• Title/Summary/Keyword: open reading frame 4

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Purification, Characterization, and Cloning of Trimethylamine Dehydrogenase from Methylophaga sp. Strain SK1

  • Kim, Hee-Gon;Kim, Yan;Lim, Heon-Man;Shin, Hyun-Jae;Kim, Si-Wouk
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.11 no.4
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    • pp.337-343
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    • 2006
  • Trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde, was purified from Methylophaga sp. strain SK1. The active TMADH was purified 12.3-fold through three purification steps. The optimal pH and temperature for enzyme activity was determined to be 8.5 and $55^{\circ}C$, respectively. The $V_{max}\;and\;K_m$ values were 7.9 nmol/min/mg protein and 1.5 mM. A genomic DNA of 2,983 bp from Methylophaga sp. strain SK1 was cloned, and DNA sequencing revealed the open reading frame (ORF) of the gene coding for TMADH. The ORF contained 728 amino acids with extensive identity (82%) to that of Methylophilus methylotrophus $W_3A_1$.

Aspergillus niger로 부터 $\alpha$-glucosidase 발현억제 형질전환체의 분리

  • 이동건;이진영;서영배
    • Microbiology and Biotechnology Letters
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    • v.24 no.4
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    • pp.427-429
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    • 1996
  • We have already cloned an extracellular $\alpha$-glucosidase gene from Aspergillus niger with oligonucleotide probe synthesized on the basis of the peptide sequences determined previously. The DNA sequence revealed an open reading frame of 895 amino acids split by three introns. We are attempting to construct an A. niger strain deficient in the $\alpha$-glucosidase enzyme activity, which would be useful for the glucoamylase production without contamination by the industrially undesirable $\alpha$-glucosidase. For destruction of the $\alpha$-glucosidase gene, we try to make transformations. A cloned partial $\alpha$-glucosidase gene was introduced into Aspergillus niger, and transformants with suppressed $\alpha$-glucosidase activity were isolated. The transformants were cultured on YPD medium which contained Hygromycin B at 30$\circ$C. The activity of $\alpha$-glucosidase of the suppressed transformants was compared to that of wild type activity. As shown by southern-hybridization, we detected that the transformant was a heterocaryon.

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Amino Acid Sequence Homology of Hybrid Poplar O-methyltransferuse Involved in Lignin Biosynthesis

  • Park, Young-Goo;Sul, Ill-Whan;Shin, Dong-Ill;Park, Jang-Won;Park, Hee-Sung
    • Journal of Plant Biotechnology
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    • v.3 no.3
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    • pp.131-134
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    • 2001
  • In $\lambda$-Zap II vector system, a cDNA library was constructed for the developing secondary xylem mRNA from hybrid poplar, Populus nigra x maximowiczii. A cDNA clone of 1.5 kb in size, pOMTB1.4 encoding a lignin-bispecific O-methyltransferase was screened by plaque hybridization using a probe of 540 bp cDNA amplified by polymerase chain reaction from the cDNA library and identified by nucleotide sequencing. Its nucleotide sequence contains one open reading frame of 366 amino acids. The deduced amino acid sequence in comparison with that of Populus tremuloides showed the differences of 9 amino acids and revealed 85-99% homology among alfalfa, poplar and aspen.

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Inositol(1,4,5)triphosphate 3-Kinase의 유전자 재조합과 CCL39 Hamster Lung Fibroblasts에서 발현

  • 김재웅;최관용
    • The Korean Journal of Food And Nutrition
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    • v.9 no.2
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    • pp.123-136
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    • 1996
  • IPSKCDNA gene(1.8 kbp) encoding rat brain IP3K enzyme contained Not I restric site in open reading frame. The Not I sequence, GCGGCCGC, was converted to GCAGCCGC by site-directed mutagenesis. The mutated IP3KcDNA was digested with EcoR I and ligated with EcoR I-restricted psp72·Not2 vector. The resulting psp72 · Not2-IP3KCDNA was digested with the Not I restriction enzyme and then subcloned into the Not I -digested PZIP · NeoSV(X) mammalian expression vector. The PZIP · NeoSV(X) -IPSKCDNA was transfected into CCL39 hamster lung fibroblast cells. The efficiency of the expressed IPSKCDNA gene was significantly higher than expected generally, not only a mean 5-fold increase in the amount of enzyme, but also 16-fold increase in enzyme activity from tractsfected CCL39 cells by the method of Western blot using anti-lP3K antibodies. Both distribution of IPSK in various rat tissues and biochemical properties were discussed.

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Isolation and Characterization of Brain-Derived Neurotrophic Factor Gene from Flounder (Paralichthys olivaceus)

  • LEE JAE HYUNG;CHOI TAE-JIN;NAM SOO WAN;KIM YOUNG TAE
    • Journal of Microbiology and Biotechnology
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    • v.15 no.4
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    • pp.838-843
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    • 2005
  • Brain-derived neurotrophic factor (BDNF) is a small secretory protein and a member of the nerve growth factor (NGF) gene family. We cloned the flounder BDNF gene from a flounder brain cDNA library. The nucleotide sequence of the cloned gene showed an open reading frame (ORF) consisting of 810 bp, corresponding to 269 amino acid residues. The tissue distribution of flounder BDNF was determined by reverse transcription-polymerase chain reaction (RT-PCR) in brain, embryo, and muscle tissues. To express fBDNF using a eukaryotic expression system, we constructed the vector mpCTV-BDNF containing the fBDNF gene and transformed this vector into Chlorella ellipsoidea. Stable integration of introduced DNA was confirmed by PCR analysis of genomic DNA, and mRNA expression in C. ellipsoidae was confirmed by RT-PCR analysis.

