DNA polymerase III is required for DNA repair in Saccharomyces cerevisiae

We have studied the role of DNA polymerase III, encoded in S. cerevisiae by the CDC2 gene, in the repair of yeast nuclear DNA. It was found that the repair of MMS-induced single-strand breaks is defective in the DNA polymerase III temperature-sensitive mutant cdc2-1 at the restrictive temperature (37 °C), but is not affected at the permissive temperature (23 °C). Under conditions where only a small number of lesions was introduced into DNA (80% survival), the repair of MMS-induced damage could also be observed in the mutant at the restrictive temperature, although with low efficiency. When the quantity of lesions increased (50% survival or less), the repair of single-strand breaks was blocked. At the same time we observed a high rate of reversion in the meth, his and trp loci of the cdc2-1 mutant under restrictive conditions. The results presented suggest that DNA polymerase III is involved in the repair of MMS-induced lesions in yeast DNA and that the cdc2-1 mutation affects the proofreading activity of this polymerase.     

The isolation of osmotic-remedial conditional lethal mutants of Candida albicans

Summary We have developed a method for the isolation of a broad range of conditional lethal mutants of the pathogenic fungus Candida albicans. The method substantially alleviates the problems posed by the diploid nature, and the biased mutant spectrum, exhibited by most strains of this organism. We have used the method to isolate 560 temperature-sensitive mutants which grew at 30°C but not at 42°C. We identified amongst them a number of osmotic-remedial strains which, at the restrictive temperature, show phenotypes indicating defects in cell wall biosynthesis.     

Complementation of Saccharomyces cerevisiae mutations in genes involved in translation and protein folding (EFB1 and SSB1) with Candida albicans cloned genes

We have demonstrated that the expression of Candida albicans genes involved in translation and protein folding (EFB1 and SSB1) complements the phenotype of Saccharomyces cerevisiae mutants. The elongation factor 1beta (EF-1beta) is essential for growth and efb1 S. cerevisiae null mutant cells are not viable; however, viable haploid cells, carrying the disrupted chromosomal allele of the S. cerevisiae EFB1 gene and pEFB1, were isolated upon sporulation of a diploid strain which was heterozygous at the EFB1 locus and transformed with pEFB1 (a pEMBLYe23 derivative plasmid containing an 8-kb DNA fragment from the C. albicans genome which contains the EFB1 gene). This indicates that the C. albicans EFB1 gene encodes a functional EF-1beta. Expression of the SSB1 gene from C. albicans, which codes for a member of the 70-kDa heat shock protein family, in S. cerevisiae ssb1 ssb2 double mutant complements the mutant phenotype (poor growth particularly at low temperature, and sensitivity to certain protein synthesis inhibitors, such as paromomycin). This complementation indicates that C. albicans Ssbl may function as a molecular chaperone on the translating ribosomes, as described in S. cerevisiae. Northern blot analysis showed that SSB mRNA levels increased after mild cold shift (28 degrees C to 23 degrees C) and rapidly decreased after mild heat shift (from 28 degrees C to 37 degrees C, and particularly to 42 degrees C), indicating that SSB1 expression is regulated by temperature. Therefore, Ssb1 may be considered as a molecular chaperone whose pattern of expression is similar to that found in ribosomal proteins, according to its common role in translation.     

Molecular cloning of Rab-related genes in the yeast Yarrowia lipolytica. Analysis of RYL1, an essential gene encoding a SEC4 homologue

Small GTP-binding proteins of the Rab family are involved in the vesicular traffic inside eukaryotic cells. A gene library from the yeast Yarrowia lipolytica was screened with an oligonucleotide deduced from a highly conserved sequence in the Rab family. Four different genes were isolated. One of them, RYL1, was shown to be essential for cell viability. RYL1p displayed a high similarity with and tight phylogenetic relationships to SEC4p. When placed under the control of the GAL10 promoter, RYL1 was able to specifically relieve the thermosensitivity of a sec4–8 mutant of Saccharomyces cerevisiae. Therefore, it is proposed that RYL1 is a functional homologue of the S. cerevisiae SEC4 gene and is involved in the fusion of secretory vesicles with the plasma membrane in the general protein secretion pathway.     

