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PubMed2003

[Detection of bcr/abl fusion gene in chronic myeloid leukemia by fluorescence in situ hybridization and nested reverse transcription polymerase chain reaction].

OBJECTIVE: To detect the bcr/abl fusion gene in chronic myeloid leukemia (CML) for assisting in clinical diagnosis. METHODS: Fluorescence in situ hybridization (FISH) and nested reverse transcription polymerase chain reaction (RT-PCR) were used. RESULTS: Out of 16 CML patients, the results of FISH was not consistent with that of nested RT-PCR in 3 cases. When compared with the results of Northern blot, it was found that nested RT-PCR was more sensitive than FISH, but might give false positive results. Moreover, FISH was the easier method for detecting rarer types of bcr/abl transcripts. For detecting the latter occasions nested RT-PCR have to design several specific primers. CONCLUSION: It could take their advantages of applying FISH and nested RT-PCR at the same time to make the results more accurate and reliable for the diagnosis, prognosis and monitor of minimal residual disease.

Adolescent↗

PubMed1999

Expression of transitional cell-specific genes, uroplakin Ia and II, in bladder cancer: detection of circulating cancer cells in the peripheral blood of metastatic patients.

BACKGROUND: Uroplakins (UP), urothelium-specific transmembrane proteins, are present only in urothelia and may be good candidates as tumor markers specific for transitional cell carcinomas (TCC). We investigated the expression of UP-Ia and UP-II genes in the tissues and peripheral blood of patients with TCC. METHODS: We investigated UP-Ia and UP-II gene expression in tissues from 12 patients with TCC by reverse transcription-polymerase chain reaction (RT-PCR). HT1197, a TCC cell line, was used as an indicated cell line to assess a detection system for the UP-II gene-expressing cancer cells by nested RT-PCR. We also investigated UP-II gene expression in the peripheral blood of 12 other patients with TCC by nested RT-PCR. RESULTS: Prior to the investigation of UP-Ia and UP-II gene expression, a partial nucleotide sequence of human UP-II gene cDNA was determined to prepare the primers for RT-PCR. Uroplakin genes were expressed in both cancerous and non-cancerous urothelia taken from all patients examined by RT-PCR. The detection sensitivity of our assay showed that one cancer cell could be detected in 5 mL peripheral blood. UP-II gene-expression was detected in the peripheral blood from all three patients with metastatic TCC but not from the nine patients with non-metastatic TCC nor the three healthy volunteers. CONCLUSIONS: Uroplakins may be employed as tumor markers for transitional cell cancer, because they are highly conserved and well expressed, not only in non-cancerous cells but also in cancer cells. Furthermore, detection of UP-II gene expression in blood by nested RT-PCR may provide helpful information in the diagnosis and management of TCC.

Adult↗

PubMed1996

Upstream regulatory elements controlling expression of the Entamoeba histolytica lectin.

Entamoeba histolytica genomic organization and putative promoter elements appear to be distinct from both metazoan and better characterized protozoan organisms. The recent development of DNA-mediated transfection for E. histolytica enabled characterization of cis-acting promoter elements required for gene expression. A deletion and replacement analysis was conducted on the promoter of an E. histolytica gene encoding the heavy subunit of the N-acetyl-beta-D-galactosamine-specific adhesin (hgl5). Deletion of the DNA from -1000 bases to -272 bases upstream from the start of transcription of hgl5 did not decrease reporter gene expression. Subsequent nested deletions and 10-bp replacement mutagenesis identified four positive upstream regulatory elements between bases -219 to -200, -189 to -160, -69 to -60, and -49 to -40. A negative upstream regulatory element between bases -89 to -80 was conserved upstream of three other E. histolytica genes. Mutation of the previously unidentified 'GAAC' element conserved within the putative core promoter decreased reporter gene expression by 75%. Site directed mutagenesis of the putative TATA element decreased reporter gene expression by greater than 50%, while mutation of the putative initiator element resulted in a more modest decrease. This analysis suggests that E. histolytica promoters are unlike other protozoan promoters, with AT-rich upstream regulatory elements, a non-consensus TATA element, the "GAAC' element, and an unusual initiator element.

Animals↗

PubMed1993

Localization of seed protein genes on flow-sorted field bean chromosomes.

