• BMB Reports
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• 제28권5호
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• pp.458-463
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• 1995

Two apparent rho-independent transcription terminator structures within the coding sequence of aceK have been destroyed to access their roles in the differential expression between aceA and aceK in the glyoxylate bypass operon of E. coli. The effect of mutations on the expression of aceK was evaluated in two different ways: one by maxicell labeling and the other by lacZ fusion gene construction. The maxicell labeling experiment with the mutant operon clones has failed, like that of the wild type operon clone, to visibly show isocitrate dehrogenase (IDH) kinase/phosphatase, the product of aceK, on the autoradiogram of a protein gel. When the same mutations were introduced into an aceK::lacZ fusion gene to quantitatively evaluate the mutational effect, the activity of ${\beta}-galactosidase$ in neither of the mutant versions of the fusion gene was elevated significantly enough to explain the degree of polarity observed in this region. Thus, we conclude that neither of these intragenic, apparent rho-independent transcription terminator structures, which have long been suspected as a major determinant in the down regulation of aceK, really act as a premature transcriptional terminator.

• 미생물학회지
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• 제37권4호
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• pp.259-264
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• 2001

S. typhimurium cadBA operon의 발현에는 산성 pH와 고농도의 lysine 등 적어도 두 가지의 세포외부 신호가 요구된다. pH와 lysine신호에 따른 cadBA 발현 조절 기작을 이해하기 위하여, Tn10삽입, 자발적 돌연변이, EMS돌연변이 등을 수행하여 JF2238과 cadA-lacZ의 발현특성에 차이를 보이는 돌연변이체를 선별하였다. cadBA의 양성적 조절자의 유전자인 cadC내의 돌연변이 중 cadC4 돌연변이는 pH-비의존성, lysine-의존성 발현을, cadC6 돌연변이는 pH-비의존성, lysine-비의존성 cadA-lacZ 발현을 나타내었다. 산성 pH, lysine이 없는 조건에서 cadA-lacZ 발현을 유도하는 cadR::Tn10과 cadR3 돌연변이체를 분리하여 cadR이 lysine이 없는 조건에서 cadBA 발현을 음성적으로 조절하는 음성적 조절자의 유전자임을 알 수 있었다. cadR 돌연변이체는 lysine의 독성 유사체인 thiosine에 대해 내성($Ts^{r}$)을 나타냈으며, E. coli의 lysP 클론을 갖고 있는 pLYSP에 의해 돌연변이 형질이 보상되었다. 또한 lysine decarboxylase (CadA)의 반응산물인 cadaverine은 cadC+ 균주에서 cadA-lacZ 발현에 억제효과를 나타냈으나, cadC 돌연변이에는 억제효과가 나타나지 않았다.

• BMB Reports
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• 제40권1호
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• pp.119-124
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• 2007

Adenoviral vectors are among the most promising vectors available for human gene therapy. However, the use of recombinant adenoviral vectors, including replicationcompetent adenovirus (RCA), raises a variety of safety concerns in relation to the development of new therapies based on gene therapy. To examine how organic compounds change in rat plasma following the injection of adenovirus, $\beta$-galactosidase expressing recombinant adenovirus (designated rAdLacZ) or RCA, we investigated the content of fatty acids (FAs), which are important biochemical indicators in pathological conditions. Pattern recognition analysis on the level of FAs in rat plasma is described for the visual discrimination of adenovirus infection groups from normal controls. Plasma FAs from four control rats (normal group), and from four rats with rAdLacZ infection and six rats with RCA infection (the two abnormal groups), were examined by gas chromatography-mass spectrometry in selected ion monitoring modes as their tert-butyldimethylsilyl derivatives. In total, 20 FAs were positively detected and quantified. The results of the Student's t-test on the normal mean of two abnormal groups, the levels of three FAs (p<0.05) from rAdLacZ group and eleven FAs (p<0.05) from RCA group were significantly different. When star symbol plotting was applied to the group mean values of 20 FAs after normalization to the corresponding normal mean values, the resulting eicosagonal star patterns of the two infected groups were distorted into similar shapes, but were distinguishable from each other. Thus, these approaches will be useful for screening and monitoring of diagnostic markers for the effects of infection following the use of adenoviral vectors in gene therapy.

