Analysis of DNA interband regions 3A5/A6, 3C5-6/c7 and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes]

Using electron microscopic (EM) data on the formation of a novel band from the P-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes were cloned and sequenced. EM analysis of the 3C region have shown that the formation of the full-size 3C5-6/C7 interband requires a 880-bp DNA sequences removed by deletion Df(1)faswb. A comparison of DNA sequences of six bands, two of which were obtained in the present work and four were described earlier, demonstrated the uniqueness of each of them in the Drosophila genome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19 transcribed throughout the development, in particular in salivary glands. In the other interbands examined 5' and 3' nontranslated gene regions are located. These results suggest that Drosophila interbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.     

Molecular and genetic organization of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>polytene chromosomes: evidence for two types of interband regions

The 3A and 60E regions of Drosophila melanogaster polytene chromosomes containing inserted copies of the P{lArB} transposon have been subjected to an electron microscopic (EM) analysis. We show that both inserts led to formation of new bands within the interband regions 3A4/A6 and 60E8-9/E10. This allowed us to clone DNA of these interbands. Their sequences, as well as those of DNA from other four interbands described earlier, have been analyzed. We have found that, with the exception of 60E8-9/E10 interband, all other five regions under study corresponded to 5' or 3' ends of genes. We have further obtained the evidence for 60E8-9/E10 interband to harbor the 'housekeeping' RpL19 gene, which is transcribed in many tissues, including salivary glands. Based upon the genetic heterogeneity of the interbands observed a revised model of polytene chromosome organization is discussed.     

Structural features of 3C6/C7 interband chromatin organization in Drosophila melanogaster polytene chromosomes

Mechanisms of chromatin decompaction in interbands of Drosophila polytene chromosomes have been studied. Using the example of interband 3C6/C7 of the X chromosome, we investigated the ability of different DNA segments to form an interband in a new genetic environment. We applied site-specific FLP recombination between two transposons with FRT-sites that allows introducing the DNA fragments from the interband 3C6/C7 into pICon(dv) transposon located in cytologically well-characterized 84F region of chromosome 3 followed by electron microscopic analysis of changes in the region caused by insertion of the DNA fragments into the transposon. It was shown that the insertion of a 276-bp DNA fragment from the 3C6/C7 region into the pICon(dv) transposon leads to the formation of a new interband between two thin bands represented by the transposon material. This DNA fragment is the known minimal sequence that is necessary and sufficient for interband generation. In addition, the sequence containing three copies repeated in tandem of 0.9 kb DNA from the interband 3C6/C7, including the 276-bp fragment, were integrated in the transposon. The presence of introduced DNA fragments did not change the morphology of the resulting interband. It was shown that the sites of DNase I hypersensitivity were saved in transposon sequences introduced into a new genetic environment. The data obtained allow analysis to be started of specific factors (proteins, DNA motifs, etc.) that determine the formation of decompacted chromatin in a certain interband region and chromomeric organization of interphase chromosomes in Drosophila as a whole.     

Cloning and molecular genetic analysis of Drosopbila melanogaster interband DNA

Interband DNA of Drosophila melanogaster polytene chromosomes was studied using a novel approach based on the electron microscopic (EM) analysis of chromosome regions carrying DNA fragements of known molecular genetic composition, inserted by P element-mediated transformation. Insertion of such fragments predominantly into interbands makes it possible to clone interband DNA by constructing genomic libraries from transformed strains and probing them with the insert DNA. The transformed strain P[H-sp70:Adh](61C) has insertion in the 61 C7-8 interband on the left arm of chromosome 3. This DNA consists of part of the hsp70 gene promoter fused to the coding region of the Adh gene, and is flanked on either side by P element sequences. We constructed a genomic library from DNA of this strain and isolated a clone containing the insert and the interband DNA. Subsequently the genomic library of wild-type strain was probed with a subclone composed of interband DNA only. We have thus isolated a clone containing the entire native interband. 1289 by of interband DNA was sequenced and found to be AT-rich (53.4%) with numerous regions of overlapping direct and inverted repeats, regulatory sites, and two overlapping open reading frames (ORFs).     

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.     

