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STRUCTURE OF THE HUMAN CALMODULIN GENE AND EXPRESSION

人类钙调蛋白基因的结构和表达

基本信息

批准号：
3383153
负责人：
FELIX FRIEDBERG
金额：
$ 7.62万
依托单位：
HOWARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-09-30 至 1994-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3383153
关键词：
RNA splicing calmodulin genetic library genetic promoter element genetic transcription human tissue molecular cloning natural gene amplification northern blottings nucleic acid probes regulatory gene reporter genes restriction mapping tissue /cell culture transcription factor transposon /insertion element

项目摘要

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.

在确定负责调节的组件之前，为了分离和表征每个人钙调蛋白基因，染色体特异性库和基因组库。这将允许分析两个或更多个活性基因，从而阐明这些基因之间的区别和共同特征，特别是在非编码片段。因为至少有4种不同的钙调素在人类基因组中，可能有超过2个真正的基因序列，基因是存在的我们现在从测序数据中得到的证据表明从17号染色体特异性文库中分离的是假基因。氨基该假基因中的酸取代是激进的和保守的。基因结构的知识将允许检查的安排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经被被证明是保守的。人类基因结构将揭示这是否是这个基因的进化特征本研究的第二部分涉及的表达和调控，个体基因。目前尚不清楚这两个或更多的活动是如何以及何时进行的。基因被转录。寡核苷酸探针或基因片段将使用代表每种基因的独特核苷酸序列的术语。通过使用来自各种人类细胞系或组织的多聚腺苷酸化RNA，北方印迹，溶液杂交。RNA斑点印迹和引物延伸试验，将有可能确定是否存在两个或两个以上活性基因的表达差异。组织将阐明转录物的特异性、水平和大小。为确定可能的调节顺式作用序列、载体含有“报告”基因并由人钙调蛋白基因驱动启动子或其部分将被允许复制不同类型的培养的人类细胞。