Polytene Chromosomes: 70 Years of Genetic Research

Polytene Chromosomes: 70 Years of Genetic Research I. F. Zhimulev,* E. S. Belyaeva,* V. F. Semeshin,* D. E. Koryakov,{ S. A. Demakov,* O. V. Demakova,* G. V. Pokholkova,* and E. N. Andreyeva* *Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, 630090, Russia Novosibirsk State University, Novosibirsk, 630090, Russia

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Polytene chromosomes were described in 1881 and since 1934 they have served as an outstanding model for a variety of genetic experiments. Using the polytene chromosomes, numerous biological phenomena were discovered. First the polytene chromosomes served as a model of the interphase chromosomes in general. In polytene chromosomes, condensed (bands), decondensed (interbands), genetically active (puffs), and silent (pericentric and intercalary heterochromatin as well as regions subject to position effect variegation) regions were found and their features were described in detail. Analysis of the general organization of replication and transcription at the cytological level has become possible using polytene chromosomes. In studies of sequential puff formation it was found for the first time that the steroid hormone (ecdysone) exerts its action through gene activation, and that the process of gene activation upon ecdysone proceeds as a cascade. Namely on the polytene chromosomes a new phenomenon of cellular stress response (heat shock) was discovered. Subsequently chromatin boundaries (insulators) were discovered to flank the heat shock puffs. Major progress in solving the problems of dosage compensation and postion effect variegation phenomena was mainly related to studies on polytene chromosomes. This review summarizes the current status of studies of polytene chromosomes and of various phenomena described using this successful model. KEY WORDS: Polytene chromosomes, Puffs, Ecdysone, Heat shock, Heterochromatin, Intercalary heterochromatin, Position effect variegation, Telomeres. ß 2004 Elsevier Inc.

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Copyright 2004, Elsevier Inc. All rights reserved.

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I. Introduction Polytene chromosomes were discovered by E. G. Balbiani (College de France, Paris) in 1881 in the larval salivary glands, Malpighian tubules, intestine, hypoderm, and muscles of Chironomus plumosus as a cylindrical cord that repeatedly unraveled and filled the nucleus. This structure was termed a ‘‘permanent spireme.’’ Nine years later, in 1890, Balbiani found a permanent spireme in the macronucleus anlage of the infusorian, Loxophyllum meleagris (Balbiani, 1881, 1890). At the end of the nineteenth century giant spireme nuclei were found in plant cells (Osterwalder, 1898; Strasburger, 1887). However, for a long time it was unclear what kind of structure this permanent spireme represented. Rambousek (1912) was the first to propose that it corresponded to mitotic interphase chromosomes, but his paper, written in Czech, did not attract attention. In 1933–1934, three groups of researchers (Heitz and Bauer, 1933; King and Beams, 1934; Painter, 1933, 1934), using a method of squashed preparations, showed that the ‘‘spireme’’ is not continuous but consists of separate elements whose number is close to the haploid number of the mitotic chromosomes. Each element is formed as a result of the tight synapsis of the homologous chromosomes. Each chromosome has a definite and constant morphology and is composed of segments, each showing a distinctive transverse-banding pattern (Fig. 1). In the same year Painter (1934) obtained conclusive evidence concerning the chromosomal nature of the ‘‘spireme.’’ Using a series of chromosomal rearrangements with break points in known regions of chromosomes, he mapped 22 genes and demonstrated the complete linear correspondence of their order on the genetic and chromosome maps, both mitotic and ‘‘spiremic.’’ KoltzoV (1934) proposed that the giant size of the salivary gland chromosomes was a consequence of multistrandedness. The term ‘‘polytene’’ was proposed by Koller (1935) and finally adopted by Darlington (1942). Since 1934 the polytene chromosomes have become one of the most important models in genetic research and, therefore, in 2004 we celebrate the 70-year anniversary of the rediscovery of polytene chromosomes. The characteristics of polytene chromosomes were so impressive and promising for geneticists that Painter wrote: ‘‘it was clear that we had within our grasp the material of which everyone had been dreaming. . . the highway led to the lair of the gene.’’ Polytene chromosomes are gigantic interphase chromosomes, which represent very important models for the analysis of the genetic organization of chromosomes and the genome as a whole. At first, data obtained from polytene chromosomes provided final proof for the chromosome theory of heredity. The first time gene activity was observed directly under the

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FIG. 1 First picture of a Drosophila melanogaster polytene in comparison with mitotic chromosomes of this species chromosomes. (From Painter, 1934, with minor changes.)

microscope was the puV formation. Later, polytene chromosomes were used to analyze hormonal action on gene activity and diVerential gene (puYng) activity. Using polytene chromosomes the heat shock phenomenon was discovered in the early 1960s and gene insulators in the 1980s. Data on polytene chromosomes were very important for understanding the organization of specific structures of interphase chromosomes, bands and interbands, dosage compensation, telomere organization, heterochromatin structure, and position eVect variegation phenomenon. Polytene chromosomes are indispensable for gene mapping by means of chromosome rearrangements and nucleic acid in situ hybridization, and indirect immunofluorescence antibodies against numerous proteins. Among other advantages of Drosophila as a model organism, use of polytene chromosomes proved to be decisive in the rapid development of cloning methods (e.g., chromosome walking and jumping) as well as genomic projects. A great step forward in understanding the organization and function of the chromosome and genome came with the development of methods to microclone chromosome regions, making it possible to choose a chromosome region, dissect it out with a micromanipulator, and finally generate a library of DNA clones from the region.

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In this review the main characterisitics of polytene chromosomes as well as discoveries concerning gene, chromosome, and genome organization made with the use of polytene chromosomes will be described. Abundant information on the structure and organization of polytene chromosomes can be found in reviews by Beermann (1962, 1972), Ashburner (1970), Kiknadze (1972), Berendes (1973), Ashburner and Berendes (1978), Nagl (1985), Richards (1985), Sorsa (1988a,b), Zhimulev (1996, 1998, 1999), Zybina and Zybina (1996), Michailova (1998), Daneholt (2001), Henderson (2004), and Ashburner et al. (2004).

II. Organization of Polytene Chromosomes A. General Description Cells with polytene chromosomes diVer in the following features from those of mitotically dividing cells and those undergoing endomitosis. First, the formation of polytene chromosomes is associated with the elimination of the entire mechanism of mitosis after each DNA doubling, so that the cell cycle consists of just two alternating periods, synthetic (S) and intersynthetic (G). The polytenization cell cycle is set during mid embryogenesis in D. melanogaster. Second, at the end of each replication period, sister chromatids do not segregate; rather, they remain paired to each other to diVerent degrees. It is known that the escargot gene encoding for the snail family of transcription factors in D. melanogaster is needed to maintain the imaginal disk cells in the diploid cell cycle. Its expression in such tissues inhibits polyteny. This gene does not function in larval tissues having polytene chromosomes. Similarly, the mouse gene mSna that is most homologous to escargot is downregulated during trophoblast diVerentiation and polytenization (Edgar and Orr-Weaver, 2001). Third, the polytene chromosomes formed are incapable of being involved in mitosis. Fourth, the nuclear membrane and nucleolus remain intact during consecutive DNA replication cycles.

1. Polyteny Polyteny arises and attains high levels in tissues, organs, and at developmental stages when rapid development of an organ at an unaltered high level of function is needed. Organs containing cells with polytene chromosomes are, as a rule, involved in intense secretory functions accomplished during a short time against a background of rapid growth. The features of polyteny provide the condition necessary to accomplish these functions.

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Inasmuch as the entire mechanism of nuclear and cell division is completely blocked during the cell cycle the process of chromosome replication is maximally simplified and accelerated. This reduction certainly confers significant advantages in terms of energy and time. As a consequence, the mass of an organ increases at a much higher rate as a result of polytenization than it would do by mitotic division of diploid cells. It is also obvious that the cycle of polytene cells ensures the maintenance of high functional activity by the organ, there being discontinuities due to mitosis. More details are in Zhimulev (1996). 2. Morphology of Polytene Chromosomes The morphology of polytene chromosomes varies subsubstantially due to diVering degrees of synapsis of each chromosome element (chromatid). The polytene chromosomes develop from the chromosomes of diploid nuclei by successive duplication of chromatids. If homologous chromatid conjugation is maximal, classic polytene chromosomes, i.e., cylindrical cable with a distinct banding pattern, such as those described for Chironomus tentans or D. melanogaster, are formed. But the degree of the chromatid conjugation can vary widely. Tight alignment side by side of the chromatids results in formation of classic polytene chromosomes, cable-like structures with clear banding patterns. If it is minimal, a polyploid nucleus with a reticular structure is formed (cryptic polyteny). The state of chromatid conjugation can be transient. The disintegration, by fibrillation, of polytene chromosomes resulting in typical reticular endopolyploid nuclei is normal for salivary gland nuclear development in many Cecidomyiid larvae. Sharp diVerences in the degree of chromatid conjugation may be related to tissue-specific features of cells. In many dipteran species, polytene chromosomes of the classic type are detected in salivary gland cells. However, in ovarian nurse cells they are identified in only a few species. In other species ovarian nurse cell nuclei are highly polyploid and reticular, i.e., they show cryptic polyteny. The role of genetic variation of the degree of chromatid conjugation has been clearly demonstrated by Ribbert (1979), who has succeeded in obtaining classic polytene chromosomes of ovarian nurse cells by inbreeding Calliphora erythrocephala. After establishing the stocks with classic polytene chromosomes in ovarian nurse cells, he mated these stocks with each other and in the first generation all nuclei were of reticular structure. Very often excellent polytene chromosomes develop in the ovarian nurse cells of Anopheles mosquitos after bloodsucking (Zhimulev, 1996). Classic polytene chromosomes in nurse cells of D. melanogaster form in mutants such as ovarian tumor (otu) and fs(2)B, while in normal development of this species nurse cell nuclei are reticular (King et al., 1981; Koryakov et al., 2004).

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As a rule, polytene chromosome bands become more distinct at low temperatures, even in the case of classic polytene chromosomes. The role of other environmental eVects on polytene chromosome formation is best demonstrated by Aphiohaeta xanthina. When larvae develop under normal nutrient conditions classic polytene chromosomes are seen in their salivary glands; when their food contains little protein and fat, the nuclei become reticular (Zhimulev, 1996). Striking changes in the general morphology of the polytene chromosomes take place in beans (Phaseolus coccineus and Ph. vulgaris). Under the eVect of low temperature, polytene chromosomes
developing at 20–22 C look like irregular bundles of chromatids, whereas in plants developing at low temperatures (12
C in the day and 8
C in the night), the chromosomes become shorter and assume a distinct banding pattern, i.e., they are a classic type (Nagl, 1969). Viewed broadly, the morphology of polytene chromosomes in females and males appears to be the same. The X chromosome of Drosophila males, however, has a diVerent appearance. Although the polytene X chromosome of the male, as expected, contains half as much DNA as the female, it occupies almost the same area in cytological preparations as the two female X chromosomes (Belote and Lucchesi, 1980; Berendes, 1966; Tan, 1935). These diVerences are due to the dosage compensation phenomenon (see below). In some cases conjugation of chromatids is disturbed only in one or several chromosomes of the set. This polytene chromosome then completely loses its banding pattern and looks diVuse, the so-called ‘‘pompon’’like chromosome (Pavan et al., 1969). The pompon-like chromosomes are formed more frequently from the male X chromosome and less frequently from female X chromosomes and autosomes. They occur during normal development and various physiological conditions. Among the last it is worth mentioning diVerent mutations in Drosophila, eVects of intracellular infections by microorganisms, and the very long life of cells out of the host organism: in vitro cultivation or continuous cultivation of the transplanted organs in the recipient’s organism. Incorporation of both [3H]uridine and [3H]thymidine in pompons does not diVer from those in normal chromosomes. All references and details are in the review by Zhimulev (1996). Recently new approaches appeared for the analysis of pompon-like chromosomes. The male X chromosome of the ISWI mutant in D. melanogaster converts into pompon. The DNA sequence of this gene shares strong homology with the SWI/SNF family of genes encoding ATPases, which are involved in chromatin remodeling. The ISWI gene codes for the catalytic subunit of three chromatin remodeling complexes: NURF, CHRAC, and ACF (Deuring et al., 2000). The specific pompon-like structure is likely related to interactions of the ISWI protein with the complex of dosage compensation proteins and acetylated histones of male X polytene chromosome (Corona et al., 2002).

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The mutation of the Su(var)3–7 gene, which is a suppressor of position eVect variegation (see below), also induces a pompon-like structure of the male X chromosome and loss of activity of mle—one of the genes encoding for proteins of the dosage compensation complex results in improvements of chromosome integrity (Spierer et al., 2003). 3. Occurrence of Polyteny Polytene chromosomes have been found in many tissues of the representatives of two orders of insects: Diptera and Collembola, in the macronuclear anlagen of Infusoria, in certain organs and tissues of mammals, and also in the cells of the synergids, antipods, and endosperm of angiospermous plants (Table I). 4. Multistrandedness of Polytene Chromosomes Growth resulting from an increase in size of relatively few cells, rather than an increase in cell number through cell division, is a phenomenon well known in the Insecta. Such growth is, of course, accompanied by a parallel increase in nuclear size and DNA content. At present, there is numerous and varied evidence for polytene chromosomes being a bundle of individual chromatids. Beermann and Pelling (1965) were the first to provide autoradiographic evidence that the polytene chromosome of Chironomus tentans consists of unit chromatids, apparently extending the entire length of the polytene structure. Later, DuPraw and Rae (1966) proposed a ‘‘folded fiber’’ model of polytene chromosome structure according to which unit chromatids join each other and bands were posited to be regions of chromatid coiling separated by interbands, regions of low DNA density. Using quantitative Southern blot hybridization to measure DNA levels across a contiguous 315-kb stretch, Spierer and Spierer (1984) found similar levels of DNA between 13 pairs of bands and interbands in the region 87D5–E6. In the interband regions, where chromosome material is more decompacted, fibrils distributed along the chromosome axis can be easily seen under the electron microscope and can be counted (Ananiev and Barsky, 1985). All these data demonstrate that there is an equal level of polyteny along the euchromatic arms, excluding several types of chromosome regions such as pericentric and intercalary heterochromatin. Polyteny levels (i.e., the number of chromatids in one polytene chromosome—C) diVer considerably in diVerent cells within an organ, between organs, and between organisms and species, as can be clearly seen from the data presented in Table II. Table II lists some of the quantitative studies done on tissues of diVerent organisms, most of which used Feulgen

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TABLE I Occurrence of Polytene Chromosomes in Animals and Plantsa Organism Insects, Diptera

Organ

Type of polyteny

Larva Salivary glands, gut, midgut, hindgut, gastric caeca, Malpighian tubules, fat bodies, epidermal cells, hypoderm, ring gland

Classic type

Pupa Malpighian tubules, cardiac wall, fat body, rectum, foot pad cells, bristle-forming cells

Classic type

Adults Malpighian tubules, hindgut, midgut, fat body

Classic type

Ovarian nurse cells

Classic, or cryptic types; in the last case can be transformed in classic type at the expense of mutations or inbreeding

Insects, Collembola

Salivary glands

Cryptic, cryptic-classic, classic types

Protozoa, Infusoria

Macronuclear anlage

Classic type

Mammals

Trophoblast cells

Cryptic, semicryptic

Tumor cells

Cryptic type

Plants

Antipods, suspensors, endosperm, synergids, endosperm haustorium, tissue culture, callus culture

Cryptic, can be transformed in classic, e.g., at low temperature

a

Modified from Zhimulev (1996).

cytophotometry to measure DNA amounts (Henderson, 2004; Zhimulev, 1996). Astonishing polyteny levels are achieved in salivary gland cells of Rhynchosciara angelae, infected with microsporidians, where, judging by cytophotometric data, the DNA content can be 2, 4, 8, 16, 32, 64, and 128 higher than in usual normal chromosomes. The chromosomes may contain up to 512 and even 1024 thousand DNA strands (Pavan et al., 1975) and are seen in squash preparations with an unarmed eye (C. Pavan, unpublished observations, 1996).

