Isolation and characterization of six different chicken actin genes.

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.     

Cardiac actin is the major actin gene product in skeletal muscle cell differentiation in vitro.

We examined the expression of alpha-skeletal, alpha-cardiac, and beta- and gamma-cytoskeletal actin genes in a mouse skeletal muscle cell line (C2C12) during differentiation in vitro. Using isotype-specific cDNA probes, we showed that the alpha-skeletal actin mRNA pool reached only 15% of the level reached in adult skeletal muscle and required several days to attain this peak, which was then stably maintained. However, these cells accumulated a pool of alpha-cardiac actin six times higher than the alpha-skeletal actin mRNA peak within 24 h of the initiation of differentiation. After cells had been cultured for an additional 3 days, this pool declined to 10% of its peak level. In contrast, over 95% of the actin mRNA in adult skeletal muscle coded for alpha-actin. This suggests that C2C12 cells express a pattern of sarcomeric actin genes typical of either muscle development or regeneration and distinct from that seen in mature, adult tissue. Concurrently in the course of differentiation the beta- and gamma-cytoskeletal actin mRNA pools decreased to less than 10% of their levels in proliferating cells. The decreases in beta- and gamma-cytoskeletal actin mRNAs are apparently not coordinately regulated.     

Expression of actin mRNAs in denervated chicken skeletal muscle

The expression of actin genes in chicken pectoralis muscle denervated 1 week after hatching was examined 1-8 weeks after the operation by RNA blot hybridization using a generic actin cDNA probe and DNA probes specific for alpha-skeletal and alpha-cardiac actin genes. Total and alpha-skeletal actin mRNAs/microgram total RNA decreased to about half of the levels found in contralateral control muscle, while the expression of alpha-cardiac actin mRNA was up-regulated. Consequently, alpha-cardiac actin mRNA formed about 15% of the total actin mRNA as compared to less than 1% found in control muscle. The expression of actin genes in the denervated muscle was similar to that in the late embryonic muscle. These results suggest that innervation is required to show the expression pattern of striated muscle actin genes found in mature muscle.     

Sequential expression of chicken actin genes during myogenesis

Embryonic muscle development permits the study of contractile protein gene regulation during cellular differentiation. To distinguish the appearance of particular actin mRNAs during chicken myogenesis, we have constructed DNA probes from the transcribed 3' noncoding region of the single-copy alpha-skeletal, alpha-cardiac, and beta-cytoplasmic actin genes. Hybridization experiments showed that at day 10 in ovo (stage 36), embryonic hindlimbs contain low levels of actin mRNA, predominantly consisting of the alpha-cardiac and beta-actin isotypes. However, by day 17 in ovo (stage 43), the amount of alpha-skeletal actin mRNA/microgram total RNA increased more than 30-fold and represented approximately 90% of the assayed actin mRNA. Concomitantly, alpha-cardiac and beta-actin mRNAs decreased by 30% and 70%, respectively, from the levels observed at day 10. In primary myoblast cultures, beta-actin mRNA increased sharply during the proliferative phase before fusion and steadily declined thereafter. alpha-Cardiac actin mRNA increased to levels 15-fold greater than alpha-skeletal actin mRNA in prefusion myoblasts (36 h), and remained at elevated levels. In contrast, the alpha-skeletal actin mRNA remained low until fusion had begun (48 h), increased 25-fold over the prefusion level by the completion of fusion, and then decreased at later times in culture. Thus, the sequential accumulation of sarcomeric alpha-actin mRNAs in culture mimics some of the events observed in embryonic limb development. However, maintenance of high levels of alpha-cardiac actin mRNA as well as the transient accumulation of appreciable alpha-skeletal actin mRNA suggests that myoblast cultures lack one or more essential components for phenotypic maturation.     

Human cytoplasmic actin proteins are encoded by a multigene family

We characterized nine human actin genes that we isolated (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981) from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and alpha-, beta-, and gamma-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria we show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken beta-actin cDNA. We conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.     

