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PubMed1998

Definition and distribution analysis of glycoprotein B gene alleles of human herpesvirus 7.

As for other herpesviruses, glycoprotein B (gB) of human herpesvirus 7 (HHV-7) is believed to play a major role in virus infection and as a target of the host immunogenic response. Using nested PCR, we amplified the whole HHV-7 gB gene from 108 human peripheral blood mononuclear cell samples and studied its variability. By means of restriction fragment length polymorphism (RFLP) analysis, three distinct patterns, designated I, II, and III, were defined and detected at frequencies of 93, 5, and 2%, respectively. Determination of the nucleotide sequence allowed us to recognize five critical positions in the gB gene with six specific combinations of point changes at these positions. These combinations were gB alleles A, B, C, D, E, and F. Alleles D and E corresponded to RFLP patterns II and III, respectively, while the other four alleles corresponded to RFLP pattern I. Identical gB alleles were detected in serial samples as well as in paired samples of blood and saliva from the same individuals, except for one case. In contrast, the distribution of gB alleles differed according to the geographical origin of the human samples: C was the most frequent allele in both African and Caribbean samples, whereas F was the most frequent allele in European ones. Although none of the allele-specific nucleotide changes induced any modification at the protein level, the definition of gB alleles provided convenient viral markers for the study of both HHV-7 infections and human population genetics.

Africa↗
PubMed2003

Genotyping of human cytomegalovirus using non-radioactive single-strand conformation polymorphism (SSCP) analysis.

Genetic variation in the glycoprotein B (gB) gene may play a role in human cytomegaloviruses (HCMVs) pathogenesis. Using restriction analysis of the gB gene product (PCR-RFLP), amplified by the nested polymerase chain reaction, the HCMV strains can be compared and classified into at least four HCMV groups. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques used to identify a mutant sequence or a polymorphism in a known gene. SSCP analysis has the advantage over RFLP analysis on detection of DNA polymorphisms and point mutations at a variety of positions in DNA fragments. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis for detection are labour intensive and time-consuming. A simplified SSCP protocol is described to identify HCMV strains and the gB genotype, allowing the detection of sequence variations not residing in the endonuclease recognition sites.

Bone Marrow Transplantation↗
PubMed2001

Phylogeny of Pneumocystis carinii from 18 primate species confirms host specificity and suggests coevolution.

Primates are regularly infected by fungal organisms identified as Pneumocystis carinii. They constitute a valuable population for the confirmation of P. carinii host specificity. In this study, the presence of P. carinii was assessed by direct examination and nested PCR at mitochondrial large subunit (mtLSU) rRNA and dihydropteroate synthetase (DHPS) genes in 98 lung tissue samples from captive or wild nonhuman primates. Fifty-nine air samples corresponding to the environment of different primate species in zoological parks were also examined. Cystic forms of P. carinii were detected in smears from 7 lung tissue samples corresponding to 5 New World primate species. Amplifications at the mtLSU rRNA gene were positive for 29 lung tissue samples representing 18 different primate species or subspecies and 2 air samples corresponding to the environment of two simian colonies. Amplifications at the DHPS gene were positive for 8 lung tissue samples representing 6 different primate species. Direct sequencing of nested PCR products demonstrated that a specific mtLSU rRNA and DHPS sequence could be attributed to each primate species or subspecies. No nonhuman primate harbored the human type of P. carinii (P. carinii f. sp. hominis). Genetic divergence in primate-derived P. carinii organisms varied in terms of the phylogenetic divergence existing among the corresponding host species, suggesting coevolution.

Air Microbiology↗
PubMed2004

Interaction between genetic variations in DNA repair genes and plasma folate on breast cancer risk.

