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.     

Tissue restricted and stage specific transcription is maintained within 411 nucleotides flanking the 5'' end of the chicken alpha-skeletal actin gene.

alpha-skeletal actin message levels have been shown to be tightly regulated in chicken primary myoblast cultures. To test for gene elements required for muscle cell specific expression, DNA sequences containing the 5'-flanking regions of the chicken alpha-skeletal actin, beta-cytoplasmic actin, and the histone H2b genes were linked to the coding sequences of the chloramphenicol acetyltransferase gene and transfected into myogenic and non-myogenic cells. In contrast to beta-actin CAT hybrids, the alpha-skeletal actin CAT constructions displayed restricted CAT expression in transfected non-myogenic cells. We showed that a 411 nucleotide fragment flanking the 5' end of of the alpha-skeletal actin gene was responsible for a 9-15 fold increase in CAT enzymatic activity during myoblast fusion, versus only a transient 2 fold rise for the beta-actin and histone flanking sequences. These results indicate that DNA sequences within 411 bp of the 5' terminus of the alpha-skeletal actin gene influenced its cell type and stage specific expression.     

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.     

Angiotensin II stimulates α-skeletal actin expression in cadiomyocytes in vitro and in vivo in the absence of hypertension

Using a specific alpha-skeletal actin antibody, we have previously shown, that during hypertension-associated cardiac hypertrophy in the rat, the expression of alpha-skeletal actin in the myocardium is increased, but maintains focal distribution, compared to normotensive animals. In the present study, we have investigated whether alpha-skeletal actin expression can be induced in the absence of hypertension. For this purpose, we have examined transgenic mice overexpressing angiotensinogen exclusively in the heart. These animals are characterized by high cardiac angiotensin II levels and cardiac hypertrophy accompanied or not by high blood pressure depending on their genetic background, i.e. presence of one or two renin genes. Alpha-skeletal actin levels were highly increased in transgenic compared to wild-type myocardium independently of the number of renin genes, indicating that angiotensin II can stimulate alpha-skeletal actin expression in normotensive animals. Additional in vitro experiments using cultured mouse and rat cardiomyocytes showed that angiotension II not only increases alpha-skeletal actin expression but also induces an increase of its incorporation within II-bands compared to control cardiomyocytes. Angiotensin II increases also the expression of alpha-smooth muscle actin in sarcomeres of cardiomyocytes as well as in fibroblastic cells present within the culture.     

Differentiation of cardiac myocytes after mitogen withdrawal exhibits three sequential states of the ventricular growth response

During cardiac myogenesis, ventricular muscle cells lose the capacity to proliferate soon after birth. It is unknown whether this developmental block to mitotic division and DNA replication might involve irreversible repression of the cellular oncogene c-myc. Ventricular myocytes from 2 d-old rats continued to differentiate in vitro during 15 d of mitogen withdrawal, as shown by the formation of cross-striations, increased proportion of the muscle isoenzyme of creatine kinase, stable expression of alpha-cardiac actin and myosin heavy chain mRNAs, and appropriate down-regulation of alpha-skeletal actin mRNA. After mitogen withdrawal for 2 d, serum evoked both DNA synthesis and mitotic division; after 7 d, DNA replication was uncoupled from cell division; after 15 d, DNA synthesis itself was markedly attentuated. These three distinct phenotypic states resemble the sequential properties of growth found in the neonatal rat heart in vivo. Despite failure to induce DNA replication or division after 15 d of mitogen withdrawal, serum elicited both c-myc and alpha-skeletal actin as found during hypertrophy of the intact heart. The results agree with previous evidence that one or more functional pathways that transduce the effects of serum factors may persist in older cardiac muscle cells, and indicate that irreversible down-regulation of c-myc cannot be the basis for the loss of growth responses.     

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.     

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.     

A new repetitive element of the CR1 family downstream of the chicken vitellogenin gene.

We have analyzed a repetitive DNA sequence found in the 3'-flanking region of the chicken vitellogenin gene. By its sequence, the repetitive DNA has been identified as a hitherto unreported member of the chicken CR1 family of repetitive elements. The CR1 sequence displays the structural characteristics of a long terminal repeat located at the 3' end of an avian retrovirus. The CR1 element lies 2.2 kb downstream of the vitellogenin gene and 'points' away from the gene rather than toward it. In this respect, this element differs from other CR1 repeats. The CR1 element is embedded in a region showing changes in chromatin structure implying a potential role for this sequence in determining the structural state of the local chromatin.     

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.     

A CR1 element is embedded in a novel tandem repeat (HinfI repeat) within the chicken genome.

Highly repetitive DNA sequences constitute a significant portion of most eukaryotic genomes, raising questions about their evolutionary origins and amplification dynamics. In this study, a novel chicken repetitive DNA family, the HinfI repeat, was characterized. The basic repeating unit of this family displays a uniform length of 770 bp, which was defined by the recognition site of HinfI. The HinfI repeat was specifically localized in the pericentric region of chromosome 4 by fluorescence in situ hybridization and constitutes 0.51% of the chicken genome. Interestingly, a chicken repeat 1 (CR1) element has been identified within this basic repeating unit. Like other CR1 elements, this CR1 element also displays typical retrotransposition characteristics, including a highly conserved 3' region and a badly truncated 5' end. This direct evidence from sequence analysis, together with our Southern blot results, suggests that the HinfI repeat may originate from a unique region containing a retrotransposed CR1 element.     

Human actin genes are single copy for alpha-skeletal and alpha-cardiac actin but multicopy for beta- and gamma-cytoskeletal genes: 3'' untranslated regions are isotype specific but are conserved in evolution.

We have constructed isotype-specific subclones from the 3' untranslated regions of alpha-skeletal, alpha-cardiac, beta-cytoskeletal, and gamma-cytoskeletal actin cDNAs. These clones have been used as hybridization probes to assay the number and organization of these actin isotypes in the human genome. Hybridization of these probes to human genomic actin clones (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981; Engel et al., Mol. Cell. Biol. 2:674-684, 1982) has allowed the unambiguous assignment of the genomic clones to isotypically defined actin subfamilies. In addition, only one isotype-specific probe hybridizes to each actin-containing gene, with a single exception. This result suggests that the multiple actin genes in the human genome are not closely linked. Genomic DNA blots probed with these subclones under stringent conditions demonstrate that the alpha-skeletal and alpha-cardiac muscle actin genes are single copy, whereas the cytoskeletal actins, beta and gamma, are present in multiple copies in the human genome. Most of the actin genes of other mammals are cytoplasmic as well. These observations have important implications for the evolution of multigene families.     

Gene targeting restricted to mouse striated muscle lineage.

Spatially and temporally regulated somatic mutations can be achieved by using the Cre/LoxP recombination system of bacteriophage P1. In order to develop gene knockouts restricted to striated muscle, we generated a transgenic mouse line expressing Cre recombinase under the control of the human alpha-skeletal actin promoter. Specific excision of a loxP-flanked gene was demonstrated in striated muscle, heart and skeletal muscle, in a pattern very similar to the expression of the endogenous alpha-skeletal actin gene. Therefore, the reported transgenic line can be used to target inactivation or activation of a given gene to the skeletal muscle lineage.