Diagnosis of mycobacterial infection is dependent upon the isolation and identification of causative agents. The procedures involved are time consuming and technically demanding. To improve the laborious identification process mycobacterial systematics supported by gene analysis is feasible, being particularly useful for slowly growing or uncultivable mycobacteria. To complement genetic analysis for the differentiation and identification of mycobacterial species, an alternative marker gene, rpoB encoding the B subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 52 reference strains of mycobacteria including Mycobacterium tuberculosis H37Rv (ATCC 27294) and clinical isolates by the PCR. The nucleotide sequences were directly determined (306 bp) and aligned using the multiple alignment algorithm in the MegAlign package (DNASTAR) and MEGA program. A phylogenetic tree was constructed with a neighborhood joining method. Comparative sequence analysis of rpoB DNA provided the basis for species differentiation. By being grouped into species-specific clusters with low sequence divergence among strains belonging to same species, all the clinical isolates could be easily identified. Furthermore RFLP analysis enabled rapid identification of clinical isolates.
Base Sequence ; Classification ; Complement System Proteins ; Diagnosis ; DNA ; DNA-Directed RNA Polymerases ; Mycobacterium tuberculosis ; Polymerase Chain Reaction* ; Polymorphism, Restriction Fragment Length* ; Residence Characteristics ; Sequence Analysis*

No abstract available.
Antibodies, Monoclonal* ; Glycoproteins* ; Humans*

No abstract available.
Epithelial Cells* ; Salmonella typhimurium* ; Salmonella*

The gene encoding the 66 kilodalton (kDa) protein of Borrelia hermsii HS1 was cloned and sequenced. Chromosomal DNA was prepared from purified B. hermsii and used in construction of genomic library. The library was screened for positive clones by 314 bp DIG-labeled probe synthesized on the basis of the part of the sequence of B. hermsii. Positive clone was subcloned into p2ErO vector and was designated as pBH11. pBH11 were subcloned into pBluscript vector and were designated as pBH11-1 (500 bp), pBH11-2 (800 bp), pBH11-3 (600 bp) and pBH11-4 (800 bp). The plasmids were sequenced and determined the nucleotide sequence of p66. The open reading frame of the p66 consisted of 1803 base pairs coding for 600 amino acid protein. The basic information on the p66 gene of B. hermsii HS1 obtained from this study will be useful for further analysis and experiment of pathogenesis of the borrelia.
Base Pairing ; Base Sequence ; Borrelia* ; Clinical Coding ; Clone Cells* ; Cloning, Organism* ; DNA ; Genomic Library ; Open Reading Frames ; Plasmids

Human cytomegalovirus (HCMV) isolated from Korean patients is different in the antigenic and genomic structure of gB from the laboratory-adapted strain. To dissect the reactivity to HCMV glycoprotein B (gB) domains, each domain gene of gB of HCMV SNUCH1, Korean isolate, was amplified from the extracted DNA of the virus-infected fibroblasts with the specific primers by polymerase chain reaction (PCR). Amplified DNA was cloned into pcDNA3. Immunofluorescent staining and western blot analysis revealed that the expressed gB in mammalian cells was immunoreactive and equivalent to the naturally expressed gB in virus-infected fibroblasts. The antigenic component reactive with monoclonal antibodies, MCMVA 57, 88, and 98 appeared at the D3 domain of gB molecule, and that with MCMVA 66 and 135 at the D2b domain. Antibody titer was measured with HCMV-infected fibroblasts and the domains of gB expressed in mammalian cells. There was no correlation between the antibody titer to the whole HCMV and neutralizing antibody titer, and between the antibody titer to whole HCMV and whole gB. It was more reasonable to use whole gB than whole HCMV in the comparison with the neutralizing antibody titer. D3 was representative domain in gB molecule in the anti-gB reactivity. Conclusively it is highly recommendable to use the representing isolates in Korea and its domains for the detection of antibody or the analysis of antigen in the aspect of immunological properties and molecular structures.
Antibodies, Monoclonal ; Antibodies, Neutralizing* ; Blotting, Western ; Clone Cells ; Cytomegalovirus* ; DNA ; Epitopes* ; Fibroblasts ; Glycoproteins ; Humans* ; Korea* ; Molecular Structure ; Polymerase Chain Reaction

No abstract available.
Antibodies, Monoclonal* ; Herpesvirus 3, Human*

No abstract available.
Cytomegalovirus* ; Humans*

No abstract available.
Humans* ; Hybridomas*

No abstract available.
Clone Cells* ; Humans* ; Mycobacterium tuberculosis* ; Mycobacterium* ; T-Lymphocytes*

No abstract available.
Salmonella*