Effects of CMV Enhancer on Activity and Specificity of Bovine MyoG Gene Promoter

Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL3-CMV, pGL3MyoGpro, pGL3-CMV-MyoGpro, and pGL3-Basic which contained CMV promoter, MyoG promoter, CMV-MyoG synthesis promoter, and a promoterless negative control, respectively. Then the four vectors and internal control Renilla luciferase report gene vector phRL-TK were transfected into bovine skeletal muscle satellite cells, mouse C2C12 cells and bovine fetal fibroblast cells to detect the promoter activity with dual luciferase report system. The results showed that CMV enhancer could significantly improve the transcription activity of bovine MyoG gene promoter in muscle satellite cells and mouse C2C12 cells, and it had certain specificity. This study provided experimental materials for increasing the high expression of exogenous gene in bovine muscle cells, and also laid the molecular theoretical basis for obtaining the high specific promoter of bovine muscle and the transgenic beef cattle.

A Preliminary Study on a Specifically Expressed Arabidopsis Promoter in Vascular Bundle

From a population of about 3500 single plants in Arabidopsis promoter trapping bank, one plant whose GUS-gene had been specifically expressed in vascular bundle, was screened by the method of gus tissue staining. The T-DNA flanking sequence was amplified using TAIL-PCR. This band will be purified and connected to TA cloning vector. After sequencing and searching in the genebank, its function will be demonatrated through transformation.

Construction of Smac gene-containing and human prostate specific antigen promoter-regulated vector and its expression

To construct an eukaryotic expression vector containing Smac gene and study the expression efficiency and specificity of prostate specific antigen（PSA） enhancer/promoter in a possible targeted gene therapy scheme for prostate cancer. Methods： PSA enhancer （PSAE） and promoter （PSAP） sequences were amplified using PCR method. CMV and T7 promoters were deleted from pcDNA3.1-Smac and replaced by the two specific fragments to generate pPSAE-PSAP-Smac. After transfection into different cell lines, the status of cells was observed. And then, we determined the relative concentration of Smac mRNA in RT-PCR. Results： The recombinant plasmid of pPSAE-PSAP-Smac was successfully constructed. And only the prostate cancer cell line PC-3 was suppressed after transfection with pPSAE-PSAP-Smac. However, other nonprostate lines were not. Moreover, the concentration of Smac mRNA regulated by PSA promoter and enhancer was higher in comparison to the CMV promoter-driven control vectors. Conclusion： An expression vector containing the Smac gene （based on elements of the PSA gene regulatory sequences） has been developed and shown to function in prostate cancer cell lines which provides a solid platform for launching clinical studies.

Identification and validation of root-specific promoters in rice

Novel promoters that confer root-specific expression would be useful for engineering resistance against problems of nutrient and water absorption by roots. In this study, the reverse transcriptase polymerase chain reaction was used to identify seven genes with root-specific expression in rice. The isolation and characterization of upstream promoter regions of five selected genes rice root-specific promoter （rRSP） 1 to 5 （rRSP1-rRSP5） and A2P （the promoter of OsAct2） revealed that rRSP1, rRSP3, and rRSP5 are particularly important with respect to root-specific activities. Furthermore, rRSP1, rRSP3, and rRSP5 were observed to make different contributions to root activities in various species. These three promoters could be used for root-specific enhancement of target gene（s）.

Identification of an intestine-specific promoter and inducible expression of bacterial α-galactosidase in mammalian cells by a lac operon system

Background： o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors （such as o-galactoside） in feed. Intestine-specific and substrate inducible expression of a-galactosidase would be highly beneficial for transgenic animal production. Methods： To achieve the intestine-specific and substrate inducible expression of o-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified lac operon system was constructed to investigate the induction of o-galactosidase expression and enzyme activity by isopropyl p-D-]-thiogalactopyranoside （IPTG） and an a-galactosidase substrate, a-lactose. We declared that the research carried out on human （Zhai Yafeng） was in compliance with the Helsinki Declaration and experimental research on animals also followed internationally recognized guidelines. Results： The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the/ac operon system, the repressor significantly decreased （P 〈 0.05） luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein （GFP） by approximately 2-fold. In addition, the expression level of o-galactosidase mRNA was decreased by 6-fold and a-galactosidase activity was reduced by 8-fold. in line with our expectations, IPTG and a-lactose supplementation reversed （P 〈 O.O5） the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in o-galactosidase mfiNA abundance （by about 5-fold） and o-galactosidase activity （by about 7-fold）. Conclusions： We have successfully constructed a high specificity inducible lac operon system in an intestine-derived cell line, which could be of great value for gene therapy applications and transgenic animal production.

