• Journal of Sericultural and Entomological Science
• /
• v.39 no.2
• /
• pp.180-185
• /
• 1997

To develope the novel baculovirus transfer vector, the p10 gene was cloned from the Bombyx mori nuclear polygedrosis virus (BmNPV) vB2 strain isolated from the B. mori larvae of sericultural farms. The novel transfer vector was constructed by using the p10 gene of BmNPV vB2 strain was 210 bp. The TAAG sequence at the -71 bp of upstream from translation initiator ATG and two polyadenylation signal site at the downstream from terminator TAA were also detected in the p10 gene. The 5' and 3' flanking region of the p10 gene amplified by PCR was cloned into pBluescriptII SK(+) and then transfer vector pBm10 was construceted. The 7.9 kb pBm10 was analysed by restriction enzymes and the map was confirmed. In order to determine the expression of foreign gene of pBm10, $\beta$-galactosidase gene was inserted in the SmaI site of foreign gene cloning site of pBm10. The pBm10 containing $\beta$-galactosidase gene was cotranfected wth genomic DNA of BmNPV vB2 into BmN-4 cells. The recombinant baculovirus expressing $\beta$-galactosidase was also produced polygedra in the infected cells. The results indicated that pBm10 is functional, suggesting that in the baculovirus expression vector system, the recombinant virus produced by pBm10 was effective by oral infection for the producing recombinant proteins in in vivo expression.

• Toxicological Research
• /
• v.20 no.2
• /
• pp.109-116
• /
• 2004

For the safety evaluation of adenovirus-mediated gene transfer, we investigated differential gene expressions after transfecting adenoviral vector containing p16 tumor suppressor gene (Ad5CMV-p16) into human non-small cell lung cancer cells. In the previous study, we showed adenovirus-mediated $p16^{INK4a}$ gene transfer resulted in significant inhibition of cancer cell growth. We investigated gene expression changes after transfecting Ad5CMV-p16, Ad5CMV (null type, a mock vector) into A549 cells by using cDNA chip and oligonucleotide microarray chip (1200 genes) which carries genes related with signal transduction pathways, cell cycle regulations, oncogenes and tumor suppressor genes. We found that $p16^{INK4a}$ gene transfer down regulated 5 genes (cdc2, cyclin D3, cyclin B, cyclin E, cdk2) among 26 genes involved in cell cycle regulations. Compared with serum-free medium treated cells, Ad5CMV-p16 changed 27 gene expressions, two fold or more on oligonucleotide chip. In addition, Ad5CMV-p16 did not seem to increase the tumorigenicity-related gene expression in A549 cells. Further studies will be needed to investigate the effect of Ad5CMV-p16 on normal human cells and tissues for safety evaluation.

• Applied Biological Chemistry
• /
• v.35 no.4
• /
• pp.227-231
• /
• 1992

The plasmids pSUPBT and pSUPBTR were constructed with a vector pSUP2021 and the BT toxin gene in the plasmid pES 1. The plasmids constructed were introduced into the antagonistic rhizobacteria P. fluorescens KR164 by conjugation and P. fluorescens having pSUPBT and pSUPBTR were named P. fluorescens KR164(pSUPBT)#2, KR164(pSUPBT)#3, KR164(pSUPBTR)#2 and KR164(pSUPBTR)#3, respectively. The BT toxin gene were identified in all transformants by Southern hybridization and the final product of BT toxin gene was identified only in P. fluorescens KR164(pSUPBTR)#3 by SDS-PAGE. This crystal toxin protein were also observed in electron microscopy.

• Microbiology and Biotechnology Letters
• /
• v.23 no.3
• /
• pp.275-281
• /
• 1995

From the plasmid pYA300 carring a CMCase of Rhizobium fredii USDA193 plasmid was subcloned into pBluescript II KS(+)/pBluescript II SK(+) vectors and designated pYA500 and pYA600, respectively. Escherchia coli cells transformed with pYA500 porduced the CMCase more than with pYA600. The orientation of the cloned fragment in pBluescript vector had the effect on gene expression in E. coli background. When the 1.7 kb CMCase gene fragment of R. fredii USDA193 was hybridized to EcoRI-digested total DNA from R. meliloti and R. fredii USDA 191 the unique bands hybridized respectively, indicating that some genetic diversity exists in the EcoRI restriction enzyme site for CMCase gene in Rhizobium strains. The optimum pH of enzyme activity was 7 and the optimum temperature of that was nearly 37$\circ$C. The cellulase-minus derivatives of pYA500 were constructed by Tn5 insertional mutation. Among 6000 transconjugants, two mutant plasmids (designated pYA500::Tn5a and pYA500::Tn5b) were detected from the cellulase- negative transconjugants. The product of CMCase gene was analyzed by one dimensional SDS- PAGE of the cell extracts. About 45 kDa protein was considered to be a product of CMCase gene.

• BMB Reports
• /
• v.38 no.1
• /
• pp.89-96
• /
• 2005

Earlier, we reported that the bacteriophage $\lambda$ P gene product is lethal to Escherichia coli, and the E. coli rpl mutants are resistant to this $\lambda$ P gene-mediated lethality. In this paper, we show that under the $\lambda$ P gene-mediated lethal condition, the host DNA synthesis is inhibited at the initiation step. The rpl8 mutation maps around the 83 min position in the E. coli chromosome and is 94% linked with the dnaA gene. The rpl8 mutant gene has been cloned in a plasmid. This plasmid clone can protect the wild-type E. coli from $\lambda$ P gene-mediated killing and complements E. coli dnaAts46 at $42^{\circ}C$. Also, starting with the wild-type dnaA gene in a plasmid, the rpl-like mutations have been isolated by in vitro mutagenesis. DNA sequencing data show that each of the rpl8, rpl12 and rpl14 mutations has changed a single base in the dnaA gene, which translates into the amino acid changes N313T, Y200N, and S246T respectively within the DnaA protein. These results have led us to conclude that the rpl mutations, which make E. coli resistant to $\lambda$ P gene-mediated host lethality, are located within the DNA initiator gene dnaA of the host.

