• Title/Summary/Keyword: ORF analysis

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Study on Norovirus Genotypes in Busan, Korea (부산지역에서 분리된 norovirus 유전자형 연구)

  • Kim, Nam-Ho;Park, Eun-Hee;Park, Yon-Koung;Min, Sang-Kee;Jin, Seong-Hyeon;Park, So-Hyun
    • Journal of Life Science
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    • v.21 no.6
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    • pp.845-850
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    • 2011
  • Norovirus (NoV) causes major acute non-bacterial gastroenteritis in humans. NoV genus is a member of the family Caliciviridae, which is transmitted by contaminated food and water or from human to human. Many genotypes of genogroups I and II have been reported because of their high genetic diversity. To obtain molecular epidemiological information on gastroenteritis sporadic cases in Busan, Korea, we analyzed the nucleotide sequences of NoV strains detected during 2008~2010. We performed one step RT-PCR amplifying the open reading frame (ORF) 2 (capsid region) followed by semi-nested PCR. Fecal samples were collected from 4,071 acute gastroenteritis, and genotypes of the 421 positive samples were determined by sequence analysis. Based on partial sequence of capsid region, 7 NoV were categorized into genogroup I and 13 into genogroup II. Prevalent genotypes among gastroenteritis patients within Busan were GII.4, GI.6, GII.5 in 2008~2010. The results of this study will contribute to the currently available epidemiological data and improve public health and hygiene via development of diagnostic methods and sustainable surveillance.

Comparative Genomics Profiling of Clinical Isolates of Helicobacter pylori in Chinese Populations Using DNA Microarray

  • Han, Yue-Hua;Liu, Wen-Zhong;Shi, Yao-Zhou;Lu, Li-Qiong;Xiao, Shudong;Zhang, Qing-Hua;Zhao, Guo-Ping
    • Journal of Microbiology
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    • v.45 no.1
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    • pp.21-28
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    • 2007
  • In order to search for specific genotypes related to this unique phenotype, we used whole genomic DNA microarray to characterize the genomic diversity of Helicobacter pylori (H. pylori) strains isolated from clinical patients in China. The open reading frame (ORF) fragments on our microarray were generated by PCR using gene-specific primers. Genomic DNA of H. pylori 26695 and J99 were used as templates. Thirty-four H. pylori isolates were obtained from patients in Shanghai. Results were judged based on In(x) transformed and normalized Cy3/Cy5 ratios. Our microarray included 1882 DNA fragments corresponding to 1636 ORFs of both sequenced H. pylori strains. Cluster analysis, revealed two diverse regions in the H. pylori genome that were not present in other isolates. Among the 1636 genes, 1091 (66.7%) were common to all H. pylori strains, representing the functional core of the genome. Most of the genes found in the H. pylori functional core were responsible for metabolism, cellular processes, transcription and biosynthesis of amino acids, functions that are essential to H. pylori's growth and colonization in its host. In contrast, 522 (31.9%) genes were strain-specific genes that were missing from at least one strain of H. pylori. Strain-specific genes primarily included restriction modification system components, transposase genes, hypothetical proteins and outer membrane proteins. These strain-specific genes may aid the bacteria under specific circumstances during their long-term infection in genetically diverse hosts. Our results suggest 34 H. pylori clinical strains have extensive genomic diversity. Core genes and strain-specific genes both play essential roles in H. pylori propagation and pathogenesis. Our microarray experiment may help select relatively significant genes for further research on the pathogenicity of H. pylori and development of a vaccine for H. pylori.

