FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079897132> ?p ?o ?g. }

• W2079897132 endingPage "34369" @default.
• W2079897132 startingPage "34358" @default.
• W2079897132 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 ∼15 distinct DNA fragments complementary to α-tubulin cDNA probes, which suggests that the α-tubulins of this cold-adapted fish are encoded by a substantial multigene family. From an N. coriicepstesticular DNA library, we isolated a 13.8-kilobase pair genomic clone that contains a tightly linked cluster of three α-tubulin genes, designated NcGTbαa, NcGTbαb, andNcGTbαc. Two of these genes, NcGTbαa andNcGTbαb, are linked in head-to-head (5′ to 5′) orientation with ∼500 bp separating their start codons, whereasNcGTbαa and NcGTbαc are linked tail-to-tail (3′ to 3′) with ∼2.5 kilobase pairs between their stop codons. The exons, introns, and untranslated regions of the three α-tubulin genes are strikingly similar in sequence, and the intergenic region between the αa and αbgenes is significantly palindromic. Thus, this cluster probably evolved by duplication, inversion, and divergence of a common ancestral α-tubulin gene. Expression of the NcGTbαc gene is cosmopolitan, with its mRNA most abundant in hematopoietic, neural, and testicular tissues, whereas NcGTbαa andNcGTbαb 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 α- and β-tubulin gene families to ensure efficient synthesis of tubulin polypeptides. 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 ∼15 distinct DNA fragments complementary to α-tubulin cDNA probes, which suggests that the α-tubulins of this cold-adapted fish are encoded by a substantial multigene family. From an N. coriicepstesticular DNA library, we isolated a 13.8-kilobase pair genomic clone that contains a tightly linked cluster of three α-tubulin genes, designated NcGTbαa, NcGTbαb, andNcGTbαc. Two of these genes, NcGTbαa andNcGTbαb, are linked in head-to-head (5′ to 5′) orientation with ∼500 bp separating their start codons, whereasNcGTbαa and NcGTbαc are linked tail-to-tail (3′ to 3′) with ∼2.5 kilobase pairs between their stop codons. The exons, introns, and untranslated regions of the three α-tubulin genes are strikingly similar in sequence, and the intergenic region between the αa and αbgenes is significantly palindromic. Thus, this cluster probably evolved by duplication, inversion, and divergence of a common ancestral α-tubulin gene. Expression of the NcGTbαc gene is cosmopolitan, with its mRNA most abundant in hematopoietic, neural, and testicular tissues, whereas NcGTbαa andNcGTbαb 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 α- and β-tubulin gene families to ensure efficient synthesis of tubulin polypeptides. million years ago base pairs kilobase pairs polymerase chain reaction untranslated region. Subjected to an increasingly severe thermal environment as the Southern Ocean began to cool approximately 25–40 million years ago (mya)1 (1Eastman J.T. Antarctic Fish Biology: Evolution in a Unique Environment. Academic Press, San Diego1993Google Scholar), the coastal fishes of the Antarctic diverged from temperate fishes (2DeWitt H.H. Bushnell V.C. Antarctic Map Folio Series, Folio 15. American Geographical Society, New York1971: 1-10Google Scholar) and evolved compensatory molecular, cellular, and physiological adaptations that maintain metabolic efficiency and preserve macromolecular function in their now chronically cold marine environment (−1.86 to approximately 1 °C). The translational machinery of these fishes, for example, shows clear evidence of cold adaptation (3Smith M.A.K. Haschemeyer A.E.V. Physiol. Zool. 1980; 53: 373-382Crossref Google Scholar, 4Haschemeyer A.E.V. Mathews R.W. Biol. Bull. 1982; 162: 18-27Crossref Google Scholar), 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 (5Swezey R.R. Somero G.N. Biochemistry. 1982; 21: 4496-4503Crossref PubMed Scopus (83) Google Scholar) and the ATPase activities of their skeletal myosins (6Johnston I.A. Walesby N.J. J. Comp. Physiol. 1977; 119: 195-206Crossref Scopus (66) Google Scholar) 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 (7Williams Jr., R.C. Correia J.J. DeVries A.L. Biochemistry. 