A microsatellite sequence in the fifth intron provides a broad-spectrum SSR marker for multiple alleles of the <Emphasis Type="Italic">er1</Emphasis>/<Emphasis Type="Italic">PsMLO1</Emphasis> powdery mildew resistance gene in <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

Powdery mildew caused by the biotrophic ascomycete fungus Erysiphe pisi Syd. is one the most devastating diseases of peas (Pisum sativum L.) with enormous impact in seed production. The most efficient genetic resistance to this disease, so far identified, is conferred by the naturally occurring or experimentally induced by chemical mutagenesis recessive state of the locus er1. Genetically mapped over 2 decades ago, this gene was recently identified as a homolog of the barley (Hordeum sativum L.) powdery mildew resistance gene MLO, and renamed as PsMLO1. The broad wide resistance conferred by the er1/PsMLO1 locus was found to be a consequence of the loss of function of the encoded PsMLO1 protein. After the publication of the expressed sequence of this gene by another research group, we published the genomic sequences of this gene which harbors a relatively long (TA) microsatellite sequence (SSR) in the fifth intron. SSR markers based on this highly polymorphic microsatellite can be used for marker-assisted selection in multiple pea powdery mildew resistance breeding programs involving the er1/PsMLO1 resistance, except in the rare circumstances where the progenitor lines are monomorphic for the microsatellite sequence.     

Identification of a complete set of functional markers for the selection of er1 powdery mildew resistance in Pisum sativum L.

Powdery mildew is the most widespread disease of pea (Pisum sativum L.) and causes severe economic losses worldwide. Recessively inherited er1 powdery mildew resistance, successfully used for decades in pea breeding programs, has recently been shown to originate from the loss of function of the PsMLO1 gene. Five er1 alleles, each corresponding to a different PsMLO1 null mutation, have been characterized to date in pea germplasm. In order to aid er1 selection, we aimed to identify functional markers which target PsMLO1 polymorphisms directly responsible for the resistant phenotype. Highly informative cleaved amplified polymorphic sequence (CAPS), derived cleaved amplified polymorphic sequence (dCAPS), sequence tagged site (STS) and high-resolution melting (HRM) markers were developed which enable the selection of each of the five er1 alleles. Taken together, the results described here provide a powerful tool for breeders, overcoming limitations of previously reported er1-linked markers due to the occurrence of recombination with the resistance locus and/or the lack of polymorphism between parental genotypes. The HRM marker er1-5/HRM54 reported here, targeting a mutagenesis-induced er1 allele recently described by us, does not require manual processing after PCR amplification, and is therefore suitable for large-scale breeding programs based on high-throughput automated screening.     

Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1

Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea.     

Pea powdery mildew <Emphasis Type="Italic">er1</Emphasis> resistance is associated to loss-of-function mutations at a <Emphasis Type="Italic">MLO</Emphasis> homologous locus

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60 years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F₂ individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection.     

加拿大豌豆品种（系）抗白粉病表型和基因型鉴定

对36个引自加拿大的豌豆品种(系)进行抗白粉病表型和标记基因型鉴定,明确了豌豆品种Cooper和Tara白粉病抗性等位基因。苗期接种了2个不同地理来源的豌豆白粉病菌分离物,32个品种(系)对2个分离物均表现为免疫;品系MP1818-2对云南白粉菌分离物EPYN免疫,但对北京分离物EPBJ感病;其余3个品种对2个分离物均感病。4个与豌豆抗白粉病基因er1连锁的SCAR标记将36个豌豆品种(系)区分为5个标记基因型。与野生型PsMLO1基因序列比较发现,豌豆品种Cooper和Tara的PsMLO1候选基因均在680 bp处发生C变G的单核苷酸突变。     

Gene conversion in nonsense suppressors of Schizosaccharomyces pombe. I. The influence of the genetic background and of three mutant genes (rad2, mut1 and mut2) on the frequency of the post-meiotic segregation.

