Evolution, Organization, and Expression of α-Tubulin Genes in the Antarctic Fish Notothenia coriiceps

Abstract

To assess the organization and expression of tubulin genes in ectothermic vertebrates, we have chosen the Antarctic yellowbelly rockcod, Notothenia coriiceps, as a model system. The genome of N. coriiceps contains ϳ15 distinct DNA fragments complementary to ␣-tubulin cDNA probes, which suggests that the ␣-tubulins of this cold-adapted fish are encoded by a substantial multigene family. From an N. coriiceps testicular DNA library, we isolated a 13.8-kilobase pair genomic clone that contains a tightly linked cluster of three ␣-tubulin genes, designated NcGTb␣a, NcGTb␣b, and NcGTb␣c. Two of these genes, NcGTb␣a and NcGTb␣b, are linked in head-to-head (5 to 5) orientation with ϳ500 bp separating their start codons, whereas NcGTb␣a and NcGTb␣c are linked tail-to-tail (3 to 3) with ϳ2.5 kilobase pairs between their stop codons. The exons, introns, and untranslated regions of the three ␣-tubulin genes are strikingly similar in sequence, and the intergenic region between the ␣a and ␣b genes is significantly palindromic. Thus, this cluster probably evolved by duplication, inversion, and divergence of a common ancestral ␣-tubulin gene. Expression of the NcGTb␣c gene is cosmopolitan, with its mRNA most abundant in hematopoietic, neural, and testicular tissues, whereas NcGTb␣a and NcGTb␣b transcripts accumulate primarily in brain. The differential expression of the three genes is consistent with distinct suites of putative promoter and enhancer elements. We propose that cold adaptation of the microtubule system of Antarctic fishes is based in part on expansion of the ␣-and ␤-tubulin gene families to ensure efficient synthesis of tubulin polypeptides.Subjected to an increasingly severe thermal environment as the Southern Ocean began to cool approximately 25-40 million years ago (mya) 1 (1), the coastal fishes of the Antarctic diverged from temperate fishes (2) and evolved compensatory molecular, cellular, and physiological adaptations that maintain metabolic efficiency and preserve macromolecular function in their now chronically cold marine environment (Ϫ1.86 to approximately 1°C). The translational machinery of these fishes, for example, shows clear evidence of cold adaptation (3, 4), with rates of polypeptide chain elongation more than 10-fold greater than those measured in temperate fishes cooled to comparable temperatures. Similarly, the polymerization energetics of the actins of Antarctic fishes (5) and the ATPase activities of their skeletal myosins (6) support efficient myofibrillar assembly and function at their low habitat temperatures. Our goal is to determine the molecular adaptations, both qualitative and quantitative, that maintain the efficient expression of the tubulin genes and the polymerization capacity of the tubulin polypeptides of these extreme psychrophiles.We and others (7,8) have shown previously that the critical concentration for microtubule formation by the brain tubulins of Antarctic fishes (ϳ1 mg/ml) is comparable to those of temperate poikilotherms and homeotherms at their much higher body temp...

Cold Adaptation of Microtubule Assembly and Dynamics

“…cDNA Library Construction and Screening-The production of two N. coriiceps brain cDNA libraries, the first primed with oligo(dT) and constructed in gt10 and the second primed with random hexanucleotides/oligo(dT) (75:25%) and cloned in ZAP II (Stratagene), has been described previously (13,14). The C. rastrospinosus brain cDNA library was constructed in gt10 by similar methods, with cDNA synthesis primed by a 50:50 mixture of random hexanucleotides and oligo(dT).…”

“…Libraries were screened for recombinant clones containing ␣-and ␤-tubulin coding sequences as described by Parker and Detrich (14). One hundred fifty nine candidate ␣-tubulin cDNA isolates and 55 prospective ␤-tubulin cDNAs were obtained from a total of six screens of the two N. coriiceps libraries (632,000 total recombinant phage used in three ␣-screens, 385,000 for three ␤-screens).…”

“…NcTb␣3, -␣5, and -␣6) or contained an artifactual internal deletion (NcTb␣1). NcTb␣7, -␣2, and -␣8 are encoded by a tightly linked cluster of three ␣-tubulin genes, NcGTb␣a (A), NcGTb␣b (B), and NcGTb␣c (C) (14). The vertebrate ␣-tubulin consensus (Con␣Tb) was established using isotypes from chicken (chains ␣3-␣5, ␣8; GenBank TM / Swiss-Prot accession numbers P09642-P09645), human (␣1, ␣4; P04687, P05215), mouse (␣1-␣3, ␣6; P02551, P05213, P05214, P05216), pig (␣-chain, P02550), Chinese hamster ovary cells (␣1, P05209), Xenopus laevis (␣-chain, P08537), three salmonid fishes (Oncorhynchus mykiss testis-specific ␣, P18288; O. keta ␣, P30436; Notophthalmus viridescens ␣, Q91060), and the electric ray (Torpedo marmorata ␣T6, P36220).…”

Cold-adapted tubulins in the glacier ice worm, Mesenchytraeus solifugus

“…Psychrophilic ciliates (Pucciarelli and Miceli, 2002) contain tubulin genes with relatively high A/T contents (α-55.7%, β-57.7%), which may facilitate DNA strand separation and favor DNA replication and transcription; similar observations have been made in other coldadapted taxa (e.g., Parker and Detrich, 1998;Zhao et al, 1998), but note that others have observed the opposite trend (e.g., Feller and Gerday, 2003). Ice worm tubulin genes were relatively G/C rich (i.e., ∼55%), which runs counter to other ice worms genes (e.g., ∼45% G/C content in a random sampling of ice worm genes; Farrell et al, 2004).…”

“…Posttranslational modifications such as polyglutamylation may also confer some cold stability to microtubules by reducing electrostatic repulsions between tubulin dimers (Redeker et al, 2004). Additionally, the presence of multigene tubulin families in N. coriiceps (nine α-and four β-isotypes) appears to facilitate the synthesis and maintenance of tubulin subunits at low physiological temperatures (Parker and Detrich, 1998).…”