• Title/Summary/Keyword: ORF

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Cloning and Characterization of the Paraquat Resistance-Related Genes from Ochrobactrum anthropi JW-2 (Ochrobactrum anthropi JW-2 유래의 Paraquat 내성유전자 PqrA의 주변 유전자군 분석)

  • Bae Eun-Kyung;Lee Hyo-Shin;Won Sung-Hye;Lee Byung-Hyun
    • Microbiology and Biotechnology Letters
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    • v.34 no.1
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    • pp.15-22
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    • 2006
  • A 4,971 bp chromosomal DNA fragment containing the pqrA, paraquat resistance gene, was cloned from Ochrobactrum anthropi JW-2, and the complete nucleotide sequence was determined. Nucleotide and deduced amino acid sequences of the fragment revealed the presence of 4 complete ORFs (orf2, pqrA, orf3, orf4) and two incomplete ORFs(orf1, orf5). Orf1, pqrA, orf4 and orf5 exists at the direct strand but orf2 and orf3 exists at the reverse complementary strand. Orf1 which of incomplete sequences without start codon shares homology with ATP binding region of the response regulator receiver. Orf2 shares high homology with members of the tetR family of transcriptional repressor which have a helix-turn-helix (H-T-H) motif. Therefore, the orf2 is predicted as a transcriptional repressor of pqrA and is designated as pqrR2. Orf3 shares high homology with the members of the lysR family acting as a transcriptional activator which have both of a H-T-H motif at the N-terminal region and substrate binding domain at the C-terminal region. Therefore, the orf3 is predicted as a transcriptional activator of pqrA and is designated as pqrR1. Orf4 shows homology with the periplasmic substrate-binding protein of amino acid ABC transporter. Orf5 which of incomplete sequences without stop codon revealed the homology with the permeases protein of amino acid ABC transporter.

Molecular Cloning and Analysis of the Genes in the Vicinity of Streptomyces griseus Trypsin (SGT) Gene from Streptomyces griseus ATCC10137 (Streptomyces griseus ATCC10137에서 Trypsin 유전자 sprT의 주변 유전자군 분석)

  • Chi Won-Jae;Kim Mi-Soon;Kim Jong-Hee;Kang Dae-Kyung;Hong Soon-Kwang
    • Korean Journal of Microbiology
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    • v.41 no.4
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    • pp.255-261
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    • 2005
  • A 6.7kb DNA fragment containing the sprT gene encoding Streptomyces griseus trypsin (SGT) was cloned from Streptomyces griseus ATCC 10137, and the complete nucleotide sequence was determined. Nucleotide sequence and deduced amino acid or the EcoRI-HindIII fragment revealed the presence or the six complete ORFs containing the sprT gene and one incomplete ORF, which were named ORF1, SGT, ORF2, ORF3, ORF4, ORF5, and ORF6, respectively. ORF1 has homology with the oxidoreductases from several organisms. ORF2 and ORF3 show similarity with unknown proteins and transcription regulator that belongs to the ArsR family, respectively. ORF4 and ORF5 show homology with the peptidoglycan bound protein with LPXTG motif from Listeria monocytogenes and the membrane protein with transmembrane helix from several organisms, respectively. The last ORF, ORF6, shows homology with the lipoprotein from Streptomyces avermitilis.

Analysis of the orf 282 Gene and Its Function in Rhodobacter sphaeroide 2.4.1 (R. sphaeroides 에서의 orf282 유전자의 분석과 이들의 기능)

