Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H.

PubMed

Schultz, Sharon J; Zhang, Miaohua; Champoux, James J

2010-03-19

The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3'-end-directed, and RNA 5'-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions -1 and +1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3'-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions +1, -2, -4, -5, -10, and -14 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions -7 and -12 for HIV-1 and positions -4, -9, and -11 for M-MuLV had more modest effects. DNA 3'-end-directed cleavage was influenced substantially by positions +1, -2, -4, and -5 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions +1 and -2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Development and customization of a color-coded microbeads-based assay for drug resistance in HIV-1 reverse transcriptase.

PubMed

Gu, Lijun; Kawana-Tachikawa, Ai; Shiino, Teiichiro; Nakamura, Hitomi; Koga, Michiko; Kikuchi, Tadashi; Adachi, Eisuke; Koibuchi, Tomohiko; Ishida, Takaomi; Gao, George F; Matsushita, Masaki; Sugiura, Wataru; Iwamoto, Aikichi; Hosoya, Noriaki

2014-01-01

Drug resistance (DR) of HIV-1 can be examined genotypically or phenotypically. Although sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more appropriate for epidemiological studies and public health research. We used a Japanese database to design and synthesize sequence-specific oligonucleotide probes (SSOP) for the detection of wild-type sequences and 6 DR mutations in the clade B HIV-1 reverse transcriptase region. We coupled SSOP to microbeads of the Luminex 100 xMAP system and developed a GRT based on the polymerase chain reaction (PCR)-SSOP-Luminex method. Sixteen oligoprobes for discriminating DR mutations from wild-type sequences at 6 loci were designed and synthesized, and their sensitivity and specificity were confirmed using isogenic plasmids. The PCR-SSOP-Luminex DR assay was then compared to direct sequencing using 74 plasma specimens from treatment-naïve patients or those on failing treatment. In the majority of specimens, the results of the PCR-SSOP-Luminex DR assay were concordant with sequencing results: 62/74 (83.8%) for M41, 43/74 (58.1%) for K65, 70/74 (94.6%) for K70, 55/73 (75.3%) for K103, 63/73 (86.3%) for M184 and 68/73 (93.2%) for T215. There were a number of specimens without any positive signals, especially for K65. The nucleotide position of A2723G, A2747G and C2750T were frequent polymorphisms for the wild-type amino acids K65, K66 and D67, respectively, and 14 specimens had the D67N mutation encoded by G2748A. We synthesized 14 additional oligoprobes for K65, and the sensitivity for K65 loci improved from 43/74 (58.1%) to 68/74 (91.9%). We developed a rapid high-throughput assay for clade B HIV-1 DR mutations, which could be customized by synthesizing oligoprobes suitable for the circulating viruses. The assay could be a useful tool especially for public health research in both resource-rich and resource-limited settings.

copia-like retrotransposons are ubiquitous among plants.

PubMed Central

Voytas, D F; Cummings, M P; Koniczny, A; Ausubel, F M; Rodermel, S R

1992-01-01

Transposable genetic elements are assumed to be a feature of all eukaryotic genomes. Their identification, however, has largely been haphazard, limited principally to organisms subjected to molecular or genetic scrutiny. We assessed the phylogenetic distribution of copia-like retrotransposons, a class of transposable element that proliferates by reverse transcription, using a polymerase chain reaction assay designed to detect copia-like element reverse transcriptase sequences. copia-like retrotransposons were identified in 64 plant species as well as the photosynthetic protist Volvox carteri. The plant species included representatives from 9 of 10 plant divisions, including bryophytes, lycopods, ferns, gymnosperms, and angiosperms. DNA sequence analysis of 29 cloned PCR products and of a maize retrotransposon cDNA confirmed the identity of these sequences as copia-like reverse transcriptase sequences, thereby demonstrating that this class of retrotransposons is a ubiquitous component of plant genomes. Images PMID:1379734

Preliminary investigation of bottlenose dolphins (Tursiops truncatus) for hfe gene-related hemochromatosis.

PubMed

Phillips, Brianne E; Venn-Watson, Stephanie; Archer, Linda L; Nollens, Hendrik H; Wellehan, James F X

2014-10-01

Hemochromatosis (iron storage disease) has been reported in diverse mammals including bottlenose dolphins (Tursiops truncatus). The primary cause of excessive iron storage in humans is hereditary hemochromatosis. Most human hereditary hemochromatosis cases (up to 90%) are caused by a point mutation in the hfe gene, resulting in a C282Y substitution leading to iron accumulation. To evaluate the possibility of a hereditary hemochromatosis-like genetic predisposition in dolphins, we sequenced the bottlenose dolphin hfe gene, using reverse transcriptase-PCR and hfe primers designed from the dolphin genome, from liver of affected and healthy control dolphins. Sample size included two case animals and five control animals. Although isotype diversity was evident, no coding differences were identified in the hfe gene between any of the animals examined. Because our sample size was small, we cannot exclude the possibility that hemochromatosis in dolphins is due to a coding mutation in the hfe gene. Other potential causes of hemochromatosis, including mutations in different genes, diet, primary liver disease, and insulin resistance, should be evaluated.

Primer design for a prokaryotic differential display RT-PCR.

PubMed Central

Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H

1997-01-01

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR. PMID:9108168

Primer design for a prokaryotic differential display RT-PCR.

PubMed

Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H

1997-05-01

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR.

Novel Codon Insert in HIV Type 1 Clade B Reverse Transcriptase Associated with Low-Level Viremia During Antiretroviral Therapy

PubMed Central

Gianella, Sara; Vazquez, Homero; Ignacio, Caroline; Zweig, Adam C.; Richman, Douglas D.; Smith, Davey M.

2014-01-01

Abstract We investigated the pol genotype in two phylogenetically and epidemiologically linked partners, who were both experiencing persistent low-level viremia during antiretroviral therapy. In one partner we identified a new residue insertion between codon 248 and 249 of the HIV-1 RNA reverse transcriptase (RT) coding region (HXB2 numbering). We then investigated the potential impact of identified mutations in RT and antiretroviral binding affinity using a novel computational approach. PMID:24020934

The LINEs and SINEs of Entamoeba histolytica: comparative analysis and genomic distribution.

PubMed

Bakre, Abhijeet A; Rawal, Kamal; Ramaswamy, Ram; Bhattacharya, Alok; Bhattacharya, Sudha

2005-07-01

Autonomous non-long terminal repeat retrotransposons are commonly referred to as long interspersed elements (LINEs). Short non-autonomous elements that borrow the LINE machinery are called SINES. The Entamoeba histolytica genome contains three classes of LINEs and SINEs. Together the EhLINEs/SINEs account for about 6% of the genome. The recognizable functional domains in all three EhLINEs included reverse transcriptase and endonuclease. A novel feature was the presence of two types of members-some with a single long ORF (less frequent) and some with two ORFs (more frequent) in both EhLINE1 and 2. The two ORFs were generated by conserved changes leading to stop codon. Computational analysis of the immediate flanking sequences for each element showed that they inserted in AT-rich sequences, with a preponderance of Ts in the upstream site. The elements were very frequently located close to protein-coding genes and other EhLINEs/SINEs. The possible influence of these elements on expression of neighboring genes needs to be determined.

First report of Cocksfoot mottle virus infecting wheat (Triticum aestivum) in Ohio

USDA-ARS?s Scientific Manuscript database

Cocksfoot mottle virus (CfMV) was discovered in Ohio wheat during a 2016 field survey utilizing RNA-Seq to identify virus-like sequences. Virus sequences were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Sanger sequencing, and CfMV was transmitted to orchardgrass and pas...

Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

PubMed Central

Schuster, W; Brennicke, A

1987-01-01

We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration. PMID:14650433

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in the architecture of eukaryotic genomes and are the evolutionary origin of retroviruses, including human immunodeficiency virus (HIV).

The Role of eIF4E Activity in Breast Cancer

DTIC Science & Technology

2010-08-01

ORF, open reading frame; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase ; uORF, upstream ORF; UTR...were also performed using template lacking RT ( reverse transcriptase ): products were either undetectable or greatly reduced (>30000-fold less product...have previously shown that a 5’UTR expressed from the human AXIN2 gene contains a sixty nucleotide sequence that is predicted to form a stable stem

miR-128 inhibits telomerase activity by targeting TERT mRNA

PubMed Central

Guzman, Herlinda; Sanders, Katie; Idica, Adam; Bochnakian, Aurore; Jury, Douglas; Daugaard, Iben; Zisoulis, Dimitrios G; Pedersen, Irene Munk

2018-01-01

Telomerase is a unique cellular reverse transcriptase (RT) essential for maintaining telomere stability and required for the unlimited proliferation of cancer cells. The limiting determinant of telomerase activity is the catalytic component TERT, and TERT expression is closely correlated with telomerase activity and cancer initiation and disease progression. For this reason the regulation of TERT levels in the cell is of great importance. microRNAs (miRs) function as an additional regulatory level in cells, crucial for defining expression boundaries, proper cell fate decisions, cell cycle control, genome integrity, cell death and metastasis. We performed an anti-miR library screen to identity novel miRs, which participate in the control of telomerase. We identified the tumor suppressor miR (miR-128) as a novel endogenous telomerase inhibitor and determined that miR-128 significantly reduces the mRNA and protein levels of Tert in a panel of cancer cell lines. We further evaluated the mechanism by which miR-128 regulates TERT and demonstrated that miR-128 interacts directly with the coding sequence of TERT mRNA in both HeLa cells and teratoma cells. Interestingly, the functional miR-128 binding site in TERT mRNA, is conserved between TERT and the other cellular reverse transcriptase encoded by Long Interspersed Elements-1 (LINE-1 or L1), which can also contribute to the oncogenic phenotype of cancer. This finding supports the novel idea that miRs may function in parallel pathways to inhibit tumorigenesis, by regulating a group of enzymes (such as RT) by targeting conserved binding sites in the coding region of both enzymes. PMID:29568354

Antimicrobial peptide evolution in the Asiatic honey bee Apis cerana.

PubMed

Xu, Peng; Shi, Min; Chen, Xue-Xin

2009-01-01

The Asiatic honeybee, Apis cerana Fabricius, is an important honeybee species in Asian countries. It is still found in the wild, but is also one of the few bee species that can be domesticated. It has acquired some genetic advantages and significantly different biological characteristics compared with other Apis species. However, it has been less studied, and over the past two decades, has become a threatened species in China. We designed primers for the sequences of the four antimicrobial peptide cDNA gene families (abaecin, defensin, apidaecin, and hymenoptaecin) of the Western honeybee, Apis mellifera L. and identified all the antimicrobial peptide cDNA genes in the Asiatic honeybee for the first time. All the sequences were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). In all, 29 different defensin cDNA genes coding 7 different defensin peptides, 11 different abaecin cDNA genes coding 2 different abaecin peptides, 13 different apidaecin cDNA genes coding 4 apidaecin peptides and 34 different hymenoptaecin cDNA genes coding 13 different hymenoptaecin peptides were cloned and identified from the Asiatic honeybee adult workers. Detailed comparison of these four antimicrobial peptide gene families with those of the Western honeybee revealed that there are many similarities in the quantity and amino acid components of peptides in the abaecin, defensin and apidaecin families, while many more hymenoptaecin peptides are found in the Asiatic honeybee than those in the Western honeybee (13 versus 1). The results indicated that the Asiatic honeybee adult generated more variable antimicrobial peptides, especially hymenoptaecin peptides than the Western honeybee when stimulated by pathogens or injury. This suggests that, compared to the Western honeybee that has a longer history of domestication, selection on the Asiatic honeybee has favored the generation of more variable antimicrobial peptides as protection against pathogens.

The Role of elF4E Activity in Breast Cancer

DTIC Science & Technology

2011-08-01

protein; ORF, open reading frame; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase ; uORF, upstream ORF; UTR...Reactions were also performed using template lacking RT ( reverse transcriptase ): products were either undetectable or greatly reduced (>30000-fold less...that a 5’UTR expressed from the human AXIN2 gene contains a sixty nucleotide sequence that is predicted to form a stable stem-loop structure6. This

Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases.

PubMed

Kjaersgård, I V; Jespersen, H M; Rasmussen, S K; Welinder, K G

1997-03-01

cDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP 1a and ATP 2a, have been identified by searching the Arabidopsis database of expressed sequence tags (dbEST). They represent a novel branch of hitherto uncharacterized plant peroxidases which is only 35% identical in amino acid sequence to the well characterized group of basic plant peroxidases represented by the horseradish (Armoracia rusticana) isoperoxidases HRP C, HRP E5 and the similar Arabidopsis isoperoxidases ATP Ca, ATP Cb, and ATP Ea. However ATP 1a is 87% identical in amino acid sequence to a peroxidase encoded by an mRNA isolated from cotton (Gossypium hirsutum). As cotton and Arabidopsis belong to rather diverse families (Malvaceae and Crucifereae, respectively), in contrast with Arabidopsis and horseradish (both Crucifereae), the high degree of sequence identity indicates that this novel type of peroxidase, albeit of unknown function, is likely to be widespread in plant species. The atp 1 and atp 2 types of cDNA sequences were the most redundant among the 28 different isoperoxidases identified among about 200 peroxidase encoding ESTs. Interestingly, 8 out of totally 38 EST sequences coding for ATP 1 showed three identical nucleotide substitutions. This variant form is designated ATP 1b. Similarly, six out of totally 16 EST sequences coding for ATP 2 showed a number of deletions and nucleotide changes. This variant form is designated ATP 2b. The selected EST clones are full-length and contain coding regions of 993 nucleotides for atp 1a, and 984 nucleotides for atp 2a. These regions show 61% DNA sequence identity. The predicted mature proteins ATP 1a, and ATP 2a are 57% identical in sequence and contain the structurally and functionally important residues, characteristic of the plant peroxidase superfamily. However, they do show two differences of importance to peroxidase catalysis: (1) the asparagine residue linked with the active site distal histidine via hydrogen bonding is absent; (2) an N-glycosylation site is located right at the entrance to the heme channel. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify mRNAs coding for ATP 1a/b and ATP 2a/b in germinating seeds, seedlings, roots, leaves, stems, flowers and cell suspension culture using elongation factor 1alpha (EF-1alpha) for the first time as a positive control. Both mRNAs were transcribed at levels comparable to EF-1alpha in all plant tissues investigated which were more than two days old, and in cell suspension culture. In addition, the mRNA coding for ATP 1a/b was found in two day old germinating seeds. The abundant transcription of ATP 1a/b and ATP 2a/b is in line with their many entries in dbEST, and indicates essential roles for these novel peroxidases.

Demonstration of retrotransposition of the Tf1 element in fission yeast.

PubMed

Levin, H L; Boeke, J D

1992-03-01

Tf1, a retrotransposon from fission yeast, has LTRs and coding sequences resembling the protease, reverse transcriptase and integrase domains of retroviral pol genes. A unique aspect of Tf1 is that it contains a single open reading frame whereas other retroviruses and retrotransposons usually possess two or more open reading frames. To determine whether Tf1 can transpose, we overproduced Tf1 transcripts encoded by a plasmid copy of the element marked with a neo gene. Approximately 0.1-4.0% of the cell population acquired chromosomally inherited resistance to G418. DNA blot analysis demonstrated that such strains had acquired both Tf1 and neo specific sequences within a restriction fragment of the same size; the size of this restriction fragment varied between different isolates. Structural analysis of the cloned DNA flanking the Tf1-neo element of two transposition candidates with the same regions in the parent strain showed that the ability to grow on G418 was due to transposition of Tf1-neo and not other types of recombination events.

Sequence quality analysis tool for HIV type 1 protease and reverse transcriptase.

PubMed

Delong, Allison K; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W; Kantor, Rami

2012-08-01

Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( http://hivdb.Stanford.edu ). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or <0.5% or >15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.

Mutations in the S gene and in the overlapping reverse transcriptase region in chronic hepatitis B Chinese patients with coexistence of HBsAg and anti-HBs.

PubMed

Ding, Feng; Miao, Xi-Li; Li, Yan-Xia; Dai, Jin-Fen; Yu, Hong-Gang

2016-01-01

The mechanism underlying the coexistence of hepatitis B surface antigen and antibodies to HBsAg in chronic hepatitis B patients remains unknown. This research aimed to determine the clinical and virological features of the rare pattern. A total of 32 chronic hepatitis B patients infected by HBV genotype C were included: 15 carrying both HBsAg and anti-HBs (group I) and 17 solely positive for HBsAg (group II). S gene and reverse transcriptase region sequences were amplified, sequenced and compared with the reference sequences. The amino acid variability within major hydrophilic region, especially the "a" determinant region, and within reverse transcriptase for regions overlapping the major hydrophilic region in group I is significantly higher than those in group II. Mutation sI126S/T within the "a" determinant was the most frequent change, and only patients from group I had the sQ129R, sG130N, sF134I, sG145R amino acid changes, which are known to alter immunogenicity. In chronic patients, the concurrent HBsAg/anti-HBs serological profile is associated with an increased aa variability in several key areas of HBV genome. Additional research on these genetic mutants are needed to clarify their biological significance for viral persistence. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

High Degree of Interlaboratory Reproducibility of Human Immunodeficiency Virus Type 1 Protease and Reverse Transcriptase Sequencing of Plasma Samples from Heavily Treated Patients

PubMed Central

Shafer, Robert W.; Hertogs, Kurt; Zolopa, Andrew R.; Warford, Ann; Bloor, Stuart; Betts, Bradley J.; Merigan, Thomas C.; Harrigan, Richard; Larder, Brendon A.

2001-01-01

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself. PMID:11283081

Superior ab initio identification, annotation and characterisation of TEs and segmental duplications from genome assemblies.

PubMed

Zeng, Lu; Kortschak, R Daniel; Raison, Joy M; Bertozzi, Terry; Adelson, David L

2018-01-01

Transposable Elements (TEs) are mobile DNA sequences that make up significant fractions of amniote genomes. However, they are difficult to detect and annotate ab initio because of their variable features, lengths and clade-specific variants. We have addressed this problem by refining and developing a Comprehensive ab initio Repeat Pipeline (CARP) to identify and cluster TEs and other repetitive sequences in genome assemblies. The pipeline begins with a pairwise alignment using krishna, a custom aligner. Single linkage clustering is then carried out to produce families of repetitive elements. Consensus sequences are then filtered for protein coding genes and then annotated using Repbase and a custom library of retrovirus and reverse transcriptase sequences. This process yields three types of family: fully annotated, partially annotated and unannotated. Fully annotated families reflect recently diverged/young known TEs present in Repbase. The remaining two types of families contain a mixture of novel TEs and segmental duplications. These can be resolved by aligning these consensus sequences back to the genome to assess copy number vs. length distribution. Our pipeline has three significant advantages compared to other methods for ab initio repeat identification: 1) we generate not only consensus sequences, but keep the genomic intervals for the original aligned sequences, allowing straightforward analysis of evolutionary dynamics, 2) consensus sequences represent low-divergence, recently/currently active TE families, 3) segmental duplications are annotated as a useful by-product. We have compared our ab initio repeat annotations for 7 genome assemblies to other methods and demonstrate that CARP compares favourably with RepeatModeler, the most widely used repeat annotation package.

Superior ab initio identification, annotation and characterisation of TEs and segmental duplications from genome assemblies

PubMed Central

Zeng, Lu; Kortschak, R. Daniel; Raison, Joy M.

2018-01-01

Transposable Elements (TEs) are mobile DNA sequences that make up significant fractions of amniote genomes. However, they are difficult to detect and annotate ab initio because of their variable features, lengths and clade-specific variants. We have addressed this problem by refining and developing a Comprehensive ab initio Repeat Pipeline (CARP) to identify and cluster TEs and other repetitive sequences in genome assemblies. The pipeline begins with a pairwise alignment using krishna, a custom aligner. Single linkage clustering is then carried out to produce families of repetitive elements. Consensus sequences are then filtered for protein coding genes and then annotated using Repbase and a custom library of retrovirus and reverse transcriptase sequences. This process yields three types of family: fully annotated, partially annotated and unannotated. Fully annotated families reflect recently diverged/young known TEs present in Repbase. The remaining two types of families contain a mixture of novel TEs and segmental duplications. These can be resolved by aligning these consensus sequences back to the genome to assess copy number vs. length distribution. Our pipeline has three significant advantages compared to other methods for ab initio repeat identification: 1) we generate not only consensus sequences, but keep the genomic intervals for the original aligned sequences, allowing straightforward analysis of evolutionary dynamics, 2) consensus sequences represent low-divergence, recently/currently active TE families, 3) segmental duplications are annotated as a useful by-product. We have compared our ab initio repeat annotations for 7 genome assemblies to other methods and demonstrate that CARP compares favourably with RepeatModeler, the most widely used repeat annotation package. PMID:29538441

Prevalence of HIV-1 Subtypes and Drug Resistance-Associated Mutations in HIV-1-Positive Treatment-Naive Pregnant Women in Pointe Noire, Republic of the Congo (Kento-Mwana Project).

PubMed

Bruzzone, Bianca; Saladini, Francesco; Sticchi, Laura; Mayinda Mboungou, Franc A; Barresi, Renata; Caligiuri, Patrizia; Calzi, Anna; Zazzi, Maurizio; Icardi, Giancarlo; Viscoli, Claudio; Bisio, Francesca

2015-08-01

The Kento-Mwana project was carried out in Pointe Noire, Republic of the Congo, to prevent mother-to-child HIV-1 transmission. To determine the prevalence of different subtypes and transmitted drug resistance-associated mutations, 95 plasma samples were collected at baseline from HIV-1-positive naive pregnant women enrolled in the project during the years 2005-2008. Full protease and partial reverse transcriptase sequencing was performed and 68/95 (71.6%) samples were successfully sequenced. Major mutations to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 4/68 (5.9%), 3/68 (4.4%), and 2/68 (2.9%) samples, respectively. Phylogenetic analysis of HIV-1 isolates showed a high prevalence of unique recombinant forms (24/68, 35%), followed by CRF45_cpx (7/68, 10.3%) and subsubtype A3 and subtype G (6/68 each, 8.8%). Although the prevalence of transmitted drug resistance mutations appears to be currently limited, baseline HIV-1 genotyping is highly advisable in conjunction with antiretroviral therapy scale-up in resource-limited settings to optimize treatment and prevent perinatal transmission.

Occurrence of Cucumber mosaic virus on vanilla (Vanilla planifolia Andrews) in India.

PubMed

Madhubala, R; Bhadramurthy, V; Bhat, A I; Hareesh, P S; Retheesh, S T; Bhai, R S

2005-06-01

Cucumber mosaic virus (CMV) causing mosaic, leaf distortion and stunting of vanilla (Vanilla planifolia Andrews) in India was characterized on the basis of biological and coat protein (CP) nucleotide sequence properties. In mechanical inoculation tests, the virus was found to infect members of Chenopodiaceae, Cucurbitaceae, Fabaceae and Solanaceae. Nicotiana benthamiana was found to be a suitable host for the propagation of CMV. The virus was purified from inoculated N. benthamiana plants and negatively stained purified preparations contained isometric particles of about 28 nm in diameter. The molecular weight of the viral coat protein subunits was found to be 25.0 kDa. Polyclonal antiserum was produced in New Zealand white rabbit, immunoglobulin G (IgG) was purified and conjugated with alkaline phosphatase enzyme. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) method was standardized for the detection of CMV infection in vanilla plants. CP gene of the virus was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR), cloned and sequenced. Sequenced region contained a single open reading frame of 657 nucleotides potentially coding for 218 amino acids. Sequence analyses with other CMV isolates revealed the greatest identity with black pepper isolate of CMV (99%) and the phylogram clearly showed that CMV infecting vanilla belongs to subgroup IB. This is the first report of occurrence of CMV on V. planifolia from India.

Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine.

PubMed Central

Medlin, H K; Zhu, Y Q; Remington, K M; Phillips, T R; North, T W

1996-01-01

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis. PMID:8849258

Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

PubMed

AboElkhair, M; Iwamoto, T; Clark, K F; McKenna, P; Siah, A; Greenwood, S J; Berthe, F C J; Casey, J W; Cepica, A

2012-01-01

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences. Copyright © 2011 Elsevier Inc. All rights reserved.

Dynamics of drug resistance-associated mutations in HIV-1 DNA reverse transcriptase sequence during effective ART.

PubMed

Nouchi, A; Nguyen, T; Valantin, M A; Simon, A; Sayon, S; Agher, R; Calvez, V; Katlama, C; Marcelin, A G; Soulie, C

2018-05-29

To investigate the dynamics of HIV-1 variants archived in cells harbouring drug resistance-associated mutations (DRAMs) to lamivudine/emtricitabine, etravirine and rilpivirine in patients under effective ART free from selective pressure on these DRAMs, in order to assess the possibility of recycling molecules with resistance history. We studied 25 patients with at least one DRAM to lamivudine/emtricitabine, etravirine and/or rilpivirine identified on an RNA sequence in their history and with virological control for at least 5 years under a regimen excluding all drugs from the resistant class. Longitudinal ultra-deep sequencing (UDS) and Sanger sequencing of the reverse transcriptase region were performed on cell-associated HIV-1 DNA samples taken over the 5 years of follow-up. Viral variants harbouring the analysed DRAMs were no longer detected by UDS over the 5 years in 72% of patients, with viruses susceptible to the molecules of interest found after 5 years in 80% of patients with UDS and in 88% of patients with Sanger. Residual viraemia with <50 copies/mL was detected in 52% of patients. The median HIV DNA level remained stable (2.4 at baseline versus 2.1 log10 copies/106 cells 5 years later). These results show a clear trend towards clearance of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA after a long period of virological control, free from therapeutic selective pressure on these DRAMs, reflecting probable residual replication in some reservoirs of the fittest viruses and leading to persistent evolution of the archived HIV-1 DNA resistance profile.

A de novo transcriptome and valid reference genes for quantitative real-time PCR in Colaphellus bowringi.

PubMed

Tan, Qian-Qian; Zhu, Li; Li, Yi; Liu, Wen; Ma, Wei-Hua; Lei, Chao-Liang; Wang, Xiao-Ping

2015-01-01

The cabbage beetle Colaphellus bowringi Baly is a serious insect pest of crucifers and undergoes reproductive diapause in soil. An understanding of the molecular mechanisms of diapause regulation, insecticide resistance, and other physiological processes is helpful for developing new management strategies for this beetle. However, the lack of genomic information and valid reference genes limits knowledge on the molecular bases of these physiological processes in this species. Using Illumina sequencing, we obtained more than 57 million sequence reads derived from C. bowringi, which were assembled into 39,390 unique sequences. A Clusters of Orthologous Groups classification was obtained for 9,048 of these sequences, covering 25 categories, and 16,951 were assigned to 255 Kyoto Encyclopedia of Genes and Genomes pathways. Eleven candidate reference gene sequences from the transcriptome were then identified through reverse transcriptase polymerase chain reaction. Among these candidate genes, EF1α, ACT1, and RPL19 proved to be the most stable reference genes for different reverse transcriptase quantitative polymerase chain reaction experiments in C. bowringi. Conversely, aTUB and GAPDH were the least stable reference genes. The abundant putative C. bowringi transcript sequences reported enrich the genomic resources of this beetle. Importantly, the larger number of gene sequences and valid reference genes provide a valuable platform for future gene expression studies, especially with regard to exploring the molecular mechanisms of different physiological processes in this species.

Evolutionary Conservation of a Coding Function for D4Z4, the Tandem DNA Repeat Mutated in Facioscapulohumeral Muscular Dystrophy

PubMed Central

Clapp, Jannine ; Mitchell, Laura M. ; Bolland, Daniel J. ; Fantes, Judy ; Corcoran, Anne E. ; Scotting, Paul J. ; Armour, John A. L. ; Hewitt, Jane E. 

2007-01-01

Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions within the polymorphic DNA tandem array D4Z4. Each D4Z4 repeat unit has an open reading frame (ORF), termed “DUX4,” containing two homeobox sequences. Because there has been no evidence of a transcript from the array, these deletions are thought to cause FSHD by a position effect on other genes. Here, we identify D4Z4 homologues in the genomes of rodents, Afrotheria (superorder of elephants and related species), and other species and show that the DUX4 ORF is conserved. Phylogenetic analysis suggests that primate and Afrotherian D4Z4 arrays are orthologous and originated from a retrotransposed copy of an intron-containing DUX gene, DUXC. Reverse-transcriptase polymerase chain reaction and RNA fluorescence and tissue in situ hybridization data indicate transcription of the mouse array. Together with the conservation of the DUX4 ORF for >100 million years, this strongly supports a coding function for D4Z4 and necessitates re-examination of current models of the FSHD disease mechanism. PMID:17668377

A model of directional selection applied to the evolution of drug resistance in HIV-1.

PubMed

Seoighe, Cathal; Ketwaroo, Farahnaz; Pillay, Visva; Scheffler, Konrad; Wood, Natasha; Duffet, Rodger; Zvelebil, Marketa; Martinson, Neil; McIntyre, James; Morris, Lynn; Hide, Winston

2007-04-01

Understanding how pathogens acquire resistance to drugs is important for the design of treatment strategies, particularly for rapidly evolving viruses such as HIV-1. Drug treatment can exert strong selective pressures and sites within targeted genes that confer resistance frequently evolve far more rapidly than the neutral rate. Rapid evolution at sites that confer resistance to drugs can be used to help elucidate the mechanisms of evolution of drug resistance and to discover or corroborate novel resistance mutations. We have implemented standard maximum likelihood methods that are used to detect diversifying selection and adapted them for use with serially sampled reverse transcriptase (RT) coding sequences isolated from a group of 300 HIV-1 subtype C-infected women before and after single-dose nevirapine (sdNVP) to prevent mother-to-child transmission. We have also extended the standard models of codon evolution for application to the detection of directional selection. Through simulation, we show that the directional selection model can provide a substantial improvement in sensitivity over models of diversifying selection. Five of the sites within the RT gene that are known to harbor mutations that confer resistance to nevirapine (NVP) strongly supported the directional selection model. There was no evidence that other mutations that are known to confer NVP resistance were selected in this cohort. The directional selection model, applied to serially sampled sequences, also had more power than the diversifying selection model to detect selection resulting from factors other than drug resistance. Because inference of selection from serial samples is unlikely to be adversely affected by recombination, the methods we describe may have general applicability to the analysis of positive selection affecting recombining coding sequences when serially sampled data are available.

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases

PubMed Central

Zajac, Pawel; Islam, Saiful; Hochgerner, Hannah; Lönnerberg, Peter; Linnarsson, Sten

2013-01-01

Reverse transcriptases derived from Moloney Murine Leukemia Virus (MMLV) have an intrinsic terminal transferase activity, which causes the addition of a few non-templated nucleotides at the 3´ end of cDNA, with a preference for cytosine. This mechanism can be exploited to make the reverse transcriptase switch template from the RNA molecule to a secondary oligonucleotide during first-strand cDNA synthesis, and thereby to introduce arbitrary barcode or adaptor sequences in the cDNA. Because the mechanism is relatively efficient and occurs in a single reaction, it has recently found use in several protocols for single-cell RNA sequencing. However, the base preference of the terminal transferase activity is not known in detail, which may lead to inefficiencies in template switching when starting from tiny amounts of mRNA. Here, we used fully degenerate oligos to determine the exact base preference at the template switching site up to a distance of ten nucleotides. We found a strong preference for guanosine at the first non-templated nucleotide, with a greatly reduced bias at progressively more distant positions. Based on this result, and a number of careful optimizations, we report conditions for efficient template switching for cDNA amplification from single cells. PMID:24392002

Characterization of the synthesis and expression of the GTA-kinase from transformed and normal rodent cells.

PubMed

Kerr, M; Fischer, J E; Purushotham, K R; Gao, D; Nakagawa, Y; Maeda, N; Ghanta, V; Hiramoto, R; Chegini, N; Humphreys-Beher, M G

1994-08-02

The murine transformed cell line YC-8 and beta-adrenergic receptor agonist (isoproternol) treated rat and mouse parotid gland acinar cells ectopically express cell surface beta 1-4 galactosyltransferase during active proliferation. This activity is dependent upon the expression of the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones for the protein coding region of the kinase were isolated by reverse transcriptase-PCR cloning. DNA sequence analysis failed to show sequence differences with the normal homolog from mouse cells although Southern blot analysis of YC-8, and a second cell line KI81, indicated changes in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The rat cDNA clone from isoproterenol-treated salivary glands showed a high degree of protein and nucleic acid sequence homology to the GTA-kinase from both murine and human sources. Northern blot analysis of YC-8 and a control cell line LSTRA revealed the synthesis of a major 3.0 kb mRNA from both cell lines plus the unique expression of a 4.5 kb mRNA in the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirmed the increased steady state levels of the GTA-kinase mRNA in YC-8. In the mouse, induction of cell proliferation by isoproterenol resulted in a 50-fold increase in steady state mRNA levels for the kinase over the low level of expression in quiescent cells. Expression of the rat 3' untranslated region in rat parotid cells in vitro led to an increased rate of DNA synthesis, cell number an ectopic expression of cell surface galactosyltransferase in the sense orientation. Antisense expression or vector alone did not alter growth characteristics of acinar cells. A polyclonal antibody monospecific to a murine amino terminal peptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. However, upon growth stimulation, kinase was detected primarily in a perinuclear and nuclear immunostaining pattern. Western blot analysis confirmed a translocation from a cytoplasmic localization in both LSTRA and quiescent salivary cells to a membrane-associated localization in YC-8 and proliferating salivary cells.

HIV type 1 diversity in the Seychelles.

PubMed

Razafindratsimandresy, Richter; Hollanda, Justina; Soares, Jean-Louis; Rousset, Dominique; Chetty, Agnes P; Reynes, Jean-Marc

2007-06-01

Subtype determination and drug resistance-associated mutations (DRM) detection were performed on 40 HIV-1 Western blot-positive sera detected, obtained from consecutive patients resident in the Seychelles and consulting the Communicable Disease Control Unit, HIV reference center, in Victoria Hospital (Mahe) from October 2005 to June 2006. Amplification and sequencing of at least two of the partial reverse transcriptase, protease, and partial envelope genes were successful for all strains. All three genes sequences were obtained for 39 strains. A high degree of subtype or circulating recombinant forms (CRF) was observed for these 39 strains: A-A1 (17 cases), C (10 cases), B (8 cases), CRF02_AG (2 cases), D (1 case) and CRF01_AE (1 case). According to the ANRS 2006 DRM list and algorithm, none of the 40 isolates was found to be resistant to any protease or reverse transcriptase inhibitors.

Isolation and characterization of the pea cytochrome c oxidase Vb gene.

PubMed

Kubo, Nakao; Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Kadowaki, Koh-Ichi; Hirai, Masashi

2006-11-01

Three copies of the gene that encodes cytochrome c oxidase subunit Vb were isolated from the pea (PscoxVb-1, PscoxVb-2, and PscoxVb-3). Northern Blot and reverse transcriptase-PCR analyses suggest that all 3 genes are transcribed in the pea. Each pea coxVb gene has an N-terminal extended sequence that can encode a mitochondrial targeting signal, called a presequence. The localization of green fluorescent proteins fused with the presequence strongly suggests the targeting of pea COXVb proteins to mitochondria. Each pea coxVb gene has 5 intron sites within the coding region. These are similar to Arabidopsis and rice, although the intron lengths vary greatly. A phylogenetic analysis of coxVb suggests the occurrence of gene duplication events during angiosperm evolution. In particular, 2 duplication events might have occurred in legumes, grasses, and Solanaceae. A comparison of amino acid sequences in COXVb or its counterpart shows the conservation of several amino acids within a zinc finger motif. Interestingly, a homology search analysis showed that bacterial protein COG4391 and a mitochondrial complex I 13 kDa subunit also have similar amino acid compositions around this motif. Such similarity might reflect evolutionary relationships among the 3 proteins.

Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine.

PubMed Central

Gu, Z; Gao, Q; Li, X; Parniak, M A; Wainberg, M A

1992-01-01

We have used the technique of in vitro selection to generate variants of human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxyinosine (ddI) and cross-resistant to 2',3'-dideoxycytidine (ddC). The complete reverse transcriptase (RT)-coding regions, plus portions of flanking sequences, of viruses possessing a ddI-resistant phenotype were cloned and sequenced by polymerase chain reaction (PCR)-based methods. We observed that several of these viruses possessed mutations at amino acid sites 184 (Met-->Val; ATG-->GTG) and 294 (Pro-->Ser; CCA-->TCA). These mutations were introduced in the pol gene of infectious, cloned HXB2-D DNA by site-directed mutagenesis. Viral replication assays confirmed the importance of site 184 with regard to resistance to ddI. The recombinant viruses thus generated displayed more than fivefold-greater resistance to ddI than parental HXB2-D did. Moreover, more than fivefold-greater resistance to ddC was also documented; however, the recombinant viruses continued to be inhibited by zidovudine (AZT). No resistance to ddI, ddC, or AZT was introduced by inclusion of mutation site 294 in the pol gene of HXB2-D. PCR analysis performed on viral samples obtained from patients receiving long-term ddI therapy confirmed the presence of mutation site 184 in five of seven cases tested. In three of these five positive cases, the wild-type codon was also detected, indicating that mixtures of viral quasispecies were apparently present. Viruses possessing a ddI resistance phenotype were isolated from both subjects whose viruses contained only the mutated rather than wild-type codon at position 184 as well as from a third individual, whose viruses appeared to be mostly of the mutated variety. Images PMID:1279198

Emergence of a replicating species from an in vitro RNA evolution reaction

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Joyce, G. F.

1994-01-01

The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.

Molecular Characterization of the Human Immunodeficiency Virus Type 1 in Women and Their Vertically Infected Children.

PubMed

Vaz, Sara Nunes; Giovanetti, Marta; Rego, Filipe Ferreira de Almeida; Oliveira, Tulio de; Danaviah, Siva; Gonçalves, Maria Luiza Freire; Alcantara, Luiz Carlos Junior; Brites, Carlos

2015-10-01

Approximately 35 million people worldwide are infected with human immunodeficiency virus (HIV) around 3.2 million of whom are children under 15 years. Mother-to-child-transmission (MTCT) of HIV-1 accounts for 90% of all infections in children. Despite great advances in the prevention of MTCT in Brazil, children are still becoming infected. Samples from 19 HIV-1-infected families were collected. DNA was extracted and fragments from gag, pol, and env were amplified and sequenced directly. Phylogenetic reconstruction was performed. Drug resistance analyses were performed in pol and env sequences. We found 82.1% of subtype B and 17.9% of BF recombinants. A prevalence of 43.9% drug resistance-associated mutations in pol sequences was identified. Of the drug-naive children 33.3% presented at least one mutation related to protease inhibitor/nucleoside reverse transcriptase inhibitor/nonnucleoside reverse transcriptase inhibitor (PI/NRTI/NNRTI) resistance. The prevalence of transmitted drug resistance mutations was 4.9%. On env we found a low prevalence of HR1 (4.9%) and HR2 (14.6%) mutations.

High level of APOBEC3F/3G editing in HIV-2 DNA vif and pol sequences from antiretroviral-naive patients.

PubMed

Bertine, Mélanie; Charpentier, Charlotte; Visseaux, Benoit; Storto, Alexandre; Collin, Gilles; Larrouy, Lucile; Damond, Florence; Matheron, Sophie; Brun-Vézinet, Françoise; Descamps, Diane

2015-04-24

In HIV-1, hypermutation introduced by APOBEC3F/3G cytidine deaminase activity leads to defective viruses. In-vivo impact of APOBEC3F/3G editing on HIV-2 sequences remains unknown. The objective of this study was to assess the level of APOBEC3F/3G editing in HIV-2-infected antiretroviral-naive patients. Direct sequencing of vif and pol regions was performed on HIV-2 proviral DNA from antiretroviral-naive patients included in the French Agence Nationale de Recherches sur le SIDA et les hépatites virales CO5 HIV-2 cohort. Hypermutated sequences were identified using Hypermut2.0 program. HIV-1 proviral sequences from Genbank were also assessed. Among 82 antiretroviral-naive HIV-2-infected patients assessed, 15 (28.8%) and five (16.7%) displayed Vif proviral defective sequences in HIV-2 groups A and B, respectively. A lower proportion of defective sequences was observed in protease-reverse transcriptase region. A higher median number of G-to-A mutations was observed in HIV-2 group B than in group A, both in Vif and protease-reverse transcriptase regions (P = 0.02 and P = 0.006, respectively). Compared with HIV-1 Vif sequences, a higher number of Vif defective sequences was observed in HIV-2 group A (P = 0.00001) and group B sequences (P = 0.013). We showed for the first time a high level of APOBEC3F/3G editing in HIV-2 sequences from antiretroviral-naive patients. Our study reported a group effect with a significantly higher level of APOBEC3F/3G editing in HIV-2 group B than in group A sequences.

Phylogenetic analysis of rubella viruses in Vietnam during 2009-2010.

PubMed

Tran, Dinh Nguyen; Pham, Ngan Thi Kim; Tran, Thi Thuy Trinh; Khamrin, Pattara; Thongprachum, Aksara; Komase, Katsuhiro; Hayakawa, Satoshi; Mizuguchi, Masashi; Ushijima, Hiroshi

2012-04-01

Rubella virus (RV) usually causes a mild disease. However, infection during the first trimester of pregnancy often leads to severe birth defects known as congenital rubella syndrome (CRS). Although wild-type RVs exist and circulate worldwide, their genotypes remain unknown in many countries. The aim of this study was to identify the molecular characteristics of RVs found in Vietnam during the years 2009-2010 and to provide the first data concerning RV genotypes in this country. Throat swab samples were collected between 2009 and 2010 from four CRS cases and nine rubella infection cases visiting one Children's Hospital and one outpatient clinic in Ho Chi Minh City. The 739-nucleotide coding region of the RV E1 gene recommended by the World Health Organization was amplified by reverse transcriptase PCR, and the resulting DNA fragments were then sequenced. Sequences were assigned to genotypes by phylogenetic analysis with RV reference strains. RV RNA was detected in 11 clinical specimens. Phylogenetic analysis of the sequences showed that all 11 strains belonged to 2B genotype. Several variations in amino acids were found, among which five changes were involved in the B and T cell epitopes. These data indicate that viruses of genotype 2B were circulating in Vietnam. The increasing information about RV genotype in Vietnam should aid in the control of rubella infection and CRS in this country. Copyright © 2012 Wiley Periodicals, Inc.

Chickpea chlorotic stunt virus: A New Polerovirus Infecting Cool-Season Food Legumes in Ethiopia.

PubMed

Abraham, A D; Menzel, W; Lesemann, D-E; Varrelmann, M; Vetten, H J

2006-05-01

ABSTRACT Serological analysis of diseased chickpea and faba bean plantings with yellowing and stunting symptoms suggested the occurrence of an unknown or uncommon member of the family Luteoviridae in Ethiopia. Degenerate primers were used for reverse transcriptase-polymerase chain reaction amplification of the viral coat protein (CP) coding region from both chickpea and faba bean samples. Cloning and sequencing of the amplicons yielded nearly identical (96%) nucleotide sequences of a previously unrecognized species of the family Luteoviridae, with a CP amino acid sequence most closely related (identity of approximately 78%) to that of Groundnut rosette assistor virus. The complete genome (5,900 nts) of a faba bean isolate comprised six major open reading frames characteristic of polero-viruses. Of the four aphid species tested, only Aphis craccivora transmitted the virus in a persistent manner. The host range of the virus was confined to a few species of the family Fabaceae. A rabbit antiserum raised against virion preparations cross-reacted unexpectedly with Beet western yellows virus-like viruses. This necessitated the production of murine monoclonal antibodies which, in combination with the polyclonal antiserum, permitted both sensitive and specific detection of the virus in field samples by triple-antibody sandwich, enzyme-linked immunosorbent assay. Because of the characteristic field and greenhouse symptoms in chickpea, the name Chickpea chlorotic stunt virus is proposed for this new member of the genus Polerovirus (family Luteoviridae).

Lentin, a novel and potent antifungal protein from shitake mushroom with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase and proliferation of leukemia cells.

PubMed

Ngai, Patrick H K; Ng, T B

2003-11-14

From the fruiting bodies of the edible mushroom Lentinus edodes, a novel protein designated lentin with potent antifungal activity was isolated. Lentin was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. The N-terminal sequence of lentin manifested similarity to endoglucanase. Lentin, which had a molecular mass of 27.5 kDa, inhibited mycelial growth in a variety of fungal species including Physalospora piricola, Botrytis cinerea and Mycosphaerella arachidicola. Lentin also exerted an inhibitory activity on HIV-1 reverse transcriptase and proliferation of leukemia cells.

Varied prevalence of Borna disease virus infection in Arabic, thoroughbred and their cross-bred horses in Iran.

PubMed

Bahmani, M K; Nowrouzian, I; Nakaya, T; Nakamura, Y; Hagiwara, K; Takahashi, H; Rad, M A; Ikuta, K

1996-11-01

Borna disease virus (BDV) naturally infects horses and sheep and induces progressive poliomeningoencephalomyelitis. Here, BDV recombinant proteins of the first open reading frame (ORF-I; coding for p40 nucleoprotein) and the second ORF-II (coding for p24 polymerase cofactor) were immunoblotted with plasma derived from 72 healthy (28 Arabic, 17 thoroughbred and 27 cross-bred) race horses at Tehran in Iran to detect anti-BDV antibodies. In addition, their peripheral blood mononuclear cells (PBMCs) were also examined for BDV RNA by a nested reverse transcriptase-polymerase chain reaction (RT-PCR) at ORF-II. The prevalence of BDV antibodies and/or RNA was 41.2% in Arabic, 23.5% in thoroughbred, and 33.3% in cross-bred horses, but only 17.9, 5.9, and 11.1% of them, respectively, showed positive signals for both BDV antibodies and RNA. Especially, cross-bred horses showed a higher prevalence for BDV RNA, which was detected only in females. In addition, significantly higher prevalence for BDV RNA was observed in Arabic males and thoroughbred females. The BDV prevalence did not increase with aging of the horse. Sequencing at the region of BDV derived from Iranian horses revealed a slight difference from those of Japanese horse- and European horse-derived BDVs even in the amino acid residues, although those in the three groups of Iranian horses were quite similar. Thus, the varied prevalence of BDV was observed with the horse strain or sex in Iranian horses, although BDV sequences were very similar among all three groups in Iran compared with those derived from other countries.

Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros.

PubMed

Yang, J; Yamamoto, M; Ishibashi, J; Taniai, K; Yamakawa, M

1998-08-01

An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

PubMed

Foord, A J; Heine, H G; Pritchard, L I; Lunt, R A; Newberry, K M; Rootes, C L; Boyle, D B

2006-07-01

To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

Characterization of Nucleoside Reverse Transcriptase Inhibitor-Associated Mutations in the RNase H Region of HIV-1 Subtype C Infected Individuals.

PubMed

Ngcapu, Sinaye; Theys, Kristof; Libin, Pieter; Marconi, Vincent C; Sunpath, Henry; Ndung'u, Thumbi; Gordon, Michelle L

2017-11-08

The South African national treatment programme includes nucleoside reverse transcriptase inhibitors (NRTIs) in both first and second line highly active antiretroviral therapy regimens. Mutations in the RNase H domain have been associated with resistance to NRTIs but primarily in HIV-1 subtype B studies. Here, we investigated the prevalence and association of RNase H mutations with NRTI resistance in sequences from HIV-1 subtype C infected individuals. RNase H sequences from 112 NRTI treated but virologically failing individuals and 28 antiretroviral therapy (ART)-naive individuals were generated and analysed. In addition, sequences from 359 subtype C ART-naive sequences were downloaded from Los Alamos database to give a total of 387 sequences from ART-naive individuals for the analysis. Fisher's exact test was used to identify mutations and Bayesian network learning was applied to identify novel NRTI resistance mutation pathways in RNase H domain. The mutations A435L, S468A, T470S, L484I, A508S, Q509L, L517I, Q524E and E529D were more prevalent in sequences from treatment-experienced compared to antiretroviral treatment naive individuals, however, only the E529D mutation remained significant after correction for multiple comparison. Our findings suggest a potential interaction between E529D and NRTI-treatment; however, site-directed mutagenesis is needed to understand the impact of this RNase H mutation.

Murine Leukemia Virus Reverse Transcriptase: Structural Comparison with HIV-1 Reverse Transcriptase

PubMed Central

Coté, Marie L.; Roth, Monica J.

2008-01-01

Recent X-ray crystal structure determinations of Moloney murine leukemia virus reverse transcriptase (MoMLV RT) have allowed for more accurate structure/function comparisons to HIV-1 RT than were formerly possible. Previous biochemical studies of MoMLV RT in conjunction with knowledge of sequence homologies to HIV-1 RT and overall fold similarities to RTs in general, provided a foundation upon which to build. In addition, numerous crystal structures of the MoMLV RT fingers/palm subdomain had also shed light on one of the critical functions of the enzyme, specifically polymerization. Now in the advent of new structural information, more intricate examination of MoMLV RT in its entirety can be realized, and thus the comparisons with HIV-1 RT may be more critically elucidated. Here, we will review the similarities and differences between MoMLV RT and HIV-1 RT via structural analysis, and propose working models for the MoMLV RT based upon that information. PMID:18294720

HIV type 1 genotypic variation in an antiretroviral treatment-naive population in southern India.

PubMed

Balakrishnan, Pachamuthu; Kumarasamy, Nagalingeswaran; Kantor, Rami; Solomon, Suniti; Vidya, Sundararajan; Mayer, Kenneth H; Newstein, Michael; Thyagarajan, Sadras P; Katzenstein, David; Ramratnam, Bharat

2005-04-01

Most studies of HIV-1 drug resistance have examined subtype B viruses; fewer data are available from developing countries, where non-B subtypes predominate. We determined the prevalence of mutations at protease and reverse transcriptase drug resistance positions in antiretroviral drug-naive individuals in southern India. The pol region of the genome was amplified from plasma HIV-1 RNA in 50 patients. All sequences clustered with HIV-1 subtype C. All patients had at least one protease and/or RT mutation at a known subtype B drug resistance position. Twenty percent of patients had mutations at major protease inhibitor resistance positions and 100% had mutations at minor protease inhibitor resistance positions. Six percent and 14% of patients had mutations at nucleoside reverse transcriptase inhibitor and/or nonnucleoside reverse transcriptase inhibitor resistance positions, respectively. Larger scale studies need to be undertaken to better define the genotypic variation of circulating Indian subtype C viruses and their potential impact on drug susceptibility and clinical outcome in treated individuals.

Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

PubMed

Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

2016-11-01

Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

PubMed

Kilian, A; Bowtell, D D; Abud, H E; Hime, G R; Venter, D J; Keese, P K; Duncan, E L; Reddel, R R; Jefferson, R A

1997-11-01

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Comparative analysis of drug resistance mutations in the human immunodeficiency virus reverse transcriptase gene in patients who are non-responsive, responsive and naive to antiretroviral therapy.

PubMed

Misbah, Mohammad; Roy, Gaurav; Shahid, Mudassar; Nag, Nalin; Kumar, Suresh; Husain, Mohammad

2016-05-01

Drug resistance mutations in the Pol gene of human immunodeficiency virus 1 (HIV-1) are one of the critical factors associated with antiretroviral therapy (ART) failure in HIV-1 patients. The issue of resistance to reverse transcriptase inhibitors (RTIs) in HIV infection has not been adequately addressed in the Indian subcontinent. We compared HIV-1 reverse transcriptase (RT) gene sequences to identify mutations present in HIV-1 patients who were ART non-responders, ART responders and drug naive. Genotypic drug resistance testing was performed by sequencing a 655-bp region of the RT gene from 102 HIV-1 patients, consisting of 30 ART-non-responding, 35 ART-responding and 37 drug-naive patients. The Stanford HIV Resistance Database (HIVDBv 6.2), IAS-USA mutation list, ANRS_09/2012 algorithm, and Rega v8.02 algorithm were used to interpret the pattern of drug resistance. The majority of the sequences (96 %) belonged to subtype C, and a few of them (3.9 %) to subtype A1. The frequency of drug resistance mutations observed in ART-non-responding, ART-responding and drug-naive patients was 40.1 %, 10.7 % and 20.58 %, respectively. It was observed that in non-responders, multiple mutations were present in the same patient, while in responders, a single mutation was found. Some of the drug-naive patients had more than one mutation. Thymidine analogue mutations (TAMs), however, were found in non-responders and naive patients but not in responders. Although drug resistance mutations were widely distributed among ART non-responders, the presence of resistance mutations in the viruses of drug-naive patients poses a big concern in the absence of a genotyping resistance test.

DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.

PubMed

Zubradt, Meghan; Gupta, Paromita; Persad, Sitara; Lambowitz, Alan M; Weissman, Jonathan S; Rouskin, Silvi

2017-01-01

Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.

The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus

PubMed Central

Figueiredo, Luisa M.; Rocha, Eduardo P. C.; Mancio-Silva, Liliana; Prevost, Christine; Hernandez-Verdun, Danièle; Scherf, Artur

2005-01-01

Telomerase replicates chromosome ends, a function necessary for maintaining genome integrity. We have identified the gene that encodes the catalytic reverse transcriptase (RT) component of this enzyme in the malaria parasite Plasmodium falciparum (PfTERT) as well as the orthologous genes from two rodent and one simian malaria species. PfTERT is predicted to encode a basic protein that contains the major sequence motifs previously identified in known telomerase RTs (TERTs). At ∼2500 amino acids, PfTERT is three times larger than other characterized TERTs. We observed remarkable sequence diversity between TERT proteins of different Plasmodial species, with conserved domains alternating with hypervariable regions. Immunofluorescence analysis revealed that PfTERT is expressed in asexual blood stage parasites that have begun DNA synthesis. Surprisingly, rather than at telomere clusters, PfTERT typically localizes into a discrete nuclear compartment. We further demonstrate that this compartment is associated with the nucleolus, hereby defined for the first time in P.falciparum. PMID:15722485

Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

PubMed Central

Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

2016-01-01

Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage. PMID:26613339

Secondary structure model of the RNA recognized by the reverse transcriptase from the R2 retrotransposable element.

PubMed Central

Mathews, D H; Banerjee, A R; Luan, D D; Eickbush, T H; Turner, D H

1997-01-01

RNA transcripts corresponding to the 250-nt 3' untranslated region of the R2 non-LTR retrotransposable element are recognized by the R2 reverse transcriptase and are sufficient to serve as templates in the target DNA-primed reverse transcription (TPRT) reaction. The R2 protein encoded by the Bombyx mori R2 can recognize this region from both the B. mori and Drosophila melanogaster R2 elements even though these regions show little nucleotide sequence identity. A model for the RNA secondary structure of the 3' untranslated region of the D. melanogaster R2 retrotransposon was developed by sequence comparison of 10 species aided by free energy minimization. Chemical modification experiments are consistent with this prediction. A secondary structure model for the 3' untranslated region of R2 RNA from the R2 element from B. mori was obtained by a combination of chemical modification data and free energy minimization. These two secondary structure models, found independently, share several common sites. This study shows the utility of combining free energy minimization, sequence comparison, and chemical modification to model an RNA secondary structure. PMID:8990394

Nucleotide Sequence Analysis of RNA Synthesized from Rabbit Globin Complementary DNA

PubMed Central

Poon, Raymond; Paddock, Gary V.; Heindell, Howard; Whitcome, Philip; Salser, Winston; Kacian, Dan; Bank, Arthur; Gambino, Roberto; Ramirez, Francesco

1974-01-01

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as template for in vitro synthesis of 32P-labeled RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. Several fragments were long enough to fit uniquely with the α or β globin amino-acid sequences. These data demonstrate that the cDNA was copied from globin mRNA and contained no detectable contaminants. Images PMID:4139714

The molecular epidemiology of HIV-1 in the Comunidad Valenciana (Spain): analysis of transmission clusters.

PubMed

Patiño-Galindo, Juan Ángel; Torres-Puente, Manoli; Bracho, María Alma; Alastrué, Ignacio; Juan, Amparo; Navarro, David; Galindo, María José; Ocete, Dolores; Ortega, Enrique; Gimeno, Concepción; Belda, Josefina; Domínguez, Victoria; Moreno, Rosario; González-Candelas, Fernando

2017-09-14

HIV infections are still a very serious concern for public heath worldwide. We have applied molecular evolution methods to study the HIV-1 epidemics in the Comunidad Valenciana (CV, Spain) from a public health surveillance perspective. For this, we analysed 1804 HIV-1 sequences comprising protease and reverse transcriptase (PR/RT) coding regions, sampled between 2004 and 2014. These sequences were subtyped and subjected to phylogenetic analyses in order to detect transmission clusters. In addition, univariate and multinomial comparisons were performed to detect epidemiological differences between HIV-1 subtypes, and risk groups. The HIV epidemic in the CV is dominated by subtype B infections among local men who have sex with men (MSM). 270 transmission clusters were identified (>57% of the dataset), 12 of which included ≥10 patients; 11 of subtype B (9 affecting MSMs) and one (n = 21) of CRF14, affecting predominately intravenous drug users (IDUs). Dated phylogenies revealed these large clusters to have originated from the mid-80s to the early 00 s. Subtype B is more likely to form transmission clusters than non-B variants and MSMs to cluster than other risk groups. Multinomial analyses revealed an association between non-B variants, which are not established in the local population yet, and different foreign groups.

Sequence diversity among badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin and Nigeria.

PubMed

Eni, A O; Hughes, J d'A; Asiedu, R; Rey, M E C

2008-01-01

We analysed the sequence diversity in the reverse transcriptase (RT)/ribonuclease H (RNaseH) coding region of 19 badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin, and Nigeria. Phylogenetic analysis of the deduced amino acid sequences revealed that the isolates are broadly divided into two distinct species, each clustering with Dioscorea alata bacilliform virus (DaBV) and Dioscorea sansibarensis bacilliform virus (DsBV). Fourteen isolates had 90-96% amino acid identity with DaBV, while four isolates had 83-84% amino acid identity with DsBV. One isolate from Benin, BN4Dr, was distinct and had 77 and 75% amino acid identity with DaBV and DsBV, respectively, and may be a member of a new badnavirus species infecting yam in West Africa. Viruses of the two main species were present in Ghana, Togo and Benin and were observed to infect both D. alata and D. rotundata indiscriminately. This is the first confirmed report of DsBV infection in yam in Ghana and Togo. The results of this study demonstrate that members of two distinct species of badnaviruses infect yam in the West African yam zone and suggest a putative new species, BN4Dr. We also conclude that these species are not confined to limited geographic regions or specific for yam host species. However, the three badnavirus species are serologically related. The sequence information obtained from this study can be used to develop PCR-based diagnostics to detect members of the various species and/or strains of badnaviruses infecting yam in West Africa.

HIV drug resistance in infants increases with changing prevention of mother-to-child transmission regimens.

PubMed

Poppe, Lisa K; Chunda-Liyoka, Catherine; Kwon, Eun H; Gondwe, Clement; West, John T; Kankasa, Chipepo; Ndongmo, Clement B; Wood, Charles

2017-08-24

The objectives of this study were to determine HIV drug resistance (HIVDR) prevalence in Zambian infants upon diagnosis, and to determine how changing prevention of mother-to-child transmission (PMTCT) drug regimens affect drug resistance. Dried blood spot (DBS) samples from infants in the Lusaka District of Zambia, obtained during routine diagnostic screening, were collected during four different years representing three different PMTCT drug treatment regimens. DNA extracted from dried blood spot samples was used to sequence a 1493 bp region of the reverse transcriptase gene. Sequences were analyzed via the Stanford HIVDRdatabase (http://hivdb.standford.edu) to screen for resistance mutations. HIVDR in infants increased from 21.5 in 2007/2009 to 40.2% in 2014. Nonnucleoside reverse transcriptase inhibitor resistance increased steadily over the sampling period, whereas nucleoside reverse transcriptase inhibitor resistance and dual class resistance both increased more than threefold in 2014. Analysis of drug resistance scores in each group revealed increasing strength of resistance over time. In 2014, children with reported PMTCT exposure, defined as infant prophylaxis and/or maternal treatment, showed a higher prevalence and strength of resistance compared to those with no reported exposure. HIVDR is on the rise in Zambia and presents a serious problem for the successful lifelong treatment of HIV-infected children. PMTCT affects both the prevalence and strength of resistance and further research is needed to determine how to mitigate its role leading to resistance.

Antiretroviral treatment sequencing strategies to overcome HIV type 1 drug resistance in adolescents and adults in low-middle-income countries.

PubMed

De Luca, Andrea; Hamers, Raphael L; Schapiro, Jonathan M

2013-06-15

Antiretroviral treatment (ART) is expanding to human immunodeficiency virus type 1 (HIV-1)-infected persons in low-middle income countries, thanks to a public health approach. With 3 available drug classes, 2 ART sequencing lines are programmatically foreseen. The emergence and transmission of viral drug resistance represents a challenge to the efficacy of ART. Knowledge of HIV-1 drug resistance selection associated with specific drugs and regimens and the consequent activity of residual drug options are essential in programming ART sequencing options aimed at preserving ART efficacy for as long as possible. This article determines optimal ART sequencing options for overcoming HIV-1 drug resistance in resource-limited settings, using currently available drugs and treatment monitoring opportunities. From the perspective of drug resistance and on the basis of limited virologic monitoring data, optimal sequencing seems to involve use of a tenofovir-containing nonnucleoside reverse-transcriptase inhibitor-based first-line regimen, followed by a zidovudine-containing, protease inhibitor (PI)-based second-line regimen. Other options and their consequences are explored by considering within-class and between-class sequencing opportunities, including boosted PI monotherapies and future options with integrase inhibitors. Nucleoside reverse-transcriptase inhibitor resistance pathways in HIV-1 subtype C suggest an additional reason for accelerating stavudine phase out. Viral load monitoring avoids the accumulation of resistance mutations that significantly reduce the activity of next-line options. Rational use of resources, including broader access to viral load monitoring, will help ensure 3 lines of fully active treatment options, thereby increasing the duration of ART success.

Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2008-01-01

Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

Isolation and characterization of an AGAMOUS homolog from Fraxinus pennsylvanica

Treesearch

Ningxia Du; Paula M. Pijut

2010-01-01

An AGAMOUS homolog (FpAG) was isolated from green ash (Fraxinus pennsylvanica) using a reverse transcriptase polymerase chain reaction method. Southern blot analysis indicated that FpAG was present as a single-copy sequence in the genome of green ash. RNA accumulated in the reproductive tissues (female...

Recombination Creates Novel L1 (Line-1) Elements in Rattus Norvegicus

PubMed Central

Hayward, B. E.; Zavanelli, M.; Furano, A. V.

1997-01-01

Mammalian L1 (long interspersed repeated DNA, LINE-1) retrotransposons consist of a 5' untranslated region (UTR) with regulatory properties, two protein encoding regions (ORF I, ORF II, which encodes a reverse transcriptase) and a 3' UTR. L1 elements have been evolving in mammals for >100 million years and this process continues to generate novel L1 subfamilies in modern species. Here we characterized the youngest known subfamily in Rattus norvegicus, L1(mlvi2), and unexpectedly found that this element has a dual ancestry. While its 3' UTR shares the same lineage as its nearest chronologically antecedent subfamilies, L1(3) and L1(4), its ORF I sequence does not. The L1(mlvi2) ORF I was derived from an ancestral ORF I sequence that was the evolutionary precursor of the L1(3) and L1(4) ORF I. We suggest that an ancestral ORF I sequence was recruited into the modern L1(mlvi2) subfamily by recombination that possibly could have resulted from template strand switching by the reverse transcriptase during L1 replication. This mechanism could also account for some of the structural features of rodent L1 5' UTR and ORF I sequences including one of the more dramatic features of L1 evolution in mammals, namely the repeated acquisition of novel 5' UTRs. PMID:9178013

Molecular cloning and expression analysis of annexin A2 gene in sika deer antler tip.

PubMed

Xia, Yanling; Qu, Haomiao; Lu, Binshan; Zhang, Qiang; Li, Heping

2018-04-01

Molecular cloning and bioinformatics analysis of annexin A2 ( ANXA2 ) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer ( Cervus Nippon hortulorum ) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus . Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration.

PubMed

Kantor, Rami; Katzenstein, David A; Efron, Brad; Carvalho, Ana Patricia; Wynhoven, Brian; Cane, Patricia; Clarke, John; Sirivichayakul, Sunee; Soares, Marcelo A; Snoeck, Joke; Pillay, Candice; Rudich, Hagit; Rodrigues, Rosangela; Holguin, Africa; Ariyoshi, Koya; Bouzas, Maria Belen; Cahn, Pedro; Sugiura, Wataru; Soriano, Vincent; Brigido, Luis F; Grossman, Zehava; Morris, Lynn; Vandamme, Anne-Mieke; Tanuri, Amilcar; Phanuphak, Praphan; Weber, Jonathan N; Pillay, Deenan; Harrigan, P Richard; Camacho, Ricardo; Schapiro, Jonathan M; Shafer, Robert W

2005-04-01

The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.

Molecular characterization and genomic distribution of Isis: a new retrotransposon of Drosophila buzzatii.

PubMed

García Guerreiro, M P; Fontdevila, A

2007-01-01

A new transposable element, Isis, is identified as a LTR retrotransposon in Drosophila buzzatii. DNA sequence analysis shows that Isis contains three long ORFs similar to gag, pol and env genes of retroviruses. The ORF1 exhibits sequence homology to matrix, capsid and nucleocapsid gag proteins and ORF2 encodes a putative protease (PR), a reverse transcriptase (RT), an Rnase H (RH) and an integrase (IN) region. The analysis of a putative env product, encoded by the env ORF3, shows a degenerated protein containing several stop codons. The molecular study of the putative proteins coded by this new element shows striking similarities to both Ulysses and Osvaldo elements, two LTR retrotransposons, present in D. virilis and D. buzzatii, respectively. Comparisons of the predicted Isis RT to several known retrotransposons show strong phylogenetic relationships to gypsy-like elements, particulary to Ulysses retrotransposon. Studies of Isis chromosomal distribution show a strong hybridization signal in centromeric and pericentromeric regions, and a scattered distribution along all chromosomal arms. The existence of insertional polymorphisms between different strains and high molecular weight bands by Southern blot suggests the existence of full-sized copies that have been active recently. The presence of euchromatic insertion sites coincident between Isis and Osvaldo could indicate preferential insertion sites of Osvaldo element into Isis sequence or vice versa. Moreover, the presence of Isis in different species of the buzzatii complex indicates the ancient origin of this element.

The origin and early evolution of nucleic acid polymerases

NASA Technical Reports Server (NTRS)

Lazcano, A.; Cappello, R.; Valverde, V.; Llaca, V.; Oro, J.

1992-01-01

The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA-polymerase beta-prime subunit and its homologues is discussed. It is shown that, in the DNA-dependent RNA polymerases from three cellular lineages, a very conserved sequence of eight amino acids, also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein, is present. The optimal conditions for the replicase activity of the avian-myeloblastosis-virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is discussed.

Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kato, Yukinari

2018-05-03

Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

PubMed

Pal, Arnab; Srivastava, Tapasya; Sharma, Manish K; Mehndiratta, Mohit; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

2010-11-01

Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration. Real-time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter-alu PCR profile of DNA from cells cultured under 6-week hypoxia, its 4-week revert back to normoxia and 6-week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells. © 2009 The Authors Journal compilation © 2010 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

A recombination hot spot in HIV-1 contains guanosine runs that can form a G-quartet structure and promote strand transfer in vitro.

PubMed

Shen, Wen; Gao, Lu; Balakrishnan, Mini; Bambara, Robert A

2009-12-04

The co-packaged RNA genomes of human immunodeficiency virus-1 recombine at a high rate. Recombination can mix mutations to generate viruses that escape immune response. A cell-culture-based system was designed previously to map recombination events in a 459-bp region spanning the primer binding site through a portion of the gag protein coding region. Strikingly, a strong preferential site for recombination in vivo was identified within a 112-nucleotide-long region near the beginning of gag. Strand transfer assays in vitro revealed that three pause bands in the gag hot spot each corresponded to a run of guanosine (G) residues. Pausing of reverse transcriptase is known to promote recombination by strand transfer both in vivo and in vitro. To assess the significance of the G runs, we altered them by base substitutions. Disruption of the G runs eliminated both the associated pausing and strand transfer. Some G-rich sequences can develop G-quartet structures, which were first proposed to form in telomeric DNA. G-quartet structure formation is highly dependent on the presence of specific cations. Incubation in cations discouraging G-quartets altered gel mobility of the gag template consistent with breakdown of G-quartet structure. The same cations faded G-run pauses but did not affect pauses caused by hairpins, indicating that quartet structure causes pausing. Moreover, gel analysis with cations favoring G-quartet structure indicated no structure in mutated templates. Overall, results point to reverse transcriptase pausing at G runs that can form quartets as a unique feature of the gag recombination hot spot.

Generation and Characterization of a Defective HIV-1 Virus as an Immunogen for a Therapeutic Vaccine

PubMed Central

García-Pérez, Javier; García, Felipe; Blanco, Julia; Escribà-García, Laura; Gatell, Jose Maria; Alcamí, Jose; Plana, Montserrat; Sánchez-Palomino, Sonsoles

2012-01-01

Background The generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals. Results Viral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors. Conclusions We propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles. PMID:23144996

DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo

PubMed Central

Zubradt, Meghan; Gupta, Paromita; Persad, Sitara; Lambowitz, Alan M.; Weissman, Jonathan S.; Rouskin, Silvi

2017-01-01

Coupling structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structural studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduces biases and necessitates population-average assessments of RNA structure. Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase (TGIRT). DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in non-canonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs to their mature isoforms. These applications illustrate DMS-MaPseq’s capacity to dramatically expand in vivo analysis of RNA structure. PMID:27819661

The short interspersed repetitive element of Trypanosoma cruzi, SIRE, is part of VIPER, an unusual retroelement related to long terminal repeat retrotransposons

PubMed Central

Vázquez, Martín; Ben-Dov, Claudia; Lorenzi, Hernan; Moore, Troy; Schijman, Alejandro; Levin, Mariano J.

2000-01-01

The short interspersed repetitive element (SIRE) of Trypanosoma cruzi was first detected when comparing the sequences of loci that encode the TcP2β genes. It is present in about 1,500–3,000 copies per genome, depending on the strain, and it is distributed in all chromosomes. An initial analysis of SIRE sequences from 21 genomic fragments allowed us to derive a consensus nucleotide sequence and structure for the element, consisting of three regions (I, II, and III) each harboring distinctive features. Analysis of 158 transcribed SIREs demonstrates that the consensus is highly conserved. The sequences of 51 cDNAs show that SIRE is included in the 3′ end of several mRNAs, always transcribed from the sense strand, contributing the polyadenylation site in 63% of the cases. This study led to the characterization of VIPER (vestigial interposed retroelement), a 2,326-bp-long unusual retroelement. VIPER's 5′ end is formed by the first 182 bp of SIRE, whereas its 3′ end is formed by the last 220 bp of the element. Both SIRE moieties are connected by a 1,924-bp-long fragment that carries a unique ORF encoding a complete reverse transcriptase-RNase H gene whose 15 C-terminal amino acids derive from codons specified by SIRE's region II. The amino acid sequence of VIPER's reverse transcriptase-RNase H shares significant homology to that of long terminal repeat retrotransposons. The fact that SIRE and VIPER sequences are found only in the T. cruzi genome may be of relevance for studies concerning the evolution and the genome flexibility of this protozoan parasite. PMID:10688909

Deep sequencing analysis of HIV-1 reverse transcriptase at baseline and time of failure in patients receiving rilpivirine in the phase III studies ECHO and THRIVE.

PubMed

Van Eygen, Veerle; Thys, Kim; Van Hove, Carl; Rimsky, Laurence T; De Meyer, Sandra; Aerssens, Jeroen; Picchio, Gaston; Vingerhoets, Johan

2016-05-01

Minority variants (1.0-25.0%) were evaluated by deep sequencing (DS) at baseline and virological failure (VF) in a selection of antiretroviral treatment-naïve, HIV-1-infected patients from the rilpivirine ECHO/THRIVE phase III studies. Linkage between frequently emerging resistance-associated mutations (RAMs) was determined. DS (llIumina®) and population sequencing (PS) results were available at baseline for 47 VFs and time of failure for 48 VFs; and at baseline for 49 responders matched for baseline characteristics. Minority mutations were accurately detected at frequencies down to 1.2% of the HIV-1 quasispecies. No baseline minority rilpivirine RAMs were detected in VFs; one responder carried 1.9% F227C. Baseline minority mutations associated with resistance to other non-nucleoside reverse transcriptase inhibitors (NNRTIs) were detected in 8/47 VFs (17.0%) and 7/49 responders (14.3%). Baseline minority nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs M184V and L210W were each detected in one VF (none in responders). At failure, two patients without NNRTI RAMs by PS carried minority rilpivirine RAMs K101E and/or E138K; and five additional patients carried other minority NNRTI RAMs V90I, V106I, V179I, V189I, and Y188H. Overall at failure, minority NNRTI RAMs and NRTI RAMs were found in 29/48 (60.4%) and 16/48 VFs (33.3%), respectively. Linkage analysis showed that E138K and K101E were usually not observed on the same viral genome. In conclusion, baseline minority rilpivirine RAMs and other NNRTI/NRTI RAMs were uncommon in the rilpivirine arm of the ECHO and THRIVE studies. DS at failure showed emerging NNRTI resistant minority variants in seven rilpivirine VFs who had no detectable NNRTI RAMs by PS. © 2015 Wiley Periodicals, Inc.

Sequence diversity among badnavirus isolates infecting black pepper and related species in India.

PubMed

Bhat, A I; Sasi, Shina; Revathy, K A; Deeshma, K P; Saji, K V

2014-01-01

The badnavirus, piper yellow mottle virus (PYMoV) is known to infect black pepper (Piper nigrum), betelvine (P. betle) and Indian long pepper (P. longum) in India and other parts of the world. Occurrence of PYMoV or other badnaviruses in other species of Piper and its variability is not reported so far. We have analysed sequence variability in the conserved putative reverse transcriptase (RT)/ribonuclease H (RNase H) coding region of the virus using specific badnavirus primers from 13 virus isolates of black pepper collected from different cultivars and regions and one isolate each from 23 other species of Piper. Of these, four species failed to produce expected amplicon while amplicon from four other species showed more similarities to plant sequences than to badnaviruses. Of the remaining, isolates from black pepper, P. argyrophyllum, P. attenuatum, P. barberi, P. betle, P. colubrinum, P. galeatum, P. longum, P. ornatum, P. sarmentosum and P. trichostachyon showed an identity of >85 % at the nucleotide and >90 % at the amino acid level with PYMoV indicating that they are isolates of PYMoV. On the other hand high sequence variability of 21-43 % at nucleotide and 17-46 % at amino acid level compared to PYMoV was found among isolates infecting P. bababudani, P. chaba, P. peepuloides, P. mullesua and P. thomsonii suggesting the presence of new badnaviruses. Phylogenetic analyses showed close clustering of all PYMoV isolates that were well separated from other known badnaviruses. This is the first report of occurrence of PYMoV in eight Piper spp and likely occurrence of four new species in five Piper spp.

Molecular cloning, sequence identification and tissue expression profile of three novel sheep (Ovis aries) genes - BCKDHA, NAGA and HEXA.

PubMed

Liu, G Y; Gao, S Z

2009-01-01

The complete coding sequences of three sheep genes- BCKDHA, NAGA and HEXA were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR), based on the conserved sequence information of the mouse or other mammals. The nucleotide sequences of these three genes revealed that the sheep BCKDHA gene encodes a protein of 313 amino acids which has high homology with the BCKDHA gene that encodes a protein of 447 amino acids that has high homology with the Branched chain keto acid dehydrogenase El, alpha polypeptide (BCKDHA) of five species chimpanzee (93%), human (96%), crab-eating macaque (93%), bovine (98%) and mouse (91%). The sheep NAGA gene encodes a protein of 411 amino acids that has high homology with the alpha-N-acetylgalactosaminidase (NAGA) of five species human (85%), bovine (94%), mouse (91%), rat (83%) and chicken (74%). The sheep HEXA gene encodes a protein of 529 amino acids that has high homology with the hexosaminidase A(HEXA) of five species bovine (98%), human (84%), Bornean orangután (84%), rat (80%) and mouse (81%). Finally these three novel sheep genes were assigned to GenelDs: 100145857, 100145858 and 100145856. The phylogenetic tree analysis revealed that the sheep BCKDHA, NAGA, and HEXA all have closer genetic relationships to the BCKDHA, NAGA, and HEXA of bovine. Tissue expression profile analysis was also carried out and results revealed that sheep BCKDHA, NAGA and HEXA genes were differentially expressed in tissues including muscle, heart, liver, fat, kidney, lung, small and large intestine. Our experiment is the first to establish the primary foundation for further research on these three sheep genes.

Impact of HIV-1 Subtype and Antiretroviral Therapy on Protease and Reverse Transcriptase Genotype: Results of a Global Collaboration

PubMed Central

Kantor, Rami; Katzenstein, David A; Efron, Brad; Carvalho, Ana Patricia; Wynhoven, Brian; Cane, Patricia; Clarke, John; Sirivichayakul, Sunee; Soares, Marcelo A; Snoeck, Joke; Pillay, Candice; Rudich, Hagit; Rodrigues, Rosangela; Holguin, Africa; Ariyoshi, Koya; Bouzas, Maria Belen; Cahn, Pedro; Sugiura, Wataru; Soriano, Vincent; Brigido, Luis F; Grossman, Zehava; Morris, Lynn; Vandamme, Anne-Mieke; Tanuri, Amilcar; Phanuphak, Praphan; Weber, Jonathan N; Pillay, Deenan; Harrigan, P. Richard; Camacho, Ricardo; Schapiro, Jonathan M; Shafer, Robert W

2005-01-01

Background The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. Methods and Findings To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Conclusion Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations. PMID:15839752

A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.

PubMed

Waugh, Caryll; Cromer, Deborah; Grimm, Andrew; Chopra, Abha; Mallal, Simon; Davenport, Miles; Mak, Johnson

2015-04-09

Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles. Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses. We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts. Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies.

PubMed

Nagel, Inga; Szczepanowski, Monika; Martín-Subero, José I; Harder, Lana; Akasaka, Takashi; Ammerpohl, Ole; Callet-Bauchu, Evelyne; Gascoyne, Randy D; Gesk, Stefan; Horsman, Doug; Klapper, Wolfram; Majid, Aneela; Martinez-Climent, José A; Stilgenbauer, Stephan; Tönnies, Holger; Dyer, Martin J S; Siebert, Reiner

2010-08-26

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis.

Partial androgen insensitivity syndrome caused by a deep intronic mutation creating an alternative splice acceptor site of the AR gene.

PubMed

Ono, Hiroyuki; Saitsu, Hirotomo; Horikawa, Reiko; Nakashima, Shinichi; Ohkubo, Yumiko; Yanagi, Kumiko; Nakabayashi, Kazuhiko; Fukami, Maki; Fujisawa, Yasuko; Ogata, Tsutomu

2018-02-02

Although partial androgen insensitivity syndrome (PAIS) is caused by attenuated responsiveness to androgens, androgen receptor gene (AR) mutations on the coding regions and their splice sites have been identified only in <25% of patients with a diagnosis of PAIS. We performed extensive molecular studies including whole exome sequencing in a Japanese family with PAIS, identifying a deep intronic variant beyond the branch site at intron 6 of AR (NM_000044.4:c.2450-42 G > A). This variant created the splice acceptor motif that was accompanied by pyrimidine-rich sequence and two candidate branch sites. Consistent with this, reverse transcriptase (RT)-PCR experiments for cycloheximide-treated lymphoblastoid cell lines revealed a relatively large amount of aberrant mRNA produced by the newly created splice acceptor site and a relatively small amount of wildtype mRNA produced by the normal splice acceptor site. Furthermore, most of the aberrant mRNA was shown to undergo nonsense mediated decay (NMD) and, if a small amount of aberrant mRNA may have escaped NMD, such mRNA was predicted to generate a truncated AR protein missing some functional domains. These findings imply that the deep intronic mutation creating an alternative splice acceptor site resulted in the production of a relatively small amount of wildtype AR mRNA, leading to PAIS.

Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

PubMed

Haendeler, Judith; Dröse, Stefan; Büchner, Nicole; Jakob, Sascha; Altschmied, Joachim; Goy, Christine; Spyridopoulos, Ioakim; Zeiher, Andreas M; Brandt, Ulrich; Dimmeler, Stefanie

2009-06-01

The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.

Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods.

PubMed

Chahorm, Kanchana; Prakitchaiwattana, Cheunjit

2018-01-02

The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 10 2 to 10 5 CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 10 2 CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

PubMed Central

Chang, G J; Trent, D W; Vorndam, A V; Vergne, E; Kinney, R M; Mitchell, C J

1994-01-01

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay. PMID:7512096

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

PubMed

Chang, G J; Trent, D W; Vorndam, A V; Vergne, E; Kinney, R M; Mitchell, C J

1994-02-01

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay.

Mutations in FLVCR1 Cause Posterior Column Ataxia and Retinitis Pigmentosa

PubMed Central

Rajadhyaksha, Anjali M.; Elemento, Olivier; Puffenberger, Erik G.; Schierberl, Kathryn C.; Xiang, Jenny Z.; Putorti, Maria L.; Berciano, José; Poulin, Chantal; Brais, Bernard; Michaelides, Michel; Weleber, Richard G.; Higgins, Joseph J.

2010-01-01

The study of inherited retinal diseases has advanced our knowledge of the cellular and molecular mechanisms involved in sensory neural signaling. Dysfunction of two specific sensory modalities, vision and proprioception, characterizes the phenotype of the rare, autosomal-recessive disorder posterior column ataxia and retinitis pigmentosa (PCARP). Using targeted DNA capture and high-throughput sequencing, we analyzed the entire 4.2 Mb candidate sequence on chromosome 1q32 to find the gene mutated in PCARP in a single family. Employing comprehensive bioinformatic analysis and filtering, we identified a single-nucleotide coding variant in the feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), a gene encoding a heme-transporter protein. Sanger sequencing confirmed the FLVCR1 mutation in this family and identified different homozygous missense mutations located within the protein's transmembrane channel segment in two other unrelated families with PCARP. To determine whether the selective pathologic features of PCARP correlated with FLVCR1 expression, we examined wild-type mouse Flvcr1 mRNA levels in the posterior column of the spinal cord and the retina via quantitative real-time reverse-transcriptase PCR. The Flvcr1 mRNA levels were most abundant in the retina, followed by the posterior column of the spinal cord and other brain regions. These results suggest that aberrant FLVCR1 causes a selective degeneration of a subpopulation of neurons in the retina and the posterior columns of the spinal cord via dysregulation of heme or iron homeostasis. This finding broadens the molecular basis of sensory neural signaling to include common mechanisms that involve proprioception and vision. PMID:21070897

Temporal and Spatial Expression of a Polygalacturonase during Leaf and Flower Abscission in Oilseed Rape and Arabidopsis1

PubMed Central

González-Carranza, Zinnia Haydé; Whitelaw, Catherine Ann; Swarup, Ranjan; Roberts, Jeremy Alan

2002-01-01

During leaf abscission in oilseed rape (Brassica napus), cell wall degradation is brought about by the action of several hydrolytic enzymes. One of these is thought to be polygalacturonase (PG). Degenerate primers were used to isolate a PG cDNA fragment by reverse transcriptase-polymerase chain reaction from RNA extracted from ethylene-promoted leaf abscission zones (AZs), and in turn a full-length clone (CAW471) from an oilseed rape AZ cDNA library. The highest homology of this cDNA (82%) was to an Arabidopsis sequence that was predicted to encode a PG protein. Analysis of expression revealed that CAW471 mRNA accumulated in the AZ of leaves and reached a peak 24 h after ethylene treatment. Ethylene-promoted leaf abscission in oilseed rape was not apparent until 42 h after exposure to the gas, reaching 50% at 48 h and 100% by 56 h. In floral organ abscission, expression of CAW471 correlated with cell separation. Genomic libraries from oilseed rape and Arabidopsis were screened with CAW471 and the respective genomic clones PGAZBRAN and PGAZAT isolated. Characterization of these PG genes revealed that they had substantial homology within both the coding regions and in the 5′-upstream sequences. Fusion of a 1,476-bp 5′-upstream sequence of PGAZAT to β-glucuronidase or green fluorescent protein and transformation of Arabidopsis revealed that this fragment was sufficient to drive expression of these reporter genes in the AZs at the base of the anther filaments, petals, and sepals. PMID:11842157

[Study of human immunodeficiency virus transmission chains in Andalusia: analysis from baseline antiretroviral resistance sequences].

PubMed

Pérez-Parra, Santiago; Chueca-Porcuna, Natalia; Álvarez-Estevez, Marta; Pasquau, Juan; Omar, Mohamed; Collado, Antonio; Vinuesa, David; Lozano, Ana Belen; García-García, Federico

2015-11-01

Protease and reverse transcriptase HIV-1 sequences provide useful information for patient clinical management, as well as information on resistance to antiretrovirals. The aim of this study is to evaluate transmission events, transmitted drug resistance, and to georeference subtypes among newly diagnosed patients referred to our center. A study was conducted on 693 patients diagnosed between 2005 and 2012 in Southern Spain. Protease and reverse transcriptase sequences were obtained for resistance to cART analysis with Trugene(®) HIV Genotyping Kit (Siemens, NAD). MEGA 5.2, Neighbor-Joining, ArcGIS and REGA were used for subsequent analysis. The results showed 298 patients clustered into 77 different transmission events. Most of the clusters were formed by pairs (n=49), of men having sex with men (n=26), Spanish (n=37), and below 45 years of age (73.5%). Urban areas from Granada, and the coastal areas of Almeria and Granada showed the greatest subtype heterogeneity. Five clusters were formed by more than 10 patients, and 15 clusters had transmitted drug resistance. The study data demonstrate how the phylogenetic characterization of transmission clusters is a powerful tool to monitor the spread of HIV, and may contribute to design correct preventive measures to minimize it. Copyright © 2015 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Tyrosine Recombinase Retrotransposons and Transposons.

PubMed

Poulter, Russell T M; Butler, Margi I

2015-04-01

Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from the slime mold Dictyostelium discoideum. The YR elements are bordered by terminal repeats related to their replication via free circular dsDNA intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. Recently a large number of YR retrotransposons have been described, including elements from fungi (mucorales and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish, amphibia and reptiles. YR retrotransposons can be divided into three major groups: the DIRS elements, PAT-like and the Ngaro elements. The three groups form distinct clades on phylogenetic trees based on alignments of reverse transcriptase/ribonuclease H (RT/RH) and YR sequences, and also having some structural distinctions. A group of eukaryote DNA transposons, cryptons, also carry tyrosine recombinases. These DNA transposons do not encode a reverse transcriptase. They have been detected in several pathogenic fungi and oomycetes. Sequence comparisons suggest that the crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. This acquisition must have occurred at a very early point in the evolution of eukaryotes.

Ferrate oxidation of murine leukemia virus reverse transcriptase: identification of the template-primer binding domain.

PubMed

Reddy, G; Nanduri, V B; Basu, A; Modak, M J

1991-08-20

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with potassium ferrate, an oxidizing agent known to oxidize amino acids involved in phosphate binding domains of proteins, results in the irreversible inactivation of both the DNA polymerase and the RNase H activities. Significant protection from ferrate-mediated inactivation is observed in the presence of template-primer but not in the presence of substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme loses template-primer binding activity as judged by UV-mediated cross-linking of radiolabeled DNA. Comparative tryptic peptide mapping by reverse-phase HPLC of native and ferrate-oxidized enzyme indicated the presence of two new peptides eluting at 38 and 57 min and a significant loss of a peptide eluting at 74 min. Purification, amino acid composition, and sequencing of these affected peptides revealed that they correspond to amino acid residues 285-295, 630-640, and 586-599, respectively, in the primary amino acid sequence of MuLV RT. These results indicate that the domains constituted by the above peptides are important for the template-primer binding function in MuLV RT. Peptide I is located in the polymerase domain whereas peptides II and III are located in the RNase H domain. Amino acid sequence analysis of peptides I and II suggested Lys-285 and Cys-635 as the probable sites of ferrate action.

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

PubMed

Silas, Sukrit; Makarova, Kira S; Shmakov, Sergey; Páez-Espino, David; Mohr, Georg; Liu, Yi; Davison, Michelle; Roux, Simon; Krishnamurthy, Siddharth R; Fu, Becky Xu Hua; Hansen, Loren L; Wang, David; Sullivan, Matthew B; Millard, Andrew; Clokie, Martha R; Bhaya, Devaki; Lambowitz, Alan M; Kyrpides, Nikos C; Koonin, Eugene V; Fire, Andrew Z

2017-07-11

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium- Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis , a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic. Copyright © 2017 Silas et al.

Transmitted drug resistance is still low in newly diagnosed human immunodeficiency virus type 1 CRF06_cpx-infected patients in Estonia in 2010.

PubMed

Avi, Radko; Huik, Kristi; Pauskar, Merit; Ustina, Valentina; Karki, Tõnis; Kallas, Eveli; Jõgeda, Ene-Ly; Krispin, Tõnu; Lutsar, Irja

2014-03-01

The presence of transmitted drug resistance (TDR) in treatment-naive HIV-1-positive subjects is of concern, especially in the countries of the former Soviet Union in which the number of subjects exposed to antiretrovirals (ARV) has exponentially increased during the past decade. We assessed the rate of TDR among newly diagnosed subjects in Estonia in 2010 and compared it to that in 2008. The study included 325 subjects (87% of all subjects tested HIV positive from January 1 to December 31, 2010). Of the 244 sequenced viral genomic RNA in the reverse transcriptase (RT) region 214 were CRF06_cpx, nine were subtype A1, three (one each) were subtype B and subtype C, CRF02_AG, and CRF03_AB; 15 viruses remained unclassified as putative recombinant forms between CRF06_cpx and subtype A1. HIV-1 TDR mutations in 2010 and 2008 (n=145) occurred at similar frequency in 4.5% (95% CI 2.45; 7.98) and 5.5% (95% CI 1.8; 9.24) of the patients, respectively. In 2010, 2.5% (6/244) of the sequences harbored nonnucleoside reverse transcriptase inhibitor (NNRTI) (K103N and K101E), 1.6% (4/244) nucleoside reverse transcriptase inhibitor (NRTI) (M41L, M184I, and K219E), and 0.4% (1/244) protease inhibitor (PI) (V82A) mutations. Our findings indicate that in spite of the increased consumption of ARVs the rate of TDR in Estonia has remained unchanged over the past 3 years. Similar stabilizing or even decreasing trends have been described in Western Europe and North America albeit at higher levels and in different socioeconomic backgrounds.

Etravirine and Rilpivirine Drug Resistance Among HIV-1 Subtype C Infected Children Failing Non-Nucleoside Reverse Transcriptase Inhibitor-Based Regimens in South India.

PubMed

Saravanan, Shanmugam; Kausalya, Bagavathi; Gomathi, Selvamurthi; Sivamalar, Sathasivam; Pachamuthu, Balakrishnan; Selvamuthu, Poongulali; Pradeep, Amrose; Sunil, Solomon; Mothi, Sarvode N; Smith, Davey M; Kantor, Rami

2017-06-01

We have analyzed reverse transcriptase (RT) region of HIV-1 pol gene from 97 HIV-infected children who were identified as failing first-line therapy that included first-generation non-nucleoside RT inhibitors (Nevirapine and Efavirenz) for at least 6 months. We found that 54% and 65% of the children had genotypically predicted resistance to second-generation non-nucleoside RT inhibitors drugs Etravirine (ETR) and Rilpivirine, respectively. These cross-resistance mutations may compromise future NNRTI-based regimens, especially in resource-limited settings. To complement these investigations, we also analyzed the sequences in Stanford database, Monogram weighted score, and DUET weighted score algorithms for ETR susceptibility and found almost perfect agreement between the three algorithms in predicting ETR susceptibility from genotypic data.

The "grep" command but not FusionMap, FusionFinder or ChimeraScan captures the CIC-DUX4 fusion gene from whole transcriptome sequencing data on a small round cell tumor with t(4;19)(q35;q13).

PubMed

Panagopoulos, Ioannis; Gorunova, Ludmila; Bjerkehagen, Bodil; Heim, Sverre

2014-01-01

Whole transcriptome sequencing was used to study a small round cell tumor in which a t(4;19)(q35;q13) was part of the complex karyotype but where the initial reverse transcriptase PCR (RT-PCR) examination did not detect a CIC-DUX4 fusion transcript previously described as the crucial gene-level outcome of this specific translocation. The RNA sequencing data were analysed using the FusionMap, FusionFinder, and ChimeraScan programs which are specifically designed to identify fusion genes. FusionMap, FusionFinder, and ChimeraScan identified 1017, 102, and 101 fusion transcripts, respectively, but CIC-DUX4 was not among them. Since the RNA sequencing data are in the fastq text-based format, we searched the files using the "grep" command-line utility. The "grep" command searches the text for specific expressions and displays, by default, the lines where matches occur. The "specific expression" was a sequence of 20 nucleotides from the coding part of the last exon 20 of CIC (Reference Sequence: NM_015125.3) chosen since all the so far reported CIC breakpoints have occurred here. Fifteen chimeric CIC-DUX4 cDNA sequences were captured and the fusion between the CIC and DUX4 genes was mapped precisely. New primer combinations were constructed based on these findings and were used together with a polymerase suitable for amplification of GC-rich DNA templates to amplify CIC-DUX4 cDNA fragments which had the same fusion point found with "grep". In conclusion, FusionMap, FusionFinder, and ChimeraScan generated a plethora of fusion transcripts but did not detect the biologically important CIC-DUX4 chimeric transcript; they are generally useful but evidently suffer from imperfect both sensitivity and specificity. The "grep" command is an excellent tool to capture chimeric transcripts from RNA sequencing data when the pathological and/or cytogenetic information strongly indicates the presence of a specific fusion gene.

A Laccase with Antiproliferative and HIV-I Reverse Transcriptase Inhibitory Activities from the Mycorrhizal Fungus Agaricus placomyces

PubMed Central

Sun, Jian; Chen, Qing-Jun; Cao, Qing-Qin; Wu, Ying-Ying; Xu, Li-Jing; Zhu, Meng-Juan; Ng, Tzi-Bun; Wang, He-Xiang; Zhang, Guo-Qing

2012-01-01

A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7′-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, Km values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC50 of 1.8 μM, 1.7 μM, and 1.25 μM, respectively, signifying that it is an antipathogenic protein. PMID:23093860

The impact of HIV-1 reverse transcriptase polymorphisms on responses to first-line nonnucleoside reverse transcriptase inhibitor-based therapy in HIV-1-infected adults.

PubMed

Mackie, Nicola E; Dunn, David T; Dolling, David; Garvey, Lucy; Harrison, Linda; Fearnhill, Esther; Tilston, Peter; Sabin, Caroline; Geretti, Anna M

2013-09-10

HIV-1 genetic variability may influence antiretroviral therapy (ART) outcomes. The study aim was to determine the impact of polymorphisms in regions known to harbor major nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations (codons 90-108, 135-138, 179-190, 225-348) on virologic responses to first-line NNRTI-based ART. Reverse transcriptase sequences from ART-naive individuals who commenced efavirenz (EFV) or nevirapine (NVP) with at least two nucleos(t)ide reverse transcriptase inhibitors (NRTIs) without major drug resistance mutations were analyzed. The impact of polymorphisms on week 4 viral load decrease and time to virologic failure was measured over a median 97 weeks. Among 4528 patients, most were infected with HIV-1 subtype B (67%) and commenced EFV-based ART (84%). Overall, 2598 (57%) had at least one polymorphism, most frequently at codons 90, 98, 101, 103, 106, 135, 138, 179, and 238. Virologic failure rates were increased in patients with two (n = 597) or more than two (n = 72) polymorphisms [adjusted hazard ratio 1.43; 95% confidence interval (CI) 1.07-1.92; P = 0.016]. Polymorphisms associated with virologic failure occurred at codons 90 (mostly V90I), 98 (mostly A98S), and 103 (mostly K103R), with adjusted hazard ratios of 1.78 (1.15-2.73; P = 0.009), 1.55 (1.16-2.08; P = 0.003), and 1.75 (1.00-3.05: P = 0.049), respectively. Polymorphisms at codon 179, especially V179D/E/T, predicted reduced week 4 responses (P = 0.001) but not virologic failure. The occurrence of multiple polymorphisms, though uncommon, was associated with a small increase in the risk of NNRTI treatment failure; significant effects were seen with polymorphisms at codon 90, 98, and 103. The mechanisms underlying the slower suppression seen with V179D/E/T deserve further investigation.

Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase.

PubMed Central

Allawi, H T; Dong, F; Ip, H S; Neri, B P; Lyamichev, V I

2001-01-01

A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta-globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility. PMID:11233988

The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons.

PubMed Central

Xiong, Y; Eickbush, T H

1988-01-01

Two types of insertion elements, R1 and R2 (previously called type I and type II), are known to interrupt the 28S ribosomal genes of several insect species. In the silkmoth, Bombyx mori, each element occupies approximately 10% of the estimated 240 ribosomal DNA units, while at most only a few copies are located outside the ribosomal DNA units. We present here the complete nucleotide sequence of an R1 insertion from B. mori (R1Bm). This 5.1-kilobase element contains two overlapping open reading frames (ORFs) which together occupy 88% of its length. ORF1 is 461 amino acids in length and exhibits characteristics of retroviral gag genes. ORF2 is 1,051 amino acids in length and contains homology to reverse transcriptase-like enzymes. The analysis of 3' and 5' ends of independent isolates from the ribosomal locus supports the suggestion that R1 is still functioning as a transposable element. The precise location of the element within the genome implies that its transposition must occur with remarkable insertion sequence specificity. Comparison of the deduced amino acid sequences from six retrotransposons, R1 and R2 of B. mori, I factor and F element of Drosophila melanogaster, L1 of Mus domesticus, and Ingi of Trypanosoma brucei, reveals a relatively high level of sequence homology in the reverse transcriptase region. Like R1, these elements lack long terminal repeats. We have therefore named this class of related elements the non-long-terminal-repeat (non-LTR) retrotransposons. Images PMID:2447482

Ancient DNA sequence revealed by error-correcting codes.

PubMed

Brandão, Marcelo M; Spoladore, Larissa; Faria, Luzinete C B; Rocha, Andréa S L; Silva-Filho, Marcio C; Palazzo, Reginaldo

2015-07-10

A previously described DNA sequence generator algorithm (DNA-SGA) using error-correcting codes has been employed as a computational tool to address the evolutionary pathway of the genetic code. The code-generated sequence alignment demonstrated that a residue mutation revealed by the code can be found in the same position in sequences of distantly related taxa. Furthermore, the code-generated sequences do not promote amino acid changes in the deviant genomes through codon reassignment. A Bayesian evolutionary analysis of both code-generated and homologous sequences of the Arabidopsis thaliana malate dehydrogenase gene indicates an approximately 1 MYA divergence time from the MDH code-generated sequence node to its paralogous sequences. The DNA-SGA helps to determine the plesiomorphic state of DNA sequences because a single nucleotide alteration often occurs in distantly related taxa and can be found in the alternative codon patterns of noncanonical genetic codes. As a consequence, the algorithm may reveal an earlier stage of the evolution of the standard code.

Ancient DNA sequence revealed by error-correcting codes

PubMed Central

Brandão, Marcelo M.; Spoladore, Larissa; Faria, Luzinete C. B.; Rocha, Andréa S. L.; Silva-Filho, Marcio C.; Palazzo, Reginaldo

2015-01-01

A previously described DNA sequence generator algorithm (DNA-SGA) using error-correcting codes has been employed as a computational tool to address the evolutionary pathway of the genetic code. The code-generated sequence alignment demonstrated that a residue mutation revealed by the code can be found in the same position in sequences of distantly related taxa. Furthermore, the code-generated sequences do not promote amino acid changes in the deviant genomes through codon reassignment. A Bayesian evolutionary analysis of both code-generated and homologous sequences of the Arabidopsis thaliana malate dehydrogenase gene indicates an approximately 1 MYA divergence time from the MDH code-generated sequence node to its paralogous sequences. The DNA-SGA helps to determine the plesiomorphic state of DNA sequences because a single nucleotide alteration often occurs in distantly related taxa and can be found in the alternative codon patterns of noncanonical genetic codes. As a consequence, the algorithm may reveal an earlier stage of the evolution of the standard code. PMID:26159228

Multihormonal induction of hepatic α2u-globulin mRNA as measured by hybridization to complementary DNA

PubMed Central

Kurtz, David T.; Feigelson, Philip

1977-01-01

A procedure is presented for the preparation of a 3H-labeled complementary DNA (cDNA) specific for the mRNA coding for α2u-globulin, a male rat liver protein under multihormonal control that represents approximately 1% of hepatic protein synthesis. Rat liver polysomes are incubated with monospecific rabbit antiserum to α2u-globulin, which binds to the nascent α2u-globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit IgG that is covalently linked to p-aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% α2u-globulin mRNA. A labeled cDNA is made to this α2u-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-α2u-globulin sequences, this cDNA preparation is hybridized to an RNA concentration × incubation time (R0t) of 1000 mol of ribonucleotide per liter × sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no α2u-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for α2u-globulin mRNA by several criteria. Good correlation was found in all endocrine states studied between the hepatic level of α2u-globulin, the level of functional α2u-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of α2u-globulin mRNA sequences as measured by hybridization to the α2u-globulin cDNA. Thus, the hormonal control of hepatic α2u-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of α2u-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis. PMID:73184

Reverse transcription polymerase chain reaction protocols for cloning small circular RNAs.

PubMed

Navarro, B; Daròs, J A; Flores, R

1998-07-01

A protocol is described for general application for cloning small circular RNAs which requires only minimal amounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-mer primer whose six 3'-terminal positions are totally degenerate in two consecutive reactions catalyzed by reverse transcriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with the non-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of one or more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is also described.

Two-terminal video coding.

PubMed

Yang, Yang; Stanković, Vladimir; Xiong, Zixiang; Zhao, Wei

2009-03-01

Following recent works on the rate region of the quadratic Gaussian two-terminal source coding problem and limit-approaching code designs, this paper examines multiterminal source coding of two correlated, i.e., stereo, video sequences to save the sum rate over independent coding of both sequences. Two multiterminal video coding schemes are proposed. In the first scheme, the left sequence of the stereo pair is coded by H.264/AVC and used at the joint decoder to facilitate Wyner-Ziv coding of the right video sequence. The first I-frame of the right sequence is successively coded by H.264/AVC Intracoding and Wyner-Ziv coding. An efficient stereo matching algorithm based on loopy belief propagation is then adopted at the decoder to produce pixel-level disparity maps between the corresponding frames of the two decoded video sequences on the fly. Based on the disparity maps, side information for both motion vectors and motion-compensated residual frames of the right sequence are generated at the decoder before Wyner-Ziv encoding. In the second scheme, source splitting is employed on top of classic and Wyner-Ziv coding for compression of both I-frames to allow flexible rate allocation between the two sequences. Experiments with both schemes on stereo video sequences using H.264/AVC, LDPC codes for Slepian-Wolf coding of the motion vectors, and scalar quantization in conjunction with LDPC codes for Wyner-Ziv coding of the residual coefficients give a slightly lower sum rate than separate H.264/AVC coding of both sequences at the same video quality.

Occurrence of transmitted HIV-1 drug resistance among Drug-naïve pregnant women in selected HIV-care centres in Ghana.

PubMed

Martin-Odoom, Alexander; Adiku, Theophilus; Delgado, Elena; Lartey, Margaret; Ampofo, William K

2017-03-01

Access to antiretroviral therapy in Ghana has been scaled up across the country over the last decade. This study sought to determine the occurrence of transmitted HIV-1 drug resistance in pregnant HIV-1 positive women yet to initiate antiretroviral therapy at selected HIV Care Centres in Ghana. Plasma specimens from twenty-six (26) HIV seropositive pregnant women who were less than 28weeks pregnant with their first pregnancy and ART naïve were collected from selected HIV care centres in three (3) regions in Ghana. Genotypic testing was done for the reverse transcriptase gene and the sequences generated were analyzed for HIV-1 drug resistance mutations using the Stanford University HIV Drug Resistance Database. Resistance mutations associated with the reverse transcriptase gene were detected in 4 (15.4%) of the participants. At least one major drug resistance mutation in the reverse transcriptase gene was found in 3 (11.5%) of the women. The detection of transmitted HIV-1 drug resistance in this drug-naïve group in two regional HIV care sites is an indication of the need for renewed action in monitoring the emergence of transmitted HIV-1 drug resistance in Ghana. None declared.

Telomerase Mechanism of Telomere Synthesis

PubMed Central

Wu, R. Alex; Upton, Heather E.; Vogan, Jacob M.; Collins, Kathleen

2017-01-01

Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines. PMID:28141967

Variable coding sequence protein A1 as a marker for erectile dysfunction.

PubMed

Tong, Yuehong; Tar, Moses; Davelman, Felix; Christ, George; Melman, Arnold; Davies, Kelvin P

2006-08-01

To investigate whether variable coding sequence protein A1 (Vcsa1) is down-regulated in rat models of diabetes and ageing, and to investigate the role of Vcsa1 in erectile function, as Vcsa1 is the most down-regulated gene in the corpora of a rat model of neurogenic erectile dysfunction (ED). Quantitative reverse-transcriptase polymerase-chain reaction was used to determine Vcsa1 expression in the corpora of rats in three models of ED, i.e. streptozotocin-induced diabetes, retired breeder (old), and neurogenic (bilaterally ligated cavernosal nerves), and in control rats. To confirm a physiological role of Vcsa1 in erectile function, we carried out gene transfer studies using a plasmid in which Vcsa1 was expressed from a cytomegalovirus promoter (pVAX-Vcsa1). This plasmid was injected intracorporally into old rats, and the effect on physiology of corporal tissue was analysed by intracorporal/blood pressure (ICP/BP) measurement and histological analysis, and compared with the effects of a positive control plasmid (pVAX-hSlo, which we previously reported to restore erectile function in diabetic and ageing rats) and a negative control plasmid (pVAX). In each rat model of ED there was a significant down-regulation of the Vcsa1 transcript of at least 10-fold in corporal tissue. Remarkably, intracorporal injection with 80 microg pVAX-Vcsa1 caused priapism, as indicated by visible prolonged erection, histological appearance, and elevated resting ICP/BP. Lower doses of pVAX-Vcsa1 (5 and 25 microg) increased ICP/BP over that in untreated controls. These results show that Vcsa1 has a role in erectile function and might be a molecular marker for organic ED. The role of Vcsa1 in erectile function suggests that it could represent a novel therapeutic target for treating ED.

WHO Collaborating Centre for Acquired Immunodeficiency Syndrome for the Eastern Mediterranean Regional Office, Faculty of Medicine, Kuwait University, Kuwait.

PubMed

Altawalah, Haya; Al-Nakib, Widad

2014-01-01

In the early 1980s, the World Health Organization (WHO) designated the Virology Unit of the Faculty of Medicine, Health Sciences Centre, Kuwait University, Kuwait, a collaborating centre for AIDS for the Eastern Mediterranean Regional Office (EMRO), recognizing it to be in compliance with WHO guidelines. In this centre, research integral to the efforts of WHO to combat AIDS is conducted. In addition to annual workshops and symposia, the centre is constantly updating and renewing its facilities and capabilities in keeping with current and latest advances in virology. As an example of the activities of the centre, the HIV-1 RNA viral load in plasma samples of HIV-1 patients is determined by real-time PCR using the AmpliPrep TaqMan HIV-1 test v2.0. HIV-1 drug resistance is determined by sequencing the reverse transcriptase and protease regions on the HIV-1 pol gene, using the TRUGENE HIV-1 Genotyping Assay on the OpenGene® DNA Sequencing System. HIV-1 subtypes are determined by sequencing the reverse transcriptase and protease regions on the HIV-1 pol gene using the genotyping assays described above. A fundamental program of Kuwait's WHO AIDS collaboration centre is the national project on the surveillance of drug resistance in human deficiency virus in Kuwait, which illustrates how the centre and its activities in Kuwait can serve the EMRO region of WHO. © 2014 S. Karger AG, Basel.

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

PubMed Central

Hwang, Jung-Shan; Yamada, Kazunori; Honda, Ayae; Nakade, Kohji; Ishihama, Akira

2000-01-01

Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni2+-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris. PMID:10756019

Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer.

PubMed

Nisa-Martínez, Rafael; Jiménez-Zurdo, José I; Martínez-Abarca, Francisco; Muñoz-Adelantado, Estefanía; Toro, Nicolás

2007-01-01

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase-maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (DeltaORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic DeltaORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature.

A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines

PubMed Central

Sciamanna, Ilaria; Gualtieri, Alberto; Cossetti, Cristina; Osimo, Emanuele Felice; Ferracin, Manuela; Macchia, Gianfranco; Aricò, Eleonora; Prosseda, Gianni; Vitullo, Patrizia; Misteli, Tom; Spadafora, Corrado

2013-01-01

LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the ‘normal’ small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells. PMID:24345856

The Reverse Transcriptase of the Tf1 Retrotransposon Has a Specific Novel Activity for Generating the RNA Self-Primer That Is Functional in cDNA Synthesis▿

PubMed Central

Hizi, Amnon

2008-01-01

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5′ end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3′ end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs. PMID:18753200

The reverse transcriptase of the Tf1 retrotransposon has a specific novel activity for generating the RNA self-primer that is functional in cDNA synthesis.

PubMed

Hizi, Amnon

2008-11-01

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5' end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3' end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs.

Detection of SYT-SSX mutant transcripts in formalin-fixed paraffin-embedded sarcoma tissues using one-step reverse transcriptase real-time PCR.

PubMed

Norlelawati, A T; Mohd Danial, G; Nora, H; Nadia, O; Zatur Rawihah, K; Nor Zamzila, A; Naznin, M

2016-04-01

Synovial sarcoma (SS) is a rare cancer and accounts for 5-10% of adult soft tissue sarcomas. Making an accurate diagnosis is difficult due to the overlapping histological features of SS with other types of sarcomas and the non-specific immunohistochemistry profile findings. Molecular testing is thus considered necessary to confirm the diagnosis since more than 90% of SS cases carry the transcript of t(X;18)(p11.2;q11.2). The purpose of this study is to diagnose SS at molecular level by testing for t(X;18) fusion-transcript expression through One-step reverse transcriptase real-time Polymerase Chain Reaction (PCR). Formalin-fixed paraffin-embedded tissue blocks of 23 cases of soft tissue sarcomas, which included 5 and 8 cases reported as SS as the primary diagnosis and differential diagnosis respectively, were retrieved from the Department of Pathology, Tengku Ampuan Afzan Hospital, Kuantan, Pahang. RNA was purified from the tissue block sections and then subjected to One-step reverse transcriptase real-time PCR using sequence specific hydrolysis probes for simultaneous detection of either SYT-SSX1 or SYT-SSX2 fusion transcript. Of the 23 cases, 4 cases were found to be positive for SYT-SSX fusion transcript in which 2 were diagnosed as SS whereas in the 2 other cases, SS was the differential diagnosis. Three cases were excluded due to failure of both amplification assays SYT-SSX and control β-2-microglobulin. The remaining 16 cases were negative for the fusion transcript. This study has shown that the application of One-Step reverse transcriptase real time PCR for the detection SYT-SSX transcript is feasible as an aid in confirming the diagnosis of synovial sarcoma.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

A Novel Laccase with Potent Antiproliferative and HIV-1 Reverse Transcriptase Inhibitory Activities from Mycelia of Mushroom Coprinus comatus

PubMed Central

Zhao, Shuang; Rong, Cheng-Bo; Kong, Chang; Liu, Yu; Xu, Feng; Miao, Qian-Jiang; Wang, Shou-Xian; Wang, He-Xiang

2014-01-01

A novel laccase was isolated and purified from fermentation mycelia of mushroom Coprinus comatus with an isolation procedure including three ion-exchange chromatography steps on DEAE-cellulose, CM-cellulose, and Q-Sepharose and one gel-filtration step by fast protein liquid chromatography on Superdex 75. The purified enzyme was a monomeric protein with a molecular weight of 64 kDa. It possessed a unique N-terminal amino acid sequence of AIGPVADLKV, which has considerably high sequence similarity with that of other fungal laccases, but is different from that of C. comatus laccases reported. The enzyme manifested an optimal pH value of 2.0 and an optimal temperature of 60°C using 2,2′-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) as the substrate. The laccase displayed, at pH 2.0 and 37°C, K m values of 1.59 mM towards ABTS. It potently suppressed proliferation of tumor cell lines HepG2 and MCF7, and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC50 value of 3.46 μM, 4.95 μM, and 5.85 μM, respectively, signifying that it is an antipathogenic protein. PMID:25540778

The mRNA and miRNA transcriptomic landscape of Panax ginseng under the high ambient temperature.

PubMed

Jung, Inuk; Kang, Hyejin; Kim, Jang Uk; Chang, Hyeonsook; Kim, Sun; Jung, Woosuk

2018-03-19

Ginseng is a popular traditional herbal medicine in north-eastern Asia. It has been used for human health for over thousands of years. With the rise in global temperature, the production of Korean ginseng (Panax ginseng C.A.Meyer) in Korea have migrated from mid to northern parts of the Korean peninsula to escape from the various higher temperature related stresses. Under the high ambient temperature, vegetative growth was accelerated, which resulted in early flowering. This precocious phase change led to yield loss. Despite of its importance as a traditional medicine, biological mechanisms of ginseng has not been well studied and even the genome sequence of ginseng is yet to be determined due to its complex genome structure. Thus, it is challenging to investigate the molecular biology mechanisms at the transcript level. To investigate how ginseng responds to the high ambient temperature environment, we performed high throughput RNA sequencing and implemented a bioinformatics pipeline for the integrated analysis of small-RNA and mRNA-seq data without a reference genome. By performing reverse transcriptase (RT) PCR and sanger sequencing of transcripts that were assembled using our pipeline, we validated that their sequences were expressed in our samples. Furthermore, to investigate the interaction between genes and non-coding small RNAs and their regulation status under the high ambient temperature, we identified potential gene regulatory miRNAs. As a result, 100,672 contigs with significant expression level were identified and 6 known, 214 conserved and 60 potential novel miRNAs were predicted to be expressed under the high ambient temperature. Collectively, we have found that development, flowering and temperature responsive genes were induced under high ambient temperature, whereas photosynthesis related genes were repressed. Functional miRNAs were down-regulated under the high ambient temperature. Among them are miR156 and miR396 that target flowering (SPL6/9) and growth regulating genes (GRF) respectively.

Characteristics of a group of nonnucleoside reverse transcriptase inhibitors with structural diversity and potent anti-human immunodeficiency virus activity.

PubMed

Yang, S S; Fliakas-Boltz, V; Bader, J P; Buckheit, R W

1995-10-01

Current thrust in controlling the Acquired Immune Deficiency Syndrome (AIDS) focuses on antiviral drug development targeting the infection and replication of the human immunodeficiency virus (HIV), the causative agent of AIDS. To date, treatment of AIDS has relied on nucleoside reverse transcriptase inhibitors such as AZT, ddI, and ddC, which eventually become ineffective upon the emergence of resistant mutants bearing specific nucleotide substitutions. The Anti-AIDS Drug Screening Program of the NCI conducts and coordinates a high-capacity semi-robotic in vitro screening of synthetic or natural compounds submitted by academic, research and pharmaceutical institutions world-wide. About 10,000 synthetic compounds are screened annually for anti-HIV activity. Confirmed active agents are subjected to in-depth studies on range and mechanism of action. Emerging from this intense screening activity were a number of potentially promising categories of nonnucleoside reverse transcriptase inhibitors (NNRTI) with structural diversity but strong and reproducible anti-HIV activity. Over 2500 active compounds were evaluated for their inhibitory activity against a panel of both laboratory and clinical virus isolates in the appropriate established cell line or fresh human peripheral blood leukocyte and macrophage preparations. Out of these, 40 agents could be placed structurally in nine categories with an additional 16 unique compounds that share the characteristics of NNRTI. These NNRTIs were shown to inhibit reverse transcriptase enzymatically using homopolymeric or ribosomal RNA as templates. NNRTIs demonstrated similarity in their inhibitory pattern against the HIV-1 laboratory strains IIIB and RF, and an AZT-resistant strain; all were inactive against HIV-2. These compounds were further tested against NNRTI-resistant HIV-1 isolates. NNRTI-resistant HIV-1 isolates were selected and characterized with respect to the change(s) in the viral reverse transcriptase nucleotide sequence. Also, differential cross-resistance or sensitivity patterns to NNRTIs were studied in detail among NNRTI-resistant mutants. When tested in combination with AZT, all of the NNRTI's uniformly exhibited synergistic inhibition of HIV-1, suggesting that combination antiviral therapy of NNRTIs with AZT may be therapeutically promising for AIDS treatment.

Novel HBV recombinants between genotypes B and C in 3'-terminal reverse transcriptase (RT) sequences are associated with enhanced viral DNA load, higher RT point mutation rates and place of birth among Chinese patients.

PubMed

Liu, Baoming; Yang, Jing-Xian; Yan, Ling; Zhuang, Hui; Li, Tong

2018-01-01

As one of the major global public health concerns, hepatitis B virus (HBV) can be divided into at least eight genotypes, which may be related to disease severity and treatment response. We previously demonstrated that genotypes B and C HBV, with distinct geographical distribution in China, had divergent genotype-dependent amino acid polymorphisms and variations in reverse transcriptase (RT) gene region, a target of antiviral therapy using nucleos(t)ide analogues. Recently recombination between HBV genotypes B and C was reported to occur in the RT region. However, their frequency and clinical significance is poorly understood. Here full-length HBV RT sequences from 201 Chinese chronic hepatitis B (CHB) patients were amplified and sequenced, among which 31.34% (63/201) were genotype B whereas 68.66% (138/201) genotype C. Although no intergenotypic recombination was detected among C-genotype HBV, 38.10% (24/63) of B-genotype HBV had recombination with genotype C in the 3'-terminal RT sequences. The patients with B/C intergenotypic recombinants had significantly (P<0.05) higher serum HBV DNA level than the "pure" B-genotype cohort did. Moreover, the B/C intergenotypic recombinants were prone to more substitutions at several specific residues in the RT region than genotype B or C. Besides, unlike their parental genotypes, the recombinant HBV appeared to display an altered geographic distribution feature in China. Our findings provide novel insight into the virological, clinical and epidemiological features of new HBV B/C intergenotypic recombinants at the 3' end of RT sequences among Chinese CHB patients. The highly complex genetic background of the novel recombinant HBV carrying new mutations affecting RT protein may contribute to an enhanced heterogeneity in treatment response or prognosis among CHB patients. Published by Elsevier B.V.

Emergence of uncommon HIV-1 non-B subtypes and circulating recombinant forms and trends in transmission of antiretroviral drug resistance in patients with primary infection during the 2013-2015 period in Marseille, Southeastern France.

PubMed

Tamalet, Catherine; Tissot-Dupont, Hervé; Motte, Anne; Tourrès, Christian; Dhiver, Catherine; Ravaux, Isabelle; Poizot-Martin, Isabelle; Dieng, Thérèse; Tomei, Christelle; Bregigeon, Sylvie; Zaegel-Faucher, Olivia; Laroche, Hélène; Aherfi, Sarah; Mokhtari, Saadia; Chaudet, Hervé; Ménard, Amelie; Brouqui, Philippe; Stein, Andreas; Colson, Philippe

2018-05-24

Primary HIV-1 infections (PHI) with non-B subtypes are increasing in developed countries while transmission of HIV-1 harboring antiretroviral resistance-associated mutations (RAMs) remains a concern. This study assessed non-B HIV-1 subtypes and RAMs prevalence among patients with PHI in university hospitals of Marseille, Southeastern France, in 2005-2015 (11 years). HIV-1 sequences were obtained by in-house protocols from 115 patients with PHI, including 38 for the 2013-2015 period. On the basis of the phylogenetic analysis of the reverse transcriptase region, non-B subtypes were identified in 31% of these patients. They included 3 different subtypes (3A, 1C, 4F), 23 circulating recombinant forms (CRFs) (CRF02_AG, best BLAST hits being CRF 36_cpx and CRF30 in 7 and 1 cases, respectively), and 5 unclassified sequences (U). Non-B subtypes proportion increased significantly, particularly in 2011-2013 vs in 2005-2010 (P = .03). CRF02_AG viruses largely predominated in 2005-2013 whereas atypical strains more difficult to classify and undetermined recombinants emerged recently (2014-2015). The prevalence of protease, nucleos(t)ide reverse transcriptase, and first-generation nonnucleoside reverse transcriptase inhibitors-associated RAMs were 1.7% (World Health Organization [WHO] list, 2009/2.6% International AIDS Society [IAS] list, 2017), 5.2%/4.3%, and 5.2%/5.2%, respectively. Etravirine/rilpivirine-associated RAM (IAS) prevalence was 4.3%. Men who have sex with men (MSM) were more frequently infected with drug-resistant viruses than other patients (26% vs 7%; P = .011). The recent increase of these rare HIV-1 strains and the spread of drug-resistant HIV-1 among MSM in Southeastern France might be considered when implementing prevention strategies and starting therapies. © 2018 Wiley Periodicals, Inc.

Improving performance of DS-CDMA systems using chaotic complex Bernoulli spreading codes

NASA Astrophysics Data System (ADS)

Farzan Sabahi, Mohammad; Dehghanfard, Ali

2014-12-01

The most important goal of spreading spectrum communication system is to protect communication signals against interference and exploitation of information by unintended listeners. In fact, low probability of detection and low probability of intercept are two important parameters to increase the performance of the system. In Direct Sequence Code Division Multiple Access (DS-CDMA) systems, these properties are achieved by multiplying the data information in spreading sequences. Chaotic sequences, with their particular properties, have numerous applications in constructing spreading codes. Using one-dimensional Bernoulli chaotic sequence as spreading code is proposed in literature previously. The main feature of this sequence is its negative auto-correlation at lag of 1, which with proper design, leads to increase in efficiency of the communication system based on these codes. On the other hand, employing the complex chaotic sequences as spreading sequence also has been discussed in several papers. In this paper, use of two-dimensional Bernoulli chaotic sequences is proposed as spreading codes. The performance of a multi-user synchronous and asynchronous DS-CDMA system will be evaluated by applying these sequences under Additive White Gaussian Noise (AWGN) and fading channel. Simulation results indicate improvement of the performance in comparison with conventional spreading codes like Gold codes as well as similar complex chaotic spreading sequences. Similar to one-dimensional Bernoulli chaotic sequences, the proposed sequences also have negative auto-correlation. Besides, construction of complex sequences with lower average cross-correlation is possible with the proposed method.

Trends of drug-resistance-associated mutations in the reverse transcriptase gene of HIV type 1 isolates from North India.

PubMed

Azam, Mohd; Malik, Abida; Rizvi, Meher; Rai, Arvind

2014-04-01

A major cause of failure of antiretroviral therapy (ART) is the presence of drug-resistance-associated mutations in the polymerase gene of HIV-1. The paucity of data regarding potential drug resistance to reverse transcriptase inhibitors (RTIs) prompted us to carry out this study. This information will shed light on the extent of drug resistance already present in HIV strains and will give future directions in patient treatment and in drug design. Drug resistance genotyping of a partial reverse transcriptase gene was done in 103 HIV-1-infected patients, including the ART-naive and ART-experienced population. The drug resistance pattern was analyzed using the Stanford HIV-DR database, the IAS-USA mutation list and the REGA algorithm-v8.0. Subtyping was done using the REGA HIV-1 subtyping tool-v2.01. The majority of our sequences (96 %) were found to be subtype C, and four (3.8 %) were subtype A1. Significant prevalence of DR mutations (28 %) was observed in the RT gene. Major amino acid substitutions were seen at positions 41, 90, 98, 103, 106, 108, 138, 181, 184, 190, 215, and 219, which confer high/intermediate levels of resistance to most RTIs, independently or together. Our results show that there is an urgent need to tailor ART drug regimens to the individual to achieve optimum therapeutic outcome in North India.

Lower genetic variability of HIV-1 and antiretroviral drug resistance in pregnant women from the state of Pará, Brazil.

PubMed

Machado, Luiz Fernando Almeida; Costa, Iran Barros; Folha, Maria Nazaré; da Luz, Anderson Levy Bessa; Vallinoto, Antonio Carlos Rosário; Ishak, Ricardo; Ishak, Marluisa Oliveira Guimarães

2017-04-12

The present study aimed to describe the genetic diversity of HIV-1, as well as the resistance profile of the viruses identified in HIV-1 infected pregnant women under antiretroviral therapy in the state of Pará, Northern Brazil. Blood samples were collected from 45 HIV-1 infected pregnant to determine the virus subtypes according to the HIV-1 protease (PR) gene and part of the HIV-1 reverse transcriptase (RT) gene by sequencing the nucleotides of these regions. Drug resistance mutations and susceptibility to antiretroviral drugs were analyzed by the Stanford HIV Drug Resistance Database. Out of 45 samples, only 34 could be amplified for PR and 30 for RT. Regarding the PR gene, subtypes B (97.1%) and C (2.9%) were identified; for the RT gene, subtypes B (90.0%), F (6.7%), and C (3.3%) were detected. Resistance to protease inhibitors (PI) was identified in 5.8% of the pregnant, and mutations conferring resistance to nucleoside reverse transcriptase inhibitors were found in 3.3%, while mutations conferring resistance to non-nucleoside reverse transcriptase inhibitors were found in 3.3%. These results showed a low frequency of strains resistant to antiretroviral drugs, the prevalence of subtypes B and F, and the persistent low transmission of subtype C in pregnant of the state of Pará, Brazil.

Characterization of potential antiviral resistance mutations in hepatitis B virus reverse transcriptase sequences in treatment-naïve Chinese patients.

PubMed

Liu, Bao-Ming; Li, Tong; Xu, Jie; Li, Xiao-Guang; Dong, Jian-Ping; Yan, Ping; Yang, Jing-Xian; Yan, Ling; Gao, Zhi-Yong; Li, Wen-Peng; Sun, Xie-Wen; Wang, Yu-Hua; Jiao, Xiu-Juan; Hou, Chun-Sheng; Zhuang, Hui

2010-03-01

Full-length hepatitis B virus (HBV) reverse transcriptase (RT) sequences were amplified and sequenced among 192 nucleos(t)ide analogue (NA)-naïve Chinese patients with chronic hepatitis B. Deduced amino acids (AAs) at 42 previously reported potential NA resistance (NAr) mutation positions in RT region were analyzed. Patients were found with either B-genotype (28.65%) or C-genotype (71.35%) infections. Rt53, rt91, rt124, rt134, rt221, rt224, rt238 and rt256 were identified as B- and C-genotype-dependent polymorphic AA positions. AA substitutions at 11 classical NAr mutation positions, i.e. rt80, rt169, rt173, rt180, rt181, rt184, rt194, rt202, rt204, rt236 and rt250, were not detected. However, potential NAr mutations were found in 30.73% (59/192) isolates, which involved 18 positions including rt53, rt207, rt229, rt238 and rt256, etc. The concomitant AA changes of HBsAg occurred in 16.67% (32/192) isolates including sG145R mutation. One-third of mutation positions were located in functional RT domains (e.g. rt207 and rt233), A-B interdomains (overlapping HBsAg 'a' determinant and showing most concomitant immune-associated mutations) and non-A-B interdomains (e.g. rt191 and rt213), respectively. Genotypes B and C each showed several preferred positions to mutate. These results might provide insights into understanding the evolution and selection basis of NAr HBV strains under antiviral therapy.

[Transposition errors during learning to reproduce a sequence by the right- and the left-hand movements: simulation of positional and movement coding].

PubMed

Liakhovetskiĭ, V A; Bobrova, E V; Skopin, G N

2012-01-01

Transposition errors during the reproduction of a hand movement sequence make it possible to receive important information on the internal representation of this sequence in the motor working memory. Analysis of such errors showed that learning to reproduce sequences of the left-hand movements improves the system of positional coding (coding ofpositions), while learning of the right-hand movements improves the system of vector coding (coding of movements). Learning of the right-hand movements after the left-hand performance involved the system of positional coding "imposed" by the left hand. Learning of the left-hand movements after the right-hand performance activated the system of vector coding. Transposition errors during learning to reproduce movement sequences can be explained by neural network using either vector coding or both vector and positional coding.

Informational structure of genetic sequences and nature of gene splicing

NASA Astrophysics Data System (ADS)

Trifonov, E. N.

1991-10-01

Only about 1/20 of DNA of higher organisms codes for proteins, by means of classical triplet code. The rest of DNA sequences is largely silent, with unclear functions, if any. The triplet code is not the only code (message) carried by the sequences. There are three levels of molecular communication, where the same sequence ``talks'' to various bimolecules, while having, respectively, three different appearances: DNA, RNA and protein. Since the molecular structures and, hence, sequence specific preferences of these are substantially different, the original DNA sequence has to carry simultaneously three types of sequence patterns (codes, messages), thus, being a composite structure in which one had the same letter (nucleotide) is frequently involved in several overlapping codes of different nature. This multiplicity and overlapping of the codes is a unique feature of the Gnomic, language of genetic sequences. The coexisting codes have to be degenerate in various degrees to allow an optimal and concerted performance of all the encoded functions. There is an obvious conflict between the best possible performance of a given function and necessity to compromise the quality of a given sequence pattern in favor of other patterns. It appears that the major role of various changes in the sequences on their ``ontogenetic'' way from DNA to RNA to protein, like RNA editing and splicing, or protein post-translational modifications is to resolve such conflicts. New data are presented strongly indicating that the gene splicing is such a device to resolve the conflict between the code of DNA folding in chromatin and the triplet code for protein synthesis.

Genetic Code Analysis Toolkit: A novel tool to explore the coding properties of the genetic code and DNA sequences

NASA Astrophysics Data System (ADS)

Kraljić, K.; Strüngmann, L.; Fimmel, E.; Gumbel, M.

2018-01-01

The genetic code is degenerated and it is assumed that redundancy provides error detection and correction mechanisms in the translation process. However, the biological meaning of the code's structure is still under current research. This paper presents a Genetic Code Analysis Toolkit (GCAT) which provides workflows and algorithms for the analysis of the structure of nucleotide sequences. In particular, sets or sequences of codons can be transformed and tested for circularity, comma-freeness, dichotomic partitions and others. GCAT comes with a fertile editor custom-built to work with the genetic code and a batch mode for multi-sequence processing. With the ability to read FASTA files or load sequences from GenBank, the tool can be used for the mathematical and statistical analysis of existing sequence data. GCAT is Java-based and provides a plug-in concept for extensibility. Availability: Open source Homepage:http://www.gcat.bio/

SEQassembly: A Practical Tools Program for Coding Sequences Splicing

NASA Astrophysics Data System (ADS)

Lee, Hongbin; Yang, Hang; Fu, Lei; Qin, Long; Li, Huili; He, Feng; Wang, Bo; Wu, Xiaoming

CDS (Coding Sequences) is a portion of mRNA sequences, which are composed by a number of exon sequence segments. The construction of CDS sequence is important for profound genetic analysis such as genotyping. A program in MATLAB environment is presented, which can process batch of samples sequences into code segments under the guide of reference exon models, and splice these code segments of same sample source into CDS according to the exon order in queue file. This program is useful in transcriptional polymorphism detection and gene function study.

Correlation approach to identify coding regions in DNA sequences

NASA Technical Reports Server (NTRS)

Ossadnik, S. M.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Mantegna, R. N.; Peng, C. K.; Simons, M.; Stanley, H. E.

1994-01-01

Recently, it was observed that noncoding regions of DNA sequences possess long-range power-law correlations, whereas coding regions typically display only short-range correlations. We develop an algorithm based on this finding that enables investigators to perform a statistical analysis on long DNA sequences to locate possible coding regions. The algorithm is particularly successful in predicting the location of lengthy coding regions. For example, for the complete genome of yeast chromosome III (315,344 nucleotides), at least 82% of the predictions correspond to putative coding regions; the algorithm correctly identified all coding regions larger than 3000 nucleotides, 92% of coding regions between 2000 and 3000 nucleotides long, and 79% of coding regions between 1000 and 2000 nucleotides. The predictive ability of this new algorithm supports the claim that there is a fundamental difference in the correlation property between coding and noncoding sequences. This algorithm, which is not species-dependent, can be implemented with other techniques for rapidly and accurately locating relatively long coding regions in genomic sequences.

Molecular Epidemiology of Norovirus Outbreaks in Norway during 2000 to 2005 and Comparison of Four Norovirus Real-Time Reverse Transcriptase PCR Assays

PubMed Central

Vainio, Kirsti; Myrmel, Mette

2006-01-01

During the period from January 2000 to August 2005 a total of 204 outbreaks of norovirus gastroenteritis were diagnosed at the Norwegian Institute of Public Health. A clear increase in the norovirus activity was seen in healthcare institutions during the winter seasons. Polymerase sequence analysis of norovirus strains from 122 outbreaks showed that 112 were caused by GII strains (91.8%). Two norovirus variants seen during the study period—GIIb and GII.4—were predominant between January 2000 and September 2002, whereas GII.4 was predominant from September 2002 onward. The highest norovirus activity was seen during the 2002-2003 and 2004-2005 seasons with the emergence of new GII.4 variants. This study describes the molecular epidemiology of norovirus strains circulating in Norway during the five previous seasons and compares four norovirus real-time reverse transcriptase PCR assays. A suitable assay for routine diagnostics is suggested. PMID:17021099

The ORF1 Protein Encoded by LINE-1: Structure and Function During L1 Retrotransposition

PubMed Central

Martin, Sandra L.

