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PubMed2000

Direct amplification of Entamoeba histolytica DNA from amoebic liver abscess pus using polymerase chain reaction.

An important and serious complication of intestinal infection with Entamoeba histolytica is the involvement of the liver (hepatic amoebiasis). Hepatic amoebiasis is usually diagnosed by the clinical picture (pain in the right upper quadrant and fever), ultrasound examination and positive serology. However, none of these tests are definitive and the picture overlaps with pyogenic liver abscess caused by bacteria. It is for this reason that the feasibility of using polymerase chain reaction (PCR) for the detection of E. histolytica DNA in liver abscess pus was investigated. A comparative study was done to verify the sensitivity of ten pairs of primers specific for detecting E. histolytica in stools. Samples of liver abscess pus from 22 serology-positive patients were collected under ultrasound guidance; and these were used directly in PCR assays without any prior pre-treatment of the samples. Of the ten pairs of previously published primers tested, two pairs of primers (PI + P2 and P11 + P12) were found to give 100% sensitivity. Based on these results, we recommend that PCR assay can be successfully used to confirm the diagnosis of amoebic liver abscess with the primers identified.

Animals↗
PubMed1995

Cosmid assembly and anchoring to human chromosome 21.

A human chromosome 21-specific cosmid library from the Lawrence Livermore National Laboratory has been analyzed by two complementary methods, fingerprinting and hybridization; 40% coverage of the entire chromosome 21 has been achieved. To prepare a contig pool, approximately 9300 cosmid clones randomly selected from the library were fingerprinted and automatically assembled into 467 overlapping sets by the fluorescence-tagged restriction fragment method. The average size of the overlapping sets was 9.5 cosmids with minimal tiling paths consisting of 5.4 cosmids with a 10-kb extension each. However, as many as 10% of overlaps within members were estimated to be false. For regional localization, we hybridized gridded arrays of cosmids with inter-Alu-PCR probes obtained from YAC clones and somatic cell hybrids and assigned 592 cosmids to 26 subregions of 21q. Of these, 371 clones were incorporated into 139 contigs, anchoring the total 1864 cosmids to the subregion. The remaining 221 clones were mapped as orphans. To correlate the cytogenetic, YAC, and cosmid maps on 21q, the translocation breakpoints of the chromosomes contained in the somatic cell hybrids were mapped with respect to the STS content of the YACs. From the gene cluster regions, 176 ribosomal and 25 alphoid clones were isolated by hybridization. Together, these sets of anchored contigs and cosmids will provide a valuable resource for construction of a high-resolution map and for isolation of genes of interest from chromosome 21.

Animals↗
PubMed1997

Amplification, cloning and sequencing of the sigmaC-encoded gene of avian reovirus.

The sigmaC-encoding cDNA of avian reovirus (ARV) 1733 strain was amplified, cloned and sequenced using double nested polymerase chain reaction (PCR). The ARV sigmaC protein is a minor component of the outer capsid that induces type-specific neutralization antibodies. Four overlapping sigmaC-encoding cDNA fragments were obtained. Together, the four fragments represented the whole coding sequence. The nucleotide and deduced amino acid sequences of sigmaC-encoded gene of U.S. (S1133 and 1733) and Australian isolates (RAM-1 and SOM-4) were compared. The U.S. isolates were closely related, but different from Australian isolates. The degree of differences between the U.S. and Australian isolates was over 44.89% at both the nucleotide and deduced amino acid levels and suggested that the virus is evolving separately in different continents. The deduced amino acid sequences of ARV sigmaC indicated a heptapeptide repeat in the N-terminal region of ARV sigmaC existed in all ARVs. The results suggested that ARV sigmaC is structurally related to mammalian reovirus (MRV) sigma1.

Amino Acid Sequence↗
PubMed2000

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes.

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT-PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.

Amino Acid Sequence↗
PubMed2005

Effect of common B-RAF and N-RAS mutations on global gene expression in melanoma cell lines.

