MOLECULAR MECHANISMS OF CELLULAR DIFFERENTIATION IN ASCIDIAN EMBRYOS

从分子生物学水平探讨了海鞘胚胎肌肉细胞分化的机制。首先，我们生产了针对肌肉细胞发育的单克隆抗体。其次，利用从尾芽胚中分离的POLY(A)+RNA构建cDNA文库。将得到的cDNA插入表达载体gt11中，用抗体筛选文库。选择阳性克隆1个，插入1.6 kb。利用反义探针原位杂交发现，这些转录片段仅局限于尾芽胚胎的肌肉细胞。对cDNA进行序列分析，得到氨基酸序列。所得序列与啮齿类动物肌球蛋白重链蛋白具有广泛的同源性。Southern blot杂交分析表明，肌球蛋白重链基因序列以单拷贝形式存在于单倍体盐藻基因组中。Northern blot杂交结果显示，孕前期未检测到肌球蛋白重链基因转录本，孕前期后肌球蛋白重链基因转录本迅速积累。我们正试图获得cDNA探针来研究肌动蛋白和乙酰胆碱酯酶基因的表达。我们还打算分离出在腹膜胚胎中负责肌肉细胞分化的基因。我们已经获得了特异性识别肌浆成分的单克隆抗体，肌浆被认为含有肌肉决定因素。利用抗体，我们现在正在分离编码肌浆特异性多肽的基因。海鞘胚胎中的myod1样基因也在研究中。

THE MECHANISMS INVOLVED IN MUSCLE CELL DIFFERENTIATION IN ASCIDIAN EMBRYOS WERE INVESTIGATED AT MOLECULAR BIOLOGICAL LEVEL. FIRST, WE PRODUCED MONOCLONAL ANTIBODIES SPECIFIC TO DEVELOPING MUSCLE SELLS. SECOND, WE MADE UP cDNA LIBRARY FROM POLY(A)+RNA ISOLATED FROM TAILBUD EMBRYOS. THE RESULTING cDNA WERE INSERTED INTO THE EXPRESSION VECTOR gt11, AND THEN THE LIBRARY WAS SCREENED WITH THE ANTIBODY. ONE OF THE POSITIVE CLONES, CONTAINING 1.6-KB INSERT, WAS SELECTED. IN SITU HYBRIDIZATION USING ANTISENSE PROBE SHOWED THAT THE TRANSCRIPTSWERE RESTRICTED TO MUSCLE CELLS OF THE TAILBUD EMBRYOS. SEQUENCE ANALYSES OF THE cDNA ALLOWED THE AMINO ACID SEQUENCE. THE DEDUCED SEQUENCE SHOWED EXTENSIVE HOMOLOGY TO RODENT MYOSIN HEAVY CHAIN PROTEIN. SOUTHERN BLOT HYBRIDIZATION ANALYSIS SUGGESTED THAT MYOSIN HEAVY CHAIN GENE SEQUENCE IS PRESENT AS A SINGLE COPY PER HAPLOID HALOCYNTHIA GENOME. NORTHERN BLOT HYBRIDIZATION SHOWED THAT THE MYOSIN HEAVY CHAIN GENE TRANSCRIPTS WERE NOT DETECTED AT PREGASTRULA STAGES, AFTER WHICH THEY ACCUMULATED RAPIDLY.WE ARE TRYING TO OBTAIN cDNA PROBES FOR INVESTIGATING ACTIN AND ACETYLCHOLINESTERASE GENE EXPRESSION. WE ARE ALSO INTENDING TO ISOLATE GENES WHICH HAVE THE RESPONSIBILITY FOR MUSCLE CELL DIFFERENTLATION IN ASCDIAIN EMBRYOS. WE HAVE ALREADY OBTAINED MONOCLONAL ANTIBODIES WHICH SPECIFICALLY RECOGNIZE COMPONENTS OF MYOPLASM, WHICH IS THOUGHT TO CONTAIN MUSCLE DETERMINANTS. UTLLIZING THE ANTIBODY WE ARE NOW ISOLATING THE GENE ENCODING THE MYOPLASM-SPECIFIC POLYPEPTIDE. MYOD1-LIKE GENES IN ASCIDIAN EMBRYOS ARE ALSO UNDER INVESTIGATIONS.