TOOLS FOR GENETIC MAPPING IN YEAST

酵母基因图谱工具

• 批准号: 2900920
• 负责人: David J Stillman
• 金额: $ 9.17万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900920
• 关键词: Saccharomyces cerevisiae; alleles; chromosome deletion; fungal genetics; genetic mapping; genetic markers; genetic techniques; method development; molecular cloning; plasmids; transfection /expression vector

Dr. David Stillman proposed to develop a portable, rapid mapping system for the yeast Saccharomyces cerevisiae. The primary use for this system will be map mutations that have proved difficult to clone by complementation; it would provide the information necessary for positional mapping. The development of the system will proceed in two phases. The first phase involves the construction of sixteen plasmids each of which will be targeted to integrate near the centromere of one of the yeast chromosomes. These plasmids will carry a GAL1 promoter that when activated would transcribe through the centromere, inactivating it and causing chromosome loss. The plasmids also will contain a dominant marker that would allow easy scoring for such a loss. Using these plasmids, a researcher could construct sixteen strains with a specific genetic background, each carrying one of these plasmids in a different chromosome. Each strain could then be mated with the mutant strain, and the loss of the plasmid containing chromosome in each diploid would be induced. In one of the diploid strains, the recessive mutant phenotype would be uncovered when the chromosome carrying the wildtype allele was lost, thus mapping the mutation to that chromosome. The second phase of development involves the construction of a set of 35 chromosome fragmentation vectors. These plasmids will allow a researcher to divide the larger chromosomes into two or three fragments to map the mutation of interest to a subchromosomal segment. Each plasmids will carry a centromere joined to a GAL1 promoter, suitable selectable markers, and a region to which integration is targeted, all contained between telomeric sequences. The restriction enzyme SceI flanked by sites for this enzyme will be inserted between the telomeric sequences opposite the rest of the vector. The integration of this vector at the targeted site will generate an unstable dicentromeric chromosome. The instability will be resolved through cleavage by SceI (which does not cleave nuclear DNA), cutting the chromosome in two between the centromeres. The presence of the telomeric sequences will generate proper ends. Thus, the targeted chromosome will be cut in two at the chosen site. One of the new chromosomes will contain the centromere that can be inactivated when the GAL1 promoter is induced, causing its loss and exposing the recessive mutation if the wildtype allele were contained within that chromosomal segment. Thus, once a mutation is mapped to a particular chromosome, that chromosome could be fragmented to determine which fragment contains the mutation. Once a gene is mapped to a region of a chromosome, complementation could be tested using clones from that region.

大卫斯蒂尔曼博士建议开发一种便携式快速绘图系统 酿酒酵母 该系统的主要用途是 将是已经证明很难克隆的地图突变， 补充;它将提供必要的信息， 位置映射 该系统的开发将分两个阶段进行。 第一 阶段涉及16个质粒的构建，每个质粒将 被靶向整合到一个酵母的着丝粒附近， 染色体 这些质粒将携带GAL 1启动子， 激活后会通过着丝粒转录，使其失活， 导致染色体丢失。 质粒也将包含一个显性的 这样的损失很容易得分的标记。 使用这些 质粒，研究人员可以构建16个菌株， 基因背景，每个携带这些质粒中的一个在不同的 染色体 然后每个菌株可以与突变菌株交配， 每个二倍体中含有染色体的质粒的丢失将是 诱导。在其中一个二倍体菌株中，隐性突变表型 当携带野生型等位基因的染色体 丢失，从而将突变映射到该染色体。 第二阶段 发育的一个重要步骤是构建一套35条染色体 碎片化载体。 这些质粒可以让研究人员 将较大的染色体分成两到三个片段， 与亚染色体片段相关。 每一个质粒都会携带一个 连接到GAL 1启动子的着丝粒，合适的选择标记，和 整合的目标区域，所有包含在 端粒序列 限制性内切酶SceI的两侧是 该酶将插入相对的端粒序列之间 剩下的向量。 该载体在靶向 位点将产生不稳定的双着丝粒染色体。 的 不稳定性将通过SceI的裂解来解决（SceI不 切割核DNA），将染色体在两个细胞之间切割成两个。 着丝粒 端粒序列的存在会产生 适当的结束。因此，靶向染色体将在染色体的末端被切成两半。 选择的网站 其中一条新染色体将包含着丝粒 当GAL 1启动子被诱导时，它可以被灭活， 如果野生型等位基因是 包含在染色体片段中。因此，一旦突变发生， 映射到一个特定的染色体上， 以确定哪个片段含有突变。 一旦一个基因被定位到染色体的某个区域，互补作用就可以 使用该地区的克隆进行测试。