• Title/Summary/Keyword: Open Frame Structure

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Cloning, Sequencing and Baculovirus-based Expression of Fusion-Glycoprotein D Gene of Herpes Simplex Virus Type 1 (F)

  • Uh, Hong-Sun;Choi, Jin-Hee;Byun, Si-Myung;Kim, Soo-Young;Lee, Hyung-Hoan
    • BMB Reports
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    • v.34 no.4
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    • pp.371-378
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    • 2001
  • The Glycoprotein D (gD) gene of the HSV-1 strain F was cloned, sequenced, recombinated into the HcNPV (Hyphantria cunea nuclear polyhedrosis virus) expression vector and expressed in insect cells. The gD gene was located in the 6.43 kb BamHI fragment of the strainF. The open reading frame (ORF) of the gD gene was 1,185 by and codes 394 amino acid residues. Recombinant baculoviruses, GD-HcNPVs, expressing the gD protein were constructed. Spodoptera frugiperda cells, infected with the recombinant virus, synthesized a matured gX-gD fusion protein with an approximate molecular weight of 54 kDa and secreted the gD proteins into the culture media by an immunoprecipitation assay The fusion gD protein was localized on the membrane of the insect cells, seen by using an immunofluorescence assay The deduced amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. These results indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins.

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Molecular Cloning of Seven-band Grouper (Epinephelus septemfasciatus) Growth Hormone cDNA and Its Expression in Escherichia coli

  • Lee Jehee;Munasinghe Helani;Song Choon Bok
    • Fisheries and Aquatic Sciences
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    • v.6 no.3
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    • pp.116-124
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    • 2003
  • Isolation and cloning of seven-band grouper (Epinephelus septemfasciatus) growth hormone cDNA from pituitary gland revealed an open reading frame of 612 bp coding for a pre-growth hormone of 204 amino acids with a 17 amino acid putative signal peptide. Deduced amino acid sequence showed that there was one possible N-glycosylation site at $Asn^{l84}$ and four cysteine residues $(Cys^{52},\;Cys^{160},\;Cys^{177},\;Cys^{185})$ on t e same positions as in some other species where they were involved in the stabilization of the tertiary structure. The seven-band grouper growth hormone (sbgGH) presented a $99.5\%$ amino acid sequence identity with the growth hormone of Epinephelus coioides and contained the conserved hormone domain region. Comparison of growth hormone sequences from evolutionarily diverse species revealed 25 amino acid residues conserved in jawless fishes to modern mammals. It also revealed an evolutionary trend to retain the same polypeptide sequence even in the distantly related animals while allowing alterations to occur in polypeptides of the closely related species. In order to create a recombinant system to produce high levels of the growth hormone, it was expressed in Escherichia coli (BL21) cells. The gel analysis revealed theoretically expected molecular weights for both mature and pre-sbgGHs.

Cloning of a pore-forming subunit of ATP-sensitive potassium channel from Clonorchis sinensis

  • Hwang, Seung-Young;Han, Hye-Jin;Kim, So-Hee;Park, Sae-Gwang;Seog, Dae-Hyun;Kim, Na-Ri;Han, Jin;Chung, Joon-Yong;Kho, Weon-Gyu
    • Parasites, Hosts and Diseases
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    • v.41 no.2
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    • pp.129-133
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    • 2003
  • A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-senstive potassium channel in the adult worm, Clonorchis sinensis, termed CsKir6.2, was isolated from an adult CDNA library. The cDNA contained a single open-reading frame of 333 amino acids, which has a structural motif (a GFG-motif) of the putative pore-forming loop of the Kir6.2. Peculiarly, the Cskir6.2 shows a lack-sequence structure, which deleted 57 amino acids were deleted from its N-terminus. The predicted amino acid sequence revealed a highly conserved sequence as other known other Kir6.2 subunits. The mRNA was weekly expressed in the adult worm.

