RESUMO
An increase in the dose of the Su(var)3-7 locus of Drosophila augments heterochromatin-promoted variegated silencing. The deduced protein sequence of Su(var)3-7 reveals seven widely spaced zinc fingers. We found that Su(var)3-7 has affinity for DNA in vitro and that the minimal protein sequence requirement for DNA binding is any module containing two zinc fingers and the interval between them. As Su(var)3-7 is a heterochromatin-associated protein, we tested its affinity for various satellite DNA sequences in vitro. The AATAT and 353-bp elements have the highest affinity. If affinity for satellite DNAs contributes to the presence of Su(var)3-7 in heterochromatin, a general affinity for DNA, or sequences yet to be determined, suggests a function in the genomic silencing of position-effect variegation: expansion of heterochromatin, whether continuous by spreading or discontinuous by pairing with sequence elements scattered through euchromatin, could use the affinity of Su(var)3-7 for DNA.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Regulação da Expressão Gênica , Ordem dos Genes/genética , Sequência Rica em At/genética , Animais , Sequência de Bases , Sítios de Ligação , Western Blotting , Sequência Consenso , DNA/genética , DNA Satélite/genética , DNA Satélite/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Genes de Insetos/genética , Deleção de Sequência/genética , Dedos de ZincoRESUMO
An increase in the dose of the Su(var)3-7 locus of Drosophila melanogaster enhances the genomic silencing of position-effect variegation caused by centromeric heterochromatin. Here we show that the product of Su(var)3-7 is a nuclear protein which associates with pericentromeric heterochromatin at interphase, whether on diploid chromosomes from embryonic nuclei or on polytene chromosomes from larval salivary glands. The protein also associates with the partially heterochromatic chromosome 4. As these phenotypes and localizations resemble those described by others for the Su(var)2-5 locus and its heterochromatin-associated protein HP1, the presumed co-operation of the two proteins was tested further. The effect of the dose of Su(var)3-7 on silencing of a number of variegating rearrangements and insertions is strikingly similar to the effect of the dose of Su(var)2-5 reported by others. In addition, the two loci interact genetically, and the two proteins co-immunoprecipitate from nuclear extracts. The results suggest that SU(VAR)3-7 and HP1 co-operate in building the genomic silencing associated with heterochromatin.
Assuntos
Padronização Corporal/genética , Proteínas Cromossômicas não Histona/isolamento & purificação , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/química , Proteínas Nucleares/isolamento & purificação , Proteínas Repressoras/isolamento & purificação , Animais , Compartimento Celular , Divisão Celular , Centrômero/química , Homólogo 5 da Proteína Cromobox , Drosophila/embriologia , Imunofluorescência , Dosagem de Genes , Proteínas de Insetos/isolamento & purificação , Larva , Testes de PrecipitinaRESUMO
An increase in the number of copies of the Drosophila locus Suvar(3)7 enhances position-effect variegation, i.e. the inactivation in some cells of genes brought close to heterochromatin by a chromosomal rearrangement. The locus produces two transcripts of 5047 and 4203 nt that differ solely by the length of their 3' untranslated region. That these transcripts encode the modifier of variegation Suvar(3)7 is demonstrated by genetic transformation with the corresponding cDNAs. The deduced protein is 1169 amino acids long and contains seven widely spaced zinc fingers. These fingers are each preceded at 11-16 amino acids before the N-terminal cysteine by a tryptophan-containing motif. The transcripts are maternally transmitted, but are also found throughout development. The ubiquitous distribution of transcripts in embryos and the different sequence motifs support our speculation that the locus encodes a chromosomal protein implicated in heterochromatin-mediated DNA silencing.