The Effect of Interleukin $1-{\beta}$, Platelet Derived Growth Factor-BB and Transforming Growth $Factor-{\beta}$ on the expression of PDLs17 mRNA in the Cultured Human Periodontal Ligament Fibroblasts (($IL-1{\beta}$), PDGF-BB 그리고 $TGF-{\beta}$가 사람 배양 치주인대 섬유모세포의 PDLs17 mRNA의 발현에 미치는 영향)

  • Lirn, Ki-Jung;Han, Kyung-Yoon;Kirn, Byung-Ock;Yeorn, Chang-Yeob;Park, Joo-Cheol
    • Journal of Periodontal and Implant Science
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    • v.31 no.4
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    • pp.787-801
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    • 2001
  • The molecular mechanisms control the function of PDL(periodonta1 ligament) cells and/or fibroblasts remain unclear. PDLsl7, PDL-specific gene, had previousely identified the cDNA for a novel protein from cultured PDL fibroblasts using subtraction hybridization between gingival fibroblasts and PDL fibroblasts. The purpose of this study was to determine the regulation by growth factors and cytokines on PDLsl7 gene expression in cultured human periodontal ligament cells and observe the immunohistochemical localization of PDLsl7 protein in various tissues of mouse. Primary PDL fibroblasts isolated by scraping the root of the extracted human mandibular third molars. The cells were incubated with various concentration of human recombinant $IL-1{\beta}$, PDGF-BB and TGF\;${\beta}$ for 48h nd 2 weeks. At each time point total RNA was extracted and the levels of transcription ere assessed by reverse transcription-polymerase chain reaction (RT-PCR assay). polyclonal antiserum raised against PDLsl7 peptides, CLSVSYNRSYQINE and SEAVHETDLHDGC, were made, and stained the tooth, periodontium, developing bone, bone marrow and mid-palatal suture of the mouse. The results were as follows. 1. PDLsl7 mRNA levels were increased in response to PDGF (10ng/ml) and $TGF\;{\beta}$(20ng/ml) after treatment of the $IL-1{\beta}$, PDGF-BB and $TGF{\beta}$for 48 h. 2. PDLsl7 was up-regulated only by $TGF{\beta}$(20 ng/ml) after treatment of the $IL-1{\beta}$, PDGF-BB and $TGF\;{\beta}$ for 2 weeks and unchanged by the other stimulants. 3. PDLsl7 was a novel protein coding the 142 amino acid peptides in the ORF and the nucleotide sequences of the obtained cDNA from RT-PCR was exactly same as the nucleotides of the database. 4. Immunohistochemical analysis showed that PDLsl7 is preferentially expressed in the PDL, differentiating osteoblast-like cells and stromal cells of the bone marrow in the adult mouse. 5. The expression of PDLsl7 protein was barely detectable in gingival fibroblasts, hematopoetic cells of the bone marrow and mature osteocytes of the alveolar bone. These results suggest that PDLsl7 might upregulated by PDGF-BB or $TGF{\beta}$ and acts at the initial stage of differentiation when the undifferentiated mesenchymal cells in the bone marrow and PDL differentiate into multiple cell types. However, more research needs to be performed to gain a better understanding of the exact function of PDLsl7 during the differentiation of bone marrow mesenchymal and PDL cells.

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Characterization and Cloning of a Phytase from Escherichia coli WC7. (Escherichia coli WC7가 생산하는 Phytase의 효소특성과 그 유전자의 클로닝)

  • 최원찬;오병철;김형권;강선철;오태광
    • Microbiology and Biotechnology Letters
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    • v.30 no.1
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    • pp.1-7
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    • 2002
  • Phytase from Escherichia coli WC7 was purified from cell extracts and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for phytate hydrolysis was 6$0^{\circ}C$ and pH 5.0, respectively. The enzyme was stable up to 6$0^{\circ}C$ and over broad pH range (pH 2-12). The enzyme had higher affinity for sodium phytate than p-nitrophenylphosphate (pNPP). That is, the apparent Km value for sodium phytate and pNPP were $0.15\pm$0.02 mM and 2.82$\pm$0.05 mM, respectively. The gene encoding the phytase was cloned in E. coli XL1-Blue. Sequence analysis showed an open reading frame of 1241 Up encoding a signal peptide (22 aa) and a mature enzyme (410 aa). WC7 phytase was expressed up to 17.5 U/ml in the transformed E. coli XL1-Blue/pUEP, which was 23-fold higher than the activity from wild strain.