1985; 24: 2790-2798Crossref PubMed Scopus (49) Google Scholar, 8Detrich III, H.W. Johnson K.A. Marchese-Ragona S.P. Biochemistry. 1989; 28: 10085-10093Crossref PubMed Scopus (60) Google Scholar) have shown previously that the critical concentration for microtubule formation by the brain tubulins of Antarctic fishes (∼1 mg/ml) is comparable to those of temperate poikilotherms and homeotherms at their much higher body temperatures. Conservation of the critical concentration by Antarctic fishes probably results from structural changes, both in primary sequences and in posttranslational modifications, intrinsic to their α- and β-tubulin subunits (9Detrich III, H.W. Overton S.A. J. Biol. Chem. 1986; 261: 10922-10930Abstract Full Text PDF PubMed Google Scholar, 10Detrich III, H.W. Prasad V. Ludueña R.F. J. Biol. Chem. 1987; 262: 8360-8366Abstract Full Text PDF PubMed Google Scholar, 11Detrich III, H.W. Neighbors B.W. Sloboda R.D. Williams Jr., R.C. Cell Motil. Cytoskeleton. 1990; 17: 174-186Crossref PubMed Scopus (17) Google Scholar, 12Detrich III, H.W. Parker S.P. Cell Motil. Cytoskeleton. 1993; 24: 156-166Crossref PubMed Scopus (26) Google Scholar). The primary sequence of class II β-tubulin from the yellowbelly rockcod Notothenia coriiceps, for example, contains unique residue substitutions that increase both the hydrophobicity and the flexibility of the polypeptide chain (12Detrich III, H.W. Parker S.P. Cell Motil. Cytoskeleton. 1993; 24: 156-166Crossref PubMed Scopus (26) Google Scholar, 13Detrich III, H.W. Comp. Biochem. Physiol. 1997; 118: 501-513Crossref Scopus (24) Google Scholar), two factors that should favor microtubule formation in an energy-poor environment. Similar alterations have been observed in other α- and β-tubulin chains of this species. 2Detailed analysis of the structural changes unique to Antarctic fish tubulins will be presented elsewhere (S. K. Parker, E. Nogales, K. H. Downing, and H. W. Detrich III, manuscript in preparation). A second, related challenge confronting Antarctic fishes is the synthesis of sufficient quantities of the α- and β-tubulins to attain the critical concentration of tubulin dimers in their cells. The abundant expression of tubulin in the brains of Antarctic fishes is likely to require compensatory adjustments in gene transcription to offset the rate-depressing effects of low temperature. Potential adaptations include increases in tubulin gene number, organization of tubulin genes into efficient transcription units, evolution of more efficient gene promoters, enhancers, transcription factors, and/or RNA polymerases, and enhancement of mRNA stabilization. To evaluate these possibilities, we have initiated analysis of the structure, genomic organization, and expression of the tubulin genes of N. coriiceps. Our results suggest that several of these modes of adaptation may be exploited by these cold-living vertebrates. In higher vertebrates, the α- and β-tubulins are encoded by small gene families (∼6–7 functional genes for α and a similar number for β), each member of which yields a structurally distinct polypeptide (14Sullivan K.F. Annu. Rev. Cell Biol. 1988; 4: 687-716Crossref PubMed Scopus (427) Google Scholar, 15Murphy D.B. Curr. Opin. Cell Biol. 1991; 3: 43-51Crossref PubMed Scopus (40) Google Scholar, 16Ludueña R.F. Mol. Biol. Cell. 1993; 4: 445-457Crossref PubMed Scopus (217) Google Scholar). These genes are generally thought to be unlinked and dispersed throughout the genome (17Cleveland D.W. Hughes S.H. Stubblefield E. Kirschner M.W. Varmus H.E. J. Biol. Chem. 1981; 256: 3130-3134Abstract Full Text PDF PubMed Google Scholar). In a study of the chicken α-tubulin gene family, for example, Pratt and Cleveland (18Pratt L.F. Cleveland D.W. EMBO J. 1988; 7: 931-940Crossref PubMed Scopus (37) Google Scholar) found that four of five genomic clones contained single α-tubulin genes; the fifth contained two α-tubulin genes, one functional and the second a pseudogene. The genomes of lower, nonvertebrate eukaryotes, by contrast, frequently contain tightly linked tubulin genes. Protozoan parasites possess tubulin gene ensembles, either as separate tandem groupings of α- or β-tubulin genes (Leishmania spp. (19Landfear S.M. McMahon-Pratt D. Wirth D.F. Mol. Cell. Biol. 1983; 3: 1070-1076Crossref PubMed Scopus (79) Google Scholar,20Huang P.L. Roberts B.E. McMahaon Pratt D. David J.R. Miller J.S. Mol. Cell. Biol. 1984; 4: 1372-1383Crossref PubMed Google Scholar)) or as linked α/β tandem repeats (Trypanosoma brucei(21Kimmel B.E. Samson S. Wu J. Hirschberg R. Yarbrough L.R. Gene (Amst.). 