The isolation of a mammalian myoglobin gene.

• Main Author: Wood, David
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: Genetics 2015
• Subjects: harbour seal; Sperm whale
• Online Access: http://hdl.handle.net/2381/34373

Myoglobin is the major haemoprotein of vertebrate muscle where it facilitates oxygen diffusion from the blood to the mitochondria and acts as an oxygen store. Elevated levels of myoglobin are found in diving mammals and birds. Myoglobins and haemoglobins form part of the globin superfamily and are related evolutionarily. The ancestral globin gene underwent a duplication between 500 and 800 million years ago to give rise to monomeric myoglobins and the haemoglobin lines. The globin superfamily of proteins has been extensively characterised and sperm whale myoglobin was the first protein whose three-dimensional structure was determined. In addition the haemoglobin genes represent the best characterised multigene family. However the myoglobin gene has not been investigated. In order to understand the relationship of myoglobin genes in relation to other globin genes from an evolutionary and structural aspect, a mammalian myoglobin gene has been isolated. After unsuccessful attempts to clone myoglobin messenger RNA from human muscle, grey seal muscle containing high levels of myoglobin was used to prepare polyadenylated mRNA. cDNA was prepared by reverse transcription of polyadenylated mRNA and cloned into a plasmid vector. 4% of cDNA recombinants contained myoglobin cDNA inserts. One clone, shown to contain only 3' non-translated mRNA sequences, was used to determine that grey seal myoglobin is coded by a single gene which is transcribed to give a 1400 nucleotide mRNA considerably longer than related haemoglobin mRNAs. This cDNA clone was also used to isolate a myoglobin genomic clone from grey seal DNA cloned into a bacteriophage vector. The 3' end of this gene has been completely sequenced and the gene is interrupted by an intervening sequence at codon 105, a position precisely homologous to that in alpha- and beta-globin genes. The 3' exon of the grey seal myoglobin gene is remarkably long and comprises codons 106-153, which give an amino acid sequence identical to that of the harbour seal myoglobin, and 548 bp of untranslated sequence. This untranslated sequence contains a typical poly- adenylation signal and is much longer than the corresponding sequences of alpha- and beta-globin genes.