gen. Mimobiol. 138, 2045-2049. Sommer, T., Chambers, J. A. A., Eberle, J., Lauter, F. R., and Russo, V. E. A. (1989). Fast lightregulated genes of Neurospora cram. Nuckic Acids Res. 14,5713-5723. Springer, M. L. (1993). Genetic control of fungal differentiation: The three sporulation pathways of Neurospora craw. BioEssays 15, 365-374. Stuart, W. D., Koo, K., and Vollmer, S. 1. (1988). Cloning of ma, an amino acid transport gene of Neurospora crassa. Genome 30, 198-203. Wagner, D., and Quail, P. H. (1995). Mutational analysis of phytochrome B identifies a small COOH-terminal-domain region critical for regulatory activity. Proc. Natl. Acad Sci. USA 92, 8596-8600. Wagner, D., Koloszvari, M., and Quail, P. H. (1996). Two small spatially distinct regions of phytochrome B are required for efficient signaling rates. Plant Cell 8, 859-871. Zeng, H., Qian, Z., Myers, M. P., and Rosbach, M. (1996). A light-entrainment mechanism for the Drosophila circadian clock. Nature 380, 129- 135.

Retardation ~

Giovanni Neri* Istituto di Genetica Medica Facolth di Medicina e Chirurgia “A. Gemelli” Universith Cattolica del Sacro Cuore 00168 Roma, Italy

Pietro Chiurani Centro Ricerche per la Disabilith Mentale e Motoria Associazione Anni Verdi 00168 Roma, Italy

I. Introduction

11. Syndromal XLMR

A. Fragile X Syndrome B. Simpson-Golabi- Behmel Syndrome C. ATR-X Syndrome D. Opitz/G-BBB Syndrome E. The Aarskog-Scott Syndrome F. The Coffin-Lowry Syndrome 111. Nonsyndromal XLMR (MRX) A. FMR2 B. GDll C. OPHNZ D. PAK3 IV. Conclusion References *To whom correspondence should be addressed. E-mail: [email protected]: +39063054449. Fax: +39-063050031. Advances in Genetics, VoI. 41

Copyright 0 1999 by Academic Press All rights of reproduction in any form reserved. 0065-2660/99$30.00

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G. Neri and P. Chiuraui

I. INTRODUCTION It has been known for a long time that there is an excess of males among the mentally retarded, especially if one considers mental retardation (MR) of mildto-moderate degree. In his famous “Colchester Survey,” conducted in an institution for the mentally retarded, Penrose (1938) estimated this excess to be as high as 25%, attributing it mainly to ascertainment bias. In preparing his doctoral thesis approximately 30 years later, Lehrke analyzed a sample of mentally retarded individuals that included a number of familial cases with X-linked inheritance. This analysis led him to formulate the hypothesis that the excess of MR among males is due to the existence of a number of conditions caused by X-linked mutant genes, and therefore MR is bound to be more, if not exclusively, expressed in hemizygous males. This concept of X-linked MR (XLMR) was formally defined by Lehrke in a later publication (Lehrke, 1974) and is now largely accepted. However, the idea that extrinsic factors may also contribute to the excess of MR among males, should not be totally dismissed. Although accurate epidemiologic data prospectively collected from sufficiently large populations are virtually nonexistent, one can still calculate, based on available data, that XLMR represents approximately 5% of all MR, corresponding to a prevalence in the general population of about 1.8 per 1000 (Herbst and Miller, 1980). In 1991, Neri et al. published the first of a series of XLMR gene updates, thus establishing a catalog of the corresponding clinical conditions, which contained, in the first edition, 39 entries. The most recent edition, published in 1999, contains 179 entries (Lubs et al., 1999), the large increase being due in part to the discovery of new conditions in the intervening years and in part to the adoption of more inclusive criteria. All listed disorders are subdivided into two major groups, one for the syndromal forms of XLMR and the other for the nonsyndromal ones. The former group is composed of those conditions that are clinically recognizable because of a specific pattern of physical, neurological, or metabolic abnormalities. The latter includes all those disorders whose only consistent clinical manifestation is MR. These disorders can be distinguished from each other only on the basis of the different regional assignment of the corresponding locus on the X chromosome. Table 3.1 provides a summary of the current status of XLMR genes-disorders and also indicates the number of genes cloned and/or regionally mapped.

57

3. X-Linked Mental Retardation

Table 3.1. Counts of XLMR Conditions

Syndromal XLMR Malformation syndromes Metabolic disorders Neuromuscular diseases Dominant conditions (lethal in males) Total Nonsyndromal XLMR (MRX) Total Entries

Total count

Mapped

Cloned

68 13 32 7 120 59 179

30 2 15 5 52 55 107

6 10 6 1 23

4 27

Owing to the practical impossibility of giving a detailed description of all known

XLMR syndromes, only some will be reported here based on their relatively higher frequency and better characterization. Those that are also fairly common and well known, such as Duchenne muscular dystrophy and Hunter syndrome, are extensively treated in specialized books (e.g., Scriver et al., 1995). Others that are very rare and sometimes reported in a single family are summarized in the recent review by Lubs et al. (1999). Cloned and mapped genes are graphically displayed in Figure 3.1 which makes it apparent that many regional assignments are quite extended and largely overlapping. However, given the phenotypic differences among the various clinical conditions, it can be safely assumed that even overlapping loci correspond to distinct conditions until proven otherwise. The fragile X syndrome will be treated more extensively than other syndromes because of its importance as an archetypal model of XLMR.

A. Fragile X syndrome The fragile X syndrome is the prototype of a growing list of disorders known to be caused by the so-called dynamic mutations resulting from the instability and expansion of triplet repeats (Djian, 1998). The mutant gene, FMRl , is located in Xq27.3 and harbors a repeated CGG triplet in its 5' untranslated region (Verkerk et al., 1991). The syndrome derives its name from the fragile site FEIAXA, which is colocalized with the CGG repeat in Xq27.3 and was first observed by Lubs (1969) in four mentally retarded males and three obligate carrier females of the same family. The expression of the fragile X site is best induced when cells are cultured with low folate concentration and when either

58

6. Neri and P. Chiurani

-H

pyruvate DH deficiency

cop-Lo.1.y

Oprtzc/BBB

k-:j,IXZ

+

Fned

22.1

MAO-A deficiency+ N o d e disease --t therbd NRXS3

AarsLog-Scott

syndrome -11.21

::”

ATR-X Menkes disease PGK Mciency

21.1

1

MobTranebjaerg PeIizaeus-Menbaeher

Lowe syndrome SGBsyndmme Leseh-Nyhan

Hunter

Hamel fJL IGHD) CowChoek-FLshbeek

25

27 FRAxA--t

wD= +

WAS (LlCAM)

Wilson/MRXSL

U

Walsman-Lpxova Bdlons dystrophy Incontin, pigmenti Dyskeretosiacow. PPM-X, BPNH Schwa&,

Pa

Amfield, L u h

Figure 3.1. X chromosome ideogram with the known localizations of genes responsible for syn-

dromal XLMR. The bars on the right indicate the locus assignment for those putative genes that have been regionally mapped. The arrows on the left indicate the position of the cloned genes.


59

fluorodeoxyuridine or an excess of thymidine is added (Jacky et al., 1991).The fragile site is usually expressed in 30 to 50% of the cells examined. However, a lower expression is not unusual, and, in fact, it can be sometimes as low as 4 or 5%, especially in carrier females. It appears as a decondensed chromatin gap between Xq28 and the rest of the X chromosome. I t has been shown that DNA replication is delayed well after the S phase in the region containing the expanded CGG repeat and could be incomplete at mitosis, thus determining the chromosomal “fragility” (Hansen et al., 1993). The first large family with mental retardation and macroorchidism in males transmitted in an X-linked fashion and later confirmed to have fragile X syndrome was described over 50 years ago by Martin and Bell (1943), and their names have been often used as an eponym for the syndrome.

1. Clinical phenotype The clinical phenotype of the fragile X syndrome can be quite variable. In typical cases there is tall stature and relative macrocephaly, a long and narrow face with prominent forehead and mandible, and midface hypoplasia with hypoteloric, sunken eyes. The ears are large and the palate is highly arched. Testes are generally large, with volumes up to 100 ml. Generalized muscular hypotonia is a virtually constant finding and is usually accompanied by joint laxity. These latter findings might be caused by an underlying connective tissue dysplasia, which could also be responsible for the frequently observed mitral valve prolapse. MR is usually of moderate degree and behavior tends to be introverted, with poor eye contact and avoidance of new and unexpected situations. In extreme cases this behavior can be described as autistic. The phenotype is usually more subtle in newborns and children, in whom facial traits tend to be less pronounced and macroorchidism is less obvious. Increased birthweight and generalized congenital hypotonia may be the only significant findings. Hyperactivity and attention deficit disorder have been described in children. Seizures may also occur during infancy and a characteristic EEG pattern of trains of medium-high voltage spikes discharging from the temporal regions during sleep has been reported (Musumeci et al., 1991). Epileptic seizures, if present, generally disappear before puberty and tend to respond well to treatment. Brain MRI shows volume conservation of brain tissue with a diminished white-to-gray matter ratio and a relatively enlarged caudate nucleus and hippocampus, while cerebrospinal fluid is increased, especially in the lateral ventricles (Reiss et at., 1995). The fourth ventricle is also enlarged in correspondence to a smaller posterior cerebellar vermis (Reiss et al., 1991). Among nontypical cases of the syndrome a subgroup that, because of obesity and short stature bore some resemblance to the Prader- Willi syndrome,

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G. Nerl and P. Chiuraal

was identified (de Vries et al., 1993). Although this is nothing more than a superficial similarity, it is a good reminder of the pitfalls of a purely clinical diagnosis and justifies the view that every mentally retarded person should be tested for fragile X syndrome in the absence of another reasonable diagnosis. The affected females, who represent about one-third of all females carrying a full mutation, usually do not demonstrate a characteristic physical phenotype. They are mildly retarded or may present only a learning disability and have a shy and introverted personality.

2. Diagnosis and prevalence Molecular diagnosis of the CGG amplification, which constitutes greater than 95% of the fragile X mutations, has been available since the cloning of the FMRl gene in 1991 and fundamentally relies on Southern blotting and hybridization of probes specific for the promoter region, whereas PCR is employed to measure the length of the CGG repeat tract in the normal and premutation range. Screening for full and premutations should thus combine both PCR and Southern blotting, possibly using a pooling-reanalysis strategy as in Rousseau et al. (1995). Cytogenetic testing can still be considered in looking for full mutations in males, although positive cases should be checked with DNA analysis and may lead to the identification of a few FRAXE individuals (Knight et. al., 1993). A rapid method based on antibody detection of the FMRl protein in cells of blood smears has been described and validated by Willemsen et al. (1995) and is useful in screening affected males. In our opinion, prenatal diagnosis still depends on the availability of sufficient DNA to perform a Southern blotting after double digestion that includes a methylation-sensitive enzyme (usually EagI or BssHII). The sex of the fetus can be determined with a standard karyotype or by Y-specific PCR analysis. Both false positives, due to suboptimal amplification, and false negatives, due to the possible presence of reverted alleles in the wild-type range, can occur when PCR alone is performed on a sample from a male fetus. Furthermore, only direct DNA analysis after digestion with a methylation-sensitive enzyme can demonstrate the actual methylation status of the FMRl CpG island, especially in the presence of a full mutation. Given the occurrence of unmethylated full mutations in unaffected transmitting males (Rousseau et al., 1994; Smeets et al., 1995) and the evidence that in extraembryonic tissues, such as chorionic villi, a full mutation may remain largely undermethylated until 1011 weeks of gestation (Sutcliffe et al., 1992; Luo et al., 1993; Castellvi-Be1 et al., 1995), CVS might not display the hypermethylation already present in the embryonic tissues and may need confirmation with amniocentesis. Detection of the FMRl protein is also possible in amniocytes (Willemsen et al., 1997) and chorionic villi (Willemsen et al., 1996a), but given the semiquantitative nature


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of the assay, the role of the protein test can only be viewed as confirmatory of the DNA analysis. Although the fragile X syndrome is still believed to account for the majority of XLMR cases, it seems that its prevalence is not as high as initially estimated. A recent reevaluation of the the same population that yielded the much-quoted figure of 1:1300 males (Webb et al., 1986) led to the conclusion that 1:4000 males is probably a more realistic figure (Turner et al., 1996). Most likely, this apparent discrepancy can be explained by the use in the restudy of the molecular test, which is more accurate and specific than the cytogenetic test previously available. No general population screening has been done on unselected populations, such as consecutive newborns. Surveys have concentrated on children with MR or learning disabilities or institutionalized patients, where the prevalence of the fragile X syndrome is approximately 5% (van den Ouweland et al., 1994). Some data on the prevalence of healthy female carriers have been provided by a French-Canadian study that screened 10,624 unselected women and found 41 (1:259) carriers of FMRl premutated alleles with 55- 101 CGG repeats (Rousseau et al., 1995). Additional similar studies are needed to establish whether this unexpectedly high prevalence of premutation carriers is unique to the specific population studied or applies to other populations as well, as seems more likely (Sherman et al., 1995). Evidence that expansion to full mutation upon transmission from a premutated mother is more likely to occur in male than in female fetuses has been provided by Loesch and co-workers (1995) and may explain a relative lack of premutated males in the general population (Rousseau et al., 1996). Large population studies on unselected series of newborns would be useful to settle the question of the true prevalence of affected (fully mutated) and normal transmitting (premutated) males and of full-mutation and premutation carrier females. Although very few fragile X cases have been reported without amplification of the CGG repeat and with either point mutations or deletions in other parts of the FMRl gene (Gronskov et al., 1998)) it is worth considering that the prevalence of these “nondynamic” mutations might be underestimated because most molecular diagnostic strategies test only the status of the CGG repeat and its flanking sequences.

3. Genetics a. Gene structure and protein isoforms The FMRl gene structure has been determined in detail. The 17 exons of the gene are embedded in 38 kb of genomic sequence in Xq27.3 (Eichler et al., 1993). The polymorphic CGG repeat is located in the 5’ untranslated region of exon 1 and is included in all FMRl transcripts (Verkerk et al., 1993). FMRi

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was shown to be ubiquitously transcribed during murine and human embryogenesis (Hinds et al., 1993) with the highest level of expression in differentiated neurons of the hippocampus and basal ganglia (Abitbol et al., 1993). In adult mice, FMRl is expressed only in neurons and in spermatogonia. FMRl protein has been detected in synapses, dendritic spines, and the soma of rat neurons but not in the nucleus or axon, and active FMRl protein production has been demonstrated near synapses in response to neurotransmitter activation (Weiler et al., 1997). FMRl action is probably required for normal maturation of synaptic connections, which appear immature and reduced in number in fragile X brains (Hinton et al., 1991). The 4.4-kb full-length mRNA can code for a protein with a maximum length of 632 amino acids and an apparent molecular weight of 70-80 kDa (Verheij et al., 1993; Devys et al., 1993), and although 20 different transcripts might be produced by alternative splicing (Verkerk et al., 1993; Ashley e t al., 1993), only 4 to 5 of them and their corresponding protein products are actually detected in various tissues. Isoform 7 (IS07), which lacks only the 21 amino acids of exon 12, makes up almost all the FMRl protein with an approximate molecular weight of 80 kDa, (Sittler et al. 1996). Two KH domains (KH1 and KH2) and one RGG box, common to several RNA-binding proteins, have been identified in exons 8, 10, and 15, respectively (Siomi et al. 1993). It was shown that FMRl could bind synthetic RNAs in vimo, and the importance of KH domains was underscored by the description of a severely retarded fragile X patient with a point mutation changing a highly conserved isoleucine of KH2 into asparagine (Ile304Asn) (De Boulle et al., 1993), which impaired the RNAbinding activity of FMRl (Siomi et al., 1994). It is still not clear whether FMRl binds mRNAs and participates in mRNP (ribonucleoprotein) particle formation (Corbin et al., 1997; Feng et al., 1997) or if it binds to ribosomal RNA 1996; Siomi et al. 1996). (Tamanini et d., The major isoform (IS07) is localized in the cytoplasm (Verheij et al., 1993; Devys et d.,1993), whereas the minor isoforms, lacking exon 14 and with a different C-terminus (IS06 or IS012), are confined to the nucleus (Sittler et al., 1996). The N-terminus of the protein (exons 1-5) seems to contain a putative nuclear localization signal (NLS) (Sittler et al., 1996), whereas exon 14 contains sequences capable of acting like the nuclear export signal (NES) of the HIV-1 Rev regulatory protein (Fridell et al., 1996). A shuttling between cytoplasm and nucleus was therefore envisaged and, in fact, FMRl has been detected by electron microscopy free and associated with ribosomes in the cytoplasm, but also in the nucleoli (Willemsen et al., 1996b). The serendipitous discovery of a fragile-X-relatedprotein, FXRl (Siomi et al., 1995), led to the search for other FXR proteins possibly interacting with FMRl or complementing its functions. FXR2 was identified by using the yeast two-hybrid system (Zhang et d . , 1995) and is also able to bind FXR1. The


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FXRl and FXRZ genes have been mapped to 3q28 and 17~13.1,respectively, and an intronless form of FXRl has been localized to 12q13 (Coy e t al., 1991). Both FXRs and FMRl are highly homologous in the N-terminal portion, including the KH domains, the RGG box, and the first half of exon 14 (the ribosome-binding site coinciding with the NES) (Zhang e t al., 1995), and their genes possibly evolved from a common ancestor. All three proteins can interact with each other and form hetero- as well as homodimers in vitro (Zhang e t al., 1995). One isoform of FXRl is highly expressed in skeletal muscle and postmeiotic spermatids and absent in differentiated neurons and in spermatogonia; other isoforms of FXRI and FXRZ are transcribed in neurons in both the cerebellum and the cortex (Coy et al., 1995).

b. Amplification mechanisms

It is now known that in more than 95% of cases, the fragile X syndrome is caused by a single type of mutation (“full mutation”), i.e., the expansion and hypermethylation of a potentially unstable CGG trinucleotide repeat in the 5’ UTR of the FMRl gene. Depending on the length of the CGG repeat, three general classes of alleles are observed in the FMRl gene: wild-type alleles (650 repeats), premutations (50-200 repeats), and full mutations (200- 1000 repeats and more). However, the boundaries between these classes are not absolute, and the initial instability depends not only on the total length but also on the repeat configuration (Hirst e t al., 1995). Detailed analysis of over 400 wild-type alleles showed that the CGG repeat stretch is commonly interrupted by AGG triplets, usually two, occurring every 9 to 10 CGGs (Eichler e t al., 1994; Hirst e t al., 1994; Kunst et al., 1996), which apparently have a stabilizing effect by preventing replication slippage (Heale and Petes, 1995). In vitro replication studies of expanded CTG and CGG repeats demonstrated that DNA polymerase pauses after copying 29 to 3 1 pure repeat units (Kang e t al., 1995). This is likely to allow the formation of secondary structures on the nascent strand, including unimolecular hairpins (reviewed by Darlow and Leach, 1998), which results in a more substantial increase in repeat length (Wells, 1996). Subsequently, when the so-called expansion threshold (about 70 pure CGG repeats) has been reached (Eichler e t al., 1994), multiple hairpins and/or stem-and-loop structures can form. This can also happen because Okazaki fragments exclusively composed of CGG repeats may slip at both ends (Richards and Sutherland, 1994). Such structures are extremely unstable and, after inappropriate repair, may result in a variety of expanded full mutations that are frequently accompanied by smaller or even deleted alleles. Actually, fragile X patients are often mosaics for full mutations and premutations (Chiurazzi et al., 1994a), alleles of normal size (van den Ouweland et al., 1994), or deletions of the entire CGG stretch and part of its flanking sequences (de Graaff et al., 1995; Mila et al., 1996).


