Molecular Analysis of Chlamydomonas Mating-Type Locus

衣藻交配型基因座的分子分析

• 批准号: 8917050
• 负责人: Ursula Goodenough
• 金额: $ 29.84万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-03-15 至 1994-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8917050&HistoricalAwards=false
• 关键词: Molecular; Analysis; Chlamydomonas; Mating; Type

The mating-type (mt) locus of Chlamydomonas reinhardtii is a key regulatory unit which appears to govern life-cycle transitions in this simple eukaryote. It consists of both regulatory and structural genes maintained as a unit by recombinational suppression. Possibly it shares functional homologies with loci that govern embryological transitions in the metazoa and/or higher plants. The long-term objective of this research is to understand how mt works. As a first step, it is critical that the locus be defined at a molecular level. A cloned cDNA has been isolated which exhibits tight genetic linkage to the mating-type locus and is uniquely expressed within 10 min after gametes undergo cell fusion to form zygotes. Genomic clones homologous to this probe have been identified, and a "chromosome walk" has identified-300 kb of cloned DNA to either side of the gene. Three lines of experimentation are proposed. The first entails generation of deletions in diploid strains that cover known recessive mutations linked to mt; such deletions will provide physical landmarks in the walk and may also identify the loci of particular genes. The second entails an attempt to overcome the recombinational suppression in the region by sporulating diploids homozygous at mt; any recombinants between the mt-linked markers will again provide physical landmarks and provide a long-sought genetic map of the region. Finally, the recent cloning of homologous selectable genes and their successful use in Chlamydomonas transformation (reported by 3 independent labs) will permit us to attempt co-transformation of the mt-linked mutations using sectors of the existing walk and, if necessary, extensions of the walk. Putative transformants will be verified by genetic and molecular analysis. Clones certifiably transformed for interesting genes related to mating-type functions will be further analyzed: the relevant sector of the transforming DNA will be identified and sequenced to look for sequence motifs suggestive of DNA binding proteins, kinases, nucleases, and so on. The presence/absence/modification of each gene in the chromosome of the opposite mt can also be ascertained. In future years, the activities of these genes in gamete differentiation and zygote development can be assessed in detail. This study of basic reproduction in the model organism, Chlamydomonas, will provide information about genetic recombination and regulation. Chlamydomonas is of particular interest to plant biologists because it carries out photosynthesis. Understanding of fundamental processes in this organism can frequently be transferred and applied to higher plants.

莱茵衣藻的交配型（mt）位点是一个关键 监管单位，似乎管理生命周期的过渡， 这个简单的真核生物 它包括监管和 结构基因通过重组维持为一个单位 镇压 可能它与基因座具有功能同源性 控制后生动物和/或更高级动物的胚胎转变 植物 这项研究的长期目标是了解 MT如何工作 作为第一步，关键的是， 在分子水平上定义。 克隆的cDNA已被分离， 其表现出与交配型基因座的紧密遗传连锁， 在配子经历细胞后10分钟内独特表达 融合形成合子。 与该探针同源的基因组克隆 已经确定，“染色体漫步”已经确定-300 将克隆的DNA插入基因的两侧。 三行 实验提出。 第一个需要产生 二倍体菌株中的缺失覆盖了已知的隐性突变 链接到mt;此类删除将在 也可以识别特定基因的位点。 的 第二个需要尝试克服重组 在该区域通过孢子形成在mt纯合的二倍体抑制; MT连锁标记之间的任何重组体将再次提供 物理地标，并提供了长期寻求的基因图谱 地区 最后，最近同源选择基因的克隆 以及它们在衣原体转化中的成功应用（报道 由3个独立的实验室）将允许我们尝试共转化 的MT连锁突变使用现有的步行和部门， 如有必要，延长步行距离。 推定的转化体将 通过基因和分子分析进行验证。 可证明的克隆 转化与交配型功能相关的感兴趣的基因 将进一步分析：转型的相关部门 DNA将被识别和测序，以寻找序列基序 暗示DNA结合蛋白、激酶、核酸酶等。 染色体中每个基因的存在/缺失/修饰 也可以确定相对的MT。 在未来几年， 这些基因在配子分化和合子中的活性 可以详细评估发展情况。 这项研究的基本繁殖模式生物， 衣原体，将提供有关基因重组的信息 和监管。 衣原体对植物特别有意义 因为它进行光合作用。 理解 这种生物体的基本过程经常被 转移到高等植物上。