MOLECULAR BIOLOGY OF THE HUMAN CD36 GENE

糖蛋白IV(CD36)被认为是一种血小板受体 胶原蛋白和凝血酶原蛋白。CD36还介导细胞黏附 恶性疟原虫寄生的红细胞，表明在 疟疾造成的后果。此外，CD36缺乏与 临床观察到的一种被命名为Naka阴性的输血表型。这 功能的多样性是研究CD36的基础 基因、其表达及其产物。这样做的主要目标是 建议阐明CD36在分子上的编码和加工 水平。具体目标有四个： 1.人CD36基因的分离和测序CD36基因将被用于 筛选与CD36编码对应的基因组文库和纯化克隆 序列及其邻近区域。与已知外显子的同源性 边界和监管要素将由完整的 测序和序列分析。 2.确定RNA加工中间体和选择性剪接产物 CD36基因。将对cDNA进行分离、测序和分析 CD36mRNAs前体和选择性剪接的证据。稀有的mRNA 将通过聚合酶链式反应进行扩增，以便进行鉴定。S_1核酸酶的消化 将使用mRNA/DNA杂交体来确认在 CD36表达细胞类型。 3.鉴定CD36基因的启动子和其他调控序列。 转录起始点将由引物延伸和S1决定 核酸酶分析与序列分析相结合。疑似监管机构 将在报告基因构建中对区域进行评估，并将其导入 表达CD36的细胞系。反式作用因子将通过凝胶进行检测 迁移率改变分析与CD36基因DNase I足迹相结合 从表达CD36的细胞类型中提取核提取物。 4.鉴定Naka阴性表型的分子基础。CD36 Naka阴性的血小板似乎有缺陷，尽管相关程度较低 检测到分子量种类和CD36基因的表达。的起源 这种缺陷将与为其定义的调控途径相关 正常CD36蛋白的表达。 这些研究将阐明CD36在正常人中的表达机制 巨核细胞和其他细胞，识别任何CD36变体并定义 Naka阴性表型的分子基础。

Glycoprotein IV (CD36) has been identified as a platelet receptor for collagen and thrombospondin. CD36 also mediates cytoadherence of Plasmodium falciparum-parasitized erythrocytes, indicating a role in malariasequestration. In addition, CD36 deficiency has been correlated to a clinically observed transfusion phenotype designated Naka-negative. This diversity of function constitutes a basis for investigation of the CD36 gene, its expression, and its products. The principal goal of this proposal is to elucidate the coding and processing of CD36 on the molecular level. There are four specific aims: 1. Isolate and sequence the human CD36 gene. CD36 cDNA will be used to screen genomic libraries and purify clones corresponding to the CD36 coding sequence and its neighboring regions. Homologies to known exonintron boundaries and regulatory elements will be delineated by complete sequencing and sequence analysis. 2. Identify RNA processing intermediates and alternatively spliced products of the CD36 gene. cDNAs will be isolated, sequenced, and analyzed for evidence of precursor and alternatively spliced CD36 mRNAs. Rare mRNAs will be amplified by PCR to allow identification. S1 nuclease digestion of mRNA/DNA hybrids will be used to confirm alternative splicing events in CD36 expressing cell types. 3. Characterize promoter and other regulatory sequences for the CD36 gene. Transcriptional start sites will be determined by primer extension and S1 nuclease analysis coupled with sequence analysis. Suspected regulatory regions will be evaluated in reporter gene constructs transfected into CD36-expressing cell lines. Trans-acting factors will be detected by gel mobility shift assay and DNase I footprinting of the CD36 gene combined with nuclear extracts from CD36-expressing cell types. 4. Identify the molecular basis of the Naka-negative phenotype. CD36 appears deficient in Naka-negative platelets, although related lower molecular weight species are detected as well as CD36 mRNA. The origin of the deficiency will be correlated with the regulatory pathways defined for expression of normal CD36 protein. These studies will elucidate mechanisms of CD36 expression in normal megakaryocytes and other cells, identify any CD36 variants and define the molecular basis of the Naka-negative phenotype.