Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “third generation sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

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

At least 451 records · Page 25Linked to original sources
PubMed2006

Aromatase inhibitors and breast cancer.

Oestrogens are involved in risk to, and progression of, breast cancer. Drugs that inhibit the production of oestrogens (aromatase inhibitors, AIs), are therefore extremely attractive strategies both to prevent the disease and to treat established tumours. AIs now have a central role in the treatment of established breast cancer and are being considered for prevention. Third generation agents have been derived from rational drug design. They are able to block oestrogen production and reduce oestrogen levels to a degree that has not been observed previously and without affecting levels of other hormones. Such selective reduction of endogenous oestrogen provides targeted therapy for hormone-dependent breast cancer. This has led to improved clinical benefits in patients with these tumours. Anastrozole, letrozole and exemestane all have impressive antitumour effects in postmenopausal women with breast cancer and they are at least as beneficial as or better than other established endocrine agents when used to treat hormone-sensitive cancer in the advanced setting or as an adjuvant to surgery in earlier stages of the disease; ongoing trials are exploring the use of AIs in the preventative setting. Third generation inhibitors are well tolerated, having no greater side effects than might be expected from oestrogen suppression. Important differences in endocrinological and molecular effects exist between AIs and SERMs. These have implications for the preferred drug sequence and setting in which AIs are used. Since the major obstacle to more widespread use is primary/acquired resistance, discovery of the mechanisms by which resistance occurs offers hope for the future. More detailed study of AIs will yield important information about the involvement of oestrogen on the development and progression of breast cancer. Consequently AIs offer major clinical benefits to patients with breast cancer and the promise of relatively nontoxic intervention in women at high risk of the disease.

Adipose Tissue↗
PubMed1997

Differential distribution of hepatitis C virus subtypes in Asia: comparative study among Thailand, Indonesia, the Philippines and Japan.

Hepatitis C virus (HCV) is currently classified into at least six major genotypes, each of which is further divided into a number of subtypes. It has been reported that prevalence of each subtype varies among different geographical regions of the world and that severity of liver disease and sensitivity to interferon treatment varies with different subtypes. The purpose of this study was to determine and compare the prevalence of each subtype among HCV isolates in different areas in Asia such as southern (Hat Yai) and northern (Chiang Mai) parts of Thailand, Indonesia (Surabaya), the Philippines (Manila) and Japan (Kobe). Sera were obtained from various groups of patients and tested for antibodies against HCV using second and/or third generation ELISA kits. RNA was extracted from anti-HCV-positive sera and reverse-transcribed into cDNA. The cDNA-preparations were subjected to nested PCR to amplify NS5B and 5'-untranslated region (5'UTR) sequences. Amplified fragments were sequenced and subtypes of the isolates were determined based on sequence similarities with reported sequences. In Chiang Mai and Hat Yai, Thailand, HCV-3a, HCV-1a and HCV-1b were common in various populations. HCV type 6 variants were commonly found among blood donors and drug addicts in Chiang Mai, but not in Hat Yai. In Surabaya, Indonesia, HCV-2a was frequently detected in blood donors, but less frequently in patients with chronic liver disease. In blood donors, HCV-1a, HCV-1b and HCV-1d were more strongly associated with elevation of serum aminotransferase levels than HCV-2a. HCV-1a was significantly more common in patients on maintenance hemodialysis than in blood donors or patients with chronic liver disease. HCV-1d was detected exclusively in Indonesia. Another unique subtype HCV-3g was found also in Indonesia, though less frequently than HCV-1d. In the Philippines, a vast majority of the isolates were either HCV-1a or HCV-1b. Thus, HCV subtype prevalence varies among different regions of Asia.

Hepacivirus↗
PubMed2003

Functional genomic analysis of the 61D-61F region of the third chromosome of Drosophila melanogaster.

