A knockdown of the herpes simplex virus type-1 gene in all-in-one CRISPR vectors.

INTRODUCTION: Herpes simplex virus type 1 (HSV-1) is a virus that causes serious human disease and establishes a long-term latent infection. The latent form of this virus has shown to be resistant to antiviral drugs. Clustered Regularly Interspace Short Palindromic Repeats (CRISPR), is an important tool in genome engineering and composed of guide RNA (gRNA) and Cas9 nuclease that makes an RNA-protein complex to digest exclusive target sequences implementation of gRNA. Moreover, CRISPR-Cas9 system effectively suppresses HSV-1 infection by knockout of some viral genes. MATERIALS AND METHODS: To survey the efficacy of Cas9 system on HSV-1 genome destruction, we designed several guide RNAs (gRNAs) that all packaged in one vector. Additionally, we performed a one-step restriction using BamHI and Esp3I enzymes. RESULTS: CRISPR/Cas9 system targeted against the gD gene of HSV-1 was transfected into HEK-AD cells that showed a significant reduction of HSV-1 infection by plaque assay and real-time PCR. CONCLUSION: The pCas-Guide-EF1a-GFP CRISPR vector can create a fast and efficient method for gRNA cloning by restriction enzymes (Esp3I (BsmBI) and BamHI). Therefore, the CRISPR/Cas9 system may be utilized for the screening of genes critical for the HSV-1 infection and developing new strategies for targeted therapy of viral infections caused by HSV-1.

Epstein-Barr Virus (EBV)-BamHI-A Rightward Transcript (BART)-6 and Cellular MicroRNA-142 Synergistically Compromise Immune Defense of Host Cells in EBV-Positive Burkitt Lymphoma.

BACKGROUND This study was designed to explore the molecular mechanism underlying the effect of cellular miRNAs and EBV miRNA upon the expression of targets such as PTEN, and their involvement in the pathogenesis of Burkitt lymphoma. MATERIAL AND METHODS In this study, we examined several differentially expressed cellular miRNAs in EBV-positive versus EBV-negative Burkett lymphoma tissue samples, and confirmed PTEN as targets of cellular miR-142 by using a bioinformatics tool, luciferase reporter system, oligo transfection, real-time PCR, and Western blot analysis. RESULTS We further confirmed the binding site of miR-142 in the 3'UTR of the target genes, and established the negative regulatory relationship between miRNA and mRNAs with luciferase activity assay. To verify the regulatory relationship between the miRNAs and PTEN, we evaluated the expression of PTEN in the tissue samples, and found that PTEN was downregulated in EBV- positive Burkett lymphoma. Additionally, lymphoma cells were transfected with EBV-BART-6-3p and miR-142 and we found that EBV-BART-6-3p and miR-142 synergistically reduced expression of IL-6R and PTEN. Furthermore, we also examined viability of the cells in each treatment group, and showed that EBV-BART-6-3p and miR-142 synergistically promoted proliferation of the cells. CONCLUSIONS These findings improve our knowledge about the role of miR-142/EBV-BART-6-3p and their target, PTEN, in the development of Burkett lymphoma; they could be novel therapeutic targets for the treatment of EBV-positive Burkett lymphoma.

Evaluation of the risk of lymphomagenesis in xenografts by the PCR-based detection of EBV BamHI W region in patient cancer specimens.

Establishment of patient-derived tumor xenografts (PDXs) is hampered by lymphomagenesis mostly caused by the latently-infected Epstein-Barr virus (EBV) contained in patient cancer tissues. However, the character of patient tissues that result in lymphomagenesis after xenotransplantation is not elucidated. In this study, we analyzed the patient colorectal cancer (CRC) tissues and the PDXs established by their xenotransplantation. We found that 2 of 9 (22%) PDX tumors were EBV-associated human diffuse large B cell lymphoma which was formed by clonal proliferation of human B-cell lymphocytes, were strongly positive for EBER-ISH, and were classified as type III latency. Expression of EBV genes and RNAs, such as EBNAs, LMP1, EBER and EBV-associated microRNAs in patient CRC tissues were unlikely to be associated with lymphomagenesis in PDXs. In contrast, the positive PCR-based amplification of BamHI W region, a major internal repeat in EBV genome, in the patient CRC tissues was correlated with lymphomagenesis in PDXs. These results suggest that the detection of the EBV BamHI W region in the patient surgical specimens will be an effective way to predict the risk of lymphomagenesis in PDXs before xenotransplantation.

NF-κB Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma.

UNLABELLED: Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-κB activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC. IMPORTANCE: Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system.

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.

