• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.57-62
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• 1999

Light-regulated translation of chloroplast mRNAs requires nuclear-encoded trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. A set of four proteins (60, 55, 47, and 38 kDa) that bind to the 5'-UTR of the psbA mRNA had been identified in C. reinhardtii. 47 kDa protein (RB47) was found to encode a chloroplast poly (A)-binding protein (cPABP) that specifically binds to the 5'-UTR of the psbA mRNA, and essential for translation of this mRNA, cDNA encoding 60 kDa protein (RB60) was isolated, and the amino acid sequence of the encoded protein was highly homologous to plants and mammalian protein disulfide isomerases (PDI), normally found in the endoplasmic reticulum (ER). Immunoblot analysis of C. reinhardtii proteins showed that anti-PDI recognized a distinct protein of 56 kDa in whole cell extract, whereas anti-rRB60 detected a 60 kDa protein. The ER-PDI was not retained on heparin-agarose resin whereas RB60 was retained. In vitro translation products of the RB60 cDNA can be transported into C. reinhardtii chloroplast in vitro. Immunoblot analysis of isolated pea chloroplasts indicated that higher plant also possess a RB60 homolog. In vitro RNA-binding studies showed that RB60 modulates the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP using redox potential or ADP-dependent phosphorylation. Site-directed mutagenesis of -CGHC- catalytic site in thioredoxin-like domain of RB60 is an unique PDI located in the chloroplast of C. reinhardtii, and suggest that the chloroplast PDI may have evolved to utilize the redox-regulated thioredoxin like domain as a mechanism for regulating the light-activated translation of the psbA mRNA.

• Proceedings of the Botanical Society of Korea Conference
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• 1985.08b
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• pp.7-18
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• 1985

Light-regulated translation of chloroplast mRNAs requires nuclear-encoded trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. A set of four proteins (60, 55, 47, and 38 kDa) that bind to the 5'-UTR of the psbA mRNA had been identified in C. reinhardtii. 47 kDa protein (RB47) was found to encode a chloroplast poly (A)-binding protein (cPABP) that specifically binds to the 5'-UTR of the psbA mRNA, and essential for translation of this mRNA, cDNA encoding 60 kDa protein (RB60) was isolated, and the amino acid sequence of the encoded protein was highly homologous to plants and mammalian protein disulfide isomerases (PDI), normally found in the endoplasmic reticulum (ER). Immunoblot analysis of C. reinhardtii proteins showed that anti-PDI recognized a distinct protein of 56 kDa in whole cell extract, whereas anti-rRB60 detected a 60 kDa protein. The ER-PDI was not retained on heparin-agarose resin whereas RB60 was retained. In vitro translation products of the RB60 cDNA can be transported into C. reinhardtii chloroplast in vitro. Immunoblot analysis of isolated pea chloroplasts indicated that higher plant also possess a RB60 homolog. In vitro RNA-binding studies showed that RB60 modulates the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP using redox potential or ADP-dependent phosphorylation. Site-directed mutagenesis of -CGHC- catalytic site in thioredoxin-like domain of RB60 is an unique PDI located in the chloroplast of C. reinhardtii, and suggest that the chloroplast PDI may have evolved to utilize the redox-regulated thioredoxin like domain as a mechanism for regulating the light-activated translation of the psbA mRNA.

• BMB Reports
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• v.35 no.3
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• pp.273-282
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• 2002

Sp3 is a bifunctional transcription factor that has been reported to stimulate or repress the transcription of numerous genes. Although the size of Sp3 mRNA is 4.0kb, the size of the known Sp3 cDNA sequence is 3.6kb. Thus, Sp3 functional studies have been performed with an artificially introduced start codon, and thus an amino-terminus that differs from the wild-type. Ideally, full-length cDNA expression vectors with the appropriate start codon should be utilized for these studies. Using 5'rapid amplification of cDNA ends, a full-length Sp3 cDNA clone was generated and the sequence verified in nine cell lines. No AUG initiation codon was present. However, stop codons were present in all three frames 5' to the known coding sequence. In vitro translation of this full-length cDNA clone produced the expected three isoforms-one at 100 kDa and two in the mid 60 kDa range. Electrophoretic mobility shift assays showed that the protein products had the ability to bind to the Sp1/3 consensus sequence. In vitro studies, using our Sp3 clone and site directed mutagenesis, identified the translation initiation site for the larger isoform as AUA. AUA has not been previously described as an endogenous initiation codon in eukaryotes.

• Journal of Animal Environmental Science
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• v.19 no.1
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• pp.63-68
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• 2013

In vitro culture and radiation techniques were used for obtaining mutants tin tall fescue. Endophyte free and friendly tall fescue cultivars Kentucky-31 and Jesup were used for induction of genetic variability through in-vitro mutagenesis. Mature seeds was used for callus induction on 6 mg/L 2,4-D. Actively growing and compact callus was treated with three different doses of gamma rays (10 Gy, 30 Gy and 50 Gy). Maximum proliferation and plantlets regeneration growth was observed in control and minimum at 10 Gy. Furthermore, the maximum number of tiller in the irradiated population was observed in 10 Gy. The treatments 30 Gy and 50 Gy exhibited negative impact on the tillering potential of the tall fescue plant. The object of this study was to develop protocols for mutation breeding in tall fescue through radiation techniques.

