• Journal of Chest Surgery
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• 제36권11호
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• pp.852-857
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• 2003

배경: 중피종은 일반적 치료에 대하여 큰 효과가 없다고 알려져 있다. 저자들은 Adenoviral p53에 민감하게 반응하는 중피종 세포주인 염증 및 표피세포 아유형(subtype)에 adenovirus유전자 핵산전달감염(transfection)으로 중피종 치료의 새로운 방법에 대하여 평가하고자 하였다. 대상 및 방법: 두 쌍의 adenoviral PTEN와 LacZ (Ad/GT-LacZ와 Ad/GV16) 매개체(vectors)에 REN (p53 sensitive)인 중피종 세포주(methothelioma cell lines)의 형질을 도입(transduction)하였으며, 단백질 함량은 Western blotting 분석을 이용하여 측정하였다. 세포사멸은 fluorescence-activated cell sorter analysis of subdiploid populations에 의하여 평가하였으며, 세포 생존력은 XTT 분석에 의하여 결정하였다. 통계 분석은 analysis of variance와 Student t test를 이용하여 하였다. 결과: Adenoviral PTEN 유전자로 처치된 세포사는 72시간 후에 MOI of 20에서 대조군 2.5%에 비하여 REN군 32.9%로 상대적으로 높게 나타났다. 또한 REN cell에서의 전구세포사멸 단백질(proapoptotic protein)인 BAX 발현 증가를, BCL-2에서 발현 감소를 나타내었으나, BCL-XL, BAK 및 BAD 단백질은 변화가 없었다. 결론: Adenovirus PTEN을 매개로 한 BAX 발현 증가는 세포사멸을 유도하고 p53에 민감한 중피종 세포들(p53-sensitive methothelioma cells)에서 세포 생존력을 감소시킨다. 이러한 결과는 PTEN 유전자 핵산전달감염하는 것은 중피종 치료의 새로운 대안적 방법이 될 수 있다는 것을 암시한다.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.670-676
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• 2000

Several clones obtained from Serratia marcescens stimulated E. coli TP2139 (${\Delta}lac, \;{\Delta} crp$) cells to use maltose as a carbon source. The crp gene clone, pCKB12, was confirmed to stimulate the $\beta$-galactosidase activity, by Southern hybridization [31]. The nucleotide sequence of the crp region consisting of 1,979 bp was determined. The sequencing of the fragment led to the identification of two open reading frames: One of these, the crp gene, encoded 210 amino acid and the other encoded a truncated protein. The S. marcescens and E. coli crp genes showed a higher degree of divergence in their nucleotide sequence with 120 changes, however, the corresponding amino acid sequences showed only two amino acid differences. Yet, an analysis of the amino acid divergence revealed that the catabolite gene activator protein, the crp gene product, was the most conserved protein observed so far. Using a crp-lac protein fusion, it was demonstrated that S. marcescens CRP could repress its own expression, probably via a mechanism similar to that previously described for the E. coli crp gene.

• Journal of Microbiology
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• 제44권6호
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• pp.671-673
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• 2006

A new 4.87 kb Escherichia-Pseudomonas shuttle vector has been constructed by inserting a 1.27 kb DNA fragment with a replication origin of a Pseudomonas plasmid pRO1614 into the 3.6 kb E. coli plasmid pBGS18. This vector, designated pJH1, contains an aminogly-coside phosphotransferase gene (aph) from Tn903, a lacZ' gene for $\alpha$-complementation and a versatile multiple cloning site possessing unique restriction sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, BspMI, PstI, SphI, and HindIII. When pJH1 was transformed into E. coli DHS${\alpha}$ and into P. putida HK-6, it was episomally and stably maintained in both strains. In addition, the enhanced green fluorescent protein (EGFP) gene which was transcriptionally cloned into pJH1 rendered E. coli cells fluorescence when its transformants were illuminated at 488 nm.

