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Results for “Nested Genes”

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At least 235 records · Page 13Linked to original sources

PubMed1999

The common 18-base pair deletion at codons 418-423 of the E-cadherin gene in differentiated-type adenocarcinomas and intramucosal precancerous lesions of the stomach with the features of gastric foveolar epithelium.

Alterations of the E (epithelial)-cadherin gene are often involved in undifferentiated-type carcinomas of the stomach, but not in the differentiated type. Recently, it was recognized that certain differentiated-type carcinomas possess the features of gastric foveolar epithelium. Such carcinomas are closely linked with undifferentiated-type carcinomas. Nineteen lesions (ten differentiated-type carcinomas and nine precancerous lesions) with a predominant gastric foveolar phenotype, as determined not only by mucin histochemistry and immunohistochemistry but also by morphology, were analysed for mutations of the E-cadherin gene using the nested polymerase chain reaction single-strand conformation polymorphism method. E-cadherin gene mutation was detected in 4 of 19 lesions (21 per cent) (three precancerous and one cancerous). Sequencing revealed an 18 bp deletion corresponding to codons 418-423 of exon 9 common to all four lesions. This mutation was previously reported in an advanced carcinoma of undifferentiated type. The results suggest that these tumours progress to undifferentiated-type carcinomas through loss of E-cadherin function.

Adenocarcinoma↗

PubMed1997

Identification of a gene within the tandem array of red and green color pigment genes.

The tandem array of color pigment genes on chromosome Xq28 contains nested exons of a distinct gene. This gene (termed TEX28) is composed of five exons that span almost the entire distance between the protein-coding regions of the color pigment genes and a transketolase-related gene. Although most of the TEX28 gene is repeated within the color pigment gene array, the exclusion of exon 1 from the array is predicted to restrict transcription to a single copy of the gene. The TEX28 gene encodes a polypeptide of 410 amino acid residues. This polypeptide does not display significant homology with any known proteins in public databases. Transcripts of the gene (1.8 kb) were detected in testes, but not in any other tissue examined. Color vision disorders that result from the deletion of color pigment genes should be reappraised for associated phenotypes that may derive from disruption of the TEX28 gene.

Amino Acid Sequence↗

PubMed1991

Activation of tyrosinase kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins.

Chronic myelogenous leukemia and one type of acute lymphoblastic leukemia are characterized by a 9;22 chronosome translocation in which 5' sequences of the bcr gene become fused to the c-abl proto-oncogene. The resulting chimeric genes encode bcr/abl fusion proteins which have deregulated tyrosine kinase activity and appear to play an important role in induction of these leukemias. A series of bcr/abl genes were constructed in which nested deletions of the bcr gene were fused to the c-abl gene. The fusion proteins encoded by these genes were assayed for autophosphorylation in vivo and for differences in subcellular localization. Our results demonstrate that bcr sequences activate two functions of c-abl; the tyrosine kinase activity and a previously undescribed microfilament-binding function. Two regions of bcr which activate these functions to different degrees have been mapped: amino acids 1 to 63 were strongly activating and amino acids 64 to 509 were weakly activating. The tyrosine kinase and microfilament-binding functions were not interdependent, as a kinase defective bcr/abl mutant still associated with actin filaments and a bcr/abl mutant lacking actin association still had deregulated kinase activity. Modification of actin filament functions by the bcr/abl tyrosine kinase may be an important event in leukemogenesis.

Actin Cytoskeleton↗

PubMed2006

No evidence of persisting measles virus in peripheral blood mononuclear cells from children with autism spectrum disorder.

