A single amino-acid substitution in the beta-tubulin gene of Neurospora confers both carbendazim resistance and diethofencarb sensitivity

Summary Two MBC-resistant mutants of Neurospora crassa, F914 and F939, were sensitive to diethofencarb at a concentration of 0.1 g/ml, while the wild-type strain and other MBC-resistant mutants showed resistance to diethofencarb at a concentration of 100 g/ml. Genetic analysis suggested that the mutations in these two strains were closely linked to the Bml locus which codes for beta-tubulin. When the wild-type strain was transformed by the cloned beta-tubulin gene of the F914 strain, the transformants showed both MBC resistance and diethofencarb sensitivity. On the other hand, the diethofencarb sensitivity of the F914 strain was cancelled by transformation with the wild-type beta-tubulin gene. DNA sequencing of F914 beta-tubulin revealed that glycine was substituted for glutamic acid at position 198 in the F914 strain. Therefore, a single base change in the betatubulin gene was proved to confer both MBC resistance and diethofencarb sensitivity.     

The gene for cadmium metallothionein from a cadmium-resistant yeast appears to be identical to CUP 1 in a copper-resistant strain

Summary A cadmium-resistant strain of Saccharomyces cerevisiae produces a cadmium metallothionein with the same characteristics as the copper metallothionein that is encoded by CUP 1 in a copper-resistant strain. The structural gene for metallothionein from the cadmium-resistant strain resembles CUP 1 in terms of the fragmentation patterns generated by restriction enzymes. Furthermore, the gene may be amplified as 2.0 kb repeating units in both the cadmium-resistant and the copperresistant strains. However, transformants with a plasmid that carried the metallothionein gene from the cadmiumresistant strain were resistant to copper but not to cadmium. It appears that the same metallothionein gene, CUP 1, is amplified in both cadmium- and copper-resistant yeasts. However, the mechanism for the cadmiumspecific inducibility of the gene may be restricted to the cadmium-resistant strain.     

The cadmium-resistant gene, CAD2, which is a mutated putative copper-transporter gene (PCA1), controls the intracellular cadmium-level in the yeast S. cerevisiae

Yeast cells carrying the CAD2 gene exhibit a resistance to cadmium. We cloned this gene and demonstrated that it was a mutated form derived from the gene of a putative copper-transporting ATPase (PCA1). By site-directed mutagenesis, it appeared that the mutation conferring cadmium resistance was a R970G-substitution in the C-terminal region of Pca1 protein. The intracellular cadmium level of cells carrying CAD2 was lower than that of cells carrying either PCA1 or Δcad2. Furthermore, cells with overexpression of CAD2 showed a much lower intracellular cadmium level than that of cells with a single-copy CAD2. From these results, we conclude that the Cad2 protein controls the intracellular cadmium level through an enhanced cadmium efflux system. Received: 3 September / 9 November 1999     

Cloning of maltase regulatory genes in Saccharomyces cerevisiae

Summary By hybridization with a putative MAL2p regulatory sequence we have identified a 19 kb long BamH1 DNA fragment to contain the MALp sequence in a MAL4 strain. A mixture of recombinant plasmids was prepared by ligation of purified 19 kb BamH1 fragments partially digested with Sau3A into the multicopy vector YEp1357. The source of DNA was a strain carrying the MAL4 locus. Yeast maltose non-fermenting strains were transformed with the plasmid mixture. A recombinant plasmid, pRM-4, containing the MAL4p regulatory gene was isolated that complements the maltose-negative phenotype. The plasmid was shown to confer the ability to synthesize maltase to recipient strains grown under inducing as well as under repressing conditions.The MAL4p regulatory sequence cloned was used as a probe in hybridization experiments to study the degrees of homology between the different MAL regulatory genes. The results showed that the sequence from MAL4 strains is strongly homologous to that of MAL3 strains whereas it shows significant differences to the ones of MAL1 and MAL2 strains.Southern analysis of the segregants of crosses between maltose-positive strains and ma10 strains allowed us to localize the maltase regulatory sequence of each MAL locus within a characteristic BamH1 fragment of genomic DNA hybridizing to the isolated sequence.     

