Polytene Chromosomes

 

General Description of Polyteny

Polytene chromosomes were discovered by Balbiani (1881) in larval salivary glands, Malpighian tubules, intestine, hypoderm and muscles of Chironomus plumosus as a cylindrical cord that repeatedly unravelled and filled the nucleus. He called this structure a ‘permanent spireme’. Nine years later, in 1890, he found a permanent spireme in the macronucleus anlage of the infusorian Loxophyllum meleagris (Balbiani, 1890; Figure 1).

Figure 1. First drawings of polytene chromosome made by Balbiani in (1881) (a) and (1890) (b). (a) Salivary gland cells of Chironomus plumosus and (b) Macronucleus (anlagen?) of Loxophyllum meleagris.

In 1933–1934, three groups of researchers (Painter, 1933; Heitz and Bauer, 1933; King and Beams, 1934), using 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 (joining together) of homologous chromosomes. Each chromosome has a definite and constant morphology and is composed of segments, displaying a distinctive transverse-banding pattern (Figure 2).

Figure 2. Drawing of a polytene chromosome set of Drosophila melanogaster. The chromosomes have been spread out by squashing them on a microscopic slide. Each parental chromosome is tightly paired with its homologue (somatic synapsis). There are regions where two homologous chromosomes are separated (asynapsis). All the chromosomes are linked together by the pericentromeric regions to create a single chromocentre. In the left lower corner mitotic chromosomes from ovarian tissue are shown at the same magnification. From Painter T (1934) Salivary chromosomes and the attack on the gene. Journal of Heredity 25: 465–476.

The relationship between polytene chromosomes (known as ‘giant chromosomes’ at that time) and regular mitotic chromosomes was first proposed by Rambousek (1912) and then by Kostoff (1930), Koltzoff (1934) and Bauer (1935).

Conclusive evidence for the chromosomal nature of the ‘spireme’ was obtained by Painter (1933). Using a series of chromosomal rearrangements with breakpoints 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’. The giant size of the salivary gland chromosomes was explained by Koltzoff (1934) as being the consequence of multistrandedness. The term ‘polytene’ was proposed by Koller (1935) and adopted by Darlington (1937).

Polytene chromosomes are now considered to be very important objects for the analysis of numerous features of interphase chromosome organization and the genome as a whole. Moreover, according to Ashburner (1970, p. 2), polytene chromosomes are a ‘system in which differential gene activity and its control can be analysed directly at the level of the genes themselves’.

 

Peculiarities of Polyteny

Cells with polytene chromosomes differ in the following ways from 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 deoxyribonucleic acid (DNA) doubling, as a result of which the cell cycle consists of just two periods, synthetic (S) and intersynthetic (G). The polytenization cell cycle is set during midembryogenesis in Drosophila melanogaster. Second, at the end of each replication period, DNA strands do not segregate; rather, they remain paired to each other to different degrees. It is known that the escargot gene in D. melanogaster is needed to maintain the imaginal disk cells in the diploid cell cycle. This gene does not function in larval tissues with polytene chromosomes. Its artificial expression in such tissues, as in the salivary glands, inhibits polyteny. 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. See also Cell Cycle, and Mitosis

Polyteny arises and attains high levels in tissues, organs and at developmental stages when there is need for the rapid development of an organ at an unaltered high level of function. 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 conditions necessary to accomplish these functions. 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 by mitotic division of diploid cells. It is also obvious that the polytene cell cycle ensures the maintenance of high functional activity by the organ, avoiding the discontinuities due to mitosis.