Cellular localization of muscle and nonmuscle actin mRNAs in chicken primary myogenic cultures: the induction of alpha-skeletal actin mRNA is regulated independently of alpha-cardiac actin gene expression

Specific DNA fragments complementary to the 3' untranslated regions of the beta-, alpha-cardiac, and alpha-skeletal actin mRNAs were used as in situ hybridization probes to examine differential expression and distribution of these mRNAs in primary myogenic cultures. We demonstrated that prefusion bipolar-shaped cells derived from day 3 dissociated embryonic somites were equivalent to myoblasts derived from embryonic day 11-12 pectoral tissue with respect to the expression of the alpha-cardiac actin gene. Fibroblasts present in primary muscle cultures were not labeled by the alpha-cardiac actin gene probe. Since virtually all of the bipolar cells express alpha-cardiac actin mRNA before fusion, we suggest that the bipolar phenotype may distinguish a committed myogenic cell type. In contrast, alpha-skeletal actin mRNA accumulates only in multinucleated myotubes and appears to be regulated independently from the alpha-cardiac actin gene. Accumulation of alpha- skeletal but not alpha-cardiac actin mRNA can be blocked by growth in Ca2+-deficient medium which arrests myoblast fusion. Thus, the sequential appearance of alpha-cardiac and then alpha-skeletal actin mRNA may result from factors that arise during terminal differentiation. Finally, the beta-actin mRNA was located in both fibroblasts and myoblasts but diminished in content during myoblast fusion and was absent from differentiated myotubes. It appears that in primary myogenic cultures, an asynchronous stage-dependent induction of two different alpha-striated actin mRNA species occurs concomitant with the deinduction of the nonmuscle beta-actin gene.     

Quantitative changes in cytoskeletal beta- and gamma-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos

Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice. Cytoskeletal beta-actin appears to be the major component, followed by gamma-actin, but the synthesis of alpha-actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (beta- and gamma-) and sarcomeric (alpha-cardiac and alpha-skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform-specific RNA probes. The results show that there are about 2 x 10(4) beta-actin mRNA molecules in the fully grown oocyte; this number drops to about one-half in the egg and less than one-tenth in the late two-cell embryo but increases rapidly during cleavage to about 3 x 10(5) molecules in the late blastocyst. The amount of gamma-actin mRNA is similar to that of beta-actin in oocytes and eggs but only about 40% as much in late blastocysts, indicating a differential accumulation of these mRNAs during cleavage. The developmental pattern of beta- and gamma-actin mRNA provides a striking example of the transition from maternal to embryonic control that occurs at the two-cell stage and involves the elimination of most or all of the maternal actin mRNA. There was no detectable alpha-cardiac or alpha-skeletal mRNA (i.e., less than 1,000 molecules per embryo) at any stage from oocyte to late blastocyst, suggesting that the sarcomeric actin genes are silent during preimplantation development.     

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

BALB/c mice possess a 5' duplication of the alpha-cardiac actin gene which is associated with abnormal levels of alpha-cardiac and alpha-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 alpha-cardiac actin mRNA declines, whereas that of alpha-skeletal actin increases. In mutant mice, these trends are exaggerated so that whereas normal mice have 95.8% alpha-cardiac mRNA and 4.2% alpha-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 alpha-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.     

Isolation and characterization of human actin genes cloned in phage lambda vectors

Using Drosophila and chicken actin probes, we have selected 14 human actin lambda recombinants from a genomic library. We present a restriction maps indicating the positions of the sequences homologous to actin and to an Alu probe. Restriction mapping has revealed that nine out of ten of these clones are distinct, indicating that actin is a multigene family. Hybrid elution of HeLa cell mRNA from filters containing the recombinant DNA, followed by in vitro translation and immunoprecipitation, as well as one- or two-dimensional protein analysis, shows that these recombinants code for actin. Hybridization back to human DNA digested with restriction enzymes shows that the EcoRI fragments of at least one of the lambda recombinants (lambda HA-5) result in similar-sized human DNA fragments in the intact genome. In nuclei, a 4.5-kb mRNA precursor to the cytoplasmic 1.9-kb mRNA can be detected by hybridization with genomic or cDNA probes, indicating the presence of additional sequences and RNA processing.     

A complex gene superfamily encodes actin in petunia.

We have shown by several independent criteria that actin is encoded by a very large and complex superfamily of genes in Petunia. Several cDNA and genomic probes encoding actins from diverse organisms (Dictyostelium, Drosophila, chicken and soybean) hybridize to hundreds of restriction fragments in the petunia genome. Actin-hybridizing sequences were isolated from a petunia genomic library at a rate of at least 200 per genome equivalent. Twenty randomly selected actin-hybridizing clones were characterized in more detail. DNA sequence data from four representative and highly divergent clones, PAc2, PAc3, PAc4 and PAc7, demonstrate that these actin-like sequences are related to functional actin genes. Intron positions typical of other known plant actin genes are conserved in these clones. Four of six clones analyzed (PAc1, PAc2, PAc3, PAc4) hybridize to leaf mRNA of the same size (1.7 kb) as that reported for other plant actin mRNAs and to a slightly smaller mRNA species (1.5 kb). Five distinct subfamilies of actin-related genes were characterized which varied in size from a few members to several dozen members. It is clear from our data that other actin gene subfamilies must also exist within the genome. Possible mechanisms of actin gene amplification and genome turnover are discussed.     