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POLYTENE CHROMOSOMES TABLE II Degree of Polyteny in Polytene Chromosomes of Some Organismsa Species

Organ

Degree of polyteny (C)

Chironomus plumosus

Larval salivary glands

1024–4096

Ch. tentans

Larval salivary glands

8192–32768

Drosophila melanogaster

Larval salivary glands

1024–2048

Larval midgut Imaginal Malpighian tubules Larval fat bodies Larval prothoracic gland Ovarian nurse cells Rhynchosciara angelae

Larval salivary glands Larval salivary glands after intracellular microsporidial infection

Mammals, diVerent species

Trophoblast

Plants, diVerent species

Suspensor, haustorium, antipods, and synergids

512–1024 2– 256 16–512 64–512 512–8192 4000–16,000 512,000–1,024,000

64–4096 2–8192

a

Modified from Zhimulev (1996).

Not all DNA fragments in an individual chromatid polytenize to the same extent. Local underreplication of DNA during polytenization is most evident in the pericentric heterochromatin, and in intercalary and telomeric heterochromatin (see below). Overreplication of regions of polytene chromosomes, the ‘‘DNA puVs,’’ has been described in the polytene chromosomes of Sciaridae (see below). 5. Banding Pattern A diVerent extent of coiling of the DNA and its associated proteins along the linear axis of each chromatid leads to local variations in chromatin quantity. Regions of high concentration are known as chromomeres and those with low concentration as interchromomeres or interbands (Fig. 2). For each chromatid the pattern of chromomeres is highly specific, so that in the polytene chromosome homologous chromomeres align alongside one another exactly and usually appear to fuse as a band across the polytene element. The banding of polytene chromosome is generally such a stable and specific feature of their organization that the individual bands can be recognized, mapped, and assigned reference numbers.

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FIG. 2 Electron microscopic view of two fragments of Drosophila melanogaster salivary gland polytene chromosomes demonstrating the presence of condensed material (bands, some of them are indicated with arrowheads) and loose material between them (interbands). (a) Region 84E1–2 in wild-type chromosomes. (b) The same region in the transgenic strain containing insertion of transposon HBd-194(84E). This transposon contains 18 kb comprising rosy and E. coli b-gal genes. The last gene is under the promoter of the heat shock gene hsp70. The material forms a new band (indicated by arrow). The position of insertion in the wild type is indicated by a white arrow (Semeshin et al., 1989).

The pattern of bands and interbands of each polytene chromosome is specific for the species, and in general is characteristic of that particular chromosome in diVerent tissues or developmental stages. A thorough analysis of the banding of the chromosomes of four organs of Chironomus tentans led W. Beermann in 1950 to the 1970s to the conclusion that a given banding pattern is, to a large extent, the same and all the visible diVerences are due to technical diYculties such as apparent or real fusion of neighboring bands. The numerous species and diverse organs, whose banding patterns have been compared, support the conclusion that the patterns of most prominent bands are reproducible. However, variations in banding pattern are found and there is evidence indicating that these diVerences correspond to those Beermann (1952–1972) has described, that is, fusion or fission of neighboring bands

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and lengthening of interbands. There are data indicating considerable diVerences in banding patterns of the chromosomes from cells of normally functioning organs. A comparison of polytene chromosome banding patterns of trichogen cells and ovarian nurse cells failed to reveal any clear homology between the banding patterns despite their stability within each tissue (Ribbert, 1979); however, for the nurse cells and salivary gland polytene chromosomes of Anopheles Redfern (1981) demonstrated clear homologies of banding. Numerous cases of dramatic changes of general banding patterns in diVerent mutants and in diVerent experimental conditions were reviewed by Zhimulev (1996, 1999). The phenomenon of seasonal changes in the length and banding patterns of polytene chromosomes described by Ilyinskaya in the end of the 1970s for some species of Chironomus is another good example of considerable variations in the banding patterns of polytene chromosomes. Before the cold season, the chromosomes become much shorter, the number of puVs is minimal, and many of the easily recognized neighboring bands fuse to form blocks of chromatin. The chromosomes start to lengthen in January– February, and there are three to four times more bands in March than in September: the ‘‘September’’ blocks of chromatin split into separate bands and interbands (Ilyinskaya, 1994; Zhimulev, 1996). Thus, from this survey the following inferences may be drawn: the chromomeric pattern in the cells of normally functioning organs is relatively stable. The structure of the chromomeres is flexible and the general banding pattern is highly variable when intracellular and/or extracellular conditions change. Large discrepancies in polytene band numbers in diVerent Dipterans were found, constituting approximately 3000 on average (Zhimulev, 1996). According to the revised Bridges maps of polytene chromosomes, in D. melanogaster there are 5059 bands (Lefevre, 1976). However, it should be noted that when revising the polytene chromosome maps Bridges (1935) used stretched chromosomes that were squashed in 45% acetic acid. As a result, a total of 1207 bands were presented as doublets on revised maps. Subsequently, these were shown to be fixation artifacts (Berendes, 1970; Zhimulev et al., 1981), and, as a rule, every doublet was nothing but a single band. It is pertinent to mention that the number of bands identified by diVerent authors depends not only on the fixation protocol, but also on the criteria used for the interpretation of the images obtained. Thus, assuming the doublets as single bands, the total number of bands in D. melanogaster polytene chromosomes equals 3727. Use of electron microscopy analysis along with the elimination of possible fixation artifacts result in very close estimates of total band number in polytene chromosomes of three diVerent species; Chironomus tentans, D. melanogaster, and D. hydei, being about 3500 (Grond and Derksen, 1983; ten Tusscher and Derksen, 1982; Zhimulev,

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1996). This value corresponds well to the initial Bridges (1935) estimate, before he found the ‘‘doublets.’’ 6. Somatic Synapsis of Homologous Chromosomes The phenomenon of somatic synapsis is based on the association of homologous polytene chromosomes. Both homologues synapse band to band with the highest precision, resulting in the impression that the chromosome is single. As a consequence, in Drosophila or Chironomus the number of polytene chromosomes in the nucleus is reduced to the haploid chromosome number. The somatic synapsis is not an obligatory feature of polytene chromosomes: homologous chromosomes consistently conjugate to various degrees in dipteran insects; synapsis is normally absent in plants or Collembolan insects. It is not quite clear whether polytene homologues synapse in Infusoria and mammals, as the data available are controversial. Very often, e.g., in Simuliidae, synapsis is incomplete: homologues chromosomes are in physical contact with each other; the contact, however, is restricted to some of the regions, while elsewhere the homologues are partly separated from each other (Fig. 1). The frequency of salivary gland nuclei showing partial synapsis in any of the chromosomes during normal development of D. melanogaster varies between 6.5% and 45% in diVerent studies (Zhimulev, 1996). Of particular interest is Balbiani’s (1881) and Bauer’s (1936) discovery of specific asynapsis: in Chironomus plumosus only the fourth chromosomes of the set do not conjugate and are separated. As was discovered later, synapsis is disturbed to a diVerent degree in the fourth chromosomes of closely related members of the plumosus species group and in diVerent populations of Chironomus plumosus (Zhimulev, 1996). Enhancement of asynapsis occurs in hybrids from the crosses between representatives of various forms or races. Whereas in some interspecific hybrids, for example, in Drosophila of the willistoni group, chromosome pairing is just as complete as in the parent species, the synapsis in hybrids between D. insularis and D. tropicalis is retained only at the chromocenter (Dobzhansky, 1957).

B. Molecular and Genetic Organization 1. Bands The lengths of bands along the chromosome axis vary in a wide range from 0.057 to 0.42 mm, or, according to the calculations, from 5 to 195 kb (Zhimulev, 1996). In Drosophila melanogaster the calculated average band

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size is 30 kb (Beermann, 1972), which corresponds very well with new estimates obtained using genomic data. According to the Drosophila genome release 3.1, the euchromatic part of the genome comprises 116 Mb DNA (Celniker et al., 2002), so an average band can contain 31 kb (assuming a ‘‘fine’’ band count of 3700). Big diVerences in the degree of DNA packaging in bands ranging between 8 and 380 can be due to mistakes in the cytological method of measurement and determination of DNA content in the bands. Analysis of the bands formed from transgenic DNA of transposons (Fig. 2) with a known amount of DNA showed that 5 kb is the minimal DNA length necessary for creating a band discernible under the electron microscope at the site of the insertion of transposon. Longer transposons form bigger bands. The packing ratio of DNA in the transgenic bands is about 30, which corresponds to the super bead level of DNA packing in the nucleus (Semeshin et al., 1989). The problem of the genetic content of polytene chromosome bands arose at the same time with rediscovery of polytene chromosomes in 1933–1934. Numerous hypotheses were developed concerning the genetic organization of bands and puVs. Beermann’s idea that a band is the ‘‘morphological equivalent of a Mendelian gene’’ (Beermann, 1967) became most popular and bands began to be considered as units of all genetic functions: transcription and replication, etc. The conclusions with respect to the correspondence of genes and bands were mainly based on the cytological data showing that a puV develops from a band as well as the results of genetic ‘‘saturation’’ (Alikhanyan, 1937) of chromosome regions with lethal mutations. The latter method was used widely in the 1960s to 1980s and now more than 60 regions have been studied (Zhimulev, 1999). After the first famous paper by Judd et al. (1972) a 1:1 correspondence between genes and bands was considered as ‘‘dogma.’’ However, application of more complex methods of genetic analysis and extensive use of molecular approaches permitted new data to be obtained shedding light on band organization and showing that there are several types of bands. a. Polygenic Bands In several chromosome regions significant deviation from the 1:1 ratio was found in favor of genes. Molecular and genetic analysis shows that there are bands comprising numerous genes, e.g., the block of 160–200 identical sequences, of the 5S rRNA genes, 385 bp long, occupies a group of four bands. Hundreds of copies of the 18S, 5.8S, and 28S ribosomal RNA genes are located in the nucleolar organizer, which in many dipteran species looks like a single band. The bands containing repeated histone genes are arranged similarly. b. Oligogenic Bands The 10A1–2 band (190 kb) contains 22 genes and RNA coding sequences (Kozlova et al., 1994, 1997; Zhimulev et al., 1981).

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Numerous bands contain three to seven genes. Among them are 67B harboring seven heat shock genes; several genes and RNA coding regions were found in the 87E1–2 band (Gausz et al., 1986), the 89E1–4 band contains the BXC-Complex with several more genes in the region (Lewis et al., 1995), and the 11A6–9 band, which in males looks like a solid singlet band, contains at least 18 genes according to the Drosophila genome annotation 3.1. Similarly 27 genes are found in the three bands in chromosome region 19E1–5 (Belyakin et al., 2004). Various DNA fragments were used as parts of transformation constructs: the mobile P element, marker genes (the rosy, Adh, and Escherichia coli b-galactosidase genes), and expressed genes carried by plasmids. According to electron microscopic data (Fig. 2), a new band is formed upon transformation in 32 of 45 transformed regions studied, which indicates that all these diVerent DNAs in the transposons are capable of forming a single band (Semeshin et al., 1986, and unpublished). c. Simple Bands At the same time there are ‘‘simple’’ bands containing DNA amounts suYcient for localization of a single gene (i.e., the bands may be simple in terms of information). The organization of the 3AB region in Drosophila melanogaster X chromosome containing a series of very thin bands strongly supports the ‘‘one band–one gene’’ hypothesis (Judd et al., 1972). Data cited demonstrate that despite the existence of bands encompassing the only gene, this principle cannot be universal. It is appropriate to turn to a statement Lefevre and Watkins (1986, p. 869) made when viewing the results of their extensive studies on the organization of the Drosophila X chromosome: ‘‘We conclude that the one-band, one-gene hypothesis, in its literal sense, is not true; furthermore, it is diYcult to support, even approximately. The question of the total gene number in Drosophila will no doubt, eventually be solved by molecular analysis not by statistical analysis of mutation data or saturation studies.’’ Results of analysis of average gene density in the whole genome indicate that it is higher than the DNA content in an average band (30 kb): e.g., in the Achaete-Scute and Broad complexes it is one gene per 8.0 kb. Sequencing about 2700 kb DNA from the 34C4–36A2 region of D. melanogaster (Ashburner et al., 1999) indicated 1 gene per 13.7 kb. So, on average, one 30-kb band may contain from three to five genes. According to release 3.1 of the Drosophila melanogaster genomic project the genome of this species contains 13,647 genes (Celniker et al., 2002), therefore we can assume 2.7–4.5 genes per band in the whole Drosophila genome.

