Studies on replication of the yeast linear DNA plasmids.

本工作旨在阐明乳酸克鲁维酵母线性DNA载体pGKL1和pGKL2复制和维持的遗传机制，并构建酵母中基于pGKL的人工线性载体。在一株乳酸克雷伯菌突变株中发现了两个新的线性质粒(pK192S和pK192L)。PK192S是pGKL1的缺失突变体，来源于ORF1，具有发夹结构，而pK192L是pK192S的反向二聚体。遗传分析表明，pK192S/L的复制依赖于pGKL1和pGKL2，ORF1编码pGKL1自身复制所必需的蛋白质(可能是DNA聚合酶)。酿酒酵母突变体携带了来源于pGKL2的ORFs1-3的两个新的线性质粒pKA209S和pKA209L。它们也是发夹及其倒置二聚体pl…较多的阿米德分别存在，并存在于数百个拷贝数的异常高。携带pKA209S/L的细胞形态异常，生长迟缓，提示高拷贝数质粒阻碍了细胞代谢。与细胞质pGKL不同，得到的线形载体pLS1和pLB1在其两端都有染色体端粒序列，并在细胞核内复制，线形pGKL在酿酒酵母单倍体中稳定复制，但在二倍体中不稳定且经常丢失。对MATA等位基因突变体(Mata1)的遗传分析表明，二倍体中pGKL复制的不稳定可归因于由MATA/MATAlpha基因座编码的二倍体特异阻遏因子(A1-α2)对pGKL复制的负调控。在pGKL2-ORF2和其他可能编码DNA聚合酶和一些复制pGKL质粒所必需的蛋白质的ORF序列中，实际上发现了A1-α2抑制物识别位点。较少

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 … More 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