Heinisch J
Mol Gen Genet. 1986 Jan;202(1):75-82. doi: 10.1007/BF00330520.
Yeast phosphofructokinase is an octamer composed of two 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 to 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.
酵母磷酸果糖激酶是一种由两种不同亚基组成的八聚体。通过在pfk1 pfk2双突变体中进行功能互补，分离出了编码这些亚基的基因。作为DNA来源，使用了在酵母多拷贝载体YEp13中构建的Nasmyth和Tatchell（1980）的基因组文库。通过回转化到pfk单突变体（pfk1 PFK2、PFK1 pfk2）中，鉴定出含有其中一个或另一个基因信息的质粒。提供了各个插入片段的限制性图谱。通过Southern分析证实了基因组结构。Northern分析表明，这两个基因均与约3.6 kb的mRNA杂交，这与蛋白质亚基的分子量相对应。用其中一个质粒进行转化并未导致磷酸果糖激酶活性增加。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后，与野生型相比，比活性提高了3.5倍。通过考马斯亮蓝染色的粗提物的SDS-聚丙烯酰胺凝胶电泳证实了该转化体中蛋白质亚基的过量表达。但这并未伴随着乙醇产量的增加。编码这两个亚基的序列显示出同源性。