2006-01-01

LINE-1, or L1 is an autonomous non-LTR retrotransposon in mammals. Retrotransposition requires the function of the two, L1-encoded polypeptides, ORF1p and ORF2p. Early recognition of regions of homology between the predicted amino acid sequence of ORF2 and known endonuclease and reverse transcriptase enzymes led to testable hypotheses regarding the function of ORF2p in retrotransposition. As predicted, ORF2p has been demonstrated to have both endonuclease and reverse transcriptase activities. In contrast, no homologs of known function have contributed to our understanding of the function of ORF1p during retrotransposition. Nevertheless, significant advances have been made such that we now know that ORF1p is a high affinity RNA binding protein that forms a ribonucleoprotein particle together with L1 RNA. Furthermore, ORF1p is a nucleic acid chaperone and this nucleic acid chaperone activity is required for L1 retrotransposition. PMID:16877816

Statistical properties of DNA sequences

NASA Technical Reports Server (NTRS)

Peng, C. K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Mantegna, R. N.; Simons, M.; Stanley, H. E.

1995-01-01

We review evidence supporting the idea that the DNA sequence in genes containing non-coding regions is correlated, and that the correlation is remarkably long range--indeed, nucleotides thousands of base pairs distant are correlated. We do not find such a long-range correlation in the coding regions of the gene. We resolve the problem of the "non-stationarity" feature of the sequence of base pairs by applying a new algorithm called detrended fluctuation analysis (DFA). We address the claim of Voss that there is no difference in the statistical properties of coding and non-coding regions of DNA by systematically applying the DFA algorithm, as well as standard FFT analysis, to every DNA sequence (33301 coding and 29453 non-coding) in the entire GenBank database. Finally, we describe briefly some recent work showing that the non-coding sequences have certain statistical features in common with natural and artificial languages. Specifically, we adapt to DNA the Zipf approach to analyzing linguistic texts. These statistical properties of non-coding sequences support the possibility that non-coding regions of DNA may carry biological information.

Cellulases and coding sequences

DOEpatents

Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

2001-02-20

The present invention provides three fungal cellulases, their coding sequences, recombinant DNA molecules comprising the cellulase coding sequences, recombinant host cells and methods for producing same. The present cellulases are from Orpinomyces PC-2.

Cellulases and coding sequences

DOEpatents

Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

2001-01-01

The present invention provides three fungal cellulases, their coding sequences, recombinant DNA molecules comprising the cellulase coding sequences, recombinant host cells and methods for producing same. The present cellulases are from Orpinomyces PC-2.

Pectinases From Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae): Putative Accessory Digestive Enzymes

PubMed Central

Evangelista, Danilo Elton; de Paula, Fernando Fonseca Pereira; Rodrigues, André; Henrique-Silva, Flávio

2015-01-01

The cell wall in plants offers protection against invading organisms and is mainly composed of the polysaccharides pectin, cellulose, and hemicellulose, which can be degraded by plant cell wall degrading enzymes (PCWDEs). Such enzymes are often synthesized by free living microorganisms or endosymbionts that live in the gut of some animals, including certain phytophagous insects. Thus, the ability of an insect to degrade the cell wall was once thought to be related to endosymbiont enzyme activity. However, recent studies have revealed that some phytophagous insects are able to synthesize their own PCWDEs by endogenous genes, although questions regarding the origin of these genes remain unclear. This study describes two pectinases from the sugarcane weevil, Sphenophorus levis Vaurie, 1978 (Sl-pectinases), which is considered one of the most serious agricultural pests in Brazil. Two cDNA sequences identified in a cDNA library of the insect larvae coding for a pectin methylesterase (PME) and an endo-polygalacturonase (endo-PG)—denominated Sl-PME and Sl-endoPG, respectively—were isolated and characterized. The quantitative real-time reverse transcriptase polymerase chain reaction expression profile for both Sl-pectinases showed mRNA production mainly in the insect feeding stages and exclusively in midgut tissue of the larvae. This analysis, together Western blotting data, suggests that Sl-pectinases have a digestive role. Phylogenetic analyses indicate that Sl-PME and Sl-endoPG sequences are closely related to bacteria and fungi, respectively. Moreover, the partial genomic sequences of the pectinases were amplified from insect fat body DNA, which was certified to be free of endosymbiotic DNA. The analysis of genomic sequences revealed the existence of two small introns with 53 and 166 bp in Sl-endoPG, which is similar to the common pattern in fungal introns. In contrast, no intron was identified in the Sl-PME genomic sequence, as generally observed in bacteria. These data support the theory of horizontal gene transfer proposed for the origin of insect pectinases, reinforcing the acquisition of PME genes from bacteria and endo-PG genes from fungi. PMID:25673050

Distemper outbreak and its effect on African wild dog conservation.

PubMed

van de Bildt, Marco W G; Kuiken, Thijs; Visee, Aart M; Lema, Sangito; Fitzjohn, Tony R; Osterhaus, Albert D M E

2002-02-01

In December 2000, an infectious disease spread through a captive breeding group of African wild dogs (Lycaon pictus) in Tanzania, killing 49 of 52 animals within 2 months. The causative agent was identified as Canine distemper virus (CDV) by means of histologic examination, virus isolation, reverse transcriptase-polymerase chain reaction analysis, and nucleotide sequencing. This report emphasizes the importance of adequate protection against infectious diseases for the successful outcome of captive breeding programs of endangered species.

Microaspiration of esophageal gland cells and cDNA library construction for identifying parasitism genes of plant-parasitic nematodes.

PubMed

Hussey, Richard S; Huang, Guozhong; Allen, Rex

2011-01-01

Identifying parasitism genes encoding proteins secreted from a plant-parasitic nematode's esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Parasitism genes have been cloned by directly microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages of cyst or root-knot nematodes to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. cDNA clones are sequenced and deduced protein sequences with a signal peptide for secretion are identified for high-throughput in situ hybridization to confirm gland-specific expression.

Triazole-linked DNA as a primer surrogate in the synthesis of first-strand cDNA.

PubMed

Fujino, Tomoko; Yasumoto, Ken-ichi; Yamazaki, Naomi; Hasome, Ai; Sogawa, Kazuhiro; Isobe, Hiroyuki

2011-11-04

A phosphate-eliminated nonnatural oligonucleotide serves as a primer surrogate in reverse transcription reaction of mRNA. Despite of the nonnatural triazole linkages in the surrogate, the reverse transcriptase effectively elongated cDNA sequences on the 3'-downstream of the primer by transcription of the complementary sequence of mRNA. A structure-activity comparison with the reference natural oligonucleotides shows the superior priming activity of the surrogate containing triazole-linkages. The nonnatural linkages also protect the transcribed cDNA from digestion reactions with 5'-exonuclease and enable us to remove noise transcripts of unknown origins. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of three genotypes of sugarcane yellow leaf virus causing yellow leaf disease from India and their molecular characterization.

PubMed

Viswanathan, R; Balamuralikrishnan, M; Karuppaiah, R

2008-12-01

Sugarcane yellow leaf virus (SCYLV) that causes yellow leaf disease (YLD) in sugarcane (recently reported in India) belongs to Polerovirus. Detailed studies were conducted to characterize the virus based on partial open reading frames (ORFs) 1 and 2 and complete ORFs 3 and 4 sequences in their genome. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on 48 sugarcane leaf samples to detect the virus using a specific set of primers. Of the 48 samples, 36 samples (field samples with and without foliar symptoms) including 10 meristem culture derived plants were found to be positive to SCYLV infection. Additionally, an aphid colony collected from symptomatic sugarcane in the field was also found to be SCYLV positive. The amplicons from 22 samples were cloned, sequenced and acronymed as SCYLV-CB isolates. The nucleotide (nt) and amino acid (aa) sequence comparison showed a significant variation between SCYLV-CB and the database sequences at nt (3.7-5.1%) and aa (3.2-5.3%) sequence level in the CP coding region. However, the database sequences comprising isolates of three reported genotypes, viz., BRA, PER and REU, were observed with least nt and aa sequence dissimilarities (0.0-1.6%). The phylogenetic analyses of the overlapping ORFs (ORF 3 and ORF 4) of SCYLV encoding CP and MP determined in this study and additional sequences of 26 other isolates including an Indian isolate (SCYLV-IND) available from GenBank were distributed in four phylogenetic clusters. The SCYLV-CB isolates from this study lineated in two clusters (C1 and C2) and all the other isolates from the worldwide locations into another two clusters (C3 and C4). The sequence variation of the isolates in this study with the database isolates, even in the least variable region of the SCYLV genome, showed that the population existing in India is significantly different from rest of the world. Further, comparison of partial sequences encoding for ORFs 1 and 2 revealed that YLD in sugarcane in India is caused by at least three genotypes, viz., CUB, IND and BRA-PER, of which a majority of the samples were found infected with Cuban genotype (CUB) and lesser by IND and BRA-PER genotypes. The genotype IND was identified as a new genotype from this study, and this was found to have significant variation with the reported genotypes.

Familial 46,XY sex reversal without campomelic dysplasia caused by a deletion upstream of the SOX9 gene

PubMed Central

Layman, Lawrence C.; Ullmann, Reinhard; Shen, Yiping; Ha, Kyungsoo; Rehman, Khurram; Looney, Stephen; McDonough, Paul G.; Kim, Hyung-Goo; Carr, Bruce R.

2014-01-01

Background 46,XY sex reversal is a rare disorder and familial cases are even more rare. The purpose of the present study was to determine the molecular basis for a family with three affected siblings who had 46,XY sex reversal. Methods DNA was extracted from three females with 46,XY sex reversal, two normal sisters, and both unaffected parents. All protein coding exons of the SRY and NR5A1 genes were subjected to PCR-based DNA sequencing. In addition, array comparative genomic hybridization was performed on DNA from all seven family members. A deletion was confirmed using quantitative polymerase chain reaction. Expression of SOX9 gene was quantified using reverse transcriptase polymerase chain reaction. Results A 349kb heterozygous deletion located 353kb upstream of the SOX9 gene on the long arm of chromosome 17 was discovered in the father and three affected siblings, but not in the mother. The expression of SOX9 was significantly decreased in the affected siblings. Two of three affected sisters had gonadoblastomas. Conclusion This is the first report of 46,XY sex reversal in three siblings who have a paternally inherited deletion upstream of SOX9 associated with reduced SOX9 mRNA expression. PMID:24907458

Cloning and characterization of the fatty acid-binding protein gene from the protoscolex of Taenia multiceps.

PubMed

Nie, Hua-Ming; Xie, Yue; Fu, Yan; Yang, Ying-Dong; Gu, Xiao-Bin; Wang, Shu-Xian; Peng, Xi; Lai, Wei-Ming; Peng, Xue-Rong; Yang, Guang-You

2013-05-01

Taenia multiceps (Cestoda: Taeniidae), a worldwide cestode parasite, is emerging as an important helminthic zoonosis due to serious or fatal central nervous system disease commonly known as coenurosis in domestic and wild ruminants including humans. Herein, a fatty acid-binding protein (FABP) gene was identified from transcriptomic data in T. multiceps. This gene, which contains a complete coding sequence, was amplified by reverse-transcriptase polymerase chain reaction. The corresponding protein, which was named TmFABP, had a molecular weight of 14 kDa, and subsequently was recombinantly expressed in Escherichia coli. The fusion protein was purified on Ni-NTA beads (Bio-Rad). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses showed that the purified recombinant protein caused immunogenicity. Immunohistochemical studies showed that TmFABP was expressed at the tegumental level in the protoscolices and in the cells between the body wall and parenchyma layer of the cestode. In sections from gravid proglottids, intense staining was detected in the uterus and eggs. Based on this, TmFABP could be switched on during differentiation of germinative layers to protoscoleces and from metacestodes to adult worms. Taken together, our results already reported for T. multiceps suggest the possibility of TmFABP developing a vaccine to control and prevent coenurosis.

The baculovirus-integrated retrotransposon TED encodes gag and pol proteins that assemble into viruslike particles with reverse transcriptase.

PubMed Central

Lerch, R A; Friesen, P D

1992-01-01

TED is a lepidopteran retrotransposon found inserted within the DNA genome of the Autographa californica nuclear polyhedrosis virus mutant, FP-D. To examine the proteins and functions encoded by this representative of the gypsy family of retrotransposons, the gag- and pol-like open reading frames (ORFs 1 and 2) were expressed in homologous lepidopteran cells by using recombinant baculovirus vectors. Expression of ORF 1 resulted in synthesis of an abundant TED-specific protein (Pr55gag) that assembled into viruslike particles with a diameter of 55 to 60 nm. Expression of ORF 2, requiring a -1 translational frameshift, resulted in synthesis of a protease that mediated cleavage of Pr55gag to generate p37, the major protein component of the resulting particles. Expression of ORF 2 also produced reverse transcriptase that associated with these particles. Both protease and reverse transcriptase activities mapped to domains within ORF 2 that contain sequence similarities with the corresponding functional domains of the pol gene of the vertebrate retroviruses. These results indicated that TED ORFs 1 and 2 functionally resemble the retrovirus gag and pol genes and demonstrated for the first time that an invertebrate member of the gypsy family of elements encodes active forms of the structural and enzymatic functions necessary for transposition via an RNA intermediate. TED integration within the baculovirus genome thus represents one of the first examples of transposon-mediated transfer of host-derived genes to an eukaryotic virus. Images PMID:1371168

An analysis of mobile genetic elements in three Plasmodium species and their potential impact on the nucleotide composition of the P. falciparum genome.

PubMed

Durand, Pierre M; Oelofse, Andries J; Coetzer, Theresa L

2006-11-04

The completed genome sequences of the malaria parasites P. falciparum, P. y. yoelii and P. vivax have revealed some unusual features. P. falciparum contains the most AT rich genome sequenced so far--over 90% in some regions. In comparison, P. y. yoelii is approximately 77% and P. vivax is approximately 55% AT rich. The evolutionary reasons for these findings are unknown. Mobile genetic elements have a considerable impact on genome evolution but a thorough investigation of these elements in Plasmodium has not been undertaken. We therefore performed a comprehensive genome analysis of these elements and their derivatives in the three Plasmodium species. Whole genome analysis was performed using bioinformatic methods. Forty potential protein encoding sequences with features of transposable elements were identified in P. vivax, eight in P. y. yoelii and only six in P. falciparum. Further investigation of the six open reading frames in P. falciparum revealed that only one is potentially an active mobile genetic element. Most of the open reading frames identified in all three species are hypothetical proteins. Some represent annotated host proteins such as the putative telomerase reverse transcriptase genes in P. y. yoelii and P. falciparum. One of the P. vivax open reading frames identified in this study demonstrates similarity to telomerase reverse transcriptase and we conclude it to be the orthologue of this gene. There is a divergence in the frequencies of mobile genetic elements in the three Plasmodium species investigated. Despite the limitations of whole genome analytical methods, it is tempting to speculate that mobile genetic elements might have been a driving force behind the compositional bias of the P. falciparum genome.

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

PubMed

Mohr, Sabine; Ghanem, Eman; Smith, Whitney; Sheeter, Dennis; Qin, Yidan; King, Olga; Polioudakis, Damon; Iyer, Vishwanath R; Hunicke-Smith, Scott; Swamy, Sajani; Kuersten, Scott; Lambowitz, Alan M

2013-07-01

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor.

PubMed

Gong, Qingguo; Menon, Lakshmi; Ilina, Tatiana; Miller, Lena G; Ahn, Jinwoo; Parniak, Michael A; Ishima, Rieko

2011-01-01

HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors. © 2010 John Wiley & Sons A/S.

Nucleotide sequence determination of guinea-pig casein B mRNA reveals homology with bovine and rat alpha s1 caseins and conservation of the non-coding regions of the mRNA.

PubMed Central

Hall, L; Laird, J E; Craig, R K

1984-01-01

Nucleotide sequence analysis of cloned guinea-pig casein B cDNA sequences has identified two casein B variants related to the bovine and rat alpha s1 caseins. Amino acid homology was largely confined to the known bovine or predicted rat phosphorylation sites and within the 'signal' precursor sequence. Comparison of the deduced nucleotide sequence of the guinea-pig and rat alpha s1 casein mRNA species showed greater sequence conservation in the non-coding than in the coding regions, suggesting a functional and possibly regulatory role for the non-coding regions of casein mRNA. The results provide insight into the evolution of the casein genes, and raise questions as to the role of conserved nucleotide sequences within the non-coding regions of mRNA species. Images Fig. 1. PMID:6548375

DNA barcode goes two-dimensions: DNA QR code web server.

PubMed

Liu, Chang; Shi, Linchun; Xu, Xiaolan; Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

2012-01-01

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, "DNA barcode" actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications.

Clinical comparison of branched DNA and reverse transcriptase-PCR and nucleic acid sequence-based amplification assay for the quantitation of circulating recombinant form_BC HIV-1 RNA in plasma.

PubMed

Pan, Pinliang; Tao, Xiaoxia; Zhang, Qi; Xing, Wenge; Sun, Xianguang; Pei, Lijian; Jiang, Yan

2007-12-01

To investigate the correlation between three viral load assays for circulating recombinant form (CRF)_BC. Recent studies in HIV-1 molecular epidemiology, reveals that CRF_BC is the dominant subtype of HIV-1 virus in mainland China, representing over 45% of the HIV-1 infected population. The performances of nucleic acid sequence-based amplification (NASBA), branched DNA (bDNA) and reverse transcriptase polymerase chain reaction (RT-PCR) were compared for the HIV-1 viral load detection and quantitation of CRF_BC in China. Sixteen HIV-1 positive and three HIV-1 negative samples were collected. Sequencing of the positive samples in the gp41 region was conducted. The HIV-1 viral load values were determined using bDNA, RT-PCR and NASBA assays. Deming regression analysis with SPSS 12.0 (SPS Inc., Chicago, Illinois, USA) was performed for data analysis. Sequencing and phylogenetic analysis of env gene (gp41) region of the 16 HIV-1 positive clinical specimens from Guizhou Province in southwest China revealed the dominance of the subtype CRF_BC in that region. A good correlation of their viral load values was observed among three assays. Pearson's correlation between RT-PCR and bDNA is 0.969, Lg(VL)RT-PCR = 0.969 * Lg(VL)bDNA + 0.55; Pearson's correlation between RT-PCR and NASBA is 0.968, Lg(VL)RT-PCR = 0.968 * Lg(VL)NASBA + 0.937; Pearson's correlation between NASBA and bDNA is 0.980, Lg(VL)NASBA = 0.980 * Lg(VL)bDNA - 0.318. When testing with 3 different assays, RT-PCR, bDNA and NASBA, the group of 16 HIV-1 positive samples showed the viral load value was highest for RT-PCR, followed by bDNA then NASBA, which is consistent with the former results in subtype B. The three viral load assays are highly correlative for CRF_BC in China.

Lichenase and coding sequences

DOEpatents

Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

2000-08-15

The present invention provides a fungal lichenase, i.e., an endo-1,3-1,4-.beta.-D-glucanohydrolase, its coding sequence, recombinant DNA molecules comprising the lichenase coding sequences, recombinant host cells and methods for producing same. The present lichenase is from Orpinomyces PC-2.

Evaluation of Cytokine Synthesis in Human Whole Blood by Enzyme Linked Immunoassay (ELISA), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), and Flow Cytometry

DTIC Science & Technology

2007-05-08

deoxynucleotide triphosphates, from Sigma. Sequences for glyceraldehyde-3-phosphate dehydrogenase ( G3PDH ), IL-8,and TNF-a were amplified with primer...This was accomplished by normalizing all samples to the mRNA for the moderately expressed housekeeping function glyceraldehyde-3 -phosphate...without and with isolation of cells before reverse transcription and PCR. G3PDH mRNA target amplifies at 983 base pairs. The 630 base pair band is the

Distemper Outbreak and Its Effect on African Wild Dog Conservation

PubMed Central

van de Bildt, Marco W.G.; Kuiken, Thijs; Visee, Aart M.; Lema, Sangito; Fitzjohn, Tony R.

2002-01-01

In December 2000, an infectious disease spread through a captive breeding group of African wild dogs (Lycaon pictus) in Tanzania, killing 49 of 52 animals within 2 months. The causative agent was identified as Canine distemper virus (CDV) by means of histologic examination, virus isolation, reverse transcriptase-polymerase chain reaction analysis, and nucleotide sequencing. This report emphasizes the importance of adequate protection against infectious diseases for the successful outcome of captive breeding programs of endangered species. PMID:11897078

FRAGS: estimation of coding sequence substitution rates from fragmentary data

PubMed Central

Swart, Estienne C; Hide, Winston A; Seoighe, Cathal

2004-01-01

Background Rates of substitution in protein-coding sequences can provide important insights into evolutionary processes that are of biomedical and theoretical interest. Increased availability of coding sequence data has enabled researchers to estimate more accurately the coding sequence divergence of pairs of organisms. However the use of different data sources, alignment protocols and methods to estimate substitution rates leads to widely varying estimates of key parameters that define the coding sequence divergence of orthologous genes. Although complete genome sequence data are not available for all organisms, fragmentary sequence data can provide accurate estimates of substitution rates provided that an appropriate and consistent methodology is used and that differences in the estimates obtainable from different data sources are taken into account. Results We have developed FRAGS, an application framework that uses existing, freely available software components to construct in-frame alignments and estimate coding substitution rates from fragmentary sequence data. Coding sequence substitution estimates for human and chimpanzee sequences, generated by FRAGS, reveal that methodological differences can give rise to significantly different estimates of important substitution parameters. The estimated substitution rates were also used to infer upper-bounds on the amount of sequencing error in the datasets that we have analysed. Conclusion We have developed a system that performs robust estimation of substitution rates for orthologous sequences from a pair of organisms. Our system can be used when fragmentary genomic or transcript data is available from one of the organisms and the other is a completely sequenced genome within the Ensembl database. As well as estimating substitution statistics our system enables the user to manage and query alignment and substitution data. PMID:15005802

Visual pattern image sequence coding

NASA Technical Reports Server (NTRS)

Silsbee, Peter; Bovik, Alan C.; Chen, Dapang

1990-01-01

The visual pattern image coding (VPIC) configurable digital image-coding process is capable of coding with visual fidelity comparable to the best available techniques, at compressions which (at 30-40:1) exceed all other technologies. These capabilities are associated with unprecedented coding efficiencies; coding and decoding operations are entirely linear with respect to image size and entail a complexity that is 1-2 orders of magnitude faster than any previous high-compression technique. The visual pattern image sequence coding to which attention is presently given exploits all the advantages of the static VPIC in the reduction of information from an additional, temporal dimension, to achieve unprecedented image sequence coding performance.

[Influence of "prehistory" of sequential movements of the right and the left hand on reproduction: coding of positions, movements and sequence structure].

PubMed

Bobrova, E V; Liakhovetskiĭ, V A; Borshchevskaia, E R

2011-01-01

The dependence of errors during reproduction of a sequence of hand movements without visual feedback on the previous right- and left-hand performance ("prehistory") and on positions in space of sequence elements (random or ordered by the explicit rule) was analyzed. It was shown that the preceding information about the ordered positions of the sequence elements was used during right-hand movements, whereas left-hand movements were performed with involvement of the information about the random sequence. The data testify to a central mechanism of the analysis of spatial structure of sequence elements. This mechanism activates movement coding specific for the left hemisphere (vector coding) in case of an ordered sequence structure and positional coding specific for the right hemisphere in case of a random sequence structure.

Differential expression of genes encoding anti-oxidant enzymes in Sydney rock oysters, Saccostrea glomerata (Gould) selected for disease resistance.

PubMed

Green, Timothy J; Dixon, Tom J; Devic, Emilie; Adlard, Robert D; Barnes, Andrew C

2009-05-01

Sydney rock oysters (Saccostrea glomerata) selectively bred for disease resistance (R) and wild-caught control oysters (W) were exposed to a field infection of disseminating neoplasia. Cumulative mortality of W oysters (31.7%) was significantly greater than R oysters (0.0%) over the 118 days of the experiment. In an attempt to understand the biochemical and molecular pathways involved in disease resistance, differentially expressed sequence tags (ESTs) between R and W S. glomerata hemocytes were identified using the PCR technique, suppression subtractive hybridisation (SSH). Sequencing of 300 clones from two SSH libraries revealed 183 distinct sequences of which 113 shared high similarity to sequences in the public databases. Putative function could be assigned to 64 of the sequences. Expression of nine ESTs homologous to genes previously shown to be involved in bivalve immunity was further studied using quantitative reverse-transcriptase PCR (qRT-PCR). The base-line expression of an extracellular superoxide dismutase (ecSOD) and a small heat shock protein (sHsP) were significantly increased, whilst peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine factor (IK) were significantly decreased in R oysters. From these results it was hypothesised that R oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H(2)O(2)) faster and to higher concentrations during respiratory burst due to the differential expression of genes for the two anti-oxidant enzymes of ecSOD and Prx6. To investigate this hypothesis, protein extracts from hemolymph were analysed for oxidative burst enzyme activity. Analysis of the cell free hemolymph proteins separated by native-polyacrylamide gel electrophoresis (PAGE) failed to detect true superoxide dismutase (SOD) activity by assaying dismutation of superoxide anion in zymograms. However, the ecSOD enzyme appears to generate hydrogen peroxide, presumably via another process, which is yet to be elucidated. This corroborates our hypothesis, whilst phylogenetic analysis of the complete coding sequence (CDS) of the S. glomerata ecSOD gene is supportive of the atypical nature of the ecSOD enzyme. Results obtained from this work further the current understanding of the molecular mechanisms involved in resistance to disease in this economically important bivalve, and shed further light on the anomalous oxidative processes involved.

Discrete Cosine Transform Image Coding With Sliding Block Codes

NASA Astrophysics Data System (ADS)

Divakaran, Ajay; Pearlman, William A.

1989-11-01

A transform trellis coding scheme for images is presented. A two dimensional discrete cosine transform is applied to the image followed by a search on a trellis structured code. This code is a sliding block code that utilizes a constrained size reproduction alphabet. The image is divided into blocks by the transform coding. The non-stationarity of the image is counteracted by grouping these blocks in clusters through a clustering algorithm, and then encoding the clusters separately. Mandela ordered sequences are formed from each cluster i.e identically indexed coefficients from each block are grouped together to form one dimensional sequences. A separate search ensues on each of these Mandela ordered sequences. Padding sequences are used to improve the trellis search fidelity. The padding sequences absorb the error caused by the building up of the trellis to full size. The simulations were carried out on a 256x256 image ('LENA'). The results are comparable to any existing scheme. The visual quality of the image is enhanced considerably by the padding and clustering.

DNA Barcode Goes Two-Dimensions: DNA QR Code Web Server

PubMed Central

Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

2012-01-01

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, “DNA barcode” actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications. PMID:22574113

Terminal oxidase diversity and function in "Metallosphaera yellowstonensis": gene expression and protein modeling suggest mechanisms of Fe(II) oxidation in the sulfolobales.

PubMed

Kozubal, M A; Dlakic, M; Macur, R E; Inskeep, W P

2011-03-01

"Metallosphaera yellowstonensis" is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowstonensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEFGHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b(558/566), several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that the fox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance of fox genes (e.g., foxA and foxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex. An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores.

Cloning of cDNAs for H1F0, TOP1, CLTA and CDK1 and the effects of cryopreservation on the expression of their mRNA transcripts in yak (Bos grunniens) oocytes.

PubMed

Niu, Hui-Ran; Zi, Xiang-Dong; Xiao, Xiao; Xiong, Xian-Rong; Zhong, Jin-Cheng; Li, Jian; Wang, Li; Wang, Yong

2014-08-01

We cloned and sequenced four pivotal cDNAs involved in DNA structural maintenance (H1F0 and TOP1) and the cell cycle (CLTA and CDK1) from yak oocytes. In addition, we studied the consequences of freezing-thawing (F/T) processes on the expression of their mRNA transcripts in yak immature and in vitro matured (MII) oocytes. H1F0, TOP1, CLTA and CDK1 cDNAs were cloned from yak oocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) strategy. The expression of their mRNA transcript analyses were performed upon fresh and frozen-thawed immature germinal vesicle (GV) and MII yak oocytes following normalization of transcripts with GAPDH by real-time PCR. The yak H1F0, TOP1, CLTA and CDK1 cDNA sequences were found to consist of CDK1 585, 2539, 740, and 894 bp, respectively. Their coding regions encoded 195, 768, 244, and 298 amino acids, respectively. The homology with that of cattle was very high (95.2%, 98.8%, 93.6%, and 89.5%, respectively nucleotide sequence level, and 94.3%, 98.2%, 87.7%, and 90.9%, respectively at the deduced amino acid level). The overall mRNA expression levels of these four transcripts were reduced by F/T process, albeit at different levels. TOP1 in GV-oocytes, and H1F0 and CDK1 in MII-oocytes of the yak were significantly down-regulated (P<0.05). This is the first isolation and characterization of H1F0, TOP1, CLTA, and CDK1 cDNAs from yak oocytes. The lower fertility and developmental ability of yak oocytes following fertilization after cryopreservation may be explained by the alterations to their gene expression profiles. Copyright © 2014 Elsevier Inc. All rights reserved.

cDNA cloning and initial characterization of CYP3A43, a novel human cytochrome P450.

PubMed

Domanski, T L; Finta, C; Halpert, J R; Zaphiropoulos, P G

2001-02-01

The RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression. This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43. Interestingly, the majority of the cDNAs identified were characterized by alternative splicing events such as exon skipping and complete or partial intron inclusion. CYP3A43 expression was detected in liver, kidney, pancreas, and prostate. The amino acid sequence is 75% identical to that of CYP3A4 and CYP3A5 and 71% identical to CYP3A7. CYP3A43 differs from CYP3A4 at six amino acid residues, found within the putative substrate recognition sites of CYP3A4, that are known to be determinants of substrate selectivity. The N terminus of CYP3A43 was modified for efficient expression of the protein in Escherichia coli, and a 6X histidine tag was added at the C terminus to facilitate purification. CYP3A43 gave a reduced carbon monoxide difference spectra with an absorbance maximum at 450 nm. The level of heterologous expression was significantly lower than that observed for CYP3A4 and CYP3A5. Immunoblot analyses revealed that CYP3A43 comigrates with CYP3A4 in polyacrylamide gel electrophoresis but does separate from CYP3A5. Monooxygenase assays were performed under a variety of conditions, several of which yielded reproducible, albeit low, testosterone hydroxylase activity. The findings from this study demonstrate that there is a novel CYP3A member expressed in human tissues, although its relative contribution to drug metabolism has yet to be ascertained.

The mechano-chemistry of a monomeric reverse transcriptase

PubMed Central

Malik, Omri; Khamis, Hadeel; Rudnizky, Sergei

2017-01-01

Abstract Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex. PMID:29165701

Telomeres and telomerase.

PubMed Central

Chan, Simon R W L; Blackburn, Elizabeth H

2004-01-01

Telomeres are the protective DNA-protein complexes found at the ends of eukaryotic chromosomes. Telomeric DNA consists of tandem repeats of a simple, often G-rich, sequence specified by the action of telomerase, and complete replication of telomeric DNA requires telomerase. Telomerase is a specialized cellular ribonucleoprotein reverse transcriptase. By copying a short template sequence within its intrinsic RNA moiety, telomerase synthesizes the telomeric DNA strand running 5' to 3' towards the distal end of the chromosome, thus extending it. Fusion of a telomere, either with another telomere or with a broken DNA end, generally constitutes a catastrophic event for genomic stability. Telomerase acts to prevent such fusions. The molecular consequences of telomere failure, and the molecular contributors to telomere function, with an emphasis on telomerase, are discussed here. PMID:15065663

CRITICA: coding region identification tool invoking comparative analysis

NASA Technical Reports Server (NTRS)

Badger, J. H.; Olsen, G. J.; Woese, C. R. (Principal Investigator)

1999-01-01

Gene recognition is essential to understanding existing and future DNA sequence data. CRITICA (Coding Region Identification Tool Invoking Comparative Analysis) is a suite of programs for identifying likely protein-coding sequences in DNA by combining comparative analysis of DNA sequences with more common noncomparative methods. In the comparative component of the analysis, regions of DNA are aligned with related sequences from the DNA databases; if the translation of the aligned sequences has greater amino acid identity than expected for the observed percentage nucleotide identity, this is interpreted as evidence for coding. CRITICA also incorporates noncomparative information derived from the relative frequencies of hexanucleotides in coding frames versus other contexts (i.e., dicodon bias). The dicodon usage information is derived by iterative analysis of the data, such that CRITICA is not dependent on the existence or accuracy of coding sequence annotations in the databases. This independence makes the method particularly well suited for the analysis of novel genomes. CRITICA was tested by analyzing the available Salmonella typhimurium DNA sequences. Its predictions were compared with the DNA sequence annotations and with the predictions of GenMark. CRITICA proved to be more accurate than GenMark, and moreover, many of its predictions that would seem to be errors instead reflect problems in the sequence databases. The source code of CRITICA is freely available by anonymous FTP (rdp.life.uiuc.edu in/pub/critica) and on the World Wide Web (http:/(/)rdpwww.life.uiuc.edu).

The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

PubMed Central

Urano, Y; Kominami, R; Mishima, Y; Muramatsu, M

1980-01-01

Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Images PMID:6162156

Promises and pitfalls of Illumina sequencing for HIV resistance genotyping.

PubMed

Brumme, Chanson J; Poon, Art F Y

2017-07-15

Genetic sequencing ("genotyping") plays a critical role in the modern clinical management of HIV infection. This virus evolves rapidly within patients because of its error-prone reverse transcriptase and short generation time. Consequently, HIV variants with mutations that confer resistance to one or more antiretroviral drugs can emerge during sub-optimal treatment. There are now multiple HIV drug resistance interpretation algorithms that take the region of the HIV genome encoding the major drug targets as inputs; expert use of these algorithms can significantly improve to clinical outcomes in HIV treatment. Next-generation sequencing has the potential to revolutionize HIV resistance genotyping by lowering the threshold that rare but clinically significant HIV variants can be detected reproducibly, and by conferring improved cost-effectiveness in high-throughput scenarios. In this review, we discuss the relative merits and challenges of deploying the Illumina MiSeq instrument for clinical HIV genotyping. Copyright © 2016 Elsevier B.V. All rights reserved.

HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients

PubMed Central

2012-01-01

Abstract In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients. Patients and methods A total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping. Results Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C. Conclusion The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973 PMID:23044036

HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients.

PubMed

Elmi Abar, Aden; Jlizi, Asma; Darar, Houssein Youssouf; Kacem, Mohamed Ali Ben Hadj; Slim, Amine

2012-10-08

In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients. A total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping. Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C. The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973.

Flexible manipulation of terahertz wave reflection using polarization insensitive coding metasurfaces.

PubMed

Jiu-Sheng, Li; Ze-Jiang, Zhao; Jian-Quan, Yao

2017-11-27

In order to extend to 3-bit encoding, we propose notched-wheel structures as polarization insensitive coding metasurfaces to control terahertz wave reflection and suppress backward scattering. By using a coding sequence of "00110011…" along x-axis direction and 16 × 16 random coding sequence, we investigate the polarization insensitive properties of the coding metasurfaces. By designing the coding sequences of the basic coding elements, the terahertz wave reflection can be flexibly manipulated. Additionally, radar cross section (RCS) reduction in the backward direction is less than -10dB in a wide band. The present approach can offer application for novel terahertz manipulation devices.

Noncoding sequence classification based on wavelet transform analysis: part I

NASA Astrophysics Data System (ADS)

Paredes, O.; Strojnik, M.; Romo-Vázquez, R.; Vélez Pérez, H.; Ranta, R.; Garcia-Torales, G.; Scholl, M. K.; Morales, J. A.

2017-09-01

DNA sequences in human genome can be divided into the coding and noncoding ones. Coding sequences are those that are read during the transcription. The identification of coding sequences has been widely reported in literature due to its much-studied periodicity. Noncoding sequences represent the majority of the human genome. They play an important role in gene regulation and differentiation among the cells. However, noncoding sequences do not exhibit periodicities that correlate to their functions. The ENCODE (Encyclopedia of DNA elements) and Epigenomic Roadmap Project projects have cataloged the human noncoding sequences into specific functions. We study characteristics of noncoding sequences with wavelet analysis of genomic signals.

Converting Panax ginseng DNA and chemical fingerprints into two-dimensional barcode.

PubMed

Cai, Yong; Li, Peng; Li, Xi-Wen; Zhao, Jing; Chen, Hai; Yang, Qing; Hu, Hao

2017-07-01

In this study, we investigated how to convert the Panax ginseng DNA sequence code and chemical fingerprints into a two-dimensional code. In order to improve the compression efficiency, GATC2Bytes and digital merger compression algorithms are proposed. HPLC chemical fingerprint data of 10 groups of P. ginseng from Northeast China and the internal transcribed spacer 2 (ITS2) sequence code as the DNA sequence code were ready for conversion. In order to convert such data into a two-dimensional code, the following six steps were performed: First, the chemical fingerprint characteristic data sets were obtained through the inflection filtering algorithm. Second, precompression processing of such data sets is undertaken. Third, precompression processing was undertaken with the P. ginseng DNA (ITS2) sequence codes. Fourth, the precompressed chemical fingerprint data and the DNA (ITS2) sequence code were combined in accordance with the set data format. Such combined data can be compressed by Zlib, an open source data compression algorithm. Finally, the compressed data generated a two-dimensional code called a quick response code (QR code). Through the abovementioned converting process, it can be found that the number of bytes needed for storing P. ginseng chemical fingerprints and its DNA (ITS2) sequence code can be greatly reduced. After GTCA2Bytes algorithm processing, the ITS2 compression rate reaches 75% and the chemical fingerprint compression rate exceeds 99.65% via filtration and digital merger compression algorithm processing. Therefore, the overall compression ratio even exceeds 99.36%. The capacity of the formed QR code is around 0.5k, which can easily and successfully be read and identified by any smartphone. P. ginseng chemical fingerprints and its DNA (ITS2) sequence code can form a QR code after data processing, and therefore the QR code can be a perfect carrier of the authenticity and quality of P. ginseng information. This study provides a theoretical basis for the development of a quality traceability system of traditional Chinese medicine based on a two-dimensional code.

Silent mutations at codons 65 and 66 in reverse transcriptase alleviate indel formation and restore fitness in subtype B HIV-1 containing D67N and K70R drug resistance mutations

PubMed Central

Telwatte, Sushama; Hearps, Anna C.; Johnson, Adam; Latham, Catherine F.; Moore, Katie; Agius, Paul; Tachedjian, Mary; Sonza, Secondo; Sluis-Cremer, Nicolas; Harrigan, P. Richard; Tachedjian, Gilda

2015-01-01

Resistance to combined antiretroviral therapy (cART) in HIV-1-infected individuals is typically due to nonsynonymous mutations that change the protein sequence; however, the selection of synonymous or ‘silent’ mutations in the HIV-1 genome with cART has been reported. These silent K65K and K66K mutations in the HIV-1 reverse transcriptase (RT) occur in over 35% of drug-experienced individuals and are highly associated with the thymidine analog mutations D67N and K70R, which confer decreased susceptibility to most nucleoside and nucleotide RT inhibitors. However, the basis for selection of these silent mutations under selective drug pressure is unknown. Using Illumina next-generation sequencing, we demonstrate that the D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65–67, consequently impairing viral fitness. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R reversed the error-prone DNA synthesis at codons 65–67 in RT and improved viral replication fitness, but did not impact RT inhibitor drug susceptibility. These data provide new mechanistic insights into the role of silent mutations selected during antiretroviral therapy and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses. PMID:25765644

Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors

PubMed Central

Kierczak, Marcin; Dramiński, Michał; Koronacki, Jacek; Komorowski, Jan

2010-01-01

Motivation Despite more than two decades of research, HIV resistance to drugs remains a serious obstacle in developing efficient AIDS treatments. Several computational methods have been developed to predict resistance level from the sequence of viral proteins such as reverse transcriptase (RT) or protease. These methods, while powerful and accurate, give very little insight into the molecular interactions that underly acquisition of drug resistance/hypersusceptibility. Here, we attempt at filling this gap by using our Monte Carlo feature selection and interdependency discovery method (MCFS-ID) to elucidate molecular interaction networks that characterize viral strains with altered drug resistance levels. Results We analyzed a number of HIV-1 RT sequences annotated with drug resistance level using the MCFS-ID method. This let us expound interdependency networks that characterize change of drug resistance to six selected RT inhibitors: Abacavir, Lamivudine, Stavudine, Zidovudine, Tenofovir and Nevirapine. The networks consider interdependencies at the level of physicochemical properties of mutating amino acids, eg,: polarity. We mapped each network on the 3D structure of RT in attempt to understand the molecular meaning of interacting pairs. The discovered interactions describe several known drug resistance mechanisms and, importantly, some previously unidentified ones. Our approach can be easily applied to a whole range of problems from the domain of protein engineering. Availability A portable Java implementation of our MCFS-ID method is freely available for academic users and can be obtained at: http://www.ipipan.eu/staff/m.draminski/software.htm. PMID:21234299

Global Comparison of Drug Resistance Mutations After First-Line Antiretroviral Therapy Across Human Immunodeficiency Virus-1 Subtypes

PubMed Central

Huang, Austin; Hogan, Joseph W.; Luo, Xi; DeLong, Allison; Saravanan, Shanmugam; Wu, Yasong; Sirivichayakul, Sunee; Kumarasamy, Nagalingeswaran; Zhang, Fujie; Phanuphak, Praphan; Diero, Lameck; Buziba, Nathan; Istrail, Sorin; Katzenstein, David A.; Kantor, Rami

2016-01-01

Background. Human immunodeficiency virus (HIV)-1 drug resistance mutations (DRMs) often accompany treatment failure. Although subtype differences are widely studied, DRM comparisons between subtypes either focus on specific geographic regions or include populations with heterogeneous treatments. Methods. We characterized DRM patterns following first-line failure and their impact on future treatment in a global, multi-subtype reverse-transcriptase sequence dataset. We developed a hierarchical modeling approach to address the high-dimensional challenge of modeling and comparing frequencies of multiple DRMs in varying first-line regimens, durations, and subtypes. Drug resistance mutation co-occurrence was characterized using a novel application of a statistical network model. Results. In 1425 sequences, 202 subtype B, 696 C, 44 G, 351 circulating recombinant forms (CRF)01_AE, 58 CRF02_AG, and 74 from other subtypes mutation frequencies were higher in subtypes C and CRF01_AE compared with B overall. Mutation frequency increased by 9%–20% at reverse transcriptase positions 41, 67, 70, 184, 215, and 219 in subtype C and CRF01_AE vs B. Subtype C and CRF01_AE exhibited higher predicted cross-resistance (+12%–18%) to future therapy options compared with subtype B. Topologies of subtype mutation networks were mostly similar. Conclusions. We find clear differences in DRM outcomes following first-line failure, suggesting subtype-specific ecological or biological factors that determine DRM patterns. PMID:27419147

Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors.

PubMed

Kierczak, Marcin; Dramiński, Michał; Koronacki, Jacek; Komorowski, Jan

2010-12-12

Despite more than two decades of research, HIV resistance to drugs remains a serious obstacle in developing efficient AIDS treatments. Several computational methods have been developed to predict resistance level from the sequence of viral proteins such as reverse transcriptase (RT) or protease. These methods, while powerful and accurate, give very little insight into the molecular interactions that underly acquisition of drug resistance/hypersusceptibility. Here, we attempt at filling this gap by using our Monte Carlo feature selection and interdependency discovery method (MCFS-ID) to elucidate molecular interaction networks that characterize viral strains with altered drug resistance levels. We analyzed a number of HIV-1 RT sequences annotated with drug resistance level using the MCFS-ID method. This let us expound interdependency networks that characterize change of drug resistance to six selected RT inhibitors: Abacavir, Lamivudine, Stavudine, Zidovudine, Tenofovir and Nevirapine. The networks consider interdependencies at the level of physicochemical properties of mutating amino acids, eg,: polarity. We mapped each network on the 3D structure of RT in attempt to understand the molecular meaning of interacting pairs. The discovered interactions describe several known drug resistance mechanisms and, importantly, some previously unidentified ones. Our approach can be easily applied to a whole range of problems from the domain of protein engineering. A portable Java implementation of our MCFS-ID method is freely available for academic users and can be obtained at: http://www.ipipan.eu/staff/m.draminski/software.htm.

Appearance of drug resistance-associated mutations in human immunodeficiency virus type 1 protease and reverse transcriptase derived from drug-treated Indonesian patients.

PubMed

Khairunisa, Siti Qamariyah; Kotaki, Tomohiro; Witaningrum, Adiana Mutamsari; Yunifiar M, Muhammad Qushai; Sukartiningrum, Septhia Dwi; Nasronudin; Kameoka, Masanori

2015-02-01

Although HIV-1 drug resistance is a major obstacle in Indonesia, information on drug resistance is limited. In this study, the viral subtype and appearance of drug resistance mutations in the HIV-1 protease (PR) and reverse transcriptase (RT) genes were determined among drug-treated, HIV-1-infected patients in Surabaya. HIV-1 patients who received antiretroviral therapy (ART) more than 2 years were randomly recruited regardless of the viral load or ART failure. Fifty-eight HIV-1 PR genes and 53 RT genes were sequenced. CRF01_AE viruses were identified as the predominant strain. Major drug resistance mutations were not detected in the PR genes. In contrast, 37.7% (20/53) of the participants had one or more major drug resistance mutations in the RT genes, predominantly M184V (28.3%), K103N (11.3%), and thymidine analogue mutations (TAMs) (20.8%). The high prevalence of drug resistance mutations in RT genes indicated the necessity of monitoring the effectiveness of ART in Indonesia.

A novel Met-to-Thr mutation in the YMDD motif of reverse transcriptase from feline immunodeficiency virus confers resistance to oxathiolane nucleosides.

PubMed Central

Smith, R A; Remington, K M; Lloyd, R M; Schinazi, R F; North, T W

1997-01-01

Variants of feline immunodeficiency virus (FIV) that possess a unique methionine-to-threonine mutation within the YMDD motif of reverse transcriptase (RT) were selected by culturing virus in the presence of inhibitory concentrations of (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC]. The mutants were resistant to (-)-FTC and (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and additionally exhibited low-level resistance to 2',3'-dideoxycytidine (ddC). DNA sequence analysis of the RT-encoding region of the pol gene amplified from resistant viruses consistently identified a Met-to-Thr mutation in the YMDD motif. Purified RT from the mutants was also resistant to the 5'-triphosphate forms of 3TC, (-)-FTC, and ddC. Site-directed mutants of FIV were engineered which contain either the novel Met-to-Thr mutation or the Met-to-Val mutation seen in oxathiolane nucleoside-resistant HIV-1. Both site-directed mutants displayed resistance to 3TC, thus confirming the role of these mutations in the resistance of FIV to beta-L-3'-thianucleosides. PMID:9032372

A Laccase with HIV-1 Reverse Transcriptase Inhibitory Activity from the Broth of Mycelial Culture of the Mushroom Lentinus tigrinus

PubMed Central

Xu, LiJing; Wang, HeXiang; Ng, TziBun

2012-01-01

A 59 kDa laccase with inhibitory activity against HIV-1 reverse transcriptase (IC50 = 2.4 μM) was isolated from the broth of mycelial culture of the mushroom Lentinus tigrinus. The isolation procedure involved ion exchange chromatography on DEAE-cellulose and CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was adsorbed on both types of ion exchangers. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The procedure resulted in a specific enzyme activity of 76.6 U/mg. Its N-terminal amino acid sequence was GIPDLHDLTV, which showed little similarity to other mushroom laccase and other Lentinus tigrinus strain laccase. Its characteristics were different from previously reported laccase of other Lentinus tigrinus strain. Maximal laccase activity was observed at a pH of 4 and at a temperature of 60°C, respectively. This study yielded the information about the potentially exploitable activities of Lentinus tigrinus laccase. PMID:22536022

Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus.

PubMed

Wu, Xinghai; Chen, Chanfa; Xiao, Xizhi; Deng, Ming Jun

2016-11-01

A protocol for the reverse transcription-helicase-dependent amplification (RT-HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT-HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT-HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT-HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT-HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.

Short-Term Dynamic and Local Epidemiological Trends in the South American HIV-1B Epidemic.

PubMed

Junqueira, Dennis Maletich; de Medeiros, Rubia Marília; Gräf, Tiago; Almeida, Sabrina Esteves de Matos

2016-01-01

The human displacement and sexual behavior are the main factors driving the HIV-1 pandemic to the current profile. The intrinsic structure of the HIV transmission among different individuals has valuable importance for the understanding of the epidemic and for the public health response. The aim of this study was to characterize the HIV-1 subtype B (HIV-1B) epidemic in South America through the identification of transmission links and infer trends about geographical patterns and median time of transmission between individuals. Sequences of the protease and reverse transcriptase coding regions from 4,810 individuals were selected from GenBank. Maximum likelihood phylogenies were inferred and submitted to ClusterPicker to identify transmission links. Bayesian analyses were applied only for clusters including ≥5 dated samples in order to estimate the median maximum inter-transmission interval. This study analyzed sequences sampled from 12 South American countries, from individuals of different exposure categories, under different antiretroviral profiles, and from a wide period of time (1989-2013). Continentally, Brazil, Argentina and Venezuela were revealed important sites for the spread of HIV-1B among countries inside South America. Of note, from all the clusters identified about 70% of the HIV-1B infections are primarily occurring among individuals living in the same geographic region. In addition, these transmissions seem to occur early after the infection of an individual, taking in average 2.39 years (95% CI 1.48-3.30) to succeed. Homosexual/Bisexual individuals transmit the virus as quickly as almost half time of that estimated for the general population sampled here. Public health services can be broadly benefitted from this kind of information whether to focus on specific programs of response to the epidemic whether as guiding of prevention campaigns to specific risk groups.

Telomerase activation by genomic rearrangements in high-risk neuroblastoma

PubMed Central

Peifer, Martin; Hertwig, Falk; Roels, Frederik; Dreidax, Daniel; Gartlgruber, Moritz; Menon, Roopika; Krämer, Andrea; Roncaioli, Justin L.; Sand, Frederik; Heuckmann, Johannes M.; Ikram, Fakhera; Schmidt, Rene; Ackermann, Sandra; Engesser, Anne; Kahlert, Yvonne; Vogel, Wenzel; Altmüller, Janine; Nürnberg, Peter; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Mariappan, Aruljothi; Heynck, Stefanie; Mariotti, Erika; Henrich, Kai-Oliver; Glöckner, Christian; Bosco, Graziella; Leuschner, Ivo; Schweiger, Michal R.; Savelyeva, Larissa; Watkins, Simon C.; Shao, Chunxuan; Bell, Emma; Höfer, Thomas; Achter, Viktor; Lang, Ulrich; Theissen, Jessica; Volland, Ruth; Saadati, Maral; Eggert, Angelika; de Wilde, Bram; Berthold, Frank; Peng, Zhiyu; Zhao, Chen; Shi, Leming; Ortmann, Monika; Büttner, Reinhard; Perner, Sven; Hero, Barbara; Schramm, Alexander; Schulte, Johannes H.; Herrmann, Carl; O’Sullivan, Roderick J.; Westermann, Frank; Thomas, Roman K.; Fischer, Matthias

2016-01-01

Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system1. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive2–4. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type1,2,5. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours. PMID:26466568

Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway.

PubMed

Hoffmann, N; Steinbüchel, A; Rehm, B H

2000-11-01

Various pseudomonads are capable of the synthesis of polyhydroxyalkanoate (PHA), composed of medium chain length (MCL) 3-hydroxy fatty acids (C6-C14), when grown on simple carbon sources such as, for example, gluconate or acetate. In Pseudomonas putida, the fatty acid de novo synthesis and PHA synthesis are linked by the transacylase PhaG. Southern hybridization experiments with digoxigenin-labeled phaG(Pp) from P. putida and genomic DNA from various pseudomonads indicate that phaG homologues are present in various other pseudomonads. Although P. oleovorans does not accumulate PHA(MCL) from non-related carbon sources, its genomic DNA reveals a strong hybridization signal. We employed PCR to amplify this phaG homologue. The respective PCR product comprising the coding region of phaG(Po) was cloned into pBBR1MCS-2, resulting in plasmid pBHR84. DNA sequencing revealed that putative PhaG(Po) from P. oleovorans exhibited about 95% amino acid sequence identity to PhaG(Pp) from P. putida. Reverse transcriptase-PCR analysis demonstrated that phaG(Po) was not transcribed even tinder inducing conditions, i.e. in the presence of gluconate as carbon source, whereas induction of phaG(Pp) transcription was obtained in P. putida. When octanoate was used as sole carbon source, only low levels of phaG mRNA were detected in P. putida. Plasmid pBHR84 complemented the phaG-negative mutant PhaG(N)-21 from P. putida. Interestingly, reintroduction of phaG(Po) under lac promoter control into the natural host P. oleovorans established PHA(MCL) synthesis from non-related carbon sources in this bacterium. These data indicated that phaG(Po) in P. oleovorans is not functionally expressed and does not exert its original function.

Detection of viral sequences in archival spinal cords from fatal cases of poliomyelitis in 1951-1952.

PubMed

Rekand, Tiina; Male, Rune; Myking, Andreas O; Nygaard, Svein J T; Aarli, Johan A; Haarr, Lars; Langeland, Nina

2003-12-01

Poliovirus (PV) subjected to genetic characterization is often isolated from faecal carriage. Such virus is not necessarily identical to the virus causing paralytic disease since genetic modifications may occur during replication outside the nervous system. We have searched for poliovirus genomes in the 14 fatal cases occurring during the last epidemics in Norway in 1951-1952. A method was developed for isolation and analysis of poliovirus RNA from formalin-fixed and paraffin-embedded archival tissue. RNA was purified by incubation with Chelex-100 and heating followed by treatment with the proteinase K and chloroform extraction. Viral sequences were amplified by a reverse transcriptase-polymerase chain reaction (RT-PCR), the products subjected to TA cloning and sequenced. RNA from the beta-actin gene, as a control, was identified in 13 cases, while sequences specific for poliovirus were achieved in 11 cases. The sequences from the 2C region of poliovirus were rather conserved while those in the 5'-untranslated region were variable. The developed method should be suitable also for other genetic studies of old archival material.

Structure and expression of 12-oxophytodienoate reductase (subgroup I) genes in pea, and characterization of the oxidoreductase activities of their recombinant products.

PubMed

Matsui, H; Nakamura, G; Ishiga, Y; Toshima, H; Inagaki, Y; Toyoda, K; Shiraishi, T; Ichinose, Y

2004-02-01

Recently, we observed that expression of a pea gene (S64) encoding an oxophytodienoic acid reductase (OPR) was induced by a suppressor of pea defense responses, secreted by the pea pathogen Mycosphaerella pinodes. Because it is known that OPRs are usually encoded by families of homologous genes, we screened for genomic and cDNA clones encoding members of this putative OPR family in pea. We isolated five members of the OPR gene family from a pea genomic DNA library, and amplified six cDNA clones, including S64, by RT-PCR (reverse transcriptase-PCR). Sequencing analysis revealed that S64 corresponds to PsOPR2, and the amino acid sequences of the predicted products of the six OPR-like genes shared more than 80% identity with each other. Based on their sequence similarity, all these OPR-like genes code for OPRs of subgroup I, i.e., enzymes which are not required for jasmonic acid biosynthesis. However, the genes varied in their exon/intron organization and in their promoter sequences. To investigate the expression of each individual OPR-like gene, RT-PCR was performed using gene-specific primers. The results indicated that the OPR-like gene most strongly induced by the inoculation of pea plants with a compatible pathogen and by treatment with the suppressor from M. pinodes was PsOPR2. Furthermore, the ability of the six recombinant OPR-like proteins to reduce a model substrate, 2-cyclohexen-1-one (2-CyHE), was investigated. The results indicated that PsOPR1, 4 and 6 display robust activity, and PsOPR2 has a most remarkable ability to reduce 2-CyHE, whereas PsOPR3 has little and PsOPR5 does not reduce this compound. Thus, the six OPR-like proteins can be classified into four types. Interestingly, the gene structures, expression profiles, and enzymatic activities used to classify each member of the pea OPR-like gene family are clearly correlated, indicating that each member of this OPR-like family has a distinct function.

Is a Genome a Codeword of an Error-Correcting Code?

PubMed Central

Kleinschmidt, João H.; Silva-Filho, Márcio C.; Bim, Edson; Herai, Roberto H.; Yamagishi, Michel E. B.; Palazzo, Reginaldo

2012-01-01

Since a genome is a discrete sequence, the elements of which belong to a set of four letters, the question as to whether or not there is an error-correcting code underlying DNA sequences is unavoidable. The most common approach to answering this question is to propose a methodology to verify the existence of such a code. However, none of the methodologies proposed so far, although quite clever, has achieved that goal. In a recent work, we showed that DNA sequences can be identified as codewords in a class of cyclic error-correcting codes known as Hamming codes. In this paper, we show that a complete intron-exon gene, and even a plasmid genome, can be identified as a Hamming code codeword as well. Although this does not constitute a definitive proof that there is an error-correcting code underlying DNA sequences, it is the first evidence in this direction. PMID:22649495

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

PubMed Central

Gibson, Richard M.; Meyer, Ashley M.; Winner, Dane; Archer, John; Feyertag, Felix; Ruiz-Mateos, Ezequiel; Leal, Manuel; Robertson, David L.; Schmotzer, Christine L.

2014-01-01

With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new antiretroviral drugs and, more importantly, will aid in the treatment and management of HIV-infected individuals. PMID:24468782

Pattern recognition of electronic bit-sequences using a semiconductor mode-locked laser and spatial light modulators

NASA Astrophysics Data System (ADS)

Bhooplapur, Sharad; Akbulut, Mehmetkan; Quinlan, Franklyn; Delfyett, Peter J.

2010-04-01

A novel scheme for recognition of electronic bit-sequences is demonstrated. Two electronic bit-sequences that are to be compared are each mapped to a unique code from a set of Walsh-Hadamard codes. The codes are then encoded in parallel on the spectral phase of the frequency comb lines from a frequency-stabilized mode-locked semiconductor laser. Phase encoding is achieved by using two independent spatial light modulators based on liquid crystal arrays. Encoded pulses are compared using interferometric pulse detection and differential balanced photodetection. Orthogonal codes eight bits long are compared, and matched codes are successfully distinguished from mismatched codes with very low error rates, of around 10-18. This technique has potential for high-speed, high accuracy recognition of bit-sequences, with applications in keyword searches and internet protocol packet routing.

Two Perspectives on the Origin of the Standard Genetic Code

NASA Astrophysics Data System (ADS)

Sengupta, Supratim; Aggarwal, Neha; Bandhu, Ashutosh Vishwa

2014-12-01

The origin of a genetic code made it possible to create ordered sequences of amino acids. In this article we provide two perspectives on code origin by carrying out simulations of code-sequence coevolution in finite populations with the aim of examining how the standard genetic code may have evolved from more primitive code(s) encoding a small number of amino acids. We determine the efficacy of the physico-chemical hypothesis of code origin in the absence and presence of horizontal gene transfer (HGT) by allowing a diverse collection of code-sequence sets to compete with each other. We find that in the absence of horizontal gene transfer, natural selection between competing codes distinguished by differences in the degree of physico-chemical optimization is unable to explain the structure of the standard genetic code. However, for certain probabilities of the horizontal transfer events, a universal code emerges having a structure that is consistent with the standard genetic code.

An algebraic hypothesis about the primeval genetic code architecture.

PubMed

Sánchez, Robersy; Grau, Ricardo

2009-09-01

A plausible architecture of an ancient genetic code is derived from an extended base triplet vector space over the Galois field of the extended base alphabet {D,A,C,G,U}, where symbol D represents one or more hypothetical bases with unspecific pairings. We hypothesized that the high degeneration of a primeval genetic code with five bases and the gradual origin and improvement of a primeval DNA repair system could make possible the transition from ancient to modern genetic codes. Our results suggest that the Watson-Crick base pairing G identical with C and A=U and the non-specific base pairing of the hypothetical ancestral base D used to define the sum and product operations are enough features to determine the coding constraints of the primeval and the modern genetic code, as well as, the transition from the former to the latter. Geometrical and algebraic properties of this vector space reveal that the present codon assignment of the standard genetic code could be induced from a primeval codon assignment. Besides, the Fourier spectrum of the extended DNA genome sequences derived from the multiple sequence alignment suggests that the called period-3 property of the present coding DNA sequences could also exist in the ancient coding DNA sequences. The phylogenetic analyses achieved with metrics defined in the N-dimensional vector space (B(3))(N) of DNA sequences and with the new evolutionary model presented here also suggest that an ancient DNA coding sequence with five or more bases does not contradict the expected evolutionary history.

Application of Quaternion in improving the quality of global sequence alignment scores for an ambiguous sequence target in Streptococcus pneumoniae DNA

NASA Astrophysics Data System (ADS)

Lestari, D.; Bustamam, A.; Novianti, T.; Ardaneswari, G.

2017-07-01

DNA sequence can be defined as a succession of letters, representing the order of nucleotides within DNA, using a permutation of four DNA base codes including adenine (A), guanine (G), cytosine (C), and thymine (T). The precise code of the sequences is determined using DNA sequencing methods and technologies, which have been developed since the 1970s and currently become highly developed, advanced and highly throughput sequencing technologies. So far, DNA sequencing has greatly accelerated biological and medical research and discovery. However, in some cases DNA sequencing could produce any ambiguous and not clear enough sequencing results that make them quite difficult to be determined whether these codes are A, T, G, or C. To solve these problems, in this study we can introduce other representation of DNA codes namely Quaternion Q = (PA, PT, PG, PC), where PA, PT, PG, PC are the probability of A, T, G, C bases that could appear in Q and PA + PT + PG + PC = 1. Furthermore, using Quaternion representations we are able to construct the improved scoring matrix for global sequence alignment processes, by applying a dot product method. Moreover, this scoring matrix produces better and higher quality of the match and mismatch score between two DNA base codes. In implementation, we applied the Needleman-Wunsch global sequence alignment algorithm using Octave, to analyze our target sequence which contains some ambiguous sequence data. The subject sequences are the DNA sequences of Streptococcus pneumoniae families obtained from the Genebank, meanwhile the target DNA sequence are received from our collaborator database. As the results we found the Quaternion representations improve the quality of the sequence alignment score and we can conclude that DNA sequence target has maximum similarity with Streptococcus pneumoniae.

Emergence of resistance mutations in simian immunodeficiency virus (SIV)-infected rhesus macaques receiving non-suppressive antiretroviral therapy (ART)

DOE PAGES

Policicchio, Benjamin Bruno; Sette, Paola; Xu, Cuiling; ...

2018-02-21

Two SIVmac251-infected rhesus macaques received tenofovir/emtricitabine with raltegravir intensification. Viral rebound occurred during treatment and sequencing of reverse transcriptase and integrase genes identified multiple resistance mutations. Similar to HIV infection, antiretroviral-resistance mutations may occur in SIV-infected nonhuman primates receiving nonsuppressive ART. As ART administration to nonhuman primates is currently dramatically expanding, fueled by both cure research and the study of HIV-related comorbidities, viral resistance should be factored in the study design and data interpretation

Ostertagia circumcincta: isolation of a partial cDNA encoding an unusual member of the mitochondrial processing peptidase subfamily of M16 metallopeptidases.

PubMed

Walker, J; Tait, A

1997-11-01

A reverse-transcriptase polymerase chain reaction (PCR) procedure was used to isolate an Ostertagia circumcincta partial cDNA encoding a protein with general primary sequence features characteristic of members of the mitochondrial processing peptidase (MPP) subfamily of M16 metallopeptidases. The structural relationships of the predicted protein (Oc MPPX) with MPP subfamily proteins from other species (including the model free-living nematode Caenorhabditis elegans) were examined, and Northern analysis confirmed the expression of the Oc mppx gene in adult nematodes.

fbpABC gene cluster in Neisseria meningitidis is transcribed as an operon.

PubMed

Khun, H H; Deved, V; Wong, H; Lee, B C

2000-12-01

The neisserial fbpABC locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with Neisseria meningitidis. The presence of fbpAB and fbpBC transcripts obtained by priming cDNA synthesis with an fbpC-sequence-specific oligonucleotide indicates that fbpABC is organized as a single expression unit. The ratio of fbpA to fbpABC mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR.

fbpABC Gene Cluster in Neisseria meningitidis Is Transcribed as an Operon

PubMed Central

Khun, Heng H.; Deved, Vinay; Wong, Howard; Lee, B. Craig

2000-01-01

The neisserial fbpABC locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with Neisseria meningitidis. The presence of fbpAB and fbpBC transcripts obtained by priming cDNA synthesis with an fbpC-sequence-specific oligonucleotide indicates that fbpABC is organized as a single expression unit. The ratio of fbpA to fbpABC mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR. PMID:11083849

Emergence of resistance mutations in simian immunodeficiency virus (SIV)-infected rhesus macaques receiving non-suppressive antiretroviral therapy (ART)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Policicchio, Benjamin Bruno; Sette, Paola; Xu, Cuiling

Two SIVmac251-infected rhesus macaques received tenofovir/emtricitabine with raltegravir intensification. Viral rebound occurred during treatment and sequencing of reverse transcriptase and integrase genes identified multiple resistance mutations. Similar to HIV infection, antiretroviral-resistance mutations may occur in SIV-infected nonhuman primates receiving nonsuppressive ART. As ART administration to nonhuman primates is currently dramatically expanding, fueled by both cure research and the study of HIV-related comorbidities, viral resistance should be factored in the study design and data interpretation

RNA editing: trypanosomes rewrite the genetic code.

