| Budget Amount *help |
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1987: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1986: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Research Abstract |
The aim of this work is to elucidate the genetic mechanism involved in the replication and maintenance of the linear DNA plasmids, pGKL1 and pGKL2, from the yeast Kluyveromyces lactis and also to construct pGKL-based artificial linear vectors in yeast.
Mutant pGKL plasmids were isolated spontaneously or by mutagen treatment from the pGKL killer strains of K. lactis or Saccharomyces cerevisiae. Two novel linear plasmids (pK192S and pK192L) were discovered in a mutant of K. lactis. pK192S was a deletion mutant of pGKL1, derived from the ORF1, and had a hairpin structure, while pK192L was an inverted dimer of pK192S. Genetic analysis revealed that the replication of pK192S/L is dependent on both pGKL1 and pGKL2 and that the ORF1 encodes an essential protein (probably DNA polymerase) necessary for the replication of pGKL1 itself. A mutant of S. cerevisiae carried two new linear plasmids, pKA209S and pKA209L, derived from the ORFs1-3 of pGKL2. They were also hairpin and its inverted dimer pl
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asmids, respectively, and existed in abnormally high copy number of several hundreds. Cells harboring pKA209S/L were morphologically abnormal and retarded in growth, suggesting that the high copy number plasmids hindered the cellular metabolism.
A nuclear gene marker, LEU2, was integrated into ORF2 of pGKL1 by an in vivo gene displacement technique. Unexpectedly, the resulting linear vectors, pLS1 and pLB1, possessed chromosomal telomere sequences at their both ends and replicated in nucleus, unlike the cytoplasmic pGKL plasmids.
Linear pGKL plasmids replicated stably in o haploids of S. cerevisiae, but were unstable and frequently lost in diploids. Genetic analysis with MATa allele mutant (mata1) revealed that the plasmid instability in diploids was ascribable to the negative regulation of pGKL replication by diploid specific repressor (a1-alpha 2), encoded by the MATa/MATalpha locus. The a1-alpha 2 repressor recognition sites were actually found in sequences of pGKL2-ORF2 and other ORFs which may encode DNA polymerase and some proteins necessary for the replication of pGKL plasmids. Less
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