| Project/Area Number |
09044236
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Kumamoto Institute of Technology |
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Principal Investigator |
MATSUOKA Masayoshi Kumamoto Institute of Technology, Associate Professor, 工学部, 助教授 (10121667)
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| Co-Investigator(Kenkyū-buntansha) |
FOURNIER Philippe Institute national de la Recherche Agronomique, France, Senior Scientist, Senior Sci
UCHIDA Kohji Nagahama Institute for Biochemical Science, Oriental Yeast Co.Ltd., Head Researc, 長浜生物科学研究所, 主任研究員
PHILIPPE Fou Institut National de la Recherche Agrono, Senior Sci
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1997: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | centromere / Yarrowia lipolytica / autonomous replication / plasmids / transformation / nuclear matrix / site-directed mutagenesis / 複製起点 / 染色体 |
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| Research Abstract |
Three DNA fragments displaying autonomously-replicating (ARS) activity on plasmids in the yeast Yarrowia lipolytica have been obtained and shown to harbor replication origins associated with centromeres. These fragments contain replication origins closely associated with CEN1, CEN3 and CEN5 among six chromosomes in Y lipolytica as shown by the following experiments : 1) These clones hybridized with different chromosomal bands in the pulse-field gel electrophoresis ; 2) When the CEN-containing fragments were integrated in the ectopic sites on chromosome, chromosome breakage was observed for the dicentric chromosomes ; 3) a centromeric plasmid can be used to clone many replication origins coming from several genomic locations. The replicating DNA fork was detected by 2-D gel electrophoresis.
Transformation of Y.lipolytica is therefore feasible only in the simultaneous presence of a replication origin and a centromere, the latter being necessary either for a proper partition of nuclear plasmids or for a resolution of replicated daughter plasmid DNAs. However, a simple binding to a nuclear matrix was not sufficient for a CEN function.
The minimal CEN1 region was confined within about 130 bp. A shot-gun cloning of genomic DNA on an origin-containing vector revealed three additional clones via high-frequency transformation. These new clones were CEN2, CEN4 and a fragment flanking but not overlapping with minimal CEN1. This finding could be explained if we assume that Y.lipolytica centromeres are regional structures as observed in Schizosaccharomyces pombe or higher eukaryotes. The conserved sequence elements in CEN region are investigated by means of site-directed mutagenesis.
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