Interferon-Stimulated Genes Response in Endothelial Cells Following Hantaan Virus Infection

The regulation mechanism of interferon (IFN) and IFN-stimulated genes is a very complex procedure and is dependent on cell types and virus species. We observed molecular changes related to anti-viral responses in endothelial cells during Hantaan virus (HTNV) infection. We found that there are two patterns of gene expression, the first pattern of gene expression being characterized by early induction and short action, as in that of type I IFNs,' and the other being characterized by delayed induction and long duration, as those of IRF-7, MxA, and TAP-1/2. Even though there are significant differences in their induction folds, we found that all of IFN-alpha/beta , IRF- 3/7, MxA, and TAP-1/2 mRNA expressions reached the peak when the viral replication was most active, which took place 3 days of post infection (d.p.i.). In addition, an interesting phenomenon was observed; only one gene was highly expressed in paired genes such as IFN-alpha/beta??(3/277-folds), IRF-3/7 (2.2/29.4-folds), and TAP- 1/2 (26.2/6.1-folds). Therefore, IFN-beta, IRF-7, and TAP-1 seem to be more important for the anti-viral response in HTNV infection. MxA was increased to 296-folds at 3 d.p.i. and kept continuing 207-folds until 7 d.p.i.. The above results indicate that IFN-beta works for an early anti-viral response, while IRF7, MxA, and TAP-1 work for prolonged anti-viral response in HTNV infection.

Effect of Leptin on the Expression of Chemokine Genes in THP-1 Cells / 영남의대학술지

BACKGROUND: Leptin is a 16-KDa non-glycosylated peptide hormone synthesized almost exclusively by adipocytes. The well-known function of leptin is regulation of food intake and energy expenditure. Leptin also plays a regulatory role in immune and inflammatory process including cytokine production. The purpose of this study was to investigate the effect of leptin on the expression of several chemokine genes(RANTES, IL-8, MCP-1, IP-10, Mig, MIP-1alpha, MIP-1beta, and GRO-alpha) in THP-1 cells. MATERIALS AND METHODS: Total RNA of THP-1 cells were prepared by Trizol method, and then stimulated with the leptin(250 ng/microliter) or LPS(100 ng/microliter). We examined the expression patterns of various chemokine mRNAs in THP-1 cell lines by RT-PCR and Northern blot. RESULTS: Leptin did not induce the expression of chemokine mRNAs in THP-1 cells. The expression patterns of RANTES, IL-8, MCP-1, IP-10, and Mig mRNAs in THP-1 cells stimulated with leptin and LPS simultaneously was almost same to the patterns of LPS alone-induced chemokine mRNAs. RANTES mRNA expression was independent on the concentrations of leptin. Although leptin did not have strong effect on the expression of RANTES, IL-8, MCP-1, IP-10, Mig, MIP-1alpha, MIP-1beta, and GRO-alpha mRNAs in THP-1 cells, leptin could induce the expression of long isoform of leptin receptor(OB-RL) mRNA, and its expression was elevated in simultaneous stimulation of leptin and LPS. CONCLUSION: These data suggest that leptin is able to induce OB-RL in THP-1 cells, however, leptin has little effect on the expression of pro-inflammatory chemokine genes.