• FLOWER RESEARCH JOURNAL
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• v.19 no.2
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• pp.119-125
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• 2011

Acorus calamus is an indigenous hydrophyte used as phytoremediation and water purification as well as medicinal herb. Irradiation of gamma ray was performed to investigate the effect on radioresistance of A. calamus on in vitro. Various doses (20-500 Gy) of gamma rays were irradiated to in vitro plantlets. The effect of irradiation was studied on survival rate, shoot formation and growth rate. The survival and multiplication rate was remarkably decreased with the increase of radiation dose. The growth of plantlets was significantly increased in comparison to the control at low doses. The hermetic effect of radiation was appeared in A. calamus. It was significantly decreased or interrupted above 250 Gy. The mean 50% lethal dose ($LD_{50}$) was extremely high as 240 Gy. We conclude that A. calamus had remarkably high radioresistance compared with other plant species.

• Proceedings of the Korean Society of Crop Science Conference
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• 2001.05a
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• pp.258-259
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• 2001

• Korean Journal of Plant Resources
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• v.19 no.2
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• pp.348-354
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• 2006

Homogenized prothallus of 6 species (Cyrtomium falcatum, Cyrtomium caryoptideum var. coreanum, Dryopteris varia, Asplenium incisum, Camptosous sibiricus and Phyllitis scolopendrium) were treated with gamma radiation or by chemical mutagenesis with EMS, NMU, $NaN_3$ and colchicine to assess their sensitivities for each treatments and also with the aim of inducing mutations. Generally, decrease of proliferation ratio was dose-dependent and time-dependent. Based on proliferation ratio, optimum dose of gamma irradiation was $5{\sim}10krad$ except in D. varia with 20krad. Optimum condition of EMS treatment was considered as 50mM for 3h and for NMU as $5{\sim}10mM\;for\;1{\sim}3h$. Optimum condition of $NaN_3$ treatment was considered as $0.5{\sim}1mM\;for\;1{\sim}3h$. For colchicine, there were significant differences between species as to the proliferation ratios of prothallus for each treatment.

• 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.

• Food Science and Preservation
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• v.11 no.1
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• pp.100-105
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• 2004

The objective of this study was carried out to investigate biological activities effects of Korean leaf from Rosa davurica Pall. in vitro. They were extracted with methanol, ethanol, chloroform and water. Methods of the antimutagenic used in this experiment were well-known bacterial short term tests which include Ames test and the antigenotoxic used in this experiment was DPPH radical scavenge. All extracts (ethanol, methanol, water) except chloroform extract exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity with IC$\_$50/ of 11.5 $\mu\textrm{g}$/mL, 6.4 $\mu\textrm{g}$/mL, 4.8 $\mu\textrm{g}$/mL. In Ames test, most of extracts had strong antimutagenic effects against the mutagenesis induced by 4-nitroquinoline-1-oxide (4NQO), N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indol(Trp-P-I) and benzo(${\alpha}$)pyrene(B(${\alpha}$)P). The extracts of leaves (200 $\mu\textrm{g}$/plate) showed approximately 60∼80％ inhibitory effect on the mutagenesis induced by 4NQO, Trp-P-1 and B(${\alpha}$)P against TA98 strain, whereas 60∼80％ inhibition were observed on the mutagenesis induced by MNNG, 4NQO, Trp-P-1 and B(${\alpha}$)P against TA100 strain. respectively.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.81-85
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• 2006

This study was conducted to examine the effect of IRES controlled reporter gene on screening and production of recombinant human erythropoietin (EPO) proteins from cultured CHO cells. The cDNA was cloned for EPO from human liver cDNA Using site-directed mutagenesis, we generated recombinant human EPO (rhEPO) with two additional N-glycosylations (Novel erythropoiesis-stimulating protein: NESP). Wild type hEPO and NESP were cloned into expression vectors with GFP reporter gene under regulatory control of CMV promoter and IRES so that the vectors could express both rhEPO and GFP. The expression vectors were transfected to cultured CHO-K1 cells. Under microscopy, expression of GFP was visible. Using supernatant of the culture, ELISA assay, immunocytochemistry and in vitro assay using EPO dependant cell line were performed to estimate biological activity to compare the production characteristics (secretion levels, etc.) between rhEPO and NESP. The activity of NESP protein, obtained by mutagenesis, was described and compared with its rhEPO counterpart produced under same conditions. Although NESP had less secretion level in CHO cell line, the biological activity of NESP was greater than that of rhEPO. These results are consistent with previous researches. We also demonstrated that rhEPO and GFP proteins expressed simultaneously from transfected CHO cell line. Therefore we conclude that use of GFP reporter gene under IRES control could be used to screen and produce rhEPO in cultured CHO cells.