Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 30
Filter
Add more filters










Publication year range
1.
Arch Toxicol ; 92(5): 1785-1795, 2018 05.
Article in English | MEDLINE | ID: mdl-29523930

ABSTRACT

Occupational and environmental asbestos exposure is the main determinant of malignant pleural mesothelioma (MPM), however, the mechanisms by which its fibres contribute to cell toxicity and transformation are not completely clear. Aberrant DNA methylation is a common event in cancer but epigenetic modifications involved specifically in MPM carcinogenesis need to be better clarified. To investigate asbestos-induced DNA methylation and gene expression changes, we treated Met5A mesothelial cells with different concentrations of crocidolite and chrysotile asbestos (0.5 ÷ 5.0 µg/cm2, 72 h incubation). Overall, we observed 243 and 302 differentially methylated CpGs (≥ 10%) between the asbestos dose at 5 µg/cm2 and untreated control, in chrysotile and crocidolite treatment, respectively. To examine the dose-response effect, Spearman's correlation test was performed and significant CpGs located in genes involved in migration/cell adhesion processes were identified in both treatments. Moreover, we found that both crocidolite and chrysotile exposure induced a significant up-regulation of CA9 and SRGN (log2 fold change > 1.5), previously reported as associated with a more aggressive MPM phenotype. However, we found no correlation between methylation and gene expression changes, except for a moderate significant inverse correlation at the promoter region of DKK1 (Spearman rho = - 1, P value = 0.02) after chrysotile exposure. These results describe for the first time the relationship between DNA methylation modifications and asbestos exposure. Our findings provide a basis to further explore and validate asbestos-induced DNA methylation changes, that could influence MPM carcinogenesis and possibly identifying new chemopreventive target.


Subject(s)
Asbestos/toxicity , DNA Methylation/drug effects , Transcriptome/drug effects , Antigens, Neoplasm/genetics , Asbestos/chemistry , Asbestos, Crocidolite/administration & dosage , Asbestos, Crocidolite/toxicity , Asbestos, Serpentine/administration & dosage , Asbestos, Serpentine/toxicity , Carbonic Anhydrase IX/genetics , Cell Line , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Humans , Lung Neoplasms/chemically induced , Lung Neoplasms/genetics , Mesothelioma/chemically induced , Mesothelioma/genetics , Mesothelioma, Malignant , Real-Time Polymerase Chain Reaction
2.
Cancer Lett ; 378(2): 120-30, 2016 08 10.
Article in English | MEDLINE | ID: mdl-27181379

ABSTRACT

BAP1 germline mutations predispose to a cancer predisposition syndrome that includes mesothelioma, cutaneous melanoma, uveal melanoma and other cancers. This co-occurrence suggests that these tumors share a common carcinogenic pathway. To evaluate this hypothesis, we studied 40 Italian families with mesothelioma and/or melanoma. The probands were sequenced for BAP1 and for the most common melanoma predisposition genes (i.e. CDKN2A, CDK4, TERT, MITF and POT1) to investigate if these genes may also confer susceptibility to mesothelioma. In two out of six families with both mesothelioma and melanoma we identified either a germline nonsense mutation (c.1153C > T, p.Arg385*) in BAP1 or a recurrent pathogenic germline mutation (c.301G > T, p.Gly101Trp) in CDKN2A. Our study suggests that CDKN2A, in addition to BAP1, could be involved in the melanoma and mesothelioma susceptibility, leading to the rare familial cancer syndromes. It also suggests that these tumors share key steps that drive carcinogenesis and that other genes may be involved in inherited predisposition to malignant mesothelioma and melanoma.


Subject(s)
Biomarkers, Tumor/genetics , Codon, Nonsense , Cyclin-Dependent Kinase Inhibitor p18/genetics , Germ-Line Mutation , Melanoma/genetics , Mesothelioma/genetics , Skin Neoplasms/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , Adolescent , Adult , Aged , Biomarkers, Tumor/analysis , Cyclin-Dependent Kinase Inhibitor p16 , Cyclin-Dependent Kinase Inhibitor p18/analysis , DNA Mutational Analysis , Databases, Factual , Female , Genetic Association Studies , Genetic Predisposition to Disease , Heredity , Humans , Immunohistochemistry , Italy , Male , Melanoma/chemistry , Melanoma/pathology , Mesothelioma/chemistry , Mesothelioma/pathology , Middle Aged , Pedigree , Phenotype , Risk Factors , Skin Neoplasms/chemistry , Skin Neoplasms/pathology , Tumor Suppressor Proteins/analysis , Ubiquitin Thiolesterase/analysis , Young Adult
3.
Eur J Clin Microbiol Infect Dis ; 31(3): 371-8, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21744038