We studied global gene expression in three melanoma cell lines with the most common and potent V600E mutation in the B-RAF gene-four cell lines with a common Q61R mutation in the N-RAS gene and three cell lines with no mutations using human HG-U133A 2.0 micro-arrays with 22 277 transcripts. Data analysis using stringent criteria revealed several upregulated and downregulated genes in cell lines with B-RAF and N-RAS mutations compared with cell lines without mutations. We found 29 genes specifically upregulated and 32 genes downregulated in cell lines with B-RAF mutations, whereas 70 genes were upregulated and 39 downregulated in cell lines with N-RAS mutations; 11 genes showed overlapping upregulation and 45 downregulation. The micro-array data for nine selected genes were validated by the real-time PCR technique. Expression of a large number of genes, that encode members or regulators of the RAS/RAF/MEK/ERK pathways or are involved in metastasis or invasion, was affected in cell lines with mutations in B-RAF and N-RAS. Upregulated genes in cell lines with mutations included dual-specificity phosphatase 6 (DUSP6), sprouty 2 (SPRY2), v-akt murine thymoma viral oncogene homolog 3 (AKT3) and matrix metalloproteinase 14 (MMP14); downregulated genes included interleukin 18 (IL18), Krüppel-like factor 5 (KLF5) and inhibitor of DNA binding 2 (ID2). Our results, though carried on cell lines, provide a novel insight into the effect of mutations in the B-RAF and N-RAS genes on global gene expression in melanoma and highlight the complexity of mechanisms involved in tumour initiation and maintenance.

DNA Mutational Analysis↗
PubMed2003

A sensitive transcriptome analysis method that can detect unknown transcripts.

We have developed an AFLP-based gene expression profiling method called 'high coverage expression profiling' (HiCEP) analysis. By making improvements to the selective PCR technique we have reduced the rate of false positive peaks to approximately 4% and consequently the number of peaks, including overlapping peaks, has been markedly decreased. As a result we can determine the relationship between peaks and original transcripts unequivocally. This will make it practical to prepare a database of all peaks, allowing gene assignment without having to isolate individual peaks. This precise selection also enables us to easily clone peaks of interest and predict the corresponding gene for each peak in some species. The procedure is highly reproducible and sensitive enough to detect even a 1.2-fold difference in gene expression. Most importantly, the low false positive rate enables us to analyze gene expression with wide coverage by means of four instead of six nucleotide recognition site restriction enzymes for fingerprinting mRNAs. Therefore, the method detects 70-80% of all transcripts, including non-coding transcripts, unknown and known genes. Moreover, the method requires no sequence information and so is applicable even to eukaryotes for which there is no genome information available.

Animals↗
PubMed2006

Spatial and temporal divergence of expression in duplicated barley germin-like protein-encoding genes.

Subfunctionalization is the process by which a pair of duplicated genes, or paralogs, experiences a reduction of individual expression patterns or function while still reproducing the complete expression pattern and function of the ancestral gene. Two germin-like protein (GLP)-encoding genes, GerB and GerF, are paralogs that belong to a small gene family in barley (Hordeum vulgare). Both genes share high nucleotide sequence similarity in coding and noncoding regions and encode identical apoplastic proteins. The use of RNA gel blots, coupled with single-stranded conformation polymorphism (SSCP) analysis of RT-PCR products, elucidated the developmental and tissue-specific expression patterns of each gene. Individual expression patterns provided evidence of both overlapping redundancy and early subfunctionalization. GerB is predominantly expressed in developing shoots, while GerF is predominantly expressed in seedling roots, developing spikes, and pericarp/testa. GerF promoter deletion studies located a region (-356/-97) responsible for high promoter activity and showed the ability of GerB and GerF upstream regions to drive gfp expression in coleoptiles, epicarps, and lemma/palea of developing spikes. The observed expression patterns are consistent with proposed roles in plant development and defense mechanisms for this gene family. These roles may explain why redundancy has been selectively maintained in this duplicate gene pair.

Base Sequence↗
PubMed2004

[Rapid construction of full-length MnSOD cDNA of chickens by one-step 3'RACE].

RACE (rapid amplification of cDNA ends) is a popular technique to rapidly obtain the full-length cDNA. After obtaining the 3' cDNA and 5' cDNA fragments with a overlapped region by 3' RACE and 5' RACE, the full-length cDNA could be generated by end-to-end PCR or subcloning. In this study, 3' RACE combined with touch-down PCR was successfully used for the rapid construction of full-length MnSOD cDNA of chickens. Compared with the conventional end-to-end PCR or subcloning, this method, called one-step 3' RACE, is fast, economical and highly specific. It especially fits the rapid construction of full-length cDNA by RACE method.

Animals↗
PubMed2006

[Characterization of a recombinant Listeria monocytogenes strain containing the fusion protein gene of Newcastle disease virus].

