Otu and Rif1 Double Mutant Enables Analysis of Satellite DNA in Polytene Chromosomes of Ovarian Germ Cells in Drosophila melanogaster.

Polytene chromosomes in Drosophila serve as a classical model for cytogenetic studies. However, heterochromatic regions of chromosomes are typically under-replicated, hindering their analysis. Mutations in the Rif1 gene lead to additional replication of heterochromatic sequences, including satellite DNA, in salivary gland cells. Here, we investigated the impact of the Rif1 mutation on heterochromatin in polytene chromosomes formed in ovarian germ cells due to the otu gene mutation. By the analysis of otu11; Rif11 double mutants, we found that, in the presence of the Rif1 mutation, ovarian cells undergo additional polytenization of pericentromeric regions. This includes the formation of large chromatin blocks composed of satellite DNA. Thus, the effects of the Rif1 mutation are similar in salivary gland and germ cells. The otu11; Rif11 system opens new possibilities for studying factors associated with heterochromatin during oogenesis.

Banding Pattern of Polytene Chromosomes as a Representation of Universal Principles of Chromatin Organization into Topological Domains.

Drosophila polytene chromosomes are widely used as a model of eukaryotic interphase chromosomes. The most noticeable feature of polytene chromosome is transverse banding associated with alternation of dense stripes (dark or black bands) and light diffuse areas that encompass alternating less compact gray bands and interbands visible with an electron microscope. In recent years, several approaches have been developed to predict location of morphological structures of polytene chromosomes based on the distribution of proteins on the molecular map of Drosophila genome. Comparison of these structures with the results of analysis of the three-dimensional chromatin organization by the Hi-C method indicates that the morphology of polytene chromosomes represents direct visualization of the interphase nucleus spatial organization into topological domains. Compact black bands correspond to the extended topological domains of inactive chromatin, while interbands are the barriers between the adjacent domains. Here, we discuss the prospects of using polytene chromosomes to study mechanisms of spatial organization of interphase chromosomes, as well as their dynamics and evolution.

COMPREHENSIVE APPROACH TO MAPPING LATE-REPLICATING BANDS IN POLYTHENE CHROMOSOMES OF DROSOPHILA MELANOGASTER.

Heterogeneity of chromatin structure underlies the banding pattern of Drosophila polythene chromosomes. Recently, a four-color model of chromatin has been proposed, with one chromatin type, aquamarine, largely corresponding to interbands (Zhimulev et al., 2014). In this model, most of the previously mapped regions of intercalary heterochromatin are represented by the ruby chromatin type. In the present report, we used the distal part of the chromosome arm 2R to show that all dense late-replicating bands in this region invariably encompass ruby chromatin type. We propose a comprehensive approach that combines cytology mapping data of the FlyBase-annotated genes, novel tools for predicting cytogenetic features of chromosomes based on their prothein composition and data on the sequence of replication in polythene chromosome bands. This approach allows to establish accurately the correspondence between reference late-replicating bands visible on cytology maps and the molecular map.

[Regulation of DNA replication timing].

Although distinct chromatin types have been long known to replicate at different timepoints of S phase, fine replication control has only recently become considered as an epigenetic phenomenon. It is now clear that in course of differentiation significant changes in genome replication timing occur, and these changes are intimately linked with the changes in transcriptional activity and nuclear architecture. Temporally coordinate replication is organized spatially into discrete units having specific chromosomal organization and function. Even though the functional aspects of such tight control of replication timing remain to be explored, one can confidently consider the replication program as yet another fundamental feature characteristic of the given differentiation state. The present review touches upon the molecular mechanisms of spatial and temporal control of replication timing, involving individual replication origins as well as large chromatin domains.

[Molecular combing method in the research of DNA replication parameters in isolated organs of Drosophyla melanogaster].

Methods of physical DNA mapping and direct visualization of replication and transcription in specific regions of genome play crucial role in the researches of structural and functional organization of eukaryotic genomes. Since DNA strands in the cells are organized into high-fold structure and present as highly compacted chromosomes, the majority of these methods have lower resolution at chromosomal level. One of the approaches to enhance the resolution and mapping accuracy is the method of molecular combing. The method is based on the process of stretching and alignment of DNA molecules that are covalently attached with one of the ends to the cover glass surface. In this article we describe the major methodological steps of molecular combing and their adaptation for researches of DNA replication parameters in polyploidy and diploid tissues of Drosophyla larvae.

[Molecular combing in studies of the genome organization and DNA replication].

Molecular combing (MC) yields preparations where individual DNA molecules are uniformly stretched and are parallel to each other. Fluorescence in situ hybridization on such preparations allows an exact mapping of DNA sequences, and pulsed inclusion of halogenated deoxyuridine analogs and their detection using fluorochrome-conjugated antibodies makes it possible to visualize replication. The MC technique was adapted for studying DNA replication in isolated Drosophila melanogaster organs, and it was checked whether a mutation of the Suppressor of UnderReplication (SuUR) gene directly affected the replication fork rate.

[Intercalary heterochromatin in the genome of Drosophila].

The modern concept of intercalary heterochromatin as polytene chromosome regions exhibiting a number of specific characteristics is formulated. DNA constituting these regions is replicated late in the S period; therefore, some strands of polytene chromosomes are underrepresented; i.e., they are underreplicated. Late-replicating regions account for about 7% of the genome; genes are located there in clusters of as many as 40. In general, the gene density in the clusters is substantially lower than in the main part of the genome. Late-replicating regions have an inactivating capacity: genes incorporated into these regions as parts of transposons are inactivated with a higher probability. These regions contain a specific protein SUUR affecting the rate of replication completion.

