Mutagenicity of herpes simplex virus type 1 in a shuttle vector

The shuttle vector plasmid pZ189 was used to find the kinds of mutations that are induced by herpes simplex virus type-1 (HSV-1). In cells infected by HSV-1 the frequency of mutation in supF gene, the mutagenesis marker, was increased over background by from two- to seven-fold, reaching 0.14-0.45%. No increase was induced by infection by vaccinia virus under the same conditions. Mutagenesis was an early event, showing a four-fold increase in mutation frequency at only two hours after infection, and peaking at a seven-fold increase at four hours after infection. DNA sequencing and gel electrophoresis analysis were performed on 105 HSV-1 induced mutants and 65 spontaneous mutants and provided the following information: (1) A change in plasmid size was seen in 54% of HSV-1 related mutants, compared with only 37% of spontaneous mutants. (2) Among point mutations, the predominant type was G:C to A:T transition, which accounted for 51% of point mutations in mutants isolated from cells infected with HSV-1, and 32% of point mutations in spontaneous mutants. (3) Deletions of DNA were seen in HSV-1 related mutants at a frequency of 40%, compared with 29% in spontaneous mutants. The HSV-1 related deletions were about half the length of spontaneous mutants and three contained short filler sequences. (4) Fifteen (15%) of HSV-1 induced mutants revealed the altered restriction patterns on agarose gel electrophoresis analysis and were due either to rearrangements of plasmid DNA, and/or to insertion of sequences derived from chromosomal DNA (seven plasmids). No insertions of DNA from HSV-1 were detected. Among spontaneous mutants, only 5 (7.7%) were rearrangements and none had inserted chromosomal DNA. (5) DNA sequence analysis of seven plasmids with inserted chromosomal DNA revealed that four cases had repetitive DNA sequences integrated and the other three were unidentified sequences from the GenBank database. Three repetitive DNA included $\alpha$ satellite, Alu and KpnI family sequences. The other sequence was identified as tRNA-like component. The observed mutations have implications for the mechanism of malignant transformation of cells by HSV-1. ^

Differential gene expression in multistep tumorigenesis using an {\it in vitro\/} human cell model

Using a human terato-carcinoma cell line, PA-1, the functional role of the oncogenes and tumor suppressor gene involved in the multistep process of carcinogenesis have been analyzed. The expression of AP-2 was strongly correlated with the susceptibility to ras transformation. The differential responsiveness to growth factors between stage 1 ras resistant cells and stage 2 ras susceptible cells was observed, indicating that the ability of stage 2 cells to respond to the mutated ras oncogenes in transformation correlated with the ability to be stimulated by certain growth factors. Using differential screening of cDNA libraries, a number of differentially expressed cDNA clones was isolated. One of those, clone 12, is overexpressed in ras transformed stage 3 cells. The amino acid sequence of clone 12 is almost identical to a mouse LLrep3 gene that was growth-regulated, and 78% similar to a yeast ribosomal protein S4. These results suggest that the S4 gene may be involved in regulation of growth. Clone 9 is expressed in stage 1 ras resistant cells (3.5-kb and 3.0-kb transcripts) but the expression of this clone in stage 2 ras susceptible cells and stage 3 ras-transformed cells is greatly diminished. The expression of this cDNA clone was increased to at least five fold in ras resistant cells and nontumorigenic hybrids treated with retinoic acid but not increased in retinoic acid treated ras susceptible cells, ras transformed cells and the tumorigenic segregants. Partial sequence of this clone showed no homology to the sequences in Genbank. These findings suggest that clone 9 could be a suppressor gene or the genes that are involved in the biochemical pathway of tumor suppression or neurogenic differentiation. The apparent pleiotropic effect of the loss of this suppressor gene function support Harris' proposal that tumor suppressor genes regulate differentiation. The tumor suppressor gene may act as negative regulator of tumor growth by controlling gene expression in differentiation. ^

