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).     

Extent of polyteny in the pericentric heterochromatin of polytene chromosomes of pseudonurse cells of otu (ovarian tumor) mutants of Drosophila melanogaster

In the polytene nuclei of germ-line cells (ovarian pseudonurse cells) of Drosophila melanogaster females mutant for otu ¹¹ (ovarian tumor), the pericentric heterochromatin is much more abundant than in somatic salivary gland cells. This is due to the degree of heterochromatin compaction (and consequently the level of underreplication) being lower in the nurse cells than in the salivary gland cells. The lower level of compaction probably results in a very low degree of position effect gene inactivation in the ovarian nurse cells.     

Extent of polyteny in the pericentric heterochromatin of polytene chromosomes of pseudonurse cells of otu (ovarian tumor) mutants of Drosophila melanogaster

In the polytene nuclei of germ-line cells (ovarian pseudonurse cells) of Drosophila melanogaster females mutant for otu ¹¹ (ovarian tumor), the pericentric heterochromatin is much more abundant than in somatic salivary gland cells. This is due to the degree of heterochromatin compaction (and consequently the level of underreplication) being lower in the nurse cells than in the salivary gland cells. The lower level of compaction probably results in a very low degree of position effect gene inactivation in the ovarian nurse cells.     

Transcription of intercalary heterochromatin regions in Drosophila melanogaster cell culture

Labelled RNA preparations (total newly synthesized RNA, as well as stable cytoplasmic RNA) isolated from a cell culture of D. melanogaster were hybridized in situ with polytene chromosomes. Apart from the nucleolus, in all cases the regions adjacent to the chromocentre in the polytene chromosomes and the intercalary heterochromatin regions in the X chromosome and the autosomes are the most intensively labelled. In the case of asynapsis of polytene chromosomes in heterozygotes the label is detected in a number of intercalary heterochromatin sites in one homologue only ("the asymmetrical label"). The same kind of radioactivity distribution in intercalary heterochromatin regions was observed after a hybridization of polytene chromosomes with cloned DNA fragments (Ananiev et al., 1978, 1979) coding for the abundant classes of messenger RNA (Ilyin et al., 1978) in a cultured D. melanogaster cells. In some regions of intercalary heterochromatin which do not contain these fragments the "'asymmetrical" type of label distribution is observed after hybridization with cell RNA. - These results lead one to regard the intercalary heterochromatin regions as "nests" comprising different types of actively transcribable genes, the composition of each nest varying in different stocks of D. melanogaster.     

A novel simple satellite DNA is colocalized with the Stalker retrotransposon in Drosophila melanogaster heterochromatin

In the T(1;2)dor ^var7 multibreak rearrangement the distal 1A-2B segment of the X chromosome of Drosophila melanogaster is juxtaposed to an inverted portion of the heterochromatin of chromosome 2. Analysis of mitotic chromosomes by a series of banding techniques has permitted us precisely to locate the heterochromatic breakpoint of this translocation in the h42 region of 2R. Cloning and sequencing of the eu-heterochromatic junction revealed that the translocated 1A-2B fragment is joined to (AACAC)_n repeats, which represent a previously undescribed satellite DNA in D. melanogaster. These repeated sequences have been estimated to account for about 1 Mb of the D. melanogaster genome. The repeats are located mainly in the Y chromosome and in the heterochromatin of the right arm of chromosome 2 (2Rh), where they are colocalized with the Stalker retrotransposon. Received: 3 October 1998 / Accepted: 3 December 1998     

Cytogenetic analysis of two ecdysone-regulated puffs 62C and 62E in Drosophila melanogaster

Genetic analysis has been performed to reveal vital genes around two puffs, a late 62C puff and an early-late 62E puff. Their roles in hormonal regulatory mechanisms have been estimated. A locus represented by four lethal mutations has been found in the vicinity of the 62E puff. The mutants display disturbed puffing, which suggests the involvement of this locus in hormonal regulatory mechanisms. In the 62C puff region. 26 mutations have been found that proved to be allelic to mutations in the D-Titin gene. The giant D-Titin gene is essential for the sarcomeric organization of striated muscles. According to the results of in situ hybridization with polytene chromosomes, the D-Titin gene occupies the entire 62C puff. The phenotypic characteristics of the novel mutants suggest that this protein is polyfunctional, and its role is not restricted to processes in the muscular tissue. It may also be involved in the morphogenesis of leg imaginal disks, and it is necessary for condensation and separation of sister chromatids during mitosis. Mutations in the ecdysone-induced BR-C and E74 genes cause disturbances similar to those found in this study. In addition, mutations of these genes can affect the D-Titin gene activity, which suggests that the three genes are involved in similar morphogenetic and myogenetic processes.