Formation and morphology of dark puffs in Drosophila melanogaster polytene chromosomes

The formation of unusual dark puffs in Drosophila melanogaster polytene chromosomes has been studied by electron microscopic (EM) analysis. Fly stocks transformed by the P[ry; Prat:bw] and P[hs-BRC-z1] constructs were used. In the former the bw gene is under the promoter of a housekeeping gene, Prat; in the latter the Br-C locus, mapping to the dark puff 2B, is under the promoter of a heat-shock gene, hsp70. Inserted into region 65A of the 3L chromosome, the Prat:bw copies give rise to structures which are morphologically reminiscent of the so-called "dark" puffs. In contrast, insertion of P[hs-BRC-z1] into region 99B of the 3R chromosome causes a regular "light" puff of form. Comparative analysis of the dark puffs--both transgenic and natural--suggests that there might be at least two mechanisms underlying their formation. One is a local incomplete decondensation of activated bands, characteristic of the so-called small puffs. The other is the formation of ectopic-looking contacts between the bands adjacent to the puffing zone. Transposition of the DNA, from which such a puff develops, causes a regular light puff to form at the new location. Heterochromatic regions do not appear to be directly involved in puffing.     

Modification of the DNA content in translocated regions of Drosophila polytene chromosomes

The DNA content of translocated polytene chromosome regions in Drosophila melanogaster is affected by heterochromatic position effect. Microdensitometric studies on w ^m258-21 translocation heterozygotes showed (Hartmann-Goldstein and Cowell, 1976; Cowell and Hartmann Goldstein, 1980) that band region 3D1-E2, adjacent to the breakpoint, contained less DNA than the homologous non-translocated region whereas the neighbouring 3C1-10 region contained more DNA than its non-translocated counterpart. In the nuclei selected for measurement the translocated X chromosome was morphologically euchromatic, but both regions undergo heterochromatisation in other nuclei within the same salivary gland. To explore the relationship between changes in DNA content and heterochromatisation, the effect on DNA content of two known modifiers of heterochromatisation has now been studied. Larvae cultured at 15° C, which exhibit more heterochromatisation than those grown at 25° C, have the same relative DNA contents as at the higher temperature. The addition of a Y chromosome markedly reduced heterochromatisation; in XXY larvae there was no difference between the DNA contents of translocated and non-translocated 3D1-E2 regions, and in region 3C1-10 the percentage excess of DNA in the translocated homologue was approximately double that found in XX larvae. The relationship between replication behaviour and compaction suggested by these results is discussed.     

Interchromosomal asynchrony of DNA replication in polytene chromosomes of Drosophila pseudoobscura

Analysis of ³H-thymidine autoradiograms of late third instar larval salivary glands of Drosophila pseudoobscura revealed a unique example of asynchrony of replication in the autosome complement. The two autosomal arms, 2 and 3, show similar labeling pattern during the initial phases, DD to 3C, and thereafter, the chromosome 3 has fewer labeled sites than chromosome 2 until the most terminal pattern, 1D. Detailed sitewise analysis of ³H-thymidine labeling shows that while nearly 54% of the sites examined in chromosome 2 have a labeling frequency greater than 50%, only 13% of all sites in chromosome 3 have labeling frequency at that range. The number of labeled sites on chromosome 3 plotted against that on chromosome 2 shows a hyperbolic profile rather than a linear relationship. The silver grain ratio of the 2nd to 3rd increases from 1.5 to 3.1 through different stages of the cycle. These results suggest that both chromosomes start replication simultaneously but the third chromosome appears to complete the replication earlier than the second. These data open up the possibility of separate control mechanisms for the initiation and termination of DNA replication in polytene chromosomes.This paper is dedicated to the memory of the late Prof. H. D. Berendes.     

Duplications in the polytene chromosomes of Drosophila auraria

A study of the salivary gland chromosomes of two strains of Drosophila auraria has revealed a suprisingly high number of inverted tandem duplications and one triplication. The possible origin and significance of these are discussed.     

Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes

Fragments from section 3 of the salivary gland X chromosome of D. melanogaster were dissected with a micromanipulator. The DNA was extracted, cut and ligated to a λ vector in a volume of a few nanoliters in an oil chamber monitored through a microscope. From about 10 pg of DNA we obtained 80 recombinant clones, a sample of which were analysed and shown to contain Drosophila DNA which hybridises in situ to the region of section 3 of the X chromosome. With this technique we can isolate clones from any desired region as small as 200 kb from the euchromatic arms of polytene chromosomes. This paper is dedicated to Professor W. Beermann on the occasion of his sixtieth birthday     

N-banding in polytene chromosomes of Chironomus and Drosophila

The N-banding patterns of the polytene chromosomes of Drosophila melanogaster, Chironomus melanotus, Ch. th. thummi and Ch. th. thummi x Ch. th. piger were studied. In Chironomus the polytene N-banding patterns correspond to the polytene puffing patterns. This is revealed by comparison of the puffing and N-banding patterns of identical chromosomes. Size and staining intensity of the N-bands reflect the size of the puffs as shown by puff induction. There is no evidence that the N-bands are also located in Chironomus heterochromatin or are restricted to the nucleolar organizer regions. In Drosophila the -heterochromatin is strongly N-positive, whereas the -heterochromatin, as well as the Chironomus heterochromatin is not N-banded. Contrary to Chironomus, the puffs in Drosophila polytene chromosomes do not give rise selectively to well stained N-bands. — The N-banding method is interpreted to stain specifically non-histone protein which is (1) accumulated in genetically active chromosome regions and (2) present in a specific type of heterochromatin (-heterochromatin of Drosophila).     

