• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.58-58
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• 2001

본 연구는 섬유소 분해효소 유전자 Cel D(Cellulase Digestion)가 도입된 형질전환 돼지 생산을 통하여 섬유소 함유 사료의 이용 효율을 증대시키고, 나아가 장기이식동물 및 고가의 의료용 단백질 생산가축을 개발하기 위한 원천기술 확보에 있다. 섬유소분해 유전자의 크로닝 및 조직 특이적 발현벡터를 개발하기 위하여, 우선 소의 위중 제4위 내의 미생물로부터 전체 유전자를 분리하였고, 이렇게 작성된 DNA library에서 섬유소분해 관련 유전자인 약 2.0 kb의 Cel D유전자를 크로닝하였으며, 췌장 특이적 발현 프로모터(rat elastase I: 약 200bp)를 크로닝한 후, 미세주입용 형질전환 재조합 벡터를 구축하기 위하여 rElastase I 프로모터 하류에 섬유소 분해 유전자(Cel D)를 연결하여 약 3.0 kb 크기의 재조합 벡터를 준비하였으며, 재조합 유전자를 1세포기 수정란 전핵내에 미세주입 하기 위해 Sal I과 BglII를 이용 유전자 단편을 만들었다. 구축된 유전자를 미세주입하기 위한 수정란을 회수하기위해 총68두의 돼지를 4-5두씩 분리사육하면서 발정동기화 및 과배란 유기를 위해 PG600, Altrenogest, FSH, hCG를 투여하였으며 hCG투여후 약54시간에 외과적 방법에 의해 총 1,359개의 수정란을 회수하였고, 이중 미세주입가능한 1세포기 수정란은 1,296개로 두당 평균 15.9개 였다. 1,296개의 1세포기 수정란 중에서 재조합 유전자(rE I-CelD)가 미세주입된 660개의 수정란을 32두의 수란돈에 외과적 방법에 의해 이식하였으며, 이식되어진 모돈 13두가 분만하여 40.6%의 임신율을 나타내었다. 이렇게 분만된 13두에서 총 65두(암:33두, 수:32두)의 자돈이 생산되었으며, 형질전환 여부를 판명하기 위해 자돈의 꼬리조직으로 부터 genomic DNA를 추출하고 PCR 검정을 실시하였다. PCR 검정 결과, 섬유소 분해 유전자가 도입된 자돈은 5두 이었으며, 그 결과를 Table에 나타내었다.(Table Omitted) Table 1 에서와 같이 섬유소 분해효소유전자가 형질전환된 자돈은 65마리 중 5마리로 7.69%의 형질전환율을 나타내었으며, 5마리의 자돈중 2두(암:1두, 수:1두)는 분만 후 즉시 폐사되었으며 2두(암:1두, 수:1두)는 86일령 그리고 14일령에 폐사하여 현재 1두(암)가 생존하여 섬유소 분해 사양 실험 중에 있다.

• The Korean Journal of Food And Nutrition
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• v.15 no.2
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• pp.104-111
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• 2002

The peptidyl prolyl sis-trans isomerase (PPIase, EC 5.2.1.8) from bacillus stearothermophilus was extracted from the cells treated with by lysozyme. PPIase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPIase was estimated as 18kDa by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0∼8.0. The enzyme was considerably stable after heat treatment at 60$\^{C}$ for 30minutes, and the enzyme was quite stable up to 65$\^{C}$. The presence of the PPIase in the refolding solution accelerated the isomerization rate of the assay peptide. PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-l primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPI-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus.

• The Korean Journal of Malacology
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• v.20 no.2
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• pp.165-169
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• 2004

The BLAST server for the mollusk was constructed on the basis of the Intel Server Platform SC-5250 dual Xeon 2.8 GHz cpu and Linux operating system. After establishing the operating system, we installed NCBI (National Center for Biotechnology Information) WebBLAST package after web server configuration for cgi (common gate interface) (http://chimp.kribb.re.kr/mollusks). To build up the stand alone blast, we conducted as follows: First, we downloaded the genome information (mitochondria genome information), DNA sequences, amino acid sequences related with mollusk available at NCBI. Second, it was translated into the multifasta format that was stored as database by using the formatdb program provided by NCBI. Finally, the cgi was used for the Stand Alone Blast server. In addition, we have added the vector, Escherichia coli, and repeat sequences into the server to confirm a potential contamination. Finally, primer3 program is also installed for the users to design the primer. The stand alone BLAST gave us several advantages: (1) we can get only the data that agree with the nucleotide sequence directly related with the mollusks when we are searching BLAST; (2) it will be very convenient to confirm contamination when we made the cDNA or genomic library from mollusks; (3) Compared to the current NSBI, we can quickly get the BLAST results on the mollusks sequence information.

