MAMMALIAN CELL GENETIC STUDIES OF PREMRNA SPLICING

描述(改编自申请人的摘要)：前mRNA剪接是 从基因到基因转移信息的基本和必要的步骤 哺乳动物细胞中的蛋白质，几乎每一个基因转录本都经历了 随着时间的推移，删除大量不重要的信息 变成了一个功能性的信使RNA。作为基因中不可或缺的一步 表达，剪接提供了一个干预的目标，以防止 不需要的基因的表达(例如，在癌症中)。此外，拼接 在许多遗传病病例中，这一过程已经消失。的一大部分 这项建议针对的是一个基本问题，即 前信使核糖核酸剪接；如何识别剪接位点，如何识别100个碱基 从初级基因的10,000个碱基内含子中挑选出外显子 成绩单？哺乳动物细胞遗传学将被用来干扰Pre-mRNA 结构来揭示干扰拼接过程的更改。 为此，将使用二氢叶酸还原酶(DHFR) 作为可选记者的迷你基因仅在剪接时起作用 被破坏，允许快速筛选数千个突变以选择 那些为数不多的有效的改变。在另一个项目中， 内源性腺嘌呤磷酸核糖基转移酶基因突变 将进行非常详细的分析，并记录拼接 在不同的突变体中被阻断，以及内含子的顺序 去除可能在拼接位置选择中起作用。拼接可能发生在 核的特化区域；基因位置对剪接的影响 效率将在拥有单个转染体拷贝的细胞中进行测试 Dhfr基因，每个都整合到基因组中的不同位置。这个 先前的研究发现，提前终止翻译会干扰 RNA加工将通过检查核mRNA新陈代谢和 无义突变体中核外的信使核糖核酸运输。备择 剪接是一个过程，通过这个过程，不止一个功能蛋白质可以 由单一基因产生的。选择性剪接是一个主要的决定因素 细胞分化和病毒生长。一种机制，一种 给定的基因转录产物在一种细胞类型中以一种方式拼接，在另一种细胞类型中以另一种方式拼接 在另一种细胞类型中将通过分子克隆因子进行研究 这抑制了一种剪接模式。在一个正在研究的系统中，一种基因 在甲状腺中表达为多肽荷尔蒙降钙素，它 调节钙代谢，在神经元中表达为神经肽 CGRP，它使用不同的拼接模式。克隆的cdna将是 分离出编码神经元产生的一种因子的代码，该因子抑制 降钙素剪接模式有利于CGRP模式。这段cDNA会 使用哺乳动物细胞遗传操作进行选择，涉及到 可选择的DHFR微型燃料油。将使用类似的方法来分离 导致α-原肌球蛋白选择性剪接的因子的cDNA 不同类型肌肉组织中的转录本。克隆的cDNAs分子 将作为描述以下机制的起点 选择性剪接以及这一过程在发育中的调节。

DESCRIPTION (Adapted from the applicant's abstract): Pre-mRNA splicing is a basic and essential step in the transfer of information from gene to protein in mammalian cells, where nearly every gene transcript undergoes the removal of large amounts of non-essential information as it matures into a functional messenger RNA. As an indispensable step in gene expression, splicing presents a target for intervention to prevent the expression of unwanted genes (e.g., in cancer). In addition, the splicing process has gone away in many cases of genetic disease. A major part of this proposal is directed at a basic question regarding the mechanism of pre-mRNA splicing; how are splice sites recognized, how are the 100-base exons singled out from among 10,000 bases of introns in a primary transcript? Mammalian cell genetics will be used to perturb pre-mRNA structure to reveal changes that interfere with the splicing process. Toward this end, use will be made of dilhydrofolate reductase (dhfr) minigenes as selectable reporters that function only when splicing is disrupted, allowing the rapid screening of thousands of mutations to select those few changes that are effective. In another project, the effect of mutations in an endogenous adenine phosphoribosyltransferase (aprt) gene will be analyzed in great detail, documenting the steps in splicing that are blocked in different mutants and the effect that the order of intron removal may play in splice site selection. Splicing may occur in specialized regions of the nucleus; the effect of gene position on splicing efficiency will be tested in cells harboring a single copy of a transfected dhfr gene, each integrated into a different sites in the genome. The previous finding that premature termination of translation interferes with RNA processing will be studied by examining nuclear mRNA metabolism and transport of mRNA out of the nucleus in nonsense mutants. Alternative splicing is a process by which more than one functional protein can be produced from a single gene. Alternative splicing is a major determinant of cell differentiation and of viral growth. The mechanism by which a given gene transcript is spliced one way in one cell type and another way in another cell type will be studied by the molecular cloning of factors that inhibit one splicing pattern. In one system being studied, a gene that is expressed in the thyroid as the peptide hormone calcitonin, which regulates calcium metabolism, is expressed in neurons as the neuropeptide CGRP, which uses a different splicing pattern. A cloned cDNA will be isolated that codes for a factor produced in neurons that repress the calcitonin splicing pattern in favor of the CGRP pattern. This cDNA will be selected using mammalian cell genetic manipulations involving a selectable dhfr minigene. A similar approach will be used to isolate a cDNA for a factor that causes alternative splicing of alpha-tropomyosin transcripts in different types of muscle tissue. The cloned cDNA molecules will serve as a starting point for characterizing the mechanism of alternative splicing and the regulation of this process in development.