scholarly journals Mechanisms of sod2 Gene Amplification inSchizosaccharomyces pombe

2000 ◽  
Vol 11 (3) ◽  
pp. 873-886 ◽  
Author(s):  
Elizabeth B. Albrecht ◽  
Aaron B. Hunyady ◽  
George R. Stark ◽  
Thomas E. Patterson
Keyword(s):  
Gene Amplification ◽  
Base Pair ◽  
Dna Lesions ◽  
The Novel ◽  
Wild Type ◽  
Resistant Mutants ◽  
Chromosome Ii ◽  
Chromosome I ◽  
Mutant Cells ◽  
Type Strains

Gene amplification in eukaryotes plays an important role in drug resistance, tumorigenesis, and evolution. TheSchizosaccharomyces pombe sod2 gene provides a useful model system to analyze this process. sod2 is near the telomere of chromosome I and encodes a plasma membrane Na+(Li+)/H+ antiporter. Whensod2 is amplified, S. pombe survives otherwise lethal concentrations of LiCl, and >90% of the amplifiedsod2 genes are found in 180- and 225-kilobase (kb) linear amplicons. The sequence of the novel joint of the 180-kb amplicon indicates that it is formed by recombination between homologous regions near the telomeres of the long arm of chromosome I and the short arm of chromosome II. The 225-kb amplicon, isolated three times more frequently than the 180-kb amplicon, is a palindrome derived from a region near the telomere of chromosome I. The center of symmetry of this palindrome contains an inverted repeat consisting of two identical 134-base pair sequences separated by a 290-base pair spacer. LiCl-resistant mutants arise 200–600 times more frequently in strains deficient for topoisomerases or DNA ligase activity than in wild-type strains, but the mutant cells contain the same amplicons. These data suggest that amplicon formation may begin with DNA lesions such as breaks. In the case of the 225-kb amplicon, the breaks may lead to a hairpin structure, which is then replicated to form a double-stranded linear amplicon, or to a cruciform structure, which is then resolved to yield the same amplicon.

scholarly journals Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone

2002 ◽  
Vol 46 (11) ◽  
pp. 3370-3380 ◽  
Author(s):  
Dilek Ince ◽  
Xiamei Zhang ◽  
L. Christine Silver ◽  
David C. Hooper
Keyword(s):  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Fold Increase ◽  
Single Step ◽  
The Novel ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Dual Targeting ◽  
Resistant Mutants ◽  
Selection Of

ABSTRACT We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 × 10−12 to 4.0 × 10−11) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 μg/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5′ terminus of grlB and the 3′ terminus of gyrA.

scholarly journals Barrier Activity in Candida albicans Mediates Pheromone Degradation and Promotes Mating

2007 ◽  
Vol 6 (6) ◽  
pp. 907-918 ◽  
Author(s):  
Dana Schaefer ◽  
Pierre Côte ◽  
Malcolm Whiteway ◽  
Richard J. Bennett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Cell Growth ◽  
Cell Fusion ◽  
Aspartyl Protease ◽  
Wild Type ◽  
Pheromone Activity ◽  
Important Regulator ◽  
Mutant Cells ◽  
Type Strains

ABSTRACT Mating in Candida albicans and Saccharomyces cerevisiae is regulated by the secretion of peptide pheromones that initiate the mating process. An important regulator of pheromone activity in S. cerevisiae is barrier activity, involving an extracellular aspartyl protease encoded by the BAR1 gene that degrades the alpha pheromone. We have characterized an equivalent barrier activity in C. albicans and demonstrate that the loss of C. albicans BAR1 activity results in opaque a cells exhibiting hypersensitivity to alpha pheromone. Hypersensitivity to pheromone is clearly seen in halo assays; in response to alpha pheromone, a lawn of C. albicans Δbar1 mutant cells produces a marked zone in which cell growth is inhibited, whereas wild-type strains fail to show halo formation. C. albicans mutants lacking BAR1 also exhibit a striking mating defect in a cells, but not in α cells, due to overstimulation of the response to alpha pheromone. The block to mating occurs prior to cell fusion, as very few mating zygotes were observed in mixes of Δbar1 a and α cells. Finally, in a barrier assay using a highly pheromone-sensitive strain, we were able to demonstrate that barrier activity in C. albicans is dependent on Bar1p. These studies reveal that a barrier activity to alpha pheromone exists in C. albicans and that the activity is analogous to that caused by Bar1p in S. cerevisiae.

Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene

1988 ◽  
Vol 8 (12) ◽  
pp. 5555-5560
Author(s):  
H Iida
Keyword(s):  
Heat Treatment ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Adenylate Cyclase ◽  
Uv Light ◽  
Yeast Cells ◽  
Wild Type ◽  
Coding Region ◽  
Resistant Mutants ◽  
Type Strains

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

scholarly journals Effects of Genome Position and the DNA Damage Checkpoint on the Structure and Frequency of sod2 Gene Amplification in Fission Yeast

1999 ◽  
Vol 10 (7) ◽  
pp. 2199-2208 ◽  
Author(s):  
Thomas E. Patterson ◽  
Elizabeth B. Albrecht ◽  
Paul Nurse ◽  
Shelley Sazer ◽  
George R. Stark
Keyword(s):  
Dna Damage ◽  
Gene Amplification ◽  
Genetic Background ◽  
S Phase ◽  
Checkpoint Control ◽  
Wild Type ◽  
Extrachromosomal Elements ◽  
Resistant Strains ◽  
Chromosome I ◽  
Selection Of

The Schizosaccharomyces pombe sod2 gene, located near the telomere on the long arm of chromosome I, encodes a Na+ (or Li+)/H+ antiporter. Amplification of sod2 has previously been shown to confer resistance to LiCl. We analyzed 20 independent LiCl-resistant strains and found that the only observed mechanism of resistance is amplification of sod2. The amplicons are linear, extrachromosomal elements either 225 or 180 kb long, containing bothsod2 and telomere sequences. To determine whether proximity to a telomere is necessary for sod2amplification, a strain was constructed in which the gene was moved to the middle of the same chromosomal arm. Selection of LiCl-resistant strains in this genetic background also yielded amplifications ofsod2, but in this case the amplified DNA was exclusively chromosomal. Thus, proximity to a telomere is not a prerequisite for gene amplification in S. pombe but does affect the mechanism. Relative to wild-type cells, mutants with defects in the DNA damage aspect of the rad checkpoint control pathway had an increased frequency of sod2 amplification, whereas mutants defective in the S-phase completion checkpoint did not. Two models for generating the amplified DNA are presented.

scholarly journals Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene.

1988 ◽  
Vol 8 (12) ◽  
pp. 5555-5560 ◽  
Author(s):  
H Iida
Keyword(s):  
Heat Treatment ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Adenylate Cyclase ◽  
Uv Light ◽  
Yeast Cells ◽  
Wild Type ◽  
Coding Region ◽  
Resistant Mutants ◽  
Type Strains

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

scholarly journals Echinocandins and their Activity against Aspergillus terreus Species Complex: A Novel Agar Screening Method

2021 ◽  
Author(s):  
Nina Lackner ◽  
Roya Vahedi-Shahandashti ◽  
Sonja Jähnig ◽  
Lea Schönherr ◽  
Cornelia Lass-Flörl
Keyword(s):  
Species Complex ◽  
Aspergillus Terreus ◽  
Susceptibility Testing ◽  
Screening Method ◽  
The Novel ◽  
Wild Type ◽  
Type Strains

We evaluated the newly proposed agar-screening method for echinocandin susceptibility testing of 144 Aspergillus section Terrei isolates in comparison with Etest®. Both methods defined the isolates to be wild type strains for anidulafungin and micafungin, with Etest® minimal effective concentrations (MECs) being ≤0.004 mg/L. For caspofungin, the novel agar-screening method identified 37 isolates to be caspofungin non-wild type based on their fluffy colony appearance on caspofungin agar. Etest® MECs for caspofungin for these isolates scattered widely from 0.002 to 0.750 mg/L, showing only partial accordance between the two methods.

scholarly journals SELECTIVE ABORTION OF TWO NONSISTER NUCLEI IN A DEVELOPING ASCUS OF THE hfd1—1 MUTANT IN SACCHAROMYCES CEREVISIAE

Genetics ◽  
1981 ◽  
Vol 99 (2) ◽  
pp. 197-209
Author(s):  
Susumu Okamoto ◽  
Tetsuo Iino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutant Strain ◽  
Recessive Mutation ◽  
Microscopical Examination ◽  
Wild Type ◽  
Selective Abortion ◽  
Dyad Analysis ◽  
Mutant Cells ◽  
Type Strains ◽  
Light Microscopical Examination

