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E E Strehler

Publications and source records attributed to E E Strehler.

67 records · Page 4Linked to original sources
PubMed1986

Sarcomeric myosin heavy chain gene family: organization and pattern of expression.

The sarcomeric myosin heavy chains (MHCs), which exhibit different levels of ATPase activity, are encoded by a closely related multigene family from which seven members have been identified and characterized in the rat. The MHC genes appear to map to a single chromosome, and at least two of them, alpha- and beta-cardiac, are closely linked in the genome. Each of these genes is approximately 25 kilobases long, and their coding sequences are interrupted by 40 introns. Each MHC gene displays a pattern of expression that is tissue-specific and developmentally regulated, with more than one MHC gene expressed in each muscle and developmental stage. Moreover, with the exception of the extra-ocular muscle MHC gene that has a very specific pattern of expression, the other genes are all expressed in more than one tissue. The expression of all MHC genes can be modulated by thyroid hormone. Surprisingly, however, the same myosin heavy chain gene can be regulated by thyroid hormone in highly different modes, even in opposite directions, depending on the tissue in which it is expressed. Furthermore, the skeletal embryonic and neonatal myosin heavy chain genes, so far considered specific to these two developmental stages, can be re-induced by hypothyroidism in specific adult muscles.

Animals↗
PubMed1985

Characterization of cDNA and genomic sequences corresponding to an embryonic myosin heavy chain.

We report here the isolation and characterization of cDNA and genomic sequences corresponding to a rat embryonic myosin heavy chain (MHC) protein. This gene, which is present as a single copy in the rat genome, comprises about 25 kilobase pairs of DNA and contains approximately 80% intronic sequences. The embryonic MHC gene belongs to a highly conserved multigene family, and exhibits a high degree of nucleotide and amino acid sequence conservation with other sarcomeric MHC genes from nematode to man. S1 nuclease mapping experiments using cDNA and genomic probes show that this MHC gene is transiently expressed during skeletal muscle development. Its mRNA is detected in fetal skeletal muscle during early development and persists up to 2 weeks after birth with the overlapping expression of neonatal and adult skeletal MHC mRNAs. However, this MHC is not expressed in the adult skeletal muscle with the exception of extraocular muscle fibers. The transient expression during muscle development of the isoform produced by this gene and its sequential replacement by other MHCs raises interesting questions about the mechanism controlling MHC isozyme transitions and the physiological significance of the individual MHCs in muscle fibers.

Animals↗
PubMed1985

Intron positions are conserved in the 5' end region of myosin heavy-chain genes.

We have determined the 5' end sequence of the rat embryonic skeletal muscle myosin heavy-chain (MHC) gene comprising the first three amino-terminal coding exons. Comparison with the corresponding regions of the rat ventricular alpha and the nematode Caenorhabditis elegans unc-54 MHC genes (Mahdavi, V., Chambers, A.P., and Nadal-Ginard, B. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2626-2630; Karn, J., Brenner, S., and Barnett, L. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4253-4257) shows that the degree of amino acid sequence conservation increases from the first to the third exon. Intron positions between these exons are maintained in all three genes studied, whereas size and sequence of corresponding introns are highly divergent. In contrast to the rat MHC genes where the coding region is highly split throughout its entire length, only the 5' end region is frequently interrupted by introns in the nematode gene indicating the potential importance of these introns in gene structure and expression. The occurrence of "preferential" intron positions in the MHC genes suggests the existence of a highly split ancestral MHC gene from which different evolutionary lineages removed and/or added specific sets of introns.

Amino Acid Sequence↗
PubMed1985

Myosin light-chain 1 and 3 gene has two structurally distinct and differentially regulated promoters evolving at different rates.

