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Results for “Overlapping PCR”

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PubMed2006

Expression and localization of monocarboxylate transporters and sodium/proton exchangers in bovine rumen epithelium.

Monocarboxylate-H+ cotransporters, such as monocarboxylate transporter (MCT) SLC16A, have been suggested to mediate transruminal fluxes of short-chain fatty acids, ketone bodies, and lactate. Using an RT-PCR approach, we demonstrate expression of MCT1 (SLC16A1) and MCT2 (SLC16A7) mRNA in isolated bovine rumen epithelium. cDNA sequence from these PCR products combined with overlapping expressed sequence tag data allowed compilation of the complete open reading frames for MCT1 and MCT2. Immunohistochemical localization of MCT1 shows plasma membrane staining in cells of the stratum basale, with intense staining of the basal aspects of the cells. Immunostaining decreased in the cell layers toward the rumen lumen, with weak staining in the stratum spinsoum. Immunostaining in the stratum granulosum and stratum corneum was essentially negative. Since monocarboxylate transport will load the cytosol with acid, expression and location of Na+/H+ exchanger (NHE) family members within the rumen epithelium were determined. RT-PCR demonstrates expression of multiple NHE family members, including NHE1, NHE2, NHE3, and NHE8. In contrast to MCT1, immunostaining showed that NHE1 was predominantly localized to the stratum granulosum, with a progressive decrease toward the stratum basale. NHE2 immunostaining was observed mainly at an intracellular location in the stratum basale, stratum spinosum, and stratum granulosum. Given the anatomic localization of MCT1, NHE1, and NHE2, the mechanism of transruminal short-chain fatty acid, ketone body, and lactate transfer is discussed in relation to a functional model of the rumen epithelium comprising an apical permeability barrier at the stratum granulosum, with a cell syncitium linking the stratum granulosum to the blood-facing stratum basale.

Acids↗

PubMed1994

A YAC contig of the region containing the spinal muscular atrophy gene (SMA): identification of an unstable region.

We report a 3.0-Mb YAC contig of the region 5q11.2-q13.3, which is where the spinal muscular atrophy gene has been localized. Three total genomic YAC libraries were screened by the polymerase chain reaction (PCR), and 45 YACs were recovered. These YACs were characterized for sequence tag site (STS) content, and overlaps were confirmed by vectorette PCR. Of the 45 YACs, 20 were isolated with the polymorphic marker CATT-1, which demonstrates significant allelic association with the SMA gene and maps within the 850-kb interval defined by the markers D5S557 and D5S823. Haplotyping of these YACs and their mother cell line indicates that the majority of YACs from this region contain deletions. Furthermore, a 1.9-Mb CATT-1 YAC that was negative for MAP1B and D5S435 and nonchimeric by FISH analysis provides a minimum distance between MAP1B and D5S435.

Base Sequence↗

PubMed1995

Sequence analysis of hypothalamic parathyroid hormone messenger ribonucleic acid.

PTH-like peptides and messenger RNA (mRNA) have recently been detected in neural tissues, but it is uncertain whether this reflects the transcription of the PTH gene or that of a closely related gene. This possibility has, therefore, been investigated. PTH-like complementary DNA (cDNA) moieties of predicted size were readily generated from reverse transcribed brain (hypothalamic and extrahypothalamic tissue) and pituitary RNA, using polymerase chain reaction (PCR) with three sets of overlapping oligonucleotide primers designed to amplify PTH cDNA fragments of 285, 372, and 459 base pairs (bp). PCR reamplification of the largest hypothalamic moiety with an internal set of primers also generated a cDNA fragment of the predicted size (372 bp). Restriction endonuclease digestion with BstNI cleaved the largest hypothalamic cDNA moieties into smaller fragments of 217 and 242 bp, identical to the cleavage of parathyroidal PTH cDNA. Rapid amplification of cDNA ends of the 3'-flanking cDNA sequences also produced hypothalamic and extra-hypothalamic cDNA moieties identical in size (499 bp) to parathyroidal PTH cDNA. Southern analysis of these PCR and rapid amplification of cDNA end cDNA fragments further indicated homology with PTH cDNA. This homology was subsequently confirmed by nucleotide sequencing, which demonstrated complete homology between the neural and parathyroidal cDNA fragments. This homology extended over 673 bp (spanning nucleotides 31-709 of PTH cDNA), encompassing 95% of the entire parathyroidal PTH cDNA. The mRNA for this gene, determined by Northern blotting with a riboprobe for PTH mRNA, was of identical size to the parathyroidal PTH, but its abundance in brain was less than 0.01% of that expressed in the parathyroid glands. This transcript was not, however, detected in liver. The translation of this moiety in hypothalamic tissues was indicated by the presence of a protein in the rat hypothalamus that was immunoreactive with PTH-(1-84) antiserum and of comparable size to that in parathyroidal tissue. The abundance of this protein in hypothalamic tissue was approximately 0.25% of that in the parathyroid glands, suggesting tissue-specific differences in its rate of synthesis, processing, or degradation. These results, therefore, demonstrate that the brain is an extraparathyroidal site of PTH gene expression and suggest autocrine or paracrine roles for PTH in neural function.

