Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system.
Here a method is described to identify genes encoding proteins that recognize a specific DNA sequence. A bank of random protein segments tagged with a transcriptional activation domain is screened for proteins that can activate a reporter gene containing the sequence in its promoter. This strategy was used to identify an essential protein that interacts in vivo with the yeast origin of DNA replication. Matches between its predicted amino acid sequence and peptide sequence obtained from the 50-kilodalton subunit of the yeast origin recognition complex (ORC) established that the gene isolated here, ORC6, encodes this subunit. These observations provide evidence that ORC recognizes yeast replication origins in vivo.
Li
J J
JJ
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Herskowitz
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D002453:Cell Cycle; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D051738:Origin Recognition Complex; D011401:Promoter Regions, Genetic; D012093:Replicon; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497221:ORC6 protein, S cerevisiae; D051738:Origin Recognition Complex; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins