The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA.

The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

A Botrytis cinerea emopamil binding domain protein, required for full virulence, belongs to a eukaryotic superfamily which has expanded in euascomycetes.

A previous transcriptomic analysis of 3,032 fungal genes identified the Botrytis cinerea PIE3 (BcPIE3) gene to be up-regulated early in planta (A. Gioti, A. Simon, P. Le Pêcheur, C. Giraud, J. M. Pradier, M. Viaud, and C. Levis, J. Mol. Biol. 358:372-386, 2006). In the present study, BcPIE3 was disrupted in order to determine its implication in pathogenicity. BcPIE3 was shown to be a virulence factor, since the DeltaBcPIE3 mutant was blocked during the colonization of tomato and bean leaves, giving lesions reduced in size by at least 74%. Within the emopamil binding domain (EBD), BcPIE3 shows significant structural similarities to mammalian emopamil binding proteins (EBPs). Mammalian EBPs function as sterol isomerases, but an analysis of the sterol content and the results of growth inhibition experiments with the DeltaBcPIE3 strain indicated that BcPIE3 is dispensable for ergosterol biosynthesis. The systematic identification of EBD-containing proteins included in public databases showed that these proteins constitute a protein superfamily present only in eukaryotes. Phylogenetic analysis showed that the ancestral EBD-encoding gene was duplicated in the common ancestor of animals and fungi after the split from plants. Finally, we present evidence that the EBP phylogenetic clade of this superfamily has further expanded exclusively in euascomycetes, especially in B. cinerea, which contains three copies of the EBP gene.

The hydroxyanilide fenhexamid, a new sterol biosynthesis inhibitor fungicide efficient against the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea).

Fenhexamid, a recently developed botryticide, is shown here to inhibit sterol biosynthesis. When the fungus Botryotinia fuckeliana was grown in the presence of fenhexamid, the ergosterol content was reduced, and three 3-keto compounds, 4 alpha-methylfecosterone, fecosterone and episterone, accumulated, suggesting an inhibition of the 3-keto reductase involved in C-4 demethylation. Thus, fenhexamid belongs to a new, promising class of sterol biosynthesis inhibitors not previously used in agriculture or in medicine.

The effect of the oxic cell sensitizer, beta-ara A on human fibroblasts in plateau and in exponential growth phases.

Before an oxic cell sensitizer such as beta-ara A (a DNA-dependent DNA polymerase inhibitor) can be used in cancer treatment, it is essential to know both the influence of this type of drug on certain critical normal tissues and the role of proliferation kinetics in the radiosensitizing capacity. The biological system chosen for this in vitro study was the human fibroblast cell line HF19. Cells were studied in plateau phase and in the exponential growth phase. Cells were incubated with beta-ara A for 7 hr (1 hr before and 6 hr after irradiation). beta-ara A was extremely toxic to growing cells (concentrations ranging from 200 to 1000 microM), but no detectable effect was found on plateau-phase cells (up to 4000 microM). However, for a given drug concentration, the radiosensitizing effect (Sensitizing Enhancement Ratio SER) was very similar for growing and plateau phase cells (SER measured with Ds ratio was about 1.7 for a concentration of 500 microM). The enhancement ratio depended on the radiation dose; it was relatively higher for low doses. This can be explained by a differential effect of the drug on the alpha and beta components of the survival curve. Only the alpha component was increased.

Correlation between PLD repair capacity and the survival curve of human fibroblasts in exponential growth phase: analysis in terms of several parameters.

Published data on the in vitro radiosensitivity of 46 nontransformed fibroblasts of different genetic origins studied in plateau phase with immediate or delayed plating were used to investigate to what extent potentially lethal damage repair capacity is related to intrinsic radiosensitivity (i.e., irradiated in exponential growth phase). While most of the survival curve analysis is conducted in terms of D0, Dq, and the mean inactivation dose D, some of the data are also discussed in terms of the linear-quadratic model parameter alpha. Using D it is shown that: (i) the radiosensitivity of human fibroblasts in exponential growth phase does not significantly differ from that of plateau-phase fibroblasts with immediate plating; (ii) the radiosensitivity of plateau-phase cells with delayed plating is correlated to the radiosensitivity of cells with immediate plating: the more radioresistant the cell strain in exponential growth phase, the higher its repair capacity; (iii) the repair capacity of the cell strains is related to their genetic origin. In conclusion, we suggest that the survival curve of growing cells depends on the repair capacity of the cells.

Low molecular weight thiol content in glutathione synthetase-deficient human fibroblasts.

The activity and the kinetic properties of glutathione synthetase and the concentrations of non-protein bound thiols of the gamma-glutamyl cycle were measured in 11 human fibroblast cell strains. Six of these strains were derived from patients suffering from 5-oxoprolinuria, a recessive genetic disease characterized by a deficiency in glutathione synthetase; the other cell strains were derived from healthy heterozygous or homozygous relatives of the patients. The glutathione synthetase activities of homozygous deficient strains were 1/3 of control values while those of heterozygous strains were 2/3 of control values. The total thiol concentration was lower in only 3 of the 6 deficient homozygotes and that of glutathione (GSH) was lower in only 4 of the 6 deficient homozygotes. This lower GSH level was at least partly offset by an accumulation of gamma-glutamylcysteine, a precursor of GSH, which is almost completely absent from control cells. The total quantities of thiols and GSH in plateau phase cells were about 50% and 30% respectively of the levels in growth phase cells. Approximately 80% of the GSH was in the reduced form in both quiescent and growing cells.

