Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.     

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

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

Changes in levels of actin and tubulin mRNAs upon the lectin activation of lymphocytes.

The expression of beta-actin, gamma-actin, alpha-tubulin, and beta-tubulin mRNA during the lectin activation of human peripheral blood lymphocytes was examined with specific cDNA clones. The resting lymphocyte has a low level of both alpha- and beta-tubulin mRNAs, and these increase 10-fold after 72 h of lectin stimulation in which maximum cell transformation is achieved. Although there is a slight increase in tubulin mRNA during the first 6 h, most of the increase occurs between 6 and 24 h as the cells start to increase their RNA content and progress from G0 into G1. Both beta- and gamma-actin mRNAs are more abundant than the tubulin mRNAs in resting cells, with beta-actin mRNA being the major species. Upon activation, beta-actin mRNA increases threefold, whereas gamma-actin mRNA increases almost sixfold. Both beta- and gamma-actin mRNA are elevated 2.5-fold as early as 6 h, the gamma-actin mRNA level then increasing more than beta-actin between 6 and 24 h, resulting in the reduced beta-actin/gamma-actin mRNA ratio. The lectin-stimulated lymphocyte has a similar beta-actin/gamma-actin mRNA ratio as that of the human leukemic T-lymphoblast cell line CCRF-CEM. These increases are over and above the general increase in polyadenylated RNA content upon lectin activation. On returning to a noncycling state, the levels of these cytoskeletal mRNAs decrease. There were two beta-tubulin mRNAs present in lymphocyte cytoplasm, one of 1.8 kilobases and one of 2.8 kilobases in length. The nongrowing lymphocytes had relatively lower levels of the larger sized mRNA. Upon stimulation, the relative level of the larger mRNA was increased, and at 72 h the cells had approximately equal levels of both mRNAs as did the leukemic lymphoblasts.     

Amounts and modulation of actin mRNAs in mouse oocytes and embryos

In order to measure the content of beta- and gamma-actin mRNA in mouse oocytes and ovulated eggs, Northern and slot blots were hybridized to complementary RNA probes transcribed from mouse isotype-specific cDNA sequences. The blots included samples of isotype-specific sense strand RNA standards prepared from the same cDNA sequences. Total actin mRNA content was estimated to be 40 fg per preovulatory full-grown oocyte or egg, consisting of one-third beta-actin mRNA and two-thirds gamma-actin mRNA. Ninety per cent of the actin mRNA is on polysomes in full-grown oocytes. The per cent of actin mRNA in polysomal mRNA is similar to the per cent of actin in newly synthesized proteins. Measurements on other developmental stages showed that, in mid-growth-phase oocytes, each actin mRNA reaches a level twofold higher than in full-grown oocytes. Thereafter, all modulations of the two isotypic mRNAs occur in parallel; that is, they are maintained at constant levels during the late growth phase (oocytes from females 8-14 days old); gradually degraded in oocytes that have completed their rapid growth phase (oocytes from females 15-18 days old), in maturing oocytes, and in 1- and 2-cell embryos; and deadenylated after about 7 h of progression into meiotic maturation.     

Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.     

Mouse cytoskeletal gamma-actin: analysis and implications of the structure of cloned cDNA and processed pseudogenes

The nucleotide sequence corresponding to almost the whole of a mouse gamma-cytoskeletal actin mRNA was determined from overlapping cloned DNA copies derived from brain mRNA. Several gamma-actin processed pseudogenes were isolated from a library of cloned DBA mouse genomic DNA, and the nucleotide sequences of these were determined and compared with that of the cDNA. This showed that two of these pseudogenes had arisen from a gene duplication or amplification event, and indicated that they had subsequently undergone partial correction against one another. The relative ages of the pseudogenes were estimated on the basis of their percentage divergence from the cDNA sequence and these were compared with an estimation based on the number of presumed silent mutations in the cDNA since each pseudogene had arisen. Consistent results were obtained, except in the case of one pseudogene which also showed an anomalous regional distribution of differences from the cDNA sequence. One way of accounting for the features of this anomalous pseudogene is by postulating that it is derived from a second functional gene for gamma-actin, different from that represented by the cDNA described here.     

Modulation of microfilament protein composition by transfected cytoskeletal actin genes.

HuT-14T is a highly tumorigenic fibroblast cell line which exhibits a reduced steady-state level of beta-actin due to coding mutations in one of two beta-actin alleles. The normal rate of total actin synthesis could be restored in some clones of cells following transfection of the functional beta-actin gene but not following transfection of the functional gamma-actin gene. In gamma-actin gene-transfected substrains that have increased rates of gamma-actin synthesis, beta-actin synthesis is further reduced in a manner consistent with an autoregulatory mechanism, resulting in abnormal ratios of actin isoforms. Thus, both beta- and gamma-actin proteins can apparently regulate the synthesis of their coexpressed isoforms. In addition, decreased synthesis of normal beta-actin seems to correlate with a concomitant down-regulation of tropomyosin isoforms.     

Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.

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.     

