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Three myosin heavy chain isoforms in human smooth muscle
J M o l Cell C a r d i o l 19 ( S u p p l e m e n t IV) (1987)
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THREE MYOSIN HEAVY CHAIN ISOFORMS IN HUMAN SMOOTH MUSCLE. S. Sertore*, N. Di + . .+ . . . Marzo , L. Sag~in*, L. Fabbrz and S. Schzafftno*. *Institute of General Pathology and Institute of Occupational Medicine, University of Padova, Padova, Italy. Surgical specimens of human smooth muscle, including arteries and veins, uterus, taenia ooli, bronchi and lung parenchimsl tissue, were analyzed by SDS PAGE and immunoblotting with policlonal and monoclonal antibodies reactive with smooth muscle myosin heavy chain. In 5 ~ gels the smooth muscle extracts yielded three bands, designated MHC-1, MHC-2 and MHC-3 in terms of increasing electrophoretic mobility. The three MHC isoforms were labeled on immunoblots with BF-48, a monoclonal antibody obtained from mice immunized with bovine fetal skeletal myosin. This antibody does not reset with MHCa from human skeletal and cardiac muscle and does not recognize human platelet MHC. In contrast, a polyclonal antibody raised against bovine eorta reacted only with MHC-1 and MHC-2. Degradation time course experiments confirm that MHC-3 is not a proteolytic product of MHC-1 or MHC-2. The distribution of the three MHCs was found to vary in different smooth muscles : MHC-2 was more abundant in arteries than in veins and MHC-3 was more abundant in lung parenchimal strips
compared to other t i s s u e s .
1 1 MOLECULAR CLONING ANALYSIS OF A MYOSIN HEAVY CHAIN EXPRESSED IN VASCULAR AND NON-VASCULAR SMOOTH MUSCLE TYPES. Ryozo Nagai, David M. Larson and Muthu Periasamy, Department of Physiology and Biophysics, University of Vermont, Burlington, VT 05405. We have isolated and sequenced eDNA clones encoding light meromyosin (LMM) of a rabbit smooth muscle myosin heavy chain (MHC). Comparisons of nueleotide sequences in this region with rabbit skeletal and nematode MHC showed 47.8% and 43.5% homology, suggesting that smooth and sareomeric MHC genes diverged from a common ancestor. Amino acid sequences of rabbit smooth muscle MHC share approximately 30% homology with LMM of rabbit and rat skeletal muscle. Interestingly, the L M M o f smooth muscle MHC shows very similar periodic distributions of hydrophobic and charged residues as sarcomeric MHC together with a high potential for s-helical formation, indicating an s-helical coiled-coil structure throughout the region. We have found that this smooth muscle MHC gene is expressed in abundance both in vascular and nonvascular smooth muscle tissues by SI nuclease mapping analysis. Southern blot analysis of chromosomal DNA and SI nuclease mapping indicate the smooth muscle MIIC's are encoded by a multigene family as known for sarcomerie MHC's. (Supported by PHS R29-38355)
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THE SMOOTH MUSCLE MYOSIN REGULATORY LIGHT CHAIN IS A MEMBER OF A MULTI-GENE FAMILY. James W. Grant, Mark B. Taubman and Bernardo Nadal-Ginard Departmeut of Cardiology. Children's Hospital and Howard Hughes Medical Institute, Boston Ma. The 20kD myosin regulatory light chain (RLC) is a highly conserved and essential protein for smooth muscle and non-muscle contraction. We have previously shown that a eDNA (),cSMRLC) isolated fi'om rat aortic smooth muscle cells encodes a protein which is 95% homologous to the chicken gizzard myosin regulatory light chain. and. by S1 nuclease analysis is present in non-muscle, sarcomeric and smooth muscle tissues and cell lines. In order to elucidate the genetics of this protein, a rat liver genomic library was screened with the nick-translated XcSMRLC. Overlapping clones covering approximately 40kb were isolated from a rat lix,er genomic library. Restriction mapping revealed 10kb (XG-BAMI0) and 3kb (XG-BAM3) BamHl fragments were >90% homologous to the e D N A by high stringency washes. These fi'agments are separated by approximately 15kb. Sequencing of XGBAMI0 has revealed that it contains the gene that encodes XcSMRLC. The gene is organized into 4 exons with a 4kb introo located within the 5"untranslated sequence. The amino acid coding region is divided by 2 iutrons: the COOH terminal region and the 3'untranslated region are located in a single exon. Southern blots of rat total genomic DNA hybridized to a probe generated from the the last 93 bases of the 3"untranslated sequence and 154 bases of 3" flanking DNA of XG-BAM10 demonstrates a unique band corresponding to XG-BAMI0. Hybridization of the same blots to ),cSMRLC confirmed the presence of both the 3kb and 10kb bands. This data demonstrates the presence of at least 2 genomic sequences corresponding to the SMRLC and suggests that 2 populations of m R N A could exist with differing noncoding regions. Examination of the exact sequence of the 3kb fi'agment will permit generation of specific probes to examine tissue and developmental specific expression and delineation of modes of genetic control of the smooth muscle contractile pbenotype.
