No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Abstract
The actins constitute a family of highly conserved proteins found in all eukaryotic cells. Their conservation through a very wide range of taxonomic groups and the existence of tissue-specific isoforms make the actin genes very interesting for the study of the evolution of genes and their controlling elements. On the basis of amino acid sequence data, at least six different mammalian actins have been identified (skeletal muscle, cardiac muscle, two smooth muscle actins and the cytoplasmic beta- and gamma-actins). Rat spleen DNA digested by the EcoRI restriction enzyme contains at least 12 different fragments with actin-like sequences but only one which hybridized, in very stringent conditions, with the skeletal muscle cloned cDNA probe. Here we describe the sequence of the actin gene in that fragment. The nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. There are five small introns in the coding region and a large intron in the 5'-untranslated region. Comparison of the structure of the rat skeletal muscle actin gene with available data on actin genes from other organisms shows that while the sequenced actin genes from Drosophila and yeast have introns at different locations, introns located at codons specifying amino acids 41, 121, 204 and 267 have been preserved at least from the echinoderm to the vertebrates. A similar analysis has been done by Davidson. An intron at codon 150 is common to a plant actin gene and the skeletal muscle acting gene.
... We have isolated (1) and now sequenced the entire chicken a-cardiac actin gene. The coding region of this gene was interrupted by five introns which were located in the same positions as introns previously identified in other vertebrate cardiac (13) and a-skeletal actin genes (14,15). Additionally, we have identified a sixth intron in the 5' transcribed noncoding leader of the chicken a-cardiac actin gene. ...
... Sequencing of the a-cardiac actin gene revealed the presence of 377 encoded amino acid residues, which began with a Met-Cys dipeptide (Fig. 2). This dipeptide is also found in the human a-cardiac actin gene (13) as well as the other vertebrate striated actin genes (14,15). Interestingly, this dipeptide is found in all of the Drosophila non-muscle actin genes (10) and some sea urchin actin genes (3) while the Cys codon is absent in vertebrate non-muscle actin genes (24,25). ...
... The sequences of the intron-exon junctions agree quite well with the consensus sequences compiled from the study of many genes (26). The positions of splice junction boundaries in the coding region are identical to those in the human a-cardiac actin gene (13) and the a-skeletal actin genes (14,15) isolated from chicken and rat. ...
We sequenced the entire chicken α-cardiac actin gene. A single intron was positioned 20 bp upstream from the initiation ATG
codon in the 5′ non-coding region while the coding region was interrupted by 5 introns at amino acid positions 41/42, 150,
204, 267, and 327/328.Sequencing allowed the first comparison of the α-cardiac and α-skeletal actin transcriptional promoters.
These highly G+C rich promoters share two regions of homology which are found at position −134 (10 bp) end −296 (12 bp) in
the α-cardiac actin promoter. A smaller 9 bp motif (CCGCCCCGG) homologous to the −134 sequence was detected before, between
and after the TATA and CAAT boxes of the α-cardiac actin gene. The polyadenylation signal (AATAAA) was located 156 bp downstream
from the translation termination codon. The complete length of the α-cardiac actin mRNA excluding the poly A tail is 1370
nucleotides. The 3′ noncoding transcribed portion of the chicken α-cardiac actin gene was found to be extraordinarily conserved
when compared to the human and rat α-cardiac actin mRNA sequences.
... There are 4-6 amino acid replacements between the different muscle type actins; 4 amino acid replacements between the 2 cytoplasmic actins and 25 amino acid replacements between the cytoplasmic and the skeletal muscle actins. There are no amino acid replacements between bovine, rabbit, rat and chicken skeletal muscle actins (1)(2)(3)(4)(5)(6). While the muscle actins play a major role in muscle contraction, the cytoplasmic actins are involved in many forms of cell motility and in cytoskeletal functions. ...
... The vertebrate non-muscle 8and y-actins are considered functionally and evolutionarily to be more closely related to the actins found in the lower, unicellular, eukaryotes. In previous publications we described the nucleotide sequence of the rat skeletal muscle actin gene, and the isolation and preliminary characterization of the 8-actin gene (6,7). On the basis of these data and the available data on the structure of actin genes isolated from other organisms we suggested that the separation between the skeletal muscle and cytoplasmic actin genes occurred within the deuterostome phylogenetic branch (6)(7)(8). ...
... In previous publications we described the nucleotide sequence of the rat skeletal muscle actin gene, and the isolation and preliminary characterization of the 8-actin gene (6,7). On the basis of these data and the available data on the structure of actin genes isolated from other organisms we suggested that the separation between the skeletal muscle and cytoplasmic actin genes occurred within the deuterostome phylogenetic branch (6)(7)(8). Here we present the complete nucleotide sequence of the 0-actin gene, confirming our earlier suggestion on the structure of this gene. ...
The nucleotide sequence of the rat β-actin gene was determined. The gene codes for a protein identical to the bovine β–actin.
It has a large intron in the 5’ untranslated region 6 nucleotides upstream from the initiator ATG, and 4 Introns 1n the coding
region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. Unlike the skeletal muscle actin gene and many other
actin genes, the 6-act1n gene lacks the codon for Cys between the Initiator ATG and the codon for the N-terminal amino acid
of the mature protein.
The usage of synonymous codons 1n the β–actin gene is nonrandom, and is similar to that in the rat skeletal muscle and other
vertebrate actin genes, but differs from the codon usage in yeast and soybean actin genes.
... Recently, sequence data of the chicken and rat a-actin genes and the human cardiac actin gene have also shown that the amino-terminal acidic residue is preceded in the primary translation product by a cysteine residue (6,11,29). Furthermore, two genes encoding cytoplasmiclike actins in the sea urchin have recently been shown similarly to have a cysteine codon at their amino termini (2) (W. Crain, personal communication). ...
... Sequence analysis of pHMaA-1 revealed that, like the chick and rat a-actin mRNAs (6,29), the human muscle actin cDNA encodes a cysteine Visualization of actin cDNA length heterogeneity in the human muscle library. Two 1-p.g samples of the muscle cDNA library were digested with either EcoRI (lanes A and B) or with HindlIl (lanes C and D). ...
... The problem derives from the following considerations. First, the presence of a cysteine residue at amino acid 1 (referred to as "Cys+") is unique to protostome and deuterostome actin genes (2,6,7,29), whereas all actin genes in more primitive organisms lack this residue (called "Cys-") (5,8,16). Second, the ubiquitous occurrence of both Cys+ and a mechanism to remove the aminoterminal cysteine in both protostomes and deuterostomes is too coincidental to be explained by convergent evolution. ...
cDNAclones encoding three classes ofhumanactins have been isolated and characterized. Thefirst twoclasses (yand,B, cytoplasmic actins) wereobtained froma cDNA library constructed fromsimian virus 40-transformed human fibroblast mRNA,andthethird class (a,muscle actin) wasobtained fromacDNA library constructed fromadult humanmuscle mRNA.A newapproach was developed toenrich forfull-length cDNAs.Thehumanfibroblast cDNAplasmid library waslinearized withrestriction enzymes thatdidnotcuttheinserts of interest; itwasthensize-fractionated ongels, andthechimeric molecules of
... Further reinforcing their findings on transcriptional regulation, the Yaffe lab demonstrated that DNAase1 hypersensitivity of muscle genes correlates with their expression at the time of cell fusion [8]. They also characterised muscle contractile protein genes [9] and their chromosomal location [10] and went on to look at their regulation [11]. In addition to their work on the transcriptional control of muscle cell differentiation, the Yaffe lab also examined the cell biology of the system and provided early insights into the manipulation of fusion and differentiation, for example by altered Ca 2+ levels [12]. ...