Folate status has been inversely associated with breast cancer risk. Because folate deficiency can cause DNA damage, such as uracil misincorporation, single strand breaks, and double strand breaks, genetic polymorphisms in base excision repair and double strand break repair genes may lead to variation in DNA repair proficiency and modify the effect of folate on breast cancer risk. We prospectively investigated the a priori hypothesized interaction between plasma folate levels and five nonsynonymous polymorphisms in the XRCC1, XRCC2, and XRCC3 genes on breast cancer risk in a nested case-control study within the Nurses' Health Study (712 case-control pairs). Suggestive evidence of interaction was seen for two of these polymorphisms. Compared with the reference group of non-carriers in the lowest quartile of plasma folate, the reduction in risk (66%) was statistically significant among XRCC1 194Trp carriers in the highest quartile (multivariate odds ratio, 0.34; 95% confidence interval, 0.16-0.72). The inverse association between XRCC1 194Trp and breast cancer risk was attenuated by lower plasma folate status. The inverse association between plasma folate level and breast cancer risk was stronger among 194Trp carriers (P, trend = 0.01) than non-carriers (P, trend = 0.09). We also observed that the positive association between the XRCC2 188His allele and breast cancer risk was only significant in women in the lowest plasma folate quartile (carriers versus non-carriers; multivariate odds ratio, 2.04; 95% confidence interval, 1.05-3.97), and this excess risk was abolished among those with higher plasma folate levels. Moreover, the inverse association between plasma folate level and breast cancer risk was stronger among XRCC2 188His carriers (P, trend = 0.004) than non-carriers (P, trend = 0.09). Although none of the statistical tests for interaction was significant, these data give some support for the hypothesis that genetic variations in DNA repair genes may modify the relation between plasma folate level and breast cancer risk.

Adult↗
PubMed2002

Reliability of nested PCR for detection of Chlamydia pneumoniae DNA in atheromas: results from a multicenter study applying standardized protocols.

The present multicenter study was designed to find explanations for the discrepancies in the reported rates of detection of Chlamydia pneumoniae DNA in endarterectomy specimens. Coded identical sets of (i) a C. pneumoniae DNA dilution series (panel 1; n = 10), (ii) spiked control tissue specimens (panel 2; n = 10 specimens, including 5 negative controls), and (iii) endarterectomy specimens (panel 3; 15 atheromas, 5 negative controls) were analyzed at four laboratories by three standardized DNA extraction methods in each laboratory and a nested touchdown PCR protocol targeting the ompA gene of C. pneumoniae. Panel 1 samples were correctly identified as positive to levels of 0.3 inclusion-forming units (IFU)/PCR mixture (100%) and 0.03 IFU/PCR mixture (50%). All negative controls were correctly reported as negative. Panel 2 samples were identified as C. pneumoniae positive to levels of 0.01 IFU/PCR mixture (100%) and 0.005 IFU/PCR mixture (91%), independent of the DNA extraction method used, and only one false-positive result was reported. For panel 3 samples, 5 of 240 (2%) analyses (in which DNA extractions and PCR were performed at the same laboratory) were positive; the positive specimens were from three endarterectomy specimens and two negative controls. After exchange of DNA extracts between laboratories, 13 of 15 atheroma samples were C. pneumoniae DNA positive in at least 1 of a series of 48 analyses per atheroma sample; however, the overall positivity rate did not exceed 5% (33 of 720 analyses) and therefore was lower than that for the negative controls (8%; 19 of 240 analyses). Not a single positive result could be achieved when all panel 3 extracts (n = 240 analyses) were reamplified by a 16S rRNA PCR followed by hybridization with a C. pneumoniae-specific probe. Statistical analyses demonstrated that positive results did not occur in an independent and random fashion and could most likely be explained by amplicon carryover at the nested PCR level as well as amplicon introduction during DNA extraction, but not by the patterns of distribution of very low target levels or a certain DNA extraction protocol. The results of studies by nested PCR for detection of the prevalence of C. pneumoniae will always be questionable.

Arteriosclerosis↗
PubMed2001

Preimplantation genetic diagnosis for familial amyloidotic polyneuropathy (FAP).

Preimplantation genetic diagnosis (PGD) was developed more than a decade ago to offer an alternative to prenatal diagnosis for couples at risk of transmitting an inherited disease to their offspring. Portuguese-type familial amyloidotic polyneuropathy (FAP type I), is an autosomal dominant disease presenting an inherited mutation in the gene encoding the plasma protein transthyretin (TTR). We here report the first protocol for single-cell detection of the Met30 mutation in FAP type I and its application to PGD. A nested PCR reaction for exon 2 of the TTR gene was developed. The PCR product was then analysed by restriction enzyme analysis and SSCP allowing the detection of the point mutation. Ten clinical cycles were performed in seven couples. From the 93 metaphase II (MII) injected oocytes, 82 were normally fertilized and 78 were biopsied. A positive signal in the nested PCR reaction was obtained in 61 blastomeres, corresponding to a DNA amplification efficiency of 78.2%. No allele dropout (ADO) or contamination were detected. A biochemical pregnancy was obtained in three cases and a clinical pregnancy in one couple is actually in normal evolution.

