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Results for “Overlapping PCR”

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At least 505 records · Page 28Linked to original sources

PubMed1999

Organization of the Mitochondrial Genome of a Deep-Sea Fish, Gonostoma gracile (Teleostei: Stomiiformes): First Example of Transfer RNA Gene Rearrangements in Bony Fishes.

: We determined the complete nucleotide sequence of the mitochondrial genome (except for a portion of the putative control region) for a deep-sea fish, Gonostoma gracile. The entire mitochondrial genome was purified by gene amplification using long polymerase chain reaction (long PCR), and the products were subsequently used as templates for PCR with 30 sets of newly designed, fish-universal primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products showed that the genome contained the same 37 mitochondrial structural genes as found in other vertebrates (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes), with the order of all rRNA and protein-coding genes, and 19 tRNA genes being identical to that in typical vertebrates. The gene order of the three tRNAs (tRNA(Glu), tRNA(Thr), and tRNA(Pro)) relative to cytochrome b, however, differed from that determined in other vertebrates. Two steps of tandem duplication of gene regions, each followed by deletions of genes, can be invoked as mechanisms generating such rearrangements of tRNAs. This is the first example of tRNA gene rearrangements in a bony fish mitochondrial genome.

Journal Article↗

PubMed2002

Detection procedures for neuroblastoma cells metastatic to blood and bone marrow: blinded comparison of chromogranin A heminested reverse transcription polymerase chain reaction to tyrosine hydroxylase nested reverse transcription polymerase chain reaction and to anti-GD2 immunocytology.

Specific and sensitive tumor cell detection is becoming increasingly important for diagnosing and staging as well as for the therapeutic management of neuroblastoma patients. We propose a chromogranin A heminested reverse transcription polymerase chain reaction (CgA hn RT-PCR) procedure for the detection of neuroblastoma minimal residual disease in peripheral blood and bone marrow samples. The results were checked in comparison with the presently available procedures (i.e., with the tyrosine hydroxylase nested RT-PCR [TH n RT-PCR] and with the immunocytochemical approach using anti-GD2 antibodies). Controls from healthy patients or from people with unrelated disease (12 samples of bone marrow and 23 samples of peripheral blood) and serial dilution experiments using neuroblastoma cell lines (SKNLP, SKNFI, STA6, STA8) showed CgA hn RT-PCR full specificity and sensitivity ranging from 10(3) to 10(6) (depending on the cell line). The results compared favorably with those obtained using TH n RT-PCR. Preliminary data obtained analyzing bone marrow and peripheral blood specimens from stage IV neuroblastomas showed substantially overlapping results between CgA and TH n RT-PCR procedures. Our data support the potential usefulness of CgA heminested RT-PCR as a specific and sensitive procedure for minimal disease detection in neuroblastoma. A prospective evaluation of this tool in clinical studies might be warranted.

Adolescent↗

PubMed1992

Dual asymmetric PCR: one-step construction of synthetic genes.

We have developed a one-step process for constructing synthetic genes. Four adjacent oligonucleotides 17-100 bases in length having short overlaps of 15-17 bases are used as primers in a PCR mixture. The quantity of the two internal primers is highly limited, and the resultant reaction causes an asymmetric single-stranded amplification of the two halves of the total sequence due to an excess of the two flanking primers. In subsequent PCR cycles, these dual asymmetrically amplified fragments, which overlap each other, yield a double-stranded, full-length product.

DNA-Directed DNA Polymerase↗

PubMed2005

Isolation of TIR and non-TIR NBS--LRR resistance gene analogues and identification of molecular markers linked to a powdery mildew resistance locus in chestnut rose (Rosa roxburghii Tratt).

