Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.     

Identification of an amino acid substitution in the benA, beta-tubulin gene of Aspergillus nidulans that confers thiabendazole resistance and benomyl supersensitivity

We are using molecular genetic techniques to identify sites of interaction of beta-tubulin with benzimidizole anti-microtubule agents. We have developed a marker-rescue technique for cloning mutant alleles of the benA, beta-tubulin gene of Aspergillus nidulans and have used the technique to clone two mutant benA alleles, benA16 and benA19. These are the only A. nidulans alleles known to confer resistance to the benzimidazole antimicrotubule agent thiabendazole and supersensitivity to other benzimidazole antimicrotubule agents including benomyl and its active breakdown product, carbendazim. benA16 has been shown, moreover, to reduce thiabendazole binding to beta-tubulin. We have sequenced the two mutant alleles and have found that they carry different nucleotide changes that cause the same single amino acid substitution, valine for alanine at amino acid 165. Since thiabendazole and carbendazim differ at only one side chain, the R2 group, we conclude that the region around amino acid 165 is involved in the binding of the R2 group of benzimidazole antimicrotubule agents to beta-tubulin.     

Isolation of a beta-tubulin gene from Fusarium moniliforme that confers cold-sensitive benomyl resistance.

A beta-tubulin gene from a UV-irradiated benomyl-resistant mutant of Fusarium moniliforme was isolated, cloned, and sequenced. The gene encodes a 446-amino-acid polypeptide with homology to other fungal beta-tubulins. RNA blot analysis showed expression of the gene during vegetative growth and conidial germination but no expression during conidiation. A point mutation, which likely confers benomyl resistance, has been identified in the cloned gene; this mutation results in a single amino acid substitution of asparagine for tyrosine at position 50. Expression of benomyl resistance in the mutant was also cold sensitive. Sexual crosses betweeen the mutant and a wild-type strain indicated cosegregation of benomyl resistance and cold sensitivity.     

Homologous expression of a mutated beta-tubulin gene does not confer benomyl resistance on Trichoderma virens

AIMS: To clone the beta-tubulins and to induce resistance to benzimidazoles in the biocontrol fungus Trichoderma virens through site-directed mutagenesis. METHODS AND RESULTS: Two beta-tubulin genes have been cloned using PCR amplification followed by the screening of a T. virens cDNA library. The full-length cDNA clones, coding for 445 and 446 amino acids, have been designated as T. virens tub1 and T. virens tub2. A sequence alignment of these two tubulins with tubulins from other filamentous fungi has shown the presence of some unique amino acid sequences not found in those positions in other beta-tubulins. Constitutive expression of the tub2 gene with a histidine to tyrosine substitution at position 6 (known to impart benomyl/methyl benzimadazol-2-yl carbamate resistance in other fungi), under the Pgpd promoter of Aspergillus nidulans, did not impart resistance to benomyl. CONCLUSIONS: The homologous expression of tub2 gene with a histidine to tyrosine mutation at position +6, which is known to impart benomyl tolerance in other fungi, does not impart resistance in T. virens. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike other Trichoderma spp., T. virens, has been difficult to mutate for benomyl tolerance. The present study, through site-directed mutagenesis, shows that a mutation known to impart benomyl tolerance in T. viride and other fungi does not impart resistance in this fungus. Understanding the mechanisms of this phenomenon will have a profound impact in plant-disease management, as many plant pathogenic fungi develop resistance to this group of fungicides forcing its withdrawal after a short period of use.     

The β-tubulin gene from rat and human isolates of Pneumocystis carinii

The development of new drugs for treating Pneumocystis carinii infections in AIDS patients is hampered by the lack of long-term culture systems, and by our generally limited knowledge of this organism. Recently, however, we observed significant activity of various benzimidazoles against growth of this organism in short-term cultures. Benzimidazoles inhibit microtubule polymerization; there is strong evidence that the primary target is the beta-tubulin subunit. To understand the basis for benzimidazole activity against P. carinii, and to examine the apparent relatedness of this organism to fungi, we have cloned and sequenced the single beta-tubulin gene from a rat P. carinii isolate. There was 89-91% identity at the amino acid level to beta-tubulins from filamentous fungi, but only 79-82% identity to yeast and protozoal beta-tubulins. Also, eight introns were distributed throughout the P. carinii beta-tubulin gene in a pattern characteristic of filamentous fungi. Specific residues previously implicated in benzimidazole sensitivity were conserved in P. carinii beta-tubulin. The polymerase chain reaction was used to amplify a segment of P. carinii beta-tubulin DNA from bronchoalveolar lavages obtained from two patients with AIDS. There was considerable divergence at the DNA level between the human and rat sequences, but 100% identity at the amino-acid level.     