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c. FMR1 instability and founder effects FMRl full mutations appear to be generated by a multistep process requiring the sequential action of different mechanisms (Morton and Macpherson, 1992; Richards and Sutherland, 1994; Chiurazzi et al., 1996). Thus far, no direct conversion of a wild-type to a full-mutation allele has been observed in fragile X families; all mothers of affected individuals were found to be carriers of an already expanded CGG triplet. The initial events leading to the instability of a wild-type allele are apparently much more rare than those determining the final transition from premutation to full mutation (Chiurazzi et al., 1996). Replication slippage is known to cause variation of a few repeat units in microsatellites due to a local misalignment of the template and nascent strands during a brief detachment of the DNA polymerase (Levinson and Gutman, 1987; Schloetterer and Tautz, 1992). As a consequence, both small reductions and amplifications of allele length have been detected in single sperm cells of two males carrying, respectively, a 29 and a 55 CGG repeat allele (Mornet et al., 1996). More rarely, slippage of a whole AGG(CGG), tract can determine a 10-unit increase of a normal allele (Macpherson et al., 1995). It is worth noting that the majority of alterations in repeat length occur at the 3’ end of the CGG repeat (Kunst and Warren, 1994). This polarity may derive from the different mutability of the leading and lagging strand and was shown in vim0 to depend on the local direction of replication (Wells, 1996; Hirst and White, 1998). The loss of the distal AGG, most probably due to a point mutation (A-to-C transversion), observed in many premutated alleles is implicated in a faster route to instability, as it creates a longer pure CGG tract (Kunst and Warren, 1994; Eichler et al., 1994, 1996). However, it is likely that only a few alleles can reach the instability threshold of approximately 30 uninterrupted CGG repeats. These few alleles, sometimes referred to as protomutations, must be linked to a limited number of ancestral haplotypes. Some of these founder chromosomes, which account for the linkage disequilibrium detected in different populations (reviewed in Chiurazzi et al., 1996), have apparently increased their frequency in the general population by genetic drift and constitute large pools of at-risk alleles (Mandel, 1994). These intermediate pools, which may be difficult to distinguish from wild-type alleles, most likely explain the relatively high frequency of the fragile X syndrome in spite of the low fitness of affected individuals and the limited number of founder chromosomes observed.

d. Full mutations

As the (CGG), repeat in the first exon of the FMRl gene exceeds the illdefined threshold of 200 repeats, most cytosine residues in the repeat itself and in the upstream CpG island become completely methylated, as if they were on an inactive X chromosome (Stoeger et al., 1997; Luo et al., 1993; Hansen et


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al., 1992). The FMRl gene is therefore transcriptionally silenced (Pieretti et al., 1991), and no protein is present in affected males (Verheij et al., 1993). It seems that the extreme expansion of the CGG repeat allows the formation of abnormal structures, like hairpins and tetraplex DNA, on the lagging strand during replication (reviewed by Darlow and Leach, 1998; Mitas, 1997), which in turn attract DNA methyltransferases (Bestor and Tycko, 1996; Kho et al., 1998). The hypermethylation of the CGG repeat then spreads to the surrounding CpG island, possibly after interaction with methylcytosinebinding proteins (MeCP2 and/or MBDs) (Boyes and Bird, 1992; Lewis et al., 1992) or other trinucleotide repeat-binding proteins (Deissler er al., 1996). Hypermethylation is most likely responsible not only for the transcriptional silencing (Pieretti et al., 1991; Sutcliffe et al., 1992; Hwu et al., 1993) but also for the delayed replication of the FMRl gene (Hansen et al., 1993, 1996; Samadashwily et al., 1997), which supposedly causes the cytogenetic fragility (Laird et al., 1987). The timing of pre- to full-mutation expansion and of its methylation is still being investigated. It has been observed that only premutations are present in the sperm of fragile X patients (Reyniers et al., 1993), and it has been proposed that pre- to full transition would occur only postzygotically during embryogenesis. This hypothesis would require the action of some “imprinting” signal that distinguishes maternally and paternally derived premutations, because a premutation never becomes full when transmitted from the father (Rousseau et al., 1991). On the contrary, Malter et al. (1997) have presented evidence that full-mutation alleles can be detected in oocytes and in fetal spermatogonia, although premutations seem to be selected in fetal testes. In this scenario, pre- to full-mutation transition is limited to meiosis, while postzygotic (mitotic) instability generates mosaicism within the range of full mutations as well as reduced alleles in the premutation range or even deletions (Chiurazzi et al., 1994a). In any case, premutations would be selected in fetal testes, thus explaining why all daughters of premutated males always retain a premutation and the sperm of fragile X patients only harbors premutations. As for methylation, Malter et al. (1997) showed that full mutations are unmethylated in oocytes, although they were completely methylated in all somatic tissues of a 13-week-old fetus. Methylation is therefore likely to take place after fertilization and during early embryogenesis.

e. Mutations other than CGG expansion The identification of mutations other than the expansion of the CGG repeat, even though in a minority of patients, was important in confirming that the fragile X syndrome is a single-gene disorder. Thus, point mutations (De Boulle et aE., 1993; Lugenbeel et al., 1995) or small intragenic deletions (Meijer et al., 1994) ruled out the possibility that the abnormal hypermethylation associated

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with the full mutation might not be restricted to the FMRl promoter but could affect the expression of other genes in that chromosomal region. Several larger deletions, even encompassing the entire FMRl gene (Tarleton et al., 1993), have also been reported. A review of the deletion cases has been published by Hammond et al. (1997). Careful analysis of small deletions limited to the promoter region can help define the essential regulatory sequences governing FMRl transcription (Gronskov et al., 1997). Finally, it is worth noting that some rare patients with all the phenotypic manifestations of the fragile X syndrome show no detectable alteration of the FMRl gene, which is apparently not involved in the pathogenesis of their condition (Chiurazzi et al., 199413). Considering the interactions between the FMRl and the FXR proteins, all present in neurons, it may be possible that these patients have a mutation in either the FXRl or the FXR2 gene.

f. Animal models Fmrl knockout mice have been generated by homologous recombination of a

targeting vector interrupting exon 5 in embryonic stem (ES) cells (Bakker et al., 1994). It is important to note that no reduced fertility of mutants of either

sex has been observed, and heterozygous females had a normal litter size with the expected distribution of offspring with the mutant allele. Thus, Fmrl is not necessary for spermatogenesis or oogenesis in mice, nor for normal embryonic development or postnatal viability. Fmrl knockout mice show no overt anatomical or histological abnormalities but do have macroorchidism and exhibit hyperactivity and learning deficits (Bakker et al., 1994). Apparently, an increased Sertoli cell proliferation during testicular development is responsible for the macroorchidism, although this increase does not appear to be the result of major changes in FSH signal transduction in knockout mice (SlegtenhorstEegdeman et al., 1998). Experimental designs can now be made to introduce transgenic copies of FMRl into various tissues (brain, gonads) of the knockout mice in order to dissect the pathogenetic components of the fragile X phenotype. Transgenic mouse lines with a fusion gene consisting of an Escherichia coli P-galactosidase reporter gene ( h Z ) linked to the FMRl promoter region have already been established (Hergersberg et al., 1995) and showed an expression pattern closely resembling the endogenous one, indicating that the 2.8-kb fragment 5’ of the CGG repeat contains most cis-acting elements regulating its transcription.

4. Treatment Useful guidelines for health supervision of fragile X children have been published by the American Academy of Pediatrics (1996) and include advice for both physical and behavioral aspects of the syndrome. After confirmation of the


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diagnosis with the molecular test and appropriate genetic counseling of the parents for subsequent pregnancies, a series of medical examinations can be envisaged, depending on the age of the child. Development during the first year of life may be normal, although hypotonia and irritability may be apparent. In early childhood it is important to give an ophthalmologic examination (strabismus, myopia), to perform an echocardiogram if a murmur or click is present (mitral valve prolapse), and to check for orthopedic problems (flat feet, scoliosis, and loose joints). A n inguinal hernia should also be excluded. A history of seizures or staring episodes should be reviewed and an EEG might be appropriate, though antiepileptic medication after a single seizure is not advisable given the self-limiting course of epileptic manifestations in adolescence (Musumeci et al., 1991). Hyperactive behavior (head banging, hand biting, etc.) and severe attention deficit, which are major concerns in the school-age period, can be treated pharmacologically (Hagerman, 1997). However, socialization and school integration, possibly within a mainstream program with individual support, are extremely important in helping to overcome these problems. Sports and regular physical activity (e.g., swimming) are important for counteracting the hypotonic posture and improving motor coordination. Speech, language, and occupational therapy should be goal oriented and help adolescents and young adults to attain as much autonomy as possible. Support from family organizations is extremely important, especially for the parents and sibs, because it eases the sense of isolation and helplessness that often follows the diagnosis. Treatments specifically aimed at recovering the function of the FMRl gene have been attempted with folic acid because of its action on the cytogenetic expression of the fragile site, and although a few reports indicated some effect on the behavior (Hagerman et al., 1986), others did not confirm these observations (Froster-Iskenius et al., 1986; Webb et al., 1990). It can be safely concluded that folate supplementation has no efficacy for the treatment of fragile X syndrome patients. Recent observations of intellectually normal (Rousseau et al., 1994; Smeets et al., 1995) or minimally affected (McConkie-Rose11 et at., 1993; Hagerman et al., 1994) males with an unmethylated full mutation confirmed that the abnormally amplified CGG tract per se can still be transcribed and translated. Even if translation may not be completely efficient (Feng et al., 1995), lymphoblastoid cell lines containing only unmethylated full mutations of two such males have clearly shown the presence of FMRl protein in every cell, although at a reduced level (Smeets et al., 1995). Given the observation of these exceptional individuals and knowing that the coding sequence of the mutated FMRl gene was intact, we tested the possibility of restoring its activity in vitro employing a DNA demethylation protocol. We obtained in vitro reactivation of FMRl expression after inducing DNA demethylation with 5azadeoxycytidine in the patients’ lymphoblastoid cells. Specific mRNA was detected by RT-PCR, the presence of the protein product was verified by


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immunocytochemistry, and the actual promoter demethylation was confirmed

by methylase-sensitive restriction analysis. These results clarify the clinical interpretation of the rare cases of male individuals with unmethylated full mutations and normal IQ and pave the way to future attempts at pharmacologically restoring FMRl gene activity in vivo (Chiurazzi et al., 1998). However, only less toxic drugs can be envisaged for in vivo applications and much information is still needed about the maintenance of the demethylation-reactivation effect after a time-limited treatment.

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B. Simpson Golabi -Be hmel syndrome The first report of what became later known as the Simpson-Golabi-Behmel syndrome (SGBS) was published in 1975 by Simpson et al., who described two cousins, maternally related, with macrocephaly, “coarse” face, broad hands with dysplastic fingernails, and apparently normal intelligence. In 1984, Behmel et al. described a similar condition in several males of a large family, calling attention to a number of additional findings, such as heart defects, polydactyly, and a high rate of infant mortality. They confirmed the X-linked inheritance of the trait and also noted a mild expression in carrier females. At approximately the same time, Golabi and Rosen (1984) reported yet another family in which several affected males had additional malformations of internal organs and early death. Opitz et al. (1984) also described severely affected males in a family from Michigan, although there is some question whether this instance may represent a different condition. In 1988, Neri et al., reporting on an Italian family, explicitly noted that the three affected males in this family had the same clinical condition previously reported by Simpson et al. (1975), Behmel et al. (1984), and Golabi and Rosen (1984) and coined the eponym “Simpson-GolabiBehmel syndrome.” The clinico-genetic findings in SGBS have been recently reviewed (Neri et al., 1998a).

1. Clinical phenotype SGBS is a syndrome characterized by overgrowth, multiple congenital anomalies, and dysplasia and caused by an X-linked mutant gene. The spectrum of its clinical manifestations is very broad, varying from very mild forms in carrier females to infantile lethal forms in affected males. It has been calculated that as many as 50% of affected males die neonatally (Neri et al., 1988), although the causes of this high mortality remain unknown. Overgrowth is of prenatal onset and continues postnatally. Birth measurements (height, length, head circumference) of affected males are usually well above the 97th centile, and final adult height can exceed 2.0 m, although with ample variation depending on background factors, such as average family


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height. In most patients, the facial traits are “coarse,” typically with hypertelorism, downslanting palpebral fissures with epicanthic folds, short nose, macrostomia with macroglossia, severe dental malocclusion, and central groove of the lower lip. Cleft lip and palate have been occasionally reported. Hands and feet are relatively short and broad and may display a variable combination of deformities (metatarsus varus, clubfoot), dysplasias (fingernail hypoplasia, especially of the index finger, various degrees of interdigital webbing or cutaneous syndactyly), and malformations (postaxial polydactyly). A complete transverse palmar crease is a common finding, together with striking dermatoglyphic changes, including an excess of tiradii and interdigital loops and an irregular mixture of arches, loops, and whorls on fingertips. Consistently present on the chest are supernumerary nipples. Thickened and/or darkened skin and skin tags may also be present. Genitalia are usually normal, although hypospadias and cryptorchidism have been reported in a number of patients. The internal organs may be involved in many different ways. Organomegaly is common, affecting especially the liver, spleen, and kidneys. Kidneys may be multicystic with dysplastic changes. Lung segmentation defects have been noted. A diaphragmatic defect has been reported in several patients. The heart may be affected in more than one-third of cases, with either structural defects, such as ventricular septa1 defect, patent ductus arteriosus, pulmonic stenosis, or functional defects, especially arrythmias (Lin et al., 1999). In one patient, the development of a dilated cardiomyopathy was noted, although it was impossible to tell whether this was primary or secondary to a preexisting congenital heart defect (Gurrieri et al., 1992). In any case, the heart function should be watched closely in SGBS patients because it can be a cause of early death (Konig et al., 1991). An X-ray survey of the skeleton will demonstrate, in a typical case, advanced bone age, vertebral segmentation defects such as fusion of C2/C3, cervical ribs, usually with 13 pairs of ribs, 6 lumbar vertebrae, sacral and coccygeal defects, and scoliosis. The most consistent neurological finding in SGBS is congenital muscular hypotonia, which may appear in striking contrast to the big, stocky build of the patients. Several minor anomalies can be considered a direct consequence of the congenital hypotonia: the mouth-breathing face with highly arched palate and dental malocclusion, pectus excavatum, downsloping shoulders, diastasis recti, umbilical and inguinal hernias, and cryptorchidism. The question of mental retardation in SGBS is much debated. It is possible, and even likely, that severely affected patients are mentally retarded, although in most of these cases early death prevents a formal psychometric evaluation. However, it is clearly established that the majority of patients are not mentally retarded. This is not to say that these patients do not have psychological problems; in fact, quite the opposite. The coarse appearance and

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the speech difficulties, due to macroglossia and mouth malocclusion, give the impression that these patients are mentally retarded, an impression of which they become acutely and painfully aware. There is an increased risk of neoplasia in SGBS that must be carefully considered, especially in young patients. A Wilms tumor of the kidney was diagnosed in several members of affected families in Canada (Hughes-Benzie et al., 1992; Xuan e t al., 1994), and a hepatocellular carcinoma was reported in a young child (Lapunzina et al., 1998). Because other infantile tumors can be expected, every patient should be considered at increased risk of neoplasia and consequently watched for at least the first 5 years of life.

