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Results for “Overlapping PCR”

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At least 253 records · Page 14Linked to original sources

PubMed1998

Interferon-gamma variants with deletions in the AB surface loop--flexibility is a critical point for receptor binding.

The receptor-binding AB loop of recombinant human interferon-gamma (IFN-gamma) has multiple contacts with the extracellular part of the IFN-gamma receptor a chain (IFN-gammaRalpha). We explored the possible length of truncated AB loops and their conformations by molecular modelling. Deletions of two amino acids at the tip of the loop were tolerated in the model without van der Waals collisions of the AB loop with helix F. Based on these modelling results, two deletion mutants were constructed by overlap-extension PCR mutagenesis: des-(A23, D24)-IFN-gamma and des-(N25, G26)-IFN-gamma. Both mutations were tolerated by the folding pattern of recombinant human IFN-gamma, as proved by CD spectroscopy. The stability of both mutants against cosolvent-induced unfolding was equal to that of wild-type IFN-gamma. In contrast to the biophysical similarities of wild-type and mutant IFN-gamma proteins, the biological activities of both mutants dropped significantly. Antiviral activity and human leucocyte antigen (HLA)-DR induction of des-(N25, G26)-IFN-gamma was 10% that of wild-type activity. des-(A23, D24)-IFN-gamma had only 1% remaining activity. Receptor-binding experiments confirmed that both deletions had a negative influence on the affinity of recombinant human IFN-gamma to its cellular receptor. We conclude from this combined molecular modelling and mutagenesis experiments, that the reduced flexibility of the truncated AB loop abrogates the possibility of the formation of a 3(10) helix in the receptor-bound state as observed in the X-ray structure of the IFN-gammaRalpha-IFN-gamma complex.

Antiviral Agents↗

PubMed2003

Functional relationships of the sialyltransferases involved in expression of the polysialic acid capsules of Escherichia coli K1 and K92 and Neisseria meningitidis groups B or C.

Polysialic acid (PSA) capsules are cell-associated homopolymers of alpha2,8-, alpha2,9-, or alternating alpha2,8/2,9-linked sialic acid residues that function as essential virulence factors in neuroinvasive diseases caused by certain strains of Escherichia coli and Neisseria meningitidis. PSA chains structurally identical to the bacterial alpha2,8-linked capsular polysaccharides are also synthesized by the mammalian central nervous system, where they regulate neuronal function in association with the neural cell adhesion molecule (NCAM). Despite the structural identity between bacterial and NCAM PSAs, the respective polysialyltransferases (polySTs) responsible for polymerizing sialyl residues from donor CMP-sialic acid are not homologous glycosyltransferases. To better define the mechanism of capsule biosynthesis, we established the functional interchangeability of bacterial polySTs by complementation of a polymerase-deficient E. coli K1 mutant with the polyST genes from groups B or C N. meningitidis and the control E. coli K92 polymerase gene. The biochemical and immunochemical results demonstrated that linkage specificity is dictated solely by the source of the polymerase structural gene. To determine the molecular basis for linkage specificity, we created chimeras of the K1 and K92 polySTs by overlap extension PCR. Exchanging the first 52 N-terminal amino acids of the K1 NeuS with the C terminus of the K92 homologue did not alter specificity of the resulting chimera, whereas exchanging the first 85 or reciprocally exchanging the first 100 residues did. These results demonstrated that linkage specificity is dependent on residues located between positions 53 and 85 from the N terminus. Site-directed mutagenesis of the K92 polyST N terminus indicated that no single residue alteration was sufficient to affect specificity, consistent with the proposed function of this domain in orienting the acceptor. The combined results provide the first evidence for residues critical to acceptor binding and elongation in polysialyltransferase.

Amino Acid Sequence↗

PubMed1998

Complete nucleotide sequence of an African human T-lymphotropic virus type II subtype b isolate (HTLV-II-Gab): molecular and phylogenetic analysis.