OBJECTIVES: Despite epidemiologic evidence to the contrary, claims of an association between measles-mumps-rubella vaccination and the development of autism have persisted. Such claims are based primarily on the identification of measles virus nucleic acids in tissues and body fluids by polymerase chain reaction. We sought to determine whether measles virus nucleic acids persist in children with autism spectrum disorder compared with control children. PATIENTS AND METHODS: Peripheral blood mononuclear cells were isolated from 54 children with autism spectrum disorder and 34 developmentally normal children, and up to 4 real-time polymerase chain reaction assays and 2 nested polymerase chain reaction assays were performed. These assays targeted the nucleoprotein, fusion, and hemagglutinin genes of measles virus using previously published primer pairs with detection by SYBR green I. Our own real-time assay targeted the fusion gene using novel primers and an internal fluorescent probe. Positive reactions were evaluated rigorously, and amplicons were sequenced. Finally, anti-measles antibody titers were measured by enzyme immunoassay. RESULTS: The real-time assays based on previously published primers gave rise to a large number of positive reactions in both autism spectrum disorder and control samples. Almost all of the positive reactions in these assays were eliminated by evaluation of melting curves and amplicon band size. The amplicons for the remaining positive reactions were cloned and sequenced. No sample from either autism spectrum disorder or control groups was found to contain nucleic acids from any measles virus gene. In the nested polymerase chain reaction and in-house assays, none of the samples yielded positive results. Furthermore, there was no difference in anti-measles antibody titers between the autism and control groups. INTERPRETATION: There is no evidence of measles virus persistence in the peripheral blood mononuclear cells of children with autism spectrum disorder.

Antibodies, Viral↗

PubMed2002

PCR detection of Mycobacterium paratuberculosis in Crohn's disease granulomas isolated by laser capture microdissection.

BACKGROUND AND AIMS: The uncertainty surrounding the role of Mycobacterium avium subsp paratuberculosis (Map) in Crohn's disease has been compounded by possible contamination from Map present in the lumen microflora. This study used laser capture microdissection (LCM) and polymerase chain reaction (PCR) to detect Map DNA in subepithelial granulomas, isolated from 15 surgically resected, formalin fixed specimens of granulomatous Crohn's disease and from 12 granulomatous disease controls (10 bowel, 2 non-bowel). METHODS: The effect of amplicon size on reliability of PCR from formalin fixed samples was examined by amplifying 435 bp and 133 bp sequences of the human APC gene. After this, nested primers were designed to detect a small fragment (155 bp) of the Map specific IS900 gene in Crohn's granulomas. LCM isolated granulomas from Map culture positive bovine intestine was used as positive control. PCR product specificity was confirmed by direct DNA sequencing. RESULTS: The smaller, but not the larger, fragment of the APC gene amplified reliably in all samples. Amplification of the 155 bp fragment of the IS900 gene detected Map DNA in microdissected Crohn's granulomas in 6 of 15 cases, and in 0 of 12 disease control granulomas. CONCLUSIONS: LCM can be used to detect Map DNA in granulomas in a proportion of patients with Crohn's disease. However, formalin fixation requires that comparatively short DNA fragments of the Map specific IS900 gene be targeted, to permit consistent detection. Detection of Map DNA within granulomas might suggest an infectious aetiology in a subset of patients; alternatively, a transmissible agent may not be involved but mycobacterial DNA may influence pathogenesis by modifying the local cytokine responses.

Case-Control Studies↗

PubMed2002

The detection of the meq gene in chicken infected with Marek's disease virus serotype 1.

In the genome of strains of very virulent Marek's disease virus serotype 1(vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF, termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CV1988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.

Animals↗

PubMed2000

Detection of signature sequences in overlapping genes and prediction of a novel overlapping gene in hepatitis G virus.

In viruses an increased coding ability is provided by overlapping genes, in which two alternative open reading frames (ORFs) may be translated to yield two distinct proteins. The identification of signature sequences in overlapping genes is a topic of particular interest, since additional out-of-frame coding regions can be nested within known genes. In this work, a novel feature peculiar to overlapping coding regions is presented. It was detected by analysis of a sample set of 21 virus genomic sequences and consisted in the repeated occurrence of a cluster of basic amino acid residues, encoded by a frame, combined to a stretch of acidic residues, encoded by the corresponding overlapping frame. A computer scan of an additional set of virus sequences demonstrated that this feature is common to several other known overlapping ORFs and led to prediction of a novel overlapping gene in hepatitis G virus (HGV). The occurrence of a bifunctional coding region in HGV was also supported by its extremely lower rate of synonymous nucleotide substitutions compared to that observed in the other gene regions of the HGV genome. Analysis of the amino acid sequence that was deduced from the putative overlapping gene revealed a high content of basic residues and the presence of a nuclear targeting signal; these characteristics suggest that a core-like protein may be expressed by this novel ORF.

Algorithms↗

PubMed1998

Diagnosis of kala-azar by nested PCR based on amplification of the Leishmania mini-exon gene.

