Summary
We have isolated large numbers of conditionally lethal β-tubulin mutations to provide raw material for analyzing the structure and function of β tubulin and of microtubules. We have isolated such mutations as intragenic suppressors of benA33, a heat-sensitive (hs-) β-tubulin mutation of Aspergillus nidulans. Among over 2,600 revertants isolated, 126 were cold-sensitive (cs-). In 41 of 78 cs- revertants analyzed, cold sensitivity and reversion from hs- to hs+ were due to mutations linked to benA33. In three cases reversion was due to mutations closely linked to benA33 but cold sensitivity was due to a coincidental mutation unlinked to benA33. In the remaining 34 cases reversion was due to mutations unlinked to benA33. Thirty-three of the revertants in which cold sensitivity and reversion were linked to benA33 were sufficiently cold-sensitive to allow us to select for rare recombinants between benA33 and putative suppressors in a revertant x wild-type (wt) cross. We found only one recombinant among 1,000 or more viable progeny from crosses of each of these revertants with a wt strain. Reversion is thus due to a back mutation or very closely linked suppressor in each case. We have analyzed 17 of these 33 revertants with greater precision and have found that, in each case, reversion is due to a suppressor mutation that maps to the right of benA33. The recombination frequencies between benA33 and the suppressors are very low (less than 1.2×10-4) in all cases. Five of these 33 revertants have been examined microscopically and in each of them nuclear division and nuclear migration are inhibited at a restrictive temperature. We conclude that at least some and perhaps all of these revertants carry intragenic suppressors of benA33 that, in combination with benA33, cause cold sensitivity by inhibiting the functioning of microtubules at low temperatures. Of the 17 suppressors mapped, 11 map to two clusters. These clusters are likely to define regions particularly important to the functioning of the β-tubulin molecule.
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Abraham I, Marcus M, Cabral F, Gottesman MM (1983) Mutations in α- and β-tubulin affect spindle formation in Chinese hamster ovary cells. J Cell Biol 97:1055–1061
Bandiera M, Morpurgo G, Volterra L (1970) ‘Barriers’ in intragenic mitotic crossing-over. Mutat Res 9:213–217
Cabral F, Abraham I, Gottesman MM (1982) Revertants of a Chinese hamster ovary cell mutant with an altered β-tubulin: Evidence that the altered tubulin confers both colcemid resistance and temperature sensitivity on the cell. Mol Cell Biol 2:720–729
Calef E (1957) Effect on linkage maps of selection of crossovers between closely linked markers. Heredity 11:265–279
Clutterbuck AJ (1984) Loci and linkage map of the filamentous fungus Aspergillus nidulans (Eidam) Winter (n=8). In: O'Brien SJ (ed) Genetic maps 1984, vol 3. Cold Spring Harbor Press, Cold Spring Harbor, NY, p 265–273
Dorn GL (1972) Computerized meiotic mapping in Aspergillus nidulans. Genetics 72:595–605
Elliott CG (1960) Non-localized negative interference in Aspergillus nidulans. Heredity 15:247–262
Gambino J, Bergen LG, Morris NR (1984) Effects of mitotic and tubulin mutations on microtubule architecture in actively growing protoplasts of Aspergillus nidulans. J Cell Biol 99:830–838
Hiraoka Y, Toda T, Janagida M (1984) The ND A3 gene of fission yeast encodes β-tubulin: A cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell 39:349–358
Huang B (1984) Genetic analysis of flagellar structure and motility. J Protozool 31:25–30
Luck DJL (1984) Genetic and biochemical dissection of the eukaryotic flagellum. J Cell Biol 98:789–794
Morris NR, Lai MH, Oakley CE (1979) Identification of a gene for α-tubulin in Aspergillus nidulans. Cell 16:437–442
Neff NF, Thomas JH, Grisafi P, Botstein D (1983) Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo. Cell 33:211–219
Oakley BR, Morris NR (1980) Nuclear movement is β-tubulin dependent in Aspergillus nidulans. Cell 19:255–262
Oakley BR, Morris NR (1981) A β-tubulin mutation in Aspergillus nidulans that blocks microtubule function without blocking assembly. Cell 24:837–845
Pontecorvo G, Roper JA, Hemmons LM, MacDonald KD, Bufton AJW (1953) The genetics of Aspergillus nidulans. Adv Genet 5:141–238
Pritchard RH (1955) The linear arrangement of a series of alleles of Aspergillus nidulans. Heredity 9:343–371
Pritchard RH (1960) Localized negative interference and its bearing on models of gene recombination. Genet Res (Camb) 1:1–24
Raff EC (1984) Genetics of microtubule systems. J Cell Biol 99:1–10
Thomas JH, Novick P, Botstein D (1984) Genetics of the yeast cytoskeleton. In: Borisy GG, Cleveland DW, Murphy, DB (eds) Molecular biology of the cytoskeleton. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp 153–174
Toda T, Adachi Y, Hiraoka Y, Yanagida M (1984) Identification of the pleiotropic cell division cycle gene ND A2 as one of two different α-tubulin genes in Schizosaccharomyces pombe. Cell 37:233–242
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Communicated by G.R. Fink
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Oakley, B.R., Oakley, C.E., Kniepkamp, K.S. et al. Isolation and characterization of cold-sensitive mutations at the benA, β-tubulin, locus of Aspergillus nidulans . Molec. Gen. Genet. 201, 56–64 (1985). https://doi.org/10.1007/BF00397987
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DOI: https://doi.org/10.1007/BF00397987