• Microbiology and Biotechnology Letters
• /
• v.23 no.6
• /
• pp.659-664
• /
• 1995

To improve stability of recombinant DNA pLC1 encoding endoglucanase gene and pGL1 encoding $\beta $-glucosidase gene, DNA fragments of genes coding endoglucanase and $\beta $-glucosidase were cloned within the recA gene on a pDR1453, and the pDRE10 and pDRG20 of recombinant plasmids were integrated into the recA gene on the E. coli 1100 chromosomal DNAs. The stability of inheritance was completely maintained in E. coli 1100; Transformants E. coli 1100/pDREIO and pDRG20 were expressed well by recA promoter and increased endoglucanase and $\beta $-glucosidase activities. This method can be used as a model to improve the stability of recombinant plasmid in large scale culture.

• Korean Journal of Microbiology
• /
• v.52 no.3
• /
• pp.298-305
• /
• 2016

Previously our laboratory showed that Pseudomonas alkylphenolica KL28 possesses two different lap and pcu gene clusters for p-cresol catabolism. In this study, additional gene cluster (pchACXF-pcaHG-orf4-pcaBC) has been identified to encode enzymes necessary for catabolism of p-cresol to ${\beta}$-carboxy-cis,cis-muconate. This gene cluster showed almost identical nucleotide sequence homologies to those in the plasmid of Pseudomonas putida NCIMB 9866 and 9869, British origins, indicating the possibility of a horizontal gene transfer. Through mutagenesis of each gene cluster and gfp-based promoter reporter assays, it has been shown that the three gene clusters are functionally operated and pch genes are induced by p-cresol. Furthermore, the pcu gene cluster of the three was shown to be dominantly expressed in utilization of p-cresol. Mutation of the pcu gene was defective in aerial structure formation under p-cresol vapor, indicating the utilization rate of carbon source is one of key elements for the multicellular development of this strain.

• Current Research on Agriculture and Life Sciences
• /
• v.11
• /
• pp.81-89
• /
• 1993

In order to isolate a mutant which was constitutively expressed in xylA gene, Pxyl-cat-xylA fusion gene was constructed by the insertion of cat gene between xylA promoter and xylA structural gene in pEX13 contained xylA gene. The expression of cat and xylA gene from transformants of xylA mutant DH77 with plasmid pEXC131 containing Pxyl-cat-xylA fusion gene was induced by the addition of 0.4% xylose to media. This results indicated that cat and xylA gene were expressed under control of xylA promoter the presence of xylR gene. We have also isolated constitutive mutant plasmid pEXC131-39 from pEXC131 by trementment with N-methyl-N'-nitro-N-nitrosoguanidine(NTG). cat and xylA gene from pEXC131-39 were constitutively expressed without induction of xylose regardless of xylR gene. Transformants of xylR mutant DH60 with pEXC131-39 also expressed chloramphenicol resistances and xylose isomerase without induction of xylose. This result shows that mutation in region of xylA promoter might make it possible to be constitutively expressed.

• BMB Reports
• /
• v.31 no.5
• /
• pp.503-507
• /
• 1998

To investigate the effects of the Vitreoscilla hemoglobin (VHb) gene on the production of a heterologous protein, a comparative expression system for VHb and ferritin was constructed. First, the VHb gene was inserted into the downstream and upstream regions of the ferritin gene to construct pHF2 and pHF3, respectively. Next, the two plasmids pACHB1 and pVUTFH10, having the VHb gene and the ferritin gene respectively, were constructed in order to express the two genes in different plasmids by using a coplasmid expression system. It was observed that the cell growth was improved in all strains containing the VHb gene. Furthermore, in our coplasmid expression system, the presence of the VHb gene increased production of the ferritin by 1.8 times, as much as that in a strain not having the VHb gene.

• Korean Journal Plant Pathology
• /
• v.11 no.1
• /
• pp.66-72
• /
• 1995

The coat protein (CP) gene of cucumber mosaic virus-As (CMV-As) strain was engineered for expression in the plant by using the cauliflower mosaic virus 35S transcript regulatory sequences. The CP gene was cloned into an Agrobacterium-derived binary vector. A chimeric gene was constructed by the cDNA of CMV-As CP and plant expression vector pBI121. The clone, pCMAS66, was first introduced into the phagemid vector pSPORT1 for situating sense orientation for translation and making restriction sites in order to re-introduce plant expression vector, pHI121. The resulting subclone pCASCP02 and plant expression vector pBI121 were treated with BamHI-SacI for excising the target gene and removing GUS gene, respectively. After Agrobacterium transformation by freeze-thaw technique, the clone, pCMASCP121-123 which contains sense orientation of the target gene, was selected and confirmed by restriction endonuclease analysis. The CMV-As CP gene was introduced into A. tumefaciens. The results on tobacco plant transformation with the vector system revealed that the system could be successfully introduced and showed high frequency of selection to putative transformations.