ABSTRACT

We report the results of a three-year surveillance program of Klebsiella spp. in six hospitals in Florence (Italy). A total of 172 Klebsiella isolates were identified and typed by AFLP: 122 were K. pneumoniae and 50 were K. oxytoca. Most K. pneumoniae (80%) and K. oxytoca (93%) showed unrelated AFLP profiles. Beside this heterogeneous population structure, we found five small epidemic clonal groups of K. pneumoniae. Four of these groups were involved in outbreak events, three of which occurred in neonatal ICUs. The fifth clonal group spread in three different wards of two hospitals. Only one non-epidemic clonal group of K. oxytoca was detected. The frequencies of isolates with multiple antibiotic resistances increased with time; at the end of the study period, most K. pneumoniae were resistant to all the antibiotics tested. A PCR analysis of seven ertapenem resistant isolates was unable to detect any of the major genes known to underlie carbapenem resistance in K. pneumoniae.


Subject(s)
Drug Resistance, Multiple, Bacterial , Klebsiella Infections/epidemiology , Klebsiella Infections/microbiology , Klebsiella/drug effects , Klebsiella/isolation & purification , Amplified Fragment Length Polymorphism Analysis , Carbapenems/pharmacology , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Disease Outbreaks , Drug Resistance, Multiple, Bacterial/genetics , Humans , Italy/epidemiology , Klebsiella/genetics , Molecular Typing
4.
Folia Microbiol (Praha) ; 55(6): 593-7, 2010 Nov.
Article in English | MEDLINE | ID: mdl-21253904

ABSTRACT

Saccharomyces cerevisiae as model system was used to evaluate the occurrence of resistant mutants and adaptation mechanism to mancozeb (MZ), a widespread fungicide of the dithiocarbamate class with a broad spectrum of action and multiple cell targets. We were unable to isolate mutants resistant to inhibitory concentration of MZ but found an unusually large number of mitochondrial defective petite mutants among cells incubated in the presence of subinhibitory MZ concentration. Similar results were obtained with two other dithiocarbamate fungicides. Comparison of wild type and petite mutants showed that the latter were more resistant to toxic effects of MZ, highlighting the role of mitochondria in MZ-tolerance. The data suggest that petite cells, arising by exposure to sub-inhibitory MZ concentration, are not induced by fungicides but are spontaneous mutants already present in the population before the contact with the fungicide.


Subject(s)
Fungicides, Industrial/pharmacology , Maneb/pharmacology , Saccharomyces cerevisiae/drug effects , Thiocarbamates/pharmacology , Zineb/pharmacology , Drug Resistance, Fungal , Drug Tolerance , Mitochondria/metabolism , Mutation , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism
5.
Bone Marrow Transplant ; 43(4): 335-8, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18850015

ABSTRACT

From May to October 2006, six severe Pseudomonas aeruginosa infections were diagnosed in patients undergoing SCT in the SCT unit of the Careggi hospital (Florence, Italy). Four of the infected patients were treated consecutively in the same room (room N). On the hypothesis of a possible environmental source of infection, samples were collected from different sites that had potential for cross-contamination throughout the SCT unit, including the electrolytic chloroxidant disinfectant used for hand washing (Irgasan) and the disinfectant used for facilities cleaning. Four of the environmental samples were positive for P. aeruginosa: three Irgansan soap samples and a tap swab sample from the staff cleaning and dressing room. The AFLP (amplified fragment length polymorphism) typing method employed to evaluate strain clonality showed that the isolates from the patients who had shared the same room and an isolate from Irgasan soap had a significant molecular similarity (dice index higher than 0.93). After adequate control measures, no subsequent environmental sample proved positive for P. aeruginosa. These data strongly support the hypothesis of the clonal origin of the infective strains and suggest an environmental source of infection. The AFLP method was fast enough to allow a 'real-time' monitoring of the outbreak, permitting additional preventive measures.


