ISOLATION OF GENES BY COMPLEMENTATION IN YEAST - MOLECULAR-CLONING OF A CELL-CYCLE GENE
ISOLATION OF GENES BY COMPLEMENTATION IN YEAST - MOLECULAR-CLONING OF A CELL-CYCLE GENE
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DOI：
10.1073/pnas.77.4.2119
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发表时间：
1980-01-01
期刊：
影响因子：
--
通讯作者：
REED, SI
中科院分区：
文献类型：
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作者：
NASMYTH, KA;REED, SI
cdc 28是酵母S.通过用4-碱基识别限制性内切酶Sau 3A部分消化酵母DNA以得到随机片段的等价物，在蔗糖梯度上进行大小选择，并利用Sau 3A末端与通过BamHI切割在载体中产生的那些末端的同源性将片段引入酵母载体YRp 7中，构建在含有整个酵母基因组的重组质粒库上分离酿酒酵母。通过转化cdc 28 ts菌株并选择能够在限制性温度下生长的克隆来分离能够补充cdc 28突变的重组质粒。负责补充cdc 28 ts表型的质粒显示与染色体cdc 28位点特异性重组，证实了克隆序列的同一性。此外，其中一个重组质粒能够补充与cdc 28基因连锁的tyr 1基因的突变。这种基因分离和鉴定的方法应该适用于所有酵母基因，对于这些基因有容易评分的突变体。
One of several genes required for cell division, cdc28, in the yeast S. cerevisiae was isolated on recombinant plasmid pool containing the entire yeast genome was constructed by partial digestion of yeast DNA with the 4-base recognition restriction endonuclease Sau3A to give the equivalent of random fragments, size selection on sucrose gradients, and introduction of the fragments into the yeast vector YRp7 by use of the homology of Sau3A ends with those generated in the vector by cleavage with BamHI. Recombinant plasmids capable of complementing cdc28 mutations were isolated by transformation of a cdc28ts strain and selection for clones capable of growth at the restrictive temperature. Plasmids responsible for complementing the cdc28ts phenotype were shown to recombine specifically with the chromosomal cdc28 locus confirming the identity of the cloned sequences. In addition, one of the recombinant plasmids was capable of complementing a mutation in tyr1, a gene genetically linked to cdc28. This method of gene isolation and identification should be applicable to all yeast genes for which there are readily scorable mutants.