 

Morphology of Polytene Chromosomes

The morphology of polytene chromosomes can vary widely due to the differing degrees of synapsis of the DNA strands. Below, each strand within polytene chromosome will be conditionally designated as chromatid. Polytene chromosomes develop from the chromosomes of diploid nuclei by successive duplication of each chromosome element (chromatid). If homologous chromatid conjugation is maximal, classic polytene chromosomes, i.e. cylindrical cables with a distinct banding pattern, such as those described for Chironomus tentans or D. melanogaster, are formed (Figure 3a). However, the degree of chromatid conjugation can vary widely. If it is minimal, a polyploid nucleus with a reticular structure is formed (cryptic polyteny) (Figure 3b). In some cases, conjugation of chromatids is disturbed in only one chromosome of the set. This polytene chromosome then completely loses its banding pattern and looks diffuse, becoming a so-called pompon-like chromosome (Figure 3c). See also Chromosome Structure

Figure 3. Arrangement and degree of conjugation of chromatids in classic polytene chromosomes (a), cryptic polyteny (b) and pompon-like chromosomes (c). (a) Individual chromatids with chromomeres (shown as black rectangles) contact each other tightly, the chromomeres forming bands. (b) The chromatids contact each other only in some of the chromomeres, forming a broom-like structure. (c) The conjugation of the chromatids is completely disturbed and a ‘pompon’ is formed. Open circles indicate the centromeric region.

The state of chromatid conjugation can be transient. The disintegration, by fibrillation, of polytene chromosomes resulting in typical reticular endopolyploid nuclei is normal to salivary gland nuclear development in many Cecidomyiid larvae. Sharp differences 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 the salivary gland cells. However, they are identified in the ovarian nurse cells 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 was clearly demonstrated by Ribbert (1979), who succeeded in obtaining classic polytene chromosomes of the ovarian nurse cells by inbreeding Calliphora erythrocephala. Often, excellent polytene chromosomes develop in the ovarian nurse cells of Anopheles mosquitoes after bloodsucking. Classic polytene chromosomes in the nurse cells of D. melanogaster form in mutants such as ovarian tumour (otu) and fs(2)B, whereas in normal development of this species nurse cell nuclei are reticular.

The role of environmental effects on polytene chromosome formation is demonstrated by the case of Aphiohaeta xanthina. When larvae develop under normal conditions, classic polytene chromosomes are seen in their salivary glands; when their food contains little protein and fat, the nuclei become reticular.

Striking changes take place in the general morphology of the polytene chromosomes of beans (Phaseolus coccineus and Phaseolus vulgaris) when they develop in low temperature conditions: polytene chromosomes developing at 20–22°C look like ‘pompons’, whereas in plants developing at low temperatures (12°C day time, 8°C at night) the chromosomes become shorter and assume a distinct banding pattern, i.e. they are of classic type. In general, it is found that polytene chromosome bands become more distinct at low temperatures, even in the case of classic polytene chromosomes. See also Ploidy Variation in Plants

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 different appearance. Although the polytene X-chromosome of the male, as expected, contains half as much DNA as that of the female, it occupies almost the same area as the two female X-chromosomes. It is more loosely packed, twice as much ribonucleic acid (RNA) is synthesized in it as in a single female chromosome and it contains more nonhistone proteins than the female X-chromosome. The Sxl, mle and msl genes that regulate dosage compensation in Drosophila X-chromosome are of special significance in forming the peculiar morphology of the male chromosome. The proteins coded by mle and msl genes and special acetylated forms of histones take part in the process of loosening the structure of the male X-chromosome. See also Chromosomes: Nonhistone Proteins

In many polytene nuclei, nonhomologous chromosomes are joined by their centromeric regions to form a common chromocentre (Figure 2). Chromocentres in polytene chromosomes correspond to heterochromatin in mitotic chromosomes. There are two types of heterochromatin in Drosophila species: and heterochromatin. The former consist mainly of satellite DNA (i.e. short nucleotide sequences repeated hundred thousand or even million times) and the latter comprises mainly middle repeated mobile elements. The heterochromatic regions contain special proteins: HP1, the product of the Su(var)205 gene and the proteins coded by the genes Su(var)3-7, Su(var)3-9 and SuUR. These are probably members of a family of proteins compacting heterochromatin. In many other species a common chromocentre does not form (Figure 4). See also Developmentally Programmed DNA Rearrangements, and Heterochromatin and Euchromatin

Figure 4. The polytene chromosome set of Chironomus thummi. The chromosomes of this species lie separately from each other; they do not have a common chromocentre. BR, Balbiani rings; NU, nucleolus; CEN, pericentromeric regions. Courtesy of LI Gunderina, unpublished.

