Isolation, sequence and analysis of the ovine homologue of myoD1
Abstract (Summary)Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan. MyoD1 was first detected in mouse and is the founding member of a family of transcription factors essential for the formation of skeletal muscle. MyoD1 is known to bind DNA directly at an enhancer sequence (E-box) and thereby stimulate the expression of a battery of muscle-specific genes (reviewed by Weintraub et al., 1991a). In this work the sheep homologue of myoD1 was isolated as a single clone from a cDNA library constructed from skeletal muscle poly A+ RNA. Sequence analysis indicated that the recombinant was incomplete at its 5'terminus. A genomic library was therefore constructed and a clone containing the missing region was characterized. The sheep myoD1 open reading frame codes for a putative protein of 319 amino acids which shares 90% homology with that of mouse. Most homology spans a motif, known to be essential for inducing myogenesis, consisting of a basic set of residues adjacent to a region capable of forming a helix-loop-helix (bHLH) structure. Sheep myoD1 was shown by RT-PCR to be expressed in foetal and adult skeletal muscle tissue, but not cardiac or smooth muscle tissue. Southern analysis suggests that myoD1 is most likely present in the sheep genome as a single copy gene with restriction fragment length polymorphisms for the restriction enzymes Hincll and Pvull. These polymorphisms may have arisen by DNA rearrangement or differential methylation of the restriction enzyme sites within each myoD1 allele. Sequence from the 5' terminus of the sheep genomic myoD1 clone has identified several putative promoter and regulatory elements. An unusual TATA and CCAAT box region, thought to stimulate tissue-specific expression, is shared between sheep, mouse and Xenopus myoD1. Once induced, mouse MyoD1 is known to stimulate its own expression via an autoregulatory loop, possibly by binding to E-box sequences upstream of its gene (Thayer et al., 1989). In accordance with the role of MyoD1 as an autoregulator, several consensus E-box sequences (CANNTG) have been found in the 5'terminus of sheep myoD1. One of these is also present in the same location in mouse myoD1. Attempts were made to identify preferential MyoD1 binding sites within the genes upstream region using an in vitro DNA-protein binding assay. MyoD1 was purified as a fusion protein with Glutathione-S-transferase and shown to require the bHLH region for DNA binding. Binding appeared to be restricted to DNA fragments harbouring the consensus E-box sequence CANNTG. Manipulation of binding conditions did not reveal nucleotide preferences for MyoD1-DNA binding outside the consensus sequence, suggesting that other factors might be contributing to the ability of MyoD1 to bind specifically to certain E-box sequences.
School Location:New Zealand
Source Type:Master's Thesis
Date of Publication:01/01/1994