SEARCH · PubMed Health

Results for “second generation sequencing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,261 records · Page 70Linked to original sources

PubMed1996

Deletions in the extracellular domain of rat trkA lead to an altered differentiative phenotype in neurotrophin responsive cells.

We have investigated the role(s) of conserved sequences in the extracellular domain of rat trkA by generating 5' and 3' deletions and assaying changes in neurotrophin binding, tyrosine kinase activity, and neurite outgrowth. Essential sequences required for both nerve growth factor (NGF) and neurotrophin-3 (NT-3) binding were mapped to the immunoglobulin-like domains. Small deletions in the second immunoglobulin-like domain and in the juxtamembrane region abolished neurotrophin binding. Dose-response curves on cells expressing full-length trkA were identical for NGF and NT-3 (0.1 ng/ml-50 ng/ml) while cells expressing leucine rich motif (LRM) minus receptors required high concentrations of NT-3 (50 ng/ml). Scatchard analysis revealed a loss of high-affinity NT-3, but not NGF, binding to the LRM minus receptor consistent with the neurite dose-response curves. Moreover, cells expressing the LRM minus receptors failed to fasciculate and showed delayed arborization in comparison to cells expressing wild-type trkA, suggesting a possible role for the LRM's in neurotrophin-induced differentiation and in high-affinity NT-3 binding.

Animals↗

PubMed1989

Restriction analysis of chromosomal sequences homologous to single-copy fragments cloned from small polydisperse circular DNA (spcDNA).

Restriction fragments from the fraction of small polydisperse circular DNA (spcDNA) were cloned in pBR322. The spcDNA was prepared from cell cultures derived from an angiofibroma of a patient with tuberous sclerosis (TS). Such cultures have been shown previously to contain increased amounts of spcDNA. Four cloned spcDNA fragments containing single-copy sequences were chosen to characterize the homologous chromosomal DNA segments by restriction analysis. When used as hybridization probes, these four fragments generate well-defined nonvariable patterns in the chromosomal DNA from healthy donors. The restriction patterns obtained with one of the fragments (D-C4) can best be interpreted by assuming the presence of two copies of the homologous sequences in chromosomal DNA. A second sequence, A-B4, occurs at least 30-50 times in the haploid human genome. In both cases the duplicated regions span relatively large segments of DNA.

Base Sequence↗

PubMed2006

Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development.

We report a novel gene tagging, identification and mutagenicity ('gene-breaking') method for the zebrafish, Danio rerio. This modular approach consists of two distinct and separable molecular cassettes. The first is a gene-finding cassette. In this study, we employed a 3' gene-tagging approach that selectively 'traps' transcripts regardless of expression status, and we show that this cassette identifies both known and novel endogenous transcripts in transgenic zebrafish. The second is a transcriptional termination mutagenicity cassette assembled from a combination of a splice acceptor and polyadenylation signal to disrupt tagged transcripts upon integration into intronic sequence. We identified both novel and conserved loci as linked phenotypic mutations using this gene-breaking strategy, generating molecularly null mutations in both larval lethal and adult viable loci. We show that the Histone 2a family member z (H2afza) variant is essential for larval development through the generation of a lethal locus with a truncation of conserved carboxy-terminal residues in the protein. In principle this gene-breaking strategy is scalable for functional genomics screens and can be used in Sleeping Beauty transposon and other gene delivery systems in the zebrafish.

Animals↗

PubMed2006

Methods for studying mutagenesis and checkpoints in Schizosaccharomyces pombe.

