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. 1988 Jun 1;106(6):2035–2046. doi: 10.1083/jcb.106.6.2035

The alpha-tubulin gene family expressed during cell differentiation in Naegleria gruberi

PMCID: PMC2115128  PMID: 2838492

Abstract

Genes that direct the programmed synthesis of flagellar alpha-tubulin during the differentiation of Naegleria gruberi from amebae to flagellates have been cloned, and found to be novel with respect to gene organization, sequence, and conservation. The flagellar alpha- tubulin gene family is represented in the genome by about eight homologous DNA segments that are exceptionally similar and yet are neither identical nor arrayed in a short tandem repeat. The coding regions of three of these genes have been sequenced, two from cDNA clones and one from an intronless genomic gene. These three genes encode an identical alpha-tubulin that is conserved relative to the alpha-tubulins of other organisms except at the carboxyl terminus, where the protein is elongated by two residues and ends in a terminal glutamine instead of the canonical tyrosine. In spite of the protein conservation, the Naegleria DNA sequence has diverged markedly from the alpha-tubulin genes of other organisms, a counterexample to the idea that tubulin genes are conserved. alpha-Tubulin mRNA homologous to this gene family has not been detected in amebae. This mRNA increases markedly in abundance during the first hour of differentiation, and then decreases even more rapidly with a half-life of approximately 8 min. The abundance of physical alpha-tubulin mRNA rises and subsequently falls in parallel with the abundance of translatable flagellar tubulin mRNA and with the in vivo rate of flagellar tubulin synthesis, which indicates that flagellar tubulin synthesis is directly regulated by the relative rates of transcription and mRNA degradation.

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Selected References

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  1. Alexandraki D., Ruderman J. V. Evolution of alpha q- and beta-tubulin genes as inferred by the nucleotide sequences of sea urchin cDNA clones. J Mol Evol. 1983;19(6):397–410. doi: 10.1007/BF02102315. [DOI] [PubMed] [Google Scholar]
  2. Alexandraki D., Ruderman J. V. Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins. Mol Cell Biol. 1981 Dec;1(12):1125–1137. doi: 10.1128/mcb.1.12.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ares M., Jr, Howell S. H. Cell cycle stage-specific accumulation of mRNAs encoding tubulin and other polypeptides in Chlamydomonas. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5577–5581. doi: 10.1073/pnas.79.18.5577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baker E. J., Schloss J. A., Rosenbaum J. L. Rapid changes in tubulin RNA synthesis and stability induced by deflagellation in Chlamydomonas. J Cell Biol. 1984 Dec;99(6):2074–2081. doi: 10.1083/jcb.99.6.2074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blin N., von Gabain A., Bujard H. Isolation of large molecular weight DNA from agarose gels for further digestion by restriction enzymes. FEBS Lett. 1975 Apr 15;53(1):84–86. doi: 10.1016/0014-5793(75)80688-0. [DOI] [PubMed] [Google Scholar]
  6. Breitling F., Little M. Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL1/34, DM1A and DM1B. J Mol Biol. 1986 May 20;189(2):367–370. doi: 10.1016/0022-2836(86)90517-6. [DOI] [PubMed] [Google Scholar]
  7. Brunke K. J., Young E. E., Buchbinder B. U., Weeks D. P. Coordinate regulation of the four tubulin genes of Chlamydomonas reinhardi. Nucleic Acids Res. 1982 Feb 25;10(4):1295–1310. doi: 10.1093/nar/10.4.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Callahan R. C., Shalke G., Gorovsky M. A. Developmental rearrangements associated with a single type of expressed alpha-tubulin gene in Tetrahymena. Cell. 1984 Feb;36(2):441–445. doi: 10.1016/0092-8674(84)90237-x. [DOI] [PubMed] [Google Scholar]
  9. Cleveland D. W., Hughes S. H., Stubblefield E., Kirschner M. W., Varmus H. E. Multiple alpha and beta tubulin genes represent unlinked and dispersed gene families. J Biol Chem. 1981 Mar 25;256(6):3130–3134. [PubMed] [Google Scholar]
  10. Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J., Kirschner M. W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell. 1980 May;20(1):95–105. doi: 10.1016/0092-8674(80)90238-x. [DOI] [PubMed] [Google Scholar]