Chromosomes from reconstructed field bean (Vicia faba L.) karyotypes were flow-sorted and the DNA was used for the physical localization of seed storage and nonstorage (USP) protein genes using PCR with sequence specific primers. The data were confirmed and refined by using DNA of microisolated chromosomes of other karyotypes as the target for PCR. The specificity of the PCR products was proved by restrictase digestion into fragments of predicted length or by reamplification using 'nested' primers. The genes are located within defined regions of chromosome I (USP = unknown seed protein genes), II (vicilin genes, legumin B3 genes), III (legumin B4 genes), IV (pseudogenes psi 1) and V (legumin A genes and pseudogenes psi 1). Except for the pseudogene derived from the sequence of legumin B4 gene, all members of each gene family are located in one chromosome region exclusively. This approach proved to be useful for localizing genes that cannot be mapped genetically (due to the lack of allelic variants) and might be applied to integrate physical and genetic maps.

Base Sequence↗

PubMed1990

Detection of HIV-1 DNA in crude cell lysates of peripheral blood mononuclear cells by the polymerase chain reaction and nonradioactive oligonucleotide probes.

The aim of this study was to detect HIV-1 proviral DNA in lysates of peripheral blood mononuclear cells (PBMCs) by the polymerase chain reaction (PCR) and hybridization with a nonradioactive probe. PBMCs were lysed in 1% Triton X-100. PCR was then carried out using primers complementary to a conserved region of the HIV-1 pol gene. Bracket and nested amplification protocols were used. Products were identified by dot-blot hybridization or agarose gel electrophoresis and Southern hybridization, using an alkaline phosphatase-linked oligonucleotide probe specific for amplified sequences. Colorimetric and chemiluminescent substrates were used. HIV-1 DNA was detected in PBMCs of 57/59 HIV-1-seropositive individuals, 8 of which were positive only following the use of nested primers. Of 12 seropositive samples that were negative by other HIV-1 diagnostic tests (PBMC coculture and serum p24 antigen detection), 11 were positive by PCR. PCR using PBMC lysates is a very sensitive method of detecting HIV-1 proviral sequences. The use of nested primers appears to increase the sensitivity of the procedure.

Base Sequence↗

PubMed1997

[The expression of MLC2-chymase fusion gene in cultured neonatal myocardial cells].

A series of MLC2-chymase fusion genes consisting of nested 5' deletion of the chymase promoter region were constructed and transfected into primary cultured neonatal Wistar rat myocardial cells according to the modified calcium phosphate precipitation method. Total RNA Dot-blot hybridization and analysis of densitometric scanning demonstrated that MLC2 promoter is enough to direct human heart chymase gene transcription in cultured neonatal rat myocardial cells. The highest level of expression was found with the MLC2-chymase construct containing 6 bp of the chymase promoter region. More sequence of the chymase promoter region between MLC2 promoter and the ATG of chymase gene resulted in a subsequent decrease in the expression of human heart chymase gene.

Animals↗

PubMed1991

Detection of Escherichia coli heat-stable enterotoxin genes in pig stool specimens by an immobilized, colorimetric, nested polymerase chain reaction.

A combination of selective enrichment by using immunomagnetic separation of F4 (K88)-positive Escherichia coli and a nested colorimetric polymerase chain reaction (PCR) was used on crude clinical and spiked samples for determination of genes encoding heat-stable enterotoxins (STs) Ia (ST Ia) and Ib (ST Ib). The combination increased the sensitivity of the nested PCR compared with that of application onto crude samples. Dead cells were also enriched by use of this technology, giving results that are not available by traditional cultivation as enrichment before PCR. The second step in the PCR was modified to be able to differentiate between ST Ia and ST Ib genes. The colorimetric PCR was performed in a microtiter format, making it useful for automation in clinical laboratories and for the screening of large numbers of samples.

Animals↗

PubMed1989

Structure, organization, and expression of the rat cardiac myosin light chain-2 gene. Identification of a 250-base pair fragment which confers cardiac-specific expression.

