ADVANCED MESSENGER-RNA DIFFERENTIAL DISPLAY - ISOLATION OF A NEW DIFFERENTIALLY REGULATED MYOSIN HEAVY CHAIN-ENCODING GENE IN AMPHIBIAN LIMB REGENERATION

In an effort to make mRNA differential display more amenable as a mole cular screen, we have optimized the technology for the isotopic and no n-isotopic detection of differentially regulated mRNAs. The number of amplification rounds in the displays was significantly reduced, result ing in the semi-quantitative detection of expression patterns of both low- and high-abundance transcripts. Moreover, we extended the method beyond the display of mRNAs by introducing a direct sequencing approac h for the fast molecular analysis of isolated cDNAs. Applying this imp roved technique to the regenerating amphibian limb system, we have ide ntified cDNA PCR products with a temporal difference in expression. Th is differential regulation was confirmed by Northern analysis, and DNA sequencing uncovered a novel newt differentiation-specific transcript encoding a skeletal myosin heavy chain (MHC).