Summary The frequency of gene conversion was assessed at the anticodon site of the sup3 gene of Schizosaccharomyces pombe. In order to detect a possible influence of the genetic background on the relative frequency of post-meiotic segregations amongst conversions, three similar crosses were analyzed which differed as follows: In cross I the two parents were derived by spontaneous mutation from one and the same strain. The heterogeneity of the genetic background between these two parent strains is assumed to be at a minimum level. In cross II the crossing partners were strains of the Bernese stock collection and differ probably in their genetic background to some extent. Last, in cross III, one of the parent strains was ten times repeatedly mutagenized with nitrosoguanidine in order to introduce cryptic mutations in the genome. A maximum degree of background heterogeneity between parents is expected for this cross. Neither the total conversion frequency nor the frequency of post-meiotic segregations amongst conversions were found to be significantly different in these three crosses.In addition, no effect of the radiation-sensitive mutation, rad2-44, and of two mutator mutations, mut1-4 and mut2-9, on the conversion pattern could be detected.     

Involvement of histidine permease (Hip1p) in manganese transport in Saccharomyces cerevisiae

In a search for components involved in Mn²⁺ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn²⁺ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1–272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn²⁺ resistance, whereas the corresponding null mutation did not. Both hip1–272 cells and the null mutant exhibited low tolerance to divalent cations such as Co²⁺, Ni²⁺, Zn²⁺, and Cu²⁺. The Mn²⁺ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn²⁺ content of the hip1–272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn²⁺ efflux. We propose that Hip1p is involved in Mn²⁺ transport, carrying out a function related to Mn²⁺ export.     

Mutators in SACCHAROMYCES CEREVISIAE: MUT1–1, MUT1–2 and MUT2–1

The purpose of this study was to characterize two mutator stocks of yeast which were induced and selected on the basis of high spontaneous reversion rates of the suppressible "ochre" nonsense allele lys1-1. In the mutator stock VA-3, a single mutation, designated mut1-1, is responsible for the increase in the reversion rate of the ochre alleles lys1-1 and arg4-17. In stock VA-105, there are two separate mutator mutations. Tetrad analysis data showed these two loci are loosely linked. Based on complementation data, one of these mutations is at the same locus as mut1-1 and designated mut1-2. The second mutator of stock VA-105 was designated mut2-1. All three mutators are recessive. Both mut1-1 and mut1-2 give a high mutation rate for ochre nonsense suppressor (SUP) loci, but not for the ochre nonsense alleles. On the contrary, the mutation rates of the ochre alleles are greatly reduced. With the mutant mut2-1 there were mutations at both the lys1-1 site and its suppressors; mut2-1 is as effective as mut1-2 but not as effective as mut1-1 in inducing reversions of a missense mutant, his1-7. Neither mut1-1, mut1-2 nor mut2-1 were effective in inducing reversions of a putative frameshift mutation, hom3-10, or in inducing forward mutations to canavanine resistance.     

Studies on the bacteriophage MS2: XLI. Nature of the azure mutation

Azure (or reverse amber) mutants grow normally on wild-type Escherichia coli but not on host strains harbouring a strong UAG suppressor mutation. Three different bacteriophage MS2 azure mutants obtained by treatment with nitrous acid have been characterized at the nucleotide sequence level. The 3′-end fragment of the ³²P-labelled mutant genomes was isolated by DNA:RNA hybridization and treatment with nuclease S₁, and was analyzed by mini-fingerprinting of the RNA. It is known that the wild-type MS2 polymerase gene ends with a UAG codon, followed seven triplets further by an in-phase UAA triplet. All three azure mutants contained an A → G transition in this UAA second stop codon of the polymerase gene, resulting in a second suppressible UAG (amber) codon. Analysis of revertants demonstrated that the azure mutation can be counteracted either by a true site reversion at the second stop or by the creation of a new stop signal for the polymerase gene, either UAA (ochre) or UGA (opal), before or at the first stop, or beyond the second stop. On the basis of these results, a mechanism for the azure mutation is proposed. Silent mutations (one in the coding region, three in the untranslated 3′-terminal sequence) have also been observed in these phage stocks.     