Cloning and molecular genetic analysis of Drosopbila melanogaster interband DNA

Interband DNA of Drosophila melanogaster polytene chromosomes was studied using a novel approach based on the electron microscopic (EM) analysis of chromosome regions carrying DNA fragements of known molecular genetic composition, inserted by P element-mediated transformation. Insertion of such fragments predominantly into interbands makes it possible to clone interband DNA by constructing genomic libraries from transformed strains and probing them with the insert DNA. The transformed strain P[H-sp70:Adh](61C) has insertion in the 61 C7-8 interband on the left arm of chromosome 3. This DNA consists of part of the hsp70 gene promoter fused to the coding region of the Adh gene, and is flanked on either side by P element sequences. We constructed a genomic library from DNA of this strain and isolated a clone containing the insert and the interband DNA. Subsequently the genomic library of wild-type strain was probed with a subclone composed of interband DNA only. We have thus isolated a clone containing the entire native interband. 1289 by of interband DNA was sequenced and found to be AT-rich (53.4%) with numerous regions of overlapping direct and inverted repeats, regulatory sites, and two overlapping open reading frames (ORFs).     

Structural paradox of polytene chromosomes

The observation of thick chromatin fibers in interbands of Dipteran polytene chromosomes suggests that there should be 5 to 10 times more mass and DNA in interbands than is commonly thought to be present. To resolve this paradox, the chromatin content of interbands was estimated, using whole-mounted polytene chromosomes from Drosophila melanogster. Densitometry of high voltage electron microscopic negatives provides an estimate of less than 4:1 for the average ratio of cross-sectional dry mass (or mass per unit chromosome length) of bands relative to interbands. This ratio, combined with an estimate of the length of chromosome composed of interbands, indicates that at least 26% of chromosome mass is contributed by interband chromatin. Since DNA comprises a similar proportion of chromatin mass in bands and interbands (Laird et al., 1980b), these data imply that DNA sequences in interbands represent at least 26% of the euchromatic genome of D. melanogaster. This result calls for reinterpretation of some of the genetic and molecular data from Diptera. The discrepancy between this higher estimate of interband mass and DNA, and previous estimates of 3-5%, is discussed. One possibility is that previous measurements were made on prominent interbands, which are proposed here to be in regions that are delayed in DNA replication. Such interbands would be reduced in polyteny and DNA content compared with the average interband region. The concept of local variations in polyteny is also used here to explain major differences in the cross-sectional mass of bands. This leads to a revised model of polytene chromosomes in which at least three levels of polyteny, rather than one or two levels, can be present within one euchromatic region.     

DNA from Drosophila melanogaster Interband 61C7/C8 Evolves at a High Rate

The results of a comparative study of cloned DNA fragments ofDrosophila simulans, D. mauritiana, D. teissieri, and D. erecta are presented. The fragments were amplified in PCR with primers specified to the region of D. melanogaster interband 61C7/C8. The uniqueness of all cloned fragments in the genomes of these species was confirmed. A comparative analysis of nucleotide sequences revealed that the rate of evolution of DNA from D. melanogaster interband 61C7/C8 is close to the rate of neutral evolution in the genusDrosophila.     

Ubiquitin genes and ubiquitin protein location in polytene chromosomes of Drosophila

Ubiquitin genes are found in Drosophila either as a repeat block or as gene fusions with ribosomal proteins. Here is described the location of a new repeat block in the X chromosome that is present in the strain Canton S but absent in Vallecas. There are also two ubiquitin-ribosomal protein fusion genes located at regions 97A of chromosome 3R and 31E of 2L. Using an anti-ubiquitin antibody in Drosophila polytene chromosomes it is shown that ubiquitin is mainly associated with the compact and stabilized structure that forms the bands rather than with the more decondensed and destabilized protein-DNA structure that forms interbands and puffs.     

Electron microscopic band-interband pattern of the X chromosome in Drosophila hydei

The band-interband pattern of the salivary gland X chromosome in Drosophila hydei was studied by electron microscopy (EM) using the technique of surface-spread polytene (SSP) chromosome preparation. We observed 526 chromosome bands, i.e. 135 additional bands as compared with the original light microscopic chromosome map (Berendes 1963). Individual interband lengths and band thicknesses were measured for the entire X chromosome in electron micrographs of ten SSP chromosome preparations. Average values were used to plot an EM chromosome map. The average interband had an axial length of 0.38 m. Depending upon the extension of the DNA packing ratio in interbands, this indicates 1.1 kb of totally extended DNA or 3.8 kb, if a DNA packing ratio of 0.10 m/kb is assumed for SSP chromosomes (Kress et al. 1985).