Identical functional organization of nonpolytene and polytene chromosomes in Drosophila melanogaster.

Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes.

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.

Localization and characteristics of DNA underreplication zone in the 75C region of intercalary heterochromatin in Drosophila melanogaster polytene chromosomes.

In Drosophila polytene chromosomes, regions of intercalary heterochromatin are scattered throughout the euchromatic arms. Here, we present data on the first fine analysis of the individual intercalary heterochromatin region, 75C1-2, located in the 3L chromosome. By using electron microscopy, we demonstrated that this region appears as three closely adjacent condensed bands. Mapping of the region on the physical map by means of the chromosomal rearrangements with known breakpoints showed that the length of the region is about 445 kb. Although it seems that the SUUR protein binds to the whole 75C1-2 region, the proximal part of the region is fully polytenized, so the DNA underreplication zone is asymmetric and located in the distal half of the region. Finally, we speculate that intercalary heterochromatin regions of Drosophila polytene chromosomes are organized into three different types with respect to the localization of the underreplication zone.

Microdissection and sequence analysis of pericentric heterochromatin from the Drosophila melanogastermutant Suppressor of Underreplication.

In the Suppressor of Underreplication( SuUR) mutant strain of Drosophila melanogaster, the heterochromatin of polytene chromosomes is not underreplicated and, as a consequence, a number of beta-heterochromatic regions acquire a banded structure. The chromocenter does not form in these polytene chromosomes, and heterochromatic regions, normally part of the chromocenter, become accessible to cytological analysis. We generated four genomic DNA libraries from specific heterochromatic regions by microdissection of polytene chromosomes. In situ hybridization of individual libraries onto SuUR polytene chromosomes shows that repetitive DNA sequences spread into the neighboring euchromatic regions. This observation allows the localization of eu-heterochromatin transition zones on polytene chromosomes. We find that genomic scaffolds from the eu-heterochromatin transition zones are enriched in repetitive DNA sequences homologous to those flanking the suppressor of forked gene [ su(f) repeat]. We isolated and sequenced about 300 clones from the heterochromatic DNA libraries obtained. Most of the clones contain repetitive DNA sequences; however, some of the clones have unique DNA sequences shared with parts of unmapped genomic scaffolds. Hybridization of these clones onto SuUR polytene chromosomes allowed us to assign the cytological localizations of the corresponding genomic scaffolds within heterochromatin. Our results demonstrate that the SuUR mutant renders possible the mapping of heterochromatic scaffolds on polytene chromosomes.