Lund T, Grosveld F G, Flavell R A
Proc Natl Acad Sci U S A. 1982 Jan;79(2):520-4. doi: 10.1073/pnas.79.2.520.
A procedure has been developed to allow the recovery of an integrated plasmid genome from a transformed cell, together with large areas of the flanking DNA sequences. DNA from Saccharomyces cerevisiae BAS2, in which the pBR322--ura 3 plasmid (Y1p5) is integrated at the yeast histone H2A and H2B locus, was used to generate a cosmid library, using a new cosmid vector (pTL5) that is ampicillin sensitive and tetracycline resistant. Colonies were selected for ampicillin resistance, which was conferred by the incorporation of the integrated pBR322 beta-lactamase gene into the recombinant cosmid. Restriction enzyme and blot hybridization analyses show that the rescued clones contain the yeast histone genes in addition to the Y1p5 sequences; a total of approximately 50 kilobase pairs of DNA sequences flanking the plasmid was recovered as a series of overlapping cosmids. This approach should allow the recovery of most genes that can be linked to a marker pBR322 sequence and for which a specific phenotype can be selected in a recipient eukaryotic cell.
已开发出一种程序，可从转化细胞中回收完整的质粒基因组以及大片段侧翼DNA序列。来自酿酒酵母BAS2的DNA（其中pBR322-ura 3质粒（Y1p5）整合在酵母组蛋白H2A和H2B基因座处）被用于构建黏粒文库，使用的是一种新的对氨苄青霉素敏感且对四环素耐药的黏粒载体（pTL5）。选择对氨苄青霉素具有抗性的菌落，这是通过将整合的pBR322β-内酰胺酶基因掺入重组黏粒中来实现的。限制性内切酶和印迹杂交分析表明，获救的克隆除了含有Y1p5序列外，还包含酵母组蛋白基因；作为一系列重叠黏粒，总共回收了约50千碱基对的质粒侧翼DNA序列。这种方法应能回收大多数可与标记pBR322序列相连且能在受体真核细胞中选择特定表型的基因。
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