Segregant-defective heterokaryons of Candida albicans

Summary Heterokaryons (hets) of the asexual, pathogenic yeast Candida albicans obtained by fusing protoplasts of complementing auxotrophic strains generate large numbers of parental-type auxotrophic monokaryons by random assortment of single nuclei into blastospores, and smaller numbers of monokaryons bearing hybrid nuclei formed through either karyogamy or the transfer of genetic material from one het nucleus to another. Het populations grown at 30 °C or 37 °C contain high frequencies (approx. 5%–10%) of two kinds of stable variants peculiar specifically for segregation of parental-type monokaryons: NS variants produce inviable auxotrophic monokaryons of one or both parental classes while AT variants yield parental-type monokaryons which grow very slowly. Variant frequencies are not affected by the wild-type strain background of hets, or the auxotrophies used to force heterokaryosis. However, both kinds of variants are induced by growth at 25 °C or by treatments with certain chemical or physical metabolic inhibitors. Evidence is presented that variant nuclei of independent origins carry different nutritionally irreparable recessive lethal (NS) or debilitating (AT) defects acquired in the course of actual or potential internuclear transfers of genetic material within het cells. The high incidence of variants, therefore, indicates considerable intrinsic genetic instability among het nuclei. Significances of these observations for parasexual genetic analyses of C. albicans and other yeasts through protoplast fusions are considered.     

The mitochondrial genome of the aquatic phycomycete Allomyces macrogynus. Physical mapping and mitochondrial DNA instability

Summary A physical map of the mitochondrial genome of the aquatic phycomycete Allomyces macrogynus strain Burma 3–35 (35°C) has previously been published (Borkhardt and Delius 1983). This map has been extended in this study by locating 37 additional recognition sites for five new restriction enzymes in the mitochondrial genome. Homologous regions for the genes coding for cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, ATPase subunits 6 and 9, the small and large ribosomal RNA, URF1, URF5, and perhaps urfa, a presumptive gene hitherto found only in the mitochondrial genome of the fission yeast Schizosaccharomyces pombe, were located in the mitochondrial genome of A. macrogynus by heterologous hybridizations with specific, mitochondria) gene probes from Saccharomyces cerevisiae, Aspergillus nidulans, Neurospora crassa, and S. pombe. The mitochondrial gene order in A. macrogynus was found to be identical to that of A. arbuscula; a gene order hitherto found only among members of the family Blastocladiaceae. Spontaneous insertion mutations have been found to occur quite frequently in the mitochondrial genome of A. macrogynus. In all mutated mitochondrial genomes so far studied, insertions have been located in a specific region located between the genes coding for the ATPase subunit 9 and the large ribosomal RNA. In two of the mutated mitochondrial genomes the insertional event(s) resulted in the presence of mitochondrial DNA molecules differing in size by multiples of approximately 70 base pairs.     

The <Emphasis Type="Italic">Candida albicans</Emphasis> Kar2 protein is essential and functions during the translocation of proteins into the endoplasmic reticulum

Since the secretory pathway is essential for Candida albicans to transition from a commensal organism to a pathogen, an understanding of how this pathway functions may be beneficial for identifying novel drug targets to prevent candidiasis. We have cloned the C. albicans KAR2 gene, which performs many roles during the translocation of proteins into the endoplasmic reticulum (ER) during the first committed step of the secretory pathway in many eukaryotes. Our results show that C. albicans KAR2 is essential, and that the encoded protein rescues a temperature-sensitive growth defect found in a Saccharomyces cerevisiae strain harboring a mutant form of the Kar2 protein. Additionally, S. cerevisiae containing CaKAR2 as the sole copy of this essential gene are viable, and ER microsomes prepared from this strain exhibit wild-type levels of post-translational translocation during in vitro translocation assays. Finally, ER microsomes isolated from a C. albicans strain expressing reduced amounts of KAR2 mRNA are defective for in vitro translocation of a secreted substrate protein, establishing a new method to study ER translocation in this organism. Together, these results suggest that C. albicans Kar2p functions during the translocation of proteins into the ER during the first step of the secretory pathway.     

