GENES FOR HYDROXYPROLINE-RICH GLYCOPROTEINS

细胞-细胞/细胞-基质识别现象对许多 生物过程(正常和致病)。我们希望确定在 详细说明管理特定大分子缔合的分子规则 参与细胞黏附和细胞外基质组装。衣藻 是这类研究的极好的实验生物，因为它的设备 生化、结构和遗传分析。识别/粘连 分子(凝集素)和细胞外基质(细胞壁)成分 从衣藻属分离出来，并被证明是一个家庭的成员 富含羟脯氨酸的糖蛋白(HRGP‘s)可能具有共同的 进化论遗产。每一类HRGP都参与特定的结合 以模仿其体内功能的方式在体外发挥作用。什么 构成一个特定的结合域？这些域之间有何不同 同源(正负)凝集素分子？是同源域 负责细胞壁组件的自组装？是凝集素和 Matrix HRGP是多基因家族的成员吗？CDNA文库将被 由每次交配的营养细胞和配子细胞的聚A+RNA构建 衣藻(Mt+和Mt-)型，在表达载体Lambdagtll中， 或者在Lambdagt10中。将使用抗体(Lambdagtll)筛选文库 或合成寡核苷酸(Lambdagt10)探针以识别编码的插入片段 对于MT+和MT-凝集素和GPL，晶状层的一种成分 似乎与凝集素相关的细胞壁的 标准。将确定凝集素的候选结合序列 通过差异杂交。产生的抗体对应于 融合蛋白，将用于抑制试验和表位绘制。 我们将使用合成肽进一步研究功能域。 抗肽抗体。GPL结合域，在同一 方式，将被绘制并与凝集素序列进行比较以作为证据 同源性。

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.