Identification of the <Emphasis Type="Italic">het-r</Emphasis> vegetative incompatibility gene of <Emphasis Type="Italic">Podospora anserina</Emphasis> as a member of the fast evolving <Emphasis Type="Italic">HNWD</Emphasis> gene family

In fungi, vegetative incompatibility is a conspecific non-self recognition mechanism that restricts formation of viable heterokaryons when incompatible alleles of specific het loci interact. In Podospora anserina, three non-allelic incompatibility systems have been genetically defined involving interactions between het-c and het-d, het-c and het-e, het-r and het-v. het-d and het-e are paralogues belonging to the HNWD gene family that encode proteins of the STAND class. HET-D and HET-E proteins comprise an N-terminal HET effector domain, a central GTP binding site and a C-terminal WD repeat domain constituted of tandem repeats of highly conserved WD40 repeat units that define the specificity of alleles during incompatibility. The WD40 repeat units of the members of this HNWD family are undergoing concerted evolution. By combining genetic analysis and gain of function experiments, we demonstrate that an additional member of this family, HNWD2, corresponds to the het-r non-allelic incompatibility gene. As for het-d and het-e, allele specificity at the het-r locus is determined by the WD repeat domain. Natural isolates show allelic variation for het-r. Sequence data reported here are available in the Genbank database under accession numbers FJ269240 and FJ269239 for het-r and het-R, respectively.     

Identification and characterization of a novel <Emphasis Type="Italic">Tritimovirus</Emphasis> species isolated from wild <Emphasis Type="Italic">Trisetum flavescens</Emphasis> L., family <Emphasis Type="Italic">Poaceae</Emphasis>

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3′ end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> and <Emphasis Type="Italic">cagA</Emphasis> and <Emphasis Type="Italic">vacA</Emphasis> gene status in children from Brazil with chronic gastritis

Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1-16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

Association analysis of <Emphasis Type="Italic">SLC22A4</Emphasis>, <Emphasis Type="Italic">SLC22A5</Emphasis> and <Emphasis Type="Italic">DLG5</Emphasis> in Japanese patients with Crohn disease

Crohn disease (CD) is an inflammatory bowel disease characterized by chronic transmural, segmental, and typically granulomatous inflammation of the gut. Recently, two novel candidate gene loci associated with CD, SLC22A4 and SLC22A5 on chromosome 5 known as IBD5 and DLG5 on chromosome 10, were identified through association analysis of Caucasian CD patients. We validated these candidate genes in Japanese patients with CD and found a weak but possible association with both SLC22A4 (P=0.028) and DLG5 (P=0.023). However, the reported genetic variants that were indicated to be causative in the Caucasian population were completely absent in or were not associated with Japanese CD patients. These findings imply significant differences in genetic background with CD susceptibility among different ethnic groups and further indicate some difficulty of population-based studies.     

Repeat induced point mutation in two asexual fungi, <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Penicillium </Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis>

Repeat induced point mutation (RIP) is a gene silencing mechanism present in fungal genomes. During RIP, duplicated sequences are efficiently and irreversibly mutated by transitions from C:G to T:A. For the first time, we have identified traces of RIP in transposable elements of Aspergillus niger and Penicillium chrysogenum, two biotechnologically relevant fungi. We found that RIP in P. chrysogenum has affected a large set of sequences, which also contain other mutations. On the other hand, RIP in A. niger is limited to only few sequences, but literally all mutations are RIP-like. Surprisingly, RIP occurred only in transposon sequences that have disrupted open reading frames in A. niger, a phenomenon not yet reported for other fungi. In both fungal species, we identified two sequences with strong sequence similarity to Neurospora crassa RID. RID is a putative DNA methyltransferase and the only known enzyme involved in the RIP process. Our findings suggest that both A. niger and P. chrysogenum either had a sexual past or have a sexual potential. These findings have important implications for future strain development of these fungi.     

<Emphasis Type="Italic">MECP2 </Emphasis>and <Emphasis Type="Italic">CDKL5</Emphasis> gene mutation analysis in Chinese patients with Rett syndrome