Effect of Leptin on the Expression of Chemokine Genes in THP-1 Cells / 영남의대학술지

BACKGROUND: Leptin is a 16-KDa non-glycosylated peptide hormone synthesized almost exclusively by adipocytes. The well-known function of leptin is regulation of food intake and energy expenditure. Leptin also plays a regulatory role in immune and inflammatory process including cytokine production. The purpose of this study was to investigate the effect of leptin on the expression of several chemokine genes(RANTES, IL-8, MCP-1, IP-10, Mig, MIP-1alpha, MIP-1beta, and GRO-alpha) in THP-1 cells. MATERIALS AND METHODS: Total RNA of THP-1 cells were prepared by Trizol method, and then stimulated with the leptin(250 ng/microliter) or LPS(100 ng/microliter). We examined the expression patterns of various chemokine mRNAs in THP-1 cell lines by RT-PCR and Northern blot. RESULTS: Leptin did not induce the expression of chemokine mRNAs in THP-1 cells. The expression patterns of RANTES, IL-8, MCP-1, IP-10, and Mig mRNAs in THP-1 cells stimulated with leptin and LPS simultaneously was almost same to the patterns of LPS alone-induced chemokine mRNAs. RANTES mRNA expression was independent on the concentrations of leptin. Although leptin did not have strong effect on the expression of RANTES, IL-8, MCP-1, IP-10, Mig, MIP-1alpha, MIP-1beta, and GRO-alpha mRNAs in THP-1 cells, leptin could induce the expression of long isoform of leptin receptor(OB-RL) mRNA, and its expression was elevated in simultaneous stimulation of leptin and LPS. CONCLUSION: These data suggest that leptin is able to induce OB-RL in THP-1 cells, however, leptin has little effect on the expression of pro-inflammatory chemokine genes.

Effect of Interleukin-10 on Lipopolysaccahride/Interferon-gamma- Induced Chemokine Mig Gene Expression

Interferon-gamma (IFN-gamma) is well known as a potent inducer in monokine induced by IFN-gamma (Mig) mRNA expression. Although lipopolysaccharide (LPS) alone is weakly effective on Mig mRNA expression. the stimulation of LPS and IFN-gamma (LPS/IFN-gamma simultaneously has been shown to synergize to produce a high level of Mig mRNA in mouse peritoneal macrophages. In this study, interleukin-10 (IL-10) was found to suppress the LPS/IFN-gamma- induced Mig mRNA expression in cell type- and mouse strain-specific fashion, but IFN-gamma alone-induced Mig mRNA was unaffected by IL-10 under identical experimental conditions. The IL-10-mediated suppression of LPS/IFN-gamma-stimulated Mig mRNA expression was dependent on the concentration of IL-10, and was prevented when the agent was added 2 hours after LPS/IFN-gamma treatment. The suppressive action of IL-10 was dependent on a protein synthesis. However, IL-10 did not reduce the stability of LPS/IFN-gamma-induced Mig mRNA. These data may have important implications for a previously unrecognized role for IL-10 as a regulator of synergistic effect of LPS on the IFN-gamma-induced expression of the Mig gene in macrophages.

Microbial Genome Analysis and Application to Clinical Bateriology / 영남의대학술지

With the establishment of rapid sequence analysis of 16S rRNA and the recognition of its patential to determine the phylogenetic position of any prokaryotic organism, the role of 16S rRNA similarities in the present species definition in bacteriology need to be clarified. Comparative studies clearly reveal the limitations of the sequence analysis of this conserved gene and gene product in the determination of relationship at the pathogenic strain level for which DNA-DNA reassociation exprements still constitute the superior method. Since today the primary structure of 16S rRNA is easier to determine than hybridization between DNA strands, the strength of the sequence analysis is to recognize the level at which DNA pairing studies need to be performed, which certainly applies to similarities of 97% and higher.

Microbial Genome Analysis and Application to Clinical Bateriology / 영남의대학술지

With the establishment of rapid sequence analysis of 16S rRNA and the recognition of its patential to determine the phylogenetic position of any prokaryotic organism, the role of 16S rRNA similarities in the present species definition in bacteriology need to be clarified. Comparative studies clearly reveal the limitations of the sequence analysis of this conserved gene and gene product in the determination of relationship at the pathogenic strain level for which DNA-DNA reassociation exprements still constitute the superior method. Since today the primary structure of 16S rRNA is easier to determine than hybridization between DNA strands, the strength of the sequence analysis is to recognize the level at which DNA pairing studies need to be performed, which certainly applies to similarities of 97% and higher.

