• 약학회지
• /
• 제47권3호
• /
• pp.184-189
• /
• 2003

Peptide deformylase (PDF) is essential and unique to bacteria, thus making it an attractive target for the discovery of novel antibacterial drugs. PDF deformylates the N-formylmethionine of newly synthesized polypeptides in prokaryotes. In this study, a pdf gene from Staphylococcus aureus 6538p was cloned in pET-14b vector and PDF protein was over-produced in Escherichia coli BL21 (DE3). NH$_2$-terminal His-tagged PDF protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography. Enzymatic activity of purified 6xHis-tagged PDF was tested on the substrate (formyl-Methionine-Alanine-Serine) by formate dehydrogenase-coupled spectrometric assay of peptide deformylase. For the discovery of new PDF inhibitors from chemical libraries and culture broths of soil bacteria, a target-oriented screening system using a 96-well plate was developed. About 3,000 commercial chemical libraries were tested in this screening system, and 2 chemicals (0.07％) among them showed an inhibitory activity against PDF enzyme. This result showed that a new screening system can be used for the discovery of new PDF inhibitors.

• KSBB Journal
• /
• 제28권3호
• /
• pp.213-215
• /
• 2013

A high-throughput screening strategy was developed to simplify the selection process for improved mannitol producing strain after chemical mutagenesis. Ethylmethyl sulfonate (EMS) was used as a chemical mutagen to alter the fructokinase-I gene which is an essential enzyme to metabolize fructose for growth. Leuconostoc mesenteroides treated with EMS were plated on the modified MRS solid medium containing fructose as a sole carbon source. Strains showing inhibited growth were primarily selected to evaluate the mannitol producing ability. By applying this strategy, L. mesenteroides ATCC 8293 M1, L. mesenteroides ATCC 9135 M3 and L. mesenteroides D1 M3 showed improvement in mannitol production.

• 한국미생물·생명공학회지
• /
• 제26권6호
• /
• pp.562-565
• /
• 1998

Nisin-producing and nisin resistant L. lactis subsp. lactis ATCC7962 harbored six plasmids. To find a plasmid containing a nisin resistant gene, these plasmids were transformed into L lactis LM0230 of plasmid-free and nisin sensitive strain. After screening on nisin selection media containing nisin (150 $\mu\textrm{g}$/$m\ell$), several nisin resistant transformants were obtained and the level of nisin resistance was very similar to that of wild type L lactis subsp. lactis ATCC7962. A 26.5 kb plasmid, named as pCS100, which confers resistance to nisin, was identified in transformants. The pCS100 was digested with EcoRI and Southern blot hybridization was done with nisI probe to localize the nisin resistant gene. A 4 kb EcoRI fragment showed a strong positive signal, and it was cloned into pBluescript for the potential selection marker.

• 한국연초학회지
• /
• 제25권2호
• /
• pp.87-94
• /
• 2003

Recently, many researches for plant alkaloids, one of the largest groups of natural products, are reported because of their various pharmacological activity. This study was carried out to clone putrescine N-methyltransferase (PMT) gene which is a key enzyme in diverting polyamine metabolism towards the biosynthesis of nicotine and related alkaloids from Burley tobacco. To induce expression of PMT gene in tobacco plant, the floral meristem was removed and then mRNA was purified from root. cDNA encoding PMT gene was isolated by RT PCR and cloned. Three different groups of clones were screened by PCR and restriction enzyme digestion analysis and were characterized. The data of these screening revealed that three types of PMT are present in Burley tobacco. Comparison of the nucleotide sequence of this three genes encoding putative PMT with those of other tobaccos revealed that two types of PMT are newly discovered from Nicotiana tabacum cv. Br21 tobacco and they were same as PMT2, PMT3 of N. tabacum cv. Xanthi.

