Functional analysis of Candida albicans genes whose Saccharomyces cerevisiae homologues are involved in endocytosis

PCR-based techniques for directed gene alterations have become standard tools in Candida albicans. To help to increase the speed of functional analysis of Candida albicans genes, we previously constructed and updated a modular set of pFA-plasmid vectors for PCR-based gene targeting in C. albicans. Here we report the functional analyses of C. albicans ORFs whose homologues in S. cerevisiae are involved in endocytosis, to explore their potential involvement in polarized cell growth. Three C. albicans genes, ABP1, BZZ1 and EDE1, were found to be non-essential. Yeast and hyphal morphogenesis were not affected by the individual deletions and the mutant strains appeared wild-type-like under the different growth conditions tested. On the other hand, deletion of both alleles of the C. albicans PAN1 homologue was not feasible. Promoter shut-down experiments using a MET3p-PAN1/pan1 strain indicated severe growth defects and abolished endocytosis, indicating that PAN1 is an essential gene. Subcellular distribution of CaAbp1 and CaPan1 was analysed via GFP-tagged proteins. Both proteins were found to localize at the cortex and at hyphal tips in a patch-like manner, supporting their role in endocytosis. Localization patterns of Abp1 and Pan1, however, were distinct from that of the FM4-64 stained Spitzenk?rper.     

Constitutive promoter modules for PCR-based gene modification in Saccharomyces cerevisiae

Initial steps in investigating gene function often include deleting and overexpressing the gene of interest and identifying the subcellular location of the gene product. To facilitate these procedures, a number of new PCR modules, which contain selectable markers and in some cases other genetic elements (e.g promoter elements, epitope tags, and reporter genes) have been developed. These modules are used as PCR substrates to create products that can be targeted to specified locations in the yeast genome, thus modifying that genomic locus. We describe here a series of plasmids that contain a truncated version of the strong ADH1 promoter with and without amino-terminal 3HA and GST tags. Because these plasmids contain the same vector sequences as the GAL1 promoter plasmids, a constitutive and an inducible promoter can now be integrated with a minimal number of primers.     

Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4⁺ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe. © 1998 John Wiley & Sons, Ltd.     

GFP as a quantitative reporter of gene regulation in Candida albicans

A system has been developed for the quantitative analysis of gene expression within individual Candida albicans cells in infected tissue. The system is based on the plasmid pGFP, which contains the codon-optimized yeast enhanced green fluorescent protein (yEGFP; Cormack et al., 1997) cloned between a basal CaADH1 promoter and the ScCYC1 terminator on an integrating vector. Promoters were inserted into pGFP and GFP levels measured in individual cells by quantitative fluorescence microscopy. Analysis of pPCK1-GFP and pMET3-GFP fusions revealed that GFP folds rapidly following gene induction, and is turned over rapidly following gene repression. Hence, single cell fluorescence measurements are likely to reflect ongoing gene expression levels with reasonable accuracy. pACT1-GFP expression levels were relatively constant during growth of C. albicans in both yeast and hyphal forms, and during growth in vivo in the mouse model of systemic infection. Therefore, pACT1-GFP provides a useful control for this quantitative GFP-based system in future analyses of C. albicans molecular responses during fungal infections.     

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5⁺ or Escherichia coli kan^r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. © 1998 John Wiley & Sons, Ltd.     

Cassettes for the PCR-mediated construction of regulatable alleles in Candida albicans

The recent availability of genome sequence information for the opportunistic pathogen Candida albicans has greatly facilitated the ability to perform genetic manipulations in this organism. Two important molecular tools for studying gene function are regulatable promoters for generating conditional mutants and fluorescent proteins for determining the subcellular localization of fusion gene products. We describe a set of plasmids containing promoter-GFP cassettes (P(MET3)-GFP, P(GAL1)-GFP, and P(PCK1)-GFP), linked to a selectable nutritional marker gene (URA3). PCR-mediated gene modification generates gene-specific promoter, or gene-specific promoter-GFP, fusions at the 5'-end of the gene of interest. One set of primers can be used to generate three strains expressing a native protein of interest, or an amino-terminal GFP-tagged version, from three different regulatable promoters. Thus, these promoter cassette plasmids facilitate construction of conditional mutant strains, overexpression alleles and/or inducible amino-terminal GFP fusion proteins.     

Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans.

We have developed a set of plasmids containing fluorescent protein cassettes for use in PCR-mediated gene tagging in Candida albicans. We engineered YFP and CFP variants of the GFP sequence optimized for C. albicans codon usage. The fluorescent protein sequences, linked to C. albicans auxotrophic marker sequences, were amplified by PCR and transformed directly into yeast. Gene-specific sequence was incorporated into the PCR primers, such that the tag-cassette integrates by homologous recombination at the 3'-end of the gene of interest. This technique was used to tag Cdc3 and Tub1 with GFP, YFP and CFP, which were readily visualized by fluorescence microscopy and localized as expected. In addition, Tub1-YFP and Cdc3-CFP were visualized in the same cells. Thus, this technique directs one-step construction of multiple fluorescent protein fusions, facilitating the study of protein co-expression and co-localization in C. albicans cells in vivo.     