The evolution of paired domains

Pax genes are important developmental control genes. They are involved in nervous system development, organogenesis and oncogenesis. A DNA specific binding domain called the paired domain, which is well conserved during evolution, defines Pax genes. Furthermore, Pax genes are also conserved in terms of their functions. For example, the Pax-6 gene has been showed to be one of the master control genes for eye development both in Drosophila and vertebrates. All of these properties of Pax genes make them an excellent model for studying the evolution of gene function. ^ Molecular evolutionary studies of paired domain are carried out in this study. Five Pax genes from cnidarians, which are the most primitive organisms possessing a nervous system, were isolated and characterized for their DNA binding properties. By combining data obtained from Genbank and this study, the phylogenetic relationship between Pax genes was studied. It was found that Pax genes could be divided into five groups: Pax-1/9, Pax-3 /7, Pax-A, Pax-2/5/ 8/B, and Pax- 4/6. Furthermore, Pax-2/5/8/ B, Pax-A and Pax-4/6 could be clustered into a supergroup I, while Pax-1/9 and Pax-3/7 could be clustered into supergroup II. The phylogeny was also supported by studies on DNA binding properties of paired domains from different groups. A statistical method was applied to infer the critical amino acid residue substitutions between two supergroups and within the supergroup I. It was found that two amino acid residues were mainly responsible for the difference of DNA binding between two supergroups, while only one amino acid was critical for the evolution of novel DNA binding properties of Pax-4/6 group from ancestor. Evolutionary implications of these data are also discussed. ^

Dehydrin expression as a potential diagnostic tool for cold stress in white clover

Cold acclimation is important for crop survival in environments undergoing seasonal low temperatures. It involves the induction of defensive mechanisms including the accumulation of different cryoprotective molecules among which are dehydrins (DHN). Recently several sequences coding for dehydrins were identified in white clover (Trifolium repens). This work aimed to select the most responsive to cold stress DHN analogues in search for cold stress diagnostic markers. The assessment of dehydrin transcript accumulation via RT-PCR and immunodetection performed with three antibodies against the conserved K-, Y-, and S-segment allowed to outline different dehydrin types presented in the tested samples. Both analyses confirmed that YnKn dehydrins were underrepresented in the controls but exposure to low temperature specifically induced their accumulation. Strong immunosignals corresponding to 37–40 kDa with antibodies against Y- and K-segment were revealed in cold-stressed leaves. Another ‘cold-specific’ band at position 52–55 kDa was documented on membranes probed with antibodies against K-segment. Real time RT-qPCR confirmed that low temperatures induced the accumulation of SKn and YnSKn transcripts in leaves and reduced their expression in roots. Results suggest that a YnKn dehydrin transcript with GenBank ID: KC247805 and the immunosignal at 37–40 kDa, obtained with antibodies against Y- and K-segment are reliable markers for cold stress in white clover. The assessment of SKn (GenBank ID: EU846208) and YnSKn (GenBank ID: KC247804) transcript levels in leaves could serve as additional diagnostic tools.

A genomic perspective on a new bacterial genus and species from the Alcaligenaceae family, Basilea psittacipulmonis

