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Results for “second generation sequencing”

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At least 289 records · Page 16Linked to original sources

PubMed2003

Identification and characterization of a new class of human immunodeficiency virus type 1 recombinants comprised of two circulating recombinant forms, CRF07_BC and CRF08_BC, in China.

We identified a new class of human immunodeficiency virus type 1 (HIV-1) recombinants (00CN-HH069 and 00CN-HH086) in which further recombination occurred between two established circulating recombinant forms (CRFs). These two isolates were found among 57 HIV-1 samples from a cohort of injecting drug users in eastern Yunnan Province of China. Informative-site analysis in conjunction with bootscanning plots and exploratory tree analysis revealed that these two strains were closely related mosaics comprised of CRF07_BC and CRF08_BC, which are found in China. The genotype screening based on gag-reverse transcriptase sequences of 57 samples from eastern Yunnan identified 47 CRF08_BC specimens (82.5%), 5 CRF07_BC specimens (8.8%), and 3 additional specimens with the novel recombinant structure. These new "second-generation" recombinants thus constitute a substantial proportion (5 of 57; 8.8%) of HIV-1 strains in this population and may belong to a new but yet-undefined class of CRF. This might be the first example of CRFs recombining with each other, leading to the evolution of second-generation inter-CRF recombinants.

Base Sequence↗

PubMed1981

Primary structure of the Fc region of human immunoglobulin D: implications for evolutionary origin and biological function.

We have determined the complete amino acid sequence of a tryptic Fc delta fragment generated from an intact human IgD (WAH); it is 226 residues long and includes the second (C delta 2) and the third (C delta 3) constant domains of the delta chain. Comparison of the homology of the Fc sequence of the five human immunoglobulin classes suggests that either the delta-chain gene evolved from the alpha-chain gene soon after the divergence of a mu-alpha common ancestor or it evolved from an ancestral gene distinct from both the mu-alpha and the gamma-epsilon common ancestors. Comparative study using a spatial model of the Fc region indicates that the structure of the C delta 3 domain differs extensively from that of the carboxy-terminal domains of other heavy chain classes; this, together with the unique hinge region structure, probably reflects the biological role of IgD as a receptor molecule on the B-lymphocyte surface.

Amino Acid Sequence↗

PubMed1986

Programs to produce high quality dichotic tapes for central auditory testing.

Dichotic stimulation, the simultaneous presentation of two different acoustic signals to the right and left ears, respectively, is used routinely in the clinical assessment of speech lateralization as well as in other central auditory testing procedures in the clinic and laboratory. At present, most researchers and clinicians depend on a few commercial sources for dichotic tapes, because there are a limited number of facilities which are able to produce such tapes. Moreover, some of the commercial tapes currently offered for sale contain important stimulus errors. In the present paper, we describe a general set of programs for the PDP 11 series of computers, which permits sequences of dichotic stimuli to be generated and output with a high degree of precision. These programs therefore allow researchers to generate any desired sequence of dichotic stimulation, for recording and taping, for use in their research. The programs operate in two stages to generate the required dichotic sequences. In the first stage, the constrained random ordering of the stimuli is generated as specified by the user. In the second stage, after the preliminary stimulus preparation has been completed (for example, using a waveform editing package; cf., D.G. Jamieson and D. A. Naugler, Comput. Biomed. Res., 1985, 18, 480), an audio tape is generated with stimuli presented dichotically, with timing and sequencing precisely as specified.

Auditory Cortex↗

PubMed1984

The light-harvesting polypeptides of Rhodospirillum rubrum. I. The amino-acid sequence of the second light-harvestng polypeptide B 880-beta (B 870-beta) of Rhodospirillum rubrum S 1 and the carotenoidless mutant G-9+. carotenoidless mutant G-9+.