Mutation at tyrosine in AMLRY (GILRY like) motif of yeast eRF1 on nonsense codons suppression and binding affinity to eRF3

Termination translation in Saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. Two regions in human eRF1, position at 281-305 and position at 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed and characterized yeast eRF1 mutant at position 410 (correspond to 415 human eRF1) from tyrosine to serine residue resulting eRF1(Y410S). The mutations did not affect the viability and temperature sensitivity of the cell. The stop codons suppression of the mutant was analyzed in vivo using PGK-stop codon-LACZ gene fusion and showed that the suppression of the mutant was significantly increased in all of codon terminations. The suppression on UAG codon was the highest increased among the stop codons by comparing the suppression of the wild type respectively. In vitro interaction between eRF1 (mutant and wild type) to eRF3 were carried out using eRF1-(His)6 and eRF1(Y410S)-(His)6 expressed in Escherichia coli and indigenous Saccharomyces cerevisiae eRF3. The results showed that the binding affinity of eRF1(Y410S) to eRF3 was decreased up to 20% of the wild type binding affinity. Computer modeling analysis using Swiss-Prot and Amber version 9.0 programs revealed that the overall structure of eRF1(Y410S) has no significant different with the wild type. However, substitution of tyrosine to serine triggered the structural change on the other motif of C-terminal domain of eRF1. The data suggested that increasing stop codon suppression and decreasing of the binding affinity of eRF1(Y410S) were probably due to the slight modification on the structure of the C-terminal domain.     

Identification and functional characterization of isocitrate dehydrogenase 1 (IDH1) mutations in thyroid cancer

Mutations in the genes for isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) have been recently identified in glioblastoma. In the present study, we investigated IDH1 and IDH2 mutations in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC), with the latter, like glioblastoma, having a rapidly aggressive and lethal clinical course. By direct genomic DNA sequencing, we analyzed exon 4 of the IDH1 and IDH2 genes that harbored the mutation hot spots codon 132 and 172 of the two genes in glioblastoma, respectively, in 12 thyroid cancer cell lines, 20 FTC, and 18 ATC tumor samples. A novel homozygous G367A IDH1 mutation, resulting in a G123R amino acid change in codon 123, was identified in a case of ATC. A previously described IDH1 V71I mutation was found in a case of FTC and a case of ATC and no mutations were found in the cell lines. The overall prevalence of mutations was thus 1/20 (5%) in FTC and 2/18 (11%) in ATC. We did not find mutation in the IDH2 gene in these thyroid cancer cell lines and tumor samples. Sequence alignment analysis of 16 species revealed that the novel IDH1 G123R mutation was located in a highly conserved region, raising the possibility of a serious functional consequence as could also be predicted by the occurrence of a positively charged amino acid from this mutation. To test this, we created a G123R mutant by site-directed mutagenesis and demonstrated a decreased enzymatic activity of IDH1, similar to the expected reduction in the enzymatic activity of the previously described R132H IDH1 mutant measured as a control. Thus, functionally relevant IDH1 mutations can also occur in thyroid cancer, particularly ATC, suggesting a potential tumorigenic role of the IDH1 system that could represent a new therapeutic target for thyroid cancer.     

Biochemical Characterization of Pathogenic Mutations in Human Mitochondrial Methionyl-tRNA Formyltransferase

N-Formylation of initiator methionyl-tRNA (Met-tRNA^Met) by methionyl-tRNA formyltransferase (MTF) is important for translation initiation in bacteria, mitochondria, and chloroplasts. Unlike all other translation systems, the metazoan mitochondrial system is unique in using a single methionine tRNA (tRNA^Met) for both initiation and elongation. A portion of Met-tRNA^Met is formylated for initiation, whereas the remainder is used for elongation. Recently, we showed that compound heterozygous mutations within the nuclear gene encoding human mitochondrial MTF (mt-MTF) significantly reduced mitochondrial translation efficiency, leading to combined oxidative phosphorylation deficiency and Leigh syndrome in two unrelated patients. Patient P1 has a stop codon mutation in one of the MTF genes and an S209L mutation in the other MTF gene. P2 has a S125L mutation in one of the MTF genes and the same S209L mutation as P1 in the other MTF gene. Here, we have investigated the effect of mutations at Ser-125 and Ser-209 on activities of human mt-MTF and of the corresponding mutations, Ala-89 or Ala-172, respectively, on activities of Escherichia coli MTF. The S125L mutant has 653-fold lower activity, whereas the S209L mutant has 36-fold lower activity. Thus, both patients depend upon residual activity of the S209L mutant to support low levels of mitochondrial protein synthesis. We discuss the implications of these and other results for whether the effect of the S209L mutation on mitochondrial translational efficiency is due to reduced activity of the mutant mt-MTF and/or reduced levels of the mutant mt-MTF.     