Induction of multiple germ tubes in Neurospora crassa by antitubulin agents

The antitubulin fungicide benomyl suppressed the linear growth of Neurospora crassa wild type strain St. Lawrence 74 at micromolar concentrations. The rate of germination of macroconidia was not affected. Macroconidia exposed to 1.7 microM benomyl for 5 h formed multiple germ tubes. When germlings incubated for 4 h were exposed to 1.7 microM benomyl for 3 h, their germ tube stopped growing, swelled and emitted several branches. Normal linear growth was restored after removal of the fungicide. Linear growth of N. crassa was resistant up to 16 microM nocodazole. This drug induced multipolar germination at 8 microM, and griseofulvin only at 140 microM. The microtubule (MT) cytoskeleton of N. crassa could be revealed by indirect immunofluorescence with the monoclonal antibody YOL 1/34 directed against yeast alpha-tubulin. We detected no striking effects of the benomyl treatments on MT organization. The MT-stabilizing agents deuterium oxide (D2O) and cAMP have no antagonistic effects on the benomyl-induced multipolar germination. The positioning of nuclei and mitochondria was determined from the DAPI and Rhodamine 123 fluorescence patterns, respectively. Benomyl inhibited nuclear migration into multiple germ tubes. Quantitative scanning cytophotometry revealed a peak in the intensity of the mitochondria-associated Rhodamine 123 fluorescence near the apex of untreated germlings. This peak disappeared in multiple germ tubes. Benomyl-resistant mutant bml 511 (r), mutated in its beta-tubulin gene, germinated normally in the presence of the fungicide. This strongly suggests that multiple germ tube formation was due to the effect of benomyl on beta-tubulin. Benomyl-resistant strain 74-3, constructed by reintroducing the cloned mutant N. crassa beta-tubulin gene into the cells by transformation, displayed a partial resistance to benomyl with respect to multipolar germination. Its rate of germination was slow (50% germination reached after 4 h at 37 degrees C as compared to 2.5 h for the wild type). In contrast to N. crassa, the other ascomycete Aspergillus nidulans is nocodazole-sensitive (linear growth suppressed at 1.6 microM). It did not respond to the MT inhibitors benomyl and nocodazole with respect to the pattern of germ tube emergence. Our results suggest that microtubule or membrane beta-tubulin is involved in the maintenance of developmental polarity during germ tube emergence and growth of N. crassa.     

Isolation and sequence analysis of a beta-tubulin gene from Aspergillus flavus and its use as a selectable marker

An altered beta-tubulin gene that confers resistance to benomyl [whose active ingredient is 2-(methoxycarbonylamino)benzimidazole (MBC)] was isolated from a DNA library of Aspergillus flavus and used as a selectable marker for transformation. The beta-tubulin gene was cloned into a plasmid vector containing the pyr-4 gene of Neurospora crassa, and transformants were selected either for uracil prototrophy or MBC resistance. Transformants selected for uracil prototrophy were of three phenotypic classes: sensitive, intermediate, and resistant to MBC. Transforming DNA appeared to integrate at several sites in the genome, with the more resistant phenotypes having more copies of the altered beta-tubulin gene than the sensitive and intermediate phenotypes. Transformants were also selected on medium containing MBC. The average frequency of transformation (1 to 3 transformants per micrograms of transforming DNA) was lower than that obtained by selection for uracil prototrophy, presumably because of failure to select transformants that contained few copies of the altered beta-tubulin gene. The sequence of the beta-tubulin gene was determined and compared with the published sequence of the benA gene of A. nidulans; the beta-tubulin gene was found to be highly conserved between the two Aspergillus species. Notable differences were that the beta-tubulin gene of A. flavus lacks intron 6 present in benA and has an additional leucine at position 148. This is the first gene sequence reported from an aflatoxin-producing fungus and adds to the growing body of knowledge of the beta-tubulin genes and their use as selectable markers for transformation of filamentous fungi.     

