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">P</Emphasis>. <Emphasis Type="Italic">aeruginosa</Emphasis> Biofilms in CF Infection

Pseudomonas aeruginosa is an opportunistic pathogen of immunocompromised hosts. In cystic fibrosis (CF), P. aeruginosa causes acute and chronic lung infections that result in significant morbidity and mortality. P. aeruginosa possesses several traits that contribute to its ability to colonize and persist in acute and chronic infections. These include high resistance to antimicrobials, ability to form biofilms, plethora of virulence products, and metabolic versatility. In P. aeruginosa, a cell-to-cell communication process termed quorum sensing (QS) regulates many of these factors that contribute to its pathogenesis. Recent evidence suggests that the CF lung environment presents a specialized niche for P. aeruginosa. The relationship of P. aeruginosa QS, biofilm formation, and the CF lung environment is discussed.     

How heterologously expressed <Emphasis Type="Italic">Escherichia coli</Emphasis> genes contribute to understanding DNA repair processes in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

DNA-damaging agents constantly challenge cellular DNA; and efficient DNA repair is therefore essential to maintain genome stability and cell viability. Several DNA repair mechanisms have evolved and these have been shown to be highly conserved from bacteria to man. DNA repair studies were originally initiated in very simple organisms such as Escherichia coli and Saccharomyces cerevisiae, bacteria being the best understood organism to date. As a consequence, bacterial DNA repair genes encoding proteins with well characterized functions have been transferred into higher organisms in order to increase repair capacity, or to complement repair defects, in heterologous cells. While indicating the contribution of these repair functions to protection against the genotoxic effects of DNA-damaging agents, heterologous expression studies also highlighted the role of the DNA lesions that are substrates for such processes. In addition, bacterial DNA repair-like functions could be identified in higher organisms using this approach. We heterologously expressed three well characterized E. coli repair genes in S. cerevisiae cells of different genetic backgrounds: (1) the ada gene encoding O⁶-methylguanine DNA-methyltransferase, a protein involved in the repair of alkylation damage to DNA, (2) the recA gene encoding the main recombinase in E. coli and (3) the nth gene, the product of which (endonuclease III) is responsible for the repair of oxidative base damage. Here, we summarize our results and indicate the possible implications they have for a better understanding of particular DNA repair processes in S. cerevisiae.     

Functional analysis of <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> carboxylic acids permeases: heterologous expression of <Emphasis Type="Italic">KlJEN1</Emphasis> and <Emphasis Type="Italic">KlJEN2</Emphasis> genes

The present work describes a detailed physiological and molecular characterization of the mechanisms of transport of carboxylic acids in Kluyveromyces lactis. This yeast species presents two homologue genes to JEN1 of Saccharomyces cerevisiae: KlJEN1 encodes a monocarboxylate permease and KlJEN2 encodes a dicarboxylic acid permease. In the strain K. lactis GG1888, expression of these genes does not require an inducer and activity for both transport systems was observed in glucose-grown cells. To confirm their key role for carboxylic acids transport in K. lactis, null mutants were analyzed. Heterologous expression in S. cerevisiae has been performed and chimeric fusions with GFP showed their proper localization in the plasma membrane. S. cerevisiae jen1Δ cells transformed with KlJEN1 recovered the capacity to use lactic acid, as well as to transport labeled lactic acid by a mediated mechanism. When KlJEN2 was heterologously expressed, S. cerevisiae transformants gained the ability to transport labeled succinic and malic acids by a mediated mechanism, exhibiting, however, a poor growth in malic acid containing media. The results confirmed the role of KlJen1p and KlJen2p as mono and dicarboxylic acids permeases, respectively, not subjected to glucose repression, being fully functional in S. cerevisiae. O. Queirós and L. Pereira contributed equally to this work.     

Age-Dependent Stability of Sensorimotor Functions in the Life-Extended <Emphasis Type="Italic">Drosophila</Emphasis> mutant <Emphasis Type="Italic">Methuselah</Emphasis>

Methuselah is a Drosophila mutant with a 35% increased lifespan. We examined the robustness of methuselah’s sensorimotor abilities in tethered flight as a function of age in experiments designed to test visuomotor synchronization and phototaxis in simulated flight. A total of 282 flies from different age groups (4 hours to 70 days) and genotypes (mth and w1118) were individually tethered under an infrared laser-sensor system that digitally recorded wing-beat frequency (WBF). We found that mth has a higher average WBF throughout most of its lifespan compared to parental control flies (w1118) and develops flight ability at a younger age. Its WBF at late life, however, is not significantly different than that of its parental control line. We further found that mth entrains during flight to motion of a visual grating significantly better than its parental line. These findings suggest that the mth gene not only delays chronological aging but enhances sensorimotor abilities critical to survival during early and middle, but not late life. Edited by Yong-Kyu Kim     

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.     

