scholarly journals Expression of transfected mutant beta-actin genes: transitions toward the stable tumorigenic state.

1987 ◽  
Vol 7 (7) ◽  
pp. 2467-2476 ◽  
Author(s):  
J Leavitt ◽  
S Y Ng ◽  
M Varma ◽  
G Latter ◽  
S Burbeck ◽  
...  
Keyword(s):  
Human Fibroblasts ◽  
Actin Gene ◽  
Wild Type ◽  
Transfected Cells ◽  
Actin Genes ◽  
Beta Actin ◽  
Normal Human ◽  
Actin Mrna ◽  
Elevated Rate ◽  
Actin Expression

Mutant human beta-actin genes were introduced into normal human (KD) fibroblasts and the derivative cell line HuT-12, which is immortalized but nontumorigenic, to test their ability to promote conversion to the tumorigenic state. Transfected substrains of HuT-12 fibroblasts that expressed abundant levels of mutant beta-actin (Gly-244----Asp-244) produced subcutaneous tumors in athymic mice after long latent periods (1.5 to 3 months). However, transfected substrains of KD fibroblasts retained their normal finite life span in culture and consequently were incapable of producing tumors. Substrains of HuT-12 cells transfected with the wild-type beta-actin gene and some transfected strains that expressed low or undetectable levels of mutant beta-actin did not produce tumors. Cell lines derived from transfectant cell tumors always exhibited elevated synthesis of the mutant beta-actin, ranging from 145 to 476% of the level expressed by the transfected cells that were inoculated to form the tumor. In general, primary transfectant cells that expressed the highest levels of mutant beta-actin were more tumorigenic than strains that expressed lower levels. The tumor-derived strains were stable in tumorigenicity and produced tumors with shortened latent periods of only 2 to 4 weeks. These findings imply that the primary transfectant strains develop subpopulations of cells that are selected to form tumors because of their elevated rate of exogenous mutant beta-actin synthesis. Actin synthesis and accumulation of gamma-actin mRNA from the endogenous beta- and gamma-actin genes were diminished in tumor-derived strains, apparently to compensate for elevated mutant beta-actin synthesis and maintain the normal cellular concentration of actin. Synthesis of the transformation-sensitive tropomyosin isoforms was decreased along with mutant beta-actin expression. Such modulations in tropomyosin synthesis are characteristically seen in transformation of avian, rodent, and human fibroblasts. Our results suggest that this mutant beta-actin contributes to the neoplastic phenotype of immortalized human fibroblasts by imposing a cytoarchitectural defect and inducing abnormal expression of cytoskeletal tropomyosins.

Expression of transfected mutant beta-actin genes: transitions toward the stable tumorigenic state

1987 ◽  
Vol 7 (7) ◽  
pp. 2467-2476
Author(s):  
J Leavitt ◽  
S Y Ng ◽  
M Varma ◽  
G Latter ◽  
S Burbeck ◽  
...  
Keyword(s):  
Human Fibroblasts ◽  
Actin Gene ◽  
Wild Type ◽  
Transfected Cells ◽  
Actin Genes ◽  
Beta Actin ◽  
Normal Human ◽  
Actin Mrna ◽  
Elevated Rate ◽  
Actin Expression

Mutant human beta-actin genes were introduced into normal human (KD) fibroblasts and the derivative cell line HuT-12, which is immortalized but nontumorigenic, to test their ability to promote conversion to the tumorigenic state. Transfected substrains of HuT-12 fibroblasts that expressed abundant levels of mutant beta-actin (Gly-244----Asp-244) produced subcutaneous tumors in athymic mice after long latent periods (1.5 to 3 months). However, transfected substrains of KD fibroblasts retained their normal finite life span in culture and consequently were incapable of producing tumors. Substrains of HuT-12 cells transfected with the wild-type beta-actin gene and some transfected strains that expressed low or undetectable levels of mutant beta-actin did not produce tumors. Cell lines derived from transfectant cell tumors always exhibited elevated synthesis of the mutant beta-actin, ranging from 145 to 476% of the level expressed by the transfected cells that were inoculated to form the tumor. In general, primary transfectant cells that expressed the highest levels of mutant beta-actin were more tumorigenic than strains that expressed lower levels. The tumor-derived strains were stable in tumorigenicity and produced tumors with shortened latent periods of only 2 to 4 weeks. These findings imply that the primary transfectant strains develop subpopulations of cells that are selected to form tumors because of their elevated rate of exogenous mutant beta-actin synthesis. Actin synthesis and accumulation of gamma-actin mRNA from the endogenous beta- and gamma-actin genes were diminished in tumor-derived strains, apparently to compensate for elevated mutant beta-actin synthesis and maintain the normal cellular concentration of actin. Synthesis of the transformation-sensitive tropomyosin isoforms was decreased along with mutant beta-actin expression. Such modulations in tropomyosin synthesis are characteristically seen in transformation of avian, rodent, and human fibroblasts. Our results suggest that this mutant beta-actin contributes to the neoplastic phenotype of immortalized human fibroblasts by imposing a cytoarchitectural defect and inducing abnormal expression of cytoskeletal tropomyosins.