Subject(s)
Disease Outbreaks , Pseudomonas Infections/epidemiology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/genetics , Stem Cell Transplantation , Adult , Amplified Fragment Length Polymorphism Analysis/methods , Female , Humans , Italy/epidemiology , Male , Microbial Sensitivity Tests , Middle Aged , Molecular Epidemiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/isolation & purification , Serotyping
6.
Folia Microbiol (Praha) ; 52(1): 35-8, 2007.
Article in English | MEDLINE | ID: mdl-17571793

ABSTRACT

The effect of colony density on the dimorphic switch was determined in natural strains of Saccharomyces cerevisiae. In some strains invasiveness and pseudohyphal (PH) growth were highly sensitive to colony density; moreover, strains constitutively able to invade the substrate with PH formation positively influenced the invasiveness but not the PH growth of a different strain less prone to the dimorphic switch.


Subject(s)
Gene Expression Regulation, Fungal , Hyphae/growth & development , Saccharomyces cerevisiae/growth & development , Colony Count, Microbial , Culture Media , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
7.
Folia Microbiol (Praha) ; 49(5): 534-8, 2004.
Article in English | MEDLINE | ID: mdl-15702541

ABSTRACT

Two new plasmids, pEC3 and pECkan, were constructed and their use in yeast transformation described. Both plasmids are derivative of the pRS416 vector, in which the URA3 auxotrophic marker was replaced by the LEU4* gene (pEC3) or the kanMX4 gene (pECkan). pEC3 and pECkan plasmids transformed natural and commercial Saccharomyces cerevisiae strains to 5,5,5-trifluoro-DL-leucine and G418 (aminoglycoside related to gentamicin) resistance, respectively, with efficiency ranging from 10(-5) to 10(-7) transformants per number of viable cells. pEC3 transformed the Leu- laboratory strain, carrying the mutations leu4 leu9, to leucine prototrophy with efficiency of approximately 10(-4).


Subject(s)
Leucine/analogs & derivatives , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Base Sequence , DNA, Fungal/genetics , Drug Resistance, Fungal/genetics , Genes, Fungal , Genetic Vectors , Gentamicins/pharmacology , Leucine/pharmacology , Mutagenesis, Site-Directed , Plasmids/genetics , Species Specificity , Transformation, Genetic
8.
Biochem J ; 351 Pt 2: 341-6, 2000 Oct 15.
Article in English | MEDLINE | ID: mdl-11023819

ABSTRACT

Glutathione S-transferases (GSTs) normally use hydroxy-group-containing residues in the N-terminal domain of the enzyme for stabilizing the activated form of the co-substrate, glutathione. However, previous mutagenesis studies have shown that this is not true for Beta class GSTs and thus the origin of the stabilization remains a mystery. The recently determined crystal structure of Proteus mirabilis GST B1-1 (PmGST B1-1) suggested that the stabilizing role might be fulfilled in Beta class GSTs by one or more residues in the C-terminal domain of the enzyme. To test this hypothesis we mutated His(106) and Lys(107) of PmGST B1-1 to investigate their possible role in the enzyme's catalytic activity. His(106) was mutated to Ala, Asn and Phe, and Lys(107) to Ala and Arg. The effects of the replacement on the activity, thermal stability and antibiotic-binding capacity of the enzyme were examined. The results are consistent with the involvement of His(106) and Lys(107) in interacting with glutathione at the active site but these residues do not contribute significantly to catalysis, folding or antibiotic binding.


Subject(s)
Glutathione Transferase/chemistry , Alanine/chemistry , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Arginine/chemistry , Asparagine/chemistry , Binding Sites , Catalysis , Conserved Sequence , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Guanidine/pharmacology , Histidine/chemistry , Lysine/chemistry , Molecular Sequence Data , Mutagenesis, Site-Directed , Phenylalanine/chemistry , Protein Binding , Protein Conformation , Protein Folding , Protein Structure, Secondary , Proteus mirabilis/enzymology , Rifamycins/pharmacology , Spectrometry, Fluorescence , Temperature , Time Factors
9.
Yeast ; 16(6): 539-45, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10790691

ABSTRACT

The function of the open reading frame (ORF) YOR108w of Saccharomyces cerevisiae has been analysed. The deletion of this ORF from chromosome XV did not give an identifiable phenotype. A mutant in which both ORF YOR108w and LEU4 gene have been deleted proved to be leucine auxotrophic and alpha-isopropylmalate synthase (alpha-IPMS)-negative. This mutant recovered alpha-IPMS activity and a Leu(+) phenotype when transformed with a plasmid copy of YOR108w. These data and the sequence homology indicated that YOR108w is the structural gene for alpha-IPMS II, responsible for the residual alpha-IPMS activity found in a leu4Delta strain. The leu4Delta strain appeared to be very sensitive to the leucine analogue trifluoroleucine. In the absence of leucine, its growth was not much impaired in glucose but more on non-fermentable carbon sources.