Toll and interleukin-1 receptor (TIR) and non-TIR nucleotide binding site-leucine rich repeat (NBS-LRR) resistance gene analogues (RGAs) were obtained from chestnut rose (Rosa roxburghii Tratt) by two PCR-based amplification strategies (direct amplification and overlap extension amplification) with degenerate primers designed to the conserved P-loop, kinase-2, and Gly-Leu-Pro-Leu (GLPL) motifs within the NBS domain of plant resistance gene (R gene) products. Thirty-four of 65 cloned PCR fragments contained a continuous open reading frame (ORF) and their predicted protein products showed homology to the NBS-LRR class R proteins in the GenBank database. These 34 predicted protein sequences exhibited a wide range (19.5--99.4%) of sequence identity among them and were classified into two distinct groups by phylogenetic analysis. The first group consisted of 23 sequences and seemed to belong to the non-TIR NBS-LRR RGAs, since they contained group specific motifs (RNBS-A-non-TIR motif) that are often present in the coiled-coil domain of the non-TIR NBS-LRR class R genes. The second group comprised 11 sequences that contained motifs found in the TIR domain of TIR NBS-LRR class R genes. Restriction fragment length polymorphic (RFLP) markers were developed from some of the RGAs and used for mapping powdery mildew resistance genes in chestnut rose. Three markers, RGA 22 C, RGA 4 A, and RGA 7 B, were identified to be linked to a resistance gene locus, designated CRPM 1 for chestnut rose powdery mildew resistance 1, which accounted for 72% of the variation in powdery mildew resistance phenotype in an F1 segregating population. To our knowledge, this is the first report on isolation, phylogenetic analysis and potential utilization as genetic markers of RGAs in chestnut rose.

Amino Acid Sequence↗

PubMed1992

Assessment of iduronate-2-sulfatase mRNA expression in Hunter syndrome (mucopolysaccharidosis type II).

Eight unrelated patients with Hunter syndrome were investigated for expression of iduronate-2-sulfatase (IDS) mRNA by reverse transcription (RT) linked to polymerase chain reaction (PCR), or RT-PCR. The entire coding region was studied by amplification of two overlapping segments of 0.7 and 1.1 kb. Seven children with Hunter syndrome had PCR products indistinguishable in size from normal. One patient, with clinically severe disease, did not produce either IDS product although mRNA for a control gene was readily amplified. This method rapidly identifies patients having absent or qualitatively abnormal IDS mRNA and may be useful in investigating genotype-phenotype relationships.

Base Sequence↗

PubMed2005

High-throughput detection of multiple genetic polymorphisms influencing drug metabolism with mismatch primers in allele-specific polymerase chain reaction.

Because of genetic polymorphisms of drug-metabolizing enzyme genes, the activities of the enzymes in humans vary widely and alter the metabolism of commonly used clinical agents. Severe adverse effects or resistance to therapy may result. We have developed a rapid and high-throughput genotyping method for detecting polymorphisms of the drug-metabolizing enzyme genes CYP2C9*3, CYP2C19*2, *3, CYP2D6*2, *4, *10, *14, *21, NAT2*5, *6, *7, and TPMT*3 using allele-specific polymerase chain reaction (PCR) with mismatch primers (ASPCR-MP) and CYP2D6*5, *36, and CYP2D6xN using stepdown PCR with detection by SYBR Green I. We analyzed genomic DNA from 139 Japanese volunteers. Identical genotyping results were obtained by using ASPCR-MP, stepdown PCR, and conventional PCR. We found that the methods clearly differentiate three specific profiles with no overlap in the signals. Moreover, both ASPCR-MP and stepdown PCR for genotyping took less than 3-4h. To our knowledge, this is the first report of successful simultaneous detection of multiple genetic polymorphisms with point mutations using ASPCR-MP or multiple genetic polymorphisms with large structural alterations using stepdown PCR. In conclusion, ASPCR-MP and stepdown PCR appear to be suitable for large clinical and epidemiological studies as methods that enable highly sensitive genotyping and yield a high-throughput.

Alleles↗

PubMed1991

Complete sequence of human cardiac alpha-myosin heavy chain gene and amino acid comparison to other myosins based on structural and functional differences.