1985; 35: 237-248Crossref PubMed Scopus (94) Google Scholar, 22Thomashow LS. Milhausen M. Rutter W.J. Agabian N. Cell. 1983; 32: 35-43Abstract Full Text PDF PubMed Scopus (176) Google Scholar)). Similarly, some of the tubulin genes of the sea urchinLytechinus pictus are organized in distinct α or β clusters (23Alexandraki D. Ruderman J.V. Mol. Cell. Biol. 1981; 1: 1125-1137Crossref PubMed Google Scholar). The regulation of tubulin gene expression occurs at both transcriptional and translational levels. The tissue-specific and hormonally regulated expression of the β-tubulin genes ofDrosophila is controlled both by upstream promoter elements and by negative and positive regulatory elements (silencers and enhancers) generally located within the first introns (24Michiels F. Wolk A. Renkawitz-Pohl R. Nucleic Acids Res. 1991; 19: 4515-4521Crossref PubMed Scopus (20) Google Scholar, 25Bruhat A. Tourmente S. Chapel S. Sobrier M.L. Couderc J.L. Dastugue B. Nucleic Acids Res. 1990; 18: 2861-2867Crossref PubMed Scopus (36) Google Scholar, 26Gasch A. Hinz U. Renkawitz-Pohl R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3215-3218Crossref PubMed Scopus (59) Google Scholar, 27Hinz U. Wolk A. Renkawitz-Pohl R. Development. 1992; 116: 543-554PubMed Google Scholar, 28Buttgereit D. J. Cell Sci. 1993; 105: 721-727PubMed Google Scholar). Less is known regarding regulation of tubulin gene expression in vertebrates. TATA boxes are generally present in vertebrate α- and β-tubulin promoters (29Sullivan K.F. Machlin P.S. Ratrie III, H. Cleveland D.W. J. Biol. Chem. 1986; 261: 13317-13322Abstract Full Text PDF PubMed Google Scholar, 30Hair A. Morgan G.T. Mol. Cell. Biol. 1993; 13: 7925-7934Crossref PubMed Scopus (11) Google Scholar), and high level expression of a Xenopusα-tubulin gene, XαT14, in oocytes is regulated by three CCAAT boxes, a “heat-shock-like” element (all located 60–200 bp upstream of the transcription start site), and their corresponding transcription factors (31Middleton K.M. Morgan G.T. Nucleic Acids Res. 1989; 17: 5041-5055Crossref PubMed Scopus (9) Google Scholar). Cotranslational regulation of tubulin mRNA stability also contributes to control of cellular tubulin levels (32Cleveland D.W. Trends Biochem. Sci. 1988; 13: 339-343Abstract Full Text PDF PubMed Scopus (126) Google Scholar). When the pool of tubulin dimers is high, β-tubulin mRNAs are targeted for degradation by binding of a cellular factor to the ribosome-bound amino-terminal β-tubulin tetrapeptide (33Yen T.J. Machlin P.S. Cleveland D.W. Nature. 1988; 334: 580-585Crossref PubMed Scopus (247) Google Scholar,34Theodorakis N.G. Cleveland D.W. Mol. Cell. Biol. 1992; 12: 791-799Crossref PubMed Google Scholar). Here we report the first example of clustered tubulin genes in a vertebrate, the Antarctic rockcod N. coriiceps. Three α-tubulin genes, designated NcGTbαa,NcGTbαb, and NcGTbαc, are tightly linked in an ∼10-kb segment of DNA, with αa and αblinked head-to-head (5′ to 5′) and αa and αctail-to-tail (3′ to 3′). The similarity of the nucleotide sequences of these genes, strikingly illustrated by an ∼480-bp palindrome linkingαa and αb, suggests that the cluster evolved approximately 7–31 million years ago by duplication, inversion, and divergence of a common ancestral α-tubulin gene. The neurally restricted expression of the αa/αb gene pair and the widespread expression of αc appear to be governed by distinct sets of promoter and enhancer elements. We have also identified a 285-bp element from the αa/αcintergenic region that is distributed widely in