BACKGROUND A novel Gram-negative, non-haemolytic, non-motile, rod-shaped bacterium was discovered in the lungs of a dead parakeet (Melopsittacus undulatus) that was kept in captivity in a petshop in Basel, Switzerland. The organism is described with a chemotaxonomic profile and the nearly complete genome sequence obtained through the assembly of short sequence reads. RESULTS Genome sequence analysis and characterization of respiratory quinones, fatty acids, polar lipids, and biochemical phenotype is presented here. Comparison of gene sequences revealed that the most similar species is Pelistega europaea, with BLAST identities of only 93% to the 16S rDNA gene, 76% identity to the rpoB gene, and a similar GC content (~43%) as the organism isolated from the parakeet, DSM 24701 (40%). The closest full genome sequences are those of Bordetella spp. and Taylorella spp. High-throughput sequencing reads from the Illumina-Solexa platform were assembled with the Edena de novo assembler to form 195 contigs comprising the ~2 Mb genome. Genome annotation with RAST, construction of phylogenetic trees with the 16S rDNA (rrs) gene sequence and the rpoB gene, and phylogenetic placement using other highly conserved marker genes with ML Tree all suggest that the bacterial species belongs to the Alcaligenaceae family. Analysis of samples from cages with healthy parakeets suggested that the newly discovered bacterial species is not widespread in parakeet living quarters. CONCLUSIONS Classification of this organism in the current taxonomy system requires the formation of a new genus and species. We designate the new genus Basilea and the new species psittacipulmonis. The type strain of Basilea psittacipulmonis is DSM 24701 (= CIP 110308 T, 16S rDNA gene sequence Genbank accession number JX412111 and GI 406042063).

Anaplasma phagocytophilum DNA amplified from lesional skin of seropositive dogs.

Canine granulocytic anaplasmosis (CGA) is caused by the rickettsial microorganism Anaplasma phagocytophilum. CGA is typically characterized by fever, thrombocytopenia, lethargy, anorexia, arthropy, and other nonspecific clinical signs. Skin lesions have been described in naturally infected lambs and humans. The pathophysiology of CGA is not entirely clear, and the persistence of the organism after the resolution of clinical signs has been described. The aim of the study was to investigate if A. phagocytophilum can be detected in canine lesional skin biopsies from A. phagocytophilum-seropositive dogs with etiologically unclear skin lesions that improved after the treatment with doxycycline. Paraffin-embedded lesional skin biopsies were allocated into separate groups: biopsies from A. phagocytophilum-seropositive dogs responsive to treatment with doxycycline (n=12), biopsies from A. phagocytophilum-seronegative dogs (n=2), and biopsies in which skin lesions histopathologically resembled a tick bite (n=10). The serological status of the latter group was unknown. Histology of the seropositive and seronegative dog skin lesions did not indicate an etiology. DNA was extracted, and a conventional PCR for partial 16S rRNA gene was performed. Anaplasma phagocytophilum DNA was amplified from 4/12 seropositive dogs' skin biopsies. All sequences were 100% identical to the prototype A. phagocytophilum human strain (GenBank accession number U02521). Anaplasma phagocytophilum was not amplified from the 2 seronegative and 10 suspected tick bite dogs. Serum antibody titers of the PCR-positive dogs ranged from 1:200 to 1:2048. Histopathologically, a mild-to-moderate perivascular to interstitial dermatitis composed of a mixed cellular infiltrate and mild-to-moderate edema was seen in all seropositive dogs. In 8/12 seropositive dogs, vascular changes as vasculopathy, fibrinoid necrosis of the vessel walls, and leukocytoclastic changes were observed. In summary, our results support the hypothesis that the persistence of A. phagocytophilum in the skin may be causative for otherwise unexplained skin lesions in seropositive dogs.

Genetic diversity of bovine Neospora caninum determined by microsatellite markers

Neospora caninum is one of the most significant parasitic organisms causing bovine abortion worldwide. Despite the economic impact of this infection, relatively little is known about the genetic diversity of this parasite. In this study, using Nc5 and ITS1 nested PCR, N. caninum has been detected in 12 brain samples of aborted fetuses from 298 seropositive dairy cattle collected from four different regions in Tehran, Iran. These specimen (Nc-Iran) were genotyped in multilocus using 9 different microsatellite markers previously described (MS4, MS5, MS6A, MS6B, MS7, MS8, MS10, MS12 and MS21). Microsatellite amplification was completely feasible in 2 samples, semi-completely in 8 samples, and failed in 2 samples. Within the two completely performed allelic profiles of Nc-Iran strains, unique multilocus profiles were obtained for both and novel allelic patterns were found in the MS8 and MS10 microsatellite markers. The Jaccard's similarity index showed significant difference between these two strains and from other standard isolates derived from GenBank such as Nc-Liv, Nc-SweB1, Nc-GER1, KBA1, and KBA2. All samples originating from the same area showed identical allelic numbers and a correlation between the number of repeats and geographic districts was observed.