  11. Cleveland D. W., Sullivan K. F. Molecular biology and genetics of tubulin. Annu Rev Biochem. 1985;54:331–365. doi: 10.1146/annurev.bi.54.070185.001555. [DOI] [PubMed] [Google Scholar]
  12. Cowan N. J., Dobner P. R., Fuchs E. V., Cleveland D. W. Expression of human alpha-tubulin genes: interspecies conservation of 3' untranslated regions. Mol Cell Biol. 1983 Oct;3(10):1738–1745. doi: 10.1128/mcb.3.10.1738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Elliott E. M., Henderson G., Sarangi F., Ling V. Complete sequence of three alpha-tubulin cDNAs in Chinese hamster ovary cells: each encodes a distinct alpha-tubulin isoprotein. Mol Cell Biol. 1986 Mar;6(3):906–913. doi: 10.1128/mcb.6.3.906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  15. Fulton C. Cell differentiation in Naegleria gruberi. Annu Rev Microbiol. 1977;31:597–629. doi: 10.1146/annurev.mi.31.100177.003121. [DOI] [PubMed] [Google Scholar]
  16. Fulton C., Cheng K. L., Lai E. Y. Two calmodulins in Naegleria flagellates: characterization, intracellular segregation, and programmed regulation of mRNA abundance during differentiation. J Cell Biol. 1986 May;102(5):1671–1678. doi: 10.1083/jcb.102.5.1671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fulton C., Kowit J. D. Programmed synthesis of flagellar tubulin during cell differentiation in Naegleria. Ann N Y Acad Sci. 1975 Jun 30;253:318–332. doi: 10.1111/j.1749-6632.1975.tb19210.x. [DOI] [PubMed] [Google Scholar]
  18. Gergen J. P., Stern R. H., Wensink P. C. Filter replicas and permanent collections of recombinant DNA plasmids. Nucleic Acids Res. 1979 Dec 20;7(8):2115–2136. doi: 10.1093/nar/7.8.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Geuens G., Gundersen G. G., Nuydens R., Cornelissen F., Bulinski J. C., DeBrabander M. Ultrastructural colocalization of tyrosinated and detyrosinated alpha-tubulin in interphase and mitotic cells. J Cell Biol. 1986 Nov;103(5):1883–1893. doi: 10.1083/jcb.103.5.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ginzburg I., Behar L., Givol D., Littauer U. Z. The nucleotide sequence of rat alpha-tubulin: 3'-end characteristics, and evolutionary conservation. Nucleic Acids Res. 1981 Jun 25;9(12):2691–2697. doi: 10.1093/nar/9.12.2691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gundersen G. G., Kalnoski M. H., Bulinski J. C. Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell. 1984 Oct;38(3):779–789. doi: 10.1016/0092-8674(84)90273-3. [DOI] [PubMed] [Google Scholar]
  22. Hall J. L., Cowan N. J. Structural features and restricted expression of a human alpha-tubulin gene. Nucleic Acids Res. 1985 Jan 11;13(1):207–223. doi: 10.1093/nar/13.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Helftenbein E. Nucleotide sequence of a macronuclear DNA molecule coding for alpha-tubulin from the ciliate Stylonychia lemnae. Special codon usage: TAA is not a translation termination codon. Nucleic Acids Res. 1985 Jan 25;13(2):415–433. doi: 10.1093/nar/13.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  25. Howley P. M., Israel M. A., Law M. F., Martin M. A. A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes. J Biol Chem. 1979 Jun 10;254(11):4876–4883. [PubMed] [Google Scholar]
  26. Kemphues K. J., Kaufman T. C., Raff R. A., Raff E. C. The testis-specific beta-tubulin subunit in Drosophila melanogaster has multiple functions in spermatogenesis. Cell. 1982 Dec;31(3 Pt 2):655–670. doi: 10.1016/0092-8674(82)90321-x. [DOI] [PubMed] [Google Scholar]
  27. Kimmel B. E., Samson S., Wu J., Hirschberg R., Yarbrough L. R. Tubulin genes of the African trypanosome Trypanosoma brucei rhodesiense:nucleotide sequence of a 3.7-kb fragment containing genes for alpha and beta tubulins. Gene. 1985;35(3):237–248. doi: 10.1016/0378-1119(85)90002-2. [DOI] [PubMed] [Google Scholar]
  28. Kowit J. D., Fulton C. Programmed synthesis of tubulin for the flagella that develop during cell differentiation in Naegleria gruberi. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2877–2881. doi: 10.1073/pnas.71.7.2877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Krämmer G., Singhofer-Wowra M., Seedorf K., Little M., Schedl T. A plasmodial alpha-tubulin cDNA from Physarum polycephalum. Nucleotide sequence and comparative analysis. J Mol Biol. 1985 Jun 25;183(4):633–638. doi: 10.1016/0022-2836(85)90176-7. [DOI] [PubMed] [Google Scholar]
