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. 1986 Jun 1;102(6):2076–2087. doi: 10.1083/jcb.102.6.2076

A microtubule-associated protein in Drosophila melanogaster: identification, characterization, and isolation of coding sequences

PMCID: PMC2114242  PMID: 3086324

Abstract

Microtubules and microtubule-associated proteins (MAPs) have been isolated from cultured cells of Drosophila melanogaster by a taxol- dependent polymerization procedure. The principal MAPs are a group of four polypeptides with similar electrophoretic mobilities corresponding to approximately Mr 205,000 (the 205K MAP). These proteins are resistant to precipitation by boiling. One mouse monoclonal antibody and one polyclonal rabbit antiserum specific for the Mr 205,000 MAP were produced and characterized by immunoblotting and indirect immunofluorescence. Both antibody preparations stain the Mr 205,000 molecules and an Mr 255,000 molecule in immunoblots of Drosophila cell homogenates; the rabbit antiserum also stains an Mr 150,000 triplet. Both preparations stain the microtubules of the mitotic spindle, and the rabbit antiserum stains the cytoplasmic microtubules as well. Experiments using affinity-purified rabbit antiserum demonstrate that it is the Mr 205,000 species that is located in the mitotic apparatus and on cytoplasmic microtubules. A random shear genomic library was produced in the expressing vector lambda gt11 and screened with the rabbit antiserum to isolate the DNA sequences encoding these polypeptides. Several cross-hybridizing clones were recovered, shown to encode antigenic determinants in the Mr 205,000 MAP, and characterized by hybridization to Northern blots of mRNA and Southern blots of genomic DNA. Analysis by in situ hybridization reveals that the gene encoding the 205K MAP is located in polytene region 100EF.

