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Differential expression of duplicated LDH-A genes during temperature acclimation of weatherfish Misgurnus fossilis: functional consequences for the enzyme

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Zakhartsev, M. , Lucassen, M. , Kulishova, L. , Deigweiher, K. , Smirnova, Y. , Zinov'eva, R. , Mugue, N. , Baklushinskaya, I. , Pörtner, H. O. and Ozernyuk, N. (2007): Differential expression of duplicated LDH-A genes during temperature acclimation of weatherfish Misgurnus fossilis: functional consequences for the enzyme , FEBS journal, 274 (6), pp. 1503-1513 . doi: 10.1111/j.1742-4658.2007.05692.x
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Abstract:

Temperature acclimation of poikilotherms entails metabolic rearrangements provided through the variation of enzyme properties. However, in most cases the underlying molecular mechanisms resulting in structural changes of enzymes are obscure. The present study reports that acclimation to low (5°C) and high (18°С) temperatures causes differential expression of alternative forms of the LDH-A gene in white skeletal muscle of weatherfish Misgurnus fossilis. Two isoforms of LDH-A mRNA were isolated and characterised: a short (1332 bp) and long (1550 bp) form, which differ in the length of 3'UTR, despite identical ORF lengths. These two mRNAs have 44 nucleotide point mismatches along the sequence resulting in three amino acid mismatches (Gly214Val; Val304Ile and Asp312Glu) between protein products from short and long forms of mRNA. Correspondingly, LDH-A subunit synthesized from short form of mRNA has been denoted as S-subunit, whereas the other one is L-subunit. It is expected that the L-subunit is more aliphatic due to properties of the mismatched amino acids. According to molecular modelling of M.fossilis LDH-A the Val304Ile mismatch is located in the subunit contacts of the tetramer, whereas the remaining two surround the contact area, which should manifest in kinetic and thermodynamic properties of the assembled tetramer. In warm acclimated fish the relative ratio between short and long isoforms of the LDH-A mRNA expression is around 5:1, whereas in cold acclimated fish expression of long mRNA is reduced to almost zero. This indicates that at low temperature the pool of overall tetrameric LDH-A is more homogeneous in terms of subunit composition. The temperature acclimation pattern of proportional pooling of subunits with different kinetic and thermodynamic properties of the tetrameric enzyme could result in the fine tuning of properties of overall skeletal LDH-A, which is in line with previously observed kinetic and thermodynamic differences between cold and warm LDH-A purified from weatherfish. Additionally, an irregular pattern of nucleotide mismatches indicates that these mRNAs are products of two independently evolving genes, i.e. paralogs. Karyotype analysis has revealed that the experimental population of M.fossilis is tetraploid (2n=100), therefore gene duplication possibly through tetraploidy may contribute to the adaptability towards temperature variation.

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