New tools for phenotypic analysis in Candida albicans: the WAR1 gene confers resistance to sorbate

Availability of the complete sequence of the Candida albicans genome allows for global gene analysis. We designed a gene deletion method to facilitate such studies. First, we constructed C. albicans strains that are both Deltaura3 and Deltatrp1. Second, we designed a system that relies on in vitro recombination, using the Gateway((R)) technology, for efficient generation of deletion cassettes. They are generated in two steps: (a) upstream and downstream DNA fragments of the chromosomal region to be deleted are amplified by PCR and introduced into two separate entry vectors; (b) the second step involves a quadruple recombination event including the two entry vectors, a plasmid bearing a marker of interest and a destination vector, in order to generate a plasmid containing the deletion cassette. The deletion plasmid contains very rare restriction sites for convenient excision of the knockout cassette. Selection in C. albicans can be performed with one of the following markers: the C. albicans URA3 gene, a modified S. cerevisiae TRP1 gene or the mycophenolic acid resistance (MPA(R)) gene. Upon integration into the genome, these markers can be removed by the use of 5-fluoroorotic acid (URA3), 5-fluoroanthranilic acid (TRP1) or the FLP recombinase (MPA(R)). Using this approach, we show that removal of the C. albicans orf19.1035 gene results in sensitivity to the weak acid sorbate, while its overexpression increases resistance to this compound. We named it WAR1, in analogy to its S. cerevisiae orthologue.     

Induced expression of the Candida albicans multidrug resistance gene CDR1 in response to fluconazole and other antifungals

The Candida albicans CDR1 gene encodes a member of the ABC-type family of multidrug transporters which has been shown to be involved in azole resistance. Using an in-frame gene fusion between the CDR1 open reading frame and the green fluorescent protein allele yEGFP3, an optimized derivative for its use in C. albicans, we show here how the CDR1-yEGFP3 gene expression is induced in response to azoles as well as to other structurally unrelated drugs like cycloheximide. Moderate increases were observed for calcofluor, canavanine, 5′-fluorcytosine, cilofungin and caffeine, while no induction was found for the antifungals benomyl and amphotericin B or hydrogen peroxide at subinhibitory concentrations. The use of confocal microscopy enabled us to localize the Cdr1p fusion protein at the cell periphery, thus suggesting a cytoplasmic membrane localization. These results suggest deregulation of CDR1 gene as a putative mechanism for the generation of azole resistance in this clinically important pathogenic fungus. © 1998 John Wiley & Sons, Ltd.     

Candida albicans homologue of GGP1/GAS1 gene is functional in Saccharomyces cerevisiae and contains the determinants for glycosylphosphatidylinositol attachment

The GGP1/GAS1/CWH52 gene of Saccharomyces cerevisiae encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gp115 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. PHR1 gene isolated from Candida alibicans is homologous to the GGP1 gene. In this report we have analysed the ability of PHR1 to complement a ggp1Δ mutation in S. cerevisiae. The expression of PHR1 controlled by its natural promoter or by the GGP1 promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that PHR1 in budding yeast gives rise to a 75–80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. albicans gene product is functional in S. cerevisiae.     

Isolation and characterization of Candida albicans homologue of RAP1, a repressor and activator protein gene in Saccharomyces cerevisiae

To study the function of RAP1, a Candida albicans gene (CaRAP1) that shows sequence similarity to RAP1 of Saccharomyces cerevisiae was isolated by colony hybridization. DNA sequencing predicted an open reading frame of 429 amino acids with an overall identity of 24% to the ScRap1p. The DNA binding domain (DBD) was highly conserved, and EMSA using a GST-CaRap1p fusion protein confirmed its binding ability to the RPG-box of S. cerevisiae ENO1. In contrast, the N-terminus was less conserved and a moderate homology was observed in the BRCT domain. Interestingly, CaRap1p did not contain the C-terminal activation/repression region of ScRap1p.     

Use of specific PCR-based molecular markers for discrimination, rapid analysis of purity and identification of six fragrant rice varieties

Fragrant rice is high in quality and hence price. Therefore, fragrant rice is often mixed with non-fragrant or low grade rice. The quality of fragrant rice depends on many factors such as variety, region in which it is cultivated, handling practices, etc. In this study, two specific primer pairs, including external sense primer, external antisense primer, internal non-fragrant sense primer and internal fragrant antisense primer were used to differentiate fragrant and non-fragrant rice based on the detection of 8-bp deletion in betaine aldehyde dehydrogenase 2 gene. The purity of the fragrant rice can be detected at 1% and 2% level of adulteration with homogeneous and heterogeneous non-fragrant rice varieties, respectively, using only one primer pair (internal non-fragrant sense primer and external antisense primer). Furthermore, six particular fragrant rice varieties were identified using specific RM 1 microsatellite markers. Further analysis should be conducted to verify the discriminating power of this method of analysis for the traceability of fragrant rice varieties.     

Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans

A role for the cAMP-dependent pathway in regulation of the cell wall in the model yeast Saccharomyces cerevisiae has recently been demonstrated. In this study we report the results of a phenotypic analysis of a Candida albicans mutant, characterized by a constitutive activation of the cAMP pathway due to deletion of PDE2, the gene encoding the high cAMP-affinity phosphodiesterase. Unlike wild-type strains, this mutant has an increased sensitivity to cell wall and membrane perturbing agents such as SDS and CFW, and antifungals such as amphotericin B and flucytosine. Moreover, the mutant is characterized by an altered sensitivity and a significantly reduced tolerance to fluconazole. The mutant's membrane has around 30% higher ergosterol content and the cell wall glucan was 22% lower than in the wild-type. These cell wall and membrane changes are manifested by a considerable reduction in the thickness of the cell wall, which in the mutant is on average 60-65 nm, compared to 80-85 nm in the wild-type strains as revealed by electron microscopy. These results suggest that constitutive activation of the cAMP pathway affects cell wall and membrane structure, and biosynthesis, not only in the model yeast S. cerevisiae but also in the human fungal pathogen C. albicans.     

A rapid method for the discrimination of genes encoding classical Shiga toxin (Stx) 1 and its variants, Stx1c and Stx1d, in Escherichia coli

Subtyping of Shiga toxin (Stx)-encoding genes by conventional polymerase chain reaction (PCR) is time-consuming. We developed a single step real-time fluorescence PCR with melting curve analysis to distinguish rapidly stx1 from its variants, stx1c and stx1d. Melting temperatures (Tm) of 206 Stx-producing Escherichia coli (STEC) identified to harbor stx1 or stx1c were analyzed using a specific hybridization probe over the variable region. 170 of 171 stx1-harboring STEC displayed Tm of 69 degrees C to 70 degrees C, whereas 34 of 35 strains containing stx1c had Tm of 65 degrees C-66 degrees C. This constant and reproducible difference of 4 degrees C demonstrated that melting curve analysis is a reliable technique to differentiate stx1 from stx1c. Two isolates displayed atypical Tm. Sequence analysis showed that one of them was 100% identical to stx1d within a 511 bp DNA stretch. Our data demonstrate that real-time PCR is a rapid and reliable tool to differentiate stx1 from stx1c and stx1d and to detect new stx1 variants. Because stx1-harboring STEC cause diarrhoea and hemolytic-uremic syndrome, whereas those containing stx1c are often shed asymptomatically, a rapid differentiation between stx1 and its variants using the procedure developed here has both clinical implications and a direct significance for the risk assessment analysis of STEC isolated from foods.     

Determination of unprocessed genetically modified soybean in foods using simplex and duplex real-time PCR with an internal standard

We developed a method using an internal standard to determine the amount of unprocessed genetically modified organisms (GMO) in foods as GMO weight per weight of total food (w/w_tf) using real-time PCR. Food samples were spiked with an internal standard, a ColE1 plasmid, and DNA was extracted from the samples using a silica membrane-type kit and analysed using real-time PCR. The relationship between the GMO amount and the copy number ratio of the transgene to ColE1 in 0.1–5% w/w_tf GM soybean powders was found to have a correlation coefficient ( r ) of 0.97. GMO was quantified in food samples spiked with GM soybean (final amount 0.5% w/w_tf GMO). The average GMO amount ranged from 0.35% to 0.63% w/w_tf. The results show that our method should be useful for quantifying unprocessed GMO in foods. We also developed a duplex assay, which is simpler and more accurate than the simplex assay.     

Technical note: A new and cost-effective method for detection of the bovine acyl-CoA:diacylglycerol acyltransferase 1 K232A polymorphism in cattle

A new, quick, and inexpensive method for detecting the bovine acyl-CoA:diacylglycerol acyltransferase1 (DGAT1) polymorphism (K232A) through tetra-primer amplification refractory mutation system by PCR (ARMS-PCR) was developed in the present investigation. The DGAT1 gene was recently identified as underlying variation in milk production traits. To date, PCR techniques such as PCR-RFLP have been used for detecting the DGAT1 K232A polymorphism, despite being expensive and laborious. The method proposed here, a tetra-primer ARMS-PCR, showed 100% sensitivity and specificity when compared with PCR-RFLP results obtained in a sample of 80 animals tested in a double-blind system. Our results suggest that the use of tetra-primer ARMS-PCR for DGAT1 K232A polymorphism genotyping could greatly reduce costs providing information for both research purposes and for dairy cattle breeders who perform DGAT1 genotyping for gene-assisted selection.