... Comparisons of nucleotide sequences from the protein coding regions and exon-intron arrangements of related genes provide a means of tracing their evolution pathways [17,18]. Before the advent of the era of large-scale sequencing, actin gene family has been investigated in many organisms [19][20][21][22][23][24]. Those results indicate that actin gene family is highly conserved, and the number of actin genes among these organisms is variable. ...
Actin is one of the most highly conserved proteins and plays crucial roles in many vital cellular functions. In most eukaryotes, it is encoded by a multigene family. Although the actin gene family has been studied a lot, few investigators focus on the comparison of actin gene family in relative species. Here, the purpose of our study is to systematically investigate characteristics and evolutionary pattern of actin gene family in primates. We identified 233 actin genes in human, chimpanzee, gorilla, orangutan, gibbon, rhesus monkey, and marmoset genomes. Phylogenetic analysis showed that actin genes in the seven species could be divided into two major types of clades: orthologous group versus complex group. Codon usages and gene expression patterns of actin gene copies were highly consistent among the groups because of basic functions needed by the organisms, but much diverged within species due to functional diversification. Besides, many great potential pseudogenes were found with incomplete open reading frames due to frameshifts or early stop codons. These results implied that actin gene family in primates went through "birth and death" model of evolution process. Under this model, actin genes experienced strong negative selection and increased the functional complexity by reproducing themselves.
... The most noticeable feature of the f-actin pseudogene HBAc-4l is the absence of intervening sequences. Although the structure of a functional human a-actin gene has not been determined yet this gene is likely to be split since all vertebrate actin genes studied so far, including a human cardiac muscle actin gene and a a-actin gene from chick and rat, are interrupted by several introns (29)(30)(31). In fact, with the exception of the genes in the slime mold Dictyostelium (32) and an actin gene in the fission yeast Schizosaccharomyces pombe (Mertins and Gallwitz, unpublished), the actin genes of all other eukaryotes that have been studied contain intervening sequences (26,(33)(34)(35)(36). ...
From a human genomic library we have isolated and sequenced a β-actin-related pseudogene (HBAc-φ1) which is free of intervening sequences. Several nucleotide insertions and deletions and translational stop codons generated
within the protein-coding region indicate that this gene is functionless.
... Although there are a number of different base changes between 4<* and 4 in their 3' non-coding sequences, we presume that the critical mutation is within the highly conserved poly(A) addition signal AATAAA which becomes AATGAA in ^ •> . Functional variants of AATAAA have been demonstrated in a few eukaryotic genes, in particular AGTAAA(29,30) and ATTAAA(31)(32)(33)(34). ...
Transcriptional analysis of the human pseudogene Φα globin has revealed the following features: (1) The promoter with a 23
bp deletion between the CCAAT and ATA boxes is functional both in vitro and in vivo, 3 fold and 10 fold less efficient, respectively, than α. (2) Both the Φ and α globin gene promoters are active in the absence
of transcriptional enhancers, either a gene-encoded or viral enhancer. (3) The mutated poly(A) addition signal in Φα (AATGAA)
appears to be completely nonfunctional. This result provides an explanation for the absence of Φα transcripts in human erythroid
cells.
... Codons for the Nterminal Met and Cys amino acids, which are thought to be removed by posttranscriptional processing in muscle (53), are present in the nucleotide sequence. The N-terminal Met and Cys codons are also found in other mammalian and avian muscle actins (4,12,16,17,21,47,53). The same two codons are also found in all six Drosophila actin genes (13), four nematode actin genes (10), and sea urchin actin genes (6,42). ...
A cloned quail troponin I contractile protein gene, stably transfected into a mouse myogenic cell line, exhibits appropriate developmental activation and quantitative expression during myoblast differentiation. Deletion mutagenesis analyses reveal that the troponin I gene has two distinct cis regulatory elements required for its developmental expression, as measured by mRNA accumulation and nuclear runoff transcription assays. One element in the 5' flanking region is required for maximum quantitative expression, and a second larger regulatory element (1.5 kilobases) within the first intron is responsible for differentiation-specific transcription. The upstream region is highly sensitive to negative repression by interaction with pBR322 sequences. The larger intragenic region retains some activity when moved to the 5' and 3' flanking regions and when inverted but is maximally active in its native intragenic site. The concerted activities of these two regulatory regions produce a 100- to 200-fold transcriptional activation during myoblast differentiation. The conserved 5' exon-intron organization of troponin I and other contractile protein genes suggests a possible mechanism by which intragenic control elements coordinate contractile protein gene regulation during skeletal myogenesis.
cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.
To identify the DNA sequences that regulate the expression of the sarcomeric myosin heavy-chain (MHC) genes in muscle cells, a series of deletion constructs of the rat embryonic MHC gene was assayed for transient expression after introduction into myogenic and nonmyogenic cells. The sequences in 1.4 kilobases of 5'-flanking DNA were found to be sufficient to direct expression of the MHC gene constructs in a tissue-specific manner (i.e., in differentiated muscle cells but not in undifferentiated muscle and nonmuscle cells). Three main distinct regulatory domains have been identified: (i) the upstream sequences from positions -1413 to -174, which determine the level of expression of the MHC gene and are constituted of three positive regulatory elements and two negative ones; (ii) a muscle-specific regulatory element from positions -173 to -142, which restricts the expression of the MHC gene to muscle cells; and (iii) the promoter region, downstream from position -102, which directs transcription initiation. Introduction of the simian virus 40 enhancer into constructs where subportions of or all of the upstream sequences are deleted (up to position -173) strongly increases the level of expression of such truncated constructs but without changing their muscle specificity. These upstream sequences, which can be substituted for by the simian virus 40 enhancer, function in an orientation-, position-, and promoter-dependent fashion. The muscle-specific element is also promoter specific but does not support efficient expression of the MHC gene. The MHC promoter in itself is not muscle specific. These results underline the importance of the concerted action of multiple regulatory elements that are likely to represent targets for DNA-binding-regulatory proteins.
A recombinant phage containing an actin gene (lambda Ha201) was isolated from a human DNA library and the structure of the actin gene was determined. The amino acid sequences deduced from the nucleotide sequences of lambda Ha201 were compared with those of six actin isoforms; they matched those of bovine aortic smooth muscle actin, except for codon 309, which was valine (GTC) in lambda Ha201 and alanine (GCN) in bovine aortic smooth muscle actin. Southern blot hybridization experiments showed that the gene of normal human cells did not have the TaqI-sensitive site around position 309, whereas half of the genes of HUT14 cells did. These results indicate that one allele of the aortic smooth muscle actin gene in HUT14 cells has a transition point mutation (C----T) at codon 309 and that the amino acid sequences of normal human aorta and bovine smooth muscle actins are probably identical. In addition to the five introns interrupting exons at codons 150, 204, and 267, and between codons 41 and 42 and 327 and 328, which are common to skeletal muscle and cardiac muscle actin genes, the smooth muscle actin gene has two more intron sites between codons 84 and 85 and 121 and 122. The previously unreported intron site between codons 84 and 85 is unique to the smooth muscle actin gene. The intron site between codons 121 and 122 is common to beta-actin genes but is not found in other muscle actin genes. A hypothesis is proposed for the evolutionary pathway of the actin gene family.