Biochemical and molecular characterization of a putative endoglucanase in <Emphasis Type="Italic">Magnaporthe</Emphasis><Emphasis Type="Italic">grisea</Emphasis>

Microbial pathogens secrete an array of cell wall-degrading enzymes to break down the structure of the host cell wall to facilitate colonization of the host tissue. To better understand their role in the pathogenesis, a putative endoglucanase from Magnaporthe grisea was characterized in this paper. SignalP-3.0 analysis indicates that the protein encoded by gene MGG_02532.5 in M. grisea (named MgEGL1 for M . g risea endoglucanase 1 ) contains a secretory signal peptide. Multiple alignment shows that MgEGL1 has high level of homology to endoglucanases (EC 3.1.1.4) from Aspergillus nidulans and Trichoderma reesei. The three proteins share a conserved catalytic domain, but only the one from T. reesei contains a cellulose binding module. MgEGL1 was constitutively expressed with the highest level in mycelia and the lowest in conidia. Interestingly, the MgEGL1 RNA could be alternatively processed when cultured in vitro and after infection of rice. Expression analysis confirmed that the MgEGL1 is a secreted protein. Its endoglucanase activity was assayed by Congo red plates, and further confirmed by the dinitrosalicylic acid method. The finding in this paper will provide the basis for further determination of the biochemical properties of the endoglucanase protein and its relevant function in fungal pathogenesis.     

Effect of bacterial symbionts <Emphasis Type="Italic">Xenorhabdus</Emphasis> on mortality of infective juveniles of two <Emphasis Type="Italic">Steinernema</Emphasis> species

Steinernema species are entomopathogenic nematodes associated with Xenorhabdus bacteria. The life cycle of these associations is composed of two stages: (1) a free stage in the soil, where infective juveniles (IJs), which carry bacteria in their guts, search for new insect hosts; and (2) a parasitic stage, where the IJs infect insects, release their Xenorhabdus symbionts and reproduce in order to produce new IJs. Previous studies clearly showed benefits to the association for several Steinernema species during the parasitic stage. Nevertheless, no study has so far explored, during the free stage, the existence of costs or benefits to the association for different Steinernema. Here, we compared the survival of both symbiotic and aposymbiotic IJs in two nematode species: (1) Steinernema carpocapsae-exhibiting IJs that carry a high number of Xenorhabdus cells in their guts; and (2) its closely relative species, S. scapterisci-exhibiting IJs, that carry very few Xenorhabdus cells in their guts. We showed that the bacterial symbionts were costly for S. carpocapsae by increasing IJs’ mortality but not for S. scapterisci. This difference in cost induced by bacteria to IJs during the free stage could be correlated with the difference in the numbers of bacteria carried by IJs of each nematode species.     

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.     

Characterization of <Emphasis Type="Italic">p</Emphasis>24 Gene of <Emphasis Type="Italic">Spodoptera litura</Emphasis> Multicapsid Nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) p24 gene is 753 bp long, potentially encoding 244 amino acids with a predicted molecular weight of 27.3 kDa. Homology analysis indicated that SpltMNPV P24 has 20–36% amino acid identity with that of other known baculoviruses. RT-PCR results showed that the p24 gene is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (ATAAG). Western blot analysis of extracts from SpltMNPV-infected S. litura cells detected a specific 28 kDa protein, and this protein was not N-glycosylated. Structural localization revealed that SpltMNPV P24 was associated with the nucleocapsid of occlusion-derived virus (ODV) as a complex form of 83 kDa.     

