Replacement Of Glycine Research Articles

The role of some individual amino acid substitutions in penicillin-binding protein (PBP2) of Neisseria gonorrhoeae in the emergence of resistance to ceftriaxone

The goal of the study was to identify amino acid replacements in the structure of penicillin-binding protein PBP2, which may influence on the development of resistance N. gonorhoeae to the III cephalosporins generation. The gene penA of 50 strains of N. gonorrhoeae was sequenced: 20 strains with high sensitivity to ceftriaxone (MIC, Minimum Inhibitory Concentration, = 0.002 mg/L) and 30 strains with decreased sensitivity to ceftriaxone (MIC = 0.03-0.25 mg/L). The difference of MIC sensitivity between these strains was 30-250 times. Then nucleotide sequence was transformed into the amino acid sequence of PBP2 protein. Mutations in the gene penA and amino acid replacements in the protein PBP2 were found in 16 of 20 strains (80%) with high sensitivity to ceftriaxone and in all strains with decreased sensitivity to ceftriaxone. Amino acid replacements in the PBP2 protein were compared with amino acid replacements in groups, which characterize the PBP2 structure in accordance with the international classification Ito M. The amino acid replacement of PBP2 at positions 346, 505, 511, 517, 543, 567, 575, 576 are associated with V group by Ito M and have features of resistance of N. gonorrhoeae to ceftriaxone authentically (OR = 3.9 ± 2.5; χ2 = 4.9; p < 0.05). It was shown that the replacement of glycine to serine at position 543 of PBP2 in the analyzed strains induced the multiple increase of resistance to ceftriaxone. These data may be significant as showing strong influence of amino acid replacements at positions 346, 505, 511, 517, 567, 575 and, in particular, 543 for development of resistance N. gonorrhoeae strains to ceftriaxone.

Respiratory insufficiency in a newborn with congenital hypothyroidism due to a new mutation of TTF‐1/NKX2.1 gene

NK2 homeobox-1 (NKX2.1) gene encoding the thyroid transcription factor-1 (TTF-1) plays a critical role in lung, thyroid, and central nervous system morphogenesis and function; mutations cause a rare form of progressive respiratory failure associated with alterations of surfactant synthesis, composition, and homeostasis. Molecular mechanisms are heterogeneous and poorly explored. A 28 days old male, soon after birth, presented respiratory failure requiring oxygen treatment at FiO2 27%, prolonged for 2 weeks. Routine neonatal screenings detected a high thyroid stimulating hormone concentration. On day 27 congenital hypothyroidism was confirmed and substitutive treatment was begun. Since the persistence of respiratory symptoms sweat test, CFTR mutation, lymphocyte subpopulations, and sputum cultures were tested, resulting negative. Brain and cardiac defects were also ruled out. Bronchoscopy and BAL analysis were normal. Computed tomography showed bilateral multiple ground glass attenuation, consolidative areas and diffuse bronchial wall thickening. Based on the severity of symptoms, the exclusion of other causes of respiratory disease and the CT findings of interstitial lung disease, we investigated genes affecting the surfactant homeostasis. Sequencing analysis of the three exons of the TTF1 revealed a heterozygous mutation c.334G > T that results in the replacement of glycine in position 112 with a stop codon, generating a nonsense protein that lacks the correct transactivation domain in the C-terminal region. Genetic analysis of the family showed that the father, who was asymptomatic, carried the mutation. Screening for TTF-1 deletions or mutations should always be considered in children with congenital hypothyroidism and an unexplained neonatal respiratory distress or neurodevelopmental deficits.

