Bacillus stearothermophilus No. 236 \beta-xylosidase 유전자 변이 Promoter의 Strength분석

최용진,김미동 한국미생물 · 생명공학회 2003 한국미생물·생명공학회지 Vol.31 No.2

Xylan 분해 균주인 Bacillus stearothermophilus No. 236 분리균의 $\beta$-xylosidase 생산 유전자(xylA)의 염기 서열 및 transcription start site를 결정한 이전 연구 결과에 의하면 xylA 유전자는 매우 특이하게 UUG codon에서 translation이 시작되며 initiation codon 15dp 윗쪽에는 promoter로 추정되는 염기 서열을 가지고 있는 것으로 분석되었다. 이와 같은 xylA 유전자 promoter region의 구조는 E. coli에 클로닝된 xalA 유전자를 이용한 실험 결과로도 확인되었다. xalA promoter의 -10 element는 CATAAT로서 6개의 염기 중 5개가 그리고 -35 element의 경우는 TTGTTA로서 6개의 염기 중 4개가 consensus sequence와 일치되었으나 두 hexamer 사이의 거리가 최적 거리에서 크게 벗어난 12 bp인 것으로 분석되었다. 본 연구에서는 $\beta$-xylosidase의 대량 생산을 위한 연구의 일환으로 xalA promoter sequence의 체계적 구조 변화에 의한 promoter strength에 미치는 효과를 E. coli와 B. subtilis두 숙주 세포에서 조사 분석해 본 결과, 첫째로 두 promoter elements사이의 거리를 최적거리인 17 bp로 바꾸었을 때 xalA의 발현율은 E. coli에서는 1.6배, B. subtilis에서는 2.5배 정도 증가함을 보여주었다. 그리고 -35 element는 consensus sequence와 같이 5'쪽에서 네번째 위치에 있는 T$\longrightarrow$A로 변이 시켰을 때 E. coli경우 2.3배, 특히 B. subtilis에서는 35배나 되는 가장 높은 promoter 활성의 증가를 보였다. 그러나 -10 sequence의 경우 consensus sequence와 같이 5' 쪽에서 첫번째 위치에 있는 C$\longrightarrow$T로 transition시켰을 때 예상외로 오히려 발현율이 5~15배까지 낮아지는 특이한 결과를 얻었다. 따라서 본 연구 결과 xalA promoter의 경우 -10 sequence인 CATAAT의 C와 -35 element의 두 염기가 promoter활성에 있어 가장 중요한 염기임을 알 수 있었다. The xylA gene of Bacillus stearothermophilus No. 236 encoding $\beta$-xylosidase was cloned and sequenced previously. The transcriptional start site of the xylA gene cloned in E. coli was identified to be the guanine (G) by primer extension analysis. This supports that the expression of xylA gene is also directed in the E. coli cells by the previously determined transcription initiation signals, -10 sequence (CATAAT) and -35 sequence (TTGTTA) separated by 12 bp. To increase the expression of $\beta$-xylosidase, firstly the spacer region of xylA promoter was extended from 12 to 17 bp, and then the -10 and -35 elements were converted into their respective consensus sequences. The mutant promoters thus obtained were tested for their activities in both the E. coli and B. subtilis host cells. The change of the length of the spacer region from 12 to 17 bp resulted in a 1.6- and 2.5-fold increase in promoter strength in comparison with the wild type promoter in E. coli and B. subtilis cells, respectively. Also, strength of the promoter with the fourth T to A transversion on its -35 element increased in the transcription level by about 35 times compared with that of wild-type promoter. However, surprisingly the 5' end C-to-T transition of the -10 hexamer showed a 5- to 15-fold reduction in $\beta$-xylosidase activity in both E. coli and B. subtilis. Together, the present data demonstrated that the 5' end nucleotide C of the -10 sequence CATAAT and the fourth nucleotide A of the -35 hexamer are two most critical nucleotides for the promoter activity in the context of the xylA promoter.