notothenioid genomes. We propose that expansion of the number of α-tubulin genes in theN. coriiceps genome facilitates the synthesis of α-tubulin chains at low temperature by providing additional templates for mRNA synthesis. The selective pressure favoring this expansion was probably the cooling of the Southern Ocean beginning ∼25–40 mya. A preliminary report of some of this work has appeared (35Parker S.K. Detrich III, H.W. Mol. Biol. Cell. 1996; 75: 45aGoogle Scholar). Specimens of the Antarctic yellowbelly rockcod, N. coriiceps, were collected by bottom trawling from the R/V Hero or from the R/V Polar Duke near Low and Brabant Islands in the Palmer Archipelago. They were transported alive to Palmer Station, Antarctica, where they were maintained in seawater aquaria at −1.5 to 1 °C. Tissues (testis, brain, gill, liver, spleen, blood, and muscle) were dissected, frozen in liquid nitrogen, and maintained at −70 °C until use. Frozen testis tissue from the New Zealand black cod, Notothenia angustata, was generously provided by Dr. Arthur DeVries (University of Illinois, Urbana). High molecular weight genomic DNA was purified (36Blin N. Stafford D.W. Nucleic Acids Res. 1976; 3: 2303-2308Crossref PubMed Scopus (2335) Google Scholar) from the testis tissue of one N. coriiceps male, and Southern blots (37Southern E.M. J. Mol. Biol. 1975; 98: 503-515Crossref PubMed Scopus (21490) Google Scholar) of restriction endonuclease-digested DNA samples were prepared as described previously (12Detrich III, H.W. Parker S.P. Cell Motil. Cytoskeleton. 1993; 24: 156-166Crossref PubMed Scopus (26) Google Scholar). The Southern replicas were probed for α-tubulin gene sequences by hybridization to 32P-labeled (12Detrich III, H.W. Parker S.P. Cell Motil. Cytoskeleton. 1993; 24: 156-166Crossref PubMed Scopus (26) Google Scholar, 38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) α-tubulin cDNAs from the chicken (cα1; Ref. 39Cleveland D.W. Lopata M.A. MacDonald R.J. Cowan N.J. Rutter W.J. Kirschner M.W. Cell. 1980; 20: 95-105Abstract Full Text PDF PubMed Scopus (1312) Google Scholar) or from Chlamydomonas reinhardtii (α10-2; Ref. 40Silflow C.D. Chisholm R.L. Conner T.W. Ranum L.P.W. Mol. Cell. Biol. 1985; 5: 2389-2398Crossref PubMed Scopus (133) Google Scholar). Prehybridization and hybridization of the membranes were performed at moderate stringency (3× SSC (1× SSC = 0.15 m NaCl, 0.015 m trisodium citrate), 5× Denhardt's solution (41Denhardt D. Biochem. Biophys. Res. Commun. 1966; 23: 641-652Crossref PubMed Scopus (2495) Google Scholar), 50 μg/ml sonicated, denatured Escherichia coli DNA, 0.5% (w/v) SDS, 1 mm EDTA, 68 °C) for 1 and 18–20 h, respectively, and the membranes were washed to high stringency (0.1× SSC, 25 °C, 1 h). The membranes were exposed to Kodak XAR-5 X-Omat film at −70 °C with intensification (DuPont Cronex Lightning Plus screens). A genomic library of N. coriiceps testicular DNA was constructed in the λ vector Charon 35 (42Loenen W.A.M. Blattner F.R. Gene (Amst.). 1983; 26: 171-179Crossref PubMed Scopus (120) Google Scholar). High molecular weight DNA was digested partially with MboI, and fragments of 15–20-kb, obtained by sucrose gradient centrifugation, were ligated to the BamHI sites of the vector arms. Recombinant phage DNA was packaged in vitro (Packagene; Promega). The unamplified library was screened for clones encoding α-tubulin genes by hybridization (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) of nitrocellulose replicas of bacteriophage plaque DNA to the32P-labeled chicken cDNA. Prehybridization and hybridization of the membranes were performed at moderate stringency (see “Southern Analysis of Genomic DNA”) for 1 and 18–20 h, respectively. Positive plaques were detected autoradiographically as described above. One hundred twenty candidate α-tubulin genomic isolates, obtained from a primary screen of 500,000 recombinant phage, were classified as strongly, moderately, or weakly hybridizing. One strongly hybridizing isolate, designated S2, was carried through two additional rounds of plaque purification and screening, and single plaques