Amyloid Neuropathies, Familial↗
PubMed2002

[Molecular cloning of the human CX 58 gene].

OBJECTIVE: To clone a novel human connexin gene and find out the relationship between this gene and hereditary deafness. METHODS: Through the basic local alignment search tool (BLAST) analysis against the database of expressed sequence tags (dbEST) of National Center for Biotechnology Information (NCBI) using the coding sequence of mouse Cx 57 gene, 9 novel ESTs were obtained and a contig was assembled. Nested polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) were performed using primers designed on the contig. A novel gene was obtained and was mapped by homologous analysis against human genome sequence. Mutation analysis was performed in 12 autosomal dominant hereditary deafness families. RESULTS: Nine ESTs were obtained by homologous analysis and a contig was assembled. Through nested PCR and RACE, a full length of cDNA was obtained from human liver, kidney Ready cDNA and placenta cDNA library, and was named CX 58. By comparison with human genome sequence, CX 58 was mapped at 1p32.3-p34.1. Mutation analysis of CX 58 was performed in 12 autosomal dominant hereditary deafness families, but no mutation was detected. CONCLUSION: A novel human connexin gene named CX 58 was cloned and mapped to 1p32.3-p34.1. The mutation of CX 58 may not result in autosomal dominant hereditary deafness.

Animals↗
PubMed2006

Activating mutations of Gsalpha in kidney cancer.

PURPOSE: Heterotrimeric G proteins are signal transduction proteins coupled to hormone receptors that activate intracellular second messenger systems, mainly cyclic adenosine monophosphate mediated protein kinase. Recent studies indicate that G proteins may have a major role in oncogenesis as well as in tumor invasiveness and cell proliferation. The involvement of G proteins was formerly thought to be limited to hormonal signal transduction. Activating Gsalpha mutations have been reported in tumors arising only from highly specialized endocrine tissue, such as pituitary adenomas, toxic thyroid adenomas and differentiated thyroid carcinomas, but never in other nonendocrine tumors. We hypothesized that a constitutive activation of this pathway, that is activated Gsalpha and inhibited Gialpha, could be implicated in kidney cancers. We searched for alterations on the Gsalpha gene GNAS and the Gialpha gene in renal cell carcinoma. MATERIALS AND METHODS: Using nested polymerase chain reaction, enzyme digestions, laser microdissection and direct sequencing we looked for activating mutations on GNAS codons 201 and 227, and inhibiting mutations on the Gialpha gene in 30 consecutive patients with clear cell renal cell carcinoma between January 2003 and January 2004. RESULTS: Somatic (tumor specific) activating mutations of Gsalpha were present in a significant proportion of human clear cell renal cell carcinomas. Activating mutations were identified in 5 of the 30 patient DNA preparations (16.6%) with a substitution of arginine 201 by cysteine in 3 and histidine in 2. CONCLUSIONS: These findings suggest the implication of this pathway in human oncogenesis. It may provide a potential therapeutic approach to these frequent and aggressive tumors.

Adult↗
PubMed2002

High incidence of B-cell monoclonality in follicular gastritis: a possible association between follicular gastritis and MALT lymphoma.

Many studies have indicated a close association between gastric mucosa-associated lymphoid tissue (MALT) lymphoma and Helicobacter pylori ( Hp) infection. Follicular gastritis (FG), a rare type of gastritis, is also closely associated with Hp infection and histologically similar to MALT lymphoma. However, there are few studies investigating the relationship between FG and MALT lymphoma. To clarify the issue, we examined the B-cell monoclonality in FG by immunoglobulin heavy chain (IgH) gene rearrangement. Seventy FG patients (all Hp-positive, 23 males and 47 females, median age 33 yr) were investigated; 70 age-and gender-matched non-FG Hp-positive controls and 24 non-FG Hp-negative controls were also examined. Two gastric biopsies, one from the antrum and one from the corpus, were obtained from each patient. DNA was extracted from formalin-fixed, paraffin-embedded biopsy specimens. IgH gene rearrangement was examined by semi-nested polymerase chain reaction (PCR). In the antral mucosa, monoclonal B-cell populations were detected in 19 (32%) of 60 FG, in 6 (10%) of 60 Hp-positive controls, and in none of 20 Hp-negative controls. In the corporal mucosa, monoclonal B-cell populations were detected in 14 (30%) of 47 FG, in 6 (11%) of 54 Hp-positive controls, and in none of 20 Hp-negative controls. The incidence of monoclonal B-cell populations in the FG patients was higher than in both Hp-positive and Hp-negative controls ( P<0.05). The monoclonal B-cell populations disappeared after successful Hp eradication in 8 of 8 FG patients examined. These data suggest that FG may be strongly associated with MALT lymphoma, and that Hp eradication therapy may be indicated in FG.

