RESUMO
Chromatin insulator proteins are one of the major components that determine the domain organization of the genome. According to the latest data, they can mark the boundaries of topological domains and prevent the spread of silent chromatin to adjacent areas. One approach to the analysis of the actions of these proteins is to use the ectopic involvement in the UAS>DBD(GAL4). The method allows to evaluate the effect of selected protein in chromatin organization, to establish its association with other insulator proteins and influence on the processes of transcription and replication. and influence the processes of transcription and replication. In this study, we have developed and tested the functionality of the system components in ectopic tethering of the Chromator (Chriz) to the region of intercalary heterochromatin 10A1-2. Preliminary data have been obtained showing that ectopically tethered Chromator to the band 10A1-2 can induce a partial decompactization of the band chromatin. Further use of this experimental model provides the opportunity to investigate the effect of insulator proteins on the chromatin structure.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Drosophila/metabolismo , Elementos Isolantes , Proteínas Associadas à Matriz Nuclear/metabolismo , Cromossomos Politênicos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Associadas à Matriz Nuclear/genética , Cromossomos Politênicos/genética , Fatores de Transcrição/genéticaRESUMO
About 240 specific regions that are replicated at the very end of the S-phase have been identified in D. melanogaster polytene chromosomes. These regions have a repressive chromatine state, low gene density, long intergenic distances and are enriched in tissue specific genes. In polytene chromosomes, about a quarter of these regions have no enough time to complete replication. As a result, underreplication zones represented by fewer DNA copy number, appear. We studied 60 chromosome regions that demonstrated the most pronounced under-replication. By comparing the location of these regions on a molecular map with syntenic blocks found earlier for Drosophila species by von Grotthuss et al., 2010, we have shown that across the genus Drosophila, these regions tend to have conserved gene order. This forces us to assume the existence of evolutionary mechanisms aimed at maintaining the integrity of these regions.
Assuntos
Replicação do DNA/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Cromossomos Politênicos/genética , Animais , Proteínas de Ligação a DNA , Glândulas Salivares/citologiaRESUMO
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.
Assuntos
Mapeamento Cromossômico/métodos , Replicação do DNA , DNA/química , Drosophila melanogaster/genética , Larva/citologia , Animais , Mapeamento Cromossômico/instrumentação , Reagentes de Ligações Cruzadas/química , DNA/genética , Diploide , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Vidro/química , Hibridização in Situ Fluorescente , Larva/genética , Larva/metabolismo , Sondas Moleculares/química , Desnaturação de Ácido Nucleico , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase , Poliploidia , Origem de Replicação , Sefarose/químicaRESUMO
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.