Development of positive selectable markers for the fungal pathogen Cryptococcus neoformans.
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Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningitis in approximately 10% of patients with AIDS. New selectable markers which confer resistance to G418 or phleomycin when transformed into C. neoformans were made. A hygromycin-selectable marker was modified to allow selection with a single copy of the marker. (+info)
Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes.
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Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase. (+info)
Ku is important for telomere maintenance, but not for differential expression of telomeric VSG genes, in African trypanosomes.
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Trypanosome antigenic variation, involving differential expression of variant surface glycoprotein (VSG) genes, has a strong association with telomeres and with DNA recombination. All expressed VSGs are telomeric, and differential activation involves recombination into the telomeric environment or silencing/activation of subtelomeric promoters. A number of pathogen contingency gene systems associated with immune evasion involve telomeric loci, which has prompted speculation that chromosome ends provide conditions conducive for the operation of rapid gene switching mechanisms. Ku is a protein associated with eukaryotic telomeres that is directly involved in DNA recombination and in gene silencing. We have tested the hypothesis that Ku in trypanosomes is centrally involved in differential VSG expression. We show, via the generation of null mutants, that trypanosome Ku is closely involved in telomere length maintenance, more so for a transcriptionally active than an inactive telomere, but exhibits no detectable influence on DNA double strand break repair. The absence of Ku and the consequent great shortening of telomeres had no detectable influence either on the rate of VSG switching or on the silencing of the telomeric promoters of the VSG subset that is expressed in the tsetse fly. (+info)
Inactivation of Mre11 does not affect VSG gene duplication mediated by homologous recombination in Trypanosoma brucei.
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We demonstrate, by gene deletion analysis, that Mre11 has a critical role in maintaining genomic integrity in Trypanosoma brucei. mre11(-/-) null mutant strains exhibited retarded growth but no delay or disruption of cell cycle progression. They showed also a weak hyporecombination phenotype and the accumulation of gross chromosomal rearrangements, which did not involve sequence translocation, telomere loss, or formation of new telomeres. The trypanosome mre11(-/-) strains were hypersensitive to phleomycin, a mutagen causing DNA double strand breaks (DSBs) but, in contrast to mre11(-/-) null mutants in other organisms and T. brucei rad51(-/-) null mutants, displayed no hypersensitivity to methyl methanesulfonate, which causes point mutations and DSBs. Mre11 therefore is important for the repair of chromosomal damage and DSBs in trypanosomes, although in this organism the intersection of repair pathways appears to differ from that in other organisms. Mre11 inactivation appears not to affect VSG gene switching during antigenic variation of a laboratory strain, which is perhaps surprising given the importance of homologous recombination during this process. (+info)
Solution structure of the hydroperoxide of Co(III) phleomycin complexed with d(CCAGGCCTGG)2: evidence for binding by partial intercalation.
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The bleomycins (BLMs) are natural products that in the presence of iron and oxygen bind to and cause single-strand and double-strand cleavage of DNA. The mode(s) of binding of the FeBLMs that leads to sequence-specific cleavage at pyrimidines 3' to guanines and chemical-specific cleavage at the C-4' H of the deoxyribose of the pyrimidine has remained controversial. 2D NMR studies using the hydroperoxide of CoBLM (HOO-CoBLM) have demonstrated that its bithiazole tail binds by partial intercalation to duplex DNA. Studies with ZnBLM demonstrate that the bithiazole tail binds in the minor groove. Phleomycins (PLMs) are BLM analogs in which the penultimate thiazolium ring of the bithiazole tail is reduced. The disruption of planarity of this ring and the similarities between FePLM- and FeBLM-mediated DNA cleavage have led Hecht and co-workers to conclude that a partial intercalative mode of binding is not feasible. The interaction of HOO-CoPLM with d(CCAGGCCTGG)2 has therefore been investigated. Binding studies indicate a single site with a K(d) of 16 micro M, 100-fold greater than HOO-CoBLM for the same site. 2D NMR methods and molecular modeling using NMR-derived restraints have led to a structural model of HOO-CoPLM complexed to d(CCAGGCCTGG)2. The model reveals a partial intercalative mode of binding and the basis for sequence specificity of binding and chemical specificity of cleavage. The importance of the bithiazoles and the partial intercalative mode of binding in the double-strand cleavage of DNA is discussed. (+info)
The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment.