There are about 240 regions of the so-called intercalary heterochromatin scattered throughout the genome. These manifest some of the characteristics of the centromeric heterochromatin: late DNA replication, underreplication during polytenization cycles and ectopic pairing. However, intercalary heterochromatin in contrast to centromeric contains genes, necessary only during a certain stage of development. After that those genes must be surely repressed. For example, some of the intercalary heterochromatin regions contain homeotic genes, crucial in Drosophila embryogenesis. By combining several approaches 1036 genes that are arranged in clusters were identified in 52 intercalary heterochromatin regions. These regions overlap extensively (96%) but are not completely identical with late-replicating regions of mitotically dividing Kc cells in culture. Analysis of gene expression profiles revealed that these regions include clusters of coordinately expressed genes and show a particularly common association with transcriptional territories that are expressed in testis of males but not in ovary of females or embryos (Belyakin et al., 2005). Presence of the SUUR protein is one of the most important characteristics of intercalary heterochromatin (Pindyurin et al., 2007).

 

Occurrence of Polyteny

Polyteny is found in a wide range of organisms, appearing at many stages of evolution. For example, it is seen in Infusoria, which appeared as early as the Precambrian era (more than 600 million years ago), in Collembola, which appeared in the middle of the Mesozoic era (more than 400 million years ago), in Diptera and in mammals (100 million years ago).

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 1).

 

Table 1. Occurrence of polytene chromosomes

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Organism

Organ

Type of polyteny

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Insects, Diptera

Larvae

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

Classic type

Pupae

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. Cryptic types can be transformed to the 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

Tumour cells

Cryptic type

Plants

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

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

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Multistrandedness of Polytene Chromosomes

Growth resulting from an increase in the 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 substantial evidence for chromosomes of this type being a bundle of individual chromatids. Polyteny levels (C) differ considerably in different cells within an organ, between organs and between organisms and species, as can be clearly seen from the data presented in Table 2.

 

Table 2. Degree of polyteny in some organisms

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Species	Organ	Degree of polyteny (C)
Chironomus plumosus	Larval salivary glands	1024–4096
Chironomus tentans	Larval salivary glands	8192–32 768
Drosophila melanogaster	Larval salivary glands	1024–2048
	Larval midgut	512–1024
	Imaginal Malpighian tubules	2–256
	Larval fat bodies	16–512
	Larval prothoracic gland	64–512
	Ovarian nurse cells	512–8192
Rhynchosciara angelae	Larval salivary glands	4000–16 000
	Larval salivary glands after intracellular microsporidial infection	512 000–1 024 000
Mammals, different species	Trophoblast	64–4096
Plants, different species	Suspensor, haustorium, antipods and synergids	2–8192

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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. Recently it was shown by Belyaeva that the degree of underreplication of both pericentric and intercalary heterochromatin depends on the action of the SuUR gene (Belyaeva et al., 1998). One more gene affecting DNA replication in polytene chromosomes encodes histonemethyltransferase SU(VAR)3-9. Loss or reduction of SU(VAR)3-9 enzymatic activity affects morphology, compaction state and replication status of pericentric heterochromatin (Demakova et al., 2007; Andreyeva et al., 2007).

Overreplication of regions of polytene chromosomes, the ‘DNA puffs’, have been described in the polytene chromosomes of Sciaridae.

