• Title, Summary, Keyword: ORF 3 & 3-1

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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.

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.

Molecular Characterization of a Korean Isolate of Human Norovirus, the Hu/NLV/Gunpo/2006/KO Strain (인체 노로바이러스의 한국분리주 Hu/NLV/Gunpo/2006/KO의 분자생물학적 특성)

  • Jeong, Ah-Yong;Yun, Sang-Im;Jee, Young-Mee;Kang, Yoon-Sung;Lee, Young-Min
    • Korean Journal of Microbiology
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    • v.45 no.2
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    • pp.105-111
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    • 2009
  • Norovirus (NV) with a variety of genotypes, a member of the family Caliciviridae, causes acute nonbacterial gastroenteritis in humans. We determined the nucleotide sequence of three open reading frames (ORFs) of a NV Korean strain and characterized the genetic relationship with others. The Korean strain designated Hu/NLV/Gunpo/2006/KO was isolated from the stool specimen of a 2-year-old female suffering from gastroenteritis. By performing reverse transcription and PCR amplification, three overlapping cDNAs were synthesized and used for direct sequencing. We found that like other NVs, this strain contains three ORFs: ORF1, 5,100 bp; ORF2, 1,647 bp; ORF3, 765 bp. Of 35 NVs, ORF1 had a level of genetic diversity lower than ORF2 and ORF3, of which the C-termini of the ORF2 and ORF3 showed a relatively high degree of genetic diversity. Phylogenetic analyses indicated that the Korean strain belonged to genogroup II, with Saitama U1, Gifu'96, Mc37, and Vietnam 026 being formed a single genetic cluster. The nucleotide sequence information of three ORFs of a NV Korean isolate will be useful not only for the development of a diagnostic tool and understanding of genetic relationship, but also provide important basic information for the functional analysis of their gene products.

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.

Three ORF-Containing Group I Introns in Chloroplast SSU of Caulerpa sertularioides (Ulvophyceae) and Their Evolutionary Implications

  • Lee, Jung-Ho;Manhart, James R.
    • ALGAE
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    • v.18 no.3
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    • pp.183-190
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    • 2003
  • Except for a group I intron in trnL-uaa occuring in eubacteria and plastids, group I introns are rarely documented in plastid genomes. Here, we report that a green alga, Caulerpa sertularioides, contains three group IA3 introns in the 16S gene (cpSSU), CS-cpSSU.i1, CS-cpSSU.i2 and CS-cpSSU.i3. Each intron has an open reading frame with LAGLIDADG motifs. CS-cpSSU.i1orf and CS-cpSSU.i3orf occur at Loop 6 in the intron secondary structure and CScpSSU. i2orf at Loop 8. CS-cpSSU.i1orf and CS-cpSSU.i2orf contain both LAGLI-DADG motifs but CS-cpSSU.i3orf has only one. CS-cpSSU.i1 and CS-cpSSU.i2 share the insetion sites and the ORFs at Loop 6 and 8 with CpSSU·1 and CpSSU·2 introns of Chlamydomonas pallidostigmatica (Chlorophyceae). In contrast, CS-cpSSU.i3, containing 28 copies of GAAATAT at Loop 6, is a novel intron found only in Caulerpa sertularioides. Possible scenarios of the evolution of the three introns and their possible use in systematic research are discussed.

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.

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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Identification of the+1 Ribosomal Frameshifting Site of LRV1-4 by Mutational Analysis