Divergence and differential expression of soybean actin genes

DNA sequence analysis as well as genomic blotting experiments using cloned soybean actin DNA sequences as probes show that large sequence heterogeneity exists among members of the soybean actin multigene family. This heterogeneity suggested that the members of this family might be diverged in function and/or regulation. Five of the six soybean actin gene family members examined are shown to be significantly more diverged from one another than members of other known actin gene families. This high level of divergence was utilized in the preparation of actin gene-specific probes in the analysis of the complexity and expression of these members of the soybean actin gene family. Hybridization studies indicate that the six soybean actin genes fall into three classes with a pair of genes in each class. These six genes account for all but two actin gene fragments detected in the soybean genome. We have compared the relative steady state mRNA levels of these classes of soybean actin genes in three organs of soybean. We find that actin genes SAc6 and SAc7 are most highly expressed accounting for 80% of all actin mRNA with respect to the six soybean actin genes examined. Actin genes SAc3 and SAc1 are expressed at intermediate and low levels respectively; and SAc2 and SAc4 are expressed at barely detectable levels. Four of the six soybean actin genes appear to be expressed at the same level in root, shoot and hypocotyl. SAc3 and SAc7 genes appear to be more highly expressed in shoot and 2,4-dichlorophenoxyacetic acid-induced hypocotyl than in root and hypocotyl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic organization and evolution of actin genes in the amphioxus Branchiostoma belcheri and Branchiostoma floridae

We previously described the cDNA cloning and expression patterns of actin genes from amphioxus Branchiostoma floridae (Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N.D., Holland, L.Z., 1997. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203–215). In the present paper, we report the characterization of cDNA clones for actin genes from a closely related species, Branchiostoma belcheri, and the exon–intron organization of B. floridae actin genes. Each of these two amphioxus species has two types of actin genes, muscle and cytoplasmic. The coding and non-coding regions of each type are well-conserved between the two species. A comparison of nucleotide sequences of muscle actin genes between the two species suggests that a gene conversion may have occurred between two B. floridae muscle actin genes BfMA1 and BfMA2. From the conserved positions of introns between actin genes of amphioxus and those of other deuterostomes, the evolution of deuterostome actin genes can be inferred. Thus, the presence of an intron at codon 328/329 in vertebrate muscle and cytoplasmic actin genes but not in any known actin gene in other deuterostomes suggests that a gene conversion may have occurred between muscle and cytoplasmic actin genes during the early evolution of the vertebrates after separation from other deuterostomes. A Southern blot analysis of genomic DNA revealed that the amphioxus genome contains multiple muscle and cytoplasmic actin genes. Some of these actin genes seem to have arisen from recent duplication and gene conversion. Our findings suggest that the multiple genes encoding muscle and cytoplasmic actin isoforms arose independently in each of the three chordate lineages and that gene duplications and gene conversions established the extant actin multigene family during the evolution of chordates.     

Effect of estrogen on the expression of mRNAs of different actin isoforms in immature rat uterus. Cloning of alpha-smooth muscle actin message

Cytoplasmic beta- and gamma-actin mRNAs as well as smooth muscle actin mRNAs have been shown to be transiently increased in rat uterus after treatment with the steroid hormone estradiol. A clone isolated as an estradiol-induced message from a lambda-gt10 cDNA library prepared from the mRNA of estrogen-stimulated immature rat uterus was identified as alpha-smooth muscle actin. A single-stranded RNA probe composed mainly of the 3'-untranslated region of this clone, as well as DNA probes derived from the 3'-untranslated regions of other actin genes, were used to study the induction kinetics of different actin isoforms in rat uterus after being stimulated by estradiol. The beta- and gamma-cytoskeletal actins showed an induction peak at 4 h after estradiol administration with 1.4- and 1.8-fold increases, respectively. The smooth muscle actin was maximally increased 2.1-fold at 8-12 h. Messages of alpha-skeletal and alpha-cardiac actins were neither expressed nor induced by estradiol in this tissue. The different induction kinetics of the cytoplasmic and smooth muscle actins suggest that they are regulated by different mechanisms and possibly in different cell types of the uterus.