The 5' coding and promoter regions of the four coordinately regulated tubulin genes of Chlamydomonas reinhardi have been mapped and sequenced. DNA sequencing data shows that the predicted N-terminal amino acid sequences of Chlamydomonas alpha- and beta-tubulins closely match that of tubulins of other eucaryotes. Within the alpha 1- and alpha 2-tubulin gene set and the beta 1- and beta 2-tubulin gene set, both nucleotide sequence and intron placement are highly conserved. Transcription initiation sites have been located by primer extension analysis at 140, 141, 159, and 132 base pairs upstream of the translation initiator codon for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin genes, respectively. Among the structures with potential regulatory significance, the most striking is a 16-base-pair consensus sequence [GCTC(G/C)AAGGC(G/T)(G/C)--(C/A)(C/A)G] which is found in multiple copies immediately upstream of the TATA box in each of the four genes. An unexpected discovery is the presence of pseudopromoter regions in two of the transcribed tubulin genes. One pseudopromoter region is located 400 base pairs upstream of the authentic alpha 2-tubulin gene promoter, whereas the other is located within the transcribed 5' noncoding region of the beta 1-tubulin gene.
Thymidine kinase (tk) enzyme expression is shut down when cultured skeletal muscle cells terminally differentiate. This regulation is mediated by a rapid and specific decline in the abundance of cellular tk mRNA. tk-deficient mouse myoblasts were transformed to the tk-positive phenotype by using both the cellular tk gene of the chicken and the herpesvirus tk gene. Myoblasts transformed with the cellular tk gene effectively regulate tk enzyme activity upon terminal differentiation. Conversely, myoblasts transformed with the herpesvirus tk gene continue to express tk enzyme activity in postreplicative muscle cells. A regulated pattern of expression is retained when the promoter of the cellular tk gene is replaced by the promoter of the herpesvirus tk gene. Moreover, the cellular tk gene is appropriately regulated during terminal muscle differentiation when its 3' terminus is removed and replaced by the terminus of the viral tk gene. Thus, the element of the cellular tk gene sufficient to specify its regulation is entirely intragenic.
AMP deaminase (AMPD) is an enzyme found in all eukaryotic cells. Tissue-specific and stage-specific isoforms of this enzyme are found in vertebrates, and expression of these different isoforms is determined by selective expression of the multiple genes. The AMPD1 gene is expressed predominantly in skeletal muscle, in which transcript abundance is controlled by stage-specific and fiber type-specific signals. This enzyme activity is presumed to be important in skeletal muscle because a metabolic myopathy develops in individuals with an inherited deficiency of AMPD1. In the present study, cis- and trans-acting factors that control expression of AMPD1 have been identified. Two cis-acting elements located within 100 nucleotides of the transcriptional start site are required for muscle-specific expression of AMPD1. One element (-100 to -79) behaves like a tissue-specific enhancer, and it interacts with protein(s) found predominantly in nuclei of myoblasts and myotubes. This element is similar in sequence to an MEF2 binding motif, and it contains an A/T core that is essential for enhancer activity and binding of a nuclear protein(s). The second element (-60 to -40) has properties of a stage-specific promoter in that it is essential for muscle-specific expression of the AMPD1 promoter, does not confer muscle-specific expression on a heterologous promoter construct, and interacts with a protein(s) restricted to nuclei of differentiated myotubes. Interaction between these functionally distinct elements may be required for regulating the expression of AMPD1 during myocyte differentiation and in different muscle fiber types.
We identified a novel chicken actin gene. The actin protein deduced from its nucleotide sequence very closely resembles the vertebrate cytoplasmic actins; accordingly, we classified this gene as a nonmuscle type. We adopted the convention for indicating the nonmuscle actins of the class Amphibia (Vandekerckhove et al., J. Mol. Biol. 152:413-426) and denoted this gene as type 5. RNA blot analysis demonstrated that the type 5 actin mRNA transcripts accumulate in adult tissues in a pattern indicative of a nonmuscle actin gene. Genomic DNA blots indicated that the type 5 actin is a single copy gene and a distinct member of the chicken actin multigene family. Inspection of the nucleotide sequence revealed many features that distinguished the type 5 gene from all other vertebrate actin genes examined to date. These unique characteristics include: (i) an initiation Met codon preceding an Ala codon, a feature previously known only in plant actins, (ii) a single intron within the 5' untranslated region, with no interruptions in the coding portion of the gene, and (iii) an atypical Goldberg-Hogness box (ATAGAA) preceding the mRNA initiation terminus. These unusual features have interesting implications for actin gene diversification during evolution.
The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.
Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC) gene is regulated by multiple elements in the proximal enhancer/promoter. Within this region, an M-CAT site and an A-rich site were identified as potential regulatory elements. Site-specific mutations in each site, individually, reduced activity from the wild-type promoter by approximately 85% in the adult rat heart, demonstrating that these sites were positive regulatory elements. alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts that was immunologically related to transcriptional enhancer factor 1, a factor that binds within the simian virus 40 enhancer. The factor that bound the A-rich region (ARF) was antigenically related to the RSRF family of proteins, ARF was distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of DNA-binding specificity and developmental expression. Like MEF-2, ARF DNA-binding activity was present in the heart and brain; however, no ARF activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF and ARF DNA-binding activities were developmentally regulated with peak levels in the 1- to 2-day neonatal heart. The activity of both factors increased nearly fivefold in adult rat hearts subjected to a pressure overload. By comparison, the levels of alpha-MHC binding factor 2 did not change during hypertrophy. Binding sites for MCBF and ARF are present in several genes that are upregulated during cardiac hypertrophy. Our results suggest that these factors participate in the alterations in gene expression that occur during cardiac development and hypertrophy.
The muscle-specific form of creatine kinase (MCK) is induced in differentiating myoblast cultures, and a dramatic increase in mRNA levels precedes and parallels the increase in MCK protein. To study this induction, the complete MCK gene was cloned and characterized. The transcription unit was shown to span 11 kilobases and to contain seven introns. The splice junctions were identified and shown to conform to the appropriate consensus sequences. Close homology with branchpoint consensuses was found upstream of the 3' splice sites in six of seven cases. Transcriptional regulation of the gene in differentiating myoblast cultures was demonstrated by nuclear run-on experiments; increases in transcription accounted for a major part of the increased mRNA levels. Regulated expression of a transfected MCK gene containing the entire transcription unit with 3.3 kilobases of 5'-flanking sequence was also demonstrated during differentiation of the MM14 mouse myoblast cell line. The MCK 5'-flanking region was sufficient to confer transcriptional regulation to a heterologous structural gene, since chloramphenicol acetyl transferase activity was induced during differentiation of cultures transfected with an MCK-chloramphenicol acetyl transferase fusion construct. Examination of the DNA sequence immediately upstream of the transcription start site revealed a 17-nucleotide element which occurred three times. Comparisons with other muscle-specific genes which are also transcriptionally regulated during myogenesis revealed upstream homologies in the alpha-actin and myosin heavy chain genes, but not in the myosin light-chain genes, with the regions containing these repeats. We suggest that coordinate control of a subset of muscle genes may occur via recognition of these common sequences.
A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice.
The chicken skeletal alpha-actin gene promoter region provides at least a 75-fold-greater transcriptional activity in muscle cells than in fibroblasts. The cis-acting sequences required for cell type-restricted expression within this 200-base-pair (bp) region were elucidated by chloramphenicol acetyltransferase assays of site-directed Bg/II linker-scanning mutations transiently transfected into primary cultures. Four positive cis-acting elements were identified and are required for efficient transcriptional activity in myogenic cells. These elements, conserved across vertebrate evolution, include the ATAAAA box (-24 bp), paired CCAAT-box-associated repeats (CBARs; at -83 bp and -127 bp), and the upstream T+A-rich regulatory sequence (at -176 bp). Basal transcriptional activity in fibroblasts was not as dependent on the upstream CBAR or regions of the upstream T+A-rich regulatory sequence. Transfection experiments provided evidence that positive regulatory factors required for alpha-actin expression in fibroblasts are limiting. In addition, negative cis-acting elements were detected and found closely associated with the G+C-rich sequences that surround the paired CBARs. Negative elements may have a role in restricting developmentally timed expression in myoblasts and appear to inhibit promoter activity in nonmyogenic cells. Cell type-specific expression of the skeletal alpha-actin gene promoter is regulated by combinatorial and possibly competitive interactions between multiple positive and negative cis-acting elements.