Choice of an adequate promoter for efficient complementation in Saccharomyces cerevisiae: a case study

Conservation of the function of open reading frames recently identified in fungal genome projects can be assessed by complementation of deletion mutants of putative Saccharomyces cerevisiae orthologs. A parallel complementation assay expressing the homologous wild type S. cerevisiae gene is generally performed as a positive control. However, we and others have found that failure of complementation can occur in this case. We investigated the specific cases of S. cerevisiae TBF1 and TIM54 essential genes. Heterologous complementation with Candida glabrata TBF1 or TIM54 gene was successful using the constitutive promoters TDH3 and TEF. In contrast, homologous complementation with S. cerevisiae TBF1 or TIM54 genes failed using these promoters, and was successful only using the natural promoters of these genes. The reduced growth rate of S. cerevisiae complemented with C. glabrata TBF1 or TIM54 suggested a diminished functionality of the heterologous proteins compared to the homologous proteins. The requirement of the homologous gene for the natural promoter was alleviated for TBF1 when complementation was assayed in the absence of sporulation and germination, and for TIM54 when two regions of the protein presumably responsible for a unique translocation pathway of the TIM54 protein into the mitochondrial membrane were deleted. Our results demonstrate that the use of different promoters may prove necessary to obtain successful complementation, with use of the natural promoter being the best approach for homologous complementation.     

IgE, IgA, and IgG responses to common yeasts in atopic patients

This study was undertaken to analyze the differences in exposure and sensitization to five common environmental yeasts. The responses of IgG, IgA. and IgE to Candida albicans. C. utilis, Cryptococcus albidus, Rhodotorula rubra, and Saccharomyces cerevisiae and purified S. cerevisiae enolase were analyzed by immunoblotting (IgE-1B), and the cross-reactivity of their IgE-binding components by IgE-1B inhibition. Twenty atopic subjects, with asthma, allergic rhinitis, or atopic dermatitis were included. In skin prick tests (SPT), 12 of the patients showed simultaneous reactivity to at least two of the five yeasts, four reacted to one of the yeasts, and four had no responses. Antigens run in SDS-PAGE and transferred to nitrocellulose were probed with enzyme-labeled IgA-, IgG-, and IgE-specific antibodies. The IgE immunoblotting revealed most IgE-binding bands in C. albicans (11 bands) followed by C. utilis (eight bands), S. cerevisiae (five bands), R. rubra (five bands), and Cr. albidus (four bands). Six of the IgE-binding bands of C. albicans and C. utilis shared molecular weight, and only two bands shared molecular weight with other yeasts. These were the 46-kDa band, shared by all five yeasts, and a 13-kDa band shared by four yeasts. Prominent IgE binding was seen to a 46-kDa band of C. albicans (seven patients), C. utilis (five patients), and S. cerevisiae (one patient) and to corresponding weak bands of Cr. albidus and R. rubra (one patient). The possible cross-reactivity of the 46-kDa band was analyzed by IgE-IB inhibition and densitometry, revealing clear C albicans inhibition of C. utilis (80%) and enolase (98%) (autoinhibition 100%). The strongest IgG responses were seen against S. cerevisiae and C albicans. The responses were mainly against mannans of C. albicans and S. cerevisiae, suggesting that most of the exposure is to these yeasts. Yeasts with different types of exposure, from saprophytic growth on human mucous membranes to exposure by air and food, were shown to cross-react at the allergenic level. Atopic patients primarily sensitized by C albicans and S. cerevisiae may develop allergic symptoms by exposure to other environmental yeasts due to cross-reacting IgE antibodies.     

