ISOLATION AND CHARACTERIZATION OF THE 2 STRUCTURAL GENES-CODING FOR PHOSPHOFRUCTOKINASE IN YEAST
ISOLATION AND CHARACTERIZATION OF THE 2 STRUCTURAL GENES-CODING FOR PHOSPHOFRUCTOKINASE IN YEAST
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DOI：
10.1007/bf00330520
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发表时间：
1986-01-01
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影响因子：
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通讯作者：
HEINISCH, J
中科院分区：
文献类型：
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作者：
HEINISCH, J
酵母磷酸果糖激酶在由两种不同亚基组成的八聚体中。在pfk1pfk2双突变体中，通过功能互补的方法分离了编码这些亚基的基因。用酵母多拷贝载体YEp13构建的Nasmyth和Tatchell(1980)基因组文库作为DNA的来源。通过反向转化pFK单突变体(pfk1、pfk2、pfk1、pfk2)，筛选出含有某一基因信息的质粒。提供了各个插入的限制图。Southern分析证实了其基因组结构。Northern分析显示，这两个基因的mRNAs杂交约为3.6kb，与蛋白质亚基的相对分子质量相对应。用其中一种质粒转化不会导致磷酸果糖激酶活性的增加。将这两个基因亚克隆到一个多拷贝载体(YEp13)中，并重新导入酵母细胞，比活性比野生型高3.5倍。考马斯蓝染色的粗提物经SDS-聚丙烯酰胺凝胶电泳法证实，该转化子的蛋白质亚基产量较高。这并没有伴随着乙醇产量的增加。编码这两个亚基的序列显示有同源性。
Yeast phosphofructokinase in an octamer composed o ftwo different kinds of subunit. The genes coding for these subunits have been isolated by means of functional complementation in a pfk1 pfk2 double mutant. As a source of DNA the genomic library of Nasmyth and Tatchell (1980) constructed in the yeast multicopy vector YEp13 was used. Plasmids containing the information of one or the other gene were identified by back-transformation into pfk single mutants (pfk1 PFK2, PFK1 pfk2). Restriction maps of the respective insertions are provided. The genomic organization was confirmed by Southern analysis. Northern analysis showed hybridization of mRNAs of about 3.6 kb for both genes, corresponding to the molecular weight of the protein subunits. Transformation with one of the plasmids did not lead to an increase in phosphofructokinase activity. Subcloning of both genes in one multicopy vector (YEp13) and reintroduction into the yeast cell resulted in a 3.5-fold higher specific activity compared to the wild type. Overproduction of the protein subunits in this transformant was confirmed by SDS-polyacrylamide electrophoresis of crude extracts stained with Coomassie-blue. This was not accompanied by an increased ethanol production. The sequences encoding the two subunits were shown to share homology.