Specific Expression of Maize SBEIIb Promoter Mediated by Different Promoter Region in Transgenic Tobacco Plants

Starch branching enzyme （SBE） catalyzes the biosynthesis of amylopectin. We described the isolation and characterization of SBEIIb promoter and their expression patterns in transgenic tobacco. Using the genomic DNA of maize cultivar Lunuo 1 as template, the SBEIIb promoter was isolated by PCR and was cloned into pMD18-T vector. To study SEBIIb gene regulation at the cellular level, SBEIIb promoter was fused to the ^-glucuronidase （GUS） report gene. The results of the fluorometric GUS assays indicate that the sbeⅡb-GUS fusion directed a seed-specific expression. Four series of constructs were made with the promoter and the GUS reporter gene to investigate the cis-acting analysis, showing that the four different constructs all can drive expression of the GUS gene in seed plumule and cotyledon and the GUS activity was apparently decreased with the progressive loss of promoter 5＇ end.

Molecular cloning and characterization of fruit specific promoter from <i>Cucumis sativus</i>L.

The isolation and characterization of fruit specific promoters are critical for the manipulation of nutritional value and agronomic quality of fruits by genetic engineering and also opened a new era in edible vaccine technology. Expansins are proteins that induce loosening of individual plant cells by disrupting the non-covalent interactions between cellulose and hemicellulose microfibrils and hence have role in growth programs including fruit ripening. We report the identification of an expansin gene (CsExp) from Cucumis sativus that exhibits high levels of mRNA abundance and is specifically expressed in ripened fruit. The promoter region of CsExp also contains elements responsible for its fruit specific expression. Transient expression studies of the CsExp promoter were conducted with particle bombardment, followed by GUS histochemical assay and real time PCR. CaMV35S promoter was used as the positive control in all these experiments. Clear fruit specificity was observed for CsExp promoter in all the experiments. Thus CsExp promoter from Cucumber is a good candidate to target expression of the foreign genes to engineer fruit specific traits.

Cloning and Expression of Ovarian-Specific Promoter in Rats

[Objective] To clone ovarian-specific promoter-1 （OSP-1） fragment in rats and detect the tissue-specific expression of OSP-1 at cell level. [Method] According to the known OSP-1 sequence in rats, the specific pdmers were designed. The OSP-1 fragment was amplified by PCR and compared with published OSP-1 sequence. After eukaryotic expression vector pOSP-1-EGFP-N1 was constructed by cloning OSP-1 promoter into the pEGFP-N1 vector without CMV, the recombinant plasmid was transfected into buffalo granulocytes, mammary epithelial cells and fetal fibroblast cells under mediation of liposome LipofectamineTM 2000. The green fluxorescent protein （GFP） expression was observed with the fluorescence microscope after transfection for 12, 24 and 48 h. [Result] The amplified OSP-1 fragment was 480 bp, and the homology to published rat OSP-1 sequence was 96%. The sequence analysis showed that this fragment contained a core promoter cis-element similar with TATA box and ChAT box, and multiple C/EBP beta transcription factor binding sites. The EGFP-expressing positive granulccytes were firstly observed at 12 h after transfection and they were increased. All the EGFP-expressing and non-expressing cells from granulocytes were large and round. After transfection for 48 h, the EGFP-expressing positive granulocytes were decreased. The EGFP was not expressed in buffalo mammary epithelial cell and fetal fibroblast cell. [ Conclusion] EGFP exDression controlled bv OSP-1 promoter can be found in buffao granulocvtes.