To diagnose visceral leishmaniasis (kala-azar), we have developed a nested PCR method based on amplification of the mini-exon gene, which is unique and tandomly repeated in the Leishmania genome. Nested PCR was sufficiently sensitive for the detection of DNA in an amount equivalent to a single Leishmania parasite or less. We examined the usefulness of this PCR method using bone marrow aspirates and buffy coat cells collected from kala-azar patients who had or had not received chemotherapy in northwest China. We obtained PCR positivity for all of the parasitologically positive bone marrow samples from the patients. Some ambiguities with the primary PCR results were eliminated by the subsequent nested PCR. The buffy coat samples from 7 of 12 patients with splenomegaly were positive by the nested PCR, although only 2 of them were positive for parasites by culture. However, buffy coat samples from nine children, whose splenomegaly has been reduced and clinically cured by antimony treatment, were all negative. Thus, this nested PCR method represents a new tool for the diagnosis of kala-azar with patient blood samples instead of bone marrow or spleen aspirates obtained by more invasive procedures.

Adolescent↗

PubMed2005

A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities.

The analysis of soil fungal communities by molecular fingerprinting and subsequent identification of the underlying populations require the amplification of a phylogenetically informative gene fragment. In this study we tested the reliability and suitability of the previously published fungal primer combination (NS1/FR1-GC) that amplifies almost the entire 18S rRNA gene for the DGGE analysis of fungal communities in soil samples from 36 sites. This direct PCR system failed to amplify the fragment of interest from the total DNA extracted from most of the soils tested. Thus, we developed a new semi-nested PCR system based on the initial amplification of over 1,700 bp of the 18S rRNA gene with a new primer combination, followed by a subsequent amplification with NS1/FR1-GC. By means of the PCR approach developed in this study distinct 18S rRNA gene amplicons could be reproducibly generated for all soil samples. Amplification tests with 101 soil fungal isolates showed that with the new semi-nested system 18S rRNA gene fragments could be obtained from more fungi than with the direct approach. The subsequent DGGE separation of community amplicons resulted in a high resolution and revealed reproducible complex soil fungal communities specific for each site, despite a minor variability between replicates of the same sample. The semi-nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a robust, reliable and sensitive tool for the analysis of soil fungal community structure.

Cluster Analysis↗

PubMed1997

Absence of Helicobacter pylori DNA in salivary lymphoepithelial lesions.

Helicobacter pylori is a common cause of chronic gastritis and has been implicated as the main agent responsible for the development of lymphomas of mucosa associated lymphoid tissue (MALT) in the stomach. An uncommon cause of salivary gland swelling is salivary lymphoepithelial lesion (SLEL), which shows histological features of acquired MALT and is associated with the development of MALT-type lymphomas. Since H. pylori has been identified in the oral cavity, we hypothesised that this organism might act as a potential antigen for the development of MALT in salivary glands. Routinely processed biopsies of 20 SLEL were screened for H. pylori DNA using a sensitive two-stage PCR technique to amplify the 16S ribosomal RNA gene. Immunoglobulin heavy chain gene monoclonality was determined by amplifying the VDJ gene using a nested PCR technique. All SLEL had histological features of organised MALT and 14 cases showed Ig heavy chain gene monoclonality consistent with MALT lymphoma. None of the SLEL contained H. pylori DNA. In contrast to the putative role of H. pylori as an antigenic stimulus in gastric MALT lymphomas, it appears not to play a role locally in the development of MALT or MALT lymphomas of the salivary gland.

Adult↗

PubMed2006

Association of adiponectin gene variations with risk of incident myocardial infarction and ischemic stroke: a nested case-control study.

