• Journal of Life Science
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• v.14 no.1
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• pp.21-25
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• 2004

To identify genes involved in the decolorization of both crystal violet and malachite green, we isolated random mutants generated by transposon insertion in triphenylmethane-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 14 mutants with complete defect in color removal capability of both crystal violet and malachite green. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 5 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by cmg genes were identified as follows. cmg 2 is MaIC protein in maltose transport system; cmg 6 is transcriptional regulator (LysR-type): cmg 12 is a putative oxidoreductase. The sequences deduced from two cmg genes, cmg 8 and cmg 11, showed no significant similarity to any protein with a known function. Therefore, these results indicate that these two cmg genes encode unidentified proteins responsible for decolorization of both crystal violet and malachite green.

• Journal of Microbiology
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• v.44 no.2
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• pp.192-199
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• 2006

Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene $(C_1-C_4)$, biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99 %) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.

• Journal of Animal Science and Technology
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• v.46 no.2
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• pp.155-164
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• 2004

This involves identifying and cloning trapped genes from cultured cells carrying the gene-trap constructs and generating cloned zebrafish using these cells for functional study. Gene-trapping studies in gene-trapped cells were carried out in initial and cloned zebrafish carrying gene-trap events were successfully produced based on the nuclear transplantation technique. Two kind of retroviral gene-trap constructs were adopted. The first one(SA/GFP-TP), constructed in my laboratory, carries a GFP reporter gene containing a splicing acceptor and an internal neo gene. The second one(Neo-TP), obtained from Dr. Hicks (Hicks et al., 1997), contains a promoter-less neo gene located in the LTR sequence of a retroviral vector. The infected cells were subjected to drug selection(neomycin treatment) because the two constructs carry the neomycin resistant gene. All those cells survived the neomycin treatment should carry the proviral insertions. For Neo-TP, Isolated DNA from the neomycin-resistant fibroblast cells infected by Neo-TP, was digested with EcoR1 restriction enzyme and transformed into bacteria after ligation. This procedure led to the isolation of seven clones carrying flanking cellular DNA with a typical retroviral integration signature sequence. These clones contained genomic DNA ranging from 1kb to 7kb and sequences of 300-600 bp were obtained from each of the rescued plasmids. Database searching showed that all of them share high homology to zebrafish sequences. For fish cloning using tagged cells, initially, nucleus donors directly selected from a mixture of cells(Neo-TP cells) were used. A total of 44 embryos(3.7%) out of 1179 transplants were reached blastula stage; 8 of these embryos(0.8%) hatched and 3(0.3%) of them survived to adulthood. One out of three lived cloned zebrafish has an amplified fragment and was labeled with 32P.

• 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.

• Horticultural Science & Technology
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• v.34 no.1
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• pp.112-121
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• 2016

Development of genetically-modified (GM) crops enables the introduction of new traits to the plant to confer characteristics such as disease resistance, herbicide resistance and human health-promoting bioactivity. Successful commercialization of newly developed GM crops requires stable inheritance of integrated T-DNA and newly introduced traits through the multiple generations. This study was carried out to confirm the stable inheritance of the integrated T-DNA in $T_1$ and $T_2$ transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) that was genetically modified to increase concentrations of phenylethylisothiocyanate (PEITC), which is a potential anti-carcinogenic phytochemical. For this purpose, the IGA 1-3 ($T_1$ generation) and IGA 1-3-5 ($T_2$ generation) lines were selected by PCR and a IGA 1-3 transgenic plant ($T_1$ generation) was analyzed to confirm the T-DNA insertion site in the Chinese cabbage genome by VA-TAIL PCR. The results of this study showed that the introduced T-DNA in IGA 1 line was stably inherited to the next generations without any variations in terms of the structure of the transgenes, and this line also showed the expected transgene function that resulted in increased concentration of PEITC through the multiple generations. Finally, we confirmed the increased QR activity in IGA 1 $T_1$ and $T_2$ transgenic lines, which indicates an enhanced potential anti-carcinogenic bioactivity and its stable inheritance in IGA1 $T_1$ and $T_2$ transgenic lines.

• The Korean Journal of Mycology
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• v.27 no.5 s.92
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• pp.337-340
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• 1999

The context and upper surface of Phellinus basidiocarp become blackened, rimose and woody. The basidiocarp is sessile, dimidiate and elongate. The basidiospores are pigmented and ovoid to globose. Hymenial setae are $17{\sim}35{\times}6{\sim}8{\mu}m$. Nineteen isolates of Phellinus species, including Phellinus linteus, were used for sequencing of the internal transcribed spacer (ITS) region of the nuclear rDNA. Based on these sequence data, specific primers were designed for identification of Phellinus linteus isolates in Korea. The specific primers were within the ITS1 and ITS2 regions and were nested within the universal primers flanking the spacer regions. A total of four primers (the universal primers ITS-1F and ITS-4, and the specific primers PL-F and PL-R) were used for detection of Phellinus linteus collected in Korea. The length of the four amplification products of Phellinus linteus DNA were 800 bp (ITS-1F/ITS-4), two bands of about 720 bp (ITS-1F/PL-R and PL-F/ITS-4), and 610 bp (PL-F/PL-R). Among 23 isolates of Phellinus species collected in Korea, Thirteen isolates were identified as Phellinus linteus based on the presence of the four bands. The other species produced only the single ITS-1F/ITS-4 product.