Assigning functional significance to completed genome sequences is one of the next challenges in biological science. Conventional genetic tools such as deficiency chromosomes help assign essential complementation groups to their corresponding genes. We describe an F2 genetic screen to identify lethal mutations within cytogenetic region 61D-61F of the third chromosome of Drosophila melanogaster. One hundred sixteen mutations were identified by their failure to complement both Df(3L)bab-PG and Df(3L)3C7. These alleles were assigned to 14 complementation groups and 9 deficiency intervals. Complementation groups were ordered using existing deficiencies, as well as new deficiencies generated in this study. With the aid of the genomic sequence, genetic and physical maps in the region were correlated by use of PCR to localize the breakpoints of deficiencies within a 268-kb genomic contig (GenBank accession No. AC005847). Six essential complementation groups were assigned to specific genes, including genes encoding a porphobilinogen deaminase and a Sac1-like protein.

Alleles↗
PubMed1995

Molecular characterization of nucleotide sequences encoding the extracellular glycoprotein elicitor from Phytophthora megasperma.

cDNA sequences encoding the 42 kDa glycoprotein elicitor from the oomycete, Phytophthora megasperma, that induces the defense response in parsley have been cloned and sequenced. The 5' end of the mRNA matches a consensus derived from sequences surrounding the transcription initiation sites of seven other oomycete genes. The major transcript of 1802 nucleotides contains a 529-codon open reading frame, which was predicted to encode a 57 kDa precursor protein. On the basis of peptide sequencing, the N-terminus of the mature protein is at position 163, suggesting that proteolytic processing events, in addition to signal peptide cleavage, generate the protein purified from the fungal culture filtrate. Expression studies in Escherichia coli with the cDNA as well as smaller subfragments demonstrated that a region of 47 amino acids located in the C-terminal third of the protein was sufficient to confer elicitor activity. The gene encoding the elicitor was found to be a member of a multigene family in P. megasperma. Homologous families of differing sizes were found in all eight other Phytophthora species tested, but not in other filamentous fungi including other Oomycetes. No significant similarity of the elicitor preprotein to sequences present in the databases has yet been detected.

Amino Acid Sequence↗
PubMed1996

Alternative splicing gives rise to two novel long isoforms of Zn-alpha 2-glycoprotein, a member of the immunoglobulin superfamily.

We have isolated, from a rat liver cDNA library, two cDNAs encoding novel long isoforms of Zn-alpha2-glycoprotein (Zn-alpha2-gp), a member of the immunoglobulin superfamily with a high degree of sequence similarity to class-I major histocompatibility complex (MHC) antigens. Nucleotide (nt) sequence analysis of these two novel cDNAs has revealed that they contain insertions of 138 and 123 nt between the second and third exons of Zn-alpha2-gp, resulting in in-frame insertions of 46 and 41 amino acids (aa), respectively. Analysis of the mechanism of generation of both isoforms, named Zn-alpha2-gpA and Zn-alpha2-gpB, has shown that they result from a series of alternative splicing events, including alternative use of two additional exons, and of two different 3'-splice sites present in the first of these novel exons. The occurrence of these alternative splicing events in Zn-alpha2-gp could contribute to increasing the diversity of this nonpolymorphic and soluble class-I MHC antigen.

Alternative Splicing↗
PubMed1999

T cell receptor beta-chain third complementarity-determining region gene usage is highly restricted among Sm-B autoantigen-specific human T cell clones derived from patients with connective tissue disease.

OBJECTIVE: To determine the structure of T cell receptors (TCR) used by Sm-B-reactive human T cell clones, to map T cell epitopes on the Sm-B autoantigen, and to determine the HLA restriction element used in the recognition of Sm-B by T cells. METHODS: Sm-B-reactive T cell clones were generated from patients with connective tissue disease by using either a recombinant fusion protein or synthetic peptides. The TCR structure was defined with the use of polymerase chain reaction and DNA sequencing. Synthetic peptides were used to map T cell epitopes on Sm-B. HLA restriction element usage was defined by using monoclonal antibody blocking. RESULTS: Usage of the TCR third complementarity-determining region (CDR3) was highly restricted among Sm-B autoantigen-specific human T cell clones. Only amino acids 48-96 of the Sm-B2 autoantigen were recognized by T cells, and this occurred in the context of HLA-DR. CONCLUSION: TCR CDR3 gene usage is highly conserved by Sm-B autoantigen-specific T cell clones, and this appears to be related to the recognition of a limited number of T cell epitopes on the Sm-B autoantigen presented in the context of HLA-DR.

