• The Journal of Natural Sciences
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• v.14 no.2
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• pp.83-91
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• 2004

Recently phage K11 lysozyme was cloned and characterized in our lab. The K11 lysozyme was identified to have dual functions. It not only cuts a peptidoglycan bond in bacterial cell wall but also acts as an inhibitor of K11 RNA polymerase. It has been known that the T7 lysozyme binds specifically to T7 RNA polymerase and inhibits transcription. The dual activities of K11 lysozyme are atreeable to the case of T7 phage lysozyme and RNA polymerare. In order to identify the binding magnitude of K11 lysozyme with K11 RNA polymerase, yeast two-hybrid system was used. K11 phage lysozyme gene was introduced into pLexA plasmid and used as a prey. Also, K11 phage RNA polymerase gene was introduced into pJG4-5 and used as a bait. The binding between K11 lysozyme and K11 RNA polymerase was demonstrated by expression of reporter genes such as lacZ and leu2.

• Animal cells and systems
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• v.2 no.2
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• pp.249-258
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• 1998

Obesity, one of the most common metabolic diseases in industrial countries is characterized by an increase in the number or size of adipocytes. In an effort to create transgenic mouse models for the study of obesity we developed a novel technique in which adipose tissue can be ablated genetically at will, at any specific developmental stage and/or physiological condition, by the treatment of ganciclovir. We made a series of adipocytespecific expression vectors using minimal regulatory regions of brown adipocyte-specific uncoupling protein (UCP-1) gene and adipocyte-specific aP2 gene, and then analyzed their expression characteristics in cultured cell lines. When both constructs pUCP-LacZ and paP2-LacZ were transfected transiently into differentiating 3T3-L1 (pre-while adipocytes) and HIB-1B (pre-brown adipocytes) cell lines in vitro and then monitored by X-gal staining of cells, these regulatory regions were sufficient to show proper differentiation stage-specific expression in adipocvtes. To confirm that adipocytes expressing HSV-TK controlled by these minimal requlatory elements are sufficient to kill themselves with ganciclovir treatment pUCP-TK and paP2-TK expression constructs were transfected stably into HIB-1B and 3T3-L1 cells, respectively, and their ganciclovir sensitivities were tested during in vitro differentiation of cells. As expected more than 80% of cells were dead by the 7th day of treatment with ganciclovir while negative control cells were not affected at all. The data suqqest that the constructed vectors are suitable for obtaining novel obese transqenic models based on a conditional genetic tissue ablation method.

• Journal of the Korea Organic Resources Recycling Association
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• v.11 no.3
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• pp.67-74
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• 2003

Starvation promoters can be utilized for in situ bioremediation and for the efficient bioprocessing. Previously we have cloned and characterized strong starvation promoters from envrionmentally relevant bacteria, Pseudomonas putida strains (Y. Kim, and A. Matin, J. Bacteriol. 177:1850-1859, 1995). Here we report the construction of the plasmid pYKS101 using one of the starvation promoters from P. putida MK1. The pYKS101 was found to be useful for a novel starvation promoter-driven gene expression system. Under this system, the target reporter gene, lacZ, was stably integrated into the chromosomal DNA of P. putida MK1. ${\beta}$-galactosidase activity was found to be four-fold higher upon carbon starvation than during exponential growth. The resultant recombinant strain is indigenous to the environment contaminated with various toxic materials, hence can be a good candidate for in situ bioremediation.

• Journal of Microbiology
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• v.40 no.2
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• pp.156-160
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• 2002

The whole gal/lae operon genes of Lactococcus lactis ssp. lactis 7962 were reported as follows: galA-galM-galK-galT-lacA -lacZ-galE. The galK gene encoding a galactokinase involved in one of the Leloir pathways for galactose metabolism was found to be 1,197 bp in length and encodes a protein of 43,822 Da calculated molecular mass. The deduced amino acid sequence showed over 50% homology with GaIK proteins from several other lactic acid bacteria. The galK gene was expressed in E. coli and the product was identified as a 43 kDa protein which corresponds to the estimated size from the DNA sequence. The galactokinase activity of recombinant 5. coli was about 8 times greater against that of the host strain and more than 3 times higher than the induced L. lactis 7962.

• Korean Journal of Poultry Science
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• v.26 no.2
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• pp.119-129
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• 1999

In recent years, numerous researches have been carried out in author's laboratory to develop several kinds of methods for producing transgened chicken, leaving a lot of new findings. Some of them are very useful to search for new approaches necessary to improve the efficiency of hatchability and the survival rate of developing trasgened embryos. The results obtained hitherto might be summarized as follows: (1) foreign gene(Lac Z/ Miw Z) introduced into blastodermal cells of developing embryos was successfully transferred to embryos, leading to the production of primordial germ cells(PGCs) carrying foreign DNA. However, hatched hickens failed to show the incorporation of introduced gene into the gonads. (2) When foreign gene was introduced into germinal crescent region (GCR), the gene was also efficiently incorporated into germ cells, resulting in the production of transgened chickens(offspring) which produced fruther offspring having foreign gene in the gonads. In this case, 2nd and 3rd generations of chickens were obtained through the reproduction of transgened birds. (3) In another way, the gene was injected into blood vessels of developing embryos at stage 13∼15, creating PGCs having foreign gene, and produced some transgened chickens. In this work, the PGCs were transfered between embryos, resulting in the production of transgenic chickens. (4) in these experiments, PGCs were effectively employed for producing transgenic birds, developing some kinds of chimeric chickens from homo- or hetero-sexual transfer of the PGCs from embryos. This means that the gonads from donor PGCs developed in some degree to the stage of hatching. However, these gonads showed slightly abnormal tissues similar to ovotestis like organs through histological examination. (5) Avian Leukosis Virus(ALV) induced B cell line(DT40) successfully carried foreign genes into chicken embryos, suggesting the possibility of the cells as a vector in this field of study in the future. (6) Inter-embryonic transfer of the PGCs also gave us some.

• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.871-874
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• 2010

Many studies require expression analysis of the same gene/promoter across a range of bacterial genera. However, there is currently a lack of availability of reporters based on the broad-host-range IncQ replicon, which is compatible with a popular improved IncP transfer system that is self-transfer defective. We report IncQ lacZ reporter plasmids with features including (1) compatibility with IncP, IncW, and pBHR/pBBR replicons, (2) a variety of antibiotic markers (Sp-r, Sm-r, Km-r, Cm-r), (3) convenient mobilization via a novel self-transfer-defective IncP conjugation system, and (4) GenBank DNA sequences. Utility is demonstrated using three different promoters in different Gram-negative genera.

• Journal of Microbiology
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• v.42 no.4
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• pp.353-356
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• 2004

In a previous study, a gst gene was isolated from the fission yeast Schizosaccharomyces pombe. This gene was dubbed gstI, and was characterized using the gstI -lacZ fusion plasmid pYSH2000. In this work, four additional fusion plasmids, pYSHSDl, pYSHSD2, pYSHSD3 and pYSHSD4, were constructed, in order to carry (respectively) 770, 551, 358 and 151 bp upstream regions from the translational initiation point. The sequence responsible for induction by aluminum, mercury and hydrogen peroxide was located in the range between -1,088 and -770 bp upstream of the S. pombe gstI gene. The same region was identified to contain the nucleotide sequence responsible for regulation by Papl, and has one puta­tive Papl binding site, TTACGTAT, located in the range between $-954\~-947$ bp upstream of the gstI gene. Negatively acting sequences are located between -1,088 and -151 bp. These findings imply that the Papl protein is involved in basal and inducible transcription of the gstI gene in the fission yeast S. pombe.

• Journal of Sericultural and Entomological Science
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• v.37 no.2
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• pp.181-185
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• 1995

To enhance expression of foreign gene by the novel expression vector, pBmKSK1, of Bombyx mori nuclear polyhedrosis virus, E. coli $\beta$-galactosidase gene expressing recombinant virus was infected in BmN-4 cells and various concentrations of silkworm hemolymph were added to the recombinant virus-infected BmN-4 cells containing fetal bovine serum. The expression efficiency of foreign gene was determined by $\beta$-galactosidase activity in the culture media. The results showed that the silkworm hemolymph was effective to expression of foreign gene in the BmN-4 cells, suggesting that the silkworm hemolymph could be substituted for fetal bovine serum in the BmN-4 cells to enhance expression of foreign gene.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.762-766
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• 2001

The nar promoter of Escherichia coli was known to induce maximally under anaerobic or microaerobic conditions in the presence of nitrate. In this study, the nar promoter was tested to see whether the expression level of a reporter gene which fused lacZ gene at nar promoter's downstream, in the some gram negative host strains(Agrobacterium, Pseudomonas and Rhizobium). A nar promoter system(Combination of nar promoter and gram negative strain) was grown under aerobic conditions to absorbance at 600 nm of nearly 2.0 and then, the nar promoter was induced by lowering DO to 1-2% with alternating microaerobic and aerobic condition in the fermentor cultures, using different gram negative hosts. For a wild type nar promoter (pNW61), it was possible to maintain production of ${\beta}-galactosidase$ activity per cell(specific ${\beta}-galactosidase$ activity) at 14,000, 9600, 45 Miller units in the presence of 1% nitrate. and for a nitrate - independent nar promoter (pNW618) at 12,000, 10,400 and 58 Miller units in the absence of nitrate ion, respectively.

• The Korean Journal of Zoology
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• v.38 no.1
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• pp.49-54
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• 1995

본 연구자들은 근원세포를 면역시켜 얻은 hybidoma들을 검색하여. 계배 근원세포의 분화와 관련된 단백질을 인지하여 분화를 억제하는 대과가 있는 monoclonal antibody 3H35를 선별하여 그 항원을 확인한 바 있다(Kim et af.. (1992), Korean J. Zool 35 29-36) 본 연구에서는 λZAP에 cloning된 chicken muscle CDNA library들을 lacZ fusion protein으로 발현시켜 항체 3H35로 검색하여 그 유전자를 찾아내었다. 선별한 CDNA clone 중 C59의 삽입 절편은 1.6 kb이었고, 발현시킨 facE fusion protein 은 60 kDa로, f-galactosidase에 대한 항체에 반응하며 3H35와도 반응함을 immunoaffinitv adsorbant와 immunoblot으로 확인하였다 Clone C59의 삽입 절편의 염기서열을 분석한 결과, 실제 유전자는 1.6 kb 이상이며, 알려진 어느 다른 유전자와도 관련이 없는 새로운 근특이 유전자로 판단되었다. 아미노산으로 전환시켰을 때 31개의 특이한 서열이 7차례 반복된 부분이 나타났으며 이 서열의 23개가 일정하게 보존되어있고 나머지 서열의 아미노산의 polarity도 매우 유사하게 효존되어있다. 이들의 보존성이 극히 높은 것으로 보아 독특한 기능을 수행하는 domain으로 추정된다.