MOLECULAR GENETICS OF YEAST SEXUAL AGGLUTININS

酵母性凝集素的分子遗传学

• 批准号: 3306657
• 负责人: JANET A. KURJAN
• 金额: $ 13.36万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-01 至 1995-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306657
• 关键词: Saccharomyces cerevisiae; agglutinins; binding proteins; cell adhesion molecules; cell cell interaction; cell type; fungal genetics; gene expression; genetic manipulation; glycoproteins; membrane proteins; molecular cloning; molecular genetics; nucleic acid probes; protein structure function; sporogenesis; structural genes

Glycoprotein-glycoprotein interactions play essential roles in viral-cell, cell-cell, and cell-extracellular matrix interactions in higher eukaryotes. The aim of the proposed research is to use a system in which genetic and molecular approaches are possible, i.e. Saccharomyces cerevisiae and other yeasts, to study glycoprotein-glycoprotein mediated cell-cell interactions. Haploid cells of opposite mating types (a and alpha) of S. cerevisiae mate with one another to form the a/alpha diploid. Cell-surface glycoproteins, called a- and alpha-agglutinins, mediate aggregation between a and alpha cells. Mutants that show a- or alpha-specific agglutination defects have been isolated and used to clone and characterize the alpha-agglutinin structural gene and the structural gene for one of two a-agglutinin subunits, the core or cell-surface attachment domain. Other mutants will be used to isolate the second a-agglutinin structural gene, which encodes the binding fragment. The primary function of the agglutinins is to interact with one another to mediate cell-cell interactions. One of our primary goals, therefore, is to identify and characterize the regions of the agglutinins that are involved in this interaction. Using truncation and deletion mutants, we are in progress of localizing the binding domain of alpha-agglutinin; similar experiments will be used for the a-agglutinin. After determination of the smallest region of the agglutinins sufficient for binding to the opposite agglutinin, mutagenesis of these regions will be done to determine particular amino acids and features critical for binding. Further investigation of the interactions between the two agglutinins will involve the isolation of suppressor mutants, i.e. mutations in one agglutinin that allow an interaction with a mutant agglutinin of the opposite type. A feature of both the alpha-agglutinin and a-agglutinin core structural genes suggests that glycosyl phosphatidylinositol anchors may be involved in cell-surface localization and attachment. Experiments will be done to test this hypothesis. Experiments aimed at investigating the species-specificity of agglutinin interactions will involve the isolation of agglutinin genes from other yeasts, comparison of these genes to the S. cerevisiae genes, and construction and analysis of hybrid genes. These studies will provide a detailed characterization of glycoprotein-glycoprotein interactions that mediate interactions between particular cell types.

糖蛋白-糖蛋白相互作用在病毒-细胞中起着至关重要的作用， 高等真核生物中细胞与细胞、细胞与细胞外基质的相互作用。 这项拟议研究的目的是使用一种系统，其中遗传和 分子方法是可能的，即酿酒酵母和其他 酵母菌，研究糖蛋白-糖蛋白介导的细胞-细胞相互作用。 酿酒酵母交配相反交配型(a和α)的单倍体细胞 以形成α/α二倍体。细胞表面糖蛋白， 称为α-和α-凝集素，调节α-和α-凝集素之间的聚集 细胞。出现a-或α-特异性凝集缺陷的突变体 被分离并用于克隆和鉴定α-凝集素 结构基因和两种a-凝集素中的一种的结构基因 亚单位，核心或细胞表面附着域。其他变种人也会 被用来分离第二个a-凝集素结构基因，它编码 结合片段。凝集素的主要功能是 相互作用以调节细胞与细胞之间的相互作用。我们的一个人 因此，主要目标是确定和描述 参与这种相互作用的凝集素。使用截断 和缺失突变体，我们正在对结合区域进行定位 A-凝集素；类似的实验将用于a-凝集素。 在确定了凝集素的最小区域后 为了与相反的凝集素结合，这些区域的突变将 以确定特定的氨基酸和关键的功能 有约束力的。对两者相互作用的进一步研究 凝集素将涉及到抑制突变体的分离，即 允许与突变体相互作用的一种凝集素的突变 相反类型的凝集素。α-凝集素的一个特征 而a-凝集素核心结构基因表明糖基 磷脂酰肌醇锚点可能参与细胞表面定位 和依恋。将进行实验来验证这一假设。 旨在研究凝集素的物种特异性的实验 相互作用将涉及将凝集素基因与其他 酵母，将这些基因与酿酒酵母的基因进行比较，以及 杂交种基因的构建与分析。这些研究将提供一个 糖蛋白-糖蛋白相互作用的详细特征 调节特定细胞类型之间的相互作用。