Isolation and Structural Analysis of the Seed-Specific Promoter from Soybean

The promoter region (BCSP666) of b-conglycinin a-subunit gene from the genomic DNA of soybean Jilin 43 was isolatedby PCR method. Sequencing analysis showed that the cloned fragment BCSP666 had the similar structure to the soybeanseed-specific promoter b-conglycinin a'-subunit gene promoter and b-conglycinin b-subunit gene promoter, and it alsocontains many motifs that contribute to the seed-specific promoter activity. Based on this sequencing analysis, wededuced that promoter fragment BCSP666 had the seed-sepecific promoter activity. And then we constructed the seed-specific expression vector pBMI666 with the promoter fragment BCSP666 and D6-fatty acid desaturase gene fromMortierella isabellina. The D6-fatty acid desaturase is the rate-limiting enzyme of the desaturation of linoleic acid in theproduction of a human essential fatty acid, g-linolenic acid(GLA). The production of g-linolenic acid(GLA) was observedin soybean callus cells, which were transformed with this vector. This confirmed the activity of the activity fragmentBCSP666.

Analysis of Wheat GBSS1 Promoter:Tissue Specificity and DNA Methylation

The cis-regulatory elements of promoters regulate temporal and spatial expression of genes. DNA inethylation, histone methylation and histone acetylation are the main types of epigenetic modifications, which play important roles in plant growth and development. DNA methylation could seilenco transposons, affect gene imprinting and gene expression. In this study, we found that granule bound starch synthase 1 （GBSSI） gene is expressed specifically in wheat endosperm rath- er than in the embryo. We also analyzed the cis-elements within this promoter region and found some seed-specific elements. In order to confirm the tissue specifici- ty, we cloned 4k bp sequences upstream of GBSS1 gene to link to vector with GUS and this construct was transferred to tobacco by Agrobacterium mediated transfor- marion. The results showed that wheat GBSS1 promoter mediated the seed-specific expression of GUS gene, hut not mediated expression in embryo. In addition, we found that GBSSI promoter is methylated in wheat embryo and de-methylated in wheat endosperm. Our study might provide the molecular basis for specific expres- sion of GBSSI gene.

Cloning the Promoter of BcNA1 from Brassica napus and Fad2 Gene from Arabidopsis thaliana and Construction of the Plant Expression Vector

The upstream regulatory region of a seed specific gene was isolated from the genomic DNA of Brassica napus by PCR amplification. The cloned fragment contained 1755 nucleotides, and shared a sequence homology of 99.6% with the reported data. The coding region of oleic acid desaturase gene was then cloned from Arabidopsis thaliana. The sequencing analysis indicated that the sequence of the PCR product was just the same as reported before. In addition, the plant expression vector harboring the seed specific promoter and trans Fad2 gene was constructed.

Isolation and Characterization of Ell Gene Promoter from Tomato

A fragment of 2000 bp upstream sequence of Ell clone was amplified from genomic DNA of the tomato cultivar Zhongshu- 5. Sequence analysis showed that the upstream contains the regulatory elements： TATA box （-29 - -22）, CAAT box （-193 - -189）, wound, and drought response elements. Expression vectors of Ell promoter gus fusion were constructed with the promoters of 1 200 and 2 000 bp regions, respectively. Transgenic tomato plants were obtained through Agrobacteriummediated transformation. Histochemical analysis of GUS activity in various tissues showed that the two promoters were able to direct fruit-specific gene expression. The expression driven by promoter of 2 000 bp upstream fragment could increase GUS activity with the maturation of tomato fruits. The promoter of -1 200 bp fragment could direct gus gene expression in fruits with the inductions of drought and wounding. The regulatory region for fruit-specificity was probably located in the region of 1 200 bp of 5′-flanking sequence and some positive regulatory elements or enhancers may exist in the region from -1 200 to -2000 bp.

