Dueñas E, Revuelta J L, del Rey F, Vázquez de Aldana C R
Departamento de Microbiología y Genética, Universidad de Salamanca/CSIC, Spain.
Yeast. 1999 Jan 15;15(1):63-72. doi: 10.1002/(SICI)1097-0061(19990115)15:1<63::AID-YEA338>3.0.CO;2-7.
We describe here the construction of six deletion mutants and their basic phenotypic analysis in three different backgrounds. The six genes were disrupted in three diploid strains (FY1679, W303 and CEN.PK2) by the long flanking homology (LFH) method (Wach, 1996). Transformants were selected as geneticin (G418)-resistant colonies and correct integration of the kanMX4 cassette was checked by colony PCR. Following sporulation of the heterozygous diploids, tetrads were dissected and scored for segregation of G418-resistance and auxotrophic markers. One of the six ORFs (YNL158w) corresponds to an essential gene which has no homology with other genes present in the databases and has two predicted transmembrane domains. Growth tests performed on different media at 15 degrees C, 30 degrees C or 37 degrees C with haploid deletants of the five non-essential genes revealed no apparent phenotype in any of them.
我们在此描述了六个缺失突变体的构建及其在三种不同背景下的基本表型分析。通过长侧翼同源性（LFH）方法（Wach，1996），在三个二倍体菌株（FY1679、W303和CEN.PK2）中破坏了这六个基因。将转化体选择为对遗传霉素（G418）有抗性的菌落，并通过菌落PCR检查kanMX4盒的正确整合。杂合二倍体形成孢子后，对四分体进行解剖，并对G418抗性和营养缺陷型标记的分离进行评分。六个开放阅读框（ORF）之一（YNL158w）对应于一个必需基因，该基因与数据库中存在的其他基因没有同源性，并且有两个预测的跨膜结构域。对五个非必需基因的单倍体缺失突变体在15℃、30℃或37℃的不同培养基上进行生长测试，结果显示它们中任何一个都没有明显的表型。