• The Korean Journal of Food And Nutrition
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• v.9 no.4
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• pp.452-458
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• 1996

A PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-1 primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI1-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPi-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus. The translated protein sequence of PPIase B. stearothermophilus was compared with sequence from periplasmic PPIase from Escherichina coil ; homogies of 16 and 58%, respectively, were found. The clond PPIase gene was over-expressed in E. coil cell using pUC19 as an expression vector. The enzyme was partially purified by heat treatment and colum chromatochraphy on DEAE-Sepharose CL-6B. The molecular weight of the enzyme was dermined to be about 18.0 kDal by SDS-PAGE.

• Applied Biological Chemistry
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• v.38 no.5
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• pp.382-386
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• 1995

The potato proteinase inhibitor II (PI-II) protein contains chymotrypsin and trypsin inhibitory site. Among several PI-II genes isolated from genomic library, amino acid sequence deduced from PI-IIT gene has 84% identity with that of the polypeptide chymotrypsin inhibitor (PCI). Therefore a gene fragment having homology with the PCI was cloned into a vector using polymerase chain reaction(PCR) from the potato proteinase inhibitor IIT gene. Two different primers were utilized for cloning; primer A contains NdeI restriction site and 30 nucleotides, which has AUG N-terminal methionine codon, primer B contains BclI restriction site and 28 nucleotides, which has TAG translation stop codon. After PCR, about 160 bp-long DNA fragment was cloned into pRT146, derivative of pUC118, and sequenced. The sequenced NdeI/BclI fragment was moved to pET3a, containing bacteriophage T7 promoter and terminator. The expressed proteins in E. coli BL2l(DE3) were determined on a polyacrylamide gel containing sodium dodecyl sulfate. The expected size of protein deduced from the sequenced gene fragment is about 6,500 dalton whose size was similar to the IPTG-induced protein (6,000 dalton) on a gel. However the expression level was much lower than expected.

• 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.

• The Korean Journal of Malacology
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• v.28 no.4
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• pp.377-384
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• 2012

The importance of biological resources has been gradually increasing, and mollusks have been utilized as main fishery resources in terrestrial ecosystems. But little is known about genomic and transcriptional analysis in mollusks. This is the first report on the transcriptomic profile of Meretrix lusoria. In this study, we constructed cDNA library and determined 542 of distinct EST sequences composed of 284 singletons and 95 contigs. At first, we identified 180 of EST sequences that have significant hits on protein sequences of the exclusive Mollusks database through BLASTX program and 343 of EST sequences that have significant hits on NCBI NR database. We also found that 211 of putative sequences through local BLAST (blastx, E < e-10) search against KOG database were classified into 16 functional categories. Some kinds of immune response related genes encoding allograft inflammatory factor 1 (AIF-1), B-cell translocation gene 1 (BTG1), C-type lectin A, thioester-containing protein and 26S proteasome regulatory complex were identified. To determine phylogenetic relationship, we identified partial sequences of four genes (COX1, COX2, 12S rRNA and NADH dehydrogenase) that significantly matched with the mitochondrial genomes of 3 species-Ml (Meretrix lusoria), Mp (Meretrix petechialis) and Mm (Meretrix meretrix). As a result, we found that there was a little bit of a difference between sequences of Korean isolates and other known isolates. This study will be useful to develop breeding technology and might also be helpful to establish a classification system.

• Journal of Plant Biotechnology
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• v.33 no.2
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• pp.85-91
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• 2006

Flavanone 3$\beta$-hydroxylase (FHT) is an enzyme acting in the central part of the flavonoid biosynthesis pathway. FHT catalyses the hydroxylation of flavanone to dihydroflavonols in the anthocyanin pathway. In this paper we describe the cloning and expression of the genes encoding the flavonoid-biosynthetic enzyme FHT in Gypsophila paniculata L. A heterologous cDHA probe from Dianthus cavophyllus was used to isolate FHT-encoding cDHA clones from Gypsophila paniculata L.. Inspection of the 1471 bp long sequence revealed an open reading frame 1047 bp, including a 190 bp 5' leader region and 288 bp 3' untranslated region. Comparison of the coding region of this FHT cDHA sequence including the sequences of Arabidopsis thaliana, Citrus sinensis, Dianthus caryophyllus, Ipomoea batatas, Matthiola incana, Nierembergia sp, Petunia hybrida, Solanum tuberosum, Vitis vinifera reveals a identity higher than 69% at the nucleotide level. The function of this nucleotide sequences were verified by comparison with amino acid sequences of the amino-terminus and tryptic peptides from purified plant enzyme, by northern blotting with mRHA from wild type and mutant plants, by in vitro expression yielding and enzymatically active hydroxylase, as indicated by the small dihydrokaempferol peak. Genomic southern blot analysis showed the presence of only one gene for FHT in Gypsophila paniculata.