  • Kim Se Na;Choi Jung Ho;Park Min Woo;Jeong Sun Joo;Han Kyung Sook;Kim Hong Jin
    • Archives of Pharmacal Research
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    • v.28 no.8
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    • pp.956-962
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    • 2005
  • Leishmania virus (LRV)1-4 has been reported to produce a fusion of ORF2 and ORF3 via a programmed +1 frameshift in the region where ORF2 and ORF3 overlap (Lee et a/., 1996). However, the exact frameshift site has not been identified. In this study, we compared the frameshift efficiency of a 259bp (nt. 2565-2823), frameshift region of LRV1-4, and the 71 bp (nt. 2605-2678) sub-region where ORF2 and ORF3 overlap. We then predicted the frameshift site using a new computer program (Pseudoviewer), and finally identified the specific region associated with the mechanism of the LRV1-4's+1 frameshift by means of a mutational analysis based on the predicted structure of LRV1-4 RNA. The predicted structure was confirmed by biochemical analysis. In order to measure the frameshift efficiency, constructs that generate luciferase without a frameshift or with a+1 frameshift, were generated and in vitro transcription/translation analysis was performed. Measurements of the luciferase activity generated, showed that the frameshift efficiency was about $1\%$ for both the 259bp (LRV1-4 259FS) and 71 bp region (LRV1-4 71FS). Luciferase activity was strongly reduced in a mutant (LRV1-4 NH: nt. 2635-2670) with the entire hairpin deleted and in a mutant (LRV1-4 NUS: nt. 2644-2659) with the upper stem of the hairpin deleted. These results indicate that the frameshift site in LRV1-4's is in the 71 bp region where ORF2 and ORF3 overlap, and that nt. 2644-2659 (the upward hairpin stem) playa key role in generating the +1 frameshift.

Expression of porcine reproductive and respiratory syndrome virus (PRRSV) ORF7 gene and monoclonal antibody production (돼지생식기호흡기증후군바이러스 ORF7 유전자 발현 및 단크론항체 생산)

  • Lee, Seung-Chul;Park, Ga-Hye;Lee, Kyeong-Won;Ryu, Min-Sang;Kang, Shien-Young
    • Korean Journal of Veterinary Service
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    • v.37 no.3
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    • pp.143-150
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    • 2014
  • Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiological agent of PRRS characterized by reproductive losses in sows and respiratory disorders in piglets. The PRRSV is a small enveloped virus containing a positive-sense, single-stranded RNA genome and divided into two genotype, type 1 (European) and type 2 (North American), respectively, by nucleotide identity. In this study, ORF7 gene of the type 1 and type 2 PRRSV was cloned and expressed in Baculovirus expression system. Also, monoclonal antibodies (MAbs) against ORF7 were produced and characterized. The expressed ORF7 proteins in the recombinant virus were confirmed by indirect fluorescence antibody (IFA) test using His6 and PRRSV-specific antiserum. A total of eight MAbs were produced and characterized. One (3G12) MAb was type 1 PRRSV ORF7-specific and two (6B10 and 16H8) were type 2 PRRSV ORF7-specific. Other five (1A1, 2A4, 4B4, 12C4 and 13F11) MAbs reacted with both type 1 and type 2 PRRSV. Some PRRSV ORF7-specific MAbs recognized the porcine tissues infected with PRRSV by IFA or immunohistochemistry (IHC) assay. From this experiment, it was confirmed that MAbs produced in this study were PRRSV ORF7-specific and could be used as reliable reagents for type 1/type 2 PRRSV detection.

Zoogloea ramigera 115SLR의 생고분자물질 생합성에 관여하는 pyruvyl transferase gene의 cloning 및 염기서열 결정

  • 이삼빈
    • Microbiology and Biotechnology Letters
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    • v.24 no.4
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    • pp.415-422
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    • 1996
  • A gene coding for a pyruvyl transferase enzyme involved in exopolysaccharide biosynthesis of Zoogloea ramigera 115SLR was isolated and sequenced. A 4.5 kb of BamHI DNA fragment was isolated from chromosomal DNA using a probe derived from ketal pyruvyl transferase gene of Xanthomonas campestris. The nucleotide sequence of 2.66 kb Pst1/HindIII DNA fragment which was homology with a probe revealed the existence of two complete open reading frames (ORF2 and ORF3) and two partial open reading frames (ORFI and ORF4). The deduced amino acid sequence of ORF3 was homologous to the ketalase (GumL product) of X campestris with 49.5% of similarity and 21.6% of identity. ORF2 on the other hand showed the higher identity with the ketalase (ExoV product) of Rhizobium meliloti (36%) as well as the ketalase of X campestris (23%) than that of ORF3. A gene product of ORF2 was determined with a bacteriophage T7 RNA polymerase/promoter system in E. coli. The molecular weight of protein was 33,500 dalton.

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