BACKGROUND: Adiponectin (ADIPOQ) gene variations are associated with risk of cardiovascular disease in patients with diabetes. No prospective data are available, however, on the risk of atherothrombotic disorders in persons with ADIPOQ variations who do not have diabetes. METHODS: From a group of DNA samples collected at baseline in a prospective cohort of 14 916 initially healthy American men, we assessed the presence of 5 ADIPOQ genetic variants (rs266729, rs182052, rs822396, rs2241766, and rs1501299) in samples from 600 Caucasian men who subsequently suffered an atherothrombotic event (incident myocardial infarction or ischemic stroke) and from 600 age- and smoking-matched Caucasian men who remained free of reported vascular disease during follow-up (controls). RESULTS: Genotype distributions for the variations tested were in Hardy-Weinberg equilibrium. Marker-by-marker conditional logistic regression analysis, adjusted for potential risk factors, showed an association of rs266729 [recessive: odds ratio (OR), 0.26; 95% confidence interval (CI), 0.10-0.64; P=0.004] and rs182052 (recessive: OR, 0.40; 95% CI, 0.21-0.76; P=0.006) with decreased risk of ischemic stroke. These findings remained significant after Bonferroni correction. Haplotype-based (constituted by rs266729, rs182052, and rs822396) conditional logistic regression analysis, adjusted for the same potential risk factors, showed an association of haplotype G-A-G (OR, 0.28; 95% CI, 0.09-0.87; P=0.03) with decreased risk of ischemic stroke. Prespecified analysis limited to participants without baseline diabetes showed similar significant findings. CONCLUSIONS: The present prospective investigation provides further evidence for a protective role of adiponectin gene variation in the risk of ischemic stroke that was independent of the presence of diabetes.

Adiponectin↗

PubMed1998

Detection of Mycoplasma hyopneumoniae by air sampling with a nested PCR assay.

This article describes the first successful detection of airborne Mycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 micron) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 10(4) times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and a lower air humidity.

Air Microbiology↗

PubMed2001

Promoter activity of the rat connexin 43 gene in NRK cells.

Cellular communication mediated by gap junctions plays a major role in organ function. Gap junction channels are formed by the organization of polypeptide subunits, termed connexins (Cx), on the cell surface of adjacent cells. One mechanism to regulate gap-junctional communication is by change in Cx expression. In the present study, the promoter region of the rat Cx43 gene was characterized. Nested deletions of the 5' flanking region of the first Cx43 exon were coupled to the human growth hormone gene and transfected into normal rat kidney (NRK) cells, that express this gene constitutively. The minimal region of the Cx43 gene that showed maximal promoter activity was localized within 110 bp upstream of the transcriptional initiation site. One particular subregion that contains a Sp-1 binding site (located within 98--93 bp from the transcriptional initiation site) was found to sustain Cx43 promoter activity to the same extent as that of the 110 bp promoter region. Mutations of this Sp-1 binding site abolished transcriptional activity and DNA-protein interactions. These observations suggest that the Sp-1 binding site plays a major role in the basal transcriptional activity of Cx43 gene in NRK cells.

Animals↗

PubMed2005

Detection and discrimination of Cryptosporidium parvum and C. hominis in water samples by immunomagnetic separation-PCR.

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.

Animals↗

PubMed2004

Complex nested promoters control tissue-specific expression of acetyl-CoA carboxylase genes in wheat.

Cis-acting regulatory elements of the wheat acetyl-CoA carboxylase (ACC) gene family were identified by comparing the promoter activity of 5' end gene fragments fused to a reporter gene in two transient expression systems: wheat protoplasts and epidermal cells of mature embryos. Expression of the plastid and the cytosolic ACC genes is each driven by two nested promoters responsible for the synthesis of two transcript types. The internal promoter is located in an intron removed from transcripts originating at the first promoter. These complex promoters, which are different for the cytosolic and plastid ACC genes, control tissue-specific expression of the enzymatic activity supplying cytosolic, plastid, and mitochondrial pools of malonyl-CoA. The activity of one such complex promoter, driving expression of one of the cytosolic ACC genes, was studied throughout development of transgenic wheat plants carrying a full-length promoter-reporter gene fusion. High activity of the promoter was detected in the coleoptile, in the upper sheath section of the leaf, on the top surface of the ovary, in some sections of the main veins in the lemma and glume, and in abaxial epidermis hair cells of the lemma, glume, and rachis. The findings are consistent with the developmental and environmental requirements for very-long-chain fatty acids and flavonoids, whose synthesis begins with the ACC reaction in the cytosol of these specific cell types.

Acetyl-CoA Carboxylase↗

PubMed1992

Novel DNA binding proteins participate in the regulation of human neurofilament H gene expression.

