One hundred and thirty trimethoprim-resistant R plasmids derived from of Escherichia coli isolated from clinical specimens and feces of healthy collegians were examined for incompatibility, EcoRI endonuclease restriction fragment pattern, and Southern hybridization with DHFR I, II, III, V, and VII probe. 1. Most trimethoprim-resistant R plasmids were resistant to ampicillin, tetracycline, chloramphenicol, gentamicin, and kanamycin, and showed multiple drug resistance and various antimicrobial resistance patterns. 2. Trimethoprim-resistant R plasmids ranged from 90 to 50 kilobase and 42.3% of R plasmids tested were classified to incompatibilty group Inc FI, Inc FII or Inc FIV, 3. Among 48 random selected R plasmids from various origin, 14 R plasmids (including 9 of 14 Inc FII plasmids and 3 of 14 Inc FI plasmids) hybridized with DHFR VII oligonucleotide probe but others did not respond to any of DHFR probes used. 4. Most R plasmids showed various EcoRI endonuclease fragments and different reaction sites by Southern hybridization. Six plasmids showed identical or nearly identical molecular weight, EcoRI endonuclease fragment patterns and different sites of Southern hybridization. But 2 Inc FII plasmids derived from urine and feces showed identical pattern. These findings, if confirmed by further studies, suggest that normal flora E. coli can act as reservoir of resistant genes and, consequently, as a factor in the dissemination of these genes among enteric pathogens and need to be examined further.
Ampicillin ; Chloramphenicol ; Deoxyribonuclease EcoRI ; Drug Resistance, Multiple ; Escherichia coli* ; Escherichia* ; Feces ; Gentamicins ; Immunodeficiency Virus, Feline ; Kanamycin ; Molecular Biology* ; Molecular Weight ; Plasmids ; R Factors ; Tetracycline ; Trimethoprim Resistance* ; Trimethoprim*

One hundred and twenty-two strains of E. coli isolated from urinary tract infection were examined for antibiogram, transferability of trimethoprim (Tp) resistance, incompatibility with F group plasmid and southem hybridization with DHFR I, II, and III probe of Tp-resistant R plasmids. 1. Among 172 Gram negative bacilli isolated from urinary tract infection, 122 (70.9%) were E. coli and 75 strains of them were resistant to trimethoprim (Tp). Most of Tp-resistant isolates were also resistant to penicillins (ampicillin, carbenicillin, and ticarcillin), aminoglycosides (kanamycin and gentamicin), and sulfisoxazole but almost all strains were susceptible to cephalosporins. 2. Most of Tp-resistant strains and E. coli transconjugant derived from them showed multiple drug resistance and various antimicrobial resistance patterns. 3. Thirty-three Tp-resistant strains (45.2%) transferred 35 Tp-resistant plasmids to E. coli recipients but among them 6 transconjugants did not show retransfer of resistance and plasmid DNA were not detected in 2 transconjugants after resistance transfer. 4. Tp-resistant R plasmids ranged from 157 to 67 kb and 8 R plasmids were classified to incompatibilty group IncFI or IncFII ranging from 120 to 83 kb. Three and two R plasmids belonged to IncFII showed similar molecular weight, resistance pattern, and reaction site by southern hybridization with DHFR I probe. Twenty-five plasmids specifically responded on various EcoRI endonuclease fragments to DHFR I probe but not to DHFR II or DHFR III probe. These findings suggest that most of Tp- resistant R plasmids from urine isolates of E. coli were derived from various sources but some plasmids including IncFII R plasmids were probably originated from same or similar sources.
Aminoglycosides ; Carbenicillin ; Cephalosporins ; Deoxyribonuclease EcoRI ; DNA ; Drug Resistance, Multiple ; Escherichia coli* ; Escherichia* ; Microbial Sensitivity Tests ; Molecular Weight ; Penicillins ; Plasmids ; R Factors ; Sulfisoxazole ; Trimethoprim Resistance* ; Trimethoprim* ; Urinary Tract Infections

BACKGROUND: We performed competitive nested polymerase chain reaction (PCR) to evaluate the clinical utility of quantitative measurement of HBV DNA by PCR and it's correlation with other serologic hepatits B markers. Because hepatitis markers such as HBsAg, HBeAg, anti-HBe can not accurately reflect the replication of hepatitis B virus (HBV). METHODS: The internal standard was generated from the HBV core gene by point mutation, which would result in restriction site for the restriction enzyme Eco RI and performed competitive nested PCR followed by densitometric scanning of the amplified products of agarose gel. RESULTS: The sensitivity of nested PCR was 5 molecules in direct observation of agarose gel, but because of the background effect as taking polaroid photo graph it was 50 molecules by using densitometer. When DNA pellets for original 250 microL serum were diluted with 40 microL distilled water the low detection limit was 5.0 x10(3) molecules/microL, however it could be lowered when less diluted. Lower detection limit of densitometer was 6.25 pg by twofold serial dilution of 100 pg of purified HBV DNA PCR products, and regression showed y=0.93x-0.33 (y : density, x : concentration, 6.25 pg considered as 6.25 density). The reproducibility of the densitometer from high concentration was 4.3 +/-0.6 x10(6) molecules/microL(mean +/-SD, CV 14%), and low concentration was 3.7 +/-0.7 x10(4) molecules/microL(mean +/-SD, CV : 20%) Higher concentration of HBV DNA in HBeAg positive cases comparing with HBeAg negative cases was statistically significant (p<0.01). There was no correlation between HBV DNA concentration and serum value of alanine aminotransferase. CONCLUSION: Quantification of HBV DNA should be very useful in clinical follow-up of Post-therapy Patients and in anticipating Prognosis and infectivity of the disease, especially in cases of atypical hepatitis B and hepatitis B without seroconversion of routine hepatitis B markers. The shortcoming of the method seemed to be a rough estimate of HBV concentration as measuring the ratio of specimen/internal standard of two consecutive concentration among 10 folds serially diluted internal standard.
Alanine Transaminase ; Deoxyribonuclease EcoRI ; DNA ; Follow-Up Studies ; Hepatitis B e Antigens ; Hepatitis B Surface Antigens ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis* ; Humans ; Limit of Detection ; Point Mutation ; Polymerase Chain Reaction* ; Prognosis ; Sepharose ; Water

OBJECTIVETo observe the characteristics of changes of p13E-11 labelled 4q35 EcoRI fragments and to make a gene diagnosis of facioscapulohumeral muscular dystrophy(FSHD).