• Animal Bioscience
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• v.35 no.11
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• pp.1698-1710
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• 2022

Objective: Raw potato starch (RPS) is resistant to digestion, escapes absorption, and is metabolized by intestinal microflora in the large intestine and acts as their energy source. In this study, we compared the effect of different concentrations of RPS on the intestinal bacterial community of weaned piglets. Methods: Male weaned piglets (25-days-old, 7.03±0.49 kg) were either fed a corn/soybean-based control diet (CON, n = 6) or two treatment diets supplemented with 5% RPS (RPS5, n = 4) or 10% RPS (RPS10, n = 4) for 20 days and their fecal samples were collected. The day 0 and 20 samples were analyzed using a 16S rRNA gene sequencing technology, followed by total genomic DNA extraction, library construction, and high-throughput sequencing. After statistical analysis, five phyla and 45 genera accounting for over 0.5% of the reads in any of the three groups were further analyzed. Furthermore, short-chain fatty acids (SCFAs) in the day 20 fecal samples were analyzed using gas chromatography. Results: Significant changes were not observed in the bacterial composition at the phylum level even after 20 d post feeding (dpf); however, the abundance of Intestinimonas and Barnesiella decreased in both RPS treatment groups compared to the CON group. Consumption of 5% RPS increased the abundance of Roseburia (p<0.05) and decreased the abundance of Clostridium (p<0.01) and Mediterraneibacter (p< 0.05). In contrast, consumption of 10% RPS increased the abundance of Olsenella (p<0.05) and decreased the abundance of Campylobacter (p<0.05), Kineothrix (p<0.05), Paraprevotella (p<0.05), and Vallitalea (p<0.05). Additionally, acetate (p<0.01), butyrate (p<0.05), valerate (p = 0.01), and total SCFAs (p = 0.01) were upregulated in the RPS5 treatment group Conclusion: Feeding 5% RPS altered bacterial community composition and promoted gut health in weaned piglets. Thus, resistant starch as a feed additive may prevent diarrhea in piglets during weaning.

• 대한생식의학회:학술대회논문집
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• 2001.03a
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• pp.195-205
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• 2001