We report the first complete nucleotide sequence of an African human T-cell lymphotropic virus type II. This new strain, called HTLV-II-Gab (Gab), was obtained from the uncultured peripheral blood mononuclear cells of a 44-year-old healthy Gabonese male who lived in a remote rural area, with neither history of blood transfusion nor sexual intercourse with non-Africans. Using nested PCR, 25 overlapping fragments, representing the entire proviral genome, were obtained, cloned and sequenced. The overall nucleotide sequence comparison with the four other available complete HTLV-II genomes indicated that Gab was more closely related to the HTLV-II subtype b prototypes (98.9, 99.3 and 98.2% nucleotide similarity with G12, NRA and GU respectively) than to the subtype a prototype (95.1% nucleotide similarity with Mo). Restriction profiles studies and phylogenetic analyses confirmed that Gab was a subtype b strain. However, this strain represents a newly described restriction fragment length polymorphism subtype, closely related to one of the rare partially sequenced African isolates originating from a pygmy living in Cameroon (PYGCAM). Nevertheless, the very low genetic divergence observed between this new African strain and the American strains raises several questions on the origins and level of genetic variability over time of this human retrovirus.

Adult↗

PubMed1999

Nucleotide sequences and further characterization of human papillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma cell lines.

The complete nucleotide sequences of the human papillomavirus type 16 (HPV-16) variants present in the CaSki and SiHa cervical carcinoma cell lines and the primary subgenomic HPV-18 variant present in the HeLa cervical carcinoma cell line were determined using overlapping bulk PCR products as templates. PCR-based methods were also used to characterize five previously unreported CaSki HPV-16 genomic disruptions and the 5' cellular-viral junction common to all HeLa HPV-18 subgenomic structures.

DNA, Viral↗

PubMed2004

The Human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection.

Somatic mitochondrial mutations are common in human cancers, and can be used as a tool for early detection of cancer. We have developed a mitochondrial Custom Reseq microarray as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The MitoChip contains oligonucleotide probes synthesized using standard photolithography and solid-phase synthesis, and is able to sequence >29 kb of double-stranded DNA in a single assay. Both strands of the entire human mitochondrial coding sequence (15,451 bp) are arrayed on the MitoChip; both strands of an additional 12,935 bp (84% of coding DNA) are arrayed in duplicate. We used 300 ng of genomic DNA to amplify the mitochondrial coding sequence in three overlapping long PCR fragments. We then sequenced >2 million base pairs of mitochondrial DNA, and successfully assigned base calls at 96.0% of nucleotide positions. Replicate experiments demonstrated >99.99% reproducibility. In matched fluid samples (urine and pancreatic juice, respectively) obtained from five patients with bladder cancer and four with pancreatic cancer, the MitoChip detected at least one cancer-associated mitochondrial mutation in six (66%) of nine samples. The MitoChip is a high-throughput sequencing tool for the reliable identification of mitochondrial DNA mutations from primary tumors in clinical samples.

Body Fluids↗

PubMed2005

Development of an asporogenic Bacillus licheniformis for the production of keratinase.

AIMS: Bacillus licheniformis PWD-1 is a keratin-degrading, spore-forming bacterium isolated from a poultry waste digester. A sporulation-deficient mutant of B. licheniformis PWD-1, named B. licheniformis WBG, was developed and characterized. METHODS AND RESULTS: The mutation was generated using the splicing by overlap extension PCR method (Gene SOEing) to create 256 bp deletion in the spoIIAC gene, which encodes an essential sporulation-specific sigma factor. In vivo gene replacement was accomplished with the use of a temperature-sensitive plasmid that is able to integrate and excise the nucleotide fragment 256 bp from the B. licheniformis chromosome. PCR analysis and DNA sequencing confirmed the spoIIAC gene deletion. Heat-treatment assays and electron microscopy verified the absence of spores. CONCLUSIONS: This asporogenic strain is able to express normal levels of keratinase when compared with its wild-type host. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a method of constructing a stable sporulation-defective strain was developed. It can be potentially useful as a tool to generate asporogenic strains of Bacillus that retain their industrial capabilities for production of exoproteases and other exozymes.