Mining microsatellite markers from public expressed sequence tags databases for the study of threatened plants

Background Simple Sequence Repeats (SSRs) are widely used in population genetic studies but their classical development is costly and time-consuming. The ever-increasing available DNA datasets generated by high-throughput techniques offer an inexpensive alternative for SSRs discovery. Expressed Sequence Tags (ESTs) have been widely used as SSR source for plants of economic relevance but their application to non-model species is still modest. Methods Here, we explored the use of publicly available ESTs (GenBank at the National Center for Biotechnology Information-NCBI) for SSRs development in non-model plants, focusing on genera listed by the International Union for the Conservation of Nature (IUCN). We also search two model genera with fully annotated genomes for EST-SSRs, Arabidopsis and Oryza, and used them as controls for genome distribution analyses. Overall, we downloaded 16 031 555 sequences for 258 plant genera which were mined for SSRsand their primers with the help of QDD1. Genome distribution analyses in Oryza and Arabidopsis were done by blasting the sequences with SSR against the Oryza sativa and Arabidopsis thaliana reference genomes implemented in the Basal Local Alignment Tool (BLAST) of the NCBI website. Finally, we performed an empirical test to determine the performance of our EST-SSRs in a few individuals from four species of two eudicot genera, Trifolium and Centaurea. Results We explored a total of 14 498 726 EST sequences from the dbEST database (NCBI) in 257 plant genera from the IUCN Red List. We identify a very large number (17 102) of ready-to-test EST-SSRs in most plant genera (193) at no cost. Overall, dinucleotide and trinucleotide repeats were the prevalent types but the abundance of the various types of repeat differed between taxonomic groups. Control genomes revealed that trinucleotide repeats were mostly located in coding regions while dinucleotide repeats were largely associated with untranslated regions. Our results from the empirical test revealed considerable amplification success and transferability between congenerics. Conclusions The present work represents the first large-scale study developing SSRs by utilizing publicly accessible EST databases in threatened plants. Here we provide a very large number of ready-to-test EST-SSR (17 102) for 193 genera. The cross-species transferability suggests that the number of possible target species would be large. Since trinucleotide repeats are abundant and mainly linked to exons they might be useful in evolutionary and conservation studies. Altogether, our study highly supports the use of EST databases as an extremely affordable and fast alternative for SSR developing in threatened plants.

Genetic stability of Schmallenberg virus in vivo during an epidemic, and in vitro, when passaged in the highly susceptible porcine SK-6 cell line

Schmallenberg virus (SBV), an arthropod-borne orthobunyavirus was first detected in 2011 in cattle suffering from diarrhea and fever. The most severe impact of an SBV infection is the induction of malformations in newborns and abortions. Between 2011 and 2013 SBV spread throughout Europe in an unprecedented epidemic wave. SBV contains a tripartite genome consisting of the three negative-sense RNA segments L, M, and S. The virus is usually isolated from clinical samples by inoculation of KC (insect) or BHK-21 (mammalian) cells. Several virus passages are required to allow adaptation of SBV to cells in vitro. In the present study, the porcine SK-6 cell line was used for isolation and passaging of SBV. SK-6 cells proved to be more sensitive to SBV infection and allowed to produce higher titers more rapidly as in BHK-21 cells after just one passage. No adaptation was required. In order to determine the in vivo genetic stability of SBV during an epidemic spread of the virus the nucleotide sequence of the genome from seven SBV field isolates collected in summer 2012 in Switzerland was determined and compared to other SBV sequences available in GenBank. A total of 101 mutations, mostly transitions randomly dispersed along the L and M segment were found when the Swiss isolates were compared to the first SBV isolated late 2011 in Germany. However, when these mutations were studied in detail, a previously described hypervariable region in the M segment was identified. The S segment was completely conserved among all sequenced SBV isolates. To assess the in vitro genetic stability of SBV, three isolates were passage 10 times in SK-6 cells and sequenced before and after passaging. Between two and five nt exchanges per genome were found. This low in vitro mutation rate further demonstrates the suitability of SK-6 cells for SBV propagation.

