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 Mbexpressing 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.

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SUMMARY Six of the 16 known species of Antarctic icefish (family Channichthyidae) have lost the ability to express cardiac myoglobin (Mb) via at least four independent events during radiation of these species. We report here that the lesion in Chaenocephalus aceratus Mb is a duplicated TATAAAA element that blocks transcription. This lesion is distinct from those of other icefish species that do not express cardiac Mb. The C. aceratus Mb gene is nearly identical to that of Chionodraco rastrospinosus, a closely related Mb-expressing icefish species, with one exception. A 15-bp segment is present in C. aceratus but absent from C. rastrospinosus; this insertion is located 648 bp upstream from the reference transcription start site of C. rastrospinosus and includes the sequence TATAAAA, which bound HeLa cell transcription factor IID (TFIID) and icefish nuclear proteins in gel-retardation assays. Reporter constructs containing the `full-length' C. aceratus Mb promoter were not expressed in transient expression assays in oxidative skeletal muscle of live icefish. By contrast, constructs employing the nearly identical `full-length' C. rastrospinosus Mb promoter were efficiently expressed in parallel assays in the same tissue. Truncated constructs of C. aceratus Mb that did not contain the 15-bp duplication were expressed at very low levels. These data confirm a third independent mechanism of Mb loss among channichthyid species, indicate that C. aceratus aerobic muscle is capable of expressing functional Mb genes and demonstrate that duplication of the muscle-specific TATAAAA sequence in an inappropriate context can result in loss of a gene's expression, resulting in significant physiological consequences.

We used a combined immunochemical and molecular approach to ascertain the presence and concentrations of both the intracellular oxygen-binding hemoprotein myoglobin (Mb) and its messenger RNA (mRNA) in 13 of 15 known species of Antarctic channichthyid icefishes. Mb protein is present in the hearts of eight species of icefishes: Chionodraco rastrospinosus, Chionodraco hamatus, Chionodraco myersi, Chaenodraco wilsoni, Pseudochaenichthys georgianus, Cryodraco antarcticus, Chionobathyscus dewitti and Neopagetopsis ionah. Five icefish species lack detectable Mb protein: Chaenocephalus aceratus, Pagetopsis macropterus, Pagetopsis maculatus, Champsocephalus gunnari and Dacodraco hunteri. Mb concentrations range from 0.44+/−0.02 to 0.71+/−0.08 mg Mb g(−)(1)wet mass in heart ventricle of species expressing the protein. A Mb-mRNA-specific cDNA probe was used to quantify mRNA in five Mb-expressing icefishes. Mb mRNA was found in low but detectable amounts in Champsocephalus gunnari, one of the species lacking detectable Mb. Mb mRNA concentrations in heart ventricle from Mb-expressing species ranged from 0.78+/−0.02 to 16.22+/−2.17 pg Mb mRNA microg(−)(1)total RNA). Mb protein and Mb mRNA are absent from the oxidative skeletal muscle of all icefishes. Steady-state concentrations of Mb protein do not parallel steady-state concentrations of Mb mRNA within and among icefishes, indicating that the concentration of Mb protein is not determined by the size of its mRNA pool.