Homologous recombination was utilized for construction of a recombinant strain of L. monocytogenes carrying a gene from the Newcastle diseases virus by insertional mutation targeting its listeriolysin O gene (hly). The gene encoding fusion protein of the Newcastle disease virus (NDV-F) was used as the model heterologous gene. The F gene was inserted into hly downstream to its promoter and signal sequence by overlapping extension polymerase chain reaction, which was then subcloned into the shuttle plasmid pKSV7 for allelic exchange with L. monocytogenes chromosome. PCR amplification of the target genes indicated insertion of the F gene into the chromosome DNA of L. monocytogenes. RT-PCR showed transcription of F gene from the recombinant L. monocytogenes strain. Comparisons were then made between the recombinant strain and its wild parent strain in terms of the hemolytic activity, adhesion and invasiveness to cultured HeLa cells, virulence to mice and chicken embryos, and growth kinetics in broth medium as well as its stability upon repeated subculturing and serial passages in mice. The recombinant L. monocytogenes lost its hemolytic activity on the blood agar and had no hemolytic titer from its culture supernatants as compared with the titer of 24 in the supernatant from the wild parent strain. The recombinant strain also had lower adhesiveness (P > 0.05) and significantly lower relative invasiveness to the HeLa cells than its wild type strain (P < 0.05). Such insertional mutation resulted in reduced virulence, about 3.7 logs and 6.5 logs less than its parent strain L. monocytogenes 10403S as shown by the 50% lethal dose assays in the mouse and chicken embryonated egg models respectively. The recombinant strain was relatively stable as shown by amplification of the target gene NDV-F from its genomic DNA after subculturing in BHI broth or in mice for 5 times.

Animals↗
PubMed1996

Calpain is the target antigen of a Th1 clone that transfers protective immunity against Schistosoma mansoni.

A CD4+ clone (clone B), characterized as Th1 based on its selective production of IFN-gamma and IL-2, was established from C57Bl/6 mice protectively immunized against Schistosoma mansoni by intradermal vaccination with soluble worm Ags, plus bacillus Calmette Guerin. In agreement with previous results demonstrating an IFN-gamma-dependent cell-mediated protective mechanism in this vaccination model, Ag-elicited peritoneal macrophages from syngeneic recipients of this clone were activated to kill schistosome larvae (schistosomula) in vitro. Moreover, recipients of clone B displayed significant resistance against cercarial challenge. By screening a battery of lambda(gt11) clones from an adult worm cDNA library, one recombinant (25B) was identified that stimulated clone B specifically. Analysis of the 25B cDNA insert revealed a nucleotide sequence identical with that of the large subunit of schistosome calpain, a Ca2+-activated neutral proteinase. By expressing the products of PCR subcloning, we identified a 146-amino acid region of the 25B gene containing immunologic activity equivalent to the whole polypeptide. Overlapping peptides spanning this region were synthesized, and a core epitope was identified with the sequence EWKGAWCDGS. Since clone B responds to supernatants from cultured schistosomula, we postulate that the recognition of calpain released by invading larvae and resulting induction of Th1 cytokines accounts for the protection mediated by the adoptively transferred clone. Our findings thus implicate calpain as a target of protective immunity in schistosomes and provide the first example of a candidate vaccine Ag for this parasite identified on the basis of T cell reactivity.

Adoptive Transfer↗
PubMed1989

One-sided polymerase chain reaction: the amplification of cDNA.

We report a rapid technique, based on the polymerase chain reaction (PCR), for the direct targeting, enhancement, and sequencing of previously uncharacterized cDNAs. This method is not limited to previously sequenced transcripts, since it requires only two adjacent or partially overlapping specific primers from only one side of the region to be amplified. These primers can be located anywhere within the message. The specific primers are used in conjunction with nonspecific primers targeted either to the poly(A)+ region of the message or to an enzymatically synthesized d(A) tail. Pairwise combinations of specific and general primers allow for the amplification of regions both 3' and 5' to the point of entry into the message. The amplified PCR products can be cloned, sequenced directly by genomic sequencing, or labeled for sequencing by amplifying with a radioactive primer. We illustrate the power of this approach by deriving the cDNA sequences for the skeletal muscle alpha-tropomyosins of European common frog (Rana temporaria) and zebrafish (Brachydanio rerio) using only 300 ng of a total poly(A)+ preparation. In these examples, we gained initial entry into the tropomyosin messages by using heterologous primers (to conserved regions) derived from the rat skeletal muscle alpha-tropomyosin sequence. The frog and zebrafish sequences are used in an analysis of tropomyosin evolution across the vertebrate phylogenetic spectrum. The results underscore the conservative nature of the tropomyosin molecule and support the notion of a constrained heptapeptide unit as the fundamental structural motif of tropomyosin.

