No abstract available.
DNA* ; Plasmids* ; Restriction Mapping* ; Yersinia enterocolitica* ; Yersinia*

BACKGROUND: Methicillin-resistant Staphylococcus aureus is a major etiologic agent of hospital acquired infection. Coagulase negative staphylococci (CNS) species are previously regarded as contaminants. However nowadays CNS were regarded as an important cause of bacteremia. So in this study we wanted to analyze the patterns of plasmids and antimicrobial susceptibility test of Staphylococcus species isolated from clinical specimens. METHOD: Plasmid DNA was extracted and then processed through restriction enzyme digestion for plasmid analysis of S. aureus and antimicrobial susceptibility, which was done by agar dilution method. For S. epidermidis plasmid analysis was done without enzyme digestion. RESULTS: All of MRSA have 1 to 5 plasmids. There exists 6 patterns of S. aureus plasmid without enzyme digestion. With EcoRI and HindIII digestion pattern were more distinct and clear. For S. epidermidis enzyme digestion is not needed. Antimicrobial susceptibility patterns of S. aureus are simple whereas S. epidermidis showed variable patterns. CONCLUSIONS: For the plasmid analysis of S. aureus restriction enzyme digestion is required and for the S. epidermidis, the pattern of plasmids are variable so without restriction enzyme analysis we can obtain several patterns. Plasmid analysis will be used as a good epidemilogical tool for Staphylococcus.
Agar ; Bacteremia ; Coagulase ; Digestion ; DNA ; DNA Restriction Enzymes* ; Methicillin-Resistant Staphylococcus aureus ; Plasmids* ; Restriction Mapping ; Staphylococcus aureus* ; Staphylococcus*

29 isoniazid (INH) resistant isolated strains and INH sensitive reference strain (H37Rv) of Mycobacterium tuberculosis were analysed by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and NciI restriction mapping for the detection of mutations in katG gene and inhA gene. The katG gene was divided into 3 parts (Akat, Bkat, Ckat; each part is about 800 bp) and amplified, inhA gene was amplified as a whole. Each of the amplified 800 bp DNA was digested into small fragments of less than 400 bp with restriction enzymes for the direct PCR-SSCP analysis. Firstly, 10 strains were analysed. All the 10 isolates showed clearly distinct SSCP patterns in Bkat from that of the reference strain, but only two isolates showed distinct SSCP patterns in Akat, and no isolated strain showed any distinct SSCP patterns in Ckat. 10 isolates also showed distinct SSCP patterns in inhA. NciI restriction mapping of Bkat showed mutation in codon 463 in 7 strains among 10 isolated strains. With these results an early detection strategy for the INH resistant M. tuberculosis was applied to the rest of 19 isolated INH resistant strains. Firstly, isolates were screened by Ncsl mapping in Bkat, and 13 strains showed mutations in codon 463. Secondly, the rest of 6 INH resistant isolates were analysed by PCR-SSCP with restriction enzyme digestion (PCR-SSCP-RE) in Bkat, and all the strains showed distinct SSCP patterns from that of the INH sensitive reference strain. This proved our strategy as effective and economic and time saving method in early detection of INH resistant M. tuberculosis.
Codon ; Diagnosis* ; Digestion ; DNA ; Isoniazid* ; Korea* ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymorphism, Single-Stranded Conformational ; Restriction Mapping ; Tuberculosis

BACKGROUND: In Korean population, antigen frequencies of HNA-1a, HNA-1b, and HNA-2a are determined using a serological and genotyping method. However, no study has been done to assess the gene frequencies of HNA-4a and HNA-5a. It has been reported that the antibody against HNA-4a is associated with alloimmune neutropenia and autoimune neutropenia; however, there is no confirmed clinical report on anti-HNA-5a-related disorders. The aim of this study was to determine HNA-4a and HNA-5a gene frequencies among the Korean population. METHODS: Genotyping of HNA-4a and HNA-5a genes of 110 healthy and unrelated Korean donors was performed using a polymerase chain reaction with sequence-specific primers and an allelespecific restriction enzyme analysis. RESULTS: We found that the gene frequencies of HNA-4a and HNA-5a were 0.99 (107/110) and 0.96 (3/110), respectively, among the Korean population. But only the ones of the latter was significantly higher (P<0.01) than in the one of Caucasians. CONCLUSIONS: The gene frequencies of HNA-4a and HNA-5a were determined. We also identified an individual who was the HNA-5a-negative homozygote for the granulocyte panel that could be used for anti-HNA-5a antibody identification.
Gene Frequency* ; Granulocytes ; Homozygote ; Humans* ; Neutropenia ; Neutrophils* ; Polymerase Chain Reaction ; Restriction Mapping ; Tissue Donors

OBJECTIVETo investigate a 272 base pair section of the 5'-non-coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV)-negative human subjects (not patients).