DNA fragments located 10 kilobases apart in the genome and containing, respectively, the first myosin light chain 1 (MLC1f) and the first myosin light chain 3 (MLC3f) specific exon of the rat myosin light chain 1 and 3 gene, together with several hundred base pairs of upstream flanking sequences, have been shown in runoff in vitro transcription assays to direct initiation of transcription at the cap sites of MLC1f and MLC3f mRNAs used in vivo. These results establish the presence of two separate, functional promoters within that gene. A comparison of the nucleotide sequence of the rat MLC1f/3f gene with the corresponding sequences from mouse and chicken shows that: the MLC1f promoter regions have been highly conserved up to position -150 from the cap site while the MLC3f promoter regions display a very poor degree of homology and even the absence or poor conservation of typical eucaryotic promoter elements such as TATA and CAT boxes; the exon/intron structure of this gene has been completely conserved in the three species; and corresponding exons, except for the regions encoding most of the 5' and 3' untranslated sequences, show greater than 75% homology while corresponding introns are similar in size but considerably divergent in sequence. The above findings indicate that the overall structure of the MLC1f/3f genes has been maintained between avian and mammalian species and that these genes contain two functional and widely spaced promoters. The fact that the structures of the alkali light chain gene from Drosophila melanogaster and of other related genes of the troponin C supergene family resemble a MLC3f gene without an upstream promoter and first exon strongly suggests that the present-day MLC1f/3f genes of higher vertebrates arose from a primordial alkali light chain gene through the addition of a far-upstream MLC1f-specific promoter and first exon. The two promoters have evolved at different rates, with the MLC1f promoter being more conserved than the MLC3f promoter. This discrepant evolutionary rate might reflect different mechanisms of promoter activation for the transcription of MLC1f and MLC3f RNA.
Recombinant DNA clones encoding the Drosophila melanogaster homolog of the vertebrate myosin light-chain-2 (MLC-2) gene have been isolated. This single-copy gene maps to the chromosomal locus 99E. The nucleotide sequence was determined for a 3.4-kilobase genomic fragment containing the gene and for two MLC-2 cDNA clones generated from late pupal mRNA. Comparison of these sequences shows that the gene contains two introns, the positions of which are conserved in the corresponding rat sequence. Extension of a primer homologous to the mRNA reveals two start sites for transcription 12 nucleotides apart. The sequence TATA is not present ahead of the mRNA cap site. There are two major sites of poly(A) addition separated by 356 nucleotides. The protein sequence derived from translation of the cDNA sequence shows a high degree of homology with that for the DTNB myosin light chain (MLC-2) of chicken. A lower degree of sequence homology was seen in comparisons with other evolutionarily related calcium-binding proteins. RNA blots show high levels of expression of several transcripts during the developmental time stages when muscle is being produced. In vitro translation of hybrid-selected RNA produces two polypeptides which comigrate on two-dimensional gels with proteins from Drosophila actomyosin, although the cDNA sequence reveals only one 26-kilodalton primary translation product.
We report the sequence of the single chicken triosephosphate isomerase gene and its flanking regions. The 3-kilobase-long gene is composed of seven similarly sized exons and six introns. By using crystallographic and sequence data, we argue that this ancient gene was originally assembled from the genetic antecedents of exons.
The chicken skeletal alpha-actin gene promoter region (-202 to -12) provides myogenic transcriptional specificity. This promoter contains partial dyad symmetry about an axis at nucleotide -108 and in transfection experiments is capable of directing transcription in a bidirectional manner. At least three different transcription initiation start sites, oriented toward upstream sequences, were mapped 25 to 30 base pairs from TATA-like regions. The opposing transcriptional activity was potentiated upon the deletion of sequences proximal to the alpha-actin transcription start site. Thus, sequences which serve to position RNA polymerase for alpha-actin transcription may allow, in their absence, the selection of alternative and reverse-oriented start sites. Nuclear runoff transcription assays of embryonic muscle indicated that divergent transcription may occur in vivo but with rapid turnover of nuclear transcripts. Divergent transcriptional activity enabled us to define the 3' regulatory boundary of the skeletal alpha-actin promoter which retains a high level of myogenic transcriptional activity. The 3' regulatory border was detected when serial 3' deletions bisected the element (-91 CCAAA TATGG -82) which reduced transcriptional activity by 80%. Previously we showed that disruption of its upstream counterpart (-127 CCAAAGAAGG -136) resulted in about a 90% decrease in activity. These element pairs, which we describe as CCAAT box-associated repeats, are conserved in all sequenced vertebrate sarcomeric actin genes and may act in a cooperative manner to facilitate transcription in myogenic cells.
Two nuclear factors bind to the same site in the chicken skeletal actin promoter. Mutations in the footprint sequence which eliminate detectable binding decrease expression in transfected skeletal muscle cells by a factor of 25 to 50 and do not elevate the low expression in nonmuscle cells. These results show that the factor-binding site contributes to the activation of expression in muscle cells and that it alone does not contribute significantly to repress expression in nonmuscle cells.
We have generated transgenic mouse lines that carry the promoter region of the chicken skeletal muscle alpha (alpha sk) actin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. In adult mice, the pattern of transgene expression resembled that of the endogenous alpha sk actin gene. In most of the transgenic lines, high levels of CAT activity were detected in striated muscle (skeletal and cardiac) but not in the other tissues tested. In striated muscle, transcription of the transgene was initiated at the normal transcriptional start site of the chicken alpha sk actin gene. The region from nucleotides -191 to +27 of the chicken alpha sk actin gene was sufficient to direct the expression of CAT in striated muscle of transgenic mice. These observations suggest that the mechanism of tissue-specific actin gene expression is well conserved in higher vertebrate species.
Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.
A DNA fragment of the rat embryonic myosin heavy-chain promoter (MHCemb) has been found to specifically bind a nuclear factor (NFe) present in extracts prepared from mouse C2 myoblasts, myotubes, and HeLa cells. The nucleotide sequence of the binding site (BSe) has been identified as 5'-GTGTCAGTCA-3' and was located between -93 and -84. Transient expression studies on MHCemb promoter deletion constructs in C2 myoblasts and C2 myotubes suggested that NFe is a transcriptional factor. Deletion of the NFe-binding site resulted in four- to sixfold and twofold reduction of promoter activity in C2 myotubes and C2 myoblasts, respectively. Furthermore, point mutations at the BSe not only abolished the NFe-binding activity of the MHCemb promoter but also resulted in reduction of the promoter activity to levels similar to those of the deletion constructs in C2 myotubes, myoblasts, and Hela cells (four- to sixfold). Although BSe and the binding site of the recently identified transcriptional factors AP-1 and ATF share significant homology, the results from competition binding assays indicated that NFe is different from both AP-1 and ATF.
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.