Amino Acid Sequence↗
PubMed1997

Localization, structure and expression of the gene for translation initiation factor eIF-4E from Drosophila melanogaster.

Drosophila melanogaster contains a single copy of the gene encoding translation eukaryotic initiation factor eIF-4E, which maps in the 67A2B1 region of chromosome 3L. A genomic clone containing the entire eIF-4E gene was isolated and sequenced. Comparison of this sequence with a cDNA previously obtained in our laboratory and 5'-RACE analysis revealed the existence of three mRNAs that are generated by alternative splicing of a primary transcript. All of them have different 5' untranslated leader regions. Two of the mRNAs encode the same eIF-4E polypeptide, whose sequence has been deduced from the cDNA clone. The third mRNA species contains a new open reading frame, possibly encoding another isoform of eIF-4E, which is very similar in size to the other but has a different N-terminal sequence. Several sequences which may be involved in the regulation of transcription initiation of the eIF-4E gene, except for a consensus TATA box, were found upstream of the putative transcription initiation sites. Expression of the eIF-4E gene is spatially and temporally controlled during embryonic development. It is ubiquitously expressed during embryogenesis but transcripts preferentially accumulate in certain tissues, particularly in the pole cells, at different developmental stages.

Alternative Splicing↗
PubMed1984

A fusion gene in man: DNA sequence analysis of the abnormal globin gene of hemoglobin Miyada.

An abnormal globin gene from a patient heterozygous for Hemoglobin Miyada was cloned and sequenced. The results indicated that the 5' flanking region and the 5' side of the gene were identical to those of a beta-globin gene and that the 3' side was identical to that of a delta-globin gene. The part of the gene identical to a beta-globin gene shifted to the part identical to the delta-globin gene somewhere in a homologous sequence region between the third nucleotide of the 17th codon and the second nucleotide of the 22nd codon of these two genes. Thus, results of analysis of the nucleotide sequence support the idea that the abnormal globin gene of Hemoglobin Miyada was generated as a fusion gene by unequal crossing over between a beta- and a delta-globin gene.

Amino Acid Sequence↗
PubMed1999

Analysis of T cell receptors specific for U1-70kD small nuclear ribonucleoprotein autoantigen: the alpha chain complementarity determining region three is highly conserved among connective tissue disease patients.

The U1-70kD autoantigen is a major target of B cell responses in patients with connective tissue diseases (CTD). T cell responses are important in the pathogenesis of CTD, however little is known about autoantigen-specific T cells in these diseases. We have recently proven that U1-70kD-reactive human T cells exist. To further characterize these autoreactive T cells, U1-70kD-reactive T cell clones have been generated from patients with CTD using either a recombinant fusion protein or synthetic peptides spanning the U1-70kD polypeptide. T cell receptors (TCR) isolated from the U1-70kD-reactive T cell clones were sequenced and the third complementarity-determining region (CDR3) compared to determine if a common motif was present. mAb blocking of antigen-induced proliferation was done to determine the HLA restriction element used in recognition of the U1-70kD autoantigen by T cells. The results presented here indicate that TCRAV CDR3 usage is highly restricted among U1-70kD autoantigen-specific human T cells clones derived from CTD patients with distinctive structural features. Furthermore, the recognition of the U1-70kD autoantigen occurs in the context of HLA-DR.

Amino Acid Sequence↗
PubMed1998

Functionally null mutations in patients with the cblG-variant form of methionine synthase deficiency.