2. Diagnosis SGBS belongs to the family of the overgrowth syndromes. Therefore, a question of differential diagnosis may easily arise with one or more of the clinical entities included in this family of syndromes. Conversely, a diagnosis of SGBS should be considered for any patient, especially if male, presenting with excessive growth. However, the truly critical nosologic issue is with the BeckwithWiedemann syndrome (BWS). Several patients who were reassessed and rediagnosed as SGBS after an initial diagnosis of BWS are on record (Neri et d., 1988; Punnett, 1994). Overgrowth at birth, coarse face with macroglossia, hernias, visceromegaly, congenital hypotonia, and increased incidence of tumors, especially Wilms tumor, are features common to both SGBS and BWS. Midline capillary hemangiomas, body asymmetries with hemihypertrophy, and a tendency to decelerated growth can be considered more typical of BWS. Persisting overgrowth, congenital heart defects, diaphragmatic defects, polydactyly, extra nipples, and familial occurrence with evidence of X-linkage are more typical of SGBS. However, many cases in which the clinical diagnosis will remain suspended and in which only the molecular diagnosis will be decisive are to be expected. Recently, Verloes et al. (1995) pointed out the clinical overlap between SGBS and the Perlman syndrome, an autosomal recessive overgrowth syndrome characterized by enlarged, dysplastic kidneys and a high risk of developing a Wilms tumor (Neri et al., 1984). However, the facial traits, the clinical course, and the mode of inheritance are sufficiently different in SGBS and in the Perlman syndrome to make the two conditions easily distinguishable. It should also be mentioned that a patient initially diagnosed as having Weaver syndrome (Tsukahara et al., 1984) was subsequently recognized as having SGBS (Kajii and Tsukahara, 1984). The last, and still unresolved, nosological issue concerns the possibility that SGBS is a heterogeneous condition, clinically as well as genetically. A family reported by Opitz (1984) was often questioned as being a bona fide case


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of SGBS because of the severity of the clinical presentation. Another severely affected family was recently described by Terespolsky et al. (1995). Given that X-linked inheritance was apparent in both families, future molecular studies will determine whether severe forms of SGBS are caused by allelic mutations at the same locus or by another X-linked gene. The latter hypothesis is supported by a very recent observation (Brzustowicz et al., 1998).

3. Genetics SGBS is an X-linked dominant trait with mild expression in heterozygous females and full expression in affected males. The mutant gene was initially mapped to the Xq25-q27 region by linkage analysis in a Dutch-Canadian family (Xuan et al., 1994). Close linkage to the HPRT locus in Xq26 was demonstrated by Orth et al. (1994) through the study of two European families. This location coincides with the cytogenetic breakpoint of an X; 1 translocation in the previously mentioned patient who was originally diagnosed as having BWS but who was subsequently recognized as having SGBS (Punnett, 1994). Actually, this patient became very critical for the cloning of the SGBS gene recently reported by Pilia et al. (1996). The gene, encoding an extracellular proteoglycan, designated glypican 3 (GPC3), spans more than 500 kb and contains eight exons. The cDNA measures 2.2 kb. The X;1 translocation interrupts the gene in the second intron, and another translocation, X;16, from a patient described as having the Klippel-Feil anomaly, interrupts the gene between exons 7 and 8. In the three families also analyzed by Pilia et al. (1996), three different deletions were found: one involving exon 2, one involving the last three exons, and the third one also involving the last three exons but extending further in the 3’ direction. Additional deletions were reported subsequently, although there are several bona fide patients in whom neither a deletion nor a point mutation can be found (Lindsay et al., 1997). According to Hughes-Benzie et al. (1996), lack of correlation between the extent of the deletions and the phenotypic expression of the disease suggests that “classical” cases of SGBS are likely due to the loss of function of GPC3. The gene is expressed in a number of mesoderm-derived tissues, including lung, liver, and kidney tissues, and the level of expression is higher in tissues from mouse embryos than in murine and human adult tissues. There is no expression in white blood cells. The GPC3 protein belongs to the glypican family of heparan sulfate proteoglycans (David, 1993) and can function on the cell surface as a receptor or part of a receptor complex. Most interestingly, it is capable of interacting with IGF2, the insulinlike growth factor, which has been suggested as a causal factor in BWS (Pilia et al., 1996; Weksberg et al., 1996).

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G. Neri and P. Chiurani

Treatment

There is no specific treatment for SGBS. However, symptoms should be addressed according to needs. Surgery may be indicated for congenital heart defects, diaphragmatic defects, and gastrointestinal and genitourinary malformations. Orthognathic treatment should be considered of the greatest importance. The correction of dental malocclusion and the reduction of macroglossia, if indicated, should lead to speech improvement, an essential step toward the establishment of normal social relations. This should be accompanied, when needed, by appropriate psychological support aimed at improving the self-image of the patients. It is imperative that every effort be made to eliminate the impression that SGBS patients are mentally retarded or, even worse, aggressive. Carrier females should be properly identified and adequately counseled with respect to recurrence risks and prenatal diagnosis.

C. ATR-X syndrome After the seminal paper by Weatherall et al. was published in 1981, several other reports appeared describing patients in whom MR is associated with a mild form of a-thalassemia. This combination has since been known by the acronym ATR. Analysis of the clinical phenotype and the pattern of inheritance in familial cases and molecular studies led to the identification of two distinct syndromes. A group of patients had large deletions at the tip of the short arm of chromosome 16 within band 16~13.3,including the a-globin gene complex. This explained the presence of mild (hemizygous) a-thalassemia in addition to MR and a pattern of physical anomalies whose variability likely depended on the size of the deletion (Wilkie et al., 1990a; Lamb et al., 1993). These conditions can be interpreted as typical of a contiguous gene syndrome. The other group of patients, all males, was characterized by a more specific physical phenotype, intact a-globin gene complex and familial inheritance consistent with X-linkage (Wilkie et al., 1990b; Cole et al., 1991; Donnai et al., 1991). A new condition whose main characteristics were a-thalassemia (not from deletion), MR, and X-linkage was therefore recognized and designated

ATR-X.

1. The clinical phenotype The main clinical findings of the ATR-X syndrome are a characteristic face, genital anomalies, and severe mental retardation (Gibbons et al. , 1991, 1995a; Wilkie et al., 1991). The face can be described as coarse, with hypertelorism, epicanthic folds, a flat nasal bridge, midface hypoplasia, a short nose of triangular shape with anteverted nares and flat philtrum, an inverted V shape of the


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upper lip and everted lower lip, macroglossia, and widely spaced incisors. The ears may be small, simple, low-set, and posteriorly angulated. Genitalia are usually abnormal, with small, undescended or dysgenetic testes, a shawl-like or hypoplastic scrotum, and a small penis with hypospadias. Other fairly common physical findings are microcephaly, short stature, talipes equinovams, and gastrointestinal problems, including gastroesophageal reflux and constipation. Xray investigations have shown delayed bone age, minor digital abnormalities, and kyphoscoliosis. Psychomotor development appears to be delayed from early on and is accompanied by generalized muscular hypotonia. Mental retardation is usually severe, with virtually absent speech and minimal comprehension. Seizures have been reported in some patients. Brain imaging occasionally shows cerebral atrophy. Carrier females are substantially normal, both physically and mentally, although mild midfacial anomalies have been noted in some (Donnai et al., 1991).

2. Diagnosis The phenotypic diagnosis of the ATR-X syndrome can be confirmed in the laboratory by a relatively simple blood test. The mild form of a-thalassemia in these patients is reflected in the presence of HbH inclusions in a proportion of red cells, varying from 1 to 40%. The amount of HbH detected electrophoretically is also variable, ranging from 0 to about 7% (Gibbons et al., 1991). Occasionally, a very few erythrocytes with inclusions have been noted in carrier females. Although these HbH findings can be taken as diagnostic evidence for both carriers and affected individuals, the opposite is not true. Unequivocal diagnosis is now available, based on direct mutational analysis of the responsible gene (vide infra).

3. Genetics Linkage analysis of several pedigrees segregating the ATR-X syndrome localized the corresponding locus to an interval of 11 cM in Xq12-q21.31 (Gibbons et al., 1992). This observation was followed 3 years later by the cloning of the gene. Gibbons et al. (1995b) showed that the ATR-X syndrome is caused by mutations of X H 2 , a gene belonging to the helicase superfamily, whose protein products carry out a number of regulatory functions ranging from DNA recombination and repair to control of transcription. More specifically, the protein belongs to the SNF2 subgroup, probably acting as a regulator of gene expression (Picketts et al., 1996). Analysis of several independent patients showed the existence of a variety of diverse mutations, including premature stop mutations, missense mutations, and deletions (Gibbons et al., 199%). In a subsequent report, Gibbons et al. (1997) showed the existence of a mutational hot spot in

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a segment of the gene encoding a cysteine-rich zinc-finger domain that accounts for more than 60% of known mutations. It is of the greatest interest that further recent mutational analyses of X H 2 led to the discovery that this gene can be involved in the causation of conditions that were originally described as independent entities. For example, the so-called Juberg-Marsidi syndrome, also mapped to the Xq12-q21 region and whose phenotypic manifestations include deafness in addition to mental retardation and multiple physical anomalies, was shown to be due to a mutation of the W V P gene, a different designation for the same XH2 gene (Villard et al., 1996a). Likewise, a frameshift mutation of XH2 that generated a premature stop codon was reported to segregate in a family in which the affected males had a phenotype resembling that of the ATR-X syndrome but without a-thalassemia and with male-to-female sex reversal (Ion et al., 1996b). The absence of crthalassemia was also noted in a patient with an X H 2 mutation causing a proline-to-serine transition in the helicase I1 domain (Villard et al., 1996). Taken together, these observations support the notion that XH2 mutations downregulate the expression of several genes, including the a-globin genes. This would explain the complexity of the ATR-X phenotype.

4. Treatment There is no specific treatment for the ATR-X syndrome. Female carriers have a 50% risk of their male offspring being affected. It is therefore of the greatest importance that women at risk of being carriers be properly identified by molecular tests, adequately counseled, and offered prenatal diagnosis when indicated.

D. Opitz/G-BBB syndrome The G and BBB syndromes were originally reported as two separate conditions even though both involved defects of the midline developmental field (Opitz et al., 1969a,b). The G syndrome appeared to have an autosomal dominant mode of inheritance, whereas in the case of the BBB syndrome, X-linkage seemed to be more likely, although not clearly proven. Subsequently, the striking phenotypic similarities led to the provisional conclusion that the two conditions should be considered one and the same under the comrnom designation of Opitz syndrome until proven otherwise (Cappa et al., 1987). More recently, Robin et al. (1995) performed linkage studies on several Opitz syndrome families, including the original G family, and found genetic heterogeneity: one locus was identified on the X chromosome and another one on chromosome 22. The Xlinked gene has now been cloned (Quaderi et al., 1997).


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1. Clinical phenotype Opitz syndrome can still be described phenotypically as a single entity, and it is characterized by a series of defects of the midline. The face is typical, with widely spaced eyes, broad or hypoplastic nasal sella, a large nose, and a hypoplastic philtrum or clefting of the upper lip and palate. Tracheoesophageal defects range from simple swallowing difficulties to a tracheo esophageal fistula. Pectus excavatum, umbilical hernia, and hypospadias in males are also common findings. The heart frequently has a variety of abnormalities, including septa1 and conotruncal defects. Brain imaging has demonstrated agenesis of the corpus callosum in some patients (Neri et al., 1987). Intellectual development may range from normal to mildly retarded. In familial cases one parent occasionally will show mild physical signs, suggesting variable expressivity of the mutant gene(s). This is particularly true in mothers of affected boys, supporting the notion that a partially dominant, X-linked mutation segregates in some families.

2. Genetics As already mentioned, Opitz syndrome is genetically heterogeneous, with possibly several different genes involved in different families. One of these genes is X-linked, and the corresponding locus was found to map within an 18-cM interval on band Xp22 (Robin et al., 1995). The gene has now been cloned from a pericentric inversion with breakpoints in Xp22 and Xq26 found in affected members of an Opitz syndrome family, and has been designated MIDI (Midline 1). MIDI is ubiquitously expressed as a 7-kb transcript in fetal and adult human tissues. It encodes a member of the B-box family of proteins containing a RING finger motif, which is involved in protein interaction. Mutations of MIDI were found in unrelated Opitz syndrome patients, confirming its pathogenic role in this condition. The second locus involved in Opitz syndrome, identified through linkage analysis of other families, maps to chromosome 22q in a 32-cM interval within band 22q11.2, which coincides with the velo-cardio-facial- DiGeorge syndrome region (Robin e t al., 1995). A comparison of clinical findings in Xlinked cases and in 22-linked cases did not show any significant phenotypic differences (Robin et al., 1996).

3 . Treatment Opitz syndrome patients should be treated for their anomalies or complications thereof. Surgical intervention may be indicated for the correction of a cleft lip,

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the laryngo-esophageal defects, the umbilical hernia, and the hypospadias. Swallowing difficulties may require a fundoplication of the stomach.

E. Aarskog-Scott syndrome Aarskog-Scott syndrome, also known as faciogenital dysplasia (FGDY), owes its eponym to the authors who described it independently at about the same time (Aarskog, 1970; Scott, 1971). The more descriptive acronym FGDY nicely summarizes the major componenes of the clinical phenotype, stressing the multisystemic involvement. Familial cases suggested that the syndrome is genetic in origin, with an X-linked mode of transmission (Gorlin et al., 1990). X-linkage was further proven by the observation of patients carrying an X-autosome translocation involving the p arm of the X chromosome (Bawle et al., 1984; Glover et al., 1993).

1. Clinical phenotype Growth retardation is a constant feature of Aarskog-Scott syndrome, with most patients reaching an adult height below the third centile. The hands are disproportionately short, with some degree of webbing between fingers. Most typical is the hyperextensibility of the proximal interphalangeal joints and flexion of the distal joints. A single transverse palmar crease and fifth-finger clinodactyly are often present. Similarly, feet are short and broad with splayed toes. The face is typically rounded, with a broad forehead and small chin. There are hypertelorism, epicanthic folds, downslanting of the eyes, ptosis of the upper eyelids, a short nose with anteverted nares, long, flat philtrum, and a cupid’s bow shape to the upper lip. The ears are usually low-set and posteriorly angulated and have a thick lobe. The teeth may show delayed eruption and enamel hypoplasia. Pectus excavatum and umbilical and inguinal hernias are relatively common findings. Urogenital anomalies include shawl scrotum, cryptorchidism, hypospadias, and kidney hypoplasia. X-ray studies of the skeleton have shown delayed bone age and a number of anomalies affecting mostly the hands and spine. There are hypoplasia of the terminal phalanges of the fingers in a majority of patients and cervical spina bifida occulta or other vertebral defects, such as hypoplasia of the first cervical vertebra and segmentation defects. Mental development is usually normal, with only a few cases showing mild delay. Carrier mothers may show some attenuated manifestations of the syndrome, including shortness of stature and of hands, round face, and hypertelorism. An excellent clinical description of the syndrome and a thorough


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review of the literature can be found in Syndromes of the Head and Neck (Gorlin et al., 1990).

2. Genetics The mutant gene responsible for Aarskog-Scott syndrome was mapped to the pericentromeric region of the X chromosome both by linkage analysis in informative families (Porteous er al., 1992; Stevenson er al., 1994) and by the observation of an X;8 reciprocal translocation in a mother and son showing clinical manifestations of the syndrome (Bawle et al., 1984). The X chromosome breakpoint of this translocation was subsequently localized to a region in band Xp11.21 flanked by markers ALAS2 and DXS323 (Glover et al., 1993). This finding paved the way to the cloning of FGDf, a candidate gene for the syndrome (Pasteris et al., 1994). FGDl encodes a protein of 961 amino acids that shows strong homologies to the guanine nucleotide exchange factors Rho/ Rac and contains a zinc-fingerlike region as well as two SH3-binding regions. Proteins of this family are known to be involved in growth regulation and signal transduction. In fact, FGDl was found expressed in a variety of fetal tissues, including heart, brain, lung, and kidney tissues. FGDJ is truncated by the previously mentioned X;8 translocation, and a productive mutation was found to segregate in affected members of a family by the insertion of a guanine residue at nucleotide 2122. The resulting frameshift mutation was predicted to cause a translational truncation at residue 469 (Pasteris et al., 1994). All these findings are strong evidence that FGDl is indeed the gene responsible for Aarskog-Scott syndrome, a notion supported by the recent observation of a missense mutation segregating with the disease in another affected family (Neri et al., 199813).

3 . Treatment Once again, there is no specific treatment for Aaskorg-Scott syndrome. Interventions should be directed at those anomalies that may become clinically relevant, for example, severe palpebral ptosis and hypospadias.

F. Coffin-lowry syndrome Coffin-Lowry syndrome (CLS) owes its name to the authors who independently described it 5 years apart. The first description was by Coffin et al. in 1966 and the second by Lowry et al. in 1971. However, it was Temtamy et al. (1975) who, in reporting eight patients from three different families, recognized that the patients had the same condition previously described by these authors

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and that it was indeed one and the same disorder, probably inherited as a sexlinked trait. Since then, several other patients have been described, suggesting that the syndrome may not be very rare. Thanks to these reports, especially those of Hunter et al. (1982), Gilgenkrantz et al. (1988), and Young (1988), CoffinLowry syndrome is phenotypically well delineated. Several family studies support inheritance as an X-linked dominant trait with reduced expression in the heterozygous females. The gene locus was initially mapped to the Xp22 region (Hanauer et al., 1988; Partington et al., 1988; Bird et al., 1995) and subsequently cloned (Trivier et al., 1996).