A polymorphism in GAS5 promoter impacts efficacy of cytarabine-based chemotherapy in Chinese AML patients

OBJECTIVE SNPs in lnc RNAs may alter the expression or secondary structure of lnc RNAs and then impact their functions.Whether lnc RNA SNPs affect the prognosis of acute myeloid leukemia(AML)remains unknown.To search the association between lnc RNA SNPs and AML outcomes,thirty tag SNPs in GAS5,H19,MALAT1,WT1-as and SRA were genotyped in313 AML patients.METHODS Survival analysis was performed in both AML patients recruited presently and GEO samples.The expression of GAS5 and TP63 was analyzed by real-time quantitative PCR.Dual-luciferase reporter gene assay was used to confirm the interactions between GAS5 rs55829688 and TP63.RESULTS Survival analysis indicated that rs55829688(T>C),located in GAS5 promoter,was significantly associated with the prognosis of AML.The average overall survival(OS)for patients with the rs55829688 CC genotype was significantly shorter than those carrying the rs55829688 T allele(P=0.018).Patients with rs55829688 CC genotype showed higher GAS5 expression in PBMCs than carriers of rs55829688T allele(P=0.025).Rs55829688 CC homozygotes also harbored a longer platelets recovery than those with rs55829688 T allele(P=0.040).In vitro study showed that GAS5 promoter harboring the rs55829688 C al ele showed marginal y increased reporter gene activity(P=0.054),and the promoter activity was increased by TP63 in a dose-dependent manner(P=0.001).Moreover,GAS5 expression was associated with AML OS in the GEO GSE12417 dataset,and GAS5 higher expression predict shorter OS(P=0.011).CONCLUSION Rs55829688 polymorphism could increase GAS5 expression by interacting with TP63 and was associated with worse OS in Chinese AML patients.

An <i>in silico</i>Analysis of Upstream Regulatory Modules (URMs) of Tapetum Specific Genes to Identify Regulatory <i>cis</i>-Elements and Transcription Factors

The present work presents an iin silicoi analysis of Upstream Regulatory Modules (URMs) of genes expressed in tapetum specific manner in dicotyledon and monocotyledon plants. In the current analysis, we identified several motifs conserved in these URMs of which ten were observed to be part of known icisi-elements using tools and databases like MEME, PLACE, MAST and TFSEARCH. We also identified that binding sites for two transcription factors, DOF and WRKY71 were found to be present in majority of the URMs.

Tissue Specific Expression of a Terpene Synthase in <i>Nicotiana benthamiana</i>Leaves

To study transient tissue specific linalool emission and to examine its fate, plastid targeted dual linalool/nerolidol synthase (FaNES1) was introduced into Nicotiana benthamiana leaves under LTP1, SUC2, RbcS and CaMV35 promoters. Tissue specificity of the promoters was tested with β-glucuronidase (GUS) reporter gene. Promoter::GUS/FaNES1 fusion was confirmed with colony PCR and agro-infiltrated into six weeks old N. benthamiana leaves. Eight days post inoculation (dpi), promoter::GUS constructs were examined with GUS histochemical staining at 1.5 h, 3.5 h, 5.5 h and 16 h incubation times. After 4/8 days, FaNES1 construct agro-inoculated leaves were assessed for linalool emissions and its conjugates using a dynamic headspace system and LC-MS, respectively. There was high affinity of promoters to their respective cell-types although it was not as specific as in stable transformation. This is possibly due to activations of many copies of transiently introduced promotes by few transcription factors of the respective promoter that naturally exist in untargeted cell-types. GUS staining intensity was stronger in leaf veins and injured sites compared to other plant tissues under all heterologous promoters, and it was gradually increased with increase in incubation times that could be explained by promoters wounding responses and/ or GUS leakage to unstained sites. Linalool emission was obtained in the same pattern under all promoters;it was higher at day 4 than day 8. Its concentration declined 44, 12, 4.5 and 4 folds at 8 dpi under LTP1, SUC2, RbcS and CaMV35S promoters, respectively. Conversely, linalool conjugates were significantly increased at day 8. These might be due to T-DNA degradations and/or protein modifications 4 dpi. LTP1 promoter was the least efficient to drive both GUS and FaNES1 possibly due to immature plastids in epidermal cells and/or its weak performance. Hence, to study FaNES1 activity in transient assay it is suggested to use relatively shorter duration and longer inoculation times for linalool and its conjugates, respectively.

