Search

Garcinia mangostana,popularly known as “mangosteen fruit,” originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 μg/mL] in established test assays (Comet assay, micronucleus test, andSalmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains ofSalmonella typhimurium(with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeastSaccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application.

Pathogenesis-related proteins (PRs) are induced in plants after infection by pathogens and/or abiotic stress. Among these proteins, the family 10 (PR-10) influences the biosynthesis of secondary metabolites and shows antimicrobial ribonuclease activity. TcPR-10p (Pathogenesis-related Protein 10 of Theobroma cacao) was isolated from resistant and susceptible Moniliophthora perniciosa cacao cultivars. Cell survival with Saccharomyces cerevisiae mutant lines deficient in ATP-binding cassette (ABC) transporter proteins indicated the influence on resistance to TcPR-10p. Proteins of the ABC transport type are considered important in the process of resistance to antimicrobials and toxins. Thus, the objective of this work was to observe the sensitivity of ABC transporter yeast mutants in the presence of the TcPR-10p. Chronic exposure of S. cerevisiae mitochondrial (BYatm1&Delta, and BYmdl1&Delta, ) and vacuole (BYnft1&Delta, BYvmr1&Delta, BYybt1&Delta, BYycf1&Delta, and BYbpt1&Delta, ) ABC transporter mutants to TcPR-10p (3 &mu, g/mL, 0, 6, 12 and 24 h) was performed. Two TcPR-10p sensitive strains (BYmdl1&Delta, and BYnft1&Delta, ) were submitted to a fluorescence test with the fluorogenic dihydroethidium (DHE), to visualize the presence of oxidative stress in the cells. Oxidative stress-increased sensitivity was confirmed by flow cytometry indicating induced cell death either via apoptosis or necrosis. This yeast data combined with previous data of literature (of M. perniciosa sensitivity to TcPR-10p) show that increased sensitivity to TcPR-10p in these mutants could be due to the TcPR10p-generated higher levels of intracellular reactive oxygen species (ROS), leading to increased cell death either via necrosis or apoptosis.

Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet–C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.

Pathogenesis-related proteins (PRs) are induced in plants after infection by pathogens and/or abiotic stress. Among these proteins, the family 10 (PR-10) influences the biosynthesis of secondary metabolites and shows antimicrobial ribonuclease activity. TcPR-10p (Pathogenesis-related Protein 10 of Theobroma cacao) was isolated from resistant and susceptible Moniliophthora perniciosa cacao cultivars. Cell survival with Saccharomyces cerevisiae mutant lines deficient in ATP-binding cassette (ABC) transporter proteins indicated the influence on resistance to TcPR-10p. Proteins of the ABC transport type are considered important in the process of resistance to antimicrobials and toxins. Thus, the objective of this work was to observe the sensitivity of ABC transporter yeast mutants in the presence of the TcPR-10p. Chronic exposure of S. cerevisiae mitochondrial (BYatm1Δ and BYmdl1Δ) and vacuole (BYnft1Δ, BYvmr1Δ, BYybt1Δ, BYycf1Δ and BYbpt1Δ) ABC transporter mutants to TcPR-10p (3 μg/mL, 0, 6, 12 and 24 h) was performed. Two TcPR-10p sensitive strains (BYmdl1Δ and BYnft1Δ) were submitted to a fluorescence test with the fluorogenic dihydroethidium (DHE), to visualize the presence of oxidative stress in the cells. Oxidative stress-increased sensitivity was confirmed by flow cytometry indicating induced cell death either via apoptosis or necrosis. This yeast data combined with previous data of literature (of M. perniciosa sensitivity to TcPR-10p) show that increased sensitivity to TcPR-10p in these mutants could be due to the TcPR10p-generated higher levels of intracellular reactive oxygen species (ROS), leading to increased cell death either via necrosis or apoptosis.

Cacao farmers use macerated Theobroma cacao pod husks as biofertilizer for restoration of environmentally important soil elements, i.e., N, P, K, Na, Mg, and Ca. The increasing popularity of this organic material for soil management justifies to experimentally ascertaining that its application does not produce environmental or human health risks. We therefore applied a battery of biological tests to assess possible health risks related to the extract of fermented cacao pod husks [CHE]. Minimal inhibitory concentration for selected bacteria and fungi was established and an antiviral assay (equine herpes virus – EHV-1) and insecticide assay (Aedes aegypti larvae), to observe possible environmental impact. Toxicity tests used Artemia salina, hemolytic activity and cytotoxicity were tested using HT-29 e Vero cells, respectively. Genotoxicity and anti-genotoxic activity was tested in the comet assay of leukocytes and a selection of Saccharomyces cerevisiae mutants allowed look for a possible interference of CHE with defined metabolic pathways. None of these established tests used to define toxicity and genotoxicity of chemical compounds indicated that CHE contained substances that would pose such risks. Our safety assessment on bacteria and yeasts, virus, insect larvae, and human leukocytes [3-(4,5-di-methylazol-2- yl)-2,5- diphenyltetrazolium bromide (MTT) and comet assay] thus indicated that macerated cocoa pod husk can be safely used as biofertilizer.

Theobroma cacao L. contains more than 500 different chemical compounds some of which have been traditionally used for their antioxidant, anti-carcinogenic, immunomodulatory, vasodilatory, analgesic, and antimicrobial activities. Spontaneous aerobic fermentation of cacao husks yields a crude husk extract (CHE) with antimicrobial activity. CHE was fractioned by solvent partition with polar solvent extraction or by silica gel chromatography and a total of 12 sub-fractions were analyzed for chemical composition and bioactivity. CHE was effective against the yeast Saccharomyces cerevisiae and the basidiomycete Moniliophthora perniciosa. Antibacterial activity was determined using 6 strains: Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis (Gram-positive) and Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella choleraesuis (Gram-negative). At doses up to 10 mg/mL, CHE was not effective against the Gram-positive bacteria tested but against medically important P. aeruginosa and S. choleraesuis with a minimum inhibitory concentration (MIC) of 5.0 mg/mL. Sub-fractions varied widely in activity and strongest antibacterial activity was seen with CHE8 against S. choleraesuis (MIC of 1.0 mg/mL) and CHE9 against S. epidermidis (MIC of 2.5 mg/mL). All bioactive CHE fractions contained phenols, steroids, or terpenes, but no saponins. Fraction CHE9 contained flavonoids, phenolics, steroids, and terpenes, amino acids, and alkaloids, while CHE12 had the same compounds but lacked flavonoids.

