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PubMed2005

Analysis and frequency of bovine lymphocyte antigen (BoLA-DRB3) alleles in Iranian Holstein cattle.

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune response by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. DRB3 gene has been extensively evaluated as a candidate marker for association with various bovine diseases and immunological traits. This study describes genetic variability in the BoLA-DRB3 in Iranian Holstein cattle. This is the first study of the DNA polymorphism of the BoLA-DRB3 gene in Iranian Holstein cattle. Hemi-nested PCR-RFLP method is used for identification the frequency of BoLA-DRB3 alleles. The BoLA-DRB3 locus is highly polymorphic in the studied herd (26 alleles). Almost 67% of the alleles were accounted for four alleles (BoLA-DRB3.2*8, *24, *11 and *16) in Iranian Holstein cattle. The DRB3.2*8 allele frequency (26.6%) was higher than the others. The frequencies of the DRB3.2*54, *37, *36, *28, *25, *14, *13, *10, *1 alleles were lower than 1%. Significant distinctions have been found between Iranian Holstein cattle and other cattle breeds studied. In Iranian Holstein cattle the alleles (BoLA-DRB3.2*22, *2 and *16) associated with a lower risk of cystic ovarian disease in Holstein cattle are found. The alleles associated with the resistance to mastitis and to bovine leukemia virus infection BoLA-DRB3.2*11 and *23 are detected with the frequencies 10.4% and 4.4%, respectively. Thus in the Iranian Holstein cows studied are found alleles which are associated with resistance to various diseases. The method of DNA-typing of animals can be used in agricultural practice for BoLA-DRB3 allele genotyping of cattle in order to reduce spreading of alleles providing susceptibility to mastitis or leukemia in cattle herds.

Animals↗

PubMed2000

Retrotransposable L1 elements expressed in rheumatoid arthritis synovial tissue: association with genomic DNA hypomethylation and influence on gene expression.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by a progressive destruction of joints by invasive synovial fibroblasts (SF). We searched for retroviral sequences in RA synovial fluid pellets, identified a sequence similar to that of open reading frame 2 (ORF2)/L1 retrotransposable elements, explored the expression of L1 in RA synovial tissues and cultured RA SF, and investigated the link to genomic DNA hypomethylation and the influence of functional L1 on gene expression. METHODS: RA synovial fluid pellets were screened by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerated pol primers. The sequences were identified by GenBank search. Riboprobes to ORF2/L1 and galectin-3 and antibodies to the ORF1/L1-related p40 protein were used for in situ hybridization and immunohistochemistry of synovial tissues and cultured RA SF. Real-time quantitative RT-PCR was used for detecting ORF1 messenger RNA (mRNA). Since DNA hypomethylation occurs in inflammatory diseases, we incubated cells with the methylation inhibitor 5-aza-2'-deoxycytidine (5-azaC) and compared RA SF and osteoarthritis (OA) SF. L1-negative RA SF were transfected with the functional L1.2 construct, and differential gene expression was analyzed by subtractive hybridization combined with nested PCR. RESULTS: RNA sequences similar to those of ORF2/L1 retrotransposable elements, THE1 transposon, human endogenous retrovirus (ERV)-E, human ERV-HC2, and gibbon ape leukemia virus pol genes were isolated from different RA synovial fluid pellets. In RA synovial tissues, ORF2/L1 transcripts were detected in the sublining layer and at sites of cartilage and bone destruction. Galectin-3 mRNA and L1-related ORF1/ p40 protein showed similar expression patterns. In contrast, OA synovial tissues in situ and cultures in vitro were negative. Real-time quantitative RT-PCR confirmed the presence of ORF1 mRNA in cultured RA SF (30-300-fold the amount in normal SF), demonstrating the existence of a nondegenerated and functional L1 element. In vitro, the majority of RA SF expressed ORF2/L1 mRNA. After incubation of SF with 5-azaC, L1 mRNA appeared in a time- and dose-dependent manner. Compared with OA SF, RA SF were more sensitive to 5-azaC. After transfection of RA SF with a functional L1.2 element, human stress-activated protein kinase 2 delta (SAPK2delta [or SAPK4]), met protooncogene, and galectin-3 binding protein genes were differentially expressed. The transcription of the SAPK2delta gene, favored also by DNA hypomethylation in vitro, was confirmed in RA synovial tissues. CONCLUSION: Taken together, these data suggest that L1 elements and SAPK2delta pathways play a role in the activation of RA SF.

