• Asian Pacific Journal of Cancer Prevention
• /
• 제14권11호
• /
• pp.6557-6562
• /
• 2013

Objective: Specific promoters could improve efficiency and ensure the safety of gene therapy. The aim of our study was to screen examples for lung cancer. Methods: The firefly luciferase gene was used as a reporter, and promoters based on serum markers of lung cancer were cloned. The activity and specificity of seven promoters, comprising CEACAM5 (carcinoembryonic antigen, CEA), GRP (Gastrin-Releasing Peptide), KRT19 (cytokeratin 19, KRT), SFTPB (surfactant protein B, SP-B), SERPINB3 (Squamous Cell Carcinoma Antigen, SCCA), SELP (Selectin P, Granule Membrane Protein 140kDa, Antigen CD62, GMP) and DKK1 (Dickkopf-1) promoters were compared in lung cancer cells to obtain cancer-specific examples with strong activity. Results: The CEACAM5, DKK1, GRP, SELP, KRT19, SERPINB3 and SFTPB promoters were cloned. Furthermore, we successfully constructed recombinant vector pGL-CEACAM5 (DKK1, GRP, SELP, KRT19, SERPINB3 and SFTPB) contained the target gene. After cells were transfectedwith recombinant plasmids, we found that the order of promoter activity from high to low was SERPINB3, DKK1, SFTPB, KRT19, CEACAM5, SELP and GRP and the order for promoters regarding specificity and high potential were SERPINB3, DKK1, SELP, SFTPB, CEACAM5, KRT19 and GRP. Conclusion: The approach adopted is feasible to screen for new tumour specific promoters with biomarkers. In addition, the screened lung-specific promoters might have potential for use in lung cancer targeted gene therapy research.

• BMB Reports
• /
• 제35권6호
• /
• pp.637-641
• /
• 2002

Only one natural promoter that interacts with bacteriophage K11 RNA polymerase has so far been identified. To identify more, in the present study restriction fragments of the phage genome were individually assayed for transcription activity in vitro. The K11 genome was digested with two 4-bp-recognizing restriction enzymes, and the fragments cloned in pUC119 were assayed with purified K11 RNA polymerase. Eight K11 promoter-bearing fragments were isolated and sequenced. We report that the nine K11 promoter sequences (including the one previously identified) were highly homologous from -17 to +4, relative to the initiation site at +1. Interestingly, five had -10G and -8A, while the other four had -10A and -8C. The consensus sequences with the natural -10G/-8A and -10A/-8C, and their variants with -10G/-8C and -10A/-8A, showed nearly equal transcription activity, suggesting residues at -10 and -8 do not regulate promoter activity. Using hybridization methods, physical positions of the cloned promoter-bearing sequences were mapped on SalI-and KpnI-restriction maps of the K11 genome. The flanking sequences of six cloned K11 promoters were found to be orthologous with T7 or T3 genomic sequences.

• The Plant Pathology Journal
• /
• 제21권1호
• /
• pp.13-20
• /
• 2005

Chlorella viruses are large icosahedral, plaque-forming, dsDNA viruses that infect certain unicellular, chlorellalike green algae. The genomic DNA of over 300 kb contains many useful genes and promoters. Over 40 chlorella viruses have been isolated from fresh water in Korea since 1998. The viruses were amplified initially in chlorella strain NC64A, and pure isolates were obtained by repeated plaque isolation. SDS-PAGE analysis revealed similar but distinct protein patterns, both among the group of purified viruses and in comparison with the prototype chlorella virus PBCV-1. Digestions of the 330- to 350-kb genomic DNAs with 10 restriction enzymes revealed different restriction fragment patterns among the isolates. The tRNA-coding regions of 8 chlorella viruses were cloned and sequenced. These viruses contain 14-16 tRNA genes within a 1.2- to 2-kb region, except for the SS-1 isolate, which has a 1039-bp spacer in a cluster of 11 tRNA genes. Promoter regions of several early genes were isolated and their activities were analyzed in transformed chlorella. Some promoters showed stronger activity than commonly used CaMV 35S promoter and chlorella transformation vectors for heterologous protein are beings constructed using these promoters.

• Journal of Plant Biotechnology
• /
• 제39권4호
• /
• pp.242-252
• /
• 2012

Seed storage oils are essential resources for not only human and animal diets but also industrial applications. The primary goal of this study was to increase seed oil content through comparative analysis of two seed-specific promoters, AtFAE1 from Arabidopsis Fatty Acid Elongase 1 gene and BnNapin from Brassica napus seed storage protein gene. AtWRI1 and AtDGAT1 genes encoding an AP2-type transcription factor and a Diacylglycerol Acyltransferase 1 enzyme, respectively, were expressed under the control of AtFAE1 and BnNapin promoters in Arabidopsis. The total seed oil content in all transgenic plants was increased by 8-11% compared with wild-type seeds. The increased level of oil content in AtWRI1 and AtDGAT1 transgenic lines under the control of both promoters was similar, although the activity of the BnNapin promoter is much stronger than that of AtFAE1 promoter in the mature stage of developing seeds where storage oil biosynthesis occurs at a maximum rate. This result demonstrates that the AtFAE1 promoter as well as the BnNapin promoter can be used to increase the seed oil content in transgenic plants.