Mutations in genome caretaker genes can induce genomic instability, which are potentially early events in tumorigenesis. Cells have evolved biological processes to cope with the genomic insults. One is a multifaceted response, termed checkpoint, which is a network of signaling pathways to coordinate cell cycle transition with DNA repair, activation of transcriptional programs, and induction of tolerance of the genomic perturbations. When genomic perturbations are beyond repair, checkpoint responses can also induce apoptosis or senescence to eliminate those deleterious damaged cells. Fission yeast, Schizosaccharomyces pombe (S. pombe) has served as a valuable model organism for studies of the checkpoint signaling pathways. In this chapter, we describe methods used to analyze mutagenesis and recombinational repair induced by genomic perturbations, and methods used to detect the checkpoint responses to replication stress and DNA damage in fission yeast cells. In the first section, we present methods used to analyze the mutation rate, mutation spectra, and recombinational repair in fission yeast when replication is perturbed by either genotoxic agents or mutations in genomic caretaker gene such as DNA replication genes. In the second section, we describe methods used to examine checkpoint activation in response to chromosome replication stress and DNA damage. In the final section, we comment on how checkpoint activation regulates mutagenic synthesis by a translesion DNA polymerase in generating a mutator phenotype of small sequence alterations in cells, and how a checkpoint kinase appropriately regulates an endonuclease complex to either prevent or allow deletion of genomic sequences and recombinational repair when fission yeast cells experience genomic perturbation in order to avoid deleterious mutations and maintain cell growth.

DNA Damage↗

PubMed2003

Temporal computation by synaptic signaling pathways.

Synaptic signaling comprises a complex molecular network. Such networks carry out diverse operations such as molecular logic, signal amplification, memory and other aspects of cellular decision-making. The synapse in particular encounters complex input patterns that have different temporal sequences. Different input patterns to the synapse are known to give rise to a range of synaptic responses, including facilitation, depression and various forms of short and long-term potentiation. In many cases the stimuli that generate these disparate responses are tens of seconds or more in length, much greater than the typical time-courses of calcium dynamics. In this paper I propose that the synaptic signaling network can perform temporal computation operations such as tuning for stimulus duration or interval. Using simulation methods I show that the simple time-courses of individual signaling pathways combine in the network to give rise to different temporally selective responses. Downstream pathways that exhibit temporal integration or amplitude thresholding select different input patterns and thus perform temporal computation.

Algorithms↗

PubMed1998

Molecular analysis of new subtypes of ELE/RET rearrangements, their reciprocal transcripts and breakpoints in papillary thyroid carcinomas of children after Chernobyl.

A high prevalence of RET rearrangements is found in papillary thyroid carcinomas (PTC) of children from Belarus after the Chernobyl reactor accident. The ELE/RET rearrangement (PTC3) is prevailing. Aberrant types of ELE/RET rearrangement have been found with a truncated ELE1 gene: As compared with the common form (PTC3r1) one aberrant type is shorter by one 144 bp exon (PTC3r2) (three cases); in the second atypic form (PTC3r3) the ELE1 part is 18 bp shorter than in PTC3r1. In agreement with the observation that the oncogenic RET is generated by a paracentric inversion at chromosome 10, we found not only ELE/RET, but also RET/ELE transcripts in these tumors. Sequencing of the breakpoint regions at the genomic DNA level revealed DNA modifications that might be relevant for illegitimate recombination after DNA doublestrand breaks. The high prevalence of ELE/RET rearrangements and various subtypes appears to be typical for radiation-induced thyroid carcinomas of children after the Chernobyl reactor accident.

Artificial Gene Fusion↗

PubMed1994

Complementary DNA sequencing of canine tissue factor pathway inhibitor reveals a unique nanomeric repetitive sequence between the second and third Kunitz domains.

Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent inhibitor of the factor VIIa-tissue factor complex of blood coagulation. The primary amino acid sequence of canine TFPI has been deduced from cDNA sequences obtained using the techniques of reverse transcription followed by amplification using PCR and conventional screening of a canine endothelial cell cDNA library. The open reading frame for canine TFPI encodes a signal peptide of 28 amino acids followed by a 40.7 kDa protein of 368 amino acids. Similar to human, rat and rabbit TFPI, canine TFPI contains a negatively-charged cluster of amino acids at its mature amino-terminus, followed by three Kunitz-type proteinase inhibitory domains and a cluster of positively-charged amino acids near its carboxy-terminus. In contrast to other TFPIs, following its second Kunitz-type proteinase inhibitory domain canine TFPI contains an additional amino acid insert which includes a nanomeric peptide-sequence repeated six times. Recombinant canine TFPI was expressed in both bacterial- and insect cell-expression systems for functional analysis and the generation of antibodies. The recombinant canine TFPI inhibits tissue factor-induced coagulation in an in vitro canine system. Immunoprecipitation of TFPI from canine plasma, followed by Western-blot analysis, tentatively identifies canine TFPI as an 80,000 kDa protein. Anti-peptide antibodies raised to the nanomeric peptide repeat immunoprecipitate an identical, cross-reactive, 80,000 kDa protein.

Amino Acid Sequence↗

PubMed2001

Quantitative trait loci for upper thermal tolerance in outbred strains of rainbow trout (Oncorhynchus mykiss).

The expression of three putative QTL for upper thermal tolerance (UTT) was examined in two strains of outbred rainbow trout unselected for this trait using simple-sequence repeat (SSR; microsatellite) markers associated with UTT in backcrosses of lines selected on this trait. Two-way diallel lots in the third generation of an outbred pedigree were exposed to an acute thermal challenge. QTL detection was performed separately by each second-generation parent within each diallel lot, incorporating the effects of full sib families and correlated traits. Inheritance of different alleles at the SSR Ssa20.19NUIG from the sire 93-32-1 was strongly associated with the thermal tolerance of his half sib progeny, explaining 7.5% of their phenotypic variance in this trait. A hierarchical linear model incorporating allelic inheritance from all four grandsires of the experimental diallels (in addition to family specific and covariate trait effects) was also used to detect associations between the SSR and thermal tolerance in their third-generation grandprogeny. Ssa20.19NUIG was strongly associated with thermal tolerance in the grandprogeny of the grandsire G(0)SVM2. The generally stronger marker-trait associations found in male parents may be partially due to reduced chromosomal recombination rates in male salmonids compared to females. These results indicate the effects of a QTL on a fitness-related trait in unselected populations of rainbow trout.

Alleles↗

PubMed1997

Perception of biological motion.

Boundary conditions for perception of biological motion were explored with the use of computer-generated point-light animation sequences. Perception of this unique form of structure from motion is immune to variations in dot contrast polarity, dot disparity, and spatial-frequency filtering. Biological motion is perceived in texture-defined animation sequences that presumably stimulate only second-order motion pathways, and it is undisturbed by dichoptic presentation of portions of the animation tokens separately to the two eyes.

Computer Graphics↗

PubMed1992

Primary structure, chromosomal localization, and functional expression of a voltage-gated sodium channel from human brain.

A cDNA library derived from human cerebral cortex was screened for the presence of sodium channel alpha subunit-specific clones. Ligation of three overlapping clones generated a full-length cDNA clone, HBA, that provided the complete nucleotide sequence coding for a protein of 2005 amino acids. The predicted structure suggests four homologous repeats and exhibits greatest homology and structural similarity to the rat brain sodium channel II. A second cDNA clone, HBB, that encodes a different subtype of sodium channel was isolated. Hybridization of DNA fragments from the 3' untranslated region of HBA and PCR with primers derived from HBB with human-hamster somatic cell hybrids localized these clones to human chromosome 2. In situ hybridization to human metaphase chromosomes mapped the structural genes for both HBA and HBB sodium channels to chromosome 2q23-24.3. The sodium channel HBA gene product was expressed by transfection in CHO cells. Expressed HBA currents were voltage-dependent, sodium-selective, and tetrodotoxin-sensitive and, thus, exhibit the biophysical and pharmacological properties characteristic of sodium channels.

Amino Acid Sequence↗

PubMed1988

Electronic fingerprinting of RNA.