The light-harvesting complex B 880 from Rhodospirillum rubrum S 1 (wild type) and B 870 from the carotenoidless mutant G-9+ was shown to consist mainly of an organic solvent-(chloroform/methanol-) soluble and an organic solvent-insoluble polypeptide. The isolation and separation of these two low-molecular-mass polypeptides (Mr 6101 and Mr 6079) were achieved by a two-step extraction procedure of chromatophores using in the first step chloroform/methanol containing 0.1M ammonium acetate. Following Sephadex LH-60 chromatography of this first extract a light-harvesting polypeptide (B 870-alpha) was isolated and its complete amino acid sequence was determined (R. Brunisholz et al. (1981) FEBS Lett. 129/1, 150-154, B 880-alpha: G. Gogel et al. (1983) Biochim. Biophys. Acta 746, 32-39). Upon reextraction of the chromatophore pellet with chloroform/methanol/ammonium acetate containing in addition acetic acid a second low-molecular-mass polypeptide (B 880-beta of B 870-beta) was generated. The complete amino acid sequences of the chloroform/methanol-insoluble light-harvesting polypeptide of Rs. rubrum S 1 (B 880-beta) and of Rs. rubrum G-9+ (B 870-beta) were determined. They are identical and consist of 54 amino acid residues. The conserved histidine residue within the hydrophobic stretch raises more evidence for ligand complexation of bacteriochlorophyll to this specific histidine residue which therefore possibly plays the key role in pigment-protein interactions. Both polypeptides (B 880-alpha and B 880-beta) are part of the light-harvesting complex B 880 in an apparent ratio of 1:1. Based on the primary structure data a possible arrangement of both light-harvesting polypeptides within the membrane will be discussed.

Amino Acid Sequence↗

PubMed1995

Determination of cyclic nucleotide-dependent protein kinase substrate specificity by the use of peptide libraries on cellulose paper.

An iterative approach to the a priori determination of the substrate specificity of cAMP- and cGMP-dependent protein kinases (PKA and PKG) by the use of peptide libraries on cellulose paper is described. The starting point of the investigation was an octamer library with the general structure Ac-XXX12XXX, where X represents mixtures of all 20 natural amino acids and 1 and 2 represent individual amino acid residues. The library thus contained all possible 2.56 x 10(10) octamers, divided into 400 sublibraries with defined amino acids 1 and 2 each consisting of 6.4 x 10(7) sequences. After phosphorylation with the kinases in the presence of [gamma-32P]ATP, the sublibrarys Ac-XXXRRXXX and Ac-XXXRKXXX were identified as the best substrates for PKA and PKG, respectively. The second-generation libraries had the structures Ac-XXXRR12X and Ac-XXXRK12X for PKA and PKG and resulted in the most active sequence pools Ac-XXXRRASX and Ac-XXXRKKSX. After delineation of every position in the octameric sequence and extension of the investigation to decameric peptides, the best sequences, Ac-KRAERKASIY and Ac-TQKARKKSNA, were obtained for PKA and PKG, respectively. Promising octameric and decameric peptides were assembled 5 or 10 times each and assayed in order to determine the experimental scatter inherent in the approach. The kinetic data of several octameric and decameric sequences were determined in solution and compared to data for known substrates. The recognition motif of PKA was confirmed by this approach, and a novel substrate sequence for PKG was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed2006

Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome.

BACKGROUND: The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs. RESULTS: Four positively regulated and two negatively regulated genes were identified by transposon mutagenesis including genes potentially involved in iron transport and virulence. The promoter regions of selected CepR regulated genes and site directed mutagenesis of the cepI promoter were used to predict a consensus cep box sequence for CepR binding. The first-generation consensus sequence for the cep box was used to identify putative cep boxes in the genome sequence. Eight potential CepR regulated genes were chosen and the expression of their promoters analyzed. Six of the eight were shown to be regulated by CepR. A second generation motif was created from the promoters of these six genes in combination with the promoters of cepI, zmpA, and two of the CepR regulated genes identified by transposon mutagenesis. A search of the B. cenocepacia J2315 genome with the new motif identified 55 cep boxes in 65 promoter regions that may be regulated by CepR. CONCLUSION: Using transposon mutagenesis and bioinformatics expression of twelve new genes have been determined to be regulated by the CepIR quorum sensing system. A cep box consensus sequence has been developed based on the predicted cep boxes of ten CepR regulated genes. This consensus cep box has led to the identification of over 50 new genes potentially regulated by the CepIR quorum sensing system.

4-Butyrolactone↗

PubMed1998

Practical evaluation of comparative modelling and threading methods.

