Abstract
We have isolated two cDNA clones for myosin alkali light chain (MLC) mRNA from two respective cDNA libraries of chick gizzard and fibroblast cells by cross-hybridization to the previously isolated cDNA of skeletal muscle MLC. Sequence analysis of the two cloned cDNAs revealed that both of them are homologous to but distinct from the cDNA sequence used as the probe so that they may be classified into members of the MLC family, that they are identical with each other in the 3' and 5' untranslated sequence as well as in the coding sequence with a notable exception of a 39-nucleotide insertion in the fibroblast cDNA, 26 nucleotides of which are used for encoding the C-terminal amino acid sequence, and, therefore, that they encode the identical 142-amino acid sequence with different C-terminals of nine amino acids, each specific for fibroblast and gizzard smooth muscle MLC. The position of the inserted block corresponds exactly to one of the exon-intron junctions in the other MLC genes whose structures have so far been elucidated. DNA blot analysis suggested that the two MLC mRNAs of gizzard (smooth muscle) and fibroblast cells (nonmuscle) are generated from a single gene, probably through alternative RNA splicing mechanisms. RNA blot analysis and S1 nuclease mapping analysis using RNA preparations from fibroblast and gizzard tissues showed that the fibroblast MLC mRNA is expressed predominantly in fibroblast cells, but not, or very scantily if at all, in the gizzard, whereas the reverse is true for the gizzard smooth muscle MLC mRNA.
Highlights
As an extension of the previous studies to elucidate the one of the exon-intron junctions in the MotLhCergenes structural relation among these isoforms of MLC and the whose structures haveso far been elucidated.DNA blot regulatory mechanisms underlying thetissue-specific and the analysis suggestedthat the twMoLC mRNAs of gizzard developmentallystage-specificexpression of the MLCiso
Fibroblast cDNA librariesandreport here thatthe two RNA blot analysis andS 1nuclease mappinganalysis using RNA preparations from fibroblast and gizzard tissues showed that the fibroblast MLC mRNA is expressed predominantly in fibroblast cells, but not, or veryscantily if at all, inthegizzard,whereasthe reverse is true for the gizzard smooth muscle MLC
On the other hand, type MLC mRNA is expressed only in the32-meroligonucleotideusedas a probegave a single smooth muscle tissue but. not in nonmuscle tissue, we performed SI nuclease mapping ut,ilizing the (Fig. :$A).These results show that the mRNA of MLC wit,h '"P-labeled 255-bp Ban1 fragment of pF-1 as a prohe(see the 39-hp insertis expressed specificallv in the fibroblast cells "Materials and Methods")
Summary
(smooth muscle) and fibroblast cells (nonmuscle) are forms, we have isolated and sequenced two cloned cDNAs for generated from a single gene, probably through alter- myosin alkali light chain separately from chick gizzard and native RNA splicing mechanisms. Fibroblast cDNA librariesandreport here thatthe two RNA blot analysis andS 1nuclease mappinganalysis using RNA preparations from fibroblast and gizzard tissues showed that the fibroblast MLC mRNA is expressed predominantly in fibroblast cells, but not, or veryscantily if at all, inthegizzard,whereasthe reverse is true for the gizzard smooth muscle MLC mRNA. On the basis of the sequence data, together with the result of DNA and RNA blot analyswise,suggest that thetwo mRNAs are generated fromasingle gene through alternative RNA splicing mechanisms specific to gizzard smooth muscle tissue.
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