By a combination of DNase I footprinting, methylation interference, and gel shift analyses we have identified multiple binding sites for nuclear proteins within the promoter region of the human neurofilament H gene. Two sites likely bind the transcription factor Sp1 while two others may be targets for previously unrecognized DNA binding proteins. One site, PAL, occurs within the 10 bp sequence GGGGAGGAGG. Two copies of the PAL sequence form an interrupted palindrome around one of the Sp1 sites. A second site, PROX, is found within the sequence GGTTGGACC. Nuclear extracts prepared from both neural and non-neural cell lines, mouse brain, and mouse liver contain proteins that recognize and bind to the PROX and PAL sequences indicating that proteins which bind to these target sequences are widespread. The appearance of these target sequences in the 5' upstream region of several neuron specific genes suggests that they play key roles in the transcription of neuron specific genes. The functional activity of these target DNA sequences was demonstrated by transfection assays using a reporter gene fused to nested deletions of the NF(H) promoter region. Interestingly, these assays revealed that maximal transient expression was obtained with DNA fusion genes containing the PAL, PROX and TATA sequences. Inclusion of the Sp1 sites into the fusion genes failed to enhance the expression of the reporter gene. To determine if the NF(H) promoter can be activated in a tissue specific manner during development transgenic mice containing the promoter region linked to a beta-galactosidase reporter gene were generated. In one line sporadic expression of the transgene occurred in the CNS and testis while in four other lines no expression occurred. Collectively these results suggest that the NF(H) gene promoter is active in a tissue specific manner only by interactions with regulatory elements that lie further upstream or downstream of the start site of initiation.

Animals↗

PubMed2003

Molecular detection of von Hippel-Lindau gene mutations in urine and lymph node samples in patients with renal cell carcinoma: potential biomarkers for early diagnosis and postoperative metastatic status.

PURPOSE: Organ confined renal cell carcinoma can be cured in the majority of patients, whereas more extensive lesions have a poor prognosis. Therefore, the development of a useful biomarker for early diagnosis as well as postoperative metastatic status would contribute to the appropriate therapy for renal cell carcinoma. To diagnose renal cell carcinoma preoperatively we developed a novel urinary test and detected occult lymph node micrometastasis using a molecular approach. MATERIALS AND METHODS: Urine samples were obtained preoperatively from 27 patients with renal cell carcinoma and von Hippel-Lindau (VHL) gene mutations in the tumors, and were analyzed for VHL gene mutations using a nested single strand conformational polymorphism analysis. Lymph nodes without evidence of histological metastasis were obtained from 15 patients with renal cell carcinoma and VHL gene mutations, and analyzed for VHL gene mutations using mutation specific nested reverse transcription polymerase chain reaction method. RESULTS: In urine samples 5 of 27 VHL gene mutations (18.5%) were found and each mutation pattern was the same as that detected in each renal cell carcinoma. One lymph node micrometastasis was found. CONCLUSIONS: These data indicate the presence of detectable levels of tumor derived DNA in the urine of patients with renal cell carcinoma and suggest that nested single strand conformational polymorphism analysis of VHL gene of urine samples provides a possible tool for the early detection of renal cell carcinoma. Furthermore, mutation specific nested reverse transcription polymerase chain reaction is useful to detect occult lymph node micrometastasis and may predict patients at risk for local recurrence. These 2 combined approaches using VHL gene mutations may contribute to the total therapy for and prognosis of renal cell carcinoma.

Biomarkers, Tumor↗

PubMed1999

Evaluation of a nested reverse transcription-PCR assay based on the nucleoprotein gene for diagnosis of spontaneous and experimental bovine respiratory syncytial virus infections.

The first nested reverse transcription (RT)-PCR based on the nucleoprotein gene (n RT-PCR-N) of the bovine respiratory syncytial virus (BRSV) has been developed and optimized for the detection of BRSV in bronchoalveolar lavage fluid cells of calves. This test is characterized by a low threshold of detection (0.17 PFU/ml), which is 506 times lower than that obtained by an enzyme immunosorbent assay (EIA) test (RSV TESTPACK ABBOTT). During an experimental infection of 17 immunocompetent calves less than 3 months old, BRSV RNA could be detected up to 13 days after the onset of symptoms whereas isolation in cell culture was possible only up to 5 days. Compiling results obtained by conventional techniques (serology, antigen detection, and culture isolation) for 132 field samples collected from calves with acute respiratory signs revealed that n RT-PCR-N showed the highest diagnostic sensitivity and very good specificity. This n RT-PCR-N with its long period of detection during BRSV infection thus provides a valuable tool for diagnostic and epidemiological purposes.

Animals↗