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J C Perriard

Publications and source records attributed to J C Perriard.

At least 55 records · Page 3Linked to original sources
PubMed1990

A unique chicken B-creatine kinase gene gives rise to two B-creatine kinase isoproteins with distinct N termini by alternative splicing.

In the chicken, a high degree of heterogeneity at the protein level has been reported for the creatine kinase-type B (B-CK). Here we show that the two B-CK isoproteins, Ba- and Bb-CK, are encoded by two mRNAs, which are derived from a single copy gene by a stochastic alternative splicing mechanism. The transcription of the single hnRNA is directed by a complex promoter region containing a stretch of sequences which is highly conserved among all the B-CK genes known to date. This stretch encompasses a putative binding site for the TA-rich DNA-binding protein (Hobson, G. M., Mitchell, M. T., Molloy, G. R., and Pearson, M. L. (1988) Nucleic Acids Res. 16, 8925-8944) which is located in the distal part of the promoter region, while the proximal portion containing the TATA-box used in vivo is not conserved between chicken and mammals. The two isoproteins arising from this gene contain distinct N-terminal portions. According to comparative analysis, Bb-CK is the form which is homologous to the mammalian B-CKs, whereas Ba-CK shows some sequence features unique among all other vertebrate cytosolic creatine kinases characterized so far.

Amino Acid Sequence↗
PubMed1990

Alternative ribosomal initiation gives rise to chicken brain-type creatine kinase isoproteins with heterogeneous amino termini.

In higher eukaryotes three different types of creatine kinases (CK) are expressed: the muscle-specific M-CK, the ubiquitous cytoplasmic B-CKs, and the mitochondrial Mi-CKs. They fulfill multiple tasks in cells with an intensive energy metabolism. Isolated chicken B-CK can be resolved by two-dimensional gel electrophoresis into a major acidic Ba-CK and a major basic Bb-CK protein species which are very likely produced from the unique chicken B-CK gene (Wirz, T., Hossle, J. P., Soldati, T., and Perriard, J.-C. (1989) Experientia (Basel) 45, 32 (abstr.]. However, close inspection of the gels indicates the presence of additional B-CK species. This additional heterogeneity is generated by two distinct post-transcriptional processes. Post-translational phosphorylation was shown to contribute to heterogeneity of both Ba- and Bb-CK isoproteins and appears to modulate their enzymatic activity (A. F. Q. Quest, H. M. Eppenberger, and T. Wallimann, manuscript in preparation). Alternative ribosomal initiation of Bb-CK synthesis at multiple sites was shown to occur in cell free systems as well as in vivo, resulting in proteins differing in the length of their amino termini. Using site-directed mutagenesis to "switch off" each of the first four methionine codons of a full length Bb-CK cDNA, we were able to correlate each protein product with one distinct translational start site. An additional protein species appears to be produced by initiation at a noncanonical start codon. We propose that a leucine codon may be used as a translational start site. Evidence is presented to support the role of these amino-terminal truncated subunits in the regulation of the enzyme.

Amino Acid Sequence↗
PubMed1989

Fiber type-specific distribution of M-band proteins in chicken muscle.

The functions of two myofibrillar proteins, myomesin (Mr 185,000) and M-protein (Mr 165,000), associated with the M-band are as yet unknown. To extend our knowledge of these proteins, we have examined chicken striated muscles with fast and slow contractile properties, e.g., pectoralis major, PLD, ALD, medial adductor, and lateral adductor, to determine the expression and isoform composition of myomesin and M-protein in various muscles and fiber types. The high molecular weight M-band proteins were characterized and quantitated using monoclonal antibodies in immunoblotting and double-antibody sandwich ELISA. Fiber specificity was determined by immuno- and enzyme histochemistry. In addition to the previously reported Mr 195,000 and 190,000 isoforms of myomesin in heart [Grove et al. (1985): J Cell Biol 101:1431], the Mr 185,000 myomesin in skeletal muscles may represent different isoforms in fast and slow muscles on the basis of distinctive degradation patterns. M-protein has the same molecular weight in striated chicken muscles and degradation patterns indicate only one isoform. The low quantities of M-protein in slow muscles were shown to be due to the absence of M-protein in two of the generally recognized slow fiber types, types I and III. Thus, M-protein was present only in fast type II fibers, whereas myomesin was ubiquitous in all fiber types. Whatever the causal relationship, M-protein appears to function in fast motor units composed of type II fibers.