We investigated the biodegradability of oil in mangrove sediment from Camamu Bay and measured its effect on the bacterial community. Microcosms of mangrove sediment were contaminated with 0.1, 0.5, 1, 2, and 5% (w/v) oil, and the microbial activity was compared to that in uncontaminated sediment. The evolution of CO2 and gas chromatography showed the mineralization of oil compounds, which could reach 100%. Bacterial diversity was determined by polymerase chain reaction using a set of primers for the V3 and V6-V8 regions of 16S rDNA. The band profile obtained by denaturing gradient gel electrophoresis of the amplicons that were obtained for the V3 region showed a negative correlation between band number and oil concentration, whereas that of the V6-V8 region showed a positive correlation between band numbers and oil concentration. The latter also gave similar results for microcosms that were contaminated with 2 and 5% oil. These results demonstrate the mangrove sediment's capacity to recover from oil contamination (in vitro) and suggest that native mangrove microorganisms contain enzymes necessary for the catabolism of oil.

Autophagy is defined as an intracellular system of lysosomal degradation in eukaryotic cells, and the genes involved in this process are conserved from yeast to humans. Among these genes, ATG8 encodes a ubiquitin-like protein that is conjugated to a phosphatidylethanolamine (PE) membrane by the ubiquitination system. The Atg8p-PE complex is important in initiating the formation of the autophagosome and thus plays a critical role in autophagy. In silico modeling of Atg8p of Moniliophthora perniciosa revealed its three-dimensional structure and enabled comparison with its Saccharomyces cerevisiae homologue ScAtg8p. Some common and distinct features were observed between these two proteins, including the conservation of residues required to allow the interaction of α-helix1 with the ubiquitin core. However, the electrostatic potential surfaces of these helices differ, implying particular roles in selecting specific binding partners. The proposed structure was validated by the programs PROCHECK 3.4, ANOLEA, and QMEAN, which demonstrated 100% of amino acids located in favorable regions with low total energy. Our results showed that MpAtg8p contains the same functional domains (3 α-helices and 4 β-sheets) and is similar in structure as the ScAtg8p yeast. Both proteins have many conserved sequences in common, and therefore, their proposed three-dimensional models show similar configuration.

The increasing world production of biodiesel has resulted in an accumulation of crude glycerol as the major byproduct. This could be used as carbon source for industrial microbiology, with economic and environmental advantages for the biodiesel industry. We explored an Atlantic Rainforest soil sample to search for crude glycerol-degrading microorganisms. Microcosms of this soil were established containing minimal medium + 8% crude glycerol (w/w); the biological activity was measured by respirometry. High CO2 levels were found in some of the crude glycerol microcosms, suggesting the activity of microorganisms capable of degrading this residue. In an attempt to isolate and cultivate these microorganisms in vitro, aliquots of the soil suspension were plated on minimal medium containing 10% crude glycerol (v/v). Out of 19 morphologically distinct isolates, 12 bacteria and 6 yeasts were identified by PCR from universal primers 16S and 26S rDNA, respectively. Optical density readings revealed growth differences among cultures. Two yeasts and three bacteria with distinct growth profiles stood out and appeared to have potential for liquid fermentation of crude glycerol. The yeasts adapted rapidly, but produced relatively little biomass. Opposite tendencies were found in the bacteria. Amplicon sequencing placed the bacterial isolates as close to Staphylococcus arlettae, Pseudomonas citronellolis, and Bacillus megaterium, and the yeasts to Trichosporon moniliiforme and Meyerozyma guilliermondii. We concluded that these species have potential for use in crude glycerol bioreactors and for bioremediation processes.

Biodiesel is a promising alternative energy source to diesel fuel and determining its biodegradability is an important aspect of addressing potential environmental spills. The aim of this study was to compare the biodegradability of biodiesel and its effect on soil microbial diversity with that of normal diesel using blends of biodiesel with diesel in microcosm experiments with soil from the Atlantic Rain Forest. Degradation was monitored by respirometry and GC analysis of the substrates. The highest respiration rates were observed in soil contaminated with the B50 (41.47 mg CO 2 /kg) and B100 (42.35 mg CO 2 /kg) blends. Soil contaminated with blends B5, B20, B50 and B100 showed higher numbers of culturable heterotrophic microbes than control soil. Chromatographic analyses showed that microcosms contaminated with diesel fuel and the B5 blend had less biodegradation than soil contaminated with the B20, B50 and B100 blends (80%, 62% and 84% of biodegradation, respectively). DGGE analysis showed that samples contaminated with the B5, B20, B50 blends and D100 showed changes in microbial community after the incubation period. Interestingly, soil contaminated with B100 decreased the microbial community compared with the other contaminated samples. While the degradation of pure biodiesel (B100) was efficient, microbial diversity of soil was reduced, which could compromise the overall activity of microorganisms involved in the bioremediation of contaminated soils.

Tin or stannous (Sn 2+ ) compounds are used as catalysts, stabilizers in plastic industries, wood preservatives, agricultural biocides and nuclear medicine. In order to verify the Sn 2+ up-take and toxicity in yeast cells we utilized a multi-elemental analysis known as particle-induced X-ray emission (PIXE) along with cell survival assays and quantitative real-time PCR. The detection of Sn 2+ by PIXE was possible only in yeast cells in stationary phase of growth (STAT cells) that survive at 25 mM Sn 2+ concentration. Yeast cells in exponential phase of growth (LOG cells) tolerate only micro-molar Sn 2+ concentrations that result in intracellular concentration below of the method detection limit. Our PIXE analysis showed that STAT XV185-14c yeast cells demonstrate a significant loss of intracellular elements such as Mg, Zn, S, Fe and an increase in P levels after 1 h exposure to SnCl 2 . The survival assay showed enhanced tolerance of LOG yeast cells lacking the low-affinity iron and zinc transporters to stannous treatment, suggesting the possible involvement in Sn 2+ uptake. Moreover, our qRT-PCR data showed that Sn 2+ treatment could generate reactive oxygen species as it induces activation of many stress-response genes, including SOD1 , YAP1 , and APN1 .

