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Changes in the myosin phenotype of differentiated muscle are a prominent feature of the adaptation of the tissue to a variety of physiological stimuli. In the present study the molecular basis of changes in the proportion of myosin isoenzymes in rat skeletal muscle which occur during compensatory hypertrophy caused by the combined removal of synergist muscles and spontaneous running exercise was investigated. The relative amounts of sarcomeric myosin heavy (MHC)- and light (MLC)-chain mRNAs in the plantaris (fast) and soleus (slow) muscles from rats was assessed with cDNA probes specific for different MHC and MLC genes. Changes in the proportion of specific MHC mRNA levels were in the same direction as, and of similar magnitude to, changes in the proportion of myosin isoenzymes encoded for by the mRNAs. No significant changes in the proportion of MLC proteins or mRNA were detected. However, high levels of MLC3 mRNA were measured in both normal and hypertrophied soleus muscles which contained only trace amounts of MLC3 protein. Small amounts of embryonic and neonatal MHC mRNAs were induced in both muscles during hypertrophy. We conclude that the change in the pattern of myosin isoenzymes during skeletal-muscle adaptation to work overload is a consequence of changes in specific MHC mRNA levels.  相似文献   

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The myosin heavy-chain (Mhc) gene of Drosophila is a single-copy gene from which four messenger RNAs are transcribed. Two of these mRNAs, CA-1 and CA-2, are expressed in all stages of development when Mhc mRNA is detected. The 3' ends of these mRNAs differ by alternate choice of poly(A) addition sites. Two additional Mhc mRNAs, CBA-1 and CBA-2, are detected only in midpupal to adult stages of development. The 3' ends of these mRNAs are alternately polyadenylated as the above mRNAs; however, these mRNAs contain an additional alternately spliced exon. We have used in situ hybridization to tissue sections to determine the tissue-specific expression of the alternately processed Mhc mRNAs. Four probes were used in the in situ hybridization experiments: one that detects all Mhc mRNAs, one that is specific for mRNA molecules polyadenylated at the downstream site 2, one that is specific for alternately spliced mRNAs containing the B exon, and one that is specific for Mhc mRNAs Ca-1 and CA-2. This last probe is an oligodeoxynucleotide, while the others are single-stranded RNA molecules synthesized in vitro. Our results demonstrate that the alternate splicing of Mhc mRNAs is muscle-cell-type-specific during pupal development, while the polydenylation site usage at the downstream site 2 is not muscle-cell-type-specific during either embryonic or pupal development.  相似文献   

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A high molecular-weight protein from Escherichia coli sharing structural homology at the protein level with a yeast heavy-chain myosin encoded by the MYO1 gene is described. This 180 kD protein (180-HMP) can be enriched in cell fractions following the procedure normally utilized for the purification of non-muscle myosins. In Western blots this protein cross-reacts with a monoclonal antibody against yeast heavy-chain myosin. Moreover, antibodies raised against the 180 kD protein cross-react with the yeast myosin and with a myosin heavy chain from chicken. Recognition by anti-180-HMP antibodies of an overexpressed fragment of yeast myosin encoded by MYO1 allows the localization of one of the shared epitopes to a specific region around the ATP binding site of the yeast myosin heavy chain. The existence of a high molecular-weight protein with structural similarity to myosin in E. coli raises the possibility that such a protein might generate the force required for movement in processes such as nucleoid segregation and cell division.  相似文献   

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The stoichiometry of the two heavy chains of myosin in smooth muscle was determined by electrophoresing extracts of native myosin and of dissociated myosin on sodium dodecyl sulfate (SDS) 4%-polyacrylamide gels. The slower migrating heavy chain was 3.6 times more abundant in toad stomach, 2.3 in rabbit myometrium, 2.0 in rat femoral artery, 1.3 in guinea pig ileum, 0.93 in pig trachea and 0.69 in human bronchus, than the more rapidly migrating chain. Both heavy chains were identified as smooth muscle myosin by immunoblotting using antibodies to smooth muscle and non-muscle myosin. The unequal proportion of heavy chains suggested the possibility of native isoforms of myosin comprised of heavy-chain homodimers. To test this, native myosin extracts wer electrophoresed on non-dissociating (pyrophosphate) gels. When each band was individually analysed on SDS-polyacrylamide gel the slowest was found to be filamin and the other bands were myosin in which the relative proportion of the heavy chains was unchanged from that found in the original tissue extracts. Since this is incompatible with either a heterodimeric or a homodimeric arrangement it suggests that pyrophosphate gel electrophoresis is incapable of separating putative isoforms of native myosin.  相似文献   