Six protein pairs, all with known 3D-structures, were used to evaluate different protein structure prediction tools. Firstly, alignments between a target sequence and a template sequence or structure were obtained by sequence alignment with QUANTA or by threading with THREADER, 123D and PHD Topits. Secondly, protein structure models were generated using MODELLER. The two protein structure assessment tools used were the root mean square deviation (RMSD) compared with the experimental target structure and the total 3D profile score. Also the accuracy of the active sites of models built in the absence and presence of ligands was investigated. Our study confirms that threading methods are able to yield more accurate models than comparative modelling in cases of low sequence identity (< 30%). However, a gap of 2 A (RMSD) exists between the theoretically best model and the models obtained by threading methods. For high sequence identities (> 30%) comparative modelling using MODELLER resulted in accurate models. Furthermore, the total 3D profile score was not always able to distinguish correct from incorrect folds when different alignment methods were used. Finally, we found it to be important to include possible ligands in the model-building process in order to prevent unrealistic filling of active site areas.

Algorithms↗

PubMed2026

Employing Metagenomics Capture targeted next-generation sequencing for the etiological diagnosis of bloodstream infections.

BACKGROUND: Bloodstream infections (BSIs) represent a significant public health concern. Metagenomic Capture targeted next-generation sequencing technology, as a newly emerging method for pathogen detection, has been applied in the etiological diagnosis of various infectious diseases and demonstrates good diagnostic efficacy. However, there is relatively limited research on the diagnostic value of this technology for the etiological diagnosis of BSIs. METHODS: A comprehensive retrospective analysis was performed on patients suspected of having BSIs who were admitted to the Affiliated Guangdong Second Provincial General Hospital of Jinan University in 2024. These patients underwent both blood culture analysis and Metagenomic Capture targeted next-generation sequencing technology for diagnostic testing, and a detailed comparison of the results was conducted. RESULTS: It was found that the Metagenomic Capture-targeted next-generation sequencing method has a shorter time to result [1.33 (1.18 - 1.69) vs 2.73 (1.89 - 3.84) days, p < 0.001], more pathogenic microbial species detected, higher positive detection rate and higher sensitivity than blood culture. CONCLUSIONS: Metagenomic Capture targeted next-generation sequencing technology is a promising tool for pathogen identification in BSIs, offering substantial methodological advantages in terms of turnaround time, detection breadth, and sensitivity. These diagnostic performance characteristics support its potential utility in clinical microbiology practice.

Humans↗

PubMed2000

Development of a second generation linkage map for almond using RAPD and SSR markers.

Fifty-four RAPD (random amplified polymorphic DNA) markers and 6 SSRs (simple sequence repeats) were included in a molecular marker map with 120 RFLPs (restriction fragment length polymorphisms) and 7 isozyme genes previously constructed using the offspring of a cross between the almond (Prunus amygdalus) cultivars 'Ferragnès' and 'Tuono'. Only highly reproducible RAPDs segregating 1:1 were used. To identify these markers, a total of 325 primers were screened, from which 41 produced RAPDs useful for mapping. Polymorphism was detected in six of the eight Prunus SSRs (simple sequence repeats) studied, thus enabling these to be mapped. All markers were placed on the 8 linkage groups previously identified. The number of new markers included in the map of 'Ferragnès' was 33 for a total of 126, and 30 in the map of 'Tuono' for a total of 99. The sizes of the maps of 'Ferragnès' (415 cM) and 'Tuono' (416 cM) were similar, representing a 5% increase over the maps constructed solely with isozymes and RFLPs. The estimated total size of the almond map was of 457 cM. Some markers were placed in zones with low density of markers and others in the extreme of linkage groups. The use of RAPD markers to complete genetic maps constructed with transferable markers is discussed.

Chromosome Mapping↗

PubMed2006

A second expressed kininogen gene in mice.

We isolated PCR, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE-PCR)-, and RT-PCR-generated clones from mouse kininogen family transcripts. DNA sequencing indicated that the clones were from two distinct genes. One set (K1) is from the previously reported mouse kininogen gene. The second set (K2) has an open reading frame, is 93% identical to K1 in the overlapping nucleotide sequence, and, unlike T-kininogens in the rat, encodes a bradykinin motif identical to K1. We discovered that K2 exists with two different 5' ends. We used RT-PCR to determine the distribution and relative abundance of K1 and K2 mRNA in mouse tissues. K2 is transcribed and K1 and K2 are generally both expressed in the same tissues; however, they differ in their regulation of the alternative splicing event that yields either low-molecular-weight kininogen (LMWK) or high-molecular-weight kininogen (HMWK). For example, in the liver K1 is expressed as both HMWK and LMWK, whereas K2 is only expressed as LMWK. Conversely, in the kidney K2 is strongly expressed as both HMWK and LMWK, whereas K1 is not expressed as HMWK and expressed only very weakly as LMWK.

