A. Minty

Comparison of three actin-coding sequences in the mouse; evolutionary relationships between the actin genes of warm-blooded vertebrates.

SummaryWe have determined the sequences of three recombinant cDNAs complementary to different mouse actin mRNAs that contain more than 90% of the coding sequences and complete or partial 3′ untranslated regions (3′UTRs): pAM 91, complementary to the actin mRNA expressed in adult skeletal muscle (αsk actin); pAF 81, complementary to an actin mRNA that is accumulated in fetal skeletal muscle and is the major transcript in adult cardiac muscle (αc actin); and pAL 41, identified as complementary to a β nonmuscle actin mRNA on the basis of its 3′UTR sequence. As in other species, the protein sequences of these isoforms are highly (>93%) conserved, but the three mRNAs show significant divergence (13.8–16.5%) at silent nucleotide positions in their coding regions. A nucleotide region located toward the 5′ end shows significantly less divergence (5.6–8.7%) among the three mouse actin mRNAs; a second region, near the 3′ end, also shows less divergence (6.9%), in this case between the mouse β and αsk actin mRNAs. We propose that recombinational events between actin sequences may have homogenized these regions. Such events distort the calculated evolutionary distances between sequences within a species. Codon usage in the three actin mRNAs is clearly different, and indicates that there is no strict relation between the tissue type, and hence the tRNA precursor pool, and codon usage in these and other muscle mRNAs examined. Analysis of codon usage in these coding sequences in different vertebrate species indicates two tendencies: increases in bias toward the use of G and C in the third codon position in paralogous comparisons (in the order αc), and in orthologous comparisons (in the order chicken < rodent < man). Comparison of actin-coding sequences between species was carried out using the Perler method of analysis. As one moves backward in time, changes at silent sites first accumulate rapidly, then begin to saturate after −(30–40) million years (MY), and actually decrease between −400 and −500 MY. Replacements or silent substitutions therefore cannot be used as evolutionary clocks for these sequences over long periods. Other phenomena, such as gene conversion or isochore compartmentalization, probably distort the estimated divergence time.

A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA

We compare a recombinant cDNA plasmid (pAF81) complementary to a fetal skeletal muscle actin mRNA with a plasmid (pAM91) complementary to the actin mRNA expressed in adult skeletal muscle. The two mRNAs are significantly diverged in silent nucleotide positions; they are coexpressed in fetal skeletal muscle, and in differentiating muscle cell cultures their accumulation begins coordinately. The sequence of pAF81 shows that the amino acid sequence of mouse fetal skeletal muscle actin is almost identical to that of adult bovine cardiac actin. Hybridization of pAF81 to RNA from different mouse tissues shows that fetal skeletal muscle actin mRNA is very homologous or identical to fetal and adult cardiac actin mRNA. Only one gene homologous to pAF81 is detected on blots of restricted mouse DNA. We conclude that this gene must be expressed both in fetal skeletal muscle and in fetal heart. Whereas mRNA transcribed from this gene is the major actin mRNA species in adult heart, it is present in low amounts, if at all, in adult skeletal muscle.

Coding potential of non-polyadenylated messenger RNA in mouse Friend cells

When total cytoplasmic RNA from mouse Friend cells is fractionated using oligo(dT)-cellulose or poly(U)-Sepharose chromatography, approximately 20% of the messenger RNA activity (as measured in the reticulocyte lysate cell-free system) remains in the unbound fraction, even though this contains < 0.5% of the poly(A) (as measured by titration with poly(U)). This RNA, operationally defined as poly(A)−, is found almost entirely in polysome structures in vivo. Its major translation products, as shown by one-dimensional sodium dodecyl sulphate-containing gels, are the histones and actin. Two-dimensional gels (isoelectric focusing: sodium dodecyl sulphate/gel electrophoresis) show that, with the exception of the mRNAs coding for histones, poly(A)− mRNA encodes similar proteins to poly(A)+ mRNA, though in very different abundances. This is directly confirmed by the arrest of the translation of the abundant poly(A)− mRNAs after hybridization with a complementary DNA transcribed from poly(A)+ RNA. RNA sequences which are rare in the poly(A)+ RNA are also found in poly(A)− RNA, as shown by hybridizing a cDNA transcribed from poly(A)+ RNA to total and poly(A)− polysomal RNA. That this does not simply represent a flow-through of poly(A)+ RNA is indicated by (i) the lack of poly(A) by hybridizing to poly(U) in this fraction, (ii) the fact that further passage through poly(U)-Sepharose does not remove the hybridizing sequences, (iii) the very different quantitative distribution of proteins encoded by poly(A)+ and poly(A)− RNAs. We also think that it does not result from removal of poly(A) from polyadenylated RNAs during extraction because RNAs prepared using the minimum of manipulations give similar results. The distribution of both total mRNA and α and β globin mRNAs between poly(A)+ and poly(A)− RNA does not change significantly during the dimethyl sulphoxide-induced differentiation of Friend cells.

The Actin and Myosin Multigene Families

The Actin and Myosin Multigene Families: a) a study of the accumulation of their RNA transcripts demonstrates different developmental strategies during skeletal muscle formation, b) a genetic analysis of their chromosomal organization indicates gene dispersion and permits some precise localizations on the genetic map of the mouse.

Actin and Myosin Genes and Their Expression During Skeletal Muscle Myogenesis

The differentiation of skeletal muscle cells is characterized morphologically by the fusion of myoblasts to form multinucleated muscle fibres. This process takes place gradually during skeletal muscle development in vivo. It can also be followed in tissue culture. Mammalian myoblasts will grow in monolayers, either in primary culture or as established cell lines, and will fuse spontaneously when the culture becomes confluent (for review see Yaffe 1968, Buckingham 1977). The formation of muscle fibres is characterized biochemically by the increased synthesis of contractile proteins (e.g. Devlin and Emerson 1978, Garreis 1979) and their organization into sarcomeric structures (Fischman 1970), by the accumulation of enzymes important in muscle metabolism (e.g. Caravatti et al. 1979), and by the appearance of membrane components such as the acetylcholine receptor (e.g. Merlie et al. 1975), essential for nerve-muscle interaction.