We studied sequence-dependent retention properties of synthetic 5'-terminal phosphate absent trinucleotides containing adenine, guanine and thymine through reversed-phase liquid chromatography (RPLC) and QSRR modelling. We investigated the influence of separation conditions, namely mobile phase composition (ion interaction agent content, pH and organic constituent content), on sequence-dependent separation by means of ion-interaction RPLC (II-RPLC) using two types of models: experimental design-artificial neural networks (ED-ANN), and linear regression based on molecular dynamics data. The aim was to determine those properties of the above-mentioned analytes responsible for the retention dependence of the sequence. Our results show that there is a deterministic relation between sequence and II-RPLC retention properties of the studied trinucleotides. Further, we can conclude that the higher the content of ion-interaction agent in the mobile phase, the more prominent these properties are. We also show that if we approximate the polar component of solvation energy in QSRR by the electrostatic work in transferring molecules from vacuum to water, and the non-polar component by the solvent accessible surface area, these parameters best describe the retention properties of trinucleotides. There are some exceptions to this finding, namely sequences 5'-NAN-3', 5'-ANN-3', 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' (N stands for generic nucleotide). Their role is still unknown, but since linear regression including these specific constellations showed a higher observable variance coverage than the model with only the basic descriptors, we may assume that solvent-analyte interactions are responsible for the exceptional behaviour of 5'-NAN-3' & 5'-ANN-3' trinucleotides and some intramolecular interactions of neighbouring nucleobases for 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' trinucleotides.
- MeSH
- Adenine analogs & derivatives isolation & purification MeSH
- Chromatography, Reverse-Phase MeSH
- Guanine analogs & derivatives isolation & purification MeSH
- Quantitative Structure-Activity Relationship MeSH
- Neural Networks, Computer MeSH
- Oligonucleotides isolation & purification MeSH
- Solvents MeSH
- Molecular Dynamics Simulation MeSH
- Static Electricity MeSH
- Thymine analogs & derivatives isolation & purification MeSH
- Water MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
Viral RNA dependent polymerases (vRdPs) are present in all RNA viruses; unfortunately, their sequence similarity is too low for phylogenetic studies. Nevertheless, vRdP protein structures are remarkably conserved. In this study, we used the structural similarity of vRdPs to reconstruct their evolutionary history. The major strength of this work is in unifying sequence and structural data into a single quantitative phylogenetic analysis, using powerful a Bayesian approach. The resulting phylogram of vRdPs demonstrates that RNA-dependent DNA polymerases (RdDPs) of viruses within Retroviridae family cluster in a clearly separated group of vRdPs, while RNA-dependent RNA polymerases (RdRPs) of dsRNA and +ssRNA viruses are mixed together. This evidence supports the hypothesis that RdRPs replicating +ssRNA viruses evolved multiple times from RdRPs replicating +dsRNA viruses, and vice versa. Moreover, our phylogram may be presented as a scheme for RNA virus evolution. The results are in concordance with the actual concept of RNA virus evolution. Finally, the methods used in our work provide a new direction for studying ancient virus evolution.
- MeSH
- Species Specificity MeSH
- Phylogeny MeSH
- Evolution, Molecular * MeSH
- Models, Molecular MeSH
- Molecular Sequence Data MeSH
- RNA-Dependent RNA Polymerase chemistry genetics MeSH
- RNA Viruses classification enzymology genetics MeSH
- Protein Structure, Secondary MeSH
- Amino Acid Sequence MeSH
- Sequence Homology, Amino Acid MeSH
- Protein Structure, Tertiary * MeSH
- Binding Sites genetics MeSH
- Viral Proteins chemistry genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Construction of functionalized nucleic acids (DNA or RNA) via polymerase incorporation of modified nucleoside triphosphates is reviewed and selected applications of the modified nucleic acids are highlighted. The classical multistep approach for the synthesis of modified NTPs by triphosphorylation of modified nucleosides is compared to the novel approach consisting of direct aqueous cross-coupling reactions of unprotected halogenated nucleoside triphosphates. The combination of cross-coupling of NTPs with polymerase incorporation gives an efficient and straightforward two-step synthesis of modified nucleic acids. Primer extension using biotinylated templates followed by separation using streptavidine-coated magnetic beads and DNA duplex denaturation is used for preparation of modified single stranded oligonucleotides. Examples of using this approach for electrochemical DNA labelling and bioanalytical applications are given.