PubMed

Stuart, K

1998-01-01

The understanding of how genetic information is stored and expressed has advanced considerably since the "central dogma" asserted that genetic information flows from the nucleotide sequence of DNA to that of messenger RNA (mRNA) which in turn specifies the amino acid sequence of a protein. It was found that genetic information can be stored as RNA (e.g. in RNA viruses) and can flow from RNA to DNA by reverse transcriptase enzyme activity. In addition, some genes contain introns, nucleotide sequences that are removed from their RNA (by RNA splicing) and thus are not represented in the resultant protein. Furthermore, alternative splicing was found to produce variant proteins from a single gene. More recently, the study of trypanosome parasites revealed an unexpected and indeed counter-intuitive genetic complexity. Genetic information for a single protein can be dispersed among several (DNA) genes in these organisms. One of these genes specifies an encrypted precursor mRNA that is converted to a functional mRNA by a process called RNA editing that inserts and deletes uridylate nucleotides. The sequence of the edited mRNA is specified by multiple small RNAs, named guide RNAs, (gRNAs) each of which is encoded in a separate gene. Thus, edited mRNA sequences are assembled from multiple genes by the transfer of information from one type of RNA to another. The existence of editing was surprising but has stimulated the discovery of other types of RNA editing. The Stuart laboratory has been exploring RNA editing in trypanosomes from the time of its discovery. They found dramatic differences between the mitochondrial gene sequences and those of the corresponding mRNAs, which indicated editing by the insertion and deletion of uridylates. Some editing was modest; simply eliminating shifts in sequence register of minimally extending the protein coding sequence. However, editing of many mRNAs was startingly extensive. The RNA sequence was essentially entirely remodeled with its sequence more the result of editing than the gene sequence. The identities of genes for such extensively edited RNA were not recognizable from the DNA sequence but they were readily identifiable from the edited mRNA sequence. Thus, despite the complex and extensive editing the resultant mRNA sequence is precise. Characterization of partially edited RNAs indicated that editing proceeds in the direction opposite to that used to specify the protein which reflects the use of the gRNAs. The numerous gRNAs that are used for editing are encoded in the DNA molecules whose role was previously a mystery. Using information gained in our earlier studies, the Stuart group developed an in vitro system that reproduces the fundamental process of editing in order to resolve the mechanism by which it occurs. They determined that editing entails a series of enzymatic steps rather than the mechanism used in RNA splicing. They also showed that chimeric gRNA-mRNA molecules are aberrant by-products of editing rather than intermediates in the process as had been proposed. Additional studies are exploring precisely how the number of added and deleted uridylates is specified by the gRNA. The Stuart laboratory showed that editing is performed by an aggregation of enzymes that catalyze the separate steps of editing. It also developed a method to purify this multimolecule complex that contains several, perhaps tens of, proteins. This will allow the study of its composition and the functions of its component parts. Indeed, the gene for one component has been identified and its detailed characterization begun. These studies are developing tools to explore related processes. An early finding in the lab was that the various mRNAs are differentially edited during the life cycle of the parasite. The pattern of this editing indicates that editing serves to regulate the alternation between two modes of energy generation. This regulation is coordinated with other events that are occurring during the life c

Circular codes revisited: a statistical approach.

PubMed

Gonzalez, D L; Giannerini, S; Rosa, R

2011-04-21

In 1996 Arquès and Michel [1996. A complementary circular code in the protein coding genes. J. Theor. Biol. 182, 45-58] discovered the existence of a common circular code in eukaryote and prokaryote genomes. Since then, circular code theory has provoked great interest and underwent a rapid development. In this paper we discuss some theoretical issues related to the synchronization properties of coding sequences and circular codes with particular emphasis on the problem of retrieval and maintenance of the reading frame. Motivated by the theoretical discussion, we adopt a rigorous statistical approach in order to try to answer different questions. First, we investigate the covering capability of the whole class of 216 self-complementary, C(3) maximal codes with respect to a large set of coding sequences. The results indicate that, on average, the code proposed by Arquès and Michel has the best covering capability but, still, there exists a great variability among sequences. Second, we focus on such code and explore the role played by the proportion of the bases by means of a hierarchy of permutation tests. The results show the existence of a sort of optimization mechanism such that coding sequences are tailored as to maximize or minimize the coverage of circular codes on specific reading frames. Such optimization clearly relates the function of circular codes with reading frame synchronization. Copyright © 2011 Elsevier Ltd. All rights reserved.

Automated conserved non-coding sequence (CNS) discovery reveals differences in gene content and promoter evolution among grasses

PubMed Central

Turco, Gina; Schnable, James C.; Pedersen, Brent; Freeling, Michael

2013-01-01

Conserved non-coding sequences (CNS) are islands of non-coding sequence that, like protein coding exons, show less divergence in sequence between related species than functionless DNA. Several CNSs have been demonstrated experimentally to function as cis-regulatory regions. However, the specific functions of most CNSs remain unknown. Previous searches for CNS in plants have either anchored on exons and only identified nearby sequences or required years of painstaking manual annotation. Here we present an open source tool that can accurately identify CNSs between any two related species with sequenced genomes, including both those immediately adjacent to exons and distal sequences separated by >12 kb of non-coding sequence. We have used this tool to characterize new motifs, associate CNSs with additional functions, and identify previously undetected genes encoding RNA and protein in the genomes of five grass species. We provide a list of 15,363 orthologous CNSs conserved across all grasses tested. We were also able to identify regulatory sequences present in the common ancestor of grasses that have been lost in one or more extant grass lineages. Lists of orthologous gene pairs and associated CNSs are provided for reference inbred lines of arabidopsis, Japonica rice, foxtail millet, sorghum, brachypodium, and maize. PMID:23874343

Insights into the strategies used by related group II introns to adapt successfully for the colonisation of a bacterial genome

PubMed Central

Martínez-Rodríguez, Laura; García-Rodríguez, Fernando M; Molina-Sánchez, María Dolores; Toro, Nicolás; Martínez-Abarca, Francisco

2014-01-01

Group II introns are self-splicing RNAs and site-specific mobile retroelements found in bacterial and organellar genomes. The group II intron RmInt1 is present at high copy number in Sinorhizobium meliloti species, and has a multifunctional intron-encoded protein (IEP) with reverse transcriptase/maturase activities, but lacking the DNA-binding and endonuclease domains. We characterized two RmInt1-related group II introns RmInt2 from S. meliloti strain GR4 and Sr.md.I1 from S. medicae strain WSM419 in terms of splicing and mobility activities. We used both wild-type and engineered intron-donor constructs based on ribozyme ΔORF-coding sequence derivatives, and we determined the DNA target requirements for RmInt2, the element most distantly related to RmInt1. The excision and mobility patterns of intron-donor constructs expressing different combinations of IEP and intron RNA provided experimental evidence for the co-operation of IEPs and intron RNAs from related elements in intron splicing and, in some cases, in intron homing. We were also able to identify the DNA target regions recognized by these IEPs lacking the DNA endonuclease domain. Our results provide new insight into the versatility of related group II introns and the possible co-operation between these elements to facilitate the colonization of bacterial genomes. PMID:25482895

Insights into the strategies used by related group II introns to adapt successfully for the colonisation of a bacterial genome.

PubMed

Martínez-Rodríguez, Laura; García-Rodríguez, Fernando M; Molina-Sánchez, María Dolores; Toro, Nicolás; Martínez-Abarca, Francisco

2014-01-01

Group II introns are self-splicing RNAs and site-specific mobile retroelements found in bacterial and organellar genomes. The group II intron RmInt1 is present at high copy number in Sinorhizobium meliloti species, and has a multifunctional intron-encoded protein (IEP) with reverse transcriptase/maturase activities, but lacking the DNA-binding and endonuclease domains. We characterized two RmInt1-related group II introns RmInt2 from S. meliloti strain GR4 and Sr.md.I1 from S. medicae strain WSM419 in terms of splicing and mobility activities. We used both wild-type and engineered intron-donor constructs based on ribozyme ΔORF-coding sequence derivatives, and we determined the DNA target requirements for RmInt2, the element most distantly related to RmInt1. The excision and mobility patterns of intron-donor constructs expressing different combinations of IEP and intron RNA provided experimental evidence for the co-operation of IEPs and intron RNAs from related elements in intron splicing and, in some cases, in intron homing. We were also able to identify the DNA target regions recognized by these IEPs lacking the DNA endonuclease domain. Our results provide new insight into the versatility of related group II introns and the possible co-operation between these elements to facilitate the colonization of bacterial genomes.

Protease inhibitors as potential therapeutic agents for AIDS.

PubMed

Jamjoom, G A

1991-09-01

A decade since the epidemic of the acquired immunodeficiency syndrome (AIDS) was first recognized, a wealth of information has accumulated on the molecular biology of the causative agents, the human immunodeficiency viruses (HIV). Of particular interest is knowledge of the viral enzymes involved in the formation of new virus particles. Such enzymes constitute attractive targets for efforts aimed at selecting agents that interfere with virus multiplication and subsequent spread and pathogenesis. Already, several agents that inhibit the viral reverse transcriptase (e.g., nucleoside analogs such as Zidovudine) have proved to have a beneficial effect on the course off the disease, but their prolonged use has been associated with significant toxicity and the emergence of resistant mutants. A second enzyme that has recently attracted attention is the virus-coded protease. This enzyme is involved in the cleavage of viral precursor polyproteins into the final products that constitute the mature virus particle. Protease inhibitors interfere with the process of virus maturation which is required for the formation of infective virus particles. Several custom-made inhibitors with a high selective action against HIV protease have been produced recently. They are nonhydrolyzable peptide analogs that mimic the cleavage sequences of the natural substrate of the enzyme during the transition state of the cleavage reaction. It is hoped that a similar selectivity in vivo may make protease inhibitors a promising new category of AIDS therapeutics.

A noncoding melanophilin gene (MLPH) SNP at the splice donor of exon 1 represents a candidate causal mutation for coat color dilution in dogs.

PubMed

Drögemüller, Cord; Philipp, Ute; Haase, Bianca; Günzel-Apel, Anne-Rose; Leeb, Tosso

2007-01-01

Coat color dilution in several breeds of dog is characterized by a specific pigmentation phenotype and sometimes accompanied by hair loss and recurrent skin inflammation, the so-called color dilution alopecia or black hair follicular dysplasia. Coat color dilution (d) is inherited as a Mendelian autosomal recessive trait. In a previous study, MLPH polymorphisms showed perfect cosegregation with the dilute phenotype within breeds. However, different dilute haplotypes were found in different breeds, and no single polymorphism was identified in the coding sequence that was likely to be causative for the dilute phenotype. We resequenced the 5'-region of the canine MLPH gene and identified a strong candidate single nucleotide polymorphism within the nontranslated exon 1, which showed perfect association to the dilute phenotype in 65 dilute dogs from 7 different breeds. The A/G polymorphism is located at the last nucleotide of exon 1 and the mutant A-allele is predicted to reduce splicing efficiency 8-fold. An MLPH mRNA expression study using quantitative reverse transcriptase-polymerase chain reaction confirmed that dd animals had only about approximately 25% of the MLPH transcript compared with DD animals. These results provide preliminary evidence that the reported regulatory MLPH mutation might represent a causal mutation for coat color dilution in dogs.

F4-related mutation and expression analysis of the aminopeptidase N gene in pigs.

PubMed

Goetstouwers, T; Van Poucke, M; Nguyen, V U; Melkebeek, V; Coddens, A; Deforce, D; Cox, E; Peelman, L J

2014-05-01

Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3' untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT-) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R-) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R- pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R- pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.

Second-generation sequencing of entire mitochondrial coding-regions (∼15.4 kb) holds promise for study of the phylogeny and taxonomy of human body lice and head lice.

PubMed

Xiong, H; Campelo, D; Pollack, R J; Raoult, D; Shao, R; Alem, M; Ali, J; Bilcha, K; Barker, S C

2014-08-01

The Illumina Hiseq platform was used to sequence the entire mitochondrial coding-regions of 20 body lice, Pediculus humanus Linnaeus, and head lice, P. capitis De Geer (Phthiraptera: Pediculidae), from eight towns and cities in five countries: Ethiopia, France, China, Australia and the U.S.A. These data (∼310 kb) were used to see how much more informative entire mitochondrial coding-region sequences were than partial mitochondrial coding-region sequences, and thus to guide the design of future studies of the phylogeny, origin, evolution and taxonomy of body lice and head lice. Phylogenies were compared from entire coding-region sequences (∼15.4 kb), entire cox1 (∼1.5 kb), partial cox1 (∼700 bp) and partial cytb (∼600 bp) sequences. On the one hand, phylogenies from entire mitochondrial coding-region sequences (∼15.4 kb) were much more informative than phylogenies from entire cox1 sequences (∼1.5 kb) and partial gene sequences (∼600 to ∼700 bp). For example, 19 branches had > 95% bootstrap support in our maximum likelihood tree from the entire mitochondrial coding-regions (∼15.4 kb) whereas the tree from 700 bp cox1 had only two branches with bootstrap support > 95%. Yet, by contrast, partial cytb (∼600 bp) and partial cox1 (∼486 bp) sequences were sufficient to genotype lice to Clade A, B or C. The sequences of the mitochondrial genomes of the P. humanus, P. capitis and P. schaeffi Fahrenholz studied are in NCBI GenBank under the accession numbers KC660761-800, KC685631-6330, KC241882-97, EU219988-95, HM241895-8 and JX080388-407. © 2014 The Royal Entomological Society.

Effective Identification of Similar Patients Through Sequential Matching over ICD Code Embedding.

PubMed

Nguyen, Dang; Luo, Wei; Venkatesh, Svetha; Phung, Dinh

2018-04-11

Evidence-based medicine often involves the identification of patients with similar conditions, which are often captured in ICD (International Classification of Diseases (World Health Organization 2013)) code sequences. With no satisfying prior solutions for matching ICD-10 code sequences, this paper presents a method which effectively captures the clinical similarity among routine patients who have multiple comorbidities and complex care needs. Our method leverages the recent progress in representation learning of individual ICD-10 codes, and it explicitly uses the sequential order of codes for matching. Empirical evaluation on a state-wide cancer data collection shows that our proposed method achieves significantly higher matching performance compared with state-of-the-art methods ignoring the sequential order. Our method better identifies similar patients in a number of clinical outcomes including readmission and mortality outlook. Although this paper focuses on ICD-10 diagnosis code sequences, our method can be adapted to work with other codified sequence data.

Representation of DNA sequences with virtual potentials and their processing by (SEQREP) Kohonen self-organizing maps.

PubMed

Aires-de-Sousa, João; Aires-de-Sousa, Luisa

2003-01-01

We propose representing individual positions in DNA sequences by virtual potentials generated by other bases of the same sequence. This is a compact representation of the neighbourhood of a base. The distribution of the virtual potentials over the whole sequence can be used as a representation of the entire sequence (SEQREP code). It is a flexible code, with a length independent of the sequence size, does not require previous alignment, and is convenient for processing by neural networks or statistical techniques. To evaluate its biological significance, the SEQREP code was used for training Kohonen self-organizing maps (SOMs) in two applications: (a) detection of Alu sequences, and (b) classification of sequences encoding for HIV-1 envelope glycoprotein (env) into subtypes A-G. It was demonstrated that SOMs clustered sequences belonging to different classes into distinct regions. For independent test sets, very high rates of correct predictions were obtained (97% in the first application, 91% in the second). Possible areas of application of SEQREP codes include functional genomics, phylogenetic analysis, detection of repetitions, database retrieval, and automatic alignment. Software for representing sequences by SEQREP code, and for training Kohonen SOMs is made freely available from http://www.dq.fct.unl.pt/qoa/jas/seqrep. Supplementary material is available at http://www.dq.fct.unl.pt/qoa/jas/seqrep/bioinf2002

CombAlign: a code for generating a one-to-many sequence alignment from a set of pairwise structure-based sequence alignments.

PubMed

Zhou, Carol L Ecale

2015-01-01

In order to better define regions of similarity among related protein structures, it is useful to identify the residue-residue correspondences among proteins. Few codes exist for constructing a one-to-many multiple sequence alignment derived from a set of structure or sequence alignments, and a need was evident for creating such a tool for combining pairwise structure alignments that would allow for insertion of gaps in the reference structure. This report describes a new Python code, CombAlign, which takes as input a set of pairwise sequence alignments (which may be structure based) and generates a one-to-many, gapped, multiple structure- or sequence-based sequence alignment (MSSA). The use and utility of CombAlign was demonstrated by generating gapped MSSAs using sets of pairwise structure-based sequence alignments between structure models of the matrix protein (VP40) and pre-small/secreted glycoprotein (sGP) of Reston Ebolavirus and the corresponding proteins of several other filoviruses. The gapped MSSAs revealed structure-based residue-residue correspondences, which enabled identification of structurally similar versus differing regions in the Reston proteins compared to each of the other corresponding proteins. CombAlign is a new Python code that generates a one-to-many, gapped, multiple structure- or sequence-based sequence alignment (MSSA) given a set of pairwise sequence alignments (which may be structure based). CombAlign has utility in assisting the user in distinguishing structurally conserved versus divergent regions on a reference protein structure relative to other closely related proteins. CombAlign was developed in Python 2.6, and the source code is available for download from the GitHub code repository.

Short Communication Phylogenetic Characterization of HIV Type 1 CRF01_AE V3 Envelope Sequences in Pregnant Women in Northern Vietnam

PubMed Central

Caridha, Rozina; Ha, Tran Thi Thanh; Gaseitsiwe, Simani; Hung, Pham Viet; Anh, Nguyen Mai; Bao, Nguyen Huy; Khang, Dinh Duy; Hien, Nguyen Tran; Cam, Phung Dac; Chiodi, Francesca

2012-01-01

Abstract Characterization of HIV-1 strains is important for surveillance of the HIV-1 epidemic. In Vietnam HIV-1-infected pregnant women often fail to receive the care they are entitled to. Here, we analyzed phylogenetically HIV-1 env sequences from 37 HIV-1-infected pregnant women from Ha Noi (n=22) and Hai Phong (n=15), where they delivered in 2005–2007. All carried CRF01_AE in the gp120 V3 region. In 21 women CRF01_AE was also found in the reverse transcriptase gene. We compared their env gp120 V3 sequences phylogenetically in a maximum likelihood tree to those of 198 other CRF01_AE sequences in Vietnam and 229 from neighboring countries, predominantly Thailand, from the HIV-1 database. Altogether 464 sequences were analyzed. All but one of the maternal sequences colocalized with sequences from northern Vietnam. The maternal sequences had evolved the least when compared to sequences collected in Ha Noi in 2002, as shown by analysis of synonymous and nonsynonymous changes, than to other Vietnamese sequences collected earlier and/or elsewhere. Since the HIV-1 epidemic in women in Vietnam may still be underestimated, characterization of HIV-1 in pregnant women is important to observe how HIV-1 has evolved and follow its molecular epidemiology. PMID:21936713

Complete Coding Genome Sequence for Mogiana Tick Virus, a Jingmenvirus Isolated from Ticks in Brazil

DTIC Science & Technology

2017-05-04

and capable of infecting a wide range of animal hosts (1–5). Here, we report the complete coding genome sequence (i.e., only missing portions of...segmented nature of the genome was not under- stood. Therefore, only the two genome segments with detectable sequence homolo- gies to flaviviruses were...originally reported (2). We revisited the data set of Maruyama et al. (2) and assembled the complete coding sequences for all four genome segments. We

Quantized phase coding and connected region labeling for absolute phase retrieval.

PubMed

Chen, Xiangcheng; Wang, Yuwei; Wang, Yajun; Ma, Mengchao; Zeng, Chunnian

2016-12-12

This paper proposes an absolute phase retrieval method for complex object measurement based on quantized phase-coding and connected region labeling. A specific code sequence is embedded into quantized phase of three coded fringes. Connected regions of different codes are labeled and assigned with 3-digit-codes combining the current period and its neighbors. Wrapped phase, more than 36 periods, can be restored with reference to the code sequence. Experimental results verify the capability of the proposed method to measure multiple isolated objects.

Functional interrogation of non-coding DNA through CRISPR genome editing

PubMed Central

Canver, Matthew C.; Bauer, Daniel E.; Orkin, Stuart H.

2017-01-01

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. PMID:28288828

Memorization of Sequences of Movements of the Right or the Left Hand by Right- and Left-Handers: Vector Coding.

PubMed

Bobrova, E V; Bogacheva, I N; Lyakhovetskii, V A; Fabinskaja, A A; Fomina, E V

2017-01-01

In order to test the hypothesis of hemisphere specialization for different types of information coding (the right hemisphere, for positional coding; the left one, for vector coding), we analyzed the errors of right and left-handers during a task involving the memorization of sequences of movements by the left or the right hand, which activates vector coding by changing the order of movements in memorized sequences. The task was first performed by the right or the left hand, then by the opposite hand. It was found that both'right- and left-handers use the information about the previous movements of the dominant hand, but not of the non-dom" inant one. After changing the hand, right-handers use the information about previous movements of the second hand, while left-handers do not. We compared our results with the data of previous experiments, in which positional coding was activated, and concluded that both right- and left-handers use vector coding for memorizing the sequences of their dominant hands and positional coding for memorizing the sequences of non-dominant hand. No similar patterns of errors were found between right- and left-handers after changing the hand, which suggests that in right- and left-handersthe skills are transferred in different ways depending on the type of coding.

CSTminer: a web tool for the identification of coding and noncoding conserved sequence tags through cross-species genome comparison

PubMed Central

Castrignanò, Tiziana; Canali, Alessandro; Grillo, Giorgio; Liuni, Sabino; Mignone, Flavio; Pesole, Graziano

2004-01-01

The identification and characterization of genome tracts that are highly conserved across species during evolution may contribute significantly to the functional annotation of whole-genome sequences. Indeed, such sequences are likely to correspond to known or unknown coding exons or regulatory motifs. Here, we present a web server implementing a previously developed algorithm that, by comparing user-submitted genome sequences, is able to identify statistically significant conserved blocks and assess their coding or noncoding nature through the measure of a coding potential score. The web tool, available at http://www.caspur.it/CSTminer/, is dynamically interconnected with the Ensembl genome resources and produces a graphical output showing a map of detected conserved sequences and annotated gene features. PMID:15215464

Primer development to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus.

PubMed

Agustiningsih, Agustiningsih; Trimarsanto, Hidayat; Setiawaty, Vivi; Artika, I Made; Muljono, David Handojo

2016-08-30

Influenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009. An optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested. The developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.

GATA: A graphic alignment tool for comparative sequenceanalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nix, David A.; Eisen, Michael B.

2005-01-01

Several problems exist with current methods used to align DNA sequences for comparative sequence analysis. Most dynamic programming algorithms assume that conserved sequence elements are collinear. This assumption appears valid when comparing orthologous protein coding sequences. Functional constraints on proteins provide strong selective pressure against sequence inversions, and minimize sequence duplications and feature shuffling. For non-coding sequences this collinearity assumption is often invalid. For example, enhancers contain clusters of transcription factor binding sites that change in number, orientation, and spacing during evolution yet the enhancer retains its activity. Dotplot analysis is often used to estimate non-coding sequence relatedness. Yet dotmore » plots do not actually align sequences and thus cannot account well for base insertions or deletions. Moreover, they lack an adequate statistical framework for comparing sequence relatedness and are limited to pairwise comparisons. Lastly, dot plots and dynamic programming text outputs fail to provide an intuitive means for visualizing DNA alignments.« less

Phylogenetic Network for European mtDNA

PubMed Central

Finnilä, Saara; Lehtonen, Mervi S.; Majamaa, Kari

2001-01-01

The sequence in the first hypervariable segment (HVS-I) of the control region has been used as a source of evolutionary information in most phylogenetic analyses of mtDNA. Population genetic inference would benefit from a better understanding of the variation in the mtDNA coding region, but, thus far, complete mtDNA sequences have been rare. We determined the nucleotide sequence in the coding region of mtDNA from 121 Finns, by conformation-sensitive gel electrophoresis and subsequent sequencing and by direct sequencing of the D loop. Furthermore, 71 sequences from our previous reports were included, so that the samples represented all the mtDNA haplogroups present in the Finnish population. We found a total of 297 variable sites in the coding region, which allowed the compilation of unambiguous phylogenetic networks. The D loop harbored 104 variable sites, and, in most cases, these could be localized within the coding-region networks, without discrepancies. Interestingly, many homoplasies were detected in the coding region. Nucleotide variation in the rRNA and tRNA genes was 6%, and that in the third nucleotide positions of structural genes amounted to 22% of that in the HVS-I. The complete networks enabled the relationships between the mtDNA haplogroups to be analyzed. Phylogenetic networks based on the entire coding-region sequence in mtDNA provide a rich source for further population genetic studies, and complete sequences make it easier to differentiate between disease-causing mutations and rare polymorphisms. PMID:11349229

Polymorphisms and resistance mutations in the protease and reverse transcriptase genes of HIV-1 F subtype Romanian strains.

PubMed

Paraschiv, Simona; Otelea, Dan; Dinu, Magdalena; Maxim, Daniela; Tinischi, Mihaela

2007-03-01

To evaluate the prevalence of resistance mutations in the genome of HIV-1 F subtype strains isolated from Romanian antiretroviral (ARV) treatment-naïve patients and to assess the phylogenetic relatedness of these strains with other HIV-1 strains. Twenty-nine HIV-1 strains isolated from treatment-naïve adolescents (n=15) and adults (n=14) were included in this study. Resistance genotyping was performed by using Big Dye Terminator chemistry provided by the ViroSeq Genotyping System. The sequences of the protease and reverse transcriptase genes were aligned (ClustalW) and a phylogenetic tree was built (MEGA 3 software). For subtyping purposes, all the nucleotide sequences were submitted to the Stanford database. All the studied strains were found to harbor accessory mutations in the protease gene. The most frequent mutation was M36I (29 of 29 strains), followed by L63T, K20R, and L10V. The number of polymorphisms associated with protease inhibitor resistance was different for the two age groups. Intraphylogenetic divergence was greater for adults than for adolescents infected in childhood. All the strains were found to belong to the F1 subtype. The phylogenetic analysis revealed that Romanian strains clustered together, but distinctly from F1 HIV-1 strains isolated in other parts of the world (Brazil, Finland, and Belgium). Protease secondary mutations are present with high frequency in the HIV-1 F subtype strains isolated from Romanian ARV treatment-naïve patients, but no major resistance mutations were found.

Mutations Related to Antiretroviral Resistance Identified by Ultra-Deep Sequencing in HIV-1 Infected Children under Structured Interruptions of HAART

PubMed Central

Vazquez-Guillen, Jose Manuel; Palacios-Saucedo, Gerardo C.; Rivera-Morales, Lydia G.; Garcia-Campos, Jorge; Ortiz-Lopez, Rocio; Noguera-Julian, Marc; Paredes, Roger; Vielma-Ramirez, Herlinda J.; Ramirez, Teresa J.; Chavez-Garcia, Marcelino; Lopez-Guillen, Paulo; Briones-Lara, Evangelina; Sanchez-Sanchez, Luz M.; Vazquez-Martinez, Carlos A.; Rodriguez-Padilla, Cristina

2016-01-01

Although Structured Treatment Interruptions (STI) are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM’s) at levels under limit of detection of conventional genotyping (<20% of quasispecies) could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM’s in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM’s during STI. PMID:26807922

RY-Coding and Non-Homogeneous Models Can Ameliorate the Maximum-Likelihood Inferences From Nucleotide Sequence Data with Parallel Compositional Heterogeneity.

PubMed

Ishikawa, Sohta A; Inagaki, Yuji; Hashimoto, Tetsuo

2012-01-01

In phylogenetic analyses of nucleotide sequences, 'homogeneous' substitution models, which assume the stationarity of base composition across a tree, are widely used, albeit individual sequences may bear distinctive base frequencies. In the worst-case scenario, a homogeneous model-based analysis can yield an artifactual union of two distantly related sequences that achieved similar base frequencies in parallel. Such potential difficulty can be countered by two approaches, 'RY-coding' and 'non-homogeneous' models. The former approach converts four bases into purine and pyrimidine to normalize base frequencies across a tree, while the heterogeneity in base frequency is explicitly incorporated in the latter approach. The two approaches have been applied to real-world sequence data; however, their basic properties have not been fully examined by pioneering simulation studies. Here, we assessed the performances of the maximum-likelihood analyses incorporating RY-coding and a non-homogeneous model (RY-coding and non-homogeneous analyses) on simulated data with parallel convergence to similar base composition. Both RY-coding and non-homogeneous analyses showed superior performances compared with homogeneous model-based analyses. Curiously, the performance of RY-coding analysis appeared to be significantly affected by a setting of the substitution process for sequence simulation relative to that of non-homogeneous analysis. The performance of a non-homogeneous analysis was also validated by analyzing a real-world sequence data set with significant base heterogeneity.

Palindromic repetitive DNA elements with coding potential in Methanocaldococcus jannaschii.

PubMed

Suyama, Mikita; Lathe, Warren C; Bork, Peer

2005-10-10

We have identified 141 novel palindromic repetitive elements in the genome of euryarchaeon Methanocaldococcus jannaschii. The total length of these elements is 14.3kb, which corresponds to 0.9% of the total genomic sequence and 6.3% of all extragenic regions. The elements can be divided into three groups (MJRE1-3) based on the sequence similarity. The low sequence identity within each of the groups suggests rather old origin of these elements in M. jannaschii. Three MJRE2 elements were located within the protein coding regions without disrupting the coding potential of the host genes, indicating that insertion of repeats might be a widespread mechanism to enhance sequence diversity in coding regions.

Cost-Effectiveness of the Third-Agent Class in Treatment-Naive Human Immunodeficiency Virus-Infected Patients in Portugal

PubMed Central

Aragão, Filipa; Vera, José; Vaz Pinto, Inês

2012-01-01

Introduction Current Portuguese HIV treatment guidelines recommend initiating antiretroviral therapy with a regimen composed of two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor (2NRTI+NNRTI) or two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor (2NRTI+PI/r). Given the lower daily cost of NNRTI as the third agent when compared to the average daily costs of PI/r, it is relevant to estimate the long term impact of each treatment option in the Portuguese context. Methods We developed a microsimulation discrete events model for cost-effectiveness analysis of HIV treatment, simulating individual paths from ART initiation to death. Four driving forces determine the course of events: CD4+ cell count, viral load, resistance and adherence. Distributions of time to event are conditional to individuals’ characteristics and past history. Time to event was modeled using parametric survival analysis using Stata 11®. Disease progression was structured according to therapy lines and the model was parameterized with cohort Portuguese observational data. All resources were valued at 2009 prices. The National Health Service’s perspective was assumed considering a lifetime horizon and a 5% annual discount rate. Results In this analysis, initiating therapy with two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor reduces the average number of switches by 17%, saves 19.573€ per individual and increases life expectancy by 1.7 months showing to be a dominant strategy in 57% of the simulations when compared to two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor. Conclusion This study suggests that, when clinically valid, initiating therapy with two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor is a cost-saving strategy and equally effective when compared to two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor as the first regimen. PMID:23028618

Deep Sequencing Reveals the Complete Genome and Evidence for Transcriptional Activity of the First Virus-Like Sequences Identified in Aristotelia chilensis (Maqui Berry)

PubMed Central

Villacreses, Javier; Rojas-Herrera, Marcelo; Sánchez, Carolina; Hewstone, Nicole; Undurraga, Soledad F.; Alzate, Juan F.; Manque, Patricio; Maracaja-Coutinho, Vinicius; Polanco, Victor

2015-01-01

Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1). High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs): ORFs 1 and 2 shares 66%–73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV), Petuvirus genus. ORF1 encodes a movement protein (MP); ORF2 a Reverse Transcriptase (RT) and a Ribonuclease H (RNase H) domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs), AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq). Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant. PMID:25855242

A retrotransposable element from the mosquito Anopheles gambiae .

PubMed Central

Besansky, N J

1990-01-01

A family of middle repetitive elements from the African malaria vector Anopheles gambiae is described. Approximately 100 copies of the element, designated T1Ag, are dispersed in the genome. Full-length elements are 4.6 kilobase pairs in length, but truncation of the 5' end is common. Nucleotide sequences of one full-length, two 5'-truncated, and two 5' ends of T1Ag elements were determined and aligned to define a consensus sequence. Sequence analysis revealed two long, overlapping open reading frames followed by a polyadenylation signal, AATAAA, and a tail consisting of tandem repetitions of the motif TGAAA. No direct or inverted long terminal repeats (LTRs) were detected. The first open reading frame, 442 amino acids in length, includes a domain resembling that of nucleic acid-binding proteins. The second open reading frame, 975 amino acids long, resembles the reverse transcriptases of a category of retrotransposable elements without LTRs, variously termed class II retrotransposons, class III elements or non-LTR retrotransposons. Similarity at the sequence and structural levels places T1Ag in this category. Images PMID:1689457

[The primary structure of a vaccine strain of tobacco mosaic virus V-69].

PubMed

Shiian, A N; Mil'shina, N V; Snegireva, P B; Pukhal'skiĭ, V A

1994-12-01

A random set of cDNA fragments were synthesized on genomic RNA of TMV vaccine strain V-69, using random primers and reverse transcriptase. Following synthesis of double-stranded cDNA, they were cloned into the pUC-19 plasmid; and 28 clones were sequenced (insert size 100-500 bp). High nucleotide sequence homology of V-69 (more than 95%) was shown only with tomato strain TMV-L [1]. Sequenced clones represent 54% of the genome (50% of the replicase gene, 98% of the transport protein gene, and 60% of the coat protein gene). In this genome region, 24 base substitutions were revealed, as compared to the wild-type TMV-L sequence. Six base substitutions resulted in changes in corresponding amino acid codons. No substitutions coincided with those discovered in the related TMV vaccine strain L11A [2], while two substitutions in the replicase gene were identical to those found in TMV strain Lta1 [3], which is capable of overcoming protection in tomatoes with the resistance gene Tm-1.

Structure and Distribution of Centromeric Retrotransposons at Diploid and Allotetraploid Coffea Centromeric and Pericentromeric Regions

PubMed Central

de Castro Nunes, Renata; Orozco-Arias, Simon; Crouzillat, Dominique; Mueller, Lukas A.; Strickler, Suzy R.; Descombes, Patrick; Fournier, Coralie; Moine, Deborah; de Kochko, Alexandre; Yuyama, Priscila M.; Vanzela, André L. L.; Guyot, Romain

2018-01-01

Centromeric regions of plants are generally composed of large array of satellites from a specific lineage of Gypsy LTR-retrotransposons, called Centromeric Retrotransposons. Repeated sequences interact with a specific H3 histone, playing a crucial function on kinetochore formation. To study the structure and composition of centromeric regions in the genus Coffea, we annotated and classified Centromeric Retrotransposons sequences from the allotetraploid C. arabica genome and its two diploid ancestors: Coffea canephora and C. eugenioides. Ten distinct CRC (Centromeric Retrotransposons in Coffea) families were found. The sequence mapping and FISH experiments of CRC Reverse Transcriptase domains in C. canephora, C. eugenioides, and C. arabica clearly indicate a strong and specific targeting mainly onto proximal chromosome regions, which can be associated also with heterochromatin. PacBio genome sequence analyses of putative centromeric regions on C. arabica and C. canephora chromosomes showed an exceptional density of one family of CRC elements, and the complete absence of satellite arrays, contrasting with usual structure of plant centromeres. Altogether, our data suggest a specific centromere organization in Coffea, contrasting with other plant genomes. PMID:29497436

Brain cDNA clone for human cholinesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

McTiernan, C.; Adkins, S.; Chatonnet, A.

1987-10-01

A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase. Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum.more » The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase rather than acetylcholinesterase. It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes coded for cholinesterase.« less

Functional interrogation of non-coding DNA through CRISPR genome editing.

PubMed

Canver, Matthew C; Bauer, Daniel E; Orkin, Stuart H

2017-05-15

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus.

PubMed Central

Kögel, D; Aboud, M; Flügel, R M

1995-01-01

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent. Images PMID:7544460

Telomere lengthening and other functions of telomerase.

PubMed

Rubtsova, M P; Vasilkova, D P; Malyavko, A N; Naraikina, Yu V; Zvereva, M I; Dontsova, O A

2012-04-01

Telomerase is an enzyme that maintains the length of the telomere. The telomere length specifies the number of divisions a cell can undergo before it finally dies (i.e. the proliferative potential of cells). For example, telomerase is activated in embryonic cell lines and the telomere length is maintained at a constant level; therefore, these cells have an unlimited fission potential. Stem cells are characterized by a lower telomerase activity, which enables only partial compensation for the shortening of telomeres. Somatic cells are usually characterized by the absence of telomerase activity. Telomere shortening leads to the attainment of the Hayflick limit, the transition of cells to a state of senescence. The cells subsequently enter a state of crisis, accompanied by massive cell death. The surviving cells become cancer cells, which are capable both of dividing indefinitely and maintaining telomere length (usually with the aid of telomerase). Telomerase is a reverse transcriptase. It consists of two major components: telomerase RNA (TER) and reverse transcriptase (TERT). TER is a non-coding RNA, and it contains the region which serves as a template for telomere synthesis. An increasing number of articles focussing on the alternative functions of telomerase components have recently started appearing. The present review summarizes data on the structure, biogenesis, and functions of telomerase.

Drug Susceptibility and Resistance Mutations After First-Line Failure in Resource Limited Settings

PubMed Central

Wallis, Carole L.; Aga, Evgenia; Ribaudo, Heather; Saravanan, Shanmugam; Norton, Michael; Stevens, Wendy; Kumarasamy, Nagalingeswaran; Bartlett, John; Katzenstein, David

2014-01-01

Background. The development of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been associated with baseline human immunodeficiency virus (HIV)-1 RNA level (VL), CD4 cell counts (CD4), subtype, and treatment failure duration. This study describes drug resistance and levels of susceptibility after first-line virologic failure in individuals from Thailand, South Africa, India, Malawi, Tanzania. Methods. CD4 and VL were captured at AIDs Clinical Trial Group (ACTG) A5230 study entry, a study of lopinavir/ritonavir (LPV/r) monotherapy after first-line virologic failure on an NNRTI regimen. HIV drug-resistance mutation associations with subtype, site, study entry VL, and CD4 were evaluated using Fisher exact and Kruskall–Wallis tests. Results. Of the 207 individuals who were screened for A5230, sequence data were available for 148 individuals. Subtypes observed: subtype C (n = 97, 66%) AE (n = 27, 18%), A1 (n = 12, 8%), and D (n = 10, 7%). Of the 148 individuals, 93% (n = 138) and 96% (n = 142) had at least 1 reverse transcriptase (RT) mutation associated with NRTI and NNRTI resistance, respectively. The number of NRTI mutations was significantly associated with a higher study screening VL and lower study screening CD4 (P < .001). Differences in drug-resistance patterns in both NRTI and NNRTI were observed by site. Conclusions. The degree of NNRTI and NRTI resistance after first-line virologic failure was associated with higher VL at study entry. Thirty-two percent of individuals remained fully susceptible to etravirine and rilpivirine, protease inhibitor resistance was rare. Some level of susceptibility to NRTI remained; however, VL monitoring and earlier virologic failure detection may result in lower NRTI resistance. PMID:24795328

A Code Division Multiple Access Communication System for the Low Frequency Band.

DTIC Science & Technology

1983-04-01

frequency channels spread-spectrum communication / complex sequences, orthogonal codes impulsive noise 20. ABSTRACT (Continue an reverse side It...their transmissions with signature sequences. Our LF/CDMA scheme is different in that each user’s signature sequence set consists of M orthogonal ...signature sequences. Our LF/CDMA scheme is different in that each user’s signature sequence set consists of M orthogonal sequences and thus log 2 M

The problems and promise of DNA barcodes for species diagnosis of primate biomaterials

PubMed Central

Lorenz, Joseph G; Jackson, Whitney E; Beck, Jeanne C; Hanner, Robert

2005-01-01

The Integrated Primate Biomaterials and Information Resource (www.IPBIR.org) provides essential research reagents to the scientific community by establishing, verifying, maintaining, and distributing DNA and RNA derived from primate cell cultures. The IPBIR uses mitochondrial cytochrome c oxidase subunit I sequences to verify the identity of samples for quality control purposes in the accession, cell culture, DNA extraction processes and prior to shipping to end users. As a result, IPBIR is accumulating a database of ‘DNA barcodes’ for many species of primates. However, this quality control process is complicated by taxon specific patterns of ‘universal primer’ failure, as well as the amplification or co-amplification of nuclear pseudogenes of mitochondrial origins. To overcome these difficulties, taxon specific primers have been developed, and reverse transcriptase PCR is utilized to exclude these extraneous sequences from amplification. DNA barcoding of primates has applications to conservation and law enforcement. Depositing barcode sequences in a public database, along with primer sequences, trace files and associated quality scores, makes this species identification technique widely accessible. Reference DNA barcode sequences should be derived from, and linked to, specimens of known provenance in web-accessible collections in order to validate this system of molecular diagnostics. PMID:16214744

A Novel Lectin with Antiproliferative and HIV-1 Reverse Transcriptase Inhibitory Activities from Dried Fruiting Bodies of the Monkey Head Mushroom Hericium erinaceum

PubMed Central

Li, Yanrui; Zhang, Guoqing; Ng, Tzi Bun; Wang, Hexiang

2010-01-01

A lectin designated as Hericium erinaceum agglutinin (HEA) was isolated from dried fruiting bodies of the mushroom Hericium erinaceum with a chromatographic procedure which entailed DEAE-cellulose, CM-cellulose, Q-Sepharose, and FPLC Superdex 75. Its molecular mass was estimated to be 51 kDa and its N-terminal amino acid sequences was distinctly different from those of other isolated mushroom lectins. The hemagglutinating activity of HEA was inhibited at the minimum concentration of 12.5 mM by inulin. The lectin was stable at pH 1.9–12.1 and at temperatures up to 70°C, but was inhibited by Hg2+, Cu2+, and Fe3+ ions. The lectin exhibited potent mitogenic activity toward mouse splenocytes, and demonstrated antiproliferative activity toward hepatoma (HepG2) and breast cancer (MCF7) cells with an IC50 of 56.1 μM and 76.5 μM, respectively. It manifested HIV-1 reverse transcriptase inhibitory activity with an IC50 of 31.7 μM. The lectin exhibited potent mitogenic activity toward murine splenocytes but was devoid of antifungal activity. PMID:20625408

A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region.

PubMed

Kress, W John; Erickson, David L

2007-06-06

A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination.

Sequence editing by Apolipoprotein B RNA-editing catalytic component-B and epidemiological surveillance of transmitted HIV-1 drug resistance

PubMed Central

Gifford, Robert J.; Rhee, Soo-Yon; Eriksson, Nicolas; Liu, Tommy F.; Kiuchi, Mark; Das, Amar K.; Shafer, Robert W.

2008-01-01

Design Promiscuous guanine (G) to adenine (A) substitutions catalysed by apolipoprotein B RNA-editing catalytic component (APOBEC) enzymes are observed in a proportion of HIV-1 sequences in vivo and can introduce artifacts into some genetic analyses. The potential impact of undetected lethal editing on genotypic estimation of transmitted drug resistance was assessed. Methods Classifiers of lethal, APOBEC-mediated editing were developed by analysis of lentiviral pol gene sequence variation and evaluated using control sets of HIV-1 sequences. The potential impact of sequence editing on genotypic estimation of drug resistance was assessed in sets of sequences obtained from 77 studies of 25 or more therapy-naive individuals, using mixture modelling approaches to determine the maximum likelihood classification of sequences as lethally edited as opposed to viable. Results Analysis of 6437 protease and reverse transcriptase sequences from therapy-naive individuals using a novel classifier of lethal, APOBEC3G-mediated sequence editing, the polypeptide-like 3G (APOBEC3G)-mediated defectives (A3GD) index’, detected lethal editing in association with spurious ‘transmitted drug resistance’ in nearly 3% of proviral sequences obtained from whole blood and 0.2% of samples obtained from plasma. Conclusion Screening for lethally edited sequences in datasets containing a proportion of proviral DNA, such as those likely to be obtained for epidemiological surveillance of transmitted drug resistance in the developing world, can eliminate rare but potentially significant errors in genotypic estimation of transmitted drug resistance. PMID:18356601

Streamlined Genome Sequence Compression using Distributed Source Coding

PubMed Central

Wang, Shuang; Jiang, Xiaoqian; Chen, Feng; Cui, Lijuan; Cheng, Samuel

2014-01-01

We aim at developing a streamlined genome sequence compression algorithm to support alternative miniaturized sequencing devices, which have limited communication, storage, and computation power. Existing techniques that require heavy client (encoder side) cannot be applied. To tackle this challenge, we carefully examined distributed source coding theory and developed a customized reference-based genome compression protocol to meet the low-complexity need at the client side. Based on the variation between source and reference, our protocol will pick adaptively either syndrome coding or hash coding to compress subsequences of changing code length. Our experimental results showed promising performance of the proposed method when compared with the state-of-the-art algorithm (GRS). PMID:25520552

3'-terminal sequence of a small round structured virus (SRSV) in Japan.

PubMed

Utagawa, E T; Takeda, N; Inouye, S; Kasuga, K; Yamazaki, S

1994-01-01

We determined the nucleotide sequence of about 1,000 bases from the 3'-terminus of a small round structured virus (SRSV), which caused a gastroenteritis outbreak in Chiba Prefecture, Japan, in 1987. The sequence was compared with the corresponding sequence region of Norwalk virus; it consisted of a part of the open reading frame 2 (ORF2), whole ORF3, and 3'-noncoding region (NCR). The 624-base-long ORF3 had sequence homology of 68% with the corresponding region of Norwalk virus. (The amino acid sequence homology was 74%.) The 94-base-long NCR had 65% homology with Norwalk virus. We then selected two consensus-sequence portions in the above sequence between Chiba and Norwalk viruses for primers in the reverse transcriptase-polymerase chain reaction (RT-PCR). Using this primer set, we detected 669-bp bands in agarose gel electrophoresis of RT-PCR products from feces containing Chiba or Norwalk viruses. Furthermore, in Southern hybridization with Chiba probes which were labeled with digoxigenin-dUTP in PCR, the bands of the two viruses were clearly stained under a low stringency condition. Since both Chiba and Norwalk viruses were detected by the above primer set although they are geographically and chronologically different viruses, our primer-pair may be useful for detection of a broad range of SRSVs which cause gastroenteritis in different areas.

Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.

PubMed Central

Mizrahi, V; Usdin, M T; Harington, A; Dudding, L R

1990-01-01

Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity. Images PMID:1699202

Synthesis, structure-activity relationship and molecular docking of cyclohexenone based analogous as potent non-nucleoside reverse-transcriptase inhibitors

NASA Astrophysics Data System (ADS)

Nazar, Muhammad Faizan; Abdullah, Muhammad Imran; Badshah, Amir; Mahmood, Asif; Rana, Usman Ali; Khan, Salah Ud-Din

2015-04-01

The chalcones core in compounds is advantageously chosen effective synthons, which offer exciting perspectives in biological and pharmacological research. The present study reports the successful development of eight new cyclohexenone based anti-reverse transcriptase analogous using rational drug design synthesis principles. These new cyclohexenone derivatives (CDs) were synthesized by following a convenient route of Robinson annulation, and the molecular structure of these CDs were later confirmed by various analytical techniques such as 1H NMR, 13C NMR, FT-IR, UV-Vis spectroscopy and mass spectrometry. All the synthesized compounds were screened theoretically and experimentally against reverse transcriptase (RT) and found potentially active reverse transcriptase (RT) inhibitors. Of the compounds studied, the compound 2FC4 showed high interaction with RT at non-nucleoside binding site, contributing high free binding energy (ΔG -8.01 Kcal) and IC50 (0.207 μg/ml), respectively. Further results revealed that the compounds bearing more halogen groups, with additional hydrophobic character, offered superior anti-reverse transcriptase activity as compared to rest of compounds. It is anticipate that the present study would be very useful for the selection of potential reverse transcriptase inhibitors featuring inclusive pharmacological profiles.

Successful Recovery of Nuclear Protein-Coding Genes from Small Insects in Museums Using Illumina Sequencing.

PubMed

Kanda, Kojun; Pflug, James M; Sproul, John S; Dasenko, Mark A; Maddison, David R

2015-01-01

In this paper we explore high-throughput Illumina sequencing of nuclear protein-coding, ribosomal, and mitochondrial genes in small, dried insects stored in natural history collections. We sequenced one tenebrionid beetle and 12 carabid beetles ranging in size from 3.7 to 9.7 mm in length that have been stored in various museums for 4 to 84 years. Although we chose a number of old, small specimens for which we expected low sequence recovery, we successfully recovered at least some low-copy nuclear protein-coding genes from all specimens. For example, in one 56-year-old beetle, 4.4 mm in length, our de novo assembly recovered about 63% of approximately 41,900 nucleotides in a target suite of 67 nuclear protein-coding gene fragments, and 70% using a reference-based assembly. Even in the least successfully sequenced carabid specimen, reference-based assembly yielded fragments that were at least 50% of the target length for 34 of 67 nuclear protein-coding gene fragments. Exploration of alternative references for reference-based assembly revealed few signs of bias created by the reference. For all specimens we recovered almost complete copies of ribosomal and mitochondrial genes. We verified the general accuracy of the sequences through comparisons with sequences obtained from PCR and Sanger sequencing, including of conspecific, fresh specimens, and through phylogenetic analysis that tested the placement of sequences in predicted regions. A few possible inaccuracies in the sequences were detected, but these rarely affected the phylogenetic placement of the samples. Although our sample sizes are low, an exploratory regression study suggests that the dominant factor in predicting success at recovering nuclear protein-coding genes is a high number of Illumina reads, with success at PCR of COI and killing by immersion in ethanol being secondary factors; in analyses of only high-read samples, the primary significant explanatory variable was body length, with small beetles being more successfully sequenced.

Successful Recovery of Nuclear Protein-Coding Genes from Small Insects in Museums Using Illumina Sequencing

PubMed Central

Dasenko, Mark A.

2015-01-01

In this paper we explore high-throughput Illumina sequencing of nuclear protein-coding, ribosomal, and mitochondrial genes in small, dried insects stored in natural history collections. We sequenced one tenebrionid beetle and 12 carabid beetles ranging in size from 3.7 to 9.7 mm in length that have been stored in various museums for 4 to 84 years. Although we chose a number of old, small specimens for which we expected low sequence recovery, we successfully recovered at least some low-copy nuclear protein-coding genes from all specimens. For example, in one 56-year-old beetle, 4.4 mm in length, our de novo assembly recovered about 63% of approximately 41,900 nucleotides in a target suite of 67 nuclear protein-coding gene fragments, and 70% using a reference-based assembly. Even in the least successfully sequenced carabid specimen, reference-based assembly yielded fragments that were at least 50% of the target length for 34 of 67 nuclear protein-coding gene fragments. Exploration of alternative references for reference-based assembly revealed few signs of bias created by the reference. For all specimens we recovered almost complete copies of ribosomal and mitochondrial genes. We verified the general accuracy of the sequences through comparisons with sequences obtained from PCR and Sanger sequencing, including of conspecific, fresh specimens, and through phylogenetic analysis that tested the placement of sequences in predicted regions. A few possible inaccuracies in the sequences were detected, but these rarely affected the phylogenetic placement of the samples. Although our sample sizes are low, an exploratory regression study suggests that the dominant factor in predicting success at recovering nuclear protein-coding genes is a high number of Illumina reads, with success at PCR of COI and killing by immersion in ethanol being secondary factors; in analyses of only high-read samples, the primary significant explanatory variable was body length, with small beetles being more successfully sequenced. PMID:26716693

An antifungal protein from the pea Pisum sativum var. arvense Poir.

PubMed

Wang, H X; Ng, T B

2006-07-01

An antifungal protein with a molecular mass of 11 kDa and a lysine-rich N-terminal sequence was isolated from the seeds of the pea Pisum sativum var. arvense Poir. The antifungal protein was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-cellulose. It exerted antifungal activity against Physalospora piricola with an IC50 of 0.62 microM, and also antifungal activity against Fusarium oxysporum and Mycosphaerella arachidicola. It inhibited human immunodeficiency virus type 1 reverse transcriptase with an IC50 of 4.7 microM.

Isolation, nucleotide sequence and expression of a cDNA encoding feline granulocyte colony-stimulating factor.

PubMed

Dunham, S P; Onions, D E

2001-06-21

A cDNA encoding feline granulocyte colony stimulating factor (fG-CSF) was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction. The cDNA is 949 bp in length and encodes a predicted mature protein of 174 amino acids. Recombinant fG-CSF was expressed as a glutathione S-transferase fusion and purified by affinity chromatography. Biological activity of the recombinant protein was demonstrated using the murine myeloblastic cell line GNFS-60, which showed an ED50 for fG-CSF of approximately 2 ng/ml. Copyright 2001 Academic Press.

Progress towards Rapid Detection of Measles Vaccine Strains: a Tool To Inform Public Health Interventions

PubMed Central

2016-01-01

ABSTRACT Rapid differentiation of vaccine from wild-type strains in suspect measles cases is a valuable epidemiological tool that informs the public health response to this highly infectious disease. Few public health laboratories sequence measles virus-positive specimens to determine genotype, and the vaccine-specific real-time reverse transcriptase PCR (rRT-PCR) assay described by F. Roy et al. (J. Clin. Microbiol. 55:735–743, 2017, https://doi.org/10.1128/JCM.01879-16) offers a rapid, easily adoptable method to identify measles vaccine strains in suspect cases. PMID:28003421

Progress towards Rapid Detection of Measles Vaccine Strains: a Tool To Inform Public Health Interventions.

PubMed

Hacker, Jill K

2017-03-01

Rapid differentiation of vaccine from wild-type strains in suspect measles cases is a valuable epidemiological tool that informs the public health response to this highly infectious disease. Few public health laboratories sequence measles virus-positive specimens to determine genotype, and the vaccine-specific real-time reverse transcriptase PCR (rRT-PCR) assay described by F. Roy et al. (J. Clin. Microbiol. 55:735-743, 2017, https://doi.org/10.1128/JCM.01879-16) offers a rapid, easily adoptable method to identify measles vaccine strains in suspect cases. Copyright © 2017 American Society for Microbiology.

The Mitochondrial Cytochrome Oxidase Subunit I Gene Occurs on a Minichromosome with Extensive Heteroplasmy in Two Species of Chewing Lice, Geomydoecus aurei and Thomomydoecus minor

PubMed Central

Pietan, Lucas L.; Spradling, Theresa A.

2016-01-01

In animals, mitochondrial DNA (mtDNA) typically occurs as a single circular chromosome with 13 protein-coding genes and 22 tRNA genes. The various species of lice examined previously, however, have shown mitochondrial genome rearrangements with a range of chromosome sizes and numbers. Our research demonstrates that the mitochondrial genomes of two species of chewing lice found on pocket gophers, Geomydoecus aurei and Thomomydoecus minor, are fragmented with the 1,536 base-pair (bp) cytochrome-oxidase subunit I (cox1) gene occurring as the only protein-coding gene on a 1,916–1,964 bp minicircular chromosome in the two species, respectively. The cox1 gene of T. minor begins with an atypical start codon, while that of G. aurei does not. Components of the non-protein coding sequence of G. aurei and T. minor include a tRNA (isoleucine) gene, inverted repeat sequences consistent with origins of replication, and an additional non-coding region that is smaller than the non-coding sequence of other lice with such fragmented mitochondrial genomes. Sequences of cox1 minichromosome clones for each species reveal extensive length and sequence heteroplasmy in both coding and noncoding regions. The highly variable non-gene regions of G. aurei and T. minor have little sequence similarity with one another except for a 19-bp region of phylogenetically conserved sequence with unknown function. PMID:27589589

Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution.

PubMed

2004-12-09

We present here a draft genome sequence of the red jungle fowl, Gallus gallus. Because the chicken is a modern descendant of the dinosaurs and the first non-mammalian amniote to have its genome sequenced, the draft sequence of its genome--composed of approximately one billion base pairs of sequence and an estimated 20,000-23,000 genes--provides a new perspective on vertebrate genome evolution, while also improving the annotation of mammalian genomes. For example, the evolutionary distance between chicken and human provides high specificity in detecting functional elements, both non-coding and coding. Notably, many conserved non-coding sequences are far from genes and cannot be assigned to defined functional classes. In coding regions the evolutionary dynamics of protein domains and orthologous groups illustrate processes that distinguish the lineages leading to birds and mammals. The distinctive properties of avian microchromosomes, together with the inferred patterns of conserved synteny, provide additional insights into vertebrate chromosome architecture.

A Partial Least Squares Based Procedure for Upstream Sequence Classification in Prokaryotes.

PubMed

Mehmood, Tahir; Bohlin, Jon; Snipen, Lars

2015-01-01

The upstream region of coding genes is important for several reasons, for instance locating transcription factor, binding sites, and start site initiation in genomic DNA. Motivated by a recently conducted study, where multivariate approach was successfully applied to coding sequence modeling, we have introduced a partial least squares (PLS) based procedure for the classification of true upstream prokaryotic sequence from background upstream sequence. The upstream sequences of conserved coding genes over genomes were considered in analysis, where conserved coding genes were found by using pan-genomics concept for each considered prokaryotic species. PLS uses position specific scoring matrix (PSSM) to study the characteristics of upstream region. Results obtained by PLS based method were compared with Gini importance of random forest (RF) and support vector machine (SVM), which is much used method for sequence classification. The upstream sequence classification performance was evaluated by using cross validation, and suggested approach identifies prokaryotic upstream region significantly better to RF (p-value < 0.01) and SVM (p-value < 0.01). Further, the proposed method also produced results that concurred with known biological characteristics of the upstream region.

Golay sequences coded coherent optical OFDM for long-haul transmission

NASA Astrophysics Data System (ADS)

Qin, Cui; Ma, Xiangrong; Hua, Tao; Zhao, Jing; Yu, Huilong; Zhang, Jian

2017-09-01

We propose to use binary Golay sequences in coherent optical orthogonal frequency division multiplexing (CO-OFDM) to improve the long-haul transmission performance. The Golay sequences are generated by binary Reed-Muller codes, which have low peak-to-average power ratio and certain error correction capability. A low-complexity decoding algorithm for the Golay sequences is then proposed to recover the signal. Under same spectral efficiency, the QPSK modulated OFDM with binary Golay sequences coding with and without discrete Fourier transform (DFT) spreading (DFTS-QPSK-GOFDM and QPSK-GOFDM) are compared with the normal BPSK modulated OFDM with and without DFT spreading (DFTS-BPSK-OFDM and BPSK-OFDM) after long-haul transmission. At a 7% forward error correction code threshold (Q2 factor of 8.5 dB), it is shown that DFTS-QPSK-GOFDM outperforms DFTS-BPSK-OFDM by extending the transmission distance by 29% and 18%, in non-dispersion managed and dispersion managed links, respectively.

Continuous in vitro evolution of bacteriophage RNA polymerase promoters

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Banerji, A.; Joyce, G. F.

1994-01-01

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

BASiNET-BiologicAl Sequences NETwork: a case study on coding and non-coding RNAs identification.

PubMed

Ito, Eric Augusto; Katahira, Isaque; Vicente, Fábio Fernandes da Rocha; Pereira, Luiz Filipe Protasio; Lopes, Fabrício Martins

2018-06-05

With the emergence of Next Generation Sequencing (NGS) technologies, a large volume of sequence data in particular de novo sequencing was rapidly produced at relatively low costs. In this context, computational tools are increasingly important to assist in the identification of relevant information to understand the functioning of organisms. This work introduces BASiNET, an alignment-free tool for classifying biological sequences based on the feature extraction from complex network measurements. The method initially transform the sequences and represents them as complex networks. Then it extracts topological measures and constructs a feature vector that is used to classify the sequences. The method was evaluated in the classification of coding and non-coding RNAs of 13 species and compared to the CNCI, PLEK and CPC2 methods. BASiNET outperformed all compared methods in all adopted organisms and datasets. BASiNET have classified sequences in all organisms with high accuracy and low standard deviation, showing that the method is robust and non-biased by the organism. The proposed methodology is implemented in open source in R language and freely available for download at https://cran.r-project.org/package=BASiNET.

The Number, Organization, and Size of Polymorphic Membrane Protein Coding Sequences as well as the Most Conserved Pmp Protein Differ within and across Chlamydia Species.

PubMed

Van Lent, Sarah; Creasy, Heather Huot; Myers, Garry S A; Vanrompay, Daisy

2016-01-01

Variation is a central trait of the polymorphic membrane protein (Pmp) family. The number of pmp coding sequences differs between Chlamydia species, but it is unknown whether the number of pmp coding sequences is constant within a Chlamydia species. The level of conservation of the Pmp proteins has previously only been determined for Chlamydia trachomatis. As different Pmp proteins might be indispensible for the pathogenesis of different Chlamydia species, this study investigated the conservation of Pmp proteins both within and across C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci. The pmp coding sequences were annotated in 16 C. trachomatis, 6 C. pneumoniae, 2 C. abortus, and 16 C. psittaci genomes. The number and organization of polymorphic membrane coding sequences differed within and across the analyzed Chlamydia species. The length of coding sequences of pmpA,pmpB, and pmpH was conserved among all analyzed genomes, while the length of pmpE/F and pmpG, and remarkably also of the subtype pmpD, differed among the analyzed genomes. PmpD, PmpA, PmpH, and PmpA were the most conserved Pmp in C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci, respectively. PmpB was the most conserved Pmp across the 4 analyzed Chlamydia species. © 2016 S. Karger AG, Basel.

A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region

PubMed Central

Kress, W. John; Erickson, David L.

2007-01-01

Background A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Methodology/Principal Findings Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. Conclusions/Significance A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination. PMID:17551588

A common class of transcripts with 5'-intron depletion, distinct early coding sequence features, and N1-methyladenosine modification.

PubMed

Cenik, Can; Chua, Hon Nian; Singh, Guramrit; Akef, Abdalla; Snyder, Michael P; Palazzo, Alexander F; Moore, Melissa J; Roth, Frederick P

2017-03-01

Introns are found in 5' untranslated regions (5'UTRs) for 35% of all human transcripts. These 5'UTR introns are not randomly distributed: Genes that encode secreted, membrane-bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 5'UTR introns tend to harbor specific RNA sequence elements in their early coding regions. To model and understand the connection between coding-region sequence and 5'UTR intron status, we developed a classifier that can predict 5'UTR intron status with >80% accuracy using only sequence features in the early coding region. Thus, the classifier identifies transcripts with 5 ' proximal- i ntron- m inus-like-coding regions ("5IM" transcripts). Unexpectedly, we found that the early coding sequence features defining 5IM transcripts are widespread, appearing in 21% of all human RefSeq transcripts. The 5IM class of transcripts is enriched for non-AUG start codons, more extensive secondary structure both preceding the start codon and near the 5' cap, greater dependence on eIF4E for translation, and association with ER-proximal ribosomes. 5IM transcripts are bound by the exon junction complex (EJC) at noncanonical 5' proximal positions. Finally, N 1 -methyladenosines are specifically enriched in the early coding regions of 5IM transcripts. Taken together, our analyses point to the existence of a distinct 5IM class comprising ∼20% of human transcripts. This class is defined by depletion of 5' proximal introns, presence of specific RNA sequence features associated with low translation efficiency, N 1 -methyladenosines in the early coding region, and enrichment for noncanonical binding by the EJC. © 2017 Cenik et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Comparison of an In Vitro Diagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing.

PubMed

Tzou, Philip L; Ariyaratne, Pramila; Varghese, Vici; Lee, Charlie; Rakhmanaliev, Elian; Villy, Carolin; Yee, Meiqi; Tan, Kevin; Michel, Gerd; Pinsky, Benjamin A; Shafer, Robert W

2018-06-01

The ability of next-generation sequencing (NGS) technologies to detect low frequency HIV-1 drug resistance mutations (DRMs) not detected by dideoxynucleotide Sanger sequencing has potential advantages for improved patient outcomes. We compared the performance of an in vitro diagnostic (IVD) NGS assay, the Sentosa SQ HIV genotyping assay for HIV-1 genotypic resistance testing, with Sanger sequencing on 138 protease/reverse transcriptase (RT) and 39 integrase sequences. The NGS assay used a 5% threshold for reporting low-frequency variants. The level of complete plus partial nucleotide sequence concordance between Sanger sequencing and NGS was 99.9%. Among the 138 protease/RT sequences, a mean of 6.4 DRMs was identified by both Sanger and NGS, a mean of 0.5 DRM was detected by NGS alone, and a mean of 0.1 DRM was detected by Sanger sequencing alone. Among the 39 integrase sequences, a mean of 1.6 DRMs was detected by both Sanger sequencing and NGS and a mean of 0.15 DRM was detected by NGS alone. Compared with Sanger sequencing, NGS estimated higher levels of resistance to one or more antiretroviral drugs for 18.2% of protease/RT sequences and 5.1% of integrase sequences. There was little evidence for technical artifacts in the NGS sequences, but the G-to-A hypermutation was detected in three samples. In conclusion, the IVD NGS assay evaluated in this study was highly concordant with Sanger sequencing. At the 5% threshold for reporting minority variants, NGS appeared to attain a modestly increased sensitivity for detecting low-frequency DRMs without compromising sequence accuracy. Copyright © 2018 American Society for Microbiology.

A DS-UWB Cognitive Radio System Based on Bridge Function Smart Codes

NASA Astrophysics Data System (ADS)

Xu, Yafei; Hong, Sheng; Zhao, Guodong; Zhang, Fengyuan; di, Jinshan; Zhang, Qishan

This paper proposes a direct-sequence UWB Gaussian pulse of cognitive radio systems based on bridge function smart sequence matrix and the Gaussian pulse. As the system uses the spreading sequence code, that is the bridge function smart code sequence, the zero correlation zones (ZCZs) which the bridge function sequences' auto-correlation functions had, could reduce multipath fading of the pulse interference. The Modulated channel signal was sent into the IEEE 802.15.3a UWB channel. We analysis the ZCZs's inhibition to the interference multipath interference (MPI), as one of the main system sources interferences. The simulation in SIMULINK/MATLAB is described in detail. The result shows the system has better performance by comparison with that employing Walsh sequence square matrix, and it was verified by the formula in principle.

The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes.