Development of a Mycobacterial Gene Knock-out System using Sequence-specific Recombinase FLP/FRT and its Application to the Construction of a Rhamnose Biosynthetic Gene rmlD Deletion Mutant

Development of a new and effecient tuberculosis vaccine is very important since the efficacy of the only available vaccine against tuberculosis, BCG, is variable among races and different ages. Attempts to develop attenuated vaccines by disrupting virulence gene(s) specifically in Mycobacterium tuberculosis are now actively being tried after the release of whole genome sequence of M. tuberculosis in 1998. However, disruption of specific genes in M. tuberculosis is still very difficult due to the lack of effective gene knock-out system(s) in mycobacteria. In this study, we developed a novel method to delete specific genes in both Escherichia coli and mycobacteria. This knock-out system is operated by a sequence-specific recombinase FLP and its recognition sequence FRT (FLP/FRT system). Two shuttle vectors (an FLP expressing vector and a gene targeting vector) between Escherichia coli and Mycobacteria were developed. The gene targeting vector contains a kanamycin resistance gene (KmR) flanked by two neighboring genes and two FRTs (FLPrecognition targets). We applied this system to knock-out the rhamnose biosynthetic gene rmlD of Escherichia coli. The upstream and downstream genes of rmlD, rmlB and rmlA, were cloned into the gene targeting vector. After and allelic exchange of E. coli chromosomal rmlB, rmlD, rmlA with vectoral rmlB, FRT-KmR-FRT, rmlA by homologous recombination, FLP-expressing plasmid was introduced to induce the excision of KmR cassette remaining one FRT sequence between rmlB and rmlA. We also demonstrated our shuttle vector could disrupt a target gene (kanamycin resistance gene) in M. smegmatis. These results suggest that our gene knock-out system can be used for the development of an attenuated tuberculosis vaccines and for the functional genomic study of mycobacteria.

Effect of Lipopolysaccharide on the Expression of Chemokine Mig Gene in Mouse Peritoneal Macrophages

No abstract available.

IS6110 - RFLP Analysis using Mycobacterium tuberculosis Isolates from Taegu - Kyungpook Region

No abstract available.

Overexpression, purification and truncation analysis of RmlC protein of Mycobacterium tuberculosis

dTDP-rhamnose provides L-rhamnose to the bridge-like structure between mycolyl arabinogalactan and peptidoglycan of the mycobacterial cell wall. dTDP-rhamnose is composed of glucose-1-phosphate and dTTP by four enzymes encoded by rmlA-D. To determine the region(s) of RmlC protein essential for its dTDP-4-keto-6-deoxyglucose epimerase activity, we overexpressed both whole (202 amino acids) and three different truncated (N-terminal 106 or 150 or C-terminal 97 amino acids) RmlC proteins of Mycobacterium tuberculosis. The RmlC enzyme activity in the soluble lysates of DELTArmlC E. coli strain SPHI874 (DE3 PlysS) expressing the wild type or truncated rmlC genes was initially analyzed by three sequential reactions from dTDP-glucose to dTDP-rhamnose in the presence of purified RmlB and RmlD. All three soluble lysates containing the truncated RmlC proteins showed no enzyme activity, while that containing the wild type RmlC was active. This wild type RmlC was then overexpressed and purified. The incubation of the purified RmlC enzyme so obtained with dTDP-4-keto-6-deoxyglucose resulted in the conversion of dTDP-4-keto-rhamnose. The results show that the truncated regions of the RmlC protein are important for the RmlC enzyme activity in M. tuberculosis.

