Abstract
An internal 1.4-kb Bst EII fragment was used to disrupt the benA gene and establish heterokaryons. The heterokaryons demonstrated that the molecular disruption of benA results in a recessive benA null mutation. Conidia from a heterokaryon swell and germinate but cannot undergo nuclear division and are thus inviable. A chimeric beta-tubulin gene was constructed with the benA promoter driving the tubC structural gene. This chimeric gene construction was placed on a plasmid containing a selectable marker for Aspergillus transformation and the gene disrupting fragment of benA. Integration of this plasmid at benA by the internal gene disrupting fragment of benA simultaneously disrupts the benA gene and replaces it with the chimeric beta-tubulin gene, rescuing the benA null generated by the integration. Strains generated by this procedure contain only tubC beta-tubulin for all beta- tubulin functions. Strains having only tubC beta-tubulin are viable and exhibit no detectable microtubule dysfunction though they are more sensitive than wild-type strains to the antimicrotubule drug benomyl. It is concluded that the two beta-tubulin genes of Aspergillus nidulans, though highly divergent, are interchangeable.
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- Adachi Y., Toda T., Niwa O., Yanagida M. Differential expressions of essential and nonessential alpha-tubulin genes in Schizosaccharomyces pombe. Mol Cell Biol. 1986 Jun;6(6):2168–2178. doi: 10.1128/mcb.6.6.2168. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bond J. F., Fridovich-Keil J. L., Pillus L., Mulligan R. C., Solomon F. A chicken-yeast chimeric beta-tubulin protein is incorporated into mouse microtubules in vivo. Cell. 1986 Feb 14;44(3):461–468. doi: 10.1016/0092-8674(86)90467-8. [DOI] [PubMed] [Google Scholar]
- 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]
- Cleveland D. W. The multitubulin hypothesis revisited: what have we learned? J Cell Biol. 1987 Mar;104(3):381–383. doi: 10.1083/jcb.104.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cove D. J. The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta. 1966 Jan 11;113(1):51–56. doi: 10.1016/s0926-6593(66)80120-0. [DOI] [PubMed] [Google Scholar]
- Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
- Gard D. L., Kirschner M. W. A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line. J Cell Biol. 1985 Mar;100(3):764–774. doi: 10.1083/jcb.100.3.764. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gasch A., Hinz U., Leiss D., Renkawitz-Pohl R. The expression of beta 1 and beta 3 tubulin genes of Drosophila melanogaster is spatially regulated during embryogenesis. Mol Gen Genet. 1988 Jan;211(1):8–16. doi: 10.1007/BF00338387. [DOI] [PubMed] [Google Scholar]
- Gu W., Lewis S. A., Cowan N. J. Generation of antisera that discriminate among mammalian alpha-tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function. J Cell Biol. 1988 Jun;106(6):2011–2022. doi: 10.1083/jcb.106.6.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Huber G., Matus A. Differences in the cellular distributions of two microtubule-associated proteins, MAP1 and MAP2, in rat brain. J Neurosci. 1984 Jan;4(1):151–160. doi: 10.1523/JNEUROSCI.04-01-00151.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kimble M., Incardona J. P., Raff E. C. A variant beta-tubulin isoform of Drosophila melanogaster (beta 3) is expressed primarily in tissues of mesodermal origin in embryos and pupae, and is utilized in populations of transient microtubules. Dev Biol. 1989 Feb;131(2):415–429. doi: 10.1016/s0012-1606(89)80014-4. [DOI] [PubMed] [Google Scholar]
- L'Hernault S. W., Rosenbaum J. L. Reversal of the posttranslational modification on Chlamydomonas flagellar alpha-tubulin occurs during flagellar resorption. J Cell Biol. 1985 Feb;100(2):457–462. doi: 10.1083/jcb.100.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis S. A., Gu W., Cowan N. J. Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules. Cell. 1987 May 22;49(4):539–548. doi: 10.1016/0092-8674(87)90456-9. [DOI] [PubMed] [Google Scholar]
- 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]
- May G. S., Morris N. R. Developmental regulation of a conidiation specific beta-tubulin in Aspergillus nidulans. Dev Biol. 1988 Aug;128(2):406–414. doi: 10.1016/0012-1606(88)90302-8. [DOI] [PubMed] [Google Scholar]
- May G. S., Morris N. R. The unique histone H2A gene of Aspergillus nidulans contains three introns. Gene. 1987;58(1):59–66. doi: 10.1016/0378-1119(87)90029-1. [DOI] [PubMed] [Google Scholar]
- May G. S., Tsang M. L., Smith H., Fidel S., Morris N. R. Aspergillus nidulans beta-tubulin genes are unusually divergent. Gene. 1987;55(2-3):231–243. doi: 10.1016/0378-1119(87)90283-6. [DOI] [PubMed] [Google Scholar]
- Natzle J. E., McCarthy B. J. Regulation of Drosophila alpha- and beta-tubulin genes during development. Dev Biol. 1984 Jul;104(1):187–198. doi: 10.1016/0012-1606(84)90047-2. [DOI] [PubMed] [Google Scholar]
- Oakley B. R., Morris N. R. A beta-tubulin mutation in Aspergillus nidulans that blocks microtubule function without blocking assembly. Cell. 1981 Jun;24(3):837–845. doi: 10.1016/0092-8674(81)90109-4. [DOI] [PubMed] [Google Scholar]
- Oakley B. R., Morris N. R. Nuclear movement is beta--tubulin-dependent in Aspergillus nidulans. Cell. 1980 Jan;19(1):255–262. doi: 10.1016/0092-8674(80)90407-9. [DOI] [PubMed] [Google Scholar]
- Osmani S. A., Engle D. B., Doonan J. H., Morris N. R. Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene. Cell. 1988 Jan 29;52(2):241–251. doi: 10.1016/0092-8674(88)90513-2. [DOI] [PubMed] [Google Scholar]
- Osmani S. A., May G. S., Morris N. R. Regulation of the mRNA levels of nimA, a gene required for the G2-M transition in Aspergillus nidulans. J Cell Biol. 1987 Jun;104(6):1495–1504. doi: 10.1083/jcb.104.6.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raff E. C. Genetics of microtubule systems. J Cell Biol. 1984 Jul;99(1 Pt 1):1–10. doi: 10.1083/jcb.99.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schatz P. J., Solomon F., Botstein D. Genetically essential and nonessential alpha-tubulin genes specify functionally interchangeable proteins. Mol Cell Biol. 1986 Nov;6(11):3722–3733. doi: 10.1128/mcb.6.11.3722. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weatherbee J. A., May G. S., Gambino J., Morris N. R. Involvement of a particular species of beta-tubulin (beta 3) in conidial development in Aspergillus nidulans. J Cell Biol. 1985 Sep;101(3):706–711. doi: 10.1083/jcb.101.3.706. [DOI] [PMC free article] [PubMed] [Google Scholar]