Directed mutagenesis in an asporogenous methylotrophic yeast: cloning, sequencing, and one-step gene disruption of the 3-isopropylmalate dehydrogenase gene (LEU2) of Candida boidinii to derive doubly auxotrophic marker strains.

A model system for one-step gene disruption for an asporogenous methylotrophic yeast, Candida boidinii, is described. In this system, the 3-isopropylmalate dehydrogenase gene (C. boidinii LEU2) was selected as the target gene for disruption to derive new host strains for transformation. First, the C. boidinii LEU2 gene was cloned, and its complete nucleotide sequence was determined. Next, the LEU2 disruption vectors, which had the C. boidinii URA3 gene as the selectable marker, were constructed. Of the Ura+ transformants obtained with these plasmids, more than half showed a Leu- phenotype. Finally, the double-marker strains of C. boidinii were derived. When vectors with repeated flanking sequences of the C. boidinii URA3 gene were used for gene disruption, Leu- Ura+ transformants changed spontaneously to a Leu- Ura- phenotype ca. 100 times more frequently than they did when plasmids without the repeated sequences were used. Southern analysis showed that these events included a one-step gene disruption and a subsequent popping out of the C. boidinii URA3 sequence from the transformant chromosome.     

High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as in monomeric forms in C. boidinii cells. The C. boidinii URA3 gene was overexpressed in C. boidinii on these CARS vectors. CARS1 and CARS2 were found to function as an autonomous replicating element in Saccharomyces cerevisiae as well. Different portions of the CARS1 sequence were needed for autonomous replicating activity in C. boidinii and S. cerevisiae. C. boidinii could also be transformed with vectors harboring a CARS fragment and the S. cerevisiae URA3 gene.     

Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants.

A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura- by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confirmed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms.     

Development of an integrative DNA transformation system for the yeast Candida tropicalis.

We developed the alkane and fatty-acid utilizing yeast Candida tropicalis as a host for DNA transformations. The system is based on an auxotrophic mutant host of C. tropicalis which is defective in orotidine monophosphate decarboxylase (ura3). The ura3 host was isolated by mutagenesis and a double-selection procedure that combined nystatin enrichment selection and 5-fluoro-orotic acid resistance selection. As a selectable marker, we isolated and characterized the C. tropicalis URA3 gene. Plasmid vectors that contained the C. tropicalis URA3 gene transformed the C. tropicalis mutant host at a frequency of 10(3) to 10(4) transformants per micrograms of plasmid DNA. Vectors that contained the Saccharomyces cerevisiae URA3 gene could not transform C. tropicalis. DNA transfer was accomplished by modified versions of either spheroplast generation (CaCl2-polyethylene glycol)-fusion or cation (LiCl) procedures developed for S. cerevisiae. Plasmid vectors that had been cut within the C. tropicalis URA3 fragment integrated by homologous recombination at the URA3 locus.     

Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation.

A cDNA encoding Cryptococcus neoformans orotidine monophosphate pyrophosphorylase (OMPPase) has been isolated by complementation of the cognate Escherichia coli pyrE mutant. The cDNA was used as a probe to isolate a genomic DNA fragment encoding the OMPPase gene (URA5). By using electroporation for the introduction of plasmid DNA containing the URA5 gene, C. neoformans ura5 mutants could be transformed at low efficiency. Ura+ transformants obtained with supercoiled plasmids containing the URA5 gene showed marked mitotic instability and contained extrachromosomal URA5 sequences, suggesting limited ability to replicate within C. neoformans. Transformants obtained with linear DNA were of two classes: stable transformants with integrated URA5 sequences, and unstable transformants with extrachromosomal URA5 sequences.     

High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2.

This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.     