We have obtained the 5820 nucleotide sequence encoding all 1939 amino acids of the human cardiac alpha-myosin heavy chain (alpha-MHC), as established by dideoxy sequencing of cloned cDNA, genomic DNA and polymerase chain reaction (PCR) amplification products. This sequence represents overlapping fragments of the entire coding sequence. Amino acid sequence comparison of the human cardiac alpha-MHC with the published human cardiac beta-MHC have demonstrated that there are, at least, 7 isoform-specific divergent regions, including functionally important binding protein-related sites such as ATP, actin and myosin light chain. It has been reported that in the rat, there are 8 isoform-specific divergent regions. The 7th divergent area (residue area 1633-1657, which is thought to mediate thick filament formation) in the light meromyosin region in the rat is not apparent in the human. The amino acid compositions of cardiac alpha- and beta-MHCs in the human and the rat, and human embryonic skeletal muscle and chicken gizzard smooth muscles were compared. Amino acid sequences in cardiac alpha- and beta-MHCs in the human and the rat are well conserved. In the head portion, the amino acid composition divergence of human cardiac alpha-MHC is ranked between rat cardiac alpha-MHC and human cardiac beta- or rat cardiac beta-MHC; human skeletal muscle MHC is the most divergent of the myosin isoform examined. These data predict that human cardiac alpha-MHC may have undergone evolutionary changes toward obtaining the biochemical and physiological properties of cardiac beta-MHC.

Amino Acid Sequence↗

PubMedDate not supplied

In vivo assessment of mitochondrial functionality in human gastrocnemius muscle by 31P MRS. The role of pH in the evaluation of phosphocreatine and inorganic phosphate recoveries from exercise.

In this study we compared the kinetics of phosphocreatine (PCr) and P(i) recovery, and their dependency on cytosolic pH in 38 normal individuals. Spectra were acquired during rest, work and recovery. A time resolution of 10 s was used to obtain detailed information. The kinetics of PCr and P(i) recovery almost overlapped when the lowest value of cytosolic pH reached during recovery (termed the minimum pH) was < 6.95, while they were completely dissociated when the minimum pH was > 6.95. This result is interpreted as indirect in vivo evidence of the kinetic control exerted by ADP on mitochondrial oxidation. Our results represent a rationale for new experimental conditions to be used in clinical routine studies of pathologies due to primary or secondary mitochondrial malfunction.

Adult↗

PubMed1995

The biochemically and immunologically distinct CSPG of notochord is a product of the aggrecan gene.

Using the monoclonal antibody S103L, which reacts specifically with an epitope in the chondroitin sulfate-rich domain of the chick cartilage chondroitin sulfate proteoglycan (CSPG) core protein, we have identified the predominant CSPG expressed by notochord. This large notochord CSPG is first detected immunohistochemically as early as stage 16, long before chondrogenesis occurs, and is expressed continuously during the time of active neural crest migration and through the onset of sclerotomal differentiation. Because of the cross-reactivity of both notochord and cartilage CSPGs with the S103L antibody, extensive molecular and biochemical analysis of the two CSPGs was carried out. Striking differences distinguish the notochord and cartilage (aggrecan) CSPGs at the level of posttranslational modification. Notably, cartilage aggrecan carries a significant content of keratan sulfate (KS) chains, while the notochord CSPG is devoid of KS. In contrast, cartilage aggrecan lacks the HNK-1 epitope, while the notochord CSPG has a high content of HNK-1. Three different approaches were used to establish the relationship of the two CSPGs at the molecular level. Northern blot analysis, using aggrecan probes, detected same-sized messages from notochord and cartilage RNA. Overlapping fragments, generated by RT-PCR using primers covering 98% of the entire coding sequence from the known cartilage structure, were of identical size in notochord and cartilage. Taking advantage of our recent studies, which demonstrated a single base change in the aggrecan gene resulting in conversion of Glu to a STOP codon in exon 12 of chick aggrecan as the molecular basis of the defect nanomelia, we demonstrated that the same mutation was present in notochord mRNA from nanomelic chicks. These results provide evidence that the chick aggrecan gene is expressed very early in development in notochord and confirm that the core proteins expressed in chick notochord and cartilage are derived from the same gene. These findings strongly support the hypothesis that the final structural characteristics of each proteoglycan are determined not only by the core protein but also by tissue-specific, developmentally regulated posttranslational mechanisms, functioning within the context of the requirement for specific extracellular matrices.

