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A.J. Kiss, A.L. DeVries, C.–H.C. Cheng; Molecular Diversity and Genome Organisation of the Cold Stable , ß and Eye Lens Crystallins from the Antarctic Toothfish (Dissostichus mawsoni) . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2566.
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© ARVO (1962-2015); The Authors (2016-present)
The eye lens of the Antarctic toothfish, which lives in the –2°C Southern Ocean, is cold–stable and remains transparent to temperatures as low as –12°C. To investigate the molecular basis of this cold stability, we isolated, cloned and sequenced 22 full length crystallin cDNAs. To evaluate the αßγ crystallin gene organisation, we characterised positive clones isolated from a bacterial artificial chromosomal (BAC) library.
Messenger RNA from toothfish lenses was used to construct a cDNA library. A combination of Southern blotting and PCR was used to screen 800 clones. Gamma isoforms were isolated by Southern blotting of cDNA clones using a toothfish γ crystallin probe. The probe was generated using PCR primers based on the γM2 crystallin isoform from carp. Alpha and ß crystallin cDNAs were obtained by screening single clones in a 96–well plate format using isoform specific primers. The αß primers were designed based on previously reported zebrafish, chicken and cow α and ß cDNA sequences. Genomic organisation was assesed by fingerprint analysis of positive clones obtained by screening a BAC library with toothfish α, ß, and γ radio–labelled probes.
We found two α crystallins (αA, αB), six ß crystallins (ßA1, ßA2, ßA4, ßB1, ßB2, ßB3) and 14 γ crystallins (γN, γS1, γS2, γM1, γM2b, γM2c, γM5a, γM5b, γM5c, γM7, γM8a, γM8b, γM8c, and γM8d). Alignments of toothfish α and ß with mammalian and other fish sequences indicate that these are relatively conserved orthologues with other vetrabrate taxa. However, in phylogenetic analyses, the toothfish and other fish γM crystallins form a unique clade that are not orthologuous to mammalian γ crystallins. Screening of a toothfish BAC library for crystallin genes has revealed that α and ß crystallins are clustered in a very small region, while γ crystallins span a larger region of the genome.
The Antarctic toothfish is the first cold–adapted, ectothermic vertebrate species to have its crystallin cDNAs isolated, cloned and sequenced, as well as the organisation of its αßγ gene regions evaluated. The large number (11) of γM crystallins differs from the current mammalian paradigm. The size and number of the α, ß and γ genes within the toothfish genome correlates with the cDNA and protein abundance we previously reported. Thus, the long lived toothfish is a promising model system to study long–term crystallin stability, thermal protein adaptation and crystallin evolution.
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