Subject(s)
2-Isopropylmalate Synthase/genetics , Genes, Fungal/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , 2-Isopropylmalate Synthase/metabolism , Cell Division/drug effects , Cell Division/genetics , Gene Deletion , Genes, Fungal/physiology , Isoenzymes/genetics , Isoenzymes/metabolism , Leucine/analogs & derivatives , Leucine/pharmacology , Multigene Family , Mutation , Open Reading Frames , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/metabolism
10.
FEMS Microbiol Lett ; 180(2): 229-33, 1999 Nov 15.
Article in English | MEDLINE | ID: mdl-10556716

ABSTRACT

The resistance to 5,5,5-trifluoro-DL-leucine, encoded by the dominant allele LEU4-1, was used as a selectable marker to transform laboratory and natural Saccharomyces cerevisiae strains by the lithium acetate procedure. Results of transformation of S. cerevisiae laboratory and wine natural strains showed that trifluoroleucine resistance is a very effective selection marker and can be widely used to transform prototrophic S. cerevisiae strains. The LEU4-1 gene could also be exploited to improve wine flavour, as indicated by the higher isoamyl alcohol content of the transformants compared to the parental strains.


Subject(s)
Leucine/analogs & derivatives , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/drug effects , Transformation, Genetic , 2-Isopropylmalate Synthase/genetics , Drug Resistance, Microbial/genetics , Fermentation , Fungal Proteins/genetics , Genes, Dominant , Genetic Markers , Leucine/metabolism , Leucine/pharmacology , Pentanols/metabolism , Plasmids/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Wine/microbiology
11.
Yeast ; 15(15): 1691-701, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10572265

ABSTRACT

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN. PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids.


Subject(s)
Open Reading Frames/genetics , Saccharomyces cerevisiae/genetics , DNA Primers/chemistry , DNA, Fungal/chemistry , Gene Deletion , Mutagenesis, Insertional , Polymerase Chain Reaction , Saccharomyces cerevisiae/growth & development , Sequence Analysis, DNA , Transformation, Genetic
12.
FEBS Lett ; 445(2-3): 347-50, 1999 Feb 26.
Article in English | MEDLINE | ID: mdl-10094487

ABSTRACT

The role of the evolutionarily conserved residue Pro-53 in Proteus mirabilis glutathione transferase B1-1 has been examined by replacing it with a serine residue using site-directed mutagenesis. The effect of the replacement on the activity, thermal stability and antibiotic binding capacity of the enzyme was examined. The results presented support the view that Pro-53 participates in the maintenance of the proper conformation of the enzyme fold rather than playing a direct role in the catalytic reaction. Furthermore, this residue appears to be an important determinant of the antibiotic binding to the enzyme. Experiments with wild type and mutated enzymes provide evidence that glutathione transferases may play an important role in antibiotic resistance exhibited by bacteria.


Subject(s)
Glutathione Transferase/physiology , Proline/physiology , Proteus mirabilis/enzymology , Amino Acid Sequence , Conserved Sequence , Evolution, Molecular , Glutathione/metabolism , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Mutagenesis, Site-Directed , Proline/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/physiology , Spectrophotometry/methods
13.
Mol Gen Genet ; 261(1): 152-60, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10071221

ABSTRACT

Seven spontaneous Saccharomyces cerevisiae mutants that express dominant resistance to 5,5,5-trifluoro-DL-leucine have been characterised at the molecular level. The gene responsible for the resistance was cloned from one of the mutants (FSC2.4). Determination of its nucleotide sequence showed that it was an allele of LEU4 (LEU4-1), the gene that encodes alpha-isopropyl malate synthase I (alpha-IPM synthase I), and that the mutation involved a codon deletion localised close to the 3' end of the LEU4 ORF. Six different point mutations--four transitions and two transversions--were found in the remaining mutants. Alpha-IPM synthase activity was found to be insensitive to feedback inhibition by leucine in five of the strains. In the other two the enzyme was resistant to Zn2+-mediated inactivation by Coenzyme A, a previously postulated control mechanism in energy metabolism; as far as we know, this represents the first direct in vivo evidence for this mechanism. The seven mutations define a region, the R-region, involved in both leucine feedback inhibition and in Zn2+-mediated inactivation by CoA. Deletion experiments involving the R-region showed that it is also necessary for enzyme activity.