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Selected References

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  1. Batteiger B., Newhall W. J., 5th, Jones R. B. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods. 1982 Dec 30;55(3):297–307. doi: 10.1016/0022-1759(82)90089-8. [DOI] [PubMed] [Google Scholar]
  2. Berkowitz S. A., Katagiri J., Binder H. K., Williams R. C., Jr Separation and characterization of microtubule proteins from calf brain. Biochemistry. 1977 Dec 13;16(25):5610–5617. doi: 10.1021/bi00644a035. [DOI] [PubMed] [Google Scholar]
  3. Bloom G. S., Luca F. C., Vallee R. B. Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules. J Cell Biol. 1984 Jan;98(1):331–340. doi: 10.1083/jcb.98.1.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bloom G. S., Schoenfeld T. A., Vallee R. B. Widespread distribution of the major polypeptide component of MAP 1 (microtubule-associated protein 1) in the nervous system. J Cell Biol. 1984 Jan;98(1):320–330. doi: 10.1083/jcb.98.1.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bloom G. S., Vallee R. B. Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells. J Cell Biol. 1983 Jun;96(6):1523–1531. doi: 10.1083/jcb.96.6.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bonner J. J., Pardue M. L. Ecdysone-stimulated RNA synthesis in imaginal discs of Drosophila melanogaster. Assay by in situ hybridization. Chromosoma. 1976 Oct 12;58(1):87–99. doi: 10.1007/BF00293443. [DOI] [PubMed] [Google Scholar]
  7. Bulinski J. C., Borisy G. G. Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo. J Cell Biol. 1980 Dec;87(3 Pt 1):792–801. doi: 10.1083/jcb.87.3.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bulinski J. C., Borisy G. G. Microtubule-associated proteins from cultured HeLa cells. Analysis of molecular properties and effects on microtubule polymerization. J Biol Chem. 1980 Dec 10;255(23):11570–11576. [PubMed] [Google Scholar]
  9. Bulinski J. C., Borisy G. G. Self-assembly of microtubules in extracts of cultured HeLa cells and the identification of HeLa microtubule-associated proteins. Proc Natl Acad Sci U S A. 1979 Jan;76(1):293–297. doi: 10.1073/pnas.76.1.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bulinski J. C., Borisy G. G. Widespread distribution of a 210,000 mol wt microtubule-associated protein in cells and tissues of primates. J Cell Biol. 1980 Dec;87(3 Pt 1):802–808. doi: 10.1083/jcb.87.3.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cathala G., Savouret J. F., Mendez B., West B. L., Karin M., Martial J. A., Baxter J. D. A method for isolation of intact, translationally active ribonucleic acid. DNA. 1983;2(4):329–335. doi: 10.1089/dna.1983.2.329. [DOI] [PubMed] [Google Scholar]
  12. Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Immunoflourescent staining of cytoplasmic and spindle microtubules in mouse fibroblasts with antibody to tau protein. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2437–2440. doi: 10.1073/pnas.74.6.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence. J Cell Biol. 1978 Mar;76(3):781–786. doi: 10.1083/jcb.76.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Drubin D. G., Caput D., Kirschner M. W. Studies on the expression of the microtubule-associated protein, tau, during mouse brain development, with newly isolated complementary DNA probes. J Cell Biol. 1984 Mar;98(3):1090–1097. doi: 10.1083/jcb.98.3.1090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Duerr A., Pallas D., Solomon F. Molecular analysis of cytoplasmic microtubules in situ: identification of both widespread and specific proteins. Cell. 1981 Apr;24(1):203–211. doi: 10.1016/0092-8674(81)90516-x. [DOI] [PubMed] [Google Scholar]
  16. Erickson P. F., Minier L. N., Lasher R. S. Quantitative electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: a method for their re-use in immunoautoradiographic detection of antigens. J Immunol Methods. 1982 Jun 11;51(2):241–249. doi: 10.1016/0022-1759(82)90263-0. [DOI] [PubMed] [Google Scholar]
  17. Helfman D. M., Feramisco J. R., Fiddes J. C., Thomas G. P., Hughes S. H. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A. 1983 Jan;80(1):31–35. doi: 10.1073/pnas.80.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Izant J. G., Weatherbee J. A., McIntosh J. R. A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells. J Cell Biol. 1983 Feb;96(2):424–434. doi: 10.1083/jcb.96.2.424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Izant J. G., Weatherbee J. A., McIntosh J. R. A microtubule-associated protein in the mitotic spindle and the interphase nucleus. Nature. 1982 Jan 21;295(5846):248–250. doi: 10.1038/295248a0. [DOI] [PubMed] [Google Scholar]
  20. Kwan S. P., Wood T. G., Lingrel J. B. Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells. Proc Natl Acad Sci U S A. 1977 Jan;74(1):178–182. doi: 10.1073/pnas.74.1.178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Lengyel J., Spradling A., Penman S. Methods with insect cells in suspension culture. II. Drosophila melanogaster. Methods Cell Biol. 1975;10:195–208. doi: 10.1016/s0091-679x(08)60738-4. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Murphy D. B., Borisy G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2696–2700. doi: 10.1073/pnas.72.7.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Neville D. M., Jr Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J Biol Chem. 1971 Oct 25;246(20):6328–6334. [PubMed] [Google Scholar]
  26. Nigg E. A., Walter G., Singer S. J. On the nature of crossreactions observed with antibodies directed to defined epitopes. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5939–5943. doi: 10.1073/pnas.79.19.5939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Olmsted J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem. 1981 Dec 10;256(23):11955–11957. [PubMed] [Google Scholar]
  28. Olmsted J. B., Cox J. V., Asnes C. F., Parysek L. M., Lyon H. D. Cellular regulation of microtubule organization. J Cell Biol. 1984 Jul;99(1 Pt 2):28s–32s. doi: 10.1083/jcb.99.1.28s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pallas D., Solomon F. Cytoplasmic microtubule-associated proteins: phosphorylation at novel sites is correlated with their incorporation into assembled microtubules. Cell. 1982 Sep;30(2):407–414. doi: 10.1016/0092-8674(82)90238-0. [DOI] [PubMed] [Google Scholar]
  30. Parysek L. M., Asnes C. F., Olmsted J. B. MAP 4: occurrence in mouse tissues. J Cell Biol. 1984 Oct;99(4 Pt 1):1309–1315. doi: 10.1083/jcb.99.4.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Parysek L. M., Wolosewick J. J., Olmsted J. B. MAP 4: a microtubule-associated protein specific for a subset of tissue microtubules. J Cell Biol. 1984 Dec;99(6):2287–2296. doi: 10.1083/jcb.99.6.2287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Scholey J. M., Neighbors B., McIntosh J. R., Salmon E. D. Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs. J Biol Chem. 1984 May 25;259(10):6516–6525. [PubMed] [Google Scholar]
  33. Sloboda R. D., Dentler W. L., Rosenbaum J. L. Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry. 1976 Oct 5;15(20):4497–4505. doi: 10.1021/bi00665a026. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vallee R. B. A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs). J Cell Biol. 1982 Feb;92(2):435–442. doi: 10.1083/jcb.92.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vallee R. B., Bloom G. S. Isolation of sea urchin egg microtubules with taxol and identification of mitotic spindle microtubule-associated proteins with monoclonal antibodies. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6259–6263. doi: 10.1073/pnas.80.20.6259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vallee R. B., Bloom G. S., Theurkauf W. E. Microtubule-associated proteins: subunits of the cytomatrix. J Cell Biol. 1984 Jul;99(1 Pt 2):38s–44s. doi: 10.1083/jcb.99.1.38s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  40. Weatherbee J. A., Luftig R. B., Weihing R. R. Purification and reconstitution of HeLa cell microtubules. Biochemistry. 1980 Aug 19;19(17):4116–4123. doi: 10.1021/bi00558a033. [DOI] [PubMed] [Google Scholar]
  41. Weatherbee J. A., Sherline P., Mascardo R. N., Izant J. G., Luftig R. B., Weihing R. R. Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules. J Cell Biol. 1982 Jan;92(1):155–163. doi: 10.1083/jcb.92.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Weisenberg R. C., Borisy G. G., Taylor E. W. The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry. 1968 Dec;7(12):4466–4479. doi: 10.1021/bi00852a043. [DOI] [PubMed] [Google Scholar]
  44. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]

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