해양의 Pseudomonas sp. 로부터 분리한 alginate lyase 유전자의 promoter에 의한 대장균 내에서의 \beta-agarase 유전자의 발현과 catabolite repression의 변화

공인수,박제현,한정현,최윤혁,이종희,진철호,이정기 한국미생물·생명공학회 2001 한국미생물·생명공학회지 Vol.29 No.2

Strong promoter로 밝혀진 alginate lyase 유전자의 promoter 부위에 대한 특성을 검토하기 위해 alginate lyase 유전자의 46개 N-terminal amino acid가 포함된 promoter 부분과, 같은 균으로부터 분리한 $\beta$-agarase의 유전자를 연결시켜 agarase의 activity를 평판배지상에서 보다 쉽게 확인하는 방법으로 promoter의 활성을 측정한 결과 alginate lyase 유전자 promoter에 의해서 $\beta$-agarase 유전자의 대량발현이 유도되고 있었으며 glucose의 존재하에서 $\beta$-agarase 유전자 발현이 일어나지 않는 catabolite repression 양상을 나타내고 있다. PCR로써 alginate lyase의 46개 N-terminal amino acid 부분이 순차적으로 제거된 plasmid를 제조하여 대량발현을 조사한 결과 46개의 아미노산이 제거된 후에도 $\beta$-agarase의 활성에는 변화가 없어 46개의 N-말단이 정상적인 상태에서 발현에는 영향을 미치고 있지 않음을 확인할 수 있었다. 또한 alginate lyase 유전자의 promoter region에 존재하는 가능한 2개의 promoter consensus sequence PI, PII를 subcloning한 결과 promoter PII만이 존재할 때도 대량발현이 유도되고 있음을 확인할 수 있었으며 동시에 glucose가 존재할 때 catabolite repression이 역시 나타나고 있어 이 부분이 발현 및 glucose에 의한 regulation에 매우 중요하게 작용하는 부분이라는 것을 확인할 수 있었다. Expression of f3 ~agarase Gene and Catabolite Repression in Escherichia coli by the Promoter of Alginate Lyase Gene Isolated from Marine Pseudomonas sp. Jin, Cheal~Ho, J~Hyeon Park, Jeong-Hyun Han, YoonM Hyeok Chae, Jong~Hee Lee, Jung-Kee Lee!, and In-800 Kong*. Faculty of Food Science and Biotechnology, Pukyong National UniversitYt Pusan 608-737, Korea, llnBioNet Co. 1690-3 Taejon 306-230, Korea - Promoter is a key factor for expression of the recombinant protein. There are many promoters for overexpression of protein in various organisms. The aly promoter of Pseudomonas sp. W7 isolated from marine environment was known to be a constitutive expression promoter of the alginate lyase gene, and it's promoter activity is repressed by glucose in Escherichia coli. To investigate the catabolite repression of the aly promoter ~md association between the promoter mutants, f3 agarase gene, which was also cloned from Pseudomonas sp. W7 was connected to the aly promoter with the sequence the coding 46 N-terminal amino acids ofthe alginate lyase gene. The constructed plasmid was introduced into E. coli and the agarase activity was measured. Fourty six amino acids of the alginate lyase gene was serially deleted using peR to the direction of 5' upstream region and subcloned. The agarase was overexpressed by the aly promoter and the production of agarase was repressed by the addition of glucose into culture media. Fourty six amino acids of alginate lyase did not affect the production of agarase at all. The deletion of a putative stem-loop structure in the aly promoter induced the decrease of f3 -agarase productivity.

Functional analysis of a cryptic promoter from Arabidopsis thaliana reveals bidirectionality

Sujatha. T. Parvathy,R. Srinivasan 한국식물생명공학회 2016 Plant biotechnology reports Vol.10 No.4

Cryptic promoter elements play a significant role in evolution of plant gene expression patterns and are prospective tools for creating gene expression systems in plants. In a previous report, a 452 bp promoter fragment designated as cryptic root-specific promoter (AY601849) was identified immediately upstream to T-DNA insertion, in the intergenic region between divergent genes SAHH1 and SHMT4, in T-DNA tagged mutant M57 of Arabidopsis thaliana. In silico analysis of 452 bp promoter revealed typical eukaryotic promoter architecture, presence of rootspecific motifs and other cis-regulatory motifs responsible for the spatial and temporal expression. GUS expression driven by 452 bp in M57 was developmentally as well as light-regulated. The AT-rich 452 bp promoter does not show homology to any known sequences. The 452 bp promoter was further proved cryptic and detailed molecular characterization of the promoter carried out through serial 50 and 30 deletion analysis, by cloning the promoter fragments upstream to promoter-less GUS vector. A 279 bp fragment obtained by deleting 173 bp from 50 end of 452 bp was capable of driving root-specific expression, similar to that of full-length promoter. Further, root tipspecific, root-specific and core-regulatory motifs for rootspecific expression were identified at positions 173–227, 251–323 and 408–452 bp, respectively, from the 50 end of 452 bp. The 452 bp promoter was equally functional in inverse orientation, hence bidirectional and symmetric. In heterologous systems, such as Brassica juncea and Oryza sativa, the promoter activity was not significant since GUS was not visually detected in transient assays.

해양의 Pseudomonas sp. 로부터 분리한 alginate lyase 유전자의 promoter에 의한 대장균 내에서의 β-agarase 유전자의 발현과 catabolite repression의 변화

진철호,박제현,한정현,최윤혁,이종희,이정기,공인수 한국산업미생물학회 2001 한국미생물·생명공학회지 Vol.29 No.2

Strong promoter로 밝혀진 alginate lyase유전자의 promoter부위에 대한 특성을 검토하기 위해 alginate lyase유전자의 46개 N-terminal amino acid가 포함된 promoter부분과, 같은 균으로부터 분리한 β-agarase의 유전자를 연결시켜 agarase의 activity를 평판배지상에서 보다 쉽게 확인하는 방법으로 promoter의 활성을 측정한 결과 alginate lyase유전자 promoter에 의해서 β-agarase유전자의 대량발현이 유도되고 있었으며 glucose의 존재하에서 β-agarase유전자 발현이 일어나지 않는 catabolite repression양상을 나타내고 있다. PCR로써 alginate lyase 유전자의 promoter하류에 존재하는 alginate lyase의 46개 N-terminal amino acid 부분이 순차적으로 제거된 plasmid를 제조하여 대량발현을 조사한 결과 46개의 아미노산이 제거된 후에도 β-agarase의 활성에는 변화가 없어 46개의 N-말단이 정상적인 상태에서 발현에는 영향을 미치고 있지 않음을 확인할 수 있었다. 또한 alginate lyase유전자의 promoter region에 존재하는 가능한 2개의 promoter consensus sequence PⅠ, PⅡ를 subcloning한 결과 promoter PⅡ만이 존재할 때도 대량발현이 유도되고 있음을 확인할 수 있었으며 동시에 glucose가 존재할때 catabolite repression이 역시 나타나고 있어 이 부분이 발현 및 glucose에 의한 regulation에 매우 중요하게 작용하는 부분이라는 것을 확인할 수 있었다. Promoter is a key factor for expression of the recombinant protein. There many promoters for overexpression of protein in various organisms. The aly promoter of Pseudomonas sp. W7 isolated from marine environment was known to be a constitutive expression promoter of the alginate lyase gene, and it's promoter activity is repressed by glucose in Escherichia coli. To investigate the catabolite repression of the aly promoter and association between the promoter mutants, β-agarase gene, which was also cloned from Pseudomonas sp. W7 was connected to the aly promoter with the sequence the coding 46 N-terminal amino acids of the alginate lyase gene. The constructed plasmid was introduced into E. coli and the agarase activity was measured. Fourty six amino acids of the alginate lyase gene was serially deleted using PCR to the direction of 5' upstream region and subcloned. The agarase was overexpressed by the aly promoter and the production of agarase was repressed by the addition of glucose into culture media. Fourty six amino acids of alginate lyase did not affect the production of agarase at all. The deletion of a putative stem-loop structure in the aly promoter induced the decrease of β-agarase productivity.