• 자연과학논문집
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• 제11권1호
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• pp.89-94
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• 1999

본 연구에서는 다양한 신호에 의해 면역세포를 세포사멸로 유도하는 22 kDa의 calcium-binding 단백질인 ALG-2 (apoptosis linked gene-2) 에 대해 LexA DNA blinding domain (DBD) 과 융합시킨 Lex/ALG-2 융합단백질을 이용하여 효모에서의 전사활성화 능력을 측정하였다. hALG-2의 전사활성화 능력을 측정한 결과 전체 ALG-2 (아미노산 1-191) 와 N-말단 (아미노산 1-98) 에서는 전사활성화 능력은 보이지 않았으나, C-말단 (아미노산 93-191) 에서는 reporter 유전자인 LacZ를 전사활성화 시킴을 확인하였다. hALG-2 C-말단의 전사활성화 능력은 양성 대조구로 사용한 Lex-B42에 비하여 2.7배 강한 것으로 나타났다. 본 연구의 결과는 hALG-2의 N-말단에 전사 억제 조절 신호가 존재할 가능성을 시사한다고 보여진다 .

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.157-161
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• 2002

A lactic acid bacteria with ${\beta}$-gal activity was isolated from Kimchi, a traditional fermented vegetable food in Korea. The isolate was identified as a Lactococcus lactis strain and named L. lactis A2. The gene encoding ${\beta}$-gal of L. lactis A2 was cloned as a 5.8 kb PstI fragment. DNA sequencing identified the complete lacA (galactoside acetyltransferase)-lacZ (${\beta}$-galactosidase) genes together with the 3' part of upstream galT (galactose-1-phosphate uridyltransferase), and the 5'region of downstream galE (UDP-galactose-4-epimerase) genes. L. lactis A2 had the same gal/lac operon structure as in L. lactis subsp. lactis 7962. Other genes of the Leloir pathway are most likely to be located in the 5'upstream of the 5.8 kb fragment on the A2 chromosome. Sequences downstream of galE were different from those of L. lactis subsp. lactis 7962.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제27권2호
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• pp.103-109
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• 2005

In spite of the ongoing advances, standard therapies for oral cancer still has some limitations in efficacy and in ability to prolong survival rate of advanced disease and result in significant functional defect and severe cosmetic deformity. Currently gene therapy using tumor suppressor gene is considered as a potent candidate for new therapeutic approaches that can improve efficacy and reduce complications. The purpose of this research is to identify the role of adenoviral vector to transfer HCCS-1 tumor suppressor gene in oral cancer cells and to find out whether there is a possibility for it to serve in the field of gene therapy. The human SCC-25 cell line was used for transfection. To determine the efficiency of the adenovirus as a gene delivery vector cell line was transduced with LacZ gene and analysed with X-gal staining. Northern blot was performed to confirm the tranfection with HSCC-1 gene and cell viability was assessed by cell cytotoxicity assay. We had successfully construct the recombinant HSCC-1 adenovirus(Ad5CMV-HCCS-1). DNA extracted from Ad5CMV-HCCS-1 revealed HCCS-1 gene is incorporated. The transduction efficiencies were over than 50% of SCC-25 cells with a MOI of 2 and over 95% with a MOI of 50. Northern blot analysis showed that a single 0.6kb mRNA transcript was expressed in Ad5CMV-HCCS-1 transduced SCC-25 cells. There was no or very low transcription HCCS-1 mRNA in wild and Ad5CMV-LacZ transduced SCC-25 cells. Cells transduced with Ad5CMV-HCCS-1 showed significant growth inhibition. By day 6, Ad5CMV-HCCS-1 treated cell count was decreased to 30% of mock-infected cells, while that of Ad5CMV-LacZ treated cells was 90% of mock-infected cells (p<0.05). Finally, these result suggest that the Ad5CMV-HCCS-1 has potential as a gene therapy tool for oral cancer.

