FLAGELLAR MEMBRANE ADHESION AND SEXUAL SIGNALING

本研究的长期目标是确定和分析一些 介导性分化和性别分化的关键基因和基因产物 单细胞真核鞭毛虫的受精卵发育， 莱茵衣藻。这种生物体有两种单倍体交配类型， Mt和m，以及mt/mt二倍体受精卵，由 互补配子，是唯一能够形成孢子和减数分裂的配子。 该项目将采用两种方法。在第一个中，序列 被确认位于我们最近克隆的mt基因内 来自这两种交配类型的基因，将用于转化实验 拯救在交配/发育中有缺陷的突变体。在第二个阶段，变种人 交配/发育中的缺陷将通过插入 突变，标记突变的同源物。三个基因直接 作为分析的目标。1)imp-1基因已定位于 Mt基因座的6kb限制性片段；其产物预计将 参与配子黏附，它的识别可能会导致我们 它的mt同源基因，从而理解了交配类型的特异性 实现了细胞-细胞的识别。2)iso-1基因，由一个 插入事件，仅在mt配子中起作用；预计其产品 激活mt特异性基因的表达，抑制mt基因的表达 Mt特异性基因的表达，这些基因分散在 基因组。3)位于克隆的mt基因座的imp-11基因 但尚未本地化到特定的域，预计 激活iso-1的表达，也作用于假定的mt-1 特定蛋白P开启受精卵分化程序。凝胶- 迟滞分析将补充我们的标准分子生物学 IMP-11(和ISO-1)分析的方法，期望是 IMP-11和P都可以通过它们与克隆的结合能力来鉴定 受精卵特异基因上游的序列。身份识别和 对这些基因的分析可能会对评估和操作有所启发 性别分化的结果。

The long-term objective of this research is to identify and analyze some of the key genes and gen products that mediate sexual differentiation and zygote development in the unicellular eukaryotic flagellate, Chlamydomonas reinhardtii. The organism has two haploid mating types, mt and m, and mt/mt diploid zygotes, created by the fusion of complementary gametes, are uniquely capable of sporulation and meiosis. The project will employ two approaches. In the first, sequences identified as lying within the mt locus, which we have recently cloned from both mating types, will be used in transformation experiments to rescue mutants defective in mating/development. In the second, mutants defective in mating/development will be generated by insertional mutagenesis, which tags the mutated homologue. Three genes are directly targeted for analysis. 1) The imp-1 gene has been localize to within a 6 kb restriction fragment of the mt locus; its product is anticipated to participate in gametic adhesion, and its identification may lead us to its mt homologue and hence an understanding of how mating type-specific cell-cell recognition is achieved. 2) the iso-1 gene, tagged by an insertion event, functions only in mt gametes; its product is anticipated to activate the expression of mt-specific genes, and repress the expression of mt-specific genes, that are dispersed throughout the genome. 3) The imp-11 gene, which resides within the cloned mt- locus but has yet been localized to a specific domain, is anticipated to activate the expression of iso-1 and also to act with the postulated mt- specific protein P to switch on the zygote differentiation program. Gel- retardation assays will supplement our standard molecular-biology approaches to imp-11 (and iso-1) analysis, the expectation being that both imp-11 and P can be identified by their ability to bind to cloned sequences upstream of zygote-specific genes. The identification and analysis of these genes may illuminate the evaluation and the operation of sexual differentiation.