Local DNA underreplication correlates with accumulation of phosphorylated H2Av in the Drosophila melanogaster polytene chromosomes.

DNA in Drosophila melanogaster polytene chromosomes is known to be locally underreplicated in both pericentric and intercalary heterochromatin. When the SuUR gene is mutant, complete and partial suppression of underreplication are observed in intercalary and pericentric heterochromatin, respectively; in contrast, overexpression of SuUR results in stronger underreplication. Using antibodies against phosphorylated histone H2Av and flies with different levels of SuUR expression, we demonstrated a clear correlation between the extent of underreplication in specific chromosome regions and the accumulation of H2Av phosphorylated at S137 (gamma-H2AX) at the same sites. Phosphorylated H2Av is a well-established marker of DNA double-stranded breaks (DSB). Our data thus argue that DNA underreplication leads to DSBs and that DSBs accumulate as salivary gland cells progress throughout repeated endocycles. We speculate that ligation of free double-stranded DNA termini causes the formation of ectopic contacts between the underreplicated regions in heterochromatin.

[Contribution of the SuUR gene to the organization of epigenetically repressed regions of Drosophila melanogaster chromosomes].

A significant portion of a eukaryotic genome is silent (epigenetically repressed). In Drosophila melanogaster, this portion includes mainly regions of pericentric and intercalary heterochromatin and euchromatin regions subject to position-effect variegation. Detailed study of the organization of intercalary heterochromatin regions of Drosophila melanogaster polytene chromosomes started from the discovery of the SuUR gene (Suppressor of UnderReplication). The ability of the SuUR mutation to suppress underreplication in intercalary heterochromatin regions was used for molecular tagging of these regions. We showed that underreplicated intercalary heterochromatin regions contained silent unique genes and retained the features of late replication and transcriptionally inactive chromatin state in various cell types. Over 50% of these regions contain unique genes clustered on the base of coordinated expression. The origin of clusters and putative mechanisms of their gene expression are discussed. Data on the SuUR gene, its expression, and effect on polytene chromosome structure and replication are summarized.

Functional dissection of the Suppressor of UnderReplication protein of Drosophila melanogaster: identification of domains influencing chromosome binding and DNA replication.

The Suppressor of UnderReplication (SuUR) gene controls the DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster salivary gland polytene chromosomes. In the present work, we investigate the functional importance of different regions of the SUUR protein by expressing truncations of the protein in an UAS-GAL4 system. We find that SUUR has at least two separate chromosome-binding regions that are able to recognize intercalary and pericentric heterochromatin specifically. The C-terminal part controls DNA underreplication in intercalary heterochromatin and partially in pericentric heterochromatin regions. The C-terminal half of SUUR suppresses endoreplication when ectopically expressed in the salivary gland. Ectopic expression of the N-terminal fragments of SUUR depletes endogenous SUUR from polytene chromosomes, causes the SuUR- phenotype and induces specific swellings in heterochromatin.

Ectopic expression of the Suppressor of Underreplication gene inhibits endocycles but not the mitotic cell cycle in Drosophila melanogaster.

The Suppressor of Underreplication ( SuUR) gene contributes to the regulation of DNA replication in regions of intercalary heterochromatin in salivary gland polytene chromosomes. In the SuUR mutant these regions complete replication earlier than in wild type and, as a consequence, undergo full polytenization. Here we describe the effects of ectopic expression of SuUR using the GAL4-UAS system. We demonstrate that ectopically expressed SuUR exerts qualitatively distinct influences on polyploid and diploid tissues. Ectopic expression of SuUR inhibits DNA replication in polytene salivary gland nuclei, and reduces the degree of amplification of chorion protein genes that occurs in the follicle cell lineage. Effects caused by ectopic SuUR in diploid tissues vary considerably; there is no obvious effect on eye formation, but apoptosis is observed in the wing disc, and wing shape is distorted. The effect of ectopic SuUR expression is enhanced by mutations in the genes E2F and mus209 ( PCNA). Differential responses of polyploid and diploid cells to ectopic SuUR may reflect differences in the mechanisms underlying mitotic cell cycles and endocycles.

[Interline differences in morphology of the precentromeric region of polytene X-chromosome in Drosophila melanogaster salivary glands]. / Mezhlineinye razlichiia v morfologii pritsentromernogo raiona politennoi X-khromosomy v sliunnykh zhelezakh Drosophila melanogaster.

Morphology of the Drosophila melanogaster polytene X chromosome section 20 in normal flies, in strains carrying inversions that break pericentric heterochromatin at different points, and at the background of the Su(UR)ES mutation has been examined. In all of the strains carrying the Su(UR)ES mutation section 20 displayed a distinct banding pattern till to the section 20F, while in the wild-type strains this region was represented by beta-heterochromatin. The strains carrying different inversions substantially differed in the number and morphology of bands forming section 20. In the Su(UR)ES mutants the most proximal X chromosome euchromatin gene, su(f), is mapped to the boundary between sections 20E and F, while rDNA forming the middle part of the X chromosome mitotic heterochromatin is located in the proximal part of section 20F. All large bands observed in section 20 of the w; Su(UR)ES strain were also present in In(1)sc4; Su(UR)ES, which breaks heterochromatin in the distal part. Hence, the bands of polytene chromosome section 20 are virtually devoid of mitotic heterochromatin.