OBJECTIVES: To test the performance of an oral cancer prognostic 13-gene signature for the prediction of survival of patients diagnosed with HPV-negative and p16-negative oral cavity cancer. MATERIALS AND METHODS: Diagnostic formalin-fixed paraffin-embedded oral cavity cancer tumor samples were obtained from the Fred Hutchinson Cancer Research Center/University of Washington, University of Calgary, University of Michigan, University of Utah, and seven ARCAGE study centers coordinated by the International Agency of Research on Cancer. RNA from 638 Human Papillomavirus (HPV)-negative and p16-negative samples was analyzed for the 13 genes using a NanoString assay. Ridge-penalized Cox regressions were applied to samples randomly split into discovery and validation sets to build models and evaluate the performance of the 13-gene signature in predicting 2-year oral cavity cancer-specific survival overall and separately for patients with early and late stage disease. RESULTS: Among AJCC stage I/II patients, including the 13-gene signature in the model resulted in substantial improvement in the prediction of 2-year oral cavity cancer-specific survival. For models containing age and sex with and without the 13-gene signature score, the areas under the Receiver Operating Characteristic Curve (AUC) and partial AUC were 0.700 vs. 0.537 (p < 0.001), and 0.046 vs. 0.018 (p < 0.001), respectively. Improvement in predicting prognosis for AJCC stage III/IV disease also was observed, but to a lesser extent. CONCLUSIONS: If confirmed using tumor samples from a larger number of early stage oral cavity cancer patients, the 13-gene signature may inform personalized treatment of early stage HPV-negative and p16-negative oral cavity cancer patients.
- MeSH
- Survival Analysis MeSH
- Adult MeSH
- Tissue Fixation MeSH
- Cyclin-Dependent Kinase Inhibitor p16 metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- Human papillomavirus 16 isolation & purification MeSH
- Young Adult MeSH
- Biomarkers, Tumor genetics MeSH
- Mouth Neoplasms genetics metabolism pathology MeSH
- Area Under Curve MeSH
- Sequence Analysis, RNA MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Carcinoma, Squamous Cell genetics metabolism pathology MeSH
- Neoplasm Staging MeSH
- Gene Expression Profiling methods MeSH
- Paraffin Embedding MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Wood-inhabiting taxa of the Sordariomycetidae comprise several distantly related monotypic or small genera, which lack familial or ordinal affiliation and share a simple and inconspicuous morphology of dark ascomata with carbonaceous walls and long necks, stipitate asci and hyaline ellipsoidal, fusiform to cylindrical ascospores. Recent collections of an undescribed fungus and of Ceratosphaeria abietis reveal two additional evolutionary lineages characterized by this simple and indistinct teleomorph morphology. Phylogenetic analysis of three genes, small and large subunit nuclear ribosomal DNA (nc28S and nc18S rDNA) combined with the second largest subunit of RNA polymerase II (rpb2), supports the recognition of two new genera, Ceratolenta and Platytrachelon for C. abietis. Platytrachelon abietis is redescribed and illustrated based on additional collections. In culture it produced a dematiaceous hyphomycetous anamorph with blastic conidiogenesis and ellipsoidal, septate, pale brown conidia. It was associated with a synanamorph producing cylindrical, strongly curved hyaline conidia. Molecular data suggest a relationship of Platytrachelon with the Papulosaceae, while Ceratolenta forms a monophylum on a separate branch. Both taxonomic novelties possess striking morphological similarities with Ceratosphaeria, Lentomitella and Rhodoveronaea, which recently were reinstated based on DNA sequence data. A key to morphologically similar wood-inhabiting fungi classified in the Sordariomycetidae is provided.