A two-step protocol for efficient deletion of genes in the filamentous ascomycete Podospora anserina

Deletion of genes in Podospora anserina via conventional methods is an inefficient and time-consuming process since homologous recombination occurs normally only at low frequency (about 1%). To improve the efficiency of replacement, we adopted the two-step protocol developed for Aspergillus nidulans (Chaveroche et al. in Nucleic Acids Res 28:E97, 2000). As a prerequisite, a vector was generated containing a blasticidin resistance cassette for selection in the Escherichia coli host strain KS272 (pKOBEG) and a phleomycin resistance cassette for selection in P. anserina. A derivative of this vector, into which short (∼250 bp) PCR-generated sequences flanking the gene to be deleted have been integrated, is introduced into the E. coli host strain which contains a cosmid with the gene of interest and long 5′ and 3′ flanking sequences. Subsequently, a cosmid is reisolated from E. coli in which the gene of interest is replaced by the resistance cassette. This construct is used to transform P. anserina. The long stretches flanking the resistance cassette facilitate recombination with homologous sequences in the fungal genome and increase the efficiency of gene deletion up to 100%. The procedure is not dependent on the availability of specific auxotrophic mutant strains and may be applicable to other fungi.     

Gene deletion and allelic replacement in the filamentous fungus <Emphasis Type="Italic">Podospora anserina</Emphasis>

Gene replacement via homologous recombination is a fundamental tool for the analysis of gene function. However, this event is rare in organisms like the filamentous fungus Podospora anserina. We show here that deletion of the PaKu70 gene is an efficient strategy for improving gene manipulation in this organism. By using the ΔPaKu70 strain, it is now possible (1) to produce deletion mutants with an efficiency of 100%, (2) to achieve allelic exchange by introducing a mutated allele associated with a selection cassette at the locus, (3) to introduce a mutation in a gene without co-insertion of a selectable marker and without any modification of the target locus.     

Chromosome mapping ofSoa,a gene influencing gustatory sensitivity to sucrose octaacetate in mice

Strain distribution patterns among recombinant inbred strains suggested that a locus influencing taste sensitivity to sucrose octaacetate was on chromosome 6. A location forSoa was established by linkage analysis of behavioral and electrophoretic data from outbred and congenic strains and from test-cross progeny. Haplotyping of 41 outbred CFW-Cr animals with a cDNA probe showed perfect cosegregation ofSoa andPrp, a gene for salivary proline-rich proteins. Five of twelve B6. SW-Soa ^a strains were found to retainLdr-1, lactate dehydrogenase regulator-1, on chromosome 6 as an allelic passenger from the SWR/J donor strain (source of theSoa ^a Taster allele). Centimorgan distance was estimated using the ABP/Le linkage-testing strain (non-Taster,Soa ^b) and the SWR/J strain (Taster,Soa ^a) in a testcross breeding system. The data are consistent with a position for theSoa locus on mouse chromosome 6, 62 cM from the centromere.This research was supported in part by Grants DC00150 (G. W.) and DE003658 (E.A.A.). This paper is based on a thesis submitted to the Florida State University by the first author in partial fulfillment of the requirements for the Master of Sciences degree.     

Isolation and structure of an acetolactate synthase gene from Schizosaccharomyces pombe and complementation of the ilv2 mutation in Saccharomyces cerevisiae

A gene encoding a functional acetolactate synthase (ALS) subunit has been isolated from the fission yeast Schizosaccharomyces pombe, and has been structurally and genetically characterized. The approximate 5-kbp cloned DNA segment was found to contain a 2007-bp open reading frame capable of encoding a 669 aminoacid polypeptide which exhibited 57.1% similarity to the corresponding ALS subunit from Saccharomyces cerevisiae. The putative ilv1 isolated from S. pombe was shown to encode a functional subunit of acetolactate synthase by complementation of an S. cerevisiae strain deleted for the ILV2 locus.     

Mapping of four ras superfamily genes by physical and genetic means in Schizosaccharomyces pombe

Summary Four ras superfamily genes, namely ypt1, ypt2, ypt3 and ryh1, have been located on the S. pombe linkage map. This was achieved by constructing strains carrying a new NotI cutting site and the S. cerevisiae LEU2 gene integrated next to the respective gene. The physical location of these genes of the chromosomes was then determined by NotI restriction analysis of the DNA prepared from each strain. Fine genetic mapping was carried out by conventional tetrad analysis using the integrated LEU2 gene as a marker. The results indicated that ypt1 is tightly linked to top1 on the right arm of chromosome II; that ypt2 is 2.5 cM apart from ura2 on the right arm of chromosome I; that ypt3 is tightly linked to arg3 on the left arm of chromosome I; and that rhy1 is located approximately 20 cM from ade3 on the left arm of chromosome I.     

