MOLECULAR BIOLOGY OF THE HUMAN CD36 GENE

人类 CD36 基因的分子生物学

• 批准号: 3365589
• 负责人: JOHN F MILL
• 金额: $ 17.14万
• 依托单位: AMERICAN NATIONAL RED CROSS
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3365589
• 关键词: CD antigens; DNA footprinting; RNA directed DNA polymerase; RNA splicing; clone cells; complementary DNA; gel mobility shift assay; gene expression; gene mutation; genetic enhancer element; genetic library; genetic mapping; genetic promoter element; genetic regulatory element; human genetic material tag; messenger RNA; molecular cloning; nucleic acid sequence; phenotype; platelets; polymerase chain reaction; reporter genes; restriction fragment length polymorphism; tissue /cell culture; transcription factor

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.

糖蛋白IV（CD 36）已被鉴定为血小板受体， 胶原蛋白和血小板反应蛋白。 CD 36还介导了 恶性疟原虫寄生的红细胞，表明在 疟疾 此外，CD 36缺陷与以下因素相关： 临床观察到的输血表型指定为Naka阴性。 这 功能的多样性构成了研究CD 36的基础。 基因及其表达和产物。 其主要目标是 目的是阐明CD 36在分子水平上的编码和加工， 水平 有四个具体目标： 1.分离并测序人CD 36基因。 CD 36 cDNA将用于 筛选基因组文库并纯化对应于CD 36编码的克隆， 序列及其邻近区域。 与已知外显子内含子同源 边界和监管要素将由完整的 测序和序列分析。 2.识别RNA加工中间体和可变剪接产物 CD 36基因 cDNA将被分离、测序并分析， 前体和选择性剪接的CD 36 mRNA的证据。 稀有mRNA 将通过PCR扩增以进行鉴定。 S1核酸酶消化 mRNA/DNA杂合体将用于确认选择性剪接事件， 表达CD 36的细胞类型。 3.鉴定CD 36基因的启动子和其他调控序列。 转录起始位点将通过引物延伸和S1 核酸酶分析结合序列分析。 疑似监管 将在转染到 表达CD 36的细胞系。 将通过凝胶检测反式作用因子 CD 36基因的迁移率变动分析和DNA酶I足迹法结合 用来自表达CD 36的细胞类型的核提取物。 4.确定Naka阴性表型的分子基础。 CD36 似乎缺乏Naka阴性血小板，尽管相关的较低 检测分子量种类以及CD 36 mRNA。 的起源 该缺陷将与定义的调节途径相关， 正常CD 36蛋白表达。 这些研究将阐明CD 36表达的机制，在正常人， 巨核细胞和其他细胞，鉴定任何CD 36变体并确定 Naka阴性表型的分子基础。