Cell Type Specific Expression of CD80 （B7-1） Gene in Murine

The CD80 （B7-1） costimulatory molecule is expressed on the surface of B cells and its expression is transcriptionally upregulated upon stimuli. To identify the region of murine CD80 promoter that direct cell type specific gene expression, four promoters construct of CD80 gene were generated with DNA fragments fused to the GFP reporter gene. In the present study, significant promoter activity was detected with all four promoter constructs only in the murine B lymphocyte. Further, the CD80 promoter region extending from -3,005 bp to ＋273 bp in relation to the previously reported transcription start site, was identified as tissue specific region. Interestingly, the shortest 700 bp （-427/＋273） of promoter fragment was sufficient to direct the CD80 gene expression. The level of CD80 expression was also found to be modulated by exogenous stimuli in B lymphocyte. Additionally, it was demonstrated that the CD80 gene expression is regulated at the level of transcription where the inducible CD80 gene transcript was detected in B lymphocyte with increasing time.

Transcription Activity of Ectogenic Human Carcinoembryonic Antigen Promoter in Lung Adenocarcinoma Cells A549

The transcription activity of ectogenic human carcinoembryonic antigen （CEA） promoter in lung adenocarcinoma cells A549 was investigated for the further gene-targeting therapy. The reporter gene green fluorescent protein （GFP） driven by CEA promoter and human cytomegalovirus （CMV） promoter were relatively constructed and named plasmid pCEA-EGFP and pCMV-GFP respectively. The intensity of fluorescence was detected by fluorescence microscope and flow cytometry analysis after the pCEA-GFP and pSNAV-GFP plasmids were transfected into A549 cells through liposome respectively. The results showed （4,08±0.63） % of the A549 cells transfected with pCEA-AFP plasmid expressed, significantly lower than that of the A549 cells transfected with pCMV-GFP [（43.27±3.54） %]. It was suggested that ectogenic human CEA promoter in lung adenocarcinoma cells A549 was weakly expressed. The distinct specificity of CEA promoter in CEA high expression cells was regarded as a tool in selective gene therapy, but the transcription activity of ectogenic human CEA promoter was needed to increase in the future.

Expression of ipt gene driven by tomato fruit specific promoter and its effects on fruit development of tomato

Fruit specific promoter (2A12) from Lycopersi-com esculentum and cDNA of isopentenyl-transferase (ipt) from Ti plasmid of Agrobacterium tumerfaciens C58 were cloned by PCR procedure respectively. Two plant expression vectors with 2A12/gus or 2A12/ipt were respectively constructed. These two chimeric genes were transferred into tomato by Agrobacterium mediated procedure. The results of Southern hybridization showed that the fusion genes had been integrated into tomatoes. The result of gus histochemi-cal staining showed that 2A12 had high fruit specific expressive capability in transgenic tomato. The ipt expression resulted in accumulation of high level of cytokinins (CTKs) in fruit lead to developmental changes in fruits and seeds. The fruit of ipt transformed tomato had the hyperplastic placenta with very few seeds or even seedless. The shelf life of transgenic fruits elongated for 1-2 weeks. The ratio of fruit set, the dry weight of fruit and the crude protein content in fruit were increased, while

Downregulation of orosomucoid 2 acts as a prognostic factor associated with cancer-promoting pathways in liver cancer