Isolation and Characterization of a Novel Flavonoid 3'-Hydroxylase (F3'H) Gene from a Chrysanthemum (Dendranthema grandiflorum) and Its Gamma-ray Irradiated Mutants (감마선 처리에 의한 스프레이형 국화 화색변이체로부터 Flavonoid 3'-Hydroxylase(F3'H) 유전자의 분리 및 특성 구명)

  • Chung, Sung-Jin;Lee, Geung-Joo;Kim, Jin-Baek;Kim, Dong-Sub;Kim, Sang-Hoon;Kang, Si-Yong
    • Horticultural Science & Technology
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    • v.30 no.2
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    • pp.162-170
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    • 2012
  • The objectives of this study were to isolate and the sequence of novel $F3'H$ gene related to an anthocyanin pathway, and to confirm the expression patterns of the gene involved in the flower color variations of chrysanthemum mutants. In this study, we isolated the full-length cDNAs and the genomic DNAs of an $F3'H$ gene from a wild type (WT) chrysanthemum (cv. Argus) and its three color mutants. The sequence analysis revealed a putative open reading frame of 1,527 bp that encodes a polypeptide of 509 amino acids. Sequence homology ranged from 97% to 99% between 'Argus' and its three color mutants. The sequence analysis from the genomic DNA revealed that the chrysanthemum $DgF3'H$ gene consisted of three exons and two introns spanning a 3,830 bp length. The sizes of the gene for three mutants ranged from a shorter size of 3,828 bp to a longer size of 3,838 bp when compared to the size of WT. The total size of the two introns was 2,157 bp for WT, but those for three color mutants ranged from 2,154 bp to 2,159 bp. A result of an RT-PCR analysis indicated that the color variations of the mutants AM1 and AM2 can be partly explained by the structural modification derived from the sequencial changes in the gene caused by gamma ray. A Southern blot analysis revealed that the $DgF3'H$ gene existing as multiple copies in the chrysanthemum genome. A systemic study will be further needed to provide a genetic mechanism responsible for the color mutation and to uncover any involvement of genetic elements for the expression of the $DgF3'H$ gene for the color variation in chrysanthemum.

Amino Acid Biosynthesis and Gene Regulation in Seed (종자내 아미노산 합성 조절 유전자에 관한 연구)