Bacillus↗

PubMed1993

The deduced amino acid sequences of human platelet and frontal cortex monoamine oxidase B are identical.

Monoamine oxidases (MAOs) A and B play important roles in the metabolism of neuroactive, vasoactive amines. Human platelets contain only MAO B, often used as an indicator of brain MAO B. The validity of this model remained to be evaluated. This report describes the molecular cloning of human MAO B from frontal cortex and platelets. Two overlapping PCR-amplified clones of human platelet MAO B and four PCR-amplified clones of human frontal cortex MAO B covering the entire coding region were sequenced using five internal oligomers and M13 reverse and forward primers. The nucleotide sequences of human MAO B cDNA from platelet and frontal cortex were identical to that of human liver MAO B except for three nucleotides that differed in frontal cortex: nucleotides 440 A-->G, 794 C-->T, and 825 C-->T. Whether or not these differences are artifactual, all three represent silent mutations, which would not alter the amino acid of the encoded polypeptides. Thus, the deduced amino acid sequences of MAO B from frontal cortex, platelet, and liver are identical. These findings indicate the validity of using platelet MAO B mRNA as a marker for brain MAO B and provide a new approach to study the role of brain MAO B in humans.

Amino Acid Sequence↗

PubMed2002

Expression, autoregulation, and DNA binding properties of the Mycobacterium tuberculosis TrcR response regulator.

The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis. Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M. tuberculosis growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli. Upon induction of the TrcR protein, there was a >500-fold increase in beta-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR. This report indicates that the M. tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter.

Base Sequence↗

PubMed2006

GeneChip resequencing of the smallpox virus genome can identify novel strains: a biodefense application.

We developed a set of seven resequencing GeneChips, based on the complete genome sequences of 24 strains of smallpox virus (variola virus), for rapid characterization of this human-pathogenic virus. Each GeneChip was designed to analyze a divergent segment of approximately 30,000 bases of the smallpox virus genome. This study includes the hybridization results of 14 smallpox virus strains. Of the 14 smallpox virus strains hybridized, only 7 had sequence information included in the design of the smallpox virus resequencing GeneChips; similar information for the remaining strains was not tiled as a reference in these GeneChips. By use of variola virus-specific primers and long-range PCR, 22 overlapping amplicons were amplified to cover nearly the complete genome and hybridized with the smallpox virus resequencing GeneChip set. These GeneChips were successful in generating nucleotide sequences for all 14 of the smallpox virus strains hybridized. Analysis of the data indicated that the GeneChip resequencing by hybridization was fast and reproducible and that the smallpox virus resequencing GeneChips could differentiate the 14 smallpox virus strains characterized. This study also suggests that high-density resequencing GeneChips have potential biodefense applications and may be used as an alternate tool for rapid identification of smallpox virus in the future.

Bioterrorism↗

PubMed1997

Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity.

The Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which contain overlapping amino acid sequences. They are required for viral replication and preferential integration of the AAV genome into a region of human chromosome 19. During the terminal resolution process of AAV DNA replication, these proteins make a site-specific and strand-specific endonuclease cut within the AAV inverted terminal repeat DNA. The Rep68 and Rep78 proteins also have helicase and DNA-binding activities. The endonuclease activity is believed to involve the covalent attachment of Rep68 or Rep78 at the cut site via a phosphotyrosine linkage. In an attempt to identify the active-site tyrosine residue of Rep78 and Rep68, tyrosine residues were site specifically mutated to phenylalanines by overlap extension PCR, and the resulting PCR fragments were cloned into a maltose binding protein-Rep68 fusion (MBP-Rep68delta) expression vector. The mutant MBP-Rep68delta proteins were expressed in Escherichia coli cells, purified with amylose resin, and assayed in vitro for Rep68-specific activities. Although several of the mutations disrupted the endonuclease activity, only the mutation of tyrosine 152 abrogated the endonuclease activity with no discernible effect on the helicase or DNA-binding activities. Our data therefore suggest that there are distinct active sites for the helicase and endonuclease activities.

