This study examined the diversity of Na' channel gene expression in intact cardiac tissue and purified myocardial cells. The screening of neonatal rat myocardial cell cDNA libraries with a conserved rat brain Na' channel cDNA probe, resulted in the isolation and characterization of a putative rat cardiac Na' channel cDNA probe (pCSC-1). The deduced amino acid sequence of pCSC-1 displayed a striking degree of homology with the eel, rat brain-i, and rat brain-2 Na' channel, thereby identifying pCSC-1 as a related member of the family of Na' channel genes. Northern blot analysis revealed the expression of a 7-kb CSC-1 transcript in rat cardiac tissue and purified myocardial cells, but little or no detectable expression of CSC-1 in rat brain, skeletal muscle, denervated skeletal muscle, or liver. Using RNase protection and Northern blot hybridization with specific rat brain Na' channel gene probes, expression of the rat brain-i Na' channel was observed in rat myocardium, but no detectable expression of the rat brain-2 gene was found. This study provides evidence for the expression of diverse Na' channel mRNAs in rat myocardium and presents the initial characterization of a new, related member of the family of Na' channel genes, which appears to be expressed in a cardiac-specific manner.
Previous studies on two Escherichia coli rpoB mutants, carrying single amino acid substitutions at approximate amino acid positions 736 and 906 in the beta subunit, showed that these alterations in the RNA polymerase resulted in an apparent reduced response to valine-induced amino acid starvation in vivo and prevented ppGpp-mediated inhibition of transcriptional initiation at stable RNA promoters in vitro. These observations suggested that the mutations had altered either the ppGpp binding site or the promoter selectivity of the enzyme. The in vivo analysis presented here indicates that these mutants encode an RNA polymerase that responds normally to changes in the level of ppGpp; their apparent relaxedness is due to a reduced accumulation of ppGpp during isoleucine starvation. Thus, there is no indication that the mutations have altered ppGpp binding sites. These observations and the difference between in vitro and in vivo results can be explained by the assumption that the mutations produce an extended ppGpp-dependent pausing of RNA polymerase during the transcription of unstable RNA. Comparison of the vivo and in vitro effects of ppGpp on rrn transcription further suggests that these reflect different phenomena, although in both cases ppGpp inhibits rrn transcription.
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