Antigens, Differentiation↗

PubMed1995

Nested polymerase chain reaction study of 53 cases with Turner's syndrome: is cytogenetically undetected Y mosaicism common?

Turner's syndrome patients with Y mosaicism face a high risk of developing gonadoblastoma. Cytogenetic analysis can fail to detect rare cells bearing a normal or structurally abnormal Y chromosome (low level Y mosaicism). We screened 53 individuals with Turner's syndrome for presence of sex-determining region Y (SRY), the testis-specific protein, Y encoded, gene, and the Y centromeric DYZ3 repeat using nested polymerase chain reaction (PCR). Thirty girls (57%) had the 45,X karyotype, determined through standard analysis of blood lymphocytes. The remaining 23 girls (43%) were mosaics and/or had structural abnormalities in 1 X-chromosome. Genomic DNA from blood leukocytes was amplified using 2 rounds of PCR. This method was sensitive enough to detect 0.0001% male DNA on a female background. None of 53 Turner's syndrome cases was positive for Y-specific loci after the first round of PCR. After the second round, 2 of 53 Turner's syndrome cases were positive for SRY mapping to the distal short arm of chromosome Y. In 1 SRY-positive subject, the karyotype was 45,X, and in the other, it was 46,Xi(Xq). None of 53 Turner's syndrome individuals, including the 2 SRY-positive subjects, were positive for the testis-specific protein, Y encoded, gene on the proximal short arm of chromosome Y or the centromeric DYZ3 repeat. These data exclude low level Y mosaicism in almost all Turner's syndrome cases tested.

Base Sequence↗

PubMed2002

A new strategy for the diagnosis of MAGE-expressing cancers.

The expression of melanoma antigen gene (MAGE), coding for tumor antigens recognized by cytotoxic T cell, is highly specific to cancer cells, but their use in the detection of a few cancer cells by reverse transcription-polymerase chain reaction (RT-PCR) has been limited by the low frequency of expression of individual MAGE genes. In order to increase MAGE detection rate in RT-PCR assay, here, we designed multiple MAGEs recognizing primers (MMRPs) that can bind to the sequences of cDNA of MAGE-1, -2, -3, -4a, -4b, -5a, -5b and-6 (MAGE 1-6) together. The nested RT-PCR assay using MMRPs, MAGE 1-6 assay, detected MAGE messages of 1 to 5 SNU484 cells in a background of 10(7) SNU638 cells. MAGE detection rate of MAGE 1-6 assay in cancers was higher than that of nested RT-PCR that detects single MAGE gene expression. The expressions of MAGE genes was detected by MAGE 1-6 assay in 70.4% (19/27) of head and neck cancer tissues, 91.7% (11/12) of breast cancer tissues, 75% (9/12) of lung cancer tissues. However, they were not detected in 18 benign lesions and 20 normal head and neck tissues and 30 blood samples from healthy donor. In conclusions, MAGE 1-6 assay can detect any cancer cells that express at least one of eight MAGE subtype genes, and this method may be very useful for the diagnosis of MAGE-expressing cancers.

Antigens, Neoplasm↗

PubMed2003

[Hepatitis C virus nonstructural 5A protein inhibits tumor necrosis factor alpha mediated apoptosis of HepG2 cells].

OBJECTIVE: To study the suppressive effect of HCV nonstructural 5A (NS5A) protein on tumor necrosis factor alpha (TNF alpha) mediated apoptosis of HepG2 cells. METHODS: NS5A gene fragment was amplified by reverse transcription and nested polymerase chain reaction from serum samples positive for anti-HCV. The PCR product was directly cloned using TA cloning kit, and 2 independent clones were isolated, digested and sequenced. Then we constructed HCV NS5A expression plasmid (pcDNA3.1-NS5A), stably transfected into HepG2 cells with lipofectamine. Successful transfection of NS5A gene and expression of NS5A protein were confirmed by Western blot. Transfected cells were incubated with TNFalpha for 48 h, then labeled with Annexin V and visualized by fluorescence microscopy. To examine the effects of NS5A protein on the apoptotic signaling pathway, caspase-3 cleavages and release of cytochrome C were investigated in the transfectant treated with TNF alpha for 48 h and the cell cytosol was subjected to SDS-PAGE and Western blot analysis. RESULTS: The stable transfectant of HepG2 cells lines for HCV NS5A protein expression was achieved. The NS5A protein blocked the activation of caspase-3 and the release of cytochrome C in the TNF alpha treated cells. CONCLUSION: HCV NS5A protein inhibits TNF alpha mediated apoptosis of HepG2 cells in vitro.