Congenital hypothyroidism caused by a novel mutation of the dual oxidase 2 (DUOX2) gene

The dual oxidase 2 (DUOX2) mutation results in an impairment of the hydrogen peroxidase-generating system and is identified as a dyshormonogenic cause of congenital hypothyroidism (CH). Here, we describe two unrelated Japanese girls with CH due to a novel DUOX2 mutation. They had high serum thyrotropin levels and low free thyroxine/thyroxine concentrations during the neonatal period. A novel missense mutation with a transversion of G to A at position 1462 in exon 12 of the DUOX2 gene that caused a replacement of glycine (G) with arginine (R) at codon 488 of the protein (c.1462G>A, p.[G488R]) was identified. One patient was a compound heterozygote for p.[L479SfsX3]+[G488R]. The other was homozygous for p.[G488R]. This p.G488R substitution occurred in a highly conserved glycine residue of the mammalian DUOX2 protein. The two patients had different haplotypes, suggesting that the p.G488R alleles were the result of independent, recurrent mutations. Later in life, both patients were still euthyroid even after discontinuing thyroid hormone therapy. We conclude that this p.G488R missense mutation in the DUOX2 gene of the patients is associated with thyroid dysfunction that presents during the neonatal period.

Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes

Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.

Serum stability of selected decapeptide agonists of KISS1R using pseudopeptides

Metastin/kisspeptin, a 54-amino acid peptide, is the ligand of the G-protein-coupled receptor KISS1R which plays a key role in pathways that regulate reproduction and cell migration in many endocrine and gonadal tissues. The N-terminally truncated decapeptide, metastin(45–54), has 3–10 times higher receptor affinity and intracellular calcium ion-mobilizing activity but is rapidly inactivated in serum. In this study we designed and synthesized stable KISS1R agonistic decapeptide analogs with selected substitutions at positions 47, 50, and 51. Replacement of glycine with azaglycine (azaGly) in which the α-carbon is replaced with a nitrogen atom at position 51 improved the stability of amide bonds between Phe50-Gly51 and Gly51-Leu52 as determined by in vitro mouse serum stability studies. Substitution for tryptophan at position 47 with other amino acids such as serine, threonine, β-(3-pyridyl)alanine, and d-tryptophan (d-Trp), produced analogs that were highly stable in mouse serum. d-Trp47 analog 13 showed not only high metabolic stability but also excellent KISS1R agonistic activity. Other labile peptides may have increased serum stability using amino acid substitution.

Evolutionary analysis of the segment from helix 3 through helix 5 in vertebrate progesterone receptors

The interaction between helix 3 and helix 5 in the human mineralocorticoid receptor [MR], progesterone receptor [PR] and glucocorticoid receptor [GR] influences their response to steroids. For the human PR, mutations at Gly-722 on helix 3 and Met-759 on helix 5 alter responses to progesterone. We analyzed the evolution of these two sites and the rest of a 59 residue segment containing helices 3, 4 and 5 in vertebrate PRs and found that a glycine corresponding to Gly-722 on helix 3 in human PR first appears in platypus, a monotreme. In lamprey, skates, fish, amphibians and birds, cysteine is found at this position in helix 3. This suggests that the cysteine to glycine replacement in helix 3 in the PR was important in the evolution of mammals. Interestingly, our analysis of the rest of the 59 residue segment finds 100% sequence conservation in almost all mammal PRs, substantial conservation in reptile and amphibian PRs and divergence of land vertebrate PR sequences from the fish PR sequences. The differences between fish and land vertebrate PRs may be important in the evolution of different biological progestins in fish and mammalian PR, as well as differences in susceptibility to environmental chemicals that disrupt PR-mediated physiology.

Effects of mutations in the WD40 domain of α-COP on its interaction with the COPI coatomer in Saccharomyces cerevisiae

Replacement of glycine 227 in the fifth WD40 motif of α-COP/Ret1p/Soo1p by charged or aromatic amino acids is responsible for the temperature-dependent osmo-sensitivity of Saccharomyces cerevisiae, while truncations of WD40 motifs exerted a reduction in cell growth rate and impairment in assembly of cell-wall associated proteins such as enolase and Gas1p. Yeast two-hybrid analysis revealed that the ret1-1/soo1-1 mutation of α-COP abolished the interaction with β- and ɛ-COP, respectively, and that the interaction between α-COP and β-COP relied on the WD40 domain of α-COP. Furthermore, although the WD40 domain is dispensable for interaction of α-COP with ɛ-COP, structural alterations in the WD40 domain could impair the interaction.

