Jacoby J J, Nilius S M, Heinisch J J
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Mol Gen Genet. 1998 Apr;258(1-2):148-55. doi: 10.1007/s004380050717.
We employed the constitutive BCK1-20 allele of the gene for the MAP kinase kinase kinase (MAP-KKK) in the yeast Pkc signal transduction pathway to develop a genetic screen for mutants in genes encoding upstream components. Transposon mutagenesis yielded a mutant that was completely dependent on the active allele in the absence of osmotic stabilization. The transposon had integrated at the yeast SLG1 (HCS77) locus. This gene encodes a putative membrane protein. Haploid slg1 deletion strains are sensitive to caffeine, as expected for mutants in the Pkc pathway, as well as a variety of other drugs. The response to elevated temperatures and the dependence on osmotic stabilization depends on the genetic background. Thus, in the strain used for mutagenesis, disruption of SLG1 causes the cells to become non-viable in the absence of osmotic stabilization at both 30 degrees C and 37 degrees C. In a different genetic background this phenotype was not observed. Sensitivity of the haploid deletion mutants to caffeine can be partially suppressed by overexpression of genes for other components of the Pkc pathway, such as PKC1, SLT2, ROM2, and STE20. In addition, a SLG1-lacZ reporter construct shows higher expression in the presence of caffeine or magnesium chloride in a wild-type diploid background.
我们利用酵母Pkc信号转导途径中促分裂原活化蛋白激酶激酶激酶（MAP-KKK）基因的组成型BCK1-20等位基因，开发了一种针对编码上游组分的基因突变体的遗传筛选方法。转座子诱变产生了一个突变体，在没有渗透稳定剂的情况下，该突变体完全依赖于活性等位基因。转座子整合到了酵母SLG1（HCS77）基因座。该基因编码一种假定的膜蛋白。单倍体slg1缺失菌株对咖啡因敏感，这与Pkc途径中的突变体预期一致，对多种其他药物也敏感。对高温的反应以及对渗透稳定剂的依赖性取决于遗传背景。因此，在用于诱变的菌株中，SLG1的破坏导致细胞在30℃和37℃没有渗透稳定剂的情况下无法存活。在不同的遗传背景下未观察到这种表型。单倍体缺失突变体对咖啡因的敏感性可通过Pkc途径其他组分的基因（如PKC1、SLT2、ROM2和STE20）的过表达得到部分抑制。此外，在野生型二倍体背景下，SLG1-lacZ报告构建体在存在咖啡因或氯化镁的情况下表达更高。