• Microbiology and Biotechnology Letters
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• v.32 no.3
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• pp.199-205
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• 2004

The nucleotide sequence of 8.6-kb EcoRI fragment containing sucrose phosphorylase gene isolated from Bifidobacterium longum SJ32 was determined. It was found that the fragment contained five open reading frames including the gene cluster for sucrose utilization such as a sucrose phosphorylase (ScrP), a sucrose transporter (ScrT), and a GalR-LacI-type transcriptional regulator (ScrR) identified by amino acid homology. Each gene showed over 94% amino acid homology among various B. longum strains. Genomic organization of the gene cluster is the same as those of other strains of B. longum but different from that of B. lactis. In spite of high homology of each gene among B. longum strains, the difference of flanking sequences of the gene cluster between strains SJ32 and NCC2705 insinuates the horizontal transfer of scrPTR between B. longum strains. The increase of sucrose phosphorylase activity in heterologous E. coli system by the co-expression of scrT with scrP against the single expression of scrP was measured. It seems to be the result of sucrose uptake increment by scrT in the host and is an indirect evidence that scrT is the gene for sucrose transport. The existence of multiple sucrose uptake systems in B. longum is supposed from the findings of several genes besides scrPTR involved in sucrose uptake in the genome of B. longum NCC2705.

• Journal of Animal Science and Technology
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• v.49 no.1
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• pp.1-8
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• 2007

This study was focused on discriminating the molecular sexes of red deer and elk by duplex polymerase chain reaction(PCR) using two primer sets. Sex differentiation of mammals is primarily dependent on the presence or absence of sex determining region Y(SRY) gene encoded on Y chromosome which plays a key role for male development. Zinc finger X-Y(ZFX-ZFY) gene, one of X-Y homology gene group was found on X- and Y- chromosomes, respectively. At first, the nucleotide sequences were characterized for the intron 9 flanking region of ZFX-ZFY genes. The intron 9 of ZFX and ZFY is 529-bp and 665-bp in length, respectively. A transposable element sequence similar to bovine SINE element Bov-tA was detected only in ZFY gene of Cervidae. Sexing analysis was conducted by duplex PCR assay for amplification of SRY and ZFX-ZFY genes. Two differentially amplified patterns were found: one for females has a common band amplified only from ZFX as a template, and another for males had three bands(a common ZFX and two male-specific ZFY and SRY). On the separate tests using each gene, the results was identical to those from duplex PCR assay. Moreover, the results from PCR assays provide also identical information to phenotypic investigation of individuals of red deer, elk as well as their hybridized progenies collected from two isolated farms. These results suggest that it may be a rapid and precise method for determining the sexes by duplex PCR amplification using Y-chromosome specific SRY and X- and Y- homologous ZFX-ZFY genes showing sexual dimorphism in red deer and elk without any other controls.

• Microbiology and Biotechnology Letters
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• v.31 no.1
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• pp.25-31
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• 2003

Delftia acidovorans 51-A isolated from river water degrades aniline. In order to clone genes involved in aniline degradation, transposon Tn5-B20 was inserted into the strain 51-A to generate a mutant strain 10-4-2 that cannot utilize aniline as a carbon source. The mutant strain was not an auxotroph but could not degrade aniline. Southern hybridization analysis indicated that the transposon was inserted into the mutant bacterial DNA as a single copy. Flanking DNA fragment of Tn5-B2O insertion was cloned and sequenced. DNA sequence analysis revealed three ORFs encoding TdnQ, TdnT, and TdnA 1 that arc responsible for catechol formation from aniline through oxidative deamination. The analysis also confirmed that Tn5-B2O was inserted at the immediate downstream of tdnA1. The result suggests that the transposon insertion behind tdirA1 disrupted the pathway of the catechol formation from aniline, resulting in the mutant phenotype, which cannot degrade aniline. A large plasmid over 100-kb in size was detected from D. acidovorans 51-A and Southern hybridization analysis with Tn5-B2O probe showed that the transposon was inserted on the plasmid named pTDN51. Our results indicated that the tdn genes on pTDN51 of D. acidovorans 51-A are involved in aniline degradation.

• The Korean Journal of Pharmacology
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• v.24 no.1
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• pp.1-10
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• 1988

To obtain information about the structure of the human phenylethanolamin N-methyltransferase (PNMT) and to further define the extent of the evolutionary relationships among PNMT molecules of several spesies, a full length cDNA clone for bovine adrenal PNMT was used to screen a charon 4A genomic library. One phage was isolated and identified, which included the entire PNMT gene. The length of inserted genomic DNA was 13.1-Kilobase (Kb) containing two internal EcoRI sites. Construction of a restriction map and subsequent Southern and dot blot analysis with 5'-and3'-specific cDNA probes allowed the identification of exon-containing fragments. This is the first report of the cloning of gene for human epinephrine synthesizing enzyme.