Profiling of EBV-Encoded microRNAs in EBV-Associated Hemophagocytic Lymphohistiocytosis.

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening complication of EBV infection. MicroRNAs (miRNAs) were small non-coding RNA, and EBV could encode miRNAs that are involved in the progression of infection. However, the profiles of EBV-miRNAs in EBV-HLH were unknown. Here, we aimed to profile the expression of EBV-miRNAs in children with EBV-HLH by analyzing 44 known EBV-miRNAs, encoded within the BamHI fragment H rightward open reading frame 1 (BHRF1) and the BamHI-A region rightward transcript (BART), in plasma and cellular targets by real-time quantitative PCR. The study included 15 children with EBV-HLH, 15 children with infectious mononucleosis (IM), and 15 healthy controls. CD8(+) T cells were found to be the cellular target of EBV infection in EBV-HLH, while CD19(+) B cells were infected with EBV in IM. We also found the greater levels of several miRNAs encoded by BART in EBV-HLH, compared to those in IM and healthy controls, whereas the levels of BHRF1 miRNAs were lower than those in IM. The profile and pattern of EBV-miRNAs in EBV-HLH indicated that EBV could display type II latency in EBV-HLH. Importantly, the level of plasma miR-BART16-1 continued decreasing during the whole chemotherapy, suggesting that plasma miR-BART16-1 could be a potential biomarker for monitoring EBV-HLH progression. The pathogenesis of EBV-HLH might be attributed to the abundance of EBV-miRNAs in EBV-HLH. These findings help elucidate the roles of EBV miRNAs in EBV-HLH, enabling the understanding of the basis of this disease and providing clues for its treatment.

Polymorphisms in the vitamin D receptor gene and multiple sclerosis risk: a meta-analysis of case-control studies.

BACKGROUND: The vitamin D receptor (VDR) polymorphisms have been reported to be associated with multiple sclerosis (MS), however, evidence remains conflicting. A meta-analysis was conducted to investigate this association. METHODS: We searched Pubmed, Medline and Embase databases for case-control studies evaluating the association between the VDR Apa-I, Bsm-I, Fok-I, Taq-I polymorphisms and MS risk. Data were extracted using standardized forms and odds ratios (OR) with 95% confidence intervals (CI) were calculated. RESULTS: 11 case-control studies involving a total of 2599 cases and 2816 controls were included in this meta-analysis. Available data did not suggest an association between any of the VDR polymorphisms and the risk for MS. For Taq-I, which is the most investigated VDR polymorphism with 8 studies (2472 cases and 2446 controls), the combined OR was 1.12 (95% CI: 1.00-1.26) for the dominant model (tt+Tt vs. TT), 1.03(95% CI: 0.88-1.20) for the recessive model (tt vs. Tt+TT), and 1.04 (95% CI: 0.78-1.38) for the homozygote model (tt vs. TT). ORs for other VDR polymorphisms were similar. CONCLUSION: The VDR Apa-I, Bsm-I, Fok-I and Taq-I polymorphisms are not associated with MS risk.

The association of genomic variation of Epstein-Barr virus BamHI F fragment with the proliferation of nasopharyngeal carcinoma.

To investigate the f variant of Epstein-Barr virus (EBV) in nasopharyngeal carcinogenesis, we detected the f variant in primary nasopharyngeal carcinoma (NPC), metastatic carcinoma of the lymph node (LN), and chronic inflammation of the nasopharynx from the Guangdong region. Meanwhile, we analyzed the relationship between the f variant of EBV and LMP1, Fascin, pStat3, p53, Bcl-2, and Ki-67 expression in NPC. The results showed that the f variant of EBV was found in 11 cases of primary NPCs with LN metastasis, 12 LN metastases, and 18 primary NPCs without LN metastasis. However, only one demonstrated the F/f variant in 50 cases of chronic inflammation of the nasopharynx. The expression rate of LMP1, Fascin, pStat3, p53, Bcl-2, and Ki-67 in NPC with the f or F/f variant was higher than that with the F prototype. Furthermore, there was a significantly positive correlation between the f variant of EBV and Ki-67 expression (p < 0.05). Our study suggests that the f variant of EBV may be closely related to nasopharyngeal carcinogenesis.

Photochemical regulation of restriction endonuclease activity.

Removal by the light: The photochemical regulation of restriction endonucleases, which are important enzymes in molecular biology, has been investigated. Photolabile protecting groups have been installed on DNA substrates and have been demonstrated to inhibit restriction endonuclease activity until removed by UV light irradiation. Interestingly, these groups do not appear to dramatically affect initial binding of the enzyme to the DNA substrate, but rather prevent recognition of the specific cleavage site.