• 미생물학회지
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• 제30권5호
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• pp.371-376
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• 1992

플라스미드 pKM101 과 이의 돌연변이체 pSL4 는 UV 및 MMS 에 대해 높은 치사 저항성과 돌연변이률을 나타낸다. pKM101 과 pSLA 의 mucB 유전자의 일부분과 mucA 유전자를 lacZ fusion 벡터인 pMC874 에 subcloning 시켜 플라스미드 pBH31 과 pBH30 을 선별하였고 이 플라스미드들을 $recA^{+}lexA^{-}$, $recA^{-}와lexA^{+}$, 균주에 각각 도입하였다. $recA^{+}lexA^{+}$ 균주에서 pSLA 의 muc 유전자를 포함하는 pBH30 의 $\beta$-galactosidase활성은 pKM101 의 muc 유전자가 연결된 pBH31 보다 높았고 $recA^{-}$ 와 $lexA^{-}$ 돌연변이주에서는 UV 조사의 유무에 관계없이 $\beta$-galactosidase를 유도하지 못하였지만 $recA^{-}$ 균주에서는 UV 조사를 하지 않았을 때 pBH30 의 $\beta$-galactosidase가 pBH31 보다 약간 높게 유도되었다. 이러한 결과는 pKM101 과 pSLA 의 기능적 차이가 두 플라스미드의 Muc 단백질의 구조적 차이라고 생삭할 수 있지만, muc 유전자의 조절부위가 돌연변이되너 LexA repessor 가 작용하는 부위의 변화에 의한 것이라고도 생각햐ㄹ 수 있었다. 또한, umuC-lacZ 유전자를 가지는 pBH100 를 construction 하여 umu oeron 의 발현은 UV 조사에 의해 유도되며 recA 와 lexA 유전자에 의해 조절됨을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.84-91
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• 2006

A Saccharomyces cerevisiae pgs1 nulI mutant, which is deficient with phosphatidyl glycerol (PG) and cardiolipin (CL) biosynthesis, grows well on most fermentable carbon sources, but fails to grow on non-fermentable carbon sources such as glycerol, ethanol, and lactate. This mutant also cannot grow on galactose medium as the sole carbon source. We found that the incorporation of $[^{14}C]-galactose$, which is the first step of the galactose metabolic pathway (Leloir pathway), into the pgs 1 null mutant cell was extremely repressed. Exogenously expressed PGS1 (YCpPGS1) under indigenous promoter could completely restore the pgs1 growth defect on non-fermentable carbon sources, and dramatically recovered $[^{14}C]-galactose$ incorporation into the pgs1 mutant cell. However, PGS1 expression under the GALl promoter $(YEpP_{GAL1}-PGS1myc)$ could not complement pgs1 mutation, and the GAL2-lacZ fusion gene $(YEpP_{GAL2}-lacZ)$ also did not exhibit its $\beta-galactosidase$ activity in the pgs1 mutant. In wild-type yeast, antimycin $A(1\;{\mu}g/ml)$, which inhibits mitochondrial complex III, severely repressed not only the expression of the GAL2-lacZ fusion gene, but also uptake of $[^{14}C]-galactose$. However, exogenously expressed PGS1 partially relieved these inhibitory effects of antimycin A in both the pgs1 mutant and wild-type yeast, although it could not basically restore the growth defect on galactose by antimycin A. These results suggest that the PGSI gene product has an important role in utilization of galactose by Gal genes, and that intact mitochondrial function with PGS1 should be required for galactose incorporation into the Leloir pathway. The PGS1 gene might provide a clue to resolve the historic issue about the incapability of galactose with deteriorated mitochondrial function.

• BMB Reports
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• 제43권7호
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• pp.451-460
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• 2010

Starting with the first publication of lacZ gene fusion in 1980, reporter genes have just entered their fourth decade. Initial studies relied on the simple fusion of a promoter or gene with a particular reporter gene of interest. Such constructs were then used to determine the promoter activity under specific conditions or within a given cell or organ. Although this protocol was, and still is, very effective, current research shows a paradigm shift has occurred in the use of reporter systems. With the advent of innovative cloning and synthetic biology techniques and microfluidic/nanodroplet systems, reporter genes and their proteins are now finding themselves used in increasingly intricate and novel applications. For example, researchers have used fluorescent proteins to study biofilm formation and discovered that microchannels develop within the biofilm. Furthermore, there has recently been a "fusion" of art and science; through the construction of genetic circuits and regulatory systems, researchers are using bacteria to "paint" pictures based upon external stimuli. As such, this review will discuss the past and current trends in reporter gene applications as well as some exciting potential applications and models that are being developed based upon these remarkable proteins.