Molecular Characterization of a New Hantaan Virus Howang Strain (한탄바이러스 호왕균주의 M, S 유전자 절편의 염기서열 및 분자생물학적 특성)

  • Chu, Yong-Kyu;Lee, Ho-Wang
    • The Journal of Korean Society of Virology
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    • v.27 no.1
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    • pp.59-68
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    • 1997
  • Hantaan virus Howang strain which isolated from the blood of severe case of Korean hemorrhagic fever is more virulent than HTN 76/118 and showed different RFLP from partial PCR amplifed M genome segment to established Hantaan serotype viruses. We have determined the nucleotide sequence of the M and S genome segments and compared to HTN 76/118. The M and S segment of Howang strain has 3615 and 1696 nucleotides long, respectively. The M segment sequence of Howang strain is one mucleotide shorter than HTN 76/118. The sequence data of Howang strain shows 93.5% homology to HTN 76/118. One long open reading frame, which strats from 41nt. to 3448nt. of the M segment and from 37nt. to 1326nt. of the S segment, exist to on complementary sense of the virus genome. There are no significant difference between HTN 76/118 and Howang strain on hydrophobicity of deduced polypeptides, but has slight difference on secondary structure.

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Isolation of N-Acetylmuramoyl-L-Alanine Amidase Gene (amiB) from Vibrio anguillarum and the Effect of amiB Gene Deletion on Stress Responses

  • Ahn Sun-Hee;Kim Dong-Gyun;Jeong Seung-Ha;Hong Gyeong-Eun;Kong In-Soo
    • Journal of Microbiology and Biotechnology
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    • v.16 no.9
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    • pp.1416-1421
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    • 2006
  • We identified a gene encoding the N-acetylmuramoyl L-alanine amidase (amiB) of Vibrio anguillarum, which catalyzes the degradation of peptidoglycan in bacteria. The entire open reading frame (ORF) of the amiB gene was composed of 1,722 nucleotides and 573 amino acids. The deduced amino acid sequence of AmiB showed a modular structure with two main domains; an N-terminal region exhibiting an Ami domain and three highly conserved, continuously repeating LysM domains in the C-terminal portion. An amiB mutant was constructed by homologous recombination to study the biochemical function of the AmiB protein in V. anguillarum. Transmission electron microscopy (TEM) revealed morphological differences, and that the mutant strain formed trimeric and tetrameric unseparated cells, suggesting that this enzyme is involved in the separation of daughter cells after cell division. Furthermore, inactivation of the amiB gene resulted in a marked increase of sensitivity to oxidative stress and organic acids.

Sequencing of the RSDA Gene Encoding Raw Starch-Digesting $\alpha$-Amylase of Bacillus circulans F-2: Identification of Possible Two Domains for Raw Substrate-Adsorption and Substrate-Hydrolysis

  • Kim, Cheorl-Ho
    • Journal of Microbiology and Biotechnology
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    • v.2 no.1
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    • pp.56-65
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    • 1992
  • The complete nucleotide sequence of the Bacillus circulans F-2 RSDA gene, coding for raw starch digesting a-amylase (RSDA), has been determined. The RSDA structure gene consists of an open reading frame of 2508 bp. Six bp upstream of the translational start codon of the RSDA is a typical gram-positive Shine-Dalgarno sequence and the RSDA encodes a preprotein of 836 amino acids with an Mr of 96, 727. The gene was expressed from its own regulatory region in E. coli and two putative consensus promoter sequences were identified upstream of a ribosome binding site and an ATG start codon. Confirmation of the nucleotide sequence was obtained and the signal peptide cleavage site was identified by comparing the predicted amino acid sequence with that derived by N-terminal analysis of the purified RSDA. The deduced N-terminal region of the RSDA conforms to the general pattern for the signal peptides of secreted prokaryotic proteins. The complete amino acid sequence was deduced and homology with other enzymes was compared. The results suggested that the Thr-Ser-rich hinge region and the non-catalytic domain are necessary for efficient adsorption onto raw substrates, and the catalytic domain (60 kDa) is necessary for the hydrolysis of substrates, as suggested in previous studies (8, 9).