were picked for clone stock preparation. By using testicular DNA from N. angustata, we constructed a genomic library of 15–20-kb fragments in the vector LambdaGEM-11 (Promega). DNA was digested partially with MboI; fragments were ligated to phage arms containing XhoI half-sites, and recombinant phage DNA was packaged in vitro (Packagene; Promega). The library (titer = 1 × 106) was screened for candidate α-tubulin clones by hybridization to anN. coriiceps α-tubulin cDNA, NcTbα1, essentially as described for α-globin genes by Zhao et al. (43Zhao Y. Ratnayake-Lecamwasam M. Parker S.K. Cocca E. Camardella L. di Prisco G. Detrich III, H.W. J. Biol. Chem. 1998; 273: 14745-14752Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Twelve candidate α-tubulin genomic isolates were obtained from a primary screen of 200,000 recombinant phage. Six of these isolates were carried through two additional rounds of plaque purification and screening, and single plaques were picked for clone stock preparation. Total RNA was isolated from brain tissues of N. coriiceps (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 44Puissant C. Houdebine L.-M. BioTechniques. 1990; 8: 148-149Crossref PubMed Google Scholar), and poly(A)+ RNA was selected by oligo(dT)-cellulose affinity chromatography (45Aviv H. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 1408-1412Crossref PubMed Scopus (5183) Google Scholar). Two different libraries were made. Oligo(dT)-primed cDNA synthesis and construction of the first library in λgt10 followed the procedures described by Huynh et al. (46Huynh T.V. Young R.A. Davis R.W. Glover D.M. DNA Cloning. IRL Press at Oxford University Press, Oxford1985: 49-78Google Scholar). The second library was constructed in λZAP II (Stratagene); cDNA synthesis was primed with a mixture of random hexanucleotides (75%) and oligo(dT) (25%). The libraries were screened for recombinant clones bearing α-tubulin coding sequences by hybridization of nitrocellulose or nylon (MagnaLift, MSI, Westboro, MA) replicas of bacteriophage plaque DNA to the probe,32P-labeled by nick translation (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) or by random priming (47Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16651) Google Scholar). In early screens cα1 was used as probe, whereas in later screens α-tubulin cDNAs from N. coriiceps were employed. Hybridization and washing of the membranes were performed as described (12Detrich III, H.W. Parker S.P. Cell Motil. Cytoskeleton. 1993; 24: 156-166Crossref PubMed Scopus (26) Google Scholar), and positive plaques were detected autoradiographically. A total of 159 candidate α-tubulin cDNA isolates were obtained from three screens (632,000 total recombinant phage) of the two libraries, and 80 of these were carried through tertiary plaque purification/screening. Three cDNA clones (designated NcTbα2, NcTbα7, and NcTbα8) from the second library that corresponded to the three α-tubulin genes (αb,αa, and αc, respectively) of the genomic cluster (Fig. 1) were sequenced (see below). The nucleotide sequence of NcTbα2 downstream of codon 168 was used to complete the sequence of the partial αb gene. Two cDNAs (NcTbα1 and NcTbα3) from the first library were also characterized. Parental clones and restriction fragment or deletion (48Henikoff S. Gene (Amst.). 1984; 28: 351-359Crossref PubMed Scopus (2832) Google Scholar) subclones were sequenced manually on both strands by use of the dideoxynucleotide chain termination method (49Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52769) Google Scholar) and T4 DNA polymerase (Sequenase II; U. S. Biochemical Corp.). Portions of the sequence were established by use of the PRISM Ready Reaction Dye Deoxy Termination Cycle Sequencing Kit (Applied Biosystems), and the products were electrophoresed on an Applied Biosystems 373A automated DNA sequencer (University of Maine DNA Sequencing Facility). Nucleotide and amino acid sequence analyses of the N. coriiceps α-tubulin genes, cDNAs, and their encoded proteins were performed by use of the Clustal method provided by DNASTAR MegAlign. DNA sequence