RESUMO
REB1 is a DNA-binding protein that recognizes sites within both the enhancer and the promoter of rRNA transcription as well as upstream of many genes transcribed by RNA polymerase II. We report here the cloning of the gene for REB1 by screening a yeast genomic lambda gt11 library with specific oligonucleotides containing the REB1 binding site consensus sequence. The REB1 gene was sequenced, revealing an open reading frame encoding 809 amino acids. The predicted protein was highly hydrophilic, with numerous OH-containing amino acids and glutamines, features common to many of the general DNA-binding proteins of Saccharomyces cerevisiae, such as ABF1, RAP1, GCN4, and HSF1. There was some homology between a portion of REB1 and the DNA-binding domain of the oncogene myb. REB1 is an essential gene that maps on chromosome II. However, the physiological role that it plays in the cell has yet to be established.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Proteínas de Ligação a DNA/isolamento & purificação , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myb , Mapeamento por Restrição , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Fatores de TranscriçãoRESUMO
Restriction endonucleases EcoR1 and BamH1 are used to produce fragments pBR322C (375 bp) and pBR322B (3987 bp) from pBR322, and to produce lambda F2A (65.6--71.3% of lambda DNA, 2679 bp) and lambda F2B (71.3--81% of lambda DNA, 4559 bp) from EcoR1 restriction fragment lambda F2 (65.6--81% of lambda DNA) of lambda cI857S7 DNA. By recombining pBR322B and lambda F2B in vitro, a new plasmid called pCB2 carrying promoters and structural genes cI and cro is constructed. The desired strain with pCB2 is selected from 338 transformants for its AprTcs and for its immunity to lambda infection. The length of the pCB2 DNA molecule is 2.66 +/- 0.33 micrometers and its MW is 5.51 +/- 0.68 x 10(6)d, as determined by electron microscope and agarose gel electrophoresis. The lengths of single strands and the double strands of the heteroduplex formed between lambda F2 and linear pCB2 (EcoR1 digested) agree well with the original design for its construction, From the above data, we come to the conclusion that pCB2 we constructed is a new plasmid with cI and/or cro gene expressed in E. coli.