Clinical usefulness of serum EBV DNA levels of BamHI W and LMP1 for Nasal NK/T-cell lymphoma.

Quantitative real-time polymerase chain reaction (PCR) was utilized to measure serum EBV DNA levels of BamHI W fragment and latent membrane protein 1 (LMP1) in 20 nasal natural killer (NK)/T-cell lymphoma patients. Both serum EBV DNAs were detected at high levels in all patients, but the levels were below the limit of detection in all healthy controls. The BamHI Z fragment, Epstein-Barr-replication activator (ZEBRA) expression was detected in a small proportion (0.1-3%) of lymphoma cells from 10 (50%) of the patients. Patients with ZEBRA expression showed significantly higher DNA levels of BamHI W and LMP1 (P = 0.0081, P = 0.004), suggesting that EBV DNA may be caused by EBV replication from lymphoma cells. Kaplan-Meier and univariate analyses revealed that high DNA levels of BamHI W and LMP1 at pre-treatment and high BamHI W DNA level at post-treatment were associated with short disease-free survival and overall survival (P < 0.05, each). Although the DNA levels of BamHI W and LMP1 correlated significantly, their dynamics were not always parallel. Patients with low pre-treatment level of both EBV DNAs showed a favorable course, in contrast to patients with high pre-treatment level of both EBV DNAs who showed an aggressive course (P = 0.0085). More importantly, the high pre-treatment level of both EBV DNAs was determined as the only independent prognostic factor among various prognostic factors. These data suggest that simultaneous measurement of serum levels of both BamHI W and LMP1 DNAs may be useful for diagnosis, disease monitoring, and prediction of prognosis for nasal NK/T-cell lymphoma patients.

Infection of Epstein-Barr virus in colorectal cancer in Chinese.

BACKGROUND & OBJECTIVE: Except for the tight correlation to nasopharyngeal carcinoma, accumulating evidences show that Epstein-Barr virus (EBV) is correlated to other carcinomas. This study was to investigate the correlation of EBV to colorectal carcinoma in Chinese. METHODS: EBV DNA was detected by polymerase chain reaction (PCR) in 90 specimens of primary colorectal carcinoma and 25 specimens of corresponding adjacent non-cancerous tissue, with the primers covering 2 different regions of EBV genome, BamH I W fragment and latent membrane protein 1 (LMP1) exon 3. The expression of EBV nuclear antigen 1 (EBNA1) and LMP1 were determined by immunohistochemistry, and the expression of EBV-encoded RNAs (EBERs) was detected by in situ hybridization. RESULTS: EBV LMP1 exon 3 and W fragment were detected in 27.7% and 32.2% of the 90 colorectal carcinoma specimens, which were significantly higher than the positive rate of EBV gene in the 25 adjacent non-cancerous tissues (4.0%, P<0.001). Of the 29 W fragment-positive tumors, 23 (79.3%) were EBNA1-positive, 1 (3.4%) was EBERs-positive; most EBNA1-positive cells were tumor cells with positive signals gathered in the nuclei. No expression of EBNA1 and EBERs were detected in the 8 W fragment-negative tumors. No expression of LMP1 was detected in all tumor specimens. CONCLUSION: EBV infection might be associated with colorectal carcinoma in Chinese.

Chromosomal localization, copy number assessment, and transcriptional status of BamHI repeat fractions in water buffalo Bubalus bubalis.

Higher eukaryotes contain a wide variety of repetitive DNA, although their functions often remain unknown. We describe cloning, chromosomal localization, copy number assessment, and transcriptional status of 1378- and 673-bp repeat fractions in the buffalo genome. The pDS5, representing the 1378-bp fragment, showed FISH signals in the centromeric region of acrocentric chromosomes only, whereas pDS4, corresponding to 673 bp, detected signals in the centromeric regions of all the chromosomes. Crosshybridization studies of pDS5 and pDS4 with genomic DNA from different sources showed signals only in buffalo, cattle, goat, and sheep. Real-time PCR analysis uncovered 1234 and 3420 copies of pDS5 and pDS4 fragments per the haploid genome, corresponding to 30 and 68 copies per chromosome, respectively. Analysis of cDNA from different tissues of buffalo with Real-time PCR showed maximum expression of pDS5 and pDS4 in the spleen and liver, respectively. Phylogenetic analysis of these sequences showed a close relationship between buffalo and cattle. The prospect of this approach in comparative genomics is highlighted.

A comparison study of different PCR assays in measuring circulating plasma epstein-barr virus DNA levels in patients with nasopharyngeal carcinoma.