• Bulletin of the Korean Chemical Society
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• 제30권4호
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• pp.791-793
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• 2009

Escherichia coli RNase P is composed of a large RNA subunit (M1 RNA) and a small protein subunit (C5 protein). Since both subunits are assembled in a 1:1 ratio, expression of M1 RNA and C5 protein should be coordinately regulated for RNase P to be efficiently synthesized in the cell. However, it is not known yet how the coordination occurs. In this study, we investigated how overexpression of C5 protein affects expression of the rnpB gene encoding M1 RNA, using a lysogenic strain, which carries an rnpB-lacZ transcription fusion. Primer extension analysis of rnpB-lacZ fusion transcripts showed that the overexpression of C5 protein increased the amount of the fusion transcripts, suggesting that rnpB expression increases with the increase of intracellular level of C5 protein.

• 한국미생물·생명공학회지
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• 제33권4호
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• pp.241-247
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• 2005

선별마커로써 Lactococcus lactis ssp. lactis ATCC 7962 유래의 $\beta$-galactosidase 유전자를 포함하는 Lactococcus용 셔틀/발현 벡터 pWgall3T를 제조하여 Escherichia coli DH5$\alpha$와 고. Lactis MG1363내로 도입하였다. 이들 형질 전환체들은 X-gal을 포함하는 배지에서 파란색의 표현형을 보임으로써 쉽게 확인할 수 있었다. 또한, L. lactis MG1363 형질전환체로부터 $\beta$-galactosidase 활성을 측정한 결과 기존에 $\beta$-galactosidase를 활성을 지닌 L. lactis ATCC 7962에 비해 glucose를 포함하는 M17배지에서 4배정도 높은 활성을 보임으로써 선별마커로써의 효율성을 나타내었다. pWgal13T는 $\beta$-galactosidase 유전자 외에 L. lactis Wg2유래의 replicon과 외래 유전자의 발현을 위한 L. lactis ssp. cremoris LM0230의 promoter P13C, terminator를 포함하고 있다. 이 벡터의 이용가능성을 확인하기 위하여 외래 유전자 EGFP유전자를 P13C 아래에 삽입하여 E. coli와 L. iactis에서 발현을 확인하였다. 이 연구에서 제조된 Lactococcus용 발현 벡터 pWgal13T는 E. coli와 L. lactis에서 외래 유용 유전자를 생산을 위해 이용 할 수 있을 것이다.

• 생명과학회지
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• 제8권3호
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• pp.263-271
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• 1998

전사조절인자로서 잘 알려져 있는 cAMP receptor protein(CRP)은 cAMP와 DNA에 결합하는 특별한 활성을 가지고 있으며, cAMP-CRP complex를 형성하여 수많은 유전자의 발현조절에 관여한다. 이러한 측면에서 cAMP-CRP의 조절은 어떤 면에서 총체적 조절체계라고까지 한다.본 연구는 Serratia 균주에서 crp 유전자의 분자적 특성 및 cAMP에 의한 발현조절을 받는 분자기구를 해석하고자 유전자를 클로닝하고 발현을 확인하였다. MacConkey 배지에서 maltose를 탄소원으로 충분히 이용하지 못하는 대장균 TP2139(${\Delta}crp$,${\Delta}lac$를 숙주로 이용하고, 염색체 DNA를 library로 작성하여 얻은 형질전환체 약 일만개의 콜로니에서 red colony를 나타내는 5종류의 양성 클론을 얻었다. 이들 클론을 Southern 방법으로 확인한 결과 3kh의 단편을 가진 pCKB12클론이 crp유전자를 coding하고 있음을 확인하였다. glpD-lacZ 융합 plasmid인 pLDC6의 BamHI부위에 pCKB12의 3kb 단편을 삽입시킨 재조합 plasmid pLDC6-Scrp를 작성하여, 클로닝된 Serratia의 crp유전자가 대장균에서 유전자 전사조절에 미치는 영향을 확인한 결과 cAMP-CRP 복합체 형성에 의한 전사조절 기능이 확인되어졌다.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.355-362
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• 2000

The Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) gene in the pHLA-21 plasmid was inserted into a baculovirus (Hyphantria cunea nuclear polyhedrosis virus) expression vector (lacZ-HcNPV) to construct a recombinant virus gB-HcNPV expressing gB. Spodoptera frugiperda cells infected with this recombinant virus synthesized and processed gB of approximately 120 kDa, which cross-reacted with the monoclonal antibody to gB. The recombinant gB was identified on the membrane of the insect cells using an immunofluorescence assay. Antibodies to this recombinant raised in mice recognize the viral gB and neutralized the infectivity of the HSV-1 in vitro. These results show that the gB gene has the potential to be expressed in insect cells. They also demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the lacZ-HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV. Accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.