• Korean Journal of Microbiology
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• v.36 no.1
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• pp.1-7
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• 2000

The genes involved in riboflavin synthesis (ribI, II, III, and IV) were found immediately downstream of luxG in the lux operon from Photobacterium species. The single stranded DNA containing the intergenic region of lux genes and rib genes from Photobacterium phosphoreum was fully protected by P. phosphoreum mRNA from the S1 nuclease mapping assay suggesting that a transcriptional terminator was not present in the region. In addition, the levels of riboflavin synthase activity in P. phosphoreum was increased during the development of bacterial bioluminescence in the same fashion as the luciferase and fatty acid reductase activities. Insertion of the Photobacterium leiognathi DNA extending from luxB to ribII, between a strong lux promoter and a reporter gene (chloramphenicol acetyltransferase, CAT) and transferred by conjugation into P. leiognathi, did not affect expression of reporter gene. Moreover the CAT gene was not expressed in an analogous construct missing the lux promoter indicating that a promoter was not present in this region. Based on the data here, it can be concluded that the lux genes and rib genes in Photobacterium species are under common regulation.

• Journal of Plant Biology
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• v.35 no.3
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• pp.229-235
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• 1992

Differential expression of the three chlorophyll afb binding (cab) protein gene (cabl, cab2, and cab3) promoters of Arabidopsis thaliana was studied in tobacco plants transformed with cab-CAT (chloramphenicol acetyltransferase) translational fusions. CAT activity was measured to monitor the activities of the cab promoters. The activity of cabi promoter was higher than the other two in transformed tobacco leaves and also in calli and shoots derived from the leaves. Their activities were organ-specific and were the lowest in roots, medium in stems, and the highest in leaves. The relative activity of cabi promoter in stems comparing to it activity in leaves was, however, much higher than the values of cab2 and cab3. When the cab promoter activity was expressed as CAT activity per unit chlorophyll instead of CAT activity per unit protein, the relative cab] promoter activity (stem/leaf) became almost unity. This result suggests that cab2 and cab3 show photosynthetic organ-specificity but cabl does not. Similar result was obtained in the differentiation process of stems and leaves from shoots derived from the transgenic tobacco leaves.leaves.

• BMB Reports
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• v.29 no.2
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• pp.156-162
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• 1996

Transcription of hepatitis B viral pregenomic promoter is known to be regulated mainly by the combined interaction of enhancers I, II and the intervening regulatory sequences between the two enhancers. A positive regulatory element was identified by serial deletion and measuring the linked chloramphenicol acetyltransferase (CAT) activities, which overlapped with the 5' region of the X open reading frame. When the positive regulatory element was inserted upstream of the SV40 early promoter, it elevated SV40 promoter activity in HepG2 cells. Two cellular proteins of 110 (p110) and 33 (p33) kDa interacted with the positive element and both of them were present in the nucleus, but p110 also existed in the cytoplasm in phosphorylated form. Dephosphorylation of p110 by acid phosphatase enhanced the DNA-binding activity of p110. The p33 could bind to single-strand DNA specifically as well as to double-strand DNA.

• BMB Reports
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• v.30 no.4
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• pp.269-273
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• 1997

Heptocvte-specific expression induced by Hepatitis B virus (HBV) enhancer 2-core gene promoter was examined in various hepatocyte and non-hepatocyte cell lines. using non-viral and retroviral vector systems in which chloramphenicol acetyltransferase (CAT) is used as a reporter. The non-viral plasmid containing the HBV enhancer 2-core promoter exhibited 22 and 66% of CAT activities in hepatoma cell lines. HepG2 and Hep3B, respectively when compared with CAT activity expressed by CMV promoter. The CAT activities, however. were found to be marginal in other tested hepatoma cell lines as well as mouse primary hepatocytes and non-hepatocytes. The HBV enhancer 2 located upstream the CMV promoter did not affect the CMV promoter activity nor provided hepatocyte-specific expression. Transfection of retroviral plasmid DNA containing the HBV enhancer 2-core promoter as an internal promoter exhibited high and specific CAT expression in HepG2 and Hep3B cell lines but the activity value was 5 to 10 fold lower than the non-viral plasmid with identical promoter. These results suggest that the usage of HBV enhancer 2-core promoter for liver specific expression is limited to certain vectors and hepatocyte cell lines.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.420-424
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• 2006

A simple method for depleting E. coliS30 extracts of endogenous tRNA has been developed. An $ethanolamine-Sepharose^{(R)}$ column equilibrated with water selectively captured the tRNA molecules in E. coli S30 extracts. As a result, S30 extracts filtered through this column became completely dependent upon the addition of exogenous tRNA to mediate cell-free protein synthesis reactions. We anticipate that the procedures developed and described will be particularly useful for in vitro suppression reaction studies designed to introduce unnatural amino acids into protein molecules.