  • ;;;;;Fumio Takaiwa
    • Proceedings of the Botanical Society of Korea Conference
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    • 1996.07a
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    • pp.61-74
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    • 1996
  • Human and monogastric animals can not synthesize 10 out of the 20 amino asids and therefor need to obtain these from their diet. The plant seed is a major source of dietary protein. It is particular important in their study to increase nutritional quality of the seed storage proteins. The low contents of lysine, asparagine and threonenein various cereal seeds and of cystein and methionine. In legume seeds is due to the low proportions of these amino acids in the major storage proteins, we have tried to apply the three strategies; (1) mutagenesis and selection of specific amino acid analogue resistance, (2) cloning and expression study of lysine biosynthesis related gene, (3) transfomation of lysine rich soybean glycinin gene. The 5-methyltryptophan (5MT) resistant cell lines, SAR1, SAR2 and SAR3 were selected from anther derived callus of rice (Oryza sativa L. "Sasanishiki"). Among these selected cell lines, two (SAR1 and SAR3) were able to grow stably at 200 mg/L of 5MT. Analysis of the freed amino acids in callus shows that 5MT resistant cells (SAR3) accumulated free tryptophan at least up to 50 times higher than those that of the higher than of SAS. These results indicated that the 5MT resistant cell lines are useful in studies of amino acid biosynthesis. Tr75, a rice (Oryza sativa L., var. Sasanishiki) mutant resistant to 5MT was segregated from the progenies of its initial mutant line, TR1. The 5MT resistant of TR75 was inherited in the M8 generations as a single dominant nuclear gene. The content of free amino acids in the TR75 homozygous seeds increased approximately 1.5 to 2.0 fold compared to wild-type seeds. Especially, the contents of tryptophan, phenylalanine and aspartic acid were 5.0, 5.3 and 2.7 times higher than those of wild-type seeds, respectively. The content of lysine is significantly low in rice. The lysine is synthesized by a complex pathway that is predominantly regulated by feedback inhibition of several enzymes including asparginase, aspatate kinase, dihydrodipicolinat synthase, etc. For understanding the regulation mechanism of lysine synthesis in rice, we try to clone the lysine biosynthetic metabolism related gene, DHPS and asparaginase, from rice. We have isolated a rice DHPS genomic clone which contains an ORF of 1044 nucleotides (347 amino acids, Mr. 38, 381 daltons), an intron of 587 nucleotides and 5'and 3'-flanking regions by screening of rice genomic DNA library. Deduced amino acid sequence of mature peptide domain of GDHPS clone is highly conserved in monocot and dicot plants whereas that of transit peptide domain is extremely different depending on plant specie. Southern blot analysis indicated that GDHPS is located two copy gene in rice genome. The transcripts of a rice GDHPS were expressed in leaves and roots but not detected in callus tissues. The transcription level of GDHPS is much higher in leaves indicating enormous chloroplast development than roots. Genomic DNA clones for asparaginase genes were screened from the rice genomic library by using plaque hybridization technique. Twelve different genomic clones were isolated from first and second screening, and 8 of 12 clones were analyzed by restriction patterns and identified by Southern Blotting, Restriction enzyme digestion patterns and Southern blot analysis of 8 clones show the different pattern for asparaginase gene. Genomic Southern blot analysis from rice were done. It is estimated that rice has at least 2-3 copy of asparaginase gene. One of 8 positive clones was subcloned into the pBluescript SK(+) vector, and was constructed the physical map. For transformation of lysine rich storage protein into tobacco, soybean glycinin genes are transformed into tobacco. To examine whether glycinin could be stably accumulated in endosperm tissue, the glycinin cDNA was transcriptionally fused to an endosperm-specific promotor of the rice storage protein glutelin gene and then introduced into tobacco genomic via Agrobacterium-mediated transformation. Consequently the glycinin gene was expressed in a seed-and developmentally-specific manner in transgenic tobacco seeds. Glycinin were targeted to vacuole-derived protein bodies in the endosperm tissue and highly accumulated in the matrix region of many transgenic plant (1-4% of total seed proteins). Synthesized glycinin was processed into mature form, and assembled into a hexamer in a similar manner as the glycinin in soybean seed. Modified glycinin, in which 4 contiguous methionine residues were inserted at the variable regions corresponding to the C - teminal regions of the acidic and basic polypeptides, were also found to be accumulated similarly as in the normal glycinin. There was no apparent difference in the expression level, processing and targeting to protein bodies, or accumulation level between normal and modified glycinin. glycinin.

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Studies on Host-Virus Interaction of Poxviruses 1. Cytochemical, Autoradiographic and Immunocytological Analysis in Cowpox Virus-FL Cell System (Poxvirus 감염(感染)에 있어서의 Virus-숙주세포(宿主細胞)의 상호관계(相互關係) 1. Cowpox Virus-FL 세포계(細胞系)의 세포화학적(細胞化學的) Autoradiography 및 세포면역학적해석(細胞免疫學的解析))

