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

• Journal of Plant Biology
• /
• v.33 no.4
• /
• pp.303-307
• /
• 1990

A genomic clone carrying a proteinase inhibitor I sequence was isolated and characterized. The clone contained a 0.7 kb EcoRI fragment hybridized with tomato inhibitor I cDNA. The nucleotide sequence of the EcoRI fragment revealed presence of a truncated form of a proteinase inhibitor I gene of potato. The truncated gene contained the 5' flanking region and the first exon of a functional proteinase inhibitor I gene. Although the 5' flanking region contained the regulatory sequences TATAAA and CCACT, a deletion of 40 bp occurred between them.

• Proceedings of the Korean Information Science Society Conference
• /
• 2001.04a
• /
• pp.751-753
• /
• 2001

지금까지 인간이나 다른 생물체의 전체 유전체 염기서열을 밝혀내는 작업은 크게 세가지 방법으로 진행되었다. Clone-by-clone approach, sequence tagged connector approach, random shotgun approach[1]가 그것인데 마지막의 random shotgun approach는 fragment assembly problem을 비롯한 여러 가지 전산학적인 문제들을 수반한다. 미생물체의 전체 염기서열을 random shotgun approach를 이용하여 밝혀낼 때 몇 가지 전산학적인 문제가 테크닉이 필요하며 그 중에서도 서열간의 forward, reverse의 mating 정보를 이용하는 것이 중요하다. 본 논문은 이러한 mating 작업을 한 눈에 볼 수 있게 하는 소프트웨어 페키지 “Mater”에 대해 소개하고자 하며 그 의미에 대해 논하고자 한다.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.10
• /
• pp.1735-1740
• /
• 2008

Clone PERV-C (A3) env was isolated from the genomic DNA of domestic pig (Sus scrofa domesticus) in Korea to investigate the molecular properties of PERV-C. The nucleic acid homologies between the PERV-MSL (type C) reference and the PERV-C(A3) clone was 99% for env, but a single base pair deletion was found in the transmembrane (TM) region of the env open reading frame. To examine the functional characteristics of truncated PERV-C env, we constructed a replication-incompetent retroviral vector by replacing the env gene of the pCL-Eco retrovirus vector with PERV-C env. A retroviral vector bearing PERV-C/A chimeric envelopes was also created to complement the TM defect. Our results indicated that truncated PERV-C env was not infectious in human cells as expected. Interestingly, however, the vector with the PERV-C/A envelope was able to infect 293 cells. This observation suggests that recombination within PERV-C TM could render PERV-C infectious in humans. To further characterize PERV-C/A envelopes, we constructed an infectious molecular clone by using a PCR-based technique. This infectious molecular clone will be useful to examine more specific regions that are critical for human cell tropism.

• Korean journal of applied entomology
• /
• v.36 no.1
• /
• pp.96-104
• /
• 1997

A silkworm Bombyx mori genomic DNA library was constructed from polyphagous J111 strain and unpolyphagous $C_3$ strain to develop the genomic study by DNA makers. Genomic DNAs of two strains were digested with restriction enzyme EcoRI and ligated into pUC18. The ligated plasmids were transferred into E. coli host strain DH5$\alpha$. When the genomic DNAs were hybridized with insert DNAs from transformant, could be categorized from hybridization patterns to three groups as high repetitive sequence, moderately repetitive sequence, and low-copy number sequences. A total of 219 clones containing single or low-copy number sequence inserts were examined for any polymorphisms between two strains of J111 and $C_3$. Forty six clones showed RFLPs and 10 of these clones were used as a probe of analysis of $F_2$ population derived from crossing between J111 and $C_3$ strain. The genetic inheritance tested with each clones will be important tools to construct the genetic map of the silkworm, Bombyx mori.