Subject(s)
2-Isopropylmalate Synthase/metabolism , Leucine/analogs & derivatives , Saccharomyces cerevisiae/enzymology , Alleles , Amino Acid Sequence , Blotting, Northern , Cloning, Molecular , Coenzyme A/metabolism , DNA Mutational Analysis , Drug Resistance, Microbial/genetics , Gene Deletion , Genes, Fungal/genetics , Leucine/metabolism , Leucine/pharmacology , Molecular Sequence Data , Mutation , Plasmids/genetics , RNA, Fungal/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Analysis , Sequence Homology, Amino Acid , Transformation, Genetic , Zinc/pharmacology
14.
FEBS Lett ; 423(2): 122-4, 1998 Feb 20.
Article in English | MEDLINE | ID: mdl-9512342

ABSTRACT

In order to investigate the roles of near N-terminus Tyr, Cys, and Ser residues in the activity of bacterial glutathione transferase (GSTB1-1) site-directed mutagenesis was used to replace the following residues: Tyr-4, Tyr-5, Ser-9, Cys-10, Ser-11, and Ser-13. The results presented here show that, unlike all other alpha, mu, pi, theta and sigma classes of glutathione transferases so far investigated, GSTB1-1 does not utilise any Tyr, Ser or Cys residue to activate glutathione. These results also suggest that the bacterial glutathione transferases may require classification into their own class.


Subject(s)
Bacterial Proteins/genetics , Glutathione Transferase/genetics , Proteus mirabilis/genetics , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Proteus mirabilis/enzymology
15.
Gene ; 197(1-2): 9-17, 1997 Sep 15.
Article in English | MEDLINE | ID: mdl-9332345

ABSTRACT

The HIS6 gene from Saccharomyces cerevisiae strain YNN282 is able to complement both the S. cerevisiae his6 and the Escherichia coli hisA mutations. The cloning and the nucleotide sequence indicated that this gene encodes a putative phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxiamide isomerase (5' Pro-FAR isomerase, EC 5.3.1.16) of 261 amino acids, with a molecular weight of 29,554. The HIS6 gene product shares a significant degree of sequence similarity with the prokaryotic HisA proteins and HisF proteins, and with the C-terminal domain of the S. cerevisiae HIS7 protein (homologous to HisF), indicating that the yeast HIS6 and HIS7 genes are paralogous. Moreover, the HIS6 gene is organized into two homologous modules half the size of the entire gene, typical of all the known prokaryotic hisA and hisF genes. The structure of the yeast HIS6 gene supports the two-step evolutionary model suggested by Fani et al. (J. Mol. Evol. 1994; 38: 489-495) to explain the present-day hisA and hisF genes. According to this idea, the hisF gene originated from the duplication of an ancestral hisA gene which, in turn, was the result of an earlier gene elongation event involving an ancestral module half the size of the extant gene. Results reported in this paper also suggest that these two successive paralogous gene duplications took probably place in the early steps of molecular evolution of the histidine pathway, well before the diversification of the three domains, and that this pathway was one of the metabolic activities of the last common ancestor. The molecular evolution of the yeast HIS6 and HIS7 genes is also discussed.


Subject(s)
Aldose-Ketose Isomerases/genetics , Evolution, Molecular , Genes, Fungal/genetics , Histidine/biosynthesis , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Aminohydrolases/genetics , Base Sequence , Cloning, Molecular , Molecular Sequence Data , Multienzyme Complexes/genetics , Phylogeny , Restriction Mapping , Saccharomyces cerevisiae/enzymology , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transferases/genetics
16.
Res Microbiol ; 148(7): 613-23, 1997.
Article in English | MEDLINE | ID: mdl-9765846

ABSTRACT

Eighteen mutants resistant to 5',5',5'-trifluoroleucine (TFL), a leucine analog, were isolated in Saccharomyces cerevisiae strains YNN281 and YNN282. The mutants were characterized genetically and clustered in two groups, one comprising all the dominant (TFL1) and the other one all the recessive (tfl2) mutations. Genetic and biochemical data suggested that the dominant mutations are located on the LEU4 gene, coding for alpha-isopropylmalate synthase I. These mutations resulted in accumulation of leucine as a consequence of the synthesis of an enzyme insensitive to the feedback inhibition by leucine. Leucine excretion in the TFL1 mutants appeared to be affected by the genetic background of the strain and was greatly influenced by lysine metabolism. The measurement of intra- and extracellular amino acid concentrations in prototrophic strains carrying TFL1 or tfl2 genes showed that both were leucine overproducers. Some of the TFL-resistant mutants were tested in alcoholic fermentation of grape must: analysis of the fermentation secondary metabolites showed that the major effect of the TFL-resistant strains was an increased production of isoamyl alcohol compared to that of the parental strain.