Effects of interleukin-10 on chemokine KC gene expression by mouse peritoneal macrophages in response to Candida albicans

Chemokine KC has been considered to be a murine homologue of human GRO/MGSA and was identified as chemoattractant for monocytes and neutrophils. This study examined the expression of KC mRNA in thioglycollate-elicited mouse peritoneal macrophages that were stimulated in vitro with Candida albicans (CA). Also examined were the inhibitory effects of IL-10 on the CA-induced expression of KC gene by Northern blot analysis. CA was found to induce chemokine gene expression in a gene-specific manner, CXC chemokine IP-10 mRNA expression was not detected in CA-stimulated macrophages. Maximum KC mRNA expression was observed approximately 2 hr after adding CA. The inhibitory action of IL-10 to CA-induced KC mRNA expression on mouse peritoneal macrophages was independent on concentration and stimulation time of IL-10 and was observed approximately one hour after adding IL-10 and CA simultaneously. IL-10 produced a decrease in the stability of KC mRNA, and CA-stimulated macrophages with cycloheximide blocked the suppressive effect of IL-10. These results suggest that CA also induces chemokine KC from macrophages, and IL-10 acts to destabilize CA-induced KC mRNA and de novo synthesis of an intermediate protein is a part of the IL-10 suppressive mechanism.

The Supressive Effects of Integrin Antibodies on the Infection of Hantaan Virus in Fibroblasts / 영남의대학술지

Pathophysiological mechanism of hemorrhagic fever with renal syndrome (HFRS) is not fully understood. Major clinical findings of HFRS patients are widespread hemorrhage, acute renal failure and shock. Basic lesion is vascular injury with microvascular hemorrhage and relatively little inflammation. According to autopsy findings, renal medulla shows focal hemorrhage, tubular necrosis and interstitial mononuclear infiltrates. The predominant cell type in the renal and pulmonary interstitium is a fibroblast and it participates in the healing process at the injury site by secreting a large amount of extracellular matrix proteins. Cultured human lung fibroblasts and Mongolian gerbil fibroblasts were known to be good host cells for the hantaan virus. It is possible that not only the endothelial cell but also the fibroblast is a target of Hantaan virus and the fibroblast might be involved in the pathogenesis and the healing process in HFRS. Integrins are adhesion molecules, and act as receptors for many extracellular matrix proteins. Recently, there are many reports that cell surface integrins influence on some viral infections or reversely viruses influence on the expression of integrins. The alpha5beta1 integrin is a major receptor for the fibronectin which is an important extracellular matrix protein secreted by fibroblasts. In this study, the role of alpha5beta1 integrin in the infection of Hantaan virus was examined by using anti-alpha5beta1 integrin, anti-alpha5 integrin and anti-beta1 integrin antibodies in chicken embryo fibroblasts (CEF) and Mongolian gerbil fibroblasts(MGF). The treatment of anti-alpha5beta1 integrin antibody in CEF reduced the virion titers 26.8% and the amount of nucleocapsid N protein 32.6% when compared with control CEF. When MGF were treated with anti-alpha5, anti-beta1 and anti-alpha5beta1 integrin antibodies, virion titers were reduced by 26.5%, 29.4% and 28.7% and the amount of nucleocapsid N protein were reduced by 65.2%, 59.7% and 72.6%. These results suggested that alpha5beta1 integrin might act as a receptor for the Hantaan virus or blocking of alpha5beta1 integrin influences on the viral replication in CEF and MGF. It is also possible that the blocking of only one subunit of integrin represents similar results in that of whole molecule.

Effects of Hantaan Virus and IFN-gammaon Induction of Surface ICAM-1 in Primary Cultured Buman Nasal Epithelial Cells and Human Lung Fibroblasts

The primary culture of human nasal epithelial cells was performed using the inferior nasal turbinate tissues, and infected with Hantaan virus to examine the hypothesis of airborne transmission of Hantaan virus in humans. The primary culture cells were identified as epithelial cells by morphologic and immunologic analyses. The viral antigens were detected in the primary human nasal epithelial cells infected with Hantaan virus by immunofluorescence staining. The ICAM-1 induction by Hantaan virus or IFN-gammawas examined in the primary human nasal epithelial cells and human lung fibroblasts (WI-38). Hantaan virus induced the surface ICAM-1 in Wl-38 cells in a time-dependent manner, and IFN-gammainduced the surface ICAM-1 in a dose-dependent manner in HNEC and WI-38 cells. These results revealed that the human nasal epithelial cells are susceptible to Hantaan viral infection supporting the hypothesis of airborne transmission of Hantaan virus in humans. The human lung fibroblasts also might have an important role in the pathogenesis of Hantaan virus through the induction of ICAM-1.