Molecular cloning and characterization of mutant and wild-type human beta-actin genes

1984 ◽  
Vol 4 (10) ◽  
pp. 1961-1969
Author(s):  
J Leavitt ◽  
P Gunning ◽  
P Porreca ◽  
S Y Ng ◽  
C S Lin ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Human Fibroblasts ◽  
Actin Gene ◽  
Actin Genes ◽  
Beta Actin ◽  
Gene Libraries ◽  
Actin Mrna ◽  
Dna Fragment ◽  
Specific Sequences

There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.

scholarly journals Molecular cloning and characterization of mutant and wild-type human beta-actin genes.

1984 ◽  
Vol 4 (10) ◽  
pp. 1961-1969 ◽  
Author(s):  
J Leavitt ◽  
P Gunning ◽  
P Porreca ◽  
S Y Ng ◽  
C S Lin ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Human Fibroblasts ◽  
Actin Gene ◽  
Actin Genes ◽  
Beta Actin ◽  
Gene Libraries ◽  
Actin Mrna ◽  
Dna Fragment ◽  
Specific Sequences

There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.

scholarly journals Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

1992 ◽  
Vol 117 (4) ◽  
pp. 787-797 ◽  
Author(s):  
C Lloyd ◽  
G Schevzov ◽  
P Gunning
Keyword(s):  
Actin Cytoskeleton ◽  
Feedback Regulation ◽  
Cytochalasin D ◽  
Actin Gene ◽  
Down Regulation ◽  
Actin Genes ◽  
Beta Actin ◽  
Actin Mrna ◽  
Actin Protein

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.

An amphibian cytoskeletal-type actin gene is expressed exclusively in muscle tissue

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 393-402 ◽  
Author(s):  
T.J. Mohun ◽  
N. Garrett
Keyword(s):  
Nucleotide Sequence ◽  
Protein Isoforms ◽  
Actin Gene ◽  
Promoter Regions ◽  
Conserved Sequence ◽  
Beta Actin ◽  
Genes Encoding ◽  
Actin Mrna ◽  
Ccaat Box ◽  
Equivalent Region

The complete nucleotide sequence of two Xenopus actin genes encoding cytoskeletal protein isoforms has been determined. Transcripts from these genes are remarkably similar in nucleotide sequence throughout their length and code for type-5 and type-8 cytoskeletal actins. Both share some sequence homology with human gamma-actin mRNA within the 3′ untranslated region but none with the equivalent region of any vertebrate beta-actin transcript. The promoter regions of the two Xenopus genes are virtually identical from the cap site to the CCAAT box and show extensive homology further upstream. Despite such similarity, the two genes are divergently expressed during embryonic development. The type-5 actin gene is expressed in all regions of the developing embryo whilst the type-8 gene is coregulated with the muscle-specific skeletal actin gene. In common with mammalian and avian cytoskeletal actin counterparts, the Xenopus genes possess a conserved sequence within their promoter that has previously been identified as a transcription-factor-binding site.

scholarly journals Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation.

1988 ◽  
Vol 107 (6) ◽  
pp. 2575-2586 ◽  
Author(s):  
D L Ruzicka ◽  
R J Schwartz
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Cardiac Cells ◽  
Actin Gene ◽  
Heart Tube ◽  
Specific Expression ◽  
Actin Genes ◽  
Beta Actin ◽  
Smooth Muscle Alpha Actin ◽  
Alpha Actin

The expression of cytoplasmic beta-actin and cardiac, skeletal, and smooth muscle alpha-actins during early avian cardiogenesis was analyzed by in situ hybridization with mRNA-specific single-stranded DNA probes. The cytoplasmic beta-actin gene was ubiquitously expressed in the early chicken embryo. In contrast, the alpha-actin genes were sequentially activated in avian cardiac tissue during the early stages of heart tube formation. The accumulation of large quantities of smooth muscle alpha-actin transcripts in epimyocardial cells preceded the expression of the sarcomeric alpha-actin genes. The accumulation of skeletal alpha-actin mRNAs in the developing heart lagged behind that of cardiac alpha-actin by several embryonic stages. At Hamburger-Hamilton stage 12, the smooth muscle alpha-actin gene was selectively down-regulated in the heart such that only the conus, which subsequently participates in the formation of the vascular trunks, continued to express this gene. This modulation in smooth muscle alpha-actin gene expression correlated with the beginning of coexpression of sarcomeric alpha-actin transcripts in the epimyocardium and the onset of circulation in the embryo. The specific expression of the vascular smooth muscle alpha-actin gene marks the onset of differentiation of cardiac cells and represents the first demonstration of coexpression of both smooth muscle and striated alpha-actin genes within myogenic cells.