Software has been developed to assist RNA fingerprinting analysis. One program generates, from a DNA sequence data file, the oligonucleotides resulting from digestion of an RNA transcript labeled with any specified nucleotide(s). Oligonucleotides are sorted according to their position on the fingerprint. Expected molar yields and products of secondary redigestion are also indicated. A second program facilitates calculation of experimental molar yields of oligonucleotides.

Base Sequence↗

PubMed2005

Characterization of a second gene encoding gamma-glutamyl transpeptidase from Schizosaccharomyces pombe.

The first gene encoding gamma-glutamyl transpeptidase (GGTI) of the fission yeast has previously been characterized, and its expression was found to be regulated by various oxidative stress-inducing agents. In this work, a second gene, encoding GGTII, was cloned and characterized from the fission yeast Schizosaccharomyces pombe. The structural gene encoding GGTII was amplified from the genomic DNA of the fission yeast and ligated into the shuttle vector pRS316 to generate the recombinant plasmid pPHJ02. The determined sequence contains 3040 bp and is able to encode the putative 611 amino acid sequence of GGTII, which resembles the counterparts of Saccharomyces cerevisiae, Homo sapiens, Rattus norvegicus, and Escherichia coli. The DNA sequence also contains 940-bp upstream and 289-bp downstream regions of the GGTII gene. The Schizosaccharomyces pombe cells harboring plasmid pPHJ02 showed about 4-fold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGTII gene is functional. The S. pombe cells containing the cloned GGTII gene were found to contain higher levels of both intracellular glutathione (GSH) content and GSH uptake. The S. pombe cells harboring plasmid pPHJ02 showed increased survival on solid media containing hydrogen peroxide, diethylmaleate, aluminum chloride, cadmium chloride, or mercuric chloride. The GGTII mRNA level was significantly elevated by treatment with GSH-depleting diethylmaleate. These results imply that the S. pombe GGTII gene produces functional GGTII protein and is involved in the response to oxidative stresses in S. pombe cells.

Amino Acid Sequence↗

PubMed1991

Hepatitis B virus and hepatocellular carcinoma: a possible role for the viral transactivators.

Several epidemiological studies have demonstrated a link between chronic B virus infection and primary hepatocellular carcinoma (PHC). HBV DNA sequence integrations into the host cell genome have often been observed in hepatocarcinoma tissues. However, since only in a few cases of PHC the target of HBV-DNA insertion has been identified, alternative mechanisms for HBV-induced hepatocyte transformation have been investigated. Like many other DNA viruses, the hepatitis B virus bears a transactivational potential. Both full length and truncated versions of HBV X protein are able to influence the expression of cellular nuclear protooncogenes c-fos and c-myc. A second transcriptional activator is encoded by the PreS/S region of HBV, but its activity on viral and cellular genes become evident only after dislocations from its downstream sequences. Thus, HBV is able to influence infected cell growth and differentiation using both native proteins, newly generated truncated proteins and virus-cell fusion polypeptides.

Carcinoma, Hepatocellular↗

PubMed1990

Kinetics and amplification in neutrophil activation and adaptation.

Neutrophil activation and adaptation are beginning to be understood in quantitative and mechanistic detail. Neutrophil responses to chemoattractants involve cell surface receptors, guanine nucleotide binding (G) proteins and intracellular second messengers which are generated within a few seconds of the presentation of the ligand. Novel real-time fluorescent methods have made it possible to examine the dynamics of assembly and disassembly of receptors and G proteins during cell activation and to model, by computer, the complex sequence of ligand-receptor events. Rapid amplification cascades of signals are observed in both stimulatory and inhibitory receptor pathways. The adaptation of neutrophils involves uncoupling between receptors and G proteins, both by sequestering receptors from the transduction sequence and by reducing the activatibility of the G proteins.