The promoter of tomato HISTIDINE DECARBOXYLASE A is fruit-specific, and its expression is stably maintained in fruits during ripening

김아영,김현민,마상훈,박서영,MAI THANH DAT,장규필,정영희 한국식물생명공학회 2019 Plant biotechnology reports Vol.13 No.1

Identifying novel promoters with specific temporal and spatial expression patterns is crucial for crop biotechnology. In this study, we isolated a fruit-specific promoter in tomato, HISTIDINE DECARBOXYLASE A (SlHDC-A) promoter. Through RNA-seq and RT-PCR analysis, we found that SlHDC-A was predominantly expressed in fruits and that its expression was stable in fruits during ripening. These results suggest that the promoter of SlHDC-A might have the ability to determine fruit-specific gene expression. To test this possibility, we generated transgenic tomato transformed with SlHDC-A::GUS and 35S::GUS. Unlike 35S::GUS transgenic tomato with constitutive expression in various tissues, SlHDC-A::GUS transgenic plants showed fruit-specific expression of GUS. The intensity of GUS activity in fruits of SlHDC-A::GUS transgenic plants was approximately tenfold higher than that in fruits of 35S::GUS transgenic plants. The core region responsible for its fruit-specific expression was identified by promoter deletion analyses. Removal of the − 880 to − 577 region abolished the fruit-specific expression of SlHDC-A promoter. This suggests that the − 880 to − 577 region is the core region responsible for the fruit-specific expression of SlHDC-A. This finding was further supported by analysis of chimeric fusion promoter. Unlike 35S minimal promoter which had no activity to express GUS, the chimeric fusion promoter of the core region and 35S minimal promoter showed fruit-specific expression similar to intact SlHDC-A promoter. Collectively, these findings indicate that the promoter of SlHDC-A is fruit-specific and the − 880 to − 577 region is the core region of SlHDC-A promoter.

Identification of Endothelial Specific Region in the Intracellular Adhesion Molecule-2 (ICAM2) Promoter of Miniature Pig

Jang, Hoon,Jang, Won-Gu,Kim, Dong Un,Kim, Eun-Jung,Hwang, Sung Soo,Oh, Keon Bong,Lee, Jeong-Woong The Korean Society of Animal Reproduction 2012 Reproductive & developmental biology Vol.36 No.3

The shortage of human organs for transplantation has induced the research on the possibility of using animal as porcine. However, pig to human transplantation as known as xeno-transplantation has major problem as immunorejection. Recently, the solutions of pig to human xenotransplantation are commonly mentioned as having a genetically modification which include alpha 1, 3 galatosyl transferase knockout (GTKO) and immune-suppressing gene transgenic model. Unfortunately, the expression level of transgenic gene is very low activity. Therefore, development of gene overexpression system is the most urgent issue. Also, the tissue specific overexpression system is very important. Because most blood vessels are endothelial cells, establishment of the endothelial-specific promoter is attractive candidates for the introduction of suppressing immunorejection. In this study, we focus the ICAM2 promoter which has endothelial-specific regulatory region. To detect the regulatory region of ICAM2 promoter, we cloned 3.7 kb size mini-pig ICAM2 promoter. We conduct serial deletion of 5' flanking region of mini-pig ICAM2 promoter then selected promoter size as 1 kb, 1.5 kb, 2 kb, 2.5 kb, and 3 kb. To analyze promoter activity, luciferase assay system was conducted among these vectors and compare endothelial activity with epithelial cells. The reporter gene assay revealed that ICAM2 promoter has critical activity in endothelial cells (CPAE) and 1 kb size of ICAM2 promoter activity was significantly increased. Taken together, our studies suggest that mini-pig ICMA2 promoter is endothelial cell specific overexpression promoter and among above all size of promoters, 1 kb size promoter is optimal candidate to overcome the vascular immunorejection in pig to human xenotransplantation.