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2. PuVs Painter (1935) described a series of relatively achromatic swollen segments as the salient features of the third chromosome of D. melanogaster. There were no thickenings in the given regions in the other larvae in the tube; they contained the usual bands. Bridges (1935) called some of the swellings, for example, those in the X chromosome region 2B, ‘‘puVs.’’ Other kinds of puVs were long known as well, e.g., the largest were detected by Balbiani (1881) in Chironomus plumosus as muV-like thickenings, of the chromosome regions, subsequently called Balbiani rings by Erhard (1910) and Alverdes (1912). The detailed studies of the morphology of the puVs performed by Beermann (1952), Pavan and Breuer (1952, 1955), Mechelke (1953) resulted in the suggestion that structural changes in the chromosomes, namely loosening of band material and the formation of a specific puV-like swelling in it, are morphological manifestations of gene activation and that these changes correlate with a definite state of diVerentiation. Thus, the notion that the puVs of polytene chromosomes are indicators of gene activity was generally accepted. Very soon after this the transcriptional activity of puVs and Balbiani rings was proven (see above). Somewhat later the relation of puVs to specific cell functions was documented. As early as in 1912 Rambousek suggested that the giant formations in the salivary gland nuclei of Chironomus larvae must be regarded as chromosomes unusually changed to perform a secretory function, to release a secretion by which detritus is glued. In 1961 Beermann found a strong cytogenetic correlation between the appearance of a specific secretion in a particular lobe of the salivary gland of Chironomus pallidivittatus and the activity of an additional Balbiani ring in the cells of the lobe. Thus, a relationship between the activity of a puV and one of the products of the cell was observed for the first time (Beermann, 1961). Subsequently, many authors demonstrated that Balbiani rings of the main lobe of the salivary gland coded for the major polypeptides of the secretion of this organ (Grossbach, 1969, 1974). In Drosophila the relationship between the function of several puVs and the synthesis of salivary gland secretion was demonstrated with the use of cytogenetic methods (Zhimulev, 1999). It was commonly believed that the puV originates from the decompacted material of the single band (Fig. 3) (Beermann, 1956, 1962; Kiknadze, 1972; Pelling, 1966, 1972). However, from the earliest descriptions of the puVs, the idea that many bands may be included in the region of the active puV gained increasing support (Poulson and Metz, 1938). Beermann (1962, 1967) paid great attention to this issue. His thorough studies of Balbiani ring development in representatives of various Chironomus

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FIG. 3 Electron microscopic view of ecdysone-inducible 63E2–3 (a) and heat shock-inducible 63B9 (c) puVs in polytene chromosomes of Drosophila melanogaster (V. F. Semeshin, unpublished observations). (b) Chromosome before puYng.

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species demonstrated that with the exception of the two to three nearest adjacent bands at either side, the rest of the bands remains unpuVed. Beermann introduced two notions, the ‘‘appearance zone,’’ which is the locus at which puYng starts (Entstehungsort), and the ‘‘activity zone’’ (Aktivitatsort), which encompasses all the fully developed puVs. Beermann emphasized that in Chironomus the development of even giant puVs, such as Balbiani rings, starts with the decondensation of the single band, and a long active region composed of several bands results from their secondary involvement in puYng. In the course of development of D. hydei, movement of puV maxima was described in many puVs (Berendes, 1965). A similar movement of puV maxima was found in the pulvilli cells of Sarcophaga bullata (Whitten, 1969), in the bristle-forming cells of Calliphora erythrocephala (Ribbert, 1972), and in the salivary gland cells of D. melanogaster (Korge, 1977; Semeshin et al., 1985; Zhimulev, 1974). The major evidence obtained so far indicates that complex puVs originate from a series of puVs that are adjacent, although functionally independent. New data on the activation of band material and puV formation were obtained from the experiments with bands that appeared from transgenic constructs. If a transposon insert contains a single gene, its activation leads to the decompactization of the band (constructs harboring the genes hsp70Adh or Sgs3). When a transposon is composed of two genes (such as ry and hsp28 or ry and hsp70-lacZ), a newly formed structure still appears as a single band (Fig. 2). However, heat shock causes decompaction of only a part of this band, which obviously encompasses the gene being activated by heat shock. The rest of the band, corresponding to the ry gene, remains compact. If the hsp70 promoter is located in the middle of the construct, i.e., between the ry and lacZ genes, the brief heat shock results in the band splitting in two parts. At early stages of puV formation this new structure appears loose and looks like a small puV. Upon longer heat shocks, a large puV is formed, the remainder of the construct staying compact. The analysis of well-developed puVs in transformed regions allowed identification of a direct correlation between the size of the decompacted region and the length of the transcribed region of the inserted DNA fragment: genes of 1–2 kb in size form structures resembling long interbands (e.g., the genes Sgs3 and hsp28); those about 3 kb long tend to form average-sized puVs (such as the chimeric gene hsp70-Adh), whereas genes 9–11 kb long produce large puVs (hsp70-lacZ). Comparison of the known DNA fragment length with the axial length of the puV indicates that the compaction ratio is 1.4–3.5 (Semeshin et al., 1989).

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3. Localization of Transcriptionally Active Regions in Polytene Chromosomes An overwhelming amount of literature exists on the localization of sites of transcriptional activity in polytene chromosomes (Zhimulev, 1999). Here we discuss the main points only. By the 1960s, preferential RNase-sensitive accumulation of various RNAspecific dyes was localized mainly in nucleoli, Balbiani rings, and puVs (Beermann, 1952, 1956; Beermann and Clever, 1964; Pelling, 1959, 1964). RNA was detected not only in these easily recognized morphological structures; according to Pelling’s (1964) data, the diameter of the chromosome increased (by puYng) only in a small part of the 272 staining regions in Chironomus tentans polytene chromosomes. The remaining RNA-containing regions were represented by diVuse bands and by light regions reminiscent of interbands. The polytene chromosomes of D. virilis stained for pyronine at the end of the third larval instar in 348–360 regions and only a minority were mapped to puVs. The chromocenter did not stain for RNA (Kress, 1993). It can be concluded that RNase-sensitive incorporation of [3H]uridine occurs over all the chromosomes, notably over puVs, Balbiani rings, and nucleoli after incubation of the cells of salivary glands or other organs with the radioactive precursors of RNA synthesis followed by the examination of autoradiographs (Pelling, 1959, 1964). There is also evidence that radioactive uridine is incorporated into more than just puVs, Balbiani rings, and the nucleolus (Fujita and Takamoto, 1963). Thus, of the total number of loci incorporating [3H]uridine into D. hydei chromosomes, only 35–50% showed swelling of these chromosome regions into the puVed structure (Berendes, 1965). In C. tentans the label was diVusely distributed all over the chromosomes, not just confined to puVs and Balbiani rings, after the salivary glands were incubated for 30 min with the radioactive precursor (Clever and Romball, 1966). In D. melanogaster, no more than 50% of the total number of grains are detected in typical puVs of the chromosomes of zero-hour prepupae (Zhimulev and Belyaeva, 1975). Taking into account that the functioning puVs reach their maximum number at this particular stage, the percentage for unpuVed labeled regions would be even higher at the other stages. Among unpuVed regions loose bands might be regarded as loci at the early or late stages of puV development (and, consequently, decompaction); then their intense incorporation of [3H]uridine would not be unexpexted. Interbands could thus be considered as the second type of chromosome regions active in transcription (Zhimulev and Belyaeva, 1975). Indirect immunofluorescence oVers advantages in studies of the localizaion of RNA-synthesizing regions in chromosomes. DNA–RNA hybrid molecules containing single-stranded DNA are formed after denaturation–renaturation

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of chromosomal DNA, while the other strand of the double helix is being represented by the cell’s endogenous RNA located at its transcription site. Subsequent localization of the hybrid molecule allows mapping of the transcriptionally active chromosome regions (Alcover et al., 1982; Rudkin and Stollar, 1977; Stollar, 1970). Using the indirect immunofluorescence assay, many hybridization sites were revealed in polytene chromosomes: about 200 fluorescent bands in the chromosomes of C. pallidivittatus (Diez and Barettino, 1984) and about 350 sites in zero-hour prepupae of D. melanogaster. In this species all the regions of polytene chromosomes decompacted at varying degrees were revealed by indirect immunofluorescence. They were the puVs (prominent, medium-sized, and small) showing bright fluorescence and quite intense incorporation of [3H]uridine. Taken together, these regions constituted 50% of the bright fluorescent bands. The other 50% represented the chromosome regions weakly incorporating [3H]uridine and sites showing no swelling and incorporating little or no [3H]uridine; these were mostly loosened bands, which must probably be referred to as the small puVs that formerly passed unnoticed. The compact dense bands that start Bridges’ subdivisions hardly ever occurred in the bright fluorescent regions (Vlassova et al., 1985). Although Alcover et al. (1982) believe that fluorescence is mainly detected in the interbands, this does not appear plausible because the ‘‘true’’ interbands are actually visible only in the electron microscope. Correct judgments concerning the presence of antibodies against DNA–RNA hybrids can be based on the analysis of those interbands, which are located between the large compact nonfluorescent bands and do not contain minibands according to electron microscopic observations. The large interband between the two dense 100B3 and 100B4–5 bands meets these requirements. The binding of DNA–RNA hybrids in the 100B3/100B4–5 interbands (Zhimulev, 1996), in conjunction with the results obtained with the incorporation of [3H]uridine (Semeshin et al., 1979) and with the immunoelectron microscopic localization of RNA polymerase on polytene chomosomes (Sass and Bautz, 1982), provides convincing evidence for the transcriptional activity of these regions. Intense hybridization was detected in the regions of centromeric heterochromatin. A direct correlation between the degree of chromatin packaging and fluorescence intensity has not been consistently observed. To identify the transcriptionally active regions of polytene chromosomes, it is important to map proteins involved in transcription, processing, and packaging of the transcribed RNA. RNA polymerase I has been mapped to the nucleolus by the indirect immunofluorescence technique (Jamrich et al., 1977, 1978). With this technique, RNA polymerase II has also been detected in all the decompacted chromosome regions, e.g., in Balbiani rings, puVs, loose bands, and interbands. The results of autoradiographic analysis and of

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the localization of RNA polymerase II in polytene chromosomes are in agreement. The coincidence of indirect immunofluorescence with the incorporation of [3H]uridine was most conspicuous in the stretched salivary gland chromosomes (Sass, 1982). Electron microscopy using antibodies directed against RNA polymerase II demonstrated its presence in most, if not all interbands in C. tentans chromosomes. It was concluded that the interbands of polytene chromosomes are the binding sites for the enzyme and the start points for transcriptional activity (Sass and Bautz, 1982). RNA polymerase III was localized in about 30 regions of polytene chromosomes to which the transfer RNA genes and several small nuclear RNAs were assigned either cytogenetically or by in situ hybridization (Kontermann et al., 1989). Several other proteins specific for polytene chromosome interbands will be considered below. Localization of antibodies against proteins that bind to the synthesized mRNA and, presumably, are included in ribonuleoprotein (RNP) particles, generally agrees with their localization in puVs and interbands. These proteins rarely occur in chromocenters and not at all in densely packed bands (Bauren and Wieslander, 1994; Saumweber et al., 1980). There is a variety of small nuclear ribonucleoproteins, or snRNPs, that are essential for the assembly of a multicomponent complex, termed the spliceosome. The spliceosome is involved in processing events resulting in the production of the mature RNA transcripts and especially in splicing of mRNA precursors (pre-mRNA). At least 13 distinct snRNA species have so far been detected. The U3, U8, and U13 are localized in the nucleolus where they may be engaged in the splicing procedure of preribosomal RNA. The U1 and U2 snRNPs additionally contain seven diVerent proteins. It might be expected that antibodies against the proteins of spliceosomes would be primarily localized in the puVs of polytene chromosomes where the introncontaining genes are actively transcribed. However, in D. melanogaster, these antibodies were detected in the heat shock 87A and 87C puVs, where the hsp70 genes devoid of introns are located (Martin et al., 1987). Antibodies against the chironomid U1 and U2 snRNPs were localized in the major sites of transcription, namely Balbiani rings and puVs (Sass and Pederson, 1984; Vazquez-Nin et al., 1990). It is noteworthy that indirect fluorescence with specific snRNP antibodies at Balbiani rings of C. tentans chromosomes correlated with rates of [3H]uridine labeling. Changes in transcriptional activity in Balbiani rings and puVs occurred in parallel with those in intensity of U1 and U2 RNP antibody immunofluorescence (Sass and Pederson, 1984). Observations on the fine structure of the Balbiani ring in Chironumus have shown the presence of specific granules (Beermann and Bahr, 1954), which are considered transcription products of the gene located in this site (Daneholt, 2001).

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RNP granules were found to be present not only in Balbiani rings; they were also identified in all the chromosome structures decompacted at least to some extent: puVs, interbands, and loose areas of centromeric heterochromatin (Table III). It has been estimated that RNP granules occur in about 30% of the interbands, thereby indicating that more than 1500 sites are transcriptionally active at any one time (Skaer, 1977). The data considered provide evidence indicating that the gene products (RNP particles) occur in all the chromosome regions decompacted to diVerent degrees. Localization of the RNA-synthesizing regions in the polytene chromosomes, judging on localization of RNP particles, cannot be completely accurate inasmuch as the granules can migrate into the spacing between the bands of the polytene chromosomes after they have been synthesized in other regions, such as the puVs, for example (Perov and Chentsov, 1971). 4. PuVs in Development and Hormonal Control of PuYng In species of the Dipteran order, larval development takes several days and during this time dramatic and readily observable changes in puYng occur. In D. melanogaster, which is the most thoroughly studied species, puVs during the last 24 h of larval and during 12 h of prepupal development have been described in detail. These puVs can be grouped into two categories: the large puVs, i.e., the swellings proper, and the small loosenings of chromosome structure not associated with an increase in chromosome diameter, the small puVs. At a particular developmental stage each larva or prepupa possesses a characteristic set of large puVs. In the young larvae, whose polytene chromosomes are already large enough to permit their analysis (about 100–110 h after egg laying or somewhat after the mid-third larval instar), several large intermolt puVs are present in the chromosomes. At the very end of larval development, about 6 h before puparium formation, these puVs become inactivated against the background of a sharp increase in ecdysterone titer, and the large puVs start to form; the early puVs appear fast, followed by the late puYng. The late puVs could be divided into two classes based on their inducibility by ecdysone. The early late puVs are induced more quickly and require the continuous presence of the hormone. The late late puVs are induced later and are prematurely induced after ecdysone withdrawal (Ashburner, 1967; Ashburner and Richards, 1976; Ashburner et al., 1974). In zero-hour prepupae, the activity of the puVs is highest, that is, their number and the size of the puVs are highest. During the next 2–3 h, their activity sharply falls, and several mid-prepupal puVs appear. At the end of the prepupal stage a wave of the late and the early puVs passes. The total number of the large puVs, whose activities change during this period of development,

TABLE III Interband-Specific Proteins of Dipteran Polytene Chromosomes, as Identified by Indirect Immunofluorescence Protein

Comment on localization pattern

References

Histones H2A2.2

Predominantly in interbands

Donahue et al., 1986

H2A2.Z

Interbands, bands and chromocenter; very specific chromosome distribution distinct from the banding pattern