 

Banding Pattern

Along the linear axis of each chromatid, variation in the extent of coiling of the DNA and its associated proteins leads to variation in the concentration of the chromatin. Regions of high concentrations are known as chromomeres (Figure 5). For each chromatid the pattern of chromomeres is highly specific so that in the polytene chromosome homologous chromomeres align alongside each other 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. In the interchromomeric or interband regions, the DNA and protein concentration is lower than in the bands. See also DNA Topology: Fundamentals, Karyotype Analysis and Chromosome Banding, and Supercoiled DNA: Structure

Figure 5. Electron microscopic view of part of the 3R chromosome of Drosophila melanogaster. Polytene chromosome bands and interbands are seen as black and white transverse stripes. Courtesy of VF Semeshin, unpublished.

The pattern of bands and interbands in each polytene chromosome is specific for the species, and in general is characteristic of that particular chromosome in different tissues or at different developmental stages.

A thorough analysis of the banding of the chromosomes of four organs of C. tentans led Beermann in 1950–1970 to the conclusion that a given banding pattern is, to a large extent, the same and ‘all the visible differences are due to technical difficulties such as seeming or real fusion of neighboring bands’ (Beermann, 1950, 1952, 1961, 1972). Studies on the banding patterns of numerous species and diverse organs support the conclusion that the most prominent bands are stable. However, variations in banding pattern are found and there is evidence that those differences correspond to the fusion–fission of neighbouring bands and lengthening of interbands, as described by Beermann (1962). Some studies have noted considerable differences in the banding patterns of chromosomes from cells of normally functioning organs. A comparison of polytene chromosome banding patterns between trichogen cells and ovarian nurse cells failed to reveal any clear homology between the banding patterns, despite their stability within each tissue (Ribbert, 1979).

The phenomenon of seasonal changes in the length of polytene chromosomes described by Ilyinskaya in 1977–1980 for some species of Chironomus is another good example of variation in the banding patterns of polytene chromosomes (Ilyinskaya, 1977, 1980). Before the cold season, the chromosomes become much shorter, the number of puffs is minimal and many of the easily recognized neighbouring bands fuse to form blocks of chromatin. The chromosomes start to lengthen in January–February, and there are 3–4 times more bands in March than in September: the ‘September’ blocks of chromatin split into separate bands and interbands.

These bandings suggest that the chromomeric pattern in the cells of normally functioning organs is relatively stable, but that the structure of the chromomeres is flexible and the general banding pattern is highly variable when intra- and/or extracellular conditions change.

 

Somatic Synapsis of Homologous Chromosomes

Somatic synapsis occurs when two homologous polytene chromosomes fuse. The chromosomes synapse band to band with high precision, giving the impression that a single chromosome is produced. As a consequence, in Drosophila or Chironomus the number of polytene chromosomes decreases to haploid in the nucleus (Figure 2 and Figure 3).

Somatic synapsis is not an obligatory feature of polytene chromosomes: homologous chromosomes consistently conjugate to various degrees in dipteran insects, but in plants or Collembolan insects synapsis is normally absent. It is not quite clear whether polytene homologues synapse in Infusoria and mammals, because the available data are controversial. Synapsis is often incomplete, e.g. in Simuliidae, homologous chromosomes are in physical contact with each other, but the contact is restricted to just some of the regions, the homologues being partly separated from each other in other areas. See also Homologous DNA Interactions in Interphase: Spatial Organization of Interphase Nucleus

The frequency of salivary gland nuclei showing disturbed synapsis (see Figure 2) in any of the chromosomes during normal development of D. melanogaster varies between 6.5% and 45% according to different authors.

Of particular interest is Balbiani's (1881) and Bauer's (1945) discovery of specific asynapsis: in C. plumosus only one chromosome of the set (the fourth chromosomes) do not conjugate and are separated. As was later discovered, synapsis is disturbed to a different degree in the fourth chromosomes of closely related members of the plumosus species group.

Enhancement of asynapsis occurs in hybrids from crosses between representatives of various forms or races. Although in some interspecific hybrids, e.g. in Drosophila of the guarani group, chromosome pairing is just as complete as in the parent species, synapsis in hybrids between Drosophila insularis and Drosophila tropicalis is retained only in the chromocentre.

