RESUMO
BACKGROUND: Chromosome four of Drosophila melanogaster, known as the dot chromosome, is largely heterochromatic, as shown by immunofluorescent staining with antibodies to heterochromatin protein 1 (HP1) and histone H3K9me. In contrast, the absence of HP1 and H3K9me from the dot chromosome in D. virilis suggests that this region is euchromatic. D. virilis diverged from D. melanogaster 40 to 60 million years ago. RESULTS: Here we describe finished sequencing and analysis of 11 fosmids hybridizing to the dot chromosome of D. virilis (372,650 base-pairs) and seven fosmids from major euchromatic chromosome arms (273,110 base-pairs). Most genes from the dot chromosome of D. melanogaster remain on the dot chromosome in D. virilis, but many inversions have occurred. The dot chromosomes of both species are similar to the major chromosome arms in gene density and coding density, but the dot chromosome genes of both species have larger introns. The D. virilis dot chromosome fosmids have a high repeat density (22.8%), similar to homologous regions of D. melanogaster (26.5%). There are, however, major differences in the representation of repetitive elements. Remnants of DNA transposons make up only 6.3% of the D. virilis dot chromosome fosmids, but 18.4% of the homologous regions from D. melanogaster; DINE-1 and 1360 elements are particularly enriched in D. melanogaster. Euchromatic domains on the major chromosomes in both species have very few DNA transposons (less than 0.4 %). CONCLUSION: Combining these results with recent findings about RNAi, we suggest that specific repetitive elements, as well as density, play a role in determining higher-order chromatin packaging.
Assuntos
Mapeamento Cromossômico , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Drosophila/genética , Heterocromatina/genética , Animais , DNA/genética , Proteínas de Drosophila/genética , Etiquetas de Sequências Expressas , Genoma , Hibridização In Situ , Modelos Genéticos , Modelos Estatísticos , Interferência de RNA , Sequências Repetitivas de Ácido Nucleico , Retroelementos/genética , Estatísticas não ParamétricasRESUMO
Heterochromatin Protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster localized principally in the pericentric heterochromatin, telomeres, and fourth chromosome, all regions associated with HP1. Mutations in HP2 can suppress position effect variegation, indicating a role in gene silencing and heterochromatin formation [Shaffer, C. D. et al. (2002) Proc. Natl. Acad. Sci.U.S.A. 99, 14332-14337]. In vitro coimmunoprecipitation experiments with various peptides from HP2 have identified a single HP1-binding domain. Conserved domains in HP2, including those within the HP1-binding region, have been identified by recovering and sequencing Su(var)2-HP2 from D. willistoni and D. virilis, as well as examining available sequence data from D. pseudoobscura. A PxVxL motif, shown to be an HP1-binding domain in many HP1-interacting proteins, is observed but is not well-conserved in location and sequence and does not mediate HP2 binding to HP1. The sole HP1-binding domain is composed of two conserved regions of 12 and 16 amino acids separated by 19 amino acids. Site-directed mutagenesis within the two conserved regions has shown that the 16 amino acid domain is critical for HP1 binding. This constitutes a novel domain for HP1 interaction, providing a critical link for heterochromatin formation in Drosophila.
Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Drosophila/química , Heterocromatina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Western Blotting , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Complementar/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Evolução Molecular , Heterocromatina/metabolismo , Imunoprecipitação , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Heterochromatin protein 1 (HP1), first discovered in Drosophila melanogaster, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. We report here characterization of an HP1-interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT-hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing.
