• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.153-157
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• 2006

A soil metagenomic library was constructed using an E. coli-fosmid cloning system with environmental DNAs extracted from Kwangreung forest topsoil. We targeted the genes involved in the biosynthesis of bacterial polyketides. Initially, a total of 36 clone pools (10,800 clones) were explored by the PCR-based method using the metagenomic DNAs from each pool and a degenerate primer set, which has been designed based on the highly conserved regions among ketoacyl synthase (KS) domains in actinomycete type I polyketide synthases (PKS Is). Six clone pools were tentatively selected as positive and further examined through a hybridization-based method for selecting a fosmid clone containing PKS I genes. Colony hybridization was performed against fosmid clones from the 6 positive pools, and finally 4 clones were picked out and confirmed to contain the conserved DNA fragment of KS domains. In this study, we present a simple and feasible sorting method for a desired clone from metagenomic libraries.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.236-242
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• 2003

A culture-independent and -dependent survey of the bacterial community structure in the rhizosphere and soil samples from hot pepper plants was conducted using 16S rDNA clone library and FAME analyses. Out of the 78 clones sequenced, 56% belonged to Proteobacteria, 4% to high G+C Gram- positive group, 3% to Cytophyga-Flexibacter-Bacreroides, and 32% could not be grouped with any known taxonomic division. Among the 127 FAME isolates identified, 66% belonged to low G+C Gram-positive bacteria (Baciilus spp.) and 26% to high G+C Gram-positive bacteria. In a cluster analysis, the results for both methods were found to be strikingly dissimilar. The current study is the first comparative study of FAME and 165 rDNA clonal analyses performed on the same set of soil, rhizosphere soil, and root samples.

• Korean Journal of Breeding Science
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• v.43 no.5
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• pp.448-456
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• 2011

We used suppression subtractive hybridization (SSH) combined with mirror orientation selection (MOS) method to screen differentially expressed genes from red-fleshed kiwifruit 'Hongyang'. As a result, the 288 clones were obtained by subcloning PCR product and 192 clones that showed positive clones on colony PCR analysis were selected. All the positive clones were sequenced. After comparisons with the NCBI/Genbank database using the BLAST search revealed that 30 clones showed sequence similarity to genes from other organisms; 10 clones showed significant sequence similarity to known genes. Among these clones, 3 clones (AcF21, AcF42 and AcF106) had sequence homology to 1-aminicyclopropane-carboxylic acid (ACC)-oxidase (ACO) that known to be related to fruit ripening. The expression patterns of differentially expressed genes were further investigated to validate the SSH data by reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qReal-time PCR) analysis. All the data from qReal-time PCR analysis coincide with the results obtained from RT-PCR analysis. Three clones were expressed at higher levels in 'Hongyang' than 'Hayward'. AcF21 was highly expressed in the other genes at 120 days after full bloom (DAFB) and 160 DAFB of 'Hongyang'.

• Development and Reproduction
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• v.26 no.3
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• pp.117-126
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• 2022

Bromodomain-containing protein 7 (BRD7) participates in many cellular processes and embryo development. BRD7 is down-regulated in various cancers and evidence of its tumor suppressor function has been accumulating. Here, we identified transforming stimulated clone 22 (TSC-22) as a novel BRD7 interacting protein and show its novel function as a positive regulator of BRD7. We found that TSC-22 expression potentiated the inactivation of the extracellular signal-regulate kinase (ERK) pathway by BRD7. Our data establishes TSC-22 as a modulator of BRD7 and unravels the molecular mechanisms that drive the synergistic tumor-suppressing effects of TSC-22 and BRD7. Our findings may open new avenues for developing novel molecular therapies for tumors exhibiting down-regulated BRD7 and/or TSC-22.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.159-172
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• 1998