Animals↗

PubMed1989

Computer-controlled sequencing of peptides by tandem mass spectrometry.

A fully automated computer-controlled system was used to generate series of different linked scans at constant B2E and constant neutral loss in the second field-free region. This system has been shown to be suitable for deriving the amino acid sequence of oligopeptides.

Amino Acid Sequence↗

PubMed1995

Detection of hepatitis C virus antibodies and specific hepatitis C virus ribonucleic acid sequences in cord bloods from a heterogeneous prenatal population.

OBJECTIVE: Our aim was to quantify the prevalence of at-risk pregnancies for maternal-fetal hepatitis C virus transmission in a heterogeneous prenatal population by detection of both hepatitis C virus-specific antibody and hepatitis C virus ribonucleic acid sequences in cord bloods from their deliveries. STUDY DESIGN: An anonymous serosurvey of 1432 consecutive umbilical cord blood samples were analyzed for hepatitis C virus antibodies with a second-generation enzyme immunoassay with all hepatitis C virus antibody-positive samples batched and analyzed for both human immunodeficiency virus antibodies and hepatitis C virus ribonucleic acid sequences by polymerase chain reaction. RESULTS: Forty-seven of the samples (3.2%) were positive for hepatitis C virus antibodies; seropositivity rates differed significantly by socioeconomic status but not by race. Significantly more of the antibody-positive women underwent cesarean section for delivery (31.9% vs 21.9%, p = 0.03). Three (6.4%) hepatitis C virus antibody-positive samples were also human immunodeficiency virus-antibody positive, whereas nine (19.1%) were hepatitis C virus ribonucleic acid positive. CONCLUSION: As many as 19% of hepatitis C virus antibody-positive women in this study also had hepatitis C virus ribonucleic acid isolated from their delivery cord blood samples, which may indicate an increased risk of vertical hepatitis C virus transmission in those pregnancies. Hepatitis C virus-specific antibody and ribonucleic acid detection may also be markers for other pregnancy complications that result in higher rates of cesarean section for these women.

Base Sequence↗

PubMed1997

Identification of a neutrophil chemotactic factor from Tritrichomonas foetus as superoxide dismutase.

Antibodies to a neutrophil chemotactic factor from Tritrichomonas foetus were used to screen a T. foetus cDNA expression library in lambda gt11. All positive clones were identified as homologs of iron-containing superoxide dismutase (SOD). Native gel electrophoresis showed that the antibodies indeed recognized T. foetus antigens with SOD activity. Two SOD genes were found in T. foetus, and cloned and sequenced as parts of larger genomic segments of 3844 and 4089 base pairs. Transcription initiated between the first and second methionine codons of each genomic open reading frame, generating mRNAs with 5' untranslated regions of 11-15 bases, and encoding proteins of 195 amino acids. The two SOD coding sequences lacked obvious introns. They were 79% identical at both the nucleotide and amino acid levels. Both SOD genes were inserted into a eukaryotic expression vector and stably expressed in mammalian cells; both proteins were recognized by the antibodies, and both assumed a cytosolic, extranuclear distribution in these cells. Histidine-tagged forms of both T. foetus SODs were expressed in E. coli and after purification, found to have neutrophil chemotactic activity similar to the non-recombinant factor purified from T. foetus. Identification of this neutrophil chemotactic factor as SOD provides additional insight into the host-parasite interaction.

Amino Acid Sequence↗

PubMed2001

Diagnosis of Liddle syndrome by genetic analysis of beta and gamma subunits of epithelial sodium channel--a report of five affected family members.

