Molecular Analysis of Chlamydomonas Mating-Type Locus

莱茵衣藻的交配型(Mt)基因座是一个关键的调控单位，它似乎控制着这种简单的真核生物的生命周期转变。它由调控基因和结构基因组成，通过重组抑制保持为一个单位。它可能与控制后生动物和/或高等植物胚胎转变的基因座有功能上的同源性。这项研究的长期目标是了解MT是如何工作的。作为第一步，在分子水平上定义基因座是至关重要的。已分离到一个与交配型基因紧密连锁的克隆基因，该基因在配子细胞融合形成合子后10分钟内唯一表达。与该探针同源的基因组克隆已经被鉴定，一次“染色体行走”已经鉴定了-300kb的克隆DNA到基因的两边。提出了三条实验路线。第一种需要在二倍体菌株中产生缺失，这些缺失覆盖了已知的与mt相关的隐性突变；这种缺失将在行走过程中提供物理标志，并可能识别特定基因的基因座。第二种方法是试图通过在mt产生纯合的二倍体来克服该区域的重组抑制；mt连锁标记之间的任何重组体都将再次提供物理标志，并提供该区域长期寻找的遗传图谱。最后，最近同源可选择基因的克隆和它们在衣藻转化中的成功应用(由3个独立的实验室报道)将使我们能够尝试使用现有行走的部分和必要时延长行走的部分来尝试共转化mt连锁突变。可能的转化子将通过遗传和分子分析进行验证。被证明转化为与交配型功能相关的有趣基因的克隆将被进一步分析：转化的DNA的相关部分将被鉴定和测序，以寻找暗示DNA结合蛋白、激酶、核酸酶等的序列基序。也可以确定相对mt染色体中每个基因的存在/缺失/修饰。在未来几年，这些基因在配子分化和合子发育中的活性可以得到详细的评估。这项对模式生物衣藻基本繁殖的研究将提供关于基因重组和调控的信息。衣藻是植物生物学家特别感兴趣的生物，因为它进行光合作用。对这种有机体的基本过程的了解可以经常转移到高等植物上。

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.