FLAGELLAR MEMBRANE ADHESION AND SEXUAL SIGNALING

这项研究的长期目标是确定和分析一些 介导性别分化的关键基因和性别产物， 单细胞真核鞭毛虫的合子发育， 莱茵衣原体 这种生物有两种单倍体交配类型， mt和m，以及mt/mt二倍体合子，由 互补配子是唯一能够形成孢子和减数分裂的。 该项目将采用两种方法。 首先，序列 被鉴定为位于我们最近克隆的MT基因座内， 来自两种交配类型，将用于转化实验， 挽救在交配/发育方面有缺陷的突变体。 第二种，变种人 交配/发育缺陷将通过插入产生 诱变，其标记突变的同源物。 三个基因直接 针对分析。 1)imp-1基因已经定位于 mt基因座的6 kb限制性片段;预期其产物 参与配子粘附，它的鉴定可能会导致我们 它的mt同源物，因此了解交配型特异性 实现了细胞-细胞识别。 2)iso-1基因，由一个 插入事件，仅在mt配子中起作用;预期其产物 激活线粒体特异性基因的表达，并抑制 线粒体特异性基因的表达，这些基因分散在整个细胞中， 基因组 3)imp-11基因位于克隆的mt-基因座内， 但是还没有被定位到特定的领域，预计将 激活iso-1的表达，并与假定的mt- 特异性蛋白P来启动受精卵分化程序。 凝胶- 阻滞试验将补充我们的标准分子生物学 imp-11（和iso-1）分析的方法，预期 imp-11和P都可以通过它们与克隆的 合子特异性基因上游的序列。 确定和 对这些基因的分析可为评价和操作提供依据 性别差异。

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.