I. F. Zhimulev

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

The peculiarities of compact blocks appearing as a consequence of position effect variegation were studied in male polytene chromosomes. In T(1;2)dorvar7/Y males the frequency of nuclei with a block in the 2B region was lower at all temperature and the chromosome regioninvolved in compaction was shorter than in T(1;2)dorvar7/FM6 females. The fraction of nuclei with blocks was considerably increased indorvar7/0 males, especially at 18° C when the viability of these males is sharply reduced. The following features distinguish theblocks in males from those in females: (i) compaction of the 2B region in the males results in genetic inactivation only to a very small extent; (ii) the structure of the blocks in males is diffuse; and(iii) the male blocks still maintain some transcriptional activity as indicated by 3H-uridine incorporation. The temperaturesensitive period of both block formation and geneticinactivation was found to be during the first 3 h of embryonic development.

Electron microscopical analysis of Drosophila polytene chromosomes

An electron microscopical (EM) analysis was performed on regions of polytene chromosomes which contained DNA segments of different genetic composition, inserted by P element-mediated transformation into the Drosophila melanogaster genome. In seven of ten regions examined, containing insertions of the hsp28-ry, hsp70-Adh, ryhsp 70-β-gal genes and of the ry gene tetramer, new bands appeared. Lack of new bands in three other strains is apparently connected with the fusion of the inserted material to preexisting bands. The new bands do not differ morphologically from the usual bands of polytene chromosomes, and their formation is likely due to predominant insertion of DNA segments into interbands. Among the constructs examined, the minimal length of a DNA segment which appears as a new band is about 5 kb; the DNA packing ratio in the new bands varies from 30 to 50. Activation of the inserted genes by heat shock has enabled us to observe the puffing characteristics of new bands. A sequence of some one kb forms a large interband, or micropuff; the puff size is correlated with the length of the genes being activated. If a DNA segment contains a single gene, then its activation causes the decompaction of the whole band; however, when a DNA segment consists of two genes and the promoter element of the activated gene is positioned in the middle of the sequence, the band splits and only part is decompacted and puffed. The DNA packing ratio in the puffs is 1.4–3.5. The subsequent deletion of the hsp70 promoter but retention of 23, 59, and 73 bp from the transcription start points leads to failure of puff formation. In all the transformed sites an increase in the total length of the interbands adjacent to the insert as compared with the initial interband was observed. This increase appears to be due to decompaction of the P element DNA flanking the inserted segments. It is shown that a DNA segment, consisting of four tandemly repeated ry gene copies and interspersed by material which includes P DNA, forms a complex of loose chromatin in which, however, four bands can be resolved. We also observed a lengthening of interband regions containing only the P element sequence itself. Insertion of the complete 2.9 kb P element into the large single 10A1-2 band of the X chromosome (an insertion in the region approximately 10 kb to the right of the v gene) causes splitting of the band into two parts and formation of a new interband. However, insertion of the 412 mobile genetic element from the copia family into the same region results in no such effect. These facts together with data on puffing initiation in the centre of the band, when the hsp70 promoter is inside the insert, necessitate a reappraisal of the putative unit character of polytene chromosome bands as regards decompaction.

Intercalary heterochromatin in polytene chromosomes of Drosophila melanogaster

Intercalary heterochromatin consists of extended chromosomal domains which are interspersed throughout the euchromatin and contain silent genetic material. These domains comprise either clusters of functionally unrelated genes or tandem gene duplications and possibly stretches of noncoding sequences. Strong repression of genetic activity means that intercalary heterochromatin displays properties that are normally attributable to classic pericentric heterochromatin: high compaction, late replication and underreplication in polytene chromosomes, and the presence of heterochromatin-specific proteins. Late replication and underreplication occurs when the suppressor of underreplication protein is present in intercalary heterochromatic regions. Intercalary heterochromatin underreplication in polytene chromosomes results in free double-stranded ends of DNA molecules; ligation of these free ends is the most likely mechanism for ectopic pairing between intercalary heterochromatic and pericentric heterochromatic regions. No support has been found for the view that the frequency of chromosome aberrations is elevated in intercalary heterochromatin.

Abnormal tissue-dependent polytenization of a block of chromosome 3 pericentric heterochromatin in Drosophila melanogaster

