• Title/Summary/Keyword: Gene Analysis

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Transgenic Plants Expressing an Antisense RNA of ALl-Gene from Tomato Golden Mosaic Virus(TGMV) (Tomato Golden Mosaic Virus(TGMV) AL1 -gene의 antisense RNA 발현 형질 전환 식물체)

  • 임성렬
    • Korean Journal of Plant Tissue Culture
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    • v.25 no.3
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    • pp.147-152
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    • 1998
  • AL1-gene, necessary for the replication of the genome of a gemini virus TGMV, was inserted in the opposite direction to the promoter CaMV35S resulting in the construction of a plant transformation binary vector pAR35-2. The vector pAR35-2 contains the chimeric gene cassette involving the duplicated promoter CaMV35S, opposite direction of AL1-gene fusioned with hygromycin resistant gene, and the gene cassette of the neomycin phosphotransferase II gene. The plasmid was transferred to tobacco and tomato plants by leaf disk infection via Agrobacterium. The transgenic plants were selected and grown on the MS-agar medium containing kanamycin and hygromycin. The shoots induced from the calli were regenerated to the whole transgenic plants. The antisense AL1-gene was detected in the genomic DNA isolated from the leaves by using the PCR mediated Southern blot analysis. The expression of the antisense AL1-gene was also observed using the RT-PCR mediated Southern blot analysis. The observation of chloroplasts in guard cell pair indicated that the transgenic tomato plants were diploid.

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The amino acid analysis of polyhedrin and DNA sequence of ployhedrin gene in nuclear polyhedrosis virus (Nuclear polyhedrosis virus의 polyhedrin 아미노산 및 polyhedrin gene 염기서열 분석)

  • Lee, Keun-Kwang
    • Journal of fish pathology
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    • v.8 no.1
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    • pp.37-46
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    • 1995
  • The amino acid analysis of polyhedrin protein and nucleotide sequence of polyhedrin gene in H. cunea nuclear polyhedrosis virus (HcNPV) genome have been studied. Polyhedrin had three polypeptide bands in SDS - polyactylamide gel electrophoresis. The major polypeptide had a molecular weight of 25 kd. The polyhedrin was composed of 17 different amino acids. HcNPV DNA was digested with EcoRI restriction enzyme and hybridized with ($\alpha^{32}P$) -labelled AcNPV polyhedrin gene cDNA. The polyhedrin gene was located on the fragment of EcoRI-H. The EcoRI - H fragment containing polyhedrin gene was cloned into the EcoRI site of pUC8 vector which was confirmed with southern blotting, and the recombinant plasmid containg polyhedrin gene was designated as hPE-H. The promoter region of polyhedrin genomic DNA was sequenced. The sequences identified as the TATA box was found at the 5' flanking region of the polyhedrin genomic DNA approximately -79 bp upstream from the transcriptional start site. But CAAT-like box was not shown near the TATA-like box in the polyhedrin gene. Four tandem repeats with the sequence 5' -CTAATAT-3' and 5'-TAAATAA-3' were found between -141 and -108 or -83 upstream and -52 bp downstream from the translation start site. About -141 bp region upstream from the translational start site was highly AT (78%) rich. The coding region for the polyhedrin starts and ends with ATG and TAA, respectively.

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Molecular Cloning of a Defensin Homologue Gene of a Novel Family Member from the Firefly, Pyrocoelia rufa

  • Lee, Kwang-Sik;Park, Hye-Jin;Kim, Seong-Ryul;Lee, Sang-Mong;Sohn, Hung-Dae;Jin, Byung-Rae
    • International Journal of Industrial Entomology and Biomaterials
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    • v.3 no.1
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    • pp.57-62
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    • 2001
  • A cDNA encoding the defensin homologue of a novel family member was isolated from the cDNA library of the firefly,Pyrocoelia rufa. Sequence analysis of the cDNA encoding the defensin homologue of P. rufa resulted that the 165 bp cDHA has an open reading frame of 55 amino acid residues. The deduced amino acid sequences of the defensin homologue gene from P. rufa showed identity to known mammalian defensins. Also 6 cystein residues in the P. rufa defensin homologue gene were conserved in the same position as those of known mammalian defensins. The result suggested that P. rufa defensin homologue is a novel member of the insect defensin family. Southern blot analysis suggests that there may be a single copy number of the P.rufa defensin homologue gene and their fat body-specific expression pattern at the transcriptional level was confirmed by Northern blot analysis.