Leach et al., 2000

Interbands and puVs

Cheung et al., 2000

H3 3MeK4

Predominantly in interbands

Sedkov et al., 2003

H3 PhS10 and H3 PhS10– AcK14

Interbands, two times more abundant on the male X

Wang et al., 2001

H4 AcK16

Predominantly the male X chromosome interbands and some interbands on the autosomes

Bone et al., 1994

RNA polymerase II (B)

Interbands and puVs

Jamrich et al., 1977; Sass and Bautz, 1982

Topoisomerase I

Partial colocalization with the RNA polymerase II; nucleolus

Elgin et al., 1988

Generally correlate with the chromosome distribution of RNA polymerase II; B-52 flanks the RNA polymerase localization sites in heat shock puVs; hrp36 and hrp48 are also detected in telomeres

Champlin and Lis, 1994 Matunis et al., 1993; Risau et al., 1983; Saumweber et al., 1980;

H3 AcK14 H3

Ac

K9–

Ac

K14

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Nonhistone proteins

RNP particle proteins B-36, B-52, P11, Q16, S5, T29, T7, V4, X4, P46, hrp36, hrp40, and hrp48

Proteins and protein complexes involved in transcription elongation control P-TEFb

Interbands and puVs (more than 200 sites), heat shock puVs; colocalization with the hypophosphorylated form of the RNA polymerase II

Lis et al., 2000

Spt4, Spt5, Spt6, TFIIH, dMediator, TFIIF/Elongin/dELL

Interbands and puVs; Spt4, Spt5, Spt6 colocalize with the phosphorylated RNA polymerase II form

Gerber et al., 2001; Kaplan et al., 2000; Lis et al., 2000; Park et al., 2001

FACT

Many interbands and puVs

Saunders et al., 2003

Vast majority of the male X chromosome interbands, and some interbands on the autosomes

Bone et al., 1994; Kelley et al., 1999

CHD1 (chromo-ATPase/helicaseDNA binding)

Majority of interbands and some puVs (ecdysone and heat shock puVs)

Stokes et al., 1996

JIL-1 (kinase)

Majority of both autosomal and X-specific interbands; on male X the signal is two times stronger than on the autosomes

Jin et al., 1999

RPD3 and SIN3 (deacetylases)

In interbands and telomeres (except for the 3R telomere); localization is diVerent from that of the RNA polymerase II

Pile and Wassarman, 2000

Proteins of the dosage compensation complex

225

MLE (helicase), MOF (acetyltransferase), MSL-1, -2, -3 Proteins of chromatinmodulating complexes

TRR/EcR:USP

Predominantly interlands

Sedhov et al., 2003

BRM

Majority of interbands and some puVs

Armstrong et al., 2002 (continued )

TABLE III (conitnued) Protein

Comment on localization pattern

References

Insulator proteins

226

BEAF-32, Mod(mdg4), Su(Hw), Zw5

Predominantly in interbands; BEAF-32 is also present at the band/interband borders and in some puVs

Blanton et al., 2003 Buchner et al., 2000; Dorn et al., 1993; Gerasimova and Corces, 1998; Zhao et al., 1995;

HSF and HSP70

About 200 sites in interbands, heat shock puVs, and some bands

Shopland and Lis, 1996 Velazquez et al., 1980;

Cohesin

Many interbands

Markov et al., 2003

131-kDa protein (recognized by Y55 antibodies)

Predominantly interbands; in mitosis the protein is found in the cytoplasm

Frasch et al., 1986

185-kDa, 69-kDa, and 58-kDa proteins, recognized by Bx63 antibodies

Predominantly interbands, some signal is seen in the chromocenter; in mitosis the antigen is associated with the centrosome

Frasch et al., 1986; Whitfield et al., 1988

Z4 and Chriz/Chromator

Prominent in interbands, virtually absent from puVs and chromocenter; interaction partners, showing complete colocalization

Eggert et al., 2004 Saumweber et al., 1980;

Proteins with yet unknown functions

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is about 120 (Ashburner, 1967, 1972a; Ashburner and Berendes, 1978; Becker, 1959, 1962). The activity of certain puVs replaces that of the other puVs in an ordered sequence. The developmental stage characterized by a certain puYng pattern is called the ‘‘puV stage’’ (PS) and is assigned a corresponding order number according to Becker (1959), who first described these stages. Developmental schedules for the substitution of one puV stage, assigned 10–11 of the stages to the end of the larval instar and another 10 to the prepupal period next (Ashburner, 1972a, 1975; Ashburner and Berendes, 1978). Becker (1959, 1962) was the first to provide rigorous proof that steroid hormone ecdysone induces puYng. He demonstrated that homozygotes for the l(2)gl mutation, whose ecdysone-releasing ring gland is smaller than normal, lack metamorphosis puVs. In subsequent ligature experiments, the posterior region of the salivary gland was separated from the ring gland, the source of the hormone, and puYng activity correlated with the presence or absence of the hormone. In 1960 U. Clever and P. Karlson published their now famous data showing that injection of ecdysone into C. tentans larvae directly induces metamorphosis puVs (puV I-18C appeared and another, I-19A, became inactivated within 2 h). Such an alternation of puV activity is also characteristic of normal metamorphosis (Clever and Karlson, 1960). These data demonstrated for the first time that the steroid hormone exerts its physiological action through activation of genes but not via activating enzyme molecules or by acting at the cell surface according to existing ideas (Lezzi and Richards, 1989). Later Ashburner (1972b) treated the glands with ecdysterone in vitro to reproduce the puYng cascade in late third instar larvae of D. melanogaster. The puVs occurred in the same sequence as during normal development. The same classes of puVs (the intermolt, early and late puVs) were observed during in vitro activation by the hormone (Ashburner and Richards, 1976; Ashburner et al., 1974). Clever (1964) outlined and Ashburner et al. (1974) developed a model of cascade regulation of hormone-induced genes (puVs). According to Ashburner et al. (1974) the first step is the reversible binding of ecdysone by a receptor protein molecule to form the receptor protein complex. The ecdysone–receptor complex (ER) binds to the puV sites: it induces early puVs and represses late puVs. The product (P) of early ecdysone puVs interacts with the ER complex, thereby exerting a positive control over late puVs (inductive) and a negative control over early puVs (repressive). As a consequence of inhibition of the synthesis of the P, early puVs are not inactivated at the appropriate time, and late puVs are not activated either. The ER complex disassociates on ecdysone washout, and induction of late puVs immediately commences, because P has already been synthesized by the early puVs. Disassociation of ecdysterone from its receptor results in rapid inactivation of early puVs. Disassociation of ecdysterone from its receptor results in rapid

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inactivation of early puVs. Considering all of the experimental evidence, this model explains well the processes of puV induction by the hormone. In D. melanogaster a set of mutations aVecting development of puYng patterns is known. Several mutations arrest development at the larval/pupal stage [l(2)gl, gt, dor, ecd1, grg, sta, l(1)su(f)ts67g, l(3)tl ]. Homozygotes show a preecdysone puYng pattern and normal puVs can be readily induced by addition of exogenous hormone (Zhimulev, 1999). Several patterns of changes in puYng activity demonstrate mutations of the Broad-Complex in D. melanogaster. In larvae homozygous for the so-called long BR-C alleles intermolt puVs are not inactivated, early ecdysone puVs can be induced to some extent, 2B early ecdysone puVs are active constantly, and late puVs are inactive and cannot be induced with the exogenous ecdysone (Belyaeva et al., 1981). Other mutations of the gene result in other patterns related to the arrest of development of late ecdysone puYng. Halfway (or swi) mutations result in the arrest of ecdysone-inducible wave of puVs roughly in its middle. The culture of salivary glands in an ecdysterone-containing medium resulted in the normal appearance of the early and the early late puVs. However, the late late puVs formed neither after the culturing the PS1 glands for 6 h nor after that of PS6 glands for 2.5–3.5 h. Injection of ecdysterone into PS1 larvae had no eVect as well. So, the swi (hfw) mutations disrupt the transmission of information between the early and late puVs (Belyaeva and Zhimulev, 1982). Mutations in E74A and E74B loci are lethal during prepupal and pupal development. A large number of puVs normally active at puparium formation were significantly reduced in size in the E74A mutant (Fletcher et al., 1995). Specific salivary gland overexpression of dominant-negative mutation of the EcR locus, encoding ecdysone receptor, arrests ecdysone-dependent puYng at the earliest stages when even intermoult puVs are hardly developed (Cherbas et al., 2003). DNA of numerous edysone puVs in various species was cloned and in a number of recent reviews molecular and genetic characteristics of the puV genes are described (Henrich et al., 1999; Richards, 1997; Russell and Ashburner, 1996; Segraves, 1991; Thummel, 1996, 2002; Zhimulev, 1999). In total, the DNA of about 50 puVs, both ecdysone and heat shock inducible, as well as Balbiani rings has been cloned and characterized in Dipteran species (Morimoto, 1993; Thummel, 2002; Zhimulev, 1999). These results confirm the old idea that the puVs are regions where active genes are located. A new and very eYcient tool for studies of polytene chromosome organization was derived from analysis of ecdysone-inducible puV genes. Biyasheva et al. (2001) suggested using the promoter of the Sgs3 gene, which is tissue (the salivary gland cells) and developmentally (last 20 h of larval development) specific and can be fused with yeast GAL4 activator of transcription

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(L. Cherbas and A. Andres, unpublished observations). This permits analysis of any gene, encoding chromatin protein to start its ectopic expression. In this case ectopic expression of a gene in a larval organ histolysing several hours later cannot result in any harm to the whole organism. However, its eVect is easily observable on polytene chromosomes. Using the Sgs3 promoter, very specific and unusual swellings were induced in polytene chromosomes as a result of overexpression of the SuUR gene (Zhimulev et al., 2003b). 5. Heat Shock PuVs With the realization that puVs are morphological manifestations of gene activity, numerous experiments aimed at modifying puV activities by various agents and chemical components were started. During the early 1960s Ritossa (1962, 1964) observed that several new puVs are inducible in Drosophila busckii polytene chromosomes by a brief exposure of larvae to a high temperature (heat shock) or by treatment of salivary glands with 2,4-dinitrophenol, an inhibitor of oxidative phosphorylation. Later the heat shock puVs were induced in D. hydei and D. melanogaster (Fig. 3) (Ashburner, 1970; Berendes and Holt, 1964). The discovery eventually stirred great interest and provided impetus for numerous studies. Based on the original results a new biological phenomenon has been described, the syndrome of cell response to environmental stresses. The relevant literature is voluminous (Ashburner and Bonner, 1979; Berendes, 1973; Nover et al., 1984; Schlesinger et al., 1982; Zhimulev, 1999). The heat shock puVs were found in all the studied species with polytene chromosomes. The number of puVs in the genome varies from one to nine. The optimal temperature for induction is 33–37
C. The heat shock puVs are induced not only by high temperature, but also by many chemical agents: uncouplers of oxidative phosphorylation, inhibitors of electron transport, substances that act as hydrogen acceptors, recovery from anoxia, totaling more than 60 (Ashburner, 1970; Zhimulev, 1999). The cells respond very rapidly to the inducer: puVs start to form within 1 min following the shift to the elevated temperature; the puVs then reach their maximal sizes within 20–30 min; thereafter they regress for several hours. The maximal puV size in D. melanogaster induced in the experiments depends on the severity of temperature treatment. If heat shock is extended for more than 1 h, the normally developing puVs, active at the time the temperature was raised, regress (Ashburner and Bonner, 1979; Sass, 1995). Formation of the heat shock puV requires poly(ADP)-ribose polymerase (PARP), which ribosylates chromatin borders of the 87A heat shock puV in the polytene chromosomes (Pirrotta, 2003; Tulin and Spradling, 2003).

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There are at least three types of responses to heat shock at the level of RNA synthesis. 1. RNA synthesis is induced in the heat shock puVs 2. Synthesis of RNA in most genes that do not encode the heat shock proteins—with the exception of the histone genes (Spadoro et al., 1986) or the mitochondrial genome genes (Ashburner and Bonner, 1979)—is inhibited in the heat-shocked cells. RNA synthesis virtually ceases along the entire length of the chromosome. 3. Normal processing of ribosomal 5S, 18S, and 28S RNAs is disrupted (Ellgaard and Clever, 1971; Rubin and Hogness, 1975). New polysomes containing newly synthesized mRNAs transcribed from the heat shock genes appear within 10 min after the start of temperature treatment (Ashburner and Bonner, 1979). Heat shock proteins (HSP) then appear on the electropherograms. Cessation in protein synthesis results from a very rapid decay of the preexisting polysomes (Biessmann et al., 1978; McKenzie et al., 1975; Sondermeijer and Lubsen, 1978). When cells are subjected to a very strong heat shock, the RNA released from these polysomes is degraded, and the RNA remains unaltered at the optimal temperature; however, it can be isolated and translated in an in vitro system (Mirault et al., 1978; Spradling et al., 1977). Normal protein synthesis is recovered within 1 h after the shock irrespective of the presence of actinomycin D. This indicates that mRNAs retain their ability to function in translation when freed of the ribosomes by heat shock (McKenzie, 1977; Storti et al., 1980). The ease with which the HSP can be detected made possible studies on the genomic response to agents stressing cells of organisms lacking polytene chromosomes. With this approach, it was demonstrated that the heat shock syndrome develops in all species of animals, plants, and bacteria. HSPs synthesized upon heat shock rapidly move to the nucleus or bind to cytoplasmic organelles. It is generally accepted that HSPs decrease the extent of cell damage incurred by the temperature stress (Morimoto, 1993; Schlesinger et al., 1982). To conclude, as a result of induction of the heat shock puVs a new biological phenomenon of cell stress was discovered (Lakhotia, 2001; Morimoto, 1993; Morimoto et al., 1990; Morrow and Tanguay, 2003; Villar, 2000). Many heat shock-inducible genes were studied in a variety of organisms and many peculiarities of gene organization were described, which we leave undiscussed. We would only like to mention that among other achievements is the use of the heat shock promoter (Lis et al., 1983) in a majority of transgenic constructs.