Heterochromatic DNA sequences in the polytene chromosomes of Drosophila melanogaster salivary glands are under-replicated in wild-type strains. In salivary glands of SuUR and in the nurse cells of otu mutants, under-replication is partly suppressed and a banded structure appears within the centric heterochromatin of chromosome 3. This novel banded structure in salivary gland chromosomes was called Plato Atlantis. In order to characterize the heterochromatic component of Plato Atlantis, we constructed a fine-scale cytogenetic map of deletions with break points within centric heterochromatin (Df(3L)1-16, Df(3L)2-66, Df(3R)10-65, Df(3R)4-75 and Df(3L)6B-29 + Df(3R)6B-29). Salivary gland chromosomes show that Df(3L)1-16 removes the complete Plato Atlantis, while Df(3L)2-66 deletes the most proximal 3L regions. These deletions therefore show a substantial cytological overlap. However, in the chromosomes of nurse cells, the same deficiencies remove distinct heterochromatic blocks, with the region of overlap being almost invisible. Satellite (AATAACATAG, AAGAG) and dodecasatellite DNAs mapped in a narrow interval in salivary glands but were found in three clearly distinguishable blocks in nurse cells. The 1.688 satellite was found at a single site in salivary glands but at two sites in nurse cells. We show that newly polytenized heterochromatic structures include blocks h47-h50d of mitotic heterochromatin in salivary glands, but the additional blocks h50p, h53 and h57 are also included in nurse cell chromosomes. Tissue specificity of the patterns of abnormal heterochromatic polytenization implies differential control of DNA replication in somatic and germline cells.

ALPHA AND BETA HETEROCHROMATIN IN POLYTENE CHROMOSOME 2 OF DROSOPHILA MELANOGASTER

The formation of alpha and beta heterochromatin in chromosomes ofDrosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs ofX0, XY, XYY, XX andXXY individuals the amounts of alpha heterochromatin were similar, suggesting that theY chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization withRsp sequences (Ho clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed.

Induced decondensation of heterochromatin in Drosophila melanogaster polytene chromosomes under condition of ectopic expression of the supressor of underreplication gene

Overexpression of Suppressor of Underreplication protein (SUUR) induces giant reversible swellings in intercalary and pericentric heterochromatin of salivary gland polytene chromosomes. Here, we demonstrate that morphology and extent of swellings are highly dependent on the fixation conditions used: upon glutaraldehyde fixation, we observed moderate decondensation of heterochromatic regions, which was significantly more pronounced upon acetic-acid fixation. Swellings are formed in a PARP-independent fashion. Together with data on inactive transcription in them, this indicates that the swelling-forming regions fail to acquire any features of puffs, the regions typically forming locally decondensed chromatin. Large swellings display striking re-localization of histones and SUUR protein, which are now found at the periphery of the swellings, in contrast to the DNA that fills the entirety of the swelling. We show that swelling-embedded DNA is capable of undergoing replication, however SUUR overexpression drastically alters replication timing in salivary gland cells. We speculate that swelling formation results from SUUR tipping the balance against other proteins that contribute to the organization of repressed chromatin regions.

Tethering of SUUR and HP1 proteins results in delayed replication of euchromatic regions in Drosophila melanogaster polytene chromosomes

We analyze how artificial targeting of Suppressor of Under-Replication (SUUR) and HP1 proteins affects DNA replication in the “open,” euchromatic regions. Normally these regions replicate early in the S phase and display no binding of either SUUR or HP1. These proteins were expressed as fusions with DNA-binding domain of GAL4 and recruited to multimerized UAS integrated in three euchromatic sites of the polytene X chromosome: 3B, 8D, and 18B. Using PCNA staining as a marker of ongoing replication, we showed that targeting of SUURGAL4DBD and HP1GAL4DBD results in delayed replication of appropriate euchromatic regions. Specifically, replication at these regions starts early, much like in the absence of the fusion proteins; however, replication completion is significantly delayed. Notably, delayed replication was insufficient to induce underreplication. Recruitment of SUURGAL4DBD and HP1GAL4DBD had distinct effects on expression of a mini-white reporter, found near UAS. Whereas SUURGAL4DBD had no measurable influence on mini-white expression, HP1GAL4DBD targeting silenced mini-white, even in the absence of functional SU(VAR)3-9. Furthermore, recruitment of SUURGAL4DBD and HP1GAL4DBD had distinct effects on the protein composition of target regions. HP1GAL4DBD but not SUURGAL4DBD could displace an open chromatin marker, CHRIZ, from the tethering sites.

DNA replication in nurse cell polytene chromosomes of Drosophila melanogaster otu mutants

Drosophila cell lines are used extensively to study replication timing, yet data about DNA replication in larval and adult tissues are extremely limited. To address this gap, we traced DNA replication in polytene chromosomes from nurse cells of Drosophila melanogaster otu mutants using bromodeoxyuridine incorporation. Importantly, nurse cells are of female germline origin, unlike the classical larval salivary glands, that are somatic. In contrast to salivary gland polytene chromosomes, where replication begins simultaneously across all puffs and interbands, replication in nurse cells is first observed at several specific chromosomal regions. For instance, in the chromosome 2L, these include the regions 31B-E and 37E and proximal parts of 34B and 35B, with the rest of the decondensed chromosomal regions joining replication process a little later. We observed that replication timing of pericentric heterochromatin in nurse cells was shifted from late S phase to early and mid stages. Curiously, chromosome 4 may represent a special domain of the genome, as it replicates on its own schedule which is uncoupled from the rest of the chromosomes. Finally, we report that SUUR protein, an established marker of late replication in salivary gland polytene chromosomes, does not always colocalize with late-replicating regions in nurse cells.