The present study characterized the structure, organization, and expression of the rat cardiac myosin light chain (MLC) -2 gene. The rat cardiac MLC-2 gene has seven exons which display complete conservation with the exon structure of the rat fast twitch skeletal MLC-2 gene. A 250-base pair (bp) sequence of the 5'-flanking region contains CArG motifs and additional cis elements, each greater than 10 bp in length, which were conserved in sequence and relative position with the chick cardiac MLC-2 gene. A series of MLC-2/luciferase fusion genes consisting of nested 5' deletions of the MLC-2 5'-flanking region were constructed and transfected into primary neonatal rat myocardial cells and a non-myocardial cell line (CV-1), demonstrating that this 250 bp of the MLC-2 5'-flanking region was sufficient to confer cardiac specific expression on a luciferase reporter gene. This study suggests the presence of important proximal regulatory sequences in the MLC-2 5'-flanking region which are capable of directing the cardiac specific expression of the rat cardiac myosin light chain-2 gene.

Amino Acid Sequence↗

PubMedDate not supplied

[The determination of the content of the cholera toxin gene in the composition of the DNA from Vibrio cholerae strains by means of the nested polymerase chain reaction].

The possibility of detecting cholera toxin genes in V.cholerae enterotoxigenic strains by the method of "nested" polymerase chain reaction with the use of primers on the DNA area of operon ctx of AB genes. The possibility of the detection of several V.cholerae cells by this method was shown with the use of a series of bacterial lysate dilutions. The newly developed test system for the detection of cholera toxin gene on the basis of the analysis of bacterial lysates of V.cholerae nontoxigenic strains, as well as Escherichia coli toxigenic and nontoxigenic strains, was shown to be highly specific.

Base Sequence↗

PubMed2006

Multiplex-nested RT-PCR to evaluate latent and lytic Epstein Barr virus gene expression.

This paper describes the development of two multiplex-nested RT-PCR devised to evaluate latent/immortalizing (EBNA1, EBNA2, LMP1 and LMP2) and lytic [immediate early (Zebra), early, and late (VCA), respectively] Epstein Barr virus (EBV) transcripts. Subsequently, the assays have been validated evaluating the EBV latent/lytic gene expression in peripheral blood mononuclear cells (PBMC) from immunocompetent subjects (children with primary EBV infection, past EBV infection and no EBV infection) and from immunosuppressed patients (30 asymptomatic renal transplant recipients and 4 liver transplant patients with diagnosed post-transplant lymphoproliferative disorders [PTLD]). Our two multiplex-nested RT-PCR assays provide a reliable, rapid and sensitive system, enabling the simultaneous detection and identification of seven latent/immortalizing and lytic EBV transcripts. These assays could be employed in further investigations in order to evaluate the EBV transcriptional profile in EBV-related diseases both in immunocompetent and immunocompromised hosts.

Adult↗

PubMed2001

Longer and shorter forms of Sendai virus C proteins play different roles in modulating the cellular antiviral response.

The Sendai virus (SeV) C gene codes for a nested set of four C proteins that carry out several functions, including the modulation of viral RNA synthesis and countering of the cellular antiviral response. Using mutant C genes (and in particular a C gene with a deletion of six amino acids present only in the larger pair of C proteins) and recombinant SeV carrying these mutant C genes, we find that the nested set of C proteins carry out a nested set of functions. All of the C proteins interdict interferon (IFN) signaling to IFN-stimulated genes (ISGs) and prevent pY701-Stat1 formation. However, only the larger C proteins can induce STAT1 instability, prevent IFN from inducing an antiviral state, or prevent programmed cell death. Remarkably, interdiction of IFN signaling to ISGs and the absence of pY701-Stat1 formation did not prevent IFN-alpha from inducing an anti-Vesicular stomatitis virus (VSV) state. It is possible that IFN-alpha signaling to induce an anti-VSV state can occur independently of the well-established Jak/Stat/ISGF3 pathway and that it is this parallel pathway that is targeted by the longer C proteins.

Amino Acid Sequence↗

PubMed2000

Classical swine fever virus: a second ring test to evaluate RT-PCR detection methods.

Six laboratories participated in a study to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 25 samples of random primed cDNA, synthesised from viral RNA representative of different pestiviruses. The other set comprised samples of blood and serum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols that specified the exact conditions and requirements for inclusion of control samples. Two types of test were evaluated. One amplified a part of the 5'-non coding region of the pestivirus genome by means of a closed, one-tube RT-nested PCR. The other amplified a part of the NS5B gene using non-nested RT-PCR. The results of the laboratories were compared with one another, and with those obtained earlier when similar samples were tested by the same laboratories using non-standardised methods [Paton et al., Classical swine fever virus: a ring test to evaluate RT-PCR detection methods, Vet. Microbiol., in press]. Standardisation of the protocols resulted in a more consistent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test specificity was inadequate from the results obtained with the control samples. Minimum requirements for the inclusion of adequate controls and periodic proficiency testing are proposed.