  • Son, Myung-Hwa;Lee, Sang-Joon
    • Journal of Life Science
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    • v.22 no.8
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    • pp.1009-1017
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    • 2012
  • The orf282 gene of Rhodobacter sphaeroides is located between the ccoNOQP operon encoding $cbb_3$ terminal oxidase and the fnrL gene encoding an anaerobic activator, FnrL. Its function remains unknown. In an attempt to reveal the function of the orf282 gene, we disrupted the gene by deleting a portion of the orf282 gene and constructed an orf282-knockout mutant. Two FnrL binding sites were found to be located upstream of orf282, and it was demonstrated that orf282 is positively regulated by FnrL. The orf282 gene is not involved in the regulation of spectral complex formation. The $cbb_3$ oxidase activity detected in the orf282 mutant was comparable to that in the wild-type sample, indicating that the orf282 gene is not involved in the regulation of the ccoNOQP operon and the biosynthesis of the cbb3 cytochrome c oxidase. The elevated promoter activity of the nifH and nifA genes, which are the structural genes of nitrogenase and its regulator, respectively, in the orf282 mutant, suggests that the orf282 gene product acts as a negative effector for nifH and nifA expression.

Complete Nucleotide Sequence of Tobacco Mosaic Virus Isolated from Wasabi(Eutrema wasabi Maxim.) (고추냉이에서 분리한 담배 모자이크 바이러스(TMV-W)의 전체 유전자 염기서열 분석)

  • 이귀재
    • Korean Journal of Plant Resources
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    • v.16 no.1
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    • pp.82-88
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    • 2003
  • Genomic RNA sequence of a tobamovirus infecting Eutrema wasabi plant(TMV-W) was determined. The RNA is composed 6,298 nucleotide and contains four OREs encoding the protein of 180KD(OREI), 130KD(ORE2),30KD(ORF3) and 18KD(coat protein, ORF4). ORE4, ORF 3, ORF 2 and ORF 1 are overlaped by 130, 20 and 40 nucleotides, and the overapping region can be folded into a stable hairpin styucture. This includes the 3'non-coding region of 238 nucleotides, coat protein gene(537 nucleotides,179 amino acid), 30KD movement protein gene(825 nucleotides, 275 amino acid), 13(IKD protein gene(1,896 nucleotides, 632 amino acid) and 180KD protein gene(2,958 nucleotides, 986 amino acid). The genomic RNA sequence was compared with homologous regions of eleven other tobamoviruses. TMV-WTE was similar to TMV-WSF(98.6%) in nucleotide sequence.

Subcloning and DNA Sequencing of the Phenol Regulatory Genes in Ralstonia eutropha JMP134 (Ralstonia eutropha JMP134에서 페놀분해에 관여하는 조절유전자의 Subcloning 및 염기서열 분석)

  • ;Subramanian Chitra
    • Korean Journal of Microbiology
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    • v.38 no.4
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    • pp.260-266
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    • 2002
  • In this study, chromosomal DNA fragment related to the regulation of phenol metabolism in Ralstonia eutropha JMP 134 was cloned and sequenced. The result has shown that two open reading frames (ORF1 and ORF2) exist on this regulatory region. ORF1, which initiates from 454 bp downstream of the stop codon of the phenol hydroxylase genes, was found to be composed of 501 amino acids. ORF2, whose start codon is overlapped with the stop codon of ORFl, was found to contain 232 amino acids. The comparison of amino acid sequences with other proteins has revealed that ORF1 belongs to the family of NtrC transcriptional activator, whereas ORF2 shares high homology with the family of GntR protein, which is known to be a negative regulator. ORF1 and ORF2 were designated as a putative positive regulator, phlR2 and a negative regulator phlA, respectively. Possible regulatory mechanisms of phenol metabolism in this strain was discussed.