Neonatal candidiasis: analysis of epidemiology, drug susceptibility, and molecular typing of causative isolates

A prospective observational study of invasive candidiasis was conducted in the neonatal intensive care unit of Aristotle University in Hippokration Hospital between 1994 and 2000. During this period, 59 neonates developed invasive candidiasis (58 cases of candidemia and 1 case of peritonitis), resulting in an overall incidence of 1.28% that showed a decreasing trend over the study period. Eleven (18.6%) cases developed within the first week of life and the others within a mean (±SEM) of 13.4±1.7 days after birth. The three most frequent causative species were Candida albicans (65.5%), Candida parapsilosis (15.5%), and Candida tropicalis (7%). C. albicans was the predominant species between 1994 and 1998, whereas, non-albicans Candida spp., particularly C. parapsilosis, were the most frequent species during the period 1999–2000 (P<0.001). While the overall mortality due to candidemia was 29% (17 of 59 cases), mortality associated with C. albicans and C. parapsilosis was 39.5% and 11.1%, respectively (P=0.032), and that observed in the 1999–2000 period was 0% (P=0.011). Virtually all isolates were susceptible to amphotericin B, flucytosine, fluconazole, and itraconazole, and no increases in minimal inhibitory concentrations were observed during these years. With the exception of a limited cluster of cases due to genotypically identical isolates, no clonal relation of C. albicans isolates was found. Moreover, no clonal persistence of C. albicans and no decrease in antifungal drug susceptibility occurred over the 6-year study period. Non-albicans Candida spp., mostly C. parapsilosis, have emerged as important pathogens in neonatal intensive care units, with infected patients having better outcomes as compared to patients infected with C. albicans.     

Transport properties of a C. albicans amino-acid permease whose putative gene was cloned and expressed in S. cerevisiae

Using a gene bank of C. albicans, the lysine-permease deficiency in a strain of S. cerevisiae was complemented, and the restriction map of the corresponding C. albicans DNA fragment was constructed. Its expression in S. cerevisiae showed that the permease of C. albicans actively transports arginine (K_T=18 mol/l, J_max=26 nmol/min per mg dry weight), lysine (K_T=12 mol/l, J_max=18 nmol/min per mg dry weight), histidine (K_T=37 mol/l, J_max=9.7 nmol/min per mg dry weight), as well as their toxic analogues canavanine and thialysine, with high affinity. The intracellular concentration of basic amino acids transported into S. cerevisiae by the C. albicans permease reaches more than a thousand-times-higher value compared to the external concentration in the medium. Accumulated amino acids do not leave the cells. The uptake is strongly reduced by the protonophores and inhibitors of plasma membrane H⁺-ATPase.     

Temperature-dependent internuclear transfer of genetic material in heterokaryons of Candida albicans

Summary Heterokaryons (hets) of Candida albicans are produced by fusing protoplasts of complementing auxotrophic strains and can be propagated continuously on minimal medium despite their tendency to assort nuclei into monokaryotic blastospores. Most mono-karyons have parental-type nuclei, but some are nuclear hybrids with DNA contents between one and two times that of their parental strains. Evidence is presented that hybrids arise by transfer of a portion of the genetic material of one bet nucleus to another, and that the amount of material conveyed during transfer increases with increasing het growth temperatures over the range 25°C to 41°C. This partial hybridization is a general property of hets and is not determined by the wild-type strain backgrounds of their parental components or by the kinds of auxotrophies forcing heterokaryosis. Frequencies of mitotic recombinants induced in partial hybrids by ultraviolet radiation indicate that nuclei of C. albicans are naturally diploid.     