Animals↗

PubMed2005

Putative association between a new polymorphism in exon 3 (Arg109Cys) of the pancreatic colipase gene and type 2 diabetes mellitus in two independent Caucasian study populations.

The protein encoded by the pancreatic colipase (CLPS) gene is an essential cofactor needed by pancreatic triglyceride lipase (PNLIP) for efficient dietary lipid hydrolysis. Since the inhibition of lipase activity was shown to reduce the incidence of type 2 diabetes mellitus, we tested the hypothesis that genetic variations in the CLPS and PNLIP genes are associated with type 2 diabetes; 47 unrelated subjects were screened for polymorphisms of the CLPS and PNLIP genes. A nested-case control study of 192 incident type 2 diabetes subjects and 384 sex- and age-matched controls taken from the European Prospective Investigation into Cancer and Nutrition Potsdam Cohort (EPIC) was employed for association studies. The Metabolic Intervention Cohort Kiel (MICK) consisting of 716 males was used for verification. A novel putative functional polymorphism (Arg109Cys) was identified in the CLPS gene. The frequencies of the Arg/Cys genotype were 2.6% in EPIC and 2.2% in MICK study subjects. No homozygotes for the Cys/Cys genotype were found in either study population. Logistic regression analysis showed a statistically significant association of the Arg/Cys genotype with an increased risk of type 2 diabetes. The odds ratios estimated by the model were 3.75 (95%CI = 1.13-12.49, p = 0.03) in EPIC and 4.86 (95%CI = 1.13-20.95, p = 0.03) in MICK. No comparable associations were found with other traits of the insulin-resistance syndrome (e. g.; body mass index, waist to hip ratio). In conclusion, we obtained evidence in two German Caucasian study populations that the variant of the rare CLPS Arg109Cys polymorphism might contribute to increased susceptibility of type 2 diabetes.

Aged↗

PubMed1980

[Transcription of portions of chromosomes corresponding to regions of intercalary heterochromatin in Drosophila melanogaster cell cultures].

Labelled RNA preparations (total newly synthesized RNA, as well as stable cytoplasmic RNA) isolated from a cell culture of Drosophila melanogaster were hybridized in situ with polytene chromosomes. Apart from the nucleolus, in all cases the regions adjacent to he chromocentre in the polytene chromosomes and the intercalary heterochromatin regions in the X chromosome and the autosomes are the most intensively labelled. In the case of asynapsis of polytene chromosomes in heterozygotes the label is detected in a number of intercalary heterochromatin sites in one homologous only ("the asymmetrical label"). The same kind of radioactivity distribution in intercalary heterochromatin regions was observed after a hybridization of polytene chromosomes with cloned DNA fragments (Ananiev et al.,, 1978, 1979) coding for the abundant classes of messenger RNA (Ilyin et al., 1978) in cultured D. melanogaster cells. In some regions of intercalary heterochromatin which do not contain these fragments the "asymmetrical" type of labelled distribution is observed after hybridization with cell RNA. These results lead one to regard the intercalary heterochromatin regions as "nests" comprising different types of actively transcribable genes, the composition of each nest varying in different stocks of D. melanogaster.

Animals↗

PubMedDate not supplied

Detection of alpha-fetoprotein mRNA in seminoma.

The classic testicular tumor marker alpha-fetoprotein (AFP) is associated with nonseminomatous germ cell tumors, including embryonal carcinoma, yolk sac tumor, and teratoma. AFP is not considered to be produced by pure seminoma. However, postmortem studies have demonstrated that 30 to 45% of patients who died of seminoma initially diagnosed harbored nonseminomatous metastases and had an elevated serum AFP. We analyzed AFP expression by immunohistochemistry and by nested reverse transcription-polymerase chain reaction (RT-PCR) in 10 seminomas, 3 embryonal carcinomas, and 1 immature teratoma, diagnosed by traditional clinical methods. Positive immunohistochemical staining was observed in all embryonal carcinomas and in the teratoma but not in the seminomas. AFP mRNA, however, was found in 6 of 10 seminomas, in all embryonal carcinomas, and in the teratoma. The nucleotide sequence of PCR products was identical with that of the AFP gene. We conclude that the analysis of AFP gene expression by nested RT-PCR would be useful for detecting minute quantities of nonseminomatous germ cell elements in classic seminoma. Moreover, the existence of AFP mRNA suggests the possibility that seminoma cells can differentiate into nonseminomatous germ cells.