Plant extracts have a long history to be used in folk medicine.Cassia alataextracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract ofC. alataby reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity forPseudomonas aeruginosa,Staphylococcus epidermidis,S. aureus, andBacillus subtilis. CaRP also inhibited biofilm formation ofS. epidermidisandP. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity againstS. aureusandS. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS). Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In theSalmonella/microsome assay CaRP showed weak mutagenicity (MI<3) only in strain TA98, pointing to a frameshift mutation activity. These results indicate thatC. alataleaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications.

Currently, the effect of crude oil on ammonia-oxidizing bacterium communities from mangrove sediments is little understood. We studied the diversity of ammonia-oxidizing bacteria in mangrove microcosm experiments using mangrove sediments contaminated with 0.1, 0.5, 1, 2, and 5% crude oil as well as non-contaminated control and landfarm soil from near an oil refinery in Camamu Bay in Bahia, Brazil. The evolution of CO(2) production in all crude oil-contaminated microcosms showed potential for mineralization. Cluster analysis of denaturing gradient gel electrophoresis-derived samples generated with primers for gene amoA, which encodes the functional enzyme ammonia monooxygenase, showed differences in the sample contaminated with 5% compared to the other samples. Principal component analysis showed divergence of the non-contaminated samples from the 5% crude oil-contaminated sediment. A Venn diagram generated from the banding pattern of PCR-denaturing gradient gel electrophoresis was used to look for operational taxonomic units (OTUs) in common. Eight OTUs were found in non-contaminated sediments and in samples contaminated with 0.5, 1, or 2% crude oil. A Jaccard similarity index of 50% was found for samples contaminated with 0.1, 0.5, 1, and 2% crude oil. This is the first study that focuses on the impact of crude oil on the ammonia-oxidizing bacterium community in mangrove sediments from Camamu Bay.

Green tea is one of the most consumed beverages in the world. Presently, Camellia sinensis has become a source not only for the development of several food extracts but also nutraceutical, cosmetic and medicinal purposes. The technology developed to produce these extracts aims to improve the organoleptic characteristics of the products as taste and smell, and their shelf life. But it also searches to demonstrate some medicinal attributes like antioxidant, anti-aging, anti-tumor and anti-viral activities in relation to the chemical composition of the green tea catechins, especially (-)-epigallocatechin gallate (EGCG). The target of this review is to present the various patents related to the extraction methods and their claims, and to discuss the evidence found in the literature about the pharmacological activities of green tea. It summarizes the recent progress in technology to obtain the green tea extract and in clinical studies on its applications. Health-promoting products and disease-preventing applications of green tea extract or compounds isolated from it also take part of this text.

In order to clarify the molecular mechanisms of Sn(2+) genotoxicity, we evaluated the induction of strand breaks, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (Endo III) sensitive sites, and the interference with the repair of methyl methane sulfonate (MMS)-caused DNA damage in V79 Chinese hamster lung fibroblasts exposed to stannous chloride by comet assay. A concentration-related increase in the DNA damage induced by 2 h SnCl(2) treatment at a concentration range of 50-1,000 microM was observed (r = 0.993; P0.01). Significantly elevated DNA migration in relation to the control level was detected at doses 100, 500 and 1,000 microM in normal alkaline and at doses 500 and 1,000 microM in modified (with Fpg and Endo III) comet assay. Although 50 microM SnCl(2) concentration did not increase significantly the DNA migration by itself in comet assay, it was capable to inhibit the repair of MMS-induced DNA damage during the post-treatment period of 24 h. Our results demonstrate the genotoxic and comutagenic effects of stannous chloride in V79 cells. The inhibitory effect of Sn(2+) on repair of MMS-induced DNA damage suggests that this metal can also interfere in DNA repair systems thus contributing to increased mutation by shifting the balance from error-free to error-prone repair processes.

A pathogenesis-related (PR) protein from Theobroma cacao (TcPR-10) was identified from a cacao–Moniliophthora perniciosa interaction cDNA library. Nucleotide and amino acid sequences showed homology with other PR-10 proteins having P loop motif and Betv1 domain. Recombinant TcPR-10 showed in vitro and in vivo ribonuclease activity, and antifungal activity against the basidiomycete cacao pathogen M. perniciosa and the yeast Saccharomyces cerevisiae. Fluorescein isothiocyanate-labeled TcPR-10 was internalized by M. perniciosa hyphae and S. cerevisiae cells and inhibited growth of both fungi. Energy and temperature-dependent internalization of the TcPR-10 suggested an active importation into the fungal cells. Chronical exposure to TcPR-10 of 29 yeast mutants with single gene defects in DNA repair, general membrane transport, metal transport, and antioxidant defenses was tested. Two yeast mutants were hyperresistant compared with their respective isogenic wild type: ctr3Δ mutant, lacking the high-affinity plasma membrane copper transporter and mac1Δ, the copper-sensing transcription factor involved in regulation of high-affinity copper transport. Acute exposure of exponentially growing yeast cells revealed that TcPR-10 resistance is also enhanced in the Snq2 export permease-lacking mutant which has reduced intracellular presence of TcPR-10.