PURPOSE: To compare the performance of three PCR assays in measuring circulating Epstein-Barr virus (EBV). DNA levels in nasopharyngeal carcinoma patients and to confirm its prognostic significance. EXPERIMENTAL DESIGN: Plasma from 58 newly diagnosed nasopharyngeal carcinoma patients were collected before, during, and every 3 to 6 months after radiotherapy. EBV DNA levels were determined by real-time quantitative PCR using primer/probe sets for polymerase-1 (Pol-1), latent membrane protein 2 (Lmp2), and BamHI-W. Pretreatment levels from the three assays were correlated with each other and serial measurements from the Pol-1 assay were correlated with clinical variables. RESULTS: Pol-1 was more accurate than BamHI-W in predicting EBV DNA concentrations in cell lines. Of the three assays, BamHI-W yielded the highest concentrations followed by Pol-1 in plasmas (n = 23). The correlation coefficient was 0.99 (P < 0.0001) for Pol-1 and Lmp2, 0.66 (P < 0.0001) for Pol-1 and BamHI-W, and 0.55 (P < 0.0001) for BamHI-W and Lmp2. Elevated pretreatment DNA levels as detected by Pol-1 were correlated with advanced nodal stage (P = 0.04) and overall stage (P = 0.028). There was no correlation between pretreatment EBV DNA levels and freedom-from-relapse or overall survival; however, there was a significant correlation between posttreatment levels and these variables. The 2-year freedom-from-relapse and overall survival rates were 92% and 94% for patients with undetectable, and 37% and 55% for those with detectable, posttreatment levels (P < 0.0001 and P < 0.002). CONCLUSIONS: The three PCR assays yielded similar results in detecting EBV DNA in plasmas. The Pol-1-detected posttreatment EBV DNA level was the strongest predictor for treatment outcomes.

Design and synthesis of photochemically controllable restriction endonuclease BamHI by manipulating the salt-bridge network in the dimer interface.

The strategy for the design of photochemically controllable enzymes by manipulating the dimer interface is described. Employing a restriction endonuclease BamHI, the selective incorporation of amino acids having a photoremovable 6-nitroveratryl group into the specific position (Lys132) in the dimer interface of the BamHI mutant (H133A) was performed. The activity of the photofunctionalized BamHI mutant was significantly suppressed, and the following photoirradiation induced the recovery of the activity. In addition, uncaging of the 6-nitroveratryl group introduced to Lys132 did not seriously reduce the catalytic activity and affinity for the substrate. These results indicate that the activity of the enzyme can be effectively regulated by caging and uncaging of the specific amino acid in the dimer interface using the photoremovable group.

Crystal structure of BstYI at 1.85A resolution: a thermophilic restriction endonuclease with overlapping specificities to BamHI and BglII.

We report here the structure of BstYI, an "intermediate" type II restriction endonuclease with overlapping sequence specificities to BamHI and BglII. BstYI, a thermophilic endonuclease, recognizes and cleaves the degenerate hexanucleotide sequence 5'-RGATCY-3' (where R=A or G and Y=C or T), cleaving DNA after the 5'-R on each strand to produce four-base (5') staggered ends. The crystal structure of free BstYI was solved at 1.85A resolution by multi-wavelength anomalous dispersion (MAD) phasing. Comparison with BamHI and BglII reveals a strong structural consensus between all three enzymes mapping to the alpha/beta core domain and residues involved in catalysis. Unexpectedly, BstYI also contains an additional "arm" substructure outside of the core protein, which enables the enzyme to adopt a more compact, intertwined dimer structure compared with BamHI and BglII. This arm substructure may underlie the thermostability of BstYI. We identify putative DNA recognition residues and speculate as to how this enzyme achieves a "relaxed" DNA specificity.

Stress-dependent regulation of the gene encoding gamma-glutamylcysteine synthetase from the fission yeast.

Glutathione (GSH), an important antioxidant involved in stress response, is synthesized in two sequential reactions. Gamma-glutamylcysteine synthetase (GCS) catalyzes the first step in GSH biosynthesis, which is usually known to be rate-limiting. In this work, regulatory patterns of the GCS gene from the fission yeast Schizosaccharomyces pombe have been investigated. The 607 bp upstream region from the translational initiation point was amplified by the two synthetic primers. The amplified DNA was ligated into the BamHI/HindIII site of the shuttle vector YEp367R to generate the fusion plasmid pUGCS101. The GCS-lacZ fusion gene construct was confirmed by restriction mapping and nucleotide sequencing. The GCS-lacZ fusion gene was used to study effects of various agents on the transcription of the GCS gene. The synthesis of beta-galactosidase from the fusion plasmid pUGCS101 was enhanced by metals, oxidative and nitrosative stresses, and glutathione-depleting agents. The GCS mRNA level in the wildtype S. pombe cells was significantly elevated by the treatment with sodium nitroprusside or menadione, which was detected by RT-PCR. It was also induced by low concentrations of glucose and sucrose. These results suggest that the expression of S. pombe GCS gene is regulated by various stresses and carbon sources.