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Molocular Cloning and Sequence Analysis of the Immediate Early Viral Gene, IE1, from Bombyx mori Nuclear Polyhedrosis Virus K1

  • Park, Hye-Jin;Lee, Kwang-Sik;Je, Yeon-Ho;Sohn, Hung-Dae;Jin, Byung-Rae
    • International Journal of Industrial Entomology and Biomaterials
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    • v.3 no.1
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    • pp.43-49
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    • 2001
  • We have cloned and characterized an immediate early-1 gene, iel, which is activated immediately upon entrance of the viral genome into the cell nucleus, from Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain. This gene encodes a protein 584 amino acids with a predicted molecular weight of 67 kDa. The promoter and coding regions of BmNPV-K1 ie1 showed high homology with Autographa californica nuclear polyhedrosis virus and BmNPV T3 strain. The BmNPV-K1 ie1 was different from amino acid sequence at 4 positions in BmNPV T3. The location of ie1 gene in the BmNPV-K1 genome was confirmed by Southern blot analysis and its expression patterns at the transcriptional level in the infected cells were confirmed by Nerthern hybridization analysis.

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Molecular Cloning and Characterization of DNA Repair Related Gene in Yeast

  • Kang, Seon-Ah;Park, In-Soon
    • Journal of Life Science
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    • v.10 no.1
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    • pp.40-44
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    • 2000
  • The SNF2/SW ATPase/helicase family comprises proteins form a variety of species with in vivo functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair. Here, we reported the characterization of h게2+gene which was iolated by PCR amplification using the conserved domain of SNF2 motifs. Sequence analysis of PCR product showed striking evolutionary conservation among the SNF2 family of proteins. Two transcripts of 6.7 and 3.4 Lb were detected by Northern blot analysis. furthermore, the intensities of these two bands were increased by ultraviolet(UV) irradiation. These results indicate that the hrp2+ is a novel member of the SNF2 family of proteins and is one of the UV-inducible genes in S. pombe. To determine the level of transcripts of hrp2+ gene during cellular growth, Northern blot analysis were performed. This result indicates that the level of hrp2+transcript reached its maximum before cells entered the exponential growth phase. This suggests that hrp2+ gene is experssed mainly at the early stage of cell growth.

Nucleotide sequence analysis of the 5S ribosomal RNA gene of the mushroom tricholoma matsutake

  • Hwang, Seon-Kap;Kim, Jong-Guk
    • Journal of Microbiology
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    • v.33 no.2
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    • pp.136-141
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    • 1995
  • From a cluster of structural rRNA genes which has previsouly been cloned (Hwang and Kim, in submission; J. Microbiol. Biotechnol.), a 1.0-kb Eco RI fragment of DNA which shows significant homology to the 25S and rRNA s of Tricholoma matsutake was used for sequence analysis. Nucleotide sequence was bidirectionally determined using delection series of the DNA fragment. Comparing the resultant 1016-base sequence with sequences in the database, both the 3'end of 25S-rRNA gene and 5S rRNA gene were searched. The 5S rRNA gene is 118-bp in length and is located 158-bp downstream of 3'end of the 25S rRNA gene. IGSI and IGS2 (partial) sequences are also contained in the fragment. Multiple alignment of the 5S rRNA sequences was carried out with 5S rRNA sequences from some members of the subdivision Basidiomycotina obtained from the database. Polygenetic analysis with distance matrix established by Kimura's 2-parameter method and phylogenetic tree by UPGMA method proposed that T. matsutake is closely related to efibulobasidium allbescens. Secondary structure of 5S rRNA was also hypothesized to show similar topology with its generally accepted eukaryotic counterpart.

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Analysis of a Prodigiosin Biosynthetic Gene Cluster from the Marine Bacterium Hahella chejuensis KCTC 2396

  • Kim, Doc-Kyu;Park, Yon-Kyoung;Lee, Jong-Suk;F. Kim, Ji-Hyun;Jeong, Hae-Young;Kim, Beom-Seok;Lee, Choong-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.16 no.12
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    • pp.1912-1918
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    • 2006
  • Marine bacterium Hahella chejuensis KCTC 2396 simultaneously produced red antibiotic prodigiosin and undecylprodiginine. A complete set of the prodigiosin biosynthetic gene cluster has been cloned, sequenced, and successfully expressed in a heterologous host. Sequence analysis of the gene cluster revealed 14 ORFs showing high similarity to pig and red genes from Serratia spp. and Streptomyces coelicolor A3(2), respectively, and the gene organization was almost: similar to that of pig genes. These genes were named hap for Hahella prodigiosin, and determined to be transcribed as a single operon, by RT-PCR experiment. Based on the hap gene mutagenesis experiments and comparative analysis with pig and red genes, we propose a prodigiosin-biosynthetic pathway in KCTC 2396.

