Yeast genome engineering and applications

染色体工程在基因组功能分析中发挥着越来越重要的作用。一种简单而有效的操作大染色体片段的技术是推进这些分析的关键。在这项研究中，我们开发了一种简单但创新的方法来分裂酿酒酵母中的染色体，我们称之为PCR介导的染色体分裂（PCS）。利用这种新的方法，一个单倍体细胞的染色体I（230 kb）和XV（1091 kb）被集体分裂成10个微型染色体的大小范围从29至631 kb的高效率（常规80%），偶尔会丢失在有丝分裂生长在各种组合。这种新的技术，我们称之为“染色体改组”，可以提供一种新的工具来分析表型改变所造成的替代或半合子的一个选定的染色体区域，不仅在实验室，而且在 ...更多信息 金黄色葡萄球菌对PCR方法作了进一步改进，加入了一个酵母ARS（autonomously replicating sequenc）元件，使染色体中没有ARS的区域可以转化为人工染色体，并进一步用此方法探讨了核糖体DNA（rDNA）的功能。酿酒酵母中的rDNA簇位于距离染色体XII左端450 kb和距离染色体XII右端610 kb处，由约。150个串联重复拷贝的9.1 kb rDNA单位组成。染色体XII在rDNA簇的两侧分裂，并产生了含有由450-kb、1，500-kb（仅rDNA簇）和610-kb组成的染色体XII缺失变体的菌株。在携带仅由rDNA组成的染色体XII的1，500 kb变体的菌株中，发现rDNA簇的大小减小。发现rDNA基因座内的沉默频率高于野生型菌株。核仁的定位和形态也受到影响，例如观察到单个和偶尔两个Nop 1 p病灶和圆形核仁。值得注意的是，携带仅由rDNA簇组成的分裂染色体的菌株具有比野生型更短的寿命。这些观察结果表明，染色体XII的上下文起着重要的作用，在保持一个恒定的rDNA拷贝数和在S.ae的rDNA功能相关的生理过程。总之，这种新的技术不仅可以提供一个有用的工具，以阐明基因组功能，也为生物技术。少

Chromosome engineering is playing an increasingly important role in functional analysis of genomes. A simple and efficient technology for manipulating large chromosomal segments is key to advancing these analyses. In this study, we developed a simple but innovative method to split chromosomes in Saccharomyces cerevisiae, which we call PCR-mediated chromosome splitting (PCS). Using this novel method, chromosomes I (230 kb) and XV (1091 kb) of a haploid cell were split collectively into 10 minichromosomes ranging in size from 29 to 631 kb with high efficiency (routinely 80%) that occasionally were lost during mitotic growth in various combinations.We also successfully applied this method to shuffle selected regions of chromosomes from two strains in S.cerevisiae. This novel technique, which we call 'chromosome shuffling', could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also in … More dustrial strains of S.cerevisiae. The PCR method was further improved by conferring a yeast ARS (autonomously replicating sequenc) element so that chromosome region without ARS can be converte into artificial chromosome.We have further employed this method to explore the function of ribosomal DNA (rDNA). The rDNA cluster in S.cerevisiae is located 450 kb from the left end and 610 kb from the right end of chromosome XII and consists of ca.150 tandemly repeated copies of a 9.1 -kb rDNA unit. Chromosome XII was split at both sides of the rDNA cluster and strains harboring deleted variants of chromosome XII consisting of 450-kb, 1,500-kb (rDNA cluster only) and 610-kb were created. In the strain harboring the 1,500 kb variant of chromosome XII consisting solely of rDNA, the size of the rDNA cluster was found to decrease. The frequency of silencing within the rDNA locus was found to be greater than in a wild -type strain. The localization and morphology of the nucleolus was also affected such that a single and occasionally two foci for Nop1p and a rounded nucleolus were observed. Notably, strains harboring split chromosome consisting solely of rDNA cluster had shorter life spans than wild -type. These observations suggest that the context of chromosome XII plays an important role in maintaining a constant rDNA copy number and in physiological processes related to rDNA function in S.cerevisiae. In conclusion, this novel technique could provide a useful tool not only to elucidate the genome function but also for biotechnology. Less