효모의 구성적 Promoter들에 의한 Inulinase 유전자의 발현

김연희,남수완 한국생명과학회 1999 생명과학회지 Vol.9 No.2

S. cerevisiae의 대표적인 구성적 promoter로 GAPDH, ADH1 및 ENO1를 사용하고 이들 promoter 하류에 INUl의 ORF를 in frame으로 연결한 각각의 plasmid pYIGP, pADHl-lNU 및 pENO-INU를 구축하였다. 이들 plasmid 를 함유한 재조합 S. cerevisiae SEY2102 균주들을 sucrose 함유 평판배지에서 선별한 후, 초기 포도당 농도가 2$\%$ 또는 4$\%$인 배지에서 배양했을 때, 모든 균주들은 12시간 이후부터 정지기에 들어갔으며, 정지기에서도 느리지만 균체증식과 inulinase 발현은 계속되었다. 4% 포도당 배지에서 inulinase 총발현량은 ADH1 promoter 계를 제외하고 GAPDH와 ENO1 promoter의 경우 2$\%$ 포도당 배지 때 보다 약 2배 증가한 2.0 unit/mL과 1.4 unit/mL를 각각 보였다. 단위균체농도당 inulinase 활성 즉, 비활성은 GAPDH와 ENOl promoter계의 경우 포도당 농도가 4$\%$일 때 2$\%$때보다 약 63$\%$ 정도의 비활성 증가를 나타내었다. 하지만 ADH1 promoter의 경우는 오히려 비활성이 약 40$\%$ 감소하였다. 그러나, plasmid 안정성 측면에서는 ADH1과 ENO1 promoter 발현계가 GAPDH promoter 경우의 34$\%$보다 훨씬 뛰어난 80$\%$이상을 보였다. 결론적으로 높은 포도당 농도에서 구성적 promoter의 활성 (발현능)은 GAPDH, ENO1, ADH1 promoter 순으로 나타났지만, 초기 포도당 농도가 높을 때나 에탄을 생산이 심각한 유가식 배양에서는 ENO1 promoter가 inulinase의 구성적 발현ㆍ생산에 더 적합할 것으로 사료된다. To express constitutively the inulinase gene (INUl) of Kluyveromyces marxianus in Saccharomyces cerevisiae, three yeast promoters such as GAPDH, ADH1 and ENO1 were connected upstream of INUl. The resulting plasmids, pYIGP, pADHl-INU, and pENO-INU were introduced to S. cerevisiae SEY2102 host strain, respectively, and then each transformants were selected by staining of colonies on sucrose-agar plate. When the yeast transformants were cultivated on 2$\%$ dextrose media, the total expression levels of inulinase reached to 1.11 unit/mL, 0.88 unit/mL, and 0.69 unit/mL for respective GAPDH, ADH1, and ENO1 promoter systems. On 4% dextrose media, however, the inulinase activities were observed at 2.00 unit/mL for pYIGP, 0.71 unit/mL for pADH1-INU, and 1.40 unit/mL for pENO-INU. This result indicates that the constitutive expression of INUl was significantly affected by the initial concentration of dextrose and the promoter strength was in the order GAPDH, ENO1, and ADH1 promoter at high dextrose concentration. Taking into account the plasmid stability, however, it is suggested that the ENO1 promoter system is more suitable for the INU1 expression on high dextrose medium or in the fed-batch cultivation accumulating ethanol at high level.

A Novel Bi-directional Promoter Cloned from Melon and Its Activity in Cucumber and Tobacco

( Cui Yan Wang ),( Dong Feng Ding ),( Rui Xiang Yan ),( Xiao Ju Yu ),( Wei Dong Li ),( Ming Gang Li ) 한국식물학회 2008 Journal of Plant Biology Vol.51 No.2

A bi-directional promoter, DP, was cloned by PCR amplification using the genomic DNA of melon as template. Analysis of its cis-acting elements in both directions revealed a series of inducible regulatory elements and some enhancer elements. To evaluate its transcriptional activity, DP in both directions was then cloned into vector pBI121 to replace the CaMV 35S promoter. DP in both directions also was inserted downstream of CaMV 35S to investigate whether the double promoter might affect expression of the uidA reporter gene at higher levels. Transient expression in cucumber leaves, stems, and fruits as well as in tobacco leaves and stems showed that DP in both directions drove transcription to much higher levels than did the single promoter CaMV 35S. However, activity of the double promoter was lower than the corresponding activity of the single promoter DP in both directions. These results demonstrate that DP is a natural bi-directional promoter, with much more activity than is found with the CaMV 35S promoter. Furthermore, in cucumber and tobacco, it is not suitable to insert DP in either direction downstream of the CaMV 35S promoter to form a double promoter.