Generation of new mutants of nmr,the negative-acting nitrogen regulatory gene of Neurospora crassa,by repeat induced mutation

Summary The repeat induced point mutation (RIP) phenomenon has been used to generate new mutants of nmr, the negative nitrogen regulatory gene in Neurospora crassa. The wild-type nmr gene was cotransformed along with the hygromycin B resistance gene into wild-type cells by selecting for hygromycin B resistance. Following purification of primary transformants using microconidia, crosses to wild-type. Detailed analyses of some of the progeny revealed that we had generated authentic nmr mutants at high frequency. The polymerase chain reaction was used to amplify and clone a fragment of a mutagenized nmr copy from one of the mutants. The nucleotide sequence analysis showed that 14% of the guanine residues have been converted into adenines, resulting in numerous missense and nonsense mutations. The newly created nmr mutants were found suitable for use as host strains in transformation experiments.     

A gene (Cargl) that regulates tissue resistance toCandida albicansmaps to chromosome 14 of the mouse

Tissue susceptibility and resistance to infection with the yeastCandida albicansis genetically regulated. Analysis of the strain distribution pattern of theC. albicansresistance gene (Carg1) and additional gene and DNA segment markers in the AKXL recombinant inbred (RI) set showed that 13/15 RI strains were concordant forCarg1,TcraandRib1. Therefore,Carg1is probably located within a 17 cM segment of chromosome 14, within approximately 4 cM of the other two genes.     

Shuttle vectors for <Emphasis Type="Italic">Candida albicans</Emphasis>: control of plasmid copy number and elevated expression of cloned genes

Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20–40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.     

The dominant <Emphasis Type="Italic">Hc.Sdh</Emphasis><Superscript>R</Superscript> carboxin-resistance gene of the ectomycorrhizal fungus <Emphasis Type="Italic">Hebeloma cylindrosporum</Emphasis> as a selectable marker for transformation

In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.Sdh ^R that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe–S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not affected by 2 μg l⁻¹ carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 μg l⁻¹ of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR amplification and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.Sdh ^R cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less efficient donor than LBA 1126; the former yielded ca. 0–30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60–95%. The authors Chrisse Ngari and Jean-Philippe Combier contributed equally to this work.     

Construction by one-step gene replacement of Trichoderma reesei strains that produce the glucoamylase P of Hormoconis resinae

Two one-step gene replacement vectors containing either the Hormoconis resinae glucoamylase P (gamP) genomic gene or the corresponding cDNA, each under the control of the promoter of the Trichoderma reesei cellobiohydrolase 1 gene (cbh1), were constructed and use to replace the cbh1 gene in a T. reesei strain. In both vectors the cbh1 promoter is precisely fused to the gamP protein coding region. Both the gamP cDNA and the genomic gene direct the secretion of the active glucoamylase P (GAMP) enzyme from T. reesei, which indicates that the intron sequences in the genomic gamP gene are processed in T. reesei. According to the results, a T. reesei transformant strain, in which the cbh1 gene has been replaced by a single copy of the gamP genomic gene, secretes more active GAMP than does a transformant strain having three copies of the cDNA clone in tandem orientation at the cbh1 locus.     

A recessive mutant allele of the HNM1 gene of Saccharomyces cerevisiae is responsible for hyper-resistance to nitrogen mustard

Summary A screening of haploid yeast strains for enhanced resistance to nitrogen mustard (HN2) yielded a recessive mutant allele, hnm1, that conferred hyper-resistance (HYR) to HN2. Diploids, homo- or heterozygous for the HNM1 locus, exhibit normal wild-type like resistance while homozygosity for hnm1 leads to the phenotype HYR to HN2. The hnm1 mutation could be found in yeast strains proficient or deficient in different DNA repair systems. In these mostly HN2-sensitive haploid repair-deficient mutants, hnm1 acted as a partial suppressor of HN2 sensitivity. All isolated recessive mutations conferring hyper-resistance belonged to a single complementations group. The HYR to HN2 phenotype was maximally expressed in growing cells and was associated with reduced mutability by HN2. HNM1 most probably controls uptake of HN2 which would be impaired in the hnm1 mutants.     