Rett syndrome (RTT) is a progressive neurodevelopmental disorder that is caused by mutations in the X-linked methyl-CpG-binding protein2 (MECP2) gene. In this study, the MECP2 sequences in 121 unrelated Chinese patients with classical or atypical RTT were screened for deletions and mutations. In all, we identified 45 different MECP2 mutations in 102 of these RTT patients. The p. T158M mutation (15.7%) was the most common, followed in order of frequency by p. R168X (11.8%), p. R133C (6.9%), p. R270X (6.9%), p. G269fs (6.9%), p. R255X (4.9%), and p. R306C (3.9%). In addition, we identified five novel MECP2 mutations: three missense (p. K305E, p. V122M, p. A358T), one insertion (c.45-46insGGAGGA), and one 22 bp deletion (c.881-902del22). Large deletions represented 10.5% of all identified MECP2 mutations. Conversely, mutations in exon 1 appeared to be rare (0.9%). The remaining cases without MECP2 mutations were screened for the cyclin-dependent kinase-like 5 (CDKL5) gene using denaturing high-performance liquid chromatography (DHPLC). One synonymous mutation (p. I72I) was found in exon 5, suggesting that CDKL5 is a rare cause of RTT. The overall MECP2 mutation detection rate for this patient series was 84.3:87.9% in 107 classical RTT cases and 57.1% in 14 atypical RTT cases. Moreover, there were two patients with homozygous mutations and normal female karyotypes. However, we did not pinpoint a significant relationship between genotype and phenotype in these cases.     

Functional and structural characterisation of <Emphasis Type="Italic">Ag</Emphasis>MNPV <Emphasis Type="Italic">ie1</Emphasis>

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the −492 and −357 versions contains sequence motifs important for the level of the lacZ reporter gene expression. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. The GenBank accession number of the sequence reported in this paper is AF368905.     

Behaviour of <Emphasis Type="Italic">Sinapis alba</Emphasis> chromosomes in a <Emphasis Type="Italic">Brassica napus</Emphasis> background revealed by genomic <Emphasis Type="Italic">in-situ</Emphasis> hybridization

Genomic in-situ hybridization (GISH) was applied to study the behaviour of addition chromosomes in first and second backcross (BC) progenies of hybrids between Brassica napus ssp. napus L. (AACC, 2n = 38) and Sinapis alba L. (SS, 2n = 24) produced by electrofusion. With GISH using genomic DNA of S. alba was used as probe it was possible to clearly distinguish both of the parental genomes and effectively monitor the fate of S. alba chromosomes in the BC₁ and BC₂ progenies. GISH analysis confirmed the sesquidiploid genome composition (AACCS) of the BC₁ progenies, which contained 38 chromosomes from B. napus and 12 chromosomes from S. alba. Genome painting in the pollen mother cells (PMCs) of the BC₁ plants revealed intergenomic association between B. napus and S. alba chromosomes, whereby a maximum of 4 trivalents between AC and S chromosomes were identified at metaphase I. In the BC₂ progenies, aneuploids with different numbers of additional chromosomes from S. alba, ranging from 1 to 7, were confirmed. Three putative monosomic alien addition lines were characterized, and the results are discussed with respect to the potential for intergenomic chromosome recombination.     

Effect of bioactive fractions of <Emphasis Type="Italic">Citrullus vulgaris</Emphasis> Schrad. leaf extract against <Emphasis Type="Italic">Anopheles stephensi</Emphasis> and <Emphasis Type="Italic">Aedes aegypti</Emphasis>

The benzene extract of Citrullus vulgaris was tested against Anopheles stephensi and Aedes aegypti for the larvicidal activity and ovicidal properties. The crude benzene extract was found to be more effective against A. stephensi than A. aegypti. The LC₅₀ values were 18.56 and 42.76 ppm respectively. The LC₅₀ values for silica gel fractions (bioactive fractions I, II, III and IV) were 11.32, 14.12, 14.53 and 16.02 ppm respectively. The mean per cent hatchability of the egg rafts were observed after 48 h post treatment. The crude extract of benzene exerted 100% mortality at 250 ppm against A. stephensi and at 300 ppm against A. aegypti. The silica gel fractions I and II afforded 100% mortality at 100 ppm and III and IV exerted the hatchability rate of 4.9 and 5.3% at the same concentration against A. stephensi.     

Association of positional and functional candidate genes<Emphasis Type="Italic"> FGF1</Emphasis>,<Emphasis Type="Italic"> FBN2</Emphasis>, and<Emphasis Type="Italic"> LOX</Emphasis> on 5q31 with intracranial aneurysm

We previously performed a genome-wide linkage study of intracranial aneurysm (IA) and found positive evidence of linkage at chromosomes 5q22–31, 7q11, and 14q22. In the present study, we focus on 5q31, where three candidate genes, fibroblast growth factor 1 (FGF1), fibrillin 2 (FBN2), and lysyl oxidase gene (LOX) lie, and evaluate associations with IA. Genomic DNAs were obtained from 172 IA patients and 192 controls. Association analysis was performed with ten, five, and four single-nucleotide polymorphisms (SNPs) identified in FGF1, FBN2, and LOX, respectively. A difference in allelic frequency was observed for only the SNP at intron 4 in FGF1 (χ²=4.44, df=1, P=0.035). Although a haplotype association was observed with the combination of ten SNPs in FGF1 (χ²=16.04, df=1, P=0.00006), significant haplotype associations were not observed when haplotypes were constructed with the three, two, and four SNPs in FGF1 according to the linkage disequilibrium structure. No associations of FBN2 and LOX with IA were detected in the present study.     