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Isolation and Functional Analysis of spy1 Responsible for Pristinamycin Yield in Streptomyces pristinaespiralis

  • Jin, Qingchao;Yin, Huali;Hong, Xiaowei;Jin, Zhihua
    • Journal of Microbiology and Biotechnology
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    • v.22 no.6
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    • pp.793-799
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    • 2012
  • A gene related to high pristinamycin yield in Streptomyces pristinaespiralis was selected by amplified fragment length polymorphism (AFLP) and its functions were investigated by gene disruption. First, a 561 bp polymorphic sequence was acquired by AFLP from high-yield recombinants compared with the S. pristinaespiralis ancestor ATCC25486, indicating that this approach is an effective means of screening for valuable genes responsible for antibiotic yield. Then, a 2,127 bp open reading frame of a gene designated spy1 that overlaps with the above fragment was identified and its structure and biological functions were investigated. In silico analysis of spy1 encoding a deduced 708-amino-acid-long serine/threonine protein kinase showed that it only contains a catalytic domain in the N-terminal region, which is different from some known homologs. Gene inactivation of chromosomal spy1 indicated that it plays a pleiotropic regulatory function in pristinamycin production, with a positive correlation to pristinamycin I biosynthesis and a negative correlation to pristinamycin II biosynthesis.

A Study On the Position Control System of the Small ROV Using Sonar Sensors (소나 센서를 이용한 소형 ROV의 위치제어시스템에 관한 연구)

  • Choi, Dong-Hyun;Um, Geun-Nam;Kim, Sang-Hyun
    • Journal of the Society of Naval Architects of Korea
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    • v.45 no.6
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    • pp.579-589
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    • 2008
  • In the past few years, there are many studies and researches of the underwater vehicles which are carried out its mission using sonar sensors. MSCL(Marine System Control Lab.) at Inha University developed test-bed small ROV, ISRO. ISRO is an open-frame type and has 4 thrusters. ISRO can control 4 motions i.e surge, sway, yaw and heave with sonar sensors. ISRO is developed for inspection of ship hull, marine structure, plant of lake or river and so on. When ROV ISRO inspects something, it is necessary to control the position of ROV ISRO's for the movement and anti-collision with structures in the underwater. In this paper, we deal with the development of a small ROV and verification of the position control system via simulation and experiment using sonar sensors.

Genomic Organization of Penicillium chrysogenum chs4, a Class III Chitin Synthase Gene

  • Park, Yoon-Dong;Lee, Myung-Sook;Kim, Ji-Hoon;Jun Namgung;Park, Bum-Chan;Bae, Kyung-Sook;Park, Hee-Moon
    • Journal of Microbiology
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    • v.38 no.4
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    • pp.230-238
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    • 2000
  • Class III chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class III chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5'flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.

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The Effect of MgO Content on the Preparation of Porous Hydroxyapaite Scaffolds by Polymer Sponge Method (폴리머 스펀지법을 이용한 다공성 수산화아파타이트 지지체 제조 시 MgO 첨가량에 따른 영향)

  • Jin, Hyeong-Ho;Min, Sang-Ho;Lee, Won-Ki;Park, Hong-Chae;Yoon, Seog-Young
    • Korean Journal of Materials Research
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    • v.16 no.11
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    • pp.715-718
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    • 2006
  • Porous hydroxyapatite (HAp) scaffolds have been prepared by using the slurry including HAp and magnesia based on the replication of polymer sponge substrate. The influence of MgO content in slurry on the pore morphology and size, density, porosity, and mechanical strength of porous HAp scaffolds was investigated. The obtained scaffolds with average pore sizes ranging 150 to 300 mm had open, relatively uniform, and interconnected porous structure regardless of MgO content. As the MgO content increased, the pore network frame of scaffolds became to be relatively stronger, even though the pore size was not much changed. The compressive strength of the scaffolds increased rapidly with the increase of MgO content because of increasing the pore wall thickness and density of the scaffolds. As a result, the porosity, density, and compressive strength of the porous HAp scaffolds prepared by the sponge method were significantly affected by the addition of MgO.