- MeSH
- Ascomycota classification cytology genetics isolation & purification MeSH
- DNA, Fungal chemistry genetics MeSH
- Fungal Proteins genetics MeSH
- Phylogeny MeSH
- Molecular Sequence Data MeSH
- DNA, Ribosomal chemistry genetics MeSH
- RNA Polymerase II genetics MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA MeSH
- Sequence Alignment MeSH
- Spores, Fungal cytology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
A culture of Cirrosporium novae-zelandiae, the type species of a distinctive, monotypic coelomycete genus, was isolated from a specimen collected near the holotype locality in New Zealand. Light microscopic and environmental scanning electron microscopic observations confirm the details of the unusual meristem arthric conidium ontogeny presented in the protolog. For phylogenetic analysis, a dataset of 122 species representing nine classes of euascomycetes was assembled including sequences from nuclear small and large subunits (nc18S, nc28S) and mitochondrial small subunit (mr16S) ribosomal RNA and the largest and second largest subunits of RNA polymerase II (RPB1, RPB2). A five-gene phylogeny suggests that the fungus is phylogenetically related to the Eurotiomycetes. It sits alone on a long branch as a sister to the Mycocaliciales of the Mycocaliciomycetidae. Cirrosporium exhibits several morphological characters similar to those of members of the Mycocaliciales; however, the paucity of known anamorphs in this order does not offer any further clarification on possible relationships. It is clear that the rare and broadly distributed meristem arthric ontogenetic pattern is polyphyletic, occurring in widely separate groups of anamorphs of both the Ascomycota and Basidiomycota.
- MeSH
- Ascomycota classification cytology genetics isolation & purification MeSH
- DNA, Fungal chemistry genetics MeSH
- DNA-Directed RNA Polymerases genetics MeSH
- Wood microbiology MeSH
- Fungal Proteins genetics MeSH
- Phylogeny * MeSH
- Molecular Sequence Data MeSH
- Multilocus Sequence Typing MeSH
- Mycological Typing Techniques MeSH
- DNA, Ribosomal chemistry genetics MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA MeSH
- Spores, Fungal classification cytology genetics isolation & purification MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- New Zealand MeSH
Eight Gram-positive, catalase-negative bacterial strains were isolated during screening of enterococcal populations on plants. rep-PCR fingerprinting using the (GTG)(5) primer showed that the isolates constituted a single cluster that was separate from all known enterococcal species. 16S rRNA gene sequence phylogenetic analysis of three representative strains showed that the isolates belonged to the genus Enterococcus and that they clustered with the Enterococcus faecalis species group. Sequencing of the genes for the phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) also revealed the isolates' separate taxonomic position. Application of whole-cell protein fingerprinting, automated ribotyping and extensive phenotyping demonstrated the genetic and phenotypic homogeneity of the isolates and confirmed their separate position within the E. faecalis species group. The isolates represent a novel species of the genus Enterococcus, for which the name Enterococcus plantarum sp. nov. is proposed; the type strain is CCM 7889(T) (=LMG 26214(T)=C27(T)).