relatedness was calculated as the similarity index of Dayhoff (50Dayhoff M.O. Atlas of Protein Sequence and Structure. National Biomedical Research Foundation, Washington, D. C.1979Google Scholar) as implemented by DNASTAR Align. The sequence of the N. coriiceps α-tubulin gene cluster reported in this paper has been deposited in the GenBankTM data base under the accession number AF082027. The sequence of the cluster has been scanned against the GenBankTM data base using the BLASTN program (National Center for Biotechnology Information) to identify sequences with significant relatedness. Related sequences, and their accession numbers, are presented under “Results.” Total RNAs from testis, brain, gill, liver, spleen, blood, and muscle were isolated from tissues by a modification (44Puissant C. Houdebine L.-M. BioTechniques. 1990; 8: 148-149Crossref PubMed Google Scholar) of the acid guanidinium thiocyanate/phenol/chloroform method (51Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). RNAs (5 μg/slot) were applied to nylon membranes (MagnaGraph, MSI) by vacuum aspiration using a Bio-Rad Bio-Dot slot-blot apparatus. Sets of seven RNA samples were hybridized to PCR-generated, 32P-labeled probes specific for the 3′-UTRs of the cDNAs NcTbα2 (αb gene), NcTbα3, NcTbα7 (αa gene), or NcTbα8 (αcgene). To estimate the total α-tubulin mRNA in each tissue, a control set of RNA samples was hybridized to a fragment of NcTbα1 encoding amino acid residues 1–430. Prehybridization and hybridization of the membranes were performed in 5× SSPE (1× SSPE = 0.18m NaCl, 0.01 mNa2HPO4·7H2O, 0.001 mEDTA), 5× Denhardt's solution (41Denhardt D. Biochem. Biophys. Res. Commun. 1966; 23: 641-652Crossref PubMed Scopus (2495) Google Scholar), 50% formamide, 0.2% SDS at 42 °C for 2 and 18–20 h, respectively, after which the membranes were washed sequentially with buffers of increasing stringency (final wash conditions = 0.1× SSPE, 42 °C, 15 min). The membranes were exposed to Fuji RX x-ray film at −70 °C with intensification. To determine the potential linkage of α-tubulin genes in the N. angustata genome, we employed a PCR-based strategy using as template phage DNAs purified from the six tertiary genomic clones (see “Genomic Library Construction and Screening”). Nondegenerate primers corresponding to highly conserved regions of the primary sequence of the N. coriiceps α-tubulins were synthesized as follows: 1) sense primer, 5′ CAGTTTGTGGACTGGTGC 3′ (residues 341–347, N-Gln-Phe-Val-Asp-Trp-Cys-C); 2) antisense primer, 5′ AGCTCCAGTCTCACTGAAG 3′ (reverse complement of coding sequence for residues 53–58; N-Phe-Ser-Glu-Thr-Gly-Ala-C). The primers were used in three combinations as follows: 1) sense alone to amplify tail-to-tail-linked genes; 2) antisense alone to amplify head-to-head-linked genes; and 3) sense plus antisense to establish head-to-tail linkage by difference (i.e. PCR products not shared with sense alone and antisense alone reactions). Each PCR reaction contained 3–5 ng of template DNA, 1.6 μmprimers (0.8 μm of each primer when different), andCLONTECH AdvantageTM KlenTaq polymerase mix (optimized for long distance PCR) (52Barnes W.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2216-2220Crossref PubMed Scopus (982) Google Scholar). Touchdown PCR (53Don R.H. Cox P.T. Wainwright B.J. Baker K. Mattick J.S. Nucleic Acids Res. 1991; 19: 4008Crossref PubMed Scopus (2250) Google Scholar) was performed for 29 cycles using the following parameters: 1) denaturation steps, 94 °C, 30 s; 2) annealing steps, first 9 cycles ramping the temperature from 70 to 62 °C in 1° increments followed by 20 cycles at 62 °C; and 3) extension steps, 68 °C, 6 min. PCR products were analyzed on 1% agarose gels containing 1× TBE (0.089m Tris borate, 2 mm EDTA, pH 8.0) and 0.0005% ethidium bromide. The ends of the PCR products were sequenced by the automated procedure to establish α-tubulin gene orientation. During characterization of the N. coriicepsα-tubulin gene cluster, we discovered a 285-bp repetitive element. To determine the abundance, organization, and species distribution of this fragment, we hybridized it to Southern replicas ofHindIII-digested genomic DNAs from Antarctic and temperate notothenioids, other temperate fishes, an amphibian, and a reptile. Restriction endonuclease digestion, electrophoresis, and transfer of DNAs were performed as described previously (12Detrich III, H.W. Parker S.P. Cell Motil. Cytoskeleton. 