METHODSThis sequence section bears interest because (1) it harbors several potential methylation (Cp-rich) sites, and (2) it represents the largest part of its internal ribosomal entry site. A pre-PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences.

RESULTSThe suspected HCV-specific sequence was found in the DNA of each subject tested. The pre-PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena.

CONCLUSIONSThe results provide formal proof that these HCV-specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences.

Unstable hemoglobins (Hb) are variants of adult Hb that tend to precipitate and form insoluble inclusions (Heinz bodies) within red blood cells (RBC) and RBC precursors. More than 100 structurally different unstable Hb variants showing broad spectrum of manifestations from asymptomatic to severe hemolytic anemia and dyserythropoiesis have been discovered. Hydroxyurea is a potent ribonucleotide reductase inhibitor and have been proposed as a new therapy for beta chain hemoglobinopathies through activation of gamma chain synthesis. We treated two patients (A : son, B : father) with highly unstable Hb diagnosed as Hb Madrid [Beta 115(G17)Ala->Pro] by direct DNA sequencing and restriction enzyme analysis. Our patients received hydroxyurea in dosages varying from 0.75g to 1.3g daily for 89 weeks. We could not show the clinical and hematological improvements after hydroxyurea therapy in thses patients. Optimization of dosage and long term side effects of hydroxyurea should be studied further.
Adult ; Anemia, Hemolytic ; Erythrocytes ; Hemoglobinopathies ; Humans ; Hydroxyurea* ; Restriction Mapping ; Ribonucleotide Reductases ; Sequence Analysis, DNA

BACKGROUND: M protein is the major virulence factor of group A streptococci (GAS). As the emm gene, which encodes the M protein, has a variable sequence at 5'-terminus by each M type, it can be classified into several restriction-fragment length polymorphisms (RFLP) by restriction enzymes. Pulsed-field gel electrophoresis (PFGE) is useful for investigating clonal outbreaks or bacterial transmission. Molecular epidemiological analysis using emm-RFLP and PFGE was performed on GAS isolated from bacteremia. METHODS: Twenty-eight strains of GAS isolated from patients with bacteremia were included. Medical records of 27 cases were reviewed retrospectively to see the prognosis of GAS bacteremia. The restriction patterns of emm gene digested with HaeIII enzyme were compared by each emm genotype. Macrorestriction patterns were produced by PFGE after digestion of chromosomal DNA with SmaI and ApaI enzymes. RESULTS: All but 2 cases with toxic streptococcal syndrome survived. The DNA fragments of emm-HaeIII restriction showed 3 to 4 bands. Each emm genotype showed a different emm-HaeIII restriction pattern, except emm49 and SP2346, of which the restriction pattern was similar to that of emm1. Thirteen different PFGE patterns were observed by SmaI, and emm6, emm12, and emm75 were not cut with SmaI. All 28 strains were cut with ApaI, which yielded 17 different PFGE patterns. All but emm13 showed a high concordance between the PFGE patterns and the emm genotypes. CONCLUSIONS: The patients with GAS bacteremia showed a relatively good prognosis. We found that the restriction enzyme analysis of emm gene was rapid, simple and inexpensive to perform, and that PFGE was useful for classifying GAS strains because it generally discriminated well between each emm genotype. Although the number of strains studied was small, GAS bacteremia was not due to a single clone.
Bacteremia* ; Clone Cells ; Digestion ; Disease Outbreaks ; DNA ; Electrophoresis, Gel, Pulsed-Field* ; Genotype ; Humans ; Medical Records ; Polymorphism, Restriction Fragment Length ; Prognosis ; Restriction Mapping ; Retrospective Studies ; Virulence

