STRUCTURE OF THE HUMAN CALMODULIN GENE AND EXPRESSION

人类钙调蛋白基因的结构和表达

• 批准号: 3383150
• 负责人: FELIX FRIEDBERG
• 金额: $ 7.61万
• 依托单位: HOWARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-30 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3383150
• 关键词: calmodulin; genetic library; genetic promoter element; genetic transcription; human tissue; molecular cloning; natural gene amplification; nucleic acid hybridization; nucleic acid probes; regulatory gene; reporter genes; restriction mapping; tissue /cell culture; transposon /insertion element

Before defining the components responsible for regulation it is necessary to isolate and characterize each of the human calmodulin genes using both chromosome-specific libraries and a genomic library. This would allow analysis of the two or more active genes thus clarifying the distinguishing and common features between these genes particularly in the non-coding segments. Since there are at least 4 different calmodulin sequences in the human genome, it is possible that more than 2 bona fide genes exist. We have evidence now from sequencing data that a clone isolated from a chromosome 17 specific library is a pseudogene. The amino acid substitutions in this pseudogene are both radical and conservative. Knowledge of gene structure would permit examination of the arrangement and organization of exons and introns. The location of intervening sequences in the Drosophila, chicken and rat gene structures have been shown to be conserved. The human gene structure would reveal if this is an evolutionary feature of this gene. The second part of this study deals with the expression and regulation of the individual genes. It is not clear how and when the two or more active genes are transcribed. Oligonucleotide probes or gene fragments representing unique nucleotide sequences of each gene will be used. By employing polyadenylated RNA from various human cell lines or tissues in Northern blots, solution hybridization. RNA dot blots and primer extension assay, it will be possible to determine whether there is a difference in the expression of the two or more active genes. Tissue specificity, the level and size of transcripts will be elucidated. For the determination of possible regulatory cis-acting sequences, vectors containing a "reporter" gene and driven by a human calmodulin gene promoter, or portions of it, will be allowed to transfect different types of human cells in culture.

在定义负责监管的组件之前，有必要 使用两种方法分离和表征每个人类钙调蛋白基因 染色体特异性文库和基因组文库。这将允许 分析两个或多个活性基因，从而阐明 这些基因之间的区别和共同特征，特别是 非编码段。因为至少有 4 种不同的钙调蛋白 人类基因组中的序列，可能有超过 2 个真实的序列 基因存在。我们现在从测序数据中得到证据表明克隆 从 17 号染色体特异性文库中分离出来的是假基因。氨基 该假基因中的酸取代既是激进的又是保守的。 基因结构的知识将允许检查排列 以及外显子和内含子的组织。干预的位置 果蝇、鸡和大鼠基因结构中的序列已被 显示为守恒。人类基因结构将揭示这是否是 该基因的进化特征。 本研究的第二部分涉及表达和调控 个体基因。目前尚不清楚两个或更多的人如何以及何时活跃 基因被转录。寡核苷酸探针或基因片段 将使用代表每个基因的独特核苷酸序列。经过 使用来自各种人类细胞系或组织的聚腺苷酸化RNA Northern 印迹、溶液杂交。 RNA 斑点印迹和引物 延伸测定，将有可能确定是否存在 两个或多个活性基因的表达差异。组织 转录本的特异性、水平和大小将被阐明。为了 确定可能的调节顺式作用序列、载体 含有“报告”基因并由人类钙调蛋白基因驱动 启动子或其部分将被允许转染不同类型 培养中的人类细胞。