In order to extend the understanding of the genetical and biochemical basis of photo-activated psoralen-induced DNA repair in the yeast Saccharomyces cerevisiae we have identified and cloned 10 pso mutants. Here, we describe the phenotypic characterization and molecular cloning of the pso10-1 mutant which is highly sensitive to photoactivated psoralens, UV(254) (nm) radiation and the alkylating agent methylmethane sulphonate. The pso10-1 mutant allele also confers a block in the mutagenic response to photoactivated psoralens and UV(254) (nm) radiation, and homoallelic diploids do not sporulate. Molecular cloning using a yeast genomic library, sequence analysis and genetic complementation experiments proved pso10-1 to be a mutant allele of gene MMS21 that encodes a SUMO ligase involved in the sumoylation of several DNA repair proteins. The ORF of pso10-1 contains a single nucleotide C--T transition at position 758, which leads to a change in amino acid sequence from serine to phenylalanine [S253F]. Pso10-1p defines a leaky mutant phenotype of the essential MMS21 gene, and as member of the Smc5-Smc6 complex, still has some essential functions that allow survival of the mutant. DNA repair via translesion synthesis is severely impaired as the pso10-1 mutant allele confers severely blocked induced forward and reverse mutagenesis and shows epistatic interaction with a rev3Delta mutant allele. By identifying the allelism of PSO10 and MMS21 we demonstrate the need of a fully functional Smc5-Smc6 complex for a WT-like adequate repair of photoactivated psoralen-induced DNA damage in yeast.

DNA damage activates several mechanisms such as DNA repair and cell cycle checkpoints. The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3 and Ddc1 subunits is an early response factor to DNA damage and activates checkpoints. This complex is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication. Growing evidence suggests that PCNA-like complexes play a major role in DNA repair as they have been shown to interact with and stimulate several proteins, including specialized DNA polymerases. With the aim of extending our knowledge concerning the link between checkpoint activation and DNA repair, we tested the possibility of a functional interaction between the Rad17/Mec3/Ddc1 complex and the replicative DNA polymerases alpha, delta and epsilon. The analysis of sensitivity response of single and double mutants to UVC and 8-MOP + UVA-induced DNA damage suggests that the PCNA-like component Mec3p of S. cerevisiae neither relies on nor competes with the third subunit of DNA polymerase delta, Pol32p, for lesion removal. No enhanced sensitivity was observed when inactivating components of DNA polymerases alpha and epsilon in the absence of Mec3p. The hypersensitivity of pol32Delta to photoactivated 8-MOP suggests that the replicative DNA polymerase delta also participates in the repair of mono- and bi-functional DNA adducts. Repair of UVC and 8-MOP + UVA-induced DNA damage via polymerase delta thus occurs independent of the Rad17/Mec3/Ddc1 checkpoint clamp.

The PSO3 gene of Saccharomyces cerevisiae was molecularly cloned by complementing the cold-sensitivity phenotype of a pso3-1 mutant and was found to be allelic to RNR4, encoding one of the two DNA damage-inducible small subunits of the ribonucleotide reductase (RNR) complex. Compared to a rnr4Delta mutant that allows only very little mutation induction at very low doses of 254(nm) ultraviolet light (UVC), the pso3-1 mutant allele confers leakiness in that it permits some DNA damage-induced mutagenesis at low doses of UVC. Similarly, the pso3 mutant is slightly less sensitive to UVC than an rnr4Delta mutant. Cloning and sequencing of the RNR4 locus of the pso3-1 mutant revealed that its intermediate phenotype is attributable to a G --A transition at nucleotide 352, leading to replacement of glycine by arginine [G118R] in the mutant's protein. Both RNR4 mutant alleles confer significantly less sensitivity to UVC than mutant alleles of non-UVC-mutable REV3, indicating that, apart from nucleotide excision repair, RAD6-dependent error-free DNA repair may still be functional. The phenotype of a strongly reduced UVC-induced mutagenesis for rnr4 mutant alleles has not yet been described; it suggests the importance of this gene for a fully functional RNR providing correct amounts of DNA precursor molecules, thereby, allowing translesion synthesis (error-prone) of UVC-damaged DNA. Stationary phase cells of the rnr4Delta mutant, but not of the original pso3-1 mutant, are swollen with a fourfold to eightfold increase in volume. The central role of RNR in DNA precursor metabolism and its complex regulation allow for several modes of suppression that may influence the phenotypes of RNR4 mutants, especially those containing the leaky pso3-1 mutant allele.

The main goal of this work was to determine the concentration of Sn 2+ ions in cells of the yeast Saccharomyces cerevisiae and to correlate their quantity with the genotoxicity of intracellularly accumulated metal ions. The intracellular metal content of yeast cells was determined by PIXE (particle-induced X-ray emission) after cell exposure to SnCl 2 . To that end, a thick target protocol was developed for PIXE analysis. The samples were irradiated with a 2 MeV proton beam, while the induced X-rays were detected with a high-purity germanium detector. The results of the toxicity of SnCl 2 and the PIXE analysis performed with two different yeast strains (haploid and diploid) suggest that the exposure of haploid and diploid yeast to Sn 2+ induces DNA lesions and that the absorption depends on the genetic background of each strain.

Resistance to stannous chloride (SnCl(2)) of the yeast Saccharomyces cerevisiae is a product of several metabolic pathways of this unicellular eukaryote. Sensitivity testing of different null mutants of yeast to SnCl(2) revealed that DNA repair contributes to resistance, mainly via recombinational (Rad52p) and error-prone (Rev3p) steps. Independently, the membrane transporter Atr1p/Snq1p (facilitated transport) contributed significantly to Sn(2+)-resistance whereas absence of ABC export permease Snq2p did not enhance sensitivity. Sensitivity of the superoxide dismutase mutants sod1 and sod2 revealed the importance of these anti-oxidative defence enzymes against Sn(2+)-imposed DNA damage while a catalase-deficient mutant (ctt1) showed wild type (WT) resistance. Lack of transcription factor Yap1, responsible for the oxidative stress response in yeast, led to 3-fold increase in Sn(2+)-sensitivity. While loss of mitochondrial DNA did not change the Sn(2+)-resistance phenotype in any yeast strain, cells with defect cytochrome c oxidase (CcO mutants) showed gradually enhanced sensitivities to Sn(2+) and different spontaneous mutation rates. Highest sensitivity to Sn(2+) was observed when yeast was in exponential growth phase under glucose repression. During diauxic shift (release from glucose repression) Sn(2+)-resistance increased several hundred-fold and fully respiring and resting cells were sensitive only at more than 1000-fold exposure dose, i.e. they survived better at 25 mM than exponentially growing cells at 25 microM Sn(2+). This phenomenon was observed not only in WT but also in already Sn(2+)-sensitive rad52 as well as in sod1, sod2 and CcO mutant strains. The impact of metabolic steps in contribution to Sn(2+)-resistance had the following ranking: Resting WT cells > membrane transporter Snq1p > superoxide dismutases > transcription factor Yap1p >or= DNA repair >> exponentially growing WT cells.