Adolescent↗
PubMed2000

Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR.

A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-microm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the alpha- and gamma-subdivisions of the division Proteobacteria (alpha- and gamma-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, alpha-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.

Alphaproteobacteria↗
PubMed2001

Establishment and characterization of a new human myeloma cell line, KYdelta-1, producing the delta/kappa type immunoglobulin.

We describe the establishment and characterization of a new multiple myeloma (MM) cell line, KYdelta-1, which expressed delta/kappa type immunoglobulin (Ig). The patient was a 65-year-old woman with MM, who presented extramedullary dissemination, lymphadenopathy and short survival. The KYdelta-1 cell line was derived from the pleural fluid obtained in the terminal phase of the disease. The cells expressed delta/kappa Ig in the cytoplasm, and CD10, CD29, CD33, CD38, CD44, CD54, and HLA-DR antigens on the cell surface. Chromosomal analysis revealed two independent translocations, t(3;14)(p21;q32) and t(3;11)(p21;q13), which were confirmed by fluorescence in situ hybridization using chromosome painting probes. Reverse transcriptase-mediated polymerase chain reaction (PCR) and Northern blot analyses demonstrated overexpression of the CCND1 gene, suggesting alteration of the BCL1-CCND1 locus. We thus performed long-distance inverse PCR using nested primers for the Calpha constant region of immunoglobulin heavy chain gene (IGH) and obtained a clone that encompassed the 11q13/IGH fusion. Nucleotide sequencing determined that the fusion occurred at the Salpha2 switch region and at the centromeric side of the major translocation cluster of BCL1. The other IGH allele consisted of a VDJ complex that was adjacent to the Cdelta constant gene, indicating that a class switch-like mechanism from the C(mu) to Cdelta was involved in the production of the Ig delta heavy chain. Point mutations within the P53 and N-RAS genes were presumably related to the rapidly progressive disease in this particular MM patient.

Aged↗
PubMed2004

Truncated hepatitis C virus core protein encoded in hepatocellular carcinomas.

Studies have suggested that a truncated form of the hepatitis C virus (HCV) core protein can enter hepatocyte nuclei and might play a role in HCV-associated hepato-carcinogenesis. In the present study, the HCV core gene from hepatocellular carcinomas (HCC) and/or adjacent non-tumorous liver tissues from eight patients was amplified by nested RT-PCR and sequenced. Mutations in the HCV core gene that would encode a truncated core protein were found in 4 of the 8 patients. Since truncated core proteins have been shown to be capable of entry into the hepatocyte nucleus (unlike HCV itself, which is an exclusively cytoplasmic virus), the detection of mutated sequences encoding them in these four HCC patients suggests that these mutations may have played a role in the development of these HCCs.

Adult↗
PubMed1992

Mapping of an inducible element in the T cell receptor V beta 2 promoter.

The murine V beta 2 promoter was analyzed for an element regulating phorbol ester inducibility of the TCR beta chain gene. In transient expression analysis of 5' nested deleted fragments of the V beta 2 promoter, the TPA-inducible element mapped between -85 and -42. The -85 to -62 oligo conferred 12-0-tetradecanoylphorbol-13-acetate (TPA) inducibility to the heterologous TPA-uninducible thymidine kinase promoter. The -85 to -62 region contained an AP-1 site (-85 to -72) and inverted repeat motif (-72 to -62). The AP-1 site required the 3' flanking inverted repeat region for conferring optimal inducibility. In vitro transcribed and translated jun/fos heterodimers bind to the V beta 2 AP-1 motif with a 16-fold lower affinity as compared to the collagenase AP-1 motif. This explains the inability of the V beta 2 AP-1 motif to confer optimal TPA inducibility by itself. The affinity of jun/fos heterodimers for the V beta 2 AP-1 motif was not increased by the presence in cis of the inverted repeat motif. The 3' flanking inverted repeat binds the ets transactivator but not jun/fos heterodimers. The demonstrated cooperativity between the AP-1 and the 3' flanking sequence to confer TPA inducibility can thus be explained by the individual contributions of jun/fos and ets transactivators.