PubMed Central

Fletcher, Simon P; Ali, Iraj K; Kaminski, Ann; Digard, Paul; Jackson, Richard J

2002-01-01

Classical swine fever virus (CSFV) is a member of the pestivirus family, which shares many features in common with hepatitis C virus (HCV). It is shown here that CSFV has an exceptionally efficient cis-acting internal ribosome entry segment (IRES), which, like that of HCV, is strongly influenced by the sequences immediately downstream of the initiation codon, and is optimal with viral coding sequences in this position. Constructs that retained 17 or more codons of viral coding sequence exhibited full IRES activity, but with only 12 codons, activity was approximately 66% of maximum in vitro (though close to maximum in transfected BHK cells), whereas with just 3 codons or fewer, the activity was only approximately 15% of maximum. The minimal coding region elements required for high activity were exchanged between HCV and CSFV. Although maximum activity was observed in each case with the homologous combination of coding region and 5' UTR, the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the 5' UTR and coding sequences. On the other hand, inversion of the coding sequences resulted in low IRES activity, particularly with the HCV coding sequences. RNA structure probing showed that the efficiency of internal initiation of these chimeric constructs correlated most closely with the degree of single-strandedness of the region around and immediately downstream of the initiation codon. The low activity IRESs could not be rescued by addition of supplementary eIF4A (the initiation factor with ATP-dependent RNA helicase activity). The extreme sensitivity to secondary structure around the initiation codon is likely to be due to the fact that the eIF4F complex (which has eIF4A as one of its subunits) is not required for and does not participate in initiation on these IRESs. PMID:12515388

Detecting the borders between coding and non-coding DNA regions in prokaryotes based on recursive segmentation and nucleotide doublets statistics

PubMed Central

2012-01-01

Background Detecting the borders between coding and non-coding regions is an essential step in the genome annotation. And information entropy measures are useful for describing the signals in genome sequence. However, the accuracies of previous methods of finding borders based on entropy segmentation method still need to be improved. Methods In this study, we first applied a new recursive entropic segmentation method on DNA sequences to get preliminary significant cuts. A 22-symbol alphabet is used to capture the differential composition of nucleotide doublets and stop codon patterns along three phases in both DNA strands. This process requires no prior training datasets. Results Comparing with the previous segmentation methods, the experimental results on three bacteria genomes, Rickettsia prowazekii, Borrelia burgdorferi and E.coli, show that our approach improves the accuracy for finding the borders between coding and non-coding regions in DNA sequences. Conclusions This paper presents a new segmentation method in prokaryotes based on Jensen-Rényi divergence with a 22-symbol alphabet. For three bacteria genomes, comparing to A12_JR method, our method raised the accuracy of finding the borders between protein coding and non-coding regions in DNA sequences. PMID:23282225

Nonspatial Sequence Coding in CA1 Neurons

PubMed Central

Allen, Timothy A.; Salz, Daniel M.; McKenzie, Sam

2016-01-01

The hippocampus is critical to the memory for sequences of events, a defining feature of episodic memory. However, the fundamental neuronal mechanisms underlying this capacity remain elusive. While considerable research indicates hippocampal neurons can represent sequences of locations, direct evidence of coding for the memory of sequential relationships among nonspatial events remains lacking. To address this important issue, we recorded neural activity in CA1 as rats performed a hippocampus-dependent sequence-memory task. Briefly, the task involves the presentation of repeated sequences of odors at a single port and requires rats to identify each item as “in sequence” or “out of sequence”. We report that, while the animals' location and behavior remained constant, hippocampal activity differed depending on the temporal context of items—in this case, whether they were presented in or out of sequence. Some neurons showed this effect across items or sequence positions (general sequence cells), while others exhibited selectivity for specific conjunctions of item and sequence position information (conjunctive sequence cells) or for specific probe types (probe-specific sequence cells). We also found that the temporal context of individual trials could be accurately decoded from the activity of neuronal ensembles, that sequence coding at the single-cell and ensemble level was linked to sequence memory performance, and that slow-gamma oscillations (20–40 Hz) were more strongly modulated by temporal context and performance than theta oscillations (4–12 Hz). These findings provide compelling evidence that sequence coding extends beyond the domain of spatial trajectories and is thus a fundamental function of the hippocampus. SIGNIFICANCE STATEMENT The ability to remember the order of life events depends on the hippocampus, but the underlying neural mechanisms remain poorly understood. Here we addressed this issue by recording neural activity in hippocampal region CA1 while rats performed a nonspatial sequence memory task. We found that hippocampal neurons code for the temporal context of items (whether odors were presented in the correct or incorrect sequential position) and that this activity is linked with memory performance. The discovery of this novel form of temporal coding in hippocampal neurons advances our fundamental understanding of the neurobiology of episodic memory and will serve as a foundation for our cross-species, multitechnique approach aimed at elucidating the neural mechanisms underlying memory impairments in aging and dementia. PMID:26843637

Functional analysis of the interactions between reovirus particles and various proteases in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sargent, M.D.; Long, D.G.; Borsa, J.

1977-01-01

The digestion of purified reovirus particles by various proteases including chymotrypsin, trypsin, pronase, papain, bromelain, proteinase K, and fibrinolysin has been examined as it relates to virion transcriptase activation and alteration of infectivity. In every case uncoating to the level of active transcriptase proceeds via two mechanistically distinct steps. All the proteases tested serve to mediate only the first of the two steps, converting intact virions to intermediate subviral particles (ISVP) in which the transcriptase is retained in a latent state. The second step of the uncoating process is mediated by a K/sup +/ ion-triggered, endogenous mechanism and results inmore » conversion of ISVP to cores, concomitant with transcriptase activation and loss of infectivity. All of the tested enzymes, except trypsin, reversibly block the second step of uncoating. These results indicate the generality, with respect to protease employed, of the two-step process for reovirus uncoating and transcriptase activation demonstrated previously with chymotrypsin.« less

Repeats of base oligomers as the primordial coding sequences of the primeval earth and their vestiges in modern genes.

PubMed

Ohno, S

1984-01-01

Three outstanding properties uniquely qualify repeats of base oligomers as the primordial coding sequences of all polypeptide chains. First, when compared with randomly generated base sequences in general, they are more likely to have long open reading frames. Second, periodical polypeptide chains specified by such repeats are more likely to assume either alpha-helical or beta-sheet secondary structures than are polypeptide chains of random sequence. Third, provided that the number of bases in the oligomeric unit is not a multiple of 3, these internally repetitious coding sequences are impervious to randomly sustained base substitutions, deletions, and insertions. This is because the recurring periodicity of their polypeptide chains is given by three consecutive copies of the oligomeric unit translated in three different reading frames. Accordingly, when one reading frame is open, the other two are automatically open as well, all three being capable of coding for polypeptide chains of identical periodicity. Under this circumstance, a frame shift due to the deletion or insertion of a number of bases that is not a multiple of 3 fails to alter the down-stream amino acid sequence, and even a base change causing premature chain-termination can silence only one of the three potential coding units. Newly arisen coding sequences in modern organisms are oligomeric repeats, and most of the older genes retain various vestiges of their original internal repetitions. Some of the genes (e.g., oncogenes) have even inherited the property of being impervious to randomly sustained base changes.

Novel splice-site and missense mutations in the ALDH1A3 gene underlying autosomal recessive anophthalmia/microphthalmia.

PubMed

Semerci, C Nur; Kalay, Ersan; Yıldırım, Cem; Dinçer, Tuba; Olmez, Akgün; Toraman, Bayram; Koçyiğit, Ali; Bulgu, Yunus; Okur, Volkan; Satıroğlu-Tufan, Lale; Akarsu, Nurten A

2014-06-01

This study aimed to identify the underlying genetic defect responsible for anophthalmia/microphthalmia. In total, two Turkish families with a total of nine affected individuals were included in the study. Affymetrix 250 K single nucleotide polymorphism genotyping and homozygosity mapping were used to identify the localisation of the genetic defect in question. Coding region of the ALDH1A3 gene was screened via direct sequencing. cDNA samples were generated from primary fibroblast cell cultures for expression analysis. Reverse transcriptase PCR (RT-PCR) analysis was performed using direct sequencing of the obtained fragments. The causative genetic defect was mapped to chromosome 15q26.3. A homozygous G>A substitution (c.666G>A) at the last nucleotide of exon 6 in the ALDH1A3 gene was identified in the first family. Further cDNA sequencing of ALDH1A3 showed that the c.666G>A mutation caused skipping of exon 6, which predicted in-frame loss of 43 amino acids (p.Trp180_Glu222del). A novel missense c.1398C>A mutation in exon 12 of ALDH1A3 that causes the substitution of a conserved asparagine by lysine at amino acid position 466 (p.Asn466Lys) was observed in the second family. No extraocular findings-except for nevus flammeus in one affected individual and a variant of Dandy-Walker malformation in another affected individual-were observed. Autistic-like behaviour and mental retardation were observed in three cases. In conclusion, novel ALDH1A3 mutations identified in the present study confirm the pivotal role of ALDH1A3 in human eye development. Autistic features, previously reported as an associated finding, were considered to be the result of social deprivation and inadequate parenting during early infancy in the presented families. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome.

PubMed

Onozawa, Masahiro; Zhang, Zhenhua; Kim, Yoo Jung; Goldberg, Liat; Varga, Tamas; Bergsagel, P Leif; Kuehl, W Michael; Aplan, Peter D

2014-05-27

We used the I-SceI endonuclease to produce DNA double-strand breaks (DSBs) and observed that a fraction of these DSBs were repaired by insertion of sequences, which we termed "templated sequence insertions" (TSIs), derived from distant regions of the genome. These TSIs were derived from genic, retrotransposon, or telomere sequences and were not deleted from the donor site in the genome, leading to the hypothesis that they were derived from reverse-transcribed RNA. Cotransfection of RNA and an I-SceI expression vector demonstrated insertion of RNA-derived sequences at the DNA-DSB site, and TSIs were suppressed by reverse-transcriptase inhibitors. Both observations support the hypothesis that TSIs were derived from RNA templates. In addition, similar insertions were detected at sites of DNA DSBs induced by transcription activator-like effector nuclease proteins. Whole-genome sequencing of myeloma cell lines revealed additional TSIs, demonstrating that repair of DNA DSBs via insertion was not restricted to experimentally produced DNA DSBs. Analysis of publicly available databases revealed that many of these TSIs are polymorphic in the human genome. Taken together, these results indicate that insertional events should be considered as alternatives to gross chromosomal rearrangements in the interpretation of whole-genome sequence data and that this mutagenic form of DNA repair may play a role in genetic disease, exon shuffling, and mammalian evolution.

Photoaffinity labeling of the primer binding domain in murine leukemia virus reverse transcriptase.

PubMed

Tirumalai, R S; Modak, M J

1991-07-02

We have labeled the primer binding domain of murine leukemia virus reverse transcriptase (MuLV RT) by covalently cross-linking 5' end labeled d(T)8 to MuLV RT, using ultraviolet light energy. The specificity and the functional significance of the primer cross-linking reaction were demonstrated by the fact that (i) other oligomeric primers, tRNAs, and also template-primers readily compete with radiolabeled d(T)8 for the cross-linking reaction, (ii) under similar conditions, the competing primers and template-primer also inhibit the DNA polymerase activity of MuLV RT to a similar extent, (iii) substrate deoxynucleotides have no effect, and (iv) the reaction is sensitive to high ionic strength. In order to identify the primer binding domains/sites in MuLV RT; tryptic digests prepared from the covalently cross-linked MuLV RT and [32P]d(T)8 complexes were resolved on C-18 columns by reverse-phase HPLC. Three distinct radiolabeled peptides were found to contain the majority of the bound primer. Of these, peptide I contained approximately 65% radioactivity, while the remainder was associated with peptides II and III. Amino acid composition and sequence analyses of the individual peptides revealed that peptide I spans amino acid residues 72-80 in the primary amino acid sequence of MuLV RT and is located in the polymerase domain. The primer cross-linking site appears to be at or near Pro-76. Peptides II and III span amino acid residues 602-609 and 615-622, respectively, and are located in the RNase H domain. The probable cross-linking sites in peptides II and III are suggested to be at or near Leu-604 and Leu-618, respectively.

Transmitted Antiretroviral Drug Resistance in Newly HIV-Infected and Untreated Patients in Ségou and Bamako, Mali

PubMed Central

Fofana, Djeneba Bocar; Maiga, Aichatou Chehy; Diallo, Fodie; Ait-Arkoub, Zaina; Daou, Fatoumata; Cisse, Mamadou; Sarro, Yaya dit Sadio; Oumar, Aboubacar Alassane; Sylla, Aliou; Katlama, Christine; Taiwo, Babafemi; Murphy, Robert; Tounkara, Anatole; Marcelin, Anne-Genevieve; Calvez, Vincent

2013-01-01

Abstract The WHO recommends regular surveillance for transmitted antiretroviral drug-resistant viruses in HIV antiretroviral treatment (ART)-naive patients in resource-limited settings. This study aimed to assess the prevalence of mutations associated with resistance in ART-naive patients newly diagnosed with HIV in Bamako and Ségou in Mali. HIV-positive patients who never received ART were recruited in Bamako and Ségou, Mali. The reverse transcriptase (RT) and protease (PR) genes of these patients were sequenced by the “ViroSeq” method. Analysis and interpretation of the resistance were made according to the WHO 2009 list of drug resistance mutations. In all, 51/54 (94.4%) sample patients were sequenced. The median age (IQR) of our patients was 24 (22–27) years and the median CD4 count was 380 (340–456) cells/mm3. The predominant subtype was recombinant HIV-1 CRF02_AG (66.7%) followed by CRF06_cpx (12%) and CRF09_cpx (4%). Four patients had mutations associated with resistance, giving an overall prevalence of resistance estimated at 7.9%. There were two (4%) patients with nucleoside reverse transcriptase inhibitor (NRTI) mutations (one M184V and one T215Y), two (4%) with non-NRTI mutations (two K103N), and one (2%) with a protease inhibitor mutation (one I54V). The prevalence of primary resistance in newly infected patients in Mali is moderate (7.9%). This indicates that the standard NNRTI-based first-line regimen used in Mali is suboptimal for some patients. This study should be done regularly to inform clinical practice. PMID:22823755

Detection of EML4-ALK fusion gene in Chinese non-small cell lung cancer by using a sensitive quantitative real-time reverse transcriptase PCR technique.

PubMed

Fu, Sha; Wang, Fang; Shao, Qiong; Zhang, Xu; Duan, Li-Ping; Zhang, Xiao; Zhang, Li; Shao, Jian-Yong

2015-04-01

Anaplastic lymphoma kinase (ALK) rearrangement is present in approximately 5% of lung adenocarcinoma. Clinical trials on ALK inhibitor phase I to III have shown an interesting disease control rate and acceptable tolerability in ALK rearrangement patients. In clinical application, the precise diagnostic strategy for identifying ALK rearrangements remains to be determined. In this study, ALK rearrangement was screened by using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), direct sequencing, 2 fluorescence in situ hybridization (FISH) assays, and immunohistochemistry in 173 lung adenocarcinomas. We identified 18 cases (10.4%) with EML4-ALK fusion-positive by qRT-PCR, and all were positive for EML4-ALK fusion gene validated by direct sequencing. The result was consistent with that of other methods. Furthermore, of the 18 EML4-ALK fusion-positive cases, 16 (9.2%) were positive by using EML4-ALK fusion probe FISH, and 15 (8.7%) were positive by using ALK break-apart probe FISH and immunohistochemistry staining. Of the 18 ALK fusion-positive lung adenocarcinomas, 8 cases (44.4%) were histologically diagnosed as subtypes of cribriform adenocarcinoma, 7 cases (38.9%) as cribriform adenocarcinoma mixed with papillary and/or mucinous pattern, 2 cases (11.1%) as papillary adenocarcinoma, and 1 case (5.6%) as mucinous adenocarcinoma. In the present study, the ALK rearrangement frequency detected by qRT-PCR in Chinese NSCLC patients was higher than that in the western populations. QRT-PCR is a rapid, sensitive technology that could be used as a screening tool for identifying EML4-ALK fusion-positive NSCLC patients who would be sensitive for receiving ALK inhibitor therapy.

Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus.

PubMed Central

Maurer, B; Bannert, H; Darai, G; Flügel, R M

1988-01-01

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae. Images PMID:2451755

New Subtypes and Genetic Recombination in HIV Type 1-Infecting Patients with Highly Active Antiretroviral Therapy in Peru (2008–2010)

PubMed Central

Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

2012-01-01

Abstract HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country. PMID:22559065

Recurrent TERT promoter mutations identified in a large-scale study of multiple tumour types are associated with increased TERT expression and telomerase activation.

PubMed

Huang, Dong-Sheng; Wang, Zhaohui; He, Xu-Jun; Diplas, Bill H; Yang, Rui; Killela, Patrick J; Meng, Qun; Ye, Zai-Yuan; Wang, Wei; Jiang, Xiao-Ting; Xu, Li; He, Xiang-Lei; Zhao, Zhong-Sheng; Xu, Wen-Juan; Wang, Hui-Ju; Ma, Ying-Yu; Xia, Ying-Jie; Li, Li; Zhang, Ru-Xuan; Jin, Tao; Zhao, Zhong-Kuo; Xu, Ji; Yu, Sheng; Wu, Fang; Liang, Junbo; Wang, Sizhen; Jiao, Yuchen; Yan, Hai; Tao, Hou-Quan

2015-05-01

Several somatic mutation hotspots were recently identified in the telomerase reverse transcriptase (TERT) promoter region in human cancers. Large scale studies of these mutations in multiple tumour types are limited, in particular in Asian populations. This study aimed to: analyse TERT promoter mutations in multiple tumour types in a large Chinese patient cohort, investigate novel tumour types and assess the functional significance of the mutations. TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumour types and 799 tumour tissues from Chinese cancer patients. Thymic epithelial tumours, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and promoter activity by the luciferase reporter assay. TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%) and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in gastrointestinal stromal tumour (GIST), thymic epithelial tumours, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. TERT promoter mutations are frequent in multiple tumour types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumourigenesis, making them potential therapeutic targets. Copyright © 2015 Elsevier Ltd. All rights reserved.

New subtypes and genetic recombination in HIV type 1-infecting patients with highly active antiretroviral therapy in Peru (2008-2010).

PubMed

Yabar, Carlos Augusto; Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

2012-12-01

HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country.

Minority Human Immunodeficiency Virus Type 1 Variants in Antiretroviral-Naive Persons with Reverse Transcriptase Codon 215 Revertant Mutations▿ †

PubMed Central

Mitsuya, Yumi; Varghese, Vici; Wang, Chunlin; Liu, Tommy F.; Holmes, Susan P.; Jayakumar, Prerana; Gharizadeh, Baback; Ronaghi, Mostafa; Klein, Daniel; Fessel, W. Jeffrey; Shafer, Robert W.

2008-01-01

T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method—ultradeep pyrosequencing (UDPS; 454 Life Sciences)—to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants (“revertant” samples) compared with samples from untreated persons that lack such revertants (“control” samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P = 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants. PMID:18715933

Tenofovir-based regimens associated with less drug resistance in HIV-1-infected Nigerians failing first-line antiretroviral therapy.

PubMed

Etiebet, Mary-Ann A; Shepherd, James; Nowak, Rebecca G; Charurat, Man; Chang, Harry; Ajayi, Samuel; Elegba, Olufunmilayo; Ndembi, Nicaise; Abimiku, Alashle; Carr, Jean K; Eyzaguirre, Lindsay M; Blattner, William A

2013-02-20

In resource-limited settings, HIV-1 drug resistance testing to guide antiretroviral therapy (ART) selection is unavailable. We retrospectively conducted genotypic analysis on archived samples from Nigerian patients who received targeted viral load testing to confirm treatment failure and report their drug resistance mutation patterns. Stored plasma from 349 adult patients on non-nucleoside reverse transcriptase inhibitor (NNRTI) regimens was assayed for HIV-1 RNA viral load, and samples with more than 1000 copies/ml were sequenced in the pol gene. Analysis for resistance mutations utilized the IAS-US 2011 Drug Resistance Mutation list. One hundred and seventy-five samples were genotyped; the majority of the subtypes were G (42.9%) and CRF02_AG (33.7%). Patients were on ART for a median of 27 months. 90% had the M184V/I mutation, 62% had at least one thymidine analog mutation, and 14% had the K65R mutation. 97% had an NNRTI resistance mutation and 47% had at least two etravirine-associated mutations. In multivariate analysis tenofovir-based regimens were less likely to have at least three nucleoside reverse transcriptase inhibitor (NRTI) mutations after adjusting for subtype, previous ART, CD4, and HIV viral load [P < 0.001, odds ratio (OR) 0.04]. 70% of patients on tenofovir-based regimens had at least two susceptible NRTIs to include in a second-line regimen compared with 40% on zidovudine-based regimens (P = 0.04, OR = 3.4). At recognition of treatment failure, patients on tenofovir-based first-line regimens had fewer NRTI drug-resistant mutations and more active NRTI drugs available for second-line regimens. These findings can inform strategies for ART regimen sequencing to optimize long-term HIV treatment outcomes in low-resource settings.

HIP1-ALK, a novel fusion protein identified in lung adenocarcinoma.

PubMed

Hong, Mineui; Kim, Ryong Nam; Song, Ji-Young; Choi, So-Jung; Oh, Ensel; Lira, Maruja E; Mao, Mao; Takeuchi, Kengo; Han, Joungho; Kim, Jhingook; Choi, Yoon-La

2014-03-01

The most common mechanism underlying overexpression and activation of anaplastic lymphoma kinase (ALK) in non-small-cell lung carcinoma could be attributed to the formation of a fusion protein. To date, five fusion partners of ALK have been reported, namely, echinoderm microtubule associated protein like 4, tropomyosin-related kinase-fused gene, kinesin family member 5B, kinesin light chain 1, and protein tyrosine phosphatase, nonreceptor type 3. In this article, we report a novel fusion gene huntingtin interacting protein 1 (HIP1)-ALK, which is conjoined between the huntingtin-interacting protein 1 gene HIP1 and ALK. Reverse-transcriptase polymerase chain reaction and immunohistochemical analysis were used to detect this fusion gene's transcript and protein expression, respectively. We had amplified the full-length cDNA sequence of this novel fusion gene by using 5'-rapid amplification of cDNA ends. The causative genomic translocation t(2;7)(p23;q11.23) for generating this novel fusion gene was verified by using genomic sequencing. The examined adenocarcinoma showed predominant acinar pattern, and ALK immunostaining was localized to the cytoplasm, with intense staining in the submembrane region. In break-apart, fluorescence in situ hybridization analysis for ALK, split of the 5' and 3' probe signals, and isolated 3' signals were observed. Reverse-transcriptase polymerase chain reaction revealed that the tumor harbored a novel fusion transcript in which exon 21 of HIP1 was fused to exon 20 of ALK in-frame. The novel fusion gene and its protein HIP1-ALK harboring epsin N-terminal homology, coiled-coil, juxtamembrane, and kinase domains, which could play a role in carcinogenesis, could become diagnostic and therapeutic target of the lung adenocarcinoma and deserve a further study in the future.

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses.

PubMed

Alves-Costa, Fernanda A; Wasko, A P

2010-03-01

Differentially expressed genes in males and females of vertebrate species generally have been investigated in gonads and, to a lesser extent, in other tissues. Therefore, we attempted to identify sexually dimorphic gene expression in the brains of adult males and females of Leporinus macrocephalus, a gonochoristic fish species that presents a ZZ/ZW sex determination system, throughout a comparative analysis using differential display reverse transcriptase-PCR and real-time PCR. Four cDNA fragments were characterized, representing candidate genes with differential expression between the samples. Two of these fragments presented no significant identity with previously reported gene sequences. The other two fragments, isolated from male specimens, were associated to the gene that codes for the protein APBA2 (amyloid beta (A4) precursor protein-binding, family A, member 2) and to the Rab 37 gene, a member of the Ras oncogene family. The overexpression of these genes has been associated to a greater production of the beta-amyloid protein which, in turns, is the major factor that leads to Alzheimer's disease, and to the development of brain-tumors, respectively. Quantitative RT-PCR analyses revealed a higher Apba2 gene expression in males, thus validating the previous data on differential display. L. macrocephalus may represent an interesting animal model to the understanding of the function of several vertebrate genes, including those involved in neurodegenerative and cancer diseases.

Comparison of simple sequence repeats in 19 Archaea.

PubMed

Trivedi, S

2006-12-05

All organisms that have been studied until now have been found to have differential distribution of simple sequence repeats (SSRs), with more SSRs in intergenic than in coding sequences. SSR distribution was investigated in Archaea genomes where complete chromosome sequences of 19 Archaea were analyzed with the program SPUTNIK to find di- to penta-nucleotide repeats. The number of repeats was determined for the complete chromosome sequences and for the coding and non-coding sequences. Different from what has been found for other groups of organisms, there is an abundance of SSRs in coding regions of the genome of some Archaea. Dinucleotide repeats were rare and CG repeats were found in only two Archaea. In general, trinucleotide repeats are the most abundant SSR motifs; however, pentanucleotide repeats are abundant in some Archaea. Some of the tetranucleotide and pentanucleotide repeat motifs are organism specific. In general, repeats are short and CG-rich repeats are present in Archaea having a CG-rich genome. Among the 19 Archaea, SSR density was not correlated with genome size or with optimum growth temperature. Pentanucleotide density had an inverse correlation with the CG content of the genome.

Association of Amine-Receptor DNA Sequence Variants with Associative Learning in the Honeybee.

PubMed

Lagisz, Malgorzata; Mercer, Alison R; de Mouzon, Charlotte; Santos, Luana L S; Nakagawa, Shinichi

2016-03-01

Octopamine- and dopamine-based neuromodulatory systems play a critical role in learning and learning-related behaviour in insects. To further our understanding of these systems and resulting phenotypes, we quantified DNA sequence variations at six loci coding octopamine-and dopamine-receptors and their association with aversive and appetitive learning traits in a population of honeybees. We identified 79 polymorphic sequence markers (mostly SNPs and a few insertions/deletions) located within or close to six candidate genes. Intriguingly, we found that levels of sequence variation in the protein-coding regions studied were low, indicating that sequence variation in the coding regions of receptor genes critical to learning and memory is strongly selected against. Non-coding and upstream regions of the same genes, however, were less conserved and sequence variations in these regions were weakly associated with between-individual differences in learning-related traits. While these associations do not directly imply a specific molecular mechanism, they suggest that the cross-talk between dopamine and octopamine signalling pathways may influence olfactory learning and memory in the honeybee.

Coherent direct sequence optical code multiple access encoding-decoding efficiency versus wavelength detuning.

PubMed

Pastor, D; Amaya, W; García-Olcina, R; Sales, S

2007-07-01

We present a simple theoretical model of and the experimental verification for vanishing of the autocorrelation peak due to wavelength detuning on the coding-decoding process of coherent direct sequence optical code multiple access systems based on a superstructured fiber Bragg grating. Moreover, the detuning vanishing effect has been explored to take advantage of this effect and to provide an additional degree of multiplexing and/or optical code tuning.

FOURTH SEMINAR TO THE MEMORY OF D.N. KLYSHKO: Algebraic solution of the synthesis problem for coded sequences

NASA Astrophysics Data System (ADS)

Leukhin, Anatolii N.

2005-08-01

The algebraic solution of a 'complex' problem of synthesis of phase-coded (PC) sequences with the zero level of side lobes of the cyclic autocorrelation function (ACF) is proposed. It is shown that the solution of the synthesis problem is connected with the existence of difference sets for a given code dimension. The problem of estimating the number of possible code combinations for a given code dimension is solved. It is pointed out that the problem of synthesis of PC sequences is related to the fundamental problems of discrete mathematics and, first of all, to a number of combinatorial problems, which can be solved, as the number factorisation problem, by algebraic methods by using the theory of Galois fields and groups.

Evaluating the protein coding potential of exonized transposable element sequences

PubMed Central

Piriyapongsa, Jittima; Rutledge, Mark T; Patel, Sanil; Borodovsky, Mark; Jordan, I King

2007-01-01

Background Transposable element (TE) sequences, once thought to be merely selfish or parasitic members of the genomic community, have been shown to contribute a wide variety of functional sequences to their host genomes. Analysis of complete genome sequences have turned up numerous cases where TE sequences have been incorporated as exons into mRNAs, and it is widely assumed that such 'exonized' TEs encode protein sequences. However, the extent to which TE-derived sequences actually encode proteins is unknown and a matter of some controversy. We have tried to address this outstanding issue from two perspectives: i-by evaluating ascertainment biases related to the search methods used to uncover TE-derived protein coding sequences (CDS) and ii-through a probabilistic codon-frequency based analysis of the protein coding potential of TE-derived exons. Results We compared the ability of three classes of sequence similarity search methods to detect TE-derived sequences among data sets of experimentally characterized proteins: 1-a profile-based hidden Markov model (HMM) approach, 2-BLAST methods and 3-RepeatMasker. Profile based methods are more sensitive and more selective than the other methods evaluated. However, the application of profile-based search methods to the detection of TE-derived sequences among well-curated experimentally characterized protein data sets did not turn up many more cases than had been previously detected and nowhere near as many cases as recent genome-wide searches have. We observed that the different search methods used were complementary in the sense that they yielded largely non-overlapping sets of hits and differed in their ability to recover known cases of TE-derived CDS. The probabilistic analysis of TE-derived exon sequences indicates that these sequences have low protein coding potential on average. In particular, non-autonomous TEs that do not encode protein sequences, such as Alu elements, are frequently exonized but unlikely to encode protein sequences. Conclusion The exaptation of the numerous TE sequences found in exons as bona fide protein coding sequences may prove to be far less common than has been suggested by the analysis of complete genomes. We hypothesize that many exonized TE sequences actually function as post-transcriptional regulators of gene expression, rather than coding sequences, which may act through a variety of double stranded RNA related regulatory pathways. Indeed, their relatively high copy numbers and similarity to sequences dispersed throughout the genome suggests that exonized TE sequences could serve as master regulators with a wide scope of regulatory influence. Reviewers: This article was reviewed by Itai Yanai, Kateryna D. Makova, Melissa Wilson (nominated by Kateryna D. Makova) and Cedric Feschotte (nominated by John M. Logsdon Jr.). PMID:18036258

RAMICS: trainable, high-speed and biologically relevant alignment of high-throughput sequencing reads to coding DNA

PubMed Central

Wright, Imogen A.; Travers, Simon A.

2014-01-01

The challenge presented by high-throughput sequencing necessitates the development of novel tools for accurate alignment of reads to reference sequences. Current approaches focus on using heuristics to map reads quickly to large genomes, rather than generating highly accurate alignments in coding regions. Such approaches are, thus, unsuited for applications such as amplicon-based analysis and the realignment phase of exome sequencing and RNA-seq, where accurate and biologically relevant alignment of coding regions is critical. To facilitate such analyses, we have developed a novel tool, RAMICS, that is tailored to mapping large numbers of sequence reads to short lengths (<10 000 bp) of coding DNA. RAMICS utilizes profile hidden Markov models to discover the open reading frame of each sequence and aligns to the reference sequence in a biologically relevant manner, distinguishing between genuine codon-sized indels and frameshift mutations. This approach facilitates the generation of highly accurate alignments, accounting for the error biases of the sequencing machine used to generate reads, particularly at homopolymer regions. Performance improvements are gained through the use of graphics processing units, which increase the speed of mapping through parallelization. RAMICS substantially outperforms all other mapping approaches tested in terms of alignment quality while maintaining highly competitive speed performance. PMID:24861618

Scarabaecin, a novel cysteine-containing antifungal peptide from the rhinoceros beetle, Oryctes rhinoceros.

PubMed

Tomie, Tetsuya; Ishibashi, Jun; Furukawa, Seiichi; Kobayashi, Satoe; Sawahata, Ryoko; Asaoka, Ai; Tagawa, Michito; Yamakawa, Minoru

2003-07-25

A novel antifungal peptide, scarabaecin (4080Da), was isolated from the coconut rhinoceros beetle, Oryctes rhinoceros. Scarabaecin cDNA was cloned by reverse transcriptase-polymerase chain reactions (RT-PCR) using a primer based on the N-terminal amino acid sequence. The amino acid sequence deduced from scarabaecin cDNA showed no significant similarity to those of reported proteins. Chemically synthesized scarabaecin indicated antifungal activity against phytopathogenic fungi such as Pyricularia oryzae, Rhizoctonia solani, and Botrytis cinerea, but not against phytopathogenic bacteria. It showed weak activity against Bauberia bassiana, an insect pathogenic fungus, and Staphylococcus aureus, a pathogenic bacterium. Scarabaecin showed chitin binding property and its K(d) was 1.315 microM. A comparison of putative chitin-binding domains among scarabaecin, invertebrate, and plant chitin-binding proteins suggests that scarabaecin is a new member of chitin-binding antimicrobial proteins.

Breast-milk shedding of drug-resistant HIV-1 subtype C in women exposed to single-dose nevirapine.

PubMed

Lee, Esther J; Kantor, Rami; Zijenah, Lynn; Sheldon, Wayne; Emel, Lynda; Mateta, Patrick; Johnston, Elizabeth; Wells, Jennifer; Shetty, Avinash K; Coovadia, Hoosen; Maldonado, Yvonne; Jones, Samuel Adeniyi; Mofenson, Lynne M; Contag, Christopher H; Bassett, Mary; Katzenstein, David A

2005-10-01

Single-dose nevirapine reduces intrapartum human immunodeficiency virus 1 type (HIV-1) transmission but may also select for nonnucleoside reverse-transcriptase inhibitor (NNRTI) resistance in breast milk (BM) and plasma. Among 32 Zimbabwean women, median 8-week postpartum plasma and BM HIV-1 RNA levels were 4.57 and 2.13 log(10) copies/mL, respectively. BM samples from women with laboratory-diagnosed mastitis (defined as elevated BM Na(+) levels) were 5.4-fold more likely to have HIV-1 RNA levels above the median. BM RT sequences were not obtained for 12 women with BM HIV-1 RNA levels below the lower limit of detection of the assay used. In 20 paired BM and plasma samples, 65% of BM and 50% of plasma RT sequences had NNRTI-resistance mutations, with divergent mutation patterns.

Partial 16S rRNA primary structure of five Actinomyces species: phylogenetic implications and development of an Actinomyces israelii-specific oligonucleotide probe.

PubMed

Stackebrandt, E; Charfreitag, O

1990-01-01

The intra- and intergeneric relationships of the genus Actinomyces were determined by comparing long 16S rRNA sequences, generated by reverse transcriptase. All species formed a phylogenetically coherent cluster in which Actinomyces bovis, A. viscosus, A. naeslundii, A. odontolyticus and A. israelii constituted genetically well defined species. A. israelii DSM 43322 (serotype 2) was not closely related to three other strains of this species (serotype 1) and, as judged from phylogenetic distances, could be accommodated within A. naeslundii, or represent a new species. In contrast to previous findings, members of the genus Actinomyces appear to be related to Bifidobacterium bifidum. Sequence information was used to develop an oligonucleotide probe for the A. israelii serotype 1 strains, which did not react with the serotype 2 strain or with rRNA from strains of eight Actinomyces species.

Genomic Sequence around Butterfly Wing Development Genes: Annotation and Comparative Analysis

PubMed Central

Conceição, Inês C.; Long, Anthony D.; Gruber, Jonathan D.; Beldade, Patrícia

2011-01-01

Background Analysis of genomic sequence allows characterization of genome content and organization, and access beyond gene-coding regions for identification of functional elements. BAC libraries, where relatively large genomic regions are made readily available, are especially useful for species without a fully sequenced genome and can increase genomic coverage of phylogenetic and biological diversity. For example, no butterfly genome is yet available despite the unique genetic and biological properties of this group, such as diversified wing color patterns. The evolution and development of these patterns is being studied in a few target species, including Bicyclus anynana, where a whole-genome BAC library allows targeted access to large genomic regions. Methodology/Principal Findings We characterize ∼1.3 Mb of genomic sequence around 11 selected genes expressed in B. anynana developing wings. Extensive manual curation of in silico predictions, also making use of a large dataset of expressed genes for this species, identified repetitive elements and protein coding sequence, and highlighted an expansion of Alcohol dehydrogenase genes. Comparative analysis with orthologous regions of the lepidopteran reference genome allowed assessment of conservation of fine-scale synteny (with detection of new inversions and translocations) and of DNA sequence (with detection of high levels of conservation of non-coding regions around some, but not all, developmental genes). Conclusions The general properties and organization of the available B. anynana genomic sequence are similar to the lepidopteran reference, despite the more than 140 MY divergence. Our results lay the groundwork for further studies of new interesting findings in relation to both coding and non-coding sequence: 1) the Alcohol dehydrogenase expansion with higher similarity between the five tandemly-repeated B. anynana paralogs than with the corresponding B. mori orthologs, and 2) the high conservation of non-coding sequence around the genes wingless and Ecdysone receptor, both involved in multiple developmental processes including wing pattern formation. PMID:21909358

Speech processing using conditional observable maximum likelihood continuity mapping

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hogden, John; Nix, David

A computer implemented method enables the recognition of speech and speech characteristics. Parameters are initialized of first probability density functions that map between the symbols in the vocabulary of one or more sequences of speech codes that represent speech sounds and a continuity map. Parameters are also initialized of second probability density functions that map between the elements in the vocabulary of one or more desired sequences of speech transcription symbols and the continuity map. The parameters of the probability density functions are then trained to maximize the probabilities of the desired sequences of speech-transcription symbols. A new sequence ofmore » speech codes is then input to the continuity map having the trained first and second probability function parameters. A smooth path is identified on the continuity map that has the maximum probability for the new sequence of speech codes. The probability of each speech transcription symbol for each input speech code can then be output.« less

SequenceL: Automated Parallel Algorithms Derived from CSP-NT Computational Laws

NASA Technical Reports Server (NTRS)

Cooke, Daniel; Rushton, Nelson

2013-01-01

With the introduction of new parallel architectures like the cell and multicore chips from IBM, Intel, AMD, and ARM, as well as the petascale processing available for highend computing, a larger number of programmers will need to write parallel codes. Adding the parallel control structure to the sequence, selection, and iterative control constructs increases the complexity of code development, which often results in increased development costs and decreased reliability. SequenceL is a high-level programming language that is, a programming language that is closer to a human s way of thinking than to a machine s. Historically, high-level languages have resulted in decreased development costs and increased reliability, at the expense of performance. In recent applications at JSC and in industry, SequenceL has demonstrated the usual advantages of high-level programming in terms of low cost and high reliability. SequenceL programs, however, have run at speeds typically comparable with, and in many cases faster than, their counterparts written in C and C++ when run on single-core processors. Moreover, SequenceL is able to generate parallel executables automatically for multicore hardware, gaining parallel speedups without any extra effort from the programmer beyond what is required to write the sequen tial/singlecore code. A SequenceL-to-C++ translator has been developed that automatically renders readable multithreaded C++ from a combination of a SequenceL program and sample data input. The SequenceL language is based on two fundamental computational laws, Consume-Simplify- Produce (CSP) and Normalize-Trans - pose (NT), which enable it to automate the creation of parallel algorithms from high-level code that has no annotations of parallelism whatsoever. In our anecdotal experience, SequenceL development has been in every case less costly than development of the same algorithm in sequential (that is, single-core, single process) C or C++, and an order of magnitude less costly than development of comparable parallel code. Moreover, SequenceL not only automatically parallelizes the code, but since it is based on CSP-NT, it is provably race free, thus eliminating the largest quality challenge the parallelized software developer faces.

ANN modeling of DNA sequences: new strategies using DNA shape code.

PubMed

Parbhane, R V; Tambe, S S; Kulkarni, B D

2000-09-01

Two new encoding strategies, namely, wedge and twist codes, which are based on the DNA helical parameters, are introduced to represent DNA sequences in artificial neural network (ANN)-based modeling of biological systems. The performance of the new coding strategies has been evaluated by conducting three case studies involving mapping (modeling) and classification applications of ANNs. The proposed coding schemes have been compared rigorously and shown to outperform the existing coding strategies especially in situations wherein limited data are available for building the ANN models.

The primitive code and repeats of base oligomers as the primordial protein-encoding sequence.

PubMed Central

Ohno, S; Epplen, J T

1983-01-01

Even if the prebiotic self-replication of nucleic acids and the subsequent emergence of primitive, enzyme-independent tRNAs are accepted as plausible, the origin of life by spontaneous generation still appears improbable. This is because the just-emerged primitive translational machinery had to cope with base sequences that were not preselected for their coding potentials. Particularly if the primitive mitochondria-like code with four chain-terminating base triplets preceded the universal code, the translation of long, randomly generated, base sequences at this critical stage would have merely resulted in the production of short oligopeptides instead of long polypeptide chains. We present the base sequence of a mouse transcript containing tetranucleotide repeats conserved during evolution. Even if translated in accordance with the primitive mitochondria-like code, this transcript in its three reading frames can yield 245-, 246-, and 251-residue-long tetrapeptidic periodical polypeptides that are already acquiring longer periodicities. We contend that the first set of base sequences translated at the beginning of life were such oligonucleotide repeats. By quickly acquiring longer periodicities, their products must have soon gained characteristic secondary structures--alpha-helical or beta-sheet or both. PMID:6574491

Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis.

PubMed

Smola, Matthew J; Rice, Greggory M; Busan, Steven; Siegfried, Nathan A; Weeks, Kevin M

2015-11-01

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPE probing and to construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles and entire transcriptomes.

Phylogenetic analysis of rubella virus strains during an outbreak in São Paulo, 2007-2008.

PubMed

Figueiredo, C A; Oliveira, M I; Curti, S P; Afonso, A M S; Frugi Yu, A L; Gualberto, F; Durigon, E L

2012-10-01

Rubella virus (RV) is an important human pathogen that causes rubella, an acute contagious disease. It also causes severe birth defects collectively known as congenital rubella syndrome when infection occurs during the first trimester of pregnancy. Here, we present the phylogenetic analysis of RV that circulated in São Paulo during the 2007-2008 outbreak. Samples collected from patients diagnosed with rubella were isolated in cell culture and sequenced. RV RNA was obtained from samples or RV-infected cell cultures and amplified by reverse transcriptase-polymerase chain reaction. Sequences were assigned to genotypes by phylogenetic analysis using RV reference sequences. Seventeen sequences were analyzed, and three genotypes were identified: 1a, 1G, and 2B. Genotypes 1a and 1G, which were isolated in 2007, were responsible for sporadic rubella cases in São Paulo. Thereafter, in late 2007, the epidemiological conditions changed, resulting in a large RV outbreak with the clear dominance of genotype 2B. The results of this study provide new approaches for monitoring the progress of elimination of rubella from São Paulo, Brazil. Copyright © 2012 Wiley Periodicals, Inc.

Insights about minority HIV-1 strains in transmitted drug resistance mutation dynamics and disease progression.

PubMed

Leda, Ana Rachel; Hunter, James; Oliveira, Ursula Castro; Azevedo, Inacio Junqueira; Sucupira, Maria Cecilia Araripe; Diaz, Ricardo Sobhie

2018-04-19

The presence of minority transmitted drug resistance mutations was assessed using ultra-deep sequencing and correlated with disease progression among recently HIV-1-infected individuals from Brazil. Samples at baseline during recent infection and 1 year after the establishment of the infection were analysed. Viral RNA and proviral DNA from 25 individuals were subjected to ultra-deep sequencing of the reverse transcriptase and protease regions of HIV-1. Viral strains carrying transmitted drug resistance mutations were detected in 9 out of the 25 patients, for all major antiretroviral classes, ranging from one to five mutations per patient. Ultra-deep sequencing detected strains with frequencies as low as 1.6% and only strains with frequencies >20% were detected by population plasma sequencing (three patients). Transmitted drug resistance strains with frequencies <14.8% did not persist upon established infection. The presence of transmitted drug resistance mutations was negatively correlated with the viral load and with CD4+ T cell count decay. Transmitted drug resistance mutations representing small percentages of the viral population do not persist during infection because they are negatively selected in the first year after HIV-1 seroconversion.

A conserved segmental duplication within ELA.

PubMed

Brinkmeyer-Langford, C L; Murphy, W J; Childers, C P; Skow, L C

2010-12-01

The assembled genomic sequence of the horse major histocompatibility complex (MHC) (equine lymphocyte antigen, ELA) is very similar to the homologous human HLA, with the notable exception of a large segmental duplication at the boundary of ELA class I and class III that is absent in HLA. The segmental duplication consists of a ∼ 710 kb region of at least 11 repeated blocks: 10 blocks each contain an MHC class I-like sequence and the helicase domain portion of a BAT1-like sequence, and the remaining unit contains the full-length BAT1 gene. Similar genomic features were found in other Perissodactyls, indicating an ancient origin, which is consistent with phylogenetic analyses. Reverse-transcriptase PCR (RT-PCR) of mRNA from peripheral white blood cells of healthy and chronically or acutely infected horses detected transcription from predicted open reading frames in several of the duplicated blocks. This duplication is not present in the sequenced MHCs of most other mammals, although a similar feature at the same relative position is present in the feline MHC (FLA). Striking sequence conservation throughout Perissodactyl evolution is consistent with a functional role for at least some of the genes included within this segmental duplication. © 2010 The Authors, Journal compilation © 2010 Stichting International Foundation for Animal Genetics.

Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes

PubMed Central

Lee, Michael; Hills, Mark; Conomos, Dimitri; Stutz, Michael D.; Dagg, Rebecca A.; Lau, Loretta M.S.; Reddel, Roger R.; Pickett, Hilda A.

2014-01-01

Telomeres are terminal repetitive DNA sequences on chromosomes, and are considered to comprise almost exclusively hexameric TTAGGG repeats. We have evaluated telomere sequence content in human cells using whole-genome sequencing followed by telomere read extraction in a panel of mortal cell strains and immortal cell lines. We identified a wide range of telomere variant repeats in human cells, and found evidence that variant repeats are generated by mechanistically distinct processes during telomerase- and ALT-mediated telomere lengthening. Telomerase-mediated telomere extension resulted in biased repeat synthesis of variant repeats that differed from the canonical sequence at positions 1 and 3, but not at positions 2, 4, 5 or 6. This indicates that telomerase is most likely an error-prone reverse transcriptase that misincorporates nucleotides at specific positions on the telomerase RNA template. In contrast, cell lines that use the ALT pathway contained a large range of variant repeats that varied greatly between lines. This is consistent with variant repeats spreading from proximal telomeric regions throughout telomeres in a stochastic manner by recombination-mediated templating of DNA synthesis. The presence of unexpectedly large numbers of variant repeats in cells utilizing either telomere maintenance mechanism suggests a conserved role for variant sequences at human telomeres. PMID:24225324

Genomics dataset of unidentified disclosed isolates.

PubMed

Rekadwad, Bhagwan N

2016-09-01

Analysis of DNA sequences is necessary for higher hierarchical classification of the organisms. It gives clues about the characteristics of organisms and their taxonomic position. This dataset is chosen to find complexities in the unidentified DNA in the disclosed patents. A total of 17 unidentified DNA sequences were thoroughly analyzed. The quick response codes were generated. AT/GC content of the DNA sequences analysis was carried out. The QR is helpful for quick identification of isolates. AT/GC content is helpful for studying their stability at different temperatures. Additionally, a dataset on cleavage code and enzyme code studied under the restriction digestion study, which helpful for performing studies using short DNA sequences was reported. The dataset disclosed here is the new revelatory data for exploration of unique DNA sequences for evaluation, identification, comparison and analysis.

CDSbank: taxonomy-aware extraction, selection, renaming and formatting of protein-coding DNA or amino acid sequences.

PubMed

Hazes, Bart

2014-02-28

Protein-coding DNA sequences and their corresponding amino acid sequences are routinely used to study relationships between sequence, structure, function, and evolution. The rapidly growing size of sequence databases increases the power of such comparative analyses but it makes it more challenging to prepare high quality sequence data sets with control over redundancy, quality, completeness, formatting, and labeling. Software tools for some individual steps in this process exist but manual intervention remains a common and time consuming necessity. CDSbank is a database that stores both the protein-coding DNA sequence (CDS) and amino acid sequence for each protein annotated in Genbank. CDSbank also stores Genbank feature annotation, a flag to indicate incomplete 5' and 3' ends, full taxonomic data, and a heuristic to rank the scientific interest of each species. This rich information allows fully automated data set preparation with a level of sophistication that aims to meet or exceed manual processing. Defaults ensure ease of use for typical scenarios while allowing great flexibility when needed. Access is via a free web server at http://hazeslab.med.ualberta.ca/CDSbank/. CDSbank presents a user-friendly web server to download, filter, format, and name large sequence data sets. Common usage scenarios can be accessed via pre-programmed default choices, while optional sections give full control over the processing pipeline. Particular strengths are: extract protein-coding DNA sequences just as easily as amino acid sequences, full access to taxonomy for labeling and filtering, awareness of incomplete sequences, and the ability to take one protein sequence and extract all synonymous CDS or identical protein sequences in other species. Finally, CDSbank can also create labeled property files to, for instance, annotate or re-label phylogenetic trees.

Variability and transmission by Aphis glycines of North American and Asian Soybean mosaic virus isolates.

PubMed

Domier, L L; Latorre, I J; Steinlage, T A; McCoppin, N; Hartman, G L

2003-10-01

The variability of North American and Asian strains and isolates of Soybean mosaic virus was investigated. First, polymerase chain reaction (PCR) products representing the coat protein (CP)-coding regions of 38 SMVs were analyzed for restriction fragment length polymorphisms (RFLP). Second, the nucleotide and predicted amino acid sequence variability of the P1-coding region of 18 SMVs and the helper component/protease (HC/Pro) and CP-coding regions of 25 SMVs were assessed. The CP nucleotide and predicted amino acid sequences were the most similar and predicted phylogenetic relationships similar to those obtained from RFLP analysis. Neither RFLP nor sequence analyses of the CP-coding regions grouped the SMVs by geographical origin. The P1 and HC/Pro sequences were more variable and separated the North American and Asian SMV isolates into two groups similar to previously reported differences in pathogenic diversity of the two sets of SMV isolates. The P1 region was the most informative of the three regions analyzed. To assess the biological relevance of the sequence differences in the HC/Pro and CP coding regions, the transmissibility of 14 SMV isolates by Aphis glycines was tested. All field isolates of SMV were transmitted efficiently by A. glycines, but the laboratory isolates analyzed were transmitted poorly. The amino acid sequences from most, but not all, of the poorly transmitted isolates contained mutations in the aphid transmission-associated DAG and/or KLSC amino acid sequence motifs of CP and HC/Pro, respectively.

Closed Genome Sequence of Chryseobacterium piperi Strain CTMT/ATCC BAA-1782, a Gram-Negative Bacterium with Clostridial Neurotoxin-Like Coding Sequences

PubMed Central

Wentz, Travis G.; Muruvanda, Tim; Thirunavukkarasu, Nagarajan; Hoffmann, Maria; Allard, Marc W.; Hodge, David R.; Pillai, Segaran P.; Hammack, Thomas S.; Brown, Eric W.

2017-01-01

ABSTRACT Clostridial neurotoxins, including botulinum and tetanus neurotoxins, are among the deadliest known bacterial toxins. Until recently, the horizontal mobility of this toxin gene family appeared to be limited to the genus Clostridium. We report here the closed genome sequence of Chryseobacterium piperi, a Gram-negative bacterium containing coding sequences with homology to clostridial neurotoxin family proteins. PMID:29192076

Improving the genome annotation of the acarbose producer Actinoplanes sp. SE50/110 by sequencing enriched 5'-ends of primary transcripts.

PubMed

Schwientek, Patrick; Neshat, Armin; Kalinowski, Jörn; Klein, Andreas; Rückert, Christian; Schneiker-Bekel, Susanne; Wendler, Sergej; Stoye, Jens; Pühler, Alfred

2014-11-20

Actinoplanes sp. SE50/110 is the producer of the alpha-glucosidase inhibitor acarbose, which is an economically relevant and potent drug in the treatment of type-2 diabetes mellitus. In this study, we present the detection of transcription start sites on this genome by sequencing enriched 5'-ends of primary transcripts. Altogether, 1427 putative transcription start sites were initially identified. With help of the annotated genome sequence, 661 transcription start sites were found to belong to the leader region of protein-coding genes with the surprising result that roughly 20% of these genes rank among the class of leaderless transcripts. Next, conserved promoter motifs were identified for protein-coding genes with and without leader sequences. The mapped transcription start sites were finally used to improve the annotation of the Actinoplanes sp. SE50/110 genome sequence. Concerning protein-coding genes, 41 translation start sites were corrected and 9 novel protein-coding genes could be identified. In addition to this, 122 previously undetermined non-coding RNA (ncRNA) genes of Actinoplanes sp. SE50/110 were defined. Focusing on antisense transcription start sites located within coding genes or their leader sequences, it was discovered that 96 of those ncRNA genes belong to the class of antisense RNA (asRNA) genes. The remaining 26 ncRNA genes were found outside of known protein-coding genes. Four chosen examples of prominent ncRNA genes, namely the transfer messenger RNA gene ssrA, the ribonuclease P class A RNA gene rnpB, the cobalamin riboswitch RNA gene cobRS, and the selenocysteine-specific tRNA gene selC, are presented in more detail. This study demonstrates that sequencing of enriched 5'-ends of primary transcripts and the identification of transcription start sites are valuable tools for advanced genome annotation of Actinoplanes sp. SE50/110 and most probably also for other bacteria. Copyright © 2014 Elsevier B.V. All rights reserved.

(PCG) Protein Crystal Growth HIV Reverse Transcriptase

NASA Technical Reports Server (NTRS)

1992-01-01

HIV Reverse Transcriptase crystals grown during the USML-1 (STS-50) mission using Commercial Refrigerator/Incubator Module (CR/IM) at 4 degrees C and the Vapor Diffusion Apparatus (VDA). Reverse transcriptase is an enzyme responsible for copying the nucleic acid genome of the AIDS virus from RNA to DNA. Studies indicated that the space-grown crystals were larger and better ordered (beyond 4 angstroms) than were comparable Earth-grown crystals. Principal Investigators were Charles Bugg and Larry DeLucas.

Identification and characterization of jute LTR retrotransposons:

PubMed Central

Ahmed, Salim; Shafiuddin, MD; Azam, Muhammad Shafiul; Islam, Md. Shahidul; Ghosh, Ajit

2011-01-01

Long Terminal Repeat (LTR) retrotransposons constitute a significant part of eukaryotic genomes and play an important role in genome evolution especially in plants. Jute is an important fiber crop with a large genome of 1,250 Mbps. This genome is still mostly unexplored. In this study we aimed at identifying and characterizing the LTR retrotransposons of jute with a view to understanding the jute genome better. In this study, the Reverse Transcriptase domain of Ty1-copia and Ty3-gypsy LTR retrotransposons of jute were amplified by degenerate primers and their expressions were examined by reverse transcription PCR. Copy numbers of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy elements were determined by dot blot analysis. Sequence analysis revealed higher heterogeneity among Ty1-copia retrotransposons than Ty3-gypsy and clustered each of them in three groups. Copy number of RT genes in Ty1-copia was found to be higher than that of Ty3-gypsy elements from dot blot hybridization. Cumulatively Ty1-copia and Ty3-gypsy may constitute around 19% of the jute genome where two groups of Ty1-copia were found to be transcriptionally active. Since the LTR retrotransposons constitute a large portion of jute genome, these findings imply the importance of these elements in the evolution of jute genome. PMID:22016842

Prevalence and resistance mutations of non-B HIV-1 subtypes among immigrants in Southern Spain along the decade 2000-2010

PubMed Central

2011-01-01

Background Most of the non-B HIV-1 subtypes are predominant in Sub-Saharan Africa and India although they have been found worldwide. In the last decade, immigration from these areas has increased considerably in Spain. The objective of this study was to evaluate the prevalence of non-B subtypes circulating in a cohort of HIV-1-infected immigrants in Seville, Southern Spain and to identify drug resistance-associated mutations. Methods Complete protease and first 220 codons of the reverse transcriptase coding regions were amplified and sequenced by population sequencing. HIV-1 subtypes were determined using Stanford University Drug Resistance Database, and phylogenetic analysis was performed comparing multiple reported sequences. Drug resistance mutations were defined according to the International AIDS Society-USA. Results From 2000 to 2010 a total of 1,089 newly diagnosed HIV-1-infected patients were enrolled in our cohort. Of these, 121 were immigrants, of which 98 had ethical approval and informed consent to include in our study. Twenty-nine immigrants (29/98, 29.6%) were infected with non-B subtypes, of which 15/29 (51.7%) were CRF02-AG, mostly from Sub-Saharan Africa, and 2/29 (6.9%) were CRF01-AE from Eastern Europe. A, C, F, J and G subtypes from Eastern Europe, Central-South America and Sub-Saharan Africa were also present. Some others harboured recombinant forms CRF02-AG/CRF01-AE, CRF2-AG/G and F/B, B/C, and K/G, in PR and RT-coding regions. Patients infected with non-B subtypes showed a high frequency of minor protease inhibitor resistance mutations, M36I, L63P, and K20R/I. Only one patient, CRF02_AG, showed major resistance mutation L90M. Major RT inhibitor resistance mutations K70R and A98G were present in one patient with subtype G, L100I in one patient with CRF01_AE, and K103N in another patient with CRF01_AE. Three patients had other mutations such as V118I, E138A and V90I. Conclusions The circulation of non-B subtypes has significantly increased in Southern Spain during the last decade, with 29.6% prevalence, in association with demographic changes among immigrants. This could be an issue in the treatment and management of these patients. Resistance mutations have been detected in these patients with a prevalence of 7% among treatment-naïve patients compared with the 21% detected among patients under HAART or during treatment interruption. PMID:21871090

Motion Detection in Ultrasound Image-Sequences Using Tensor Voting

NASA Astrophysics Data System (ADS)

Inba, Masafumi; Yanagida, Hirotaka; Tamura, Yasutaka

2008-05-01

Motion detection in ultrasound image sequences using tensor voting is described. We have been developing an ultrasound imaging system adopting a combination of coded excitation and synthetic aperture focusing techniques. In our method, frame rate of the system at distance of 150 mm reaches 5000 frame/s. Sparse array and short duration coded ultrasound signals are used for high-speed data acquisition. However, many artifacts appear in the reconstructed image sequences because of the incompleteness of the transmitted code. To reduce the artifacts, we have examined the application of tensor voting to the imaging method which adopts both coded excitation and synthetic aperture techniques. In this study, the basis of applying tensor voting and the motion detection method to ultrasound images is derived. It was confirmed that velocity detection and feature enhancement are possible using tensor voting in the time and space of simulated ultrasound three-dimensional image sequences.

ISOLATION OF THE REGULATORY REGIONS AND GENOMIC ORGANIZATION OF THE PORCINE α1,3-GALACTOSYLTRANSFERASE GENE1

PubMed Central

Koike, Chihiro; Friday, Robert P.; Nakashima, Izumi; Luppi, Patrizia; Fung, John J.; Rao, Abdul S.; Starzl, Thomas E.; Trucco, Massimo

2010-01-01

Background α1,3-galactosyltransferase (α1,3GT) is an enzyme that produces carbohydrate chains termed αGal epitopes found in most mammals, although some species of higher primates, including human, are notable exceptions. The evolutionary origin of the lost α1,3GT enzyme activity is not yet known, although it has been suggested that the promoter activity of this gene in the ancestors of higher primates was inactivated. Methods We used 5′-or 3′-RACE, GenomeWalking, reverse transcriptase polymerase chain reaction (RT-PCR) and dual Luciferase reporter assay for identification of the full-length cDNA, which includes the transcription initiation site and the promoter region of porcine α1,3GT gene. Results The region around exon 1 is guanine and cytosine (GC)-rich (about 70%), comprising a CpG island spanning more than 1.5 kbp. The 5′-flanking region of exon 1 contains multiple transcription factor consensus motifs, including GC-box, SP1, AP2, and GATA-box sites, in the absence of TATA or CAAT-box sequences. The entire gene consists of three 5′ noncoding and six coding region exons spanning more than 52 kbp. Detailed analysis of α1,3GT transcripts revealed two major alternative splicing patterns in the 5′-untranslated region (5′-UTR) and evidence for minor splicing activity that occurs in a tissue-specific manner. Interspecies comparison of 5′-UTR shows minimal homology between porcine and murine sequences except for exon 2, which suggests that the regulatory regions differ among species. Conclusions These observations have important implications for experiments involving genetic manipulation of the α1,3GT gene in transgenic animals in terms of promoter utilization, and particularly in genetically engineering cells for the animal cloning technology by nuclear transfer. PMID:11087141

Novel Gene Expression Profile of Women with Intrinsic Skin Youthfulness by Whole Transcriptome Sequencing

PubMed Central

Xu, Jin; Spitale, Robert C.; Guan, Linna; Flynn, Ryan A.; Torre, Eduardo A.; Li, Rui; Raber, Inbar; Qu, Kun; Kern, Dale; Knaggs, Helen E.; Chang, Howard Y.; Chang, Anne Lynn S.

2016-01-01

While much is known about genes that promote aging, little is known about genes that protect against or prevent aging, particularly in human skin. The main objective of this study was to perform an unbiased, whole transcriptome search for genes that associate with intrinsic skin youthfulness. To accomplish this, healthy women (n = 122) of European descent, ages 18–89 years with Fitzpatrick skin type I/II were examined for facial skin aging parameters and clinical covariates, including smoking and ultraviolet exposure. Skin youthfulness was defined as the top 10% of individuals whose assessed skin aging features were most discrepant with their chronological ages. Skin biopsies from sun-protected inner arm were subjected to 3’-end sequencing for expression quantification, with results verified by quantitative reverse transcriptase-polymerase chain reaction. Unbiased clustering revealed gene expression signatures characteristic of older women with skin youthfulness (n = 12) compared to older women without skin youthfulness (n = 33), after accounting for gene expression changes associated with chronological age alone. Gene set analysis was performed using Genomica open-access software. This study identified a novel set of candidate skin youthfulness genes demonstrating differences between SY and non-SY group, including pleckstrin homology like domain family A member 1 (PHLDA1) (p = 2.4x10-5), a follicle stem cell marker, and hyaluronan synthase 2-anti-sense 1 (HAS2-AS1) (p = 0.00105), a non-coding RNA that is part of the hyaluronan synthesis pathway. We show that immunologic gene sets are the most significantly altered in skin youthfulness (with the most significant gene set p = 2.4x10-5), suggesting the immune system plays an important role in skin youthfulness, a finding that has not previously been recognized. These results are a valuable resource from which multiple future studies may be undertaken to better understand the mechanisms that promote skin youthfulness in humans. PMID:27829007

microRNAs involved in auxin signalling modulate male sterility under high-temperature stress in cotton (Gossypium hirsutum).

PubMed

Ding, Yuanhao; Ma, Yizan; Liu, Nian; Xu, Jiao; Hu, Qin; Li, Yaoyao; Wu, Yuanlong; Xie, Sai; Zhu, Longfu; Min, Ling; Zhang, Xianlong

2017-09-01

Male sterility caused by long-term high-temperature (HT) stress occurs widely in crops. MicroRNAs (miRNAs), a class of endogenous non-coding small RNAs, play an important role in the plant response to various abiotic stresses. To dissect the working principle of miRNAs in male sterility under HT stress in cotton, a total of 112 known miRNAs, 270 novel miRNAs and 347 target genes were identified from anthers of HT-insensitive (84021) and HT-sensitive (H05) cotton cultivars under normal-temperature and HT conditions through small RNA and degradome sequencing. Quantitative reverse transcriptase-polymerase chain reaction and 5'-RNA ligase-mediated rapid amplification of cDNA ends experiments were used to validate the sequencing data. The results show that miR156 was suppressed by HT stress in both 84021 and H05; miR160 was suppressed in 84021 but induced in H05. Correspondingly, SPLs (target genes of miR156) were induced both in 84021 and H05; ARF10 and ARF17 (target genes of miR160) were induced in 84021 but suppressed in H05. Overexpressing miR160 increased cotton sensitivity to HT stress seen as anther indehiscence, associated with the suppression of ARF10 and ARF17 expression, thereby activating the auxin response that leads to anther indehiscence. Supporting this role for auxin, exogenous Indole-3-acetic acid (IAA) leads to a stronger male sterility phenotype both in 84021 and H05 under HT stress. Cotton plants overexpressing miR157 suppressed the auxin signal, and also showed enhanced sensitivity to HT stress, with microspore abortion and anther indehiscence. Thus, we propose that the auxin signal, mediated by miRNAs, is essential for cotton anther fertility under HT stress. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

MpSaci is a widespread gypsy-Ty3 retrotransposon highly represented by non-autonomous copies in the Moniliophthora perniciosa genome.

PubMed

Pereira, Jorge F; Araújo, Elza F; Brommonschenkel, Sérgio H; Queiroz, Casley B; Costa, Gustavo G L; Carazzolle, Marcelo F; Pereira, Gonçalo A G; Queiroz, Marisa V

2015-05-01

Transposons are an important source of genetic variation. The phytopathogen Moniliophthora perniciosa shows high level of variability but little is known about the role of class I elements in shaping its genome. In this work, we aimed the characterization of a new gypsy/Ty3 retrotransposon species, named MpSaci, in the M. perniciosa genome. These elements are largely variable in size, ranging from 4 to 15 kb, and harbor direct long terminal repeats (LTRs) with varying degrees of similarity. Approximately, all of the copies are non-autonomous as shifts in the reading frame and stop codons were detected. Only two elements (MpSaci6 and MpSaci9) code for GAG and POL proteins that possess functional domains. Conserved domains that are typically not found in retrotransposons were detected and could potentially impact the expression of neighbor genes. Solo LTRs and several LARDs (large retrotransposon derivative) were detected. Unusual elements containing small sequences with or without interruptions that are similar to gag or different pol domains and presenting LTRs with different levels of similarities were identified. Methylation was observed in MpSaci reverse transcriptase sequences. Distribution analysis indicates that MpSaci elements are present in high copy number in the genomes of C-, S- and L-biotypes of M. perniciosa. In addition, C-biotype isolates originating from the state of Bahia have fragments in common with isolates from the Amazon region and two hybridization profiles related to two chromosomal groups. RT-PCR analysis reveals that the gag gene is constitutively expressed and that the expression is increased at least three-fold with nutrient depravation even though no new insertion were observed. These findings point out that MpSaci collaborated and, even though is primarily represented by non-autonomous elements, still might contribute to the generation of genetic variability in the most important cacao pathogen in Brazil.

Molecular cloning and localization of a novel cotton annexin gene expressed preferentially during fiber development.

PubMed

Wang, Li Ke; Niu, Xiao Wei; Lv, Yan Hui; Zhang, Tian Zhen; Guo, Wang Zhen

2010-10-01

Annexins constitute a family of multifunction and structurally related proteins. These proteins are ubiquitous in the plant kingdom, and are important calcium-dependent membrane-binding proteins that participate in the polar development of different plant regions such as rhizoids, root caps, and pollen tube tips. In this study, a novel cotton annexin gene (designated as GhFAnnx) was isolated from a fiber cDNA library of cotton (Gossypium hirsutum). The full-length cDNA of GhFAnnx comprises an open reading frame of 945 bp that encodes a 314-amino acid protein with a calculated molecular mass of 35.7 kDa and an isoelectric point of 6.49. Genomic GhFAnnx sequences from different cotton species, TM-1, Hai7124 and two diploid progenitor cottons, G. herbaceum (A-genome) and G. raimondii (D-genome) showed that at least two copies of the GhFAnnx gene, each with six exons and five introns in the coding region, were identified in the allotetraploid cotton genome. The GhFAnnx gene cloned from the cDNA library in this study was mapped to the chromosome 10 of the A-subgenome of the tetraploid cotton. Sequence alignment revealed that GhFAnnx contained four repeats of 70 amino acids. Semi-quantitative reverse transcriptase-polymerase chain reaction revealed that GhFAnnx is preferentially expressed in different developmental fibers but its expression is low in roots, stems, and leaves. Subcellular localization of GhFAnnx in onion epidermal cells and cotton fibers suggests that this protein is ubiquitous in the epidermal cells of onion, but assembles at the edge and the inner side of the apex of the cotton fiber tips with brilliant spots. In summary, GhFAnnx influences fiber development and is associated with the polar expansion of the cotton fiber during elongation stages.

