de Graaff L, van den Broek H, Visser J
Department of Genetics, Agricultural University, The Netherlands.
Curr Genet. 1988 Apr;13(4):315-21. doi: 10.1007/BF00424425.
The Aspergillus nidulans pyruvate kinase gene was isolated by heterologous hybridization using the corresponding yeast gene as a probe. A 2.9 kb EcoRI/BamHI fragment, which exclusively hybridized to the yeast gene, was subcloned in pBR322. This clone was used to transform an A. nidulans pkiA deletion mutant to PKI+. The analysis of transformants with respect to the kind of integration revealed about 80% homologous integration--55% by a double cross-over event (type III integration), 25% by a single cross-over event (type I integration). Type II transformants (20%) that arise by non-homologous integration have not been further characterized with respect to the sites of integration. A direct correlation between the number of copies of the gene integrated into the genome and the measured pyruvate kinase activity was found after growth ona glycolytic carbon source. From this, it was concluded that the 2.9 kb EcoRI/BamHI fragment contains the complete pyruvate kinase structural gene, including the promoter region. However, after growth on a gluconeogenic carbon source, the regulation of gene expression was found to be disturbed. On acetate an increase in activity per gene copy (0.2 IU) was found in the transformants, as compared with wild-type levels. It is suggested that the pyruvate kinase gene is regulated by negative control, and that some sequences involved in this regulation are missing in the cloned fragment.
利用相应的酵母基因作为探针，通过异源杂交分离出构巢曲霉丙酮酸激酶基因。一个2.9 kb的EcoRI/BamHI片段，它只与酵母基因杂交，被亚克隆到pBR322中。这个克隆用于将构巢曲霉pkiA缺失突变体转化为PKI+。关于整合类型对转化体的分析显示，约80%为同源整合——55%通过双交换事件（III型整合），25%通过单交换事件（I型整合）。通过非同源整合产生的II型转化体（20%）尚未就整合位点进行进一步表征。在糖酵解碳源上生长后，发现整合到基因组中的基因拷贝数与测得的丙酮酸激酶活性之间存在直接相关性。由此得出结论，2.9 kb的EcoRI/BamHI片段包含完整的丙酮酸激酶结构基因，包括启动子区域。然而，在糖异生碳源上生长后，发现基因表达的调控受到干扰。在乙酸盐上，与野生型水平相比，转化体中每个基因拷贝的活性增加（0.2 IU）。有人提出丙酮酸激酶基因受负调控，并且在克隆片段中缺少一些参与这种调控的序列。
