A novel vertebrate myoglobin gene containing three A+T-rich introns is conserved among Antarctic teleost species which differ in myoglobin expression.

This report presents the first teleost myoglobin (Mb) genomic DNA sequence, several features of which are distinct from mammalian Mb genes. We have isolated and compared genomic clones of three closely related Antarctic icefish: Chionodraco rastrospinosus, which expresses Mb mRNA and functional polypeptide; Champsocephalus gunnari, which transcribes the Mb gene but does not produce polypeptide due to a 5-base pair (bp) frameshift insertion; and Chaenocephalus aceratus, which lacks both Mb protein and mRNA. The single-copy icefish Mb gene contains three introns: two at positions identical to those found in mammalian Mb and a novel intron located in the 5' untranslated region three nucleotides upstream from the initiator codon. All three introns are shorter than those found in mammalian Mb genes and exhibit a considerably higher A+T content. The entire Mb transcriptional unit is intact in C. aceratus, indicating that the failure to express this gene is not due to aberrations in the coding region, splice junctions, polyadenylation signals, or core promoter elements. The three icefish Mb sequences display an extreme degree of identity in the transcriptional unit and putative promoter region. In contrast, sequences 65 bp downstream from the polyadenylation site bear no homology among the three species, demonstrating that rapid sequence change has occurred in the 1 million years since the divergence of these species.

Conservation of the myoglobin gene among Antarctic notothenioid fishes.

We determined the myoglobin cDNA sequence for seven Antarctic notothenioid fish species. These data identify mutations in the myoglobin gene for Champsocephalus gunnari and Pagetopsis macropterus, two icefish species that lack detectable quantities of the polypeptide but express myoglobin mRNA. a third species lacking myoglobin polypeptide, Chaenocephalus aceratus, is devoid of myoglobin mRNA and accordingly failed to produce myoglobin products on polymerase chain reaction (PCR) amplification. Myoglobin cDNA sequences were highly conserved among the species the express the protein, particularly in the coding region. Sequence variation among the myoglobin-expressing channichthyid species was 2.0% to 2.9% in the coding region and 2.6% to 3.3% over the entire cDNA. The same extent of variation, 1.6% to 3.2% in the coding sequence and 2.8% to 3.7% overall, was observed between the icefishes and more distantly related, red-blooded nototheniid species. The two species expressing mutant myoglobin mRNA, C. gunnari and P. macropterus, exhibited the highest degree of sequence variation among the fish myoglobins examined. Drift in the myoglobin sequence in these two species, and conservation of myoglobin cDNA among fishes from two distinct families, suggest that a selective pressure operates to maintain myoglobin in the species that express the protein.

Variable expression of myoglobin among the hemoglobinless Antarctic icefishes.

The important intracellular oxygen-binding protein, myoglobin (Mb), is thought to be absent from oxidative muscle tissues of the family of hemoglobinless Antarctic icefishes, Channichthyidae. Within this family of fishes, which is endemic to the Southern Ocean surrounding Antarctica, there exist 15 known species and 11 genera. To date, we have examined eight species of icefish (representing seven genera) using immunoblot analyses. Results indicate that Mb is present in heart ventricles from five of these species of icefish. Mb is absent from heart auricle and oxidative skeletal muscle of all species. We have identified a 0.9-kb mRNA in Mb-expressing species that hybridizes with a Mb cDNA probe from the closely related red-blooded Antarctic nototheniid fish, Notothenia coriiceps. In confirmation that the 0.9-kb mRNA encodes Mb, we report the full-length Mb cDNA sequence of the ocellated icefish, Chionodraco rastrospinosus. Of the eight icefish species examined, three lack Mb polypeptide in heart ventricle, although one of these expresses the Mb mRNA. All species of icefish retain the Mb gene in their genomic DNA. Based on phylogeny of the icefishes, loss of Mb expression has occurred independently at least three times and by at least two distinct molecular mechanisms during speciation of the family.