  • Kim, Uh Ho
    • Korean Journal of Veterinary Research
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    • v.15 no.1
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    • pp.57-67
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    • 1975
  • The poxvirus group is considered to be a typical cytoplasmic inclusion forming virus. Every poxvirus has been reported to produce only one kind of inclusion in the infected tissues. A vague concept that inclusions of poxviruses are eosinophilic or acidophilic has prevailed. Although many papers and theories about the nature of the inclusion have been presented, most of them are not quite convincing on the point of the relations with virus multiplication, and an analysis of papers published showed that there seem to be many discrepancies in the descriptions of the nature of the poxvirus inclusions. Comparative studies on host-virus interaction with cowpox, orf, swinepox and fowlpox viruses which selected from each Group (I-IV) of poxviruses were performed from the morphological and virological standpoints. At first, in cowpox virus-FL cell system, as a comparative model, cytoplasmic inclusion, nucleic acid metabolism by autoradiography and detection of viral antigen by immunofluorescence were studied and obtained the results as follows: 1. The focus-like cytopathic effect (CPE) at early stage developed to entire culture at terminal stage of infection, and also the developing status of CPE was correlated to viral doses for inoculation. Two kinds of cytoplasmic inclusions which named A and B type were easily observed by Giemsa, hematoxylin-eosin (H & E) and May-Greenwald Giemsa (MGG) stainings in the infected cells. The B type inclusions were formed at early stage of infection and the A type inclusions were produced subsequently the B type formation. The B type which common type inclusion in poxviruses was a small compact or aggregate at early stage and developed to a large diffuse body at terminal stage of infection. On the other hand, the A type inclusion which depend upon the kind of virus was appeared as round and discrete shape, and its size and number was increased gradually during the culture period. It was characteristic to form distinct halos around the both types of inclusions in acid fixed, H & E stained preparations of infected cultures. The B type inclusion was always positive in Feulgen reaction and showed as DNA containing body but the A type inclusion was not. 2. In the relationship between inclusion and DNA metabolism of infected cells by the qualitative autoradiography using 3H-thymidine, the appearance of silver grains was coincided with B type inclusion but not with A type inclusion. This showed that the DNA synthesis was proceeded in all B type inclusions except those in the terminal stage with a diffuse form. This suggested that the B type inclusions are only sites of DNA synthesis and this was proceeded after the cell infection independently. The activity of DNA synthesis of the inclusions was nearly the same as that of the nucleic of normal cells and non-inclusion bearing cells. and non-inclusion bearing cells. Regardless of the size of the degree of DNA synthesis of the B type inclusion, inclusion bearing cells all showed remarkable suppression of nuclear DNA synthesis. 3. By the direct fluorescent antibody technique viral antigen in infected cells was detected. The B type inclusions have been proved to contain a great deal of viral antigen, whereas the basic substance of A type inclusion did not show antigenicity except the round edge. It was suggested that the round edge fluorescence might be caused by the glare of cytoplasmic viral antigen which pushed out and concentrated by the A type inclusion development. 4. Hemorrhagic red pock formations on chorioallantoic membrane of embryonated chicken egg had proved the characteristic of used viral strain. 5. By the above studies on the nature of two types of inclusions and the role they play in virus multiplication, it was concluded that the B type inclusion must be the site of the synthesis of viral DNA and protein as well as the site of the virus.

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Identification of a Potexvirus in Korean Garlic Plants (한국 마늘 Potexvirus의 cDNA 유전자 분리 및 분포에 관한 연구)

  • Song, Jong-Tae;Choi, Jin-Nam;Song, Sang-Ik;Lee, Jong-Seob;Choi, Yang-Do
    • Applied Biological Chemistry
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    • v.38 no.1
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    • pp.55-62
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    • 1995
  • To understand the molecular structure of Korean garlic viruses, cDNA cloning of virus genomic RNA was attempted. Virus particles were isolated from virus-infected garlic leaves and a cDNA library was constructed from garlic virus RNA. One of these clones, S81, selected by random sequencing has been identified as a member of potexvirus group other than potyvirus and carlavirus. The clone is 873 bp long contains most of the coat protein (CP) coding region and 3'-noncoding region including poly(A) tail. A putative polyadenylation signal sequence (AAUAAA) and the hexanucleotide motif (ACUUAA), a replicational cis-acting element conserved in the 3'-noncoding region of potexvirus RNAs are noticed. The clone S81 shows about 30-40% identity in both nucleotide and amino acid sequences with CPs of potexviruses. The genome size of the virus was analysed to be 7.46 knt by Northern blot analysis, which was longer than those of other potexviruses. The open reading frame encoding CP was expressed as a fusion protein (S81CP) in Escherichia coli and the recombinant protein was purified by immobilized metal binding affinity chromatography. Polyclonal antibody was raised against S81CP in rabbit to examine the occurrence of garlic potexvirus in Korean garlic plants by immunoblot analysis. Two virus protein bands of Mr 27,000 and 29,000 from garlic leaf extract of various cultivars reacted with the antibody. It was shown that Mr 27,000 band might not be a degradation product of Mr 29,000 band, suggesting that two types of potexvirus different in size of coat protein could exist in Korean garlic plants.