Modifiers of position-effect variegation (temperature, quantity of Y-chromosome heterochromatin) influence the degree of asynapsis.

 

Molecular Organization of Bands and Interbands

The band number in the D. melanogaster genome can be estimated at 3500–5000. According to genome sequence, euchromatin contains approximately 120 megabase pairs of DNA and about 14 thousand genes. Simple estimations, confirmed by previous photometric data, show that a medium-sized band contains 30 kilobase (kb) pairs of DNA. Analysis of gene and complementary DNA (cDNA) clone distribution along the length of the DNA molecule shows that genes are situated over a shorter range. The band 10A1–2, occupying about 190 kb contains about 25 genes and cDNAs, i.e. 1 gene per 7.6 kb. The average gene density in the Achaete-Scute and Broad-Complexes is 1 gene per 8.0 kb. Sequencing about 2700 kb DNA from the 34C4–36A2 region of D. melanogaster (Rubin, 1998) gave a figure of 1 gene per 13.7 kb. Average value for the whole euchromatic part of the genome is 1 gene per 8.6 kb DNA. So, in general, one 30 kb band may contain between 3 and 5 genes.

Molecular and genetic analyses show that there are bands with very many genes, e.g. the block of 160–200 identical sequences, 385 bp long, of the 5S ribosomal RNA (rRNA) genes occupy a group of four bands. Hundreds of copies of the 18S, 5.8S and 28S rRNA genes are located in the nucleolar organizer, which in many dipteran species looks like a single band. The bands containing repeated histone genes are similarly arranged.

‘Artificial bands’, i.e. those that appear at the sites of transposon insertions in the polytene chromosome, are even more complex. The transposon contains DNA of different kinds: fragments of mobile element, marker genes derived from D. melanogaster, parts of Escherichia coli plasmids and -gal gene. As shown in the electron microscopic studies of Semeshin et al. (1986), all these fragments are united in a single band. See also DNA Transposition: Classes and Mechanisms, and Transposons: Eukaryotic

The regions of polytene chromosome lying between two bands are called interbands or interchromomeres. Because interband chromatin is more decondensed than band chromatin, when the stained chromosomes are viewed in light, phase-contrast or electron microscopes in the interbands appear lighter than the bands. The precise identification of the interbands in polytene chromosomes is difficult because of their small size. According to data obtained for many dipteran species, interbands vary between 0.05 and 0.38 m in size, most frequently being 0.1–0.2 m, and the molecular size of the interband is 0.3–3.8 kb. Using antibodies, many nonhistone proteins can be seen located specifically in the interbands. Among them are RNA polymerase II, proteins-binding RNA in ribonucleoprotein particles, protein included in heterogeneous RNA complexes. Cloning and sequencing the DNA of several interbands by Demakov and colleagues has shown that: (1) they are unique and have no common consensus; (2) a large number of purine, pyrimidine and AT-rich (>80%) fragments have been found as well as several putative Z-DNA-forming sequences, autonomous replication sites (ARS) and binding sites for nuclear matrix protein and (3) they contain several short open reading frames (<550 bp) (Demakov et al., 1993). Codon frequencies in these frames differ significantly from those characteristic of the protein-coding genes of Drosophila. Organization of the D. melanogaster interband DNA is similar to that of scaffold/matrix attachment regions (S/MAR). See also DNA Structure: A-, B- and Z-DNA Helix Families

With respect to their genetic organization interbands could presumably be divided into two classes. Interbands of the first type (I) correspond to the regulatory regions of genes inactive in salivary glands. However, correlation of the regulatory regions of genes with this type of interbands is not obligatory. Interbands of second type (II) correspond to the genes constantly active in this tissue, i.e. contain housekeeping genes (Demakov et al., 2004).

 

Puffs

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; they contained the usual bands. Bridges (1935) called some of the swellings, e.g. those in the X-chromosomal 2B region, ‘puffs’.