Unique example of amyloid aggregates stabilized by main chain H-bond instead of the steric zipper: molecular dynamics study of the amyloidogenic segment of amylin wild-type and mutants

Most proteins do not aggregate while in their native functional states. However, they may be disturbed from their native conformation by certain change in the environment, and form unwanted oligomeric or polymeric aggregates. Recent experimental data demonstrate that soluble oligomers of amyloidogenic proteins are responsible for amyloidosis and its cytotoxicity. Human islet amyloid polypeptide (IAPP or amylin) is a 37-residue hormone found as fibrillar deposits in pancreatic extracts of nearly all type II diabetics. In this study we performed in silico mutation analysis to examine the stability of the double layer five strand aggregates formed by heptapeptide NNFGAIL segment from amyline peptide. This segment is one of the shortest fragments that can form amyloid fibrils similar to those formed by the full length peptide. The mutants obtained by single glycine replacement were also studied to investigate the specificity of the dry self-complementary interface between the neighboring β-sheet layers. The molecular dynamics simulations of the aggregates run for 20 ns at 330 K, the degree of the aggregate disassembly was investigated using several geometry analysis tools: the root mean square deviations of the C(α) atoms, root mean square fluctuations per residue, twist angles, interstrand distances, fraction of the secondary structure elements, and number of H-bonds. The analysis shows that most mutations make the aggregates unstable, and their stabilities were dependent to a large extent on the position of replaced residues. Our mutational simulations are in agreement with the pervious experimental observations. We also used free binding energy calculations to determine the role of different components: nonpolar effects, electrostatics and entropy in binding. Nonpolar effects remained consistently more favorable in wild type and mutants reinforcing the importance of hydrophobic effects in protein-protein binding. While entropy systematically opposed binding in all cases, there was no clear trend in the entropy difference between wildtype and glycine mutants. Free energy decomposition shows residues situated at the interface were found to make favorable contributions to the peptide-peptide association. The study of the wild type and mutants in an explicit solvent could provide valuable insight into the future computer guided design efforts for the amyloid aggregation inhibitor.

Can molecular dynamics simulations assist in design of specific inhibitors and imaging agents of amyloid aggregation? Structure, stability and free energy predictions for amyloid oligomers of VQIVYK, MVGGVV and LYQLEN

The aggregation modes of hexapeptide fragments of Tau, Insulin and Aβ peptide (VQIVYK, MVGGVV and LYQLEN) were found from their microcrystalline structures that had been recently resolved by X-ray analysis. The atomic structures reveal a dry self-complementary interface between the neighboring β-sheet layers, termed "steric zipper". In this study we perform several all-atom molecular dynamics simulations with explicit water to analyze stability of the crystalline fragments of 2-10 hexapeptides each and their analogs with single glycine replacement mutations to investigate the structural stability, aggregation behavior and thermodynamic of the amyloid oligomers. Upon comparing single and double layer models, our results reveal that additional strands contribute significantly to the structural stability of the peptide oligomers for double layer model, while in the case of single layer model the stability decreases (or remains the same in the case of LYQLEN). This is in agreement with the previous studies performed on different types of amyloid models. We also replaced the side-chains participating in the steric zipper interfaces with glycine. None of the mutants were structurally stable compared to the respective wild type model, except for mutants V2G and V6G in MVGGVV2 case. The exception can be explained by structural features of this particular polymorph. The double layer decamer and dodecamer aggregates of the wild type hexapeptides appear to be stable at 300K, which is confirmed by the conservation of high anti-parallel β-sheet content throughout the whole simulation time. Deletions of the side chains resulted in decline of secondary structure content compared to corresponding wild type indicating that the role of the replaced amino acid in stabilizing the structure. Detailed analysis of the binding energy reveals that stability of these peptide aggregates is determined mainly by the van der Waals and hydrophobic forces that can serve as quantitative measure of shape complementarities between the side chains. This observation implies that interactions among side chains forming the dehydrated steric zipper, rather than among those exposed to water, are the major structural determinant. The electrostatic repulsion destabilizes the studied double layer aggregates in two cases, while stabilizes the other two. Negative total binding free energy indicates that both wild type and mutants complex formation is favorable. However, the mutants complexation is less favorable than the wild type's. The present study provides the atomic level understanding of the aggregation behavior and the driving force for the amyloid aggregates, and could be useful for rational design of amyloid inhibitors and amyloid-specific biomarkers for diagnostic purposes.