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Molecular Characterization of Cinnamate 4-Hydroxylase gene in Red Hot Pepper (Capsicum annuum L.) (고추에서 분리한 Cinnamate 4-Hydroxylase 유전자의 분자생물학적 특성)

  • Kim Kye-Won;Ha Sun-Hwa;Cho Kang-Jin;Kim Eun-Ju;Lee Min-Kyung;Yu Jae-Ju;Kim Jong-Guk;Lee Shin-Woo
    • Journal of Plant Biotechnology
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    • v.32 no.3
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    • pp.167-173
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    • 2005
  • Three different cDNAS for cinnamate 4-hydroxylase (C4H) which are involved in the second step of the general phenylpropanoid pathway were isolated and designated as pc4h1 (1,755 bp), pc4h2 (1,655 bp), and pc4h3 (1,316 bp), respectively. The nucleotide sequence analysis revealed that both pc4h1 and pc4h2 clones encode polypeptides of 505 amino acids frame but pc4h3 clone was truncated at the 5'-end of coding region. The alignment of the deduced amino acid sequences showed that PC4H1 and PC4H2 are highly homologous (95.8% identical) with each other and contain three conserved domains which are typical in cytochrome P450 monooxygenase: proline-rich region, threonine-containing binding pocket for the oxygen molecule, and heme binding region. In addition, result of the phylogenic tree analysis revealed that both pepper C4Hs belong to Class 1. pc4h2 transcription was strongly induced in wounded fruit (400%) and root (200%) relative to its very low basal level but not in leaf or stem tissue. In case of pc4h1, the basal level of transcription was higher than pc4h2 but induction by wounding was lower in fruit and root while leaf and stem tissues did not respond to wounding. The basal level of pc4h3 transcripts was not, if any, detectable and response to wounding was not observed.

Cloning and Characterization of a 5-Enolpyruvyl Shikimate 3-Phosphate Synthase (EPSPS) Gene from Korean Lawn Grass (Zoysia japonica) (들잔디 5-Enolpyruvyl Shikimate 3-Phosphate Synthase(EPSPS) 유전자 클로닝 및 특성)

  • Lee, Hye-Jung;Lee, Geung-Joo;Kim, Dong-Sub;Kim, Jin-Beak;Ku, Ja-Hyeong;Kang, Si-Yong
    • Horticultural Science & Technology
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    • v.28 no.4
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    • pp.648-655
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    • 2010
  • This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn grass ($Zoysia$ $japonica$). The full length cDNA of the EPSPS from Korean lawn grass ($zj$EPSPS) obtained from a 3' and 5' RACE method was 1540 bp, containing a 1176 bp ORF, a 144 bp leader sequence (5' UTR) and a 220 bp 3' UTR, which was eventually decoded 391 amino acid residues with a molecular mass of 41.74 kDa. The Southern blot detection of the $zj$EPSPS showed that the gene exists as a single copy in the Korean lawn grass genome. Sequence comparison of the $zj$EPSPS gene demonstrated that the glyphosate-tolerant mutant (GT) having a Pro-53 to Ser substitution in the gene seems to have a preferred binding activity of the enzyme to phosphoenol pyruvate(PEP) over glyphosate, which allows the continuous synthesis of aromatic amino acids in the shikimate pathway. From the Northern blotting analysis, the $zj$EPSPS was found to be highly expressed, with continuous increase until 36 hours after 0.5% glyphosate treatment in both wild and mutant samples, but 1.5-fold higher EPSP synthase activity was observed in the tolerant mutant when exposed to the glyphosate treatment. The molecular information of the $zj$EPSPS gene obtained from this study needs to be further dissected to be more effectively applied to the development of gene-specific DNA markers and zoysiagrass cultivars; nevertheless, the glyphosate-tolerant mutant having the featured $zj$EPSPS gene can be provided to turfgrass managers for weed problems with timely adoptable management options.