• Plant Resources
• /
• 제7권2호
• /
• pp.116-122
• /
• 2004

We tried to isolating a noble gene from cucumber library with heterogeneouse RNA probe labeled DIG of Arabidopsis PIN3 gene. Two kinds of RNA probes which had no significant homology each others, were designed from the 5'- and 3'- prime nucleotides of the AtPIN3 gene. In the first and second screenings of the cDNA library of cucumber with the probes, two positive clones were identified with specific duplicate signals. However, we isolated cDNA fragments homologous with putative nucleases from Nicotiana, Arabidopsis, Cordialis, and Oryza sativa, there was no significant homology with any other PIN family genes.

• Genomics & Informatics
• /
• 제11권4호
• /
• pp.200-210
• /
• 2013

Studying biological networks, such as protein-protein interactions, is key to understanding complex biological activities. Various types of large-scale biological datasets have been collected and analyzed with high-throughput technologies, including DNA microarray, next-generation sequencing, and the two-hybrid screening system, for this purpose. In this review, we focus on network-based approaches that help in understanding biological systems and identifying biological functions. Accordingly, this paper covers two major topics in network biology: reconstruction of gene regulatory networks and network-based applications, including protein function prediction, disease gene prioritization, and network-based genome-wide association study.

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

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제5권1호
• /
• pp.7-12
• /
• 2000

The expression of the mouse $\alpha-amylase$ gene in the methylotrophic yeast, P pastoris was investigated. The mouse $\alpha-amylase$ gene was inserted into the multi-cloning site of a Pichi a expression vector, pPIC9, yielding a new expression vector pME624. The plasmid pME624 was digested with SalI or BglII, and was introduced into P. pastoris strain GSl15 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested with SaiII or BglII into the HIS4locus $(38\;of\;Mut^+\;clone)$ or into the AOX1 locus $(15\;of\;Mut^s\;clone)$. Southern blot was carried out in 11 transformants, which showed that the mouse $\alpha-amylase$ gene was integrated into the Pichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest $\alpha-amylase$ activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse $\alpha-amylase$ gene is compared with that in recombinant Saccharomyces cerevisiae harboring a plasmid encoding the same mouse $\alpha-amylase$ gene, the specific enzyme activity is eight fold higher than that of the recombinant S. cerevisiae.

• Journal of Microbiology
• /
• 제45권1호
• /
• pp.69-74
• /
• 2007

Escherichia coli is a clonal species, and occurs as both commensal and pathogenic strains, which are normally classified on the basis of their O, H, and K antigens. The O-antigen (O-specific polysaccharide), which consists of a series of oligosaccharide (O-unit) repeats, contributes major antigenic variability to the cell surface. The O-antigen gene cluster of E. coli O66 was sequenced in this study. The genes putatively responsible for the biosynthesis of dTDP-6-deoxy-L-talose and GDP-mannose, as well as those responsible for the transfer of sugars and for O-unit processing were identified based on their homology. The function of the wzy gene was confirmed by the results of a mutation test. Genes specific for E. coli O66 were identified via PCR screening against representatives of 186 E. coli and Shigella O type strains. The comparison of intergenic sequences located between galF and the O-antigen gene cluster in a range of E. coli and Shigella showed that this region may perform an important function in the homologous recombination of the O-antigen gene clusters.

• Journal of Microbiology and Biotechnology
• /
• 제15권3호
• /
• pp.491-496
• /
• 2005

The biosynthetic gene cluster for the aminoglycoside antibiotic kasugamycin was isolated and characterized from the kasugamycin producing strain, Streptomyces kasugaensis KACC 20262. By screening a fosmid library using kasA, the gene encoding aminotransferase, we isolated a 22 kb DNA fragment. The fragment contained seventeen complete open reading frames (ORFs); one of these ORFs, kasD, was identified as the gene for dNDP-glucose 4,6-dehydratase, which catalyzes the conversion of dNDP-glucose to 4-keto-6-deoxy-dNDP-glucose. The enzyme showed a broad spectrum of substrate specificity. In addition, ksR was overexpressed in E. coli BL21 and proved to be a self-resistance gene against kasugamycin. These findings suggest that the isolated gene cluster is highly likely responsible for the biosynthesis of kasugamycin.