A frameshift mutation of the chloroplast <Emphasis Type="Italic">mat</Emphasis>K coding region is associated with chlorophyll deficiency in the <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> virescent mutant <Emphasis Type="Italic">Wogon</Emphasis>-Sugi

Wogon-Sugi has been reported as a cytoplasmically inherited virescent mutant selected from a horticultural variety of Cryptomeria japonica. Although previous studies of plastid structure and inheritance indicated that at least some mutations are encoded by the chloroplast genome, the causative gene responsible for the primary chlorophyll deficiency in Wogon-Sugi, has not been identified. In this study, we identified this gene by genomic sequencing of chloroplast DNA and genetic analysis. Chloroplast DNA sequencing of 16 wild-type and 16 Wogon-Sugi plants showed a 19-bp insertional sequence in the matK coding region in the Wogon-Sugi. This insertion disrupted the matK reading frame. Although an indel mutation in the ycf1 and ycf2 coding region was detected in Wogon-Sugi, sequence variations similar to that of Wogon-Sugi were also detected in several wild-type lines, and they maintained the reading frame. Genetic analysis of the 19 bp insertional mutation in the matK coding region showed that it was found only in the chlorophyll-deficient sector of 125 full-sibling seedlings. Therefore, the 19-bp insertion in the matK coding region is the most likely candidate at present for a mutation underlying the Wogon-Sugi phenotype. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An autophagy gene, <Emphasis Type="Italic">MgATG5</Emphasis>, is required for cell differentiation and pathogenesis in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Autophagy is a conserved degradation pathway that is involved in the maintenance of normal cell differentiation and development. The Saccharomyces cerevisiae ATG5 gene is an important component of the autophagy process. In this study, we identified MgATG5 as an autophagy-related gene in Magnaporthe oryzae that is homologous to ATG5. Using targeted gene replacement, an Mgatg5∆ mutant was generated and fungal autophagy was blocked. Cytological analysis revealed that the mutant had poor fungal morphogenic development, including a shortened aerial hyphae lifespan, decreased conidiation and perithecia formation, delayed conidial germination and appressorial formation, postponement of conidial cytoplasm transfer during appressorium formation, and reduction in formation of the penetration peg. Turnover of endogenous matter in the Mgatg5∆ mutant was also affected, as demonstrated by defects in the formation of conidial lipid droplets, and in the degradation of conidial glycogen deposits during appressorium formation. Lipid droplets and glycogen are necessary to generate adequate turgor in appressoria for invading the host surface. As a result of the decreased appressorium turgor and differentiation in the penetration peg, Mgatg5∆ pathogenicity was deficient in two host plants tested. The developmental and pathogenic phenotypes were restored by the introduction of an intact copy of MgATG5 into Mgatg5∆, demonstrating that the MgATG5 deletion was responsible for the cellular defects. Taken together, these findings suggest that autophagy promotes cell differentiation through turnover of endogenous matter during fungal development, and is thus essential for the pathogenicity of the rice blast fungus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J.-P. Lu and X.-H. Liu contributed equally to this work and are regarded joint first authors.     

Tracing origin of serrated adenomas with <Emphasis Type="Italic">BRAF</Emphasis> and <Emphasis Type="Italic">KRAS</Emphasis> mutations

Serrated neoplasm of the colorectum raised many as-yet unanswered issues. To characterize serrated neoplasia pathway, we investigated BRAF and KRAS mutations in 35 traditional serrated adenomas. BRAF exons 11 and 15, and KRAS exon 2 were amplified by polymerase chain reaction and directly sequenced. BRAF V599E mutation was found in 27 serrated adenomas (77.1%), and KRAS mutations were found in 3 (8.6%) of 35 traditional serrated adenomas. In 13 cases, mixed polyps composed of traditional serrated adenomas and hyperplastic (serrated) polyps were observed, and seven of them showed the same BRAF mutations in both components. Somatic mutations of BRAF and KRAS genes were mutually exclusive. These findings suggest that BRAF mutations are early and a critical event in the serrated adenomas, and most serrated adenomas in both sides of colon may progress from microvesicular hyperplastic polyps via BRAF mutations, and some left-sided serrated adenomas develop via KRAS mutations.