Moore P A, Bettany A J, Brown A J
Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, UK.
J Gen Microbiol. 1990 Dec;136(12):2359-66. doi: 10.1099/00221287-136-12-2359.
The Saccharomyces cerevisiae pyruvate kinase gene (PYK1) was transformed into yeast using the multicopy vector pJDB207. Growth rates and PYK1 gene expression levels varied considerably amongst the transformants. Yeast transformants expressing the PYK1 gene at high levels formed small colonies compared with those expressing the gene at relatively low levels. Slow-growing transformants 'reverted' at high frequency to more rapid growth, and this correlated with decreases in PYK1 gene copy number and PYK1 mRNA abundance. This apparent selection against PYK1 over-expression was disrupted by the introduction of a stop codon at the 5'-end of the PYK1 coding region, thus confirming that the growth effects were mediated by the PYK1 gene. However, massive overproduction of pyruvate kinase in yeast, using multiple copies of a PGK:PYK gene fusion, had no significant effect upon cell growth. This suggests that the deleterious effect upon the host yeast cell is mediated by abnormally high levels of the wild-type gene or PYK1 mRNA, rather than by increased pyruvate kinase levels.
使用多拷贝载体pJDB207将酿酒酵母丙酮酸激酶基因（PYK1）转化到酵母中。各转化子的生长速率和PYK1基因表达水平差异很大。与那些相对低水平表达该基因的转化子相比，高水平表达PYK1基因的酵母转化子形成小菌落。生长缓慢的转化子高频“回复”为生长更快的状态，这与PYK1基因拷贝数和PYK1 mRNA丰度的降低相关。通过在PYK1编码区5'端引入一个终止密码子，这种对PYK1过表达的明显选择被破坏，从而证实生长效应是由PYK1基因介导的。然而，使用PGK:PYK基因融合的多个拷贝在酵母中大量过量生产丙酮酸激酶，对细胞生长没有显著影响。这表明对宿主酵母细胞的有害作用是由野生型基因或PYK1 mRNA的异常高水平介导的，而不是由丙酮酸激酶水平的增加介导的。
