RESUMO
BACKGROUND: Despite many efforts, little is known about distribution and interactions of chromatin proteins which contribute to the specificity of chromomeric organization of interphase chromosomes. To address this issue, we used publicly available datasets from several recent Drosophila genome-wide mapping and annotation projects, in particular, those from modENCODE project, and compared molecular organization of 13 interband regions which were accurately mapped previously. RESULTS: Here we demonstrate that in interphase chromosomes of Drosophila cell lines, the interband regions are enriched for a specific set of proteins generally characteristic of the "open" chromatin (RNA polymerase II, CHRIZ (CHRO), BEAF-32, BRE1, dMI-2, GAF, NURF301, WDS and TRX). These regions also display reduced nucleosome density, histone H1 depletion and pronounced enrichment for ORC2, a pre-replication complex component. Within the 13 interband regions analyzed, most were around 3-4 kb long, particularly those where many of said protein features were present. We estimate there are about 3500 regions with similar properties in chromosomes of D. melanogaster cell lines, which fits quite well the number of cytologically observed interbands in salivary gland polytene chromosomes. CONCLUSIONS: Our observations suggest strikingly similar organization of interband chromatin in polytene chromosomes and in chromosomes from cell lines thereby reflecting the existence of a universal principle of interphase chromosome organization.
Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Drosophila melanogaster/genética , Cromossomos Politênicos/genética , Animais , Histonas/genética , InterfaseRESUMO
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.
Assuntos
Replicação do DNA , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , Animais , Sequência de Bases , Southern Blotting , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/ultraestrutura , Heterocromatina/genética , Heterocromatina/ultraestrutura , Mapeamento Físico do Cromossomo , Transporte ProteicoRESUMO
Telomeres are generally considered heterochromatic. On the basis of DNA composition, the telomeric region of Drosophila melanogaster contains two distinct subdomains: a subtelomeric region of repetitive DNA, termed TAS, and a terminal array of retrotransposons, which perform the elongation function instead of telomerase. We have identified several P-element insertions into this retrotransposon array and compared expression levels of transgenes with similar integrations into TAS and euchromatic regions. In contrast to insertions in TAS, which are silenced, reporter genes in the terminal HeT-A, TAHRE, or TART retroelements did not exhibit repressed expression in comparison with the same transgene construct in euchromatin. These data, in combination with cytological studies, provide evidence that the subtelomeric TAS region exhibits features resembling heterochromatin, while the terminal retrotransposon array exhibits euchromatic characteristics.
Assuntos
Cromossomos/genética , Drosophila melanogaster/genética , Telômero/genética , Animais , Sequência de Bases , Southern Blotting , Cromossomos/ultraestrutura , Biologia Computacional , Primers do DNA , Componentes do Gene , Biblioteca Genômica , Heterocromatina/genética , Hibridização In Situ , Microscopia Eletrônica , Dados de Sequência Molecular , Retroelementos/genética , Análise de Sequência de DNA , Sequências Repetidas Terminais/genética , Transgenes/genéticaAssuntos
Bandeamento Cromossômico/métodos , Cromossomos/ultraestrutura , Drosophila melanogaster/genética , Glândulas Salivares/ultraestrutura , Cromossomo X/ultraestrutura , Animais , Autorradiografia/métodos , Mapeamento Cromossômico/métodos , Cromossomos/genética , Drosophila melanogaster/ultraestrutura , Imuno-Histoquímica , Hibridização In Situ/métodos , Indicadores e Reagentes , Microscopia Eletrônica/métodos , Reprodutibilidade dos Testes , Cromossomo X/genéticaRESUMO
We studied whether interbands can be ectopically formed in Drosophila melanogaster polytene chromosomes. For comparative purposes, two types of P-element constructs were used. The first type was represented by P-element based insertions into compact bands. Sequences of these insertions or adjacent genomic sequences could be activated ectopically either by GAL4 or by dosage compensation machinery. In the second type, the DNA from transcriptionally silent interbands was positioned between the FRT sites, and was flanked by DNA sequences of genes that were also inactive in salivary glands. Electron microscopy analysis of salivary gland polytene chromosomes demonstrated that both types of constructs formed distinct, yet morphologically similar interbands. Notably, the second class of transposon insertions appeared in polytene chromosomes as two bands separated by one interband. Excision of interband material from such insertions resulted in fusion of newly appeared bands into a single band. We were able to confirm by molecular means that the DNA sequences in integrated constructs were intact, that chromatin organization of this DNA mimicked that of native interbands, and that it was accurately excised from the constructs by FLP. Thus, we demonstrate that transfer of interband DNA into a silent genetic environment does not compromise interband formation. Our results do not support the idea of the existence of distinct cytogenetic "band + interband" units, furthermore, they suggest the autonomy of the decompacted state of interbands.
Assuntos
Bandeamento Cromossômico , Cromossomos/genética , Drosophila melanogaster/genética , Animais , Animais Geneticamente Modificados , Cromatina/genética , Cromossomos/ultraestrutura , DNA/análise , DNA/genética , Elementos de DNA Transponíveis , DNA Mitocondrial , Diploide , Evolução Molecular , Genes de Insetos , Heterocromatina , Hibridização In Situ , Masculino , Filogenia , Glândulas Salivares/citologia , Análise de Sequência de DNARESUMO
The 3A and 60E regions of Drosophila melanogaster polytene chromosomes containing inserted copies of the P[1ArB] transposon have been subjected to an electron microscopic (EM) analysis. We show that both inserts led to formation of new bands within the interband regions 3A4/A6 and 60E8-9/E10. This allowed us to clone DNA of these interbands. Their sequences, as well as those of DNA from other four interbands described earlier, have been analyzed. We have found that, with the exception of 60E8-9/E10 interband, all other five regions under study corresponded to 5' or 3' ends of genes. We have further obtained the evidence for 60E8-9/E10 interband to harbor the 'housekeeping' RpL19 gene, which is transcribed in many tissues, including salivary glands. Based upon the genetic heterogeneity of the interbands observed a revised model of polytene chromosome organization is discussed.
Assuntos
Cromossomos/genética , Drosophila melanogaster/genética , Animais , Bandeamento Cromossômico , Cromossomos/fisiologia , Cromossomos/ultraestrutura , Clonagem Molecular , Elementos de DNA Transponíveis , Microscopia Eletrônica , Análise de Sequência de DNARESUMO
The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.