Apoptosis↗

PubMed1985

DNA sequence of a 3.8 kilobase pair region controlling Drosophila chorion gene amplification.

During Drosophila oogenesis, two clusters of chorion genes and their flanking DNA sequences undergo amplification in the ovarian follicle cells. Amplification results from repeated rounds of initiation and bidirectional replication within the chorion gene regions, possibly from a single origin, producing nested replication forks. Previously we have shown that following reintroduction into the Drosophila genome, a specific 3.8 kilobase pair DNA segment from the amplified third chromosome domain could induce developmentally regulated amplification at its site of insertion. Here we present the complete nucleotide sequence of this "amplification control element" and of genes encoding the chorion structural proteins s18-1 and s15-1, which are contained within it. Sequences that may be involved in the regulation of chorion gene amplification and expression are identified.

Animals↗

PubMed1993

A distal activation domain is critical in the regulation of the rat androgen receptor gene promoter.

The far upstream region of the rat androgen receptor (AR) gene has been cloned, and the nucleotide sequence up to -2656 bp established. Nested deletion mutants of rat AR 5' flanking sequences were ligated to the luciferase reporter gene, and their promoter activities were examined in transfected COS1 cells. Results show a critical cis-acting domain located between positions -960 and -940. Deletion of this cis element resulted in a greater than 90% decrease in the promoter activity. A nuclear protein that specifically binds to this 21-nucleotide sequence was identified by gel mobility shift analysis. The -960/-940 cis element has no identify to the binding sequence of any known transcription factor. Furthermore, the cognate binding protein is present in both rat and human (HeLa) cell nuclear extracts. We conclude that a novel trans-activator interacting at the -960/-940 region plays a critical role in the regulation of AR gene expression.

Animals↗

PubMed2004

Targeted disruption of the 3p12 gene, Dutt1/Robo1, predisposes mice to lung adenocarcinomas and lymphomas with methylation of the gene promoter.

The DUTT1 gene is located on human chromosome 3, band p12, within a region of nested homozygous deletions in breast and lung tumors. It is therefore a candidate tumor suppressor gene in humans and is the homologue (ROBO1) of the Drosophila axonal guidance receptor gene, Roundabout. We have shown previously that mice with a targeted homozygous deletion within the Dutt1/Robo1 gene generally die at birth due to incomplete lung development: survivors die within the first year of life with epithelial bronchial hyperplasia as a common feature. Because Dutt1/Robo1 heterozygous mice develop normally, we have determined their tumor susceptibility. Mice with a targeted deletion within one Dutt1/Robo1 allele spontaneously develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls: invasive lung adenocarcinomas are by far the predominant carcinoma. In addition to the mutant allele, loss of heterozygosity analysis indicates that these tumors retain the structurally normal allele but with substantial methylation of the gene's promoter. Substantial reduction of Dutt1/Robo1 protein expression in tumors is observed by Western blotting and immunohistochemistry. This suggests that Dutt1/Robo1 is a classic tumor suppressor gene requiring inactivation of both alleles to elicit tumorigenesis in these mice.

Adenocarcinoma↗

PubMed2002

Korean Multi-center Cancer Cohort Study including a Biological Materials Bank (KMCC-I).

Cohort studies of human populations have provided the most convincing evidence of links between exposure to specific agents and cancer. The Korean Multi-center Cancer Cohort (KMCC-II) is a multi-center prospective cohort designed to investigate the relationship between exposures to environmental factors, lifestyle factors, host factors and the risk of cancer in Korea. Data on general lifestyle, physical activity, diet, reproductive factors, and agricultural exposures have been collected by direct interview since 1993. Anthropometric measurements and some clinical laboratory findings have also been recorded. This cohort is characterized by the exclusion of cancer cases at the time of recruitment through cancer screening by physicians. The number of cancer-free subjects in the cohort at present totals some 35,692 men and women aged over 35 (121,856 person-years as of December 2000). Of the KMCC-II, 11,045 subjects who have donated their blood and/or urine sample were classified as the KMCC-I. In order to provide an opportunity to incorporate various biomarkers of exposure and the effect of exposure, as well as information on genetic susceptibility, a biological materials bank with blood (plasma, or serum buffy coat, packed erythrocytes) at -70oC and urine supernatants at -20oC has been established for future studies on cancer etiology. The total number of the KMCC-I is 10,694 for the blood bank and 8,907 for the urine bank (total number of observation in person-year = 47,002). Follow-up for cancer incidence has commenced based on an active surveillance system conducted mainly through telephone interview by health personnel or through diagnosis by physicians in each district, and a passive surveillance system through record linkages between the national cancer registry, the national death certificate system, and health insurance medical records databases in Korea. Moreover, this cohort study is expected to play a significant role in assessing the role of genes in the etiology of cancer. These include the determination of the cost-efficiency and reliability of the nested case-control approach, using the cohort, for exploring gene-disease associations and gene-environmental interactions using reduced sample sizes. Along with other cancer cohorts, the KMCC study could eventually provide convincing evidence on new etiologies of cancer in the Asian-Pacific region.