BACKGROUND: Yersinia pseudotuberculosis, a member of genus Enterobactericeae, is a main etiologic organism of diarrhea in childhood. Because a mouse and a unchlorinated spring water are main reservoirs of Y. pseudotuberculosis, the strains from a contaminated spring water and mouse could be involved in human epidemic. The purpose of this study was to investigate a clonality between the strains from patients and those from an unchlorinated spring water and a mouse by restriction enzyme analysis of plasmid DNA and pulsed-field gel electrophoresis (PFGE). METHOD: We isolated 15 Y. pseudotuberculosis strains including 8 isolates from patients (S1-S8), 6 isolates from mountain water (W1-W6), 1 isolate from a mouse (M1) in northeast area of Seoul. Plasmid and chromosomal DNA of all strains were analyzed by REAP with Bam H1 restriction and by PFGE with Xba I restriction , respectively. RESULTS: Restriction enzyme analysis of plasmid DNA was classified into type B and type D. All 7 strains of serotype 15 were classified as type B and 8 strains of serotype 4b were classified as type D. PFGE were classified into 6 different types. Among them, strains of PFGE type I, II, III, IV belong to Y. pseudotuberculosis serotype 15 and Y. pseudotuberculosis 4b strains were classified into PFGE type V, VI. S1 and W1 were classified into PFGE type I . S8, W6 and M1 were classfied into PFGE type VI. CONCLUSIONS: PFGE revealed clonality among strains from patients, a water and a mouse. PFGE was more discriminative than REAP to characterize the Y. pseudotuberculosis outbreaks in Korea.
Animals ; Diarrhea ; Disease Outbreaks ; DNA* ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Korea* ; Mice ; Plasmids* ; Restriction Mapping* ; Seoul ; Yersinia pseudotuberculosis* ; Yersinia*

Among the fifty-three clinical isolates of Haemophilus influenzae, nineteen isolates including eight isolates of each biotype I-VIII, six of serotype b (Hib) strains and five of nontypeable strains were characterized by SDS-PAGE about outer membrane protein (OMP), restriction enzyme analysis (REA) and rRNA gene restriction pattems. OMP patterns showed to common band patterns in each H. influenzae isolate. Based on the two major proteins, 31KDa-38KDa, isolated strains were classified into 7 subtypes. In the OMP patterns about biotype and serotype, the specific pattern of each biotype was not distinguishable, but all of the serotype b strains were shown identical unique pattern, therefore it made distinctive difference with nontypeable strains. The digested genomic DNAs with EcoRI were identical result with rRNA gene restriction. It was more subdivided into 10 ribotypes. The most common ribotype I and serotype 1 accounted for 6 strains (31.6%) and 7 strains (36.8%) of the 19 clinical isolates, respectively. Hib isolates that were both OMP subtype 1 and ribotype I accounted for 2 strains (10.5%). In the epidemiologically unrelated strains, the putative association between the subtypes could not be confirmed. According to these results, the three methods were discriminatory and appropriate techniques for epidemiological studies of H. influenzae.
DNA ; Electrophoresis, Polyacrylamide Gel* ; Genes, rRNA* ; Haemophilus influenzae type b ; Haemophilus influenzae* ; Haemophilus* ; Influenza, Human ; Membrane Proteins ; Restriction Mapping* ; Ribotyping

We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F2 population of 107 plants with 320 RFLP, 136 AFLP, and 46 SSR markers. The resulting linkage map consists of 15 linkage groups covering 1,720 cM with an average map distance of 3.7 cM between framework markers. Most RFLP markers (80%) were pepper-derived clones and these markers were evenly distributed all over the genome. Genes for defense and biosynthesis of carotenoids and capsaicinoids were mapped on this linkage map. By using 30 primer combinations, AFLP markers were generated in the F2 population. For development of SSR markers in Capsicum, microsatellites were isolated from two small-insert genomic libraries and the GenBank database. This combined map provides a starting point for high-resolution QTL analysis, gene isolation, and molecular breeding.
Capsicum ; Carotenoids ; Chromosome Mapping* ; Clone Cells ; Databases, Nucleic Acid ; DNA Shuffling ; Genome* ; Genomic Library ; Microsatellite Repeats ; Polymorphism, Restriction Fragment Length