Subject(s)
Genes, Fungal/genetics , Glycogen Synthase/metabolism , Leucine/analogs & derivatives , Pentanols/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Chromosome Mapping , Drug Resistance, Microbial/genetics , Feedback , Fermentation , Genotype , Leucine/metabolism , Lysine/metabolism , Phenotype , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/isolation & purification
17.
Biochem J ; 318 ( Pt 1): 157-62, 1996 Aug 15.
Article in English | MEDLINE | ID: mdl-8761466

ABSTRACT

The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421-425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli sigma 70 consensus promoter and to promoters of P. mirabilis cat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-beta-D-thiogalactopyranoside and growth at 37 degrees C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.


Subject(s)
Cloning, Molecular , Genes, Bacterial , Glutathione Transferase/genetics , Proteus mirabilis/enzymology , Amino Acid Sequence , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Base Sequence , Cell Division/drug effects , DNA Primers , Dinitrochlorobenzene/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Gene Expression , Glutathione Transferase/chemistry , Glutathione Transferase/metabolism , Kinetics , Molecular Sequence Data , Proteus mirabilis/drug effects , Recombinant Proteins/biosynthesis , Substrate Specificity
18.
Res Microbiol ; 146(7): 587-94, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8578000

ABSTRACT

Random amplified polymorphic DNA (RAPD) analysis was applied to genomic DNA from nineteen yeast strains belonging to the genera Saccharomyces and Zygosaccharomyces. Results obtained with five primers indicated that this technique is a powerful tool for yeast differentiation and identification. The data were consistent with those derived from restriction fragment length polymorphism (RFLP) using two S. cerevisiae DNA probes. We conclude that RAPD fingerprinting, combined with the analysis of RFLP, can provide unambiguous type assignment in yeasts.


Subject(s)
DNA, Bacterial/chemistry , Polymorphism, Restriction Fragment Length , Random Amplified Polymorphic DNA Technique , Saccharomyces/classification , DNA, Bacterial/genetics , Electrophoresis, Agar Gel , In Vitro Techniques , Polymorphism, Genetic , Saccharomyces/genetics , Saccharomyces/isolation & purification
19.
Yeast ; 10(8): 1101-10, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7992510

ABSTRACT

In this paper we describe the cloning and sequencing of the gene (SUL1) responsible for sulphite resistance in a Saccharomyces cerevisiae mutant (Casalone et al., 1992). The deduced amino acid sequence predicted that the gene codes for a zinc-finger protein with five fingers. Comparison of wild-type and mutant gene sequences demonstrated that the mutation event was a transversion from C to G; as a consequence of the mutation a histidine substituted an aspartic acid, affecting directly the fourth finger structure. The SUL1 gene sequence corresponds to that of FZF1 gene (Breitwieser et al., 1993) to which no function was attributed.


Subject(s)
Fungal Proteins/genetics , Genes, Fungal/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Sulfites/pharmacology , Transcription Factors , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Drug Resistance, Microbial/genetics , Gene Dosage , Genome, Fungal , Molecular Sequence Data , Point Mutation , Restriction Mapping , Saccharomyces cerevisiae/drug effects , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Zinc Fingers
20.
Curr Genet ; 22(6): 435-40, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1473174

ABSTRACT

Growth inhibition and cell killing caused by sulphite were reduced in seven Saccharomyces cerevisiae sulphite-resistant independent mutants, compared to their parental strains. Genetic analysis showed that in the seven mutants resistance was inherited as a single-gene dominant mutation and that all the analyzed mutations were allelic, thus identifying a major gene responsible for sulphite resistance in S. cerevisiae. Two of the mutants, MBS20-9 and MBS30, were further characterized. 35S-sulphite uptake experiments showed that the ability to accumulate sulphite was markedly reduced in the two resistant strains. No difference between resistant and sensitive strains with respect to glyceraldehyde-3-phosphate dehydrogenase sensitivity to sulphite, or to intracellular glutathione content, were revealed. In contrast, the extracellular acetaldehyde concentration was higher in the resistant mutants, both in the presence and in the absence of sulphite.


Subject(s)
Saccharomyces cerevisiae/genetics , Sulfites/pharmacology , Drug Resistance, Microbial/genetics , Fungal Proteins/metabolism , Genes, Fungal , Glutathione/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/antagonists & inhibitors , Kinetics , Mutation , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/growth & development , Sulfites/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...