CTGF/Hcs24 interacts with the cytoskeletal protein actin in chondrocytes

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) displays multiple functions in several types of mesenchymal cells, including the promotion of proliferation and differentiation of chondrocytes. Recently, the internalization and intracellular function of CTGF/Hcs24 were indicated as well. In this study, a binding protein for this factor was purified from the cytosolic fraction of human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by CTGF/Hcs24-affinity chromatography. The apparent molecular weight of the protein was 42kDa and determination of the internal amino acid sequence revealed this protein to be beta- or gamma-actin. An in vitro competitive binding assay of 125I-labeled recombinant CTGF/Hcs24 with cold-rCTGF/Hcs24 showed that the binding between actin and 125I-CTGF/Hcs24 was specific. Immunoprecipitation analysis also showed that CTGF/Hcs24 bound to actin in HCS-2/8 cells. However, rCTGF/Hcs24 had no effects on the expression level of gamma-actin mRNA or total actin protein. These findings suggest that a significant portion of intracellular CTGF/Hcs24 may regulate certain cell biological events in chondrocytes through the interaction with this particular cytoskeletal protein.     

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

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.     

Absence of gamma-actin expression in the mouse fibroblast cell line, L

Cytoplasmic isoactins of mouse fibroblast L-cells were examined by two-dimensional gel electrophoresis. In contrast to other cultured cell lines, which contain both beta- and gamma-actin, L-cells contained only beta-actin species. This unique phenomenon was due neither to the transformed status of this cell line nor to the characteristic nature of adipose tissue fibroblasts of C3H mice. When RNA of L-cells was translated in a nuclease-treated reticulocyte lysate system, actin synthesized in vitro also appeared as only one species, beta-actin. Therefore, the genetic information for gamma-actin is absent at the level of translatable mRNA in L-cells.     

A unique structure of a mouse gamma-actin processed pseudogene

We have isolated several gamma-actin-related genes from a mouse genomic library. One of these has been shown to be a gamma-actin processed pseudogene (Tokunga, K., Yoda, K. and Sakiyama, S. (1985) Nucleic Acids Res. 13, 3031-3042). Here, we report the structure of another pseudogene (pMA131). pMA131 contained the sequences corresponding to the carboxyl half of a cytoskeletal actin in which random point mutations as well as insertion and deletion events took place. This region was flanked at its 5' end by the sequences related to mouse repetitive sequences, including the MIF-1 family, and was interrupted by the sequence homologous to the R family which is also a mouse repetitive sequence. The coding region was followed by the sequence corresponding to 3' untranslated region of gamma-actin mRNA.     

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

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.     

Expression of transfected mutant beta-actin genes: alterations of cell morphology and evidence for autoregulation in actin pools.

Two different mutant human beta-actin genes have been introduced into normal diploid human (KD) fibroblasts and their immortalized derivative cell line, HuT-12, to assess the impact of an abnormal cytoskeletal protein on cellular phenotypes such as morphology, growth characteristics, and properties relating to the neoplastic phenotype. A mutant beta-actin containing a single mutation (Gly-244----Asp-244) was stable and was incorporated into cytoskeletal stress fibers. Transfected KD cells which expressed the stable mutant beta-actin in excess of normal beta-actin were morphologically altered. In contrast, a second mutant beta-actin gene containing two additional mutations (Gly-36----Glu-36 and Glu-83----Asp-83, as well as Gly-244----Asp-244) did not alter cell morphology when expressed at high levels in transfected cells, but the protein was labile and did not accumulate in stress fibers. In both KD and HuT-12 cells, endogenous beta- and gamma-actin decreased in response to high-level expression of the stable mutant beta-actin, in a manner consistent with autoregulatory feedback of actin concentrations. Since the percent decreases in the endogenous beta- and gamma-actins were equal, the ratio of net beta-actin (mutant plus normal) to gamma-actin was significantly increased in the transfected cells. Antisera capable of distinguishing the mutant from the normal epitope revealed that the mutant beta-actin accumulated in stress fibers but did not participate in the formation of the actin filament-rich perinuclear network. These observations suggest that different intracellular locations differentially incorporate actin into cytoskeletal microfilaments. The dramatic impact on cell morphology and on beta-actin/gamma-actin ratios in the transfected diploid KD cells may be related to the acquisition of some of the characteristics of cells that underwent the neoplastic transformation event that originally led to the appearance of the beta-actin mutations.     

