Bach M L
Laboratoire de Génétique Physiologique, I.B.M.C. du C.N.R.S., Strasbourg, France.
Curr Genet. 1987;12(7):527-34. doi: 10.1007/BF00419562.
URA4, the gene coding for orotidine monophosphate decarboxylase (OMPdecase), has been cloned from the fission yeast by homologous complementation and restricted in an Escherichia coli-Schizosaccharomyces pombe (E. coli-S. pombe) replicative plasmid to a 1.76 kb HindIII fragment. This plasmid is maintained at a high copy number in S. pombe and allows OMPdecase expression in Saccharomyces cerevisiae (S. cerevisiae) as well as in E. coli. After characterisation by restriction mapping and Southern hybridisation, the cloned gene was used as a probe to measure URA4 transcription and to examine its regulation. Messenger RNA levels were measured by DNA/RNA filter-hybridisation with pulse labelled RNAs during 6-azauridine (6-AUR) inhibited growth in wild type and 6-AUR sensitive strains. We found that in S. pombe the OMP analogue 6-AUR does not regulate the level of OMPdecase formation as it does in S. cerevisiae but rather modifies the ratio of total polyA+ to polyA- RNAs in the cell. Based on these results and on corresponding enzyme activities this study demonstrates divergent pyrimidine pathway regulation in the two yeasts S. cerevisiae and S. pombe. Finally, we propose the use of the URA4 gene as a convenient selective marker for genetic engineering in S. pombe.
URA4基因编码乳清苷单磷酸脱羧酶（OMP脱羧酶），已通过同源互补从裂殖酵母中克隆出来，并在大肠杆菌 - 粟酒裂殖酵母（E. coli - S. pombe）复制质粒中被限制在一个1.76 kb的HindIII片段上。该质粒在粟酒裂殖酵母中以高拷贝数维持，并允许OMP脱羧酶在酿酒酵母（S. cerevisiae）以及大肠杆菌中表达。通过限制性图谱分析和Southern杂交进行鉴定后，克隆的基因被用作探针来测量URA4转录并检查其调控。在野生型和对6 - 氮尿苷（6 - AUR）敏感的菌株中，在6 - AUR抑制生长期间，通过与脉冲标记的RNA进行DNA/RNA滤膜杂交来测量信使RNA水平。我们发现，在粟酒裂殖酵母中，OMP类似物6 - AUR并不像在酿酒酵母中那样调节OMP脱羧酶的形成水平，而是改变了细胞中总polyA +与polyA - RNA的比例。基于这些结果和相应的酶活性，本研究证明了酿酒酵母和粟酒裂殖酵母这两种酵母中嘧啶途径调控的差异。最后，我们提议将URA4基因用作粟酒裂殖酵母基因工程中方便的选择标记。