Journal Article↗

PubMed1996

Genotype determination of hepatitis C virus from northern India: identification of a new subtype.

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the non-structural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5'-noncoding (5' NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India.

Amino Acid Sequence↗

PubMed2000

Further evidence of the absence of measles virus genome sequence in full thickness intestinal specimens from patients with Crohn's disease.

Specimens of macroscopically inflamed and normal intestine along with mesenteric lymph nodes were obtained at resection from patients with Crohn's disease. The samples were systematically examined by RT-PCR-nested PCR targeting N, M and H gene regions of the measles virus genome. None of the samples examined gave any evidence of the persistence of measles virus in the intestine of Crohn's disease patients. The study supports previous findings produced by this laboratory and others using highly sensitive measles virus specific PCR diagnostic technology.

Adult↗

PubMed1993

Investigation of bcr-abl transcription by Ph-positive chronic myeloid leukemia progenitors.

It is now feasible to investigate bcr-abl transcription by the progeny of Ph-positive (Ph+) early and committed hemopoietic progenitor cells from patients with chronic myeloid leukemia (CML). Cells from individual colonies can be bisected and each half analyzed by cytogenetics or the reverse transcriptase-polymerase chain reaction (RT-PCR) method to detect the bcr-abl transcript using internal nested oligonucleotide primers that flank the chimeric gene junction. We previously showed that some Ph+ colonies have undetectable PCR products for bcr-abl. When colonies are generated in the presence of alpha interferon (IFN-alpha) bcr-abl transcripts are undetectable in the majority of Ph+ colonies. These data suggest a potential mechanism for the action of IFN-alpha in Ph+ CML and indicate the need for a combined approach with cytogenetics and RT-PCR in analyzing the bcr-abl gene.

DNA, Neoplasm↗

PubMed1998

Molecules, morphology, fossils, and the relationship of angiosperms and Gnetales.

Morphological analyses of seed plant phylogeny agree that Gnetales are the closest living relatives of angiosperms, but some studies indicate that both groups are monophyletic, while others indicate that angiosperms are nested within Gnetales. Molecular analyses of several genes agree that both groups are monophyletic, but differ on whether they are related. Conflicts among morphological trees depend on the interpretation of certain characters; when these are analyzed critically, both groups are found to be monophyletic. Conflicts among molecular trees may reflect the rapid Paleozoic radiation of seed plant lines, aggravated by the long branches leading to extant taxa. Trees in which angiosperms are not related to Gnetales conflict more with the stratigraphic record. Even if molecular data resolve the relationships among living seed plant groups, understanding of the origin of angiosperm organs will require integration of fossil taxa, necessarily using morphology.

Evolution, Molecular↗

PubMed1998

Expression of SRY gene transcripts with a longer 3' untranslated sequence in adult human male lymphocytes and tumor cells.

Using 3' RACE PCR and the repeated nested-PCR method, the expression of transcripts of the sex-determining gene SRY was investigated in single lymphocytes from a human adult male and in male tumor cell lines. The gene is functionally transcribed in the early stages of embryogenesis and mRNA is also expressed in adult testes. However, in this study, SRY gene transcripts were also detected in somatic cells of adult male and in tumor cells. Moreover, this mRNA possessed a longer additional untranslated exon. Although expression of the transcripts might not have any functional meaning in these cells, these new findings support the hypothesis that any given human cell can contain illegitimate mRNAs.

Adult↗

PubMed1993

Application of the polymerase chain reaction to the diagnosis of human toxoplasmosis.