태반 영양배엽 (trophoblast)은 포유동물의 발생과정 중 가장 먼저 분화되는 세포로서, 자궁환경내에서 배아가 착상, 발생, 및 분화하기 위해서 반드시 필요한 태반을 형성하는 색심적인 세포이다. 영양배엽 세포의 분화과정중의 결함은 배아의 사산이나 임신질환 등의 치명적 결과를 초래한다. 하지만, 영양배엽 세포의 분화를 조절하는 분자생물학적인 메카니즘은 아직 규명되지 않고 있다. 영양배엽 세포의 분화를 조절하는 경로를 규경하기 위한 선결과제는 분화된 영양배엽 세포에서만 발현하는 많은 유전자들이 밝혀져야만 한다. 본 연구팀은 최근에 분화된 영양배엽 세포에서만 발현하는 두 종류의 새로운 유전자들을 찾았다. 한 종류는 homeobox를 보유하고 있는 조절 유전자 Psx이고, 다른 한 종류는 임신호르몬인 태반 프로락틴 라이크 단백질 유전자 PLP-C${\beta}$이다. 본 연구과제의 목표는 이들 유전자의 기능과 조절 메카니즘을 규명함으로써, 영양배엽 세포의 분화를 조절하는 조절경로를 밝히는 것이다. 이를 위하여 다음과 같은 일련의 연구를 수행할 것이다. 1) Psx 유전자가 분화된 영양배엽 세포에서만 발현케 하는 조절 메카니즘을 규명하기 위해 functional assays, in vitro footprinting, gel mobility shift assays, 생쥐형질전화, UV crosslinking, Southwestern blot 등의 방법을 통해 Psx 유전자의 cis-acting 요인과 trans-acting factor를 밝혀 분석한다. 2) 영양배엽 세포의 분화조절 경로를 규명하기 위해 random oligonuclotide library screening, DD-PCR, subtractive screening 등의 방법을 이용하여 Psx 유전자에 의해 조절되는 하부유전자를 밝힌다. 3) Psx 유전자를 knock-out시켜 영양배엽 세포가 발달 및 분화하는데 미치는 역할을 밝힌다. 4) Yeast two-hybrid screening방법을 이용하여 태반 프로락틴 유전자의 수용체를 찾아 이들의 신호전달 기전을 밝힌다. 제1차년 연구결과로서, mouse와 rat으로부터 각각 Psx 유전자의 genomic DNA를 클로닝하여, 유전자 구조를 비교한 결과, mouse Psx (mPsx2)는 4개의 exons으로 이루어져 있는 반면에, rat Psx (Psx3)는 3개의 exons으로 구성되어 있었다. 즉, rPsx3는 mPsx2의 exon1이 없었다. Notrhern blot과 in situ hybridization 분석에 의해 mouse와 rat에서 Psx 유전자가 다르게 발현 조절되는 현상을 밝혔다. 실제로 mPsx2와 rPsx3의 5'-flanking지역을 클로닝하여 염기서열 분석 결과 전혀 homology를 찾을 수 없었다. 또한, 이들 각각 promoter의 activity를 luciferase reporter를 이용하여 조사한 결과 Rcho-1 trophoblast cells에서 각기 다른 activity를 보여 주는 것을 발견하였다. Psx 유전자의 transcription start sites는 Primer extension에 의해 밝혔다. 또한 Psx2 유전자를 knock-out 시키기 위해 targeting vector를 Osdupde1에 제작하였다. 본 과제를 시작할 때 새로운 프로락틴 유전자 하나를 클로닝하여 이 유전자를 PLP-I라고 이름을 붙였다. 이 후 이 유전자 (PLP-I)는 PLP-C${\beta}$라고 이름을 붙이게 되었다. Mouse PLP-C${\beta}$ 유전자의 counterpart를 rat에서 찾아 염기서열을 비교한 결과 mouse와 rat에서 PLP-C${\beta}$유전자의 homology는 약 79% (amino acid level)였다. 본 연구과정을 통해 또 하나의 새로운 PLP-C subfamily member를 mouse로부터 클로닝 하였고, 이 유전자를 PLP-C${\gamma}$라 하였다. PLP-C${\beta}$와 PLP-C${\gamma}$의 발현 유형은 Northern blot과 in 냐셔 hybridization 분석에 의해 태반의 제한된 spongitrophoblast와 trophoblast giant cells에서만 발현하는 것을 밝혔다. 놀랍게도 이들 두 새로운 유전자는 alternative splicing에 의해 두 종류의 isoform이 있음을 밝혔다. PLP family member 유전자로서 splicing에 의한 isoforms을 보여 주는 유전자로는 PLP-C${\beta}$와 PLP-C${\gamma}$가 최초이다. 이들 isoform mRNAs의 발현 유형은 RT-PCR 방법을 이용하여 규명하였다. 또 하나의 새로운 발견은 PLP-C${\beta}$와 PLP-C${\gamma}$가 독특한 유전자 구조를 갖고 있었다. 즉, PLP-C${\beta}$는 exon3의 alternative splicing에 의해 5개 혹은 6개의 exons을 갖는 two isoforms이 생긴다. 반면에 PLP-C${\gamma}$는 exon2가 alternative splcing이 되면서 7개의 exons을 갖거나 6개의 exons을 갖는 isoforms을 만든다. 그리고, PLP-C${\gamma}$의 promoter activity를 trophoblast Rcho-l${\gamma}$ 세포주를 이용하여 PLP-C${\gamma}$ 의 1.5 kb 5'-flanking 지역이 trophoblast-specific promoter activity를 갖고 있음을 밝혔다. PLP-C${\gamma}$ 유전자의 transcription start site는 Primer extension에 의해 밝혔다. 제 1차 년도의 연구결과를 토대로, 2차년에서는 다음단계의 연구를 수행하고자 한다. 즉, 1) mPsx2와 rPsx3의 promoter를 비교분석 함으로서 mouse와 rat에서 Psx 유전자가 다르게 조절되는 메카니즘 규명, 2) Psx와 PLP-C 유전자의 promoter에 있는 cis-acting elements 탐색, 3) Psx2와 Psx3의 단백질을 이용하여 이들이 binding하는 target sequence 규명, 4) 제작한 Psx2 targeting vector를 이용하여 ES cells에서 Psx2 유전자 knock-out, 5) Psx 유전자를 과발현시키는 세포주를 만들고 Psx에 의해 조절되는 유전자 탐색, 6) 새로 밝히 PLP-C members 유전자들의 조절기전을 Rcho-1 세포주를 이용하여 여러 거지 성장인자와 다른 호르몬에 대한 반응을 탐색, 7) Psx와 PLP-C${\gamma}$ 유전자의 chromosomal mapping 등을 밝힐 것이다.