OBJECT: The aim of this study is to determine whether exposure to chlorpromazine causes mutagenicity and genetic disorders. METHOD: Ames (Salmonella typhimurium) test and Rec assay (Bacillus subtilis) were used as indicators for DNA damage. Furthermore, the levels of umu operon expression by measuring the beta-galactosidase activity were monitered with the SOS umu test using S. typhimurium 1535 containing plasmid pSK1002. And the host-mediated assay was used to investigate the muta-genicity of chlorpromazine after the activation with in vivo metabolic systems. RESULTS: From the results, chlorpromazine did not affect DNA of S. typhimurium and B. subtilis strains and showed no mutagenicity at the all concentrations tested. These phenomena was also similar to that after metabolic activation of chlorpromazine in in vivo system. CONCLUSION: These results suggested that chlorpromazine did not show the mutagenicity and genotoxicity by four different methods used in this study.
beta-Galactosidase ; Biotransformation ; Chlorpromazine* ; DNA ; DNA Damage ; Operon ; Plasmids

BACKGROUND: Aspergillus species are second only to Candida species as the most commonly isolated fungi from clinical specimens. As well as the identification of the Aspergillus species, it has been necessary for epidemiological studies to differentiate between strains of the same species. We performed genotypic identification and characterization of species and strains within the genus Aspergillus by using RAPD. METHODS: A total of 63 clinical strains of Aspergillus species (including 21 A. fumigatus, 12 A. flavus, 12 A. niger, 12 A. terreus, 3 A. nidulans, and 3 A. sydowii) from 63 patients was analyzed. For RAPD alanysis, M13 primer (5'GAGGGTGGCGGTTCT3') and five random 10-mer primers (OPC-6, 7, 10, 18 and 20; Operon Technologies, USA) were used. RESULTS: The RAPD patterns by M13 primer appeared to be identical when the isolates of the same Aspergillus species were compared. Distinctive and reproducible sets of amplification products by primer M13 were observed for different Aspergillus species: 60 of 63 (95%) isolates were correctly identified by the RAPD analysis using primer M13. RAPD patterns obtained from different strains of the same Aspergillus species by five OPC primers were far more similar than those derived from different Aspergillus species, but the RAPD profiles with some OPC primers showed polymorphism among isolates of the same Aspergillus species. The application of some OPC primers made it possible to cluster the isolates of the same Aspergillus species into several groups. CONCLUSION: These results indicate that RAPD can be useful for the rapid identification of Aspergillus species and for strain typing in the epidemiological investigations.
Aspergillus* ; Candida ; DNA* ; Epidemiologic Studies ; Fungi ; Genotype ; Humans ; Niger ; Operon

OBJECTIVE: The transfection efficiencies of gynecologic cancer cell lines were investigated by different mediated transfection methods using recombinant LacZ plasmid (pRcCMVLacZ and pAAVCMVLacZ). METHODS: In this study, the gynecologic cancer cell lines were used CaSki, SiHa (cervical, HPV16+, wild type p53 gene), HeLa, HeLa S3 (cervical, HPV18+, wild type p53 gene), C33A, HT3 (cervical, HPV-, p53 mutant), HckE6/E7 (cervical, HPV16 immortalized keratocyte), PA-1 (ovary, wild type p53), SKOV-3, A2774 (ovary, p53del) and OVCAR-3 (ovary, p53 mutant). The pRcCMVLacZ and pAAVCMVLacZ plasmid transfection were performed by using liposome system such as Ca2+-phosphate, Fugen6(TM), Lipofection(TM), Lipogen(TM) and N-stearyl lactobionamide (N-SLBA) with X-gal staining. The LacZ gene was used the reporter gene for the transfection efficiencies evaluation. RESULTS: Each of cell lines were showed different transfection efficiencies by Ca2+-phosphate, Fugen6(TM), Lipofectin(TM), Lipogen(TM) and N-SLBA. Each of cell were revealed that HeLa S3, HT3 and A2774 were high transfection efficiency using the pRcCMVLacZ by the Lipogen(TM), SiHa, HeLa, QGU, OVCAR-3 and PA-1 were high efficiency using the pAAVCMVLacZ by Lipofectin(TM), CaSki was high efficiency using the pRcCMVLacZ by the Lipogen(TM), A2774 and Cx16.2 were high efficiency using the pRcCMVLacZ by the Lipofectin(TM), SKOV-3 and HkcE6/E7 were high efficiency using pAAVCMVLacZ by the Lipogen(TM). CONCLUSION: As a result, We proved that each of cell lines differed trasnfection efficiencies according to mediated transfection and recombinant LacZ plasmid style. Above all, Lipofectin(TM) mediated transfection was showed high efficiency at the most of cell lines.
Cell Line* ; Genes, Reporter ; Humans* ; Lac Operon* ; Liposomes ; Plasmids* ; Transfection*

OBJECTIVETo examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell.

METHODSTwo series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed. The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments. The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments. The expression level of desired protein was assayed by SDS-PAGE and laser density scanning. Plasmid copy number was determined by incorporation with (3)H-thymidine ((3)H-TdR).

RESULTSNo influence of the tandem-joined operons on host growth and plasmid stability was observed. Upon induction, the desired protein accumulations in the CW11 series were 44.9% +/- 3.9%, 51.3% +/- 4.1%, 54.8% +/- 3.3% and 58.2% +/- 3.4% of total cell protein. In the CW12 series, the yields were 32.2% +/- 5.0%, 42.8% +/- 4.1% and 46.9% +/- 4.0% of total cell protein. As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P < 0.01). Further calculation showed that the total amount of plasmid DNA per cell was not significantly different in each series (P > 0.05) and restricted to some extent.

CONCLUSIONSIncreasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein. Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E. coli strains under identical incubation condition.

DNA ; analysis ; Escherichia coli ; genetics ; Gene Dosage ; Operon ; Plasmids

Vibrio vulnificus causes fatal infections in susceptible individuals. Group 1 capsular polysaccharide (CPS) operon is responsible for CPS expression, which plays an essential role in the pathogenesis of this pathogen. Cyclic AMP (cAMP) and cAMP receptor protein (crp) complex, which responds to glucose availability and functions as a global regulator, has been known to affect CPS production in this pathogen. This study was undertaken to experimentally verify whether cAMP-Crp directly or indirectly affects CPS production. A mutation in cyaA encoding adenylate cyclase, which is required for cAMP biosynthesis, inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing cyaA or by adding exogenous cAMP. A mutation in crp encoding Crp also inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing crp. Moreover, the crp or cyaA mutation decreased the susceptibility of V. vulnificus against NaOCl. The crp mutation reduced the transcription levels of group 1 CPS operon on a per cell basis. Glucose addition in the absence of Crp stimulated V. vulnificus growth, changed translucent colonies to opaque colonies, and increased the transcription levels of group 1 CPS operon. These results indicate that cAMP or Crp is indirectly involved in optimal CPS production by positively affecting metabolism or V. vulnificus growth rather than by directly controlling the expression of group 1 CPS operon.
Adenylyl Cyclases ; Complement System Proteins ; Cyclic AMP Receptor Protein ; Cyclic AMP* ; Glucose ; Metabolism ; Operon ; Vibrio vulnificus

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.
Base Sequence ; Clone Cells* ; Cloning, Organism* ; DNA ; Enterobacteriaceae* ; Escherichia coli ; Klebsiella pneumoniae ; Operon*

BACKGROUND: There are three kinds of diseases caused by dematiaceous fungi: chromoblastomycosis, phaeohyphomycosis, and eumycotic mycetoma. The dematiaceous fungi have been identified and classified by morphological, biochemical and physiological tests. Recently molecular analysis has been introduced to the field of medical mycology. OBJECTIVE: We investigated the genetic diversity of dematiaceous fungi using random amplified polymorphic DNA (RAPD). METHODS: The dematiaceous fungal strains studied were eight clinical isolates of chromoblastomycosis and phaeohyphomycosis agents (3 strains of Fonsecaea pedrosoi, 2 strains of Exophiala dermatitidis, 1 strain of Exophiala jeanselmei, 1 strain of Phialophora verrucosa, 1 strain of Rhinocladiella aquaspersa) and 4 standard strains (F. pedrosoi IFM 4889, E. dermatitidis IFM 4828, P. verrucosa IFM 4928, R. aquaspersa IFM 4930). Total twelve strains of dematiaceous fungi were cultured on Sabouraud's dextrose broth and their DNA were extracted by bead-beating method. RESULTS: The optimal condition for PCR was template DNA 0.025 mg and annealing temperature 39 degrees C. The RAPD analysis using OPA 10 primer (5'-GTGATCGCAG-3') of Operon kit showed different patterns among dematiaceous fungi. But one clinical isolate of F. pedrosoi showed intra-specific variability. CONCLUSION: The RAPD analysis is considered a rapid and reliable method for identification and classification of dematiaceous fungi if the procedure is carefully standardized with adequate primer.
Chromoblastomycosis ; Classification ; DNA* ; Exophiala ; Fungi* ; Genetic Variation* ; Glucose ; Mycetoma ; Mycology ; Operon ; Phaeohyphomycosis ; Phialophora ; Polymerase Chain Reaction

β-carotene belongs to carotenoids family, widely applied in pharmaceuticals, neutraceuticals, cosmetics and food industries. In this study, three key genes (dxs, idi, and crt operon) within β-carotene synthetic pathway in recombinant Escherichia coli strain CAR005 were modulated with RBS Library to improve β-carotene production. There were 7%, 11% and 17% increase of β-carotene yield respectively after modulating dxs, idi and crt operon genes with RBS Library, demonstrating that modulating gene expression with regulatory parts libraries would have more opportunities to obtain optimal production of target compound. Combined modulation of crt operon, dxs and idi genes led to 35% increase of β-carotene yield compared to parent strain CAR005. The optimal gene expression strength identified in single gene modulation would not be the optimal strength when used in combined modulation. Our study provides a new strategy for improving production of target compound through modulation of gene expression.
Escherichia coli ; metabolism ; Gene Expression ; Gene Library ; Operon ; beta Carotene ; biosynthesis

OBJECTIVETo understand the mechanism of invasion by Legionella dumoffii.

METHODSThe L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903dIIlacZ. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in HeLa and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR.

RESULTSThe transposon insertion was in a gene homologous to Salmonella typhi traC, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the traC gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a traC deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain..

CONCLUSIONOur results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells.

A549 Cells ; Animals ; Genes, Bacterial ; HeLa Cells ; Humans ; Legionella ; genetics ; physiology ; Male ; Mice ; Mutation ; Operon

Bacterial Proteins/metabolism* ; Operon/genetics* ; Phosphotransferases/metabolism* ; Quorum Sensing/genetics* ; Vibrio cholerae/genetics*