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The previously described rabbit 2.3-kilobase smooth muscle myosin haevy-chain (SMHCwt) promoter targets gene expression in transgenic animals to vascular smooth muscle cells (SMCs), including coronary arteries. Therefore, SMHCwt is thought to provide a promising tool for human gene therapy. In the present study, we examined tissue specificity and expression levels of wild-type and mutated SMHC promoters within the system of high-capacity adenoviral (hcAd) vectors. SMHCwt and a series of SMHC promoter deletion mutants, a triple promoter as well as a cytomegalovirus-SMHC hybrid promoter driving the enhanced green fluorescence protein (EGFP) reporter gene were transiently transfected into aortic SMCs. Fluorescence intensity was measured by flow cytometric analysis. Consecutively, hcAd vectors were constructed with the SMHCwt and the mutant promoter with the highest fluorescence activity. Levels of EGFP expression were determined after transduction of SMCs derived from human coronary arteries. For analysis of tissue specificity, embryonic stem (ES) cell-derived SMCs (ESdSMHCs) and cardiomyocytes, (ESdCMs) were used. In comparison with SMHCwt, only the SMHCdel94 mutant lacking a 94-bp GC-rich element revealed a 1.5-fold increased fluorescence activity. Transduction of primary SMCs of human coronary arteries with hcAd vectors confirmed an increased EGFP expression driven by the SMHCdel94 promoter. In ES-cell-derived embryoid bodies, SMHCwt was exclusively active in transduced ESdSMCs. In contrast, expression of SMHCdel94 was also found in ESdCMs and other nontarget cells of the embryoid body. The tissue-specific rabbit SMHCwt promoter seems to be suitable for adenoviral gene transfer in SMCs of human coronary arteries and deletion of a 94-bp negative cis-acting GC-rich element results in loss of specificity. These authors contributed equally to the study.  相似文献   

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A cDNA clone, labeled pFOD5, isolated from a fetal-rat skeletal-muscle cDNA library, has been characterized and found to contain sequences corresponding to a perinatal-specific skeletal myosin heavy-chain (MHC) mRNA. This MHC cDNA demonstrates a high degree of nucleotide- and amino acid-sequence conservation with other MHC genes, but its carboxyl-terminal peptide and 3'-untranslated region are highly divergent and specific for this gene. S1 nuclease mapping experiments have shown that the perinatal MHC gene represented by this cDNA clone is only transiently expressed during skeletal-muscle development. Perinatal MHC mRNA is first detected late in fetal life, reaches maximal levels of expression at the end of the first postnatal week, and is de-induced thereafter. Its levels are almost undetectable at 28 days of postnatal life. During fetal and early postnatal life, the expression of this perinatal gene in skeletal muscle overlaps with the expression of the embryonic MHC gene. After the first week of extrauterine life, this gene is coexpressed with two adult MHC genes. The transient expression of this perinatal MHC gene raises interesting questions about the physiological significance of the MHC transitions and offers an interesting model for the study of MHC gene regulation.  相似文献   

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A human myosin heavy-chain gene, cloned in gamma Charon 4A phage (and as a clone designated lambda gMHC-1), was shown to code for a cardiac myosin heavy chain of the beta-type. The 5' end of the 14,200-base-pair genomic DNA clone is located in the head region of the myosin chain. The 3' end was shown to extent to the COOH terminus and includes the 3'-nontranslated sequence of the corresponding mRNA. The identification of lambda gMHC-1 as coding for a cardiac beta-myosin heavy chain was achieved by heteroduplex mapping using genomic cardiac myosin heavy-chain DNA of rabbit as a probe and, furthermore, by DNA sequence analysis of three selected subregions of the clones DNA including the 3'-nontranslated sequence. It was demonstrated by the S1 nuclease protection technique that the beta-myosin heavy-chain gene is transcribed in human heart muscle. In addition, we have found by the same technique that it is also expressed in human skeletal muscle.  相似文献   

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The erect wing (ewg) locus of Drosophila melanogaster encodes a vital function important for the development of the nervous system and the indirect flight muscles. In order to understand the ewg function at a molecular level, cDNA clones were isolated. Sequence analysis of cDNAs revealed a single open reading frame (ORF) encoding a protein of 733 residues. The translational start for this ORF is a CTG codon. A 225-amino-acid region of this protein is 71% identical to the DNA binding region of the Strongylocentrotus purpuratus P3A2 DNA binding protein. Additionally, the ORF contains large acidic and basic domains characteristic of those in proteins involved in nuclear regulatory functions. Immunoblot analysis using polyclonal anti-EWG antisera generated against a bacterial fusion protein reveals a single, 116-kDa protein present throughout development, beginning at approximately stage 12 of embryogenesis, which is enriched in adult heads and absent from embryos carrying certain ewg alleles. Additionally, we show that EWG is localized specifically to the nuclei of virtually all embryonic neurons. Finally, a minigene consisting of an ewg cDNA under control of the hsp70 promoter can provide the ewg function in transgenic ewg mutant flies.  相似文献   