Methionine synthase (MS) catalyses the methylation of homocysteine to methionine and requires the vitamin B12 derivative, methylcobalamin, as cofactor. We and others have recently cloned cDNAs for MS and described mutations associated with the cblG complementation group that correspond to MS deficiency. A subset of cblG, known as "cblG variant," shows no detectable MS activity and failure of [57Co]CN cobalamin to incorporate into MS in patient fibroblasts. We report the mutations responsible for three cblG-variant patients, two of them siblings, who presented with neonatal seizures, severe developmental delay, and elevated plasma homocysteine. Cell lines from all three patients were negative by northern blotting, though trace MS mRNA could be detected by means of phosphorimage analysis. Reverse transcriptase-PCR, SSCP, and nucleotide sequence analysis revealed four mutations. All were functionally null, creating either a frameshift with a downstream stop codon or an insert containing an internal stop codon. Of the two mutations found in the siblings, one of them, intervening sequence (IVS)-166A-->G, generates a cryptic donor splice site at position -166 of an intron beginning after Leu113, resulting in a 165-bp insertion of intronic sequence at junction 339/340. The second is a 2-bp deletion, 2112delTC. Mutations in the third patient include a G-->A substitution, well within the intron after Lys203, which results in intronic inserts of 128 or 78 bp in the mRNA. The second mutation is a 1-bp insertion, 3378insA. We conclude that the absence of MS protein in these cblG variants is due to mutations causing premature translation termination and consequent mRNA instability.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1997

Marek's disease virus latency-associated transcripts belong to a family of spliced RNAs that are antisense to the ICP4 homolog gene.

Marek's disease virus (MDV) latency-associated transcripts include at least two MDV small RNAs (MSRs) and a 10-kb RNA which map antisense to the ICP4 homolog gene and are relatively abundant in MDV-transformed lymphoblastoid cells. This report further describes the biological and structural properties of these RNAs. First, these RNAs were detected in primary lymphomas isolated from chickens infected with several oncogenic MDV strains. Second, the MSRs are nonpolyadenylated, whereas, the 10-kb RNA is predominantly polyadenylated. Third, MSRs localize to the nuclei of both lymphoblastoid cells and cytolytically infected chicken embryo fibroblasts. Fourth, the 3'-region splice junctions of the MSRs during latent and productive infection were determined by sequencing RNA-PCR products generated with primers that flank the 3' splice region. The MSRs contain at least three introns, the largest of which overlaps the ICP4 putative translational start site. Fifth, the 5' end of the MSRs initiates approximately 5 kb upstream from the main body of the RNA. The extreme 5' exon is approximately 251 nucleotides (nt) long and is joined to the main body of the transcript upon removal of a 4,852-nt intron. Finally, the 10-kb RNA lies entirely within the repeats flanking the unique short region of the genome. We believe that the MSRs and 10-kb RNA belong to a family of spliced RNAs that map antisense to the ICP4 gene and comprise a complex transcriptional unit expressed during MDV-induced T-cell transformation.

Animals↗
PubMed2005

GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs.

We have recently demonstrated that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. A functional receptor for human GnRH-II has not yet been identified. In this study, we have generated a polyclonal antiserum to the putative human GnRH-II receptor using a peptide (YSPTMLTEVPPC) corresponding to the third extracellular domain coupled to keyhole limpet haemocyanin via the Cys residue. A database search showed no identical peptide sequences in any other human gene. To avoid cross-reactions against two similar amino acid sequences the antiserum was pre-absorbed using these peptides. Immune histological sections of human placenta and human endometrial, ovarian and prostate cancers using rabbit anti-human GnRH-II receptor antiserum showed GnRH-II receptor-like staining. Western blot analysis of cell membrane preparations of human endometrial and ovarian cancer cell lines yielded a band at approximately 43 kDa whereas Western blot analysis of cell membrane preparations of ovaries obtained from the marmoset monkey (Callithrix jacchus) yielded a band at approximately 54 kDa. To identify the GnRH-II receptor-like antigen we used the photo-affinity labelling technique. Photochemical reaction of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II (10(-9) M) with cell membrane preparations of human endometrial and ovarian cancer cells yielded a band at approximately 43 kDa. In competition experiments, the GnRH-I agonist Triptorelin (10(-7) M) showed a weak decrease of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II binding to its binding site. The GnRH-I antagonist Cetrorelix (10(-7) M) showed a clearly stronger decrease, whereas GnRH-II agonist [d-Lys(6)]-GnRH-II (10(-7) M) was the most potent competitor. Western blot analysis of the same gel using rabbit anti-human GnRH-II receptor antiserum identified this band as GnRH-II receptor-like antigen.