It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and nature. A native of Israel, David grew up in the historic years that preceded the birth of the State of Israel. He was a member of the group that established Kibbutz Revivim in the Negev desert, and in 1948 participated in Israel’s War of Independence. David and Ruth eventually joined Kibbutz Givat Brenner by Rehovot, permitting David to be both a kibbutz member and a life-long researcher at the Weizmann Institute of Science, where David received his PhD in 1959. David returned to the Institute after his postdoc at Stanford. Here, after several years of researching a number of tissues as models for studying the process of differentiation, David entered the myogenesis field and stayed with it to his last day. With his dedication to the field of myogenesis and his commitment to furthering the understanding of the People and the Land of Israel throughout the international scientific community, David organized the first ever myogenesis meeting that took place in Shoresh, Israel in 1975. This was followed by the 1980 myogenesis meeting at the same place and many more outstanding meetings, all of which brought together myogenesis, nature and scenery. Herein, through the preparation and publication of this current manuscript, we are meeting once again at a “David Yaffe myogenesis meeting". Some of us have been members of the Yaffe lab, some of us have known David as his national and international colleagues in the myology field. One of our contributors has also known (and communicates here) about David Yaffe’s earlier years as a kibbutznick in the Negev. Our collective reflections are a tribute to Professor David Yaffe. We are fortunate that the European Journal of Translational Myology has provided us with tremendous input and a platform for holding this 2020 distance meeting "Farwell to Professor David Yaffe - A Pillar of the Myogenesis Field".
It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and nature. A native of Israel, David grew up in the historic years that preceded the birth of the State of Israel. He was a member of the group that established Kibbutz Revivim in the Negev desert, and in 1948 participated in Israel’s War of Independence. David and Ruth eventually joined Kibbutz Givat Brenner by Rehovot, permitting David to be both a kibbutz member and a life-long researcher at the Weizmann Institute of Science, where David received his PhD in 1959. David returned to the Institute after his postdoc at Stanford. Here, after several years of researching a number of tissues as models for studying the process of differentiation, David entered the myogenesis field and stayed with it to his last day. With his dedication to the field of myogenesis and his commitment to furthering the understanding of the People and the Land of Israel throughout the international scientific community, David organized the first ever myogenesis meeting that took place in Shoresh, Israel in 1975. This was followed by the 1980 myogenesis meeting at the same place and many more outstanding meetings, all of which brought together myogenesis, nature and scenery. Herein, through the preparation and publication of this current manuscript, we are meeting once again at a “David Yaffe myogenesis meeting". Some of us have been members of the Yaffe lab, some of us have known David as his national and international colleagues in the myology field. One of our contributors has also known (and communicates here) about David Yaffe’s earlier years as a kibbutznick in the Negev. Our collective reflections are a tribute to Professor David Yaffe. We are fortunate that the European Journal of Translational Myology has provided us with tremendous input and a platform for holding this 2020 distance meeting "Farwell to Professor David Yaffe - A Pillar of the Myogenesis Field".
Sea urchins began to be used over a century ago for studies of fertilization and development, and they are now among the best understood experimental models for early embryogenesis. The use of sea urchin embryos can be attributed in part to practical considerations. The adult animals are abundant, widely distributed, and easy to collect. Gametes are available in relatively large quantities during long breeding seasons, and under appropriate laboratory regimens they can be obtained all year round (1,2). Embryonic development is rapid and synchronous, occurs reliably under laboratory conditions, and in most species is complete within a few days. Unlike most invertebrates commonly utilized for research, echinoderms belong to the same great branch of the Animal Kingdom as do the vertebrates, i.e., and they are deuterostomes. Thus to the advantage of experimental accessibility may be added the attraction of a developmental system that shares with the chordates a common if remote evolutionary ancestry. Definitive homologies in the morphogenesis of chordate and echinoderm embryos were noted by classical observers [reviewed in (3)], and in recent years specific molecular homologies have been reported as well.
In vitro myogenesis offers and experimental model for the study of the molecular mechanisms involved in gene expression during cell differentiation. Terminal differentiation of muscle cells is characterized by the fusion of mononucleated myoblasts into multinucleated fibers. The morphological changes are accompanied by biochemical modifications, including the onset or a great increase in the synthesis of the major muscle contractile proteins, their regulatory polypeptides and enzymes needed to produce the energy for muscle contraction (reviewed in ref. 1). Several of these proteins (actin, myosin heavy chain, tropomyosin, creatine kinase) have been shown to be members of families of closely related isoforms, some of which are muscle-specific and are synthesized during terminal differentiation, others are present in many cell types. The capacity of myogenic cells (as well as other precursor cells) to proliferate during extended periods without expressing the genes involved in terminal differentiation (2, 3), indicate the existence of mechanisms which retain the latent program of gene expression, and mechanisms of gene activation which recognize these genes.
In certain respects the fetal heart resembles a small version of the adult heart. It fulfills similar functions in that it is required to fill, develop pressure, expel blood, and relax, thereby ensuring continuous circulation of blood. At the cellular and molecular level there are, however, significant differences. It can be demonstrated, for example, that muscle from adult heart has a greater ability to generate tension compared to fetal;1,2 adult and fetal cardiac muscle fibers show differing responses to intracellular calcium,3 differing resistance to acidosis4,5 and differing responses to adrenergic stimulation.6,7 Such developmental differences in contractility have important clinical implications in the understanding and treatment of cardiac disease in neonates and infants.
Restriction site analysis of polymerase chain reaction (PCR) pro-ducts from a conserved region of the alpha-actin gene has been used for the specific identification of wreck fish (Poly-prion americanus) and Nile perch (Lates niloticus)fillets. PCR amplification was carried out using a set of primers designed from the DNA nucleotide sequences reported for alpha-actins from humans and various animals. Restriction endonuclease analysis based on sequence data of the PCR products of each fish species revealed the presence of species-specific polymorphic sites for Rsal and Hinfl endonucleases. Electrophoretic analysis of the amplicons digested with these endonucleases produced species-specific profiles that allowed the genetic identification of wreck fish and Nile perch.
In the heart, mRNA accumulations for sarcomeric actins and myosin heavy chains (MHC) are subject to diverse regulatorial processes. To study cardiac contractile protein transcriptional regulations, an in vitro transcription system using nonenzymatically isolated rat cardiac nuclei was characterized. Transcription was shown to be rapid and continuous during the first 20 min of incubation and 5.4-fold less than that seen from comparably isolated hepatocyte nuclei. Neither RNase nor DNase activities were detectable. Direct transcriptional analyses of the alpha- and beta-MHC and cardiac and skeletal alpha-actin genes from cardiac nuclei were performed. In 23-24-day-old rats, significant levels of transcription were seen for alpha-MHC and for the sarcomeric alpha-actins. Beta-MHC was just detectable, and no positive signals were ever seen for fibronectin. We then compared the percentages of MHC and sarcomeric alpha-actin expressions determined from 1) the transcriptional assays and 2) total isolated RNA (alpha-MHC: 90.1 +/- 4.8% (transcription), 93.0 +/- 4.7% (accumulation); beta-MHC: 9.9 +/- 4.8%, 7.0 +/- 4.7%; cardiac alpha-actin: 84.0 +/- 2.5%, 84.9 +/- 2.5%; skeletal alpha-actin: 16.1 +/- 2.5%. 15.0 +/- 2.5%). The results support the conclusion that the primary mechanisms controlling the accumulations of these gene products are transcriptional. Additionally, we show that an anti-sense mRNA showing strong homology or identity with the 5' end of the beta-MHC gene is transcribed in cardiac nuclei but not in hepatocyte nuclei.
The control of expression of muscle-specific genes introduced into myogenic cells and transgenic mice was studied. The expression of chimeric genes containing the 5′ regions and flanking DNA of the genes coding for rat skeletal muscle actin and myosin light chain 2 was greatly increased during differentiation of stably transfected myogenic clones and in transiently transfected myogenic cells. Likewise, the expression of the chicken skeletal muscle actin gene was developmentally regulated in several stably transfected rat myogenic clones, indicating that the mechanisms controlling muscle-specific gene expression have been conserved for at least 300 million years. In contrast, the expression of a fused mouse/human β-globin gene, and of a chimeric gene containing the 5′ region and flanking cDNA of the rat β-actin gene did not Increase during differentiation of transfected myogenic clones. The expression of the rat MLC2 gene was tissue-specific and developmentally regulated in transgenic mice.
