Molecular cloning and expression of the chicken smooth muscle γ‐actin mRNA

Abstract

We have investigated the expression of chicken smooth muscle γ-actin mRNA by isolation and characterization of cDNAs representing this actin isoform and utilizing the cDNA to probe RNA from adult and developing cells. Nucleotide sequence elucidated from an apparent full length smooth muscle γ-actin cDNA revealed that it contained 94 bp of 5′ non-translated sequence, an open reading frame of 1131 bp, and 97 bp of 3′ non-translated sequence. Within the 376 amino acid sequence deduced from the chicken cDNA were diagnostic amino acids at the NH₂- and COOH-terminal regions which provided unequivocal identification of the γ-enteric smooth muscle actin isoform. In addition, the chicken γ-enteric actin deduced from our cDNA clones was found to differ from the sequence reported in earlier protein studies [J. Vandekerckhove and K. Weber, FEBS Lett. 102:219, 1979] by containing a proline rather than a glutamine at position 359 of the protein, indicating that the avian γ-enteric actin isoform is identical to its mammalian counterpart. Comparison of the 5′ and 3′ non-translated sequence determined from the chicken cDNA to that elucidated for rat, mouse, and human showed that there is not a high degree of cross-species sequence conservation outside of the coding regions among these mRNAs. Northern hybridization analyses demonstrated that the γ-enteric actin mRNA is expressed in adult aorta and oviduct tissues but not in adult skeletal muscle, cardiac muscle, liver, brain, and spleen tissues. The γ-enteric actin mRNA was first observed in measurable quantities in gizzard tissue from 4–5 day embryos and increased in content in developing smooth muscle cells through 16–17 embryonic days. Following this initial increase during embryonic development, the γ-enteric actin mRNA exhibits a decline in content until ∼7 days posthatching, after which there is an increase in content to maximal levels found in adult gizzard tissue. In general, the developmental appearance of the γ-enteric mRNA parallels that observed for this protein in previous studies indicating that the developmental expression of smooth muscle γ-actin is regulated, in part, by an increased content of mRNA in chicken visceral smooth muscle cells during myogenesis.