ATP-Binding Cassette Transporters↗

PubMed1999

Analysis of the effects of charge cluster mutations in adeno-associated virus Rep68 protein in vitro.

The Rep78 and Rep68 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which are required for viral replication, regulation of AAV promoters, and preferential integration of the AAV genome into a region of human chromosome 19. These proteins bind the hairpin structures formed by the AAV inverted terminal repeat (ITR) origins of replication, make site- and strand-specific endonuclease cuts within the AAV ITRs, and display nucleoside triphosphate-dependent helicase activities. Additionally, several mutant Rep proteins display negative dominance in helicase and/or endonuclease assays when they are mixed with wild-type Rep78 or Rep68, suggesting that multimerization may be required for the helicase and endonuclease functions. Using overlap extension PCR mutagenesis, we introduced mutations within clusters of charged residues throughout the Rep68 moiety of a maltose binding protein-Rep68 fusion protein (MBP-Rep68Delta) expressed in Escherichia coli cells. Several mutations disrupted the endonuclease and helicase activities; however, only one amino-terminal-charge cluster mutant protein (D40A-D42A-D44A) completely lost AAV hairpin DNA binding activity. Charge cluster mutations within two other regions abolished both endonuclease and helicase activities. One region contains a predicted alpha-helical structure (amino acids 371 to 393), and the other contains a putative 3,4 heptad repeat (coiled-coil) structure (amino acids 441 to 483). The defects displayed by these mutant proteins correlated with a weaker association with wild-type Rep68 protein, as measured in coimmunoprecipitation assays. These experiments suggest that these regions of the Rep molecule are involved in Rep oligomerization events critical for both helicase and endonuclease activities.

ATP-Binding Cassette Transporters↗

PubMed1993

Sequencing of cDNA from 50 unrelated patients reveals that mutations in the triple-helical domain of type III procollagen are an infrequent cause of aortic aneurysms.

Detailed DNA sequencing of the triple-helical domain of type III procollagen was carried out on cDNA prepared from 54 patients with aortic aneurysms. The 43 male and 11 female patients originated from 50 different families and five different nationalities. 43 patients had at least one additional blood relative who had aneurysms. Five overlapping asymmetric PCR products, covering all the coding sequences of the triple-helical domain of type III procollagen, were sequenced with 28 specific sequencing primers. Analysis of the sequencing gels revealed only two nucleotide changes that altered the structure of the protein. One was a substitution of threonine for proline at amino acid position 501 and its functional importance was not clearly established. The other was a substitution of arginine for an obligatory glycine at amino acid position 136. In 40 of the 54 patients, detection of a polymorphism in the mRNA established that both alleles were expressed. The results indicate that mutations in type III procollagen are the cause of only about 2% of aortic aneurysms.

Adult↗

PubMed2001

Molecular cloning of the porcine acid-labile subunit (ALS) of the insulin-like growth factor-binding protein complex and detection of ALS gene expression in hepatic and non-hepatic tissues.

