STRUCTURE OF THE HUMAN CALMODULIN GENE AND EXPRESSION

在定义负责监管的组成部分之前，有必要 使用这两种方法分离和鉴定每个人的钙调蛋白基因 染色体特异库和基因组文库。这将允许 对两个或更多活性基因的分析，从而澄清 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种不同的钙调蛋白 在人类基因组中，有可能有超过2个真正的 基因是存在的。我们现在从测序数据中有证据表明，克隆人 从17号染色体特异文库中分离出来的是一个假基因。氨基 这种假基因中的酸取代既是自由的，又是保守的。 对基因结构的了解将允许检查这种安排 以及外显子和内含子的组织。干预的地点 果蝇、鸡和大鼠基因结构中的序列已经被 被证明是保守的。人类的基因结构将揭示如果这是 这种基因的一种进化特征。 本研究的第二部分涉及到基因的表达和调控。 个体基因。目前尚不清楚两人或更多人如何以及何时活跃 基因是转录的。寡核苷酸探针或基因片段 代表每个基因的唯一核苷酸序列将被使用。通过 使用来自不同人类细胞系或组织的多腺苷化RNA Northern杂交，溶液杂交。RNA斑点印迹和引物 延伸化验，将有可能确定是否存在 两个或两个以上活性基因表达的差异。组织 具体情况、抄本的水平和大小将被阐明。为 确定可能的调控顺式作用序列、载体 含有“报告”基因，并由人类钙调蛋白基因驱动 启动子或部分启动子将被允许转染不同类型 培养中的人类细胞。

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.