CHARACTERIZATION OF THE MURINE COMPLEMENT RECEPTORS

拟议的研究将描述该基因的结构、表达和功能 一组相关的小鼠蛋白，其共同特征是结合 C3的碎片。这些被称为补体受体的蛋白质具有 与免疫反应的多个角色有关，包括 加强哺乳动物补体激活级联反应的调控 体液反应包括对补体激活的控制 级联增强哺乳动物的体液反应并启动 免疫反应中细胞亚群的增殖。的关注点 这一应用是为了继续分析基因和基因产物 并将这项研究扩展到 包括它们在表达它们的细胞的生物学中的作用。 具体地说，这将包括CRY的组织特异性表达 基因(这是小鼠与人CR1的同源物)和分析 由Crry转录本产生的两种变异蛋白质。使用转染法 分析，CRY蛋白的结构域在这一推测的作用中很重要 补体活化和免疫吞噬功能的调控 复合体将通过引入突变来定位和仔细检查 正常基因。此外，包含序列的基因组序列 与Crry基因同源的基因将被分离并进行编码检查 序列与Crry的序列相似。我们对小鼠CR Will的分析 也包括Cr2基因，它似乎能产生更多种类的基因 产品比人类同源基因CR2。供养机制 这种多样性(组织特异性转录和选择性剪接) 将通过基因产品的功能进行分析 转基因分析。这些蛋白质在细菌生物学中的作用 将通过创造突变的小鼠品系对该动物进行进一步研究 不再将Crry或Cr2基因表达到植物的Crry或Cr2基因中 一种ES细胞，能够发育成小鼠。任何表型 在这些老鼠身上可以检测到的变化可以用来对人类进行筛查 在人类的表达中寻找类似的缺陷 补体受体。GRANT=R01AI23483 许多参与免疫和炎症的造血细胞 反应[包括肥大细胞、嗜碱性粒细胞、自然杀伤细胞、细胞毒 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 细胞内分泌颗粒中的一个独特的蛋白多糖家族 车厢。因为这些蛋白多糖存在的范围如此之广 免疫反应的效应细胞，因为编码 这些蛋白多糖的肽核心在很早的时候就表达出来 对造血细胞的分化，现在已经很清楚了 这种特殊的基因对于人类的发育和 这些造血细胞的功能。研究的目标是 在本申请中提出的是确定序列的主要序列 分泌颗粒蛋白多糖肽核心基因在两个进化中的作用 ，然后了解这个基因是如何在 造血细胞。大小约18千碱基的基因组片段已经被 含有编码这本小说的多肽核心的基因的分离株 蛋白多糖家族。双链核苷酸测序 化学合成的寡核苷酸引物将用于检测 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-N的核苷酸序列 肽核心的末端在进化过程中高度保守，就像 猪瘟病毒5‘和3’非翻译区的特异核苷酸序列 他们的信使核糖核酸转录。同样，当人类的5‘侧翼区域 将该基因与相应的类似物的5‘侧翼区进行比较 小鼠基因，紧接在转录启动之前的区域 网站被发现几乎完全相同。据观察，后者 序列甚至比任何相应的区域都更加保守 翻译成蛋白质的基因意味着这个5‘侧翼区域 包含顺式作用调节元件，这些元件对于表达 这种基因存在于造血细胞中。造血细胞和非造血细胞 将用嵌合构建体导入造血细胞以 阐明该基因的哪些区域对ITS很重要 转录调控。将产生识别该病毒的抗体 小鼠多肽核心蛋白多糖的翻译和研究 糖胺多聚糖在转基因细胞中对该蛋白多糖的修饰。

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.