1993; 24: 156-166Crossref PubMed Scopus (26) Google Scholar). Prehybridization of the membrane and subsequent hybridization to the 285-bp probe (labeled with 32P by random priming (47Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16651) Google Scholar)) were performed as described by Detrich and Parker (12Detrich III, H.W. Parker S.P. Cell Motil. Cytoskeleton. 1993; 24: 156-166Crossref PubMed Scopus (26) Google Scholar) with the following exceptions: 1) prehybridization was for 2 h; 2) the prehybridization/hybridization temperature was 63 °C; and 3) the membranes were washed to final stringencies of 0.1–1× SSC, 63 °C, for 15–40 min. The membranes were exposed to Fuji RX x-ray film as described above. Genomic DNA from the zebrafish (Danio rerio) was prepared from total body tissues (36Blin N. Stafford D.W. Nucleic Acids Res. 1976; 3: 2303-2308Crossref PubMed Scopus (2335) Google Scholar). Samples of genomic DNAs from the African lungfish (Protopterus aethiopicus), the clearnose skate (Raja eglanteria), the goldfish (Carassius auratus), the horned shark (Heterdontus francisci), the sea lamprey (Petromyzon marinus), the spotted ratfish (Hydrolagus colliei), the sturgeon (Acipenser fulvescens), the clawed frog (Xenopus mulleri), and the snapping turtle (Chelydra serpentina) were generously provided by Dr. Chris Amemiya (Boston University School of Medicine). To estimate the number of α-tubulin genes possessed by N. coriiceps, we probed its genome for sequences complementary to α-tubulin cDNAs from the chicken (cα1) and fromChlamydomonas (α10–2). Fig.1 shows that the α-tubulin probes hybridized to 10–15 different fragments in each restriction digest of the fish DNA. Furthermore, the hybridization patterns generated by the two heterologous cDNAs were virtually identical. These results suggest that the α-tubulins of N. coriiceps, like its β-tubulins (12Detrich III, H.W. Parker S.P. Cell Motil. Cytoskeleton. 1993; 24: 156-166Crossref PubMed Scopus (26) Google Scholar), are encoded by a multigene family that is larger than those of higher vertebrates (14Sullivan K.F. Annu. Rev. Cell Biol. 1988; 4: 687-716Crossref PubMed Scopus (427) Google Scholar, 16Ludueña R.F. Mol. Biol. Cell. 1993; 4: 445-457Crossref PubMed Scopus (217) Google Scholar, 39Cleveland D.W. Lopata M.A. MacDonald R.J. Cowan N.J. Rutter W.J. Kirschner M.W. Cell. 1980; 20: 95-105Abstract Full Text PDF PubMed Scopus (1312) Google Scholar). Of particular note, the strong hybridization signals observed for some of the fragments raised the possibility that they contain multiple, linked α-tubulin genes. To investigate the organization of the α-tubulin genes of N. coriiceps, we selected a strongly hybridizing clone, S2, that carried an insert of ∼13.8 kb. Preliminary restriction mapping and Southern hybridization analysis suggested that the insert contained two or more α-tubulin genes in a segment of ∼10 kb. Subsequent sequence analysis revealed that S2 contains two complete α-tubulin genes, designated NcGTbαa and NcGTbαc, and one partial gene, NcGTbαb, that abuts one end of the genomic fragment. Fig. 2 presents the organization and salient features of this gene complex. Two of the genes, αa and αb, a" @default.
• W2079897132 created "2016-06-24" @default.
• W2079897132 creator A5016895478 @default.
• W2079897132 creator A5045110261 @default.
• W2079897132 date "1998-12-01" @default.
• W2079897132 modified "2023-10-01" @default.
• W2079897132 title "Evolution, Organization, and Expression of α-Tubulin Genes in the Antarctic Fish Notothenia coriiceps" @default.
• W2079897132 cites W108613487 @default.
• W2079897132 cites W1491713847 @default.
• W2079897132 cites W1504598037 @default.
• W2079897132 cites W1523398066 @default.
• W2079897132 cites W1606427077 @default.