Animals↗
PubMed1995

Variation in the hypervariable region of cucumber mosaic virus satellite RNAs is affected by the helper virus and the initial sequence context.

The D satellite RNA (sat RNA) of cucumber mosaic virus (CMV) was previously shown to contain a region of hypervariability around nucleotide 230, in wild-type populations and in cDNA clones and progeny of one such clone (pDsat4) after passage with the subgroup I strain Fny-CMV. This hypervariable region (HVR) consists of a series of consecutive A and/or U residues. We found that variability is also generated in the HVR of transcript derived from pDsat4 after passage with the subgroup II strain LS-CMV and with tomato aspermy virus (TAV). However, the progeny differ with respect to the sequence of the HVR after passage with both LS-CMV and TAV. Another D-sat RNA cDNA clone that contains a C residue in the HVR, pDsat1, was previously shown not to develop variability in the HVR upon passage with Fny-CMV. However, when the C (position 231) was changed to an A residue, variability developed by the third passage with Fny-CMV. An additional cDNA clone derived from the B1-sat RNA, pBsat5, also contains a C residue in the region analogous to the D-sat RNA HVR and did not develop variability upon passage with either Fny- or LS-CMV. Changing this C to a U residue did not result in the development of hypervariability in the progeny of transcript from this mutant. Models to explain the generation of hypervariability are discussed.

Base Sequence↗
PubMed1991

Anterior and posterior association cortex contributions to the somatosensory P300.

A P300 (P3)-evoked response is generated in a variety of mammalian species upon detection of significant environmental events. The P3 component has been proposed to index a neural system involved in attention and memory capacity. We investigated the contribution of anterior and posterior association cortex to somatosensory P3 generation. Somatosensory event-related potentials (ERPs) were recorded in controls (n = 10) and patients with unilateral lesions in temporal-parietal junction (n = 8), lateral parietal cortex (n = 8), or dorsolateral frontal cortex (n = 10). Subjects pressed a button to mechanical taps of the fifth finger (targets; p = 0.12), randomly interposed in sequences of taps to the second (standards; p = 0.76) and the third or fourth finger (tactile novels; p = 0.06). Occasional shock stimuli were delivered to the wrist (shock novels; p = 0.06). The scalp-recorded P3 was differentially affected by anterior and posterior association cortex lesions. Subjects with temporal-parietal lesions showed markedly reduced P3s to all types of stimuli at all scalp locations. The reductions were largest at the parietal electrode site over the lesioned hemisphere. Parietal patients had normal P3s for all stimulus types except for contralateral shock novels, which generated reduced P3s. Frontal lesions had reductions of the novelty P3 over frontal sites with minimal changes in the target P3. The data support the existence of multiple intracranial P3 sources. The data further indicate that association cortex in the temporal-parietal junction is critical for generating the scalp-recorded target and novelty P3s, whereas dorsolateral frontal cortex contributes preferentially to novelty P3 generation. The N2 component was reduced by parietal and frontal lesions in patients who had intact target P3s, suggesting that different neural systems underlie N2 and P3 generation.

Aged↗
PubMed2006

Antimicrobial susceptibility of Neisseria gonorrhoeae isolated in Jiangsu Province, China, with a focus on fluoroquinolone resistance.

In this study, the phenotypic and genotypic resistance to fluoroquinolones in Neisseria gonorrhoeae isolated in Jiangsu Province, China, was analysed. In vitro susceptibility testing of eight antimicrobial agents, including ciprofloxacin and levofloxacin, against 95 clinical isolates was carried out. Detection of mutations in the gyrA and parC genes was performed by sequence analysis. The clinical isolates demonstrated 100% resistance to ciprofloxacin and 98.9% non-susceptibility to levofloxacin. All of the isolates were susceptible to cefotaxime and ceftriaxone. For cefepime, spectinomycin and tetracycline, 98.9, 94.7 and 1.1% of the isolates were susceptible, respectively. None of the isolates was susceptible to penicillin. Five types based on gyrA mutations could be categorized among 54 isolates with seven different mutation sites found on their parC gene. Analysis of sequence results showed that the gyrA mutation Asp-95-->Ala and the parC mutations Ser-87-->Arg and Ser-87-->Asn made a significant contribution to the resistance to fluoroquinolones, in addition to double mutations found in each gene. Therefore, the use of fluoroquinolones in the treatment of N. gonorrhoeae infections in Jiangsu Province is not recommended, while the use of third- and fourth-generation cephalosporins and spectinomycin is recommended.

