MOLECULAR MECHANISMS OF CELLULAR DIFFERENTIATION IN ASCIDIAN EMBRYOS

从分子生物学水平探讨了蛔虫胚胎肌细胞分化的机制。首先，我们生产了特异性的单克隆抗体，以抑制肌肉的生长。第二，我们利用分离的聚（A）~+RNA构建了cDNA文库。将重组cDNA克隆到表达载体gt 11中，用抗体筛选文库。选择了一个阳性克隆，含有1.6-KB cDNA。用反义探针进行的原位杂交表明，该基因仅限于尾芽胚的肌细胞。cDNA的序列分析允许氨基酸序列。推导出的序列与啮齿动物肌球蛋白重链蛋白具有广泛的同源性。Southern杂交分析表明，肌球蛋白重链基因序列在单倍体盐蟹基因组中是以单拷贝形式存在的。北方杂交结果表明，肌球蛋白重链基因转录子在消化前期未检测到，消化前期肌球蛋白重链基因转录子迅速积累，我们正试图获得抑制肌动蛋白和乙酰胆碱酯酶基因表达的cDNA探针。我们也打算分离出在ASCDIAIN胚胎中负责肌细胞分化的基因。我们已经获得了单克隆抗体，它可以特异性识别肌浆的成分，而肌浆被认为含有肌肉毒素。利用抗体，我们现在分离编码肌浆特异性多肽的基因。ASCIDIAN胚胎中的MYOD 1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.