DNA fragments located 10 kilobases apart in the genome and containing, respectively, the first myosin light chain 1 (MLC1f) and the first myosin light chain 3 (MLC3f) specific exon of the rat myosin light chain 1 and 3 gene, together with several hundred base pairs of upstream flanking sequences, have been shown in runoff in vitro transcription assays to direct initiation of transcription at the cap sites of MLC1f and MLC3f mRNAs used in vivo. These results establish the presence of two separate, functional promoters within that gene. A comparison of the nucleotide sequence of the rat MLC1f/3f gene with the corresponding sequences from mouse and chicken shows that: the MLC1f promoter regions have been highly conserved up to position -150 from the cap site while the MLC3f promoter regions display a very poor degree of homology and even the absence or poor conservation of typical eucaryotic promoter elements such as TATA and CAT boxes; the exon/intron structure of this gene has been completely conserved in the three species; and corresponding exons, except for the regions encoding most of the 5' and 3' untranslated sequences, show greater than 75% homology while corresponding introns are similar in size but considerably divergent in sequence. The above findings indicate that the overall structure of the MLC1f/3f genes has been maintained between avian and mammalian species and that these genes contain two functional and widely spaced promoters. The fact that the structures of the alkali light chain gene from Drosophila melanogaster and of other related genes of the troponin C supergene family resemble a MLC3f gene without an upstream promoter and first exon strongly suggests that the present-day MLC1f/3f genes of higher vertebrates arose from a primordial alkali light chain gene through the addition of a far-upstream MLC1f-specific promoter and first exon. The two promoters have evolved at different rates, with the MLC1f promoter being more conserved than the MLC3f promoter. This discrepant evolutionary rate might reflect different mechanisms of promoter activation for the transcription of MLC1f and MLC3f RNA.

Amino Acid Sequence↗
PubMed1984

Fast skeletal muscle myosin light chains 1 and 3 are produced from a single gene by a combined process of differential RNA transcription and splicing.

Fast myosin light chains (MLCf) 1 and 3 are proteins associated with the thick filament in vertebrate fast muscle fibers. MLC1f and MLC3f have complete sequence homology for the first 141 amino acids from their COO- end, but they differ in length and amino acid sequence at their NH+3 ends (MLC1f = 49 amino acids, MLC3f = 8 amino acids), and they are translated from different mRNAs. To elucidate the structural relationship between mammalian fast myosin light chain 1 and 3 isoforms, cDNA clones have been isolated from rat skeletal muscle and their primary nucleotide sequences were determined. MLC1f and MLC3f mRNAs display complete sequence identity in the 3' untranslated sequences (285 base pairs) and in the codons specifying the 142 carboxyl-terminal amino acids. In contrast, the sequences encoding the amino-terminal 49 and 8 amino acids of MLC1f and MLC3f, respectively, as well as their 5' untranslated regions, are highly divergent. Using the cloned cDNAs as probes we have isolated the single gene locus encoding both MLC1f and MLC3f in four overlapping genomic clones spanning over approximately 25 kilobase pairs (kb) of DNA. The sequences encoding the common body of MLC1f and MLC3f are distributed in 4 separate exons at the 3' end of the gene. The start of transcription site and 5' untranslated region of MLC3f is found 5 kb upstream from the common body while the MLC1f mRNA transcription start site and the exon specifying the 5' untranslated region and amino acid 1-40 of MLC1f lies about 10 kb further upstream. Strikingly, a mini-exon that specifies amino acids 41-49 of MLC1f is located downstream from the two MLC3f-specific exons (5' untranslated and amino acids 1-8) and is separated from the upstream MLC1f exons by 12 kb of DNA. This bizarre gene organization implies a novel form of alternative promoter utilization and RNA splicing that is tissue-specific and developmentally regulated in order to generate the mature MLC1f and MLC3f mRNAs from a single gene.

Amino Acid Sequence↗
PubMed1983

Ultrastructural localization of M-band proteins in chicken breast muscle as revealed by combined immunocytochemistry and ultramicrotomy.

