CHARACTERIZATION OF THE MURINE COMPLEMENT RECEPTORS

本研究将描述其结构、表达和功能 一组相关的小鼠蛋白质，其共同特征是结合 C3的碎片。 这些蛋白质被称为补体受体， 参与了免疫反应的许多作用，包括 控制补体级联的激活，增强哺乳动物的免疫应答， 包括补体激活控制的体液应答 级联增强哺乳动物的体液反应和启动 免疫应答中细胞亚群的增殖。 的焦点 本申请是为了继续分析基因和基因产物， 的小鼠补体受体，并扩大这项调查， 包括它们在表达它们的细胞的生物学中的作用。 具体地，这将包括Crry蛋白的组织特异性表达。 基因（其是人CR 1的鼠同源物）和对CR 1基因的分析。 由Crry转录物产生的两种变体蛋白质。 使用转染 分析，Crry蛋白的结构域在这种假定的作用中很重要， 控制补体激活和免疫细胞的吞噬作用 复合物将通过引入到细胞中的突变进行定位和检查。 正常基因 此外，含有序列的基因组序列 将分离与Crry基因同源的基因，并检查其编码 与Crry相似的序列。 我们对鼠CR的分析将 还包括Cr2基因，它似乎产生更广泛的基因， 比人类同源物CR2的产物。 提供资金的机制 这种多样性（组织特异性转录和选择性剪接） 将被分析，因为将基因产物的功能，通过 转染分析。 这些蛋白质在生物学中的作用 动物将进一步研究通过创建突变品系的小鼠 其不再表达Crry或Cr2基因，转化为 一种能够发育成小鼠的胚胎干细胞。 任何表型 在这些小鼠中可以检测到的变化可以用于筛选人类 人群中寻找类似的缺陷，在人类的表达 补体受体 授权= R 01 AI 23483 许多造血细胞参与免疫和炎症反应， 反应[包括肥大细胞、嗜碱性粒细胞、自然杀伤细胞、细胞毒性 T淋巴细胞、嗜酸性粒细胞、中性粒细胞、巨噬细胞和血小板]储存 细胞内分泌颗粒中蛋白多糖的一个独特家族 车厢 因为这些蛋白聚糖存在于如此广泛的范围内， 免疫反应的效应细胞，因为编码 这些蛋白聚糖的肽核心在细胞中很早就表达， 造血细胞的分化，现在很清楚，调节 这种特殊基因的存在对人类的发育和 这些造血细胞的功能。 研究的目标 在本申请中提出的是确定所述化合物的一级序列。 分泌颗粒蛋白聚糖肽核心基因在两个进化上 然后了解这个基因是如何在 造血细胞 已发现大小约为18个内切酶的基因组片段， 分离的含有编码这种新的肽核心的基因 蛋白聚糖家族。 双链核苷酸测序 化学合成的寡核苷酸引物将用于确定 人类和小鼠基因的完整核苷酸序列。 数据具有 已经获得了编码N- 肽核心的末端在进化过程中高度保守， 5'和3'非翻译区内的特定核苷酸序列 它们的mRNA转录本。 同样地，当人免疫球蛋白的5'侧翼区 将该基因与类似物的相应5'侧翼区进行比较， 小鼠基因，即转录起始前的区域 发现位置几乎相同。 观察到，后者 序列甚至比任何相应的区域更高度保守。 翻译成蛋白质的基因意味着这个5'侧翼区 含有顺式作用调节元件，其对于表达 这个基因在造血细胞中。 造血细胞和非造血细胞 造血细胞将用嵌合构建体转染， 阐明基因的区域，这是重要的， 转录调控 抗体将被提高， 小鼠蛋白聚糖的肽核心，以研究其翻译和 在转染的细胞中该蛋白聚糖的糖胺聚糖修饰。

The proposed research will describe the structure, expression and function of a set of related murine proteins whose common feature is the binding of fragments of C3. These proteins, known as the complement receptors, have been implicated in a number of roles of the immune response including the control of the activation of the complement cascade enhancing the mammalian humoral response including the control of the activation of the complement cascade enhancing the mammalian humoral response and priming the proliferation of subsets of cells within the immune response. The focus of this application is to continue the analysis of the genes and gene products of the murine complement receptors and to extend this investigation to include their roles in the biology of the cells which express them. Specifically, this will include the tissue specific expression of the Crry gene (which is the murine homologue to human CR1) and the analysis of the two variant proteins produced form Crry transcripts. Using transfection analyses, the domains of the Crry protein important in this presumed roles of the control of complement activation and phagocytosis of immune complexes will localized and scrutinized via mutations introduced into the normal gene. In addition, a genomic sequence which contains sequences homologous to the Crry gene will be isolated and examined for coding sequences similar to those of Crry. our analysis of the murine CR will also include the Cr2 gene which appears to produce a wider variety of gene products than the human homologue, CR2. The mechanisms of providing for this diversity (tissue specific transcription and alternative splicing) will be analyzed as will the functions of the gene products via transfection analyses. The role of these proteins in the biology of the animal will be further investigated by creating mutant strains of mice which no longer express the Crry or Cr2 genes into the Crry or Cr2 genes of an ES cell, which is capable of developing into a mouse. Any phenotypic change which can be detected in these mice can be used to screen the human population to look for similar defects in the expression of the human complement receptors. GRANT=R01AI23483 Many of the hematopoietic cells that participate in immune and inflammatory responses [including mast cells, basophils, natural killer cells, cytotoxic T lymphocytes, eosinophils, neutrophils, macrophages, and platelets] store a unique family of proteoglycans in an intracellular secretory granule compartment. Because these proteoglycans are present in such a wide range of effector cells of the immune response and because the gene that encodes the peptide core of these proteoglycans is expressed very early in the differentiation of hematopoietic cells, it is now clear that the regulation of this particular gene is extremely important for the development and function of these hematopoietic cells. The objectives of the research proposed in this application are to determine the primary sequence of the secretory granule proteoglycan peptide core gene in two evolutionarily distant species, and then to understand how this gene is regulated in hematopoietic cells. Genomic fragments of ~18 kilobases in size have been isolated that contain the gene that encodes the peptide core of this novel family of proteoglycans. Double stranded nucleotide sequencing using chemically synthesized oligonucleotide primers will be used to determine the complete nucleotide sequence of the human and mouse gene. Data have already been obtained that the nucleotide sequences that encode the N- terminus of the peptide core are highly conserved through evolution, as are specific nucleotide sequences within the 5' and 3' untranslated regions of their mRNA transcripts. Likewise, when the 5' flanking region of the human gene was compared to the corresponding 5' flanking region of the analogous mouse gene, a region that immediately precedes the transcription-initiation site was found to be nearly identical. The observation that this latter sequence is even more highly conserved than any corresponding region of the gene that is translated into protein implies that this 5' flanking region contains cis acting regulatory elements that are critical for expression of this gene in hematopoietic cells. Hematopoietic cells and non- hematopoietic cells will be transfected with chimeric constructs to elucidate the regions of the gene which are important for its transcriptional regulation. Antibodies will be raised that recognize the peptide core of the mouse proteoglycan to study the translation and glycosaminoglycan modification of this proteoglycan in transfected cells.