Aggrecans↗

PubMed1994

Use of an intron polymorphism to localize the tropoelastin gene to mouse chromosome 5 in a region of linkage conservation with human chromosome 7.

The complete coding sequence for mouse tropoelastin was obtained from overlapping reverse transcriptase polymerase chain reaction (PCR) amplimers. These cDNA fragments were derived from mouse tropoelastin mRNA using PCR oligomers complementary to conserved domains within rat tropoelastin mRNA. A comparison of coding domains of mouse and rat tropoelastin mRNA revealed a greater than 93% homology at the nucleotide level and over 96% similarity in the predicted amino acid sequence. PCR primers complementary to regions of the mouse tropoelastin mRNA were used to define a novel intron length polymorphism (ILP) within intron 8 of the mouse tropoelastin gene (Eln). This ILP proved to be informative in an interspecific backcross in which genomic DNA samples from 75 backcross mice were used to map the tropoelastin gene to a position in the distal half of mouse chromosome 5. The linkage and genetic distances between Eln and the closest molecular markers used in this study are centromere-D5Mit95, D5Mit96-6.7 cM-Gus, Eln-4.0 cM-Zp3-telomere.

Amino Acid Sequence↗

PubMed1995

Atypical phytochrome gene structure in the green alga Mesotaenium caldariorum.

The phytochrome photoreceptor in the green alga Mesotaenium caldariorum is encoded by a small family of highly related genes. DNA sequence analysis of two of the algal phytochrome genes indicates an atypical gene structure with numerous long introns. The two genes, termed mesphy1a and mesphy1b, encode polypeptides which differ by one amino acid in the region of overlap that was sequenced. RT-PCR studies have established the intron-exon junctions of both genes and show that both are expressed. RNA blot analysis indicates a single transcript of ca. 4.1 kb in length. The deduced amino acid sequence of the mesphy1b gene reveals that the photoreceptor consists of 1142 amino acids, with an overall structure similar to other phytochromes. Phylogenetic analyses indicate that the algal phytochrome falls into a distinct subfamily with other lower plant phytochromes. Profile analysis of an internal repeat found within the central hinge region of the phytochrome polypeptide indicates an evolutionary relatedness to the photoactive yellow protein from the purple bacterium Ectothiorhodospira halophila, to several bacterial sensor kinase family members, and to a family of eukaryotic regulatory proteins which includes the period clock (per) and single-minded (sim) gene products of Drosophila. Since mutations which alter phytochrome activity cluster within the region delimited by these direct repeats (P.H. Quail et al., Science 268 (1995): 675-680), this conserved motif may play an important role in the signal transducing function of these disparate protein families.

Amino Acid Sequence↗

PubMed2006

High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression.

Human beta-defensin-4 (hBD4) is a cationic 50-amino acid antimicrobial peptide with three conserved cysteine disulfide bonds. It exhibits a broad antimicrobial spectrum. This study describes the synthesis of hBD4 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released hBD4. A PCR-based gene SOEing (splicing by overlap extension) synthesis method was used in the synthesis of the hBD4 gene with optimized codons. By constructing the expression plasmid (pET32-smhBD4), high concentration of soluble hBD4 fusion protein (1.9 g/l) can be obtained in E. coli. Further optimization studies showed that the expression system was very efficient to produce soluble target protein, and the solubility of the target protein could attain more than 99% even when the culture temperature was as high as 37 degrees C. The highest productivity (2.68 g/l) of the hBD4 fusion protein was achieved by cultivating the E. coli (pET32-smhBD4) in MBL medium at 34 degrees C, inducing the culture at the mid-exponential phase with 0.4-mM isopropyl beta-D-galactopyranoside (IPTG), and collecting the broth after 6-h expression. The soluble target protein accounted for 64.6% of the total soluble proteins, and the mature hBD4 expression level was stoichiometrically estimated to be 0.689 g/l. This fusion protein was then purified and cleaved to get the mature hBD4 peptide that showed antimicrobial activity against E. coli and Pseudomonas aeruginosa.