  30. Lai E. Y., Walsh C., Wardell D., Fulton C. Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria. Cell. 1979 Aug;17(4):867–878. doi: 10.1016/0092-8674(79)90327-1. [DOI] [PubMed] [Google Scholar]
  31. Laird C. D., McConaughy B. L., McCarthy B. J. Rate of fixation of nucleotide substitutions in evolution. Nature. 1969 Oct 11;224(5215):149–154. doi: 10.1038/224149a0. [DOI] [PubMed] [Google Scholar]
  32. Landfear S. M., McMahon-Pratt D., Wirth D. F. Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii. Mol Cell Biol. 1983 Jun;3(6):1070–1076. doi: 10.1128/mcb.3.6.1070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lemischka I. R., Farmer S., Racaniello V. R., Sharp P. A. Nucleotide sequence and evolution of a mammalian alpha-tubulin messenger RNA. J Mol Biol. 1981 Sep 5;151(1):101–120. doi: 10.1016/0022-2836(81)90223-0. [DOI] [PubMed] [Google Scholar]
  34. Lemischka I., Sharp P. A. The sequences of an expressed rat alpha-tubulin gene and a pseudogene with an inserted repetitive element. Nature. 1982 Nov 25;300(5890):330–335. doi: 10.1038/300330a0. [DOI] [PubMed] [Google Scholar]
  35. Lewis S. A., Lee M. G., Cowan N. J. Five mouse tubulin isotypes and their regulated expression during development. J Cell Biol. 1985 Sep;101(3):852–861. doi: 10.1083/jcb.101.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lis J. T., Prestidge L., Hogness D. S. A novel arrangement of tandemly repeated genes at a major heat shock site in D. melanogaster. Cell. 1978 Aug;14(4):901–919. doi: 10.1016/0092-8674(78)90345-8. [DOI] [PubMed] [Google Scholar]
  37. Mar J., Lee J. H., Shea D., Walsh C. J. New poly(A)+RNAs appear coordinately during the differentiation of Naegleria gruberi amebae into flagellates. J Cell Biol. 1986 Feb;102(2):353–361. doi: 10.1083/jcb.102.2.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. May G. S., Gambino J., Weatherbee J. A., Morris N. R. Identification and functional analysis of beta-tubulin genes by site specific integrative transformation in Aspergillus nidulans. J Cell Biol. 1985 Sep;101(3):712–719. doi: 10.1083/jcb.101.3.712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Monteiro M. J., Cox R. A. Primary structure of an alpha-tubulin gene of Physarum polycephalum. J Mol Biol. 1987 Feb 5;193(3):427–438. doi: 10.1016/0022-2836(87)90257-9. [DOI] [PubMed] [Google Scholar]
  40. Montgomery D. L., Hall B. D., Gillam S., Smith M. Identification and isolation of the yeast cytochrome c gene. Cell. 1978 Jul;14(3):673–680. doi: 10.1016/0092-8674(78)90250-7. [DOI] [PubMed] [Google Scholar]
  41. Nader W. F., Edlind T. D., Huettermann A., Sauer H. W. Cloning of Physarum actin sequences in an exonuclease-deficient bacterial host. Proc Natl Acad Sci U S A. 1985 May;82(9):2698–2702. doi: 10.1073/pnas.82.9.2698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Neff N. F., Thomas J. H., Grisafi P., Botstein D. Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo. Cell. 1983 May;33(1):211–219. doi: 10.1016/0092-8674(83)90350-1. [DOI] [PubMed] [Google Scholar]
  43. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  44. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ponstingl H., Krauhs E., Little M., Kempf T. Complete amino acid sequence of alpha-tubulin from porcine brain. Proc Natl Acad Sci U S A. 1981 May;78(5):2757–2761. doi: 10.1073/pnas.78.5.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ponstingl H., Little M., Krauhs E., Kempf T. Carboxy-terminal amino acid sequence of alpha-tubulin from porcine brain. Nature. 1979 Nov 22;282(5737):423–425. doi: 10.1038/282423a0. [DOI] [PubMed] [Google Scholar]
  47. Pratt L. F., Okamura S., Cleveland D. W. A divergent testis-specific alpha-tubulin isotype that does not contain a coded C-terminal tyrosine. Mol Cell Biol. 1987 Jan;7(1):552–555. doi: 10.1128/mcb.7.1.552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ricciardi R. P., Miller J. S., Roberts B. E. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. doi: 10.1073/pnas.76.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sackett D. L., Bhattacharyya B., Wolff J. Tubulin subunit carboxyl termini determine polymerization efficiency. J Biol Chem. 1985 Jan 10;260(1):43–45. [PubMed] [Google Scholar]
