OBJECTIVETo develop a simple method for genotyping of hepatitis E virus (HEV) and to investigate HEV genotype distribution in Nanjing area.

METHODSTwenty-seven full HEV sequences currently-available in GenBank were analyzed with MegAlign and MapDraw programs of DNA STAR software. Degenerate primers were designed and applied to amplify a fragment in HEV ORF1 region. HEV genotypes were determined by the size of the PCR products and by single restriction endonuclease analysis.

RESULTSThe PCR products of HEV genotype 1 and 2 were 275 bp and 269 bp in size. Distinctively, the PCR products of genotype 3 and 4 were 317 bp and 314 bp in size. Moreover, the PCR products of genotype 1 could be digested by Nae 1, but the products of genotype 2 could not. Distinctively, the PCR products of HEV genotype 3 could be digested by Not 1, but the products of genotype 4 could not. Six HEV reference strains standing for different HEV genotypes were clustered into their own types as predicted. Within 43 HEV IgM-positive clinical specimens collected in Nanjing, 19 were HEV PCR-positive and identified as genotype 4.

CONCLUSIONA simple method of PCR combined with single restriction endonuclease analysis is developed for HEV genotyping. This assay allows rapid identification of a large number of HEV isolates directly from clinical specimens. Among patients with hepatitis E in Nanjing, most were infected with HEV genotype 4.

DNA Restriction Enzymes ; metabolism ; DNA, Complementary ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Genotype ; Hepatitis E ; blood ; genetics ; immunology ; Hepatitis E virus ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo clone the prtH gene from Porphyromonas gingivalis (P.g) ATCC 33277 and analyze the polymorphism of prtH gene from 5 strains of P.g in order to explore the relationship between P.g and periodontitis.

METHODSUsing PCR, the prtH was amplified and cloned into pGEM-T vector. To illustrate the prtH polymorphism among P.g strains, the genomic DNAs were extracted and screened by PCR with three pairs of specific primers, dot blot and Southern blot hybridization using the biotin-labeled prtH sequence as probe.

RESULTSRecombinant DNA pGEM-T- prtH was verified by restriction endonuclease and sequence assay. Strain W 381 and ATCC 33277 showed the identical results in PCR and hybridization assays, whereas strain ATCC 49417 and 14-3-2 revealed individual hybridization patterns. Strain 47A-1 seemed even not to contain prtH gene.

CONCLUSIONSDifferent prtH gene sequences exist in different P.g strains. This polymorphism may indicate various potential virulent effects during the infection and pathogenesis. Established PCR protocol is sensitive for identification of prtH gene.

Bacterial Proteins ; Blotting, Southern ; Cloning, Molecular ; Cysteine Endopeptidases ; genetics ; DNA, Bacterial ; genetics ; metabolism ; Deoxyribonuclease BamHI ; metabolism ; Deoxyribonuclease HindIII ; metabolism ; Polymorphism, Genetic ; Porphyromonas gingivalis ; genetics ; Species Specificity

No abstract available.
DNA Restriction Enzymes* ; DNA* ; Ribotyping* ; Staphylococcus aureus* ; Staphylococcus*

OBJECTIVETo investigate the genomic variation of Epstein-Barr virus (EBV) and its significance in nasopharyngeal carcinogenesis.

METHODSForty nasopharyngeal carcinoma (NPC) biopsy tissues were used for detection of EBV BamHI f variant and LMP1 XhoI-loss by polymerase chain reaction (PCR), nested PCR, and RFLP (restriction fragment length polymorphism). Forty-eight samples of peripheral blood mononuclear cells (PBMC) taken from apparently healthy adult individuals were used for detection of LMP1 XhoI-loss. Three samples of amplified LMP1 exon 1 DNA from B95-8 cell line and 2 NPC tissues (one having XhoI-loss and the other having Wt-XhoI/XhoI-loss) were sequenced.