Induction of heterothallic strains and their genetic and physiological characterization in a homothallic strain of the yeast Saccharomyces exiguus

Summary We isolated heterothallic strains from a homothallic strain of S. exiguus by mutagenization with UV or ethylmethanesulfonate (EMS). A gene, not linked to the mating-type locus, was found to control homothallism in the yeast, as in S. cerevisiae. Pheromone of S. exiguus (^se pheromone) induced formation of large pear-shaped cells (shmooing) in a strains of S. exiguus, S. cerevisiae, and S. kluyveri, and sexual agglutinability of an inducible a strain of S. cerevisiae. ^se Pheromone is a peptidyl substance a little different from pheromone of S. cerevisiae. a Pheromone of S. exiguus acts only on a cells of S. exiguus. Contrary to the above results, neither sexual agglutination nor zygote formation occurred among these three Saccharomyces yeasts.     

Crossing the barrier: Evolution and spread of a major class of mosaic pbp2x in Streptococcus pneumoniae, S. mitis and S. oralis

A paradigm for Streptococcus interspecies gene transfer is represented by the mosaic pbp genes encoding the target enzymes for beta-lactam antibiotics, the penicillin-binding proteins, in Streptococcus pneumoniae. We investigated a collection of oral streptococci from three continents by comprehensive multi-locus sequence typing analysis in order to trace the origin of a mosaic block belonging to a dominant family of mosaic pbp2x implicated in penicillin resistance of S. pneumoniae. One widespread family of mosaic pbp2x occurred in all three distinct clusters of S. pneumoniae, Streptococcus mitis and Streptococcus oralis, documenting independent inter- and intraspecies recombination events. Moreover, potential ancestor genes of this mosaic block could be identified in two penicillin-susceptible S. mitis strains from South Africa and Spain, facilitating the identification of pbp2x mutations relevant for resistance development.     

Isolation and characterization of Pioneer1, a novel Chlamydomonas transposable element

During the course of this study a novel family of Chlamydomonas mobile elements has been identified in natural isolate strain 224. The first member of this class to be characterized, a 2.8-kb element named Pioneer1, was trapped in an intron of the nitrate reductase structural gene, NIT1. This element has been cloned and completely sequenced and found to be unusual in structure. Pioneer elements are present in a very low-copy number of three per genome in strain 224. The copy number increased by one upon transposition of Pioneer1. Hybridization of Pioneer1 to a variety of Chlamydomonas strains confirmed that this element differed from previously described Chlamydomonas transposons. It also indicated that related elements are present in low-copy number in natural isolate strains 356 and S1D2, but not in the most commonly used laboratory strains 137c and 21 gr. For these reasons, members of the Pioneer family might prove useful as insertional mutagens.     

Physical mapping of genetic determinants on yeast mitochondrial DNA affecting the apparent size of the Var 1 polypeptide

Summary The var1 locus on yeast mitochondria) DNA is defined by a set of alleles that affect the apparent size (M_r 40,000–44,000) of a mitochondria) translation product (var1), a protein of the mitochondria) 38S ribosomal subunit. Using petite deletion and restriction endonuclease site mapping, we have defined the physical location of all var1 alleles to a specific restriction fragment of roughly 2.1 kbp located between the antibiotic resistance loci ery1 and olil. Surprisingly, from base sequence studies of this region by Tzagoloff et al. (1980), it appears that the DNA fragment we have mapped contains little or none of the structural gene for the var1 protein since the fragment is composed primarily of long stretches of dA + dT interspersed with short clusters high in dG + dC. Nevertheless, by a type of complementation test termed zygotic gene rescue (Strausberg and Butow, 1977), we show that with petites retaining that restriction fragment and short flanking sequences, var1 polypeptide characteristic of the strain from which the petite was derived, is expressed in zygotes formed between the petite and a wild-type tester. Thus the ability of the var1 determinant to act in trans suggests that control of expression of different var1 species involves intermolecular interactions, perhaps at the level of RNA splicing. Our results are discussed in terms of several possible models for the organization and control of the var1 structural gene.