Characterization of a novel CDC gene (ORC1) partly homologous to CDC6 of Saccharomyces cerevisiae

Yuji Hori, Katsuhiko Shirahige, Chikashi Obuse, Toshiki Tsurimoto, Hiroshi Yoshikawa

Research output: Contribution to journalArticle

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Abstract

A novel cell cycle gene was identified by a computer search for genes partly homologous to known CDC genes, CDC6 of Saccharomyces cerevisiae and CDC18 of Schizosaccharomyces pombe, using the nucleotide sequence data base for S. cerevisiae produced by the Yeast Sequencing Project. The protein sequence coded by the cloned gene was found to be identical to that of purified ORC1 protein. Disruption of the gene and subsequent tetrad analysis revealed that the gene was essential for growth. The function of the gene product was analyzed by depleting the protein from the cell using a mutant haploid strain containing the disrupted ORC1 gene on the chromosome and a galactose-inducible gene coding for HA-tagged ORC1 protein on a single copy plasmid. The HA-tagged protein was expressed during growth in the presence of galactose but began to decrease rapidly upon depletion of galactose. Analysis of the cell cycle progression of the mutant cells by FACS after the removal of galactose from the medium, and microscope observations of cells and their nuclei revealed that the normal progression of 2N cells was immediately impeded as the ORC1 protein started to decrease. This was blocked completely in the cells that had progressed to the S phase under conditions deficient in ORC1 protein followed by cell death. Two-dimensional gel analysis of the replication intermediates after the galactose removal revealed that the depletion of ORC1 protein caused a decrease in the frequency of initiation of chromosomal replication, eventually resulting in the inhibition of replication as a whole. The function of the ORC1 protein in the cell cycle progression of S. cerevisiae is discussed in light of current information on ORC.

Original languageEnglish
Pages (from-to)409-418
Number of pages10
JournalMolecular Biology of the Cell
Volume7
Issue number3
DOIs
Publication statusPublished - Jan 1 1996
Externally publishedYes

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Origin Recognition Complex
Centers for Disease Control and Prevention (U.S.)
Saccharomyces cerevisiae
Galactose
Genes
Cell Cycle
cdc Genes
Proteins
Schizosaccharomyces
Haploidy
Essential Genes
Growth
Cell Nucleus
S Phase
Plasmids
Cell Death
Chromosomes
Yeasts
Gels
Databases

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

Characterization of a novel CDC gene (ORC1) partly homologous to CDC6 of Saccharomyces cerevisiae. / Hori, Yuji; Shirahige, Katsuhiko; Obuse, Chikashi; Tsurimoto, Toshiki; Yoshikawa, Hiroshi.

In: Molecular Biology of the Cell, Vol. 7, No. 3, 01.01.1996, p. 409-418.

Research output: Contribution to journalArticle

Hori, Yuji ; Shirahige, Katsuhiko ; Obuse, Chikashi ; Tsurimoto, Toshiki ; Yoshikawa, Hiroshi. / Characterization of a novel CDC gene (ORC1) partly homologous to CDC6 of Saccharomyces cerevisiae. In: Molecular Biology of the Cell. 1996 ; Vol. 7, No. 3. pp. 409-418.
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