Candida spp.- Induced Cytokine Gene Expression on Mouse Peritoneal Macrophages and NIH 3T3 Fibroblasts

Interaction with Candida albicans (C. albicans) causes secretion of a variety of cytokines by macrophages. Although macrophages and neutrophils are thought to be major effector cell types in inflammation, fibroblasts have also been shown to participate in a wide array of inflammatory reactions. The patterns of cytokine gene (IL-4, IL-6, IL-10) expression have been examined in NIH 3T3 fibroblasts (NIH 3T3) and thioglycollate-elicited murine peritoneal macrophages (macrophages) in response to Candida spp. (C. albicans and C. tropicalis) and compared with NIH 3T3 and macrophage cells stimulated with lipopolysaccharide (LPS) by using RT-PCR. Active and heat inactivated (100'C, 30min) C. albicans and C. tropicalis were used at 1:10 (macrophages or NIH 3T3: yeasts ratio) concentration as a stimulators. Active and inactivated forms of Candidn spp. induced same patterns of cytokine gene expression on macrophages or NIH 3T3 cells. IL-6 mRNA was induced on both macrophages and fibroblasts, but IL-4 mRNA was not induced on both of them. Howerver, IL-10 mRNA expression was observed differently in that it was expressed in macrophages but not in fibroblasts. C. albicans-induced cytokine mRNA expression were same to C. tropicalis-induced one. C. albicans or C. tropicalis-induced cytokines gene expression (IL-4, IL-6, IL-10) showed same patterns to LPS-induced one. These observation provide that, although C. albicans and C. tropicalis have different pathogenic activity, they can induce the expression of Th2 cell-derived cytokine genes with same patterns, and IL-10 gene expression may be in a cell type specific manner. But further experiment with various kinds of fibroblasts is needed to determine cell type-specific manner in C. albicans or C. tropicalis-induced IL-10 gene expression.

Dose effect of LPS on Cytokine mRNA Expressions by Mouse Peritoneal Macrophages

Many studies for LPS-induced cytokines, chemokines gene expressions have been reported, but results are highly diverse. The dose of LPS used, cell type studied or other technical factors may contribute to the differences. We investigated the cytokine mRNA expressions, interleukin-2 (IL-2), IL-6, IL-10 and IFNr, on thioglycollate (TG) elicited-mouse peritoneal macrophages stimulated with various concentrations, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1 ug/ml and 10 ug/ml, of LPS used by reverse transcription polymerase chain reaction (RT-PCR) and northern blot analysis. TG- elicited peritoneal macrophages expressed IL-6 and IFN-r mRNA in a dose-dependent manner. The expressions of IL-2 and IL-10 mRNA were dose-independent manner. In northern blot analysis, IL-6 mRNA expression was detected only in 10 ug/ml LPS concentration for short stimulation time (0.5 h), while IL-10 mRNAs were expressed evenly in all LPS concentrations. The expression of IL-6 mRNA was sustained until 72 h at 10 ug/ml concentration only, but IL- 10 mRNAs of all cases of LPS concentrations were sustained evenly until then. These results suggest that the concentration of LPS required for cytokine induction must be determined differently from gene to gene types, and although LPS concentration 10 ug/ml has not been used as an ordinary concentration in vitro cytokine study, 10 ug/ml of LPS might be more appropriate concentration in the study of IL-6 expression on mouse peritoneal macrophages.