The unicellular green alga Chlamydomonas reinhardtii has two actin genes, one encoding a conventional actin and the other an unconventional actin called novel actin-like protein (NAP). These actins apparently differ in their ability to polymerize, but their specific functions in the cell are unknown. To understand their evolutionary relationship, we investigated the molecular phylogeny of the actin/NAP family in Volvocales, using fully identified sequences (Chlamydomonas moewusii, Gonium pectorale, and Volvox carteri) and newly determined partial sequences (Eudorina elegans, and Volvulina steinii). Although the origin of the NAP clade remains ambiguous, the inferred phylogenetic trees strongly support the monophyly of NAP genes and show that only the genomes of volvocine species contain NAP genes. The nonsynonymous substitution rate of the NAP gene is, in consistence with its long branch length in the phylogeny, relatively high compared with that of the actin gene. NAP is thus apparently unique to volvocine species and most likely performs a cellular function with fewer constraints.
Exon-intron structures of eukaryotic genes were examined closely in their relation to primary and tertiary structures of the proteins they encode. Specific attention was given to the introns of genes encoding proteins having no repeats in their amino acid sequences. such introns have been shown to be located at sites corresponding to inter-domain or inter-module junctions of proteins identified in their three dimensional structures. “Modules,” compact structural units in globular domains of proteins, are identified by drawing a distance map. Intron positions are found to correspond to inter-module junctions in various proteins whose X-ray crystallographic data are available: the glogin family, CEWL, ovomucoid, cytochrome c, ADH, and trypsin-like serine proteinases.The good correspondence between intron positions and inter-module junctions excludes a mechanism of random insertion of introns, because the probability of intron insertion at each inter-module junction is extraordinarily small. Intron positions have been very stable and well conserved during evolution. However, at some inter-module junctions no introns are found.Modules in small proteins having no core modules buried in their interior have a character suitable for recruitment through their assembly into a stable domain; one side of them is rich in hydrophobic residues and the other in hydrophilic residues. Functionally important residues are scattered on different modules in the proteins examined. Based on these observations, the role of modules in the precellular period was conjectured: some of them might be functionally active by themselves but most modules might be only segments who could functions as an active protein only in an assembly. The origin of introns might be traced back prior to the divergence of prokaryotes and eukaryotes.
The authors determined the actin isotypes encoded by 30 actin cDNA clones previously isolated from an adult human muscle cDNA library. Using 3' untranslated region probes, derived from ..cap alpha.. skeletal, ..beta..- and ..gamma..-actin cDNAs and from an ..cap alpha..-cardiac actin genomic clone, they showed that 28 of the cDNAs correspond to ..cap alpha..-skeletal actin transcripts. Unexpectedly, however, the remaining two cDNA clones proved to derive from ..cap alpha..-cardiac actin mRNA. Sequence analysis confirmed that the two skeletal muscle ..cap alpha..-cardiac actin cDNAs are derived from transcripts of the cloned ..cap alpha..-cardiac actin gene. Comparison of total actin mRNA levels in adult skeletal muscle and adult heart revealed that the steady-state levels in skeletal muscle are about twofold greater, per microgram of total cellular RNA, than those in heart. Thus, in skeletal muscle and in heart, both of the sarcomeric actin mRNA isotypes are quite abundant transcripts. They conclude that ..cap alpha..-skeletal and ..cap alpha..-cardiac actin genes are coexpressed as an actin pair in human adult striated muscles. Since the smooth-muscle actins (aortic and stomach) and the cytoplasmic actins (..beta.. and ..gamma..) are known to be coexpressed in smooth muscle and nonmuscle cells, respectively, they postulate that coexpression of actin pairs may be a common feature of mammalian actin gene expression in all tissues.
ABSTRACF Pressure overload on the heart is known to produce hypertrophy of cardiomyocytes and distinct changes in protein phenotype, including reduced expression of the gene for the sarcoplasmic reticuhun (SR) Ca2'ATPase (SERCA2). In this study we have shown that the decrease in SERCA2 gene expression (normalized by poly(A)+ mRNA or 18 S rRNA) in rats with 8 wk of aortic constriction was prevented by treatment with etomoxir, an inhibitor of carnitine pahnitoyltransferase 1. The reduction in steady-state niRNA levels for SR phospholamban (PLP) and Ca2 release channel (CRC) in the pres- sure-overloaded animals was also prevented without any reduction in the extent of cardiac hypertrophy by treatment with etomoxir. Although no changes in inRNA levels for GAPDH were evident in rats with pressure overload, the expression of the a-skeletal actin was increased; this change was prevented by etomoxir. Similar beneficial effects of etomoxir treatment were also evident when the gene expres- sion for SR SERCA2, PLP, and CRC in the hyper- trophied heart was normalized with respect to mRNA for GAPDH. These results support the view that drugs such as etomoxir may increase the abundance of. the mRNA for SR proteins in the hypertrophied heart and thus may prevent the transition of cardiac hypertrophy into heart failure.-Zarain-Herzberg, A., Rupp, H., Elimban, V., Dhalla, N. S. Modification of sarcoplasmic reticulum gene expression in pres-
A human actin cDNA clone pGF3 isolated from a fetal skeletal muscle cDNA library is described. The insert cDNA is homologous to skeletal muscle a-actin as judged by restriction mapping and nucleotide sequencing. The recombinant contains a substantial portion of the coding and the complete 3'-untranslated region. Comparison of the 3' ends of human and rat skeletal muscle and human cardiac a-actins reveals little homology between different types of actin genes in man but marked conservation of this region in the skeletal muscle actins of man and rat.
In isolating skeletal muscle satellite cells, sometimes a problem is encountered in removing contaminating nonmyogenic cells. In the present study, we constructed a novel vector, pSKA-EGFP, which achieves the expression of enhanced green fluorescent protein (EGFP) exclusively in myogenic cells under the control of skeletal a-actin promoter when transfected to primary cultured cells from skeletal muscle. Cells from rat skeletal muscle positive for EGFP after transfecting with pSKA-EGFP were all positive for desmin and none of the nonmyogenic cells expressed EGFP, indicating that the expression of EGFP is specific to myogenic cells. Among the cells positive for EGFP were proliferating cells, presumably satellite cells. In addition, EGFP positive cells derived from horse skeletal muscle after transfecting pSKA-EGFP in vitro formed multinuclear myotubes, indicating that myogenic expression of EGFP driven by skeletal cc-actin was achieved also in the equine cells. These results indicated that pSKA-EGFP vector will be useful in identifying and following up the satellite cells in real time, and also permit us to isolate satellite cells in combination with fluorescence-activated cell sorting (FACS).