Animals↗

PubMed1998

Mitochondrial DNA rearrangements, including partial duplications, occur in young and old rat tissues.

Using polymerase chain reaction (PCR) with back-to-back primers, 85 different mitochondrial DNA (mtDNA) rearrangements, consisting of partial duplications or mini-circles, were detected in brain, liver, and heart tissue from Fischer 344 rats. The regions around the mitochondrial tRNALeu(UUR) gene, the cluster of three tRNA genes [His, Ser(AGY), Leu(UUC)], as well as the region of the displacement loop were analyzed separately with different primer sets. Rearrangements were detected in all regions analyzed in samples taken throughout the animal life span, ranging from 1 day old to 33 months of age (senescent). Two-thirds of the rearrangements terminated at short (3-9-bp) direct repeats. Three of the different rearrangements were detected in more than one animal; the most common rearrangement was found in nine different template preparations. Two loci (hot spots) were found to be particularly susceptible to rearrangement, and both were located at sequences that exhibited highly conserved potential for secondary structure formation. The displacement loop region of 10 samples exhibited the presence of multiple tandem duplications ranging between 324 and 449 bp in length. One of these consisted of heterologous, but overlapping, repeating units. Identical PCR protocols were carried out in control experiments using a cloned fragment of mtDNA that encompassed the most common hot spot sequence. The results showed that this fragment did not artifactually generate a rearrangement junction under our PCR conditions and suggested that this sequence does not promote rearrangement mutations in bacteria during the cloning process.

Aging↗

PubMed1991

Cloning the cDNA encoding the AmbtV allergen from giant ragweed (Ambrosia trifida) pollen.

Ragweed (Ambrosia) pollens contain a number of proteins that cause allergic disease in ragweed-sensitive people. The cloning of the AmbtV cDNA is important, since the 4.4-kDa AmbtV, one of the allergens in giant ragweed (Ambrosia trifida) pollen, serves as a simple model system to study the basic structural requirements for immune recognition of foreign protein allergens. We report the cloning of the AmbtV cDNA by means of the polymerase chain reaction (PCR) using degenerate primers. We generated three sets of overlapping cDNA clones by a combination of PCR and anchored-PCR, and determined the complete nucleotide (nt) sequence. From the nt sequence, the amino acid (aa) sequence of the protein was confirmed and the leader sequence was deduced. This general approach can be used to clone allergen and other cDNAs from complex biological sources provided partial aa sequence information is available. It may be the best available approach in cases where the isolation of clones from a cDNA library is difficult, which proved to be the case for AmbtV.

Allergens↗

PubMed2000

Sequence and phylogenetic analysis of squid myosin-V: a vesicle motor in nerve cells.