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MEC1 and TEL1 encode ATR- and ATM-related proteins in the budding yeast Saccharomyces cerevisiae, respectively. Phleomycin is an agent that catalyzes double-strand breaks in DNA. We show here that both Mec1 and Tel1 regulate the checkpoint response following phleomycin treatment. MEC1 is required for Rad53 phosphorylation and cell-cycle progression delay following phleomycin treatment in G1, S or G2/M phases. The tel1Delta mutation confers a defect in the checkpoint responses to phleomycin treatment in S phase. In addition, the tel1Delta mutation enhances the mec1 defect in activation of the phleomycin-induced checkpoint pathway in S phase. In contrast, the tel1Delta mutation confers only a minor defect in the checkpoint responses in G1 phase and no apparent defect in G2/M phase. Methyl methanesulfonate (MMS) treatment also activates checkpoints, inducing Rad53 phosphorylation in S phase. MMS-induced Rad53 phosphorylation is not detected in mec1Delta mutants during S phase, but occurs in tel1Delta mutants similar to wild-type cells. Finally, Xrs2 is phosphorylated after phleomycin treatment in a TEL1-dependent manner during S phase, whereas no significant Xrs2 phosphorylation is detected after MMS treatment. Together, our results support a model in which Tel1 contributes to checkpoint control in response to phleomycin-induced DNA damage in S phase. (+info)
Mammalian promoter element function in the fission yeast Schizosaccharomyces pombe.
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We have analyzed the function of several mammalian promoter elements in the fission yeast, Schizosaccharomyces pombe. Mutants of the human HSP70 promoter were introduced into S. pombe as single copy integrants at a specific location. Transcription initiation sites utilized in S. pombe with the HSP70 TATA element were similar to those used in mammalian cells. Of three mammalian TATA elements tested, only the HSP70 TATA element functioned in S. pombe. The adenovirus Ella TATA element had little or no activity in S. pombe, indicating that S. pombe is deficient in the factor(s) necessary for recognition of this element. Of upstream promoter elements tested, the CCAAT, Sp1 binding, ATF binding and heat shock elements were functional in S. pombe. Strains containing mutant promoters fused to the ble gene were used to demonstrate that phleomycin can be used as a graded selection in S. pombe. These data demonstrate that S. pombe should provide a useful system in which to characterize and isolate mammalian factors involved in initiation site determination and transcriptional regulation. (+info)
The Mre11 nuclease is not required for 5' to 3' resection at multiple HO-induced double-strand breaks.
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Current hypotheses suggest the Mre11 nuclease activity could be directly involved in double-strand break (DSB) resection in the presence of a large number of DSBs or limited to processing abnormal DNA ends. To distinguish between these possibilities, we used two methods to create large numbers of DSBs in Saccharomyces cerevisiae chromosomes, without introducing other substrates for the Mre11 nuclease. Multiple DSBs were created either by expressing the HO endonuclease in strains containing several HO cut sites embedded within randomly dispersed Ty1 elements or by phleomycin treatment. Analysis of resection by single-strand DNA formation in these systems showed no difference between strains containing MRE11 or the mre11-D56N nuclease defective allele, suggesting that the Mre11 nuclease is not involved in the extensive 5' to 3' resection of DSBs. We postulate that the ionizing radiation (IR) sensitivity of mre11 nuclease-defective mutants results from the accumulation of IR-induced DNA damage that is normally processed by the Mre11 nuclease. We also report that the processivity of 5' to 3' DSB resection and the yield of repaired products are affected by the number of DSBs in a dose-dependent manner. Finally, we show that the exonuclease Exo1 is involved in the processivity of 5' to 3' resection of an HO-induced DSB at the MAT locus. (+info)