Adaptation, Physiological↗

PubMed1997

Trypsin- and SLIGRL-induced vascular relaxation and the inhibition by benzamidine derivatives.

Serine proteinases are involved in several physiological processes and elicit profound cellular effects in a variety of tissues. Besides the thrombin receptor a second receptor, activated by trypsin, the proteinase-activated receptor 2 (PAR-2), was cloned and characterized. Both enzymes generate a new extracellular N-terminus by limited proteolytic cleavage which functions as tethered ligand to activate the receptor. Synthetic peptides corresponding to the sequences of the newly generated N-terminus are able to mimic the effects of the enzymes. In porcine pulmonary arteries trypsin and the receptor-derived peptide SLIGRL elicited an endothelium-dependent transient relaxation of PGF2alpha-precontracted vessels. The EC50 values for trypsin and SLIGRL amounted to 1.1 +/- 0.2 nM and 5.4 +/- 0.6 microM, respectively. Trypsin and SLIGRL caused a homologous desensitization but thrombin and the thrombin receptor-activating peptide SFLLRN were still able to elicit pronounced relaxant effects. The trypsin- and SLIGRL-induced relaxant responses were markedly diminished after blockade of the nitric oxide synthesis by N(G)-nitro-L-arginine methyl ester (200 microM) and were absent in endothelium-denuded vessels. Indomethacin and hirudin did not influence the relaxant effects. The effect of trypsin but not that of SLIGRL was blocked by the proteinase inhibitor aprotinin suggesting that only proteolytically active trypsin activates the receptor. Benzamidine derivatives of the 3-amidinophenylalanine type with different affinity for trypsin and thrombin inhibited the vascular effects of trypsin (IC50 0.007-0.7 microM) correlating with its antitrypsin activity. The data suggest that the vascular effects of trypsin and SLIGRL are mediated through activation of PAR-2 which differs from the thrombin receptor.

Animals↗

PubMed1990

The T----C substitution at -198 of the A gamma-globin gene associated with the British form of HPFH generates overlapping recognition sites for two DNA-binding proteins.

Defects in the developmental changes of human hemoglobin production characterized by the continued expression of fetal globin during adult life are classified as hereditary persistence of fetal hemoglobin (HPFH). Among the various molecular lesions associated with this phenotype, the non-deletion forms with point mutations in the promoter region are thought to provide mechanistic clues for gamma-globin gene regulation. The natural occurrence of four different base substitutions mapping within six nucleotides of a homopurine.homopyrimidine motif in the upstream promoter region demarcate a potential control element. To assess its importance for transcriptional activity, we compared the -202 (C----G), -198 (T----C) and -196 (C----T) HPFH mutations with the normal sequence in binding studies with nuclear proteins from erythroid and non-erythroid cells. Wildtype DNA and HPFH mutations at -202 or -196 showed only a weak protein interaction of unclear functional significance. In contrast, -198 (T----C) generated overlapping, high-affinity binding sites for two ubiquitous nuclear proteins. One cognate protein was identified as the transcription factor Sp1. The second one was termed NF-G.C as it interacted strongly with the homopolymer poly(dG).poly(dC). The generation of additional recognition sites for trans-acting factors by the -198 HPFH mutation correlated with a modest increase in promoter activity in vitro specifically with nuclear extracts from erythroid cells. The activation appears to be mediated by binding of Sp1, but it requires interaction with an erythroid-specific factor, most likely GF-1. Templates containing the -196 HPFH mutation showed a transcriptional activity identical to wildtype. This suggests that despite the topological proximity of the mutations, the HPFH phenotype may be established by different mechanisms.

Base Composition↗

PubMed2004

Rectal cancer: MR imaging in local staging--is gadolinium-based contrast material helpful?