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6. Boundary Elements/Insulators In the course of studying the heat shock puVs and genes, specific genomic elements were found: two hsp genes located within the heat shock puV 87A7 are flanked on both the proximal and distal sides by novel chromatin structures, scs and scs0 (specialized chromatin structures). Each element contains two nuclease-hypersensitive sites flanking a nuclease-resistant core and topoisomerase II cleavage sites that redistribute upon heat shock (Kellum and Schedl, 1991; Udvardy et al., 1985). It has been proposed that chromatin is folded into a series of discrete domains and scs and scs0 have been regarded as the best candidates for elements flanking genes, and were named boundary elements or insulators, i.e., regulatory elements that establish independent domains of transcriptional activity within eukaryotic genomes. Insulators are regulatory elements that establish domains of transcriptional activity within eukaryotic genomes and demonstrate two properties: they block enhancer– promoter communication and when placed at the borders of heterochromatic loci of chromosomes can limit the spread of silenced chromatin (Kuhn and Geyer, 2003). Nevertheless, more detailed studies have shown that the scs and scs0 elements reside within the puV and do not delimit the border of the heat shock puV, nor do they regulate the structural domain of decondensation at the 87A puV and the role of these insulators may be restricted to controlling regulatory interactions in the puV region (Kuhn et al., 2004). After their discovery in polytene chromosome puVs, the boundary elements (i.e., those sequences harboring hypersensitive sites and marking periphery of chromosomal domains) were found in several species: yeast, Xenopus, and several new elements in Drosophila. These insulators have been identified in a variety of ways, such as by eVects on chromosome structure, transcriptional regulation, or the presence of binding sites for an insulator protein. The properties of these diverse insulators suggest that the regulatory isolation may be established in mechanistically distinct ways (Bell et al., 2001; Cuvier et al., 2002; Gerasimova and Corces, 2001; Kuhn and Geyer, 2003; West et al., 2002). Insulators or boundary elements are proposed to generate topologically independent structural domains by assembling protein complexes. Several proteins have been identified: two proteins involved in the insulator eVects of scs and scs0 . The zinc finger Zeste-white 5 (Zw5) protein binds scs, while the boundary element-associated factor (BEAF) proteins bind scs0 (Zhao et al., 1995). Zw5 and BEAF localize at the opposite borders of the 87A heat shock puV in the polytene chromosomes, with additional sites of association throughout the chromosome. Suppressor of Hairy wing [Su(Hw)], Scs-binding protein (SBP), and CCCTC-binding factor (CTCF) were shown to interact with boundary elements as well (Cuvier et al., 2002; Kuhn et al., 2004). Insulators found in Drosophila are widely distributed, which is consistent with the proposal that eukaryotic chromosomes are divided into independent

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functional domains that are important for the correct elaboration of transcriptional programs. 7. Balbiani Rings The Balbiani rings are exceptionally large chromosome puVs in the salivary gland chromosomes of some species. Poulson and Metz (1938) first made the distinction between the puVs and the larger swellings (bulbs) in Sciara. The bulbs seemed to be bursting the chromosome regions, whereas the usual puVs were readily recognized by an even and diVuse increase in diameter. The two similar types of changes in the chromosomes also occur in Chironomus species: puVs and Balbiani rings (Beermann, 1962). A local chromosome region becomes decompacted when certain regions are activated; it is further converted into a small swelling rapidly increasing in diameter. When the Balbiani ring is forming the chromosome unravels into individual filamentous elements or threads: in the region of maximal decompaction chromatin forms loops and chromatid fibers. Drawings of such thickenings were already presented in the first descriptions of polytene chromosomes (Alverdes, 1912; Balbiani, 1881). Several aspects of the organization of Balbiani ring morphology have been amply reviewed (Beermann, 1962; Daneholt, 1974; Panitz, 1972; Pelling, 1972). The Balbiani rings occur in the salivary gland cells of the Chironomidae larvae, except for the parasitic species (Balbiani, 1881; Beermann, 1962; Belyanina, 1993; Kiknadze et al., 1990; Poulson and Metz, 1938), in certain species of Drosophila (particularly in species of the montium subgroup) (Kastritsis and Grossfield, 1971), and in apterigotan insects of the Collembola order (Cassagnau, 1971). The number of Balbiani rings per haploid set varies from one in Cryptochironomus vulneratus or Chironomus pilicornis to six in Acricotopus lucidus, the number being most frequently three (Beermann, 1963; Konstantinov and Nesterova, 1971; Mechelke, 1953). In dipterans Balbiani rings occur only in salivary gland cells during most of the larval period when polytene chromosomes can be observed (Beermann, 1962). Based on the presence of a certain set of Balbiani rings, lobes are distinguished in the salivary gland. The highest level of such diVerentiation was described in Acricotopus lucidus, whose salivary gland has three lobes: the main, anterior, and posterior. A set of Balbiani rings is specific to each lobe. Genes of Balbiani rings 1 and 2 in Chironomus tentans are 35–40 kb, contain four short introns, and encode salivary polypeptides of one million Da molecular weight (Daneholt, 2001). Analysis of nucleotide sequences of DNA sequences from diVerent Balbiani rings revealed that a substantial portion (15–25 kb) of each gene consists of repetitive sequences, the so-called core block. A tandem repeat of 200–270 bp composed of unique

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and repetitive parts is common to all Balbiani rings; the repetitive portion is composed of subrepeats of a smaller size. It is represented 75–100 times in the genome. The unique portion remains relatively unaltered; it shows 94% homology in nucleotides to the counterpart regions in the other Balbiani rings. The ‘‘subrepeat’’ region is known to evolve rapidly; the diVerences in nucleotide sequences, length, and subrepeat number are very large. In contrast, the sequences at the 30 -terminal exon and the 30 -untranslated region are highly conserved. It was suggested that the giant Balbiani ring gene has evolved through duplication and divergence from a short 110–120 bp primordial sequence (Sumegy et al., 1982). These very long genes are internally repetitive, resemble each other, and belong to a single gene family (Daneholt, 2001; Wieslander, 1994). Each gene contains four small introns (Daneholt, 2001). The morphology of Balbiani rings is characteristic because the transcription of the genes they harbor is exceptionally intense. According to the autoradiographic data, up to 15% of [3H]uridine incorporated into the nucleus of C. tentans is accounted for by one Balbiani ring (Pelling, 1972). The chromosome I in this species contains as much RNA as one Balbiani ring, approximately 20 pg (Edstrom and Beermann, 1962). Enormously high levels of transcription in Balbiani rings permitted the analyses of the processes of transcription and translation of Balbiani ring RNA. DNA content in the transcription unit is 2.28  103 kb. The total RNA content in Balbiani ring 2 is 10.8 pg or 1.96  107 kb (Edstrom et al., 1978). Therefore, the total number of transcription units represented in Balbiani ring 2 is 8600 (1.96  107 kb/2.28  103 kb), which is in close agreement with the polyteny level of the chromosomes (8200–16,400). This indicates that the BR2 gene is present in one copy in the chromatid (Lamb and Daneholt, 1979). As estimated, the number of RNA polymerase molecules per 1 mm of gene length is 16–82 (Lamb and Daneholt, 1979; Masich, 1992). The average distance between two traversing RNA polymerase molecules is 300 bp during normal development (Lamb and Daneholt, 1979), so they form lampbrushlike structures (Stevens and Swift, 1966). The number of polymerases starting to traverse the whole gene is six per minute (Lamb and Daneholt, 1979) or 10–15 (Trepte, 1993). The rate of RNA chain elongation is 21–41 (31, on average) nucleotides per second (Lamb and Daneholt, 1979). Complete
transcription of the Balbiani ring 2 gene takes 20–30 min at 18 C (Edstrom et al., 1978). Giant polysomes contain Balbiani ring RNA templates (Clever and Storbeck, 1970; Franke et al., 1982). The average number of ribosomes in the giant polysomes of C. tentans is about 100 (Franke et al., 1982) and varies from 79 to 146 in C. thummi; its average number is 97 (Kiseleva and Masich, 1991). Protein is presumably synthesized on relatively long-lived mRNA

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templates. Glandular protein synthesis proceeds at a rate similar to the rate before the addition of actinomycin D and after RNA synthesis has been inhibited by the drug for 48 h (Clever, 1969). From the very first electron microscopic studies it was evident that the Balbiani ring system was very convenient for the visualization of the assembly and transport of specific messenger RNP particles, products of the Balbiani ring genes. Observations on the fine structure of Balbiani ring (BR) in Chironomus (Beermann and Bahr, 1954) have shown the presence of specific granules. Later they were found in a variety of species. Their diameter varies in species mainly between 25 and 50 nm (Zhimulev, 1999). The granules were found to be either freely dispersed in the nucleoplasm, or are located within Balbiani rings, or pass through the pores in the nuclear envelope. As was shown in intensive work of B. Daneholt and his colleagues, a BR transcript is packed with proteins into a thick RNP ribbon bent into a compact ring-like structure. Upon passage through the nuclear pore the ribonuclein particle specifically orients at the entrance, straigthens out, and reenters the pore at the 50 end of mRNA. On the cytoplasm side, the RNA becomes immediately engaged in protein synthesis. Several proteins, which are added to the Balbiani ring particle during transcription of the BR RNA, have been identified and characterized. These proteins behave diVerently during the ensuing RNA transport, some remaining in the nucleus, others entering the cytoplasm coupled to RNA (Daneholt, 2001). 8. DNA PuVs In Sciarid species certain puVs are loci involved in DNA amplification as discovered by Breuer and Pavan (1955). They diVer in this respect from the great majority of puVs. As a result of decompaction and, consequently, a decrease in DNA concentration in puV volume, the usual RNA puVs become lightly staining or transparent in appearance. In contrast the DNA puVs remain darkly stained because of the accumulation of extra DNA. Such DNA puVs arise only in the salivary glands at the late stages of larval development and are involved in coding for proteins of salivary gland secretion. DNA puV amplification provides for additional DNA templates for the burst of active trancription. Alongside the DNA amplification the DNA puVs are very active in transcription. In the salivary gland cells of Sciara coprophila there are nine major DNA puVs and nine minor ones. Activity of this kind of puV is induced by ecdysone. During normal development of S. coprophila there are about four rounds of ‘‘extra’’ DNA replication, resulting in DNA amplification at the DNA puV loci, the first occurring before the DNA puVs start to develop (Crouse, 1968; Ficq and Pavan, 1957; Mok et al., 2001; Poulson and Metz, 1938; Stocker et al., 1997; Yokosawa et al., 1999).

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9. Interbands The most unstudied structures of the polytene chromosome are interbands— the chromosome regions separating bands. Chromosome material in the interbands is more decondensed than in the band (Fig. 2). For this reason, when the stained chromosomes are viewed in light, phase-contrast, or electron microscopes the interbands appear lighter than the bands. The precise cytological identification of the interbands in polytene chromosomes is diYcult because of their small size. According to data obtained from many Dipteran species, the size of the interbands varies between 0.05 and 0.38 mm, most frequently being 0.1–0.2 mm and the molecular size of the interband is 0.3–3.8 kb (Beermann, 1962; Kalisch et al., 1986; Sorsa, 1984; Zhimulev, 1996). Reliable mapping of ‘‘genuine’’ interbands is possible only with electron microscopy, upon following certain rules (Semeshin et al., 1985). However, such analysis is often omitted and the features detected at decompact chromosome regions, which are in fact formed by both faint bands and interbands, are assigned to interbands (Zhimulev, 1996). Thus, these inaccuracies must be taken into account when treating the data described below. Using high-resolution microscopy, interbands appear to be composed of fibrils, whose sizes, according to diVerent reports, vary widely from 5 to 25–30 nm, and even more (Ananiev and Barsky, 1985; de Grauw et al., 1998; Sass, 1980; Sorsa, 1984). As exemplified by the studies of the 61C7/ C8 interband, it was clearly demonstrated that interband DNA is organized into nucleosomes (Schwartz et al., 2001). The idea that interbands are transcriptionally active is rather popular. It is largerly based on the data of [3H]uridine incorporation (Semeshin et al., 1979; Zhimulev and Belyaeva, 1975); however, the resolution of autoradiography is not high enough to be conclusive. By using the method of indirect immunofluorescence detection of DNA/RNA hybrids (see above) in polytene chromosomes of zero-hour prepupae of D. melanogaster, about 350 fluorescence sites were found, many interbands demonstrating the fluorescence signal, including those interbands that were mapped under electron microscope. In particular, these requirements are met by the interband found between the two dense bands 100B3 and 100B4/5. Taken together, localization of DNA/RNA hybrids (Vlassova et al., 1985), RNA polymerase IIspecific antibodies within this interband (Sass and Bautz, 1982), and the data on [3H]uridine incorporation (Semeshin et al., 1979) might point to its transcriptional activity. Specific proteins are localized in interbands (Table III). Interband localization of RNA polymerase II and respective associated proteins, along with the topoisomerase II and RNP-specific proteins (see above), also suggests that the interbands are involved in transcription (Jamrich et al., 1977; Sass

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and Bautz, 1982). According to Sass and Bautz (1982), the interbands are the sites of RNA polymerase II binding. RNP granules representing the products of the transcription (see above) were found in the interband regions as well (Mott et al., 1980; Skaer, 1977). Antibodies against dCHD1 (chromo-ATPase/helicase-DNA-binding protein), tandem kinase JIL-1, Brahma, and modified histones are also localized in interbands (Table III). These proteins may take part in the processes of gene activity regulation through remodeling of the chromatin structure in given regions. Indirect immunofluorescence data on H2A2 histone variant distribution (Donahue et al., 1986), absense of histone H1 (Jamrich et al., 1977), and possible existence of the left-handed Z-form of DNA (Lancillotti et al., 1987; Nordheim et al., 1981) might indicate its specific interaction with the interband regions. At present, little is known regarding the genetic functions of interbands. Interband-specific localization of a number of insulator proteins (see above) suggested the correspondence of the polytene chromosome banding pattern to the separation of the interphase chromosome onto functionally independent domains. It was demonstrated that the Notch locus, controlling many processes of D. melanogaster early development, maps to the 3C7 band (Keppy and Welshons, 1977). Visible recessive mutation faswb is caused by a deletion of 880 bp from the 50 untranscribed region of this gene (Ramos et al., 1989), which removes the interband 3C5–6/7, and possibly some of the material of the neighboring bands, so that these bands join in one (Keppy and Welshons, 1977). As judged from in situ hybridization data, the DNA clone Ah1.6 harboring the promoter region of the Notch gene and the region deleted in faswb is localized in the interband 3C5–6/3C7, whereas the fragments encompassing the coding part of the gene maps to band 3C7 (Rykowski et al., 1988). Electron microscopy data provide evidence that the majority of transposons integrate in interband regions of polytene chromosomes (see above and Fig. 2). Therefore, using the DNA of transposons as a probe, the interband DNA can be cloned (Demakov et al., 1993, 2004). Analysis of nucleotide sequences in DNA fragments located 2 kb on either side of the integration sites of six interbands revealed the following: 1. The interband DNA is unique in the Drosophila genome. 2. In all regions studied a significant number of short open reading frames (more than 10, as a rule) is observed, the codon usage frequencies being diVerent from the Drosophila average in known coding DNA sequences (Ashburner, 1989). It was only in the case of the interband 60E8–9/E10 that the open reading frame identified matched that of the gene RpL19, coding for a ribosomal protein.