Insights into the structural stability and possible aggregation pathways of the LYQLEN peptides derived from human insulin

The LYQLEN peptide from the A chain (residues 13–18) of insulin has been shown to form amyloid-like fibrils. Recently, the structural characterization of this peptide has revealed a general feature of amyloid-like fibers, the so-called “steric zipper”, which is constituted by a tight side-chain complementation of the opposing β-sheet layers. In this study, several molecular dynamics simulations with all-atom explicit water at low pH condition were conducted to investigate the structural stability and aggregation behavior of the LYQLEN peptide of various sizes and its single glycine replacement mutations. Our simulations show that the structural stability of the LYQLEN oligomers increases tremendously with increasing number of β-strands and that the minimal nucleus seed for LYQLEN fibril formation could be as small as a trimer or tetramer. The mutation results also indicate that the hydrophobic interaction between Y2 and E5 plays a crucial role in stabilizing the adjacent β-strands within the same β-sheet layer; while the steric zipper formed via the side chains of L1, Q3, L4, and N6 holds two neighboring β-sheet layers together. Based on the average twist angle between the adjacent β-strands (nearly 9.6°) of the stable LYQLEN oligomers observed in this study, two twist models of the LYQLEN assemblies (SH1-ST60 and SH2-ST60), were built. Finally, two possible aggregation pathways for the LYQLEN peptide were also proposed. Our results provide detailed insights into the stabilization of the LYQLEN oligomers and a thorough understanding of the aggregation pathways of this peptide, which may contribute to imparting further knowledge for designing new or modified capping peptides to inhibit the fibrillization of the LYQLEN peptide and/or insulin.

G708E Mutation in the Androgen Receptor Results in Complete Loss of Androgen Function

End-organ resistance to androgens, called androgen insensitivity syndrome (AIS), is a rare disorder. The most common cause of AIS is mutations(s) in the androgen receptor (AR) gene; however, a significant number of these mutations have not been functionally analyzed. In the present study, we analyzed a case of complete AIS for mutations in the AR gene. Sequencing of the entire coding region of the AR gene revealed a 2650G>A mutation (mRNA sequence reference) in exon 4 of the gene, resulting in replacement of glycine with glutamate at codon 708 in the ligand-binding domain of the AR protein. The mutation was absent in 200 normal male individuals analyzed to look at its occurrence in general population. In vitro androgen-binding and transactivation assays showed that the mutation resulted in approximately 65% loss of ligand binding and almost complete loss of transactivation function. Complete AIS in this individual was due to a G708E substitution in the AR protein.

Single, double and triple alanine to glycine replacements in the AxAxAxA motif of alkaliphilic Bacillus pseudofirmus OF4 c-subunits affect c-ring stability, change both monomer and c-ring mobility in SDS-PAGE and lead to deficits in ATP synthesis

Single, double and triple alanine to glycine replacements in the AxAxAxA motif of alkaliphilic Bacillus pseudofirmus OF4 c-subunits affect c-ring stability, change both monomer and c-ring mobility in SDS-PAGE and lead to deficits in ATP synthesis

Structural stability and aggregation behavior of the VEALYL peptide derived from human insulin: A molecular dynamics simulation study