Regulation of Endotoxin - Induced TNF-alpha Gene Expression

It is well known that tumor necrosis factor (TNF-a), interleukin-1, platelet-activating factor (PAF) and arachidonic acid metabolites, such as thromboxane and leukotriens, are major mediators involved in the pathogenesis of endotoxic shock. In this study, we have investigated the effect of pentoxifylline (inhibitor of TNF-a release), BN50739 (PAF antagonist), indomethacin (cyclooxygenase inhibitor) and diethylcarbamazine (lipoxygenase inhibitor) on LPS- induced lethality as well as the relationship between major mediators in endotoxic shock. All inhibitors described above except diethylcarbamazine significantly protected mice against LPS- induced lethality. BN50739 and indomethacin were also effective in protection of TNF-a-induced lethality. The elevation of circulating TNF-a by LPS was significantly blocked by BN50739, but not affected by indomethacin. Convulsion appeared shortly after LPS injection was prevented by BN50739 but not by indomethacin, whereas diarrhea and limited movement was prevented by indomethacin but not by BN50739. These results indicate that i) TNF-a, PAF and cyclooxygenase products are important mediators involved in the pathogenesis of septic shock and ii) TNF-a directly influenced the release or production of PAF as well as cyclooxygenase products, and strongly suggest that i) TNF-a and PAF stimulate the release of each other via positive feedback network but TNF-a and cyclooxygenase products do not form the network and ii) PAF and cyclooxygenase product appear not to affect the release of each other.

Effect of C . albicans on IL - 6 Gene Expression of Human Peripheral Blood Lymphocytes / 대한면역학회지

The gene expressions of Interleukin-6 (IL-6) from human peripheral blood lymphocytes (HPBL) stimulated by C. albicans were investigated by using ELISA (Enzyme linked immunosorbent assay), reverse transcription polymerase chain reaction (RT-PCR) and northern blotting. HPBL (1 X 10'/ml) obtained from normal human peripheral blood lymphocytes were cultured with live C. albicans (LCA) or heat killed C. albicans type A 311 (KCA, 3 X 10/ml) for various times (0.5, 1, 4, 8, 18, 24, 48 and 72 hours). On the purpose of this experiment, we also used lipopolysacchalide (LPS, 10 ug/ml), zymosan (1, 10, 100 ug/ml) as a polysacchaide component of the wall of yeast cells or TNFa (50, 100 ng/ml) as a IL-6 inducers. For observation of the level of IL-6 gene expression, actinomycin D (AD, 5 pg/ml) or cyclohexamide (CHX, 25 ug/ml) was added to HPBL stimulated with LCA for 0.5, 2, 4 hours and the HPBL were assessed for IL-6 mRNA. The highest value for IL-6 activity by LCA were observed at 48 hours reaction, but in the case of KCA, highest value of IL-6 activity was observed at 72 hours reaction and the value was also higher (500 pg/ml) than that of LCA (188 pg/ml). 1L-6 mRNA induced by LCA were detected up to 48 hours but in the case of KCA, the band for IL-6 mRNA were far stronger and appeared until lately than that of LCA. Therefore, the results of IL-6 gene expression agreed with that of ELISA.

Effect of Cytokines on Expression of Intercellular Adhesion Molecule - 1 in Nasal Epithelial Cells / 대한면역학회지

