• Journal of fish pathology
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• v.6 no.1
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• pp.57-64
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• 1993

The properties and DNA structures of R plasmids differ depending on the species of the fish-pathogens Aeromonas hydrophila, A. salmonicida, Edwardsiella tarda, Enterococcus seriolicida, Pasteurella piscicida and Vibrio anguillarum. However, some R plasmids with the same resistance markers in similar DNA structures were found in A. hydrophila and E. tarda, as well as in A. hydrophila and A. salmonicida. R plasmids from V. anguillarum were classified into three groups according to their DNA structures. The first group was detected before 1977, the second from 1980 to 1983, and the third from 1989 to 1991. R plasmids have been retained within P. piscieida having the same DNA structures and detected at various locations and times. E. seriolicida strains carrying the same R plasmids, which were encoded with resistance to macrolide antibiotics(MLs), lincomycin(LIM), and TC, and to MLs, LIM, and CP. were distributed in yellowtail farms in various districts. The chloramphenicol-resistance(cat) gene of the R plasmids of P. piscicida was classified as CAT type I. The cat of the R plasmids of E, tarda. A. salmonicida was classified as type II. The cat of R plasmids of V. anguillarum was classified into two types. One type detected before 1977, was classified as CAT IV and the other type, detected after 1980, was classified as CAT II. Tetracycline-resistance (tet) V. anguillarum, isolated before 1977 and after 1981, was classified as Tet B and Tet G, respectively. The class D tet gene was widely distributed in R plasmids from fish-pathogens A. hydrophila, E. tarda, P. piscicida, and also V. anguillarum isolated after 1989.

• Journal of Microbiology and Biotechnology
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• v.16 no.3
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• pp.355-359
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• 2006

The functional stability of mRNA is one of the crucial factors affecting the efficiency of in vitro translation. As the rapid degradation of mRNA in the cell extract (S30 extract) causes early termination of the translational reactions, extending the mRNA half-life will improve the productivity of the in vitro protein synthesis. Thus, a simple PCR-based method is introduced to increase the stability of mRNA in an S30 extract. The target genes are PCR-amplified with primers designed to make the ends of the transcribed mRNA molecule anneal to each other. When compared with normal mRNA, the mRNA with the annealing sequences resulted in an approximately 2-fold increase of protein synthesis in an in vitro translation reaction. In addition, sequential transcription and translation reactions in a single tube enabled direct protein expression from the PCR-amplified genes without any separate purification of the mRNA.

• Journal of Microbiology and Biotechnology
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• v.3 no.3
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• pp.146-155
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• 1993

A DNA fragment from an alkali-tolerent Bacillus sp., conferring strong promoter activity, was subcloned into the promoter probe plasmid pPL703 and the nucleotide sequence of this promoter region was determined. The sequence analysis suggested that this highly efficient promoter region containing the complex clustered promoters comprised three kinds of promoters (P1, P2 and P3), which are transcribed by $\sigma^B (formerly \sigma^{37}), \sigma^E(formerly \sigma^{29}) and \sigma^A (formerly \sigma^{43})$ RNA polymerase holoenzymes which play major rules at the onset of endospore formation, during sporulation and at the vegetative phase of growth, respectively. S1 nuclease mapping experiments showed that all three promoters had staggered transcription initiation points. The results of chloramphenicol acetyltransferase assay after the subcloning experiments also indicated that the expression of these clustered promoters was correlated with the programs of growth and endospore development. Promoter P1, P2 and P3 were preceded by 75% AT, 79% AT and 81% AT regions, respectively, and a partial deletion of AT-rich region prevented transcription from promoter P1 in vivo. Two sets of 5 -AGTGTT-3 sequences and inverted repeat sequences located around the promoter P1 were speculated as the possible cis acting sites for the catabolite repression in B. subtilis. In vivo transcripts from these sequence regions may be able to form a secondary structure, however, the possibility that a regulatory protein induced by the excess amount of glucose could be bound to such a domain for crucial action remains to be determined.

