Jaquet L, Jauniaux J C
Abteilung F0100, Unité INSERM 375, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Yeast. 1999 Jan 15;15(1):51-61. doi: 10.1002/(SICI)1097-0061(19990115)15:1<51::AID-YEA330>3.0.CO;2-1.
We describe the disruption and basic functional analysis of five novel open reading frames (ORFs) discovered during the sequencing of the Saccharomyces cerevisiae genome: YJL118w, YJL122w, YJL123c, YJL124c, YJL125c, located on chromosome X. Disruptions have been realized using the long-flanking homology-PCR replacement strategy (LFH-PCR; Wach et al., 1996) in the FY1679 diploid strain. Sporulation and tetrad analysis of these heterozygous deletants were performed, as well as a functional analysis on the haploid deleted strains: different growth conditions (complete glucose and glycerol, minimal media) at three temperatures 15, 30 and 37 degrees C were tested. Analysis revealed YJL125c as an essential gene; the four other ORFs were non-essential and showed no particular phenotype. In addition, the five kanMX4 disruption cassettes were cloned in pUG7 vector. Finally, the five ORFs with their promoter and terminator regions were cloned in the centromeric yeast vector pRS416. The vectors containing the disruption cassettes, the cognate wild-type genes, as well as the deletant strains are available at the EU EUROFAN (EUROSCARF, Frankfurt, DE) genetic and stock centre.
我们描述了在酿酒酵母基因组测序过程中发现的五个新开放阅读框（ORF）的破坏及基本功能分析：YJL118w、YJL122w、YJL123c、YJL124c、YJL125c，位于X染色体上。已在FY1679二倍体菌株中使用长侧翼同源PCR替换策略（LFH-PCR；Wach等人，1996年）实现了破坏。对这些杂合缺失体进行了孢子形成和四分体分析，以及对单倍体缺失菌株的功能分析：测试了在15、30和37摄氏度的三种温度下不同的生长条件（完全葡萄糖和甘油、基本培养基）。分析表明YJL125c是一个必需基因；其他四个ORF是非必需的，且未表现出特定表型。此外，五个kanMX4破坏盒被克隆到pUG7载体中。最后，五个带有其启动子和终止子区域的ORF被克隆到着丝粒酵母载体pRS416中。含有破坏盒、同源野生型基因以及缺失菌株的载体可在欧盟EUROFAN（EUROSCARF，德国法兰克福）遗传和菌种中心获得。