Breaking of hyphae derived from growth of the phytopathogenic fungus Crinipellis perniciosa in liquid media yielded cell aggregates that performed as "quasi single cell" in toxicity assays. When treated with the chemical mutagens 4-nitroquinoleine-1-oxide (4NQO), hydrogen peroxide (H2O2), or paraquat (PAQ) as well as with ultraviolet light (UVC), broken hyphae of C. perniciosa gave a single cell-like response, i.e., survival curves similar to those obtained when treating single-cell suspensions. C. perniciosa had significantly higher UVC resistance than haploid bakers yeast but was more sensitive to 4NQO and extremely sensitive to PAQ and H2O2 when compared to likewise-treated yeast. Haploid C. perniciosa basidiospores (monokaryotic) were significantly more UVC resistant than C. perniciosa broken hyphae and than haploid and diploid yeast wild-type strains. This suggests a high capacity in C. perniciosa for repair of UVC-induced DNA lesions or, alternatively, an efficient protection from UVC irradiation, especially in basidiospores. The pronounced sensitivity of the dikaryotic form of C. perniciosa to PAQ and H2O2 points to a weak protection from oxidative stress-inducing agents.

Complementation analysis of the pso9-1 yeast mutant strain sensitive to photoactivated mono- and bifunctional psoralens, UV-light 254 nm, and nitrosoguanidine, with pso1 to pso8 mutants, confirmed that it contains a novel pso mutation. Molecular cloning via the reverse genetics complementation approach using a yeast genomic library suggested pso9-1 to be a mutant allele of the DNA damage checkpoint control gene MEC3. Non-complementation of several sensitivity phenotypes in pso9-1/mec3Delta diploids confirmed allelism. The pso9-1 mutant allele contains a -1 frameshift mutation (deletion of one A) at nucleotide position 802 (802delA), resulting in nine different amino acid residues from that point and a premature termination. This mutation affected the binding properties of Pso9-1p, abolishing its interactions with both Rad17p and Ddc1p. Further interaction assays employing mec3 constructions lacking the last 25 and 75 amino acid carboxyl termini were also not able to maintain stable interactions. Moreover, the pso9-1 mutant strain could no longer sense DNA damage since it continued in the cell cycle after 8-MOP + UVA treatment. Taken together, these observations allowed us to propose a model for checkpoint activation generated by photo-induced adducts.

Organoselenium compounds have a potential thiol peroxidase-like activity. Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. Using TRAP assay of chemiluminescense we have shown that diphenyl diselenide clearly possesses a pro-oxidant property. For an investigation on the mechanisms of this property, we used mutant strains of Saccharomyces cerevisiae defective in antioxidant defenses, i.e. in superoxide dismutase, in biosynthesis of glutathione, and the transcription factor yAP-1-lacking yap1 mutant that cannot activate genes of the oxidative stress response. Exposure of growing cultures to the drug increased cell sensitivity to oxidizing agents. The pro-oxidant effect was independent of the metabolic condition or of the oxidative mutagen tested. N -acetylcysteine, a precursor of glutathione biosynthesis, could neutralize the pro-oxidant effects of diphenyl diselenide by stimulating an increase of endogenous glutathione biosynthesis or by directly binding to the drug. Vitamin E (Trolox™), a known antioxidant, was also able to protect S. cerevisiae against the pro-oxidant effect of diphenyl diselenide. In vitro assays showed that diphenyl diselenide interacts non-enzymatically with the thiol group of glutathione.

Stannous chloride was found genotoxic in microbial test systems of the yeast Saccharomyces cerevisiae, in one strain of Salmonella typhimurium and in the Mutoxitest of Escherichia coli. Five isogenic haploid yeast strains differing only in a particular repair-deficiency had the following ranking in Sn2+ -sensitivity: rad52deltarad6deltarad2deltarad4deltaRAD, indicating a higher relevance of recombinogenic repair mechanisms than nucleotide excision in repair of Sn2+ -induced DNA damage. Sn2+ -treated cells formed aggregates that lead to gross overestimation of toxicity when not undone before diluting and plating. Reliable inactivation assays at exposure doses of 25-75 mM SnCl2 were achieved by de-clumping with either EDTA- or phosphate buffer. Sn2+ -induced reversion of the yeast his1-798, his1-208 and lys1-1 mutant alleles, in diploid and haploid cells, respectively, and putative frameshift mutagenesis (reversion of the hom3-10 allele) was observed. In diploid yeast, SnCl2 induced intra-genic mitotic recombination while inter-genic (reciprocal) recombination was very weak and not significant. Yeast cells of exponentially growing cultures were killed to about the same extend at 0.1% of SnCl2 than respective cells in stationary phase, suggesting a major involvement of physiological parameters of post-diauxic shift oxidative stress resistance in enhanced Sn2+ -tolerance. Superoxide dismutases, but not catalase, protected against SnCl2-induced reactive oxygen species as sod1delta had a three-fold higher sensitivity than the WT while the sod2delta mutant was only slightly more sensitive but conferred significant sensitivity increase in a sod1delta sod2delta double mutant. In the Salmonella reversion assay, SnCl2 did not induce mutations in strains TA97, TA98 or TA100, while a positive response was seen in strain TA102. SnCl2 induced a two-fold increase in mutation in the Mutoxitest strain IC203 (uvrA oxyR), but was less mutagenic in strain IC188 (uvrA). We propose that the mutagenicity of SnCl2 in yeast and bacteria occurs via error-prone repair of DNA damage that is produced by reactive oxygen species.

Two genes in Saccharomyces cerevisiae, ALR1 and ALR2, encode transmembrane proteins involved in Mg2+ uptake. The present study investigates the phylogenetic relationship of Alr1p/Alr2p with bacterial CorA proteins and some proteins related to Mg2+ influx/efflux transport in mitochondrial and bacterial zinc transporters; including hydrophobic cluster analysis (HCA). The phylogenetic results indicate that the Alrp sequences of S. cerevisiae share a common carboxy-terminus with proteins related to zinc efflux transport. We also analyse the intracellular metal content by particle-induced X-ray emission (PIXE) after cell exposure to cadmium. The PIXE analysis of cadmium-exposed ALR mutants and wild-type yeast cells suggests that Alrp has a central role in cell survival in a cadmium-rich environment.

