Molecular cloning of developmentally regulated, low-abundance mRNA sequences from embryonic muscle

To study the role of low-abundance, embryonic muscle-specific gene transcripts, we have developed a method to screen cDNA clones from embryonic muscle for such sequences. The protocol involves two stages: first, partial enrichment for cDNA clones carrying possible embryo-specific sequences by selecting clones of low-abundance sequences; and second, determination, by hybridization to RNA attached to diazobenzyloxymethyl-paper, which sequences from this category are regulated in an embryonic muscle-specific manner during development. At least three different clones were obtained which hybridized to sequences present in early muscle development but absent, or present at relatively low levels, at late embryonic and adult muscle stages. Two of these clones were not muscle-specific because they hybridized to poly(A)+RNA from liver or brain or both. The third clone, 106A4, did not detectably hybridize to total poly(A)+RNA at any stage of brain or liver development tested. This sequence also was not detectable in poly(A)+RNA from embryonic muscle progenitor cells. Thus, the 106A4 sequence is a likely candidate for an embryonic muscle-specific sequence. We have demonstrated that the 106A4 sequence is a mRNA, although the specific identity and function of the translated product is unknown. The method used to identify embryonic muscle-specific cDNA clones should be generally applicable for obtaining clones for low abundance transcripts regulated in a tissue-specific or developmental stage-specific manner.