- MeSH
- DNA, Bacterial chemistry genetics MeSH
- DNA-Directed RNA Polymerases genetics MeSH
- Enterococcus classification genetics isolation & purification physiology MeSH
- Phenylalanine-tRNA Ligase genetics MeSH
- Phylogeny MeSH
- Catalase metabolism MeSH
- Molecular Sequence Data MeSH
- Molecular Typing MeSH
- DNA, Ribosomal chemistry genetics MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Plants microbiology MeSH
- Sequence Analysis, DNA MeSH
- Cluster Analysis MeSH
- Bacterial Typing Techniques MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The exact role of the central acidic domain of Mdm2 in p53 degradation remains unclear. We therefore performed a systematic and comprehensive analysis of the acidic domain using a series of short deletions and found that only a minor part of the domain was indispensable for Mdm2-mediated p53 ubiquitylation. Moreover, we identified a short stretch of acidic amino acids required for p53 degradation but not ubiquitylation, indicating that, in addition to p53 ubiquitylation, the acidic domain might be involved in a critical post-ubiquitylation step in p53 degradation. Rather than representing a single functional domain, different parts of the acidic region perform separate functions in p53 degradation, suggesting that it might be possible to therapeutically target them independently.
- MeSH
- Models, Biological MeSH
- Gene Deletion MeSH
- HEK293 Cells MeSH
- Immunoprecipitation MeSH
- Nuclear Proteins chemistry physiology MeSH
- Protein Conformation MeSH
- Humans MeSH
- Molecular Sequence Data MeSH
- DNA Mutational Analysis MeSH
- Tumor Suppressor Protein p53 chemistry metabolism MeSH
- Proto-Oncogene Proteins c-mdm2 chemistry genetics physiology MeSH
- Proto-Oncogene Proteins chemistry physiology MeSH
- Amino Acid Sequence MeSH
- Sequence Homology, Amino Acid MeSH
- Protein Structure, Tertiary MeSH
- Ubiquitin chemistry MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Microscopic diagnosis of equine piroplasmoses, caused by Theileria equi and Babesia caballi, is hindered by low parasitaemia during the latent phase of the infections. However, this constraint can be overcome by the application of PCR followed by sequencing. Out of 288 animals examined, the piroplasmid DNA was detected in 78 (27·1%). Multiplex PCR indicated that T. equi (18·8%) was more prevalent than B. caballi (7·3%), while mixed infections were conspicuously absent. Sequences of 69 PCR amplicons obtained by the 'catch-all' PCR were in concordance with those amplified by the multiplex strategy. Computed minimal adequate model analyses for both equine piroplasmid species separately showed a significant effect of host species and age in the case of T. equi, while in the B. caballi infections only the correlation with host sex was significant. Phylogenetic analyses inferred the occurrence of three genotypes of T. equi and B. caballi. Moreover, a novel genotype C of B. caballi was identified. The dendrogram based on obtained sequences of T. equi revealed possible speciation events. The infections with T. equi and B. caballi are enzootic in all ecozones of Jordan and different genotypes circulate wherever dense horse population exists.
- MeSH
- Babesia classification genetics isolation & purification MeSH
- Babesiosis epidemiology parasitology MeSH
- Equidae parasitology MeSH
- Phylogeny MeSH
- Genetic Variation * MeSH
- Genotype MeSH
- Horses MeSH
- Molecular Sequence Data MeSH
- Multiplex Polymerase Chain Reaction veterinary MeSH
- Horse Diseases epidemiology parasitology MeSH
- Parasitemia veterinary MeSH
- Prevalence MeSH
- DNA, Protozoan chemistry genetics MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA veterinary MeSH
- Cattle MeSH
- Theileria classification genetics isolation & purification MeSH
- Theileriasis epidemiology parasitology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Cattle MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Jordan MeSH
A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.
- MeSH
- Adenine chemistry metabolism MeSH
- Aptamers, Nucleotide chemical synthesis genetics MeSH
- Cytosine chemistry metabolism MeSH
- Deoxyribonucleosides chemistry genetics metabolism MeSH
- Dinucleoside Phosphates chemistry genetics metabolism MeSH
- DNA-Directed DNA Polymerase genetics metabolism MeSH
- DNA chemistry genetics metabolism MeSH
- Guanine chemistry metabolism MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Base Pairing MeSH
- Polymerase Chain Reaction MeSH
- Polymers chemical synthesis metabolism MeSH
- DNA Replication * MeSH
- Base Sequence MeSH
- Uracil chemistry metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
