Molecular Mechanisms of Muscle Cell Differentiation in Embryos Ascidian

首先，我们分离了肌球蛋白重链和肌型肌动蛋白的cDNA克隆，研究了海鞘胚胎肌肉细胞分化的分子机制。这些肌肉特异性基因的表达大约在原肠形成的时候，并且仅限于分化的SELE细胞。海鞘胚胎合成几种不同的肌动蛋白mRNAs。其中一个克隆HrcMA4含有一个1137bp的开放阅读框和一个100bp的非编码区，后面跟着一个PolyA尾巴。然后，我们分离了含有HrMA4基因的基因组片段。对该基因的分析表明，HrMA4被两个内含子打断。有几个潜在的调控元件可以准确表达该基因。此外，很明显，HrMA4和其他几个肌肉肌动蛋白基因形成了一个簇。第二，我们试图分离肌肉生成因子如MyoDl和肌肉生成因子的基因。S6到目前为止，我们已经通过PCR方法获得了该基因的候选DNA片段。最后，我们通过制备几种单克隆抗体，对被认为含有成肌因子的肌浆大分子进行了分析。其中一种抗体在注射到受精卵中时会阻止肌肉发育。然而，对该抗原性多肽的鉴定表明，它与细胞骨架蛋白有关。

Molecular mechanisms of muscle cell differentiation of ascidian embryos have been investigated with respect to an extreme potential of autonomous differentiation of the cells.First, we have isolated CDNA clones for myosin-heavy chain and museletype actins. Expression of these muscle-specific genes beans around the time of gastrulation, and is restricted to differentiating sele cells. Ascidian embryos synthesize several different actin mRNAs. One of the clones, HrcMA4 contains an open reading frame of 1137 bp and a 100 bp noncoding region followed by a poly(A)tail. Then, we have isolated genomic fragments that contain HrMA4 gene. Characterization of the gene shows that HrMA4 is interrupted by two introns. There are several potential regulatory elements for accurate expression of the gene. In addition, it becomes clear that HrMA4 and several other muscle-actin genes form a cluster.Second, we have tried to isolate genes for myogenic factors such as MyoDl and myogenic. S6 far, we have obtained by PCR method a candidate DNA-fragment for the genes.Finally, we have analyzed macromolecules of the myoplasm, which is thought to contain the myogenic factors, by producing several monoclonal antibodies. One of the antibodies blocks muscle development when it is injected into fertilized eggs. Characterization of the antigenic polypeptide, however, has shown that it is related to cytoskeletal proteins.