1. Clinical phenotype According to one of the original descriptions (Lowry et al., 1971), CLS consists of “mental retardation, small stature, retarded bone age, hypotonia, tapering fingers, a characteristic facies which includes hypertelorism, upturned nares, and prominent frontal region, and possibly arrested hydrocephalus.” Growth seems to be normal prenatally but is definitely delayed postnatally. Short stature is evident from early childhood, with adult height below the third centile in virtually all affected males and in a large proportion of carrier females. Retarded bone age was observed in nearly all reported cases. Microcephaly has been noted in only a few cases, possibly those that do not have hydrocephaly. Ventricular dilatation was reported in several patients, although it is not clear whether this is due to increased intracranial pressure or rather to cerebral atrophy (hydrocephalus ex vacuo). The face is quite distinctive and it can be described as coarse. There is a prominent forehead and thick supraorbital ridges, hypertelorism, narrow and downslanting palpebral fissures, and a broad nose with a thick septum and anteverted nares. The philtrum is high and narrow, and there is dental malocclusion with large and widely spaced upper incisors. The hands have a quite typical appearance. They are broad, soft, and puffy, with broad terminal phalanges and joint hyperlaxity. Similar findings can be observed in the feet. A characteristic horizontal crease in the hypothenar region has been noted in many patients. Genitalia are normal and pubertal development seems to occur normally. A skeletal survey in many patients has consistently shown skull hyperostosis, a drumstick aspect of the terminal phalanges of the fingers, and an involvement of the spine, including anterior webbing of the vertebral bodies and decreased intervertebral spaces resulting in severe kyphoscoliosis. Several of the clinical findings reported so far have suggested that in CLS there may be an involvement of the connective tissue. Reduced elastin and abnormal vacuolation were observed by Temtamy et al. (1975) in skin biopsies, and an abnormality of chondrocytes had already been mentioned by Coffin et al. (1966) in


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their original report. Autopsy findings of panacinar emphysema, nodular transformation of the liver, renal microcysts, and pleural calcific plaques support the concept of a generalized connective tissue disorder as part of the syndrome. A visceral neuropathy, which could have been the cause of intestinal pseudoobstructions or diverticular disease, was also observed postmortem (Machin et

al., 1987).

Severe MR is one of the hallmarks of the syndrome; IQ values are often below 20 and there is a virtual absence of speech in the majority of affected males. Generalized epileptiform seizures have been reported (Fryns et al., 1977), as well as sensorineural hearing loss and premature cataract (Hartsfield et al., 1993). In affected females the phenotype is much milder, including mild mental delay, shortness of stature, and facial changes such as a broad and prominent forehead, broad nose, and fleshy, everted lips.

2. Diagnosis The diagnosis of CLS is based on the typical facial and hand changes and can be confirmed by mutational analysis. The differential diagnosis is as with other syndromes also characterized by MR, coarse face, and short stature. BorjesonForssman -Lehmann syndrome, also X-linked, can be distinguished on the basis of obesity and hypogenitalism. Patients with Atkin-Flaitz syndrome (1985), as well as those with a similar condition reported by Clark and Baraitser (1987), have macrocephaly and macro-orchidism. In sporadic cases it is probably worth ruling out Williams syndrome, for which a simple laboratory test now exists. Differential diagnosis with the ATRX syndrome should also be considered. The two conditions can now be distinguished on the basis of molecular tests.

3. Genetics Early linkage studies assigned the CLS locus to band Xp22 in a 13-cM interval between markers DXS43 and DXS41 (Hanauer et al., 1988; Partington et al., 1988). This localization was progressively narrowed, first to a 7-cM interval between DXS207 and DXS274 (Biancalana et al., 1992) and then to a 5-cM interval in band Xp22.1. (Biancalana et al., 1994). More recently, the observation of a recombination in a carrier female from a British family has further reduced the critical region to 3.4 cM between markers AFM291wf5 and DXS365 (Bird et al., 1995). This was an important step toward the cloning of a candidate gene, which was accomplished shortly thereafter. The CLS gene encodes the 740-amino-acid protein RSK-2, a ribosomal S6 kinase belonging to a family of growth-factor-regulated serine- threonine kinases. This protein has a role in the regulation of cell proliferation and differentation. Deletions, nonsense, missense, and splice-site mutations were found in a number of patients

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(Trivier et al., 1996). All families studied so far are linked to the Xp22 locus, consistent with the notion that the CLS is genetically homogeneous.

4. Treatment There is no specific treatment for CLS. Interventions will be symptomatic for any problems that might arise. A favorable social milieu is probably helpful in minimizing the progressive deterioration, in terms of mental retardation, that has been reported in some patients. Within families, carrier females must be properly counseled, and prenatal diagnosis can be offered in informative cases.

111. NONSYNDROMAL XLMR (MRX) Nonsyndromal XLMR includes, by definition, those conditions in which MR is not accompanied by distinctive clinical signs. These conditions can be recognized only if they present as familial cases with X-linked inheritance, and can be distinguished from each other based only on linkage to different polymorphic markers of the X chromosome. Each individual entity is designated by the acronym MRX, followed by a progressive number (MRX1, MRX2, etc.). At present, the MRX count totals 59 entries (Table 3.1), but this number changes rapidly. A complete list of published MRXs can be found in the review of Lubs et al. (1999); an ideogram of the X chromosome with the localizations of each MRX is depicted in Figure 3.2. It is immediately apparent that there are large regions of overlap, suggesting that ultimately some MRXs that are now separated will be lumped, just as happened to MRX41 and MRX48 (vide infra). In fact, based on their regional assignment, no more than 10 of the currently mapped, putative loci could account for all MRXs. Only the cloning of individual genes from the affected families will allow the emergence of a clear picture and contribute to the exact count of those genes in the X chromosome that can cause MR. So far, only four genes have been cloned, FMRZ, GDll, OPHNl , and PAK3.

A. FMR2 The FMR2 gene coincides with the folate-sensitive fragile site FRAXE, approximately 600 kb distal to the FRAXA site. Actually, the first families carrying a mutation of FMR2 were ascertained as fragile X syndrome families by testing positive to the cytogenetic fragility assay. However, subsequent molecular analysis failed to show a mutation of the FMRl gene. In 1993, Knight et al. cloned FMRZ from the fragile site FRAXE and found that the mutational mechanism is essentially identical to that of FMRl . Even though in the promoter region of

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oligophrenin-l(60)

-

14 58 31

4

17

52

I

12

13

40

3

FRAXE GDll (41,48)

Figure 3.2. X chromosome ideogram with the known localizations of genes responsible for nonsyndromal XLMR (MRX). T h e bars indicate indicate the locus assignment for those putative genes that have been regionally mapped. The arrows indicate the position of the cloned genes.

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the wild-type gene there is a sequence of GCC repeats ranging in number from 6 to 25 and affected individuals have more than 200 copies, with hypermethylation of the CpG island, their physical phenotype is not distinctive and MR usually varies from mild to borderline.

B. GDll The gene GDIl encodes a-GDI, a protein highly expressed in the brain and whose function is to control the recycling of the Rab-GTPases across cell membranes (Bione et al., 1993; Wu et al., 1996), with special emphasis on its role as regulator of neurotransmitter release (Geppert et al., 1994). DAdamo e t al. (1998) found mutations in GDIl in affected individuals from families MRX41 and MRX48 that map in Xq28. In one family (MRX41) the mutation was a T + C transition at position 433 of the cDNA, resulting in substitution of a leucine with a proline in position 92, which was responsible for reduced binding and recycling of RAB3A. The mutation in the other family (MRX48) was a C + T transition at position 366 of the cDNA, causing the insertion of a premature stop codon. Lymphoblasts of affected individuals did not express crGDI, as expected.

C. OPHNl OPHNl is a newly characterized gene cloned from a mentally retarded female patient carrying an X;12 translocation with a breakpoint in Xql2 (Billuart et

al., 1998). The gene, which was found highly expressed in fetal brain, encodes a 91-kDa protein of 802 amino acids (oligophrenin-1) characterized by the presence of a domain typical of a Rho-GTPase-activating protein involved in signaling pathways that affect differentiation and migration of neurons. The pathogenic role of a loss of function of this protein was confirmed in an independent family (MRX60), in which affected individuals were shown to have a one-base-pair deletion corresponding to nucleotide 1578.

D. PAK3 The PAK3 gene, originally cloned in the mouse (Manser et al., 1995), is a member of the family of p21-activating kinase genes. It encodes PAK3, a serine-threonine kinase with a critical role in linking Rho-GTPases to the actin cytoskeleton. Allen et al. (1998) cloned the human gene and showed that it is mutated in affected individuals from family MRX30 and maps to Xq22. The mutation consists of a C + T transition that inserts a stop codon (TGA) in place of an arginine codon (CGA), corresponding to amino acid 419. This results in a truncated protein that lacks a region essential for normal kinase


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function. The pathogenic role of PAK3 is further supported by the observation that it is highly expressed in fetal brain but not in other fetal organs.

Cloning and characterizing XLMR genes will have a number of consequences. It will improve our insight into the nosology of MR; it will generate a useful model for the searching out of autosomal MR genes; it will shed light on the pathophysiology of complex clinical conditions; and it will provide new tools for prenatal diagnosis, carrier detection, and genetic counseling. Ultimately, it may lead to the development of gene therapy, at least in some cases. The importance of discovering genes whose mutations cause “pure” MR cannot be overemphasized. Two aspects appear to be particularly significant. One is that a common pathway seems to emerge through which the products of different genes operate in the central nervous system. Rho and Rab GTPases are critical factors in an intricate network of intercellular interactions and play a central role in the control of neural cell differentiation, migration, and signaling (Antonarakis and Van Aelst, 1998). Another important aspect is the obvious implication that through malfunction we may learn more about normal function, that is, that understanding mental retardation will ultimately lead us to understand normal brain functioning and the molecular bases of intelligence.

Acknowledgments The personal work quoted in this review was partially supported by a grant from Telethon, Italy (No. E-245). P.C. is the recipient of a Telethon international fellowship. The authors are indebted to Mrs. Luciana Amato for skilled secretarial assistance and for typing the manuscript.

References Abitbol, M., Menini, C., Delezoide, A. L., Rhyner, T., Vekemans, M., and Mallet, J. (1993). Nucleus basalis magnocellularis and hippocampus are the major sites of FMRl expression in the human fetal brain. Nar. Genet. 4, 147-153. Allen, K. M., Gleeson, 1. G,, Bagrodia, S., Partington, M. W., MacMillan, J. C., Cerione, R. A., Mulley, J. C., and Walsh, C. A. A. (1998). PAK3 mutation in nonsyndromic X-linked mental retardation. Nat. Genet. 20, 25-30. American Academy of Pediatrics, Committee on Genetics ( 1996). Health supervision for children with fragile X syndrome. Pediamcs 98, 297-300. Antonarakis, S. E., and Van Aelst, L. (1998). Mind the GAP, Rho, Rab and GDI. Nut. Genet. 19, 106- 108. Ashley, C. T., Sutcliffe, J. S., Kunst, C. B., Leiner, H. A,, Eichler, E. E., Nelson, D. L., and Warren,

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Musumeci, S. A., Ferri, R., Elia, M., Colognola, R. M., Bergonzi, P., and Tassinar, C. A. (1991). Epilepsy and fragile X syndrome: A follow-up study. Am. J. Med. Genet. 38, 511-513. Neri, G., Martini-Neri, M. E., Katz, B. E., and Opitz, J. M. (1984). The Perlman syndrome: Familial renal dysplasia with Wilms tumor fetal gigantism and multiple congenital anomalies. Am. J. Med. Genet. 19, 195-207. Neri, G., Genuardi, M., Natoli, G., Costa, P., and Maggioni, G. (1987). A girl with G syndrome and agenesis of the corpus callosum. Am. J. Med. Genet. 28, 287-291. Neri, G., Marini, R., Cappa, M., Borrelli, P., and Opitz, J. M. (1988). Simpson-Golabi-Behmel syndrome: An X-linked encephalo-tropho-schisissyndrome. Am. J. Med. Genet. 30,287-299. Neri, G., Gurrieri, F., Gal, A., and Lubs, H. A. (1991). XLMR genes: Update 1990. Am. J. Med. Genet. 38, 186-189. Neri, G., Gurrieri, F., Zanni, G., and Lin, A. (1998a). Clinical and molecular aspects of the Simpson-Golabi-Behmel syndrome. Am. J . Med. Genet. 79, 279-283. Neri, G., May, M., Cappa, M., Steindl, K., and Schwartz, C. (1998b). Second mutation found in the FDGl gene causing the Aarskog syndrome. Am. J. Hum. Genet. 61, A341, 1997. Opitz, J. M. (1984). The Golahi-Rosen syndrome-report of a second family. Am. J. Med. Genet. 17,359-366. Opitz, J. M., Frias, J. L., Gutenberger, J. E., and Pellet, J. R. (1969a). The G syndrome of multiple congenital anomalies. BD:OAS V(2), 95-101. Opitz, J. M., Summitt, R. L., and Smith, D. W. (1969b). The BBB syndrome: Familial telecanthus with associated congenital anomalies. BD:OAS V(2), 86-94. Opitz, J. M., Hermann, J., Gilbert, E. F., and Matalon, R. (1988). Simpson-Golabi-Behmel syndrome: Follow-up of the Michigan family. Am. J. Med. Genet. 30, 301-308. Orth, U., Gurrieri, F., Behmel, A., Genuardi, M., Cremer, M., Gal, A., and Neri, G. (1994). Gene for Simpson-Golahi-Behmel syndrome is linked to HPRT in Xq26 in two European families. Am. J. Med. Genet. 50,388-390. Partington, M. W., Mulley, J. C., Sutherland, G. R., Thode, A., and Turner, G. (1988). A family with the Coffin-Lowry syndrome revisited: Localization of CLS to Xp21-pter. Am. J. Med. Genet. 30,509-521. Pasteris, N. G., Cadle, A., Lindsday, J., Logie, L. J., Porteous, M. E. M., Schwartz, C. E., Stevenson, R. E., Glover, T. W., Wilroy, R. S., and Gorski, J. L. (1994). Isolation and characterization of the faciogenital dysplasia (Aarkog-Scott syndrome) gene: A putative Rhomac guanine nucleotide exchange factor. Cell 79, 669-678. Penrose, L. S. (1938). A clinical and genetic study of 1,280 cases of mental defects (The Colchester Survey). MRC Special Report 229, Her Majesty’s Stationery Office, London. Picketts, D. J., Higgs, D. R., Bachoo, S., Blake, D. J., Quarrell, 0. W. J., Cribbons, R. J. (1996). ATRX encodes a novel member of the SNF2 family of proteins: mutations point to a common mechanism underlying the ATRX syndrome. Hum. Moi. Genet. 12, 1899-1907. Pieretti, M., Zhang, F., Fu, Y. H., Warren, S. T., Oostra, B. A., Caskey, C. T., and Nelson, D. L. (1991). Absence of expression of the FMR-I gene in fragile X syndrome. Cell 66,817-822. Pilia, G., Hughes-Benzie, R. M., Mackenzie, A., Baybayan, P., Chen, E. Y., Huber, R., Neri, G., Cao, A., Forabosco, A., and Schlessinger, D. (1996). Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-BehmeI overgrowth syndrome. Nat. Genet. 12, 241-247. Porteous, M. E. M., Curtis, A., Lindsay, S., Williams, O., Goudie, D., Kamakari, S., and Battacharja, S. S. (1992). The gene for Aarskog syndrome is located between DXS255 and DXS566 (Xpl1.2Xq13). Genomics 14,298-301. Punnett, H. H. (1994). Simpson-Golabi-Behmel syndrome (SGBS) in a female with an Xautosome translocation. Am. J. Med. Genet. 50,391-393. Quaderi, N. A., Schweiger, S., Gaudenz, K., Franco, B., Rugarli, E. I., Berger, W., Feldman, G. J., Volta, M., Andolfi, G., Gilgenkrantz, S., Marion, R. W., Hennekam, R. C. M., Opitz, J. M.,


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Pharmaceutical Perspectives of Nonviral Gene Therapy Ram 1. Mahato* Copemicus Therapeutics, Inc. Cleveland, Ohio 44106

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

I. W h y a Gene-Based Approach for Protein Therapy? A. W h y Somatic Gene Therapy? B. Gene Therapy Approaches C. Plasmid-Based Gene Medicines D. Advantages of Gene Medicines 11. Commercialization of Gene Therapy Products A. Commercial Challenges B. Regulatory Issues C. Clinical Trials 111. Basic Components of Gene Expression Plasmids A. Bacterial Elements B. Mammalian Transcription Unit C. Promoter/Enhancer D. Untranslated Regions (UTR) E. Intron F. Poly(A) Signal G. Gene Switches

*Corresponding author: Telephone: (216) 231-0227. Fax: (216) 231-9477. E-mail: [email protected]. Advances In Genetics, Vol. 41

Copyright 0 1999 by Academic Press All rights of reproduction in any form reserved. 0065-2660/99 $30.00

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IV. Gene Delivery Systems A. Lipid-Based Gene Delivery B. Peptide-Based Gene Delivery C. Polymer-Based Gene Delivery V. Formulation Factors Influencing Gene Transfer A. DNA Topology B. DNA Condensation C. DNA Condensing Agents D. DNA Aggregation VI. Biodistribution and Pharmacokinetics of Plasmids A. Anatomical and Physiological Considerations B. Influence of (Patho)physiology on Biodistribution C. Biodistribution and Pharmacokinetics of Plasmid DNA VII. Intracellular Trafficking of Gene Medicines A. Cellular Uptake Mechanisms B. Intracellular Trafficking C. Nuclear Envelope and Nuclear Pore Complex D. Nuclear Localization Signal (NLS) Sequence

VIII. Biological Opportunities for Gene Therapy A. Systemic Gene Therapy B. Cancer Gene Therapy C. Pulmonary Gene Therapy D. Genetic Vaccines IX. Concluding Remarks

The use of nonviral plasmid-based gene medicines represents an attractive in oivo gene transfer strategy that is simple and lacks many risks that are inherent to viral systems. Commercialization of gene medicines requires a thorough analysis of business opportunities, unmet clinical needs, competitive products under development, and issues related to intellectual property. Synthetic gene delivery systems are designed to control the location of a gene within the body by affecting distribution and access of a gene expression system to the target cell, and/or recognition by a cell surface receptor and uptake followed by intracellular and nuclear translocation. Plasmid-based gene expression systems are designed to control the level, fidelity, and duration of in oioo production of a therapeutic gene product. This review will provide insights into the potentials of plasmid-based gene therapy and critical evaluation of gene delivery sciences and clinical applications of gene medicines. o 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 the body has the ability to produce thousands of different proteins that are essential for cellular structure, function, and growth. Genes are segments of deoxyribonucleic acid (DNA) and provide information needed by the cells for protein production (Berg and Singer, 1992; Drlika, 1996). The protein expressed in a particular cell may be limited to the cell itself (uutocrine or cis function), it could be secreted and act on other cells (parmine or trans function), or it could be secreted into the blood or lymph nodes (endocrine function) (Vega, 1995). Plasmid expression systems are being constructed that lead to the secretion of a therapeutic gene product into the systemic circulation for an endocrine effect. Expression plasmids are also being constructed to express genes locally at the site of administration for uutocrine or puracrine effects. The disease targets range widely, including genetic diseases (cystic fibrosis, hemophilia, Duchenne muscular dystrophy), metabolic disorders (e.g., diabetes and hyper cholesterolemia), and different forms of cancer (Rolland and Felgner, 1998).

