• 한국미생물·생명공학회지
• /
• 제18권4호
• /
• pp.406-410
• /
• 1990

토양에서 분리한 알칼리내성 Bacillus sp.의 자체 chromosomal DNA에서 유래된 strong promoter를 지닌 plasmid p-12B1을 공여균주에 삽입시키고 이 promoter의 활성을 최적화할 수 있는 배양조건을 검토하였다. 글루코오스가 고갈된 시점부터 세포의 활성은 유지되나 포자는 형성되지 않을 정도로 낮은 글루코오스 소비속도를 유지해 제한된 글루코오스를 연속적으로 공급해줌으로써 promoter의 활성을 최대로 높일 수 있었다. 또한 배지조성의 변화로 균체를 생육시킨 후 유전자 발현을 유도하는 것이 가능하였으며, 이 점에 대해서는 보다 구체적인 연구가 진행되어야 할 것이다.

• Journal of Plant Biotechnology
• /
• 제40권1호
• /
• pp.27-36
• /
• 2013

We isolated and functionally characterized a Dendrobium Actin1 (DmACT1) promoter that drives strong gene expression in the orchid genus Dendrobium. A genomic fragment containing the region 3227 bp upstream of the coding region of DmACT1 was obtained by inverse PCR. Detailed comparison of the full-length cDNA and genomic sequences revealed that DmACT1 has a 1374 bp first intron in the 5' UTR. However, the 5' flanking sequences upstream of the coding region showed no obvious sequence similarities compared to those of known promoters, including plant actin promoters. Serial deletion constructs of the 5' flanking region from the translation initiation codon were fused to the coding sequence of a GUS/luciferase fusion reporter to identify the regulatory elements necessary for promoter activity. Transient assays in the flowers of Dendrobium revealed that the 5' UTR-intron greatly enhanced promoter activity. Moreover, the DmACT1 promoter with its 5' UTR-intron yielded approximately 10-fold higher reporter activity than the rice Act1 promoter-intron. Our data suggest that the DmACT1 promoter with its 5' UTR-intron is a useful tool for strong expression of transgenes in Dendrobium orchids.

• Plant Biotechnology Reports
• /
• 제3권4호
• /
• pp.325-331
• /
• 2009

To efficiently express a gene of interest in transgenic plants, the choice of promoter is a crucial factor as it directly affects the expression of the transgene that will yield the desired phenotype. The Arabidopsis ${\beta}-carotene$ hydroxylase 1 gene (AtBch1) shows constitutive and ubiquitous expression and was thus selected as one of best candidates for constitutive promoter analysis by both in silico northern blotting and semi-quantitative RT-PCR analysis. To investigate AtBch1 promoter activity, the 1,981-bp 5'-upstream region of this gene was fused with ${\beta}-glucuronidase$ (GUS) and transformed into Arabidopsis. Through the molecular characterization of transgenic leaf tissues, the AtBch1 promoter generated strong activity that drives 1.8- and 2-fold higher GUS expression than the cauliflower mosaic virus 35S (35S) promoter at the transcriptional and translational levels, respectively. Furthermore, the GUS enzyme activity driven by the AtBch1 promoter was 2.8-fold higher than that produced by the 35S promoter. By histochemical GUS staining, the ubiquitous expression of the AtBch1 promoter was observed in all tissues of Arabidopsis. Semi-quantitative RT-PCR analysis with different tissues further showed that this promoter serves as a strong constitutive driver of transgene expression in dicot plants.