We have shown that vesicles in the axoplasm of the squid giant axon move on actin filaments and that movement is inhibited by myosin V-specific antibodies [Tabb et al., 1998]. In the study reported in this article, experiments were performed to clone and sequence the cDNA for squid brain myosin V. Five proteolytic fragments of purified squid brain myosin V were analyzed by direct protein sequencing [Tabb et al., 1998]. Based on this sequence information, degenerate primers were constructed and used to isolate cDNA clones by PCR. Five clones, representing overlapping segments of the gene, were sequenced. The sequence data and the previous biochemical characterization of the molecule support the classification of this vesicle-associated myosin as a member of the class V myosins. Motif analysis of the head, neck, and tail domains revealed that squid MyoV has consensus sequences for all the motifs found in vertebrate members of the myosin V family of motor proteins. A phylogenetic tree was constructed from a sequence alignment by the neighbor-joining method, using Megalign (DNAStar, Madison, WI); the resulting phylogenetic tree showed that squid MyoV is more closely related to vertebrate MyoV (mouse dilute, chicken dilute, rat myr6, and human myo5a) than Drosophila and yeast (myo2, and myo4) myosins V. These new data on the phylogenetic relationships of squid myosin V to vertebrate myosin V strengthens the argument that myosin V functions as a vesicle motor in vertebrate neurons.

Amino Acid Motifs↗

PubMed2003

Expression and characterization of a humanized cocaine-binding antibody.

The murine immunoglobulin G (IgG) cocaine-binding monoclonal antibody (mAb), GNC92H2, is notable for its exquisite specificity for cocaine, as opposed to chemically-related cocaine metabolites, and for its moderately high affinity (K(d) approximately 200 nM) for cocaine. Recently, we described the crystal structure of a mouse/human chimeric Fab construct at 2.3 A resolution. Herein, we report the successful framework humanization of a single-chain Fv (scFv) GNC92H2 construct without loss of affinity for cocaine. In brief, we compared the mAb GNC92H2 sequence to human antibody sequences, and used structure-based design to incorporate mutations (total = 49) that would humanize the framework region without affecting the overall shape of the binding pocket or the key cocaine-contact residues. The codons of the rationally designed sequence were optimized for E. coli expression, and the gene was synthesized by a de novo PCR reaction using 14 overlapping primers. Expression of the scFv construct was significantly improved in E. coli by fusion to thioredoxin. Intriguingly, this construct apparently refolds to form soluble active antibody in the reducing environment of the cytoplasm. Competitive ELISA and equilibrium dialysis demonstrated comparable binding activity between the humanized scFv and the whole IgG. The successful humanization of mAb GNC92H2 should enhance its potential therapeutic value by reducing its overall. immunogenicity.

Amino Acid Sequence↗

PubMed2003

Heterogeneity of the 7q36 breakpoints in the t(7;12) involving ETV6 in infant leukemia.

The t(7;12)(q36;p13) is a recurrent chromosome abnormality in infant leukemia. In these cases, the involvement of ETV6, with disruption of the gene consistently at its 5' end, has been reported by several groups. A fusion transcript between ETV6 and HLXB9 has been detected in some, but not all, reported cases of t(7;12). We report here a study based on fluorescence in situ hybridization (FISH) mapping of the translocation breakpoints in seven patients and detailed molecular studies using Southern blotting on two of these patients. The FISH studies have shown a cluster of breakpoints within a cosmid contig proximal to the HLXB9 gene. Southern blotting analysis enabled us to define two distinct breakpoints within the area covered by the cosmid contig in two patients. The analysis of an unusual case of t(7;12)(q22;p13) [full karyotype: 46,XX,der(7)t(7;12)(q22;p13)del(7)(q22q36)] also revealed a break in 7q36, although in a region proximal to the overlapping cosmids. 5' RACE PCR in one patient has shown a rearrangement involving the ETV6 allele not involved in the t(7;12), suggesting that no functional ETV6 allele might be present in this case. These data show some heterogeneity in the distribution of breakpoints in 7q36, indicating that the generation of a fusion gene might not be the mechanism responsible for leukemogenesis in the t(7;12), at least in some cases.

Base Sequence↗

PubMed1994

Metachromatic leukodystrophy in the Navajo Indian population: a splice site mutation in intron 4 of the arylsulfatase A gene.

Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder of myelin metabolism, resulting from the inability to properly degrade 3-sulfogalactosylceramide (sulfatide). This metabolic block is often due to defective functioning of the lysosomal enzyme arylsulfatase A (ARSA). Unmetabolized sulfatide accumulates in the white matter of the CNS and in the peripheral nerves, leading to progressive demyelination and death. Late infantile, juvenile and adult clinical variants of MLD have been described. A Navajo Indian child was diagnosed with late infantile MLD (LIMLD), and his ARSA gene was amplified in three overlapping regions by the PCR and sequenced. A single mutation was found: a G-->A transition in the first nucleotide of intron 4 (IVS4nt1), which abolishes the 5' splice site consensus sequence. Negligible amounts of ARSA mRNA were observed in Northern blots. However, PCR amplification and sequencing of the ARSA cDNA showed that all of the mRNA species from the patient have exon 4 deleted. A new reading frame is thus established which results in a premature stop codon within exon 5. A minority of transcripts had additional splicing errors. Both parents carry this mutation, and the father also carries the pseudodeficiency (PD) allele. Three additional unrelated Navajo LIMLD patients were found to be homozygous for the same MLD-causing mutation by allele-specific oligonucleotide (ASO) hybridization. This method could be used for carrier and patient identification in this population.

Adult↗

PubMedDate not supplied

Design and validation of a synthetic VH repertoire with tailored diversity for protein recognition.

Previous studies have indicated differences in the specificity-determining residues (SDRs) of antibodies that recognize haptens, peptides, or proteins. Here, we designed a V(H) repertoire based on the human scaffold 3-23/J(H)4 and diversification of high and medium-usage SDRs of anti-protein and anti-peptide antibodies. The repertoire was synthesized by overlapping polymerase chain reaction (PCR) and combined with the V(L) chain of the anti-hen egg-white lysozyme (HEL) antibody D1.3. The resulting chimeric single-chain Fv fragments (scFvs) phage-displayed library was panned in HEL-coated immunotubes. After two rounds of selection under non-stringent conditions, that is, trypsinization after 2 h of incubation at room temperature, 63 of 167 clones analyzed (38%) were found to express scFvs specific to HEL. Twenty clones were characterized by DNA sequencing resulting in 10 unique scFvs. Interestingly, the panel of unique scFvs was highly diverse, with V(H) sequences differing in 16 of the 17 positions variegated in the repertoire. Thus, diverse chemico-physical and structural solutions were selected from the library, even when the V(H) repertoire was constrained by the V(L) chain of D1.3 to yield binders against a definite region of HEL surface. The more often selected scFvs, namely H6-1 and B7-1, which differed in eight SDRs, showed levels of expression in E. coli TG1 strain, 6 and 10 times higher than the parental D1.3 Fv fragment, respectively. Dissociation constants (K(Ds)) measured in the BIAcore were 11 and 6.6 nM for H6-1 and B7-1, respectively. These values compared well to the K(D) of 4.7 nM measured for D1.3, indicating that the V(H) repertoire here designed is a valuable source of diverse, well-expressed and high affinity V(H) domains.

Amino Acid Sequence↗

PubMed1995

Cloning, expression, and purification of an anti-desipramine single chain antibody in NS/O myeloma cells.

Drug-specific monoclonal antibodies and their antigen-binding Fab fragments reverse acute desipramine toxicity in a rat experimental model by inducing a redistribution of drug from cardiac tissue into serum and extracellular fluid. In order to investigate the use of smaller recombinant antibody fragments such as single chain Fv (sFv) as an antidote, an efficient murine NS/O myeloma expression system was developed. The variable light (VL) and variable heavy (VH) domains of a murine anti-desipramine monoclonal antibody were cloned and sequenced. A 270 amino acid VH-(Gly4Ser)3-VL sFv was prepared by overlapping polymerase chain reaction (PCR) amplification of VH with heavy chain leader peptide, VL, and the linker. This construct was subcloned into a mammalian expression vector which utilizes the SR alpha promoter, a hybrid promoter consisting of the SV40 early promoter with portions of the human T-cell leukemia virus type I long terminal repeat and also containing the Escherichia cloi xanthine-guanine phosphoribosyltransferase gene for selection. NS/O myeloma cells were transfected by electroporation. Stable recombinant NS/O clones were screened for expression of sFv using reverse transcriptase-PCR to detect mRNA and an enzyme-linked immunosorbent assay (ELISA) to detect sFv. Secreted sFv from clones capable of growth to a cell density of 2-4 x 10(6) viable cells/mL was purified in a single step using a desipramine affinity column resulting in 12-39 mg/L of purified sFv. Affinity-purified sFv had comparable desipramine binding activity to Fab when evaluated by competitive ELISA.