A. Why somatic gene therapy? The body contains a plethora of proteins (including enzymes, hormones, and receptors) that regulate biological functions. The absence or overproduction of a specific protein can lead to a variety of clinical manifestations, depending on the structural or functional role that the protein normally plays in the body. Many severe and debilitating diseases (e.g., diabetes, hemophilia, cystic fibrosis) and several chronic diseases (i.e., hypertension, ischaemic heart disease, asthma, Parkinson’s disease, motor neuron disease, multiple sclerosis) remain inadequately treated by conventional pharmaceutical approaches (Dalgleish, 1997). Recombinant DNA technology has allowed the large-scale production and biological characterization of several therapeutic proteins, including granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO), interleukins, insulin-like growth factor-I (IGF-I), human factor VIII and IX, and tissue plasminogen activator (t-PA). However, the clinical use of many protein drugs is limited by their inappropriate concentration in blood, poor oral bioavailability, manufacturing cost, chemical and biological instability, and/or rapid hepatic metabolism and renal excretion (Tomhnson, 1992). In addition, few protein drugs can efficiently enter target cells unless administered at very high doses, which can lead to toxic side effects. These limitations lead to their frequent administration with an increased treatment cost and reduced patient compliance ( Woodley, 1994). Gene therapy is a method for the treatment or prevention of disease that uses genes to provide the patient’s somatic cells with the genetic information necessary to produce specific therapeutic proteins needed to correct or to

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modulate a disease. The promise of somatic gene therapy is to overcome limitations associated with the administration of therapeutic proteins, including low bioavailability, inadequate pharmacokinetic profiles, and high cost of manufacture. Providing a therapeutic gene as a “predrug” to a patient to allow either the production of therapeutic proteins that may be difficult to administer exogenously or the inhibition of abnormal protein production may circumvent some limitations associated with the use of recombinant therapeutic proteins (Ledley, 1996).

B. Gene therapy approaches Gene therapy approaches currently in development may be distinguished by the methods used to transfer or deliver therapeutic genes to the patient. The methods include the use of (i) cells that have been altered ex vivo (outside the body) with viruses (such as retrovirus, adenovirus, adenoassociated virus, herpes simplex virus, and vaccinia virus) or other gene transfer methods (e.g., electroporation) and (ii) in vivo (inside the body) with viruses, which have been genetically modified so that they cannot multiply and infect other cells or with synthetic formulations of plasmids (Eck and Wilson, 1996). Ex vivo approaches have significant clinical and commercial limitations. These approaches involve complex procedures whereby the target cells must be removed from the patient, modified with the therapeutic gene, expanded in number, cleansed of contaminants, and then reintroduced into the patient. In addition, most ex vivo gene therapy procedures produce a permanent genetic alteration of the cell, which generally precludes the ability to modulate treatment in response to therapeutic needs. Although a number of viral gene therapies are currently used for direct in vivo administration, safety issues may limit their further development. These include inflammation as well as cellular and humoral immune responses. There are also concerns about the possibility of integration of viral vectors into the host genome (e.g., retroviral vectors) (Miller and Vile, 1995). Nonviral methods involve the direct administration of plasmid-based gene expression systems. The plasmids contain a therapeutic gene, as well as genetic sequences, that direct the cell to transcribe and translate this gene accurately and efficiently into a therapeutic protein. In the majority of cases, plasmid-based gene therapy requires the use of a synthetic gene delivery system to control the delivery of the gene expression system from the site of administeration in the body to the nucleus of specific target cells. Nonviral gene delivery systems can be administered to patients by conventional routes, such as direct injection, inhalation, or intravenous injection, thus providing increased safety over viral gene therapy approaches. Moreover, the nonviral gene delivery systems can be degraded by the body using natural processes, allowing the gene medicine to be administered repeatedly (Mahato et al., 1997a).


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C. Plasmid-based gene medicines A gene medicine contains three components: a therapeutic gene that encodes a specific therapeutic protein, a plasmid-based gene expression system that controls the functioning of a gene within a target cell; and a gene delivery system that controls the delivery of the plasmid expression system to specific locations within the body. The gene and the gene expression system are the components of the plasmid (Mahato et al., 199713). The gene delivery system distributes the plasmid to the desired target cell, after which the plasmid is internalized into the cell by a number of mechanisms (e.g., phagocytosis, macropinocytosis, receptor-mediated endocytosis, and caveolae-mediated endocytosis) (Wolff et al., 1992; Friend et al., 1996; Labat-Moleur et al., 1996. Li and Huang, 1996). Once inside the cytoplasm, the plasmid can then translocate to the nucleus, where gene expression begins, leading to the production of a therapeutic protein through the steps of transcription and translation. The gene expression system can be engineered to control whether the resulting protein will remain within the cell for an intracellular effect or will be secreted out of the cell for either a local or systemic action. The gene expression system can also be adjusted to control the level of protein production as well as the fidelity and duration of gene expression (Figure 4.1).

Figure 4.1. Spatial and temporal modulation of gene expression. (A) Gene delivery systems are designed to control the location of a gene within the body by affecting distribution and access of a gene expression system to the target cell receptor followed by intracelM a r and nuclear translocation. (B) Plasmid-based gene expression systems are designed to control the level and duration of in vivo production of a therapeutic gene product.

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D. Advantages of gene medicines Small molecular-weight drugs usually function by interacting with proteins throughout the body. Protein drugs are large molecules that generally act as replacements for the body’s own proteins. Both small molecular-weight drugs and protein drugs are designed to act on chemical receptors on a cell’s surface. Short, normally single-strand, antisense olgonucleotides are designed to inhibit the production of aberrant proteins by hybridizing with the coding (sense) RNA. However, there is little control of the pharmacokinetic profiles of small molecular-weight drugs, protein drugs, and oligonucleotides. These molecules are widely distributed throughout the body and rapidly cleared through the kidney. The use of plasmid+basedgene medicines is intended to be analogous to conventional medicines in terms of controlled dosing, convenient systemic or local administration, and well-characterized pharmacokinetics. Plasmid expression systems can persist for a defined time in the nucleus as nonintegrated episomes before they are degraded. It should therefore be possible to use gene medicines like conventional medicines. Gene medicines could be administered repetitively to a patient according to a dosing schedule that matches the extent and severity of the disease, treating either acute or chronic diseases. They are intended to have low toxicity due to the use of synthetic carriers and nonintegrating plasmids. Although a single dose of current gene medicines generally has a low therapeutic effect, their repeated injections may be effective for several clinical targets. Compared to viral vectors, gene medicines present several potential advantages, including (i) low costs, (ii) noninfectivity, (iii) absence of immunogenicity, (iv) good compliance, (v) well-defined characteristics and (vi) possibility of repeated clinical administration (Mahato et al., 1999).

II. COMMERCIALIZATIONOF GENE THERAPY PRODUCTS Gene delivery systems need to be developed to increase and maintain an adequate level of in viva gene expression over a defined period of time. The eventual goal is to achieve cell- or tissue-specific expression and to regulate gene expression within the cells. A basic understanding of disease pathogenesis is required to define the mechanisms by which gene defects lead to disease. Furthermore, knowledge of disease (patho)physiology is crucial for better understanding of appropriate target cells for effective therapy, levels of gene expression required for clinical efficacy, and regulation of gene expression. Animal models also need to be developed to test experimental hypotheses and specific therapies prior to trials in human (Ross et al., 1996). The production of gene therapy products as well as their research and development activities are subject to regulation for safety, efficacy, and quality by governmental authorities in the United States and other countries. Safety and regulatory aspects of gene therapy can be addressed along three lines: (i)


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experimental and preclinical research, (ii) manufacturing of gene medicines, (iii) clinical trials and development. Gene therapy represents a field of daunting complexity for the regulatory authorities (Cohen-Haguenaur, 1996, 1997).

A. Commercial challenges The fundamental commercial challenges facing gene therapy products as they proceed to the market will be to provide therapeutic benefit within the confines of an acceptable safety profile. Gene therapy is a new and rapidly evolving field. Major advances in genetics and the ability to control gene delivery and expression will bring revolutionary novel therapeutic methods in the upcoming millennium. Many pharmaceutical and biotechnology companies as well as academic institutions are exploring the field of somatic gene therapy. Rapid technological development may produce potential products or technologies that could become obsolete before a company recovers its research, development, and capital expenditures. Basic information and technological advances that would normally be published in scientific journals are often delayed for incorporation into patent applications. Furthermore, numerous patents are being issued that cover the broad concepts of technology (Figure 4.2), which can inhibit the development of new technologies and products that are directly applicable to a product (Bossart and Pearson, 1995).

300 Y m

2

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Year of Issued U.S.Gene Therapy Patents Figure 4.2. Rapid growth of gene therapy intellectual property.

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There are currently no marketed gene therapy products. The existing clinical data on the safety and efficacy of potential gene therapy products are still limited. Furthermore, the results of preclinical studies do not necessarily predict safety or efficacy in humans. All of the potential products under development are in research, preclinical, or clinical development. These potential products will continue to require significant additional research and development, as well as clinical investigation efforts, prior to commercial use (Persidis and Tomczyk, 1997). Residual RNA, proteins, and bacterial DNA are considered contaminants and thus their presence should be reduced or eliminated in the product according to defined specifications. Toxic chemicals such as ethidium bromide and cesium chloride should either be avoided in plasmid production or their amount in the final product should be quantified. Gene expression is influenced by the plasmid forms and thus the percentage of supercoiled and linear DNA in the preparation should be quantified (Hermann, 1996). Aberrant expression of some proteins in nontarget organs may lead to an inappropriate activation of the immune system, resulting in acute or chronic inflammatory and immune responses and potential damage of normal tissues. Therefore, studies should be conducted over reasonably long periods of time to allow detection of potential immune reactions (Ledley, 1991).

B. Regulatory issues The marketing of a new pharmaceutical product in the United States requires Preclinical laboratory tests and in vivo preclinical studies Submission of an Investigational New Drug (IND) application to the FDA for human clinical testing Human clinical trials for establishing product safety and efficacy Submission of a New Drug Application (NDA) to the FDA for a Biologics License Application (BLA) FDA approval of the NDA or BLA prior to any commercial sale The United States is a leader in the development of safeguards for the clinical application of human somatic gene therapy, which is subject to rigorous regulation by the Food and Drug Administration (FDA) (Kessler et al., 1993; Marcel and Grausz, 1997; Ledley, 1991; Cohen-Haguenauer, 1995). The National Institutes of Health Recombinant DNA Advisory Committee (RAC) serves in an advisory function and as a public forum for many gene therapy issues rather than as a body involved in case-by-case approval. The clinical performance of gene transfer experiments is still in an early phase of development. As of June 1996, 161 clinical protocols have been approved in the United States and 46 trials in Europe (Martin and Thomas, 1998). In the United


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States, such products are regulated under the Federal Food, Drug, and Cosmetic Act. As biological products, in addition, they are subject to certain provisions of this act and are regulated under the Public Health Service Act. These laws and the regulations promulgated thereunder govern, among other things, testing, manufacturing, safety, efficacy, labeling, storage, record keeping, advertising, and promotional practices involving drugs and biological products. At the FDA, the Center for Biologics Evaluation and Research is responsible for the regulation of biological products and has regulated all gene therapy products to date. Each therapeutic product containing a particular gene will likely be regulated as a separate biological product, depending on its intended use and the FDA policies in effect at the time. To commercialize any products, the company must sponsor and file an IND application for each proposed product and will be responsible for initiating and overseeing the clinical studies to demonstrate the safety and efficacy that are necessary to obtain FDA approval of any such products. Gene therapy is a novel method of treatment and thus regulatory requirements are constantly evolving and changing. Even if regulatory approvals are obtained, they may include limitations on the indicated uses for which a product may be marketed. In addition, a marketed product is subject to continual FDA review. Later discovery of previously unknown problems may result in restrictions on the marketing of a product or withdrawal of the product from the market. Preclinical tests include laboratory evaluation of the product as well as animal studies to assess the potential safety and efficacy of the product. Compounds must be produced according to applicable current Good Manufacturing Practices (GMP), and preclinical safety tests must be conducted by laboratories that comply with FDA regulations regarding Good.Laboratory Practices (GLP). The results of the preclinical tests, together with manufacturing information and analytical data, are submitted to the FDA as part of an IND, which must become effective before human clinical trials commence.

C.

Clinical trials

The gene therapy clinical trials aim at answering the crucial questions related to the safety and efficacy of a gene therapy product (Ledley, 1991). Clinical trials involve the administration of the investigational product to healthy volunteers or to patients under the supervision of a qualified principal investigator. Clinical trials are conducted in accordance with Good Clinical Practices (GCP) under protocols that detail the objectives of the study, the parameters to be used to monitor safety, and the efficacy criteria to be evaluated. Each protocol must be submitted to the FDA as part of an IND. Further, each clinical study must be reviewed and approved by an independent institutional review board at the institution at which the study will be conducted. The institutional review

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board will consider, among other things, ethical factors and the safety of human subjects. Clinical trials typically are conducted in three sequential phases, but the phases may overlap. Phase I studies involve the very first testing of a potential gene therapy product in humans, with the aim of evaluating safety and tolerability. Phase I1 studies are moderate-scale dose-escalation studies designed to investigate efficacy in patients while continuing to accumulate safety data. Once a treatment has been shown to have a therapeutic effect in a number of patients, large-scale Phase 111 pivotal trials need to be undertaken to provide adequate statistical proof of efficacy and safety of the effect observed in Phase I1 studies and also to compare the new treatment with standard therapies, if such therapies exist. The results of the pharmaceutical development, preclinical studies, and clinical studies are submitted to the FDA in the form of an NDA or BLA for approval of the manufacture, marketing, and commercial shipment of the drug or biologic. The FDA may deny an NDA or BLA if applicable regulatory criteria are not satisfied, require additional testing or information, or require postmarketing testing and surveillance to monitor the safety or efficacy of a product. Among the conditions for NDA or BLA approval is the requirement that the prospective manufacturer’s quality control and manufacturing procedures conform to cGMP, which must be followed at all times. Foreign regulatory requirements governing human clinical trials and marketing approval for drugs may vary from those of the United States. In Europe, the approval process for the commencement of clinical trials varies from country to country (Martin and Thomas, 1998). Since the beginning of human gene therapy in 1990, a large percentage of protocols are still in Phase I. Indeed, of the 48 gene therapy trials initiated since January 1996, 77% are in Phase I and 15% in Phase 1/11. Several gene therapy trials did not produce expected results, and thus the FDA directed them to undertake further preclinical evaluation. This “back-to-the-bench” trend is apparent among the 23 existing cystic fibrosis trials. Although 75% of these trials were initiated before September 1995, they still remain in Phase I or 1/11 and none has yet reached phase 111.

111. BASIC COMPONENTS OF GENE EXPRESSION PLASMIDS Plasmids are circular double-stranded DNA molecules, which can be manufactured at high yields in a cost-effective manner. Plasmids are chemically stable under appropriate conditions for prolonged periods. Plasmid-based gene expression systems contain a cDNA sequence coding for either a full gene or a minigene and several other genetic elements, including introns, polyadenylation


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sequences, and transcript stabilizers to control transcription, translation, and protein stability, and secretion from the host cell (Brown, 1990). Optional components can be added to an expression plasmid, such as “gene switch,” which enables expression of the therapeutic protein to be turned on or off at the transcriptional level by oral administration of a specific low molecularweight drug (Wang et at., 1994).