Increasing tubC beta-tubulin synthesis by placing it under the control of a benA beta-tubulin upstream sequence causes a reduction in benA beta-tubulin level but has no effect on microtubule function

We have constructed a chimeric beta-tubulin gene that places the structural gene for the tubC beta-tubulin of Aspergillus nidulans under the control of the benA beta-tubulin promoter. Introduction of either this chimeric gene or a second wild-type benA gene into a benomyl-resistant benA22 strain causes it to become benomyl sensitive, indicating that the introduced genes are functional. Analysis of the tubulin proteins synthesized in benA22 strains into which a second wild-type benA beta-tubulin gene was transformed showed that the total amount of beta-tubulin protein was the same as in the parental strain with a single benA gene. Thus the level of beta-tubulin must be regulated. This was also true of transformants carrying an extra copy of the chimeric beta-tubulin gene. The total amount of beta-tubulin was the same as in the parental strain. Two-dimensional gel analysis showed that the endogenous benA22 and the introduced chimeric tubC gene contributed equally to the total beta-tubulin pool. The fact that one-half of the benA beta-tubulin could be replaced by tubC beta-tubulin with no effect on the growth of the cells suggests that the benA and tubC beta-tubulins are functionally interchangeable.     

acon-3, the Neurospora crassa ortholog of the developmental modifier, medA, complements the conidiation defect of the Aspergillus nidulans mutant

Aspergillus nidulans and Neurospora crassa are ascomycetes that produce asexual spores through morphologically distinct processes. MedA, a protein with unknown function, is required for normal asexual and sexual development in A. nidulans. We determined that the N. crassa ortholog of medA is acon-3, a gene required for early conidiophore development and female fertility. To test hypotheses about the evolutionary origins of asexual development in distinct fungal lineages it is important to understand the degree of conservation of developmental regulators. The amino acid sequences of A. nidulans MedA and N. crassa ACON-3 shared 37% identity and 51% similarity. acon-3 is induced at late time points of conidiation. In contrast, medA is constitutively expressed and MedA protein localizes to nuclei in all tissue types. Nonetheless, expression of acon-3 using its native promoter complemented the conidiation defects of the A. nidulans ΔmedA and medA15 mutants. We conclude that the biochemical activity of the medA orthologs is conserved for conidiation.     

Diversity among beta-tubulins: a carboxy-terminal domain of yeast beta-tubulin is not essential in vivo.

Sequences of genes for beta-tubulins from many different organisms demonstrate that they encode highly conserved proteins but that these proteins diverge considerably at their carboxyl termini. The patterns of interspecies conservation of this diversity suggest that it may have functional significance. We have taken advantage of the properties of Saccharomyces cerevisiae to test this hypothesis in vivo. The sole beta-tubulin gene of this species is one of the most divergent of all beta-tubulins and encodes 12 amino acids which extend past the end of most other beta-tubulin molecules. We have constructed strains in which the only beta-tubulin gene is an allele lacking these 12 codons. We show here that this carboxy-terminal extension is not essential. The absence of these 12 amino acids had no effect on a number of microtubule-dependent functions, such as mitotic and meiotic division and mating. It did confer dominant supersensitivity to a microtubule-depolymerizing drug.     

Cloning and sequence of a β-tubulin cDNA from Pneumocystis carinii: possible implications for drug therapy

This work describes the isolation and characterization of a full-length cDNA clone encoding beta-tubulin from the pathogen Pneumocystis carinii. P. carinii contains a single gene encoding beta-tubulin. The complete sequence of this cDNA has been determined and its inferred amino acid sequence compared with the beta-tubulins from other organisms. This analysis augments the data indicating that P. carinii should be classified as a fungal organism. Further comparisons between the P. carinii beta-tubulin and those of fungal beta-tubulins resistant to benomyl, a beta-tubulin-binding drug, indicate a difference which may be exploited in the development of a new drug therapy for P. carinii pneumonitis. These results suggest that, theoretically, a drug presently administered for treatment of nematode worm infections may be an effective agent against P. carinii, without being toxic to the mammalian host. This possibility is currently being investigated.     

