Molecular Biological Studies on Egg Cytoplasmic Factors That Are Responsible for Regulation of Gene Expression

负责基因表达调节的卵细胞质因子的分子生物学研究

基本信息

批准号：
01044077
负责人：
SATOH Noriyuki
金额：
$ 6.02万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

Presumptive muscle cells of early ascidian embryos have an extreme potential of autonomous differentiation. This potential is attributed to prelocalized egg cytoplasmic factors or determinants. The aim of our project is to understand the molecular nature of factors.The first approach to the problem includes an isolation and characterization of muscle-specific genes and analysis of regulatory factors for the gene expression. We have isolated cDNA clones for myosin-heavy chain and muscle-type actins. Expression of the muscle-specific genes begins around the time of gastrulation, and is restricted to presumptive muscle cells. It has been shown that the ascidian embryo synthesizes several different actin mRNAs. These mRNAs differ from one another in the nucleotide sequences of their 3'non-coding regions, although the sequences of the coding region are almost identical. One of the clones, HrcMA4 contains an open reading frame of 1137 bp and a 100 bp non-coding region followed by a poly (A) tail. Using a specific probe consisting mainly of 3'non-coding region, we have isolated several genomic DNA fragments containing HrMA4 gene. Characterization of the gene shows that HrMA4 is interrupted by two introns ; the position of them resembles to that of vertebrate skeletal muscle actin genes. In addition, it becomes clear that HrMA4 and several other muscle-actin genes form a cluster.The second approach is to characterize macromolecules of the myoplasm, which is thought to contain the cytoplasmic factors. We have made several monoclonal antibodies which specifically recognize the myoplasmic components. One of the antibodies blocks muscle development when it is injected into fertilized eggs. Characterization of the antigenic polypeptide has revealed that the protein is related to cytoskeletal proteins.

推测早期海鞘胚胎的肌肉细胞具有自主分化的巨大潜力。这种潜力归因于预先定位的卵细胞质因子或决定因素。我们项目的目的是了解因素的分子性质。解决该问题的第一种方法包括肌肉特异性基因的分离和表征以及基因表达调节因素的分析。我们分离了肌球蛋白重链和肌肉型肌动蛋白的 cDNA 克隆。肌肉特异性基因的表达在原肠胚形成时开始，并且仅限于假定的肌肉细胞。研究表明，海鞘胚胎合成几种不同的肌动蛋白 mRNA。这些mRNA在其3'非编码区的核苷酸序列上彼此不同，尽管编码区的序列几乎相同。其中一个克隆 HrcMA4 包含一个 1137 bp 的开放阅读框和一个 100 bp 非编码区，后面是聚腺苷酸尾。使用主要由3'非编码区组成的特异性探针，我们分离了几个含有HrMA4基因的基因组DNA片段。该基因的表征表明 HrMA4 被两个内含子打断；它们的位置类似于脊椎动物骨骼肌肌动蛋白基因的位置。此外，很明显HrMA4和其他几个肌肉肌动蛋白基因形成一个簇。第二种方法是表征肌质大分子，其被认为含有细胞质因子。我们已经制备了几种特异性识别肌浆成分的单克隆抗体。其中一种抗体被注射到受精卵中时会阻止肌肉发育。抗原多肽的表征表明该蛋白与细胞骨架蛋白相关。