Molecular Cloning and Sequencing of Cell Wall Hydrolase Gene of an Alkalophilic Bacillus subtilis BL-29

  • Kim, Tae-Ho;Hong, Soon-Duck
    • Journal of Microbiology and Biotechnology
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    • v.7 no.4
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    • pp.223-228
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    • 1997
  • A DNA fragment containing the gene for cell wall hydrolase of alkalophilic Bacillus subtilis BL-29 was cloned into E. coli JM109 using pUC18 as a vector. A recombinant plasmid, designated pCWL45B, was contained in the fragment originating from the alkalophilic B. subtilis BL-29 chromosomal DNA by Southern hybridization analysis. The nucleotide sequence of a 1.6-kb HindIII fragment containing a cell wall hydrolase-encoding gene was determined. The nucleotide sequence revealed an open reading frame (ORF) of 900 bp with a concensus ribosome-binding site located 6 nucleotide upstream from the ATG start codon. The primary amino acid sequence deduced from the nucleotide sequence revealed a putative protein of 299 amino acid residues with an M.W. of 33, 206. Based on comparison of the amino acid sequence of the ORF with amino acid sequences in the GenBank data, it showed significant homology to the sequence of cell wall amidase of the PBSX bacteriophage of B. subtilis.

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Cloning and Overexpression of the Cdd Gene Encoding Cytidine Deaminase from Salmonella typhimurium

  • Lee, Sang-Mahn
    • Korean Journal of Environmental Biology
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    • v.21 no.1
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    • pp.56-59
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    • 2003
  • The Salmonella typhimurium cdd gene encoding cytidine deaminase (cyti-dine/2'-deoxycytidine aminohydrolase; EC 3.5.4.5.) was isolated through shotgun clon-ing by complementation of the E. coli odd mutation. By subsequent deletion and sub-cloning from the original 3.7 Kb of EcoRI insert (pSAMI), the precise region of the cdd structural gene is located around the BglII site in the middle part of 1.7 Kb of NruI/PvuI segment. The 1.7 Kb containing odd gene wag subcloned to the pUC18 vector and the nucleotide sequence of the cdd gene was determined. When the putative ribosorne-binding site (Shine-Dalgarno sequence) and initiation codon were predicted to be GAGG at the position 459 and ATG at the position 470, respectively, there was an open reading frame of 885 nucleotides, encoding an 294 amino acid protein. The cdd gene expression in E. coli JF611/pSAMI was amplified about 50 fold compared to that of the wild type. The cdd gene expression was maintained in the stationary phase after rea-ching the peak in the late logarithmic phase.

Nucleotide Sequence and Characterization of ptsG Gene Encoding Glucose-specific Enzyme II of Phosphotransferase System from Brevibacterium flavum

  • Yoon, Ki-Hong
    • Journal of Applied Biological Chemistry
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    • v.48 no.4
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    • pp.218-221
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    • 2005
  • Nucleotide sequence of Brevibacterium flavum ptsG gene capable of complementing Escherichia coli ZSC113 mutations defective to glucose permease activity of phosphotransferase system was completely determined, and the gene product was compared with other glucose-specific enzyme II ($EII^{Glc}$). A ptsG gene of B. flavum consisted of open reading frame of 2,025 nucleotides putatively encoding polypeptide of 675 amino acid residues and TAA stop codon. Deduced amino acid sequence of B. flavum ($EII^{Glc}$) had high homology with ($EIIs^{Glc}$) of Corynebacterium glutamicum, C. efficiens, and B. lactofermentum. Arrangement of structural domains, IIBCA, of B. flanum ($EII^{Glc}$) protein was identical to that of EIIs belonging to glucose-phosphotransferase system.

Pseudomonas sp. strain DJ77 균주에서 extradiol dioxygenase 를 암호화하고 있는 phnE 유전자의 염기배열

  • 김영창;신명수;윤길상;박영순;김욱현
    • Korean Journal of Microbiology
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    • v.30 no.1
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    • pp.8-14
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    • 1992
  • Nucleotide Sequence of phnE Gene Encoding Extradiol Dioxygenase fromPseudomonas sp. Strain DJ77Kim, Young-Chang'.", Myeong-Su Shin1, Kil-Sang Younl, Young-Soon Park1, andUg-Hyeon Kim'.' (Department of Microbiology, C'hungbuk National University.Cheongju 360-763, KOREA. and 'Research Center for Molecular Microbiology,Seoul National University)nal University)

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DNA Sequence of the phnN Gene for Benzaldehyde Dehydrogenase from Pseudomonas sp. DJ77 and Its Substrate Preference

  • Kim, Seong-Jae;Hwang, Soon-Young;Kim, Young-Chang
    • Journal of Microbiology
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    • v.37 no.4
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    • pp.224-228
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    • 1999
  • Benzaldehyde dehydrogenase (BZDH), an enzyme involved in the degradation of toluene and xylenes, is encoded by the phnN gene of Pseudomonas sp. strain DJ77. We determined the nucleotide sequence of a DNA fragment of 1,803 base pairs which included the phnN gene. The fragment contained an open reading frame of 1,506 base pairs to accommodate th 55 kDa sized enzyme encoding BZDH. The enzyme efficiently oxidized benzaldehyde, salicylaldehyde, m-tolualdehyde and ps-tolualdehyde.

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