OBJECTIVE: To screen the gene mutation in beta and gamma subunits of the epithelial sodium channel (ENaC) of a Chinese family, some of whose members are clinically diagnosed as suffering from Liddle syndrome. METHODS: Twelve family members were recruited to the study. Among them, two brothers had been clinically diagnosed as suffering from Liddle syndrome. Peripheral blood samples were collected from all members of the family and total genomic DNA was prepared for genetic analysis. Polymerase chain reaction (PCR) was used for amplifying the last exon of beta (codon 513-673) and gamma (codon 503-632) subunits of the ENaC gene. PCR products were purified and subjected to a direct DNA sequence analysis. RESULTS: Genetic analysis of the beta ENaC gene revealed a missense mutation of CCC to CTC at codon 616 in four middle-aged men of the second generation and one young woman of the third generation. There was no mutation of the gamma ENaC gene in any of the individuals examined. CONCLUSION: Through direct DNA sequencing analysis, we diagnosed the disease present in five members of a Chinese family as Liddle syndrome, and excluded it in some other young offspring suffering from the monogenic disease. Our results provide further evidence that Pro616 is a critical amino acid that has a key role in the inhibition of sodium channel activity.

Adolescent↗

PubMed1984

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

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.

Animals↗

PubMed2006

Concise synthesis of guanidine-containing heterocycles using the Biginelli reaction.

Two general methods for the synthesis of 2-imino-5-carboxy-3,4-dihydropyrimidines were developed using the three-component Biginelli reaction. The first method utilizes pyrazole carboxamidine, a beta-ketoester, and an aldehyde in an initial Biginelli reaction. After Boc protection, these products undergo aminolysis and acidic deprotection to generate 2-imino-5-carboxy-3,4-dihydropyrimidines in a four-step sequence. The second method utilizes a triazone-protected guanidine, a beta-ketoester, and an aldehyde in a Biginelli reaction. Acidic cleavage of the triazone yields 2-imino-5-carboxy-3,4-dihydropyrimidines in a two-step sequence. We also describe the further elaboration of several of these products using a tethered Biginelli reaction to give triazaacenaphthalene structures similar to those found in crambescidin and batzelladine alkaloids.

Aldehydes↗

PubMed1996

A protein class database organized with ProSite protein groups and PIR superfamilies.

A protein class (ProClass) database is developed as a "value-added" "second-generation" database organized according to family relationships. The database collects non-redundant protein sequence entries from SwissProt and PIR databases, and classifies them in families defined collectively by the ProSite protein groups and PIR superfamilies. The major objectives of the database are to maximize family information retrieval, to provide speedy family identification, and to help organizing existing protein sequence databases. The database has two sub-databases: PCFam (ProClass Family) to define protein families and provide links to ProSite patterns and PIR superfamilies, and PCSeq (ProClass Sequence) to describe sequence entries and provide links to PCFam, SwissProt, PIR, and ProSite databases. The current ProClass release has a total of 85,165 sequence entries, about half of which are classified in 3072 ProClass families; it also contains 10,431 newly established SwissProt-PIR links. The database can help reveal domain structures of related families, define new ProSite and PIR families, and provide family assignments for unclassified sequence entries. New ProSite and PIR family members are readily identified via database cross-reference, including 9437 SwissProt entries and 8522 PIR entries. False negative family members missed by both ProSite and PIR are detected using a neural network family identification system. The newly identified superfamily memberships are being incorporated into the current PIR database releases in a collaborative effort with the PIR. The ProClass database is accessible through anonymous FTP and on-line search on the World Wide Web.

Amino Acid Sequence↗

PubMed2004

A synthetic Rev-independent bovine immunodeficiency virus-based packaging construct.

Replication competent lentivirus (RCL) has been the major safety concern associated with applications of lentivirus-based gene transfer systems for human gene therapy. Minimization and elimination of overlaps between the packaging and the transfer vector constructs are expected to reduce the potential to generate RCL. We previously developed second- and third-generation bovine immunodeficiency virus (BIV)-based gene transfer systems. However, some sequence homologies between the vector and gag/pol packaging constructs remained. In order to minimize the sequence homologies, we recoded gag/pol with codon usage optimized for expression in human cells in this report. Expression of the recoded gag/pol was Rev/RRE independent. Thus, RRE was eliminated from the packaging construct, thereby removing a 312 bp block of homology. In addition, recoding gag/pol minimized overall homologies between the packaging and transfer vector constructs. Vectors generated by the recoded packaging construct with a four plasmid system had titers greater than 1 x 10(6) transducing units per milliliter, equivalent to those of the earlier generation systems. The vectors were functional in vitro and efficiently transduced rat pigment epithelial cells in vivo. Generation of the synthetic packaging construct provides further advances to the safety of lentiviral vectors for clinical applications.

Animals↗