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3. With the exception of one interband DNA, the other five sequences harbor 50 - and 30 -untranslated regions of the genes that code diVerent types of proteins. These data allow the interbands to be subdivided into at least two groups, in terms of their genetic content. The interband from the region 60E exemplifies the first type, where a constantly active ‘‘housekeeping’’ gene RpL19 is located. Interbands of the second type (the rest five of six studied so far) are hypothesized to comprise 50 - and 30 -regulatory regions of genes (Demakov et al., 2004). The results of experiments with the DNA of interbands cloned suggest that the decompacted state of the interbands devoid of active genes might be due to their interaction with the nuclear matrix proteins, as was demonstrated for interbands 61C, 85D, and 86B. DNA sequences with strong matrix attachment regions (MAR) potential were also identified in the interband regions 3A, 3C, and 60E. Thus the interband could form the bases of chromatin loops and could serve as barriers separating the genome into a series of distinct functional domains (Schwartz et al., 1999).

III. Dosage Compensation In many species, females and males diVer in number of X chromosomes. To equalize X-linked gene expression in both sexes general regulatory mechanisms have evolved (dosage compensation). Several such systems are known now; however, the first one was discovered in Drosophila by Bridges (1922) and Muller et al. (1931) (to be noted, it was Muller who introduced the term dosage compensation) and was later developed predominantly on polytene chromosomes. The main and the best-understood pathway of dosage compensation in D. melanogaster is achieved by hypertranscription of most genes linked to the single male X chromosome. In polytene salivary gland nuclei, the increased expression of the male X was first demonstrated by means of autoradiography. The relative distribution of [3H]uridine incorporation over the two paired X chromosomes was found to be equal to that over the single X in males (Mukherjee and Beermann, 1965). Great progress in understanding the regulation of dosage compensation has been achieved due to studies of localization of the so-called MSL proteins (ms11, ms12, ms13, mle, and mof ) in male polytene X chromosome (Belote and Lucchesi, 1980; Hilfiker et al., 1997; Lucchesi et al., 1982). Loss of any of these proteins through mutation results in reduced levels of X-linked, but not autosomal, enzyme activity in males that in turn leads to male lethality during the late larval/prepupal stages. As was found, the MSLs

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are colocalized in numerous sites along the male X, this wild-type binding pattern being dependent on the mslþ function of each gene (Gorman et al., 1995; Gu et al., 1998; Lyman et al., 1997; Palmer et al., 1994). These data led to the idea that the MSL proteins associate in a complex marking predominantly the X chromosome. In wild-type males, the MSL complex is not distributed uniformly along the X. At the EM ultrastructural level, MSL binding is restricted exclusively to the band/interband borders and to loose bands (Semeshin et al., 2002). At least 30 reproducible MSL-negative regions (gaps) are determined and mapped on the X chromosome (Demakova et al., 2003). Female-specific SXL protein blocks expression of MSL2 by associating with 50 - and 30 -untranslated regions of msl2 transcripts to prevent assembly of the MSL complex in females (Kelley et al., 1997). On the contrary, the ectopic expression of msl2 from transgenes lacking these SXL binding sites is suYcient to form a functional MSL complex on both female X chromosomes (Kelley et al., 1995). To determine the order of complex assembly, the MSL binding patterns in mutant larvae were assayed. Since polytene chromosomes of dying mutant males have poor morphology, the transgenic females expressing ectopic MSL complex were used to this end. It was shown that the loss of MSL1, or MSL2 through mutations, blocks association of the remaining MSLs with two X chromosomes of transgenic females, indicating the MSL1/MSL2 dimer forms a ‘‘core’’ of the complex (Lyman et al., 1997). The loss-of-function mutations of mle, mof, or msl3 result in residual binding of ‘‘core’’ proteins in a reproducible set of 35 sites. The assembly the MSL complex appears to incorporate is first, MLE, then MOF, and, last, MSL3 (Gu et al., 1998). Recently, new important components of the MSL complex have been identified: at least two noncoding RNAs, roX1 and roX2 (RNA on X ) (Meller et al., 1997, 2000), are also required to achieve dosage compensation, since simultaneous removal of both RNAs results in a drastic reduction in male viability (Meller and Rattner, 2002). Both the roX1 and roX2 RNAs colocalize with the MSL complex along the X chromosome, this pattern depending on any one functional MSL protein (Meller et al., 2000). It was demonstrated that roX2 RNA directly binds to the MSL complex. Immunostaining of polytene chromosomes in msl loss-of-function mutants has revealed that while roX1 RNA seems to be a peripheral subunit of the complex, roX2 RNA might be incorporated into the complex at an early step, MLE function being needed for this association (Meller et al., 2000). The presence of the MSL complex is responsible for the distinct diVuse appearance of the X chromosome, which is characteristic for both wild-type males and msl2-transgenic females. These cytological observations have represented the first evidence that hypertranscription of X-linked genes

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correlates with a more open chromatin structure. Later, an important insight into understanding how the MSL complex functions to change the global chromatin structure of the X chromosome has come from the discovery that a specific isoform of histone H4 acetylated in lysine 16 (H4Ac16) also predominantly associates with the male X chromosome (Bone et al., 1994). The MOF protein was found to be a putative histone acetyltransferase (Hilfiker et al., 1997) and to acetylate specifically the histone H4 in lysine 16 in nucleosomes (Smith et al., 2000). Recently, another enzyme, the histone H3 kinase JIL-1, was also shown to be a component of the dosage compensation RNP complex (Jin et al., 1999). Thus, at least two known subunits of the complex, the acetyltransferase MOF and the kinase JIL-1, possess activities causing specific posttranslational modifications of core H4 and H3 histone tails (Akhtar and Becker, 2000; Jin et al., 2000; Smith et al., 2000). These modifications are widely considered to be involved in the chromatin remodeling essential for upregulation of transcription of the male X (Smith et al., 2000; Wang et al., 2001). One of the most intriguing issues of dosage compensation is how the MSL complex recognizes the X chromosome to mediate chromatin remodeling and, thus, to upregulate X-linked genes. Originally, it was proposed that specific enhancer-like sequences might closely link to individual X-linked genes serving as the targets for the MSL complex. (Baker et al., 1994). However, no consensus sequences have been documented to date. Recent immunofluorescent polytene chromosome studies of MSL-binding patterns in both mutant and transgenic individuals have led to a reevaluation of the longstanding model for X-specificity of dosage compensation. As mentioned above, among the hundreds of MSL-binding sites along the male X, a subset of sites is able to retain incomplete complexes in msl3, mle, or mof lossof-function mutants (Gu et al., 1998; Lyman et al., 1997). These were postulated to be chromatin entry sites (Lyman et al., 1997). In contrast, numerous additional sites on the X are bound only by functional MSL complexes and are postulated to mark genes actively transcribed in this tissue at this point in development. To date, only two chromatin entry sites localized within the roX1 and roX2 X-linked genes have been characterized (Meller and Rattner, 2002). When moved to autosomes as transgenes, both roX1 and roX2 recruit MSL complexes that occasionally spread inappropriately in cis into autosomal regions (Kelley et al., 1999; Meller et al., 2000). These findings led to the current model for X chromosome recognition by MSL proteins. It postulates that first the core MSL1/MSL2 dimer recognizes DNA targets within the chromatin entry sites that are found on the X chromosome, not the autosomes. After assembling at chromatin entry sites the complete MSL complexes may spread consecutively in cis into numerous additional sites along the X chromosome, which themselves may have no X-specific features (Kageyama et al., 2001; Kelley et al., 1999).

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Several key factors are thought to determine correct targeting of the MSL complex in a nucleus. A set of entry sites for MSL proteins may ‘‘mark’’ the X and assemble and probably spread the complex in cis (Kageyama et al., 2001; Kelley et al., 1999). The next requirement is the presence of all functional protein components and both roX RNAs in the complex, otherwise MSL binding would be restricted to a set of entry sites (Gu et al., 1998; Lyman et al., 1997; Meller and Rathner, 2002). Finally, MSL protein/roX RNA ratios are also critical for both correct MSL complex assembly and its distribution in a nucleus (Demakova et al., 2003; Meller and Rattner et al., 2002; Oh et al., 2003).

IV. Replication in Polytene Chromosomes The polytene chromosomes of single salivary glands show diVerent types of labeling patterns after exposure to [3H]thymidine: (1) no labeling of the chromosomes, they are beyond the S period; (2) the chromosomes are uniformly labeled (the phase of continuous labeling); and (3) the chromosomes show discrete spots of label (the phase of discontinuous labeling). The number of such spots varies widely from one to two to several dozen (Berendes, 1966; Pelling, 1966; Rudkin and Woods, 1959). It was found that complementary labeling types occurred among the discontinuously labeling chromosomes: puVs, not the dense bands, were labeled in only some chromosomes, while the labeling pattern was reversed in other chromosomes (Hagele and Kalisch, 1974). Based on these data, the sequential [3H]thymidine labeling of polytene chromosomes is as follows: (1) The phase of discontinuous labeling: (a) the thinnest bands, the interbands and certain puVs, are only labeled; and (b) all the puVs and the interbands are labeled. (2) The phase of continuous labeling: (a) [3H]thymidine is incorporated into all the chromosome regions, with the exception of the chromocenters; and (b) continuous labeling of all the chromosome regions. (3) The phase of discontinuous labeling: (a) certain puVs and interbands are not labeled; (b) many puVs and interbands are not labeled; and (c) toward the end of the S period only certain regions, the chromocenter in Drosophila or heterochromatin in Chironomus, still incorporate [3H]thymidine. One of the above labeling types is usually observed after incorporation of [3H]thymidine into salivary gland cells (Arcos-Teran, 1972; Arcos-Teran and Beermann, 1968; Gubenko, 1974, 1976a,b; Hagele, 1970, 1976; Keyl and Pelling, 1963; Rodman, 1968). The behavior of the X chromosome in males of Drosophila is diVerent from other chromosomes: it begins replicating some time earlier (Berendes, 1966; Gubenko, 1976b; Hagele, 1973) and terminates much earlier than the

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autosomes. Thus, in salivary glands of D. hydei, the male X chromosome already shows the discontinuous labeling pattern, whose autosomes are at the stage of continuous labeling. In the discontinuously labeled nuclei, the number of replicating regions in the male X chromosome is always smaller than in the female X chromosome (Berendes, 1966), twofold smaller, as, e.g., in D. virilis (Gubenko, 1976b). The same regions of the X chromosome were referred to late replicating in males and females (Berendes, 1966; Gubenko, 1976b). The earlier completion of replication in the male X chromosome is thought to be associated with its looser packing of chromosome material as a result of dosage compensation. So data obtained on polytene chromosomes demonstrated that the eukaryotic genome contains numerous sites of replication origins and that these origins start in a specific pattern related to the activity state of the chromosome regions. Many attempts were undertaken to find a relation between replicon length and polytene chromosome band. We will not discuss this problem in great detail but can conclude that the idea that the bands of polytene chromosomes are replication units was not supported by experimental evidence. In diVerent species of Drosophila and Chironomus one average band can contain 0.29 to 15 replicons (Zhimulev, 1999).

V. Heterochromatins A. General In higher eukaryotes pericentric regions of chromosomes and some entire chromosomes display a set of properties that classifies them as heterochromatin (Heitz, 1933). In contrast to euchromatin, heterochromatin (PH) remains condensed through most of the cell cycle, it replicates late, is gene poor, is enriched in repeated DNA sequences, and associates with distinct proteins (Gatti and Pimpinelli, 1992; Lohe and Hilliker, 1995; Richards and Elgin, 2002; Wallrath, 1998; Weiler and Wakimoto, 1995; Zhimulev, 1998). According to Back (1976), late replication was thought to be the only genuine diagnostic characteristic of heterochromatin. However, some regions, for example, centromeres, being embedded in PH, represent very special chromosome domains, and replicate in parallel to the euchromatin (Ahmad and HennikoV, 2001). One of the peculiarities of PH in polytene chromosomes is its ability to form ectopic contacts, that is, the contacts between nonhomologous chromosome regions, which in some species causes the pericentric chromosome regions to join and form a chromocenter (Fig. 1). Chromocenter formation

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can be a tissue-specific feature as well. For example, in D. melanogaster a chromocenter is always formed in salivary gland cells, whereas in fs(2)B and otu mutants, the chromocenters in oocyte nurse cells are rarely formed (King et al., 1981; Mal’ceva et al., 1995). There were two morphologically distinct structures identified in the chromocenter of D. virilis and D. melanogaster: a- and b-heterochromatin. The former represents a dense compact body in the middle of the chromocenter, whereas the latter appears as a loose reticular structure, constituting the major part of the chromocenter (Gall et al., 1971; Heitz, 1934). In D. melanogaster all chromosome arms except for the 3R possess both aand b-heterochromatin. The 3R a-heterochromatin is directly joined to the euchromatin. The third and the sixth chromosomes of D. virilis are devoid of a-heterochromatin; D. hydei lacks a-heterochromatin on the first and the sixth chromosomes (Miklos and Cotsell, 1990). Another PH peculiarity in polytene chromosomes is that it is underreplicated (Berendes and Keyl, 1967; Mulder et al., 1968; Rudkin, 1965, 1969). The majority of PH of mitotic chromosomes was supposed not to polytenize, and to form a block of a-heterochromatin, with the euchromatin to heterochromatin transition zone being partially polytenized and forming the b-heterochromatin (Gall, 1973; Yamamoto et al., 1990). Later PH was discovered to be very heterogeneous in respect to the ability to polytenize. Thus, it was shown that the two genes located within heterochromatin, light and rolled, are polytenized in salivary glands to the level of euchromatin (Devlin et al., 1990), whereas the repeated sequences AAGAG and Bari-1, which flank rolled, are significantly underrepresented (Berghella and Dimitri, 1996). DiVerent P-element-based insertions were mapped in heterochromatin, and were found to undergo a diVerent degree of polytenization, up to that of euchromatin (Zhang and Spradling, 1995). Some of the repeated sequences, such as He-T, known to reside within the PH of mitotic chromosomes, were identified as part of the partially polytenized bheterochromatin in polytene chromosomes (Traverse and Pardue, 1989). Based upon these data, a model of the polytene chromosome chromocenter organization was suggested whereby the PH is composed of alternating blocks of chromatin, showing diVerent potential/ability to polytenize. Some of these blocks do not polytenize at all; they stick together and form a-heterochromatin. Others undergo partial polytenization, loop out, and form a loose-structured b-heterochromatin (Berghella and Dimitri, 1996; Koryakov et al., 1996). Polytenization degree in distinct PH regions depends on a number of factors, such as mutations or tissue type. In fs(2)B ( fes) and otu mutants, the PH in nurse cell polytene chromosomes is polytenized to a higher extent than in salivary gland, since in nurse cells the PH underreplication is discernible from the sixth replication cycle, whereas in salivary glands PH