Amino Acid Sequence↗

PubMed1994

Nucleotide sequence of the gene coding for non-proteolytic Clostridium botulinum type B neurotoxin: comparison with other clostridial neurotoxins.

The neurotoxin gene of non-proteolytic Clostridium botulinum type B (strain Eklund 17B) was cloned as a series of overlapping polymerase chain reaction (PCR) fragments generated with primers designed to conserved regions of published botulinal toxin (BoNT) sequences. The 3' end of the gene was obtained by using primers designed to the determined sequence of non-proteolytic BoNT/B and a published downstream region of BoNT/B gene from a proteolytic strain. Translation of the nucleotide sequence derived from cloned PCR fragments demonstrated the toxin gene encodes a protein of 1291 amino acid residues. Comparative alignment of the derived BoNT/B sequence with those of other published botulinal neurotoxins revealed highest sequence relatedness with BoNT/B of proteolytic C. botulinum. The sequence identity between non-proteolytic and proteolytic BoNT/B was 97.7% for the light chain (corresponding to 10 amino acid changes) and 90.2% for the heavy chain (corresponding to 81 amino acid changes), with most differences occurring at the C-terminal end. A genealogical tree constructed from all known botulinal neurotoxin sequences revealed marked topological differences with a phylogenetic tree of C. botulinum types based upon small-subunit (16S) ribosomal RNA sequences.

Amino Acid Sequence↗

PubMed1996

Molecular characterization of carrier rabies isolates.

We compared the genomes of nine dog rabies virus isolates using two molecular methods. The viruses used in the comparison included three Ethiopian rabies strains from carrier dogs, a street strain from a rabid dog from the same geographic area, two saliva isolates made from an experimentally infected carrier dog, the virus isolated from the tonsil of this carrier dog at necropsy, and two laboratory strains. We produced overlapping polymerase chain reaction (PCR) segments spanning 97% of the genome. Restriction analysis of these PCR products with AvaII, Bcll, and BamHI detected 39 variable sites representing 668 nucleotides (nt) or 5.5% of the genome. We also compared the DNA and the deduced peptide sequences of a 200-nt segment of the 3' end of the rabies nucleoprotein gene. Previous work with these Ethiopian carrier viruses and the endemic street strain had failed to show any differences among them. Both restriction mapping and sequence analysis of 200 nt of the nucleoprotein gene allowed us to individually identify these isolates. Phylogenetic analyses of these data sets showed only the two saliva isolates of the experimentally infected carrier dog to be identical. Each of the viruses in this study, including the one isolated from the tonsil of the experimentally infected carrier dog, could be distinguished by these techniques.

Animals↗

PubMed1995

Cloning, sequencing and functional expression of a cDNA encoding a NADP-dependent malic enzyme from human liver.

This work reports the structure of a cDNA (ME) encoding a human malic enzyme (ME) (malate NADP oxidoreductase, EC 1.1.1.40) elucidated by joining several overlapping fragments amplified by PCR from human hepatic cDNA or from cDNA libraries. The full-length cDNA has an open reading frame (ORF) of 1719 bp that encodes a 572-amino-acid protein of 64 113 Da, similar to the native monomeric, cytosolic, NADP-dependent ME isolated from human liver. The comparison of the structure of this cDNA with that of the human mitochondrial NAD(P)-dependent ME (EC 1.1.1.39) shows a homology of 63%, suggesting that these two forms originated from the same gene. The expression of the cDNA in Escherichia coli as a translational fusion (glutathione S-transferase::ME) protein yielded a product of the predicted mass. The recombinant protein shows NADP-dependent malate oxidoreductase activity and is virtually inactive with NAD. It also shows other distinct features of the native cytosolic NADP-dependent ME, like Mn2+ dependence, similar substrate (Km = 117 microM) and cofactor affinity (Km = 2 microM) constants, and a lack of allosteric regulation. In human proliferative cells, the NADP-dependent ME activity is poorly expressed and barely inducible by thyroid hormones.

Amino Acid Sequence↗

PubMed1993

Sequence of the gene coding for the neurotoxin of Clostridium botulinum type A associated with infant botulism: comparison with other clostridial neurotoxins.