Mutated-gamma-actin restores growth to a yeast amino acid transport defective mutant

A mutated yeast cell 22574d lacking all three proline transporters, PUT4, UGA4, and GAP1, and incapable of growth on proline recovers its lost ability to grow on proline as sole nitrogen source when transformed with a mutagenized mouse gamma-actin cDNA (M-gamma-A). Native mouse gamma-actin cDNA is ineffective. The 3'-region of gamma-actin cDNA was mutagenized to resemble E51 cDNA previously isolated from Ehrlich tumor cells. The E51 cDNA has an extended reading frame in the 3'-region compared to that in native gamma-actin. The extension of the open reading frame in E51 cDNA, was found to be due to an additional pair of bases (TG) at position 1104 of E51 cDNA. After site-directed mutagenesis of the 3'-region of native gamma-actin cDNA to resemble that of E51 cDNA, the construct, M-gamma-A cDNA, was expressed in the 22574d yeast. While the transformation with M-gamma-A increased the uptake of both proline and gamma-amino butyric acid, the transport of five other solutes was not changed by this transformation. Northern blotting of the nontransformed and the M-gamma-A-transformed 22574d cells with gene-specific probes for the three proline transporters showed the expression of an mRNA for UGA4 in both transformed and nontransformed cells but no evidence for the expression of GAP1 or PUT4. The mRNA for UGA4 was expressed at a lower level in strain 22574d than in the parent yeast sigma1278b. Furthermore, the message in the mutated cells is smaller in size by about 15%. These results are consistent with the synthesis of a mutated transporter which requires the coexpression of M-gamma-A, but not native gamma-actin, to restore physiological function, i.e., proline or gamma-amino acid transport.     

Differences in expression of a variant actin between low and high metastatic B16 melanoma

The polypeptides of mouse B16 melanoma lines of defined metastatic potential have been analyzed by two-dimensional electrophoresis. Parent B16 melanoma and two independently isolated B16-F1 lines, which are low metastatic, exhibited a new polypeptide, Ax (pI 5.2; Mr = 43,000), comprising approximately 30% of the total actin, in addition to normal beta- and gamma-actin. The Ax is present in the Triton-insoluble fraction (cytoskeleton and nuclear matrix) as well as in the Triton-soluble fraction at a constant ratio of about 0.5 to beta- plus gamma-actin. The Ax polypeptide has been identified as a variant form of actin by immunostaining with anti-actin antibody and by a comparison of its tryptic patterns with those produced by beta- and gamma-actin polypeptides; the Ax is also identified as a component of microfilaments. On the other hand, the Ax polypeptide disappears or its expression is very low in high metastatic lines, two independently isolated B16-F10s and B16-BL6. By in vitro translation, we have identified the mRNA species that code for Ax in B16-F1, but not in B16-F10.     

Noncoding regions of the gamma-actin gene influence the impact of the gene on myoblast morphology

We have addressed the question of whether two highly conserved noncoding regions of the gamma-actin gene are of functional importance. Human gamma-actin gene constructs deleted for either the entire 3' untranslated region (UTR) and 3' flank or intron III sequences were transfected into mouse myoblasts and the resulting clones were analyzed for cell morphology and actin protein expression. Transfectants carrying the gamma-actin gene deleted for the 3' end (gamma 22) exhibited numerous long pseudopods and increased surface area. In contrast, transfectants expressing the gamma-actin gene deleted for intron III (gamma 156) were rounded with blebs over the cell surface and showed decreased surface area. The relative expression of beta- to gamma-actin protein decreased for both transfectant types. The total actin protein levels remained constant for the gamma 22 cells but decreased for the gamma 156 cells. The results indicate that alterations to transfectant cell morphology can be influenced by the presence or absence of different noncoding regions in the transfected gamma-actin gene. The mechanisms by which noncoding regions of the gamma-actin gene influence the impact of the gene are unknown. Nevertheless, these noncoding regions are isoform specific and highly conserved in evolution. We propose that the functional significance of the different actin isoforms may involve the properties of these noncoding regions in addition to the differences in protein sequence.     

Reorganization of cytoskeletal proteins of mouse oocytes mediated by integrins

To study whether integrins on cell membrane ligate with intracellular cytoskeletal proteins and mediate their reorganization in egg activation, female mice were used for superovulation. The zona-free oocytes were incubated separately with specific ligand of integrins,an active RGD peptide, in vitro for certain period of time. RGE peptide and mouse capacitated sperm were used as controls. Freshly ovulated oocytes and those treated with different factors were immunostained with FITC-labeled anti-actin antibody, then detected with confocal microscope. The results demonstrated that freshly ovulated mouse oocytes, oocytes incubated for 2 h in vitro and those treated with control RGE peptide for 15 min showed hardly visible fluorescene or only thin fluorescence in plasma membrane region. Oocytes coincubated with sperms for 15 min and those treated with active RGD peptide for 10 min, 30 min and 2 hours respectively had strong and thick fluorescence in the plasma membrane and cortical region of oocytes, and some of them showed asymmetrically fluorescent distribution. It is proved that integrins on membrane are ligated directly with cytoskeletal protein. Integrins binding with their ligands regulate reorganization of cytoskelal protein, which may be involved in transmembrane signaling in egg activation.     

Reorganization of cytoskeletal proteins of mouse oocytes mediated by integrins

Integrins, as transmembrane signalling receptors,initiate a series of events of intracellular signal trans-duction by ligating with their ligands. In the process ofintegrin-mediated transmembrane signal transduction,the roles of intracellular cytoskeletal proteins havebeen described in many types of cells[1—4]. On thebasis of the well-documented investigations, Clark andhis colleagues raised the functional pattern of integrinsmediating transmembrane signal transduction. It issuggested that th…