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Human Papillomavirus E6 Knockdown Restores Adenovirus Mediated-estrogen Response Element Linked p53 Gene Transfer in HeLa Cells

  • Kajitani, Koji;Ken-Ichi, Honda;Terada, Hiroyuki;Yasui, Tomoyo;Sumi, Toshiyuki;Koyama, Masayasu;Ishiko, Osamu
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.18
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    • pp.8239-8245
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    • 2016
  • The p53 gene is inactivated by the human papillomavirus (HPV) E6 protein in the majority of cervical cancers. Treatment of HeLa S3 cells with siRNA for HPV E6 permitted adenovirus-mediated transduction of a p53 gene linked to an upstream estrogen response element (ERE). Our previous study in non-siRNA treated HHUA cells, which are derived from an endometrial cancer and express estrogen receptor ${\beta}$, showed enhancing effects of an upstream ERE on adenovirus-mediated p53 gene transduction. In HeLa S3 cells treated with siRNA for HPV E6, adenovirus-mediated transduction was enhanced by an upstream ERE linked to a p53 gene carrying a proline variant at codon 72, but not for a p53 gene with arginine variant at codon 72. Expression levels of p53 mRNA and Coxsackie/adenovirus receptor (CAR) mRNA after adenovirus-mediated transfer of an ERE-linked p53 gene (proline variant at codon 72) were higher compared with those after non-ERE-linked p53 gene transfer in siRNA-treated HeLa S3 cells. Western blot analysis showed lower ${\beta}$-tubulin levels and comparatively higher p53/${\beta}$-tubulin or CAR/${\beta}$-tubulin ratios in siRNA-treated HeLa S3 cells after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with those in non-siRNA-treated cells. Apoptosis, as measured by annexin V binding, was higher after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with that after non-ERE-linked p53 gene transfer in siRNA-treated cells.

Expression of Bacillus thringiensis HD-1 gene in rhizobacteria Pseudomonas fluorescens KR164 (근권 길항세균 Pseudomonas fluorescens KR164에 Bacillus thuringiensis HD-1 유전자의 삽입과 발현)

  • Kim, Yeong-Yil;Rhee, Young-Hwan;Kang, Heun-Soo
    • Applied Biological Chemistry
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    • v.35 no.4
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    • pp.227-231
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    • 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.

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Construction of the Novel Baculovirus Transfer Vector Using the p10 Gene of BmNPV (BmNPV의 p10 유전자를 이용한 새로운 전이벡터 개발)

  • 강석우;진병래
    • Journal of Sericultural and Entomological Science
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    • v.39 no.2
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    • pp.180-185
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    • 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.

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Identification and Expression Analysis of Chloroplast p-psbB Gene Differentially Expressed in Wild Ginseng

  • Kim, Doo-Young;Kwon, Ki-Rok;Kang, Won-Mo;Jeon, Eun-Yi;Jang, Jun-Hyeog
    • Journal of Pharmacopuncture
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    • v.15 no.1
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    • pp.18-22
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    • 2012
  • Panax ginseng is a well-known herbal medicine in traditional Asian medicine. Although wild ginseng is widely accepted to be more active than cultivated ginseng in chemoprevention, little has actually been reported on the difference between wild ginseng and cultivated ginseng. Using suppressive subtraction hybridization, we cloned the p-psbB gene as a candidate target gene for a wild ginseng-specific gene. Here, we report that one of the clones isolated in this screen was the chloroplast p-psbB gene, a chlorophyll a-binding inner antenna protein in the photosystem II complex, located in the lipid matrix of the thylakoid membrane. Real-time results showed that the expression of the p-psbB gene was significantly up-regulated in wild ginseng as compared to cultivated ginseng. Thus, the p-psbB gene may be one of the important markers of wild ginseng.

The Mutation that Makes Escherichia coli Resistant to λ P Gene-mediated Host Lethality Is Located within the DNA Initiator Gene dnaA of the Bacterium

  • Datta, Indrani;Banik-Maiti, Sarbani;Adhikari, Lopa;Sau, Subrata;Das, Niranjan;Mandal, Nitai Chandra
    • BMB Reports
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    • v.38 no.1
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    • pp.89-96
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    • 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.

Genomic Analysis of miR-21-3p and Expression Pattern with Target Gene in Olive Flounder

  • Jo, Ara;Lee, Hee-Eun;Kim, Heui-Soo
    • Genomics & Informatics
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    • v.15 no.3
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    • pp.98-107
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    • 2017
  • MicroRNAs (miRNAs) act as regulators of gene expression by binding to the 3' untranslated region (UTR) of target genes. They perform important biological functions in the various species. Among many miRNAs, miR-21-3p is known to serve vital functions in development and apoptosis in olive flounder. Using genomic and bioinformatic tools, evolutionary conservation of miR-21-3p was examined in various species, and expression pattern was analyzed in olive flounder. Conserved sequences (5'-CAGUCG-3') in numerous species were detected through the stem-loop structure of miR-21-3p. Thus, we analyzed target genes of miR-21-3p. Among them, 3' UTR region of PPIL2 gene indicated the highest binding affinity with miR-21-3p based on the minimum free energy value. The PPIL2 gene showed high expression levels in testis tissue of the olive flounder, whereas miR-21-3p showed rather ubiquitous expression patterns except in testis tissue, indicating that miR-21-3p seems to control the PPIL2 gene expression in a complementary repression manner in various tissues of olive flounder. Taken together, this current study contributes to infer the target gene candidates for the miR-21-3p using bioinformatics tools. Furthermore, our data offers important information on the relationship between miR-21-3p and target gene for further functional study.

Differential Gene Expression after Adenovirus-Mediated p16 Gene Transfer in Human Non-Small Cell Lung Cancer Cells (폐암세포주에서 아데노바이러스 매개 p16 유전자 전달로 인한 유전자 발현의 변화)

  • 박미선;김옥희;박현신;지승완;엄미옥;염태경;강호일
    • Toxicological Research
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    • v.20 no.2
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    • pp.109-116
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    • 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.

Identification of three pathways for p-cresol catabolism and their gene expression in Pseudomonas alkylphenolica KL28 (Pseudomonas alkylphenolica KL28에 존재하는 3종류의 p-cresol 분해 경로 및 유전자 발현)

  • Sung, Jin Il;Lee, Kyoung
    • Korean Journal of Microbiology
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    • v.52 no.3
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    • pp.298-305
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    • 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.

Isolation of Constitutive Mutant of xylA Gene in Escherichia coli (대장균 xylA 유전자의 구성적 변이주의 분리)

  • Soh, Jae Hyun;Roh, Dong Hyun;Rhee, In Koo
    • Current Research on Agriculture and Life Sciences
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    • v.11
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    • pp.81-89
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    • 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.

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Construction of a Plant Expression Vector for the Coat Protein Gene of Cucumber Mosaic Virus-As Strain for Plant Transformation (오이 모자이크 바이러스 As계통 외피단백질 유전자의 식물체 형질질환을 위한 발현벡타의 구축)

  • 류기현;박원목
    • Korean Journal Plant Pathology
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    • v.11 no.1
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    • pp.66-72
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    • 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.

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