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THREE MYOSIN HEAVY CHAIN ISOFORMS IN HUMAN SMOOTH MUSCLE. S. Sertore*, N. Di + . .+ . . . Marzo , L. Sag~in*, L. Fabbrz and S. Schzafftno*. *Institute of General Pathology and Institute of Occupational Medicine, University of Padova, Padova, Italy. Surgical specimens of human smooth muscle, including arteries and veins, uterus, taenia ooli, bronchi and lung parenchimsl tissue, were analyzed by SDS PAGE and immunoblotting with policlonal and monoclonal antibodies reactive with smooth muscle myosin heavy chain. In 5 ~ gels the smooth muscle extracts yielded three bands, designated MHC-1, MHC-2 and MHC-3 in terms of increasing electrophoretic mobility. The three MHC isoforms were labeled on immunoblots with BF-48, a monoclonal antibody obtained from mice immunized with bovine fetal skeletal myosin. This antibody does not reset with MHCa from human skeletal and cardiac muscle and does not recognize human platelet MHC. In contrast, a polyclonal antibody raised against bovine eorta reacted only with MHC-1 and MHC-2. Degradation time course experiments confirm that MHC-3 is not a proteolytic product of MHC-1 or MHC-2. The distribution of the three MHCs was found to vary in different smooth muscles : MHC-2 was more abundant in arteries than in veins and MHC-3 was more abundant in lung parenchimal strips
compared to other t i s s u e s .
1 1 MOLECULAR CLONING ANALYSIS OF A MYOSIN HEAVY CHAIN EXPRESSED IN VASCULAR AND NON-VASCULAR SMOOTH MUSCLE TYPES. Ryozo Nagai, David M. Larson and Muthu Periasamy, Department of Physiology and Biophysics, University of Vermont, Burlington, VT 05405. We have isolated and sequenced eDNA clones encoding light meromyosin (LMM) of a rabbit smooth muscle myosin heavy chain (MHC). Comparisons of nueleotide sequences in this region with rabbit skeletal and nematode MHC showed 47.8% and 43.5% homology, suggesting that smooth and sareomeric MHC genes diverged from a common ancestor. Amino acid sequences of rabbit smooth muscle MHC share approximately 30% homology with LMM of rabbit and rat skeletal muscle. Interestingly, the L M M o f smooth muscle MHC shows very similar periodic distributions of hydrophobic and charged residues as sarcomeric MHC together with a high potential for s-helical formation, indicating an s-helical coiled-coil structure throughout the region. We have found that this smooth muscle MHC gene is expressed in abundance both in vascular and nonvascular smooth muscle tissues by SI nuclease mapping analysis. Southern blot analysis of chromosomal DNA and SI nuclease mapping indicate the smooth muscle MIIC's are encoded by a multigene family as known for sarcomerie MHC's. (Supported by PHS R29-38355)
12
THE SMOOTH MUSCLE MYOSIN REGULATORY LIGHT CHAIN IS A MEMBER OF A MULTI-GENE FAMILY. James W. Grant, Mark B. Taubman and Bernardo Nadal-Ginard Departmeut of Cardiology. Children's Hospital and Howard Hughes Medical Institute, Boston Ma. The 20kD myosin regulatory light chain (RLC) is a highly conserved and essential protein for smooth muscle and non-muscle contraction. We have previously shown that a eDNA (),cSMRLC) isolated fi'om rat aortic smooth muscle cells encodes a protein which is 95% homologous to the chicken gizzard myosin regulatory light chain. and. by S1 nuclease analysis is present in non-muscle, sarcomeric and smooth muscle tissues and cell lines. In order to elucidate the genetics of this protein, a rat liver genomic library was screened with the nick-translated XcSMRLC. Overlapping clones covering approximately 40kb were isolated from a rat lix,er genomic library. Restriction mapping revealed 10kb (XG-BAMI0) and 3kb (XG-BAM3) BamHl fragments were >90% homologous to the e D N A by high stringency washes. These fi'agments are separated by approximately 15kb. Sequencing of XGBAMI0 has revealed that it contains the gene that encodes XcSMRLC. The gene is organized into 4 exons with a 4kb introo located within the 5"untranslated sequence. The amino acid coding region is divided by 2 iutrons: the COOH terminal region and the 3'untranslated region are located in a single exon. Southern blots of rat total genomic DNA hybridized to a probe generated from the the last 93 bases of the 3"untranslated sequence and 154 bases of 3" flanking DNA of XG-BAM10 demonstrates a unique band corresponding to XG-BAMI0. Hybridization of the same blots to ),cSMRLC confirmed the presence of both the 3kb and 10kb bands. This data demonstrates the presence of at least 2 genomic sequences corresponding to the SMRLC and suggests that 2 populations of m R N A could exist with differing noncoding regions. Examination of the exact sequence of the 3kb fi'agment will permit generation of specific probes to examine tissue and developmental specific expression and delineation of modes of genetic control of the smooth muscle contractile pbenotype.
S.4