METHODSGenomic DNA was extracted and was digested by EcoR I /Bln I. After pulsed field gel electrophoresis, it was hybridized with probe p13E-11 by Southern blot. The illness was diagnosed as FSHD when the 4q35 EcoRI fragment was smaller than 38 kb.

RESULTSIn 26 cases of FSHD, the fragments of 20 cases were smaller than 38 kb. The positive rate was 76.92%. In 12 cases of FSHD family members, the fragments of 2 cases were smaller than 38 kb. All fragments of the 21 controls were greater than 38 kb.

CONCLUSIONIt was rather good to use <38 kb as a standard for diagnosis of FSHD. The positive rate of FSHD was similar to that from the references.

Adolescent ; Adult ; Child ; Child, Preschool ; Chromosome Mapping ; Chromosomes, Human, Pair 4 ; genetics ; DNA Fragmentation ; Deoxyribonuclease EcoRI ; metabolism ; Female ; Genes ; Humans ; Male ; Middle Aged ; Molecular Diagnostic Techniques ; Muscular Dystrophy, Facioscapulohumeral ; diagnosis ; genetics ; Restriction Mapping ; Young Adult

OBJECTIVETo elucidate the structural polymorphism of EcoR I fragment within chromosomes 4q35 and 10q26 in the Chinese population and investigate the relationship of plasticity, translocation and somatic mosaicism in these domains with deletion of D4Z4 repeated units.

METHODSOne hundred and ten unrelated healthy individuals from a random Chinese population were investigated. The genomic DNA was extracted from peripheral blood lymphocytes according to the specific procedure designed to minimize DNA shearing, then digested with EcoR I or double digested with EcoR I and Bln I. The cleaved DNA was separated by pulsed field gel electrophoresis (PFGE) and Southern blotted with the probe p13E-11. The sizes of EcoR I fragments were calculated by "curve fitting" according to the MidRange PFG marker and the alleles were assigned to their respective chromosomes based on their Bln I sensitivity. Data were analyzed using a commercially available statistical package (Version 11.0 SPSS).

RESULTSSeventy-seven point three per cent (85/110) of the unrelated healthy individuals displayed a standard configuration distribution. The mean and median of 4q35 repeat arrays are (87.9+/-3.3) kb and 78.5 kb respectively, whereas the mean and median of 10q26 homologous arrays are (90.1+/-4.1) kb and 73.0 kb. Repeat size distributions between both of them were of no significance according to the t test (P>0.05). 19.1% (21/110) of the individuals displayed a translocation repeat array configuration on chromosomes 4 and 10. No significant difference was detected between 4q-->10q translocation and 10q-->4q translocation according to Chisquare test (Chi2 test=0.053, P>0.05). Somatic mosaicism was observed in 3.6% (4/110) of the subjects and less than 35 kb 10-type array was found in 14.5% (16/110) of the individuals.

CONCLUSIONThe structural polymorphism and dynamic behaviors of EcoR I fragments within 4q35 and 10q26 were demonstrated in this study using PFGE. The occurrence of frequent translocations and somatic mosaicism between 4q35 and 10q26 subtelomeric domains in the Chinese population further confirmed that mitotic interchromosomal gene conversion or translocation might be a major mechanism relating to the deletion of D4Z4 units.

Adult ; Asian Continental Ancestry Group ; genetics ; Chromosomes, Human, Pair 10 ; Chromosomes, Human, Pair 4 ; Deoxyribonuclease EcoRI ; metabolism ; Electrophoresis, Gel, Pulsed-Field ; Female ; Humans ; Male ; Middle Aged ; Mosaicism ; Muscular Dystrophy, Facioscapulohumeral ; genetics ; Polymorphism, Genetic ; Translocation, Genetic

OBJECTIVETo provide DNA molecular marker for identification of Nelumbo nucifera by exploring the differences of nrDNA-ITS sequence of N. nucifera originated from different habitats.

METHODTo compare nrDNA-ITS base sequence using specific PCR-ITS.

RESULTThe completed sequence of ITS and 5.8 S rDNA, and the partial sequences of 18S rDNA and 26S rDNA, totally 750 bp, from N. nucifera were obtained. The differences among N. nucifera from different habitats and from different cultivars were found.

CONCLUSIONThe method can be used to identify N. nucifera among different species and to distinguish their fakes. It provided the basis for identifying N. nucifera from different geographical regions by comparison of their ITS sequences.

Base Sequence ; China ; DNA, Plant ; chemistry ; genetics ; metabolism ; DNA, Ribosomal Spacer ; classification ; genetics ; Deoxyribonuclease EcoRI ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Drug Contamination ; prevention & control ; Geography ; Nelumbo ; classification ; genetics ; Phylogeny ; Plants, Medicinal ; classification ; genetics ; Polymerase Chain Reaction ; RNA, Ribosomal ; genetics ; RNA, Ribosomal, 18S ; genetics ; RNA, Ribosomal, 5.8S ; genetics ; Sequence Analysis, DNA ; Species Specificity