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

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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.