사람 성장호르몬 수용체(hGHR) cDNA는 PCR방법에 의하여 fagment로서 보고되어진 바 있으나, liver cDNA로 부터 전장을 cloning한 보고는 없는 실정으로 본 연구에서는 기능을 가진 약 4.6kbp의 cDNA hGHR을 cloning 하는데 성공하였다. 먼저 cloning하기 위하여 human liver mRNA와 human breast cancer tissue로부터 회수한 mRNA를 RT-PCR방법에 의하여 human cDNA library와 cloning에 필요한 probe를 제작하였다. human library mRNA는 GT-PCR방법에 의하여 증폭하여 증폭되어진 산물은 λZAP Vector를 이용하여 cDNA library를 구축하였고,screeing을 위하여 임 보고 되어진 hGHR fragment native sequence를 기초로 N-terminal부분의 primer를 설계하여 950bp의 probe를 얻는데 성공하였다. 이 probe를 이용하여 준비된 human liver cDNA library로부터 2.5$\times$10 6개의 plaque로부터 6개의 positive clone을 획득하였고, 이들중 poly Asignal인 "AATAAA"를 포함하고 있는 가장 긴 약 3.8kbp의 clone을 sequencing한 결과 open reading frame을 포함하고 있었으나, 5'부분의 결손되어 있었다. 그리하여 이 부분은 human breast cancer tissue로 부터 회수한 mRNA를 RT-PCR에 의하여 증폭하였고, sequencing결과 이미 보고되어진 native hGHR와 비교한 결과 하나의 nucleotide가 silent mutation으로 판명되었다.한편 human liver cDNA library로부터 cloning한 3.8cp의 positive clone의 5'end의 결손된 부분에 silent mutation된 PCR 산물을 연결함으로써 native hGHR와 유사한 cDNA hGHR subcloning에 성공하였다. 이러한 cDNA hGHR의 clone이 function을 가지고 있는지를 검토하기 위하여 eukaryotic 발현 vector인 pCXN2에 의거 ligation한 후 chinese hamster ovary cell[CHO-KI]에 transfect를 실시하였다. Dexamethasone은 첨가하지 않고 hGH만의 존재하에서 이들 cell을 배양시키고 cell menbrane에서 발현 여부를 판정키 위하여 hGHR monocloual antibody를 사용하여 flow cytometery해석을 실시하는 한편 125I-hGH binding assay에 의하여 hGH binding activity를 측정하였다. 최종적으로 GH signal transduction의 target genedf으로 알려져 있는 serine protease inhibitor 2.1(Spi 2.1) gene의 promotor activity를 검토한 결과 hGHR을 transfect한 CHO Cell에 있어서 hGH의 농도에 의존적으로 증가되었다. 따라서 본 실험에서 cloning한 cDNA hGHR는 native hGHR와 같은 기능을 가지는 것으로 판명되었다.것으로 판명되었다.

• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.363-368
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• 1988

Molecular cloning of gene for $\alpha$-acylamino-$\beta$-lactam acylhydrolase (ALAH) III from Acetobacter turbidans has been attempted by immunochemical detection method, in which polyclonal antibody from mouse Balb/c against this enzyme was employed as a probe. As a cloning vector, λ gtll was chosen for this purpose. Two positive clones has been selected from genomic libraries of A. turbidans, which had somewhat different binding affinities on anti-ALAH III umm and anti-$\beta$-galactosidase. By restriction analysis, both clones has been turned out to lose one of EeoRI sites. From these results, it concluded that deletion of DNA between lacZ gene and inserted DNA has occurred during replication of these clones in host cells.

• The Korean Journal of Zoology
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• v.35 no.2
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• pp.203-210
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• 1992

Hsp70, a 70 kDa protein, is the maior protein expressed when cells are heat-shocked. A cDNA library from mouse ID13 cells was screened with the human hsp70 gene as a probe, and a positive clone was obtained. The positive clone was subcloned into puc19 and the precise restriction was obtained. The CDNA was sequenced by the Sanger's dideoxv termination method. Single open reading frame that codes for a protein of 70 kDa was found. The DNA sequence of the cloned mouse DNA shows great homology (66-90%) with other mouse hsp70 genes and somewhat less homology (50",) with E. coli hsp70 gene (dnak). With the exception of one amino acid, the protein sequence deduced from the CDNA is identical to the mouse that shock cognate protein 70 (hsc70) that is constitutivelv expressed at normal temperature. The result suggests that the cloned CDNA encodes a hsc70 family rather than a heatinducible family.mily.