Structure and transcription of the actin gene of Trypanosoma brucei

1988 ◽  
Vol 8 (5) ◽  
pp. 2166-2176 ◽  
Author(s):  
M F Ben Amar ◽  
A Pays ◽  
P Tebabi ◽  
B Dero ◽  
T Seebeck ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Splice Site ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Actin Gene ◽  
S1 Nuclease ◽  
Actin Genes ◽  
Gene Copies ◽  
Actin Mrna

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.

scholarly journals 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.

1988 ◽  
Vol 106 (6) ◽  
pp. 2077-2086 ◽  
Author(s):  
L J Hayward ◽  
Y Y Zhu ◽  
R J Schwartz
Keyword(s):  
Cellular Localization ◽  
Myoblast Fusion ◽  
Bipolar Cells ◽  
Gene Probe ◽  
Actin Gene ◽  
Cardiac Actin ◽  
Beta Actin ◽  
Hybridization Probes ◽  
Muscle Cultures ◽  
Actin Mrna

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.

scholarly journals High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture

1992 ◽  
Vol 117 (4) ◽  
pp. 775-785 ◽  
Author(s):  
G Schevzov ◽  
C Lloyd ◽  
P Gunning
Keyword(s):  
Cell Surface ◽  
Surface Area ◽  
Mutant Form ◽  
Actin Gene ◽  
High Level Expression ◽  
Cell Surface Area ◽  
Actin Genes ◽  
Beta Actin ◽  
High Level ◽  
Actin Protein

The impact of the human beta- and gamma-actin genes on myoblast cytoarchitecture was examined by their stable transfection into mouse C2 myoblasts. Transfectant C2 clones expressing high levels of human beta-actin displayed increases in cell surface area. In contrast, C2 clones with high levels of human gamma-actin expression showed decreases in cell surface area. The changes in cell morphology were accompanied by changes in actin stress-fiber organization. The beta-actin transfectants displayed well-defined filamentous organization of actin; whereas the gamma-actin transfectants displayed a more diffuse organization of the actin cables. The role of the beta-actin protein in generating the enlarged cell phenotype was examined by transfecting a mutant form of the human beta-actin gene. Transfectant cells were shown to incorporate the aberrant actin protein into stress-fiber-like structures. High level expression of the mutant beta-actin produced decreases in cell surface area and disruption of the actin microfilament network similar to that seen with transfection of the gamma-actin gene. In contrast, transfection of another mutant form of the beta-actin gene which encodes an unstable protein had no impact on cell morphology or cytoarchitecture. These results strongly suggest that it is the nature of the encoded protein that determines the morphological response of the cell. We conclude that the relative gene expression of beta- and gamma-actin is of relevance to the control of myoblast cytoarchitecture. In particular, we conclude that the beta- and gamma-actin genes encode functionally distinct cytoarchitectural information.

scholarly journals alpha-skeletal and alpha-cardiac actin genes are coexpressed in adult human skeletal muscle and heart.

1983 ◽  
Vol 3 (11) ◽  
pp. 1985-1995 ◽  
Author(s):  
P Gunning ◽  
P Ponte ◽  
H Blau ◽  
L Kedes
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Human Skeletal Muscle ◽  
Cdna Clones ◽  
Actin Gene ◽  
Adult Heart ◽  
Adult Human ◽  
Actin Genes ◽  
Cardiac Actin ◽  
Actin Mrna

We 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 alpha-skeletal, beta- and gamma-actin cDNAs and from an alpha-cardiac actin genomic clone, we showed that 28 of the cDNAs correspond to alpha-skeletal actin transcripts. Unexpectedly, however, the remaining two cDNA clones proved to derive from alpha-cardiac actin mRNA. Sequence analysis confirmed that the two skeletal muscle alpha-cardiac actin cDNAs are derived from transcripts of the cloned alpha-cardiac actin gene. Direct measurements of actin isotype mRNA expression in human skeletal muscle showed that alpha-cardiac actin mRNA is expressed at 5% the level of alpha-skeletal actin. Furthermore, the alpha-cardiac actin gene expressed in skeletal muscle is the same gene which produces alpha-cardiac actin mRNA in the human heart. Of equal surprise, we found that alpha-skeletal actin mRNA accounts for about half of the total actin mRNA in adult heart. 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. We conclude that alpha-skeletal and 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, we postulate that coexpression of actin pairs may be a common feature of mammalian actin gene expression in all tissues.

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