Wysocki R, Roganti T, Van Dyck E, de Kerchove D'Exaerde A, Foury F
Unité de Biochimie Physiologique, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.
Yeast. 1999 Jan 30;15(2):165-71. doi: 10.1002/(SICI)1097-0061(19990130)15:2<165::AID-YEA351>3.0.CO;2-V.
In the frame of the European Network for Functional Analysis (EUROFAN) we have deleted 18 yeast open reading frames (ORFs) from chromosomes II, X and XIV using the short flanking homology-PCR strategy. Two diploid strains were used: FY1679 and CEN.PK2. The deletion kanMX6 cassettes with long flanking homology and the cognate gene clones have also been constructed. Heterozygous diploid deletant strains have been sporulated. Tetrad analysis revealed that all the ORFs studied were non-essential. However, four deletant strains exhibited phenotypes. The YBL025w delta strain showed extremely slow cellular growth under all conditions tested. The YJL204c delta strain grew slower than wild-type at 30 degrees C and 37 degrees C, was cold-sensitive, and the homozygous diploids did not sporulate. The YNL213c delta strain did not grow on glycerol and had lost mitochondrial DNA. The deletion of YNL215w caused slower growth on all media but the defect was more pronounced on glucose-minimal and glycerol-rich media than on glucose-rich medium. All deletion mutants were complemented by the corresponding plasmid borne cognate gene. The YJL204w, YNL213c and YNL215w ORFs do not bear significant homology to proteins of known function. YBL025w has recently been identified as RRN10, a gene that encodes an RNA polymerase I-specific transcription initiator factor. The deletion of the remaining fourteen ORFs did not reveal any mutant phenotype in our basic growth tests.
在欧洲功能分析网络（EUROFAN）框架下，我们使用短侧翼同源PCR策略从酵母的第二、十和十四条染色体中删除了18个开放阅读框（ORF）。使用了两个二倍体菌株：FY1679和CEN.PK2。还构建了带有长侧翼同源序列的缺失kanMX6盒以及相关基因克隆。杂合二倍体缺失菌株已进行了孢子形成。四分体分析表明，所有研究的ORF都是非必需的。然而，四个缺失菌株表现出了表型。YBL025wΔ菌株在所有测试条件下细胞生长都极其缓慢。YJL204cΔ菌株在30℃和37℃时生长比野生型慢，对低温敏感，并且纯合二倍体不形成孢子。YNL213cΔ菌株在甘油上不生长且线粒体DNA丢失。YNL215w的缺失导致在所有培养基上生长变慢，但在葡萄糖基本培养基和富含甘油的培养基上的缺陷比在富含葡萄糖的培养基上更明显。所有缺失突变体都被相应的质粒携带的同源基因互补。YJL204w、YNL213c和YNL215w的ORF与已知功能的蛋白质没有显著同源性。YBL025w最近被鉴定为RRN10，一个编码RNA聚合酶I特异性转录起始因子的基因。在我们的基本生长测试中，其余14个ORF的缺失未显示任何突变表型。