Intexcellular adhesion molecule-10CAM- 1) is an important moleade in aehating immune and inflammatory responses. It is found on the surface of hematopoietic and nonhematopoietic cells. It can act as an adhesive ligand for integrins such as LFA-1 (CD1&/CD11a) and MAC-1 (CD18/ CD11b). ICAM-1 is basally expressed in sigaificant amount on a limited number of cell types, including monocytes and endothelial ceRs. But it is inducible or upregulated by INF-r, IL-1p and TNF-a on many cell types. IL-4, a pleiotropic cytokine and mast cell differentiation factor, is upregulated in human allergic disease and stimulates expression of vascular adhesion molecule-1 (VCAM-1) in endothelial cells. IL-4 also promotes expression of surhce ICAM-1 in human mast cells and dermal fidroblasts. So in allergic rhinitis and asthma, IL-4 may be an important cytokine implicated in the pathogenesis of inflammation. We studied the effect of INF-r and IL-4 on expression of ICAM-1 in human nasal epithelial cells (HNEC). HNEC were prepared by primary culture method of monolayer culture of dissociated cells hom human inferior nasal turbinate mucosa. Nasal mucosa were obtained by partial turbinectomy of septal deviation patients. Primary cultured cells were charaterized as an epithelial cell type by indirect immuno-fluorescence assay using antilmdies against cytokeratin-pan, cytokeratin No. 8, vimentin and von Willebrand factor. Using fluorescence activated cell sorter (Coulter EI1TE), we analyzed the quantitative expression of ICAM-1 on HNEC. Treatment of HNEC with IFN-r (1ng/ml) for 24 hours caused about 8 fold increase of the surface ICAM-1 compared with constitutive expression by mean fluorescence intensity (MIF) but IL-4 had little effect. Theses foundings suggest that IFN-r is a potent ICAM-1 inducer in HNEC and further studies are necessary for the role of IL-4 on HNEC.

Genomic analysis of Mycobacterium foruitum by pulsed-filed gel electrophoresis / 영남의대학술지

Epidemiological studies are important in both the prevention and treatment of mycobacterial infections. This study was initiated to establish the pulsed-field gel electrophoresis (PFGE) method, which are not yet extensively studied. The most apprpriate restriction endonucleases included Dral, AsnI, and XbaI. The optimal PFGE condition was different according to the enzymes used. Two stage PFGE was performed, in case of DraI first stage was performed with 10 seconds of initial pulse and 15 seconds of findA pulse, while the second stage was performed with 60 seconds of initial pulse and 70 seconds of final pu',se. The electrophoresis time for DraI-PFGE was 14 hours for each stage. Electrophoresis was performed for 22 hours, in case of XbaI, with 3 seconds of initial pulse and 12 seconds of final pulse. Electrophoresis was performed for 22 hours, in case of AsnI, with 5 seconds of initial pulse and 25 seconds of final pulse. In all cases the voltage of the electrophoresis was maintained constantly at 200 voltage. Standard mycobacterial strains, which included Mycobacterium bovis BCG, M. tuberculosis, and M. fortuitum, could not be differentiated by PFGE analysis. PFGE analysis was performed to differentiate 9 clinically isolated M. fortuitum strains using AsnI. All M. fortuitum strains showed different genotypes except 2 strains. Cluster analysis divided M. fortuitum strains into 2 large groups. PFGE analysis was performed to further differentiate M. fortuitum isolates using XbaI. The undifferentiated 2 M. fortuitum strains showed different PFGE patterns with Xba I. Cluster analysis of the XbaI-PFGE patterns showed more complex grouping than AsnI-PFGE patterns, which showed that XbaI-PFGE analysis was better than AsnI-PFGE in M. fortuitum genotyping. The top dissimilarity values of AsnI-PFGE and XbaI-PFGE were 0.74 and 0.75, respectively. This value was higher than that of arbitrarily primed polymerase chain reaction (AP-PCR) analysis and lower than that of restriction fragment length polymorphism (RFLP) analysis. This suggested that PFGE can be used as a supportive or alternative genotyping method to RFLP analysis.

Cloning and Sequencing of the phoA Gene which is Regulated by the phoP-phoQ operon in Pathogenic Enteric Bacteria / 영남의대학술지

The DNA fragment containing the phoA of Klebsiella pneumoniae was cloned into pACYC184. The size of the insert. was 4.0 kb and the restriction map showed it contained 3 Pstl sites and 4 PvuLI sites. The nucleotide sequence of the phoA region was determined, which showed strong (80%) sequence similarity with that of Escherichia coli. This suggested that these two species are phylogenetically very close to each other.