• Journal of Microbiology and Biotechnology
• /
• 제11권1호
• /
• pp.85-91
• /
• 2001

To construct an efficient Bacillus subtilis expression vector, strong promoters were isolated from the chromosomal DNA libraries of Clostridium acetobutylicum ATCC 4259, Thermoactinomyces sp. E79, and Bacillus thermoglucosidasius KCTC 3400. The $P_{C27}$ promoter cloned from the clostridial chromosmal DNA showed a 5-fold higher promoter strength than the $P_{SP02}$ promoter in the expression of the cat gene, and its sequence was estimated as an upstream region of the predicted hypothetical gene (tet-R family bacterial transcription regulator gene) in C. acetobutylicum. As a promoter element, $P_{C27}$ exhibited putative nucleotide sequences that can bind with bacterial RNAP and the 3'end of the 16S rRNA just upstream of the start codon. In addition, the promoter activity of $P_{C27}$ was distinctively repressed in the presence of glucose. Using $P_{C27}$ as the promoter element, a glucose controllable B. subtilis expression vector was constructed and the lipase gene from Staphylococcus haemolyticus KCTC 8957P was expressed in B. subtilis. When compared with the lipase expression by the T7 promoter induced by IPTG in E. coli, the $P_{C27}$ promoter showed about a 1.5-fold higher expression level in B. subtilis than that without induction.

• 한국미생물·생명공학회지
• /
• 제23권3호
• /
• pp.288-296
• /
• 1995

To investigate high-level expression of Serratia marcescens metalloprotease (SMP) in Escherichia coli and S. marcescens, we constructed various recombinant plasmids: pSP2, containing SMP gene and lac promoter; pKSP2, containing SMP gene and tac promoter; pTSP2, containing SMP gene, trc99a promoter, and lacI$^{q}$. The recombinant E. coli (pKSP2) strain expressed SMP to a high-level, about 36% of total cellular proteins but accumulated inactive SMP precursors intracellularly, which indicated that E. coli does not have activation and secretion system for SMP. To overproduce active SMP, we transformed S. marcescens with the recombinant plasmids by a modified CaCl$_{2}$ method. The recombinant S. marcescens ATCC27117 (pSP2) containing lac promoter for SMP transcription produced 530 U/ml of active SMP on LB broth, which is about 5.1 times of the SMP yield, 105 U/ml of a control strain, S. marcescens ATCC27117 (pUC19). However, S. marcescens ATCC27117 (pKSP2) containing tac promoter for SMP transcription did not grow healthy and hardly produced SMP. To overcome a harmful effect of the strong tac promoter, we constructed a regulatory plasmid pTSP2 containing a strong trc99a promoter and its repressor gene lacI$^{q}$. When S. marcescens ATCC27117 (pTSP2) was induced with 1.0 mM IPTG after 9 hr cultivation, 2,200 U/ml of SMP was obtained in LB broth, which is about 21 times of that of a control strain.

• 한국동물학회지
• /
• 제38권3호
• /
• pp.426-432
• /
• 1995

유선 조직에서 베타-락토글로불린 유전자의 발현은 프롤락틴, 글루코코르티코이드 및 인슐린등의 유촉진 호르몬들에 의해서 강력하게 유도된다. 이와 같은 호르몬 유도의 조절 기작을 규명하기 위하여, 배양 유선 세포인 HC11 세포에서 염소 베타-락토글로불린 유전자 프로모터의 유촉진 호르몬에 대한 반응을 분석하였다. 베타-락토글로불린 프로모터의 5'- 조절 부위를 연쇄적으로 제거한 발현 실험에서 호르몬 유도를 크게 변화 시키는 두 지역이 관찰되었다. 조절 부위의 -1692의 상류지역은 하류 프로모터를 강력하게 활성화 시키는 부위로, 주로 글루코코르티코이드 유도체인 덱사메타손의 작용을 매개하였다. 그러나 두번째 지역의 유도 작용은 인슐린 처리를 병행하지 않을 경우 상류 조절부위에 의해 억제되었다. 이러한 결과는, 유선세포에서 유촉진 호르몬들에 의한 베타-락토글로불린 프로모터 활성 유도가 인슐린에 의한 탈 억제화와 글루코코르티코이드 및 프롤락틴에 의한 활성화의 복합 조절에 의해서 이루어질 것이라는 점을 시사한다. 두번째 지역에 의한 덱사메타손 유도는 -700 부근의 글루코코르티코이드 수용체 결합 부위에 의해서 매개되는 것으로 추정된다.

