The mRNA products of four genes, carbonic anhydrase III (CAIII), fast myosin heavy chain (MHCf), actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were assayed by Northern and slot-blot analysis in rabbit tibialis anterior and extensor digitorum longus muscles that were subjected to electrical stimulation for periods of up to 21 days. Marked changes in mRNA levels were seen for all four genes. The mRNA for CAIII, which is specific for type 1 fibers, rose significantly, whereas the MHCf mRNA fell markedly in the stimulated muscles. Changes in GAPDH mRNA were consistent with a reduced dependence on anaerobic glycolysis as an energy source. Actin mRNA levels were noticeably depressed in the early stages of stimulation. Thus for several classes of muscle protein, the response to chronic low-frequency stimulation appears to involve changes at the level of gene transcription.
The molecular cloning of cDNA for the human muscle specific carbonic anhydrase CAIII is described. The recombinant was isolated from a human muscle cDNA library prepared in the expression vector lambda gt11, and was characterized by hybridization selection and immunoprecipitation. A comparison of insert cDNA and mRNA sizes suggests that the cDNA is full length and includes extensive untranslated sequences. Preliminary sequence data have confirmed the authenticity of this clone and Southern blotting of human and rodent DNA indicates that it will be a useful probe in the analysis of somatic cell hybrids.
Carbonic anhydrase III (CAIII) protein and mRNA amounts in fast- and slow-twitch rat muscles were examined after resection of the sciatic nerve. Striking changes occur in the fast-twitch anterior tibialis (AT) and extensor digitorum longus (EDL) muscles, where CAIII protein and mRNA are increased several-fold 16 days after denervation. The data suggest that these changes are regulated in part by changes in gene transcription and that they perhaps signal a fast-to-slow fibre type transition in these denervated muscles. AT and EDL show some differences in the effects of denervation, which are suggestive of variation in the timing of denervation-induced responses and/or the CAIII protein/mRNA turnover rates in the two muscles.
We showed that myoglobin gene transcription and the appearance of myoglobin occur very early in myogenesis, in both humans and mice. In contrast to the contractile protein genes, there is a subsequent increase of 50- to 100-fold in myoglobin mRNA and protein levels during later muscle development. Myoglobin and myoglobin mRNA are present at elevated levels in fetal heart and are also detectable at low levels in adult smooth muscle. The absolute level of myoglobin mRNA in highly myoglobinized seal muscle is very high [2.8% of the total population of poly(A)+ RNAs]. Levels of myoglobin in seal skeletal muscle and in various human muscle types appear to be determined by the size of the myoglobin mRNA pool. In contrast, low levels of myoglobin in mouse skeletal muscle are not apparently correlated with low levels of myoglobin mRNA. As expected from the early appearance of myoglobin mRNA in embryonic skeletal muscle, both rat and mouse embryonic myoblasts accumulate myoglobin mRNA on fusion and differentiation in vitro.
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