Souciet J L, Hubert J C, Lacroute F
Mol Gen Genet. 1982;186(3):385-90. doi: 10.1007/BF00729458.
Two yeast DNA pools inserted in a hybrid Escherichia coli-yeast vector pFL1 were used to transform E. coli and yeast aspartate-transcarbamylase-less strains to prototrophy. From the first pool--a BamHI yeast DNA digest--a 6.4 kb BamHI fragment was recovered that gave good complementation of the E. coli auxotrophy but poor complementation of the yeast auxotrophy. From the second pool--a partial Sau3A yeast DNA digest--five independent plasmids complementing either E. coli, yeast, or both were recovered. Each of the five plasmids possessed sequences in common with the 6.4 kb BamHI fragment. One of these plasmids, which complemented the two URA2 activities in yeast and which produced a carbamyl-phosphate synthetase, aspartate-transcarbamylase complex sensitive to UTP feedback inhibition contained the full URA2 gene. A restriction map of the URA2 gene has been constructed and seven different consecutive segments have been recloned in pBR322 to measure their hybridization with URA2 messenger RNA, allowing us to estimate the limits of the gene.
将插入到杂种大肠杆菌 - 酵母载体pFL1中的两个酵母DNA文库用于转化大肠杆菌和酵母天冬氨酸转氨甲酰酶缺陷型菌株，使其恢复为原养型。从第一个文库——BamHI酶切的酵母DNA文库中，回收了一个6.4kb的BamHI片段，它能很好地互补大肠杆菌的营养缺陷型，但对酵母营养缺陷型的互补效果较差。从第二个文库——Sau3A部分酶切的酵母DNA文库中，回收了五个分别能互补大肠杆菌、酵母或两者的独立质粒。这五个质粒中的每一个都与6.4kb的BamHI片段有共同序列。其中一个质粒能互补酵母中的两种URA2活性，并产生对UTP反馈抑制敏感的氨甲酰磷酸合成酶 - 天冬氨酸转氨甲酰酶复合物，该质粒包含完整的URA2基因。已构建了URA2基因的限制性图谱，并将七个不同的连续片段在pBR322中进行了亚克隆，以测量它们与URA2信使RNA的杂交情况，从而使我们能够估计该基因的边界。