Action mechanism of upstream open reading frame from S-adenosylmethionine decarboxylase gene as a in vivo translational inhibitor (S-Adenosylmethionine decarboxylase 유전자의 upstream open reading frame이 in vivo에서 translational inhibitor 로서의 작용 기작)

  • Choi, Yu-Jin;Park, Ky-Young
    • Journal of Plant Biotechnology
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    • v.38 no.1
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    • pp.87-93
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    • 2011
  • S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50), a key enzyme for polyamines biosynthesis, was tightly regulated for homeostatic levels. Carnation SAMDC gene (CSDC9) has an small upstream open reading frame (uORF) of 54 amino acids in 5'-leader sequence. To explore the functional mechanism of uORFs in controlling translation, we used a GUS reporter gene driven with the 35S promoter and uORF region of SAMDC gene for making transgenic tobacco plants. In our experiment, there were a translational inhibition of its downstream GUS ORF by SAMDC uORF sequence or SAMDC uORF protein. Expecially, translational inhibition was most effective in point-mutated construct, in which the start codon was changed. Therefore, this results suggested the ribosomal stalling might be involved in this translational inhibitory process. The frame shift in amino acid sequence of SAMDC uORF with start codon and stop codon resulted in a moderate increasing in GUS activity, suggesting the native amino acid sequence was important for a function as a translational inhibitor. Also, we showed that the production of GUS protein was significantly inhibited in the presence of the small uORF using histochemical analysis of GUS expression in seedlings and tobacco flowers. Importantly, the small uORF sequence induced a real peptide of 5.7 kDa, which was provided the presence of SAMDC uORF peptide band using an in vitro transcription/translation system. The peptide product of uORF might interact with other components of translational machinery as well as polyamines, which was resulted from that polyamine treatment was inhibited GUS protein band in SDS-PAGE experiment.

Nucleotide Sequence of 7.2 kb Mitochondrial Linear Plasmid DNA in Pleurotus ostreatus (Pleurotus ostreatus 미토콘드리아의 7.2 kb 선상 플라스미드 염기서열 분석)

  • 윤혜숙;구용범;노정혜
    • Korean Journal of Microbiology
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    • v.37 no.1
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    • pp.37-41
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    • 2001
  • Two linear plasmid-like DNAs, 10.2 kb and 7.2 kb were found in the mitochondria of P. ostreatus. They have covalently linked 5'-terminal proteins in both ends. Two continuous fragments of 4.7 kb and 2.3 kb from 7.2 kb DNA were cloned and sequenced. Two long open reading frames (ORF1; 2982 bp, 993 a.a and ORF2; 2703 bp, 900 a.a) and one short open reading frame(ORF3; 771 bp, 256 a.a) were found in the 7.2 kb plasmid. The putative ORF1 and ORF2 have conserved motifs of DNA polymerases and RNA polymerases, respectively, while the ORF3 has homologous regions with phosphatase from Plasmodium, and also with adhesine from Mycoplasma.

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ORF5a Protein of Porcine Reproductive and Respiratory Syndrome Virus is Indispensable for Virus Replication (PRRS 바이러스 ORF5a 단백질의기능학적역할)

  • Oh, Jongsuk;Lee, Changhee
    • Microbiology and Biotechnology Letters
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    • v.43 no.1
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    • pp.1-8
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    • 2015
  • In this study, a DNA-launched reverse genetics system was developed from a type 2 porcine reproductive and respiratory syndrome virus (PRRSV) strain, KNU-12. The complete genome of 15,412 nucleotides was assembled as a single cDNA clone and placed under the eukaryotic CMV promoter. Upon transfection of BHK-tailless pCD163 cells with a full-length cDNA clone, viable and infectious type 2 progeny PRRSV were rescued. The reconstituted virus was found to maintain growth properties similar to those of the parental virus in porcine alveolar macrophage (PAM) cells. With the availability of this type 2 PRRSV infectious clone, we first explored the biological relevance of ORF5a in the PRRSV replication cycle. Therefore, we used a PRRSV reverse genetics system to generate an ORF5a knockout mutant clone by changing the ORF5a translation start codon and introducing a stop codon at the 7th codon of ORF5a. The ORF5a knockout mutant was found to exhibit a lack of infectivity in both BHK-tailless pCD163 and PAM-pCD163 cells, suggesting that inactivation of ORF5a expression is lethal for infectious virus production. In order to restore the ORF5a gene-deleted PRRSV, complementing cell lines were established to stably express the ORF5a protein of PRRSV. ORF5a-expressing cells were capable of supporting the production of the replicationdefective virus, indicating complementation of the impaired ORF5a gene function of PRRSV in trans.

