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而创新的方法来分裂酿酒酵母的染色体，我们称之为pcr介导的染色体分裂（PCS）。使用这种新方法，单倍体细胞的染色体I （230 kb）和XV （1091 kb）以高效率（通常为80%）集体分裂成10个大小从29到631 kb的小染色体，这些小染色体在不同组合的有丝分裂生长过程中偶尔会丢失。我们还成功地将该方法应用于两株酿酒酵母的染色体洗牌区域。这种新技术，我们称之为“染色体洗牌”，可以提供一种新的工具来分析由选择的染色体区域的替换或半合子性引起的表型改变，不仅在实验室，而且在更多的工业菌株中。进一步改进了PCR方法，通过赋予酵母自主复制序列（ARS）元件，使没有ARS的染色体区域可以转化为人工染色体。我们进一步利用这种方法来探索核糖体DNA （rDNA）的功能。S.cerevisiae的rDNA集群位于第12号染色体左端450 kb和右端610 kb处，由约150个9.1 kb rDNA单元的串联重复拷贝组成。在rDNA簇的两侧分裂第XII染色体，产生含有第XII染色体缺失变体的菌株，其中包括450-kb， 1,500-kb（仅rDNA簇）和610-kb。在含有仅由rDNA组成的第XII染色体1500 kb变体的菌株中，发现rDNA簇的大小减小。rDNA位点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，因此Nop1p的单个或偶尔两个焦点和圆形核仁被观察到。值得注意的是，携带仅由rDNA簇组成的分裂染色体的菌株的寿命比野生型短。这些观察结果表明，第XII染色体在S.cerevisiae维持恒定的rDNA拷贝数和rDNA功能相关的生理过程中起着重要作用。总之，这种新技术不仅可以为阐明基因组功能，而且可以为生物技术提供有用的工具。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