Presence of copalyl diphosphate synthase gene in an actinomycete possessing the mevalonate pathway.

We have previously shown that gene clusters for biosyntheses of terpentecin and BE-40644, a diterpene antibiotic and a sesquiterpene antibiotic, respectively, were located in the adjacent mevalonate pathway gene clusters. In this study, a mevalonate pathway gene cluster was cloned from Streptomyces sp. strain KO-3988, which was known to produce furaquinocin A, employing a hybridization experiment using a 3-hydroxy-3-methyl glutaryl CoA (HMG-CoA) reductase gene, which had been previously cloned from the strain KO-3988, as a probe. By sequencing flanking regions, we found four open reading frames that could encode a putative cytochrome P450 (ORF1), an isoprenoid cyclase (ORF2), an unknown protein (ORF3), and a polyprenyl diphosphate synthase gene (ORF4) in the upstream region of the mevalonate pathway gene cluster, though we did not find any genes related to furaquinocin A biosynthesis. The two ORFs (ORF2 and 4) were expressed as recombinant enzymes in E. coli and used for studies to investigate functions of these products. The ORF4 product was confirmed to be a geranylgeranyl diphosphate (GGDP, C20) synthase. The ORF2 product proved to catalyze a conversion of GGDP into copalyl diphosphate, the first example of an enzyme with this function of prokaryotic origin. These results again showed that actinomycetes possessing the mevalonate pathway usually produce an isoprenoid and that its biosynthetic gene cluster exists in adjacent the mevalonate pathway gene cluster.

Evaluation of a whole-genome amplification method based on adaptor-ligation PCR of randomly sheared genomic DNA.

High-throughput genetic studies often require large quantities of DNA for a variety of analyses. Developing and assessing a whole-genome amplification method is thus important, especially with the current desire for large-scale genotyping in previously collected samples for which limited DNA is available. The method we have developed, called PRSG, is based on an adaptor-ligation-mediated PCR of randomly sheared genomic DNA. An unbiased representation was evaluated by performing PCR on 2,607 exons of 367 genes, which are randomly distributed throughout the genome, on PRSG products of hundreds of individuals. An infrequent loss (<1%) of the exon sequence on the PRSG products was found. Out of 307 microsatellites on various chromosomes, 258 (84%) were amplified in both the PRSG product and an original DNA, whereas 49 (16%) microsatellites were lost only in the PRSG product. Array CGH analysis of 287 loci for measuring the relative gene copy number demonstrated that a low bias was detected. Moreover, this method was validated on 100-1,000 laser-captured cells from paraffin-embedded tissues. These data show that PRSG can provide a sufficient amount of genomic sequence for a variety of genetic analyses as well as for long-term storage for future work.

[Analysis of the N-acetyltransferase 2 gene polymorphism in the patients with chronic obstructive pulmonary disease and in populations of the Volga-Ural region]. / Analiz polimorfizma gena N-atsetiltransferazy 2 u bol'nykh khronicheskimi obstruktivnymi bolezniami legkikh i v populiatsiiakh Volgo-Ural'skogo regiona.

Restriction fragment-length polymorphism of the gene coding for N-acetyltransferase 2 (NAT2) was typed in populations of the Volga-Ural region (Bashkirs, Tatars, Chuvashes, Udmurts, and Russians) as well as in patients with chronic obstructive pulmonary disease (COPD) and in healthy individuals. Rapid and slow acetylator phenotypes were determined based on the presence or absence of the KpnI, TaqI, and BamHI restriction endonuclease recognition sites. The proportion of slow acetylators in the populations examined varied from 40.00% in Bashkirs to 64.15% in Chuvashes with statistically significant difference between these two ethnic groups (chi 2 = 5.7; p = 0.02). Overall, in the Volga-Ural populations slow acetylators represented 56.25% of the subjects examined. This value was similar to those presented in other studies of Caucasoid populations. In the COPD patients a statistically significant decrease of the slow acetylator frequency to 48.28% compared to healthy individuals (62.18%) was observed (chi 2 = 4.60; p = 0.036). The data obtained suggest a possible association between the drug resistance in the COPD patients with the rapid acetylator phenotype, which can lead to the development of the chronic form of the disease.