• 한국발생생물학회지:발생과생식
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• 제2권1호
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• pp.45-52
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• 1998

강화인자 검출법을 이용 X염색체에 P[1ArB]이 형질전환되어 극세포 및 경게세포에서 표시유전자 lacZ를 발현하는 노랑초파리 (EDL 149)를 이용하여 난자형성과정 동안의 경계새포의 분화 및 이동을 조사하였다. 경계세포는 9기 난포의 선단에 위치한 난포 세포로부터 분화하여 9기와 10기에 이동하는 것을 확인하였다. 난소내 \beta -galactosidase의 활성은 우화 후 처음 4일간 급격히 증가하는 것을 확인하였으며 이 시기는 난포 내에서 경계세포가 분화하는 시기와 일치하였다. EDL149의 P[1ArB]삽입의 동형접합체의 난포 내에서 일부 경계세포의 불완전한 이동 또는 지연이 관찰되었다. 감수분열을 진행중인 정소내 세포 및 더듬이에서 확인된 lacZ 유전자의 발현양상은 P[1ArB]의 삽입부위가 난소특이 유전자부위가 아니지만 경계세포 이동의 조절에 역할을 하는 유전자임을 암시한다. 이 형질전환초파리 및 삽입위치 부근의 유전자는 발생중 진행되는 세포이동의 연구에 좋은 모델로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.872-880
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• 2003

Three vectors, pSG1, 2, and 3, which facilitate in vivo expression technology (IVET) in Gram-negative bacteria, were developed. Vectors pSG1and 2 are derivatives of ColE1, and pSG3 is a derivative of an R6K replicon. These vectors contain oriT sites that allow mobilization when the RK2 Tra functions are provided in trans. These vectors contain promoterless lacZ (pl-lacZ) and promoterless aph (pl-aph) transcriptionally fused together, which allow qualitative and quantitative measurements of the expression of genes in the genome of bacterial cells. pSG1 and 3 contain gentamicin-resistance genes, and pSG2 carries a streptomycin-/spectinomycin-resistance gene, allowing for selection of recombinants generated by a single crossover between a library fragment cloned into a pSG vector and the identical region in the genome of a bacterial species from which the library fragment originated. These vectors were successfully applied to the generation of random fusions at high rates in the genomes of four representative Gram-negative bacteria. In addition, the expression level of ${\beta}-galactosidase$ and the degree of resistance to kanamycin in cells with fusions generated by these vectors were found to be linearly correlated, proving that these vectors can be used for IVET.

• BMB Reports
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• 제34권5호
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• pp.395-401
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• 2001

Schizosaccharomyces pombe gene encoding redox enzymes, such as thioltransferase (TTase) and thioredoxin (TRX), were previously cloned and induced by oxidative stress. In this investigation, their expressions were examined using $\beta$-galactosidase fusion plasmids. The expression of the two cloned genes appeared to be growth-dependent. The synthesis of $\beta$-galactosidase from the TTase-lacZ fusion was increased in the medium with the low glucose level, whereas it was significantly decreased in the medium without glucose or with galactose. It was also decreased in the nitrogen-limited medium. The synthesis of galactosidase from the TRX-lacZ fusion was unaffected by galactose or low glucose. However, it was lowered the absence of glucose. The synthesis of $\beta$-galactosidase from the TTase-lacZ fusion was shown to be enhanced in a higher medium pH. Our findings indicate that S. pombe TTase and TRX genes may be regulated by carbon and nitrogen sources, as well as medium pH.

• 한국환경성돌연변이발암원학회지
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• 제18권1호
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• pp.15-21
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• 1998

Transgenic animal and cell line models which are recently developed and used in toxicology fields combined with molecular biological technique, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Transgenic models, which have exogenous DNA incorporated into their genome, carry recoverable shuttle vector containing reporter genes to assess endogenous effects or alteration in specific genes related to disease processes. The lac I and lac Z gnee most widely used as a mutational target in transgenic systems. The assay is performed by treatment with putative mutagenic agents, isolation of genomic DNA from cells or tissues, exposure the isolated DNA to in vitro packaging extract, plating and sequencing. The results from these processes provide not only mutant frequency as quantitative evaluation but also mutational spectrum as qualitative evaluation of various agents. Therefore we introduce and review the principle, detailed procedure and application of transgenic mutagenesis assay system in toxicology fields especially in mutagenesis and carcinogenesis.