Toxoplasmosis may cause significant damage to the developing fetus and is a life-threatening opportunistic infection in immunocompromised persons. Serological investigation is unreliable, while isolation of the parasite is time consuming and may lack sensitivity. We have developed a system for detecting Toxoplasma gondii based on the amplification of the P30 gene using sequential rounds of PCR and nested primers. The clinical value of this technique was assessed by the investigation of a range of tissues taken from pregnant women, fetuses, neonates, AIDS patients and organ graft recipients. The PCR assay produced more positive reactions than isolation of the parasite by means of cell culture or animal inoculation. Extended autoradiography was found to be more sensitive than stained agarose gels for detecting the PCR product. Systematic contamination of PCR reactions was avoided but it was not possible to exclude sporadic contamination in certain cases. Detection of specific DNA is of clinical value in the investigation of the pregnant woman in order to assess the risk of transplacental passage of infection and in the fetus and neonate to identify congenital toxoplasmosis. Even so, PCR findings must be interpreted with caution because of the risk of a sample being contaminated. PCR may be the investigation of choice when brain biopsy is performed on a patient with AIDS and when toxoplasmosis associated with bone marrow transplantation is suspected.

AIDS-Related Opportunistic Infections↗

PubMed1996

Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank.

An epidemiological investigation indicated that six patients treated in a haematology unit who developed acute hepatitis B may have been infected as a result of contamination of a liquid nitrogen bone marrow storage tank. The clinical details are described elsewhere (Tedder et al., 1995); we describe the virological methods used to support the findings. HBV DNA was amplified from sera using a nested PCR with primers for the surface gene, and a region encompassing precore, the 3' end of X, and the 5' end of core. HBV DNA was also extracted from the frozen detritus in the liquid nitrogen storage tank. After equilibration, the aqueous material was filtered, co-precipitated with albumin and polyethylene glycol and the HBV DNA extracted by phenol-chloroform and ethanol precipitation. Direct nucleotide sequence analysis indicated that four patients were infected with HBsAg subtype adw viruses which carried novel amino acid substitutions at codons 145 and 146 of the X gene. HBV DNA extracted from the storage tank detritus contained identical sequences. The samples from two other patients, subtype ayw, did not contain the novel sequence changes in X and had other sequence differences. These findings linked conclusively the four patients as a cluster and the rescue of HBV-DNA sequences from the contaminated storage tank by the method described confirmed this as the common source of infection. Two other HBsAg-positive patients were excluded from the cluster by sequence analysis. Demonstration of infection by this route has implications for the safe storage of bone marrow and other related biological materials.

Acute Disease↗

PubMed1996

Identification of Listeria species by a semi-nested polymerase chain reaction.

Three primers derived from the lap gene were used to distinguish Listeria monocytogenes from L.innocua and other Listeria species. L. monocytogenes and L. innocua yielded a PCR product of 600 and 300 bp, respectively, whereas a typical pattern of three amplimers was observed with L. ivanovii, L. seeligeri and L. welshimeri.

Electrophoresis, Agar Gel↗

PubMed2006

Prevalence of enterotoxigenic Bacteroides fragilis in hospital-acquired diarrhea.

Stool specimens from 152 hospitalized patients with diarrhea were analyzed for the presence of enterotoxigenic Bacteroides fragilis (ETBF) by a nested polymerase chain reaction (PCR) assay. ETBF gene sequences were directly detected in 14/152 (9.21%) stools of patients. The prevalence of ETBF in hospital-acquired diarrhea was statistically significant when compared to a prevalence of 2.3% in control subjects (P = 0.04). B. fragilis was cultured from 19.7% (30/152) patients with diarrhea; 4 of these isolates were enterotoxigenic. To determine whether colonization with B. fragilis is heterogeneous in nature, multiple colonies from 17 individual patients were analyzed for enterotoxin gene sequences and genotyped by arbitrarily primed PCR. Of these 17 patients, 13 harbored multiple strain types suggesting heterogeneity of colonization with both enterotoxigenic and non-enterotoxigenic strains. Identification of ETBF in the stools of 10 patients in the absence of a positive culture is likely due to the noted heterogeneity and suggests that detection of enterotoxin by PCR should be performed directly in the stool. These preliminary data indicate that ETBF may play a role in hospital-acquired diarrhea of unknown origin and suggest the need for further studies.

Bacteroides Infections↗