Cryo-ultramicrotomy and "conventional" plastic sectioning have been used in combination with extraction and immunolabeling techniques to determine the location of the two M-band proteins characterized to date, MM-creatine kinase (MM-CK: Mr, 80,000) and M-protein "myomesin" (Mr, 165,000) within the M-region of chicken pectoralis muscle. The following main results were obtained. (1) The M-band in chicken pectoralis muscle contains five major striations (M1, M4 and M4', M6 and M6' in the terminology of Sjöström & Squire, 1977a). (2) Extraction of the bulk of the electron-dense M-band with low ionic strength removes the M-striations M1, M4 and M4' while M6 and M6' are retained. Cross-sections through the M-region of such muscles lack primary M-bridges connecting the thick myosin filaments. (3) Labeling with antibodies against MM-CK enhances the M-striations M4 and M4'; sometimes the whole region between M4 and M4' is labeled. (4) Incubation with antibodies against myomesin results in the labeling of the whole M-band from M6 to M6'; no label is found in the rest of the bare zone outside M6 and M6'. (5) Incubation of low ionic strength extracted muscle fibers with antibodies against myomesin leads to an "incomplete" labeling of the M-band between M6 and M6'; lines M6 and M6' are sometimes seen to be enhanced presumably due to antibody labeling. From these results it is concluded that MM-CK is the major protein of the M4 and M4' (and possibly also of the M1) M-bridges. Myomesin is bound within the M-band along the thick filaments from M6 to M6'. Two hypothetical models for the possible location of myomesin are discussed. According to these models myomesin would either make up the M-filaments or be directly attached to and along the central bare zone of thick myosin filaments.

Animals↗
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↗
PubMed1980

Biochemical and ultrastructural aspects of Mr 165,000 M-protein in cross-striated chicken muscle.

To better understand the relationship between the Mr 165,000 M-line protein (M-protein) and H-zone structure in skeletal and in cardiac muscle, as well as the possible interaction of M-protein with another skeletal muscle M-line component, the homodimeric creatine kinase isoenzyme composed of two M subunits (MM-CK), we performed biochemical, immunological, and ultrastructural studies on myofibrils extracted by different procedures. In contrast to MM-CK, M-protein could not be completely removed from myofibrils by low ionic strength extraction. Fab-fragments of antibodies against M-protein could not release M-protein quantitatively from either breast or heart myofibrils but remained bound to the myofibrillar structure, whereas monovalent antibodies against MM-CK cause the specific release of MM-CK and the concomitant disappearance of the M-line from chicken skeletal muscle myofibrils. When MM-CK was removed from skeletal myofibrils by low ionic strength extraction or, more specifically, by incubation with anti-MM-CK Fab, M-protein was still not released quantitatively upon treatment with anti-M-protein Fab as judged from immunofluorescence data. In the ultrastructural investigation of low ionic strength extracted muscle fibers, M protein could be localized in two stripes on both sides of the former M-line, suggesting a reduced attachment to the residual H-zone structure, whereas the specific removal of MM-CK resulted in the same dense staining pattern for M-protein within the M-line as observed in untreated fibers. However, the binding of M-protein to the residual M-line structure seemed to be reduced, as a considerable amount of this protein could be identified in the supernate of sequentially incubated myofibrils. The results indicate a strong binding of M-protein within the H-zone structure of skeletal as well as heart myofibrils.

Animals↗
PubMed1979

Chicken parvalbumin. Comparison with parvalbumin-like protein and three other components (Mr = 8,000 to 13,000).

Procedures for a rapid isolation and purification of parvalbumin (Mr = 12,600), parvalbumin-like protein (Mr = 12,800), and three other polypeptides with molecular weights of 12,400 (Component 1), 11,700 (Component 2), and 8,000, respectively, from chicken leg muscle, are described. A direct comparison of parvalbumin with these other proteins showed distinct differences in the amino acid compositions, charge, and immunological behavior. Parvalbumin has two high affinity sites for Ca2+ with a KDiss less than or equal to 10(-6) M (Blum, H. E., Lehky, P., Kohler, L., Stein, E.A., and Fischer, E. H. (1977) J. Biol. Chem. 252, 2834-2838), in contrast to parvalbumin-like protein. Components 1 and 2, and the Mr = 8,000 protein, where only low affinity sites for Ca2+ could be detected (KDiss greater than 10(-3) M). From our results it is concluded that the co-extracted proteins do not constitute isoproteins of parvalbumin. The very low affinity for Ca2+ suggests that these proteins are not involved in processes of Ca2+ transport or Ca2+ regulation as proposed for parvalbumin. Parvalbumin could not be localized within isolated myofibrils and also did not accumulate in primary myogenic cell cultures together with proteins forming the myofibrillar structure. Parvalbumin was not even detected in myotubes in which myofibrils and sarcoplasmatic reticulum were already assembled and functioning. Parvalbumin (or cross-reacting material) was detected in leg muscle and brain 1 day after hatching of the chick. Possible roles for parvalbumin are discussed.

Aging↗