Studies of complementary DNAs corresponding to skeletal muscle proteins

McInnes, Colin J (1988) Studies of complementary DNAs corresponding to skeletal muscle proteins. PhD thesis, University of Glasgow.

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The initial objective of the work described in this thesis was to isolate complementary DMAs (cDNAs) corresponding to mRNAs encoding proteins expressed solely in skeletal muscle. To this end a mouse skeletal muscle cDNA library was differentially screened with radioactively-labelled single-stranded cDNA probes derived from skeletal and cardiac muscle. Of 15,000 individual colonies subjected to the mass screening procedure of colony hybridisation, 247 were selected on the basis that they gave a hybridisation signal only with the skeletal muscle probe. Southern blot analysis of plasmid DNA isolated from each of these revealed eight which continued to produce a differential hybridisation signal with the two single-stranded cDNA probes. However subsequent hybridisation of these eight radioactively-labelled cDNA clones with poly(A)+ RNA isolated from skeletal muscle, cardiac muscle, liver and brain, revealed only a single clone which appeared to represent an mRNA specific to skeletal muscle. The nucleotide sequence of the cDNA insert of this clone, which was approximately 500 base pairs (bp) in length, was determined. It was found to contain an open reading-frame, the predicted product of which corresponded to part of the sequence of the beta-isoform of rabbit tropomyosin. This isoform has only been found in skeletal muscle, although other isoforms are found in cardiac muscle, smooth muscle, and non-muscle tissues. No previous mouse beta-tropomyosin cDNA clones have been described. The skeletal muscle cDNA library was rescreened in an attempt to obtain a full-length beta-tropomyosin cDNA clone. Three clones were selected and sequenced. One of these was found to be identical to the original clone selected from the initial screening. The second, however, contained the entire beta-tropomyosin amino-acid coding region together with 72 nucleotides of the 5' non-coding region and 151 nucleotides of the 3' non-coding region. The remaining clone lacked sequence from the 3' half of the mRNA, but extended the 5' noncoding region to 95 nucleotides. From comparison with the size of the mRNA (1.3kb), revealed by Northern blotting, it is estimated that the sequence of the mouse beta-tropomyosin determined represents approximately 90% of the mRNA. The expression of tropomyosin mRNAs was examined during the differentiation of a cultured mouse cell line from the individual myoblast stage to the multinucleate cell stage. Northern blot analysis of the mRNA isolated from the cells at different stages of their development revealed that upon fusion of the cells, the expression of several different tropomyosin mRNAs is induced. In particular two species, with lengths of 1.2kb and 2.6kb, which are not found in the RNA isolated from 11-day old mouse skeletal muscle, are expressed during the differentiation of skeletal muscle cells. The two remaining species expressed upon fusion of the cells, a 1.3kb species and a 2.4kb species, correspond to the species identified in the RNA from 11-day old mice. The identities of all four tropomyosin species are unknown, except for the 1.3kb beta-tropomyosin mRNA. However comparison with published work suggests that the 1.2kb and 2.4kb species may correspond respectively to the alternatively spliced products from the primary transcripts which also give rise to the beta-tropomyosin and the a2 tropomyosin species. No previous work has reported the possible coexpression of alternatively spliced isoforms of tropomyosin. Analysis of the nucleotide sequence corresponding to mouse beta-tropomyosin mRNA revealed some unusual features. There was a deficit of the dinucleotide CpG in the codon position [2,3], but not in the [3,1] position, throughout the amino-acid coding region of beta-tropomyosin. One possible explanation of this is that there is strand-specific hemi-methylation of the corresponding germ line DNA. Previously published comparisons of a partial human beta-tropomyosin cDNA sequence with a human non-muscle tropomyosin (TM36) cDNA sequence had indicated the likelihood that beta-tropomyosin and TM36-tropomyosin are encoded by the same gene, with each of the isoforms arising from the alternative splicing of two pairs of mutually exclusive exons. Comparison of these sequences with the mouse beta-tropomyosin cDNA allowed the conclusion that none of the 5' exons not represented in the human beta-tropomyosin cDNA clone are involved in alternative splicing. Nucleotide comparisons with other beta-tropomyosin cDNA sequences reported during the completion of this work indicated that the 3' and 5' non-coding regions of the beta-tropomyosin mRNA had been subject to some selective evolutionary pressure for conservation, although not to the extent found for certain other mRNAs encoding muscle proteins (e.g. actin mRNAs).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 1988
Depositing User: Enlighten Team
Unique ID: glathesis:1988-76702
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15

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