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underreplicates from the very first cycle (Dej and Spradling, 1999). That is why blocks of heterochromatin are well detectable in PH of nurse cells, and are virtually absent in salivary gland cells (Mal’ceva et al., 1995). These blocks are formed by heterochromatin regions, which are detected by diVerential staining of mitotic chromosomes, but not by the DNA sequences within the transition zone between euchromatin and heterochromatin. Thus, in oocyte nurse cell chromosomes only the regions containing unique sequences undergo polytenization (Koryakov et al., 2003). Mutations in genes involved in DNA replication may influence the PH polytenization level. For example, in flies that are mutant for the cell-cycle control genes (cycE1672, dE2F1, and dDP), the polytenization degree is higher as compared to the wild-type (Leach et al., 2000; Lilly and Spradling, 1996; Royzman et al., 2002). Likewise, in salivary gland cells of the SuUR mutant, partial suppression of DNA underreplication in PH takes place. These result in the appearance of a new banded structure, called Plato Atlantis, in the chromocenter region of chromosome 3 (Belyaeva et al., 1998). Cytological analysis revealed that this structure is predominantly formed from the heterochromatin sequences of the 3L chromosome arm (Koryakov et al., 2003). Comparison of heterochromatin regions in salivary gland cells of the SuUR mutant and in chromosomes of nurse cells in otu mutants indicated that diVerent PH regions become polytenized in each case (Koryakov et al., 2003). Based on the observed permanent compact state of the heterochromatin, Heitz (1932) postulated that heterochromatin was genetically inert. However, many studies reported that the heterochromatin contained genes (Bridges, 1916; Gatti and Pimpinelli, 1992). It is worth noting that the gene density in heterochromatin constitutes about 1% of that in euchromatin (Hilliker et al., 1980). Still, this value is largely based upon the gene number as predicted from mutagenesis data (EMS, X-ray, P-element-mediated mutagenesis), whereas computer analysis of genome projects delivers tens of genes in heterochromatin (Carvalho et al., 2003; Dimitri et al., 2003; Hoskins et al., 2002). Rare heterochromatin genes display a number of characteristic features distinct from those of euchromatin. Genes that are located in heterochromatin possess at least one giant intron, which may be up to 20 kb long and contains numerous mobile elements (Dimitri et al., 2003). Moreover, the promoter structures are diVerent in euchromatic and heterochromatic genes. The light gene in D. melanogaster is located in heterochromatin, whereas its orthologue in D. virilis maps to the euchromatin. Interestingly, the heterochromatin version of the gene has a slippery promoter with numerous transcription initiation points, whereas the euchromatic light gene in D. virilis has a single transcription start site (Yasuhara et al., 2002). The major fraction of DNA in PH is made of repeated sequences, which are represented predominantly by satellites and mobile elements. In

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D. melanogaster, there are over 10 types of simple satellite sequences, some of which fit a consensus (AAN)n(AN)m (Lohe and Brutlag, 1986; Makunin et al., 1999). Furthermore, complex satellites with larger repeat units—10 bp (Lohe et al., 1993), 11–12 bp (Abad et al., 1992), and 359 bp (Carlson and Brutlag, 1977)—have been identified. Extensive satellite tracts are often heterogeneous in content and might have nucleotide substitutions or deletions (Lohe and Brutlag, 1987), or even large ‘‘islands’’ of the ‘‘complex’’ DNA, corresponding to numerous mobile elements (Le et al., 1995). PH comprises 11% of mobile element localization sites (Ananiev et al., 1984), their heterochromatic copies often being incomplete (Miklos et al., 1988; Shevelyov, 1993; Shevelyov et al., 1989; Vaury et al., 1989, 1994). Mobile elements and satellites form clusters on metaphase chromosomes. Some of these clusters are specific to a given chromosome. For example, the large 359-bp satellite and a G mobile element were found exclusively in heterochromatin of the X chromosome (Carmena and Gonzalez, 1995; Lohe et al., 1993; Pimpinelli et al., 1995). Heterochromatin possesses a distinct set of nonhistone heterochromatinspecific proteins, a peculiar pattern of histone modifications in nucleosomes, and more compact and regular nucleosome spacing (Sun et al., 2001), which is caused by a transcription inactivation within the PH DNA sequences (for more details, see below).

B. Position Effect Variegation Heterochromatin position eVect variegation is a form of epigenetic silencing that results from placement of euchromatic genes to PH. Relocated genes are transcriptionally silenced in a part of the cell population through successive cell divisions, causing the mosaic phenotype. This phenomenon was first described in 1930 by Muller (1930), and was subsequently termed position eVect variegation (PEV) (Lewis, 1950). Virtually any gene might undergo inactivation upon placement from euchromatin to heterochromatin; however, not all PH regions are equally capable of inducing PEV (Reuter and Spierer, 1992; Richards and Elgin, 2002; Weiler and Wakimoto, 1995; Zhimulev, 1998). The latter observation served as the basis for the hypothesis of the existence of inactivation centers where heterochromatic domains start forming. These imaginary inactivation centers, even if they do exist, may be as far as 20 kb from the target euchromatic locus (Pokholkova et al., 1993). It is noteworthy that upon inactivation, euchromatin might associate with diVerent types of heterochromatin DNA sequences, such as unique, middle repetitive sequences, or satellites, i.e., the heterochromatinization process cannot be accounted for by a specific set of DNA sequences (Cryderman et al., 1999).

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Mosaic silencing of euchromatic genes undergoing PEV is hypothesized to be caused by variable spreading of silencing from PH to the relocated euchromatin when the rearrangement removes the barrier at the heterochromatin–euchromatin boundary that normally blocks the spread of the heterochromatin state (Donze and Kamakaka, 2002; Noma et al., 2001). In Drosophila polytene chromosomes PEV can be detected cytologically as the heterochromatinization of the euchromatic region: it is possible to observe the loss of a normal banding pattern and strong compactization of the region with fusion of many nearby bands to solid blocks (HartmannGoldstein, 1967; Reuter et al., 1982; Zhimulev et al., 1986). The extent of euchromatin heterochromatinization can be very large, aVecting up to 170 bands that fuse in one huge block (Pokholkova et al., 1993). In addition to the above-described continuous chromatin compaction in PEV, a phenomenon of discontinuous compaction in chromosomes that are subject to longdistance PEV has been described as well. In this case, the compaction zones are separated by morphologically normal bands that are capable of puV formation in contrast to the compacted chromatin regions (Belyaeva and Zhimulev, 1991). Euchromatin silencing subject to PEV occurs at the transcription level. The absence of the transcripts of the genes as a result of PEV has been described for the salivary gland cells, where the chromatin compaction in polytene chromosomes is best seen (HenikoV, 1990; Zhimulev et al., 1986). Heterochromatinized euchromatin becomes late replicating, and can further be underreplicated when undergoing strong PEV (Zhimulev, 1998); moreover, it binds specific heterochromatic proteins, HP1 and SuUR (Belyaeva et al., 1993, 2003). A variety of environmental treatments and genomic elements are known to modify PEV (SpoVord, 1976; Zhimulev, 1998). Studies on the genetic modifiers of PEV, which were revealed in systematic genetic screens for dominant suppressors and enhances of PEV (Grigliatti, 1991; Reuter and Spierer, 1992; Reuter et al., 1982), played an especially important role in the development of the modern concept of the molecular mechanisms of heterochromatin-dependent PEV silencing. Certain genetic modifiers have a dose-dependent eVect on PEV, suppressing it in the presence of one dose and enhancing it in three and more doses, thereby suggesting the involvement of their products in protein complexes forming heterochromatin. The mass-action model (Locke et al., 1988) suggests that proteins encoded by PEV modifiers would be components of these complexes and reduction in this dose of any component would shift the equilibrium away from the formation of the complex and vice versa, a model that anticipated the existence of heterochromatin-specific proteins, which were predicted earlier by Zucherkandl (1974). Indeed such a class of proteins has been described; they appeared to be the products of genes modifiers of PEV.

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The function of a number of them has been established (Wallrath, 1998). Thus, Su(var)3–6 encodes protein phosphatase PP1 (Baksa et al., 1993), Su(var)2–1, histone acetylase (Dorn et al., 1986), and Su(var)3–9, the enzyme for methylation of histone H3 on lysine 9 (Czermin et al., 2001; Rea et al., 2000; Schotta et al., 2002). The missense mutations at the histone deacetylase locus (HDAC1) are suppressors on PEV (Mottus et al., 2000). Su(var)2–5 encodes the HP1 protein (Eissenberg et al., 1992; James et al., 1989) required for assembly of the protein-modifying complex (Richards and Elgin, 2002) and Su(var)3–7 encodes specific heterochromatic protein (Cleard and Spierer, 2001; Cleard et al., 1997). According to the hypothesis of the histone code (Jenuwein and Allis, 2001) modifications of histones (deacetylation and methylation) can have a crucial role in the formation of heterochromatin domains and spreading silence. Hypothetically this scenario could be as follows (Czermin et al., 2001): The multiprotein complex, containing a histone deacetylase and a histone methylase [HDAC1-Su(var)3–9], binds to chromatin and tracks along it deacetylating and methylating lysine 9 in a concerted action and generating a mark for binding compactization protein HP1. These events led to the formation and spreading of the heterochromatin state that could, once established, maintain silencing through replication cycles by a self-propagating mechanism (Czermin et al., 2001; Richards and Elgin, 2002; Turner, 2000). The role of many other heterochromatin proteins is not yet established [e.g., ORC, Su(var)3–7, HP2], and this model remains to be refined. Spreading of silencing from PH to the euchromatin region, PEV, is probably best explained by the failure of the barrier function between the euchromatin and heterochromatin, as was demonstrated for yeast (see above). It should be noted that these were the Drosophila polytene chromosomes that served as a convenient model to obtain data on chromosome material compactization, compactization spreading, localization of silencing proteins, and their behavior in diVerent mutant backgrounds, which were the basis for the investigation of the molecular mechanisms of silencing in higher eukaryotes.

C. Intercalary Heterochromatin In euchromatic parts of Drosophila melanogaster polytene chromosomes there are numerous regions of so-called intercalary heterochromatin. These regions were first described in 1939 (Kaufmann, 1939; ProkofyevaBelgovskaya and Khvostova, 1939) and named by Kaufmann (1939) ‘‘intercalary heterochromatin’’ (IH), because they frequently showed contact with centomeric heterochromatin (ectopic contacts) and demonstrated the other feature characteristic of pericentric heterochromatin, namely association

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FIG. 4 Electron microscopic view of two bands of intercalary heterochromatin, 7B1–2 (a, b) and 11A6–9 (c, d) in wild-type females (a, c) and in the SuUR mutant (b, d) (Semeshin et al., 2001). Bands 7B1–2 and 11A6–9 are broken (a, c) at the expense of local underreplication; however, the DNA in these bands is completely replicated in the mutant (b, d).

with a high rate of chromosome aberrations. The observation of IH is rather diYcult, however, and its existence was considered doubtful by a number of workers. The late replication is one of the most important characteristics of classic PH. The timing of the replication in diVerent sites of polytene chromosome is highly reproducible. IH regions replicate late compared to the bulk euchromatin part of polytene chromosome. There are about 240 such latereplicating sites in a polytene chromosome set of D. melanogaster (Zhimulev et al., 2003a). About 25% of late-replication regions demonstrate other feature of classic PH heterochromatin-noncomplete polytenization, which at a cytological level is manifested as breaks of chromosome [‘‘weak spots,’’ according to Painter’s (1934) first observation] (Fig. 4). The occurrence of DNA underreplication was confirmed by molecular methods for several regions of D. melanogaster polytene chromosomes (Lamb and Laird, 1987). Further it was shown that underreplication regions are very long [(up to 300 kb in the 89E region (Moshkin et al., 2001)]. Genome-wide microarray analysis revealed 70 underreplication regions in salivary glands of D. melanogaster ranging in length from 92 to 568 kb (Belyakin et al., 2004). The wide variations in break frequencies and, hence, in DNA underreplication include the following: 1. Sex diVerences. In males of D. melanogaster breaks do not occur in the X chromosome.

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2. Tissue diVerences. There are numerous examples of the presence of weak spots in cells of some organs and their absence in others: for example, the frequency of weak spots is very low in chromosomes of nurse cells of D. melanogaster; they are practically absent in food pad and trichogen cells of Musca domestica. 3. Low temperature considerably promotes the chromosome ‘‘fragility’’ in the weak spots. 4. Genetic factors. Removal of the Y chromosome produces a sharp increase in break frequencies (Zhimulev, 1998). Polytenization degree in both IH and PH dramatically depends on the Su(UR) gene. In the Su(UR) mutant, underreplication is suppressed completely in IH (Fig. 4) and partially in PH; the Su(UR) mutation leads to earlier completion of DNA replication in IH. In contrast, in the 4  Su(UR)þ transgenic stock carrying two additional copies of the gene the level of underreplication sharply increases, causing many late-replication sites to become underreplicated (Belyaeva et al., 1998; Zhimulev et al., 2003a). Immunostaining of polytene chromosomes showed that the SU(UR) protein is located only in late-replicating regions, suggesting direct involvement of this protein in late replication. In all cases mentioned above, diVerent degrees of underreplication are correlated with the same degree of ectopic pairing. Therefore, these correlations suggest that DNA underreplication is an important prerequisite for ectopic pairing (Ashburner, 1980; Scouras and Kastritsis, 1988; Zhimulev et al., 1982). This point of view finds confirmation in some recent data. In polytene cells heterochromatic sequences are underreplicated during heterochromatin S phase ends before replication of heterochromatin is completed. Truncated heterochromatic DNAs have been identified in polytene cells and may be the discontinuous molecules that form between fully replicated and underreplicated chromatin. Underreplication occurred during the first polytene S phase; truncation did not occur until the second polytene S phase, when the new replication fork extends to the position of the fork left unresolved in the first polytene S phase (Leach et al., 2000). It is possible that DNA molecules with double-stranded free ends are produced at sites of the stalled replication fork. On occasion, polytene cells might still attempt to repair double-stranded free ends, and in so doing might create ectopic contacts. The nonhomologous end-joining reaction between the free ends of DNA at diVerent loci would produce ectopic ligations. Ectopic ligations would also create chimeric DNA molecules as Ashburner (1980) suggested. Localization of IH regions in the same compartments on the nuclear envelope (Mathog et al., 1984) could facilitate joining of the free ends. Similarity of the features of the IH and PH regions cannot be attributed to the similarity of the DNA sequences, since the IH does not contain long