  50. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  51. Schatz P. J., Pillus L., Grisafi P., Solomon F., Botstein D. Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins. Mol Cell Biol. 1986 Nov;6(11):3711–3721. doi: 10.1128/mcb.6.11.3711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schedl T., Burland T. G., Gull K., Dove W. F. Cell cycle regulation of tubulin RNA level, tubulin protein synthesis, and assembly of microtubules in Physarum. J Cell Biol. 1984 Jul;99(1 Pt 1):155–165. doi: 10.1083/jcb.99.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Seebeck T., Whittaker P. A., Imboden M. A., Hardman N., Braun R. Tubulin genes of Trypanosoma brucei: a tightly clustered family of alternating genes. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4634–4638. doi: 10.1073/pnas.80.15.4634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Serrano L., Avila J., Maccioni R. B. Controlled proteolysis of tubulin by subtilisin: localization of the site for MAP2 interaction. Biochemistry. 1984 Sep 25;23(20):4675–4681. doi: 10.1021/bi00315a024. [DOI] [PubMed] [Google Scholar]
  55. Serrano L., Valencia A., Caballero R., Avila J. Localization of the high affinity calcium-binding site on tubulin molecule. J Biol Chem. 1986 May 25;261(15):7076–7081. [PubMed] [Google Scholar]
  56. Serrano L., de la Torre J., Maccioni R. B., Avila J. Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5989–5993. doi: 10.1073/pnas.81.19.5989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Silflow C. D., Chisholm R. L., Conner T. W., Ranum L. P. The two alpha-tubulin genes of Chlamydomonas reinhardi code for slightly different proteins. Mol Cell Biol. 1985 Sep;5(9):2389–2398. doi: 10.1128/mcb.5.9.2389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  59. Sullivan K. F., Lau J. T., Cleveland D. W. Apparent gene conversion between beta-tubulin genes yields multiple regulatory pathways for a single beta-tubulin polypeptide isotype. Mol Cell Biol. 1985 Sep;5(9):2454–2465. doi: 10.1128/mcb.5.9.2454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Sussman D. J., Sellers J. R., Flicker P., Lai E. Y., Cannon L. E., Szent-Györgyi A. G., Fulton C. Actin of Naegleria gruberi. Absence of N tau-methylhistidine. J Biol Chem. 1984 Jun 10;259(11):7349–7354. [PubMed] [Google Scholar]
  61. Sánchez F., Natzle J. E., Cleveland D. W., Kirschner M. W., McCarthy B. J. A dispersed multigene family encoding tubulin in Drosophila melanogaster. Cell. 1980 Dec;22(3):845–854. doi: 10.1016/0092-8674(80)90561-9. [DOI] [PubMed] [Google Scholar]
  62. Theurkauf W. E., Baum H., Bo J., Wensink P. C. Tissue-specific and constitutive alpha-tubulin genes of Drosophila melanogaster code for structurally distinct proteins. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8477–8481. doi: 10.1073/pnas.83.22.8477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Thomashow L. S., Milhausen M., Rutter W. J., Agabian N. Tubulin genes are tandemly linked and clustered in the genome of trypanosoma brucei. Cell. 1983 Jan;32(1):35–43. doi: 10.1016/0092-8674(83)90494-4. [DOI] [PubMed] [Google Scholar]
  64. Thompson W. C. The cyclic tyrosination/detyrosination of alpha tubulin. Methods Cell Biol. 1982;24:235–255. doi: 10.1016/s0091-679x(08)60658-5. [DOI] [PubMed] [Google Scholar]
  65. Toda T., Adachi Y., Hiraoka Y., Yanagida M. Identification of the pleiotropic cell division cycle gene NDA2 as one of two different alpha-tubulin genes in Schizosaccharomyces pombe. Cell. 1984 May;37(1):233–242. doi: 10.1016/0092-8674(84)90319-2. [DOI] [PubMed] [Google Scholar]
  66. Valenzuela P., Quiroga M., Zaldivar J., Rutter W. J., Kirschner M. W., Cleveland D. W. Nucleotide and corresponding amino acid sequences encoded by alpha and beta tubulin mRNAs. Nature. 1981 Feb 19;289(5799):650–655. doi: 10.1038/289650a0. [DOI] [PubMed] [Google Scholar]
  67. Villasante A., Wang D., Dobner P., Dolph P., Lewis S. A., Cowan N. J. Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes. Mol Cell Biol. 1986 Jul;6(7):2409–2419. doi: 10.1128/mcb.6.7.2409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Wood W. I., Gitschier J., Lasky L. A., Lawn R. M. Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1585–1588. doi: 10.1073/pnas.82.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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