Serge Alonso

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.

Genetic analysis of the interaction between cardiac and skeletal actin gene expression in striated muscle of the mouse

The two sarcomeric actin genes, encoding alpha-cardiac and alpha-skeletal actins, are co-expressed in striated muscle, but in the adult the respective isoform predominates in cardiac or skeletal muscle of the normal mouse. We have investigated the interaction between this gene pair in different genetic contexts. Northern blot analysis of alpha-actin mRNA levels in different inbred mice (129/SJ, C3H, C57BL/6) demonstrates variation of as much as threefold in skeletal muscle and eightfold in cardiac muscle. High or low-level expression is seen for both skeletal and cardiac muscle in a given line, suggesting common regulatory phenomena affecting the abundant alpha-skeletal or alpha-cardiac transcript. In the BALB/c mouse, which has a mutant cardiac actin locus, skeletal as well as cardiac actin mRNA and protein accumulate in the adult heart. We have analysed the role of the two alpha-actin genes in this phenomenon in seven recombinant inbred mouse lines (BALB/c x C57BL/6) and in a cross (BALB/c x C3H). The results demonstrate that neither alpha-actin gene alone is sufficient, and implicate other regulatory loci. DNA sequencing of the C3H and BALB/c alpha-skeletal actin gene promoters shows that they are virtually identical over 830 nucleotides. The relative levels of alpha-skeletal and alpha-cardiac actin proteins have been measured by N-terminal peptide analysis in the different mouse lines. The results point to regulatory loci affecting mRNA utilization and protein stability.

A developmental study of the abnormal expression of α-cardiac and α-skeletal actins in the striated muscle of a mutant mouse☆

Abstract BALB c mice possess a 5′ duplication of the α-cardiac actin gene which is associated with abnormal levels of α-cardiac and α-skeletal actin mRNAs in adult cardiac tissue. This mutation therefore provides a potential tool for the study of the inter-relationship between the striated muscle actins. We have examined the expression of this actin gene pair throughout the development of skeletal and cardiac muscle in BALB c mice. During embryonic and fetal development, the expression of these two genes is indistinguishable from that in normal mice, as determined by in situ hybridization. A quantitative postnatal study demonstrates that in the hearts of normal mice the level of α-cardiac actin mRNA declines, whereas that of α-skeletal actin increases. In mutant mice, these trends are exaggerated so that whereas normal mice have 95.8% α-cardiac mRNA and 4.2% α-skeletal mRNA in the adult heart, BALB c mice have 52.4 and 47.6% of these mRNAs, respectively. This difference is also reflected at the protein level. In developing skeletal muscle, the expression of these genes follows kinetics similar to that observed in the heart with a decrease in the relative level of α-cardiac mRNA as the muscle matures. Cardiac actin mRNA levels are again lower in the mutant mouse, but here the effect is less striking because skeletal actin is the predominant isoform. These results are discussed in the context of the interaction between this actin gene pair in developing and adult striated muscle.

Coexpression and evolution of the two sarcomeric actin genes in vertebrates

Actin, as a monomer, is a globular protein of 43 000 daltons called G actin. In its polymerized active form, F actin (fibrous) is composed of two chains coiled in a double helix. Actin is a major structural component of contractile systems: of the sarcomere in muscle cells and of the cytoskeleton in non-muscle cells. In the sarcomere, it is the major component of thin filaments which slide in relation to the thick filaments to produce muscle contraction [1] and in the cytoskeleton of microfilaments which are involved in a large number of cellular functions, such as morphology, movements, division, etc. [2]. Actin is found in all

Re-localization of Actsk-1 to mouse chromosome 8, a new region of homology with human chromosome 1.

We present here the genetic mapping of the α-skeletal actin locus (Actsk-1) on mouse Chromosome (Chr) 8, on the basis of the PCR analysis of a microsatellite in an interspecific backcross. Linkage and genetic distances were established for four loci by analysis of 192 (or 222) meiotic events and indicated the following gene order: (centromere)-Es-1-11.7 cM-Tat-8.3 cM-Actsk-1-0.5 cM-Aprt. Mapping of ACTSK to human Chr 1 and of TAT and APRT to human Chr 16 demonstrates the existence of a new short region of homology between mouse Chr 8 and human Chr 1. Intermingling on this scale between human and mouse chromosomal homologies that occurred during evolution creates disorders in comparative linkage studies.

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.