In vivo import of Candida tropicalis hydratase-dehydrogenase-epimerase into peroxisomes of Candida albicans

Summary We present a system for studying peroxisomal protein targeting in Candida. We have expressed the Candida tropicalis gene encoding hydratase-dehydrogenase-epimerase (HDE) in Candida albicans. Immunoblot analyses of C. albicans transformants demonstrate the presence of oleic-acid inducible HDE (100 kDa) in peroxisomes of transformed cells, but not of control cells. Peroxisomes isolated from transformed cells show increased -hydroxyacyl-CoA dehydrogenase specific activity, indicating that HDE is imported into peroxisomes of C. albicans where it is enzymatically active. C. albicans provides a useful model for the study of protein targeting to peroxisomes in vivo.     

Identification of four novel PMM2 mutations in congenital disorders of glycosylation (CDG) Ia French patients

We screened 11 unrelated French patients with congenital disorders of glycosylation (CDG) Ia for PMM2 mutations. Twenty one missense mutations on the 22 chromosomes (95%) including four novel mutations were identified: C9Y (G26A) in exon 1, L32R (TA95GC) in exon 2, and T226S (C677G) and C241S (G722C) in exon 8. We studied the PMM activity of these four novel mutant proteins and of the R141H mutant protein in an E coli expression system. The T226S, C9Y, L32R, and C241S mutant proteins have decreased specific activity (23 to 41% of normal), are all more or less thermolabile, and R141H has no detectable activity. Our results indicate that the new mutations identified here are less severe than the inactive R141H mutant protein, conferring residual PMM activity compatible with life.


Keywords: CDG; phosphomannomutase; PMM2 mutations     

Cold-sensitive mutation in Neurospora crassa affecting the production of 17S ribosomal RNA from ribosomal precursor RNA

Summary The cold-sensitive mutant strain of Neurospora crassa, crib-1 (PJ30201) has a conditional defect in ribosome biosynthesis. At 10 °C this mutant underproduces the small (37S) ribosomal subunit. Experiments were done to study rRNA synthesis in crib-1 after a shift from the permissive (25 °C) to the nonpermissive temperature and vice versa. The results showed that the primary cold-sensitive defect in crib-1 is in the function, rather than the synthesis, of a molecule needed for the production of the 17S rRNA component of the 37S subunit. Pulse-labeling experiments showed that at 10 °C crib-1 synthesizes the 2.4-Mdal pre-rRNA molecule which contains the sequences for the 17S, 5.8S and 25S rRNA species, but that processing of this molecule is defective such that relatively few stable 17S rRNA molecules accumulate.     

Effect of progesterone on Candida albicans vaginal pathogenicity

Candida albicans is responsible for the majority of cases of vulvovaginal candidiasis (VVC), an infection which occurs mainly during the luteal phase of the menstrual cycle or during the pregnancy, when levels of progesterone are elevated. One of the most important candidal virulence factors is the ability to adhere to host surfaces and form biofilms.The aim of this study was to determine the influence of progesterone on C. albicans virulence, namely biofilm formation and colonisation/invasion of a reconstituted human vaginal epithelium (RHVE). Biofilm formation on the RHVE was evaluated by enumeration of culturable cells, total mass quantification and scanning electron microscopy. The capacity of C. albicans strains to invade and colonise the tissue was examined by fluorescence microscopy using species-specific peptide nucleic acid (PNA) probe hybridisation, and quantitatively evaluated by RT-PCR Candida quantification methodology. Furthermore, gene (BCR1 and HWP1) expression of biofilm and RHVE-colonising cells was evaluated by quantitative RT-PCR. Results confirmed that progesterone reduced the capacity of C. albicans strains to form biofilms and to colonise and invade RHVE. Additionally, it was demonstrated that progesterone decreased expression of BCR1 and HWP1, which are important virulence determinants of C. albicans. In conclusion, it was evident that progesterone can have a major influence on C. albicans pathogenicity on vaginal epithelial cells and may partly explain susceptibility of women to VVC at different stages of the menstrual cycle.