• Biomolecules & Therapeutics
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• v.3 no.3
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• pp.210-216
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• 1995

Partially purified water-soluble extract mixture from Ricini and Coptidis (named as RIC) showed to be a potent inhibitor of human immunodeficiency virus-1 (HIV-1) replication. RIC was evaluated for in vitro anti-HIV activity using SupTl and H9 cells infected by a recombinant virus (pSVCAT) containing chloramphenicol acetyltransferase (CAT) gene substituted for nef gene in the HIV-1 genome. RIC inhibited syncytiaformation of SupTl cells with a half maximal effective concentration, $IC_{50}$/, of 2.5 $\mu\textrm{g}$/mι and showed marked inhibition of CAT activity in the infected H9 cells and also suppressed reverse transcriptase (RT) activity in the supernatant of the infected H9 culture. However, RIC did not inhibit the activity of reverse transcriptase directly when it was mixed with the enzyme or with viral particles. Berberine, one of components of RIC, also showed similar anti-HIV activity as RIC did. The data suggest that there are active ingredients which mediate anti-HIV activity in RIC.

• Korean Journal of Veterinary Research
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• v.39 no.4
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• pp.772-777
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• 1999

The uncaped genomic RNA of bovine viral diarrhea virus (BVDV) initiates translation by recruitment of eukaryotic translation initiation factors at the internal ribosome entry site (IRES). N-terminal protease ($N^{pro}$) is the first translation product of the open reading frame (ORF). By using the vaccinia virus SP6 RNA polymerase transient expression system, we showed previously that deletion of $N^{pro}$ region reduced translation by 21%. To better understand the biological significance of $N^{pro}$ for translation, we carried out a mutational analysis of the $N^{pro}$ region of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid. We constructed a bicistronic expression vector in which the entire 5 UTR and the mutated $N^{pro}$ region (${\Delta}386-901$, ${\Delta}415-901$ and ${\Delta}657-901$) was cloned between two reporter genes, chloramphenicol acetyltransferase (CAT) and luciferase (LUC). In vivo translation analyses showed that $N^{pro}$ region was dispensible for efficient translation. The results indicate that the $N^{pro}$ region is not essential for BVDV RNA translation and the 3' boundary of BVDV IRES is expanded into $N^{pro}$ region, suggesting that $N^{pro}$ may not play a major role in BVDV replication.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.77-77
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• 1995

Human immunodeficiency virus type 1 (HIV-1) contains several nonstructural genes which are required for the viral replication and disease pathogenesis. Among them, tat and nef genes encode an essential transactivator of HIV-1 LTR and a pluripotent protein which seems to be essential for the in vivo but not in vitro viral replication, respectively. We constructed two tat and n of defective HIV-1 and tested for their ability to replicate in several T cells. The defective viruses did not replicate in CD4$\^$+/ T cells, but rescued in the recombinant Jurkat-tat cell which also contains tat gene. The replication of tat and nef defective HIV-1 which expresses chloramphenicol acetyltransferase(CAT) gene was easily detected by a sensitive CAT assay. No revertant was identified during the passages of the mutant viruses for more than two months in Jurkat-tat cells. tat and n of defective HIV-1 could be used instead of wild type viruse for several purposes such as inhibitor screening and development of attenuated AIDS vaccine.

• Journal of Plant Biology
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• v.36 no.3
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• pp.267-273
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• 1993

Promoters of the chlorophyll a/b bidning protein genes, cab1, and cab2, of Arabidopsis thaliana were studied for their functions in differential expression during greening of etiolated shoots. The etiolated shoots were derived from leaves of transgenic tobacco plants with the cab-CAT (chloramphenicol acetyltransferase) translational fusions, and CAT activity was measured to monitor the activities of the cab promoters. Cab1 promoter activity increased rapidly and showed saturation after about 24 hours of greening, but that of cab2 increased with about 2 day-lag period and showed saturation after 6 days. Cab1 promoter activity was more sensitive to levulinic acid (LA) compared with cab2 activity. Cab2 promoter activity was inhibited more sensitively by chloramphynicol (CAP) than by inhibitors of Chl formation. Cab1 promoter activity was, however, inhibited less sensitively by CAP than by LA. The treatment of abscisic acid (ABA) did not block Chl synthesis so significantly as LA treatment did, and cab2 promoter activity was much less sensitive to ABA compared with that of cab1. These results suggest that cab1 expression is strongly related with Chl formation, possibly with $\delta$-aminolevulinic acid accumulation, and cab2 expression is suppressed more by the blockage of translation of Chl a-apoproteins than by the blockage of Chl a accumulation.

• Korean Journal of Microbiology
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• v.32 no.2
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• pp.115-119
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• 1994

Replication control region of pSBK203, a chloramphenicol acetyltransferase conferring plasmid from Staphylococus aureus was cloned and its nucleotide sequence has been determined. Base sequence homology of this copy control region with those of plasmids belonging to pT181 family was obtained and analyzed. Copy number of four copy mutants derived by addtion or deletion of nucleotides in unique XbaI recognition site in copy control region of pSBK203 was also determined.