Tanaka H, Tsuboi M
Mol Gen Genet. 1985;199(1):21-5. doi: 10.1007/BF00327504.
In order to isolate a DNA fragment able to complement a sporulation-deficient mutation in Saccharomyces cerevisiae, a simple screening procedure was devised which was based on the difference in osmotic sensitivity between protoplasts and spores. A plasmid (pHT7) containing a 13 kb DNA insert that complemented the spoT7 mutation was isolated from a yeast genomic library prepared in the vector YEp13. Gene spoT7 was linked to rna1 at 1.2 cM and to mak27 at 7.2 cM on the right arm of chromosome XIII. Mapping of the cloned gene following integration into the chromosome showed that the cloned gene was allelic to spoT7 and that a part of the RNA1 gene was also cloned into the same fragment. Gene spoT7 was localized on a 5 kb DNA fragment by further subcloning.
为了分离出能够互补酿酒酵母中孢子形成缺陷突变的DNA片段,设计了一种基于原生质体和孢子之间渗透敏感性差异的简单筛选程序。从用载体YEp13构建的酵母基因组文库中分离出一个含有13 kb DNA插入片段且能互补spoT7突变的质粒(pHT7)。基因spoT7在第十三号染色体右臂上与rna1相距1.2 cM,与mak27相距7.2 cM。将克隆基因整合到染色体后进行定位,结果表明克隆基因与spoT7等位,并且RNA1基因的一部分也被克隆到了同一个片段中。通过进一步亚克隆,将基因spoT7定位在一个5 kb的DNA片段上。