Adult↗
PubMed1996

The beta subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit.

Human and bovine rod photoreceptor cGMP-gated cation channel consists of two subunits: alpha (63 kDa) and beta (240 kDa). The human beta subunit was shown to consist partly of sequence encoded by the cDNA clone hRCNC2b. Here we present the complete sequence of the human beta subunit and demonstrate that the previously reported human GAR1 gene encoding a glutamate-rich protein (hGARP) encodes its N-terminal portion. Using PCR, RNA blot and genomic DNA analysis, we provide evidence that the beta subunit is produced from a complex locus on chromosome 16 which is also capable of generating independent transcripts corresponding to GAR1 and the C-terminal two-thirds of the beta subunit. The results indicate that the beta subunit of the cGMP-gated cation channel is produced from an unusual locus consisting of more than one transcription unit.

Alternative Splicing↗
PubMed2005

MollDE: a homology modeling framework you can click with.

UNLABELLED: Molecular Integrated Development Environment (MolIDE) is an integrated application designed to provide homology modeling tools and protocols under a uniform, user-friendly graphical interface. Its main purpose is to combine the most frequent modeling steps in a semi-automatic, interactive way, guiding the user from the target protein sequence to the final three-dimensional protein structure. The typical basic homology modeling process is composed of building sequence profiles of the target sequence family, secondary structure prediction, sequence alignment with PDB structures, assisted alignment editing, side-chain prediction and loop building. All of these steps are available through a graphical user interface. MolIDE's user-friendly and streamlined interactive modeling protocol allows the user to focus on the important modeling questions, hiding from the user the raw data generation and conversion steps. MolIDE was designed from the ground up as an open-source, cross-platform, extensible framework. This allows developers to integrate additional third-party programs to MolIDE. AVAILABILITY: http://dunbrack.fccc.edu/molide/molide.php CONTACT: rl_dunbrack@fccc.edu.

Algorithms↗
PubMed1989

Broadband proton decoupled natural abundance 13C NMR spectroscopy of humans at 1.5 T.

The feasibility of broadband proton decoupled in vivo 13C NMR spectroscopy of humans at 1.5 T was explored. A dual surface coil set-up was used, comprising a circular 13C coil and a butterfly 1H decoupling coil placed at one third of its width away from the body. A calibration procedure was introduced to evaluate the specific absorption rate (SAR) in any gram of tissue for the inhomogeneous decoupling field generated by a surface coil. For the WALTZ-4 sequence it was demonstrated that broadband decoupled spectra of both subcutaneous adipose and underlying muscle or liver tissue could be obtained at 1.5 T without exceeding recommended maximum SAR values. Broadband decoupling caused an additional resolution enhancement ascribed to the removal of (1H-13C) long range couplings. Broadband proton decoupled spectra of subcutaneous adipose tissue were obtained in less than 10 min showing highly resolved and intense signals of fully relaxed carbon spin systems of triacylglycerols. Broadband proton decoupled 13C NMR spectra of calf muscle showed several resonances for metabolites resolved from triacylglycerol signals (e.g. C1-C5 of glycogen, C4 of histidine, aromatic and carbonyl carbons of aminoacids and N linked carbons of ethanolamine, choline and creatine). With an acquisition time of 20-30 min, the C1 glycogen signal was observed with a root mean square signal-to-noise ratio of about 15. Not only the glycogen C1 signal but also its C2-C6 signals could be monitored in dynamic studies. Finally broadband proton decoupled 13C spectra were obtained with signals from liver tissue (notably the carbons of glycogen).(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