Antimicrobial Cationic Peptides↗

PubMed1998

Molecular analysis of the LYS2 gene of Candida albicans: homology to peptide antibiotic synthetases and the regulation of the alpha-aminoadipate reductase.

The unique alpha-aminoadipate pathway for lysine biosynthesis is present only in fungi and involves eight enzyme steps. alpha-Aminoadipate semialdehyde dehydrogenase, commonly called alpha-aminoadipate reductase (AAR), catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic semialdehyde by a novel mechanism. Two genes, LYS2 and LYS5, encode the heterodimeric enzyme in Saccharomyces cerevisiae. The LYS2 gene of Candida albicans was shown to be contained in the 4.8-kb insert of the plasmid pCaLYS2. This plasmid complemented lys2 mutants of both S. cerevisiae and C. albicans. The S. cerevisiae and C. albicans Lys2(+) transformants exhibited 138% and 160% of wild-type AAR activity, respectively. The DNA-sequence analysis of the 4.8-kb region in plasmid pCaLYS2 and a PCR product from genomic DNA which overlapped with the 4.8-kb insert revealed a continuous ORF of 4173 nucleotides encoding 1391 amino-acid residues. The C. albicans LYS2 ORF exhibited 63.0% identity at the nucleotide level and 56.2% identity at the amino-acid level to the LYS2 gene of S. cerevisiae. The ORF is preceded by consensus sequences for the TATA-, CAAT- and GCN4-box elements. An S. cerevesiae-type transcription termination signal is seen in the 3' flanking region. The deduced amino-acid sequence revealed a motif for an AMP-binding site and also the highly conserved core sequences common to peptide antibiotic synthetases. The LYS2 mRNA and alpha-aminoadipate reductase activity were repressed to a higher level in YEPD-grown cells than in cells grown in the presence of lysine or minimal medium. Additionally, AAR was shown to be feedback-inhibited by lysine and the lysine analog, thialysine. The results of the present report reveal the molecular characteristics of the LYS2 gene of C. albicans, its homology to peptide antibiotic synthetases, its divergence from the LYS2 gene of S. cerevisiae, and the regulation of AAR in C. albicans.

Aldehyde Oxidoreductases↗

PubMed1995

Finer mapping of neutralizing epitope(s) on the C-terminal of Japanese encephalitis virus E-protein expressed in recombinant Escherichia coli system.

In order to localize denaturation-resistant neutralizing epitope(s) in the C-terminal 180 amino acids of Japanese encephalitis (JE) virus E-protein, four recombinant clones encoding different or overlapping nucleotide sequences were constructed by PCR from a recombinant plasmid pS22. The amplified fragments were cloned into PCR 1000 vector, and then transferred into Escherichia coli expression vector pRIT2T. The inserted genes were expressed as fusion proteins with protein-A and examined for their antigenicity and immunogenicity by Western blotting and mouse immunization, respectively. Among the four recombinant fusion proteins, the highest neutralizing antibody titre was obtained by the one expressed by the recombinant clone pRIT2T-B3, which carried the coding sequence of amino acid number 373-399 of JE virus E protein. The results indicated that this short region of 27 amino acids sequence near the C-terminal of JE virus E protein possesses neutralizing epitope(s). These data should assist in the design of an efficient subunit vaccine against JE virus infection in future.

Animals↗

PubMed2004

Somatic D-loop mitochondrial DNA mutations are frequent in uterine serous carcinoma.

The mitochondria plays a role in apoptosis. Its genome is also more susceptible to mutations because of high levels of reactive oxygen species and limited repair mechanisms. The D-loop of mitochondrial DNA (mtDNA) contains essential transcription and replication elements, and mutations in this region might alter the rate of DNA replication. We examined genetic alterations in the D-loop region of mtDNA in uterine serous carcinoma (USC) samples and their paired normal adjacent endometrium. DNA was extracted after laser-capture microdissection of paraffin-embedded tissues from eight patients with USC. The entire D-loop genome was amplified using nine pairs of overlapping primers. Denatured polymerase chain reaction (PCR) products were subjected to single-strand conformation polymorphism (SSCP) analysis. Somatic mtDNA alterations were detected in five tumours (63%). Our study indicates that mtDNA D-loop sequence alterations occur at a high frequency in USC suggesting that mtDNA mutations may play a role in the development of USC.