• 미생물학회지
• /
• 제28권1호
• /
• pp.19-26
• /
• 1990

To examine the effects of sequence variations near the transcriptional start site on the rate of formation of the open complexes at bacteriophage $\lambda P_{R}$ promoter, two mutant promoters were created by site-specific mutagenesis using synthetic oligonucleotides. Mutant I coatains changes at positions -3 and -4 from TT to CC, thus having a 6-bp long G/C stretch between -10 region and transciptional start site (+1). Mutant II has changes at positions -5 and -6 from GG to AA, thereby having a 9-bp long A/T stretch between positions -11 and -3. Selective filter binding assays were performed to measure the rate of formation of the open complexes between the wild-type or two mutant $P_{R}$ promoters on 664 bp fragments and E. coli RNA polymerase at two temperatures. At 37.deg.C, the wild-type and two mutants showed similar rates for the formation of open complex. The second order rate constant $k_{a}$ and $\tau _{int}$, as determined from the .tau.-plot analysis, were $(6.0\pm0.4)\times10^{6}M^{-1}sec^{-1}$ and $11\pm5$sec, respectively. At 18.deg.C, however, the wild-type and two mutant promoters showed differences in the kinetic parameters. k for the wild-type promoter was (2.2$\pm$0.1)\times 10^{6}M^{-1}sec^{-1}$ and $\tau _{int}$ was 76$\pm$sec. Mutant I and II exhibited differences mainly in the rate of isomerization ($\tau_{int,I}=91\pm$10 sec, int,II=34$\pm$ sec), whereas the second order rate constant $k_{a}$ was similar to the wild type value. This result implies that at $18^{\circ}C$, the isomerization rate is determined by both protein conformational change and DNA melting, which are separable kinetically according to the 3-step mechanism of Roe et al.(1984,1985), and that the base changes affected mainly the rate of DNA melting as predicted.lting as predicted.

• Journal of Microbiology and Biotechnology
• /
• 제14권1호
• /
• pp.128-133
• /
• 2004

The secretion capacity of two E. coli strains (JM109 and AF1000) was evaluated through the expression of two human proinsulin fusion proteins using the translocation signal sequence from Staphylococcal protein A (SpA). Although a 7 to 11-fold difference in the expression levels was attained by the use of different promoters (SpA and malK promoters) and copy-number vectors (700 and 50 copies per cell), the maximum translocation rates for all the systems were around 140,000 amino acids $cell^{-1} min^{-1}$. Moreover, the secretion capacity was found to be independent of the size of the exiting peptide and its translational rate.

• Journal of Microbiology and Biotechnology
• /
• 제20권11호
• /
• pp.1563-1570
• /
• 2010

To construct a highly sensitive detection system for endocrine disruptors (EDs), we have compared the activity of promoters with the n-alkane-inducible cytochrome P450 gene (ALK1), isocitrate lyase gene (ICL1), ribosomal protein S7 gene (RPS7), and the translation elongation factor-1${\alpha}$ gene (TEF1) for the heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of the lacZ or hERa reporter genes, respectively, and the activity was evaluated by ${\beta}$-galactosidase assay for lacZ and Western blot analysis for hER${\alpha}$. The expression analysis revealed that the ALK1 and ICL1 promoters were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly a high level of expression in the presence of glucose and glycerol, respectively. In particular, the TEF1 promoter showed the highest ${\beta}$-galactosidase activity and a significant signal by Western blotting with the anti-estrogen receptor, compared with the other promoters. Moreover, the detection system was constructed with promoters linked to the upstream of the expression vector for the hER${\alpha}$ gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hER${\alpha}$ and CXAU1-2XERE was the most effective system for the $E_2$-dependent induction of the ${\beta}$-galactosidase activity. This system showed the highest ${\beta}$-galactosidase activity at $10^{-6}\;M\;E_2$, and the activity could be detected at even the concentration of $10^{-10}\;M\;E_2$. As a result, we have constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity, and reproducibility of the system for identifying and characterizing environmental estrogens.