PURPOSE: To determine retrospectively whether addition of gadolinium-enhanced T1-weighted magnetic resonance (MR) sequence to T2-weighted turbo spin-echo (SE) MR imaging is valuable for preoperative assessment of T stage and circumferential resection margin in patients with primary rectal cancer. MATERIALS AND METHODS: Local institutional review board approved study and waived informed patient consent. Eighty-three patients with operable primary rectal cancer underwent preoperative MR imaging. Retrospectively, two observers independently scored T2-weighted turbo SE MR images and, in a second reading, T2-weighted images combined with gadolinium-enhanced T1-weighted turbo SE MR images for tumor penetration through rectal wall and tumor extension into mesorectal fascia. A confidence level scoring system was used, and receiver operating characteristic (ROC) curves were generated. Histologic findings were standard of reference. Difference in performance of T2-weighted and combined T2-weighted plus gadolinium-enhanced T1-weighted sequences was analyzed by comparing corresponding areas under ROC curves (A(z)) for each observer. Interobserver agreement was calculated by using linear weighted kappa statistics. RESULTS: Addition of contrast-enhanced T1-weighted to T2-weighted MR imaging did not significantly improve diagnostic accuracy for prediction of tumor penetration through rectal wall (A(z) of T2-weighted vs T2-weighted plus T1-weighted images for observer 1, 0.740 vs 0.764; observer 2, 0.856 vs 0.768) and tumor extension into mesorectal fascia (A(z) for observer 1, 0.962 vs 0.902; observer 2, 0.902 vs 0.911). Diagnostic performance (A(z)) of MR and interobserver agreement were high for prediction of tumor extension into mesorectal fascia (kappa = 0.61, 0.74) but only moderate for penetration through rectal wall (kappa = 0.47, 0.45). CONCLUSION: Gadolinium-enhanced MR sequences did not improve diagnostic accuracy for assessment of tumor penetration through rectal wall and tumor extension into mesorectal fascia.

Adult↗

PubMed2000

Three different turkey luteinizing hormone receptor (tLH-R) isoforms I: characterization of alternatively spliced tLH-R isoforms and their regulated expression in diverse tissues.

Using combinations of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends, three different, alternatively spliced, partial turkey LH receptor (tLH-R) cDNA isoforms were characterized from ovarian mRNA. The first cDNA (tLH-R(intact)) showed 98% and 72-75% similarity with chicken and mammalian LH-R sequences, respectively. The second cloned cDNA isoform (tLH-R(insert)) contained an in-frame TGA stop codon within an 86-base pair insertion that was located in the extracellular domain of the seven-transmembrane region. The tLH-R(insert) isoform could encode a truncated soluble protein isoform that lacked the transmembrane region. The third cDNA isoform truncated the transmembrane region (tLH-R(trunc)) and was derived by the deletion of the last exon by incomplete splicing. Generation of multiple transcripts by alternative splicing was elucidated by partial characterization of tLH-R genomic sequences. The differentially regulated expression of the tLH-R mRNA isoforms in nongonadal tissues and ovarian stromal tissues during various reproductive stages was quantified and analyzed by Northern blot and/or RT-PCR. Alternatively spliced tLH-R isoforms were differentially expressed in a tissue-specific manner in most of the tissues examined. The steady-state levels of tLH-R mRNA isoforms were relatively high in the hypothalamus and optic nerve and relatively low in the cortex, pituitary, and cerebellum when compared to levels in ovarian follicles. In nongonadal reproductive tissues, the steady-state levels of tLH-R mRNA isoforms were relatively high in the uterus and infundibulum and relatively low in the isthmus, oviduct, and magnum. In addition, in the nongonadal peripheral tissues, the steady-state levels of tLH-R isoforms were relatively high in the thyroid gland and relatively low in the spleen, adrenal gland, kidney, skin, bursa, and muscle. The present study suggests that the alternative splicing of LH-R transcripts occurs in a tissue-specific manner and has been evolutionarily conserved (similar results were obtained in chicken and swine). These results raise fundamental questions as to the function of LH-R isoforms in nongonadal tissues.

Alternative Splicing↗