The Highly Divergent -Tubulins ofAspergiUus nidulans Are Functionally Interchangeable

An internal 1.4-kb Bst Eli 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/3-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/3-tubulin gene, rescuing the benA null generated by the integration. Strains generated by this procedure contain only tubC ~-tubulin for all/3-tubulin functions. Strains having only tubC/3-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/~-tubulin genes of Aspergillus nidulans, though highly divergent, are interchangeable. M ICROTUBULES form a variety of structures within cells and are involved in cellular shape and motility. Given the diversity of form and function for microtubules, it is surprising that in general microtubule ultrastructure is highly conserved. One possible source for functional diversity of microtubules are the c~and ~-tubulin subunits. The structural and functional diversity of microtubules and the existence of tubulin isotypes led Fulton and Simpson (1976) to formulate the multitubulin hypothesis. In its simplest form, the multitubulin hypothesis proposes that different microtubules are formed by different tubulins and that different genes encode the different tubulin isotypes found in the various microtubules of the cell. The existence of multiple tubulin genes, tubulin isotypes, and the diversity of microtubule structures and their functions in most eukaryotes has resulted in additional speculation about the significance of tubulin multigene families (Cleveland and Sullivan, 1985; Cleveland, 1987; Raft, 1984). Aspergillus nidulans has two ~-tubulin genes benA and tubC The benA gene functions during asexual growth and participates in mitosis and nuclear movement (Oaldey and Morris, 1980; 1981). The tubC gene appears to function only during asexual sporulation (conidiation), but is not essential for this process as demonstrated by the isolation of null mutants in mbC or its deliberate disruption (May et al., 1985; May and Morris, 1988; Weatherbe~ et al., 1985). Sequences of the benA and tubC genes predict proteins that are 17 % divergent at the amino acid level (May et al., 1987). This de1. Abbreviations used in this paper: DAPI, diamidino-2-phenylindole; YAG, 0.5% yeast extract, 2% glucose, 1.5% agar. gree of amino acid sequence divergence for/3-tubulins is equal to comparing either the bend or tubC polypeptides to any known/3-tubulin sequence. Thus, A. n/du/ans is an organism that allows us to test directly the functional significance of divergent/3-tubulin isotypes by constructing strains capable of producing a single/3-tubulin isotype. To develop strains producing tubC 13-tubulin, a novel one step gene disruption/replacement was used. This method should be applicable to other systems that have homologous integrative recombination. It is shown in this paper that though the benA and tubC~-tubulin genes ofA. nidulans encode highly divergent/3-tubulins, they are functionally equivalent polypeptides. Materials and Methods AspergiUus Strains and Culture Conditions The strains used in this study are listed in Table I. Strains were grown on 0.5% yeast extract, 2% glucose, 1.5% agar (YAG) I, and trace elements (Cove, 1966). Strains having the pyrG89 mutation were grown on YAG supplcmented with 5 mM uridin¢ and 10 mM uracil. Liquid media was YAG but without agar. Transformation of Aspergillvs was performed as described previously (Osmani ¢t al., 1987) except that protoplasts were plated on media made osmotically stable with 1 M sucrose. Phenotypic analysis of null mutations in an essential gene by generation of heterokaryon was as reported previously (Osmani ¢t al., 1988). Bacterial Strains and Plasmids F~cherichia coli K-12 strain TBI was used for routine plasmid propagation. Plasmid DNA was purified using the alkaline lysis method (Maniatis et al., 1982). General methods f~r plasmid construction were as described previously (May et al., 1985). © The Rockefeller University Press, 0021-9525/89/11/2267/8 $2.00 The Journal of Cell Biology, Volume 109, November 1989 2267-2274 2267 on Jne 0, 2017 D ow nladed fom Published November 1, 1989