HIGH-FREQUENCY TRANSFORMATION OF YEAST - AUTONOMOUS REPLICATION OF HYBRID DNA-MOLECULES
HIGH-FREQUENCY TRANSFORMATION OF YEAST - AUTONOMOUS REPLICATION OF HYBRID DNA-MOLECULES
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DOI：
10.1073/pnas.76.3.1035
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发表时间：
1979-01-01
影响因子：
11.1
通讯作者：
DAVIS, RW
中科院分区：
文献类型：
--
作者：
STRUHL, K;STINCHCOMB, DT;DAVIS, RW
本文描述了一组用于酿酒酵母和大肠杆菌DNA片段克隆的载体DNA （Y载体）。利用这些载体，定义了酵母转化的3种模式。含有酵母染色体DNA序列（YIp1, YIp5, type 1）的载体以低频率（1-10菌落/.mu）转化酵母细胞。G)通过同源重组整合到基因组中；这种重组是可逆的。含有内源性酵母质粒DNA序列（YEp2, YEp6, type 2）的杂交体转化酵母细胞的频率更高（5000-20,000菌落/.mu.g）。这些分子以每个酵母细胞5-10个共价闭合环的平均拷贝数自主复制，并作为染色体整合结构复制。这种DNA可以从酵母或大肠杆菌中以完整的形式分离出来，并用于高频转化这两种生物体。含有1.4千碱基酵母DNA片段(包括着丝粒连接的trp1基因（YRp7, 3型）的载体转化酵母的效率为500-5000菌落/. μ g；这些分子表现为小染色体，因为它们自主复制，但不整合到基因组中。讨论了在酵母中使用Y载体进行下列遗传操作：基因的分离；构建单倍体菌株，使其为特定DNA序列的二倍体；并指导酵母基因组的改变。介绍了这些事件的选择和分析的一般方法。
A set of vector DNA (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized. With these vectors, 3 modes of yeast transformation are defined. Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5, type 1) transform yeast cells at low frequency (1-10 colonies/.mu.g) and integrate into the genome by homologous recombination; this recombination is reversible. Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6, type 2) transform yeast cells at much higher frequency (5000-20,000 colonies/.mu.g). Such molecules replicate autonomously with an average copy number of 5-10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure. This DNA may be physically isolated in intact form from either yeast or E. coli and used to transform either organism at high frequency. Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7, type 3) transform yeast with an efficiency of 500-5000 colonies/.mu.g; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome. The use of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome. General methods for the selection and the analysis of these events were presented.