One gene in Saccharomyces cerevisiae, ALR1, encodes a transmembrane protein involved in the transport of Mg 2+ into cells. Overexpression of this gene changes the tolerance of S. cerevisiae to several metal cations. In order to study the cellular uptake mechanism of metal cations in S. cerevisiae, an accurate determination of the metals present in the cells is needed. In this work, particle-induced x-ray emission (PIXE) was employed to analyze the intracellular metal content after cell exposure to cadmium. To that end, a specific method for sample preparation was developed. The samples were irradiated with a 2 MeV proton beam, while the induced x-rays were detected with a high-purity germanium detector. The PIXE results for cadmium-exposed ALR1 mutant and wild-type yeast cells suggest that Alr1p has a central role in the cell survival process in a cadmium-rich environment.

Cashew (Anacardium occidentale) is a medicinal plant native to Brazil and also yields a nutritious fruit juice. Its large pulpy pseudo-fruit, referred to as the cashew apple, contains high concentrations of vitamin C, carotenoids, phenolic compounds and minerals. Natural and processed cashew apple juice (CAJ/cajuina) are amongst the most popular juices in Brazil, especially in the north-east. Both juices have antioxidant potential and suppress mutagenicity of hydrogen peroxide. In the present study we evaluated the inhibitory effects of CAJ/cajuina on Aflatoxin B1(AFB1)-induced mutation, using the Salmonella/microsome assay with the experimental approaches of pre-, co- and post-treatments. Both CAJ/cajuina suppress AFB1-induced mutagenesis in strain TA102 when applied in co- and in post-treatment. Possible mechanisms for anti-mutagenicity in co-treatment are (a) interaction with S9 enzymes, (b) metabolization to non-mutagenic compounds of AFB1 or (c) inactivation of S9 potential. Total suppression of AFB1 mutagenicity was observed in co-treatment with both CAJ and cajuina. Post-treatment anti-mutagenicity of both juices suggests a modulation of activity of error-prone DNA repair. CAJ/cajuina may be considered promising candidates for control of genotoxicity of AFB1 and may thus be considered as health foods with anti-carcinogenic potential. This promising characteristic warrants further evaluation with in vivo studies.

The mutagenic effects of in natura and processed (fresh and frozen) orange juices were evaluated by the Salmonella /microsome assay (Ames Test). Antioxidant potential was determined by deoxyribose degradation, as well as by the nitro blue tetrazolium reduction test. The juices inhibited degradation of deoxyribose and were able to trap the superoxide anion. At concentrations up to 1% they acted as pro-oxidants in lipid peroxidation in a concentration-dependent manner. However, this activity was less effective at 10% juice concentration. The sweetened processed juice was not able to decrease peroxidation nor to trap superoxide anions. Significant correlation between total phenolics and the inhibition of deoxyribose degradation was observed; also, a significant correlation between lipid peroxidation and total phenolics was found. Vitamin C was a pro-oxidant in tests employing transition metals. Mutagenicity was observed in the Ames test, particularly for fresh, in natura, juice samples. The highest responses were observed in strains TA97a and TA98. Fresh processed juice, which had the best antioxidant potential, was not mutagenic in any of the strains tested. Positive results for mutagenesis in TA97a, with metabolization, were correlated with total phenolics and vitamin C.

Photoactivated psoralens used in treatment of skin diseases like Psoriasis and Vitiligo cause DNA damage, the repair of which may lead to mutations and thus to higher risk to have skin cancer. The simple eukaryote Saccharomyces cerevisiae was chosen to investigate the cells’ genetic endowment with repair mechanisms for this type of DNA damage and to study the genetic consequences of such repair. Genetic studies on yeast mutants sensitive to photoactivated psoralens, named pso mutants, showed their allocation to 10 distinct loci. Cloning and molecular characterization allowed their grouping into three functional classes: (I) the largest group comprises seven PSO genes that are either generally or specifically involved in error-prone DNA repair and thus affect induced mutability and recombination; (II) one PSO gene that represents error-free excision repair, and (III) two PSO genes encoding proteins not influencing DNA repair but physiological processes unrelated to nucleic acid metabolism. Of the seven DNA repair genes involved in induced mutagenesis three PSO loci [PSO1/REV3, PSO8/RAD6, PSO9/MEC3] were allelic to already known repair genes, whereas three, PSO2/SNM1, PSO3/RNR4, and PSO4/PRP19 represent new genes involved in DNA repair and nucleic acid metabolism in S. cerevisiae. Gene PSO2 encodes a protein indispensable for repair of interstrand cross-link (ICL) that are produced in DNA by a variety of bi- and polyfunctional mutagens and that appears to be important for a likewise repair function in humans as well. In silico analysis predicts a putative endonucleolytic activity for Pso2p/Snm1p in removing hairpins generated as repair intermediates. The absence of induced mutation in pso3/rnr4 mutants indicates an important role of this subunit of ribonucleotide reductase (RNR) in regulation of translesion polymerase ζ in error-prone repair. Prp19p/Pso4p influences efficiency of DNA repair via splicing of pre-mRNAs of intron-containing repair genes but also may function in the stability of the nuclear scaffold that might influence DNA repair capacity. The seventh gene, PSO10 which controls an unknown step in induced mutagenesis is not yet cloned. Two genes, PSO6/ERG3 and PSO7/COX11, are responsible for structural elements of the membrane and for a functional respiratory chain (RC), respectively, and their function thus indirectly influences sensitivity to photoactivated psoralens.