Other kinds of swellings were long known as well; e.g. the largest were detected by Balbiani (1881) in C. plumosus as muff-like thickening of the chromosome regions called Balbiani rings by Erhard (1910) and Alverdes (1912).

The detailed studies of Beermann, Pavan and Breuer, and Mechelke in the early 1950s suggested that puffs and Balbiani rings are chromosome regions in which the genes are in an active state (Beermann, 1952; Pavan and Breuer, 1952; Mechelke, 1953). The spectrum of puffs and Balbiani rings is strictly specific to each tissue at a given stage of development (Figure 6). A detailed timetable of changes in the activity of the various puffs has been tabulated for Drosophila by Ashburner (1970). It has been observed in numerous investigations that the puffs are sites of very active transcription.

Figure 6. Electron microscopic view of ecdysone- and heat shock-induced puffs in Drosophila melanogaster. Chromosome region 63A–E is shown before (b) and after (c) heat shock, before (b) and after (a) induction of ecdysone. Inactive genes located in compact chromatin form bands. The bands shown in (b) and (c) are activated after administration of an agent inducing gene activity: ecdysone for 63E2–3 or heat shock for 63B9. As a result, the material of the bands loosens, becomes decompacted and local swelling of the chromosome region occurs. Courtesy of VF Semeshin, unpublished.

In 1961, Beermann found a 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 relation between the activity of a puff and one of the products of the cell was observed for the first time. Subsequently, many authors demonstrated that Balbiani rings of the main lobe of salivary gland code for the major polypeptides of the secretion of this organ. In Drosophila, the relation between the function of several puffs and the synthesis of salivary gland secretion has been demonstrated using cytogenetic methods. See also Cytogenetic Techniques

Studies by Clever and Karlson (1960) on Chironomus and Becker (1962) on Drosophila have demonstrated that the activity of many puffs arising at the end of larval development is induced by the hormone ecdysone, which controls metamorphosis in insects.

Somewhat later, Clever (1964) with Chironomus and Ashburner (as reviewed in Ashburner and Richards, 1976) with Drosophila demonstrated that ecdysone triggers the activation of a whole cascade of interrelated loci, with products of the activities of the puffs induced earlier being needed for the induction of puffs arising later. Ashburner and Richards demonstrated the existence of several waves of interrelated puffs induced by the hormone during larval and prepupal periods of development.

When it was realized that puffs are morphological manifestations of gene activity, numerous experiments aimed at modifying their activities by various agents and chemical components were started. Having exposed larvae of Drosophila busckii to high temperature, Ritossa (1962) revealed the induction of several new puffs (Figure 6) which also arose in response to treatment of salivary glands with poisons that blocked oxidative phosphorylation. These data led to the discovery of a new cellular–physiological system responding to various types of stress at a cellular level – the so-called heat shock syndrome. See also Heat Shock Response

In Sciarids species, certain puffs are involved in DNA amplification (Breuer and Pavan, 1954). They differ in this respect from the majority of puffs. As a result of decompaction and, consequently, a decrease in DNA concentration in puff volume, the usual RNA puffs become light-staining or transparent in appearance. In contrast the ‘DNA puffs’ remain darkly stained because of the accumulation of extra DNA. Such ‘DNA puffs’ arise only in salivary glands at the late stages of larval development and are involved in coding for proteins of salivary gland secretion. Alongside DNA amplification, they are very active in transcription.

The ribosomal DNA (rDNA) of many species consists of several dozens or even hundreds of repetitions of the sequences coding for 18S, 5.8S and 28S RNA, separated by spacer sequences which are untranscribed. Activity of this cluster of genes results in the formation of nucleoli. The morphology of the nucleolus in polytene chromosomes takes a variety of forms. In C. tentans it looks like a typical puffed chromosomal band situated in a euchromatic region of the chromosome. In Drosophila species the nucleolus is located outside the chromosome, the rRNA genes lying within the body of the nucleolus. See also Nucleolus

In the nucleolar organizers of the sciarids the rDNA spreads widely into the body of the nucleolus which is formed at the end of the X-chromosome. Some of the rDNA is found in the many micronucleoli that are scattered among the chromosomes.