• Applied Biological Chemistry
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• v.38 no.1
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• pp.49-54
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• 1995

To understand the molecular structure and pathogenesis mechanism of Korean garlic viruses, we have isolated cDNA clones for garlic viruses. The partial nucleotide sequences of 24 cDNA clones were determined and those of five clones containing poly(A) tail were compared with sequences of other plant viruses. One of these clones, V9, has a primary structure similar to the carlavirus group, suggesting that the clone V9 derived from a part of garlic latent virus (GLV). Northern blot analysis with the clone V9 as a probe demonstrated that GLV genome is 8.5 knt long and has a poly(A) tail. The clone V9 encodes coat protein (CP) of 33 kDa and nucleic acid binding protein of 10 kDa in different reading frame. The hexanucleotide motif, 5'-ACCUAA, which is conserved in the 3' noncoding region arid was proposed to be a cis-acting element involved in the production of negative strand genomic RNA was noticed. Complementary sequence to the hexanucleotide motif, 5'-TTAGGT, is also found in the positive strand of V9 RNA. The putative CP gene was cloned into the pRSET-A expression vector and expressed in E. coli BL21. The expressed recombinant V9CP protein was purified by $Ni^{2+}$ NTA affinity chromatography. The anti-V9CP antibody recognizes 34 kDa polypeptide which could be CP of GLV in infected garlic leaf extract. Immunoblot and Northern blot analysis of various cultivars shows wide occurrence of GLV in Korean garlic plants.

• Korean Journal of Agricultural Science
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• v.49 no.4
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• pp.821-831
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• 2022

American plum line pattern virus (APLPV), a member of the genus Ilarvirus in the family Bromoviridae, is one of the plant quarantine pathogens in Korea. In this study, 15 candidate primer sets were designed and examined to develop a reverse transcription polymerase chain reaction (RT-PCR) assay for plant quarantine inspection of APLPV. Using APLPV-infected and healthy samples, the primer sets were assessed for APLPV detection. To confirm the occurrence of nonspecific reactions, six ilarviruses (Apple mosaic virus, Asparagus virus 2, Blueberry shock virus, Prune dwarf virus, Prunus necrotic ringspot virus, and Tobacco streak virus) and 10 target plants (Prunus mume, P. yedoensis, P. persica, P. armeniaca, P. dulcis, P. tomentosa, P. avium, P. glandulosa, P. salicina, and P. cerasifera) were examined. Finally, two primer sets were selected. These primer sets could generate the expected amplicons even with at least 1 ng of the total RNA template in concentration-dependent amplifications. In addition, a positive clone was developed for use as a positive control in the abovementioned RT-PCR assay.

• BMB Reports
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• v.29 no.1
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• pp.52-57
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• 1996

A specific DNA fragment from Korean radish (Raphanus sativus L.) was amplified by performing PCR with oligonucleotide primers which correspond to the highly conserved regions of plant peroxidases. The size of the PCR product was ca. 400 bp, as expected from the known plant peroxidase genes. Comparison of the nucleotide and deduced amino acid sequences of the PCR product to those of other plant peroxidase-encoding genes revealed that the amplified fragment corresponded to the highly conserved region I and III of plant peroxidases. By screening a genomic library of Korean radish using the amplified fragment as a probe, two positive clones, named prxK1 and prxK2, were isolated. Restriction mapping studies indicated that the 5.2 kb Sail fragment of the prxK1 clone and the 4.0 kb EcoRI fragment of the prxK2 clone encode separate isoperoxidase genes. Analyses of the promoter region of the prxK1 clone shows that putative CAAT box, CMT box, and TGA1b binding sequence (5' TGACGT) are present 718 bp upstream from the start codon.