The acid-labile subunit (ALS) is an approximately 85 kDa N-glycoprotein that is known primarily as a component of the systemic insulin-like growth factor-binding protein (IGFBP) complex. We have amplified, using a PCR, three overlapping porcine ALS genomic DNA fragments that together encode the distal region of the signal peptide through to the COOH-terminus. The compiled sequence of 1775 nucleotides of the three overlapping DNAs and the deduced amino acid sequence of the mature porcine ALS (pALS) protein exhibited 84/81%, 79/77%, 79/78% and 84/79% identities with respect to those of the human, the rat, the mouse and the baboon respectively. Four conserved cysteine residues in the NH(2)-terminal domain and 20 leucine-rich repeats in the central domain also were identified at identical positions in the porcine ALS. By using Northern blot analysis, with a genomic DNA fragment as the probe, it was determined that a 2.2 kb ALS mRNA was induced in the liver during the late fetal stage, and hepatic ALS mRNA abundance was increased post-natally. Moreover, hepatic ALS mRNA abundance was increased by daily injection of porcine somatotropin (100 microg/kg body weight) in cross-bred market pigs each weighing approximately 100 kg. The ALS mRNA was not detected by Northern analysis in any non-hepatic tissue examined. However, results of a more sensitive solution hybridization/RNAse protection assay indicated that low levels of ALS mRNA were also present in adult muscle, spleen, ovary and uterus, but not in lung, kidney, oviduct and placenta. Taken together, the present results suggest that although liver is the primary organ that expresses the ALS gene under somatotropin stimulation, some non-hepatic tissues also express the gene at low levels in the pig.

Amino Acid Sequence↗

PubMed2004

[Development of a system for quick screening of efficient HBx-siRNA].

OBJECTIVE: To develop a system for quick screening of efficient siRNA targeted HBx mRNA. METHODS: Using recombination DNA technique, the fusion expression plasmid of HBx and EGFP was constructed, and siRNA expression cassettes (SECs) containing U6+1, H1 or tRNA(Val )promoter were prepared via one-step overlapping extension PCR. By co-transfection with recombinant plasmid and SECs into AD293 cell, the inhibition effects on the transient expression of HBx-EGFP fusion protein were analyzed by FACS and semi-quantitated RT-PCR analysis. RESULT: (1)HBx-EGFP fusion protein expression plasmid pHBx-EGFP was constructed successfully, which expressed green fluorescence in cell mainly located at plasma or the periphery of nucleus in granules. (2) Co-transfection with recombinant plasmid and SECs into AD293 cells resulted in inhibition of HBx-EGFP expression. SEC-siHBx388 showed significant inhibition effect on HBx-EGFP expression compared with SEC-siHBx271, indicating that siHBx388 is effective siRNA site and could be screened out with our screening system. In addition,the results of that U6+1-, tRNA(Val) and H1-siHBx388 reduced HBx-EGFP expression by 21.7%, 12.9% and 12.4% of control respectively indicated that both tRNAVal and H1 promoter was high efficient in driving effect of siHBx388. CONCLUSION: Combination of the HBx expression carrying reporter gene and PCR-based multi promoter SECs may develop a useful system to be applied in identification of optimal HBx- siRNA and its matching promoter.

Base Sequence↗

PubMed2006

Identification and sequence analysis of hepatitis A virus detected in market and environmental bivalve molluscs.

In Italy in 1998, hepatitis A virus (HAV) was responsible for an infectious disease transmitted by contaminated bivalve molluscs. To determine the presence of HAV in the bivalves collected during a 1-year follow-up study, hepatitis A RNA was extracted and amplified by a nested reverse transcriptase-PCR method overlapping the VP1/2A region. The HAV genome was detected in 24 (14.1%) of 170 samples: 19 clams (Tapes decussates and Tapes semidecussatus), 1 oyster (Crossostea gigas), and 4 mussels (Mytillus galloprovincialis). Eleven positive samples were collected from marketing areas, and 13 positive samples were collected from growing areas. Seventeen of the 24 positive samples had been taken from domestic products, and 7 had been imported. Sequence analysis showed the presence of genotypes IA and IB. Our results suggest significant presence of HAV in bivalves from both marketing (public consumption) and environmental (growing) areas.

Animals↗

PubMed2002

Detection of virus-like particles in the liver of black and white ruffed lemurs with hepatitis.