Differentiation of IncL and IncM Plasmids Associated with the Spread of Clinically Relevant Antimicrobial Resistance.

INTRODUCTION blaOXA-48, blaNDM-1 and blaCTX-M-3 are clinically relevant resistance genes, frequently associated with the broad-host range plasmids of the IncL/M group. The L and M plasmids belong to two compatible groups, which were incorrectly classified together by molecular methods. In order to understand their evolution, we fully sequenced four IncL/M plasmids, including the reference plasmids R471 and R69, the recently described blaOXA-48-carrying plasmid pKPN-El.Nr7 from a Klebsiella pneumoniae isolated in Bern (Switzerland), and the blaSHV-5 carrying plasmid p202c from a Salmonella enterica from Tirana (Albania). METHODS Sequencing was performed using 454 Junior Genome Sequencer (Roche). Annotation was performed using Sequin and Artemis software. Plasmid sequences were compared with 13 fully sequenced plasmids belonging to the IncL/M group available in GenBank. RESULTS Comparative analysis of plasmid genomes revealed two distinct genetic lineages, each containing one of the R471 (IncL) and R69 (IncM) reference plasmids. Conjugation experiments demonstrated that plasmids representative of the IncL and IncM groups were compatible with each other. The IncL group is constituted by the blaOXA-48-carrying plasmids and R471. The IncM group contains two sub-types of plasmids named IncM1 and IncM2 that are each incompatible. CONCLUSION This work re-defines the structure of the IncL and IncM families and ascribes a definitive designation to the fully sequenced IncL/M plasmids available in GenBank.

The Apis mellifera filamentous virus genome

A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family.

Expression cloning of a candidate prostate cancer oncogene

Genetic analysis, both karyotyping and comparative genomic hybridization, of prostate cancer cell lines and specimens have revealed multiple areas of concordant increases in DNA content. An increase of DNA in specific regions of the genome in cancer is often associated with the amplification of oncogenes. Based on these observations we have hypothesized that oncogenes are involved in the initiation or progression of prostate cancer. An expression cloning approach was utilized to identify candidate oncogenes in prostate cancer. ^ A full-length, unidirectional cDNA expression library was constructed from DU145 prostate cancer cells. The cDNA library was screened using CP12, a rat prostate epithelial cell line. In soft agarose assays, CP12 (parental or vector transfected) do not form colonies. However, upon the introduction of a number of known oncogenes CP12 becomes anchorage independent in soft agarose. Based on this in-vitro phenotypic shift, a DU145 cDNA library was stably transfected into CP12, and selected for anchorage independence. Two hundred fifty nine anchorage independent clones were isolated. Some colonies contained more than one insert, bringing the candidate oncogene pool to approximately 400. Seven inserts were sequenced at random. Using the sequences obtained, GenBank was screened, and matches were found with p53, PARG1, a mitochondrial ATPase, RNF6, and three unknown genes that mapped to Unigene clusters. As the pool of cDNA inserts appeared promising, overexpressed genes were further selected. From 259 clones, 17 clones were overexpressed more than 6-fold in DU145 compared to Normal Prostate. From the 17 clones, 12 cDNA inserts were strongly expressed in DU145 and were isolated for sequencing. ^ Two of the sequences, 1G6 and 3E9, were identical. Expression of 1G6/2G9/3E9 was tested by RT-PCR. 1G6/2G9/3E9 was not expressed in normal prostate, but was expressed in all prostate cancer cell lines tested as well as six prostate cancer samples. When retransfected into CP12, 1G6/2G9/3E9 induced the formation of foci and anchorage independent colonies. Thus, functional and expression data suggest that 1G6/2G9/3E9 may be a prostate cancer oncogene. ^

