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.     

Isolation and characterization of cold-sensitive mutations at the benA, beta-tubulin, locus of Aspergillus nidulans

Summary We have isolated large numbers of conditionally lethal -tubulin mutations to provide raw material for analyzing the structure and function of tubulin and of microtubules. We have isolated such mutations as intragenic suppressors of benA33, a heat-sensitive (hs^-) -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 -tubulin molecule.     

Identification of a gene for beta-tubulin in Aspergillus nidulans.

The tubulins of Aspergillus nidulans have been characterized in wild-type and ben A, B and C benomyl-resistant strains by two-dimensional gel electrophoresis, co-polymerization with porcine brain tubulin and peptide mapping. Four α-tubulins and at least four β-tubulins were resolved by two-dimensional gel electrophoresis of wild-type proteins. Eighteen of 26 benA mutants studied had electrophoretically abnormal β-tubulins. In these strains, one or more of the β-tubulins had either an altered isoelectric point or an altered electrophoretic mobility in the SDS gel dimension, or was diminished in amount. The a-tubulins were normal. Two-dimensional gels of protein extracts of a ben A/wild-type diploid strain demonstrated co-expression of the wild-type β-tubulins with the variant ben A tubulin. This experiment rules out post-translational modification as the source of the β-tubulin abnormalities in the benA mutants. We therefore conclude that benA must be a structural gene for β-tubulin. Due to the variety of abnormalities affecting β-tubulins in ben A mutants, and the absence of abnormalities affecting α-tubulins in any of the benomyl-resistant mutants, we also believe that the benomyl binding site must be located on the β-subunit of the tubulin dimer. The benA mutants of A. nidulans promise to be useful not only for characterizing the biochemical determinants of the benomyl binding site of tubulin but also for understanding the relationship between tubulin structure and function.     

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.     

Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.     

Identification of genes involved in terbinafine resistance in Aspergillus nidulans

AIMS: To determine the pattern and the genetic basis of resistance to terbinafine, a drug extensively used for the treatment of fungal infections in humans. METHODS AND RESULTS: Four resistant mutants from Aspergillus nidulans isolated after irradiation with ultraviolet light were crossed with the master strain F (MSF). Genetic analysis revealed that a single gene, located on chromosome IV, is responsible for resistance to terbinafine and that the alleles responsible for this resistance in these mutants are of a codominant or dominant nature at high terbinafine concentrations. Furthermore, the interaction of this mutation with another one identified on chromosome II causes the double mutant to be highly resistant. CONCLUSIONS: Periodic surveillance of antimycotic susceptibility would be an important measure in detecting the emergence and spread of resistance. Mutation in a single gene could be responsible for resistance to terbinafine and a genic interaction may be responsible for a higher level of antimycotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of the mechanisms that lead to changes in the sensitivity of a fungus to a given antifungal agent is important both in order to define strategies for the use of such agent and to guide the development of new antifungal agents.     

Transformation of Aspergillus aculeatus using the drug resistance gene of Aspergillus oryzae and the pyrG gene of Aspergillus nidulans

Transformation systems for Aspergillus aculeatus has been developed, based on the use of the pyrithiamine resistance gene of Aspergillus oryzae and the orotidine-5'-monophosphate decarboxylase gene (pyrG) of Aspergillus nidulans. An A. aculeatus mutant which can be transformed effectively by the A. nidulans pyrG gene was isolated as a transformation host. This is the first report of transformation of A. aculeatus.     

Identification and characterization of the asperthecin gene cluster of Aspergillus nidulans

The sequencing of Aspergillus genomes has revealed that the products of a large number of secondary metabolism pathways have not yet been identified. This is probably because many secondary metabolite gene clusters are not expressed under normal laboratory culture conditions. It is, therefore, important to discover conditions or regulatory factors that can induce the expression of these genes. We report that the deletion of sumO, the gene that encodes the small ubiquitin-like protein SUMO in A. nidulans, caused a dramatic increase in the production of the secondary metabolite asperthecin and a decrease in the synthesis of austinol/dehydroaustinol and sterigmatocystin. The overproduction of asperthecin in the sumO deletion mutant has allowed us, through a series of targeted deletions, to identify the genes required for asperthecin synthesis. The asperthecin biosynthesis genes are clustered and include genes encoding an iterative type I polyketide synthase, a hydrolase, and a monooxygenase. The identification of these genes allows us to propose a biosynthetic pathway for asperthecin.     

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).     

A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), encoded by the aroA locus, is a target site of glyphosate inhibition in bacteria. A glyphosate-resistant aroA allele has been cloned in Escherichia coli from a mutagenized strain of Salmonella typhimurium. Subcloning of this mutant aroA allele shows the gene to reside on a 1.3-kilobase segment of S. typhimurium DNA. Nucleotide sequence analysis of this mutant gene indicates a protein-coding region 427 amino acids in length. Comparison of the mutant and wild type aroA gene sequences reveals a single base pair change resulting in a Pro to Ser amino acid substitution at the 101st codon of the protein. A hybrid gene fusion between mutant and wild type aroA gene sequences was constructed. 5-Enolpyruvylshikimate-3-phosphate synthase was prepared from E. coli cells harboring this construct. The glyphosate-resistant phenotype is shown to be associated with the single amino acid substitution described above.