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The Sgs-4 glue protein gene of Drosophila is expressed only in third-instar larval salivary glands. Previous work suggests that a regulatory region lies 5' and remote to the gene, as indicated by a region of tissue-specific DNase I hypersensitivity and by underproducing mutants with DNA lesions in the hypersensitive region. Here we demonstrate by germ line transformation of cloned fragments containing Sgs-4 that the sequences between 840 bp 5' and 130 bp 3' to the gene are sufficient for Sgs-4 activity. When 5' sequence was removed to -392, activity was eliminated, thereby verifying the existence of essential sequences far upstream. Fragments that are active include, in addition to the capacity for normal levels of expression, three other cis-acting regulatory activities: developmental timing, tissue specificity, and dosage compensation. In contrast, the fragments tested did not specify formation of the puff with which Sgs-4 is normally associated. As shown by chromosomal rearrangements, the region required for puffing is limited to 16-19 kb surrounding the gene.  相似文献   

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A chimeric lambda DNA molecule containing the myosin alkali light-chain gene of Drosophila melanogaster was isolated. The encoded amino acid sequence was determined from the nucleic acid sequence of a cDNA homologous to the genomic clone. The identity of the encoded protein was established by two criteria: (i) sequence homology with the chicken alkali light-chain proteins and (ii) comparison of the two-dimensional gel electrophoretic pattern of the peptides synthesized by in vitro translation of hybrid-selected RNA to that of myosin alkali light-chain peptides extracted from Drosophila myofibrils. There is only one myosin alkali light-chain in D. melanogaster; its chromosomal location is region 98B . This gene is abundantly expressed during the development of larval as well as adult muscles. The Drosophila protein appears to contain one putative divalent cation-binding domain (an EF hand) as compared with the three EF hands present in chicken alkali light chains.  相似文献   

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Mesilaty-Gross S  Reich A  Motro B  Wides R 《Gene》1999,231(1-2):173-186
Drosophila STAM is a homolog of mammalian STAM genes, which encode Jak associated signal-transducing adapter molecules. A 20-kilobase stretch of genomic DNA at 32B on chromosome arm 2L, which contains Drosophila STAM, has been sequenced. By comparison to cDNAs isolated and characterized, this region contains four tightly clustered genes: ial, mitochondrial porin, and the two newly discovered genes, STAM and DNZ1. Like its mouse and human homologs, STAM bears SH3 and ITAM domains. DNZ1 is a founding member of a sub-family of proteins bearing a DHHC/NEW1 zinc finger domain. Although these four genes are contained in a defined Deficiency overlap interval, no available P-element mutations in the region disrupt any of the genes, and no other discrete mutations in the genes have been identified. Among the four genes, ial and STAM share a common 5' control region, suggesting coordinate expression. Developmental Northern data and embryonic and ovariole expression data show that STAM and ial expression are correlated. The other two genes in the cluster appear to be expressed at constitutive levels throughout development.  相似文献   

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Mutations in the Drosophila tolloid (tld) gene lead to a partial transformation of dorsal ectoderm into ventral ectoderm. The null phenotype of tld is similar to, but less severe than decapentaplegic (dpp), a TGF-beta family member required for the formation of all dorsal structures. We have cloned the tld locus by P element tagging. At the blastoderm stage, tld RNA is expressed dorsally, similar to that described for dpp. Analysis of a tld cDNA reveals three sequence motifs: an N terminal region of similarity to a metalloprotease, two EGF-like repeats, and five copies of a repeat found in human complement proteins C1r and C1s. tld sequence is 41% identical to human bone morphogenetic protein 1 (BMP-1); the closest members to dpp within the TGF-beta superfamily are BMP-2 and BMP-4, two other bone morphogenetic proteins. These findings suggest that these genes are members of a signal generating pathway that has been conserved between insects and mammals.  相似文献   

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Human heart failure is accompanied by repression of genes such as alpha myosin heavy chain (alphaMyHC) and SERCA2A and the induction of fetal genes such as betaMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human alphaMyHC promoter. We have now identified a region of the alphaMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human alphaMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases alphaMyHC mRNA expression and increases skeletal alpha-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the alphaMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human alphaMyHC promoter during heart failure.  相似文献   

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Cytochrome proteins perform a broad spectrum of biological functions ranging from oxidative metabolism to electron transport and are thus essential to all organisms. The b-type cytochrome proteins bind heme noncovalently, are expressed in many different forms and are localized to various cellular compartments. We report the characterization of the cytochrome b5 (Cyt-b) gene of Drosophila virilis and compare its structure to the Cyt-b gene of Drosophila melanogaster. As in D. melanogaster, the D. virilis gene is nuclear encoded and single copy. Although the intron/exon structures of these homologues differ, the Cyt-b proteins of D. melanogaster and D. virilis are approximately 75% identical and share the same size coding regions (1,242 nucleotides) and protein products (414 amino acids). The Drosophila Cyt-b proteins show sequence similarity to other b-type cytochromes, especially in the N-terminal heme-binding domain, and may be targeted to the mitochondrial membrane. The greatest levels of similarity are observed in areas of potential importance for protein structure and function. The exon sequences of the D. virilis Cyt-b gene differ by a total of 292 base changes. However, 62% of these changes are silent. The high degree of conservation between species separated by 60 million years of evolution in both the DNA and amino acid sequences suggests this nuclear cytochrome b5 locus encodes an essential product of the Drosophila system.Correspondence to: C.E. Rozek  相似文献   

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