FLP-mediated site-specific recombination in microinjected murine zygotes

The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous system (Escherichia coli, Drosophila, mosquito and cultured mammalian cells). In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs. Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus. The resulting event occured at the one-cell stage and deleted a chromosomal tandem array of a FRT containinglacZ expression cassette down to one or two copies. These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.     

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.     

Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole-susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild-type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild-type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP-mediated, site-specific recombination (MPAR-flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole-resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock-out mutations in any C. albicans wild-type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.     

Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions

Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes, ARG5 and ADE2, and two sequences newly identified through the Candida genome project, HRM101 and ENX3. HRM101 and ENX3 are homologous to genes in the conserved RIM101 (previously called RIM1) and PacC pathways of Saccharomyces cerevisiae and Aspergillus nidulans. We show that three independent hrm101/hrm101 mutants and two independent enx3/enx3 mutants are defective in filamentation on Spider medium. These observations argue that HRM101 and ENX3 sequences are indeed portions of genes and that the respective gene products have related functions.     

Sequential strand exchange by XerC and XerD during site-specific recombination at dif

Successful segregation of circular chromosomes in Escherichia coli requires that dimeric replicons, produced by homologous recombination, are converted to monomers prior to cell division. The Xer site-specific recombination system uses two related tyrosine recombinases, XerC and XerD, to catalyze resolution of circular dimers at the chromosomal site, dif. A 33-base pair DNA fragment containing the 28-base pair minimal dif site is sufficient for the recombinases to mediate both inter- and intramolecular site-specific recombination in vivo. We show that Xer-mediated intermolecular recombination in vitro between nicked linear dif "suicide" substrates and supercoiled plasmid DNA containing dif is initiated by XerC. Furthermore, on the appropriate substrate, the nicked Holliday junction intermediate formed by XerC is converted to a linear product by a subsequent single XerD-mediated strand exchange. We also demonstrate that a XerC homologue from Pseudomonas aeruginosa stimulates strand cleavage by XerD on a nicked linear substrate and promotes initiation of strand exchange by XerD in an intermolecular reaction between linear and supercoiled DNA, thereby reversing the normal order of strand exchanges.     

A complementation analysis by parasexual recombination of Candida albicans morphological mutants

Benomyl treatment (at 100 micrograms ml-1) of Candida albicans 1001, and other strains derived from it, determined the appearance of morphological mutants similar to those derived from UV irradiation treatment. A permanent alteration in the morphogenesis of these mutant strains determined their inability to grow by budding, to form oval yeast cells or blastospores (Y-phenotype) and their growth as long filamentous forms, mostly with the appearance of pseudomycelium, giving rise to rough colonies (R phenotype). In order to carry out a genetic complementation analysis, we isolated morphological mutants that carried other genetic markers (nutritional, conditional lethal) adequate for crosses by means of protoplast fusion. Wild-type hybrids of regular mononuclear oval yeast cells and smooth colonies were obtained by crossing pairs of complementing mutants, whereas hybrids from crosses of non-complementing mutants still retained their morphological alterations. Our results define two complementation groups, which represent two genes relevant for dimorphism, whose alteration interferes with the correct transition from blastospores to mycelium.     

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.     

Milestones in Candida albicans gene manipulation

In the United States, candidemia is one of the most common hospital-acquired infections and is estimated to cause 10,000 deaths per year. The species Candida albicans is responsible for the majority of these cases. As C. albicans is capable of developing resistance against the currently available drugs, understanding the molecular basis of drug resistance, finding new cellular targets, and further understanding the overall mechanism of C. albicans pathogenesis are important goals. To study this pathogen it is advantageous to manipulate its genome. Numerous strategies of C. albicans gene manipulation have been introduced. This review evaluates a majority of these strategies and should be a helpful guide for researchers to identify gene targeting strategies to suit their requirements.     

Infection-related gene expression in Candida albicans

Research into the major fungal pathogen, Candida albicans has firmly entered the post-genomics era. The current challenge is to apply these technologies to the analysis of C. albicans infections. Initial studies, which focused on the expression of specific virulence genes, have supported the view that secreted hydrolases and adhesins are expressed in a niche-specific fashion during infection. However, genome-wide expression profiling has revealed that most infection-related changes in C. albicans gene expression reflect environmental adaptation. Initial contacts with the host and disease progression are clearly associated with metabolic and stress adaptation. These studies, together with analyses of C. albicans mutants, indicate that physiological fitness plays a central role in the pathogenicity of this fungus, alongside virulence factors.     