• W2079897132 cites W1652542971 @default.
• W2079897132 cites W1965051773 @default.
• W2079897132 cites W1968207332 @default.
• W2079897132 cites W1968700959 @default.
• W2079897132 cites W1969772407 @default.
• W2079897132 cites W1973857059 @default.
• W2079897132 cites W1978659501 @default.
• W2079897132 cites W1978713358 @default.
• W2079897132 cites W1983184960 @default.
• W2079897132 cites W1983985678 @default.
• W2079897132 cites W1987666378 @default.
• W2079897132 cites W1990989603 @default.
• W2079897132 cites W1999966058 @default.
• W2079897132 cites W2004242813 @default.
• W2079897132 cites W2005507415 @default.
• W2079897132 cites W2006619454 @default.
• W2079897132 cites W2010335887 @default.
• W2079897132 cites W2014669590 @default.
• W2079897132 cites W2016564007 @default.
• W2079897132 cites W2019271290 @default.
• W2079897132 cites W2028082957 @default.
• W2079897132 cites W2030616232 @default.
• W2079897132 cites W2032462421 @default.
• W2079897132 cites W2034354422 @default.
• W2079897132 cites W2036714935 @default.
• W2079897132 cites W2038602948 @default.
• W2079897132 cites W2045255938 @default.
• W2079897132 cites W2045348184 @default.
• W2079897132 cites W2048227952 @default.
• W2079897132 cites W2048800885 @default.
• W2079897132 cites W2049661143 @default.
• W2079897132 cites W2054444853 @default.
• W2079897132 cites W2055613155 @default.
• W2079897132 cites W2057293309 @default.
• W2079897132 cites W2063278037 @default.
• W2079897132 cites W2063450941 @default.
• W2079897132 cites W2065123667 @default.
• W2079897132 cites W2066160869 @default.
• W2079897132 cites W2075879868 @default.
• W2079897132 cites W2076318901 @default.
• W2079897132 cites W2080183319 @default.
• W2079897132 cites W2081422634 @default.
• W2079897132 cites W2088857107 @default.
• W2079897132 cites W2090772541 @default.
• W2079897132 cites W2091306747 @default.
• W2079897132 cites W2103590067 @default.
• W2079897132 cites W2119795117 @default.
• W2079897132 cites W2121886007 @default.
• W2079897132 cites W2122385641 @default.
• W2079897132 cites W2124103214 @default.
• W2079897132 cites W2138270253 @default.
• W2079897132 cites W2141357485 @default.
• W2079897132 cites W2158512067 @default.
• W2079897132 cites W2186637226 @default.
• W2079897132 cites W2188951155 @default.
• W2079897132 cites W2263442484 @default.
• W2079897132 cites W2299692751 @default.
• W2079897132 cites W4294216491 @default.
• W2079897132 doi "https://doi.org/10.1074/jbc.273.51.34358" @default.
• W2079897132 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9852102" @default.
• W2079897132 hasPublicationYear "1998" @default.
• W2079897132 type Work @default.
• W2079897132 sameAs 2079897132 @default.
• W2079897132 citedByCount "30" @default.
• W2079897132 countsByYear W20798971322012 @default.
• W2079897132 countsByYear W20798971322013 @default.
• W2079897132 countsByYear W20798971322016 @default.
• W2079897132 countsByYear W20798971322018 @default.
• W2079897132 countsByYear W20798971322019 @default.
• W2079897132 countsByYear W20798971322020 @default.
• W2079897132 countsByYear W20798971322023 @default.
• W2079897132 crossrefType "journal-article" @default.
• W2079897132 hasAuthorship W2079897132A5016895478 @default.
• W2079897132 hasAuthorship W2079897132A5045110261 @default.
• W2079897132 hasBestOaLocation W20798971321 @default.
• W2079897132 hasConcept C104317684 @default.
• W2079897132 hasConcept C18903297 @default.
• W2079897132 hasConcept C199360897 @default.
• W2079897132 hasConcept C20418707 @default.
• W2079897132 hasConcept C2909208804 @default.
• W2079897132 hasConcept C41008148 @default.
• W2079897132 hasConcept C505870484 @default.
• W2079897132 hasConcept C54355233 @default.
• W2079897132 hasConcept C78458016 @default.
• W2079897132 hasConcept C84425145 @default.
• W2079897132 hasConcept C86803240 @default.

• next