The VEALYL peptide from B chain (residues 12-17) of insulin has been shown to form amyloid-like fibrils. Recently, the atomic structure of the VEALYL oligomer has been determined by X-ray microcrystallography and reveals a dry, tightly self-complementing structure between the neighboring beta-sheet layers, termed as "steric zipper." In this study, several molecular dynamics simulations with all-atom explicit water were conducted to investigate the structural stability and aggregation behavior of the VEALYL peptide with various sizes and its single glycine replacement mutations. The results of our single-layer models showed that the structural stability of the VEALYL oligomers increases significantly with increasing the number of beta-strands. We further suggested that the minimal nucleus seed for VEALYL fibril formation could be as small as three or four peptides. Our results also revealed that the hydrophobic interaction between E2 and Y5 plays an important role in stabilizing the adjacent beta-strands within the same layer, whereas the hydrophobic steric zipper formed via the side chains of V1, A3, L4, Y5, and L6 locks the two neighboring beta-sheet layers together. Mutation simulations showed that the substitution of a single glycine residue directly disrupts this steric zipper, resulting in the destabilization of the VEALYL oligomers. This study provides the atomic insights into understanding the aggregation behavior of the VEALYL peptide. It may also be helpful for designing new or modified capping peptides able to break the driving force for aggregation and to prevent the fibril formation of the VEALYL peptide and the insulin protein.

A Mutant Connexin50 with Enhanced Hemichannel Function Leads to Cell Death

To determine the consequences of expression of a novel connexin50 (CX50) mutant identified in a child with congenital total cataracts. The GJA8 gene was directly sequenced. Formation of functional channels was assessed by the two-microelectrode voltage-clamp Connexin protein levels and distribution were assessed by immunoblot analysis and immunofluorescence. The proportion of apoptotic cells was determined by flow cytometry. Direct sequencing of the GJA8 gene identified a 137 G>T transition that resulted in the replacement of glycine by valine at position 46 of the coding region of CX50 (CX50G46V). Both CX50 and CX50G46V induced gap junctional currents in pairs of Xenopus oocytes. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much higher than those in oocytes injected with similar amounts of CX50 cRNA. When expressed in HeLa cells under the control of an inducible promoter, both CX50 and CX50G46V formed gap junctional plaques. Induction of CX50G46V expression led to a decrease in the number of cells and an increase in the proportion of apoptotic cells. CX50G46V-induced cell death was prevented by high concentrations of extracellular calcium ions. Unlike previously characterized CX50 mutants that exhibit impaired trafficking and/or lack of function, CX50G46V traffics properly to the plasma membrane and forms functional hemichannels and gap junction channels; however, it causes cell death even when expressed at minute levels. The biochemical results indirectly suggest a potential novel mechanism by which connexin mutants could lead to cataracts: cytotoxicity due to enhanced hemichannel function.

TILLING in Lotus japonicus Identified Large Allelic Series for Symbiosis Genes and Revealed a Bias in Functionally Defective Ethyl Methanesulfonate Alleles toward Glycine Replacements

We have established tools for forward and reverse genetic analysis of the legume Lotus (Lotus japonicus). A structured population of M2 progeny of 4,904 ethyl methanesulfonate-mutagenized M1 embryos is available for single nucleotide polymorphism mutation detection, using a TILLING (for Targeting Induced Local Lesions IN Genomes) protocol. Scanning subsets of this population, we identified a mutation load of one per 502 kb of amplified fragment. Moreover, we observed a 1:10 ratio between homozygous and heterozygous mutations in the M2 progeny. This reveals a clear difference in germline genetics between Lotus and Arabidopsis (Arabidopsis thaliana). In addition, we assembled M2 siblings with obvious phenotypes in overall development, starch accumulation, or nitrogen-fixing root nodule symbiosis in three thematic subpopulations. By screening the nodulation-defective population of M2 individuals for mutations in a set of 12 genes known to be essential for nodule development, we identified large allelic series for each gene, generating a unique data set that combines genotypic and phenotypic information facilitating structure-function studies. This analysis revealed a significant bias for replacements of glycine (Gly) residues in functionally defective alleles, which may be explained by the exceptional structural features of Gly. Gly allows the peptide chain to adopt conformations that are no longer possible after amino acid replacement. This previously unrecognized vulnerability of proteins at Gly residues could be used for the improvement of algorithms that are designed to predict the deleterious nature of single nucleotide polymorphism mutations. Our results demonstrate the power, as well as the limitations, of ethyl methanesulfonate mutagenesis for forward and reverse genetic studies. (Original mutant phenotypes can be accessed at http://data.jic.bbsrc.ac.uk/cgi-bin/lotusjaponicus Access to the Lotus TILLING facility can be obtained through http://www.lotusjaponicus.org or http://revgenuk.jic.ac.uk).