Development and validation of breeder-friendly KASPar markers for <Emphasis Type="Italic">er1</Emphasis>, a powdery mildew resistance gene in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

Powdery mildew of pea is caused by Erysiphe pisi DC and is a serious threat to pea (Pisum sativum L.) production throughout much of the world. Development and utilization of genetic resistance to powdery mildew is considered an effective and sustainable strategy to manage this disease. One gene, er1, conferring powdery mildew resistance, was previously cloned and sequenced, and the functional markers for each resistance allele were reported. Allele-specific DNA markers are efficient and powerful tools to facilitate crop improvement and new cultivar development in breeding programs. However, extensive application of these markers is limited by gel-associated obstacles. In this study, eight breeder-friendly kompetitive allele-specific PCR (KASPar) markers were developed to overcome the problems of gel-based markers and increase the efficiency of genotypic screening. In order to identify additional pea germplasm with powdery mildew resistance, these KASPar markers were deployed and used to genotype a pea collection derived from the USDA pea single-plant (PSP) collection. Simultaneously, a phenotypic screening and a genotypic validation using the corresponding gel-based functional markers were conducted on the PSP collection. One pea accession, PI 142775, was identified by both phenotyping and genotyping to carry the allele er1-1 for powdery mildew resistance, indicating that the KASPar assay is an efficient and robust tool for breeding for powdery mildew resistance.     

Characterization of mutation-induced changes in the maize (Zea mays L.) ADH1-1S1108 alcohol dehydrogenase

The homodimeric alcohol dehydrogenase gene product of maize (Zea mays L.)Adh1-1S1108 mutation was purified and compared with the parentalAdh1-1S enzyme. The mutant alcohol dehydrogenase activity had pH optima and substrate specificity similar to those of the parental enzyme, but exhibited somewhat increased and decreasedK _mvalues for acetaldehyde and NADH, respectively. The mutant enzyme was also markedly less stable than the enzyme from parental tissues to temperatures as low as 50°C. Sequence analysis of a polymerase chain reaction (PCR)-generated cDNA clone revealed a G-to-C mutation at position 406 and a C-to-T mutation at position 974. These would result in residue 103 of each protein subunit being changed from an alanine to a proline and residue 292 being changed from an alanine to a valine. Whether one or both of these changes in primary sequence is responsible for the altered substrate affinities and stability is not yet understood.     

Human HSPB1 mutation recapitulates features of distal hereditary motor neuropathy (dHMN) in Drosophila

Distal hereditary motor neuropathies (dHMN) are a group of inherited peripheral nerve disorders characterized by length-dependent motor neuron weakness and subsequent muscle atrophy. Missense mutations in the gene encoding small heat shock protein HSPB1 (HSP27) have been associated with hereditary neuropathies including dHMN. HSPB1 is a member of the small heat shock protein (sHSP) family characterized by a highly conserved α-crystallin domain that is critical to their chaperone activity. In this study, we modeled HSPB1 mutant-induced neuropathies in Drosophila using a human HSPB1^S135F mutant that has a missense mutation in its α-crystallin domain. Overexpression of the HSPB1 mutant produced no significant defect in the Drosophila development, however, a partial reduction in the life span was observed. Further, the HSPB1 mutant gene induced an obvious loss of motor activity when expressed in Drosophila neurons. Moreover, suppression of histone deacetylase 6 (HDAC6) expression, which has critical roles in HSPB1 mutant-induced axonal defects, successfully rescued the motor defects in the HSPB1 mutant Drosophila model.