Molecular analysis and characterization of the Cochliobolus heterostrophus beta-tubulin gene and its possible role in conferring resistance to benomyl

Cochliobolus heterostrophus Tub1 described here is the first beta-tubulin gene characterized from a naturally occurring benomyl-resistant ascomycete plant pathogen. The gene encodes a protein of 447 amino acids. The coding region of Tub1 is interrupted by three introns, of 116, 55, and 56 nt, situated after codons 4, 12, and 53, respectively. As a result of the preference for pyrimidines in the third position of the codons when a choice exists between purines and pyrimidines, codon usage in the Tub1 gene is biased. Tub1 shows high homology with beta-tubulin genes of other ascomycete species. However, Tub1 is exceptional in having Tyr(167), compared with Phe(167), possessed by beta-tubulin genes of other ascomycetes sequenced thus far. The Tyr(167) residue has been associated with benomyl resistance in other organisms. In contrast, all other benomyl-implicated residues of Tub1 correspond to sensitivity. Based on these results, we suggest that benomyl resistance in the fungus probably is attributed to Tyr(167).     

Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function.

The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.     

Neurospora crassa ve-1 affects asexual conidiation

The velvet factor of the homothallic fungus Aspergillus nidulans promotes sexual fruiting body formation. The encoding veA gene is conserved among fungi, including the ascomycete Neurospora crassa. There, the orthologous ve-1 gene encodes a deduced protein with high similarity to A. nidulans VeA. Cross-complementation experiments suggest that both the promoter and the coding sequence of N. crassa ve-1 are functional to complement the phenotype of an A. nidulans deletion mutant. Moreover, ve-1 expression in the heterologous host A. nidulans results in development of reproductive structures in a light-dependent manner, promoting sexual development in the darkness while stimulating asexual sporulation under illumination. Deletion of the N. crassa ve-1 locus by homologous gene replacement causes formation of shortened aerial hyphae accompanied by a significant increase in asexual conidiation, which is not light-dependent. Our data suggest that the conserved velvet proteins of A. nidulans and N. crassa exhibit both similar and different functions to influence development of these two ascomycetes.     

Aspergillus contains multiple tubulin genes

Previous work with benomyl-resistant mutants of Aspergillus nidulans has demonstrated that the benA locus is a structural gene for beta-tubulin. Two of the benA mutants, benA22 and benA85, show altered electrophoretic mobilities on two-dimensional gels for two beta-tubulins (designated beta 1 and beta 2). These shifts of the beta 1- and beta 2-tubulins uncover a spot in the region where wild-type beta-tubulins migrate that is occluded on gels of wild-type extracts by the beta 1- and beta 2-tubulins. Evidence has now been obtained indicating that this spot represents an additional beta-tubulin (designated beta 3). Tubulin was partially purified from Aspergillus and run on one- and two-dimensional gels and the band or spot uncovered by the shift of the beta 1- and beta 2-tubulins was identified as a beta-tubulin by immunoblotting with monoclonal and affinity-purified polyclonal anti-tubulin antibodies and by one-dimensional peptide mapping. These observations show that Aspergillus contains at least two structural genes for beta-tubulins. Similar techniques have also been applied to a mutant showing altered alpha-tubulins to confirm and modify earlier observations suggesting that at least two structural genes for alpha-tubulins are also present.     

Identification and functional analysis of beta-tubulin genes by site specific integrative transformation in Aspergillus nidulans

We have cloned two different beta-tubulin sequences from the filamentous fungus Aspergillus nidulans. Each was used in the construction of transforming plasmids that carry the pyr4 gene of Neurospora crassa. We used these plasmids to transform a pyrG-strain of Aspergillus to uridine prototrophy. Both plasmids were shown to integrate site specifically into the homologous chromosomal sequences. We then used transformant strains in genetic crosses to demonstrate that one of the cloned beta-tubulin sequences was the benA beta-tubulin gene, which codes for the beta 1-and beta 2-tubulins. The other cloned beta-tubulin sequence was shown to be the structural gene for beta 3-tubulin by gene disruption and to participate in conidial development. This is the first report of a gene disruption by site specific, integrative recombination in Aspergillus nidulans.     

The highly divergent beta-tubulins of Aspergillus nidulans are functionally interchangeable

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.