PCR-based gene targeting in Candida albicans

PCR-based gene-targeting approaches have increased the speed of gene function analyses in ascomycetous fungi, for example, in the diploid human fungal pathogen Candida albicans. Here we describe a protocol that utilizes Rapid-PCR to amplify all cassettes available with the previously reported pFA modules. With this protocol, sufficient quantities of any cassette for use in C. albicans transformation experiments can be reliably generated in 25-50 min using either of the two alternative optimized amplification conditions; cassette amplification by standard PCR methods typically takes 3-4 h and is likely to require optimization of amplification conditions for each cassette. Transformants that appear 2-4 d after transformation can be rapidly identified using Rapid-PCR on whole cells, eliminating the need for genomic DNA extraction. In total, less than a week is required for the deletion of one allele in C. albicans. Repeating this procedure will result in the generation of homozygous mutant strains.     

FLP-mediated recombination for use in hybrid plant production

We have studied the feasibility in Arabidopsis of using a site-specific recombination system FLP/FRT, from the 2 microm plasmid of yeast, for making plant hybrids. Initially, Arabidopsis plants expressing the FLP site-specific recombinase were crossed with plants transformed with a vector containing kanamycin-resistance gene (npt) flanked by FRT sites, which also served to separate the CaMV35S promoter from a promoterless gusA. Hybrid progeny were tested for excision of the npt gene and the positioning of 35S promoter proximal to gusA. GUS activity was observed in the progeny of all crosses, but not in the progeny derived from the self-pollinated homozygous parents. We then induced male sterility in Arabidopsis plants using the antisense expression of a pollen- and tapetum-specific gene, bcp1, flanked by FRT sites. Upon cross-pollination of flowers on the same male-sterile plants with pollen from FLP-containing plants, viable seeds were produced and the progeny hybrid plants developed normally. Molecular analyses revealed that the antisense expression cassette of bcp1 had been excised in these plants. These results show for the first time that a site-specific recombinase can be used to restore fertility in male-sterile plants, providing an alternative method for the production of hybrid seeds and plants.     

Homology,disruption and phenotypic analysis of CaGS Candida albicans gene induced during macrophage infection

During macrophage infection Candida albicans expresses differentially several genes whose functions are associated with its survival strategy. Among others, we have isolated CaGS gene, which is homologous to SNF3, a glucose sensor of Saccharomyces cerevisiae. To elucidate its potential role during infection, CaGS has been disrupted and the resulting phenotype analyzed on different solid media. The null mutant lost the ability to form hyphae on a medium with low glucose concentration and serum. Furthermore, this mutant does not disrupt macrophage in in vitro infections. We believe that this putative glucose sensor is involved in hyphal development during macrophage infection.     

Homology, disruption and phenotypic analysis of CaGS Candida albicans gene induced during macrophage infection

During macrophage infection Candida albicans expresses differentially several genes whose functions are associated with its survival strategy. Among others, we have isolated CaGS gene, which is homologous to SNF3, a glucose sensor of Saccharomyces cerevisiae. To elucidate its potential role during infection, CaGS has been disrupted and the resulting phenotype analyzed on different solid media. The null mutant lost the ability to form hyphae on a medium with low glucose concentration and serum. Furthermore, this mutant does not disrupt macrophage in in vitro infections. We believe that this putative glucose sensor is involved in hyphal development during macrophage infection.     

Determination of DNA sequences essential for FLP-mediated recombination by a novel method

The yeast 2-micron circle plasmid encodes a protein, FLP, that mediates site-specific recombination across the two FLP-binding sites of the plasmid. We have used a novel technique, "exonuclease-treated substrate analysis," to determine the minimal duplex DNA sequence needed for this recombination event. A linear DNA containing two FLP sites in a direct orientation was treated with the double-strand specific 3'-exonuclease, exonuclease III, to generate molecules with a nested set of single-strand deletions that extended into one of the FLP sites. The DNA was then end-labeled at the sites of the deletions and used as a substrate for recombination in vitro. FLP-mediated recombination between two FLP sites excised a restriction endonuclease cleavage site from the DNA. Comparison of the fragments produced by restriction enzyme digestion of untreated and FLP-treated DNA showed to the nucleotide the duplex DNA sequence required for FLP-mediated recombination. To examine essential sequences in the opposite DNA strand, similar experiments were done using the 5'-exonuclease encoded by phage T7. The minimal essential duplex DNA sequence lies within the region of the FLP site that was previously shown to be protected from nuclease digestion in the presence of FLP. A modified form of this technique can be used to study the minimal sequence requirements of site-specific DNA binding proteins.