• Journal of Microbiology and Biotechnology
• /
• 제1권1호
• /
• pp.37-44
• /
• 1991

A shuttle promoter-probe vector, pEB203, was derived from pBR322, pPL703 and pUB110. Using the vector, a useful DNA fragment, 319 bp EcoRI fragment, having strong promoter activity has been cloned from Bacillus subtills chromosomal DNA. Selection was based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The nucleotide sequence of the 319 bp fragment has been determined and the putative -35 and -10 region, ribosome binding site, and ATG initiation codon were observed. This promoter was named EB promoter and the resultant plasmid which can be used as an expression vector was named pEBP313. The crystal protein gene from B. thuringiensis subsp. kurstaki HD-73 was cloned downstream from the EB promoter without its own promoter. When the resultant plasmid, pBT313, was introduced into Escherichia coli and B. subtilis, efficient synthesis of crystal protein was observed in both cells, and the cp gene expression in B. subtilis begins early in the vegetative phase. The cell extracts from both clones were toxic to Hyphantria cunea larvae.

• Journal of Plant Biology
• /
• 제37권3호
• /
• pp.371-380
• /
• 1994

To determine the patterns and the levels of expression of the cauliflower mosaic virus (CaMV 35S) promoter and the rice actin 1 (Act1) promoter in rice, transgenic rice plants containing CaMV 35S-$\beta$-glucuronidase (GUS) and Act1-GUS constructs were generated and examined by fluorometric and histochemical analyses. The fluorometric analysis of stably transformed calluses showed that the activity of the rice Act1 promoter was stronger than that of the CaMV 35S promoter in rice cells. In a histochemcial study of the transgenic rices, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in parenchymal cells of vascular tissues of leaves and roots and mesophyll cells of leaves. These results are similar to those of potato, a dicot plant. In contrast, rice plant transformed with Act1-GUS fusion construct revealed strong GUS activity in parenchymal cells of vascular tissue, mesophyll cells, epidermal cells, bulliform cells, guard subsidiary cells of leaves and most cells of the root, suggesting that the rice Act1 promoter is more constitutive than the CaMV 35S promoter. It was also confirmed that in both types of transgenic rice little or no staining was localized in metaxylen tracheary elements of vascular tissue from leaves or roots. These results indicate that the rice Act1 promoter can be utilized more successfully for expression of a variety of foreign gene in rice than the CaMV 35S promoter.

• 한국미생물·생명공학회지
• /
• 제25권6호
• /
• pp.571-579
• /
• 1997

For the overproduction of pectate lyase (PL), the recombinant plasmid pl2BS fl which has strong promoter from alkali-tolerent Bacillus sp. YA-14 was used. In order to overexpress the pectate lyase by the action of overlapping strong promoter in pl2BS$\Delta$fl, 1.6 kb of PL gene was inserted into pl2BS$\Delta$fl to form pl2BS$\delta$f1-PL and the enzyme was expressed. But decreased expression efficiency of the PL gene was observed and it was due to the presence of the transcription terminator region on the upstream of the PL gene. The transcription terminator of the PL gene in pl2BS$\delta$f1-PL was removed and the resulting plasmid p12BS$\Delta$fl$\Delta$PL was formed. Bacillus subtilis 207-25 harboring the recombinant plasmid, p12BS$\Delta$fl$\Delta$PL, revealed increased expression efficiency with chloramphenicol induction when cat-86 was used as a reporter gene.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제20권2호
• /
• pp.107-114
• /
• 2010

For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (85%) with hypothetical protein 32 gene of Lonomia obliqua. This clone, named bHp32 (B. mori hypothetical protein 32) was ubiquitously expressed in all tissues and developmental stage of fifth instar B. mori larvae. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1,200/+220) in the 5'-flanking region of bHp32 gene, which has 42-fold more intensive promoter activity than BmA3 promoter. Moreover, the bHp32 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHp32 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.