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tracts of repeated DNAs. Dense packing of chromosome material and inactivation of transcription are typical characteristics of the PH, therefore it was suggested that IH is a manifestation of silencing of the unique genes. This hypothesis finds increasing confirmation. Study of homeotic gene epigenetic silencing is most important, because two major homeotic gene complexes, BX-C and ANT-C, are located in typical IH regions, 89E and 84AB, respectively. There is functional similarity between the mechanisms responsible for PH formation and those for euchromatic gene silencing. The inactivation mechanism of the BX-C and ANT-C includes histone modification (deacetylation and methylation) and assembly of the complex of silencer proteins, encoded by Polycomb-group genes (Czermin et al., 2002). At the level of the whole chromosome set in D. melanogaster IH regions correlate with binding sites of at least seven Pc-group proteins (Zhimulev et al., 2003a). Recently new data appeared that demonstrate that HP1 protein could bind not only PH and telomeres but also 188 sites in the euchromatic regions of Drosophila polytene chromosomes. About 30% of these sites coincide with late-replicating regions (Fanti et al., 2003) and, what is more surprising, they often match the Pc-group binding sites (Zhimulev and Belyaeva, 2003). The function of the HP1 in IH is not yet established. The information content of the IH regions is especially interesting. Until recently our knowledge was restricted by sites of localization of homeotic BX-C and ANT-C genes, cluster of histone genes in the 39DE region, and 5S rRNA genes in the 56F region of polytene chromosomes. Many more genes located in IH were found using genome-wide analysis of underreplication zones of the IH in Drosophila containing two additional doses of the Su(UR) (see above). Microarray analysis of about 11,000 genes in salivary gland polytene chromosomes of SuUR and 4  SuURþ (0 and 4 doses of SuURþ) stocks revealed that approximately 1200 genes (ca. 10% of the Drosophila genome) are clustered in 70 (underreplicated in 4  SuURþ stock) IH regions, each comprising 6 to 41 genes. These clusters are enriched with coordinately expressing unique genes: 54% of these 70 regions were found to correlate with clusters of similarly expressed genes, socalled transcriptional territories (Spellman and Rubin, 2002). Moreover, the underreplicated regions are enriched in testis-specific genes, which are silenced in larval salivary gland cells: of about 550 male-specific genes (Boutanaev et al., 2002) 81% are located in underreplicating sites of IH (Belyakin et al., 2004). So, the IH regions could be considered as a large class of chromatin domains of coordinately replicated and expressed genes. So, polytene chromosomes oVer new approaches to the analysis of the genome organization.

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VI. Telomeres in Polytene Chromosomes Based on the analysis of radiation-induced rearrangments in D. melanogaster, Muller (1932) proposed the existence of special ‘‘unipolar’’ structures (telomeres) on the chromosome ends, since (1) the broken chromosome ends never joined with the most distal chromosome regions, rather they associated with other break points; and (2) the distal-most chromosome regions are virtually never transposed into internal chromosome regions as a result of the rearrangement. Later it became evident that telomeres represent special DNA–protein complexes on the termini of the linear chromosomes, and are required for complete DNA replication and chromosome integrity (Biessman and Mason, 2003; Kanoh and Ishikawa, 2003). Telomeres function in the maintenance of nuclear architecture, as they associate with the nuclear envelope in Drosophila polytene nuclei (Hari et al., 2001; Hochstrasser et al., 1986; Marshall et al., 1996) as well as in yeast cell nuclei (Hediger et al., 2002). The observations suggest that Drosophila telomeres are not defined by the primary DNA sequence, rather by some other factors, such as specific chromatin structure, the presence of specific proteins, or some other epigenetic marks (Biessmann et al., 1990). Telomeres are considered to be heterochromatinized. In contrast to the telomeres in many plants and animals, heterochromatin blocks are never observed on the tips of mitotic chromosomes in D. melanogaster. In D. melanogaster salivary gland polytene chromosomes the morphology of distal regions of the chromosomes is varying and in diVerent stocks morphology transitions from a gray net-like mass to dark dense bands can be observed (Zhimulev, 1998). The terminal DNA sequences in D. melanogaster telomeres are completely diVerent from those of other eukaryotes (Biessmann and Mason, 2003). The latter possess a specific telomerase activity, acting essentially as a reverse transcriptase, that synthesizes new DNA [TTAGGG]n stretches. Terminal DNA elongation in D. melanogaster is provided by the integration of non-long terminal repeat (LTR) retrotransposons Het-A and TART on chromosome ends via reverse transcription, recombination, or conversion (Biessmann and Mason, 2003; Kahn et al., 2000; Melnikova and Georgiev, 2002; Savitsky et al., 2003; Sheen and Levis, 1994). Thus, the distal-most parts of D. melanogaster chromosomes are made of specific non-LTR retrotransposons Het-A and TART, arranged head-to-tail (Fig. 5), subtelomeric telomere-associated sequence (TAS) repeats are located proximal, and these neighbor the euchromatic genes (Biessmann and Mason, 2003). Distal chromosome regions are capped by a special protein complex that is recruited to the chromosome ends whether they have terminal deficiencies or not (Biessmann et al., 1990).

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FIG. 5 Organization of the telomeric end in polytene chromosome of Drosophila melanogaster. (a) Functional domains of the telomeric end. (b) Localization of the specific proteins in the telomeric end. The proteins with varying localization in telomeres of diVerent chromosomes of the organism are labeled with asterisks. The localizations of the proteins are as follows: UbcD1 from Cenci et al. (1997), HOAP from Cenci et al. (2003b), HP1 from Siriaco et al. (2002), and Pc-G from Boivin et al. (2003). The other proteins were located by E. N. Andreyeva (unpublished observations).

Many aspects of the structure (Biessmann et al., 2002; Pardue and DeBaryshe, 2002), integration mechanism (Biessmann and Mason, 2003; Rashkova et al., 2003a,b), and evolution (Casacuberta and Pardue, 2002) of Het-A and TART elements have been described in detail. The length of the Het-A/TART tracts varies in wild-type flies (Mason et al., 2003; Walter et al., 1995), and is observed to be considerably larger in a mutant background for the genes involved in the control of the telomere elongation, such as Su(var)2–5 (Savitsky et al., 2002, 2003), Tel (the gene is derived from the Gaiano strain) (Siriaco et al., 2002), and E(tc) (Melnikova and Georgiev, 2002). In the Gaiano strain there are four times more Het-A copies, and two times more TART copies (Siriaco et al., 2002) in the telomeres as compared to the wild type. This causes the extended loose material to appear on the chromosome ends, and leads to an increase of frequency of ectopic intertelomere contacts (Fig. 6). Given that the full-length Het-A copies are found exclusively in telomeres, and that the truncated Het-A elements are also present in PH and in Y chromosome (Danilevskaya et al., 1993; Traverse and Pardue, 1989), it was suggested that the Het-A elements formed the heterochromatin in telomeres (Danilevskaya et al., 1998). Nonetheless, the Het-A promoter is capable of driving the transcription of the yellow gene (Kahn et al., 2000), and does not cause the repression of the reporter gene wþ when it is inserted in euchromatin (George and Pardue, 2003). TASs from diVerent chromosomes exhibit sequence similarities and some limited cross-hybridization, namely, TAS X can hybridize with the 2R and

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FIG. 6 Electron microscopic view of telomeres in Drosophila melanogaster polytene 2R and X chromosomes in Gaiano/Oregon R hybrid. Chromosomes join tail-to-tail by means of ectopic conjugation stretch (‘‘ect’’ on the figure). The ends of chromosomes in the Oregon R wild line are indicated by arrows (V. F. Semeshin, unpublished observations).

3R telomeres (Karpen and Spradling, 1992), and TAS 2L cross-hybridizes with the 3L telomere (FlyBase). TAS tract length in the X chromosome equals 4.5 kb (Karpen and Spradling, 1992), and in 2L TASs are 10–15 kb long (Mason et al., 2003). In polytene chromosomes TASs correspond to the most proximal bands. Upon integration of the reporter gene into TAS regions, or in close proximity to TASs, a telomeric position eVect (TPE) is observed (Cryderman et al., 1999; Golubovsky et al., 2001; Karpen and Spradling, 1992; Kurenova et al., 1998). An increase in the length of the Het-A/TART tracts suppresses TPE, which is manifested as an increase in expression of the reporter gene wþ (Golubovsky et al., 2001; Mason et al., 2003). DNA of the TAS repeats is shown to be underreplicated in the telomere of the Dp1187 chromosome (Karpen and Spradling, 1992), and in telomeres 2R and 3R (Cryderman et al., 1999; Wallrath et al., 1996), whereas in 2L it is polytenized completely (Wallrath et al., 1996). Based upon these data, a hypothesis whereby these are the TAS repeats that form the heterochromatin domain was formulated (Kurenova et al., 1998). It was recently shown that the ectopic expression of the SuUR protein under control of the late salivary gland-specific GAL4 driver leads to the formation of bubble-like swellings in heterochromatin; however, in telomeric regions, the swellings were observed only in 2R and 3L (Zhimulev et al., 2003b), so it is not yet clear whether TAS or Het-A/TART forms these swellings. The question of the heterochromatin properties of telomeres in the polytene chromosomes of D. melanogaster is further complicated by the fact that in close proximity to the 2R telomere there maps a region of IH (Zhimulev et al., 2003a), and in Dp1187 there is a 1-Mb-long block of PH (Karpen and Spradling, 1992), as these regions might be influencing the properties of the adjacent TAS repeats. The data on protein localization in telomeres of polytene chromosomes are diYcult to systematize, as these are rather fragmentary, and it is unclear whether the proteins are indeed the capping protein subunits, or they are just recruited by Het-A/TART or TAS repeats. In particular, the SuUR protein, being a marker of late-replicating and underreplicated regions (Belyaeva et al., 1998), can be found in telomeres (Makunin et al., 2002); however,

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the question of what complex it belongs to still remains unanswered. It is clearly demonstrated that the 1.2-kb TAS fragment from the X chromosome recruits the Pc-G proteins (Boivin et al., 2003) (see Fig. 5), whose chromosome localization sites have a 70% overlap with the sites of the intercalary heterochromatin (Zhimulev et al., 2003a). The HP1 protein, specific for the PH, does not colocalize with the Pc-G proteins in the regions of TAS repeats (Boivin et al., 2003; Cenci et al., 2003a; Siriaco et al., 2002). Chromosome capping proteins in D. melanogaster HP1, HOAP, and UbcD1 bind the chromosome ends irrespective of the underlying DNA sequence (Cenci et al., 1997, 2003a,b; Fanti et al., 1998). It might be assumed that the proteins HP2 (ShaVer et al., 2002) and Su(var)3–7 (Delattre et al., 2000), which are known HP1 counterparts, form the capping complex. A Tef protein (Queiroz-Machado et al., 2001) might be a part of the capping complex as well (Fig. 5), since in tef mutants telomere fusions, disrupting the chromosome segregation in mitosis and meiosis, are observed. As the protein encoded by the Su(var) 2–10 locus does not colocalize with HP1 and suppresses terminal deficiency-associated PEV (TDA-PEV) (Hari et al., 2001), it is most probably recruited by TAS repeats. The data on telomere proteins are summarized on the scheme. It is considered that the capping proteins and the proteins recruited by TAS repeats form two distinct repressive complexes, whereas the proteins associating with Het-A/TART elements assemble in an activating complex (Mason et al., 2003). Genes suppressors of the classic position eVect variegation do not aVect the TPE, except for some of the alleles of the Polycomb-group genes Su(z)2 and Psc (Cryderman et al., 1999), ph, Pc, PcL, Scm, Sce (Boivin et al., 2003), Su(var)2–10 (Hari et al., 2001), and Su(var)3–9 (Donaldson et al., 2002). TPE is insensitive to the addition of the Y chromosome and to the temperature eVects (Cryderman et al., 1999; Wallrath and Elgin, 1995). Possibly, diVerent protein sets account for the TPE and for the classic PEV (Donaldson et al., 2002; Kurenova et al., 1998; Mason et al., 2003). To summarize, the telomeres are composed of the three domains (see Fig. 5) with distinct functions, namely, the cap (which is close to the PH in terms of the associating proteins), the Het-A/TART elements, and the TAS repeats, each domain having specific associated proteins.

VII. Conclusion During the past seven decades polytene chromosomes have emerged as a formidable tool for the analysis of the organization of the interphase nucleus of eukaryotes. Their large size as well as the distinctive pattern of bands imparting a unique portrait to any chromosome region allow

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information to be obtained on the topography of processes occurring in the chromosomes and the distribution or transpositions of genes, chromosome regions, DNA fragments, and localization of nucleoprotein components of the chromosome. Data obtained on the polytene chromosomes strongly impacted the development of numerous genetic phenomena; however, this review has focused only on several topics related mainly to polytene chromosome structure and function: the morphology of the chromosomes, organization of transcription and replication, general description of silencing, and dosage compensation and position eVect variegation. Numerous directions of research in which polytene chromosomes have played an outstanding role have not been considered. Among them, first, are cloning the genes, walking and jumping methods, localization of genes by means of chromosomal rearrangements and by in situ hybridization, study of mobile elements of genomes where, as a result of their giant sizes, their mobility was clearly demonstrated, the voluminous literature about population heterogeneity of organisms with polytene chromosomes, precise mapping of genes and the whole genomes, and the voluminous literature on chromosome cytological mapping of a variety of species. Although the polytene chromosomes have been under analysis for the past 70 years, they provide more and more new possibilities for genetic analysis, organization of separate regions, and the whole genome. Studies of silent regions (intercalary heterochromatin), transcriptional territories, comprising gene clusters sharing coregulated replication, and transcription look especially interesting.

Acknowledgments The authors express their gratitude to Michael Ashburner for reading the manuscript and to Andrey Gorchakov and Victor Shloma for help in preparing the manuscript. The work was supported by a grant in the Molecular and Cellular Biology Program of the Russian Academy of Sciences, Program Frontiers in Genetics 2-04, RFBR 02-04-48222, Scientific Schools 918.2003.4, and grants of the Ministry of Education of the Russian Federation E02-6.0-37 and PD02-1.4-74

References Abad, J. P., Carmena, M., Baars, S., Saunders, R. D. C., Glover, D. M., Ludena, P., Sentis, C., Tyler-Smith, C., and Villasante, A. (1992). Dodeca satellite: A conserved G+C-rich satellite from the centromeric heterochromatin of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 89, 4663–4667.

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