Two young black and white ruffed lemurs (Varecia variegata variegata) died at the San Diego Zoo (San Diego, California, USA) with extensive liver lesions suggestive of acute viral infection. Immunoassays performed to detect hepatitis B virus (HBV) markers were negative. Polymerase chain reaction (PCR) primers overlapping the HBV core gene produced an amplicon of approximately 411 base pairs (bp) from serum DNA of a HBV-positive western lowland gorilla (Gorilla gorilla gorilla) but not from serum DNA of either lemur. Cesium chloride gradient fractions of liver homogenates from both lemurs contained a peak protein fraction with a density of 1.18 g/cm3. Electron microscopic analysis of fraction contents, concentrated by ultracentrifulgation, revealed numerous pleomorphic, spherical particles varying in diameter from 16-25 nm. In one of the lemurs, this peak fraction also contained a double-shelled virus-like particle 47-50 nm in diameter. The size, morphology, and density of these particles suggest they are members of the Hepadnaviridae, a group of hepatotropic DNA-genome viruses for which HBV is the prototype.

Animals↗

PubMed2001

Expression of the alpha1F calcium channel subunit by photoreceptors in the rat retina.

PURPOSE: The CACNA1F gene encodes a voltage-gated calcium channel alpha1 subunit, alpha1F, which is expressed in the human retina. Mutations in this gene cause incomplete X-linked congenital stationary night blindness (CSNB2). The aim of this study was to obtain the sequence of the rat alpha1F cDNA and localize the encoded polypeptide in the rat retina. METHODS: The full-length rat alpha1F sequence was compiled from sequencing of overlapping alpha1F PCR fragments amplified from rat retinal cDNA. Antiserum was raised against a human alpha1F peptide. It was found that the human alpha1F peptide used to generate the antiserum was conserved at only 11 out of 19 residues in the cloned rat sequence. Therefore, antibodies were affinity purified against either the human alpha1F peptide or the equivalent rat peptide and used for immunofluorescent staining of rat retina sections. RESULTS: The rat alpha1F amino acid sequence was found to be 91% and 95% identical to the human and mouse alpha1F sequences, respectively. Antibodies affinity purified against the human alpha1F peptide stained both the outer nuclear layer (ONL) and outer plexiform layer of rat retina sections. In contrast, staining with antibodies affinity purified against the corresponding rat alpha1F peptide was restricted to the ONL. CONCLUSIONS: The rat alpha1F amino acid sequence is highly homologous to the human and mouse sequences. The immunohistochemical results indicate the existence of distinct alpha1F isoforms or alpha1F-like channels, which are differentially distributed in the cell bodies and synaptic terminals of photoreceptors in the rat retina.

Amino Acid Sequence↗

PubMed2001

Construction and identification of a single stranded cDNA clone containing full-length genome of hepatitis G virus.

AIM: To construct a single cDNA clone with full-length genome of hepatitis G virus (HGV) could be transcribed and expressed in vitro. METHODS: The 5 initial HGV cDNA fragments of Iw5, Iwq2, Iwh6, Iw3 and Iw3 used in this study were amplified from serum of a Japanese non A-E hepatitis patient. These fragments overlapped and covered the entire genome from 5'-end to 3'-end of HGV cDNA. Overlap extension PCR and ligation methods were used with 12 primers for the construction of a full-length genomic HGV cDNA clone from the subgenomic fragments. RESULTS: A single HGV cDNA clone (pHGVqz) was successfully constructed, physical mapping of the generated pHGVqz found identical to what we expected, and the sequence was deposited with the GenBank under the Accession number AF081782. The analysis of the full-length sequence, which was able to be in vitro transcribed and expressed, showed that this single clone contained 9373 nucleotides (encoding 2873 amino acids), and shared high homologies with other compared HGV isolates. CONCLUSION: A full-length genomic HGV cDNA clone is generated for the first of the kind in this study, it could be expressed and transcripted. This single cDNA clone is expected to be of importance in the investigation on replication and pathogenicity of HGV.

Blotting, Western↗