• BMB Reports
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• v.29 no.5
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• pp.468-473
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• 1996

The P38 promoter of minute virus of mice (MVM) is a very weak promoter which is strongly transactivated by viral nonstructural proteins. To analyze the upstream sequence of the P38 promoter which is responsible for the transactivation by nonstructural proteins in MVM, chloramphenicol acetyltransferase (CAT) reporter plasm ids containing a series of 5' deletion and internal deletion mutants of the P38 promoter were constructed. The wild type and mutant CAT constructs of P38 promoter were cotransfected into murine A92L fibroblast cells with a plasmid expressing viral nonstructural proteins by DEAE-dextran method. Each promoter activity was analyzed by CAT assay. As previously reported (Ahn et al., 1992), the proximal DNA cis-elements required for transactivation of the MVM P38 promoter are GC box and TATA box. However, the analysis of 5' deletion mutants showed that H-l tar like sequence (MVM TAR) which is located between -143 and -122 relative to the transcription initiation site is also required for transactivation of the P38 promoter by nonstructural proteins. Interestingly, even if the MVM TAR was removed by internal deletion, the level of the transactivation is still 70% of wild type level of transactivation. We also found that, in addition to the MVM TAR motif, there are two other motifs which are similar to the MVM TAR sequence. When these TAR like motifs were further deleted, the levels of transactivation were decreased further. Taken together, the MVM TAR sequence and TAR like motifs located upstream of P38 promoter are playing an important role for the transactivation of P38 promoter by nonstructural proteins in minute virus of mice.

• The Journal of Internal Korean Medicine
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• v.20 no.1
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• pp.99-110
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• 1999

Effects of Chiyangtang(CYT) on H. pylori-induced increase of interleukin 8 and interleukin 1 gene expression was studied in Kato Ⅲ cell line, a human stomach epithelial cell line. Treatment of H. pylori to the cell culture signifant!y increased IL-8 and IL-1 mRNA synthesis. When CYT was added along with H. pylori, the increase of IL-8 and IL-1 mRNA synthesis was blocked. Activation of transcription factor $NF-{\kappa}B$ and AP-1 which were known to important in IL-8 and IL-1 gene expression was also studied using chloramphenicol acetyltransferase(CAT) assay. Treatment of H. pylori increased activation of $NF-{\kappa}B$ and AP-l and CYT effectively protected the activation. Electrophoretic mobility shift assay suggested that CYT effectively inhibited DNA binding of $NF-{\kappa}B$ and AP-l to their cognate site. These results suggested that CYT could prevent stomach diseases through the down regulation of IL -8 and IL-l gene expression which might be mediated by the inhibition of $NF-{\kappa}B$ and AP-1 activities and their binding to DNA.

• Proceedings of the Botanical Society of Korea Conference
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• 1987.07a
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• pp.261-282
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• 1987

Plants store a significant amount of their nitrogen, sulfur and carbon reserves as storage proteins in seed tissues. The major proteins present in rice seeds are the glutelins. Glutelins are initially synthesized at 4-6 days postanthesis and deposited into protein bodies via Golgi apparatus. Based on nucleic acid sequences and Southern blot analysis, the three isolated glutelin genomic clones were representative members of three gene subfamilies each containing 5 to 8 copies. A comparison of DNA sequences displayed by relevant regions of these genomic clones showed that two subfamilies, represented by clones, Gt1 and Gt2, were closely, related and probably evolved by more recent gene duplication events. The 5' flanking and coding sequences of Gt1 and Gt2 displayed at least 87% homolgy. In contrast, Gt3 showed little or no homolgy in the 5' flanking sequences upstream of the putative CAAT boxes and exhibited significant divergence in all other portions of the gene. Conserved sequences in the 5' flanking regions of these genes were identified and discussed in light of their potential regulatory role. The derived primary sequences of all three glutelin genomic clones showed significant homology to the legume 11S storage proteins indicating a common gene origin. A comparison of the derived glutelin primary sequences showed that mutations were clustered in three peptide regions. One peptide region corresponded to the highly rautable hypervariable region of legume peptide region of legume 11S storage proteins, a potential target area for protein modification. Expression studies indicated that glutelin mRNA transcripts are differentially accumulated during endosperm development. Promoterss of Gt2 and Gt3 were functional as they direct transient expression of chloramphenicol acetyltransferase in cultured plant cell.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.64 no.4
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• pp.432-440
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• 2019

Global climate change exerts adverse effects on maize production. Among abiotic stresses, drought stress during the tasseling stage (VT) can increase anthesis-silking intervals (ASI) and decrease yield. We performed an evaluation of ASI and yield using a drought-sensitive line (Ki3) and a drought-tolerant line (Ki11) to analyze the correlation with ASI and yield. Moreover, the de novo data of Ki11 were analyzed to find putative novel transcripts related todrought stress in tropical maize. A total of 182 transcripts, with a log2 ratio >1.5, were found by comparing drought conditions to a control. The top 40 transcripts of high expression levels in the de novo analysis were selected and analyzed with PCR. Of the 40 transcripts, six novel transcripts were detected by quantitative real-time PCR (qRT-PCR) using seedling and VT stage samples. Five transcripts (transcripts_1, 12, 34, 35, and 40) were up-regulated in the Ki11 shoot at seedling stage, and transcripts_1, 12, and 40 were up-regulated at the re-watering stage after 12 h of drought stress. The transcripts_32 and 34 were up-regulated at the VT stage. Hence, transcript_34 possibly plays a significant role in drought tolerance during the seedling and VT stages. The transcript_32 was identified as chloramphenicol acetyltransferase (CAT) by Pfam domain analysis. The function of the other transcripts remained unknown. Further characterization of these novel transcripts in genetic regulation will be of great value for the improvement of maize production.