Lack of CHEK2 Gene Mutations in Differentiated Thyroid Carcinoma Patients using High Resolution Melting Analysis

  • Fayaz, Shima;Fard-Esfahani, Pezhman;Torbati, Peyman Mohammadi
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.12
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    • pp.5019-5022
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    • 2014
  • Recently, mutations in the genes involved in cell cycle control, including CHEK2, are being considered as etiological factors in different kinds of cancers. The CHEK2 protein plays an important role in protecting damaged DNA from entering mitosis. In this study the potential effects of two common mutations $IVS2+1G{\rightarrow}A$ and Ile157Thr of CHEK2 gene in differentiated thyroid carcinoma (DTC) were evaluated. A total of 100 patients admitted to the Research Institute for Nuclear Medicine were diagnosed with DTC based on pathology reports of surgery samples. An additional 100 people were selected as a control group with no cancer history. PCR-HRM (high resolution melting) analysis was performed to deal with each of mutations in all case and control samples separately. During the analysis of $IVS2+1G{\rightarrow}A$ and Ile157Thr mutations of CHEK2 gene in the case and control groups, all the samples were identified as wild homozygote type. The finding suggests that $IVS2+1G{\rightarrow}A$ and Ile157Thr mutations of CHEK2 gene do not constitute a risk factor for DTC in the Iranian population. However, further studies with larger population are required to confirm the outcome.

Gene Analysis of Resistance to Bacterial Blight, Xanthomonas oryzae pv, oryzae in Korean Six Rice Cultivars (우리나라 6개 벼 품종의 흰잎마름병 저항성 유전자 분석)

  • Ryuk, Jin-Ah;Choi, Chun-Hwan;Kang, Hee-Kyoung;Choi, Jae-Eul
    • Research in Plant Disease
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    • v.10 no.1
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    • pp.73-77
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    • 2004
  • The gene analysis of resistance in rice cultivars, Daeanbyeo, Hwasunchalbyeo, Daejinbyeo, Naepungbyeo, Hwajinbyeo and Surabyeo to strains of Xanthomonas oryzae pv. oryzae was studied. F$_1$ plants and F$_2$ populations from the crosses between six cultivars and near isogenic lines carrying the single bacterial blight(BB) resistance gene were analyzed using Korean and Japanese BB races. Daeanbyeo, Hwasunchalbyeo, Daejinbyeo, Naepungbyeo, Hwajinbyeo and Surabyeo are alleic with IRBB101 but are non-alleic with IRBB104 and IRBB105. The allelic tests indicated that Daeanbyeo, Hwasunchalbyeo, Daejinbyeo, Naepungbyeo, Hwajinbyeo and Surabyeo have the Xal gene for resistance.

Plant Protein Improvement by Synthetic Gene (합성유전자를 이용한 식물단백질의 향상)

  • 김태금;양문식
    • KSBB Journal
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    • v.7 no.3
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    • pp.155-160
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    • 1992
  • To improve the nutritional quality of plant proteins, a synthetic gene, called HEAAE (high essential amino acid encoding)-DNA, was introduced and expressed in tobacco plants. The synthetic gene, which is 292 basepair-long, codes for a protein composed of about 80% essential amino acids. To improve its expression level in plants, Cauliflower Mosaic Virus (CaMV) 355 and CaMV duplicate 35S promoters which are known as strong promoters were used with Nopaline Synthase promoter as a control. Transformed and regenerated tobacco plants were subject to analysis for introduction and expression of this gene. Integration of the gene into the plant genome and its expression into mRNAs and its proteins have been demonstrated using Southern, northern blot analysis and amino acid analysis. The differences of expression levels among CaMV duplicate 35S, CaMV 35S and Nopaline Synthase promoters are significant in term of mRNAs, but not in terms of proteins.

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