Identification of C4orf32 as a Novel Type I Endoplasmic Reticulum Resident Membrane Protein (Type I 소포체 목표화 막단백질에 속하는 새로운 C4orf32 막단백질의 동정)

  • Lee, Seung-Hwan;Park, Sang-Won;Lee, Jin-A;Jang, Deok-Jin
    • Journal of Life Science
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    • v.29 no.9
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    • pp.949-954
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    • 2019
  • Membrane topology is a key characteristic of membrane proteins. We previously reported the cloning of the chromosome 4 open-reading frame 32 (C4orf32) gene as a potential membrane protein; however, the cellular localization and membrane topology of C4orf32 was as yet unknown. In this study, we found that green fluorescent protein (GFP) fused to the C-terminus of C4orf32 (C4orf32-GFP) was localized to the endoplasmic reticulum (ER). We applied three tools to identify determinants of C4orf32 topology: protease protection, fluorescence protease protection (FPP), and an inducible system using the ternary complex between FK506 binding protein 12 (FKBP), rapamycin, and the rapamycin-binding domain of mTOR (FRB) (the FRB-rapamycin-FKBP system). Using protease protection and FPP assays, we found that the GFP tag in C4orf32-GFP was localized to the cytoplasmic surface of the ER membrane of HeLa cells. Protease protection and FPP assays are useful and complimentary tools for identifying the topology of GFP fusion membrane proteins. The FRB-rapamycin-FKBP system was also used to study the topology of C4orf32. In the absence of rapamycin, a monomeric red fluorescent protein-FKBP fusion (mRFP-FKBP) and C4orf32-GFP-FRB were localized to the cytoplasm and the ER membrane, respectively. However, in the presence of rapamycin, the mRFP-FKBP was shifted from the cytoplasm to the ER and colocalized with the C4orf32-GFP-FRB. These results indicate that the FRB moiety is facing the cytoplasmic surface of ER membrane. Overall, our results clearly suggest that C4orf32 belongs to the family of type I ER resident membrane proteins.

C4orf47 is a Novel Prognostic Biomarker and Correlates with Infiltrating Immune Cells in Hepatocellular Carcinoma

  • Hye-Ran Kim;Choong Won Seo;Sang Jun Han;Jongwan Kim
    • Biomedical Science Letters
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    • v.29 no.1
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    • pp.11-25
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    • 2023
  • In hepatocellular carcinoma (HCC), chromosome 4 open-reading frame 47 (C4orf47) has not been so far investigated for its prognostic value or association with infiltrating immune cells. We performed bioinformatics analysis on HCC data and analyzed the data using online databases such as TIMER, UALCAN, Kaplan-Meier plotter, LinkedOmics, and GEPIA2. We found that C4orf47 expression in HCC was higher compared to normal tissues. High C4orf47 expression was associated with a worse prognosis in HCC. The correlation between C4orf47 and infiltrating immune cells is positively associated with CD4+T cells, B cells, neutrophils, macrophages, and dendritic cells in HCC. Moreover, high C4orf47 expression was correlated with a poor prognosis of infiltrating immune cells. Analysis of C4orf47 gene co-expression networks revealed that 12501 genes were positively correlated with C4orf47, whereas 7200 genes were negatively correlated. The positively related genes of C4orf47 are associated with a high hazard ratio in different types of cancer, including HCC. Regarding the biological functions of C4orf47 gene, it mainly regulates RNA metabolic process, DNA replication, and cell cycle. The C4orf47 gene may play a prognostic role by regulating the global transcriptome process in HCC. Our findings demonstrate that high C4orf47 expression correlates with poor prognosis and tumor-infiltrating immune cells in HCC. We suggest that C4orf47 is a novel prognostic biomarker and potential immune therapeutic target for HCC.