Increased expression of the cell proliferation-associated polo-like kinase 1 (PLK1) and apoptosis-associated BCL-2 genes has been observed in different human malignancies. Inhibition of cell proliferation and reactivation of apoptosis are basic principles in anticancer therapy. The efficiency of this approach is often limited by insuf-ficient targeting and delivery of anticancer drugs into the tumors. Phosphorothioate antisense oligodeoxynucleotides (ODNs) directed against PLK1 and BCL-2 were administered systemically via the tail vein into nude mice bearing A549, MDA-MB-435, and Detroit562 xenografts. To enhance tumor-specific uptake and to reduce systemic toxicity of antisense ODNs membrane electroporation transfer was applied in vivo. Northern and Western blot analyses were used to assess PLK1 and BCL-2 expression. Tumor mass was assessed after resection of tumors. All three cell lines and corresponding xenografts expressed high levels of PLK1 and were sensitive towards antisense PLK1 treatment. Antisense BCL-2 therapy was effective in tumors expressing high levels of BCL-2, but not in A549 cells and corresponding xenografts, which express low levels of BCL-2. Administration of antisense ODNs in a dose of 5 mg/kg, twice weekly during four weeks supported by the membrane electroporation transfer, eradicated 60-100% of the xenografted tumors. Antitumor effect in BCL-2 overexpressing MDA-MB-435 cells was synergistic for BCL-2 and PLK1 combination therapy. This study provides evidence that combined systemic administration of antisense ODNs against proliferation and pro- survival associated targets and in vivo electroporation of tumors represents a promising antitumor therapeutic approach.

Moniliophthora perniciosa is a basidiomycete fungus that causes witches' broom disease in Theobroma cacao We analyzed the morphology and survival of fungal hyphae and endoplasmic reticulum (ER) remodeling in either glucose- or glycerol-grown M. perniciosa after treatment with ER stress-inducing chemicals dithiothreitol (DTT) or tunicamycin (TM). Changes in intracellular redox potential can cause endoplasmic reticulum (ER) stress due to diminished efficiency in protein folding that could in turn reduce cell survival. Such stress diminishes protein-folding efficiency that could in turn reduce cell survival. Light microscopy revealed morphological changes in hyphae after TM but not after DTT treatment, regardless of the media carbon source. Decrease in fungal survival, after both TM and DTT treatments, was dose-dependent and glycerol-grown cells showed a higher resistance to both chemicals compared to glucose-grown cells. Electron microscopy showed TM and DDT-induced ER stress in M. perniciosa as evidenced by structural alterations of the organelle. The volume of ER structures increased as a typical consequence of unfolded protein stress, and the number of autophagosomes was higher. In glycerol-grown fungus DTT treatment slightly induced expression of molecular chaperone BiP. The TM exposure-induced expression of gene MpIRE1, involved in signaling of the unfolded protein response, was higher in glycerol than glucose-grown cells. Such difference was not observable with expression of gene MpATG8, encoding a key protein in autosome formation, that was induced 1.4-fold and 1.2-fold in glucose or glycerol-grown cells, respectively. DHE-based fluorescence assay showed M. perniciosa oxidative stress induced by H2O2, and treated cells had a higher level of oxidative stress compared to control. A comprehensive study of remodeling of ER is important in understanding M. perniciosa fungus resistance to oxidative stress and its ability to implement a successful infection in T. cacao.

The Polo-like kinase 1 (Plk1) is a highly conserved mitotic serine/threonine kinase which is commonly overexpressed in cancer cell lines. Plk1 positively regulates mitotic progression by activating the CDC25C-CDK1 amplification loop and by regulating late mitotic events, primarily the ubiquitin-dependent proteolysis. In the present study, an antisense strategy against Plk1 mRNA was developed to specifically inhibit cell proliferation of cancer cells in cell culture and in the nude-mouse tumor model. Among 41 phosphorothioate antisense oligodeoxynucleotides tested, the 20-mer JWG2000 strongly inhibited expression of Plk1 in cultured A549 cells, leading to loss of cell viability, and exhibited anti-tumor activity in nude mice A549 xenograft. JWG2000 did not inhibit growth and viability of primary human mesangial cells and human amnion fibroblasts.

Sequencing of the yeast gene that complemented the sensitivity to the photoactivated monofunctional 3-carbethoxypsoralen of the pso6-1 mutant strain revealed that the ERG3 locus, encoding sterol C-5 desaturase involved in biosynthesis of ergosterol, is allelic to PSO6. Disruption of the ERG3 gene yielded an erg3Delta mutant viable in ergosterol-containing YEPD media with the same pleiotropic mutant phenotype known for pso6-1 and erg3 mutants, including sensitivity to hydrogen peroxide and paraquat. Thus, the erg3/pso6 yeast mutant seems to be more sensitive than the WT to 3-CPs+UVA because of the oxidative damage contributed by this treatment and not because of an impaired repair of the furocoumarin-thymine monoadducts formed in the DNA. We found a significant increase of petites amongst erg3Delta and pso6-1 yeast mutant strains grown in conditions where respiration was mandatory. Mutant pso6-1, with its lowered content of ergosterol, exhibited enhanced synthesis of chitin that was maldistributed and not confined to the bud scars. Chitin overproduction in pso6/erg3 mutants resulted in hypersensitivity to Calcofluor White.

The original pso3-1 mutant isolate of the yeast Saccharomyces cerevisiae exhibits a pleiotropic mutagen-sensitivity phenotype that includes sensitivity to UVA-activated 3-carbethoxypsoralen, to UVC-light, to mono- and bi-functional nitrogen mustard, to paraquat, and to cadmium; on the other hand, it shows hyper-resistance (HYR) to nitrosoguanidine when compared to established wild-type strains. Also, the original pso3-1 mutant exhibits a low UVC-induced mutability and mitotic gene conversion and a high rate of spontaneous and UVC-induced petite mutations. Since the HYR to the nitrosoguanidine (MNNG) phenotype resembles that of low glutathione-containing yeast cells, the original pso3-1 mutant was crossed to a gsh1 knock-out mutant that lacks the enzyme for the first step in glutathione biosynthesis and the resulting diploid was tested for complementation. While there was none for HYR to nitrosoguanidine, and other low glutathione-related phenotypes, some other phenotypic characteristics of pso3-1, e.g. UVC sensitivity and UVC-induced mutability were restored to a wild-type level. Tetrad analysis of a diploid derived from a cross of the original haploid pso3-1 isolate with a repair-proficient, normal glutathione-containing, PSO3 GSH1 wild-type led to the separation of a leaky gsh1 mutation phenotype from that of the repair-deficient pso3-1 phenotype. Linkage studies by tetrad and random spore analyses indicated no linkage of the two genes. This shows that the low glutathione content in the original pso3-1 isolate is due to a second, additional, mutation in the GSH1 locus and is unrelated to the pso3-1 mutation. Thus, the original pso3-1 isolate is a pso3-1 gsh1 double mutant with most of the particular characteristics of the pleiotropic sensitivity phenotype contributed by either the pso3-1 or the gsh1-leaky mutant allele. The expression of a few phenotypic characteristics of pso3, however, were most pronounced in pso3-1 mutants with a low glutathione pool.