Animals↗
PubMed2000

The skeletal muscle alpha-actin gene of channel catfish (Ictalurus punctatus) and its association with piscine specific SINE elements.

The alpha-actin gene of channel catfish (Ictalurus punctatus) was cloned and sequenced. The gene has a similar organization and exhibited a high level of sequence similarity to those from other vertebrate animals. The upstream region of the alpha-actin gene included a TATA box, a CAAT box, three E-boxes, and a CArG box. Nested deletion segments containing these transcriptional motifs were fused to the reporter gene chloramphenicol acetyl transferase (CAT). Transfection of the clones into C2C12 cells indicated that all these motifs are required for transcriptional activities. The channel catfish alpha-actin gene is associated with two distinct short interspersed repetitive elements (SINEs). The first SINE element showed high levels of sequence similarity to the zebrafish Mermaid element, while the second SINE element is not similar to the Mermaid element except for an 8bp sequence CCCCGTGC suggesting their evolutionary linkage. However, the second SINE element appeared to co-exist with the Mermaid element in most cases and therefore was designated as the Merman element. Approximately 9000 copies and 1200 copies of the Mermaid and Merman elements exist per haploid channel catfish genome, respectively. BLAST searches indicated that both the Mermaid and the Merman elements were frequently associated with gene sequences, mostly those of aquatic animals, suggesting their evolutionary origin in association with aquatic organisms and their function in shaping the evolution of genomes in aquatic animals.

Actins↗
PubMed1999

Cohort studies for characterizing measured genes.

We describe the advantages of using established cohort studies that have collected blood samples to investigate the role of genes in the etiology of cancer. These studies include the cost-efficiency and reliability of nested case-control substudies from the cohort for exploration of gene-disease associations and gene-environment interactions as well as gene penetrance. Also, the cohort may serve as a well-defined "mini-population" from which to study population stratification and molecular markers of ethnicity. We conclude that cohort studies can play a significant role in assessing the role of genetic markers for common tumors or multiple cancer sites.

Case-Control Studies↗
PubMed2003

Identification of white spot syndrome virus latency-related genes in specific-pathogen-free shrimps by use of a microarray.

To investigate whether specific-pathogen-free (SPF) shrimps are asymptomatic carriers of white spot syndrome virus (WSSV), we used a WSSV-specific DNA microarray to measure WSSV gene expression in SPF and WSSV-infected shrimps. Three WSSV genes were found to be relatively highly expressed in SPF shrimps. Reverse transcription-PCR using nested primers as well as real-time detection confirmed that these genes have no detectable counterparts in GenBank; structural analysis of the putative proteins revealed helix-loop-helix and leucine zipper motifs. Viral sequences could be PCR amplified from genomic DNA of SPF shrimp, further supporting the suggestion that these shrimps are asymptomatic carriers.

Animals↗
PubMed2000

[A study of transgenic IFV cattle integrated with human serum albumin gene].

The mammary gland expression vector (pcDNA 3.1-GCALBm) containing the full-length sequence of human serum albumin (hALB) cDNA and intron 1 as well as the goat beta-casien gene promoter and 5' up-stream regulatory sequence was constructed. The vector was micro-injected into bovine IVF eggs. The embryos were in vitro cultured to the late stage of morulae, and then few embryo cells were aspirated for the implantation detection of target gene integration and SRY DNA sequence using nested-PCR. Afterwards, ten integrated embryos were selected to transfer into eight recipients and three were pregnant. The pregnant rate was 37.5%(3/8). However, two were miscarried in mid-trimester but one was pregnant at term to deliver a male transgenic cattle integrated with hALB mini-gene. The transgenic efficiency was 12.5% (1/8).

Animals↗