Adult↗

PubMed2006

Gene x Gene interaction between MnSOD and GPX-1 and breast cancer risk: a nested case-control study.

BACKGROUND: Germ-line mutations in genes such as BRCA1, BRCA2, and ATM can cause a substantial increase in risk of breast cancer. However, these mutations are rare in the general population, and account for little of the incidence of sporadic breast cancer in the general population. Therefore, research has been focused on examining associations between common polymorphisms and breast cancer risk. To date, few associations have been described. This has led to the hypothesis that breast cancer is a complex disease, whereby a constellation of very low penetrance alleles need to be carried to present a risk phenotype. Polymorphisms in the manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPX-1) genes have been proposed as low penetrance alleles, and have not been clearly associated with breast cancer. We investigated whether variants at both polymorphisms, while not independently associated with breast cancer risk, could influence breast cancer risk when considered together. METHODS: A case-control study nested within the Nurses' Health Study was performed comparing 1262 women diagnosed with breast cancer to 1533 disease free women. The MnSOD (Val16Ala, rs1799725) and GPX-1 (Pro198Leu, rs1050450) were genotyped via TaqMan assay. Disease risk was evaluated using logistic regression. RESULTS: While neither allele alone shows any change in breast cancer risk, an increase in the risk of breast cancer (OR 1.87, 95% CI 1.09 - 3.19) is observed in individuals who carry both the Ala16Ala genotype of MnSOD and the Leu198Leu genotype of GPX-1. CONCLUSION: Polymorphisms in the GPX-1 and MnSOD genes are associated with an increased risk of breast cancer.

Alleles↗

PubMed1995

[Molecular diagnosis of Huntington's disease: an analysis of two large families].

At present molecular techniques comprise a main resource in the early accurate diagnosis for Huntington's disease. We describe the molecular diagnosis for 8 HD patients and 39 risky family members in two large HD pedigrees by using G8 RFLP linkage analysis as well as direct detection of the expanded (CAG) repeat in IT15 gene with the nested PCR denaturing polyacrylamide gel electrophoretic autoradiography and direct DNA sequencing. The results showed that the normal IT15 alleles contained 13 to 26 CAG repeat but major in 16, while all the HD alleles had over 40 copies of (CAG)n. There was no overlap between the normal and affected alleles. The presymptomatic diagnosis of 39 family members at risk for HD disclosed that 11 subjects carried the affected alleles. All these results indicated that the unstable mutations in IT15 gene were responsible for HD in the Chinese. They also provide valuable data for HD molecular diagnosis, genetic counselling and genetic health.

Adolescent↗

PubMed1980

Transcription of intercalary heterochromatin regions in Drosophila melanogaster cell culture.

Labelled RNA preparations (total newly synthesized RNA, as well as stable cytoplasmic RNA) isolated from a cell culture of D. melanogaster were hybridized in situ with polytene chromosomes. Apart from the nucleolus, in all cases the regions adjacent to the chromocentre in the polytene chromosomes and the intercalary heterochromatin regions in the X chromosome and the autosomes are the most intensively labelled. In the case of asynapsis of polytene chromosomes in heterozygotes the label is detected in a number of intercalary heterochromatin sites in one homologue only ("the asymmetrical label"). The same kind of radioactivity distribution in intercalary heterochromatin regions was observed after a hybridization of polytene chromosomes with cloned DNA fragments (Ananiev et al., 1978, 1979) coding for the abundant classes of messenger RNA (Ilyin et al., 1978) in a cultured D. melanogaster cells. In some regions of intercalary heterochromatin which do not contain these fragments the "'asymmetrical" type of label distribution is observed after hybridization with cell RNA. - These results lead one to regard the intercalary heterochromatin regions as "nests" comprising different types of actively transcribable genes, the composition of each nest varying in different stocks of D. melanogaster.

Animals↗