The orotidine-5′-phosphate decarboxylase gene (URA3) of a methylotrophic yeast,Candida boidinii: Nucleotide sequence and its expression in Escherichia coli

Previously, we cloned a DNA fragment from a genomic library of a methylotrophic yeast, Candida boidinii. This 3.5-kb SalI fragment was capable of complementing the pyrF mutation in Escherichia coli. In this report, we identify this fragment as that harboring an orotidine-5′-phosphate decarboxylase (ODCase) gene (C. boidinii URA3); we have also determined the complete DNA sequence of the C. boidinii URA3 gene. The deduced amino acid sequence of the gene showed homology to ODCase genes from other sources, and it could complement the ura3 mutation of Saccharomyces cerevisiae. The DNA fragment, which harbored the C. boidinii URA3 gene, was able to express ODCase activity in the E. coli pyrF mutant strain without an exogenous E. coli promoter. From nested-deletion analysis, both the 5′-(136 bp) and 3′-(58 bp) flanking regions were shown to be required for pyrF-complementation of the E. coli mutant. The 5′-flanking region had sequences homologous to E. coli promoter consensus sequences (−35 and −10 regions) which may function in the expression of the C. boidinii URA3 gene in E. coli.     

Development of an integrative DNA transformation system for the yeast Hansenula anomala

A host-vector system for the yeast Hansenula anomala was developed. The system was based on an auxotrophic mutant host of H. anomala which was defective in orotidine-5′-phosphate decarboxylase (ODCase) activity. The H. anomala ODCase-negative mutant strains (ura3 strains) were isolated based on 5-fluoroorotic acid (5-FOA) resistance. A plasmid vector containing the H. anomala URA3 gene was used for transformation. Using this plasmid, all of the H. anomala ura3 strains tested could be transformed to Ura⁺ phenotypes. In all of Ura⁺ transformants, the introduced plasmid was integrated into the chromosomal URA3 locus by homologous recombination. The Ura⁺ phenotype of the transformants was stably maintained after nonselective growth.     

Isogenic Strain Construction and Gene Mapping in Candida Albicans

Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle. Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing. To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus. The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations. The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura(-) strain otherwise isogenic with the parental clinical isolate. Subsequent mutation of the Ura(-) strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker. URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene. Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura(-) phenotype. These Ura(-) segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis. To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats. Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.     

Development of a transformation system for gene knock-out in the flavinogenic yeast Pichia guilliermondii

Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5'-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib(-) Ura(+) phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3-12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3' end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8-0.9 kb sequences homologous to the target gene.     

Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene

We established a novel and convenient method to construct a ura3 strain (ura3/ura3) of the asporogenous and diploid yeast, Candida tropicalis, that produces dicarboxylic acid. One copy of the URA3 gene was disrupted using a mutated hygromycin B resistance gene (HYG#). The obtained hygromycin-resistant strain was further transformed with a URA3 disruption cassette and selected on a plate containing 5-fluoroorotic acid. The obtained strains were analyzed and the disruption of the gene was confirmed by PCR and Southern blot analysis. The results showed that the strains were obtained in which allelic URA3 genes were simultaneously disrupted. Furthermore, we established a cotransformation method for this gene disruption, using HYG# in C. tropicalis. In order to disrupt the allelic POX4 genes (encoding acyl-CoA oxidase) of dicarboxylic acid-producing strains, the ARS plasmid (which contained HYG#) and a POX4 disruption cassette (which carried the LAC4 gene encoding beta-galactosidase of Kluyveromyces lactis) were simultaneously introduced by transformation. As a result, the allelic POX4 gene was successfully disrupted.     

One-step gene disruption by cotransformation to isolate double auxotrophs in Candida albicans

Summary The Candida albicans LEU2 gene was disrupted by substituting lambda DNA for a small deletion within the LEU2 gene. Cotransformation with a selectable URA3 ARS vector was used to introduce a linear fragment containing the disruption into the genome of a C. albicans ura3 deletion mutant. Cotransformants containing the lambda DNA were identified by colony hybridization and the URA3 plasmid was subsequently cured. Leu2 disrupted heterozygotes were detected by Southern hybridization and one disruptant was subsequently treated with UV irradiation. Only one leu2 ura3 mutant (SGY-484) was isolated out of 11,000 mutagenized cells. SGY-484 was transformed to Leu⁺ with either the C. albicans or Saccharomyces cerevisiae LEU2 gene. Southern hybridization analysis revealed that the mutant is not homozygous for the disruption; the leu2 mutation reverts and is most likely a point mutation. Unexpectedly, an ade2 ura3 mutant was isolated from the same mutagenesis.     

Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata

The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.     

Cloning and characterization of complementary DNA encoding human N-acetylglucosamine-phosphate mutase protein

Endomyces fibuliger is a yeast used in the production of Chinese rice wine. It secretes enzymes such as glucoamylase, alpha-amylase and acid protease. Very little is known of the genetics of E. fibuliger. In order to develop a transformation system for this yeast, orotidine-5'-phosphate decarboxylase mutant strains were obtained and characterized. Transformation of the E. fibuliger ura3 mutant F1 with an integrative plasmid that carried the wild-type URA3 gene of E. fibuliger gave complementation of this mutation. The E. fibuliger gene encodes the orotidine-5'-phosphate decarboxylase enzyme consisting of 266 amino acid residues with a 69.4% sequence identity with orotidine-5'-phosphate decarboxylase of Saccharomyces cerevisiae. Our finding that E. fibuliger URA3 complements the ura3 mutation in S. cerevisiae confirms that the URA3 gene of E. fibuliger encodes a protein that exerts a similar function.     

Transformation of Saccharomyces cerevisiae with UV-irradiated single-stranded plasmid.

UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains. The low doses of UV used in this study (10-75 J/m2) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad+ strain, while they had no effect on transformation with double-stranded plasmids of comparable size. Neither the rev3 mutation, nor the rad18 or rad52 mutations influenced the efficiency of transformation with irradiated single-stranded plasmid. However, it was found to be decreased in the double rev3 rad52 mutant. Extracellular irradiation of plasmid that contains both URA3 and LEU2 genes (psLU) gave rise to up to 5% Leu- transformants among selected Ura+ ones in the repair-proficient strain. Induction of Leu- transformants was dose-dependent and only partially depressed in the rev3 mutant. These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged single-stranded DNA in yeast.     

Mutations in the Saccharomyces cerevisiae CDC1 gene affect double-strand-break-induced intrachromosomal recombination.

To isolate Saccharomyces cerevisiae mutants defective in recombinational DNA repair, we constructed a strain that contains duplicated ura3 alleles that flank LEU2 and ADE5 genes at the ura3 locus on chromosome V. When a HO endonuclease cleavage site is located within one of the ura3 alleles, Ura+ recombination is increased over 100-fold in wild-type strains following HO induction from the GAL1, 10 promoter. This strain was used to screen for mutants that exhibited reduced levels of HO-induced intrachromosomal recombination without significantly affecting the spontaneous frequency of Ura+ recombination. One of the mutations isolated through this screen was found to affect the essential gene CDC1. This mutation, cdc1-100, completely eliminated HO-induced Ura+ recombination yet maintained both spontaneous preinduced recombination levels and cell viability, cdc1-100 mutants were moderately sensitive to killing by methyl methanesulfonate and gamma irradiation. The effect of the cdc1-100 mutation on recombinational double-strand break repair indicates that a recombinationally silent mechanism other than sister chromatid exchange was responsible for the efficient repair of DNA double-strand breaks.     

Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.     

5-fluoro-orotic acid induces chromosome alterations in genetically manipulated strains of Candida albicans

We previously reported the occurrence of chromosome alterations in a Candida albicans prototrophic strain 3153A treated with 5-fluoro-orotic acid (5-FOA). In this study we investigated the mutagenic properties of 5-FOA with two derivatives of C. albicans strain CAF4-2 (ura3/ura3), each containing an ectopic copy of URA3 gene (ura3/ ura3 URA3) on a different chromosome. As expected, after the ura3/ura3 URA3 constructs were applied to 5-FOA containing solid medium, the "pop-outs" that lost URA3 appeared. However most of the "pop-outs" acquired various chromosome alterations. Thus constructs exposed to 5-FOA should be examined for chromosome alterations or the use of 5-FOA should be avoided.     

A System of Shuttle Vectors and Yeast Host Strains Designed for Efficient Manipulation of DNA in Saccharomyces Cerevisiae

A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.