Animals↗
PubMed1988

Distinct tissue specific mitochondrial creatine kinases from chicken brain and striated muscle with a conserved CK framework.

cDNA clones for chicken mitochondrial creatine kinase (Mi-CK) were isolated from a lambda gt11 leg muscle cDNA library and sequenced. The deduced amino acid sequence showed 6 blocks of extensive homologies with the cytosolic creatine kinases and contained twenty N-terminal amino acids, with characteristic features of part of a mitochondrial presequence. The mature enzyme contained 380 amino acids with a calculated Mr of 43'195. RNA hybridization analysis showed corresponding Mi-CK transcripts in cardiac and skeletal muscle, but not in brain RNA. Within the 30 N-terminal amino acids purified brain Mi-CK contained 10 changes with respect to cardiac Mi-CK. Thus multiple isoproteins of mitochondrial creatine kinases of brain and striated muscle are encoded by multiple mRNA's.

Amino Acid Sequence↗
PubMed1988

Intracellular targeting of isoproteins in muscle cytoarchitecture.

Part of the muscle creatine kinase (MM-CK) in skeletal muscle of chicken is localized in the M-band of myofibrils, while chicken heart cells containing myofibrils and BB-CK, but not expressing MM-CK, do not show this association. The specificity of the MM-CK interaction was tested using cultured chicken heart cells as "living test tubes" by microinjection of in vitro generated MM-CK and hybrid M-CK/B-CK mRNA with SP6 RNA polymerase. The resulting translation products were detected in injected cells with isoprotein-specific antibodies. M-CK molecules and translation products of chimeric cDNA molecules containing the head half of the B-CK and the tail half of the M-CK coding regions were localized in the M-band of the myofibrils. The tail, but not the head portion of M-CK is essential for the association of M-CK with the M-band of myofibrils. We conclude that gross biochemical properties do not always coincide with a molecule's specific functions like the participation in cell cytoarchitecture which may depend on molecular targeting even within the same cellular compartment.

Amino Acid Sequence↗
PubMed1986

Complete nucleotide and encoded amino acid sequence of a mammalian myosin heavy chain gene. Evidence against intron-dependent evolution of the rod.

The complete nucleotide sequence and exon/intron structure of the rat embryonic skeletal muscle myosin heavy chain (MHC) gene has been determined. This gene comprises 24 X 10(3) bases of DNA and is split into 41 exons. The exons encode a 6035 nucleotide (nt) long mRNA consisting of 90 nt of 5' untranslated, 5820 nt of protein coding and 125 nt of 3' untranslated sequence. The rat embryonic MHC polypeptide is encoded by exons 3 to 41 and contains 1939 amino acid residues with a calculated Mr of 223,900. Its amino acid sequence displays the structural features typical for all sarcomeric MHCs, i.e. an amino-terminal "globular" head region and a carboxy-terminal alpha-helical rod portion that shows the characteristics of a coiled coil with a superimposed 28-residue repeat pattern interrupted at only four positions by "skip" residues. The complex structure of the rat embryonic MHC gene and the conservation of intron locations in this and other MHC genes are indicative of a highly split ancestral sarcomeric MHC gene. Introns in the rat embryonic gene interrupt the coding sequence at the boundaries separating the proteolytic subfragments of the head, but not at the head/rod junction or between the 28-residue repeats present within the rod. Therefore, there is little evidence for exon shuffling and intron-dependent evolution by gene duplication as a mechanism for the generation of the ancestral MHC gene. Rather, intron insertion into a previously non-split ancestral MHC rod gene consisting of multiple tandemly arranged 28-residue-encoding repeats, or convergent evolution of an originally non-repetitive ancestral MHC rod gene must account for the observed structure of the rod-encoding portion of present-day MHC genes.

Amino Acid Sequence↗
PubMed1986

The primary structure of chicken B-creatine kinase and evidence for heterogeneity of its mRNA.

cDNA clones for chicken B-CK were isolated by immunoscreening from a gizzard cDNA library constructed in the expression vector lambda gtll. The entire coding portion in addition to the complete 3' untranslated region and 42 bp of the 5' noncoding part are represented in the clone H4. On RNA blots H4 insert DNA hybridized to a 1600 bp poly(A)+ RNA from gizzard, brain and heart but not to breast or skeletal muscle RNA. In vitro generated sense strand transcripts of H4 insert DNA were translated in vitro into a protein indistinguishable from isolated, authentic B-CK. The distinct nucleotide sequences of H4 insert DNA and M-CK cDNA were translated into 82% homologous amino acid sequences. Sequence heterogeneity among the B-CK cDNA clones within both the 3' noncoding and even in the coding region indicates the existence of multiple B-CK mRNA species.

Amino Acid Sequence↗
PubMed1985

Myomesin and M-protein: expression of two M-band proteins in pectoral muscle and heart during development.