Yeast multi-copy vectors carrying the formaldehyde-resistance markergene SFA have proved to be a valuable tool for research on industriallyused strains of Saccharomyces cerevisiae . The genetics of these strainsis often poorly understood, and for various reasons it is not possible tosimply subject these strains to protocols of genetic engineering thathave been established for laboratory strains of S. cerevisiae . We testedour vectors and protocols using 10 randomly picked baker’s and wineyeasts all of which could be transformed by a simple protocol withvectors conferring hyperresistance to formaldehyde. The applicationof formaldehyde as a selecting agent also offers the advantage of itsbiodegradation to CO 2 during fermentation, i.e., the selecting agentwill be consumed and therefore its removal during down-streamprocessing is not necessary. Thus, this vector provides an expressionsystem which is simple to apply and inexpensive to use. Correspondence M. BrendelInstitut fur MikrobiologieJ.W. Goethe-UniversitatTheodor-Stern-Kai 7, Haus 75D-60590 Frankfurt am MainGermanyResearch supported by a donationfrom Qualitatsweine D. Brendel.A. Comer was the recipient ofan Abschlusstipendium furauslandische Studenten.Received April 2, 1997Accepted September 16, 1997

Recent progress in elucidating the molecular structure of the PSSO genes PS02 to PS07 is presented. Their role in DNA repair and mutagenesis is discussed in the light of the putative proteins encoded in the respective ORFs and with the knowledge of recent progress in biological and biochemical experimentation. The role of the RecA protein in some steps of DNA repair in Saccharomyces cerevisiae is presented and discussed.

The radiation-sensitive mutant pso4-1 of Saccharomyces cerevisiae shows a pleiotropic phenotype, including sensitivity to DNA cross-linking agents, nearly blocked sporulation and reduced mutability. We have cloned the putative yeast DNA repair gene PSO4 from a genomic library by complementation of the blocked UV-induced mutagenesis and of sporulation in diploids homozygous for pso4-1. Sequence analysis revealed that gene PSO4 consists of 1512 bp located upstream of UBI4 on chromosome XII and encodes a putative protein of 56.7 kDa. PSO4 is allelic to PRP19, a gene encoding a spliceosome-associated protein, but shares no significant homology with other yeast genes. Gene disruption with a destroyed reading frame of our PSO4 clone resulted in death of haploid cells, confirming the finding that PSO4/PRP19 is an essential gene. Thus, PSO4 is the third essential DNA repair gene found in the yeast S.cerevisiae.

Two distinct N-deoxyribosyltransferases of Lactobacillus leichmannii, designated as DRTase I and DRTase II, were separated and purified almost to homogeneity by one-step affinity chromatography. DRTase I is distinguished by specifically catalyzing the direct transfer of 2-deoxyribosyl residues from purine deoxyribonucleosides to free purine bases, whereas DRTase II has a rather broad substrate specificity and is able to transfer the deoxyribosyl moiety between pyrimidines and between purines and pyrimidines. Furthermore, in addition to the different substrate spectrum, we clearly differentiated the two enzymes by comparing their varying temperature/activity and pH/activity profiles, their kinetic constants, their behaviour in Western blot analysis, and their N-terminal amino acid sequences. Denaturing and non-denaturing DISK-PAGE revealed strong evidence that both intact enzymes consist of hexamers with subunit molecular weights of approximately 20,000 for DRTase I and 18,000 for DRTase II.

The sequence of a 5653 bp DNA fragment of the right arm of chromosome II of Saccharomyces cerevisiae contains two unknown open reading frames (YBR1212 and YBR1213) next to gene CDC28. Gene disruption reveals both putative genes as non-essential. ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Sur1 protein of S. cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with a Lactococcus lactis-secreted 45 kDa protein and 24% identity with the Saccharomyces cerevisiae AGA1 gene product. The total sequence of the fragment has been submitted to the EMBL databank (accession number X80224).

By isolating putative binding partners through the two-hybrid system (THS) we further extended the characterization of the specific interstrand cross-link (ICL) repair gene PSO2 of Saccharomyces cerevisiae. Nine fusion protein products were isolated for Pso2p using THS, among them the Sak1 kinase, which interacted with the C-terminal β-CASP domain of Pso2p. Comparison of mutagen-sensitivity phenotypes of pso2Δ, sak1Δ and pso2Δsak1Δ disruptants revealed that SAK1 is necessary for complete WT-like repair. The epistatic interaction of both mutant alleles suggests that Sak1p and Pso2p act in the same pathway of controlling sensitivity to DNA-damaging agents. We also observed that Pso2p is phosphorylated by Sak1 kinase in vitro and co-immunoprecipitates with Sak1p after 8-MOP+UVA treatment. Survival data after treatment of pso2Δ, yku70Δ and yku70Δpso2Δ with nitrogen mustard, PSO2 and SAK1 with YKU70 or DNL4 single-, double- and triple mutants with 8-MOP+UVA indicated that ICL repair is independent of YKu70p and DNL4p in S. cerevisiae. Furthermore, a non-epistatic interaction was observed between MRE11, PSO2 and SAK1 genes after ICL induction, indicating that their encoded proteins act on the same substrate, but in distinct repair pathways. In contrast, an epistatic interaction was observed for PSO2 and RAD52, PSO2 and RAD50, PSO2 and XRS2 genes in 8-MOP+UVA treated exponentially growing cells.