DNA fragments located 10kilobases apartinthegenome andcontaining, respectively, thefirst myosin light chain 1 (MLC1f) andthefirst myosin light chain 3(MLC3f) specific exonoftheratmyosin light chain 1 and 3gene,together withseveral hundred basepairs ofupstream flanking sequences,havebeenshowninrunoff invitro transcription assaystodirect initiation oftranscription atthecap sites ofMLC1fandMLC3fmRNAs usedinvivo. Theseresults establish thepresenceoftwoseparate, functional promoters within that gene.A comparison ofthenucleotide sequenceoftheratMLC113f genewiththecorresponding sequencesfrommouse andchicken showsthat: (i) theMLC1fpromoter regions havebeenhighly conserved up toposition -150from thecap site while theMLC3fpromoter regions display a verypoordegree ofhomology andeventheabsence or poor conservation oftypical eucaryotic promoterelements suchas TATA andCAT boxes; (ii) the exon/intron structure ofthis genehasbeencompletely conserved inthethree species; and(iii) corresponding exons,except fortheregions encoding mostofthe5'and3'untranslated sequences,show>75% homology while corresponding introns aresimilar insize butconsiderably divergent insequence.Theabovefindings indicate thattheoverall structure oftheMLC1f/3f geneshasbeenmaintained between avian andmammalian species andthatthese genescontain twofunctional andwidely spaced promoters. Thefact thatthestructures ofthealkali light chain gene fromDrosophila melanogaster andofotherrelated genesofthetroponin C supergenefamily resemble aMLC3fgenewithout an upstream promoter andfirst exonstrongly suggests that thepresent-day MLC1l13f genesofhigher vertebrates arosefroma primordial alkali light chain genethrough theaddition ofa far-upstream MLC1rspecific promoterandfirst exon.Thetwopromoters haveevolved at different rates, withtheMLC1fpromoterbeing more conserved thantheMLC3fpromoter. Thisdiscrepant evolutionary ratemightreflect different mechanisms ofpromoter activation forthetranscription ofMLCIfand MLC3fRNA.
The safety of commercial light-water nuclear plants is highly dependent on the structural integrity of the reactor pressure vessel (RPV). In the absence of radiation damage to the RPV, fracture of the vessel is difficult to postulate. Exposure to high energy neutrons can result in embrittlement of radiation-sensitive RPV materials. The Heavy-Section Steel Irradiation (HSSI) Program at Oak Ridge National Laboratory, sponsored by the US Nuclear Regulatory Commission (USNRC), is assessing the effects of neutron irradiation on RPV material behavior, especially fracture toughness. The results of these and other studies are used by the USNRC in the evaluation of RPV integrity and regulation of overall nuclear plant safety. In assessing the effects of irradiation, prototypic RPV materials are characterized in the unirradiated condition and exposed to radiation under varying conditions. Mechanical property tests are conducted to provide data which can be used in the development of guidelines for structural integrity evaluations, while metallurgical examinations and mechanistic modeling are performed to improve understanding of the mechanisms responsible for embrittlement. The results of these investigations, in conjunction with results from commercial reactor surveillance programs, are used to develop a methodology for the prediction of radiation effects on RPV materials. This irradiation-induced degradation of the materials can be mitigated by thermal annealing, i.e., heating the RPV to a temperature above that of normal operation. Thus, thermal annealing and evaluation of reirradiation behavior are major tasks of the HSSI Program. This paper describes the HSSI Program activities by summarizing some past and recent results, as well as current and planned studies. 30 refs., 8 figs., 1 tab.
The nudeotide sequence of the chick β-actin gene was determined. The gene contains 5 introns; 4 interrupt the translated region
at codons 41/42, 120/122, 267, 327/328 and a large intron occurs in the 5′ untranslated region. The gene has a 97 nudeotide
5 ′-untrtranslated region and a 594 nudeotide 3′-untranslated region. A slight heterogeneity in the position of the poly A
addition site exists; polyadenylation can occur at either of two positions two nucleotides apart. The gene codes for an mRNA
of 1814 or 1816 nucleotides, excluding the poly(A) tail. In contrast to the chicle skeletal muscle actin gene the β-actin
gene lacks the Cys codon between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. la the
5′ flanking DNA, 15 nucleotides downstream from the CCAAT sequence, is a tract of 25 nucleotides that is highly homologous
to the sequence found in the same region of the rat β-actin gene.
We have used a modification of the Berk-Sharp technique to determine that the 5′ termini of the mouse 15 S β-globin precursor
and the mature mRNA have identical map coordinates. The modification involves the use of 5′ (or 3′) terminally labeled probes;
it allows the detection of the precursor in the presence of excess nature mRNA.
The nucleotide sequences at the 5' end of one actin cDNA and six actin genomic clones from Dictyostelium have been determined. The amino acid sequences derived from the nucleotide sequences show strong conservation for six of the seven genes relative to the NH2-terminal region of Physarum actin. The region 5' to the AUG initiating codon is greater than 90% A+T residues in all of the genes.
Plasmids p749, p106, and p150 contain cDNA inserts complementary to rat skeletal muscle actin mRNA. Nucleotide sequence analysis
indicates the following sequence relationships: p749 specifies codons 171 to 360; p150 specifies codons 357 to 374 together
with 120 nucleotides of the 3'-non-translated region; p106 specifies the last actin amino acid codon, the termination codon
and the entire 3' non-translated region. Plasmid p749 hybridized with RNA extracted from rat skeletal muscle, cardiac muscle,
smooth (stomach) muscle, and from brain. It also hybridizes well with RNA extracted from skeletal muscle and brain of dog
and chick. Plasmid p106 hybridized specifically with rat striated muscles (skeletal and cardiac muscle) mRNA but not with
mRNA from rat stomach and from rat brain. It also hybridized to RNA extracted from skeletal muscle of rabbit and dog but not
from chick. Thermal stability of the hybrids and sensitivity to S1 digestion also indicated substantial divergence between
the 3' untranslated end of rat and dog skeletal muscle actins. The investigation shows that the coding regions of actin genes
are highly conserved, whereas the 3' non-coding regions diverged considerably during evolution. Probes constructed from the
3' non-coding regions of actin mRNAs can be used to identify the various actin mRNA and actin genes.
The construction and partial characterization of recombinant bacterial plasmids carrying DNA sequences that hybridize with rat skeletal muscle actin and a myosin light chain mRNA is described. DNA of one clone hybridizes specifically with the muscle-specific alpha-actin mRNA. Three plasmid clones contain DNA inserts that hybridize with muscle as well as with nonmuscle actin mRNA. A fifth plasmid contains sequences complementary to mRNA coding for myosin light chain 2. DNA of this plasmid hybridizes specifically with RNA extracted from muscle and differentiated muscle cultures but not with RNA extracted from proliferating mononucleated myogenic cells.
Southern blots of rat genomic DNA indicate the existence of at least 12 EcoRI DNA fragments containing actin gene sequences. By using specific probes and stringent conditions of hybridization, it was found that only one of these fragments contains sequences of the skeletal muscle alpha-actin gene. Recombinant bacteriophages originating from eight different actin genes were isolated from rat genomic DNA libraries. One of them, Act 15, contains the skeletal muscle actin gene. Another clone, Act I, contains a gene coding for a cytoplasmic actin, identified tentatively as the beta-actin gene. Both genes have a large intron very close to the 5' end of their transcribed region, followed by several small introns. DNA sequence analysis and comparison with the available data on actin genes in other organisms indicated an interesting relationship between the positions of introns and the evolutionary relatedness. Several intron sites are conserved from at least the echinoderms to the vertebrates; others appear to be present in some actin genes and not in others.
The entire set of six closely related Drosophila actin genes was isolated using recombinant DNA methodology, and the structures of the respective coding regions were characterized by gene mapping techniques and by nucleotide sequencing of selected portions. Structural comparisons of these genes have resulted in several unexpected findings. Most striking is the nonconservation of the positions of intervening sequences within the protein-encoding regions of these genes. One of the Drosophila actin genes, DmA4, is split within a glycine codon at position 13; none of the remaining five genes is interrupted in the analogous position. Another gene, DmA6, is split within a glycine codon at position 307; at least two of the Drosophila actin genes are not split in the analogous position. Additionally, none of the Drosophila actin genes is split within codon four, where the yeast actin gene is interrupted. The six Drosophila actin genes encode several different proteins, but the amino acid sequence of each is similar to that of vertebrate cytoplasmic actins. None of the genes encodes a protein comparable in primary sequence to vertebrate skeletal muscle actin. Surprisingly, in each of these derived actin amino acid sequences the initiator methionine is directly followed by a cysteine residue, which in turn precedes the string of three acidic amino acids characteristic of the amino termini of mature vertebrate cytoplasmic actins. We discuss these findings in the context of actin gene evolution and function.
DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
During the late stage of adenovirus 2 infection, RNA chains are initiated at a site near coordinate 16 (Evans et al., 1977) and transcribed ∼30,000 nucleotides to the far end of the genome at coordinate 100. Late mRNAs processed from these transcripts contain a common spliced tripartite leader (Berget, Moore and Sharp, 1977; Chow et al., 1977a) encoded at ∼16, 20 and 27, and protein coding sequences which map down-stream. This report maps the late promoter and the capped 5′ end of nuclear and cytoplasmic RNAs from this transcription unit, and analyzes their structures. We show that nascent RNA chains pulse-labeled in vivo are initiated at coordinate 16.5 ± 0.5 and contain the sequences intervening between the leader segments. We map the capped 5′ terminus of late nuclear transcripts at a site between 16.4 and 16.6 by aligning T1 RNAase oligonucleotides from nuclear RNA with the DNA sequence of the promoter region. The structure of the first eleven residues of the capped 5′ terminus of late mRNA was determined by direct RNA sequencing. This structure corresponds exactly to a DNA sequence at coordinate 16.4 and precisely positions the mRNA cap template within the promoter region. These results suggest that the promoter and the cap template sites are coincident, and that the initiating residues of the primary transcript are precursors of the capped 5′ end of mRNA.
Multiple forms of actin have been found in a variety of mammalian cell lines and tissues by the use of high resolution, two-dimensional gel electrophoresis. One form (alpha actin) was found only in differentiated muscle cells, and its synthesis is induced during myogenesis in culture. Two other forms (beta and gamma actin) are present in all nonmuscle cell types examined, and they continue to be synthesized in cultured muscle cells after fusion. Tryptic peptide comparisons have shown that muscle actin is distinguished from the two "nonmuscle" actins by several peptide differences, and that the two non-muscle actins are nearly identical. All three forms contain equimolar amounts of N-methylhistidine, and extensive controls have shown no evidence of artifactual heterogeneity. In addition to the three major actins, two other proteins were identified as probably forms of actin by affinity for DNAase I-agarose. These proteins are similar in charge and molecular weight to the major actin forms, but are unstable and have lifetimes in the cell of less than 2 hr.
Pulse-labeled cytoplasmic proteins from cultured fetal calf-muscle cells at various stages of development were analyzed by one- and two-dimensional gel electrophoresis. The high resolution of the two-dimensional technique allows the determination of those protein species that begin to be synthesized after cell fusion. In addition, actin has been found to exist in three forms possessing similar biochemical properties and identical molecular weights but having slightly different isoelectric points. Two of the forms are found in prefusion dividing myoblasts and also in cultured kidney cells. The third form is the only one found in fetal muscle tissue and is predominant in cultures of fused muscle cells. Thus, it would seem that actin can exist in several isozymic forms of which one is specific to fused muscle tissue.
Actins isolated from embryonic chick brain and muscle differ in mobility when subjected to electrophoresis in gels containing urea and sodium dodecyl sulfate. Experiments were carried out to determine whether these actins are products of different structural genes and differ in primary amino acid sequence, or whether they are products of the same structural gene but are different because of post-translational modification. Messenger RNA from brain and muscle tissue was used to direct cell-free protein synthesis in wheat germ extracts. The synthesized actins were identified by conversion from globular to fibrous actin and by two-dimensional chromatographic analysis of tryptic peptides. The differences in electrophoretic mobility of brain compared to muscle actin were maintained in the cell-free protein synthetic products. Therefore, these mobility differences were not due to post-translational modification. It was concluded that brain and muscle actin are coded by different messenger RNAs and therefore arise from different structural genes. In addition, messenger RNA from 13- and 16-day embryonic thigh muscle directed the synthesis of both brain- and muscle-type actins, suggesting that muscle cell differentiation involves the regulation of at least two different actin genes.
The entire set of six closely related Drosophila actin genes was isolated using recombinant DNA methodology, and the structures of the respective coding regions were characterized by gene mapping techniques and by nucleotide sequencing of selected portions. Structural comparisons of these genes have resulted in several unexpected findings. Most striking is the nonconservation of the positions of intervening sequences within the protein-encoding regions of these genes. One of the Drosophila actin genes, DmA4, is split within a glycine codon at position 13; none of the remaining five genes is interrupted in the analogous position. Another gene, DmA6, is split within a glycine codon at position 307; at least two of the Drosophila actin genes are not split in the analogous position. Additionally, none of the Drosophila actin genes is split within codon four, where the yeast actin gene is interrupted. The six Drosophila actin genes encode several different proteins, but the amino acid sequence of each is similar to that of vertebrate cytoplasmic actins. None of the genes encodes a protein comparable in primary sequence to vertebrate skeletal muscle actin. Surprisingly, in each of these derived actin amino acid sequences in the initiator methionine is directly followed by a cysteine residue, which in turn precedes the string of three acidic amino acids characteristic of the amino termini of mature vertebrate cytoplasmic actins. We discuss these findings in the context of actin gene evolution and function.
The complete nucleotide sequence of the actin gene from Saccharomyces cerevisiae has been determined. The coding region is interrupted by a 304-base-pair intervening sequence that is located within the triplet coding for amino acid 4. DNA sequences of the intron-exon junctions are similar to those found in higher eukaryotes and can be aligned such that the intron starts with the dinucleotide 5'-G-T-3' and ends with 5'-A-G-3'. Regions fo homology within the sequences upstream from the initiation codon and those following the termination codon have been detected between the yeast iso-1-cytochrome c gene and the actin gene. As deduced from the nucleotide sequence, yeast actin has 374 amino acid residues. Its primary structure, especially the NH2-terminal third of the protein, is highly conserved during evolution.
The yeast Saccharomyces cerevisiae is known to contain the highly conserved and unbiquitous protein actin. We have used cloned actin sequences from Dictyostelium discoideum to identify and clone the actin gene in yeast. Hybridization to genomic fragments of yeast DNA suggest that there is a single actin gene in yeast. We have determined the nucleotide sequence of that gene and its flanking regions. The sequence of the gene reveals an intervening sequence of 309 base pairs in the coding sequences at the 5' end of the gene. The existence and location of the intervening sequence was verified by using the dideoxy chain termination technique to determine the sequence at the 5' terminus of the actin mRNA. The similarity of the splice junction sequences in this gene to those found in higher eukaryotes suggests that yeast must possess a similar splicing enzyme.
Soybean contains a small multigene family of actin-related sequences. We have determined the complete nucleotide sequence of a soybean actin gene carried on the recombinant plasmid pSAc3. As deduced from the nucleotide sequence, this soybean actin is composed of 376 amino acids. Compared to other eukaryotic actins, pSAc3 actin has a deletion of one amino acid between residues 118 and 122. The initiator methionine is followed by alanine, which is not found at this position in other eukaryotic actins. pSAc3 actin differs, in primary sequence, more from fungal and animal actins than any of the known nonplant actins differ from each other. pSAc3 actin appears to be related to both cytoplasmic and muscle specific actins in the location of specific NH(2)-terminal amino acids. The coding sequence is interrupted by three small introns, each less than 90 base pairs long. The splice junctions are similar to those found in other eukaryotic genes, suggesting the presence of a similar splicing apparatus in higher plants. Introns 1 and 3 interrupt the reading frame after codons 20 and 355, respectively. Intron 2 splits a glycine codon at position 151. None of these intron positions is conserved relative to the positions of introns in other actin genes examined.