A. Bacterial elements Plasmids encode two features that are important for their propagation in bacteria. One is the bacterial origin of replication, usually derived from a high-copy plasmid, such as pUC plasmid (Vieira and Messing, 1982).The second required element is a selectable marker, usually a gene that confers resistance to an antibiotic, such as kanamycin or neomycin. These “prokaryotic” plasmid segments permit the production of large quantities of a given plasmid in bacteria. The prokaryotic origin of replication is a specific DNA sequence that binds to factors that regulate replication of plasmid and, in turn, control the number of copies of plasmid per bacterium.

B. Mammalian transcription unit The minimal transcription unit that is required for the expression of a therapeutic protein consists of 5’ enhancer/promoter upstream of the gene encoding for the therapeutic protein and a poly(A) signal downstream of the gene. A heterologous intron is often inserted into the 5‘ or 3’ untranslated region (UTR) of the transcription unit. This kind of “insertion” leads to elevation in mRNA levels. A single intron inserted into the 5’ UTR of the transcription unit is the most common arrangement.

C . Promoter/En ha ncer A promoter is defined as a DNA region, usually at the 5’ end of a gene, that binds to transcription factors and RNA polymerase during the initiation of transcription of a gene at the correct nucleotide site. To date, a plethora of promoters originating from eukaryotic viruses, such as cytomegalovirus (CMV), simian virus 40 (SV40), Moloney murine leukemia virus (MoMLV), and Rous Sarcoma virus (RSV), are widely used because they are known to be strong promoters (Qin et at., 1997). However, these promoters appear to show a decrease in in vivo activity when different gene delivery systems are used. Cytokines, such as interferon-y (IFNy ) and tumor necrosis factor-a (TNF-a), have been shown to inhibit transgene expression from these promoter-based gene expression systems (Gribaudo et al., 1995; Tzen and Scott, 1993; Stein et

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al., 1993). The combination of both IF"-y and TNF-a was shown to have stronger inhibitory effects than either cytokine individually. However, these cytokines were shown not to affect the transcription of the actin promoter, which is a cellular promoter (Qin et al., 1997). Tissue-specific promoters are designed to interact with the transcription factors or other nuclear proteins that are present in the desired target cells. The chicken skeletal a-actin promoter is an attractive candidate for a musclespecific plasmid-based expression system. The a-actin promoter contains positive cis-acting elements that are required for efficient transcriptional activity in myogenic cells. Skeletal a-actin accounts for approximately 8% of the poly(A) RNA in adult chicken skeletal muscle (Petropoulos et al., 1989, Hayward and Schwartz, 1986). Therefore, an a-actin promoter could direct high expression of recombinant protein in skeletal muscle. Muscle-specific expression of insulinlike growth factor-1 (IGF-I), human growth hormone (hGH), and human factor-IX (hFIX) has been demonstrated after intramuscular administration of plasmids that encode these genes and contain skeletal a-actin (SK) promoter/ enhancer (Coleman et al., 1995; Alila et al., 1997; Anwer e t al., 1998). To generate higher levels of a gene product, several systems have been developed that can transcribe transgenes in the cytoplasm of transfected cells. One of these systems contains a reporter gene driven by the bacteriophage T7 promoter and the purified T7 RNA polymerase (Elroy-Stein and Moss, 1990). T7 RNA polymerase does not enter the nucleus. The transcriptional activity has been shown to be greater than that of the eukaryotic RNA polymerase. The level of expression increased with an increase in the amount of T7 RNA polymerase from bacteriophage T7 specifically recognizes and starts transcription at a 19-bp DNA sequence: the T7 promoter. Expression cassettes consisting of a reporter gene under transcriptional control of a T7 promoter sequence can be used to generate the reporter protein in cells that express T7 polymerase.

D. Untranslated regions (UTR) The 5 ' untranslated region (5' UTR) is the region of the mRNA transcript that is located between the cap site and the initiation codon. The linkage between methylated G residue and a 5' to 5' triphosphate bridge is known as the cap strutme, which is essential for efficient initiation of protein synthesis. The 5' UTR is known to influence mRNA translation efficiency. In eukaryotic cells, initiation factors first interact with the 5' cap structure and prepare the mRNA by unwinding its secondary structure. A n efficient 5' UTR is usually moderate in length, devoid of strong secondary structure, devoid of upstream initiation codons, and has AUG within an optimal context. Any of the following features


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that influence the accessibility of the 5‘ cap structure to initiation factors will influence mRNA translatability (Kozak, 1991, 1992): Initiation codon (AUG) appears to be best recognized when it is in the context of the sequence CCRCCAUGG with purine (R) at -3 and/or guanidine (G) at +4 (A of the AUG is numbered + l ) . If an AUG occurs alone, or an AUG in conjuction with a short open reading frame, is located between the cap site and the genuine AUG, translation will be inhibited. Secondary structures of the UTRs inhibit translation. 5’ UTR lengths that are greater than 32, but less than 100, nucleotides permit efficient recognition of the first AUG. Most naturally occuring 5‘ UTRs are 50 to 100 nucleotides in length. The 3’ UTR is defined as the mRNA sequences following the termination codon. The 3’ UTR is thought to play a potential role in mRNA stability. AU-rich motifs are commonly found in the 3’ UTR of mRNA of cytokines, growth factors, and oncogenes. These motifs are mRNA instability elements and should be eliminated for maximal levels of expression. This is usually accomplished by using standard 3’ UTR sequences in place of the one found in the cDNA. The most commonly utilized 3’ UTR sequences are from the bovine growth hormone and rabbit P-globin genes. Another approach is to minimize the length of the 3‘ UTR by placing the hexanucleotide of the poly(A) signal immediately downstream of the stop codon (Hartikka et al., 1996). Inclusion of 5’ and 3’ UTR introns may provide tissue specificity and long-term gene expression. The 3’ UTR from the chicken skeletal muscle aactin gene contains a stabilization element that improves mRNA stability and controls growth and differentiation of myoblasts. The pSK-hGH-SK expression plasmid was shown to produce -3-5 times more hGH than pSK-hGH-GH expression plasmid in the muscle (Figure 4.3) (Alila et al., 1997). Replacement of 3’ UTR from hGH gene by SK of a muscle-specific hIGF-I expression system has also been shown to produce higher accumulation and perinuclear localization of hIGF-I in the muscle after intramuscular injection (Alila et al., 1997).

E. lntron The protein-coding region in the eukaryotic gene is often interrupted by stretches of noncoding DNA called introns. Transcripts from the intronless genes are degraded rapidly in the nuclear compartment, leading to reduction in gene expression (Ryu and Mertz, 1989). Therefore, for maximal gene expression in eukaryotic cells, at least one intron should be included within the transcrip-

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Figure 4.3. Muscle-specific gene expression system. (A) Construction maps of human growth hormone (hGH) gene expression systems pSK-hGH-GH and pSK-hGH-SK (driven by chicken skeletal a-actin promoter elements), a-SKP, chicken skeletal a-actin promoter, hGH, human growth hormone genomic DNA, a-SKI, chicken skeletal a-actin intron. (B) Levels of hGH in tibialis cranalis and gastrocnemius muscle extract 21 days after the intramuclular injection of pSK-hGH-SK or pSK-hGH-GH in 5% polyvinylpyrrolidone (PVP) into hyposectomized rats. Values are mean S.E.M. (n = 5 ) (modified from Alila et al., 1997, with permission).

*

tion unit. Incorporation of introns into cDNA expression systems has been shown to enhance gene expression in cell culture up to 100-fold (Huang and Gorman, 1990).

F. Poly(A) signal The poly(A) tail is a homopolymeric stretch of A residues added to the primary transcript by a nuclear mechanism known as polyadenyiution. A poly(A) signal is required for the formation of the 3’ end of most eukaryotic mRNA. The signal directs two RNA processing reactions: site-specific endonucleolytic cleav.


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age of the RNA transcript, and stepwise addition of adenylates to the newly generated 3' end to form the poly(A) tail. The efficiency of polyadenylation is important for gene expression, as transcripts that fail to be cleaved and polyadenylated are rapidly degraded in the nuclear compartment. The poly(A) signals utilized in gene expression plasmids are chosen from a set of mammalian poly(A) signals, such as bovine growth hormone, rabbit P-globin, and SV40. These mammalian poly(A) RNAs have been extensively studied and characterized as strong (Goodwin and Rottman, 1992). The bovine growth hormone and rabbit p-globin poly( A) signals are essentially equivalent in their ability to enhance gene expression and are more effective than the SV40 late poly(A) signal (Yew et al., 1997). A modified version of the rabbit p-globin poly(A) signal yielded an approximately twofold increase in expression compared to the bovine growth hormone poly(A) signal (Hartikka et al., 1996).

G. Gene switches Many endogenous proteins are produced according to circadian rhythms. Therefore, in vivo pulsatile production of certain therapeutic proteins may be beneficial for their clinical applications. This can be achieved by including gene switches in a gene expression system to turn on or off the transcription of an administered gene. In addition, a gene switch adds another safety level in that excessive gene expression can be controlled. A gene switch is designed to be part of a gene expression system that contains both the gene switch and a therapeutic gene. In the positive system, the target gene will be inactive until the administration of an exogenous compound or ligand. Such inducing agents or drugs include progesterone antagonists (Wang et al., 1997a), tetracycline (Gossen et al., 1995), ecdysone (No e t al.,1996), and rapamycin (Wang e t al., 1997b). A common approach is that a chimeric transcription activator reversely binds to a target gene construct in response to the administered drug or ligand. Several different types of gene switches have been proposed, including one based on a modified progesterone receptor. This modified receptor has a deletion of 42 amino acid residues at its carboxy terminus and is linked to both the yeast G a l 4 DNA-binding domain and the herpes simplex virus protein VP16 transcriptional activation domain (Figure 4.4). The mutated progesterone receptor does not bind to endogenous steroids, but selectively binds to antiprogestin drugs, such as mifepristone, which act at very low concentrations (1 nM) as an agonist (Wang et al., 199713). Antiprogestins distribute to most cells in the body after oral administration and can bind the expressed gene switch protein, causing its dimerization in the cytoplasm. The activated gene switch translocates to the nucleus and then binds to the Gal4-binding sequence that is built into the gene expression system and controls the expression of the therapeutic gene. The therapeutic gene product would only be expressed when

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Figure 4.4. Mode of action of an antiprogestin gene switch. Gene expression plasmid containing promoter/enhancer transactivator GLVP linked with liver-specific transthreitin (TTR) was used to generate transactivator mice. These mice were then crossed with human growth hormone (hGH) target gene mice to generate bigenic mice harboring both transgenes (lTR-GLVP-hGH). Serum hGH was measured both prior to and 12 hr postadministration of mifepristone (250 p&g, intraperitoneally). The hGH transgene expression declined following metabolism of mifepristone ( 3 weeks later) and could be reactivated following a further mifepristone injection (adapted from Wang et al., 1997b, with permission).

the patient takes an antiprogestin drug-for instance, orally-and gene expression is turned off when the antiprogestin is eliminated from the target cells (Wang et al., 1997b). The expression of target gene hGH has been shown to be dependent on the presence of mifepristone and correlated with the relative tissue-specificexpression pattern of the transactivator GLVP (Figure 4.4).Other gene switches have been constructed based on tetracycline and rifamycin. Although very small doses of these low molecular-weight drugs are being used, their chronic administration is certainly a concern. For example, the slow clearance rate of tetracycline and the activation of ecdysone receptors by muristerone may be harmful.

Gene delivery systems are designed to control the location of a gene within the body by affecting the distribution and access of a gene expression system to the


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target cell, and/or recognition by a cell-surface receptor followed by intracellular trafficking and nuclear translocation (Rolland, 1996). Gene delivery systems should serve both to protect a gene expression system from premature degradation in the extracellular milieu and to affect the nonspecific or cell-specific delivery to a target cell. Other elements in a gene delivery system may facilitate the intracellular trafficking of a gene expression system. This section describes the development of several lipid-, peptide-, and polymer-based gene delivery systems.

A. Lipid-based gene delivery Liposomes are microscopic vesicles composed of uni- or multilamellar lipid bilayers surrounding aqueous compartments. Plasmids may be incorporated into anionic or neutral liposomes to ensure protection against degradation by nucleases in biological fluids, to control disposition profiles, and to enhance intracellular delivery (Ellens et al., 1984). However, the encapsulation efficiency of plasmids is very low. The uncondensed plasmids are large compared to the internal diameter of the vesicles. pH-sensitive liposomes are fusogenic at acidic pH and thus can be used to facilitate the endosomal disruption and subsequent release of plasmids in the cytoplasm. pH-sensitive liposomes usually consist of dioleoylphosphatidylethanolamine (DOPE) and a lipophilic anionic component containing a titratable head group. Examples are oleic acid, palmitoylhomocysteine, cholesterol hemisuccinate morpholine salt (CHEMS), and dioleoylsuccinylglycerol (DOSG) (Wang and Huang, 1987a,b, Legendre and Szoka, 1992). The in vitro transfection efficiency of pH-sensitive liposomes, composed of CHEMS:DOPE, has been compared to those of non-pH-sensitive liposomes, composed of CHEMS:dioleoylphosphatidylcholine (DOPC) and phosphatidylserine (PS):cholesterol Non-pH-sensitive liposomes were unable to transfect expression plasmids into monkey fibroblast CV- 1 cells, whereas pH-sensitive liposomes efficiently transfected plasmids into these cells (Legendre and Szoka et al., 1992). The pHsensitive immunoliposomes have been shown to mediate -6-8 times higher levels of thymidine kinase (TK) gene expression into mouse lymphoma cells compared to non-pH-sensitive immunoliposomes. Proteoliposomes, also known as virosomes or chimeraomes, have been used for plasmid delivery to cells both in vim0 and in vivo (Tikchonenko et al., 1988, Gould-Fogerite et at., 1989). Proteoliposomes incorporate viral proteins, fusogenic peptides, nuclear proteins, or nuclear localization peptides, which induce fusion of liposomes with the cell membranes and facilitate DNA release and transport through the cytoplasm. Cochleates can also be used for plasmid delivery. A negatively charged phospholipid such as phosphatidylserine, phosphatidic acid, or phosphatidyl

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~

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glycerol, in the absence or presence of cholesterol, are utilized to produce a suspension of multilamellar vesicles containing plasmids, which are then converted to small unilamellar vesicles by sonication. These vesicles are dialyzed against buffered divalent cations (e. g., calcium chloride) to produce an insoluble precipitate referred to as cochleates. Cochleates have been shown to encapsulate plasmid and enhance plasmid stability and transfection efficiency (Mannino and Gould-Fogerite, 1996). Since the introduction of the transfection reagent Lipofectin'" , a cationic liposome composed of 1:l (w/w) mixture of the cationic lipid N[1-(2,3dioleyloxy )propyl]-N,N,N-trimethylammonium chloride) (DOTMA) and the colipid DOPE (Felgner et al., 1987), many cationic lipid formulations have been tested for in vitro and in vivo transfection of plasmids. The flexibility in the design of cationic lipid structure has supported the view that cationic lipids can be used for gene tranfer in vivo (Felgner et al., 1987; Lasic and Templeton, 1996). Cationic lipids interact electrostatically with the negatively charged phosphate backbone of DNA, neutralizing the charges and promoting the condensation of DNA into a more compact structure. Usually, cationic lipids are mixed with a zwitterionic or neutral colipid such as DOPE (Farhood et al., 1995; Hui et al., 1996) or cholesterol (Bennett et al., 1995), respectively, to form liposomes or micelles. The lipid mixtures are mixed in chloroform, which is then evaporated to dryness, followed by vacuum drying. Water is added to the dried lipid film and the hydrated films then either extruded or sonicated to form cationic liposomes. Cationic liposomes have also been prepared by an ethanol injection technique (Campbell, 1995). Inclusion of a colipid is not always essential. For instance, the cationic lipid DOTAP is active in the absence of a colipid in a variety of cells in vitro (McLachlan et al., 1994).