Modulation of soleus <Emphasis Type="Italic">H</Emphasis>-reflexes during gait in healthy children

During locomotion spinal short latency reflexes are rhythmically modulated and depressed compared to rest. In adults this modulation is severely disturbed after bilateral spinal lesions indicating a role for supra-spinal control. Soleus reflex amplitudes are large in the stance phase and suppressed in the swing phase contributing to the reciprocal muscle activation pattern required for walking. In early childhood the EMG pattern during gait underlies an age-dependent process changing from co-contraction of agonists and antagonists to a reciprocal pattern at the age of 5–7 years. It is unknown whether at this stage apart from the EMG also reflexes are modulated, and if so, whether the reflex modulation is fully mature or still underlies an age-dependent development. This may give important information about the maturation of CNS structures involved in gait control. Soleus Hoffmann H-reflexes were investigated in 36 healthy children aged 7–16 years during treadmill walking at 1.2 km/h and 3.0 km/h. At 7 years old a rhythmic modulation similar to adults was observed. The H-reflex size during the stance phase decreased significantly with age while the maximum H-reflex (H _max) at rest remained unchanged. At 3.0 km/h H-reflexes were significantly larger during the stance phase and smaller during the swing phase as compared to 1.2 km/h but the age-dependent suppression was observed at both walking velocities. In conclusion H-reflex modulation during gait is already present in young children but still underlies an age-dependent process independent of the walking velocity. The finding that the rhythmic part of the modulation is already present at the age of 7 years may indicate that the supra-spinal structures involved mature earlier than those involved in the tonic reflex depression. This may reflect an increasing supra-spinal control of spinal reflexes under functional conditions with maturation.     

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.     

Mitochondrial genome sequences and comparative genomics of <Emphasis Type="Italic">Phytophthora ramorum</Emphasis> and <Emphasis Type="Italic">P. sojae</Emphasis>

The sequences of the mitochondrial genomes of the oomycetes Phytophthora ramorum and P. sojae were determined during the course of complete nuclear genome sequencing (Tyler et al., Science, 313:1261,2006). Both mitochondrial genomes are circular mapping, with sizes of 39,314 bp for P. ramorum and 42,977 bp for P. sojae. Each contains a total of 37 recognizable protein-encoding genes, 26 or 25 tRNAs (P. ramorum and P. sojae, respectively) specifying 19 amino acids, six more open reading frames (ORFs) that are conserved, presumably due to functional constraint, across Phytophthora species (P. sojae, P. ramorum, and P. infestans), six ORFs that are unique for P. sojae and one that is unique for P. ramorum. Non-coding regions comprise about 11.5 and 18.4% of the genomes of P. ramorum and P. sojae, respectively. Relative to P. sojae, there is an inverted repeat of 1,150 bp in P. ramorum that includes an unassigned unique ORF, a tRNA gene, and adjacent non-coding sequences, but otherwise the gene order in both species is identical. Comparisons of these genomes with published sequences of the P. infestans mitochondrial genome reveals a number of similarities, but the gene order in P. infestans differed in two adjacent locations due to inversions and specific regions of the genomes exhibited greater divergence than others. For example, the breakpoints for the inversions observed in P. infestans corresponded to regions of high sequence divergence in comparisons between P. ramorum and P. sojae and the location of a hypervariable microsatellite sequence (eight repeats of 24 bp) in the P. sojae genome corresponds to a site of major length variation in P. infestans. Although the overwhelming majority of each genome is conserved (81–92%), there are a number of genes that evolve more rapidly than others. Some of these rapidly evolving genes appear specific to Phytophthora, arose recently, and future evaluation of their function and the effects of their loss could prove fruitful for understanding the phylogeny of these devastating plant pathogens. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

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).     

The presence of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> in isolates of <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> impacts significantly on DNA fingerprinting results

The genetic characterization of Trichomonas vaginalis (Protista: Trichomonadidae), the causative agent of trichomoniasis in humans, is central to understanding the epidemiology, treatment, drug resistance, and virulence as well as the diagnosis and control of this parasite. Various molecular approaches, including DNA fingerprinting, have been employed for this purpose, and random amplification of polymorphic DNA (RAPD) continues to be utilized. However, little attention has been paid to the fact that some T. vaginalis populations can harbor symbiotic Mycoplasma hominis and/or other agents, which could cause artifacts in the RAPD results. In the present study, we demonstrate clearly that the presence of M. hominis from T. vaginalis isolates impacts significantly on RAPD results and on the subsequent analyses and interpretation of data sets. Moreover, symbiotic M. hominis displays an isolate-to-isolate variability in RAPD profile before elimination, suggesting a variability of M. hominis infection. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.