Investigation into the transcriptional regulation of porcine muscle fibre type

Birrell, Ronald Frederick (2006) Investigation into the transcriptional regulation of porcine muscle fibre type. PhD thesis, University of Glasgow.

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Skeletal muscle is a highly organised yet adaptive tissue, capable of changing aspects of fibre phenotype in response to demand. While the external signals mediating these changes, such as patterns of motor neuron activity or hormone signalling, are well studied the manner by which these external stimuli are converted into specific patterns of gene expression is less understood. GATA-2 and NFAT-2 are two transcription factors implicated in this process. In this study, conserved regions of the GATA and NFAT transcription factor families were used to design primers for a homology-based PCR-cloning approach to isolate the porcine orthologues of GATA-2 and NFAT-2. The pig was chosen as the target species for this investigation, given the importance of porcine muscle both as a large animal model for muscle development and as a commercially important meat animal. The long-term goal of the work was to determine what effects these transcription factors might have in the regulation of stage- and isoform-specific gene expression. A porcine orthologue of GATA-2 was cloned, and the sequence submitted to GenBank (Accession N° AY251012). Though only a truncated cDNA clone was obtained from efforts to clone porcine NFAT-2, sufficient sequence data was recovered for the design of isoform-specific primers for quantitative real-time RT-PCR. GATA-2 and NFAT-2 were found to have a wide tissue distribution. A novel finding was that GATA-2 was found to be highly expressed in uterine smooth muscle. This suggests that the segregation of GATA-family members between haemopoietic functions and mesodermal tissues may not be as distinct as was previously thought. In addition to studying trans-acting factors, this study also looked at potential targets for factors modifying fibre phenotype, in particular the isoforms of myosin heavy chain (MyHC). The different MyHC isoforms expressed in a muscle fibre greatly influence aspects of phenotype such as shortening velocity and ATPase activity, and are indicative of fibre type. One such isoform, the embryonic MyHC (emMyHC), was one of the last remaining uncharacterised isoforms in the pig. The first intron and immediate upstream region of the porcine embryonic MyHC were isolated and sequenced, and it was found that the translation start site of the porcine isoform was located on exon 2, something which had not been previously reported in any other isoforms or orthologues of the gene. A GATA-2 binding site was found, 4 kb upstream of the transcriptional start site. Reporter constructs were generated for use in conjunction with the GATA-2 clone. Through in vitro transcription-translation, the GATA-2 clone was found to encode a functional reading frame for a protein of the expected size and was deemed suitable for functional studies. Technical difficulties frustrated the efforts to quantify changes in MyHC expression in porcine primary myoblasts through quantitative real-time RT- PCR. However, a preliminary in vitro study in C2C12 murine myoblasts suggested that emMyHC reporter activity was reduced by GATA-2 overexpression. Together with developmental expression patterns showing an upregulation in GATA-2 coinciding with the developmental downregulation of emMyHC, and the presence of a putative GATA-2 binding site in the 5' UTR, this was taken as evidence of a possible role for GATA-2 in negatively regulating emMyHC. Some possibilities for studies to further explore these findings are discussed.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Chang, Dr. Kin-Chow
Date of Award: 2006
Depositing User: Mrs Monika Milewska-Fiertek
Unique ID: glathesis:2006-30872
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 08 Oct 2018 10:54
Last Modified: 08 Oct 2018 10:54
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