Molecular characterization of partial fusion gene and C-terminus extension length of haemagglutinin-neuraminidase gene of recently isolated Newcastle disease virus isolates in Malaysia

PubMed Central

2010-01-01

Background Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious disease of birds and has been one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, sporadic cases have occasionally occurred in the country and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of Malaysia between 2004 and 2007 based on sequence and phylogenetic analysis of partial F gene and C-terminus extension length of HN gene. Results The coding region of eleven NDV isolates fusion (F) gene and carboxyl terminal region of haemagglutinin-neuraminidase (HN) gene including extensions were amplified by reverse transcriptase PCR and directly sequenced. All the isolates have shown to have non-synonymous to synonymous base substitution rate ranging between 0.081 - 0.264 demonstrating presence of negative selection. Analysis based on F gene showed the characterized isolates possess three different types of protease cleavage site motifs; namely 112RRQKRF117, 112RRRKRF117 and 112GRQGRL117 and appear to show maximum identities with isolates in the region such as cockatoo/14698/90 (Indonesia), Ch/2000 (China), local isolate AF2240 indicating the high similarity of isolates circulating in the South East Asian countries. Meanwhile, one of the isolates resembles commonly used lentogenic vaccine strains. On further characterization of the HN gene, Malaysian isolates had C-terminus extensions of 0, 6 and 11 amino acids. Analysis of the phylogenetic tree revealed that the existence of three genetic groups; namely, genotype II, VII and VIII. Conclusions The study concluded that the occurrence of three types of NDV genotypes and presence of varied carboxyl terminus extension lengths among Malaysian isolates incriminated for sporadic cases. PMID:20691110

Molecular characterization of partial fusion gene and C-terminus extension length of haemagglutinin-neuraminidase gene of recently isolated Newcastle disease virus isolates in Malaysia.

PubMed

Berhanu, Ayalew; Ideris, Aini; Omar, Abdul R; Bejo, Mohd Hair

2010-08-08

Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious disease of birds and has been one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, sporadic cases have occasionally occurred in the country and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of Malaysia between 2004 and 2007 based on sequence and phylogenetic analysis of partial F gene and C-terminus extension length of HN gene. The coding region of eleven NDV isolates fusion (F) gene and carboxyl terminal region of haemagglutinin-neuraminidase (HN) gene including extensions were amplified by reverse transcriptase PCR and directly sequenced. All the isolates have shown to have non-synonymous to synonymous base substitution rate ranging between 0.081 - 0.264 demonstrating presence of negative selection. Analysis based on F gene showed the characterized isolates possess three different types of protease cleavage site motifs; namely 112RRQKRF117, 112RRRKRF117 and 112GRQGRL117 and appear to show maximum identities with isolates in the region such as cockatoo/14698/90 (Indonesia), Ch/2000 (China), local isolate AF2240 indicating the high similarity of isolates circulating in the South East Asian countries. Meanwhile, one of the isolates resembles commonly used lentogenic vaccine strains. On further characterization of the HN gene, Malaysian isolates had C-terminus extensions of 0, 6 and 11 amino acids. Analysis of the phylogenetic tree revealed that the existence of three genetic groups; namely, genotype II, VII and VIII. The study concluded that the occurrence of three types of NDV genotypes and presence of varied carboxyl terminus extension lengths among Malaysian isolates incriminated for sporadic cases.

Application of Coamplification at Lower Denaturation Temperature-PCR Sequencing for Early Detection of Antiviral Drug Resistance Mutations of Hepatitis B Virus

PubMed Central

Wong, Danny Ka-Ho; Tsoi, Ottilia; Huang, Fung-Yu; Seto, Wai-Kay; Fung, James; Lai, Ching-Lung

2014-01-01

Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR sequencing cannot detect minor variants of <20%. We developed a modified co-amplification at lower denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5 to 10% of the viral population. The performance of COLD-PCR was compared to conventional PCR sequencing and a line probe assay (LiPA) using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72%), 148 (69%), and 113 samples (53%) by LiPA, COLD-PCR, and conventional PCR sequencing, respectively. Nineteen (9%) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12%) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR sequencing. Thirty-five (16%) samples had mutations detectable by COLD-PCR but not conventional PCR sequencing, while none had mutations detected by conventional PCR sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR sequencing in detecting minor lamivudine/telbivudine resistance mutations. PMID:24951803

The Discovery of Reverse Transcriptase.

PubMed

Coffin, John M; Fan, Hung

2016-09-29

In 1970 the independent and simultaneous discovery of reverse transcriptase in retroviruses (then RNA tumor viruses) by David Baltimore and Howard Temin revolutionized molecular biology and laid the foundations for retrovirology and cancer biology. In this historical review we describe the formulation of the controversial provirus hypothesis by Temin, which ultimately was proven by his discovery of reverse transcriptase in Rous sarcoma virus virions. Baltimore arrived at the same discovery through his studies on replication of RNA-containing viruses, starting with poliovirus and then moving to vesicular stomatitis virus, where he discovered a virion RNA polymerase. Subsequent studies of reverse transcriptase led to the elucidation of the mechanism of retrovirus replication, the discovery of oncogenes, the advent of molecular cloning, the search for human cancer viruses, and the discovery and treatment of HIV/AIDS.

PpRT1: the first complete gypsy-like retrotransposon isolated in Pinus pinaster.

PubMed

Rocheta, Margarida; Cordeiro, Jorge; Oliveira, M; Miguel, Célia

2007-02-01

We have isolated and characterized a complete retrotransposon sequence, named PpRT1, from the genome of Pinus pinaster. PpRT1 is 5,966 bp long and is closely related to IFG7 gypsy retrotransposon from Pinus radiata. The long terminal repeats (LTRs) have 333 bp each and show a 5.4% sequence divergence between them. In addition to the characteristic polypurine tract (PPT) and the primer binding site (PBS), PpRT1 carries internal regions with homology to retroviral genes gag and pol. The pol region contains sequence motifs related to the enzymes protease, reverse transcriptase, RNAseH and integrase in the same typical order known for Ty3/gypsy-like retrotransposons. PpRT1 was extended from an EST database sequence indicating that its transcription is occurring in pine tissues. Southern blot analyses indicate however, that PpRT1 is present in a unique or a low number of copies in the P. pinaster genome. The differences in nucleotide sequence found between PpRT1 and IFG7 may explain the strikingly different copy number in the two pine species genome. Based on the homologies observed when comparing LTR region among different gypsy elements we propose that the highly conserved LTR regions may be useful to amplify other retrotransposon sequences of the same or close retrotransposon family.

The Status, Quality, and Expansion of the NIH Full-Length cDNA Project: The Mammalian Gene Collection (MGC)

PubMed Central

2004-01-01

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5′-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline. PMID:15489334

Presence and Expression of Microbial Genes Regulating Soil Nitrogen Dynamics Along the Tanana River Successional Sequence

NASA Astrophysics Data System (ADS)

Boone, R. D.; Rogers, S. L.

2004-12-01

We report on work to assess the functional gene sequences for soil microbiota that control nitrogen cycle pathways along the successional sequence (willow, alder, poplar, white spruce, black spruce) on the Tanana River floodplain, Interior Alaska. Microbial DNA and mRNA were extracted from soils (0-10 cm depth) for amoA (ammonium monooxygenase), nifH (nitrogenase reductase), napA (nitrate reductase), and nirS and nirK (nitrite reductase) genes. Gene presence was determined by amplification of a conserved sequence of each gene employing sequence specific oligonucleotide primers and Polymerase Chain Reaction (PCR). Expression of the genes was measured via nested reverse transcriptase PCR amplification of the extracted mRNA. Amplified PCR products were visualized on agarose electrophoresis gels. All five successional stages show evidence for the presence and expression of microbial genes that regulate N fixation (free-living), nitrification, and nitrate reduction. We detected (1) nifH, napA, and nirK presence and amoA expression (mRNA production) for all five successional stages and (2) nirS and amoA presence and nifH, nirK, and napA expression for early successional stages (willow, alder, poplar). The results highlight that the existing body of previous process-level work has not sufficiently considered the microbial potential for a nitrate economy and free-living N fixation along the complete floodplain successional sequence.

The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase.

PubMed Central

Hitzeman, R A; Hagie, F E; Hayflick, J S; Chen, C Y; Seeburg, P H; Derynck, R

1982-01-01

The DNA sequence of the gene for the yeast glycolytic enzyme, 3-phosphoglycerate kinase (PGK), has been obtained by sequencing part of a 3.1 kbp HindIII fragment obtained from the yeast genome. The structural gene sequence corresponds to a reading frame of 1251 bp coding for 416 amino acids with no intervening DNA sequences. The amino acid sequence is approximately 65 percent homologous with human and horse PGK protein sequences and is in general agreement with the published protein sequence for yeast PGK. As for other highly expressed structural genes in yeast, the coding sequence is highly codon biased with 95 percent of the amino acids coded for by a select 25 codons (out of 61 possible). Besides structural DNA sequence, 291 bp of 5'-flanking sequence and 286 bp of 3'-flanking sequence were determined. Transcription starts 36 nucleotides upstream from the translational start and stops 86-93 nucleotides downstream from the translational stop. These results suggest a non-polyadenylated mRNA length of 1373 to 1380 nucleotides, which is consistent with the observed length of 1500 nucleotides for polyadenylated PGK mRNA. A sequence TATATATAAA is found at 145 nucleotides upstream from the translational start. This sequence resembles the TATAAA box that is possibly associated with RNA polymerase II binding. Images PMID:6296791

SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

PubMed Central

Poulsen, Line Dahl; Kielpinski, Lukasz Jan; Salama, Sofie R.; Krogh, Anders; Vinther, Jeppe

2015-01-01

Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA–RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing. PMID:25805860

Generation of non-genomic oligonucleotide tag sequences for RNA template-specific PCR

PubMed Central

Pinto, Fernando Lopes; Svensson, Håkan; Lindblad, Peter

2006-01-01

Background In order to overcome genomic DNA contamination in transcriptional studies, reverse template-specific polymerase chain reaction, a modification of reverse transcriptase polymerase chain reaction, is used. The possibility of using tags whose sequences are not found in the genome further improves reverse specific polymerase chain reaction experiments. Given the absence of software available to produce genome suitable tags, a simple tool to fulfill such need was developed. Results The program was developed in Perl, with separate use of the basic local alignment search tool, making the tool platform independent (known to run on Windows XP and Linux). In order to test the performance of the generated tags, several molecular experiments were performed. The results show that Tagenerator is capable of generating tags with good priming properties, which will deliberately not result in PCR amplification of genomic DNA. Conclusion The program Tagenerator is capable of generating tag sequences that combine genome absence with good priming properties for RT-PCR based experiments, circumventing the effects of genomic DNA contamination in an RNA sample. PMID:16820068

Systematic analysis of coding and noncoding DNA sequences using methods of statistical linguistics

NASA Technical Reports Server (NTRS)

Mantegna, R. N.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Peng, C. K.; Simons, M.; Stanley, H. E.

1995-01-01

We compare the statistical properties of coding and noncoding regions in eukaryotic and viral DNA sequences by adapting two tests developed for the analysis of natural languages and symbolic sequences. The data set comprises all 30 sequences of length above 50 000 base pairs in GenBank Release No. 81.0, as well as the recently published sequences of C. elegans chromosome III (2.2 Mbp) and yeast chromosome XI (661 Kbp). We find that for the three chromosomes we studied the statistical properties of noncoding regions appear to be closer to those observed in natural languages than those of coding regions. In particular, (i) a n-tuple Zipf analysis of noncoding regions reveals a regime close to power-law behavior while the coding regions show logarithmic behavior over a wide interval, while (ii) an n-gram entropy measurement shows that the noncoding regions have a lower n-gram entropy (and hence a larger "n-gram redundancy") than the coding regions. In contrast to the three chromosomes, we find that for vertebrates such as primates and rodents and for viral DNA, the difference between the statistical properties of coding and noncoding regions is not pronounced and therefore the results of the analyses of the investigated sequences are less conclusive. After noting the intrinsic limitations of the n-gram redundancy analysis, we also briefly discuss the failure of the zeroth- and first-order Markovian models or simple nucleotide repeats to account fully for these "linguistic" features of DNA. Finally, we emphasize that our results by no means prove the existence of a "language" in noncoding DNA.

Long-range correlation properties of coding and noncoding DNA sequences: GenBank analysis.

PubMed

Buldyrev, S V; Goldberger, A L; Havlin, S; Mantegna, R N; Matsa, M E; Peng, C K; Simons, M; Stanley, H E

1995-05-01

An open question in computational molecular biology is whether long-range correlations are present in both coding and noncoding DNA or only in the latter. To answer this question, we consider all 33301 coding and all 29453 noncoding eukaryotic sequences--each of length larger than 512 base pairs (bp)--in the present release of the GenBank to dtermine whether there is any statistically significant distinction in their long-range correlation properties. Standard fast Fourier transform (FFT) analysis indicates that coding sequences have practically no correlations in the range from 10 bp to 100 bp (spectral exponent beta=0.00 +/- 0.04, where the uncertainty is two standard deviations). In contrast, for noncoding sequences, the average value of the spectral exponent beta is positive (0.16 +/- 0.05) which unambiguously shows the presence of long-range correlations. We also separately analyze the 874 coding and the 1157 noncoding sequences that have more than 4096 bp and find a larger region of power-law behavior. We calculate the probability that these two data sets (coding and noncoding) were drawn from the same distribution and we find that it is less than 10(-10). We obtain independent confirmation of these findings using the method of detrended fluctuation analysis (DFA), which is designed to treat sequences with statistical heterogeneity, such as DNA's known mosaic structure ("patchiness") arising from the nonstationarity of nucleotide concentration. The near-perfect agreement between the two independent analysis methods, FFT and DFA, increases the confidence in the reliability of our conclusion.

Long-range correlation properties of coding and noncoding DNA sequences: GenBank analysis

NASA Technical Reports Server (NTRS)

Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Mantegna, R. N.; Matsa, M. E.; Peng, C. K.; Simons, M.; Stanley, H. E.

1995-01-01

An open question in computational molecular biology is whether long-range correlations are present in both coding and noncoding DNA or only in the latter. To answer this question, we consider all 33301 coding and all 29453 noncoding eukaryotic sequences--each of length larger than 512 base pairs (bp)--in the present release of the GenBank to dtermine whether there is any statistically significant distinction in their long-range correlation properties. Standard fast Fourier transform (FFT) analysis indicates that coding sequences have practically no correlations in the range from 10 bp to 100 bp (spectral exponent beta=0.00 +/- 0.04, where the uncertainty is two standard deviations). In contrast, for noncoding sequences, the average value of the spectral exponent beta is positive (0.16 +/- 0.05) which unambiguously shows the presence of long-range correlations. We also separately analyze the 874 coding and the 1157 noncoding sequences that have more than 4096 bp and find a larger region of power-law behavior. We calculate the probability that these two data sets (coding and noncoding) were drawn from the same distribution and we find that it is less than 10(-10). We obtain independent confirmation of these findings using the method of detrended fluctuation analysis (DFA), which is designed to treat sequences with statistical heterogeneity, such as DNA's known mosaic structure ("patchiness") arising from the nonstationarity of nucleotide concentration. The near-perfect agreement between the two independent analysis methods, FFT and DFA, increases the confidence in the reliability of our conclusion.

Sequence data and association statistics from 12,940 type 2 diabetes cases and controls.

PubMed

Flannick, Jason; Fuchsberger, Christian; Mahajan, Anubha; Teslovich, Tanya M; Agarwala, Vineeta; Gaulton, Kyle J; Caulkins, Lizz; Koesterer, Ryan; Ma, Clement; Moutsianas, Loukas; McCarthy, Davis J; Rivas, Manuel A; Perry, John R B; Sim, Xueling; Blackwell, Thomas W; Robertson, Neil R; Rayner, N William; Cingolani, Pablo; Locke, Adam E; Tajes, Juan Fernandez; Highland, Heather M; Dupuis, Josee; Chines, Peter S; Lindgren, Cecilia M; Hartl, Christopher; Jackson, Anne U; Chen, Han; Huyghe, Jeroen R; van de Bunt, Martijn; Pearson, Richard D; Kumar, Ashish; Müller-Nurasyid, Martina; Grarup, Niels; Stringham, Heather M; Gamazon, Eric R; Lee, Jaehoon; Chen, Yuhui; Scott, Robert A; Below, Jennifer E; Chen, Peng; Huang, Jinyan; Go, Min Jin; Stitzel, Michael L; Pasko, Dorota; Parker, Stephen C J; Varga, Tibor V; Green, Todd; Beer, Nicola L; Day-Williams, Aaron G; Ferreira, Teresa; Fingerlin, Tasha; Horikoshi, Momoko; Hu, Cheng; Huh, Iksoo; Ikram, Mohammad Kamran; Kim, Bong-Jo; Kim, Yongkang; Kim, Young Jin; Kwon, Min-Seok; Lee, Juyoung; Lee, Selyeong; Lin, Keng-Han; Maxwell, Taylor J; Nagai, Yoshihiko; Wang, Xu; Welch, Ryan P; Yoon, Joon; Zhang, Weihua; Barzilai, Nir; Voight, Benjamin F; Han, Bok-Ghee; Jenkinson, Christopher P; Kuulasmaa, Teemu; Kuusisto, Johanna; Manning, Alisa; Ng, Maggie C Y; Palmer, Nicholette D; Balkau, Beverley; Stančáková, Alena; Abboud, Hanna E; Boeing, Heiner; Giedraitis, Vilmantas; Prabhakaran, Dorairaj; Gottesman, Omri; Scott, James; Carey, Jason; Kwan, Phoenix; Grant, George; Smith, Joshua D; Neale, Benjamin M; Purcell, Shaun; Butterworth, Adam S; Howson, Joanna M M; Lee, Heung Man; Lu, Yingchang; Kwak, Soo-Heon; Zhao, Wei; Danesh, John; Lam, Vincent K L; Park, Kyong Soo; Saleheen, Danish; So, Wing Yee; Tam, Claudia H T; Afzal, Uzma; Aguilar, David; Arya, Rector; Aung, Tin; Chan, Edmund; Navarro, Carmen; Cheng, Ching-Yu; Palli, Domenico; Correa, Adolfo; Curran, Joanne E; Rybin, Dennis; Farook, Vidya S; Fowler, Sharon P; Freedman, Barry I; Griswold, Michael; Hale, Daniel Esten; Hicks, Pamela J; Khor, Chiea-Chuen; Kumar, Satish; Lehne, Benjamin; Thuillier, Dorothée; Lim, Wei Yen; Liu, Jianjun; Loh, Marie; Musani, Solomon K; Puppala, Sobha; Scott, William R; Yengo, Loïc; Tan, Sian-Tsung; Taylor, Herman A; Thameem, Farook; Wilson, Gregory; Wong, Tien Yin; Njølstad, Pål Rasmus; Levy, Jonathan C; Mangino, Massimo; Bonnycastle, Lori L; Schwarzmayr, Thomas; Fadista, João; Surdulescu, Gabriela L; Herder, Christian; Groves, Christopher J; Wieland, Thomas; Bork-Jensen, Jette; Brandslund, Ivan; Christensen, Cramer; Koistinen, Heikki A; Doney, Alex S F; Kinnunen, Leena; Esko, Tõnu; Farmer, Andrew J; Hakaste, Liisa; Hodgkiss, Dylan; Kravic, Jasmina; Lyssenko, Valeri; Hollensted, Mette; Jørgensen, Marit E; Jørgensen, Torben; Ladenvall, Claes; Justesen, Johanne Marie; Käräjämäki, Annemari; Kriebel, Jennifer; Rathmann, Wolfgang; Lannfelt, Lars; Lauritzen, Torsten; Narisu, Narisu; Linneberg, Allan; Melander, Olle; Milani, Lili; Neville, Matt; Orho-Melander, Marju; Qi, Lu; Qi, Qibin; Roden, Michael; Rolandsson, Olov; Swift, Amy; Rosengren, Anders H; Stirrups, Kathleen; Wood, Andrew R; Mihailov, Evelin; Blancher, Christine; Carneiro, Mauricio O; Maguire, Jared; Poplin, Ryan; Shakir, Khalid; Fennell, Timothy; DePristo, Mark; de Angelis, Martin Hrabé; Deloukas, Panos; Gjesing, Anette P; Jun, Goo; Nilsson, Peter; Murphy, Jacquelyn; Onofrio, Robert; Thorand, Barbara; Hansen, Torben; Meisinger, Christa; Hu, Frank B; Isomaa, Bo; Karpe, Fredrik; Liang, Liming; Peters, Annette; Huth, Cornelia; O'Rahilly, Stephen P; Palmer, Colin N A; Pedersen, Oluf; Rauramaa, Rainer; Tuomilehto, Jaakko; Salomaa, Veikko; Watanabe, Richard M; Syvänen, Ann-Christine; Bergman, Richard N; Bharadwaj, Dwaipayan; Bottinger, Erwin P; Cho, Yoon Shin; Chandak, Giriraj R; Chan, Juliana Cn; Chia, Kee Seng; Daly, Mark J; Ebrahim, Shah B; Langenberg, Claudia; Elliott, Paul; Jablonski, Kathleen A; Lehman, Donna M; Jia, Weiping; Ma, Ronald C W; Pollin, Toni I; Sandhu, Manjinder; Tandon, Nikhil; Froguel, Philippe; Barroso, Inês; Teo, Yik Ying; Zeggini, Eleftheria; Loos, Ruth J F; Small, Kerrin S; Ried, Janina S; DeFronzo, Ralph A; Grallert, Harald; Glaser, Benjamin; Metspalu, Andres; Wareham, Nicholas J; Walker, Mark; Banks, Eric; Gieger, Christian; Ingelsson, Erik; Im, Hae Kyung; Illig, Thomas; Franks, Paul W; Buck, Gemma; Trakalo, Joseph; Buck, David; Prokopenko, Inga; Mägi, Reedik; Lind, Lars; Farjoun, Yossi; Owen, Katharine R; Gloyn, Anna L; Strauch, Konstantin; Tuomi, Tiinamaija; Kooner, Jaspal Singh; Lee, Jong-Young; Park, Taesung; Donnelly, Peter; Morris, Andrew D; Hattersley, Andrew T; Bowden, Donald W; Collins, Francis S; Atzmon, Gil; Chambers, John C; Spector, Timothy D; Laakso, Markku; Strom, Tim M; Bell, Graeme I; Blangero, John; Duggirala, Ravindranath; Tai, E Shyong; McVean, Gilean; Hanis, Craig L; Wilson, James G; Seielstad, Mark; Frayling, Timothy M; Meigs, James B; Cox, Nancy J; Sladek, Rob; Lander, Eric S; Gabriel, Stacey; Mohlke, Karen L; Meitinger, Thomas; Groop, Leif; Abecasis, Goncalo; Scott, Laura J; Morris, Andrew P; Kang, Hyun Min; Altshuler, David; Burtt, Noël P; Florez, Jose C; Boehnke, Michael; McCarthy, Mark I

2017-12-19

To investigate the genetic basis of type 2 diabetes (T2D) to high resolution, the GoT2D and T2D-GENES consortia catalogued variation from whole-genome sequencing of 2,657 European individuals and exome sequencing of 12,940 individuals of multiple ancestries. Over 27M SNPs, indels, and structural variants were identified, including 99% of low-frequency (minor allele frequency [MAF] 0.1-5%) non-coding variants in the whole-genome sequenced individuals and 99.7% of low-frequency coding variants in the whole-exome sequenced individuals. Each variant was tested for association with T2D in the sequenced individuals, and, to increase power, most were tested in larger numbers of individuals (>80% of low-frequency coding variants in ~82 K Europeans via the exome chip, and ~90% of low-frequency non-coding variants in ~44 K Europeans via genotype imputation). The variants, genotypes, and association statistics from these analyses provide the largest reference to date of human genetic information relevant to T2D, for use in activities such as T2D-focused genotype imputation, functional characterization of variants or genes, and other novel analyses to detect associations between sequence variation and T2D.

Sequence data and association statistics from 12,940 type 2 diabetes cases and controls

PubMed Central

Jason, Flannick; Fuchsberger, Christian; Mahajan, Anubha; Teslovich, Tanya M.; Agarwala, Vineeta; Gaulton, Kyle J.; Caulkins, Lizz; Koesterer, Ryan; Ma, Clement; Moutsianas, Loukas; McCarthy, Davis J.; Rivas, Manuel A.; Perry, John R. B.; Sim, Xueling; Blackwell, Thomas W.; Robertson, Neil R.; Rayner, N William; Cingolani, Pablo; Locke, Adam E.; Tajes, Juan Fernandez; Highland, Heather M.; Dupuis, Josee; Chines, Peter S.; Lindgren, Cecilia M.; Hartl, Christopher; Jackson, Anne U.; Chen, Han; Huyghe, Jeroen R.; van de Bunt, Martijn; Pearson, Richard D.; Kumar, Ashish; Müller-Nurasyid, Martina; Grarup, Niels; Stringham, Heather M.; Gamazon, Eric R.; Lee, Jaehoon; Chen, Yuhui; Scott, Robert A.; Below, Jennifer E.; Chen, Peng; Huang, Jinyan; Go, Min Jin; Stitzel, Michael L.; Pasko, Dorota; Parker, Stephen C. J.; Varga, Tibor V.; Green, Todd; Beer, Nicola L.; Day-Williams, Aaron G.; Ferreira, Teresa; Fingerlin, Tasha; Horikoshi, Momoko; Hu, Cheng; Huh, Iksoo; Ikram, Mohammad Kamran; Kim, Bong-Jo; Kim, Yongkang; Kim, Young Jin; Kwon, Min-Seok; Lee, Juyoung; Lee, Selyeong; Lin, Keng-Han; Maxwell, Taylor J.; Nagai, Yoshihiko; Wang, Xu; Welch, Ryan P.; Yoon, Joon; Zhang, Weihua; Barzilai, Nir; Voight, Benjamin F.; Han, Bok-Ghee; Jenkinson, Christopher P.; Kuulasmaa, Teemu; Kuusisto, Johanna; Manning, Alisa; Ng, Maggie C. Y.; Palmer, Nicholette D.; Balkau, Beverley; Stančáková, Alena; Abboud, Hanna E.; Boeing, Heiner; Giedraitis, Vilmantas; Prabhakaran, Dorairaj; Gottesman, Omri; Scott, James; Carey, Jason; Kwan, Phoenix; Grant, George; Smith, Joshua D.; Neale, Benjamin M.; Purcell, Shaun; Butterworth, Adam S.; Howson, Joanna M. M.; Lee, Heung Man; Lu, Yingchang; Kwak, Soo-Heon; Zhao, Wei; Danesh, John; Lam, Vincent K. L.; Park, Kyong Soo; Saleheen, Danish; So, Wing Yee; Tam, Claudia H. T.; Afzal, Uzma; Aguilar, David; Arya, Rector; Aung, Tin; Chan, Edmund; Navarro, Carmen; Cheng, Ching-Yu; Palli, Domenico; Correa, Adolfo; Curran, Joanne E.; Rybin, Dennis; Farook, Vidya S.; Fowler, Sharon P.; Freedman, Barry I.; Griswold, Michael; Hale, Daniel Esten; Hicks, Pamela J.; Khor, Chiea-Chuen; Kumar, Satish; Lehne, Benjamin; Thuillier, Dorothée; Lim, Wei Yen; Liu, Jianjun; Loh, Marie; Musani, Solomon K.; Puppala, Sobha; Scott, William R.; Yengo, Loïc; Tan, Sian-Tsung; Taylor, Herman A.; Thameem, Farook; Wilson, Gregory; Wong, Tien Yin; Njølstad, Pål Rasmus; Levy, Jonathan C.; Mangino, Massimo; Bonnycastle, Lori L.; Schwarzmayr, Thomas; Fadista, João; Surdulescu, Gabriela L.; Herder, Christian; Groves, Christopher J.; Wieland, Thomas; Bork-Jensen, Jette; Brandslund, Ivan; Christensen, Cramer; Koistinen, Heikki A.; Doney, Alex S. F.; Kinnunen, Leena; Esko, Tõnu; Farmer, Andrew J.; Hakaste, Liisa; Hodgkiss, Dylan; Kravic, Jasmina; Lyssenko, Valeri; Hollensted, Mette; Jørgensen, Marit E.; Jørgensen, Torben; Ladenvall, Claes; Justesen, Johanne Marie; Käräjämäki, Annemari; Kriebel, Jennifer; Rathmann, Wolfgang; Lannfelt, Lars; Lauritzen, Torsten; Narisu, Narisu; Linneberg, Allan; Melander, Olle; Milani, Lili; Neville, Matt; Orho-Melander, Marju; Qi, Lu; Qi, Qibin; Roden, Michael; Rolandsson, Olov; Swift, Amy; Rosengren, Anders H.; Stirrups, Kathleen; Wood, Andrew R.; Mihailov, Evelin; Blancher, Christine; Carneiro, Mauricio O.; Maguire, Jared; Poplin, Ryan; Shakir, Khalid; Fennell, Timothy; DePristo, Mark; de Angelis, Martin Hrabé; Deloukas, Panos; Gjesing, Anette P.; Jun, Goo; Nilsson, Peter; Murphy, Jacquelyn; Onofrio, Robert; Thorand, Barbara; Hansen, Torben; Meisinger, Christa; Hu, Frank B.; Isomaa, Bo; Karpe, Fredrik; Liang, Liming; Peters, Annette; Huth, Cornelia; O'Rahilly, Stephen P; Palmer, Colin N. A.; Pedersen, Oluf; Rauramaa, Rainer; Tuomilehto, Jaakko; Salomaa, Veikko; Watanabe, Richard M.; Syvänen, Ann-Christine; Bergman, Richard N.; Bharadwaj, Dwaipayan; Bottinger, Erwin P.; Cho, Yoon Shin; Chandak, Giriraj R.; Chan, Juliana CN; Chia, Kee Seng; Daly, Mark J.; Ebrahim, Shah B.; Langenberg, Claudia; Elliott, Paul; Jablonski, Kathleen A.; Lehman, Donna M.; Jia, Weiping; Ma, Ronald C. W.; Pollin, Toni I.; Sandhu, Manjinder; Tandon, Nikhil; Froguel, Philippe; Barroso, Inês; Teo, Yik Ying; Zeggini, Eleftheria; Loos, Ruth J. F.; Small, Kerrin S.; Ried, Janina S.; DeFronzo, Ralph A.; Grallert, Harald; Glaser, Benjamin; Metspalu, Andres; Wareham, Nicholas J.; Walker, Mark; Banks, Eric; Gieger, Christian; Ingelsson, Erik; Im, Hae Kyung; Illig, Thomas; Franks, Paul W.; Buck, Gemma; Trakalo, Joseph; Buck, David; Prokopenko, Inga; Mägi, Reedik; Lind, Lars; Farjoun, Yossi; Owen, Katharine R.; Gloyn, Anna L.; Strauch, Konstantin; Tuomi, Tiinamaija; Kooner, Jaspal Singh; Lee, Jong-Young; Park, Taesung; Donnelly, Peter; Morris, Andrew D.; Hattersley, Andrew T.; Bowden, Donald W.; Collins, Francis S.; Atzmon, Gil; Chambers, John C.; Spector, Timothy D.; Laakso, Markku; Strom, Tim M.; Bell, Graeme I.; Blangero, John; Duggirala, Ravindranath; Tai, E. Shyong; McVean, Gilean; Hanis, Craig L.; Wilson, James G.; Seielstad, Mark; Frayling, Timothy M.; Meigs, James B.; Cox, Nancy J.; Sladek, Rob; Lander, Eric S.; Gabriel, Stacey; Mohlke, Karen L.; Meitinger, Thomas; Groop, Leif; Abecasis, Goncalo; Scott, Laura J.; Morris, Andrew P.; Kang, Hyun Min; Altshuler, David; Burtt, Noël P.; Florez, Jose C.; Boehnke, Michael; McCarthy, Mark I.

2017-01-01

To investigate the genetic basis of type 2 diabetes (T2D) to high resolution, the GoT2D and T2D-GENES consortia catalogued variation from whole-genome sequencing of 2,657 European individuals and exome sequencing of 12,940 individuals of multiple ancestries. Over 27M SNPs, indels, and structural variants were identified, including 99% of low-frequency (minor allele frequency [MAF] 0.1–5%) non-coding variants in the whole-genome sequenced individuals and 99.7% of low-frequency coding variants in the whole-exome sequenced individuals. Each variant was tested for association with T2D in the sequenced individuals, and, to increase power, most were tested in larger numbers of individuals (>80% of low-frequency coding variants in ~82 K Europeans via the exome chip, and ~90% of low-frequency non-coding variants in ~44 K Europeans via genotype imputation). The variants, genotypes, and association statistics from these analyses provide the largest reference to date of human genetic information relevant to T2D, for use in activities such as T2D-focused genotype imputation, functional characterization of variants or genes, and other novel analyses to detect associations between sequence variation and T2D. PMID:29257133

Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

PubMed

Collins, Kathleen; Nilsen, Timothy W

2013-08-01

Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

Not All Order Memory Is Equal: Test Demands Reveal Dissociations in Memory for Sequence Information

ERIC Educational Resources Information Center

Jonker, Tanya R.; MacLeod, Colin M.

2017-01-01

Remembering the order of a sequence of events is a fundamental feature of episodic memory. Indeed, a number of formal models represent temporal context as part of the memory system, and memory for order has been researched extensively. Yet, the nature of the code(s) underlying sequence memory is still relatively unknown. Across 4 experiments that…

Complete mitochondrial genome sequence of the heart failure model of cardiomyopathic Syrian hamster (Mesocricetus auratus).

PubMed

Hu, Bo; Liu, Dong-Xing; Zhang, Yu-Qing; Song, Jian-Tao; Ji, Xian-Fei; Hou, Zhi-Qiang; Zhang, Zhen-Hai

2016-05-01

In this study we sequenced the complete mitochondrial genome sequencing of a heart failure model of cardiomyopathic Syrian hamster (Mesocricetus auratus) for the first time. The total length of the mitogenome was 16,267 bp. It harbored 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 non-coding control region.

Code-Switching to Know a TL Equivalent of an L1 Word: Request-Provision-Acknowledgement (RPA) Sequence

ERIC Educational Resources Information Center

Lucero, Edgar

2011-01-01

This article focuses on the learner's use of Code-switching to learn the TL (Target Language) equivalent of an L1 word. The interactional pattern that this situation creates defines the Request-Provision-Acknowledgement (RPA) sequence. The article explains each of the turns of the sequence under the combination of the Ethnomethodological…

Quantitative analysis of the anti-noise performance of an m-sequence in an electromagnetic method

NASA Astrophysics Data System (ADS)

Yuan, Zhe; Zhang, Yiming; Zheng, Qijia

2018-02-01

An electromagnetic method with a transmitted waveform coded by an m-sequence achieved better anti-noise performance compared to the conventional manner with a square-wave. The anti-noise performance of the m-sequence varied with multiple coding parameters; hence, a quantitative analysis of the anti-noise performance for m-sequences with different coding parameters was required to optimize them. This paper proposes the concept of an identification system, with the identified Earth impulse response obtained by measuring the system output with the input of the voltage response. A quantitative analysis of the anti-noise performance of the m-sequence was achieved by analyzing the amplitude-frequency response of the corresponding identification system. The effects of the coding parameters on the anti-noise performance are summarized by numerical simulation, and their optimization is further discussed in our conclusions; the validity of the conclusions is further verified by field experiment. The quantitative analysis method proposed in this paper provides a new insight into the anti-noise mechanism of the m-sequence, and could be used to evaluate the anti-noise performance of artificial sources in other time-domain exploration methods, such as the seismic method.

RAMICS: trainable, high-speed and biologically relevant alignment of high-throughput sequencing reads to coding DNA.

PubMed

Wright, Imogen A; Travers, Simon A

2014-07-01

The challenge presented by high-throughput sequencing necessitates the development of novel tools for accurate alignment of reads to reference sequences. Current approaches focus on using heuristics to map reads quickly to large genomes, rather than generating highly accurate alignments in coding regions. Such approaches are, thus, unsuited for applications such as amplicon-based analysis and the realignment phase of exome sequencing and RNA-seq, where accurate and biologically relevant alignment of coding regions is critical. To facilitate such analyses, we have developed a novel tool, RAMICS, that is tailored to mapping large numbers of sequence reads to short lengths (<10 000 bp) of coding DNA. RAMICS utilizes profile hidden Markov models to discover the open reading frame of each sequence and aligns to the reference sequence in a biologically relevant manner, distinguishing between genuine codon-sized indels and frameshift mutations. This approach facilitates the generation of highly accurate alignments, accounting for the error biases of the sequencing machine used to generate reads, particularly at homopolymer regions. Performance improvements are gained through the use of graphics processing units, which increase the speed of mapping through parallelization. RAMICS substantially outperforms all other mapping approaches tested in terms of alignment quality while maintaining highly competitive speed performance. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genomics dataset on unclassified published organism (patent US 7547531).

PubMed

Khan Shawan, Mohammad Mahfuz Ali; Hasan, Md Ashraful; Hossain, Md Mozammel; Hasan, Md Mahmudul; Parvin, Afroza; Akter, Salina; Uddin, Kazi Rasel; Banik, Subrata; Morshed, Mahbubul; Rahman, Md Nazibur; Rahman, S M Badier

2016-12-01

Nucleotide (DNA) sequence analysis provides important clues regarding the characteristics and taxonomic position of an organism. With the intention that, DNA sequence analysis is very crucial to learn about hierarchical classification of that particular organism. This dataset (patent US 7547531) is chosen to simplify all the complex raw data buried in undisclosed DNA sequences which help to open doors for new collaborations. In this data, a total of 48 unidentified DNA sequences from patent US 7547531 were selected and their complete sequences were retrieved from NCBI BioSample database. Quick response (QR) code of those DNA sequences was constructed by DNA BarID tool. QR code is useful for the identification and comparison of isolates with other organisms. AT/GC content of the DNA sequences was determined using ENDMEMO GC Content Calculator, which indicates their stability at different temperature. The highest GC content was observed in GP445188 (62.5%) which was followed by GP445198 (61.8%) and GP445189 (59.44%), while lowest was in GP445178 (24.39%). In addition, New England BioLabs (NEB) database was used to identify cleavage code indicating the 5, 3 and blunt end and enzyme code indicating the methylation site of the DNA sequences was also shown. These data will be helpful for the construction of the organisms' hierarchical classification, determination of their phylogenetic and taxonomic position and revelation of their molecular characteristics.

RNAcode: Robust discrimination of coding and noncoding regions in comparative sequence data

PubMed Central

Washietl, Stefan; Findeiß, Sven; Müller, Stephan A.; Kalkhof, Stefan; von Bergen, Martin; Hofacker, Ivo L.; Stadler, Peter F.; Goldman, Nick

2011-01-01

With the availability of genome-wide transcription data and massive comparative sequencing, the discrimination of coding from noncoding RNAs and the assessment of coding potential in evolutionarily conserved regions arose as a core analysis task. Here we present RNAcode, a program to detect coding regions in multiple sequence alignments that is optimized for emerging applications not covered by current protein gene-finding software. Our algorithm combines information from nucleotide substitution and gap patterns in a unified framework and also deals with real-life issues such as alignment and sequencing errors. It uses an explicit statistical model with no machine learning component and can therefore be applied “out of the box,” without any training, to data from all domains of life. We describe the RNAcode method and apply it in combination with mass spectrometry experiments to predict and confirm seven novel short peptides in Escherichia coli and to analyze the coding potential of RNAs previously annotated as “noncoding.” RNAcode is open source software and available for all major platforms at http://wash.github.com/rnacode. PMID:21357752

RNAcode: robust discrimination of coding and noncoding regions in comparative sequence data.

PubMed

Washietl, Stefan; Findeiss, Sven; Müller, Stephan A; Kalkhof, Stefan; von Bergen, Martin; Hofacker, Ivo L; Stadler, Peter F; Goldman, Nick

2011-04-01

With the availability of genome-wide transcription data and massive comparative sequencing, the discrimination of coding from noncoding RNAs and the assessment of coding potential in evolutionarily conserved regions arose as a core analysis task. Here we present RNAcode, a program to detect coding regions in multiple sequence alignments that is optimized for emerging applications not covered by current protein gene-finding software. Our algorithm combines information from nucleotide substitution and gap patterns in a unified framework and also deals with real-life issues such as alignment and sequencing errors. It uses an explicit statistical model with no machine learning component and can therefore be applied "out of the box," without any training, to data from all domains of life. We describe the RNAcode method and apply it in combination with mass spectrometry experiments to predict and confirm seven novel short peptides in Escherichia coli and to analyze the coding potential of RNAs previously annotated as "noncoding." RNAcode is open source software and available for all major platforms at http://wash.github.com/rnacode.

High compression image and image sequence coding

NASA Technical Reports Server (NTRS)

Kunt, Murat

1989-01-01

The digital representation of an image requires a very large number of bits. This number is even larger for an image sequence. The goal of image coding is to reduce this number, as much as possible, and reconstruct a faithful duplicate of the original picture or image sequence. Early efforts in image coding, solely guided by information theory, led to a plethora of methods. The compression ratio reached a plateau around 10:1 a couple of years ago. Recent progress in the study of the brain mechanism of vision and scene analysis has opened new vistas in picture coding. Directional sensitivity of the neurones in the visual pathway combined with the separate processing of contours and textures has led to a new class of coding methods capable of achieving compression ratios as high as 100:1 for images and around 300:1 for image sequences. Recent progress on some of the main avenues of object-based methods is presented. These second generation techniques make use of contour-texture modeling, new results in neurophysiology and psychophysics and scene analysis.

Complete Mitochondrial Genome of Echinostoma hortense (Digenea: Echinostomatidae).

PubMed

Liu, Ze-Xuan; Zhang, Yan; Liu, Yu-Ting; Chang, Qiao-Cheng; Su, Xin; Fu, Xue; Yue, Dong-Mei; Gao, Yuan; Wang, Chun-Ren

2016-04-01

Echinostoma hortense (Digenea: Echinostomatidae) is one of the intestinal flukes with medical importance in humans. However, the mitochondrial (mt) genome of this fluke has not been known yet. The present study has determined the complete mt genome sequences of E. hortense and assessed the phylogenetic relationships with other digenean species for which the complete mt genome sequences are available in GenBank using concatenated amino acid sequences inferred from 12 protein-coding genes. The mt genome of E. hortense contained 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 non-coding region. The length of the mt genome of E. hortense was 14,994 bp, which was somewhat smaller than those of other trematode species. Phylogenetic analyses based on concatenated nucleotide sequence datasets for all 12 protein-coding genes using maximum parsimony (MP) method showed that E. hortense and Hypoderaeum conoideum gathered together, and they were closer to each other than to Fasciolidae and other echinostomatid trematodes. The availability of the complete mt genome sequences of E. hortense provides important genetic markers for diagnostics, population genetics, and evolutionary studies of digeneans.

Complete Mitochondrial Genome of Echinostoma hortense (Digenea: Echinostomatidae)

PubMed Central

Liu, Ze-Xuan; Zhang, Yan; Liu, Yu-Ting; Chang, Qiao-Cheng; Su, Xin; Fu, Xue; Yue, Dong-Mei; Gao, Yuan; Wang, Chun-Ren

2016-01-01

Echinostoma hortense (Digenea: Echinostomatidae) is one of the intestinal flukes with medical importance in humans. However, the mitochondrial (mt) genome of this fluke has not been known yet. The present study has determined the complete mt genome sequences of E. hortense and assessed the phylogenetic relationships with other digenean species for which the complete mt genome sequences are available in GenBank using concatenated amino acid sequences inferred from 12 protein-coding genes. The mt genome of E. hortense contained 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 non-coding region. The length of the mt genome of E. hortense was 14,994 bp, which was somewhat smaller than those of other trematode species. Phylogenetic analyses based on concatenated nucleotide sequence datasets for all 12 protein-coding genes using maximum parsimony (MP) method showed that E. hortense and Hypoderaeum conoideum gathered together, and they were closer to each other than to Fasciolidae and other echinostomatid trematodes. The availability of the complete mt genome sequences of E. hortense provides important genetic markers for diagnostics, population genetics, and evolutionary studies of digeneans. PMID:27180575

SHARAKU: an algorithm for aligning and clustering read mapping profiles of deep sequencing in non-coding RNA processing.

PubMed

Tsuchiya, Mariko; Amano, Kojiro; Abe, Masaya; Seki, Misato; Hase, Sumitaka; Sato, Kengo; Sakakibara, Yasubumi

2016-06-15

Deep sequencing of the transcripts of regulatory non-coding RNA generates footprints of post-transcriptional processes. After obtaining sequence reads, the short reads are mapped to a reference genome, and specific mapping patterns can be detected called read mapping profiles, which are distinct from random non-functional degradation patterns. These patterns reflect the maturation processes that lead to the production of shorter RNA sequences. Recent next-generation sequencing studies have revealed not only the typical maturation process of miRNAs but also the various processing mechanisms of small RNAs derived from tRNAs and snoRNAs. We developed an algorithm termed SHARAKU to align two read mapping profiles of next-generation sequencing outputs for non-coding RNAs. In contrast with previous work, SHARAKU incorporates the primary and secondary sequence structures into an alignment of read mapping profiles to allow for the detection of common processing patterns. Using a benchmark simulated dataset, SHARAKU exhibited superior performance to previous methods for correctly clustering the read mapping profiles with respect to 5'-end processing and 3'-end processing from degradation patterns and in detecting similar processing patterns in deriving the shorter RNAs. Further, using experimental data of small RNA sequencing for the common marmoset brain, SHARAKU succeeded in identifying the significant clusters of read mapping profiles for similar processing patterns of small derived RNA families expressed in the brain. The source code of our program SHARAKU is available at http://www.dna.bio.keio.ac.jp/sharaku/, and the simulated dataset used in this work is available at the same link. Accession code: The sequence data from the whole RNA transcripts in the hippocampus of the left brain used in this work is available from the DNA DataBank of Japan (DDBJ) Sequence Read Archive (DRA) under the accession number DRA004502. yasu@bio.keio.ac.jp Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

HIV-1 drug resistance prevalence, drug susceptibility and variant characterization in the Jacobi Medical Center paediatric cohort, Bronx, NY, USA.

PubMed

de Mulder, M; York, V A; Wiznia, A A; Michaud, H A; Nixon, D F; Holguin, A; Rosenberg, M G

2014-03-01

With the advent of combined antiretroviral therapy (cART), perinatally HIV-infected children are surviving into adolescence and beyond. However, drug resistance mutations (DRMs) compromise viral control, affecting the long-term effectiveness of ART. The aims of this study were to detect and identify DRMs in a HIV-1 infected paediatric cohort. Paired plasma and dried blood spots (DBSs) specimens were obtained from HIV-1 perinatally infected patients attending the Jacobi Medical Center, New York, USA. Clinical, virological and immunological data for these patients were analysed. HIV-1 pol sequences were generated from samples to identify DRMs according to the International AIDS Society (IAS) 2011 list. Forty-seven perinatally infected patients were selected, with a median age of 17.7 years, of whom 97.4% were carrying subtype B. They had a mean viral load of 3143 HIV-1 RNA copies/mL and a mean CD4 count of 486 cells/μL at the time of sampling. Nineteen patients (40.4%) had achieved undetectable viraemia (< 50 copies/mL) and 40.5% had a CD4 count of > 500 cells/μL. Most of the patients (97.9%) had received cART, including protease inhibitor (PI)-based regimens in 59.6% of cases. The DRM prevalence was 54.1, 27.6 and 27.0% for nucleoside reverse transcriptase inhibitors (NRTIs), PIs and nonnucleoside reverse transcriptase inhibitors (NNRTIs), respectively. Almost two-thirds (64.9%) of the patients harboured DRMs to at least one drug class and 5.4% were triple resistant. The mean nucleotide similarity between plasma and DBS sequences was 97.9%. Identical DRM profiles were present in 60% of plasma-DBS paired sequences. A total of 30 DRMs were detected in plasma and 26 in DBSs, with 23 present in both. Although more perinatally HIV-1-infected children are reaching adulthood as a result of advances in cART, our study cohort presented a high prevalence of resistant viruses, especially viruses resistant to NRTIs. DBS specimens can be used for DRM detection. © 2013 British HIV Association.

Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study

PubMed Central

Porter, Danielle P.; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D.; White, Kirsten L.

2015-01-01

At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study. PMID:26690199

Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study.

PubMed

Porter, Danielle P; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D; White, Kirsten L

2015-12-07

At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study.

Efficient analysis of mouse genome sequences reveal many nonsense variants

PubMed Central

Steeland, Sophie; Timmermans, Steven; Van Ryckeghem, Sara; Hulpiau, Paco; Saeys, Yvan; Van Montagu, Marc; Vandenbroucke, Roosmarijn E.; Libert, Claude

2016-01-01

Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ultimately resulted in a gain of a stop codon. These genes were identified by in silico changing the C57BL/6J coding sequences to the SPRET/Ei sequences, converting them to amino acid (AA) sequences, and comparing the AA sequences. All variants and transcripts affected were also stored in a database, which can be browsed using a SPRET/Ei M. spretus variants web tool (www.spretus.org), including a manual. We validated the tool by demonstrating the loss of function of three proteins predicted to be severely truncated, namely Fas, IRAK2, and IFNγR1. PMID:27147605

Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

PubMed

Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

2010-05-07

Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

VaDiR: an integrated approach to Variant Detection in RNA.

PubMed

Neums, Lisa; Suenaga, Seiji; Beyerlein, Peter; Anders, Sara; Koestler, Devin; Mariani, Andrea; Chien, Jeremy

2018-02-01

Advances in next-generation DNA sequencing technologies are now enabling detailed characterization of sequence variations in cancer genomes. With whole-genome sequencing, variations in coding and non-coding sequences can be discovered. But the cost associated with it is currently limiting its general use in research. Whole-exome sequencing is used to characterize sequence variations in coding regions, but the cost associated with capture reagents and biases in capture rate limit its full use in research. Additional limitations include uncertainty in assigning the functional significance of the mutations when these mutations are observed in the non-coding region or in genes that are not expressed in cancer tissue. We investigated the feasibility of uncovering mutations from expressed genes using RNA sequencing datasets with a method called Variant Detection in RNA(VaDiR) that integrates 3 variant callers, namely: SNPiR, RVBoost, and MuTect2. The combination of all 3 methods, which we called Tier 1 variants, produced the highest precision with true positive mutations from RNA-seq that could be validated at the DNA level. We also found that the integration of Tier 1 variants with those called by MuTect2 and SNPiR produced the highest recall with acceptable precision. Finally, we observed a higher rate of mutation discovery in genes that are expressed at higher levels. Our method, VaDiR, provides a possibility of uncovering mutations from RNA sequencing datasets that could be useful in further functional analysis. In addition, our approach allows orthogonal validation of DNA-based mutation discovery by providing complementary sequence variation analysis from paired RNA/DNA sequencing datasets.

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity

PubMed Central

Fowler, Benjamin J.; Gelfand, Bradley D.; Kim, Younghee; Kerur, Nagaraj; Tarallo, Valeria; Hirano, Yoshio; Amarnath, Shoba; Fowler, Daniel H.; Radwan, Marta; Young, Mark T.; Pittman, Keir; Kubes, Paul; Agarwal, Hitesh K.; Parang, Keykavous A.; Hinton, David R.; Bastos-Carvalho, Ana; Li, Shengjian; Yasuma, Tetsuhiro; Mizutani, Takeshi; Yasuma, Reo; Wright, Charles; Ambati, Jayakrishna

2014-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)-derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium (RPE) in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease (GVHD), and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases. PMID:25414314

Clinical and virologic follow-up in perinatally HIV-1-infected children and adolescents in Madrid with triple-class antiretroviral drug-resistant viruses.

PubMed

Rojas Sánchez, P; de Mulder, M; Fernandez-Cooke, E; Prieto, L; Rojo, P; Jiménez de Ory, S; José Mellado, M; Navarro, M; Tomas Ramos, J; Holguín, Á

2015-06-01

Drug resistance mutations compromise the success of antiretroviral treatment in human immunodeficiency virus type 1 (HIV-1)-infected children. We report the virologic and clinical follow-up of the Madrid cohort of perinatally HIV-infected children and adolescents after the selection of triple-class drug-resistant mutations (TC-DRM). We identified patients from the cohort carrying HIV-1 variants with TC-DRM to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors according to IAS-USA-2013. We recovered pol sequences or resistance profiles from 2000 to 2011 and clinical-immunologic-virologic data from the moment of TC-DRM detection until December 2013. Viruses harbouring TC-DRM were observed in 48 (9%) of the 534 children and adolescents from 2000 to 2011, rising to 24.4% among those 197 with resistance data. Among them, 95.8% were diagnosed before 2003, 91.7% were Spaniards, 89.6% carried HIV-1-subtype B and 75% received mono/dual therapy as first regimen. The most common TC-DRM present in ≥50% of them were D67NME, T215FVY, M41L and K103N (retrotranscriptase) and L90M (protease). The susceptibility to darunavir, tipranavir, etravirine and rilpivirine was 67.7%, 43.7%, 33.3% and 33.3%, respectively, and all reported high resistance to didanosine, abacavir and nelfinavir. Despite the presence of HIV-1 resistance mutations to the three main antiretroviral families in our paediatric cohort, some drugs maintained their susceptibility, mainly the new protease inhibitors (tipranavir and darunavir) and nonnucleoside reverse transcriptase inhibitors (etravirine and rilpivirine). These data will help to improve the clinical management of HIV-infected children with triple resistance in Spain. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

High Rates of Baseline Drug Resistance and Virologic Failure Among ART-naive HIV-infected Children in Mali.

PubMed

Crowell, Claudia S; Maiga, Almoustapha I; Sylla, Mariam; Taiwo, Babafemi; Kone, Niaboula; Oron, Assaf P; Murphy, Robert L; Marcelin, Anne-Geneviève; Traore, Ban; Fofana, Djeneba B; Peytavin, Gilles; Chadwick, Ellen G

2017-11-01

Limited data exist on drug resistance and antiretroviral treatment (ART) outcomes in HIV-1-infected children in West Africa. We determined the prevalence of baseline resistance and correlates of virologic failure (VF) in a cohort of ART-naive HIV-1-infected children <10 years of age initiating ART in Mali. Reverse transcriptase and protease genes were sequenced at baseline (before ART) and at 6 months. Resistance was defined according to the Stanford HIV Genotypic Resistance database. VF was defined as viral load ≥1000 copies/mL after 6 months of ART. Logistic regression was used to evaluate factors associated with VF or death >1 month after enrollment. Post hoc, antiretroviral concentrations were assayed on baseline samples of participants with baseline resistance. One-hundred twenty children with a median age 2.6 years (interquartile range: 1.6-5.0) were included. Eighty-eight percent reported no prevention of mother-to-child transmission exposure. At baseline, 27 (23%), 4 (3%) and none had non-nucleoside reverse transcriptase inhibitor (NNRTI), nucleoside reverse transcriptase inhibitor or protease inhibitor resistance, respectively. Thirty-nine (33%) developed VF and 4 died >1 month post-ART initiation. In multivariable analyses, poor adherence [odds ratio (OR): 6.1, P = 0.001], baseline NNRTI resistance among children receiving NNRTI-based ART (OR: 22.9, P < 0.001) and protease inhibitor-based ART initiation among children without baseline NNRTI resistance (OR: 5.8, P = 0.018) were significantly associated with VF/death. Ten (38%) with baseline resistance had detectable levels of nevirapine or efavirenz at baseline; 7 were currently breastfeeding, but only 2 reported maternal antiretroviral use. Baseline NNRTI resistance was common in children without reported NNRTI exposure and was associated with increased risk of treatment failure. Detectable NNRTI concentrations were present despite few reports of maternal/infant antiretroviral use.

HIV-1 transmitted drug resistance and genetic diversity among patients from Piauí State, Northeast Brazil.

PubMed

Moura, Maria Edileuza Soares; da Guarda Reis, Mônica Nogueira; Lima, Yanna Andressa Ramos; Eulálio, Kelsen Dantas; Cardoso, Ludimila Paula Vaz; Stefani, Mariane Martins Araújo

2015-05-01

HIV-1 transmitted-drug-resistance and genetic diversity are dynamic and may differ in distinct locations/risk groups. In Brazil, increased AIDS incidence and related mortality have been detected in the Northeast region, differently from the epicenter in the Southeast. This cross-sectional study describes transmitted-dru- resistance and HIV-1 subtypes in protease/PR and reverse transcriptase/RT regions among antiretroviral naïve patients from Piauí State, Northeast Brazil. Among 96 patients recruited 89 (92.7%) had HIV-1 PR/RT regions sequenced: 44 females and 45 males, 22 self-declared as men who have sex with men. Transmitted-drug-resistance was investigated by CPR tool (Stanford HIV-1 Drug Resistance/SDRM). HIV-1 subtypes were assigned by REGA and phylogenetic inference. Overall, transmitted-drug-resistance rate was 11.2% (10/89; CI 95%: 5.8-19.1%); 22.7% among men who have sex with men (5/22; CI 95%: 8.8-43.4%), 10% in heterosexual men (2/20; CI 95%: 1.7-29.3%) and 6.8% in women (3/44; CI 95%: 1.8-17.4%). Singleton mutations to protease-inhibitor/PI, nucleoside-reverse-transcriptase-inhibitor/NRTI or non-nucleoside-reverse-transcriptase-inhibitor/NNRTI predominated (8/10): PI mutations (M46L, V82F, L90M); NRTI mutations (M41L, D67N) and NNRTI mutations (K103N/S). Dual class resistance mutations to NRTI and NNRTI were observed: T215L (NRTI), Y188L (NNRTI) and T215N (NRTI), F227L (NNRTI). Subtype B prevailed (86.6%; 77/89), followed by subtype F1 (1.1%, 1/89) and subtype C (1.1%, 1/89). B/F1 and B/C intersubtype recombinants represented 11.2% (10/89). In Piauí State extensive testing of incidence and transmitted-drug-resistance in all populations with risk behaviors may help control AIDS epidemic locally. © 2015 Wiley Periodicals, Inc.

A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins.

PubMed Central

Crawford, S; Goff, S P

1985-01-01

Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation. Images PMID:3882995

Transmitted drug resistance in patients with acute/recent HIV infection in Brazil.

PubMed

Ferreira, Ana Cristina G; Coelho, Lara E; Grinsztejn, Eduarda; Jesus, Carlos S de; Guimarães, Monick L; Veloso, Valdiléa G; Grinsztejn, Beatriz; Cardoso, Sandra W

The widespread use of antiretroviral therapy increased the transmission of antiretroviral resistant HIV strains. Antiretroviral therapy initiation during acute/recent HIV infection limits HIV reservoirs and improves immune response in HIV infected individuals. Transmitted drug resistance may jeopardize the early goals of early antiretroviral treatment among acute/recent HIV infected patients. Patients with acute/recent HIV infection who underwent resistance test before antiretroviral treatment initiation were included in this analysis. HIV-1 sequences were obtained using an in house protease/reverse transcriptase genotyping assay. Transmitted drug resistance was identified according to the Stanford HIV Database for Transmitted Drug Resistance Mutations, based on WHO 2009 surveillance list, and HIV-1 subtyping according to Rega HIV-1 subtyping tool. Comparison between patients with and without transmitted drug resistance was made using Kruskal-Wallis and Chi-square tests. Forty-three patients were included, 13 with acute HIV infection and 30 with recent HIV infection. The overall transmitted drug resistance prevalence was 16.3% (95% confidence interval [CI]: 8.1-30.0%). The highest prevalence of resistance (11.6%, 95% CI: 8.1-24.5) was against non-nucleoside reverse transcriptase inhibitors, and K103N was the most frequently identified mutation. The high prevalence of nonnucleoside reverse transcriptase inhibitors resistance indicates that efavirenz-based regimen without prior resistance testing is not ideal for acutely/recently HIV-infected individuals in our setting. In this context, the recent proposal of including integrase inhibitors as a first line regimen in Brazil could be an advantage for the treatment of newly HIV infected individuals. However, it also poses a new challenge, since integrase resistance test is not routinely performed for antiretroviral naive individuals. Further studies on transmitted drug resistance among acutely/recently HIV-infected are needed to inform the predictors of transmitted resistance and the antiretroviral therapy outcomes among these population. Copyright © 2017 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

Evidence for retrovirus infections in green turtles Chelonia mydas from the Hawaiian islands

USGS Publications Warehouse

Casey, R.N.; Quackenbush, S.L.; Work, Thierry M.; Balazs, G.H.; Bowser, P.R.; Casey, J.W.

1997-01-01

Apparently normal Hawaiian green turtles Chelonia mydas and those displaying fibropapillomas were analyzed for infection by retroviruses. Strikingly, all samples were positive for polymerase enhanced reverse transcriptase (PERT) with levels high enough to quantitate by the conventional reverse transcriptase (RT) assay. However, samples of skin, even from asymptomatic turtles, were RT positive, although the levels of enzyme activity in healthy turtles hatched and raised in captivity were much lower than those observed in asymptomatic free-ranging turtles. Turtles with fibropapillomas displayed a broad range of reverse transcriptase activity. Skin and eye fibropapillomas and a heart tumor were further analyzed and shown to have reverse transcriptase activity that banded in a sucrose gradient at 1.17 g ml-1. The reverse transcriptase activity purified from the heart tumor displayed a temperature optimum of 37??C and showed a preference for Mn2+ over Mg2+. Sucrose gradient fractions of this sample displaying elevated reverse transcriptase activity contained primarily retrovitalsized particles with prominent envelope spikes, when negatively stained and examined by electron microscopy. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of gradient-purified virions revealed a conserved profile among 4 independent tumors and showed 7 prominent proteins having molecular weights of 116, 83, 51, 43, 40, 20 and 14 kDa. The data suggest that retroviral infections are widespread in Hawaiian green turtles and a comprehensive investigation is warranted to address the possibility that these agents cause green turtle fibropapillomatosis (GTFP).

Draft Genome Sequence of Cellulolytic and Xylanolytic Paenibacillus sp. A59, Isolated from Decaying Forest Soil from Patagonia, Argentina

PubMed Central

Ghio, Silvina; Martinez Cáceres, Alfredo I.; Talia, Paola; Grasso, Daniel H.

2015-01-01

Paenibacillus sp. A59 was isolated from decaying forest soil in Argentina and characterized as a xylanolytic strain. We report the draft genome sequence of this isolate, with an estimated genome size of 7 Mb which harbor 6,424 coding sequences. Genes coding for hydrolytic enzymes involved in lignocellulose deconstruction were predicted. PMID:26494679

Gene Identification Algorithms Using Exploratory Statistical Analysis of Periodicity

NASA Astrophysics Data System (ADS)

Mukherjee, Shashi Bajaj; Sen, Pradip Kumar

2010-10-01

Studying periodic pattern is expected as a standard line of attack for recognizing DNA sequence in identification of gene and similar problems. But peculiarly very little significant work is done in this direction. This paper studies statistical properties of DNA sequences of complete genome using a new technique. A DNA sequence is converted to a numeric sequence using various types of mappings and standard Fourier technique is applied to study the periodicity. Distinct statistical behaviour of periodicity parameters is found in coding and non-coding sequences, which can be used to distinguish between these parts. Here DNA sequences of Drosophila melanogaster were analyzed with significant accuracy.

Outbreak of poliomyelitis in Finland in 1984-85 - Re-analysis of viral sequences using the current standard approach.

PubMed

Simonen, Marja-Leena; Roivainen, Merja; Iber, Jane; Burns, Cara; Hovi, Tapani

2010-01-01

In 1984, a wild type 3 poliovirus (PV3/FIN84) spread all over Finland causing nine cases of paralytic poliomyelitis and one case of aseptic meningitis. The outbreak was ended in 1985 with an intensive vaccination campaign. By limited sequence comparison with previously isolated PV3 strains, closest relatives of PV3/FIN84 were found among strains circulating in the Mediterranean region. Now we wanted to reanalyse the relationships using approaches currently exploited in poliovirus surveillance. Cell lysates of 22 strains isolated during the outbreak and stored frozen were subjected to RT-PCR amplification in three genomic regions without prior subculture. Sequences of the entire VP1 coding region, 150 nucleotides in the VP1-2A junction, most of the 5' non-coding region, partial sequences of the 3D RNA polymerase coding region and partial 3' non-coding region were compared within the outbreak and with sequences available in data banks. In addition, complete nucleotide sequences were obtained for 2 strains isolated from two different cases of disease during the outbreak. The results confirmed the previously described wide intraepidemic variation of the strains, including amino acid substitutions in antigenic sites, as well as the likely Mediterranean region origin of the strains. Simplot and bootscanning analyses of the complete genomes indicated complicated evolutionary history of the non-capsid coding regions of the genome suggesting several recombinations with different HEV-C viruses in the past.

Genetic code, hamming distance and stochastic matrices.

PubMed

He, Matthew X; Petoukhov, Sergei V; Ricci, Paolo E

2004-09-01

In this paper we use the Gray code representation of the genetic code C=00, U=10, G=11 and A=01 (C pairs with G, A pairs with U) to generate a sequence of genetic code-based matrices. In connection with these code-based matrices, we use the Hamming distance to generate a sequence of numerical matrices. We then further investigate the properties of the numerical matrices and show that they are doubly stochastic and symmetric. We determine the frequency distributions of the Hamming distances, building blocks of the matrices, decomposition and iterations of matrices. We present an explicit decomposition formula for the genetic code-based matrix in terms of permutation matrices, which provides a hypercube representation of the genetic code. It is also observed that there is a Hamiltonian cycle in a genetic code-based hypercube.

Mitochondrial genomes of the jungle crow Corvus macrorhynchos (Passeriformes: Corvidae) from shed feathers and a phylogenetic analysis of genus Corvus using mitochondrial protein-coding genes.

PubMed

Krzeminska, Urszula; Wilson, Robyn; Rahman, Sadequr; Song, Beng Kah; Seneviratne, Sampath; Gan, Han Ming; Austin, Christopher M

2016-07-01

The complete mitochondrial genomes of two jungle crows (Corvus macrorhynchos) were sequenced. DNA was extracted from tissue samples obtained from shed feathers collected in the field in Sri Lanka and sequenced using the Illumina MiSeq Personal Sequencer. Jungle crow mitogenomes have a structural organization typical of the genus Corvus and are 16,927 bp and 17,066 bp in length, both comprising 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal subunit genes, and a non-coding control region. In addition, we complement already available house crow (Corvus spelendens) mitogenome resources by sequencing an individual from Singapore. A phylogenetic tree constructed from Corvidae family mitogenome sequences available on GenBank is presented. We confirm the monophyly of the genus Corvus and propose to use complete mitogenome resources for further intra- and interspecies genetic studies.

Evolution of the alternative AQP2 gene: Acquisition of a novel protein-coding sequence in dolphins.