ABSTRACT A recessive mutation, hfd1—1, in strain SOS4 of Saccharomyces cerevisiae leads the mutant cells to produce predominantly two-spored asci. Light microscopical examination of Giemsastained cells revealed no significant differences in the meiotic figures between mutant and wild-type strains. However, only two of the four meiotic products in a developing ascus matured to ascospores in SOS4. Dyad analysis was carried out on an hfd1-1 mutant strain heterozygous for three markers, asp5, gal1 and arg4, which are closely linked to their centromeres, and for his4, which is loosely linked to its centromere. The twospored asci produced by the hfd1—1 mutant segregated dominant (+) and recessive (-) alleles of each marker in a 1:1 ratio; they generally contained one + and one - spore for any given marker. The occurrence of rare dyads with two + or two - spores can be explained quantitatively by recombination between the marker and its centromere. From the results of these cytological and genetical analyses, we infer that, in the mutant strain, one genome set is partitioned to each of the four second-meiotic division poles, but only two nonsister genomes are incorporated into mature spores. Thus, the hfd1—1 mutation in SOS4 blocks incorporation of two nonsister nuclei into mature ascospores, but does not block enclosure of the remaining two nonsister nuclei.

scholarly journals Hydrophilicity of quinolones is not an exclusive factor for decreased activity in efflux-mediated resistant mutants of Staphylococcus aureus.

1996 ◽  
Vol 40 (8) ◽  
pp. 1835-1842 ◽  
Author(s):  
T Takenouchi ◽  
F Tabata ◽  
Y Iwata ◽  
H Hanzawa ◽  
M Sugawara ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quantitative Structure Activity Relationship ◽  
Wild Type ◽  
Cellular Accumulation ◽  
Structure Activity ◽  
Carbonyl Cyanide ◽  
Elevated Expression ◽  
Resistant Mutants ◽  
Type Strains ◽  
Increment Ratio

The elevated expression of the norA gene is responsible for efflux-mediated resistance to quinolones in Staphylococcus aureus (E.Y.W. Ng, M. Trucksis, and D.C. Hooper, Antimicrob. Agents Chemother. 38:1345-1355, 1994). For S. aureus transformed with a plasmid containing the cloned norA gene, SA113(pTUS20) (H. Yoshida, M. Bogaki, S. Nakamura, K. Ubukata, and M. Konno, J. Bacteriol. 172:6942-6949, 1990), and an overexpressed mutant, SA-1199B (G.W. Kaatz, S.M. Seo, and C.A. Ruble, J. Infect. Dis. 163:1080-1086, 1991), the MICs of norfloxacin increased 16 and 64 times compared with its MICs for the recipient and wild-type strains, SA113 and SA-1199, respectively. MICs of CS-940, however, increased only two and eight times, even though these two fluoroquinolones are similarly hydrophilic (apparent logPs of approximately -1). No good correlation was found, among 15 developed and developing quinolones, between the increment ratio in MICs and hydrophobicity (r = 0.61). Analysis of the quantitative structure-activity relationship among 40 fluoroquinolones revealed that the MIC increment ratio was significantly correlated with the bulkiness of the C-7 substituent and bulkiness and hydrophobicity of the C-8 substituent of fluoroquinolones (r = 0.87) and not with its molecular hydrophobicity (r = 0.47). Cellular accumulation of norfloxacin in SA-1199B was significantly lower than that in SA-1199, and it was increased by addition of carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, accumulations of CS-940 in these strains were nearly identical, and they were not affected by addition of the protonophore.

Differential regulation of interchromosomal copies of ToxR-induced genes

2007 ◽  
Vol 53 (8) ◽  
pp. 992-999
Author(s):  
Sanjay Nag ◽  
Keya Chaudhuri
Keyword(s):  
Virulence Genes ◽  
Ph Effect ◽  
Low Ph ◽  
Insertion Mutant ◽  
Wild Type ◽  
Rt Pcr ◽  
Transcription Profiling ◽  
Environmental Signals ◽  
Chromosome Ii ◽  
Chromosome I

In Vibrio cholerae , ToxR transcriptionally activates a number of virulence genes in response to various environmental signals. In the present study, transcription profiling by macroarray was carried out with 13 pairs of genes, one copy of which is present in each chromosome under ToxR-inducing (pH 6.5, osmolarity 66 mmol/L, 30 °C) and ToxR-repressing (pH 8.5, osmolarity 300 mmol/L, 37 °C) conditions followed by high pH (8.5) and low pH (6.5) conditions to eliminate pH effect. The genes dacAII, tagEII, secDII, pmmI, pmmII, and immII showed increased expression in the ToxR-inducing conditions, but not at low pH, suggesting that the expression of these genes might be regulated by ToxR. The expression of pmmII, dacAII, tagEII, secDII, and immII genes decreased significantly in the toxR insertion mutant as determined by RT–PCR, whereas the expression of the chromosome I copy of pmm increased in toxR mutant compared with wild-type cells. Thus, the chromosome II copy of these genes, which show increased expression under ToxR-inducing conditions, are all regulated by ToxR in V. cholerae, whereas the chromosome I copy of pmm may be regulated by other factors under ToxR-inducing conditions.

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