Aged↗

PubMed1998

Squalene-hopene cyclase from Methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids.

We report the cloning and characterisation of the Methylococcus capsulatus shc gene, which encodes the squalene-hopene cyclase (SHC). This enzyme catalyses the complex cyclization of squalene to the pentacyclic triterpene skeleton of hopanoids and represents the key reaction in this biosynthesis. Using a combination of PCR amplification and DNA hybridization, two overlapping 2.6 kb PstI and 3.3 kb SalI DNA fragments were cloned bearing a 1962 bp open reading frame encoding a 74 kDa protein with 654 amino acids and a predicted isoelectric point at about pH 6.3. The deduced amino acid sequence of the M. capsulatus shc gene showed significant similarity to known prokaryotic SHCs and to a lesser degree to the related eukaryotic oxidosqualene cyclases (OSCs). Like other triterpene cyclases, the M. capsulatus SHC contains seven so-called QW-motifs as well as an aspartate-rich domain. The recombinant M. capsulatus SHC was expressed in Escherichia coli and in vitro activity of the recombinant cyclase was demonstrated using crude cell-free lysate or solubilized membrane preparation. The cyclization products hop-22-ene and hopan-22-ol (diplopterol) were identified by GC and GC-MS.

Amino Acid Sequence↗

PubMed2003

A rapid and convenient variant of fusion-PCR to construct chimeric flaviviruses.

So far, full-length cDNAs of chimeric flaviviruses have been constructed by restriction-enzyme cleavage of the gene(s) to be exchanged or by fusion-PCR of two amplified PCR fragments. The construction of a chimeric flavivirus by a faster and more convenient variant of the standard fusion-PCR is reported. A Modoc/yellow fever chimeric virus was engineered in which the structural prM and E genes of yellow fever virus 17D were replaced by the homologous genes of Modoc virus. In two PCR steps, a fusion was made between the 3' end of the C gene of yellow fever virus and the 5' end of the prM gene of Modoc virus, and between the 3' end of the E gene of Modoc virus and the 5' end of the NS1 gene of yellow fever virus. For each of the two fusions between yellow fever and Modoc virus, a standard PCR was performed to amplify a short fragment with one overlapping end that could be used as one of the primers in the subsequent (fusion) PCR.

Animals↗

PubMed1999

The screening for mutations in the thyroglobulin cDNA from six patients with congenital hypothyroidism.

The six patients described in this study were clinically diagnosed with congenital hypothyroidism. Based on clinical and pathophysiological parameters, the cause of the thyroid dyshormonogenesis was suspected to be a defect in the synthesis of thyroglobulin, the matrix protein for thyroid hormone synthesis in the thyroid gland. After RNA isolation from six goitrous tissues and control thyroid tissues, RT-PCR was used to amplify 20 overlapping thyroglobulin (TG) cDNA fragments. Two alternative splice transcripts were identified: a transcript with a deletion of nucleotides 177-274 and a transcript with a deletion of nucleotides 3430-3736 that result in frame shifts and the introduction of premature stop codons. Two alternatively spliced transcripts not changing the reading frame were also identified: a transcript containing a deletion of nucleotides 4529-4699 and a transcript with a deletion of nucleotides 7301-7561. All these transcripts were expressed in thyroid tissue of both patients and controls. Nucleotide sequence analysis and comparison to the revised TG sequence (1997) revealed one revision and eight polymorphisms that did not result in amino acid changes and four polymorphisms that did change amino acid codons. In three patients a homozygous mutation was present at nucleotide position 229, causing a glycine to serine amino acid substitution. The clinical description 'thyroglobulin synthesis defect' in this population cannot be explained by major mutations in the coding region of the TG gene. Furthermore, the presence and level of expression of the alternatively spliced transcripts do not co-segregate with thyroid dyshormonogenesis, since in normal thyroid tissue the same alternatively spliced transcripts are present.

Adolescent↗