PubMed

Kishida, Takushi; Suzuki, Miwa; Takayama, Asuka

2018-01-01

Taxon-specific de novo protein-coding sequences are thought to be important for taxon-specific environmental adaptation. A recent study revealed that bottlenose dolphins acquired a novel isoform of aquaporin 2 generated by alternative splicing (alternative AQP2), which helps dolphins to live in hyperosmotic seawater. The AQP2 gene consists of four exons, but the alternative AQP2 gene lacks the fourth exon and instead has a longer third exon that includes the original third exon and a part of the original third intron. Here, we show that the latter half of the third exon of the alternative AQP2 arose from a non-protein-coding sequence. Intact ORF of this de novo sequence is shared not by all cetaceans, but only by delphinoids. However, this sequence is conservative in all modern cetaceans, implying that this de novo sequence potentially plays important roles for marine adaptation in cetaceans. Copyright © 2017 Elsevier Inc. All rights reserved.

Complete genome sequencing of the luminescent bacterium, Vibrio qinghaiensis sp. Q67 using PacBio technology

NASA Astrophysics Data System (ADS)

Gong, Liang; Wu, Yu; Jian, Qijie; Yin, Chunxiao; Li, Taotao; Gupta, Vijai Kumar; Duan, Xuewu; Jiang, Yueming

2018-01-01

Vibrio qinghaiensis sp.-Q67 (Vqin-Q67) is a freshwater luminescent bacterium that continuously emits blue-green light (485 nm). The bacterium has been widely used for detecting toxic contaminants. Here, we report the complete genome sequence of Vqin-Q67, obtained using third-generation PacBio sequencing technology. Continuous long reads were attained from three PacBio sequencing runs and reads >500 bp with a quality value of >0.75 were merged together into a single dataset. This resultant highly-contiguous de novo assembly has no genome gaps, and comprises two chromosomes with substantial genetic information, including protein-coding genes, non-coding RNA, transposon and gene islands. Our dataset can be useful as a comparative genome for evolution and speciation studies, as well as for the analysis of protein-coding gene families, the pathogenicity of different Vibrio species in fish, the evolution of non-coding RNA and transposon, and the regulation of gene expression in relation to the bioluminescence of Vqin-Q67.

Weight distributions for turbo codes using random and nonrandom permutations

NASA Technical Reports Server (NTRS)

Dolinar, S.; Divsalar, D.

1995-01-01

This article takes a preliminary look at the weight distributions achievable for turbo codes using random, nonrandom, and semirandom permutations. Due to the recursiveness of the encoders, it is important to distinguish between self-terminating and non-self-terminating input sequences. The non-self-terminating sequences have little effect on decoder performance, because they accumulate high encoded weight until they are artificially terminated at the end of the block. From probabilistic arguments based on selecting the permutations randomly, it is concluded that the self-terminating weight-2 data sequences are the most important consideration in the design of constituent codes; higher-weight self-terminating sequences have successively decreasing importance. Also, increasing the number of codes and, correspondingly, the number of permutations makes it more and more likely that the bad input sequences will be broken up by one or more of the permuters. It is possible to design nonrandom permutations that ensure that the minimum distance due to weight-2 input sequences grows roughly as the square root of (2N), where N is the block length. However, these nonrandom permutations amplify the bad effects of higher-weight inputs, and as a result they are inferior in performance to randomly selected permutations. But there are 'semirandom' permutations that perform nearly as well as the designed nonrandom permutations with respect to weight-2 input sequences and are not as susceptible to being foiled by higher-weight inputs.

Next-generation sequencing of the Trichinella murrelli mitochondrial genome allows comprehensive comparison of its divergence from the principal agent of human trichinellosis, Trichinella spiralis.

PubMed

Webb, Kristen M; Rosenthal, Benjamin M

2011-01-01

The mitochondrial genome's non-recombinant mode of inheritance and relatively rapid rate of evolution has promoted its use as a marker for studying the biogeographic history and evolutionary interrelationships among many metazoan species. A modest portion of the mitochondrial genome has been defined for 12 species and genotypes of parasites in the genus Trichinella, but its adequacy in representing the mitochondrial genome as a whole remains unclear, as the complete coding sequence has been characterized only for Trichinella spiralis. Here, we sought to comprehensively describe the extent and nature of divergence between the mitochondrial genomes of T. spiralis (which poses the most appreciable zoonotic risk owing to its capacity to establish persistent infections in domestic pigs) and Trichinella murrelli (which is the most prevalent species in North American wildlife hosts, but which poses relatively little risk to the safety of pork). Next generation sequencing methodologies and scaffold and de novo assembly strategies were employed. The entire protein-coding region was sequenced (13,917 bp), along with a portion of the highly repetitive non-coding region (1524 bp) of the mitochondrial genome of T. murrelli with a combined average read depth of 250 reads. The accuracy of base calling, estimated from coding region sequence was found to exceed 99.3%. Genome content and gene order was not found to be significantly different from that of T. spiralis. An overall inter-species sequence divergence of 9.5% was estimated. Significant variation was identified when the amount of variation between species at each gene is compared to the average amount of variation between species across the coding region. Next generation sequencing is a highly effective means to obtain previously unknown mitochondrial genome sequence. Particular to parasites, the extremely deep coverage achieved through this method allows for the detection of sequence heterogeneity between the multiple individuals that necessarily comprise such templates. Copyright © 2010 Elsevier B.V. All rights reserved.

Cloning and sequence determination of the gene coding for the pyruvate phosphate dikinase of Entamoeba histolytica.

PubMed

Saavedra-Lira, E; Pérez-Montfort, R

1994-05-16

We isolated three overlapping clones from a DNA genomic library of Entamoeba histolytica strain HM1:IMSS, whose translated nucleotide (nt) sequence shows similarities of 51, 48 and 47% with the amino acid (aa) sequences reported for the pyruvate phosphate dikinases from Bacteroides symbiosus, maize and Flaveria trinervia, respectively. The reading frame determined codes for a protein of 886 aa.

Draft Genome Sequence of Cellulolytic and Xylanolytic Paenibacillus sp. A59, Isolated from Decaying Forest Soil from Patagonia, Argentina.

PubMed

Ghio, Silvina; Martinez Cáceres, Alfredo I; Talia, Paola; Grasso, Daniel H; Campos, Eleonora

2015-10-22

Paenibacillus sp. A59 was isolated from decaying forest soil in Argentina and characterized as a xylanolytic strain. We report the draft genome sequence of this isolate, with an estimated genome size of 7 Mb which harbor 6,424 coding sequences. Genes coding for hydrolytic enzymes involved in lignocellulose deconstruction were predicted. Copyright © 2015 Ghio et al.

Comparisons between Arabidopsis thaliana and Drosophila melanogaster in relation to Coding and Noncoding Sequence Length and Gene Expression

PubMed Central

Caldwell, Rachel; Lin, Yan-Xia; Zhang, Ren

2015-01-01

There is a continuing interest in the analysis of gene architecture and gene expression to determine the relationship that may exist. Advances in high-quality sequencing technologies and large-scale resource datasets have increased the understanding of relationships and cross-referencing of expression data to the large genome data. Although a negative correlation between expression level and gene (especially transcript) length has been generally accepted, there have been some conflicting results arising from the literature concerning the impacts of different regions of genes, and the underlying reason is not well understood. The research aims to apply quantile regression techniques for statistical analysis of coding and noncoding sequence length and gene expression data in the plant, Arabidopsis thaliana, and fruit fly, Drosophila melanogaster, to determine if a relationship exists and if there is any variation or similarities between these species. The quantile regression analysis found that the coding sequence length and gene expression correlations varied, and similarities emerged for the noncoding sequence length (5′ and 3′ UTRs) between animal and plant species. In conclusion, the information described in this study provides the basis for further exploration into gene regulation with regard to coding and noncoding sequence length. PMID:26114098

The mitochondrial genomes of the acoelomorph worms Paratomella rubra, Isodiametra pulchra and Archaphanostoma ylvae.

PubMed

Robertson, Helen E; Lapraz, François; Egger, Bernhard; Telford, Maximilian J; Schiffer, Philipp H

2017-05-12

Acoels are small, ubiquitous - but understudied - marine worms with a very simple body plan. Their internal phylogeny is still not fully resolved, and the position of their proposed phylum Xenacoelomorpha remains debated. Here we describe mitochondrial genome sequences from the acoels Paratomella rubra and Isodiametra pulchra, and the complete mitochondrial genome of the acoel Archaphanostoma ylvae. The P. rubra and A. ylvae sequences are typical for metazoans in size and gene content. The larger I. pulchra  mitochondrial genome contains both ribosomal genes, 21 tRNAs, but only 11 protein-coding genes. We find evidence suggesting a duplicated sequence in the I. pulchra mitochondrial genome. The P. rubra, I. pulchra and A. ylvae mitochondria have a unique genome organisation in comparison to other metazoan mitochondrial genomes. We found a large degree of protein-coding gene and tRNA overlap with little non-coding sequence in the compact P. rubra genome. Conversely, the A. ylvae and I. pulchra genomes have many long non-coding sequences between genes, likely driving genome size expansion in the latter. Phylogenetic trees inferred from mitochondrial genes retrieve Xenacoelomorpha as an early branching taxon in the deuterostomes. Sequence divergence analysis between P. rubra sampled in England and Spain indicates cryptic diversity.

Metal resistance sequences and transgenic plants

DOEpatents

Meagher, Richard Brian; Summers, Anne O.; Rugh, Clayton L.

1999-10-12

The present invention provides nucleic acid sequences encoding a metal ion resistance protein, which are expressible in plant cells. The metal resistance protein provides for the enzymatic reduction of metal ions including but not limited to divalent Cu, divalent mercury, trivalent gold, divalent cadmium, lead ions and monovalent silver ions. Transgenic plants which express these coding sequences exhibit increased resistance to metal ions in the environment as compared with plants which have not been so genetically modified. Transgenic plants with improved resistance to organometals including alkylmercury compounds, among others, are provided by the further inclusion of plant-expressible organometal lyase coding sequences, as specifically exemplified by the plant-expressible merB coding sequence. Furthermore, these transgenic plants which have been genetically modified to express the metal resistance coding sequences of the present invention can participate in the bioremediation of metal contamination via the enzymatic reduction of metal ions. Transgenic plants resistant to organometals can further mediate remediation of organic metal compounds, for example, alkylmetal compounds including but not limited to methyl mercury, methyl lead compounds, methyl cadmium and methyl arsenic compounds, in the environment by causing the freeing of mercuric or other metal ions and the reduction of the ionic mercury or other metal ions to the less toxic elemental mercury or other metals.

Complete mitochondrial genome of the whiter-spotted flower chafer, Protaetia brevitarsis (Coleoptera: Scarabaeidae).

PubMed

Kim, Min Jee; Im, Hyun Hwak; Lee, Kwang Youll; Han, Yeon Soo; Kim, Iksoo

2014-06-01

Abstract The complete nucleotide sequences of the mitochondrial genome from the whiter-spotted flower chafer, Protaetia brevitarsis (Coleoptera: Scarabaeidae), was determined. The 20,319-bp long circular genome is the longest among completely sequenced Coleoptera. As is typical in animals, the P. brevitarsis genome consisted of two ribosomal RNAs, 22 transfer RNAs, 13 protein-coding genes and one A + T-rich region. Although the size of the coding genes was typical, the non-coding A + T-rich region was 5654 bp, which is the longest in insects. The extraordinary length of this region was composed of 28,117-bp tandem repeats and 782-bp tandem repeats. These repeat sequences were encompassed by three non-repeat sequences constituting 1804 bp.

EDGE 2017 R&D 100 Entry with Appendix

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chain, Patrick Sam Guy; Davenport, Karen Walston; Li, Po-E

Diabetes, infertility, cancer, and Alzheimer’s disease—the key to one day preventing or even curing such afflictions and diseases (both infectious and genetically driven) may be locked in our own genetic code and the code of microorganisms that inhabit our bodies. The study of this code, known as genomics, has recently become much more promising as a result of two things: (1) vast improvements in high-throughput, nextgeneration sequencing (NSG), and (2) an exponential decrease in the cost of such sequencing. For example, it originally cost approximately $3 billion to sequence the human genome; today, this genome could be resequenced for lessmore » than $1,000.« less

HIV-1 pol mutation frequency by subtype and treatment experience: extension of the HIVseq program to seven non-B subtypes.

PubMed

Rhee, Soo-Yon; Kantor, Rami; Katzenstein, David A; Camacho, Ricardo; Morris, Lynn; Sirivichayakul, Sunee; Jorgensen, Louise; Brigido, Luis F; Schapiro, Jonathan M; Shafer, Robert W

2006-03-21

HIVseq was developed in 2000 to make published data on the frequency of HIV-1 group M protease and reverse transcriptase (RT) mutations available in real time to laboratories and researchers sequencing these genes. Because most published protease and RT sequences belonged to subtype B, the initial version of HIVseq was based on this subtype. As additional non-B sequences from persons with well-characterized antiretroviral treatment histories have become available, the program has been extended to subtypes A, C, D, F, G, CRF01, and CRF02. The latest frequency of each protease and RT mutation according to subtype and drug-class exposure was calculated using published sequences in the Stanford HIV RT and Protease Sequence Database. Each mutation was hyperlinked to published reports of viruses containing the mutation. As of September 2005, the mean number of protease sequences per non-B subtype was 534 from protease inhibitor-naive persons and 133 from protease inhibitor-treated persons, representing 13.2% and 2.3%, respectively, of the data available for subtype B. The mean number of RT sequences per non-B subtype was 373 from RT inhibitor-naive persons and 288 from RT inhibitor-treated persons, representing 17.9% and 3.8%, respectively, of the data available for subtype B. HIVseq allows users to examine protease and RT mutations within the context of previously published sequences of these genes. The publication of additional non-B protease and RT sequences from persons with well-characterized treatment histories, however, will be required to perform the same types of analysis possible with the much larger number of subtype B sequences.

HIV-1 pol mutation frequency by subtype and treatment experience

PubMed Central

Rhee, Soo-Yon; Kantor, Rami; Katzenstein, David A.; Camacho, Ricardo; Morris, Lynn; Sirivichayakul, Sunee; Jorgensen, Louise; Brigido, Luis F.; Schapiro, Jonathan M.; Shafer, Robert W.

2008-01-01

Objective HIVseq was developed in 2000 to make published data on the frequency of HIV-1 group M protease and reverse transcriptase (RT) mutations available in real time to laboratories and researchers sequencing these genes. Because most published protease and RT sequences belonged to subtype B, the initial version of HIVseq was based on this subtype. As additional non-B sequences from persons with well-characterized antiretroviral treatment histories have become available, the program has been extended to subtypes A, C, D, F, G, CRF01, and CRF02. Methods The latest frequency of each protease and RT mutation according to subtype and drug-class exposure was calculated using published sequences in the Stanford HIV RT and Protease Sequence Database. Each mutation was hyperlinked to published reports of viruses containing the mutation. Results As of September 2005, the mean number of protease sequences per non-B subtype was 534 from protease inhibitor-naive persons and 133 from protease inhibitor-treated persons, representing 13.2% and 2.3%, respectively, of the data available for subtype B. The mean number of RT sequences per non-B subtype was 373 from RT inhibitor-naive persons and 288 from RT inhibitor-treated persons, representing 17.9% and 3.8%, respectively, of the data available for subtype B. Conclusions HIVseq allows users to examine protease and RT mutations within the context of previously published sequences of these genes. The publication of additional non-B protease and RT sequences from persons with well-characterized treatment histories, however, will be required to perform the same types of analysis possible with the much larger number of subtype B sequences. PMID:16514293

Scaling features of noncoding DNA

NASA Technical Reports Server (NTRS)

Stanley, H. E.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Peng, C. K.; Simons, M.

1999-01-01

We review evidence supporting the idea that the DNA sequence in genes containing noncoding regions is correlated, and that the correlation is remarkably long range--indeed, base pairs thousands of base pairs distant are correlated. We do not find such a long-range correlation in the coding regions of the gene, and utilize this fact to build a Coding Sequence Finder Algorithm, which uses statistical ideas to locate the coding regions of an unknown DNA sequence. Finally, we describe briefly some recent work adapting to DNA the Zipf approach to analyzing linguistic texts, and the Shannon approach to quantifying the "redundancy" of a linguistic text in terms of a measurable entropy function, and reporting that noncoding regions in eukaryotes display a larger redundancy than coding regions. Specifically, we consider the possibility that this result is solely a consequence of nucleotide concentration differences as first noted by Bonhoeffer and his collaborators. We find that cytosine-guanine (CG) concentration does have a strong "background" effect on redundancy. However, we find that for the purine-pyrimidine binary mapping rule, which is not affected by the difference in CG concentration, the Shannon redundancy for the set of analyzed sequences is larger for noncoding regions compared to coding regions.

Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly

PubMed Central

Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka

2010-01-01

Background Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. Methodology We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence ∼800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. Conclusions The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches. PMID:20479877

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

PubMed

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-02-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

PubMed Central

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-01-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators. Images PMID:3257578

[Learning and Repetive Reproduction of Memorized Sequences by the Right and the Left Hand].

PubMed

Bobrova, E V; Lyakhovetskii, V A; Bogacheva, I N

2015-01-01

An important stage of learning a new skill is repetitive reproduction of one and the same sequence of movements, which plays a significant role in forming of the movement stereotypes. Two groups of right-handers repeatedly memorized (6-10 repetitions) the sequences of their hand transitions by experimenter in 6 positions, firstly by the right hand (RH), and then--by the left hand (LH) or vice versa. Random sequences previously unknown to the volunteers were reproduced in the 11 series. Modified sequences were tested in the 2nd and 3rd series, where the same elements' positions were presented in different order. The processes of repetitive sequence reproduction were similar for RH and LH. However, the learning of the modified sequences differed: Information about elements' position disregarding the reproduction order was used only when LH initiated task performing. This information was not used when LH followed RH and when RH performed the task. Consequently, the type of information coding activated by LH helped learn the positions of sequence elements, while the type of information coding activated by RH prevented learning. It is supposedly connected with the predominant role of right hemisphere in the processes of positional coding and motor learning.

Sequence Polishing Library (SPL) v10.0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oberortner, Ernst

The Sequence Polishing Library (SPL) is a suite of software tools in order to automate "Design for Synthesis and Assembly" workflows. Specifically: The SPL "Converter" tool converts files among the following sequence data exchange formats: CSV, FASTA, GenBank, and Synthetic Biology Open Language (SBOL); The SPL "Juggler" tool optimizes the codon usages of DNA coding sequences according to an optimization strategy, a user-specific codon usage table and genetic code. In addition, the SPL "Juggler" can translate amino acid sequences into DNA sequences.:The SPL "Polisher" verifies NA sequences against DNA synthesis constraints, such as GC content, repeating k-mers, and restriction sites.more » In case of violations, the "Polisher" reports the violations in a comprehensive manner. The "Polisher" tool can also modify the violating regions according to an optimization strategy, a user-specific codon usage table and genetic code;The SPL "Partitioner" decomposes large DNA sequences into smaller building blocks with partial overlaps that enable an efficient assembly. The "Partitioner" enables the user to configure the characteristics of the overlaps, which are mostly determined by the utilized assembly protocol, such as length, GC content, or melting temperature.« less

Multiple Access Interference Reduction Using Received Response Code Sequence for DS-CDMA UWB System

NASA Astrophysics Data System (ADS)

Toh, Keat Beng; Tachikawa, Shin'ichi

This paper proposes a combination of novel Received Response (RR) sequence at the transmitter and a Matched Filter-RAKE (MF-RAKE) combining scheme receiver system for the Direct Sequence-Code Division Multiple Access Ultra Wideband (DS-CDMA UWB) multipath channel model. This paper also demonstrates the effectiveness of the RR sequence in Multiple Access Interference (MAI) reduction for the DS-CDMA UWB system. It suggests that by using conventional binary code sequence such as the M sequence or the Gold sequence, there is a possibility of generating extra MAI in the UWB system. Therefore, it is quite difficult to collect the energy efficiently although the RAKE reception method is applied at the receiver. The main purpose of the proposed system is to overcome the performance degradation for UWB transmission due to the occurrence of MAI during multiple accessing in the DS-CDMA UWB system. The proposed system improves the system performance by improving the RAKE reception performance using the RR sequence which can reduce the MAI effect significantly. Simulation results verify that significant improvement can be obtained by the proposed system in the UWB multipath channel models.

Viewing multiple sequence alignments with the JavaScript Sequence Alignment Viewer (JSAV)

PubMed Central

Martin, Andrew C. R.

2014-01-01

The JavaScript Sequence Alignment Viewer (JSAV) is designed as a simple-to-use JavaScript component for displaying sequence alignments on web pages. The display of sequences is highly configurable with options to allow alternative coloring schemes, sorting of sequences and ’dotifying’ repeated amino acids. An option is also available to submit selected sequences to another web site, or to other JavaScript code. JSAV is implemented purely in JavaScript making use of the JQuery and JQuery-UI libraries. It does not use any HTML5-specific options to help with browser compatibility. The code is documented using JSDOC and is available from http://www.bioinf.org.uk/software/jsav/. PMID:25653836

Viewing multiple sequence alignments with the JavaScript Sequence Alignment Viewer (JSAV).

PubMed

Martin, Andrew C R

2014-01-01

The JavaScript Sequence Alignment Viewer (JSAV) is designed as a simple-to-use JavaScript component for displaying sequence alignments on web pages. The display of sequences is highly configurable with options to allow alternative coloring schemes, sorting of sequences and 'dotifying' repeated amino acids. An option is also available to submit selected sequences to another web site, or to other JavaScript code. JSAV is implemented purely in JavaScript making use of the JQuery and JQuery-UI libraries. It does not use any HTML5-specific options to help with browser compatibility. The code is documented using JSDOC and is available from http://www.bioinf.org.uk/software/jsav/.

The 28S–18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor

PubMed Central

Schnare, Murray N.; Collings, James C.; Spencer, David F.; Gray, Michael W.

2000-01-01

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from ∼11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an ∼55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A′ pre-rRNA processing sites within the 5′ external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5′ ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C.fasciculata and Trypanosoma brucei involves 3′-terminal addition of three A residues that are not present in the corresponding DNA sequences. PMID:10982863

Shark (Scyliorhinus torazame) metallothionein: cDNA cloning, genomic sequence, and expression analysis.

PubMed

Cho, Young Sun; Choi, Buyl Nim; Ha, En-Mi; Kim, Ki Hong; Kim, Sung Koo; Kim, Dong Soo; Nam, Yoon Kwon

2005-01-01

Novel metallothionein (MT) complementary DNA and genomic sequences were isolated from a cartilaginous shark species, Scyliorhinus torazame. The full-length open reading frame (ORF) of shark MT cDNA encoded 68 amino acids with a high cysteine content (29%). The genomic ORF sequence (932 bp) of shark MT isolated by polymerase chain reaction (PCR) comprised 3 exons with 2 interventing introns. Shark MT sequence shared many conserved features with other vertebrate MTs: overall amino acid identities of shark MT ranged from 47% to 57% with fish MTs, and 41% to 62% with mammalian MTs. However, in addition to these conserved characteristics, shark MT sequence exhibited some unique characteristics. It contained 4 extra amino acids (Lys-Ala-Gly-Arg) at the end of the beta-domain, which have not been reported in any other vertebrate MTs. The last amino acid residue at the C-terminus was Ser, which also has not been reported in fish and mammalian MTs. The MT messenger RNA levels in shark liver and kidney, assessed by semiquantitative reverse transcriptase PCR and RNA blot hybridization, were significantly affected by experimental exposures to heavy metals (cadmium, copper, and zinc). Generally, the transcriptional activation of shark MT gene was dependent on the dose (0-10 mg/kg body weight for injection and 0-20 microM for immersion) and duration (1-10 days); zinc was a more potent inducer than copper and cadmium.

The Nucleotide Sequence and Spliced pol mRNA Levels of the Nonprimate Spumavirus Bovine Foamy Virus

PubMed Central

Holzschu, Donald L.; Delaney, Mari A.; Renshaw, Randall W.; Casey, James W.

1998-01-01

We have determined the complete nucleotide sequence of a replication-competent clone of bovine foamy virus (BFV) and have quantitated the amount of splice pol mRNA processed early in infection. The 544-amino-acid Gag protein precursor has little sequence similarity with its primate foamy virus homologs, but the putative nucleocapsid (NC) protein, like the primate NCs, contains the three glycine-arginine-rich regions that are postulated to bind genomic RNA during virion assembly. The BFV gag and pol open reading frames overlap, with pro and pol in the same translational frame. As with the human foamy virus (HFV) and feline foamy virus, we have detected a spliced pol mRNA by PCR. Quantitatively, this mRNA approximates the level of full-length genomic RNA early in infection. The integrase (IN) domain of reverse transcriptase does not contain the canonical HH-CC zinc finger motif present in all characterized retroviral INs, but it does contain a nearby histidine residue that could conceivably participate as a member of the zinc finger. The env gene encodes a protein that is over 40% identical in sequence to the HFV Env. By comparison, the Gag precursor of BFV is predicted to be only 28% identical to the HFV protein. PMID:9499074

Molecular epidemiology of early and acute HIV type 1 infections in the United States Navy and Marine Corps, 2005-2010.

PubMed

Heipertz, Richard A; Sanders-Buell, Eric; Kijak, Gustavo; Howell, Shana; Lazzaro, Michelle; Jagodzinski, Linda L; Eggleston, John; Peel, Sheila; Malia, Jennifer; Armstrong, Adam; Michael, Nelson L; Kim, Jerome H; O'Connell, Robert J; Scott, Paul T; Brett-Major, David M; Tovanabutra, Sodsai

2013-10-01

The U.S. military represents a unique population within the human immunodeficiency virus 1 (HIV-1) pandemic. The last comprehensive study of HIV-1 in members of the U.S. Navy and Marine Corps (Sea Services) was completed in 2000, before large-scale combat operations were taking place. Here, we present molecular characterization of HIV-1 from 40 Sea Services personnel who were identified during their seroconversion window and initially classified as HIV-1 negative during screening. Protease/reverse transcriptase (pro/rt) and envelope (env) sequences were obtained from each member of the cohort. Phylogenetic analyses were carried out on these regions to determine relatedness within the cohort and calculate the most recent common ancestor for the related sequences. We identified 39 individuals infected with subtype B and one infected with CRF01_AE. Comparison of the pairwise genetic distance of Sea Service sequences and reference sequences in the env and pro/rt regions showed that five samples were part of molecular clusters, a group of two and a group of three, confirmed by single genome amplification. Real-time molecular monitoring of new HIV-1 acquisitions in the Sea Services may have a role in facilitating public health interventions at sites where related HIV-1 infections are identified.

Characterization and expression profiles of MaACS and MaACO genes from mulberry (Morus alba L.)*

PubMed Central

Liu, Chang-ying; Lü, Rui-hua; Li, Jun; Zhao, Ai-chun; Wang, Xi-ling; Diane, Umuhoza; Wang, Xiao-hong; Wang, Chuan-hong; Yu, Ya-sheng; Han, Shu-mei; Lu, Cheng; Yu, Mao-de

2014-01-01

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) are encoded by multigene families and are involved in fruit ripening by catalyzing the production of ethylene throughout the development of fruit. However, there are no reports on ACS or ACO genes in mulberry, partly because of the limited molecular research background. In this study, we have obtained five ACS gene sequences and two ACO gene sequences from Morus Genome Database. Sequence alignment and phylogenetic analysis of MaACO1 and MaACO2 showed that their amino acids are conserved compared with ACO proteins from other species. MaACS1 and MaACS2 are type I, MaACS3 and MaACS4 are type II, and MaACS5 is type III, with different C-terminal sequences. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) expression analysis showed that the transcripts of MaACS genes were strongly expressed in fruit, and more weakly in other tissues. The expression of MaACO1 and MaACO2 showed different patterns in various mulberry tissues. MaACS and MaACO genes demonstrated two patterns throughout the development of mulberry fruit, and both of them were strongly up-regulated by abscisic acid (ABA) and ethephon. PMID:25001221

Grasshopper, a long terminal repeat (LTR) retroelement in the phytopathogenic fungus Magnaporthe grisea.

PubMed

Dobinson, K F; Harris, R E; Hamer, J E

1993-01-01

The fungal phytopathogen Magnaporthe grisea parasitizes a wide variety of gramineous hosts. In the course of investigating the genetic relationship between pathogen genotype and host specificity we identified a retroelement that is present in some strains of M. grisea that infect finger millet and goosegrass (members of the plant genus Eleusine). The element, designated grasshopper (grh), is present in multiple copies and dispersed throughout the genome. DNA sequence analysis showed that grasshopper contains 198 base pair direct, long terminal repeats (LTRs) with features characteristic of retroviral and retrotransposon LTRs. Within the element we identified an open reading frame with sequences homologous to the reverse transcriptase, RNaseH, and integrase domains of retroelement pol genes. Comparison of the open reading frame with sequences from other retroelements showed that grh is related to the gypsy family of retrotransposons. Comparisons of the distribution of the grasshopper element with other dispersed repeated DNA sequences in M. grisea indicated that grasshopper was present in a broadly dispersed subgroup of Eleusine pathogens, suggesting that the element was acquired subsequent to the evolution of this host-specific form. We present arguments that the amplification of different retroelements within populations of M. grisea is a consequence of the clonal organization of the fungal populations.

RNAcentral: an international database of ncRNA sequences

DOE PAGES

Williams, Kelly Porter

2014-10-28

The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species.

The complete validated mitochondrial genome of the silver gemfish Rexea solandri (Cuvier, 1832) (Perciformes, Gempylidae).

PubMed

Bustamante, Carlos; Ovenden, Jennifer R

2016-01-01

The silver gemfish Rexea solandri is an important economic resource but Vulnerable to overfishing in Australian waters. The complete mitochondrial genome sequence is described from 1.6 million reads obtained via next generation sequencing. The total length of the mitogenome is 16,350 bp comprising 2 rRNA, 13 protein-coding genes, 22 tRNA and 2 non-coding regions. The mitogenome sequence was validated against sequences of PCR fragments and BLAST queries of Genbank. Gene order was equivalent to that found in marine fishes.

High rate concatenated coding systems using bandwidth efficient trellis inner codes

NASA Technical Reports Server (NTRS)

Deng, Robert H.; Costello, Daniel J., Jr.

1989-01-01

High-rate concatenated coding systems with bandwidth-efficient trellis inner codes and Reed-Solomon (RS) outer codes are investigated for application in high-speed satellite communication systems. Two concatenated coding schemes are proposed. In one the inner code is decoded with soft-decision Viterbi decoding, and the outer RS code performs error-correction-only decoding (decoding without side information). In the other, the inner code is decoded with a modified Viterbi algorithm, which produces reliability information along with the decoded output. In this algorithm, path metrics are used to estimate the entire information sequence, whereas branch metrics are used to provide reliability information on the decoded sequence. This information is used to erase unreliable bits in the decoded output. An errors-and-erasures RS decoder is then used for the outer code. The two schemes have been proposed for high-speed data communication on NASA satellite channels. The rates considered are at least double those used in current NASA systems, and the results indicate that high system reliability can still be achieved.

The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

PubMed Central

Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

2010-01-01

Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

Analyses of charophyte chloroplast genomes help characterize the ancestral chloroplast genome of land plants.

PubMed

Civaň, Peter; Foster, Peter G; Embley, Martin T; Séneca, Ana; Cox, Cymon J

2014-04-01

Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes.

Analyses of Charophyte Chloroplast Genomes Help Characterize the Ancestral Chloroplast Genome of Land Plants

PubMed Central

Civáň, Peter; Foster, Peter G.; Embley, Martin T.; Séneca, Ana; Cox, Cymon J.

2014-01-01

Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes. PMID:24682153

Frequent NF2 gene transcript mutations in sporadic meningiomas and vestibular schwannomas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deprez, R.H.L.; Groen, N.A.; Zwarthoff, E.C.

1994-06-01

The gene for the hereditary disorder neurofibromatosis type 2 (NF2), which predisposes for benign CNS tumors such as vestibular schwannomas and meningiomas, has been assigned to chromosome 22 and recently has been isolated. Mutations in the NF2 gene were found in both sporadic meningiomas and vestibular schwannomas. However, so far only 6 of the 16 exons of the gene have been analyzed. In order to extend the analysis of an involvement of the NF2 gene in the sporadic counterparts of these NF2-related tumors, the authors have used reverse transcriptase-PCR amplification followed by SSCP and DNA sequence analysis to screen formore » mutations in the coding region of the NF2 gene. Analysis of the NF2 gene transcript in 53 unrelated patients with meningiomas and vestibular schwannomas revealed mutations in 32% of the sporadic meningiomas (n = 44), in 50% of the sporadic vestibular schwannomas (n = 4), in 100% of the tumors found in NF2 patients (n = 2), and in one of three tumors from multiple-meningioma patients. Of the 18 tumors in which a mutation in the NF2 gene transcript was observed and the copy number of chromosome 22 could be established, 14 also showed loss of (parts of) chromosome 22. This suggests that in sporadic meningiomas and NF2-associated tumors the NF2 gene functions as a recessive tumor-suppressor gene. The mutations detected resulted mostly in frameshifts, predicting truncations starting within the N-terminal half of the putative protein. 23 refs., 2 figs. 3 tabs.« less

Structural and functional properties of the HIV-1 RNA-tRNA(Lys)3 primer complex annealed by the nucleocapsid protein: comparison with the heat-annealed complex.

PubMed Central

Brulé, Fabienne; Marquet, Roland; Rong, Liwei; Wainberg, Mark A; Roques, Bernard P; Le Grice, Stuart F J; Ehresmann, Bernard; Ehresmann, Chantal

2002-01-01

The conversion of the single-stranded RNA genome into double-stranded DNA by virus-coded reverse transcriptase (RT) is an essential step of the retrovirus life cycle. In human immunodeficiency virus type 1 (HIV-1), RT uses the cellular tRNA(Lys)3 to initiate the (-) strand DNA synthesis. Placement of the primer tRNA(Lys)3 involves binding of its 3'-terminal 18 nt to a complementary region of genomic RNA termed PBS. However, the PBS sequence is not the unique determinant of primer usage and additional contacts are important. This placement is believed to be achieved in vivo by the nucleocapsid domain of Gag or by the mature protein NCp. Up to now, structural information essentially arose from heat-annealed primer-template complexes (Isel et al., J Mol Biol, 1995, 247:236-250; Isel et al., EMBO J, 1999, 18:1038-1048). Here, we investigated the formation of the primer-template complex mediated by NCp and compared structural and functional properties of heat- and NCp-annealed complexes. We showed that both heat- and NCp-mediated procedures allow comparable high yields of annealing. Then, we investigated structural features of both kinds of complexes by enzymatic probing, and we compared their relative efficiency in (-) strong stop DNA synthesis. We did not find any significant differences between these complexes, suggesting that information derived from the heat-annealed complex can be transposed to the NCp-mediated complex and most likely to complexes formed in vivo. PMID:11873759

Intact coding region of the serotonin transporter gene in obsessive-compulsive disorder

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altemus, M.; Murphy, D.L.; Greenberg, B.

1996-07-26

Epidemiologic studies indicate that obsessive-compulsive disorder is genetically transmitted in some families, although no genetic abnormalities have been identified in individuals with this disorder. The selective response of obsessive-compulsive disorder to treatment with agents which block serotonin reuptake suggests the gene coding for the serotonin transporter as a candidate gene. The primary structure of the serotonin-transporter coding region was sequenced in 22 patients with obsessive-compulsive disorder, using direct PCR sequencing of cDNA synthesized from platelet serotonin-transporter mRNA. No variations in amino acid sequence were found among the obsessive-compulsive disorder patients or healthy controls. These results do not support a rolemore » for alteration in the primary structure of the coding region of the serotonin-transporter gene in the pathogenesis of obsessive-compulsive disorder. 27 refs.« less

Novel coding, translation, and gene expression of a replicating covalently closed circular RNA of 220 nt.

PubMed

AbouHaidar, Mounir Georges; Venkataraman, Srividhya; Golshani, Ashkan; Liu, Bolin; Ahmad, Tauqeer

2014-10-07

The highly structured (64% GC) covalently closed circular (CCC) RNA (220 nt) of the virusoid associated with rice yellow mottle virus codes for a 16-kDa highly basic protein using novel modalities for coding, translation, and gene expression. This CCC RNA is the smallest among all known viroids and virusoids and the only one that codes proteins. Its sequence possesses an internal ribosome entry site and is directly translated through two (or three) completely overlapping ORFs (shifting to a new reading frame at the end of each round). The initiation and termination codons overlap UGAUGA (underline highlights the initiation codon AUG within the combined initiation-termination sequence). Termination codons can be ignored to obtain larger read-through proteins. This circular RNA with no noncoding sequences is a unique natural supercompact "nanogenome."

A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

PubMed

Romero-Gómez, S; Duarte-Vázquez, M A; García-Almendárez, B E; Mayorga-Martínez, L; Cervantes-Avilés, O; Regalado, C

2008-12-01

A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.

Gene and genon concept: coding versus regulation

PubMed Central

2007-01-01

We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term “genon”. In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various pieces, as steered by the genon. It emerges finally as an uninterrupted nucleic acid sequence at mRNA level just prior to translation, in faithful correspondence with the amino acid sequence to be produced as a polypeptide. After translation, the genon has fulfilled its role and expires. The distinction between the protein coding information as materialised in the final polypeptide and the processing information represented by the genon allows us to set up a new information theoretic scheme. The standard sequence information determined by the genetic code expresses the relation between coding sequence and product. Backward analysis asks from which coding region in the DNA a given polypeptide originates. The (more interesting) forward analysis asks in how many polypeptides of how many different types a given DNA segment is expressed. This concerns the control of the expression process for which we have introduced the genon concept. Thus, the information theoretic analysis can capture the complementary aspects of coding and regulation, of gene and genon. PMID:18087760

The Use and Effectiveness of Triple Multiplex System for Coding Region Single Nucleotide Polymorphism in Mitochondrial DNA Typing of Archaeologically Obtained Human Skeletons from Premodern Joseon Tombs of Korea

PubMed Central

Oh, Chang Seok; Lee, Soong Deok; Kim, Yi-Suk; Shin, Dong Hoon

2015-01-01

Previous study showed that East Asian mtDNA haplogroups, especially those of Koreans, could be successfully assigned by the coupled use of analyses on coding region SNP markers and control region mutation motifs. In this study, we tried to see if the same triple multiplex analysis for coding regions SNPs could be also applicable to ancient samples from East Asia as the complementation for sequence analysis of mtDNA control region. By the study on Joseon skeleton samples, we know that mtDNA haplogroup determined by coding region SNP markers successfully falls within the same haplogroup that sequence analysis on control region can assign. Considering that ancient samples in previous studies make no small number of errors in control region mtDNA sequencing, coding region SNP analysis can be used as good complimentary to the conventional haplogroup determination, especially of archaeological human bone samples buried underground over long periods. PMID:26345190

Flexible and polarization-controllable diffusion metasurface with optical transparency

NASA Astrophysics Data System (ADS)

Zhuang, Yaqiang; Wang, Guangming; Liang, Jiangang; Cai, Tong; Guo, Wenlong; Zhang, Qingfeng

2017-11-01

In this paper, a novel coding metasurface is proposed to realize polarization-controllable diffusion scattering. The anisotropic Jerusalem-cross unit cell is employed as the basic coding element due to its polarization-dependent phase response. The isotropic random coding sequence is firstly designed to obtain diffusion scattering, and the anisotropic random coding sequence is subsequently realized by adding different periodic coding sequences to the original isotropic one along different directions. For demonstration, we designed and fabricated a flexible polarization-controllable diffusion metasurface (PCDM) with both chessboard diffusion and hedge diffusion under different polarizations. The specular scattering reduction performance of the anisotropic metasurface is better than the isotropic one because the scattered energies are redirected away from the specular reflection direction. For potential applications, the flexible PCDM wrapped around a cylinder structure is investigated and tested for polarization-controllable diffusion scattering. The numerical and experimental results coincide well, indicating anisotropic low scatterings with comparable performances. This paper provides an alternative approach for designing high-performance, flexible, low-scattering platforms.

Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity

USGS Publications Warehouse

House, M.L.; Kim, C.H.; Reno, P.W.

1998-01-01

Disseminated neoplasia (DN), a proliferative cell disorder of the circulatory system of bivalves, was first reported in oysters in 1969. Since that time, the disease has been determined to be transmissible through water-borne exposure, but the etiological agent has not been unequivocally identified. In order to determine if a viral agent, possibly a retrovirus, could be the causative agent of DN, transmission experiments were performed, using both a cell-free filtrate and a sucrose gradient-purified preparation of a cell-free filtrate of DN positive materials. Additionally, a PCR-enhanced reverse transcriptase assay was used to determine if reverse transcriptase was present in tissues or hemolymph from DN positive soft shell clams Mya arenaria. DN was transmitted to healthy clams by injection with whole DN cells, but not with cell-free flitrates prepared from either tissues from DN positive clams, or DN cells. The cell-free preparations from DN-positive tissues and hemolymph having high levels of DN cells in circulation exhibited positive reactions in the PCR-enhanced reverse transcriptase assay. Cell-free preparations of hemolymph from clams having low levels of DN (<0.1% of cells abnormal), hemocytes from normal soft shell clams, and normal soft shell clam tissues did not produce a positive reaction in the PCR enhanced reverse transcriptase assay.

The Need for Development of New HIV-1 Reverse Transcriptase and Integrase Inhibitors in the Aftermath of Antiviral Drug Resistance

PubMed Central

Wainberg, Mark A.

2012-01-01

The use of highly active antiretroviral therapy (HAART) involves combinations of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. There are two broad classes of reverse transcriptase inhibitors, the nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Since the first classes of such compounds were developed, viral resistance against them has necessitated the continuous development of novel compounds within each class. This paper considers the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second line therapy and describes the patterns of resistance associated with their use, as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with low genetic barrier are more commonly used in resource-limited settings. Their use results to the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings. More recently, the advent of integrase strand transfer inhibitors represents another major step forward toward control of HIV infection, but these compounds are also susceptible to problems of HIV drug resistance. PMID:24278679

Discovery of centrosomal RNA and centrosomal hypothesis of cellular ageing and differentiation.

PubMed

Chichinadze, Konstantin; Tkemaladze, Jaba; Lazarashvili, Ann

2012-01-01

In 2006, a group of scientists studying centrosomes of Spisula solidissima mollusc oocytes under the leadership of Alliegro (Alliegro, M.C.; Alliegro, M.A.; Palazzo, R.E. Centrosome-associated RNA in surf clam oocytes. Proc. Natl. Acad. Sci. USA 2006, 103(24), 9034-9038) reliably demonstrated the existence of specific RNA in centrosome, called centrosomal RNA (cnRNA). In their first article, five different RNAs (cnRNAs 11, 102, 113, 170, and 184) were described. During the process of full sequencing of the first transcript (cnRNA 11), it was discovered that the transcript contained a conserved structure-a reverse transcriptase domain located together with the most important centrosomal protein, γ-tubulin. In an article published in 2005, we made assumptions about several possible mechanisms for determining the most important functions of centrosomal structures and referred to one of them as a "RNA-dependent mechanism." This idea about participation of hypothetic centrosomal small interference RNA and/or microRNA in the process was made one year prior to the discovery of cnRNA by Alliegro's group. The discovery of specific RNA in a centrosome is indirect evidence of a centrosomal hypothesis of cellular ageing and differentiation. The presence of a reverse transcriptase domain in this type of RNA, together with its uniqueness and specificity, makes the centrosome a place of information storage and reproduction.

Follow-up on long-term antiretroviral therapy for cats infected with feline immunodeficiency virus.

PubMed

Medeiros, Sheila de Oliveira; Abreu, Celina Monteiro; Delvecchio, Rodrigo; Ribeiro, Anísia Praxedes; Vasconcelos, Zilton; Brindeiro, Rodrigo de Moraes; Tanuri, Amilcar

2016-04-01

Feline immunodeficiency virus (FIV) is a lentivirus that induces AIDS-like disease in cats. Some of the antiretroviral drugs available to treat patients with HIV type 1 are used to treat FIV-infected cats; however, antiretroviral therapy (ART) is not used in cats as a long-term treatment. In this study, the effects of long-term ART were evaluated in domestic cats treated initially with the nucleoside transcriptase reverse inhibitor (NTRI) zidovudine (AZT) over a period ranging from 5-6 years, followed by a regimen of the NTRI lamivudine (3TC) plus AZT over 3 years. Viral load, sequencing of pol (reverse transcriptase [RT]) region and CD4:CD8 lymphocyte ratio were evaluated during and after treatment. Untreated cats were evaluated as a control group. CD4:CD8 ratios were lower, and uncharacterized resistance mutations were found in the RT region in the group of treated cats. A slight increase in viral load was observed in some cats after discontinuing treatment. The data strongly suggest that treated cats were resistant to therapy, and uncharacterized resistance mutations in the RT gene of FIV were selected for by AZT. Few studies have been conducted to evaluate the effect of long-term antiretroviral therapy in cats. To date, resistance mutations have not been described in vivo. © ISFM and AAFP 2015.

Purification and Characterization of a Lectin from Green Split Peas (Pisum sativum).

PubMed

Ng, Tzi Bun; Chan, Yau Sang; Ng, Charlene Cheuk Wing; Wong, Jack Ho

2015-11-01

Lectins have captured the attention of a large number of researchers on account of their various exploitable activities, including antitumor, immunomodulatory, antifungal, as well as HIV reverse transcriptase inhibitory activities. A mannose/glucose-specific lectin was isolated from green split peas (a variety of Pisum sativum) and characterized. The purification step involved anion-exchange chromatography on a DEAE-cellulose column, cation-exchange chromatography on an SP-Sepharose column, and gel filtration by fast protein liquid chromatography (FPLC) on Superdex 200. The purified lectin had a native molecular mass of around 50 kDa as determined by size exclusion chromatography. It appeared as a heterotetramer, composed of two distinct polypeptide bands with a molecular mass of 6 and 19 kDa, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of green split pea lectin shows some degree of homology compared to lectins from other legume species. Its hemagglutinating activity was inhibited by glucose, mannose, and sucrose, and attenuated at pH values higher than 12 or lower than 3. Hemagglutinating activity was preserved at temperatures lower than 80 °C. The lectin did not show antifungal activity toward fungi including Fusarium oxysporum, Botrytis cinerea, and Mycosphaerella arachidicola. Green split pea lectin showed a mitogenic effect toward murine splenocytes and could inhibit the activity of HIV-1 reverse transcriptase.

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

PubMed

Paar, Jack; Doolittle, Mark M; Varma, Manju; Siefring, Shawn; Oshima, Kevin; Haugland, Richard A

2015-05-01

A method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for detecting interferences in RNA recovery and analysis, was developed for the direct, culture-independent detection of genetic markers from FRNA coliphage genogroups I, II & IV in water samples. Results were obtained from an initial evaluation of the performance of this method in analyses of waste water, ambient surface water and stormwater drain and outfall samples from predominantly urban locations. The evaluation also included a comparison of the occurrence of the FRNA genetic markers with genetic markers from general and human-related bacterial fecal indicators determined by current or pending EPA-validated qPCR methods. Strong associations were observed between the occurrence of the putatively human related FRNA genogroup II marker and the densities of the bacterial markers in the stormwater drain and outfall samples. However fewer samples were positive for FRNA coliphage compared to either the general bacterial fecal indicator or the human-related bacterial fecal indicator markers particularly for ambient water samples. Together, these methods show promise as complementary tools for the identification of contaminated storm water drainage systems as well as the determination of human and non-human sources of contamination. Published by Elsevier B.V.

Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.

PubMed Central

Fitzgibbon, J E; Howell, R M; Haberzettl, C A; Sperber, S J; Gocke, D J; Dubin, D T

1992-01-01

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient. Images PMID:1317143

Prevalence of Drug-Resistance Mutations and Non–Subtype B Strains Among HIV-Infected Infants From New York State

PubMed Central

Karchava, Marine; Pulver, Wendy; Smith, Lou; Philpott, Sean; Sullivan, Timothy J.; Wethers, Judith; Parker, Monica M.

2010-01-01

Summary Prevalence studies indicate that transmission of drug-resistant HIV has been rising in the adult population, but data from the perinatally infected pediatric population are limited. In this retrospective study, we sequenced the pol region of HIV from perinatally infected infants diagnosed in New York State in 2001–2002. Analyses of drug resistance, subtype diversity, and perinatal antiretroviral exposure were conducted, and the results were compared with those from a previous study of HIV-infected infants identified in 1998–1999. Eight of 42 infants (19.1%) had provirus carrying at least 1 drug-resistance mutation, an increase of 58% over the 1998–1999 results. Mutations conferring resistance to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 7.1%, 11.9%, and 2.4% of specimens, respectively. Consistent with previous results, perinatal antiretroviral exposure was not associated with drug resistance (P = 0.70). Phylogenetic analysis indicated that 16.7% of infants were infected with a non–subtype B strain of HIV. It seems that drug-resistant and non–subtype B strains of HIV are becoming increasingly common in the perinatally infected population. Our results highlight the value of resistance testing for all HIV-infected infants upon diagnosis and the need to consider subtype diversity in diagnostic and treatment strategies. PMID:16868498

A Novel Point Mutation at Position 156 of Reverse Transcriptase from Feline Immunodeficiency Virus Confers Resistance to the Combination of (−)-β-2′,3′-Dideoxy-3′-Thiacytidine and 3′-Azido-3′-Deoxythymidine

PubMed Central

Smith, Robert A.; Remington, Kathryn M.; Preston, Bradley D.; Schinazi, Raymond F.; North, Thomas W.

1998-01-01

Mutants of feline immunodeficiency virus (FIV) resistant to (−)-β-2′,3′-dideoxy-3′-thiacytidine (3TC) were selected by culturing virus in the presence of increasing stepwise concentrations of 3TC. Two plaque-purified variants were isolated from the original mutant population, and both of these mutants were resistant to 3TC. Surprisingly, these mutants were also phenotypically resistant to 3′-azido-3′-deoxythymidine (AZT) and to the combination of 3TC and AZT. Purified reverse transcriptase (RT) from one of these plaque-purified mutants was resistant to the 5′-triphosphates of 3TC and AZT. DNA sequence analysis of the RT-encoding region of the pol gene amplified from the plaque-purified mutants revealed a Pro-to-Ser mutation at position 156 of RT. A site-directed mutant of FIV engineered to contain this Pro-156-Ser mutation was resistant to 3TC, AZT, and the combination of 3TC and AZT, confirming the role of the Pro-156-Ser mutation in the resistance of FIV to these two nucleoside analogs. This represents the first report of a lentiviral mutant resistant to the combination of AZT and 3TC due to a single, unique point mutation. PMID:9499094

Endogenous New World primate type C viruses isolated from owl monkey (Aotus trivirgatus) kidney cell line.

PubMed Central

Todaro, G J; Sherr, C J; Sen, A; King, N; Daniel, M D; Fleckenstein, B

1978-01-01

A type C virus (OMC-1) detected in a culture of owl monkey kidney cells resembled typical type C viruses morphologically, but was slightly larger than previously characterized mammalian type C viruses. OMC-1 can be transmitted to bat lung cells and cat embryo fibroblasts. The virions band at a density of 1.16 g/ml in isopycnic sucrose density gradients and contain reverse transcriptase and a 60-65S RNA genome composed of approximately 32S subunits. The reverse transcriptase is immunologically and biochemically distinct from the polymerases of othe retroviruses. Radioimmunoassays directed to the interspecies antigenic determinants of the major structure proteins of other type C viruses do not detect a related antigen in OMC-1. Nucleic acid hybridization experiments using labeled viral genomic RNA or proviral cDNA transcripts to normal cellular DNA of different species show that OMC-1 is an endogenous virus with multiple virogene copies (20-50 per haploid genome) present in normal owl monkey cells and is distinct from previously isolated type C and D viruses. Sequences related to the OMC-1 genome can be detected in other New World monkeys. Thus, similar to the Old World primates (e.g., baboons as a prototype), the New World monkeys contain endogenous type C viral genes that appear to have been transmitted in the primate germ line. Images PMID:76312

Molecular epidemiological analysis of env and pol sequences in newly diagnosed HIV type 1-infected, untreated patients in Hungary.

PubMed

Mezei, Mária; Ay, Eva; Koroknai, Anita; Tóth, Renáta; Balázs, Andrea; Bakos, Agnes; Gyori, Zoltán; Bánáti, Ferenc; Marschalkó, Márta; Kárpáti, Sarolta; Minárovits, János

2011-11-01

The aim of our study was to monitor the diversity of HIV-1 strains circulating in Hungary and investigate the prevalence of resistance-associated mutations to reverse transcriptase (RT) and protease (PR) inhibitors in newly diagnosed, drug-naive patients. A total of 30 HIV-1-infected patients without prior antiretroviral treatment diagnosed during the period 2008-2010 were included into this study. Viral subtypes and the presence of RT, PR resistance-associated mutations were established by sequencing. Classification of HIV-1 strains showed that 29 (96.6%) patients were infected with subtype B viruses and one patient (3.3%) with subtype A virus. The prevalence of HIV-1 strains with transmitted drug resistance mutations in newly diagnosed individuals was 16.6% (5/30). This study showed that HIV-1 subtype B is still highly predominant in Hungary and documented a relatively high transmission rate of drug resistance in our country.

Canine distemper of vaccine origin in European mink, Mustela lutreola--a case report.

PubMed

Ek-Kommonen, C; Rudbäck, E; Anttila, M; Aho, M; Huovilainen, A

2003-04-02

Cases of canine distemper (CD) related to vaccination of exotic carnivores extend over three decades and have been described in at least nine different species. Our report describes a case of acute CD in a European mink, Mustela lutreola, vaccinated with live attenuated CD vaccine licensed for use in fur-farmed mink. The male mink died of an acute grey matter disease with an unusually long incubation period. A female vaccinated at the same time showed no obvious signs of illness. The diagnosis was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and by subsequent sequencing of the PCR products. The sequenced products of the virus isolated from the mink and of the vaccine batch showed 100% identity. This is the first report in which molecular methods were used to confirm that the disease was caused by the vaccine strain. Based on our findings, it is clearly evident that current CD vaccines cannot be safely used in exotic species.

Finding Relational Associations in HIV Resistance Mutation Data

NASA Astrophysics Data System (ADS)

Richter, Lothar; Augustin, Regina; Kramer, Stefan

HIV therapy optimization is a hard task due to rapidly evolving mutations leading to drug resistance. Over the past five years, several machine learning approaches have been developed for decision support, mostly to predict therapy failure from the genotypic sequence of viral proteins and additional factors. In this paper, we define a relational representation for an important part of the data, namely the sequences of a viral protein (reverse transcriptase), their mutations, and the drug resistance(s) associated with those mutations. The data were retrieved from the Los Alamos National Laboratories' (LANL) HIV databases. In contrast to existing work in this area, we do not aim directly for predictive modeling, but take one step back and apply descriptive mining methods to develop a better understanding of the correlations and associations between mutations and resistances. In our particular application, we use the Warmr algorithm to detect non-trivial patterns connecting mutations and resistances. Our findings suggest that well-known facts can be rediscovered, but also hint at the potential of discovering yet unknown associations.

Ependymin, a gene involved in regeneration and neuroplasticity in vertebrates, is overexpressed during regeneration in the echinoderm Holothuria glaberrima.

PubMed

Suárez-Castillo, Edna C; Medina-Ortíz, Wanda E; Roig-López, José L; García-Arrarás, José E

2004-06-09

We report the characterization of an ependymin-related gene (EpenHg) from a regenerating intestine cDNA library of the sea cucumber Holothuria glaberrima. This finding is remarkable because no ependymin sequence has ever been reported from invertebrates. Database comparisons of the conceptual translation of the EpenHg gene reveal 63% similarity (47% identity) with mammalian ependymin-related proteins (MERPs) and close relationship with the frog and piscine ependymins. We also report the partial sequences of ependymin representatives from another species of sea cucumber and from a sea urchin species. Conventional and real-time reverse transcriptase polymerase chain reaction (RT-PCRs) show that the gene is expressed in several echinoderm tissues, including esophagus, mesenteries, gonads, respiratory trees, hemal system, tentacles and body wall. Moreover, the ependymin product in the intestine is overexpressed during sea cucumber intestinal regeneration. The discovery of ependymins in echinoderms, a group well known for their regenerative capacities, can give us an insight on the evolution and roles of ependymin molecules.

Genetic Diversity of HIV-1 in Tunisia.

PubMed

El Moussi, Awatef; Thomson, Michael M; Delgado, Elena; Cuevas, María Teresa; Nasr, Majda; Abid, Salma; Ben Hadj Kacem, Mohamed Ali; Benaissa Tiouiri, Hanene; Letaief, Amel; Chakroun, Mohamed; Ben Jemaa, Mounir; Hamdouni, Hayet; Tej Dellagi, Rafla; Kheireddine, Khaled; Boutiba, Ilhem; Pérez-Álvarez, Lucía; Slim, Amine

2017-01-01

In this study, the genetic diversity of HIV-1 in Tunisia was analyzed. For this, 193 samples were collected in different regions of Tunisia between 2012 and 2015. A protease and reverse transcriptase fragment were amplified and sequenced. Phylogenetic analyses were performed through maximum likelihood and recombination was analyzed by bootscanning. Six HIV-1 subtypes (B, A1, G, D, C, and F2), 5 circulating recombinant forms (CRF02_AG, CRF25_cpx, CRF43_02G, CRF06_cpx, and CRF19_cpx), and 11 unique recombinant forms were identified. Subtype B (46.4%) and CRF02_AG (39.4%) were the predominant genetic forms. A group of 44 CRF02_AG sequences formed a distinct Tunisian cluster, which also included four viruses from western Europe. Nine viruses were closely related to isolates collected in other African or in European countries. In conclusion, a high HIV-1 genetic diversity is observed in Tunisia and the local spread of CRF02_AG is first documented in this country.

Coding visual features extracted from video sequences.

PubMed

Baroffio, Luca; Cesana, Matteo; Redondi, Alessandro; Tagliasacchi, Marco; Tubaro, Stefano

2014-05-01

Visual features are successfully exploited in several applications (e.g., visual search, object recognition and tracking, etc.) due to their ability to efficiently represent image content. Several visual analysis tasks require features to be transmitted over a bandwidth-limited network, thus calling for coding techniques to reduce the required bit budget, while attaining a target level of efficiency. In this paper, we propose, for the first time, a coding architecture designed for local features (e.g., SIFT, SURF) extracted from video sequences. To achieve high coding efficiency, we exploit both spatial and temporal redundancy by means of intraframe and interframe coding modes. In addition, we propose a coding mode decision based on rate-distortion optimization. The proposed coding scheme can be conveniently adopted to implement the analyze-then-compress (ATC) paradigm in the context of visual sensor networks. That is, sets of visual features are extracted from video frames, encoded at remote nodes, and finally transmitted to a central controller that performs visual analysis. This is in contrast to the traditional compress-then-analyze (CTA) paradigm, in which video sequences acquired at a node are compressed and then sent to a central unit for further processing. In this paper, we compare these coding paradigms using metrics that are routinely adopted to evaluate the suitability of visual features in the context of content-based retrieval, object recognition, and tracking. Experimental results demonstrate that, thanks to the significant coding gains achieved by the proposed coding scheme, ATC outperforms CTA with respect to all evaluation metrics.

Non-codingRNA sequence variations in human chronic lymphocytic leukemia and colorectal cancer.

PubMed

Wojcik, Sylwia E; Rossi, Simona; Shimizu, Masayoshi; Nicoloso, Milena S; Cimmino, Amelia; Alder, Hansjuerg; Herlea, Vlad; Rassenti, Laura Z; Rai, Kanti R; Kipps, Thomas J; Keating, Michael J; Croce, Carlo M; Calin, George A

2010-02-01

Cancer is a genetic disease in which the interplay between alterations in protein-coding genes and non-coding RNAs (ncRNAs) plays a fundamental role. In recent years, the full coding component of the human genome was sequenced in various cancers, whereas such attempts related to ncRNAs are still fragmentary. We screened genomic DNAs for sequence variations in 148 microRNAs (miRNAs) and ultraconserved regions (UCRs) loci in patients with chronic lymphocytic leukemia (CLL) or colorectal cancer (CRC) by Sanger technique and further tried to elucidate the functional consequences of some of these variations. We found sequence variations in miRNAs in both sporadic and familial CLL cases, mutations of UCRs in CLLs and CRCs and, in certain instances, detected functional effects of these variations. Furthermore, by integrating our data with previously published data on miRNA sequence variations, we have created a catalog of DNA sequence variations in miRNAs/ultraconserved genes in human cancers. These findings argue that ncRNAs are targeted by both germ line and somatic mutations as well as by single-nucleotide polymorphisms with functional significance for human tumorigenesis. Sequence variations in ncRNA loci are frequent and some have functional and biological significance. Such information can be exploited to further investigate on a genome-wide scale the frequency of genetic variations in ncRNAs and their functional meaning, as well as for the development of new diagnostic and prognostic markers for leukemias and carcinomas.

Non-codingRNA sequence variations in human chronic lymphocytic leukemia and colorectal cancer

PubMed Central

Wojcik, Sylwia E.; Rossi, Simona; Shimizu, Masayoshi; Nicoloso, Milena S.; Cimmino, Amelia; Alder, Hansjuerg; Herlea, Vlad; Rassenti, Laura Z.; Rai, Kanti R.; Kipps, Thomas J.; Keating, Michael J.

2010-01-01

Cancer is a genetic disease in which the interplay between alterations in protein-coding genes and non-coding RNAs (ncRNAs) plays a fundamental role. In recent years, the full coding component of the human genome was sequenced in various cancers, whereas such attempts related to ncRNAs are still fragmentary. We screened genomic DNAs for sequence variations in 148 microRNAs (miRNAs) and ultraconserved regions (UCRs) loci in patients with chronic lymphocytic leukemia (CLL) or colorectal cancer (CRC) by Sanger technique and further tried to elucidate the functional consequences of some of these variations. We found sequence variations in miRNAs in both sporadic and familial CLL cases, mutations of UCRs in CLLs and CRCs and, in certain instances, detected functional effects of these variations. Furthermore, by integrating our data with previously published data on miRNA sequence variations, we have created a catalog of DNA sequence variations in miRNAs/ultraconserved genes in human cancers. These findings argue that ncRNAs are targeted by both germ line and somatic mutations as well as by single-nucleotide polymorphisms with functional significance for human tumorigenesis. Sequence variations in ncRNA loci are frequent and some have functional and biological significance. Such information can be exploited to further investigate on a genome-wide scale the frequency of genetic variations in ncRNAs and their functional meaning, as well as for the development of new diagnostic and prognostic markers for leukemias and carcinomas. PMID:19926640

Probability of coding of a DNA sequence: an algorithm to predict translated reading frames from their thermodynamic characteristics.

PubMed Central

Tramontano, A; Macchiato, M F

1986-01-01

An algorithm to determine the probability that a reading frame codifies for a protein is presented. It is based on the results of our previous studies on the thermodynamic characteristics of a translated reading frame. We also develop a prediction procedure to distinguish between coding and non-coding reading frames. The procedure is based on the characteristics of the putative product of the DNA sequence and not on periodicity characteristics of the sequence, so the prediction is not biased by the presence of overlapping translated reading frames or by the presence of translated reading frames on the complementary DNA strand. PMID:3753761

mPUMA: a computational approach to microbiota analysis by de novo assembly of operational taxonomic units based on protein-coding barcode sequences.

PubMed

Links, Matthew G; Chaban, Bonnie; Hemmingsen, Sean M; Muirhead, Kevin; Hill, Janet E

2013-08-15

Formation of operational taxonomic units (OTU) is a common approach to data aggregation in microbial ecology studies based on amplification and sequencing of individual gene targets. The de novo assembly of OTU sequences has been recently demonstrated as an alternative to widely used clustering methods, providing robust information from experimental data alone, without any reliance on an external reference database. Here we introduce mPUMA (microbial Profiling Using Metagenomic Assembly, http://mpuma.sourceforge.net), a software package for identification and analysis of protein-coding barcode sequence data. It was developed originally for Cpn60 universal target sequences (also known as GroEL or Hsp60). Using an unattended process that is independent of external reference sequences, mPUMA forms OTUs by DNA sequence assembly and is capable of tracking OTU abundance. mPUMA processes microbial profiles both in terms of the direct DNA sequence as well as in the translated amino acid sequence for protein coding barcodes. By forming OTUs and calculating abundance through an assembly approach, mPUMA is capable of generating inputs for several popular microbiota analysis tools. Using SFF data from sequencing of a synthetic community of Cpn60 sequences derived from the human vaginal microbiome, we demonstrate that mPUMA can faithfully reconstruct all expected OTU sequences and produce compositional profiles consistent with actual community structure. mPUMA enables analysis of microbial communities while empowering the discovery of novel organisms through OTU assembly.

Recurrence time statistics: versatile tools for genomic DNA sequence analysis.

PubMed

Cao, Yinhe; Tung, Wen-Wen; Gao, J B

2004-01-01

With the completion of the human and a few model organisms' genomes, and the genomes of many other organisms waiting to be sequenced, it has become increasingly important to develop faster computational tools which are capable of easily identifying the structures and extracting features from DNA sequences. One of the more important structures in a DNA sequence is repeat-related. Often they have to be masked before protein coding regions along a DNA sequence are to be identified or redundant expressed sequence tags (ESTs) are to be sequenced. Here we report a novel recurrence time based method for sequence analysis. The method can conveniently study all kinds of periodicity and exhaustively find all repeat-related features from a genomic DNA sequence. An efficient codon index is also derived from the recurrence time statistics, which has the salient features of being largely species-independent and working well on very short sequences. Efficient codon indices are key elements of successful gene finding algorithms, and are particularly useful for determining whether a suspected EST belongs to a coding or non-coding region. We illustrate the power of the method by studying the genomes of E. coli, the yeast S. cervisivae, the nematode worm C. elegans, and the human, Homo sapiens. Computationally, our method is very efficient. It allows us to carry out analysis of genomes on the whole genomic scale by a PC.

A candidate gene for choanal atresia in alpaca.

PubMed

Reed, Kent M; Bauer, Miranda M; Mendoza, Kristelle M; Armién, Aníbal G

2010-03-01

Choanal atresia (CA) is a common nasal craniofacial malformation in New World domestic camelids (alpaca and llama). CA results from abnormal development of the nasal passages and is especially debilitating to newborn crias. CA in camelids shares many of the clinical manifestations of a similar condition in humans (CHARGE syndrome). Herein we report on the regulatory gene CHD7 of alpaca, whose homologue in humans is most frequently associated with CHARGE. Sequence of the CHD7 coding region was obtained from a non-affected cria. The complete coding region was 9003 bp, corresponding to a translated amino acid sequence of 3000 aa. Additional genomic sequences corresponding to a significant portion of the CHD7 gene were identified and assembled from the 2x alpaca whole genome sequence, providing confirmatory sequence for much of the CHD7 coding region. The alpaca CHD7 mRNA sequence was 97.9% similar to the human sequence, with the greatest sequence difference being an insertion in exon 38 that results in a polyalanine repeat (A12). Polymorphism in this repeat was tested for association with CA in alpaca by cloning and sequencing the repeat from both affected and non-affected individuals. Variation in length of the poly-A repeat was not associated with CA. Complete sequencing of the CHD7 gene will be necessary to determine whether other mutations in CHD7 are the cause of CA in camelids.

