APPROACHES IN YEAST TO STUDY OF ANEUPLOIDY

拟议研究的总体目标是促进 了解染色体产生的机制 非整倍体以及随后异常表型的表达。 的 建议的实验利用现有的实验方法 in S.酿酒酵母和依赖于载体和方法开发的，在我们的 人工酵母的构建和操作实验室 染色体（YAC）。 具体的实验目的是： 1)为了进一步发展YAC克隆、操作和鉴定的方法， 转移 提高单染色体遗传病发生率的方法 将开发酵母/哺乳动物细胞融合过程中YAC的转移 使用核配缺陷酵母供体菌株。 一种新颖的“重组 克隆”方法将进一步发展和用于克隆功能 C.白色念珠菌着丝粒DNA序列。 着丝粒DNA的作用 染色体断裂重组机制的序列将 要详细分析。 2)分析减数分裂Ⅰ染色体所必需的决定因子 析取 利用我们开发的2-YAC减数分裂分离系统， 将确定实验室顺式和反式作用决定因素， 表征了 缺陷的分子基础导致水平增加 早熟姐妹染色单体分离，减数分裂I不分离， 将研究减数分裂II错误分离事件。 3)分析酵母中特异的非整倍体。 的表型效应 染色体II LYS 2-远端单体性（染色体传递缺陷）将 表征并鉴定、克隆相应的基因， 表征了 4)分析染色体分离所需基因的人类同源物 忠诚 基因将通过以下功能互补来鉴定： 酵母突变体或通过氨基酸测序的进化保守性， 它们在哺乳动物细胞中的作用被确定。 对染色体非整倍体发生机制的认识， 导致表型结果的因素 与特定的非整倍体的结合是人类生物学的主要挑战。 的 开发操作染色体的方法（包括 酵母中的YAC）将有助于分析这些片段， 哺乳动物DNA导致非整倍体表型。 此外，分析 酵母中的非整倍性机制将是重要的基础 对这些过程的理解。

The general objective of the proposed research is to contribute to an understanding of the mechanisms involved in the generation of chromosomal aneuploidy and in the expression of subsequent abnormal phenotypes. The proposed experiments take advantage of experimental approaches available in S. cerevisiae and rely on vectors and methods developed in our laboratory for the construction and manipulation of Yeast Artificial Chromosomes (YACs). The specific experimental aims are: 1) To further develop methodologies for YAC cloning, manipulation, and transfer. Methodologies to increase the frequency of monochromosomal transfer of YACs during yeast/mammalian cell fusion will be developed using karyogamy deficient yeast donor strains. A novel "recombinational cloning" method will be further developed and used to clone a functional C. albicans centromere DNA sequence. The effect of centromere DNA sequences on the recombination mechanism of chromosome fragmentation will be analyzed in detail. 2) To analyze determinants necessary for Meiosis I chromosome disjunction. Using a 2-YAC meiosis segregational system developed in our laboratory, cis- and trans-acting determinants will be identified and characterized. The molecular basis of defects causing increased levels of precocious sister chromatid separation, Meiosis I nondisjunction, and Meiosis II missegregation events will be studied. 3) To analyze specific aneuploidies in yeast. The phenotypic effects of chromosome II LYS2-distal monosomy (chromosome transmission defect) will be characterize and the corresponding gene(s) identified, cloned, and characterized. 4) To analyze human homologs of genes required for chromosome disjunction fidelity. Genes will be identified by functional complementation of yeast mutants or by evolutionary conservation of amino acid sequenced and their roles in mammalian cells determined. An understanding of the mechanisms that lead to chromosal aneuploidy and the factors that contribute to the phenotypic consequences associated with specific aneuploidies are major challenges in human biology. The development of methodologies for manipulating chromosomes (including YACs) in yeast will facilitate the analysis of those segments of mammalian DNA leading to an aneuploid phenotype. Moreover, analysis of mechanisms of aneuploidy in yeast will be important to a basic understanding of these processes in general.