MOLECULAR MECHANISMS OF CELLULAR DIFFERENTIATION IN ASCIDIAN EMBRYOS

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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.