The expression of the myofibrillar M-band proteins myomesin and M-protein was studied in chicken pectoral muscle and heart during differentiation using monoclonal antibodies in a double-antibody sandwich enzyme-linked immunosorbent assay, immunoblotting, and immunocytochemistry. In presumptive pectoral muscle, myomesin accumulated first, increasing from 2% of the adult concentration at day 7 to 70% by day 16 in ovo. M-protein accumulation lagged 6-7 d behind that of myomesin attaining only 40% of the adult concentration in ovo. The molecular masses of myomesin (185 kD) and M-protein (165 kD) remained constant during embryogenesis. In cultured myogenic cells the accumulation and M-band localization of myomesin preceded that of M-protein by 1.5 d. Chicken heart was shown, in addition to M-protein, to contain unique isoforms of myomesin. In hearts of 6 d embryos, a 195-kD myomesin isoform was the major species; throughout development, however, a transition to a mixture of 195 and 190 kD was observed, the latter being the major species in the adult tissue. During heart differentiation the initial accumulation of myomesin again preceded that of M-protein, albeit on an earlier time scale than in pectoral muscle with M-protein reaching adult proportions first.

Animals↗
PubMed1985

Appearance of M-band attached MM-creatine kinase in differentiating chicken heart cells after injection of M-type isoprotein or poly (A+)-RNA enriched for M-type creatine kinase message.

Myofibrils of chicken heart cells do not contain the electron-dense material of the so-called m-bridges which transverse the sarcomere in the M-region. It has been shown that the M-isoform of creatine kinase (MM-CK) which is mainly responsible for the m-bridge material is not expressed during differentiation of chicken heart cells. No transition from the embryonic BB-CK to the muscle-specific MM-CK takes place in chicken heart, thus no MM-CK is available for m-bridge formation. Here we report on chicken heart cells microinjected with either MM-CK protein or with poly(A+)RNA enriched for M-CK message. In both cases appearance of MM-CK within the M-band of heart myofibrils could be observed by immunofluorescence, indicating translation of the injected message as well as specific binding of the translation product to the M-band of myofibrils. The M-band protein myomesin which is regularly found in heart myofibrils served as specific marker for assembled myofibrils in double immunofluorescence experiments.

Animals↗
PubMed1984

Complete cDNA-derived amino acid sequence of chick muscle creatine kinase.

cDNA clones of chick muscle creatine kinase mRNA were prepared by conventional procedures and then extended by primer extension to include the entire coding region of the mRNA. The nucleotide sequence of the cDNA clones permits, for the first time, determination of the complete sequence of the 381 amino acids comprising the muscle creatine kinase subunit of a higher vertebrate. In addition, available information regarding amino acid residues, intergral or proximal to the active site, allows tentative positioning of part of the active site within the primary sequence. Comparison of the chick muscle creatine kinase amino acid sequence with partial amino acid sequence for rat and rabbit muscle creatine kinase indicates that the primary structure of this enzyme is strongly conserved in evolution.

Amino Acid Sequence↗
PubMed1984

Complete nucleotide sequence of Torpedo marmorata mRNA coding for the 43,000-dalton nu 2 protein: muscle-specific creatine kinase.

DNA sequences coding for the muscle-specific subunit of creatine kinase have been isolated from cDNA libraries constructed from Torpedo marmorata electric organ. Clones were screened by differential in situ hybridization and hybrid-selected translation. The in vitro translation product of the selected mRNA was immunoprecipitated by anti-chicken creatine kinase antibodies and comigrated with Torpedo muscle creatine kinase on two-dimensional gels at the same position as the cytosolic 43,000-dalton protein referred to as nu 2. The cDNA inserts hybridized to a mRNA species present in adult Torpedo muscle but not in brain. The complete sequence of the mRNA was determined on one of the clones except for the 78 nucleotides of the mRNA 5' terminal sequence, which were identified by the primer extension method. The amino acid sequence of muscle-specific creatine kinase from T. marmorata was deduced and analyzed. It includes the known sequence of a peptide from the active site of rabbit muscle-specific creatine kinase.

Amino Acid Sequence↗
PubMed1984

A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies.