RESULTSThirty out of the 40 NPC cases (30/40, 75%) harbored EBV BamHI f variant and the remaining 10 (10/40, 25%) harbored BamHI F prototype. Thirty out of the 39 NPCs (30/39, 76.9%) showed single EBV LMP1 XhoI-loss, 7 (7/39, 18.0%) showed single LMP1 Wt-XhoI (presence of a XhoI site in exon 1 of LMP1 gene, as in B95-8 cell line), and 2 (2/39, 5.1%) showed both LMP1 Wt-XhoI and XhoI-loss. Thirty-eight of the 39 NPCs (97.4%) showed EBV LMP1 XhoI-loss or/and BamHI F variant. In the NPC tissue (1 case only) showing the prototype of Wt-XhoI/BamHI "f", there were several base substitutions, including 5 missense mutations and 2 silent mutations present in LMP1 exon 3, on DNA sequencing. On the other hand, 10 out of the 48 samples of PBMC taken from apparently healthy individuals could be amplified successfully by nested PCR for detection of LMP1 XhoI site. All of these 10 samples carried the prototype of EBV LMP1 Wt-XhoI.

CONCLUSIONSThe majority of EBV present in neoplastic cells of NPC is of BamHI "f" variant and/or possesses LMP1 XhoI-loss, as compared with that in healthy individuals. This genomic variation of EBV may bear some roles in the development and progression of NPC.

Adult ; Aged ; Binding Sites ; genetics ; DNA, Viral ; genetics ; metabolism ; Deoxyribonuclease BamHI ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Mutation ; Nasopharyngeal Neoplasms ; virology ; Sequence Deletion ; Viral Matrix Proteins ; genetics

Twenty-five isolates of Pythium species were identi6ed and classified on the basis of RFLP(restriction fragment length polymorphism) of ITS(internal transcribed spacer) region in ribosomal DNA. and M-13 PCR markers. Eight restriction endonucleases were used to investigate the genetic relatedness among isolates P. graminicola and P. arrhenomanes as well as P. aphanidermatum and P. delience produced identical band patterns with all restriction endonucleases used and M-13 markers. P. myriotylum and P. catenulatum also formed tight cluster on the basis of RFLP of ribosomal DNA but produced distinct band pattern with M-13 PCR markers. No intraspecitic variations were observed with RFLP of ITS region in ribosomal DNA. Molecular analysis based on M-13 marker demonstrated that each species produced distinct band patterns. Intraspecific variation of P. ultimum and P. torulosum was observed with M-13 markers.
DNA Restriction Enzymes ; DNA, Ribosomal* ; Polymerase Chain Reaction* ; Polymorphism, Restriction Fragment Length* ; Pythium*

G and P tying of group A porcine rotaviruses (P(o)RV) from field fecal samples were performed using reversetranscriptase polymerization chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis. After amplifying full length VP7 and partial length VP4 genes, restriction endonucleases were used to digest and analyze the cutting pattern of the gene products. After analysis of digests with restriction endonucleases, seven and six RFLP types were observed for VP7 and VP4, respectively. The G typing analysis of 50 fecal samples revealed that 68% (34/50) were G4, which included G4-like (22/50); 22% (11/50) were G5; 6% (3/50) were G4 and G5 mixed types. The P typing analysis of the same fecal samples revealed that 36% (18/50) were P2B, 52% (26/50) were P9, 1 sample (2%) was a mixture of P2B and P9. Combinations of G and P types, the G4P2B and G4P9 types including G4-like accounted for 26% (13/50) and 32% (16/50), respectively. The G5P2B and G5P9 type also represented 4% (2/50) and 18% (9/50) of the samples. No G3 and G11 or other new P types were identified from the samples tested. Information on the G and P types and G/P combinations in the field fecal samples is useful for developing more effective PoRV vaccines and understanding the epidemiology of PoRV infections in the field.
DNA Restriction Enzymes ; Epidemiology ; Polymerization ; Polymers ; Polymorphism, Restriction Fragment Length* ; Rotavirus* ; Vaccines