Three-dimensional structure of the two-peptide bacteriocin plantaricin JK

The three-dimensional structures of the two peptides, PlnJ and PlnK, that constitutes the two-peptide bacteriocin plantaricin JK have been solved in water/TFE and water/DPC-micellar solutions using nuclear magnetic resonance (NMR) spectroscopy. PlnJ, a 25 residue peptide, has an N-terminal amphiphilic α-helix between Trp-3 and Tyr-15. The 32 residues long PlnK forms a central amphiphilic α-helix between Gly-9 and Leu-24. Measurements of the effect on anti-microbial activity of single glycine replacements in PlnJ and PlnK show that Gly-13 and Gly-17 in both peptides are very sensitive, giving more than a 100-fold reduction in activity when large residues replace glycine. In variants where other glycine residues, Gly-20 in PlnJ and Gly-7, Gly-9, Gly-24 and Gly-25 in PlnK, were replaced, the activity was reduced less than 10-fold. It is proposed that the detrimental effect on activity when exchanging Gly-13 and Gly-17 in PlnJ and PlnK is a result of reduced ability of the two peptides to interact through the GxxxG-motifs constituting Gly-13 and Gly-17.

Discrimination of Aliphatic Substrates by a Single Amino Acid Substitution inBacillus badiusandBacillus sphaericusPhenylalanine Dehydrogenases

Replacement of glycine by serine at positions 123 and 124 of phenylalanine dehydrogenases from Bacillus badius and Bacillus sphaericus respectively strikingly decreased enzyme activity toward aromatic amino acids and resulted in an elevation of relative activity toward aliphatic amino acids. The mutant from B. badius preferentially dehydrogenated branched-chain amino acids, while that from B. sphaericus acted on amino acids with straight-chain amino acids.

Gene frequency and partial protein characterization of an allelic variant of mannan binding protein associated with low serum concentrations.

Low plasma concentration of mannan binding protein (MBP) has been shown to be the basis for a common opsonic deficiency and suggested to be caused by a single nucleotide substitution at base 230 of exon 1 in the MBP gene. This substitution causes a replacement of glycine (codon GGC) with aspartic acid (codon GAC). Of 123 healthy Danish individuals investigated by polymerase chain reaction performed on exon 1, followed by restriction fragment length polymorphism or allospecific probing, 93 were homozygous (75.6%) for GGC, 28 heterozygous (22.8%), and two homozygous for GAC (1.6%). The gene frequency of the GAC allele was found to be 0.13. DNA sequencing of the cloned exon 1 from one GAC homozygous individual revealed no other substitution. The median MBP concentration in the group containing the GAC allele was 6.4 times lower than in the GGC homozygous group (195 and 1234 micrograms/l respectively). However, the range in plasma concentrations of MBP was wide and overlapping between the groups. MBP protein was detected in both the GAC homozygotes (9 and 387 micrograms/l). Furthermore, no difference in relative mass and biological activity (mannan binding) was found when sera containing the two forms of MBP were investigated. Accordingly, it can be concluded that the GAC allele is able to produce a functional MBP protein which may be detected in serum at low concentrations.

At least two origins of fungicide resistance in grapevine downy mildew populations.

Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc1 complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.

RNR4 mutant alleles pso3-1 and rnr4Δ block induced mutation in Saccharomyces cerevisiae

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.