Animals↗
PubMed2004

Delineation of species within the Trichoderma viride/atroviride/koningii complex by UP-PCR cross-blot hybridization.

Sequences from the ribosomal DNA ITS regions have shown that there is limited variation among the species in the Trichoderma viride/atroviride/koningii species complex also known as Hypocrea rufa complex, and that infraspecies variation sometimes is larger than interspecies variation. Strains belonging to T. viride, T. atroviride, T. koningii, T. asperellum, and respective teleomorphs were analyzed using cross-blot hybridization of PCR products generated by the universally primed PCR (UP-PCR) technique. The hybridization results showed that the morphologically defined species could be delineated molecularly, suggesting that the species are more separated than indicated by ITS sequence phylogeny. However, cross-hybridization signals at infra- and interspecies level within this species complex was overlapping, again raising the question of how many species there are. Due to the heterogeneity of T. viride revealed, a further revision of this species is needed. In addition, this study shows that a macroarray (DNA chip) containing membrane-bound UP-PCR products for reference strains can be developed for routine identification of Trichoderma strains.

DNA Primers↗
PubMed1998

PCR cloning and expression of the molt-inhibiting hormone gene for the crab (Charybdis feriatus).

A PCR-based genomic DNA walking technique was used to clone the gene for the molt-inhibiting hormone of the crab, Charybdis feriatus. Several overlapping genomic clones were isolated, and the MIH gene for the crab was reconstructed. DNA sequence determination of the overlapping clone reveals that the MIH gene spans 4.3kb and consists of three exons and two introns. Exons 1 and 2 carry a coding sequence for the signal peptide, and exons 2 and 3 consist of coding sequence for the mature peptide. The exon-intron boundary of the crab MIH gene also follows the 'GT-AG rule' for the splice donor and acceptor. The deduced amino acid sequence of MIH shows the highest overall similarity to those of the crabs, Callinectes sapidus and Carcinus maenas, and the gonad-inhibiting hormone (GIH) of the lobster. The putative polyadenylation signal is approximately 1.0kb 3' downstream of the termination codon (TGA). Genomic Southern blot analysis indicates that few genomic fragments were hybridized to the cDNA probe. The 5' flanking region contains a putative promoter with several putative cis elements similar to some vertebrate neuropeptide genes. The 530-bp flanking region was subcloned separately to two promoterless reporter plasmids carrying either the Green Fluorescent Protein gene (GFP) or the Choramphenicol Acetyltransferase gene (CAT). The DNA constructs were transfected into insect cells (Sf21) and mouse pituitary cells (GH4ZR7), respectively. Green fluorescent protein was detected in some of the transfected insect cells, and expression of the CAT was detected in cells transfected with DNA constructs containing the crab promoter. By RT-PCR, MIH transcripts can be detected in the eyestalk of shrimp in intermolt, early premolt, late premolt stages and females that brood their eggs. It can also be found in the brain, but not in the ovary, hepatopancreas, muscle and epidermis. During early larval development, MIH mRNA can be detected in the pre-hatched and the newly hatched larvae. Unlike the adult, the expression of the MIH in the larvae is exclusively in the brain.

Amino Acid Sequence↗
PubMed2005

[In silicon cloning of HV126, a novel human gene related to multi drug resistance in leukemia].

OBJECTIVE: To find the novel gene related to the multi-drug resistance in leukemia and explore the molecular mechanism of multi-drug resistance. METHODS: The subtracted HL-60/VCR cDNA library was generated through the suppression subtractive hybridization using the wild HL-60 cells' cDNA as target and HL-60/ ATRA cells' as driver. A novel expression sequence tag (EST) sequence, which differentially expressed in HL-60/ ATRA cell, was screened by cDNA chip. Then a novel human gene, HV126 was assembled by the EST assembly tools. Bioinformatical databases and softwares were used to analyze and predict its function. Reverse transcription-PCR (RT-PCR) was used to detect the expression of HV-126 gene in leukemia cells before and after chemotherapy. RESULTS: The full open reading frames (ORFs) of the novel EST assembled by overlapping dbEST sequences included a 1991 bp nucleic sequence, which was named HV126. The deduced amino acid sequence consisted of 365 amino acids. The sequence of the novel gene exhibited 43% homology to a known gene, which is a possible member of the death domain-flood family implicated in apoptosis and inflammation. The expression of HV126 was proved to be related to the drug sensitivity in leukemia cells by RT-PCR. CONCLUSION: HV126, the novel gene, might have roles in regulating multi-drug resistance in leukemia. Further laboratory research should be done on cloning and making clear the gene function.