 

Utility in Genetic Analysis

As Patterson (1932) and Mackensen (1934) demonstrated, small deletions can be used for accurate gene mapping (Figure 7). With the use of this method in the 1930s the first genes were mapped in D. melanogaster polytene chromosomes with an accuracy to several bands or even part of a band. Today many hundreds of genes are precisely located in the polytene chromosomes maps.

Figure 7. View of heterozygous deletion in Drosophila melanogaster polytene chromosomes, showing normal and deleted chromosome regions. Redrawn from Painter (1934).

This cytogenetic method of gene mapping is now complemented by in situ hybridization, which allows genes to be mapped to a resolution of a few tens of kilobases. See also In Situ Hybridization

The use of Drosophila polytene chromosomes proved to be decisive in the rapid development of cloning methods (e.g. walking and jumping) and in the analysis of the structure and expression of genes. A great step forward in understanding the organization and function of the chromosome came with development of methods of microcloning chromosomes, which allow a specific chromosome region to be dissected out with a micromanipulator and a library of DNA clones to be derived from the region. See also Experimental Organisms Used in Genetics

Heterozygous inversions can be seen clearly in polytene chromosomes (Figure 8). This is important in the genetic analysis of populations.

Figure 8. The first drawing of heterozygous inversion in the X-chromosome of Drosophila melanogaster. Synapsis is complete except at the points where the chromosome fragment is inverted. From Painter (1934).

 

Summary

Polytene chromosomes are gigantic interphase chromosomes that provide an important model for the analysis of the genetic organization of chromosomes and genomes as a whole. Gene functioning can be demonstrated in the puff formation directly under the microscope. Polytene chromosomes are indispensable for gene mapping studies, using chromosome rearrangements and in situ hybridization.

 

End Notes
	Based in part on the previous version of this Encyclopedia of Life Sciences (ELS) article, Polytene Chromosomes by Igor F Zhimulev.

Glossary

Antipod

 Any of three haploid nuclei found within the egg at the apical end of the embryo sac in higher plants.

Cryptic polyteny

 In contrast to classic polyteny, when all chromatids of polytene chromosomes conjugate perfectly with each other and as a result a clear banding pattern forms, in some cases chromatid conjugation is only partial. The polytene chromosomes occupy a large volume, do not form a clear banding pattern and show a reticular structure. This is cryptic (i.e. invisible) polyteny. Changing the physical or physiological conditions results in changes in the degree of chromatid conjugation and transition of cryptic polyteny into classic polyteny.

Dosage compensation

 A regulatory mechanism for sex-linked genes which differ in dose between females and males. It operates such that the resultant phenotypes of these genes are equivalent in the two sexes.

Ectopic pairing

 The tendency of specific regions of polytene chromosomes to stick to other regions in different chromosomes.

Endopolyploid

 Cells whose chromosome number has been increased by endomitosis, i.e. within the intact nuclear envelope, and for which the degree of ploidy is proportional to the number of endomitoses which have taken place.

Haploid

 Cell or individual with a single set of chromosomes.

Macronucleus

 One of two kinds of nuclei in most ciliates. This nucleus assumes control of most synthetic activities of the cell.

Polyploid

 Individual, cell or tissue having three, four or five or more complete chromosome sets instead of two as in diploids.

Position-effect variegation

 Variegation due to the suppression of the expression of a wild-type allele when its position on the chromosome is close to a heterochromatic region.

Scaffold

 Irregular protein mass appearing when histones whose dimensions are similar to the original intact chromosome are removed from isolated metaphase chromosomes.

Synapsis

 Chromosome pairing.

Synergid

 Any of two haploid nuclei found within the egg at the micropilar end of the embryo sac in higher plants.

Z-DNA

 A left-handed double helix. It has 12 bp per turn of the helix, and presents a zigzag conformation.