• BMB Reports
• /
• v.33 no.5
• /
• pp.396-401
• /
• 2000

A differential display method is used to identify novel genes whose expression is affected by treatment with ferric ammonium citrate (FAC) or desferrioxamine (DFO), an iron chelating agent in the human hepatoblastoma cell line (HepG2). These chemicals are known to deplete or increase the intracellular concentration of iron, respectively. Initially, we isolated seventeen genes whose expressions are down- or up regulated by the treatment of the chemicals, as well as their four differentially expressed genes that are designated as clone-1, -2, -3, and -4. These are further characterized by cDNA sequencing and Northern blot analysis. Through the cDNA sequencing, as well as comparing them to genes published using the NCBI BLAST program, we identified the sequence of the clone-1 that is up-regulated by the treatment of DFO. It is identical to the human insulin-like growth factor binding protein-1 (IGFBP-1). This suggests that the IGFBP-1 gene in the HepG2 cell is up-regulated by an iron depletion condition. Also, the expression of the clone-3 and -4 is up-regulated by FAC treatment and their eDNA sequences are identical to the human ferritin-fight chain and human NADH-dehydrogenase, respectively. However, the sequence of the clone-2 has no significant homology to any other known gene. Therefore, we suggest that changes of the cellular iron level in the HepG2 cell affects the transcription of cellular genes. This includes human IGFBP-1, ferritin-fight chain, and NADH-dehydrogenase. Regulation of these gene expressions may have an important role in cellular functions that are related to cellular iron metabolism.

• Korean Journal of Plant Tissue Culture
• /
• v.24 no.2
• /
• pp.113-117
• /
• 1997

Simple sequence repeats (SSR) are widely dispersed throughout eukaryotic genomes, highly polymorphic, and easily typed using polymerase chain reaction (PCR). The objective of this study was to determine the polymorphism of different Chlamydomonas reinhartdtii strains and to determine the mode of inheritance of the SSR locus in Chlamydomonas. A genomic DNA library of C. reinhardtii was constructed and screened with a radiolabeled $(AC)_{11}$ probe for the selection of (CA/GT)n repeat clone. Selected clone was seqeuenced, and PCR primer set flanking (CA/GT)n sequence was constructed. PCR was used to specifically amplify the SSR locus from multiple isolates of C. reinhardtii. The locus was polymorphic in some of the C. reinhardtii isolates. However, the locus was amplified only 4 of 6 isolates of C. reinhardtii, not in other 2 isolates of C. reinhardtii, suggesting that this locus is not extensively conserved. A simple Mendelian inheritance pattern was found, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that this simple sequence repeat DNA polymorphism will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.

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

• Research in Plant Disease
• /
• v.12 no.3
• /
• pp.235-239
• /
• 2006

Since the cultivation of resistance cultivar is essential for organic agriculture, Phytophthora late blight resistance of 16 advanced potato clones obtained from the potato breeding laboratory at the National Institute of Highland Agriculture was evaluated. Resistance of the clone was examined through artificial inoculation in the laboratory and in the field. Dominant isolates of the pathogen in Gangwon province produced abundant sporangia on leaves of most clones showing susceptibility. The number of sporangia ranged from $10^{7{\sim}8}sporangia/ml$ in the susceptible clones at 7 days after inoculation. However, one clone is resistant in organic farming fields. Disease incidence was 2.3% using the resistant clone. The area under disease progress curve(AUDPC) was 75.5. Contrarily, disease incidence of the susceptible variety was 100% and the AUDPC was 1773.5 during the same cultivation period. The resistance clone named as 'Haryeong' is considered suitable for organic potato cultivation in Gangwon province in Korea.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.8
• /
• pp.4823-4827
• /
• 2013

Objective: To study the mechanism of effects of AZD1480 on the SKOV3 ovarian cancer cell line. Methods: The MTT method was used to assess cellular proliferation, flow cytometry for cellular apoptosis, the scratch test to determine migration, transwell chamber assays to detect cellular invasion, plate clone experiments to detect the clone forming ability and Western blotting to determine p-STAT3 protein levels. Results: The proliferation rate, migration ability, invasiveness and the clone forming ability of SKOV3 cells were reduced after treatment with AZD1480, while apoptosis rate and chemotherapeutic susceptibility were increased. After treatment with AZD1480 plus cisplatin, the apoptosis rate increased significantly while the expression level of p-STAT3 protein was decreased. Conclusion: AZD1480 can inhibit the proliferation, invasion, metastasis and clone formation of SKOV3 cells, induce cellulsar apoptosis, increase the chemotherapeutic sensitivity and reduce the expression level of p-STAT3 protein.