GENES FOR HYDROXYPROLINE-RICH GLYCOPROTEINS

富含羟脯氨酸的糖蛋白基因

• 批准号: 3292258
• 负责人: W STEVEN ADAIR
• 金额: $ 14.04万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292258
• 关键词: Chlorophyta; affinity chromatography; antibody formation; autoradiography; cell adhesion; cell cell interaction; cell wall; complementary DNA; density gradient ultracentrifugation; endonuclease; enzyme linked immunosorbent assay; extracellular matrix; gene expression; genetic library; genetic manipulation; genetic translation; high performance liquid chromatography; hydroxyproline; immunoprecipitation; messenger RNA; molecular cloning; monoclonal antibody; nucleic acid sequence; ultraviolet spectrometry

Cell-cell/cell-matrix recognition phenomena are essential to numerous biological processes (normal and pathogenic). We wish to determine in detail the molecular rules governing specific macromolecular associations involved in cell-adhesion and extracellular matrix assembly. Chlamydomonas is an excellent experimental organism for such studies due to the facility of biochemical, structural, and genetic analyses. Recognition/adhesion of molecules (agglutinins) and extracellular matrix (cell wall) components have been isolated from Chlamydomonas and shown to be members of a family of hydroxyproline-rich glycoproteins (HRGP's) that may share a common evolutionary heritage. Each class of HRGP engages in specific binding activities in vitro in a manner that mimics their in vivo function. What constitutes a specific binding domain? How do these domains differ between homologous (plus and minus) agglutinin molecules? Are homologous domains responsible for self-assembly of cell wall components? Are agglutinin and matrix HRGP's members of a multigene family? cDNA libraries will be constructed from poly A+ RNA of vegetative and gametic cells of each mating type (mt+ and mt-) of Chlamydomonas, in the expression vector Lambdagtll, or in Lambdagt10. Libraries will be screened using antibody (Lambdagtll) or synthetic oligonucleotide (Lambdagt10) probes to identify inserts coding for mt+ and mt- agglutinins and GPl, a component of the crystalline layer of the cell wall that appears to be related to the agglutinins by several criteria. Candidate binding sequences for agglutinins will be identified by differential hybridization. Antibodies produced against corresponding fusion proteins, will be used for inhibition assays and epitope-mapping. Functional domains will be examined further, using synthetic peptides anti-peptide antibodies. GPl binding domains, identified in the same manner, will be mapped and compared with agglutinin sequences for evidence of homology.

细胞-细胞/细胞-基质识别现象对于许多人来说是必不可少的 生物过程（正常和致病）。 我们希望确定在 详细说明控制特定大分子缔合的分子规则 参与细胞粘附和细胞外基质组装。 衣藻属 由于设施齐全，是此类研究的优秀实验生物体 生化、结构和遗传分析。 认识/坚持 分子（凝集素）和细胞外基质（细胞壁）成分 已从衣藻中分离出来并被证明是一个家族的成员 富含羟脯氨酸的糖蛋白（HRGP）可能具有共同点 进化遗产。 每类 HRGP 都参与特异性结合 以模仿其体内功能的方式进行体外活动。 什么 构成特异性结合域？ 这些域之间有何不同 同源（正负）凝集素分子？ 是否是同源域 负责细胞壁成分的自组装？ 是凝集素和 矩阵 HRGP 的多基因家族成员？ cDNA 文库将是 由每次交配的营养细胞和配子细胞的多聚 A+ RNA 构建 衣藻型（mt+和mt-），在表达载体Lambdagtll中， 或在 Lambdagt10 中。 将使用抗体（Lambdagtll）筛选文库 或合成寡核苷酸 (Lambdagt10) 探针来识别插入编码 对于 mt+ 和 mt- 凝集素以及 GPl，结晶层的一个组成部分 细胞壁的结构似乎与凝集素有关 标准。 将鉴定凝集素的候选结合序列 通过差异杂交。 产生针对相应的抗体 融合蛋白将用于抑制测定和表位作图。 将使用合成肽进一步检查功能域 抗肽抗体。 GPl结合域，在相同的 方式，将被绘制并与凝集素序列进行比较以获取证据 的同源性。