Expression of ventricular-type myosin light chain messenger RNA in spontaneously hypertensive rat atria.

Using cloned DNA probes specific for two isoforms of cardiac myosin light chains (MLCs), nonphosphorylatable MLC1 and phosphorylatable, regulatory MLC2, we have observed that the MLC1 messenger RNA of ventricular type does not appear in detectable amounts in atrial cells of either normotensive Wistar-Kyoto rat strain (WKY) or spontaneously hypertensive rat strain (SHR). The messenger RNA of regulatory isoform of ventricular MLC2, on the other hand, is found in threefold excess in atria of SHR relative to that of age-matched WKY. The increased level of MLC2 messenger RNA is present even in 6-week-old SHR atria where there is no established overloading of the heart. Thus, it appears that the increased expression of the regulatory MLC2 gene in SHR atrial cells is a predetermined event, which, most likely, participates in functional adaptation of the myocardium in response to pressure overload and subsequent hypertrophy.

Co-ordinate control of gene expression. Muscle-specific 7 S RNA contains sequences homologous to 3'-untranslated regions of myosin genes and repetitive DNA.

We have cloned and sequenced a complementary DNA copy (pSS48) of a novel muscle-specific, low molecular weight RNA, 7 S RNA, isolated from embryonic chick cardiac muscle cells. The hybridization pattern of plasmid pSS48 DNA to chick genomic DNA suggests that 7 S RNA is derived from the repetitive chick DNA with a repetition frequency of about 300 copies per haploid genome. Under low stringency, pSS48 DNA also hybridizes with high specificity to the single copy gene for chick myosin light chain (MLC) and to myosin heavy chain (MHC), and possibly to other co-ordinately expressed genes for chick muscle proteins. The sequence analysis of recombinant plasmids pSS48, pML10 and pMHC8, for 7 S RNA, MLC mRNA and MHC RNA, respectively, indicated that short nucleotide stretches homologous to 7 S RNA reside in the 3' untranslated regions of the respective genes. The 7 S RNA sequence appears to be highly specific for the chick muscle tissue, since RNA and DNA from several sources did not hybridize to pSS48 DNA. Furthermore, the 7 S RNA-like sequence(s) appears in chick blastodermal cells preferentially earlier than the onset of transcription of genes for major muscle proteins. These results, taken together, suggest a possible function for 7 S RNA in expression of muscle-specific genes during chick development.

Tissue specificity of 3'-untranslated sequence of myosin light chain gene: unexpected interspecies homology with repetitive DNA.

Using the 3' noncoding and coding sequences of chick heart myosin light chain mRNA cloned into Escherichia coli as probes, it was observed that, while the coding sequence shared homology with myosin light-chain mRNAs from other sources, the 3' noncoding sequence was specific for chick heart muscle. This property was used to detect chick heart-specific myosin light-chain gene activity in chick blastoderms of very early developmental stages where cells of different muscle origins cannot be distinguished morphologically. However, in spite of the tissue-specific divergence of the 3' noncoding sequence of myosin light-chain gene, which is present in a single copy in the chick genome, a surprising homology with DNA from such a diverse source like Dictyostelium discoideum was noted. The sequence homologous to chick myosin light-chain DNA was apparently present in a high repetition frequency in the Dictyostelium genome.

Quantitation of muscle-specific mRNAs by using cDNA probes during chicken embryonic muscle development in ovo.

The emergence of abundant-class mRNAs specific for contractile muscle proteins and their distribution between polysomal and free mRNP fractions were studied in skeletal muscle excised from chicken embryos during the transition from myoblasts (day 9) to myotubes (day 18). Muscle-specific cDNA was selectively prepared by hybridizing cDNA to template RNA (polysomal poly(A)+ mRNA) from day-14 embryos followed by isolation of the abundant class, which represents approximately 20% of total mRNA. The specificity of the cDNA probe for this class was confirmed by the differential degree of hybridization to cytoplasmic RNA from cultured myotube and myoblast cells and by its inability to hybridize with mRNA from nonmuscle cells such as liver. Except for muscle from day-9 embryos, the concentrations of the abundant-class muscle-specific mRNAs were higher in polysomes than in free mRNP fractions. Furthermore, the levels of these mRNAs in polysomes increased 12-fold from day 9 (myoblast) to day 14 (intermediate) with a further 3.6-fold increase from day 14 to day 18 (myotube). In contrast to this 45-fold net increase in the polysomal level of these mRNAs from day 9 to day 18, the levels in the free mRNP fraction showed only a 3-fold decrease during this period. Because the amount of mRNA lost from the mRNP fraction is much less than the net increase in the polysome fraction, mRNP does not serve as a reservoir of untranslated muscle-specific mRNA for transfer to polysomes. Consequently, the emergence of muscle-specific polysomal mRNA for contractile proteins during myogenesis in ovo appears to be regulated primarily by transcriptional control.