Chlorophyll content and temperature-dependent net photosynthesis and respiration rate in the lichen Cetraria aculeata

We studied polar and temperate samples of the lichen Cetraria aculeata to investigate whether genetical differences between photobionts are correlated with physiological properties of the lichen holobiont. Net photosynthesis and dark respiration (DR) at different temperatures (from 0 to 30 °C) and photon flux densities (from 0 to 1,200 ?mol/m**2/s) were studied for four populations of Cetraria aculeata. Samples were collected from maritime Antarctica, Svalbard, Germany and Spain, representing different climatic situations. Sequencing of the photobiont showed that the investigated samples fall in the polar and temperate clade described in Fernández-Mendoza et al. (2011, doi:10.1111/j.1365-294X.2010.04993.x). Lichens with photobionts from these clades differ in their temperature optimum for photosynthesis, maximal net photosynthesis, maximal DR and chlorophyll content. Maximal net photosynthesis was much lower in Antarctica and Svalbard than in Germany and Spain. The difference was smaller when rates were expressed by chlorophyll content. The same is true for the temperature optima of polar (11 °C) and temperate (15 and 17 °C) lichens. Our results indicate that lichen mycobionts may adapt or acclimate to local environmental conditions either by selecting algae from regional pools or by regulating algal cell numbers (chlorophyll content) within the thallus.

Soil characteristics and abundance of archaea in samples from the Ross Sea Region, Antarctica

The objective of this study was to examine the presence and diversity of Archaea within mineral and ornithogenic soils from 12 locations across the Ross Sea region. Archaea were not abundant but DNA sufficient for producing 16S rRNA gene clone libraries was extracted from 18 of 51 soil samples, from four locations. A total of 1452 clones were analysed by restriction fragment length polymorphism and assigned to 43 operational taxonomic units from which representatives were sequenced. Archaea were primarily restricted to coastal mineral soils which showed a predominance of Crenarchaeota belonging to group 1.1b (>99% of clones). These clones were assigned to six clusters (A through F), based on shared identity to sequences in the GenBank database. Ordination indicated that soil chemistry and water content determined archaeal community structure. This is the first comprehensive study of the archaeal community in Antarctic soils and as such provides a reference point for further investigation of microbial function in this environment.

Evidence of cryptic and pseudocryptic speciation in the Paracalanus parvus species complex (Crustacea, Copepoda, Calanoida), supplementary material

Introduction Many marine planktonic crustaceans such as copepods have been considered as widespread organisms. However, the growing evidence for cryptic and pseudo-cryptic speciation has emphasized the need of re-evaluating the status of copepod species complexes in molecular and morphological studies to get a clearer picture about pelagic marine species as evolutionary units and their distributions. This study analyses the molecular diversity of the ecologically important Paracalanus parvus species complex. Its seven currently recognized species are abundant and also often dominant in marine coastal regions worldwide from temperate to tropical oceans. Results COI and Cytochrome b sequences of 160 specimens of the Paracalanus parvus complex from all oceans were obtained. Furthermore, 42 COI sequences from GenBank were added for the genetic analyses. Thirteen distinct molecular operational taxonomic units (MOTU) and two single sequences were revealed with cladistic analyses (Maximum Likelihood, Bayesian Inference), of which seven were identical with results from species delimitation methods (barcode gaps, ABDG, GMYC, Rosenberg's P(AB)). In total, 10 to 12 putative species were detected and could be placed in three categories: (1) temperate geographically isolated, (2) warm-temperate to tropical wider spread and (3) circumglobal warm-water species. Conclusions The present study provides evidence of cryptic or pseudocryptic speciation in the Paracalanus parvus complex. One major insight is that the species Paracalanus parvus s.s. is not panmictic, but may be restricted in its distribution to the northeastern Atlantic.