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- Overexpression of the SPP2 Gene of Saccharomyces cerevisiae and Production of Antibodiesd to Spp2p
- Park, Kwang Hark , Lea, Ho Zoo , Woolford, John L. , Kim, Kyung Hoon
- J. Microbiol. 1995;33(3):201-207.
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Abstract
- We have previously reported that SPP2 gene product of yeast Saccharomyces cerevisiae is involved in the pre-mRNA splicing. To investigate the rol ein the splicing pathway of the Spp2p protein, the SPP2 gene was overexpressed in Escherichia coli and polyclonal antibodies to Spp2p were generated from rabbits. First, a DNA fragment containing the SPP2 GENE without its promoter was subcloned into an E. coli expression vector, pKK233-3. The resulting recombinant plasmid pBQ14 contained an IPTG inducible tac promoter and the SPP2 structural gene. Overexpression of the SPP2 gene was achieved by additionof 0.1 to 1.0 mM IPTG to a logarithmic culture of E. coli JM103(pBQ14) for 90 min at 37℃. Sequence of N-terminal 15 amino acids of the overproduced protein was well matched to the deduced one from the SPP2 reading frame. Then, polyclonal antibodies were generated from rabbits immunized with gel-purified Spp2p protein. These antibodies reacted specifically with the Spp2p protein extracted from yeast cells expressing the SPP2 gene to a great extent. The antibodies could also block the activity of yeast splicing extracts.
- Construction of genetically engineered miroorganisms for overexpression of xylE gene encoding catechol 2, 3-dioxygenase and the functional stability of the recombinant plasmid pSW3a containing xylE in aquatic environment
- Han, Hyo Yung , Kim, Chi Kyung , Park, yong Keun , Ka, Jong Ok , Lee, Byeong Jae , Min, Kyung Hee
- J. Microbiol. 1996;34(4):341-348.
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Abstract
- The regulation of xylE gene expression was examined by using vector promoter and construction of genetically engineered microorganisms (GEMs) for application in microcosm. When the xylE gene wsa subcloned into pBluscript SK(+) under the control of lac promoter (pTY1) in E. coli, and the expression was induced by IPTG, the enzyme activity of catechol 2, 3-dioxygenase was increased 4.7 times more than that of the crude extracts from transformants harboring pTY1. We suggest that the xylE gene has its own promoter at the upstream portion, because it was able to be expressed even in the absence of IPTG. A recombinant plasmid, pSW3a harboring the xylE gene under the T7 promotor, showed the activity of 14.5 units/mg protein, higher than that of parental strain, E. coli PYT1. The xylE gene in recombinant plasmid pSW3a was used as reporter gene for the application in microcosm ecosystem, since it was used for detection of xylE-positive clones by catechol spray on the agar plates. The pSW3a in E. coli was introduced into Pseudomonas putida to construct GEM strain, and examined for the expression and functional stability in microcosms.
- Overexpression of Insecticidal Protein Gene of Bacillus thuringiensis var. kurstaki HD1
- Hwang, Sung Hei , Yoo, Kwan Hee , Moon, Eui Sik , Cha Soung Chul , Lee, Hyung Hoan
- J. Microbiol. 1998;36(4):289-295.
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Abstract
- The Insecticidal protein (ICP) gene from Bacillus thuringiensis var, kurstaki HD-1 was cloned in pBluescript SK(+) vector and charicterized by overexpression in Escherichia coli XL1-blue. Total plasmids in the B. thuringiensis were isolated and digested with restriction enzyme BamHI. Then, southern blot was performed with a probe to locate the gene in the fragments. The hybridized 3.8 kb NdeI DNA fragment was cloned into the SmaI site of pBluescript SK(+) and named pHLN1-80 in forward orientation to the lacZ gene promoter and pHLN2-80 in reverse orientation to the lacZ gene promoter. Determination of 153 bp nucleotide sequence of 5'-end of the NdeI fragment in the pHLN1-80 clone revealed that there are-80 bp region of the ICP gene promoter and +73 bp region of the ICP gene at the 5'end of the ICP gene. In addition, the-80 bp promoter of the ICP gene contained transcription initiation point G at-77 bp point and BtI promoter and Shine-Dalgarno sequence at-14 to-4 bp region. The two clones showed strong insecticidal activity against 3rd the instar Bompyx mori larvae. SDS-PAGE analysis revealed that the pHLN2-80 clone clearly produces distinguishable amount (27 times more) of the 130 kDa ICP band and 100 times the insecticidal activity than that of the clone pHLN1-80. These marked differences in production and toxicity due to different orientations of the gene in the vectior provide us valuable points for further study on the ICP gene transcription at the molecular level.
- Overexpression of Fish DRG2 Induces Cell Rounding
- Jeong Jae Park , Seung Ju Cha , Myoung Seok Ko , Wha Ja Cho , Won Joon Yoon , Chang Hoon Moon , Jeong Wan Do , Sung Bum Kim , Hebok Song , Dae Kyun Chung , In Seob Han , KyuBum Kwack , Jeong Woo Park
- J. Microbiol. 2002;40(4):295-300.
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Abstract
- Previously, we reported induced expression of developmentally regulated GTP-binding protein 2 (DRG2) in fish cells at the late stage of rhabdovirus infection. To investigate the biological role of fish DRG2 (fDRG2), we transfected CHSE-214 cells with an expression vector containing complete fDRG2 fused to the N-terminal end of an enhanced green fluorescent protein (EGFP). Low level expression of fDRG2-EGFP did not induce morphological change or cell death. However, a high level expression of fDRG2-EGFP induced cell rounding and caused depletion of the cell population in FACS analysis. Several truncated fragments were fused to EGFP. FACS analysis was conducted to determine the presence of cells expressing high levels of the resulting chimera. While cells expressing a high level of N-terminus were detected, those expressing high levels of the C-terminal fragment 243-290 containing the G4 motif were absent in FACS analysis. Based on these observations, we propose that overexpression of fDRG2 may induce cell rounding, a representative cytopathic effect of virus-infected cells in the late stage of infection and the C-terminus of the fDRG2 is essential for this function.
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