Electron microscopical analysis of Drosophila polytene chromosomes

Data are presented of electron microscopic (EM) analysis of consecutive developmental stages of Drosophila melanogaster complex puffs, formed as a result of simultaneous decondensation of several bands. EM mapping principles proposed by us permitted more exact determination of the banding patterns of 19 regions in which 31 puffs develop. It is shown that 20 of them develop as a result of synchronous decondensation of two bands, 7 of three and 4 of one band. Three cases of two-band puff formation when one or both bands undergo partial decondensation are described. In the 50CF, 62CE, 63F and 71CF regions puffing zones are located closely adjacent to each other but the decondensation of separate band groups occurs at different puff stages (PS). These data are interpreted as activation of independently regulated DNA sequences. The decondensation of two or three adjacent bands during formation of the majority of the puffs occurs simultaneously in the very first stages of their development. It demonstrates synchronous activation of the material of several bands presumably affected by a common inductor. Bands adjacent to puffing centres also lose their clarity as the puff develops, probably due to "passive" decondensation connected with puff growth. The morphological data obtained suggest a complex genetic organisation of many puffs.     

Electron microscopical analysis of Drosophila polytene chromosomes

An electron microscopic (EM) analysis was performed on regions of Drosophila melanogaster polytene chromosomes that contain inserted DNA segments of 19 and 8 kb. These segments had been inserted by P-elementmediated transformation. The 19 kb segment includes both the Drosophila hsp70 gene fused to the Escherichia coli -galactosidase gene and the rosy gene (Lis et al. 1983). This insert generates a new moderate-size band at the 9D4-9E1-2 region in polytene chromosomes. Upon heat shock, a puff originates from a portion of the new band. The 8 kb segment includes the Sgs7 and Sgs3 genes (Richards et al. 1983). This insert generates very diffuse thin bands that decondense at the stage of activation of the Sgs genes to produce wide interbands or small puffs. In all of the above cases, the insertion appears to occur at interband regions, and the genetically complex DNA segments that are inserted generate only a single detectable band.     

Characteristics of structures of Drosophila polytene chromosomes formed by transposable DNA fragments

An electron microscopic analysis of regions of Drosophila melanogaster polytene chromosomes into which DNA fragments of different genetic composition were inserted by the P element-mediated transformation was performed. In 4 of 5 regions studied with integrated DNA sequences of the hsp28-ry, hsp70-Adh, ry-hsp70-beta-gal genes new bands appeared. Apparently their generation is mainly caused by integration of the DNA fragments in interbands. Absence of a new band in transformed region in one of the stocks can be explained by fusion of the insertion with a band existed in the initial untransformed stock. Among the transformants studied, the minimum length of DNA fragment revealed as a new band is about 5 kb. DNA packing ratio of such the bands varies from 30 to 50. The activation of the inserted genes by heat shock allows to trace peculiarities of the new bands puffing. The puff sizes correlate with the length of the activated genes. If the DNA of the fragment consists of the sequence of one gene, its activation will lead to decondensation of the whole band. In the case when DNA fragment consists of 2 genes and the promoter of activated gene is situated inside the sequence, the band is splitted after gene activation at the beginning and then separated portion of the band is decondensed and puffed. The data obtained evidence that a band of polytene chromosome is not a unit of decondensation. DNA packing ratio in puffs is equal to 1.5-3.5.     

Electron microscopical analysis of Drosophila polytene chromosomes

Replication studies on prophasic human Y chromosomes reveal 4 early replicating segments in the euchromatic portion. The distal segment of Yp replicates first. After replication of the euchromatic part is almost finished 3 to 5 segments start replication in the heterochromatic portion of Yq. These segments exhibit considerable intraindividual variation with respect to the origin of onset of replication. While the location of these bands — once they are differentiated — is fixed within one individual, the number of these bands varies interindividually.Dedicated to Professor Dr. Ulrich Wolf on the occasion of his 50the birthday     

Electron microscopical analysis of Drosophila polytene chromosomes

Mapping of 16 regions of polytene chromosomes in which 18 one-band puffs develop was carried out with the use of electron microscopy (EM). In most cases a uniform decondensation of the whole band was observed. However, there were examples in which only a part of the band was activated (three puffs) or its right and left parts decondensed simultaneously (three puffs). Splitting of the band into two parts with their further decondensation was also found (one puff). This suggests structural and functional complexity of the bands. On the basis of the data obtained here and those published earlier, a classification of 52 puffs by the number of bands participating in their formation is given. Four classes numbering 22, 21, 7, 2 puffs, developing from 1, 2, 3 and 4 bands, respectively, are revealed. The data show that active chromosome regions are rather diverse in both the pattern of decondensation and expansion of the decondensed region, thus providing evidence of the informational complexity of the majority of active regions.