BACKGROUND: Recent restriction enclonuclease analysis studies hsve revealed that MCV DNA can be classified into two major types, designated MCV-1 and MCV-2, by th:ir restriction enzyme cleavsge patterns. In earlier reports of MCV DNA analysis, MCV-2 was the main virus type found in genital lesions. However many recent studies cienied the relationship between virus type and anatomical distribution. OBJECTIVE: The purpose of this study was to examine the ratio of MCV-l to MCV-2 in Korean isolates of MCV DNA and the relationship between MCV subtypes and with clinical features such as anatomical location, age, sex, numiber of lesions, and atopic dermatitis. METHODS: MCV DNA extrated from 112 cases of Korean patients waa examined by restriction endonuclease analysis using Brtm HI. RESULTS: 1. MCV-1 was found in 108 of 112 (96.4%) patients and MCV-2 in of 112 (3.6%) patients. The ratio of MCV-1 to MCV-2 wss 28:1. 2. There was no significant ciprrelation between MCV subtypes and the age, sex, number of lesions, atopic dermatitis, and anatoimic loction. 3. Lesions induced by MCV-1 MCV-2 were indistinguishable on the brsis of size and form. CONCLUSION: This study showis that the ratio of MCV-1 to MCV-2 was 28:1 in Korean molluscum contagiosum patients and there was no relationship between MCV subtyies and lesional morphology or snatomical distribution.
Dermatitis, Atopic ; DNA ; DNA Restriction Enzymes ; Humans ; Molecular Epidemiology* ; Molluscum contagiosum virus* ; Molluscum Contagiosum*

To use recombinant viruses with wider host range as viral insecticides, we investigated the characteristics and pathogenicity of host range expanded recombinant viruses in insect cells. We compared host range expanded recombinant viruses, RecS-B6 and RecB-8, constructed by cotransfection of Autographa californica nuclear polyhedrosis virus (AcNPV) and Bombyx mori NPV (BmNPV), to host range expanded AcNPV, Ac-BH, by substitution of the 0.6 Kb fragment of the BmNPV helicase gene. Restriction endonuclease profiles of RecS-B6 and RecB-8 DNAs were different from those of parent viruses. Nucleotide sequence analysis of the 0.6 Kb region in the putative helicase gene of RecS-B6 and RecB-8 showed that their structures were identical to the counterpart region of BmNPV Comparison of viral replication of these recombinant viruses in Sf-21 and BmN-4 cells showed that Ac-BH, compared to wild type viruses, replicated well in BmN-4 cells but poorly in Sf-21 cells. In contrast, RecS-B6 and RecB-8 replicated relatively well in both cells compared to parent viruses. These results may imply that random genomic recombinant viruses, RecS-B6 and RecB-8, possess better potential as viral pesticides than helicase-mediated recombinant virus, Ac-BH.
Base Sequence ; Bombyx ; DNA ; DNA Restriction Enzymes ; Host Specificity* ; Humans ; Insecticides ; Insects* ; Nucleopolyhedrovirus ; Parents ; Pesticides ; Virulence*

OBJECTIVE: This study was performed to find out mitochondrial deoxyribonucleic acid mutations in preeclampsia because Mendelian models fail to explain all the patterns of inheritance in preeclampsia. METHODS: Ten preeclampsia patients and two of their related family members who have the obstetric history of preeclampsia were studied. The mitochondrial transfer ribonucleic acidleu[UUR] gene was amplified using polymerase chain reaction, cut by a restriction endonuclease (Apa , and also sequenced to see the whole gene. RESULTS: There were neither the known mutation at Nucleotide 3243 nor other mutations on the mitochondrial transfer ribonucleic acidleu[UUR] gene in these objects. CONCLUSION: It seems that the known mutation of mitochondrial transfer ribonucleic acidleu[UUR] gene is not so frequently detected in preeclampsia of South Korean, But it could not be concluded how many South Korean women with preeclampsia have the mutation.
DNA ; DNA Restriction Enzymes ; Female ; Humans ; Point Mutation* ; Polymerase Chain Reaction ; Pre-Eclampsia* ; RNA* ; Wills

Gene cloning is one of the most important and widely used technologies in molecular biology research. Generally, DNA fragment is cut with restriction enzyme, and then the product is ligated to a linearized vector with complementary sticky end or blunt end by DNA-ligase. This traditional DNA cloning method requires compatible enzyme recognition sites existing in both PCR fragment and targeted vector. Several ligase-free methods have been established to avoid the using of restriction enzyme. However, those methods are time-consuming, labor-intensive and expensive. To overcome these shortcomings, we developed an Exonuclease III based DNA cloning method that takes only 30 minutes with high cloning efficiency and significant economic advantage. Therefore, this method is suitable for large-scale gene cloning.
Cloning, Molecular ; methods ; DNA ; chemistry ; DNA Restriction Enzymes ; Exodeoxyribonucleases ; chemistry ; Genetic Vectors ; Polymerase Chain Reaction