Antineoplastic Agents↗
PubMed1993

A rapid method for site-specific mutagenesis using larger plasmids as templates.

To facilitate the introduction of specific point mutations in plasmids that are too large to be amplified efficiently by a single PCR, we have developed a method for site-directed mutagenesis by generating partial plasmid fragments, which introduces changes as simply as conventional techniques. Plasmids containing a fragment of the human immunodeficiency virus type-1 (HIV-1) envelope gene were subjected to PCR with four pairs of PCR primers for each desired point mutation. One primer in each of two of these four pairs contained the desired mutation. The four pairs of primers were designed so that four overlapping fragments were amplified from the plasmid template, two of which contained the mutation. These fragments were then reannealed and electroporated directly into Escherichia coli. The desired mutation was typically found in 66% to 83% of the resulting colonies. The technique is almost as simple as previous techniques, shows similar efficiency and is applicable to plasmids that would normally be too large for efficient site-specific mutagenesis. The entire procedure, from PCR amplification to transfection into E. coli, can be completed in one day.

Base Sequence↗
PubMed2002

A PCR-RFLP assay for the distinction between Fasciola hepatica and Fasciola gigantica.

Fascioliasis is of well-known veterinary importance and an increasing human health problem, with reported cases in the five continents. The causative agents, Fasciola hepatica and Fasciola gigantica, present geographical distributions, which overlap in many regions of Africa and Asia, and in which the differentiation of both species is usually difficult because of the many variations in their morphological characteristics. Moreover, in humans, liver fluke classification cannot be achieved by clinical, pathological, coprological or immunological methods. The differential diagnosis between F. hepatica and F. gigantica infection is very important because of their different transmission and epidemiological characteristics. A simple and rapid PCR-restriction fragment length polymorphism (RFLP) assay, using the common restriction enzymes Ava II and Dra II, is described to distinguish between both fasciolid species. It is based on a 618-bp-long sequence of the 28S rRNA gene recently obtained from liver fluke populations of South America, Europe and Africa. This sequence showed a few nucleotide differences between both fasciolids and no intraspecific variations within each species. This assay provides unambiguous results and may be useful for both individual subject diagnosis and epidemiological surveys of humans and animals in endemic regions of sympatry.

Animals↗
PubMed1999

Molecular cloning and sequence analysis of a cDNA for factor V activating enzyme.

The complete amino acid sequence of a factor V activator (VLFVA) is deduced from the nucleotide sequence of a cDNA encoding the enzyme. The cDNA was isolated by PCR screening a venomous gland cDNA library of Central Asian Vipera lebetina snake. The full-length cDNA clone, derived from two overlapping fragments, comprises 1563 basepairs which encode an open reading frame of 259 amino acids. The amino acid sequence of VLFVA (235 amino acids) shows significant homology with snake venom and mammalian serine proteinases. It contains 12 half-cysteines which form, by analogy with other serine proteinases, 6 disulfide bridges. VLFVA has the catalytic triad His43-Asp88-Ser182. The amino terminal amino acid valine is preceded by 24 amino acids: a putative signal peptide of 18, mainly hydrophobic, amino acids and an activating peptide of 6, mainly hydrophilic amino acid residues. This is the first cloned factor V activating enzyme from snake venom.

Amino Acid Sequence↗
PubMed1997

BAC and PAC contigs covering 3.5 Mb of the Down syndrome congenital heart disease region between D21S55 and MX1 on chromosome 21.

Chromosome 21 is a model for the study of human chromosomal aneuploidy, and the construction of its physical and transcriptional maps is a necessary step in understanding the molecular basis of aneuploidy-dependent phenotypes. To identify the gene(s) responsible for Down syndrome congenital heart disease (DS-CHD), we constructed a physical map of the D21S55 to MX1 region. A bacterial artificial chromosome (BAC) library was screened using several YACs spanning the interval, and a P1-derived artificial chromosome (PAC) library was screened using radiolabeled STS PCR products and whole BACs in gap-filling initiatives. FISH confirmed the location of all BAC and PAC clones to 21q22.2-q22.3. Overlaps were established using clone-to-clone Southerns and 24 new STSs, generated from the direct sequencing of BAC and PAC ends, along with 35 preexisting STSs. Approximately 3.5 Mb of the 4- to 5-Mb D21S55 to MX1 interval is covered in 85 BACs and 24 PACs, representing fourfold coverage within the contigs. These BAC and PAC contigs are valuable reagents for isolating the genes for DS-CHD.

Base Sequence↗