Grimm C, Kohli J, Murray J, Maundrell K
Institute of General Microbiology, University of Bern, Switzerland.
Mol Gen Genet. 1988 Dec;215(1):81-6. doi: 10.1007/BF00331307.
A system is described for gene disruption and replacement in Schizosaccharomyces pombe based on the homologous selectable marker, ura4, the structural gene for orotidine-5'-phosphate decarboxylase. The presence of a single copy of the wild-type gene can rescue a ura4 auxotrophic mutant. Furthermore, ura4- cells can be selected for in the presence of 5-fluoroorotic acid (5-FOA). This allows a convenient means of selecting for both forward and backward mutations. The sequence of a 1.8 kb HindIII fragment which contains the functional gene is reported. It encodes a single open reading frame of 264 amino acids which shows considerable conservation with the orotidine-5'-phosphate (OMP) decarboxylases from other organisms. The ura4 transcript is approximately 850 nucleotides long. It begins 51 bp upstream of the protein coding sequence and is unusual in that transcription termination occurs at or very close to the translational stop codon. To facilitate the use of ura4 in gene disruption experiments we have also constructed a novel strain of S. pombe called ura4-D18, in which the 1.8 kb HindIII fragment has been deleted from the chromosome. Using a combination of this strain and vectors containing ura4 as a selectable marker, we present a general method for targeting recombination events to the chromosomal locus under investigation.
描述了一种基于同源选择标记ura4（乳清苷-5'-磷酸脱羧酶的结构基因）在粟酒裂殖酵母中进行基因破坏和替换的系统。野生型基因的单拷贝存在可拯救ura4营养缺陷型突变体。此外，在5-氟乳清酸（5-FOA）存在下可以选择ura4-细胞。这提供了一种方便的方法来选择正向和反向突变。报道了包含功能基因的1.8 kb HindIII片段的序列。它编码一个264个氨基酸的单一开放阅读框，与来自其他生物体的乳清苷-5'-磷酸（OMP）脱羧酶具有相当的保守性。ura4转录本约850个核苷酸长。它在蛋白质编码序列上游51 bp处开始，其不同寻常之处在于转录终止发生在翻译终止密码子处或非常接近翻译终止密码子。为了便于在基因破坏实验中使用ura4，我们还构建了一种新的粟酒裂殖酵母菌株ura4-D18，其中1.8 kb HindIII片段已从染色体上删除。使用该菌株和含有ura4作为选择标记的载体的组合，我们提出了一种将重组事件靶向到所研究的染色体位点的通用方法。
