Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1981 Dec;1(12):1125–1137. doi: 10.1128/mcb.1.12.1125

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

D Alexandraki, J V Ruderman
PMCID: PMC369739  PMID: 6287219

Abstract

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

Full text

PDF
1125

Images in this article

1128Image
on p.1128
1129Image
on p.1129
1130Image
on p.1130
1131Image
on p.1131
1132Image
on p.1132
1133Image
on p.1133
1134Image
on p.1134

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  3. Bibring T., Baxandall J., Denslow S., Walker B. Heterogeneity of the alpha subunit of tubulin and the variability of tubulin within a single organism. J Cell Biol. 1976 May;69(2):301–312. doi: 10.1083/jcb.69.2.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bibring T., Baxandall J. Tubulin synthesis in sea urchin embryos: almost all tubulin of the first cleavage mitotic apparatus derives from the unfertilized egg. Dev Biol. 1977 Jan;55(1):191–195. doi: 10.1016/0012-1606(77)90330-x. [DOI] [PubMed] [Google Scholar]
  5. Blattner F. R., Williams B. G., Blechl A. E., Denniston-Thompson K., Faber H. E., Furlong L., Grunwald D. J., Kiefer D. O., Moore D. D., Schumm J. W. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science. 1977 Apr 8;196(4286):161–169. doi: 10.1126/science.847462. [DOI] [PubMed] [Google Scholar]
  6. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  7. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  8. Britten R. J., Graham D. E., Neufeld B. R. Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 1974;29:363–418. doi: 10.1016/0076-6879(74)29033-5. [DOI] [PubMed] [Google Scholar]
  9. Bryan R. N., Cutter G. A., Hayashi M. Separate mRNAs code for tubulin subunits. Nature. 1978 Mar 2;272(5648):81–83. doi: 10.1038/272081a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Cleveland D. W., Kirschner M. W., Cowan N. J. Isolation of separate mRNAs for alpha- and beta-tubulin and characterization of the corresponding in vitro translation products. Cell. 1978 Nov;15(3):1021–1031. doi: 10.1016/0092-8674(78)90286-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Efstratiadis A., Kafatos F. C., Maxam A. M., Maniatis T. Enzymatic in vitro synthesis of globin genes. Cell. 1976 Feb;7(2):279–288. doi: 10.1016/0092-8674(76)90027-1. [DOI] [PubMed] [Google Scholar]
  15. Feit H., Neudeck U., Baskin F. Comparison of the isoelectric and molecular weight properties of tubulin subunits. J Neurochem. 1977 Apr;28(4):697–706. doi: 10.1111/j.1471-4159.1977.tb10616.x. [DOI] [PubMed] [Google Scholar]
  16. Fujiwara K., Pollard T. D. Simultaneous localization of myosin and tubulin in human tissue culture cells by double antibody staining. J Cell Biol. 1978 Apr;77(1):182–195. doi: 10.1083/jcb.77.1.182. [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. Gilbert W., Maxam A. The nucleotide sequence of the lac operator. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3581–3584. doi: 10.1073/pnas.70.12.3581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kafatos F. C., Jones C. W., Efstratiadis A. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 1979 Nov 24;7(6):1541–1552. doi: 10.1093/nar/7.6.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kemphues K. J., Raff E. C., Raff R. A., Kaufman T. C. Mutation in a testis-specific beta-tubulin in Drosophila: analysis of its effects on meiosis and map location of the gene. Cell. 1980 Sep;21(2):445–451. doi: 10.1016/0092-8674(80)90481-x. [DOI] [PubMed] [Google Scholar]
  22. Kemphues K. J., Raff R. A., Kaufman T. C., Raff E. C. Mutation in a structural gene for a beta-tubulin specific to testis in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3991–3995. doi: 10.1073/pnas.76.8.3991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kessler S. W. Cell membrane antigen isolation with the staphylococcal protein A-antibody adsorbent. J Immunol. 1976 Nov;117(5 Pt 1):1482–1490. [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  28. Maniatis T., Jeffrey A., van deSande H. Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry. 1975 Aug 26;14(17):3787–3794. doi: 10.1021/bi00688a010. [DOI] [PubMed] [Google Scholar]
  29. Marotta C. A., Strocchi P., Gilbert J. M. Biosynthesis of heterogeneous forms of mammalian brain tubulin subunits by multiple messenger RNAs. J Neurochem. 1979 Jul;33(1):231–246. doi: 10.1111/j.1471-4159.1979.tb11725.x. [DOI] [PubMed] [Google Scholar]
  30. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  31. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  32. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Roychoudhury R., Jay E., Wu R. Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucleic Acids Res. 1976 Jan;3(1):101–116. doi: 10.1093/nar/3.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ruderman J. V., Pardue M. L. A portion of all major classes of histone messenger RNA in amphibian oocytes is polyadenylated. J Biol Chem. 1978 Mar 25;253(6):2018–2025. [PubMed] [Google Scholar]
  36. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  37. Sheir-Neiss G., Lai M. H., Morris N. R. Identification of a gene for beta-tubulin in Aspergillus nidulans. Cell. 1978 Oct;15(2):639–647. doi: 10.1016/0092-8674(78)90032-6. [DOI] [PubMed] [Google Scholar]
  38. Snyder J. A., McIntosh J. R. Biochemistry and physiology of microtubules. Annu Rev Biochem. 1976;45:699–720. doi: 10.1146/annurev.bi.45.070176.003411. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Stephens R. E. Differential protein synthesis and utilization during cilia formation in sea urchin embryos. Dev Biol. 1977 Dec;61(2):311–329. doi: 10.1016/0012-1606(77)90301-3. [DOI] [PubMed] [Google Scholar]
  41. Stephens R. E., Edds K. T. Microtubules: structure, chemistry, and function. Physiol Rev. 1976 Oct;56(4):709–777. doi: 10.1152/physrev.1976.56.4.709. [DOI] [PubMed] [Google Scholar]
  42. Stephens R. E. Major membrane protein differences in cilia and flagella: evidence for a membrane-associated tubulin. Biochemistry. 1977 May 17;16(10):2047–2058. doi: 10.1021/bi00629a001. [DOI] [PubMed] [Google Scholar]
  43. Stephens R. E. Primary structural differences among tubulin subunits from flagella, cilia, and the cytoplasm. Biochemistry. 1978 Jul 11;17(14):2882–2891. doi: 10.1021/bi00607a029. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Thireos G., Shea R. G., Kafatos F. C. Identification of chorion protein precursors and the mRNAs that encode them in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6279–6283. doi: 10.1073/pnas.76.12.6279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Villa-Komaroff L., Efstratiadis A., Broome S., Lomedico P., Tizard R., Naber S. P., Chick W. L., Gilbert W. A bacterial clone synthesizing proinsulin. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3727–3731. doi: 10.1073/pnas.75.8.3727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Wensink P. C., Finnegan D. J., Donelson J. E., Hogness D. S. A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. Cell. 1974 Dec;3(4):315–325. doi: 10.1016/0092-8674(74)90045-2. [DOI] [PubMed] [Google Scholar]
  50. Witman G. B., Carlson K., Rosenbaum J. L. Chlamydomonas flagella. II. The distribution of tubulins 1 and 2 in the outer doublet microtubules. J Cell Biol. 1972 Sep;54(3):540–555. doi: 10.1083/jcb.54.3.540. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES