DEVELOPMENTALLY REGULATED TROPONIN-C MESSENGER-RNAS OF CHICKEN SKELETAL-MUSCLE

Fast and slow/cardiac troponin C (TnC) are the two different isoforms of TnC. Expression of these isoforms is developmentally regulated in vertebrate skeletal muscle. Therefore, in our studies, the pattern of their expression was analyzed by determining the steady-state levels of both TnC mRNAs. It was also examined if mRNAs for both isoforms of TnC were efficiently translated during chicken skeletal muscle development. We have used different methods to determine the steady-state levels of TnC mRNAs. First, probes specific for the fast and slow TnC mRNAs were developed using a 390 base pair (bp) and a 255 bp long fragment, of the full-length chicken fast and slow TnC cDNA clones, respectively. Our analyses using RNA-blot technique showed that fast TnC mRNA was the predominant isoform in embryonic chicken skeletal muscle. Following hatching, a significant amount of slow TnC mRNA began to accumulate in the skeletal (pectoralis) muscle. At 43 weeks posthatching, the slow TnC mRNA was nearly as abundant as the fast isoform. Furthermore, a majority of both slow and fast TnC mRNAs was found to be translationally active. A second method allowed a more reliable measure of the relative abundance of slow and fast TnC mRNAs in chicken skeletal muscle. We used a common highly conserved 18-nucleotide-long sequence towards the 5'-end of these mRNAs to perform primer extension analysis of both mRNAs in a single reaction. The result of these analyses confirmed the predominance of fast TnC mRNA in the embryonic skeletal muscle, while significant accumulation of slow TnC mRNA was observed in chicken breast (pectoralis) muscle following hatching. In addition to primer extension analysis, polymerase chain reaction was used to amplify the fast and slow TnC mRNAs from cardiac and skeletal muscle. Analysis of the amplified products demonstrated the presence of significant amounts of slow TnC mRNA in the adult skeletal muscle.