Different evolutionary patterns of SNPs between domains and unassigned regions in human protein-coding sequences.

PubMed

Pang, Erli; Wu, Xiaomei; Lin, Kui

2016-06-01

Protein evolution plays an important role in the evolution of each genome. Because of their functional nature, in general, most of their parts or sites are differently constrained selectively, particularly by purifying selection. Most previous studies on protein evolution considered individual proteins in their entirety or compared protein-coding sequences with non-coding sequences. Less attention has been paid to the evolution of different parts within each protein of a given genome. To this end, based on PfamA annotation of all human proteins, each protein sequence can be split into two parts: domains or unassigned regions. Using this rationale, single nucleotide polymorphisms (SNPs) in protein-coding sequences from the 1000 Genomes Project were mapped according to two classifications: SNPs occurring within protein domains and those within unassigned regions. With these classifications, we found: the density of synonymous SNPs within domains is significantly greater than that of synonymous SNPs within unassigned regions; however, the density of non-synonymous SNPs shows the opposite pattern. We also found there are signatures of purifying selection on both the domain and unassigned regions. Furthermore, the selective strength on domains is significantly greater than that on unassigned regions. In addition, among all of the human protein sequences, there are 117 PfamA domains in which no SNPs are found. Our results highlight an important aspect of protein domains and may contribute to our understanding of protein evolution.

Translational resistivity/conductivity of coding sequences during exponential growth of Escherichia coli.

PubMed

Takai, Kazuyuki

2017-01-21

Codon adaptation index (CAI) has been widely used for prediction of expression of recombinant genes in Escherichia coli and other organisms. However, CAI has no mechanistic basis that rationalizes its application to estimation of translational efficiency. Here, I propose a model based on which we could consider how codon usage is related to the level of expression during exponential growth of bacteria. In this model, translation of a gene is considered as an analog of electric current, and an analog of electric resistance corresponding to each gene is considered. "Translational resistance" is dependent on the steady-state concentration and the sequence of the mRNA species, and "translational resistivity" is dependent only on the mRNA sequence. The latter is the sum of two parts: one is the resistivity for the elongation reaction (coding sequence resistivity), and the other comes from all of the other steps of the decoding reaction. This electric circuit model clearly shows that some conditions should be met for codon composition of a coding sequence to correlate well with its expression level. On the other hand, I calculated relative frequency of each of the 61 sense codon triplets translated during exponential growth of E. coli from a proteomic dataset covering over 2600 proteins. A tentative method for estimating relative coding sequence resistivity based on the data is presented. Copyright © 2016. Published by Elsevier Ltd.

Origins of Genes: "Big Bang" or Continuous Creation?

NASA Astrophysics Data System (ADS)

Kesse, Paul K.; Gibbs, Adrian

1992-10-01

Many protein families are common to all cellular organisms, indicating that many genes have ancient origins. Genetic variation is mostly attributed to processes such as mutation, duplication, and rearrangement of ancient modules. Thus it is widely assumed that much of present-day genetic diversity can be traced by common ancestry to a molecular "big bang." A rarely considered alternative is that proteins may arise continuously de novo. One mechanism of generating different coding sequences is by "overprinting," in which an existing nucleotide sequence is translated de novo in a different reading frame or from noncoding open reading frames. The clearest evidence for overprinting is provided when the original gene function is retained, as in overlapping genes. Analysis of their phylogenies indicates which are the original genes and which are their informationally novel partners. We report here the phylogenetic relationships of overlapping coding sequences from steroid-related receptor genes and from tymovirus, luteovirus, and lentivirus genomes. For each pair of overlapping coding sequences, one is confined to a single lineage, whereas the other is more widespread. This suggests that the phylogenetically restricted coding sequence arose only in the progenitor of that lineage by translating an out-of-frame sequence to yield the new polypeptide. The production of novel exons by alternative splicing in thyroid receptor and lentivirus genes suggests that introns can be a valuable evolutionary source for overprinting. New genes and their products may drive major evolutionary changes.

Protein structure and the sequential structure of mRNA: alpha-helix and beta-sheet signals at the nucleotide level.

PubMed

Brunak, S; Engelbrecht, J

1996-06-01

A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed. We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting protein. The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain. A complete search for GenBank nucleotide sequences coding for structural entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment. By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets. These signals do not originate from the clustering of rare codons, but from the similarity of codons coding for very abundant amino acid residues at the N- and C-termini of helices and sheets. No correlation between the positioning of rare codons and the location of structural units was found. The mRNA signals were also compared with conserved nucleotide features of 16S-like ribosomal RNA sequences and related to mechanisms for maintaining the correct reading frame by the ribosome.

Evolution and Diversity of the Human Hepatitis D Virus Genome

PubMed Central

Huang, Chi-Ruei; Lo, Szecheng J.

2010-01-01

Human hepatitis delta virus (HDV) is the smallest RNA virus in genome. HDV genome is divided into a viroid-like sequence and a protein-coding sequence which could have originated from different resources and the HDV genome was eventually constituted through RNA recombination. The genome subsequently diversified through accumulation of mutations selected by interactions between the mutated RNA and proteins with host factors to successfully form the infectious virions. Therefore, we propose that the conservation of HDV nucleotide sequence is highly related with its functionality. Genome analysis of known HDV isolates shows that the C-terminal coding sequences of large delta antigen (LDAg) are the highest diversity than other regions of protein-coding sequences but they still retain biological functionality to interact with the heavy chain of clathrin can be selected and maintained. Since viruses interact with many host factors, including escaping the host immune response, how to design a program to predict RNA genome evolution is a great challenging work. PMID:20204073

Novel methodologies for spectral classification of exon and intron sequences

NASA Astrophysics Data System (ADS)

Kwan, Hon Keung; Kwan, Benjamin Y. M.; Kwan, Jennifer Y. Y.

2012-12-01

Digital processing of a nucleotide sequence requires it to be mapped to a numerical sequence in which the choice of nucleotide to numeric mapping affects how well its biological properties can be preserved and reflected from nucleotide domain to numerical domain. Digital spectral analysis of nucleotide sequences unfolds a period-3 power spectral value which is more prominent in an exon sequence as compared to that of an intron sequence. The success of a period-3 based exon and intron classification depends on the choice of a threshold value. The main purposes of this article are to introduce novel codes for 1-sequence numerical representations for spectral analysis and compare them to existing codes to determine appropriate representation, and to introduce novel thresholding methods for more accurate period-3 based exon and intron classification of an unknown sequence. The main findings of this study are summarized as follows: Among sixteen 1-sequence numerical representations, the K-Quaternary Code I offers an attractive performance. A windowed 1-sequence numerical representation (with window length of 9, 15, and 24 bases) offers a possible speed gain over non-windowed 4-sequence Voss representation which increases as sequence length increases. A winner threshold value (chosen from the best among two defined threshold values and one other threshold value) offers a top precision for classifying an unknown sequence of specified fixed lengths. An interpolated winner threshold value applicable to an unknown and arbitrary length sequence can be estimated from the winner threshold values of fixed length sequences with a comparable performance. In general, precision increases as sequence length increases. The study contributes an effective spectral analysis of nucleotide sequences to better reveal embedded properties, and has potential applications in improved genome annotation.

Random digital encryption secure communication system

NASA Technical Reports Server (NTRS)

Doland, G. D. (Inventor)

1982-01-01

The design of a secure communication system is described. A product code, formed from two pseudorandom sequences of digital bits, is used to encipher or scramble data prior to transmission. The two pseudorandom sequences are periodically changed at intervals before they have had time to repeat. One of the two sequences is transmitted continuously with the scrambled data for synchronization. In the receiver portion of the system, the incoming signal is compared with one of two locally generated pseudorandom sequences until correspondence between the sequences is obtained. At this time, the two locally generated sequences are formed into a product code which deciphers the data from the incoming signal. Provision is made to ensure synchronization of the transmitting and receiving portions of the system.

Expressed gene sequence of the IFN-gamma-response chemokine CXCL9 of cattle, horses, and swine

USDA-ARS?s Scientific Manuscript database

This report describes the cloning and characterization of expressed gene sequences of bovine, equine, and swine CXCL9 from RNA obtained from peripheral blood mononuclear cell (PBMC) or other tissues. The bovine coding region was 378 nucleotides in length, while the equine and swine coding regions w...

Pyrosequencing for detection of lamivudine-resistant hepatitis B virus.

PubMed

Lindström, Anna; Odeberg, Jacob; Albert, Jan

2004-10-01

Chronic hepatitis B virus (HBV) infection can cause severe liver disease, including cirrhosis and hepatocellular carcinoma. Lamivudine is a relatively recent alternative to alpha interferon for the treatment of HBV infection, but unfortunately, resistance to lamivudine commonly develops during monotherapy. Lamivudine-resistant HBV mutants display specific mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the viral polymerase (reverse transcriptase [rt]), which is the catalytic site of the enzyme, i.e., methionine 204 to isoleucine (rtM204I) or valine (rtM204V). The latter mutation is often accompanied by a compensatory leucine-to-methionine change at codon 180 (rtL180M). In the present study, a novel sequencing method, pyrosequencing, was applied to the detection of lamivudine resistance mutations and was compared with direct Sanger sequencing. The new pyrosequencing method had advantages in terms of throughput. Experiments with mixtures of wild-type and resistant viruses indicated that pyrosequencing can detect minor sequence variants in heterogeneous virus populations. The new pyrosequencing method was evaluated with a small number of patient samples, and the results showed that the method could be a useful tool for the detection of lamivudine resistance in the clinical setting.

Isolation and Molecular Characterization of Novel Infectious Bronchitis Virus Variants from Vaccinated Broiler Flocks in Egypt.

PubMed

Abdel-Sabour, Mohammed A; Al-Ebshahy, Emad M; Khaliel, Samy A; Abdel-Wanis, Nabil A; Yanai, Tokuma

2017-09-01

The present study aimed to determine the molecular characteristics of circulating infectious bronchitis virus (IBV) strains in vaccinated broiler flocks in the Giza and Fayoum governorates. Thirty-four isolates were collected, and egg propagation revealed their ability to induce typical IBV lesions after three to five successive passages. Three selected isolates were identified as IBV using a real-time reverse transcriptase-PCR assay targeted the nucleocapsid (N) gene and further characterized by partial spike (S) gene sequence analysis. Phylogenetic analysis revealed their clustering into two variant groups. Group I consisted of one variant (VSVRI_F3), which had 99.1% nucleotide sequence identity to the Q1 reference strain. Group II consisted of variants VSVRI_G4 and VSVRI_G9, which showed 92.8%-94.3% nucleotide identity with the Egyptian variants Eg/12120S/2012, Eg/12197B/2012, and Eg/1265B/2012. Regarding the deduced amino acid sequence, the three variants had 77.1%-85.2% similarity with the vaccine strains currently used in Egypt. These findings highlight the importance of monitoring the prevalence of IBV variants in vaccinated broiler flocks as well as adopting an appropriate vaccination strategy.

Multi-OMICs and Genome Editing Perspectives on Liver Cancer Signaling Networks.

PubMed

Lin, Shengda; Yin, Yi A; Jiang, Xiaoqian; Sahni, Nidhi; Yi, Song

2016-01-01

The advent of the human genome sequence and the resulting ~20,000 genes provide a crucial framework for a transition from traditional biology to an integrative "OMICs" arena (Lander et al., 2001; Venter et al., 2001; Kitano, 2002). This brings in a revolution for cancer research, which now enters a big data era. In the past decade, with the facilitation by next-generation sequencing, there have been a huge number of large-scale sequencing efforts, such as The Cancer Genome Atlas (TCGA), the HapMap, and the 1000 genomes project. As a result, a deluge of genomic information becomes available from patients stricken by a variety of cancer types. The list of cancer-associated genes is ever expanding. New discoveries are made on how frequent and highly penetrant mutations, such as those in the telomerase reverse transcriptase (TERT) and TP53, function in cancer initiation, progression, and metastasis. Most genes with relatively frequent but weakly penetrant cancer mutations still remain to be characterized. In addition, genes that harbor rare but highly penetrant cancer-associated mutations continue to emerge. Here, we review recent advances related to cancer genomics, proteomics, and systems biology and suggest new perspectives in targeted therapy and precision medicine.

ISSYS: An integrated synergistic Synthesis System

NASA Technical Reports Server (NTRS)

Dovi, A. R.

1980-01-01

Integrated Synergistic Synthesis System (ISSYS), an integrated system of computer codes in which the sequence of program execution and data flow is controlled by the user, is discussed. The commands available to exert such control, the ISSYS major function and rules, and the computer codes currently available in the system are described. Computational sequences frequently used in the aircraft structural analysis and synthesis are defined. External computer codes utilized by the ISSYS system are documented. A bibliography on the programs is included.

Geographic and temporal trends in the molecular epidemiology and genetic mechanisms of transmitted HIV-1 drug resistance: an individual-patient- and sequence-level meta-analysis.

PubMed

Rhee, Soo-Yon; Blanco, Jose Luis; Jordan, Michael R; Taylor, Jonathan; Lemey, Philippe; Varghese, Vici; Hamers, Raph L; Bertagnolio, Silvia; Rinke de Wit, Tobias F; Aghokeng, Avelin F; Albert, Jan; Avi, Radko; Avila-Rios, Santiago; Bessong, Pascal O; Brooks, James I; Boucher, Charles A B; Brumme, Zabrina L; Busch, Michael P; Bussmann, Hermann; Chaix, Marie-Laure; Chin, Bum Sik; D'Aquin, Toni T; De Gascun, Cillian F; Derache, Anne; Descamps, Diane; Deshpande, Alaka K; Djoko, Cyrille F; Eshleman, Susan H; Fleury, Herve; Frange, Pierre; Fujisaki, Seiichiro; Harrigan, P Richard; Hattori, Junko; Holguin, Africa; Hunt, Gillian M; Ichimura, Hiroshi; Kaleebu, Pontiano; Katzenstein, David; Kiertiburanakul, Sasisopin; Kim, Jerome H; Kim, Sung Soon; Li, Yanpeng; Lutsar, Irja; Morris, Lynn; Ndembi, Nicaise; Ng, Kee Peng; Paranjape, Ramesh S; Peeters, Martine; Poljak, Mario; Price, Matt A; Ragonnet-Cronin, Manon L; Reyes-Terán, Gustavo; Rolland, Morgane; Sirivichayakul, Sunee; Smith, Davey M; Soares, Marcelo A; Soriano, Vincent V; Ssemwanga, Deogratius; Stanojevic, Maja; Stefani, Mariane A; Sugiura, Wataru; Sungkanuparph, Somnuek; Tanuri, Amilcar; Tee, Kok Keng; Truong, Hong-Ha M; van de Vijver, David A M C; Vidal, Nicole; Yang, Chunfu; Yang, Rongge; Yebra, Gonzalo; Ioannidis, John P A; Vandamme, Anne-Mieke; Shafer, Robert W

2015-04-01

Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

PubMed Central

Rhee, Soo-Yon; Blanco, Jose Luis; Jordan, Michael R.; Taylor, Jonathan; Lemey, Philippe; Varghese, Vici; Hamers, Raph L.; Bertagnolio, Silvia; de Wit, Tobias F. Rinke; Aghokeng, Avelin F.; Albert, Jan; Avi, Radko; Avila-Rios, Santiago; Bessong, Pascal O.; Brooks, James I.; Boucher, Charles A. B.; Brumme, Zabrina L.; Busch, Michael P.; Bussmann, Hermann; Chaix, Marie-Laure; Chin, Bum Sik; D’Aquin, Toni T.; De Gascun, Cillian F.; Derache, Anne; Descamps, Diane; Deshpande, Alaka K.; Djoko, Cyrille F.; Eshleman, Susan H.; Fleury, Herve; Frange, Pierre; Fujisaki, Seiichiro; Harrigan, P. Richard; Hattori, Junko; Holguin, Africa; Hunt, Gillian M.; Ichimura, Hiroshi; Kaleebu, Pontiano; Katzenstein, David; Kiertiburanakul, Sasisopin; Kim, Jerome H.; Kim, Sung Soon; Li, Yanpeng; Lutsar, Irja; Morris, Lynn; Ndembi, Nicaise; NG, Kee Peng; Paranjape, Ramesh S.; Peeters, Martine; Poljak, Mario; Price, Matt A.; Ragonnet-Cronin, Manon L.; Reyes-Terán, Gustavo; Rolland, Morgane; Sirivichayakul, Sunee; Smith, Davey M.; Soares, Marcelo A.; Soriano, Vincent V.; Ssemwanga, Deogratius; Stanojevic, Maja; Stefani, Mariane A.; Sugiura, Wataru; Sungkanuparph, Somnuek; Tanuri, Amilcar; Tee, Kok Keng; Truong, Hong-Ha M.; van de Vijver, David A. M. C.; Vidal, Nicole; Yang, Chunfu; Yang, Rongge; Yebra, Gonzalo; Ioannidis, John P. A.; Vandamme, Anne-Mieke; Shafer, Robert W.

2015-01-01

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen. PMID:25849352

HIV-1 transmission networks across Cyprus (2010-2012).

PubMed

Kostrikis, Leondios G; Hezka, Johana; Stylianou, Dora C; Kostaki, Evangelia; Andreou, Maria; Kousiappa, Ioanna; Paraskevis, Dimitrios; Demetriades, Ioannis

2018-01-01

A molecular epidemiology study of HIV-1 infection was conducted in one hundred diagnosed and untreated HIV-1-infected patients in Cyprus between 2010 and 2012, representing 65.4% of all the reported HIV-1 infections in Cyprus in this three-year period, using a previously defined enrolment strategy. Eighty-two patients were newly diagnosed (genotypic drug resistance testing within six months from diagnosis), and eighteen patients were HIV-1 diagnosed for a longer period or the diagnosis date was unknown. Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 41.0 and 19.0% respectively, followed by subtype C (7.0%), F1 (8.0%), CRF02_AG (4.0%), A2 (2.0%), other circulating recombinant forms (CRFs) (7.0%) and unknown recombinant forms (URFs) (12%). Most of the newly-diagnosed study subjects were Cypriots (63%), males (78%) with median age 39 (Interquartile Range, IQR 33-48) reporting having sex with other men (MSM) (51%). A high rate of clustered transmission of subtype B drug-sensitive strains to reverse transcriptase and protease inhibitors was observed among MSM, twenty-eight out of forty-one MSM study subjects (68.0%) infected were implicated in five transmission clusters, two of which are sub-subtype A1 and three of which are subtype B strains. The two largest MSM subtype B clusters included nine and eight Cypriot men, respectively, living in all major cities in Cyprus. There were only three newly diagnosed patients with transmitted drug resistant HIV-1 strains, one study subject from the United Kingdom infected with subtype B strain and one from Romania with sub-subtype A2 strain, both with PI drug resistance mutation M46L and one from Greece with sub-subtype A1 with non-nucleoside reverse transcriptase inhibitors (NNRTI) drug resistance mutation K103N.

HIV-1 drug resistance in antiretroviral-naive individuals with HIV-1-associated tuberculous meningitis initiating antiretroviral therapy in Vietnam.

PubMed

Thao, Vu P; Le, Thuy; Török, Estee M; Yen, Nguyen T B; Chau, Tran T H; Jurriaans, Suzanne; van Doorn, H Rogier; van Doorn, Rogier H; de Jong, Menno D; Farrar, Jeremy J; Dunstan, Sarah J

2012-01-01

Access to antiretroviral therapy (ART) for HIV-infected individuals in Vietnam is rapidly expanding, but there are limited data on HIV drug resistance (HIVDR) to guide ART strategies. We retrospectively conducted HIVDR testing in 220 ART-naive individuals recruited to a randomized controlled trial of immediate versus deferred ART in individuals with HIV-associated tuberculous meningitis in Ho Chi Minh City (HCMC) from 2005-2008. HIVDR mutations were identified by population sequencing of the HIV pol gene and were defined based on 2009 WHO surveillance drug resistance mutations (SDRMs). We successfully sequenced 219/220 plasma samples of subjects prior to ART; 218 were subtype CRF01_AE and 1 was subtype B. SDRMs were identified in 14/219 (6.4%) subjects; 8/14 were resistant to nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs; T69D, L74V, V75M, M184V/I and K219R), 5/14 to non-nucleoside reverse transcriptase inhibitors (NNRTIs; K103N, V106M, Y181C, Y188C and G190A), 1/14 to both NRTIs and NNRTIs (D67N and Y181C) and none to protease inhibitors. After 6 months of ART, eight subjects developed protocol-defined virological failure. HIVDR mutations were identified in 5/8 subjects. All five had mutations with high-level resistance to NNRTIs and three had mutations with high-level resistance to NRTIs. Due to a high early mortality rate (58%), the effect of pre-existing HIVDR mutations on treatment outcome could not be accurately assessed. The prevalence of WHO SDRMs in ART-naive individuals with HIV-associated tuberculous meningitis in HCMC from 2005-2008 is 6.4%. The SDRMs identified conferred resistance to NRTIs and/or NNRTIs, reflecting the standard first-line ART regimens in Vietnam.

Connection anonymity analysis in coded-WDM PONs

NASA Astrophysics Data System (ADS)

Sue, Chuan-Ching

2008-04-01

A coded wavelength division multiplexing passive optical network (WDM PON) is presented for fiber to the home (FTTH) systems to protect against eavesdropping. The proposed scheme applies spectral amplitude coding (SAC) with a unipolar maximal-length sequence (M-sequence) code matrix to generate a specific signature address (coding) and to retrieve its matching address codeword (decoding) by exploiting the cyclic properties inherent in array waveguide grating (AWG) routers. In addition to ensuring the confidentiality of user data, the proposed coded-WDM scheme is also a suitable candidate for the physical layer with connection anonymity. Under the assumption that the eavesdropper applies a photo-detection strategy, it is shown that the coded WDM PON outperforms the conventional TDM PON and WDM PON schemes in terms of a higher degree of connection anonymity. Additionally, the proposed scheme allows the system operator to partition the optical network units (ONUs) into appropriate groups so as to achieve a better degree of anonymity.

Novel coding, translation, and gene expression of a replicating covalently closed circular RNA of 220 nt

PubMed Central

AbouHaidar, Mounir Georges; Venkataraman, Srividhya; Golshani, Ashkan; Liu, Bolin; Ahmad, Tauqeer

2014-01-01

The highly structured (64% GC) covalently closed circular (CCC) RNA (220 nt) of the virusoid associated with rice yellow mottle virus codes for a 16-kDa highly basic protein using novel modalities for coding, translation, and gene expression. This CCC RNA is the smallest among all known viroids and virusoids and the only one that codes proteins. Its sequence possesses an internal ribosome entry site and is directly translated through two (or three) completely overlapping ORFs (shifting to a new reading frame at the end of each round). The initiation and termination codons overlap UGAUGA (underline highlights the initiation codon AUG within the combined initiation-termination sequence). Termination codons can be ignored to obtain larger read-through proteins. This circular RNA with no noncoding sequences is a unique natural supercompact “nanogenome.” PMID:25253891

Trellises and Trellis-Based Decoding Algorithms for Linear Block Codes. Part 3; The Map and Related Decoding Algirithms

NASA Technical Reports Server (NTRS)

Lin, Shu; Fossorier, Marc

1998-01-01

In a coded communication system with equiprobable signaling, MLD minimizes the word error probability and delivers the most likely codeword associated with the corresponding received sequence. This decoding has two drawbacks. First, minimization of the word error probability is not equivalent to minimization of the bit error probability. Therefore, MLD becomes suboptimum with respect to the bit error probability. Second, MLD delivers a hard-decision estimate of the received sequence, so that information is lost between the input and output of the ML decoder. This information is important in coded schemes where the decoded sequence is further processed, such as concatenated coding schemes, multi-stage and iterative decoding schemes. In this chapter, we first present a decoding algorithm which both minimizes bit error probability, and provides the corresponding soft information at the output of the decoder. This algorithm is referred to as the MAP (maximum aposteriori probability) decoding algorithm.

Automatic vehicle location system

NASA Technical Reports Server (NTRS)

Hansen, G. R., Jr. (Inventor)

1973-01-01

An automatic vehicle detection system is disclosed, in which each vehicle whose location is to be detected carries active means which interact with passive elements at each location to be identified. The passive elements comprise a plurality of passive loops arranged in a sequence along the travel direction. Each of the loops is tuned to a chosen frequency so that the sequence of the frequencies defines the location code. As the vehicle traverses the sequence of the loops as it passes over each loop, signals only at the frequency of the loop being passed over are coupled from a vehicle transmitter to a vehicle receiver. The frequencies of the received signals in the receiver produce outputs which together represent a code of the traversed location. The code location is defined by a painted pattern which reflects light to a vehicle carried detector whose output is used to derive the code defined by the pattern.

Digital data for quick response (QR) codes of alkalophilic Bacillus pumilus to identify and to compare bacilli isolated from Lonar Crator Lake, India.

PubMed

Rekadwad, Bhagwan N; Khobragade, Chandrahasya N

2016-06-01

Microbiologists are routinely engaged isolation, identification and comparison of isolated bacteria for their novelty. 16S rRNA sequences of Bacillus pumilus were retrieved from NCBI repository and generated QR codes for sequences (FASTA format and full Gene Bank information). 16SrRNA were used to generate quick response (QR) codes of Bacillus pumilus isolated from Lonar Crator Lake (19° 58' N; 76° 31' E), India. Bacillus pumilus 16S rRNA gene sequences were used to generate CGR, FCGR and PCA. These can be used for visual comparison and evaluation respectively. The hyperlinked QR codes, CGR, FCGR and PCA of all the isolates are made available to the users on a portal https://sites.google.com/site/bhagwanrekadwad/. This generated digital data helps to evaluate and compare any Bacillus pumilus strain, minimizes laboratory efforts and avoid misinterpretation of the species.

Sequence differences in the diagnostic region of the cysteine protease 8 gene of Tritrichomonas foetus parasites of cats and cattle.

PubMed

Sun, Zichen; Stack, Colin; Šlapeta, Jan

2012-05-25

In order to investigate the genetic variation between Tritrichomonas foetus from bovine and feline origins, cysteine protease 8 (CP8) coding sequence was selected as the polymorphic DNA marker. Direct sequencing of CP8 coding sequence of T. foetus from four feline isolates and two bovine isolates with polymerase chain reaction successfully revealed conserved nucleotide polymorphisms between feline and bovine isolates. These results provide useful information for CP8-based molecular differentiation of T. foetus genotypes. Copyright © 2011 Elsevier B.V. All rights reserved.

EUGENE'HOM: A generic similarity-based gene finder using multiple homologous sequences.

PubMed

Foissac, Sylvain; Bardou, Philippe; Moisan, Annick; Cros, Marie-Josée; Schiex, Thomas

2003-07-01

EUGENE'HOM is a gene prediction software for eukaryotic organisms based on comparative analysis. EUGENE'HOM is able to take into account multiple homologous sequences from more or less closely related organisms. It integrates the results of TBLASTX analysis, splice site and start codon prediction and a robust coding/non-coding probabilistic model which allows EUGENE'HOM to handle sequences from a variety of organisms. The current target of EUGENE'HOM is plant sequences. The EUGENE'HOM web site is available at http://genopole.toulouse.inra.fr/bioinfo/eugene/EuGeneHom/cgi-bin/EuGeneHom.pl.

Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations.

PubMed

Lathe, R

1985-05-05

Synthetic probes deduced from amino acid sequence data are widely used to detect cognate coding sequences in libraries of cloned DNA segments. The redundancy of the genetic code dictates that a choice must be made between (1) a mixture of probes reflecting all codon combinations, and (2) a single longer "optimal" probe. The second strategy is examined in detail. The frequency of sequences matching a given probe by chance alone can be determined and also the frequency of sequences closely resembling the probe and contributing to the hybridization background. Gene banks cannot be treated as random associations of the four nucleotides, and probe sequences deduced from amino acid sequence data occur more often than predicted by chance alone. Probe lengths must be increased to confer the necessary specificity. Examination of hybrids formed between unique homologous probes and their cognate targets reveals that short stretches of perfect homology occurring by chance make a significant contribution to the hybridization background. Statistical methods for improving homology are examined, taking human coding sequences as an example, and considerations of codon utilization and dinucleotide frequencies yield an overall homology of greater than 82%. Recommendations for probe design and hybridization are presented, and the choice between using multiple probes reflecting all codon possibilities and a unique optimal probe is discussed.

Tenebrio molitor antifreeze protein gene identification and regulation.

PubMed

Qin, Wensheng; Walker, Virginia K

2006-02-15

The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

What Information is Stored in DNA: Does it Contain Digital Error Correcting Codes?

NASA Astrophysics Data System (ADS)

Liebovitch, Larry

1998-03-01

The longest term correlations in living systems are the information stored in DNA which reflects the evolutionary history of an organism. The 4 bases (A,T,G,C) encode sequences of amino acids as well as locations of binding sites for proteins that regulate DNA. The fidelity of this important information is maintained by ANALOG error check mechanisms. When a single strand of DNA is replicated the complementary base is inserted in the new strand. Sometimes the wrong base is inserted that sticks out disrupting the phosphate backbone. The new base is not yet methylated, so repair enzymes, that slide along the DNA, can tear out the wrong base and replace it with the right one. The bases in DNA form a sequence of 4 different symbols and so the information is encoded in a DIGITAL form. All the digital codes in our society (ISBN book numbers, UPC product codes, bank account numbers, airline ticket numbers) use error checking code, where some digits are functions of other digits to maintain the fidelity of transmitted informaiton. Does DNA also utitlize a DIGITAL error chekcing code to maintain the fidelity of its information and increase the accuracy of replication? That is, are some bases in DNA functions of other bases upstream or downstream? This raises the interesting mathematical problem: How does one determine whether some symbols in a sequence of symbols are a function of other symbols. It also bears on the issue of determining algorithmic complexity: What is the function that generates the shortest algorithm for reproducing the symbol sequence. The error checking codes most used in our technology are linear block codes. We developed an efficient method to test for the presence of such codes in DNA. We coded the 4 bases as (0,1,2,3) and used Gaussian elimination, modified for modulus 4, to test if some bases are linear combinations of other bases. We used this method to analyze the base sequence in the genes from the lac operon and cytochrome C. We did not find evidence for such error correcting codes in these genes. However, we analyzed only a small amount of DNA and if digitial error correcting schemes are present in DNA, they may be more subtle than such simple linear block codes. The basic issue we raise here, is how information is stored in DNA and an appreciation that digital symbol sequences, such as DNA, admit of interesting schemes to store and protect the fidelity of their information content. Liebovitch, Tao, Todorov, Levine. 1996. Biophys. J. 71:1539-1544. Supported by NIH grant EY6234.

A Bioinformatics-Based Alternative mRNA Splicing Code that May Explain Some Disease Mutations Is Conserved in Animals.

PubMed

Qu, Wen; Cingolani, Pablo; Zeeberg, Barry R; Ruden, Douglas M

2017-01-01

Deep sequencing of cDNAs made from spliced mRNAs indicates that most coding genes in many animals and plants have pre-mRNA transcripts that are alternatively spliced. In pre-mRNAs, in addition to invariant exons that are present in almost all mature mRNA products, there are at least 6 additional types of exons, such as exons from alternative promoters or with alternative polyA sites, mutually exclusive exons, skipped exons, or exons with alternative 5' or 3' splice sites. Our bioinformatics-based hypothesis is that, in analogy to the genetic code, there is an "alternative-splicing code" in introns and flanking exon sequences, analogous to the genetic code, that directs alternative splicing of many of the 36 types of introns. In humans, we identified 42 different consensus sequences that are each present in at least 100 human introns. 37 of the 42 top consensus sequences are significantly enriched or depleted in at least one of the 36 types of introns. We further supported our hypothesis by showing that 96 out of 96 analyzed human disease mutations that affect RNA splicing, and change alternative splicing from one class to another, can be partially explained by a mutation altering a consensus sequence from one type of intron to that of another type of intron. Some of the alternative splicing consensus sequences, and presumably their small-RNA or protein targets, are evolutionarily conserved from 50 plant to animal species. We also noticed the set of introns within a gene usually share the same splicing codes, thus arguing that one sub-type of splicesosome might process all (or most) of the introns in a given gene. Our work sheds new light on a possible mechanism for generating the tremendous diversity in protein structure by alternative splicing of pre-mRNAs.

Evolutionary Dynamics of Microsatellite Distribution in Plants: Insight from the Comparison of Sequenced Brassica, Arabidopsis and Other Angiosperm Species

PubMed Central

Shi, Jiaqin; Huang, Shunmou; Fu, Donghui; Yu, Jinyin; Wang, Xinfa; Hua, Wei; Liu, Shengyi; Liu, Guihua; Wang, Hanzhong

2013-01-01

Despite their ubiquity and functional importance, microsatellites have been largely ignored in comparative genomics, mostly due to the lack of genomic information. In the current study, microsatellite distribution was characterized and compared in the whole genomes and both the coding and non-coding DNA sequences of the sequenced Brassica, Arabidopsis and other angiosperm species to investigate their evolutionary dynamics in plants. The variation in the microsatellite frequencies of these angiosperm species was much smaller than those for their microsatellite numbers and genome sizes, suggesting that microsatellite frequency may be relatively stable in plants. The microsatellite frequencies of these angiosperm species were significantly negatively correlated with both their genome sizes and transposable elements contents. The pattern of microsatellite distribution may differ according to the different genomic regions (such as coding and non-coding sequences). The observed differences in many important microsatellite characteristics (especially the distribution with respect to motif length, type and repeat number) of these angiosperm species were generally accordant with their phylogenetic distance, which suggested that the evolutionary dynamics of microsatellite distribution may be generally consistent with plant divergence/evolution. Importantly, by comparing these microsatellite characteristics (especially the distribution with respect to motif type) the angiosperm species (aside from a few species) all clustered into two obviously different groups that were largely represented by monocots and dicots, suggesting a complex and generally dichotomous evolutionary pattern of microsatellite distribution in angiosperms. Polyploidy may lead to a slight increase in microsatellite frequency in the coding sequences and a significant decrease in microsatellite frequency in the whole genome/non-coding sequences, but have little effect on the microsatellite distribution with respect to motif length, type and repeat number. Interestingly, several microsatellite characteristics seemed to be constant in plant evolution, which can be well explained by the general biological rules. PMID:23555856

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

DOE PAGES

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; ...

2015-05-12

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.« less

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.« less

NullSeq: A Tool for Generating Random Coding Sequences with Desired Amino Acid and GC Contents.

PubMed

Liu, Sophia S; Hockenberry, Adam J; Lancichinetti, Andrea; Jewett, Michael C; Amaral, Luís A N

2016-11-01

The existence of over- and under-represented sequence motifs in genomes provides evidence of selective evolutionary pressures on biological mechanisms such as transcription, translation, ligand-substrate binding, and host immunity. In order to accurately identify motifs and other genome-scale patterns of interest, it is essential to be able to generate accurate null models that are appropriate for the sequences under study. While many tools have been developed to create random nucleotide sequences, protein coding sequences are subject to a unique set of constraints that complicates the process of generating appropriate null models. There are currently no tools available that allow users to create random coding sequences with specified amino acid composition and GC content for the purpose of hypothesis testing. Using the principle of maximum entropy, we developed a method that generates unbiased random sequences with pre-specified amino acid and GC content, which we have developed into a python package. Our method is the simplest way to obtain maximally unbiased random sequences that are subject to GC usage and primary amino acid sequence constraints. Furthermore, this approach can easily be expanded to create unbiased random sequences that incorporate more complicated constraints such as individual nucleotide usage or even di-nucleotide frequencies. The ability to generate correctly specified null models will allow researchers to accurately identify sequence motifs which will lead to a better understanding of biological processes as well as more effective engineering of biological systems.

RNAcentral: A comprehensive database of non-coding RNA sequences

DOE PAGES

Williams, Kelly Porter; Lau, Britney Yan

2016-10-28

RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. Furthermore, the website has been subject to continuous improvements focusing on text and sequence similaritymore » searches as well as genome browsing functionality.« less

RNAcentral: A comprehensive database of non-coding RNA sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, Kelly Porter; Lau, Britney Yan

RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. Furthermore, the website has been subject to continuous improvements focusing on text and sequence similaritymore » searches as well as genome browsing functionality.« less

A specific indel marker for the Philippines Schistosoma japonicum revealed by analysis of mitochondrial genome sequences.

PubMed

Li, Juan; Chen, Fen; Sugiyama, Hiromu; Blair, David; Lin, Rui-Qing; Zhu, Xing-Quan

2015-07-01

In the present study, near-complete mitochondrial (mt) genome sequences for Schistosoma japonicum from different regions in the Philippines and Japan were amplified and sequenced. Comparisons among S. japonicum from the Philippines, Japan, and China revealed a geographically based length difference in mt genomes, but the mt genomic organization and gene arrangement were the same. Sequence differences among samples from the Philippines and all samples from the three endemic areas were 0.57-2.12 and 0.76-3.85 %, respectively. The most variable part of the mt genome was the non-coding region. In the coding portion of the genome, protein-coding genes varied more than rRNA genes and tRNAs. The near-complete mt genome sequences for Philippine specimens were identical in length (14,091 bp) which was 4 bp longer than those of S. japonicum samples from Japan and China. This indel provides a unique genetic marker for S. japonicum samples from the Philippines. Phylogenetic analyses based on the concatenated amino acids of 12 protein-coding genes showed that samples of S. japonicum clustered according to their geographical origins. The identified mitochondrial indel marker will be useful for tracing the source of S. japonicum infection in humans and animals in Southeast Asia.

Integrative structural annotation of de novo RNA-Seq provides an accurate reference gene set of the enormous genome of the onion (Allium cepa L.)

PubMed Central

Kim, Seungill; Kim, Myung-Shin; Kim, Yong-Min; Yeom, Seon-In; Cheong, Kyeongchae; Kim, Ki-Tae; Jeon, Jongbum; Kim, Sunggil; Kim, Do-Sun; Sohn, Seong-Han; Lee, Yong-Hwan; Choi, Doil

2015-01-01

The onion (Allium cepa L.) is one of the most widely cultivated and consumed vegetable crops in the world. Although a considerable amount of onion transcriptome data has been deposited into public databases, the sequences of the protein-coding genes are not accurate enough to be used, owing to non-coding sequences intermixed with the coding sequences. We generated a high-quality, annotated onion transcriptome from de novo sequence assembly and intensive structural annotation using the integrated structural gene annotation pipeline (ISGAP), which identified 54,165 protein-coding genes among 165,179 assembled transcripts totalling 203.0 Mb by eliminating the intron sequences. ISGAP performed reliable annotation, recognizing accurate gene structures based on reference proteins, and ab initio gene models of the assembled transcripts. Integrative functional annotation and gene-based SNP analysis revealed a whole biological repertoire of genes and transcriptomic variation in the onion. The method developed in this study provides a powerful tool for the construction of reference gene sets for organisms based solely on de novo transcriptome data. Furthermore, the reference genes and their variation described here for the onion represent essential tools for molecular breeding and gene cloning in Allium spp. PMID:25362073

Code-modulated visual evoked potentials using fast stimulus presentation and spatiotemporal beamformer decoding.

PubMed

Wittevrongel, Benjamin; Van Wolputte, Elia; Van Hulle, Marc M

2017-11-08

When encoding visual targets using various lagged versions of a pseudorandom binary sequence of luminance changes, the EEG signal recorded over the viewer's occipital pole exhibits so-called code-modulated visual evoked potentials (cVEPs), the phase lags of which can be tied to these targets. The cVEP paradigm has enjoyed interest in the brain-computer interfacing (BCI) community for the reported high information transfer rates (ITR, in bits/min). In this study, we introduce a novel decoding algorithm based on spatiotemporal beamforming, and show that this algorithm is able to accurately identify the gazed target. Especially for a small number of repetitions of the coding sequence, our beamforming approach significantly outperforms an optimised support vector machine (SVM)-based classifier, which is considered state-of-the-art in cVEP-based BCI. In addition to the traditional 60 Hz stimulus presentation rate for the coding sequence, we also explore the 120 Hz rate, and show that the latter enables faster communication, with a maximal median ITR of 172.87 bits/min. Finally, we also report on a transition effect in the EEG signal following the onset of the stimulus sequence, and recommend to exclude the first 150 ms of the trials from decoding when relying on a single presentation of the stimulus sequence.

Coexistence of BRAF V600E and TERT Promoter Mutations in Low-grade Serous Carcinoma of Ovary Recurring as Carcinosarcoma in a Lymph Node: Report of a Case.

PubMed

Tavallaee, Mahkam; Steiner, David F; Zehnder, James L; Folkins, Ann K; Karam, Amer K

2018-04-03

Low-grade serous carcinomas only rarely coexist with or progress to high-grade tumors. We present a case of low-grade serous carcinoma with transformation to carcinosarcoma on recurrence in the lymph node. Identical BRAF V600E and telomerase reverse transcriptase promoter mutations were identified in both the original and recurrent tumor. Given that telomerase reverse transcriptase promotor mutations are thought to play a role in progression of other tumor types, the function of telomerase reverse transcriptase mutations in BRAF mutated low-grade serous carcinoma deserves investigation.

Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains.

PubMed

Dumas, Laura; Dickens, C Michael; Anderson, Nathan; Davis, Jonathan; Bennett, Beth; Radcliffe, Richard A; Sikela, James M

2014-06-01

It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to contribute to the alcohol-related phenotypic differences associated with these strains.

Identification of Putative Nuclear Receptors and Steroidogenic Enzymes in Murray-Darling Rainbowfish (Melanotaenia fluviatilis) Using RNA-Seq and De Novo Transcriptome Assembly.

PubMed

Bain, Peter A; Papanicolaou, Alexie; Kumar, Anupama

2015-01-01

Murray-Darling rainbowfish (Melanotaenia fluviatilis [Castelnau, 1878]; Atheriniformes: Melanotaeniidae) is a small-bodied teleost currently under development in Australasia as a test species for aquatic toxicological studies. To date, efforts towards the development of molecular biomarkers of contaminant exposure have been hindered by the lack of available sequence data. To address this, we sequenced messenger RNA from brain, liver and gonads of mature male and female fish and generated a high-quality draft transcriptome using a de novo assembly approach. 149,742 clusters of putative transcripts were obtained, encompassing 43,841 non-redundant protein-coding regions. Deduced amino acid sequences were annotated by functional inference based on similarity with sequences from manually curated protein sequence databases. The draft assembly contained protein-coding regions homologous to 95.7% of the complete cohort of predicted proteins from the taxonomically related species, Oryzias latipes (Japanese medaka). The mean length of rainbowfish protein-coding sequences relative to their medaka homologues was 92.1%, indicating that despite the limited number of tissues sampled a large proportion of the total expected number of protein-coding genes was captured in the study. Because of our interest in the effects of environmental contaminants on endocrine pathways, we manually curated subsets of coding regions for putative nuclear receptors and steroidogenic enzymes in the rainbowfish transcriptome, revealing 61 candidate nuclear receptors encompassing all known subfamilies, and 41 putative steroidogenic enzymes representing all major steroidogenic enzymes occurring in teleosts. The transcriptome presented here will be a valuable resource for researchers interested in biomarker development, protein structure and function, and contaminant-response genomics in Murray-Darling rainbowfish.

Applications of statistical physics and information theory to the analysis of DNA sequences

NASA Astrophysics Data System (ADS)

Grosse, Ivo

2000-10-01

DNA carries the genetic information of most living organisms, and the of genome projects is to uncover that genetic information. One basic task in the analysis of DNA sequences is the recognition of protein coding genes. Powerful computer programs for gene recognition have been developed, but most of them are based on statistical patterns that vary from species to species. In this thesis I address the question if there exist universal statistical patterns that are different in coding and noncoding DNA of all living species, regardless of their phylogenetic origin. In search for such species-independent patterns I study the mutual information function of genomic DNA sequences, and find that it shows persistent period-three oscillations. To understand the biological origin of the observed period-three oscillations, I compare the mutual information function of genomic DNA sequences to the mutual information function of stochastic model sequences. I find that the pseudo-exon model is able to reproduce the mutual information function of genomic DNA sequences. Moreover, I find that a generalization of the pseudo-exon model can connect the existence and the functional form of long-range correlations to the presence and the length distributions of coding and noncoding regions. Based on these theoretical studies I am able to find an information-theoretical quantity, the average mutual information (AMI), whose probability distributions are significantly different in coding and noncoding DNA, while they are almost identical in all studied species. These findings show that there exist universal statistical patterns that are different in coding and noncoding DNA of all studied species, and they suggest that the AMI may be used to identify genes in different living species, irrespective of their taxonomic origin.

Partial sequence homogenization in the 5S multigene families may generate sequence chimeras and spurious results in phylogenetic reconstructions.

PubMed

Galián, José A; Rosato, Marcela; Rosselló, Josep A

2014-03-01

Multigene families have provided opportunities for evolutionary biologists to assess molecular evolution processes and phylogenetic reconstructions at deep and shallow systematic levels. However, the use of these markers is not free of technical and analytical challenges. Many evolutionary studies that used the nuclear 5S rDNA gene family rarely used contiguous 5S coding sequences due to the routine use of head-to-tail polymerase chain reaction primers that are anchored to the coding region. Moreover, the 5S coding sequences have been concatenated with independent, adjacent gene units in many studies, creating simulated chimeric genes as the raw data for evolutionary analysis. This practice is based on the tacitly assumed, but rarely tested, hypothesis that strict intra-locus concerted evolution processes are operating in 5S rDNA genes, without any empirical evidence as to whether it holds for the recovered data. The potential pitfalls of analysing the patterns of molecular evolution and reconstructing phylogenies based on these chimeric genes have not been assessed to date. Here, we compared the sequence integrity and phylogenetic behavior of entire versus concatenated 5S coding regions from a real data set obtained from closely related plant species (Medicago, Fabaceae). Our results suggest that within arrays sequence homogenization is partially operating in the 5S coding region, which is traditionally assumed to be highly conserved. Consequently, concatenating 5S genes increases haplotype diversity, generating novel chimeric genotypes that most likely do not exist within the genome. In addition, the patterns of gene evolution are distorted, leading to incorrect haplotype relationships in some evolutionary reconstructions.

Shared prefetching to reduce execution skew in multi-threaded systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eichenberger, Alexandre E; Gunnels, John A

Mechanisms are provided for optimizing code to perform prefetching of data into a shared memory of a computing device that is shared by a plurality of threads that execute on the computing device. A memory stream of a portion of code that is shared by the plurality of threads is identified. A set of prefetch instructions is distributed across the plurality of threads. Prefetch instructions are inserted into the instruction sequences of the plurality of threads such that each instruction sequence has a separate sub-portion of the set of prefetch instructions, thereby generating optimized code. Executable code is generated basedmore » on the optimized code and stored in a storage device. The executable code, when executed, performs the prefetches associated with the distributed set of prefetch instructions in a shared manner across the plurality of threads.« less

Elevated Human telomerase reverse transcriptase gene expression in blood cells associated with chronic and arsenic exposure in Inner Mongolia, China

EPA Science Inventory

BACKGROUND: Arsenic exposure is associated with human cancer. Telomerase containing the catalytic subunit, human telomerase reverse transcriptase (hTERT), can extend telomeres of chromosomes, delay senescence and promoting cell proliferation leading to tumorigenesis. OBJECTIVE:...

The evolution of transcriptional regulation in eukaryotes

NASA Technical Reports Server (NTRS)

Wray, Gregory A.; Hahn, Matthew W.; Abouheif, Ehab; Balhoff, James P.; Pizer, Margaret; Rockman, Matthew V.; Romano, Laura A.

2003-01-01

Gene expression is central to the genotype-phenotype relationship in all organisms, and it is an important component of the genetic basis for evolutionary change in diverse aspects of phenotype. However, the evolution of transcriptional regulation remains understudied and poorly understood. Here we review the evolutionary dynamics of promoter, or cis-regulatory, sequences and the evolutionary mechanisms that shape them. Existing evidence indicates that populations harbor extensive genetic variation in promoter sequences, that a substantial fraction of this variation has consequences for both biochemical and organismal phenotype, and that some of this functional variation is sorted by selection. As with protein-coding sequences, rates and patterns of promoter sequence evolution differ considerably among loci and among clades for reasons that are not well understood. Studying the evolution of transcriptional regulation poses empirical and conceptual challenges beyond those typically encountered in analyses of coding sequence evolution: promoter organization is much less regular than that of coding sequences, and sequences required for the transcription of each locus reside at multiple other loci in the genome. Because of the strong context-dependence of transcriptional regulation, sequence inspection alone provides limited information about promoter function. Understanding the functional consequences of sequence differences among promoters generally requires biochemical and in vivo functional assays. Despite these challenges, important insights have already been gained into the evolution of transcriptional regulation, and the pace of discovery is accelerating.

Convolutional encoding of self-dual codes

NASA Technical Reports Server (NTRS)

Solomon, G.

1994-01-01

There exist almost complete convolutional encodings of self-dual codes, i.e., block codes of rate 1/2 with weights w, w = 0 mod 4. The codes are of length 8m with the convolutional portion of length 8m-2 and the nonsystematic information of length 4m-1. The last two bits are parity checks on the two (4m-1) length parity sequences. The final information bit complements one of the extended parity sequences of length 4m. Solomon and van Tilborg have developed algorithms to generate these for the Quadratic Residue (QR) Codes of lengths 48 and beyond. For these codes and reasonable constraint lengths, there are sequential decodings for both hard and soft decisions. There are also possible Viterbi-type decodings that may be simple, as in a convolutional encoding/decoding of the extended Golay Code. In addition, the previously found constraint length K = 9 for the QR (48, 24;12) Code is lowered here to K = 8.

Gene and translation initiation site prediction in metagenomic sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyatt, Philip Douglas; LoCascio, Philip F; Hauser, Loren John

2012-01-01

Gene prediction in metagenomic sequences remains a difficult problem. Current sequencing technologies do not achieve sufficient coverage to assemble the individual genomes in a typical sample; consequently, sequencing runs produce a large number of short sequences whose exact origin is unknown. Since these sequences are usually smaller than the average length of a gene, algorithms must make predictions based on very little data. We present MetaProdigal, a metagenomic version of the gene prediction program Prodigal, that can identify genes in short, anonymous coding sequences with a high degree of accuracy. The novel value of the method consists of enhanced translationmore » initiation site identification, ability to identify sequences that use alternate genetic codes and confidence values for each gene call. We compare the results of MetaProdigal with other methods and conclude with a discussion of future improvements.« less

Discrete Ramanujan transform for distinguishing the protein coding regions from other regions.

PubMed

Hua, Wei; Wang, Jiasong; Zhao, Jian

2014-01-01

Based on the study of Ramanujan sum and Ramanujan coefficient, this paper suggests the concepts of discrete Ramanujan transform and spectrum. Using Voss numerical representation, one maps a symbolic DNA strand as a numerical DNA sequence, and deduces the discrete Ramanujan spectrum of the numerical DNA sequence. It is well known that of discrete Fourier power spectrum of protein coding sequence has an important feature of 3-base periodicity, which is widely used for DNA sequence analysis by the technique of discrete Fourier transform. It is performed by testing the signal-to-noise ratio at frequency N/3 as a criterion for the analysis, where N is the length of the sequence. The results presented in this paper show that the property of 3-base periodicity can be only identified as a prominent spike of the discrete Ramanujan spectrum at period 3 for the protein coding regions. The signal-to-noise ratio for discrete Ramanujan spectrum is defined for numerical measurement. Therefore, the discrete Ramanujan spectrum and the signal-to-noise ratio of a DNA sequence can be used for distinguishing the protein coding regions from the noncoding regions. All the exon and intron sequences in whole chromosomes 1, 2, 3 and 4 of Caenorhabditis elegans have been tested and the histograms and tables from the computational results illustrate the reliability of our method. In addition, we have analyzed theoretically and gotten the conclusion that the algorithm for calculating discrete Ramanujan spectrum owns the lower computational complexity and higher computational accuracy. The computational experiments show that the technique by using discrete Ramanujan spectrum for classifying different DNA sequences is a fast and effective method. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Evolution of Bony Vertebrate Enhancers at Odds with Their Coding Sequence Landscape.

PubMed

Yousaf, Aisha; Sohail Raza, Muhammad; Ali Abbasi, Amir

2015-08-06

Enhancers lie at the heart of transcriptional and developmental gene regulation. Therefore, changes in enhancer sequences usually disrupt the target gene expression and result in disease phenotypes. Despite the well-established role of enhancers in development and disease, evolutionary sequence studies are lacking. The current study attempts to unravel the puzzle of bony vertebrates' conserved noncoding elements (CNE) enhancer evolution. Bayesian phylogenetics of enhancer sequences spotlights promising interordinal relationships among placental mammals, proposing a closer relationship between humans and laurasiatherians while placing rodents at the basal position. Clock-based estimates of enhancer evolution provided a dynamic picture of interspecific rate changes across the bony vertebrate lineage. Moreover, coelacanth in the study augmented our appreciation of the vertebrate cis-regulatory evolution during water-land transition. Intriguingly, we observed a pronounced upsurge in enhancer evolution in land-dwelling vertebrates. These novel findings triggered us to further investigate the evolutionary trend of coding as well as CNE nonenhancer repertoires, to highlight the relative evolutionary dynamics of diverse genomic landscapes. Surprisingly, the evolutionary rates of enhancer sequences were clearly at odds with those of the coding and the CNE nonenhancer sequences during vertebrate adaptation to land, with land vertebrates exhibiting significantly reduced rates of coding sequence evolution in comparison to their fast evolving regulatory landscape. The observed variation in tetrapod cis-regulatory elements caused the fine-tuning of associated gene regulatory networks. Therefore, the increased evolutionary rate of tetrapods' enhancer sequences might be responsible for the variation in developmental regulatory circuits during the process of vertebrate adaptation to land. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Single nucleotide polymorphisms in common bean: their discovery and genotyping using a multiplex detection system

USDA-ARS?s Scientific Manuscript database

Single-nucleotide Polymorphism (SNP) markers are by far the most common form of DNA polymorphism in a genome. The objectives of this study were to discover SNPs in common bean comparing sequences from coding and non-coding regions obtained from Genbank and genomic DNA and to compare sequencing resu...

Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Shuyun; Wang, Yang; Cassidy-Amstutz, Caleb

2011-10-28

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and themore » cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.« less

Novel numerical and graphical representation of DNA sequences and proteins.

PubMed

Randić, M; Novic, M; Vikić-Topić, D; Plavsić, D

2006-12-01

We have introduced novel numerical and graphical representations of DNA, which offer a simple and unique characterization of DNA sequences. The numerical representation of a DNA sequence is given as a sequence of real numbers derived from a unique graphical representation of the standard genetic code. There is no loss of information on the primary structure of a DNA sequence associated with this numerical representation. The novel representations are illustrated with the coding sequences of the first exon of beta-globin gene of half a dozen species in addition to human. The method can be extended to proteins as is exemplified by humanin, a 24-aa peptide that has recently been identified as a specific inhibitor of neuronal cell death induced by familial Alzheimer's disease mutant genes.

Ovine mitochondrial DNA sequence variation and its association with production and reproduction traits within an Afec-Assaf flock.

PubMed

Reicher, S; Seroussi, E; Weller, J I; Rosov, A; Gootwine, E

2012-07-01

Polymorphisms in mitochondrial DNA (mtDNA) protein- and tRNA-coding genes were shown to be associated with various diseases in humans as well as with production and reproduction traits in livestock. Alignment of full length mitochondria sequences from the 5 known ovine haplogroups: HA (n = 3), HB (n = 5), HC (n = 3), HD (n = 2), and HE (n = 2; GenBank accession nos. HE577847-50 and 11 published complete ovine mitochondria sequences) revealed sequence variation in 10 out of the 13 protein coding mtDNA sequences. Twenty-six of the 245 variable sites found in the protein coding sequences represent non-synonymous mutations. Sequence variation was observed also in 8 out of the 22 tRNA mtDNA sequences. On the basis of the mtDNA control region and cytochrome b partial sequences along with information on maternal lineages within an Afec-Assaf flock, 1,126 Afec-Assaf ewes were assigned to mitochondrial haplogroups HA, HB, and HC, with frequencies of 0.43, 0.43, and 0.14, respectively. Analysis of birth weight and growth rate records of lamb (n = 1286) and productivity from 4,993 lambing records revealed no association between mitochondrial haplogroup affiliation and female longevity, lambs perinatal survival rate, birth weight, and daily growth rate of lambs up to 150 d that averaged 1,664 d, 88.3%, 4.5 kg, and 320 g/d, respectively. However, significant (P < 0.0001) differences among the haplogroups were found for prolificacy of ewes, with prolificacies (mean ± SE) of 2.14 ± 0.04, 2.25 ± 0.04, and 2.30 ± 0.06 lamb born/ewe lambing for the HA, HB, and the HC haplogroups, respectively. Our results highlight the ovine mitogenome genetic variation in protein- and tRNA coding genes and suggest that sequence variation in ovine mtDNA is associated with variation in ewe prolificacy.

Sense-antisense (complementary) peptide interactions and the proteomic code; potential opportunities in biology and pharmaceutical science.

PubMed

Miller, Andrew D

2015-02-01

A sense peptide can be defined as a peptide whose sequence is coded by the nucleotide sequence (read 5' → 3') of the sense (positive) strand of DNA. Conversely, an antisense (complementary) peptide is coded by the corresponding nucleotide sequence (read 5' → 3') of the antisense (negative) strand of DNA. Research has been accumulating steadily to suggest that sense peptides are capable of specific interactions with their corresponding antisense peptides. Unfortunately, although more and more examples of specific sense-antisense peptide interactions are emerging, the very idea of such interactions does not conform to standard biology dogma and so there remains a sizeable challenge to lift this concept from being perceived as a peripheral phenomenon if not worse, into becoming part of the scientific mainstream. Specific interactions have now been exploited for the inhibition of number of widely different protein-protein and protein-receptor interactions in vitro and in vivo. Further, antisense peptides have also been used to induce the production of antibodies targeted to specific receptors or else the production of anti-idiotypic antibodies targeted against auto-antibodies. Such illustrations of utility would seem to suggest that observed sense-antisense peptide interactions are not just the consequence of a sequence of coincidental 'lucky-hits'. Indeed, at the very least, one might conclude that sense-antisense peptide interactions represent a potentially new and different source of leads for drug discovery. But could there be more to come from studies in this area? Studies on the potential mechanism of sense-antisense peptide interactions suggest that interactions may be driven by amino acid residue interactions specified from the genetic code. If so, such specified amino acid residue interactions could form the basis for an even wider amino acid residue interaction code (proteomic code) that links gene sequences to actual protein structure and function, even entire genomes to entire proteomes. The possibility that such a proteomic code should exist is discussed. So too the potential implications for biology and pharmaceutical science are also discussed were such a code to exist.

3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

PubMed

Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

2008-09-01

We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

Origins of genes: "big bang" or continuous creation?

PubMed Central

Keese, P K; Gibbs, A

1992-01-01

Many protein families are common to all cellular organisms, indicating that many genes have ancient origins. Genetic variation is mostly attributed to processes such as mutation, duplication, and rearrangement of ancient modules. Thus it is widely assumed that much of present-day genetic diversity can be traced by common ancestry to a molecular "big bang." A rarely considered alternative is that proteins may arise continuously de novo. One mechanism of generating different coding sequences is by "overprinting," in which an existing nucleotide sequence is translated de novo in a different reading frame or from noncoding open reading frames. The clearest evidence for overprinting is provided when the original gene function is retained, as in overlapping genes. Analysis of their phylogenies indicates which are the original genes and which are their informationally novel partners. We report here the phylogenetic relationships of overlapping coding sequences from steroid-related receptor genes and from tymovirus, luteovirus, and lentivirus genomes. For each pair of overlapping coding sequences, one is confined to a single lineage, whereas the other is more widespread. This suggests that the phylogenetically restricted coding sequence arose only in the progenitor of that lineage by translating an out-of-frame sequence to yield the new polypeptide. The production of novel exons by alternative splicing in thyroid receptor and lentivirus genes suggests that introns can be a valuable evolutionary source for overprinting. New genes and their products may drive major evolutionary changes. PMID:1329098

Abstract feature codes: The building blocks of the implicit learning system.

PubMed

Eberhardt, Katharina; Esser, Sarah; Haider, Hilde

2017-07-01

According to the Theory of Event Coding (TEC; Hommel, Müsseler, Aschersleben, & Prinz, 2001), action and perception are represented in a shared format in the cognitive system by means of feature codes. In implicit sequence learning research, it is still common to make a conceptual difference between independent motor and perceptual sequences. This supposedly independent learning takes place in encapsulated modules (Keele, Ivry, Mayr, Hazeltine, & Heuer 2003) that process information along single dimensions. These dimensions have remained underspecified so far. It is especially not clear whether stimulus and response characteristics are processed in separate modules. Here, we suggest that feature dimensions as they are described in the TEC should be viewed as the basic content of modules of implicit learning. This means that the modules process all stimulus and response information related to certain feature dimensions of the perceptual environment. In 3 experiments, we investigated by means of a serial reaction time task the nature of the basic units of implicit learning. As a test case, we used stimulus location sequence learning. The results show that a stimulus location sequence and a response location sequence cannot be learned without interference (Experiment 2) unless one of the sequences can be coded via an alternative, nonspatial dimension (Experiment 3). These results support the notion that spatial location is one module of the implicit learning system and, consequently, that there are no separate processing units for stimulus versus response locations. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Adaptive decoding of convolutional codes

NASA Astrophysics Data System (ADS)

Hueske, K.; Geldmacher, J.; Götze, J.

2007-06-01

Convolutional codes, which are frequently used as error correction codes in digital transmission systems, are generally decoded using the Viterbi Decoder. On the one hand the Viterbi Decoder is an optimum maximum likelihood decoder, i.e. the most probable transmitted code sequence is obtained. On the other hand the mathematical complexity of the algorithm only depends on the used code, not on the number of transmission errors. To reduce the complexity of the decoding process for good transmission conditions, an alternative syndrome based decoder is presented. The reduction of complexity is realized by two different approaches, the syndrome zero sequence deactivation and the path metric equalization. The two approaches enable an easy adaptation of the decoding complexity for different transmission conditions, which results in a trade-off between decoding complexity and error correction performance.

Optimization of algorithm of coding of genetic information of Chlamydia

NASA Astrophysics Data System (ADS)

Feodorova, Valentina A.; Ulyanov, Sergey S.; Zaytsev, Sergey S.; Saltykov, Yury V.; Ulianova, Onega V.

2018-04-01

New method of coding of genetic information using coherent optical fields is developed. Universal technique of transformation of nucleotide sequences of bacterial gene into laser speckle pattern is suggested. Reference speckle patterns of the nucleotide sequences of omp1 gene of typical wild strains of Chlamydia trachomatis of genovars D, E, F, G, J and K and Chlamydia psittaci serovar I as well are generated. Algorithm of coding of gene information into speckle pattern is optimized. Fully developed speckles with Gaussian statistics for gene-based speckles have been used as criterion of optimization.

Simulated Assessment of Interference Effects in Direct Sequence Spread Spectrum (DSSS) QPSK Receiver

DTIC Science & Technology

2014-03-27

bit error rate BPSK binary phase shift keying CDMA code division multiple access CSI comb spectrum interference CW continuous wave DPSK differential... CDMA ) and GPS systems which is a Gold code. This code is generated by a modulo-2 operation between two different preferred m-sequences. The preferred m...10 SNR Sim (dB) S N R O ut ( dB ) SNR RF SNR DS Figure 3.26: Comparison of input S NRS im and S NROut of the band-pass RF filter (S NRRF) and

Hiding message into DNA sequence through DNA coding and chaotic maps.

PubMed

Liu, Guoyan; Liu, Hongjun; Kadir, Abdurahman

2014-09-01

The paper proposes an improved reversible substitution method to hide data into deoxyribonucleic acid (DNA) sequence, and four measures have been taken to enhance the robustness and enlarge the hiding capacity, such as encode the secret message by DNA coding, encrypt it by pseudo-random sequence, generate the relative hiding locations by piecewise linear chaotic map, and embed the encoded and encrypted message into a randomly selected DNA sequence using the complementary rule. The key space and the hiding capacity are analyzed. Experimental results indicate that the proposed method has a better performance compared with the competing methods with respect to robustness and capacity.

Physics behind the mechanical nucleosome positioning code

NASA Astrophysics Data System (ADS)

Zuiddam, Martijn; Everaers, Ralf; Schiessel, Helmut

2017-11-01

The positions along DNA molecules of nucleosomes, the most abundant DNA-protein complexes in cells, are influenced by the sequence-dependent DNA mechanics and geometry. This leads to the "nucleosome positioning code", a preference of nucleosomes for certain sequence motives. Here we introduce a simplified model of the nucleosome where a coarse-grained DNA molecule is frozen into an idealized superhelical shape. We calculate the exact sequence preferences of our nucleosome model and find it to reproduce qualitatively all the main features known to influence nucleosome positions. Moreover, using well-controlled approximations to this model allows us to come to a detailed understanding of the physics behind the sequence preferences of nucleosomes.

EUGÈNE'HOM: a generic similarity-based gene finder using multiple homologous sequences

PubMed Central

Foissac, Sylvain; Bardou, Philippe; Moisan, Annick; Cros, Marie-Josée; Schiex, Thomas

2003-01-01

EUGÈNE'HOM is a gene prediction software for eukaryotic organisms based on comparative analysis. EUGÈNE'HOM is able to take into account multiple homologous sequences from more or less closely related organisms. It integrates the results of TBLASTX analysis, splice site and start codon prediction and a robust coding/non-coding probabilistic model which allows EUGÈNE'HOM to handle sequences from a variety of organisms. The current target of EUGÈNE'HOM is plant sequences. The EUGÈNE'HOM web site is available at http://genopole.toulouse.inra.fr/bioinfo/eugene/EuGeneHom/cgi-bin/EuGeneHom.pl. PMID:12824408

An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences.

PubMed

Pollier, Jacob; González-Guzmán, Miguel; Ardiles-Diaz, Wilson; Geelen, Danny; Goossens, Alain

2011-01-01

cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

PubMed Central

Gustafson, G; Armour, S L

1986-01-01

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs. Images PMID:3754962

Colour cyclic code for Brillouin distributed sensors

NASA Astrophysics Data System (ADS)

Le Floch, Sébastien; Sauser, Florian; Llera, Miguel; Rochat, Etienne

2015-09-01

For the first time, a colour cyclic coding (CCC) is theoretically and experimentally demonstrated for Brillouin optical time-domain analysis (BOTDA) distributed sensors. Compared to traditional intensity-modulated cyclic codes, the code presents an additional gain of √2 while keeping the same number of sequences as for a colour coding. A comparison with a standard BOTDA sensor is realized and validates the theoretical coding gain.