Re-evaluation of in vitro radiosensitivity of human fibroblasts of different genetic origins.

A statistical analysis of the radiosensitivity of 204 different survival curves of nontransformed human fibroblast cell strains of different genetic origins was made using three criteria: the multi-target one-hit model (characterized by parameters n and D0), the surviving fraction for a 2 Gy dose (S2) and the mean inactivation dose (D). D is found to be the best parameter for characterization of anomalous radiosensitivity linked to a genetic disorder and for discrimination between groups of cell strains of differing radiosensitivity. Its use allows the description of a range of 'normal' radiosensitivity for control fibroblasts and the classification of the various genetic disorders as a function of their mean radiosensitivity expressed in terms of D. Nine groups of cell strains appear to exhibit radiosensitivity which differs significantly from that of the controls: seven groups are hypersensitive (ataxia-telangiectasia homozygotes and heterozygotes, Cockayne's syndrome, Gardner's syndrome, 5-oxoprolinuria homozygotes and heterozygotes, Fanconi's anaemia) and two groups are more radioresistant (fibroblasts from retinoblastoma patients and from individuals with chromosome 13 anomalies). Since the coupled parameter n and D0 failed to discriminate between the radiosensitivity of the different genetic groups, we recommend the use of D to make an intercomparison of intrinsic radiosensitivity of nontransformed human fibroblasts.

Reduced repair of potentially lethal radiation damage in glutathione synthetase-deficient human fibroblasts after X-irradiation.

Using a human fibroblast strain deficient in glutathione synthetase and a related proficient control strain, the role of glutathione (GSH) in repair of potentially lethal damage (PLD) has been investigated in determining survival by plating cells immediately or 24 h after irradiation. After oxic or hypoxic irradiation, both cell strains repair radiation-induced damage. However, under hypoxic conditions, the proficient cells repair PLD as well as under oxic conditions while the deficient cells repair less PLD after irradiation under hypoxic than under oxic conditions. Therefore, the oxygen enhancement ratio (o.e.r.) for proficient cells is similar whether the cells are plated immediately or 24 h later (2.0 and 2.13, respectively). In contrast, the o.e.r. for deficient cells is lower when the cells are plated 24 h after irradiation than when they are plated immediately thereafter (1.16 as compared to 1.55). The results indicate that GSH is involved in PLD repair and, in particular, in the repair of damage induced by radiation delivered under hypoxic conditions.

Radioprotective effect of cysteamine in glutathione synthetase-deficient cells.

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect.

Survival curves of glutathione synthetase deficient human fibroblasts: correlation between radiosensitivity in hypoxia and glutathione synthetase activity.

The role of intracellular non-protein bound sulphydryl compounds (NPSH), and in particular that of glutathione (GSH), in the response of cells to ionizing radiation under different O2 concentrations has been assessed using cell strains deficient in glutathione synthetase and exhibiting different NPSH levels. The cell strains used originated from patients with 5-oxoprolinuria and from their relatives (heterozygotes and proficient homozygotes). No correlation has been found between NPSH and GSH concentrations and radiosensitivity under oxic, aerobic and hypoxic conditions. However, a highly significant correlation has been observed between radiosensitivity under hypoxic conditions (and therefore the oxygen enhancement ratio) and the glutathione synthetase activity, suggesting that synthesis of GSH is required after irradiation. In order to explain our results we postulated, beside radical processes, the existence of a GSH-dependent enzymatic repair mechanism for N2 type damage. Hypoxic radio-sensitivity measured with survival curves would result from the interaction of both competition and biochemical repair processes.

Radiosensitizing and cytotoxic properties of misonidazole on glutathione synthetase deficient human fibroblasts.

The cytotoxic and radiosensitizing effects of misonidazole have been studied on glutathione synthetase deficient fibroblasts and on their controls. At any concentration from 0.1 to 4 mM, deficient cells are more sensitive to the cytotoxic effect of misonidazole than the control cells. The differential effect between the two cell strain concerns both the shoulder and the slope of the survival curve, thus suggesting that NPSH play a role in the determination of misonidazole cytotoxicity. Like oxygen, misonidazole clearly sensitizes deficient cells to a lesser extent than control cells. For both cell strains, the maximum sensitizing effect of misonidazole is very close to that of oxygen (1.5 and 1.5 for deficient cells, 2.8 and 2.9 for control cells, respectively). The sensitizing effect of misonidazole appears in the same concentration range for both cell strains, with a maximal effect at lower concentrations for deficient cells.

Reduced PLD repair ability in glutathione synthetase deficient human fibroblasts after UV irradiation.

Using a human cell strain deficient in glutathione synthetase and a related control, the role of glutathione in repair mechanisms has been investigated. UV light has been used in order to avoid the interaction between thiols and free radicals. When potentially lethal damage repair is completed, deficient cells in plateau phase exhibit smaller surviving fractions than do control cells. The ratio of surviving fractions in control/deficient cells is about 2 for the same radiation dose. These results indicate that thiols and especially GSH are involved in repair mechanisms.