BACKGROUND Liver cancer has a high mortality and morbidity rate throughout the world.In clinical practice,the prognosis of liver cancer patients is poor,and the complex reasons contribute to treatment failures,including fibrosis,hepatitis viral infection,drug resistance and metastasis.Thus,screening novel prognostic biomarkers is of great importance for guiding liver cancer therapy.Orosomucoid genes(ORMs)encode acute phase plasma proteins,including orosomucoid 1(ORM1)and ORM2.Previous studies showed their upregulation upon inflammation,but the specific function of ORMs has not yet been determined,especially in the development of liver cancer.AIM To determine the expression of ORMs and their potential function in liver cancer.METHODS Analysis of the expression of ORMs in different human tissues was performed on data from the HPA RNA-seq normal tissues project.The expression ratio of ORMs was determined using the HCCDB database,including the ratio between liver cancer and other cancers,normal liver and other normal tissues,liver cancer and adjacent normal liver tissues.Analysis of ORM expression in different cancer types was performed using The Cancer Genome Atlas and TIMER database.The expression of ORMs in liver tumor tissues and adjacent normal tissues were further confirmed using Gene Expression Omnibus data,including GSE36376 and GSE14520.The 10-year overall survival(OS),progression-free survival(PFS)and relapse-free survival(RFS)rates between high and low ORM expression groups in liver cancer patients were determined using the Kaplan-Meier plotter tool.Gene Set Enrichment Analysis(GSEA)was employed to explore the ORM2-associated signaling network.Correlations between ORM2 expression and tumor purity or the infiltration level of macrophages in liver tumor tissues were determined using the TIMER database.The correlation between ORM2 gene levels,tumor-associated macrophage(TAM)markers(including CD68 and TGFβ1)and T cell immunosuppression(including CTLA4 and PD-1)in liver tumor tissues and liver GTEx was determined using the GEPIA database.RESULTS ORM1 and ORM2 were highly expressed in normal liver and liver tumor tissues.ORM1 and ORM2 expression was significantly decreased in liver tumor tissues compared with adjacent normal tissues,and similar results were also noted in cholangiocarcinoma,esophageal carcinoma,and lung squamous cell carcinoma.Further analysis of the Gene Expression Omnibus Database also confirmed the downregulation of ORM1 and ORM2 in liver tumors.Survival analysis showed that the high ORM2 group had better survival rates in OS,PFS and RFS.ORM1 only represented better performance in PFS,but not in OS or RFS.GSEA analysis of ORM2 from The Cancer Genome Atlas liver cancer data identified that ORM2 positively associated with the G2/M checkpoint,E2F target signaling,as well as Wnt/β-catenin and Hedgehog signaling.Moreover,apoptosis,IFN-αresponses,IFN-γresponses and humoral immune responses were upregulated in the ORM2 high group.ORM2 expression was negatively correlated with the macrophage infiltration level,CD68,TGFβ1,CTLA4 and PD-1 levels.CONCLUSION The results showed that ORM1 and ORM2 were highly expressed specifically in liver tissues,whereas ORM1 and ORM2 were downregulated in liver tumor tissues.ORM2 is a better prognostic factor for liver cancer.Furthermore,ORM2 is closely associated with cancer-promoting pathways.

Characterization of the Tomato Prosystemin Promoter:Organ-specific Expression,Hormone Specificity and Methyl Jasmonate Responsiveness by Deletion Analysis in Transgenic Tobacco Plants

Tomato systemin is a bioactive peptide that regulates the systemic activation of wound-responsive genes. It is released from its 200 amino acid precursor called prosystemin. Initial tissue-localization and hormone-induced expression assays indicated that the tomato prosystemin gene （SIPS） accumulates mainly in floral tissues and in response to exogenous abscisic acid and methyl jasmonate （MeJA） treatments, respectively. Later, the promoter regions of the PS gene in tomato （Solanum lycopersicum L. cv. Castlemart）, pepper （Capsicum annuum） and potato （Solanum tuberosum） were isolated and an in silico analysis of the SIPS promoter revealed an over-representation of stress- and MeJA-responsive motifs. A subsequent 5＇ deletion analysis of the SIPS promoter fused to the/^-glucuronidase reporter （GUS） gene showed that the -221 to ＋40 bp proximal SIPS promoter region was sufficient to direct the stigma, vascular bundle-specific and MeJA-responsive expression of GUS in transgenic tobacco plants. Important vascular.tissue-specific, light- and MeJA-responsive cis-elements were also present in this region. These findings provide relevant information regarding the transcriptional regulation mechanisms of the SIPS promoter operating in transgenic tobacco plants. They also suggest that its Ussue-specificity and inducible nature could have wide applicability in plant biotechnology.