난소암에서 IGF-2 와 H19 gene 의 promoter 이용과 발현정도의 변화

김윤아 ( Kim Yun A ),이찬 ( Lee Chan ),김승조 ( Kim Seung Jo ),이선영 ( Lee Seon Yeong ),김인호 ( Kim In Ho ),나영정 ( Na Yeong Jeong ),정상근 ( Jeong Sang Geun ) 대한산부인과학회 2004 Obstetrics & Gynecology Science Vol.47 No.3

목적 : 난소암의 발암기전과 Insulin-like growth factor-2 (IGF-2), 그리고 H19 gene의 imprinting 상태, promoter 이용과 발현정도와의 연관성을 알아보기 위해, 본 연구는 8예의 정상난소조직, 11예의 양성난소종양, 9예의 경계성 종양과 20예의 악성종양 조직을 대상으로 실험하였다. 연구 방법 : 등록된 조직의 IGF-2와 H19을 PCR-RFLP 방법을 사용하여 이형접합성 분석을 하였으며, IGF-2의 promoter 특이발현을 살펴보고자 RT-PCR을 사용하였다. 결과 : 각 조직에서 IGF-2의 LOI (loss of imprinting)은 각각 정상 난소조직 (50%)> 양성종양 (60%) > 난소암조직 (71%) > 경계성 종양 (77%) 순으로 나타났다. H19 gene의 LOI는 정상과 양성종양의 난소조직에서는 관찰되지 않았고, 경계성 종양에서 100% (3/3), 악성종양에서 40% (2/5)로 나타났다. 각각의 난소조직에서 promoter P1, P2, P3와 P4의 이용도는 서로 다른 양상을 보였다. 암조직에서는 상대적으로 promoter P1, P2 이용이 높았으나 promoter P3 이용은 낮았다. 난소암조직에서 IGF-2의 발현은 높게 나타났으나 H19의 발현은 정상조직에 비해 낮음을 보였다. 결론 : 이상의 결과들을 보면 IGF-2의 LOI, promoter 사용의 deregulation IGF-2와 H19 발현정도의 변화가 난소암의 발생과 진행에 연관이 있으리라 생각된다. Objective : To establish the possible role of imprinting in ovarian cancer, we determined the imprinting status of both IGF-2 and H-19 genes in ovarian cancer, borderline tumors of ovary, benign ovarian tumor and normal ovarian tissues. Methods : An allelictyping assay was performed using a PCR-RFLP-based method for identification of heterozygous informative cases. The usage of Insulin-like growth factor-Ⅱ (IGF-2) promoters was examined by RT-PCR using promoter-specific primers. The mRNA expression of IGF-2 and H19 was quantified using a densitometer. Results : Loss of imprinting (LOI) of IGF-2 was observed in the order of borderline tumor (77%)>cancer (71%)>benign tumor (60%)>normal ovarian tissues (50%) respectively. And the LOI of H19 gene was not detected in the normal and benign tissues but observed in the borderline tumor and cancer tissues, respectively. The usage of promoter P1, P2, P3 and P4 were observed different pattern in normal, benign tumor, borderline tumor and cancer tissues. The activity of mRNA expression of promoter P4 was higher than other promoters. The cancer tissues predominantly used promoter P1, P2 with relative silencing of the promoter P3. The ovarian cancer tissues showed the higher expression levels of the IGF-2 but a down-regulation of the H19 relative to normal tissues. Conclusion : These results suggest that LOI, deregulation of the IGF-2 promoters, and the altered expression levels of the IGF-2 and h19 gene might be associated with progression of ovarian cancer.

B형 만성 간질환에서 Core Promoter 변이의 양상

정성택,신용준,김영수,김진홍,조성원 大韓消化器病學會 1998 大韓消化器病學會誌 Vol.31 No.6