• Korean Chemical Engineering Research
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• v.57 no.1
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• pp.85-89
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• 2019

Cell-free protein synthesis utilizes the translational machinery in a cell extract. Unlike the conventional cell-based expression methods, not being affected by the conditions for cell growth, cell-free protein synthesis enables flexible manipulation of individual factors affecting the efficiency protein biosynthesis. However, the high cost and low stability of the energy sources to regenerate ATP have limited the use of cell-free synthesis for large-scale production of recombinant proteins. One of the approaches to address this problem is to use glucose as an alternative energy source to regenerate ATP through the glucose-metabolizing pathways in a cell extract. In this study, in an attempt to improve the efficiency of ATP regeneration by reinforcing oxidative phosphorylation process, we supplemented with cellular lipids to a glucose-fueled reaction mixture for cell-free protein synthesis. As a result of the lipid supplementation, the productivity of chloramphenicol acetyltransferase in a cell-free synthesis system using glucose increased more than 6 fold compared to when the lipid was not supplemented.

• KSBB Journal
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• v.23 no.3
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• pp.231-238
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• 2008

The second family member of tissue inhibitors of matrix metalloproteinases, TIMP-2, is a 21kDa protein which inhibits matrix metalloproteinases 2 (MMP-2). Expression of mammalian proteins in E. coli often forms inclusion bodies that are made up of mis-folded or insoluble protein aggregates. The requirement for the formation of 6 disulfide bonds in the process of the TIMP-2 folding is likely to be incompatible with the reducing environment of E. coli. However, this incompatibility can be often overcome by introducing a mutagenesis that could lead to enhancement of the protein solubility. In this reason, we have attempted to express the soluble TIMP-2 in E. coli by applying a modified staggered extension process (StEP), one of the in vitro PCR-based recombinant mutagenesis methods, and error-prone PCR. C-terminally located CAT fusion protein with respect to mutated TIMP-2 proteins enables us to differentiate the soluble TIMP-2 from the insoluble in E. coli by virtue of chloramphenicol resistance. According to this scheme, E. coli harboring properly-folded CAT fused to TIMP-2 protein was selected, and some of the resulting colonies exhibited an enhanced, soluble expression of TIMP-2 compared to the wild type, implying (i) the StEP technique is successfully employed to enhance the proper folding thereby increasing the solubility of TIMP-2, and (ii) the CAT dependent screening may be a simple and effective method to differentiate the soluble protein expression in E. coli.

• Journal of Korean Society of Forest Science
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• v.84 no.1
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• pp.77-86
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• 1995

To effectively modify tree function with genetic engineering, transgenes must be expressed at the proper level in the appropriate tissues at suitable developmental stages. Toward understanding the spatial and temporal expression of transgenes in woody plants, transgene expression was evaluated in three greenhouse-grown, transgenic lines of Populus alba ${\times}$ P. grandidentata hybrid clone 'Hansen'. All transgenic poplar lines possess constructs containing the bacterial nopaline synthase(nos) promoter linked to a neomycin phosphotransferase II(NPT II) selectable marker gene. In addition, each transgenic poplar line contains one of the following gene constructs : 1) a wound-inducible potato proteinase inhibitor II (pin2) promoter linked to a chloramphenicol acetyltransferase(CAT) reporter gene. 2) a nos promoter linked to a PIN2 structural gene : or 3) a Cauliflower Mosaic Virus 35s promoter linked to a PIN2 structural gene. Polymerase chain reaction(PCR) was used to verify the presence of foreign genes in the poplar genome. Enzyme-linked immunosorbent assays(ELISAs) were used to evaluate organ specific expression of the nos-NPT II construct. NPT II expression was detected in leaves, petioles, stems, and roots of transgenic poplar, thereby indicating that the nos promoter is potentially effective for general constitutive expression of transgenes. NPT expression varied among transgenic poplar lines and among organs for one transgenic line, Tr15. With Tr15, NPT II levels were highest in older leaves and petioles. These results indicate that screening of several transgenic lines may be required to identify lines with optimal transgene expression.