1. Cationic lipid structures The general structure of a cationic lipid has three parts: (i) a hydrophobic lipid anchor group, which helps in forming liposomes (or micellar structures) and can interact with cell membranes; (ii) a linker group; and (iii) a positively charged hedpoup, which interacts with plasmid, leading to its condensation. The hydrophobic lipid anchors can be either fatty chains (e.g., derived from oleic or myristic acid) or a cholesterol group. Lipid anchors determine the physical properties of a lipid bilayer, such as membrane rigidity and rate of lipid exchange between lipid membranes. The linker group is an important component and determines the chemical stability and biodegradability of a cationic lipid. The head groups of cationic lipid appear to be critical for transfection and cytotoxicity of corresponding liposome formulations. The cationic amphiphiles differ


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markedly in structure and may be single- or multiple-charged as primary, secondary, tertiary, and/or quaternary amines. Examples are lipospermine, cationic cholesterol, cationic detergent, or lipopolysine. The physicochemical properties of plasmid/lipid complexes are strongly influenced by the relative proportions of each component and the structure of the headgroup. Many effective cationic lipids contain protonatable polyamines linked to dialkyl or cholesterol anchors. In the case of DOTMA, the hydrophobic domain is an oleoyl alcohol group that is connected to a glycerol-like, threecarbon backbone via an ether bond. A trimethyl quaternary amine is linked directly to the three-carbon backbone. 1, 2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE) is a derivative of DOTMA that contains a hydroxyethyl group attached to the quaternary amine. To increase the biodegradability of cationic lipids, a series of carbonic lipids have been synthesized in which the ether bonds were replaced with ester bonds (Felgner et al., 1994). The structure of 1,2-bis(oleoyloxy)-3-(trirnethylammonio)propane (DOTAP) is similar to DOTMA except that DOTAP contains ester bonds (McLachlan et al., 1994). 3P(N’, N’-dimethy1aminoethane)-carbamoyl] cholesterol (DC-Chol) contains a cholesterol-linked via carbamoyl bond and ethyl group to a trimethyl, quaternary amine (Gao and Huang, 1991). Several cationic lipids, including 2,3-dioleyloxy-N-[2(sperminecarboxyamido)ethyl]-N,N~ dimethyl- 1-propanaminium trifuoroacetate (DOSPA), contain a spermine group for binding to DNA (Hawley-Nelson et al., 1993). Although cationic lipid-based gene delivery systems are being extensively investigated and novel cationic lipid molecules are synthesized routinely, a definite structure-activity relationship has not clearly emerged. Lee et al. ( 1996) recently attempted to establish a structure-activity relationship by systematically analyzing a large number of different cationic lipid structures both in vitro and in viuo. Cationic lipids containing 3-P-(N%perrnine carbamoyl) cholesterol (lipid #67) and 3-P-(N+-sperrnidine carbamoyl) cholesterol (lipid #53) in a “T-shape” configuration rather than a linear configuration were found to be more effective than structures containing only a single protonatable amine (e.g., DC-Chol). However, there was a poor correlation between in vitro and in vivo results with various lipids used in that study (Lee et al., 1996). Although the cationic lipids dioctadecylamidoglyl spermine (DOGS) (Behr et al., 1989) and DOSPA also contain spermine headgroups, they were less active than the cartionic lipid &7, possibly due to the following differences in their structures: ( i ) the headgroup of lipid #67 is attached to the linker via a nitrogen atom, whereas those of DOGS and DOSPA are attached through a carbon atom; (ii) both DOGS and DOSPA contain a dialkyl chain as their lipid anchor groups, whereas lipid #67 contains a cholesterol anchor; and (iii) lipid #67 is in a free-base form, whereas DOGS and DOSPA are in salt forms.

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2. Role of colipids Although substantial attention has been paid to the functioning of cationic lipids, the role of colipids in gene transfer is less well defined. DOPE has been shown to be more effective than several other neutral co-lipids at facilitating cationic lipid-mediated transfection. The neutral colipid may facilitate escape of DNA from the endosome into the cytoplasm and increase the ability of the DNA to dissociate from the plasmid/lipid complex. The effect of the colipid on gene transfer depends on the type of cationic lipid, molar ratio of cationic lipid to colipid, and the target cell. DOPE is a phospholipid, which exhibits a high tendency to form inverted hexagonal phase at acidic pH. Dioleoylphosphatidylcholine (DOPC),a structural analog of DOPE, has no such activity (Farhood et al., 1995, Felgner et al., 1994). DOPE has been proposed to promote fusion with the endosome membrane allowing release of DNA into the cytoplasm (Fasbender et al., 1997). DOPE may be more effective in disrupting membranes because it tends to assume a nonbilayer structure, whereas DOPC tends to form a stable bilayer (Wimley and Thompson, 1991).

3. Polycation/Lipid hybrid systems Since a plasmid has a hydrodynamic diameter of -100-200 nm, depending on the number of its base pairs and topology, it is difficult to produce compact particles without efficient DNA condensation (Sternberg et al., 1994). A hybrid DNA system consisting of a polycation-condensed plasmid core and a lipid coating are being developed to allow efficient condensation of plasmid DNA. Either cationic or anionic lipids can be used in their construction. Moreover, anionic lipids may be conjugated with a targeting ligand for tissue-specific gene delivery. Gao and Huang (1996) prepared plasmid/Iipid complexes by adding DNA to the mixture of a polycation (such as poly-L-lysine or protamine) and DC-Cho1:DOPE liposomes. The resulting suspension was then subjected to sucrose density gradient ultracentrifugation to separate the complex from free cationic liposomes.

4. Interaction with biomolecules In witro transfection with cationic lipids is generally best obtained when plasmid/ lipid complexes bear a strong positive charge. However, positively charged complexes may interact with serum proteins, lipoproteins, heparin, and glycosaminoglycans in the extracellular matrix, leading to the aggregation or release of DNA from the complexes even before reaching the target cells. The poor correlation between in vitro and in vivo transfection activities of plasmid/lipid complexes may be in part due to a different biological environment encompass-


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ing the cells (Remy et al., 1994). Cationic liposomes alone or complexed with plasmid have been demonstrated to interact with plasma complement in vitro (Plank et al., 1996). Complement activation may, therefore, lead to the coating of complex with complement proteins, thereby targeting it to complement receptors present on pulmonary endothelium. Although positively charged plasmid/lipid complexes activate the complement system to a considerable degree, no significant difference was seen in biodistribution and gene expression between the complement-intact and complement-depleted mice (Barron et d., 1998). This implies that the interaction between plasmid/lipid complexes and complement proteins does not alter the properties of the injected complexes to the extent that gene delivery is altered. The preparation of negatively charged plasmid/lipid complexes or surface modification of these complexes with a steric stabilizer such as polyethylelene glycol (PEG) are likely to further minimize and possibly avoid activation of the complement system.

5. Target specificity Cationic lipid-based gene delivery systems lack target specificity, which results in low transfection efficiency in certain tissues due to the interference from cationic lipid-binding macromolecules either in the circulation or in the extracellular matrix. The electrostatic interaction between the positively charged plasmid/lipid complexes and the cell membrane usually does not provide cell specificity. To circumvent this problem, neutral plasmid/lipospermine complexes containing a trigalactolipid have been prepared and shown to efficiently transfect hepatoma HepG2 cells bearing asialoglycoprotein receptor. Addition of 25% (mol/mol) of the triantennary galactolipid increased the transfection efficiency by a thousandfold, compared to the lipid-based system with no targeting ligand (Remy et al., 1995). An efficient transfection of P-galactosidase into HeLa cells has been accomplished with the combination of transferrin and cationic liposome LipofectinTM , whereas Lipofectin" alone had low transfection efficiency (Cheng, 1995). Asialofetuin is an asialoglycoprotein containing terminal galactosyl residues that have been used to target liposomes to the liver. (Hara et al., 1995) Templeton et al. (1997) demonstrated sevenfold enhancement in CAT expression in the liver when succinylated asialofetuin was added to preformed plasmid/DOTAP:Chol complexes to provide a ligand for hepatic asiatoglycoprotein receptor.

6. Toxicity Cationic lipids may not be readily metabolized or secreted and, therefore, may accumulate in the body following administration, potentially producing undesirable side effects. Lipids containing ester or amide linkages are more likely to be

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rapidly metabolized than those with ether linkages because of the presence of high concentrations of esterases and peptidases in the body. The degree of toxicity induced by plasmid/lipid complexes has been shown to be dose dependent, which was diminished with time (San et al., 1993). At relatively low concentrations of plasmid/lipid complexes, little or no toxic effect has been reported in mice, rabbits, and pigs after systemic or local injection of the complex (Canonico et al., 1994; Stewart et al., 1992; Nabel et al., 1992). There was no evidence of autoimmunity, biochemical abnormalities, or tissue pathology in these animal models, and the gonadal tissue did not contain plasmids after intravenous and intra-arterial administration (Nabel et al., 1992). Safety studies have also been performed in nonhuman primates by once-a-week intravenous injection of plasmid/DMRIE:DOPE complexes for three weeks (San et al., 1993). The plasmid/lipid complexes did not produce autoimmunity or toxicity, and there were no or mild changes in clinical chemistries, hematology, and tissue histopathology. At high doses, acute inflammation was observed, primarily from the cationic lipid component of the plasmid/lipid complex.

B. Peptide-based gene delivery For site-specific delivery of plasmids, positively charged macromolecules such as poly(L-lysine) (PLL), histones, protamine, or poly(L-ornithine) may be linked to a cell-specific ligand and then bound to plasmids via electrostatic interaction. The resulting complexes retain their ability to interact specifically with target cell receptors, leading to receptor-mediated internalization of the complex into the cells. Receptor ligands currently being investigated include glycoproteins (Wu and Wu, 1988; Findeis et al., 1994), transferrin (Wagner et al., 1990), polymeric immunoglobulin (Ferkol et al., 1993), insulin (Huckett et al., 1990), epidermal growth factor (EGF) (Chen et al., 1994a), lectins (Cheng and Yin, 1994), folate (Gottschalk et al., 1994), malaria circumsporozoite protein (Ding et d., 1995), a*-macroglobulin (Schneider et d., 1996), CD3-T cell (Buschle et d . , 1995), sugars (Chen et d . , 199410; Erbacher et d., 1996), integrins (Hart et al., 1995), thrombomodulin (Trubetskoy et d., 1992), surfactant protein A and B (Ross et d., 1995; Baatz et d., 1995), mucin ("humher et d., 1994), and the c-kit receptor (Schwarzenberger et d., 1996). Site-specific gene delivery and expression are influenced by the extent of DNA condensation, the method of complexation, the molecular weights of both polycations and plasmid, and the number of ligand residues bound per polycation molecule (Erbacher et al., 1995).

1. Poly(L-1ysine)-basedsystems Receptor ligands usually have been conjugated to poly(L-lysine) for site-specific gene delivery. Galactosylated poly(L-lysine) (Gal-PLL) was, for instance, synthesized by reacting PLL (-2000 kDa) with a-D-galactopyranosyl phenyliso-


117

thiocyanate for delivery and expression of genes into the rat hepatocytes (Perales et al., 1994). Similarly, mannosylated poly(L-lysine) (Man-PLL), synthesized using I’LL (-2000 kDa) and a-D-mannopyranosyl phenylisothiocyanate, has been shown to express genes in murine macrophages isolated from peritoneal exudates in vitro and macrophages residing in the liver and spleen of adult animals (Ferkol et al., 1996). Poly(L-lysine) is commercially available in molecular weights ranging from approximately 1 kDa to 300 kDa. However, the preparations are heterogenous, complicating formulation and characterization of DNA condensates. Due to the high polydispersity of poly(L-lysine), the individual molecular species of the polycation interact with DNA with individually distinct kinetics, for both electrostatic and hydrophobic interactions. The extreme heterogeneity greatly confounds both the kinetics of DNA/poly( L-lysine) interaction and the thermodynamic stability of the final DNA complexes. In addition to its molecular heterogeneity, poly(L-lysine) is toxic to living cells in nM concentrations, which limits its general applicability (Smith et al., 1998).

2. Synthetic peptide-based systems To avoid high cytotoxicity, molecular heterogeneity, and possible immunogenicity of poly( L-lysine), molecularly homogenous lysine-rich synthetic peptides have been used for gene transfer. The active sites of enzymes, receptor ligands, and antibodies involve about 5 to 20 amino acids. Thus, it should be possible to use small synthetic peptides to emulate the active sites of viral proteins and fomulate synthetic DNA complexes that are as efficient as viruses, with few limitations (Tomlinson and Rolland, 1996; Duguid et al., 1998). A synthetic peptide-based gene delivery systems has the potential ability to take advantage of specific peptide sequences to overcome extra- and intracellular barriers to gene delivery. Specific sequences of interest for gene delivery include DNA binding and protecting peptides, peptide ligands for receptor-mediated uptake, peptides with endosomolytic properties to release DNA from the endosomes, and peptides that facilitate nuclear transport of DNA. Synthetic peptide-based gene delivery systems consisting of a lysinerich DNA binding motif and a pH-sensitive endosome-lytic motif have been developed for in vivo gene delivery and expression (Tomlinson and Rolland, 1996; Wadhwa et al., 1997). Molecular modeling of condensing and endosomolytic peptides is shown in Figure 4.5. One example of such a gene delivery system is composed of (i) a galactosylated peptide that both condenses the plasmid into monodisperse nanoparticles of about 100 nm in diameter and enables specific recognition and binding to asialoglycoprotein receptors, and (ii) an amphipathic, pH-selective peptide that enables the plasmid to leave the endosomes prior to their fusion with lysosomes and entry into the cytoplasm (Plank et al., 1994; Gottschalk et al., 1996).

118

R. I. Mahato, 1. C. Smith, and A. Rolland Lytic Peptide GLFEALEELWAK

Condensing Peptide KKKKKKKKWK

k

Dipalmitoylated Lytic Peptide GLFEALEELWEAK-e-(G-PamJ

Dipalmitoylated Condensing Peptide a,E-(PamJKKKKKKKKKWK

Figure 4.5. Molecular configuration of peptides. Condensing and endosomolytic peptides.

3. Lipopeptides The improved DNA binding and condensation provided by amino acids such as tryptophan suggest that the inclusion of hydrophobic interactions within DNA complexes may be beneficial. Peptides with moities that provide cooperative hydrophobic behavior of the alkyl chains of cationic lipids would improve the stability of the peptide-based DNA delivery systems. Smith and associates (1998) have constructed two general classes of lipopeptide analogs of the TyrLys-Ala-Lys,-Trp-Lys peptides by including a hydrophobic anchor. The general structures are N, N-dialkyl-Gly-Tyr-Lys-Ala-Lys,-Trp-Lys and N",N*-diacyl-LysLys,-Trp-Lys. These peptides differ from the parent structures in that they selfassociate to form micelles in aqueous solutions. The inclusion of dialkyl or diacyl chains in the cationic peptides improves the peptide ability to bind DNA and reduces aggregation of the complexes in ionic media.


119

4. Endosomolytic peptides Short synthetic peptides containing the first 23 amino acids of the HA2 subunit of influenza hemagglutinin protein (HA) are attractive because of their pHdependent lytic properties, with little activity at pH 7 but greater than or equal to a 100-fold increase in transfection efficiency at pH 5 . The lytic characteristics of the peptides are revealed as the carboxyl groups of the aspartyl and glutamyl side chains are protonated, which allows the peptides to assume an a-helical conformation that can be inserted into the membrane bilayer (Rafalski et al., 1991; Lear and De Grado, 1987). Plank et al. (1994) have used a series of these peptides derived from influenza HA to achieve endosomal rupture and thereby enhanced gene expression in vitro. Gottschalk et al. ( 1996) developed an amphipathic membrane-associating peptide, JTS-1, Gly.Ileu-Phe-Glu-Ala-Leu-Leu-Glu-Ser-Leu-Trp-Glu-LeuLeu-Leu-Glu-Ala. The hydrophobic face contains only strongly apolar amino acids, while negatively charged glutamic acid residues dominate the hydrophilic face at physiological pH. The hydrophobic face of JTS-1causes self-association and forms pores in one side of the endosomal membrane, thereby destabilizing the membrane, which leads to its rupture. The cationic DNA complex formed with the condensing peptide Tyr-Lys-Ala-LysgTrp-Lys is rapidly mixed with negatively charged JTS-1, which spontaneously incorporates through electrostatic interactions to form the tertiary complex. At a given charge ratio of condensing peptide to plasmid, the transfection efficiency has been shown to be proportional to the concentration of the endosomolytic peptide added to the complex. The pH-selective peptides form a-helices at acidic pH but not at pH 7 (Figure 4.6). This structural conformation favors partitioning of the amphipathic peptides into the endosomal membrane and promotes DNA release from the endosomal compartment into the cytoplasm. In vitro transfection efficiency was up to 10,000-fold higher than that of DNA/Tyr-Lys-Ala-Lys8-Trp-Lys complex alone (Gottschalk et al., 1996).

C. Polymer-based gene delivery 1. Noncondensing polymer-based systems Protective, interactive, noncondensing (PINC'" ) systems, such as polyvinyl polymers, have been postulated to form hydrogen bonds with DNA base pairs, resulting in a hydrophobic coating of the plasmid by the vinyl backbone (Mumper et al., 1996). Polyvinyl pyrrolidone (PVP)-based formulations are hyperosmotic and result in an improved dispersion of plasmids through the extracellular matrix of solid tissues (e.g., muscle), possibly by (i) protecting plasmids from nuclease degradation, (ii) dispersing plasmids in the muscle, and (iii) facilitating their uptake by muscle cells. By increasing the hydrophobicity of plasmids and

120


Lytic Peptide at pH 4

Lytic Peptide at pH 7

Figure 4.6. Effect of pH on the molecular configuration of lytic peptide Gly-Leu-Glu-Ala-Leu-GluGlu-Leu-Trp-Glu-Ala-Lys.

reducing their net negative surface charge, the PINC'" polymers may facilitate the uptake of plasmids by muscle cells. Intramuscular injection of PVP-based plasmid formulations in rats significantly increased the number and distribution of expressing cells, as compared to unformulated plasmid (Mumper et al., 1998). Up to a 10-fold enhancement of gene expression over unformulated plasmid has been observed in mouse and rat muscle. N-methyl-2-pyrrolidone (NMZP), which is a methylated monomer of PVP, also enhances gene expression in rat skeletal muscle. Five percent NMZP in saline containing 100 p g CMV-driven P-galactosidase (P-gal) expression plasmids has been shown to express levels of P-gal, which are approximately two-fold higher than that observed using a PVPbased formulation (Mumper et al., 1996). Kabanov and associates (1991 and 1995) have proposed the formation of condensed interpolyelectrolyte complexes between polyvinyl pyridinium, and DNA has been proposed to both protect DNA from nuclease degradation and facilitate its cellular uptake by hydrophobic interactions with cell membranes (Kabanov et al., 1991 and 1995). The increased hydrophobicity of the complex may enhance interaction with cell membranes and facilitate cell uptake.