The neurotoxin gene from a strain of Clostridium botulinum type A causing infant botulism was cloned as a series of overlapping polymerase chain reaction (PCR) fragments generated using primers designed to conserved regions of published botulinal toxin (BoNT) sequences. Translation of the nucleotide sequence derived from cloned PCR fragments demonstrated that the toxin gene encodes a protein of 1,296 amino acid residues. Comparative alignment of the derived infant BoNT/A sequence with those of other published neurotoxins revealed highest sequence relatedness with BoNT/A of classical food-borne botulism. The sequence identity between infant and classical BoNT/A was 94.9% for the light chain (corresponding to 23 amino acid changes) and 87.1% for the heavy chain (corresponding to 109 amino acid changes).

Bacterial Proteins↗

PubMed2006

Complete nucleotide sequences and gene organization of mitochondrial genome of Bufo gargarizans.

The complete mitochondrial genome of Bufo gargarizans was sequenced using overlapping polymerase chain reaction (PCR) amplicons (GenBank Accession No. DQ275350). The genome is 17,277 base pairs in length, containing 13 protein-coding genes (ATP6, ATP8, COI-III, ND1-6, ND4L, Cyt b), 2 ribosomal RNAs (12S rRNA and 16S rRNA), 22 transfer RNAs and a putative control region. We analyzed the sequence using bioinformatics methods comparing the obtained mtDNA sequence with other toads and frogs. Based on the concatenated nucleotide sequences of protein-coding genes, we constructed a phylogenetic tree with maximum likelihood (ML) and maximum parsimony (MP) methods and discussed the phylogenetic relationships among 11 species of Anura.

Animals↗

PubMed2005

Characterization of estrogen receptor betab in sea bream (Sparus auratus): phylogeny, ligand-binding, and comparative analysis of expression.

Estrogens control many physiological processes in both female and male vertebrates, mostly mediated by specific nuclear estrogen receptors (ER). Two ER subtypes (ERalpha and ERbeta) are present in most vertebrates, including the sea bream (Sparus auratus) a hermaphrodite teleost fish. In the present study several variant cDNAs encoding a second sea bream ERbeta (sbERbetab) is reported. Phylogenetic and Southern blot analysis indicate that sbERbetab and the previously cloned sbERbetaa (formerly sbERbeta) are encoded by different genes, which may have arisen by duplication of an ancestral ERbeta gene. Competitive binding assays show that sbERbetab has high affinity for 17beta-estradiol (K(d) = 1 nM) and specifically binds estrogen agonists (diethylstilbestrol and ethynylestradiol) and antagonists (ICI 182,780). In Northern blot sbERalpha, sbERbetaa, sbERbetab produce several different transcripts in a variety of tissues. RT-PCR showed a partially overlapping but differential tissue distribution in both male and female sea bream.

Amino Acid Sequence↗

PubMed2003

Lysozyme from the gut of the soft tick Ornithodoros moubata: the sequence, phylogeny and post-feeding regulation.

Sequence of a tick gut lysozyme (TGL) from the soft tick Ornithodoros moubata was determined by cloning and sequencing of overlapping polymerase chain reaction (PCR) and RACE PCR products. It is the first lysozyme sequence representing the subphylum Chelicerata. The resulting open reading frame codes for a putative signal peptide of 22 amino-acid residues and a mature protein composed of 124 amino-acids. Calculated mass of the protein is 14037.75 Da and a theoretical isoelectric point is 8.16. The phylogenetic analysis revealed that the TGL belongs to the c-type lysozymes. It forms a distinct monophyletic group together with multiple lysozyme-like sequences found in the gene products agCP6542 from Anopheles gambiae strain PEST and CG8492-PA from Drosophila melanogaster. This group is referred to as an H-branch due to a unique histidine residue at position 52 which replaces the highly conserved tyrosine present in the vast majority of c-type lysozymes. TGL seems to be an interesting case in which the features of lysozymes with anti-bacterial and digestive function are combined. Semi-quantitative RT-PCR and Northern blotting analysis demonstrated that TGL is strongly up-regulated at the transcriptional level after a bloodmeal. The maximum lysozyme mRNA level was detected 16 h post bloodmeal and the message remained stable for 5 days and then it slowly dropped down to the level of non-fed ticks within 2 weeks.

Amino Acid Sequence↗