• 한국발생생물학회지:발생과생식
• /
• 제18권1호
• /
• pp.1-11
• /
• 2014

Early growth response 1 (Egr1) is a zinc-finger transcription factor to direct second-wave gene expression leading to cell growth, differentiation and/or apoptosis. While it is well-known that Egr1 controls transcription of an array of targets in various cell types, downstream target gene(s) whose transcription is regulated by Egr1 in the uterus has not been identified yet. Thus, we have tried to identify a list of potential target genes of Egr1 in the uterus by performing multi-step in silico promoter analyses. Analyses of mRNA microarray data provided a cohort of genes (102 genes) which were differentially expressed (DEGs) in the uterus between Egr1(+/+) and Egr1(-/-) mice. In mice, the frequency of putative EGR1 binding sites (EBS) in the promoter of DEGs is significantly higher than that of randomly selected non-DEGs, although it is not correlated with expression levels of DEGs. Furthermore, EBS are considerably enriched within -500 bp of DEG's promoters. Comparative analyses for EBS of DEGs with the promoters of other species provided power to distinguish DEGs with higher probability as EGR1 direct target genes. Eleven EBS in the promoters of 9 genes among analyzed DEGs are conserved between various species including human. In conclusion, this study provides evidence that analyses of mRNA expression profiles followed by two-step in silico analyses could provide a list of putative Egr1 direct target genes in the uterus where any known direct target genes are yet reported for further functional studies.

• 유기물자원화
• /
• 제11권3호
• /
• pp.67-74
• /
• 2003

본 연구의 목적은 토양미생물의 일종인 Pseudomonas putida 에서 추출한 탄소고갈 유전자로부터 starvation promoter를 분리한 후 starvation vector를 개발하여 이를 미생물로부터 유용물질의 생산 및 오염물질의 정화등에 활용하기 위한 것이다. Starvation 유전자란 영양분의 결핍시에만 특수하게 발현되는 유전자의 일종으로 본 유전자로부터 promoter를 분리한 후 특정 물질을 분해 또는 생성하는 유전자를 분리된 promoter에 유전자 재조합 방법에 의하여 연결시키면 영양분의 공급을 최소화하고 세균의 생장을 억제 시키는 동시에 promoter에 연결된 유전자의 발현 기능은 최대화 시킴으로써 그 목적을 달성할 수 있다 하겠다. 본 연구에서는 기 분리된 starvation gene으로부터 promoter를 분리한 후 이를 적절한 벡터에 연결시켜 starvation vector인 pYKS101을 완성하였다. 본 벡터의 성능을 시험하기 위하여 reporter gene으로 lacZ유전자를 벡터내에 클로닝하여 세균의 염색체에 삽입시킨후 그 성능을 조사하였다. 삽입된 유전자는 예상한 바와같이 세균이 영양분이 결핍되었을 때 충분한 영양분이 공급되었을 때보다 약 4배의 활성도를 나타냄으로써 본 벡터의 유용성을 입증할 수 있었다.

• 한국산림과학회지
• /
• 제28권1호
• /
• pp.21-30
• /
• 1975

휴면기간(休眠期間)이 긴 주목종자(朱木種子)의 습층처리(濕層處理)에 따른 종자내(種子內) 생장(生長) 촉진물질(促進物質)과 생장(生長) 억제물질(抑制物質), 당(糖), 전분(澱粉), 단백질(蛋白質) 및 지방(脂肪) 등(等) 성분(成分)의 소장(消長)과 발아촉진(發芽促進)과의 관계(關係)를 구명(究明)코자 본(本) 실험(實驗)을 수행(遂行)하였던바 그 결과(結果)는 다음과 같다. 1. 습층처리(濕層處理)에 의(依)하여 종자내(種子內) 배육(胚肉)에 있어서의 생장촉진물질(生長促進物質)은 증가(增加)하고 생장(生長) 억제물질(抑制物質)은 급감(急減)하여, 이 양자간(兩者間)의 상호작용(相互作用)이 처리(處理) 1년 이후 11월부터 이듬해 3월사이에 이루어져서 발아(發芽)를 촉진(促進)한다고 생각되며 한편 종피(種皮)에 있어서의 생장(生長) 촉진물질(促進物質)은 배육(胚肉)에 있어서와는 달리 거의 소장(消長)에는 변동(變動)이 없고, 생장(生長) 억제물질(抑制物質)의 양(量)은 급감(急減)하여 지는 것으로 미루어, 종피(種皮)와 발아촉진(發芽促進)과의 관계(關係)는 촉진물질(促進物質)보다 억제물질(抑制物質)의 소실(消失)에 영향(影響)되며 익년(習年) 11월부터 그 다음해 3월사이에 이루어진다고 생각된다. 2. 상치종자(種子)의 발아력(發芽力) 시험결과(試驗結果) 생장(生長) 억제물질(抑制物質)이 발아(發芽)를 좌우(左右)하는 것으로 보아 주목종자(朱木種子)의 발아(發芽)는 생장촉진물질(生長促進物質)보다 생장(生長) 억제물질(抑制物質)에 영향(影響)함이 크다고 생각된다. 3. 습층처리(濕層處理) 함으로서 배육내(胚肉內) 당(糖)과 조단백질량(粗蛋白質量)은 함수율(含水率)과 함께 증가(增加)하나 조지방(粗脂肪)과 전분량(澱粉量)은 감소(減少)하며 이들 성분(成分)의 소장(消長) 또한 발아촉진(發芽促進)과 밀접(密接)한 관계(關係)가 있는 것 같다.