The M line, which transverses the center of the thick filament region of skeletal muscle sarcomeres, appears to be a complex array of multiple structural elements. To date, two proteins have definitely been shown to be associated with the M line. They are MM-CK, localized in the M 4,4' substriations, and a 165,000-dalton (164 kd) protein, referred to as both M-protein and myomesin. Here we report the positive identification of a third M-line protein of 185 kd. In the course of making monoclonal antibodies (mAbs) against a 165-kd fraction, we also obtained mAbs that bound to the M line of isolated myofibrils as detected by indirect immunofluorescence, but recognized a protein band of 185 kd in immunoblotting experiments with either the original immunogen or low ionic strength myofibril extracts as antigenic targets. The evidence that the 185- and 165-kd proteins are distinct protein species is based on the separation of the two proteins into discrete peaks by ion exchange chromatography, the distinctive patterns of their degradation products, and non-cross-reactivity of any of seven mAbs. These mAbs recognize three unique antigenic determinants on the 185-kd molecule and at least two and probably four sites on the 165-kd molecule as determined from competitive binding and immunofluorescence experiments. To resolve the problem of multiple nomenclature for the 165-kd protein, the 185-kd protein will be referred to as myomesin and the 165-kd protein as M-protein.

Animals↗
PubMed1982

Molecular cloning and expression during myogenesis of sequences coding for M-creatine kinase.

Sequences complementary to muscle poly(A)+RNA were cloned in the plasmid pBR322 and the resulting colonies were screened by colony hybridization with labeled cDNA derived from skeletal muscle and smooth muscle (gizzard). The skeletal muscle-specific clones were further screened by RNA blotting hybridization for a muscle mRNA having the size expected for a putative type M creatine kinase (M-CK) mRNA. The remaining clones with the expected hybridization properties were finally characterized by hybrid-selected translation, and a cloned sequence was shown to contain DNA hybridizing to mRNA that could be translated into M-CK. This plasmid, pMCK1, was further characterized by restriction mapping. Blot analysis of total cell RNA from differentiating myogenic cell cultures showed accumulation of M-CK mRNA in cultures older than 42 hr but not in young little-differentiated cultures.

Animals↗
PubMed1981

Turnover of the creatine kinase subunits in chicken myogenic cell cultures and in fibroblasts.

The rates of degradation of creatine kinase subunits, M-CK and B-CK subunits, were measured in cultured myogenic cells and in subcultured fibroblasts. In differentiated myogenic cells, the myotubes, both M-CK and B-CK subunits are synthesized. Their rates of degradation were compared. The M-CK subunits is slightly more stable and is degraded with an average apparent half-life of 75 h, whereas that of the B-CK subunit was shorter with 63 h. The turnover properties of M-CK subunit from soluble and of myofibril-bound MM-CK homodimeric creatine kinase isoenzyme isolated from breast muscle of young chickens were identical. The apparent half-life of the B-CK subunit was also determined in subcultured fibroblasts and 5-bromo-2'-deoxyuridine-treated cells, and found to be shorter than in myotubes (46 h and 37 h respectively). Similar observations were made for myosin heavy chain, actin and total acid-precipitable material. It appears therefore that proteins are in general degraded more slowly in differentiated myogenic cells. The differences in the stability of M-CK and B-CK subunits in myotubes probably do not reflect a major regulatory mechanism of the creatine kinase isoenzyme transition.

Actins↗
PubMed1981

The Mr 165,000 M-protein myomesin: a specific protein of cross-striated muscle cells.

The tissue specificity of chicken 165,000 M-protein, tentatively names "myomesin", a tightly bound component of the M-line region of adult skeletal and heart myofibrils, was investigated by immunological techniques. Besides skeletal and heart muscle, only thymus (known to contain myogenic cells) was found to contain myomesin. No myomesin could however, be detected in smooth muscle or any other tissue tested. This result was confirmed in vitro on several cultured embryonic cell types. Only skeletal and heart muscle cells, but not smooth muscle or fibroblast cells, showed the presence of myomesin. When the occurrence and the distribution of myomesin during differentiation of breast muscle cells in culture were studied by the indirect immunofluorescence technique, this protein was first detected in postmitotic, nonproliferating myoblasts in a regular pattern of fluorescent cross-striations. In electron micrographs of sections through young myotubes, it could be shown to be present within the forming H-zones of nascent myofibrils. In large myotubes the typical striation pattern in the M-line region of the myofibrils was observed. Synthesis of myomesin measured by incorporation of [35S]methionine into immunoprecipitable protein of differentiating cells increased sharply after approximately 48 h in culture, i.e., at the time when the major myofibrillar proteins are accumulated. No significant amounts of myomesin were, however, found in cells prevented from undergoing normal myogenesis by 5'-bromodeoxyuridine. The results indicate that myomesin (a) is a myofibrillar protein specific for cross-striated muscle, (b) represents a highly specific marker for cross-striated muscle cell differentiation and (c) might play an important role in myofibril assembly and/or maintenance.

Animals↗