121

2. Cationic polymer-based systems Cationic polymers such as polybrene and diethylaminoethyldextran (DEAEdextran) have been used for transfection of genes into cultured cells (Holter et al., 1989). However, these polymers cannot be used for in vivo application due to their poor transfection efficiency and high cytotoxicity. StarburstTM polyamidoamine (PAMAM) dendrimers are a class of highly branched spherical polymers whose surface charge and diameter are determined by the number of synthetic steps (Tomalia et al., 1990). For example, five polymerization cycles produce the 5th-generation dendrimers. The major structural differences in PAMAM dendrimers relate to the core molecules, either ammonia or ethylenediamine, with which the stepwise polymerization process begins and which dictates the overall shape, density, and surface charge of the molecule. Dendrimers can condense plasmids through electrostatic interactions of their terminal primary amines with the DNA phosphate groups. The effect of colloidal and surface characteristics of plasmid/dendrimer complexes on gene transfer has been examined (Mumper et al., 1995). These complexes were monodisperse with a mean hydrodynamic diameter of about 200 nm. The particle size, surface charge, and gene transfer efficiency of plasmidldendrimer complexes prepared with the 5th-generation of dendrimers has been shown to be influenced by dendrimer concentration in the complexes. Furthermore, covalent attachment of fusogenic peptide GALA to the dendrimer has been shown to significantly enhance gene transfer efficiency (Haensler and Szoka, 1993). Kukowska-Latalla e t al. (1996) have recently shown that DEAE-dextran facilitates the formation of small particles from the large dendrimer/plasmid aggregates and significantly improves transfection in vitro. Polyethyleneimine (PEI) is a branched cationic polymer and has been shown to condense plasmids into colloidal particles that effectively transfect genes into a variety of cells in vino (Boussif et al., 1995). In addition to enhancing cellular uptake of plasmids by nonspecific adsorptive mechanisms, PEI may also enhance the intracellular trafficking of plasmids by buffering the endosomal compartments, thus protecting plasmids against degradation and enabling endosomal release of plasmid via lysosomal osmotic swelling and disruption (Abdallah et al., 1996; Dunlap et al., 1997). Conjugation of targeting ligands, such as transferrin or anti-CD3 antibody, to PEI has recently been shown to enhance transfection efficiency by -30- 1000-fold compared to ligand-free PEI in various tumor cell lines. This activity depends on the ligand/ receptor interaction and has also been observed at low PEI/DNA charge ratios where ligand-free PEI lacks efficiency (Kircheis et al., 1997). Chitosan is a biodegradable polysaccharide composed of two subunits, D-glucosamine and N-acetyl-D-glucosamine, linked together by @(1,4) glycosidic bond (Tang and Szoka, 1997a; Richardson et al., 1997). Chitosan has been

122


shown to interact with the phosphate groups of DNA, condensing plasmids into spherical and toroidal particles. The colloidal and surface properties of plasmid/ chitosan complexes have been shown to depend on the molecular weight of chitosan, the ratio of plasmid to chitosan, and the preparation medium. Smaller nanoparticles have been observed with low molecular weight chitosan ( 2 kDa) as compared to high molecular weight chitosan (540 kDa). A number of cell lines have been transfected with plasmid/chitosan complexes (Mumper et al., 1995). Poly(2-dimethy1amino)ethyl methacrylate (PDMAEMA) has also been evaluated for transfecting plasmids encoding the P-galactosidase gene in COS7 cell lines in vitro (Chemg et al., 1996). The optimal transfection efficiency was found at a PDMAEMA/plasmid ratio of 3:l (w/w), the ratio at which homogeneous complexes of about 150 nm in diameter could be formed. Interestingly, the transfection efficiency of the complexes was not affected by the presence of serum proteins, even though the presence of serum is known to adversely affect the transfection efficiency (Zelphati et al., 1998). Poly(ethy1ene glycol)-poly(L-lysine) block co-polymers have been shown to form complexes with DNA that can transfect human embryonal kidney cells in vitro (Wolfert et al., 1996).

3, Structures of cationic polymers Poly(L-lysine) is a linear polymer, whereas dendrimers and polyethyleneimine are branched polymers. The structures of branched polymers can be further distinguished by their symmetry of branching. Dendrimers are radially branched, whereas polyethyleneimine lacks a defined center of symmetry. Dendrimers can be either intact or fractured. Intact dendrimer has two arms extending from every branch point, whereas fractured dendrimer has zero, one, or two arms extending from each branch point (Tang and Szoka, 1997a). The major differences between the cationic polymers with respect to chemical structure are the type and relative number of protonatable amines. All the polymers possess primary amines, which are predominantly protonated at neutral pH. The acidbase titration curves of dendrimers and polyethylenimine exhibit considerabIe buffer capacity over almost the entire pH range. In contrast, poly(L-lysine) shows little buffer capacity below pH 8, as shown by the nearly vertical slope of the titration curve below this point. The complex particle size in solution of poly(L-lysine) or intact dendrimer is much larger than that of the fractured dendrimer and polyethylenimine. All the polymers demonstrate their maximum transfection activities at charge ratios with an excess of primary amines to DNA phosphates. Despite the vast differences in structure of these cationic polymers, the plasmid/cationic polymer complexes have similar toroid morphology. The diameters of toroids vary slightly with the cationic polymer structure, although the differences do not appear to correlate


123

with the physical size of the cationic polymer. For example, intact dendrimers yield toroids that have a significantly smaller diameter than toroids formed from the fractured dendrimer, although the intact dendrimer has nearly twice the molecular weight of the fractured dendrimer (Tang and Szoka, 1997a).

A. DNA topology Plasmids may exist in three tertiary structures: supercoiled, open circular, and linear. An open circular molecule is formed by nicking one strand of the DNA, which relaxes the torsional stress on the supercoiled plasmid. A linear molecule is formed by breaking the double-stranded DNA sequences (Ledley, 1996). DNA topology influences both the colloidal behavior and condensation of DNA. Topologically constrained circular DNA may contribute bending energy to the condensing system through torsional elasticity. Thus, supercoiled plasmids should yield smaller toroids. For instance, Wilson and Bloomfield (1979) observed for hexamidine cobalt (111) condensates that closed circular plasmid yielded multimolecular toroids 25 -30% smaller in diameter than those made up of linearized plasmid.

B. DNA condensation The extent of DNA condensation has great implications for gene delivery and expression. Recent progress in our understanding of DNA condensation includes the observation of DNA collapse, greater insights into the intramolecular forces driving condensation, the recognition of helical structure perturbation in condensed DNA, and the increasing recognition of the likely biological consequences of condensation (Bloomfield, 1991). Unfavorable free energies associated with DNA bending, entropy of mixing, and electrostatic repulsion forces must be overcome to condense plasmid through the use of multivalent organic or inorganic cations. Although small multivalent cations bind and condense DNA, they are highly mobile. Therefore, they can easily be displaced by compounds with higher charge, and their complexes have a tendency to aggregate (Bloomfield, 1996).

C. DNA condensing agents Cations of three or more charges condense DNA in aqueous suspension primarily to toroids, although other condensation states have been observed. Divalent cations, such as MgZ+*can also condense DNA in the presence of sufficient mole fraction of alcohol (Sharp and Honig, 1995). Toroids of similar shape and size can also be formed by spermine, spermidine, hexamine cobalt (III), and

124

R. I. Mahato, 1. C. Smith, and A. Rolland

conjugated polylysines as well as various branched cationic polymers (Tang and Szoka, 199715).Flexibility in the spatial arrangement of positive charges provides many options in the design of cationic agents that can effectively condense DNA. In addition to salt-dependent electrostatic interaction, the ion atmosphere and dielectric constants are major factors in determining the stability, structure, reactivity, and binding behavior of nucleic acids (Sharp and Honig, 1995). The DNA condensate size is dependent on the type of condensing agents used. For instance, calf thymus DNA condensed by hexamine cobalt (111) has been shown to yield toroids that were substantially smaller in diameter than those of spermidine or methylated spermidine analog (Sharp and Honig, 1995). Nonelectrostatic factors, such as bridging between helices, hydration forces, or degree of hydrogen bonding may influence the contribution of cationspecific interaction to toroid size. The extent of DNA condensation depends on a number of variables, including the method of complexation, types of cationic carriers, buffers, counter-ions, and the size, sequence, and topology of plasmid.

D. DNA aggregation Cationic carriers form complexes with plasmid via electrostatic interactions. The large population of these complexes has wide particle-size distribution due to the heterogeneity of some condensing carriers (Perales et al., 1994). Nearneutral (“isoelectric”) plasmid/cationic carrier complexes usually have a strong tendency to form large aggregates over time, whereas complexes carrying a net negative or positive charge are relatively stable. Aggregation is probably a result of charge and/or hydrophobic interactions between the plasmid/cationic carrier complexes. Complexes prepared at very high ionic strength or formed at a high DNA concentration generally have a greater tendency to form aggregates over time. Insufficient or rapid vortexing of plasmid/cationic carrier complexes can also lead to aggregation. The technical difficulty in forming stable plasmid/ cationic carrier complexes at high DNA concentrations may be partially overcome by formulation using a large excess of cationic carriers. The excess in positive charge prevents rapid aggregation of the complexes during mixing. The resulting uncomplexed carriers can then be separated from the complex formulations by sucrose density gradient centrifugation (Lee and Huang, 1997). However, aggregation of purified complexes can occur following interaction with blood components.

VI. BlODlSTRlBUTlON AND PHARMACOKINETICS OF PLASMIDS Since plasmids and the carrier molecules have very different physicochemical properties, a thorough understanding of the anatomy and (patho)physiology of


125

target organs as well as the physicochemical characteristics of both active and carrier molecules is necessary. Biodistribution of plasmid DNA to either extracellular or intracellular targets is dependent on the structure of capillary walls, (path0)physiological conditions, the rate of blood and lymph supply, and the physicochemical properties of plasmid and its carrier molecules. These properties include molecular size, electrical charge, and physical forms and targeting group (if present), and an interaction with blood proteins (Tomlinson, 1987). The fate of plasmid after in vivo administration is illustrated in Figure 4.7.

A. Anatomical and physiological considerations The blood capillary walls are generally comprised of four layers, namely plasmaendothelial interface, endothelium, basal lamina, and adventia. The endothelium is a monolayer of metabolically active cells, which mediate and monitor the bidirectional exchange of fluid between the plasma and the interstitial fluid. There are several different pathways by which macromolecules can cross the endothelial barrier (Simionescu, 1983; Taylor and Granger, 1984): (i) through the cytoplasm of endothelial cells themselves; (ii) across the endothelial cell membrane vesicles; (iii) through interendothelial cell junctions; and (iv) through endothelial cell fenestrae. Based on the morphology and continuity of the endothelial layer and the basement membrane, capillary endothelium can be divided into three categories continuous, fenestrated, and discontinuous endothelium.

Figure 4.7. Fate of plasmid DNA after in vivo administration.

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R. 1. Mahato, 1. C. Smith, and A. Rolland

The continuous capillaries are found in skeletal, cardiac, and smooth muscles, as well as in lung, skin, and subcutaneous and mucous membranes. The endothelial layer of the brain microvasculature is the tightest endothelium, with no fenestrations. This endothelial barrier forms a continuous cellular layer between the blood and brain interstitiurn, which is impermeable to plasmids. Capillaries with fenestrated endothelia and a continuous basement membrane are generally found in the kidney, small intestine, and salivary glands. Most of these capillaries have diaphragmed fenestrae, which are circular openings of 40-60 nm in diameter. The discontinuous capillaries, also known as sinusoidal capillaries, are common in the liver, spleen, bone marrow, and other organs of the reticuloendothelial system. These capillaries show large interendothelial junction (fenestrations up to 150 nm). Depending on the tissue or organ, the basal membrane in sinusoidal capillaries is either absent (e.g., in liver) or present as a discontinuous membrane (e.g., in spleen and bone marrow) (Venkatachalam and Rennke, 1978). The sinusoids of the liver are lined by highly phagocytic Kupffer cells, and those of the bone marrow by flattened, phagocytic reticuloendothelial cells. In the spleen, the endothelial cells are greatly elongated and contain a large number of pinocytic vesicles (up to 100 nm in diameter). Due to their large molecular weight (greater than 1000 kDa) and hydrodynamic diameter in aqeuous suspension of 100 nm (Ledley, 1996), plasmids extravasate poorly via continuous capillaries because of tight junctions between the cells. However, plasmids can easily extravasate to sinusoidal capillaries of liver and spleen. Formulating plasmids into unimeric particles of 20-40 nm in diameter may enhance extravasation of plasmids across continuous and fenestrated capillaries.

B. Influence of (patho)physiology on biodistribution Inflammation is associated with regional changes in the structure, chemical composition, and increased permeability of the endothelium. Increase in transport of macromolecules at inflammation sites is due to openings in the endothelium at the level of postcapillary venules. Molecules greater than 50 kDa usually do not extravasate in normal tissues; in inflamed and tumor tissues this limit is significantly increased (Arfors et al., 1979). The ( patho)physiology and microanatomy of tumors are significantly different from normal tissues. A tumor contains vessels recruited from the preexisting network and vessels resulting from angiogenic response reduced by cancer cells. There is a considerable variation in the cellular composition and basement membranes and in the size of the interendothelial cell fenestrations (lain, 1989). Tumor interstitium is characterized by large interstitial volume and high diffusion rate (Takakura and Hashida, 1995). The high interstitial pressure of the tumor retards the extravasation of macromolecules, whereas large


127

vascular permeability and high interstitial diffusivity of macromolecules facilitate their migration to tumor tissues. Tumor accumulation of plasmid could result from the enhanced permeability of the tumor vasculature, combined with reduced clearance from the tumor due to the absence of the lymphatic system.

C. Biodistribution and pharmacokinetics of plasmid DNA The biodistribution of plasmid DNA can be determined by measuring the rate of disappearance of radio-labeled DNA from the bloodstream and its accumulation in tissues or by the use of fluorescence microscopy to trace the leakage of dye-labeled plasmids from the vasculature. Pharmacokinetic analysis of in vivo disposition profiles of radio-labeled plasmid DNA provides useful information on the overall distribution characteristics of systemically administered plasmids, with one critical limitation. The radio label represents both intact plasmid and its metabolites. The plasma half-life of plasmid is less than 10 min (Kawabata et al., 1995), and hence tissue distribution and pharmacokinetic parameters of plasmid DNA calculated on the basis of total radioactivity are not valid at longer time points. Thus, polymerase chain reaction (PCR) and Southem-blot analysis are required to establish the time at which the radio lable is no longer an index of plasmid distribution. Even after local administration, it is important to understand pharmacokinetics at both the organ and systemic levels because a part of injected plasmid will enter the blood circulation. Systemic disposition processes involve interaction with blood components and/or vascular endothelial cells, organ distribution, and uptake by reticuloendothelial systems (RES) before reaching the target site. In case of parenteral administration, movement in the tissues and absorption via capillary and lymphatic routes should be considered. In the early phase of distribution, the movement of plasmid DNA from the circulation to organs is roughly a unidirectional process in many organs. Thus, the disposition characteristics of plasmid DNA can be characterized using organ uptake clearance (Clorg)as an essential index of distribution to each organ. Total body clearance (Cltotal) is equal to the sum of individual organ clearance values (Figure 4.8). The deposition of plasmids after systemic administration is restricted to the intravascular space due to its low microvascular permeability in most organs with continuous capillary bed. Some organs with fenestrated capillaries, such as liver, spleen, and bone marrow, provide some opportunities for extravasation of plasmid DNA. Intravenously injected plasmids initially perfuse the pulmonary vascular beds, maximizing the potential uptake of plasmid DNA in the lung endothelial cells soon after administration. Based on the clearance concept, Kawabata et al., (1995) and Mahato et at. ( 1995a,b) determined the pharmacokinetic parameters of plasmid DNA after tail vein rejection of [32P]pCMV-CATin mice. The radioactivity was rapidly

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Organ 1

Interstitial Space

CLI yrn ph CL acm m .

- 1-+ Q

AUCp 1 Ch"t,r

=CLorg,iAUCp X; : total amount of drug accumulated in the organ i

Figure 4.8. Physiological pharmacokinetic model for evaluating biodistribution of plasmid (adapted from Mahato et al., 1997a, with permission).

eliminated from the circulation due to the extensive uptake by the lung and liver, while it was not susceptible to glomerular filtration because of the presence of the basement membrane. Pharmacokinetic analysis under conditions with minimal enzymatic degradation, derived from [32P]pCMV-CAT up to 1 min after injection, has demonstrated that the hepatic uptake clearance of pCMVCAT is almost identical to the plasma flow rate in the liver (Figure 4.9) which indicates that plasma DNA is cleared substantially on first-pass of the liver. At the later phase following intravenous injection of [32P]pCMV-CAT,the proportion of the radioactivity accumulated in the liver decreased with time, probably due to the release of degradation products into the plasma pool and accumulation of radioactivity in the kidney. In addition, pCMV-CAT was prefentially taken up by the liver nonparenchymal cells (NPC). Scavenger receptor-mediated processes are involved in the uptake of large anionic molecules (Kawabata et al., 1995; Yoshida et al., 1996). Autoradiography of mouse whole body after intravenous injection of [32P]plasmid/lipidcomplexes has shown DNA localization predominantly in the lung, with notable uptake in the liver and other tissues containing RES cells. In constrast, the autoradiograph of mouse whole body after intravenous injection of free [33P]plasmidDNA showed the highest levels of radioactivity in the

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