Sample records for aberrant gene promoter

  1. Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer.

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    Victoria Gonzalo

    Full Text Available BACKGROUND: Colorectal cancer (CRC multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect. METHODOLOGY/PRINCIPAL FINDINGS: We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2, RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008 and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047 as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006. Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17, SFRP1 (r = 0.83, 0.06, HPP1 (r = 0.64, p = 0.17, 3OST2 (r = 0.83, p = 0.06 and GATA4 (r = 0.6, p = 0.24. Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant

  2. miRNA gene promoters are frequent targets of aberrant DNA methylation in human breast cancer. (United States)

    Vrba, Lukas; Muñoz-Rodríguez, José L; Stampfer, Martha R; Futscher, Bernard W


    miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.


    Aberrant methylation in the promoter region of cancer-related genes leads to gene transcriptional inactivation and plays an integral role in lung tumorigenesis. Recent studies demonstrated that promoter methylation was detected not only in lung tumors from patients with lung canc...

  4. Aberrant CBFA2T3B gene promoter methylation in breast tumors

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    Bais Anthony J


    Full Text Available Abstract Background The CBFA2T3 locus located on the human chromosome region 16q24.3 is frequently deleted in breast tumors. CBFA2T3 gene expression levels are aberrant in breast tumor cell lines and the CBFA2T3B isoform is a potential tumor suppressor gene. In the absence of identified mutations to further support a role for this gene in tumorigenesis, we explored whether the CBFA2T3B promoter region is aberrantly methylated and whether this correlates with expression. Results Aberrant hypo and hypermethylation of the CBFA2T3B promoter was detected in breast tumor cell lines and primary breast tumor samples relative to methylation index interquartile ranges in normal breast counterpart and normal whole blood samples. A statistically significant inverse correlation between aberrant CBFA2T3B promoter methylation and gene expression was established. Conclusion CBFA2T3B is a potential breast tumor suppressor gene affected by aberrant promoter methylation and gene expression. The methylation levels were quantitated using a second-round real-time methylation-specific PCR assay. The detection of both hypo and hypermethylation is a technicality regarding the methylation methodology.

  5. Aberrant gene promoter methylation in sputum from individuals exposed to smoky coal emissions

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    Liu, Y.; Lan, Q.; Shen, M.; Jin, J.; Mumford, J.; Ren, D.X.; Keohavong, P. [University of Pittsburgh, Pittsburgh, PA (United States). Dept. of Environment and Occupational Health


    Recent studies suggested the potential for aberrant gene promoter methylation in sputum as a predictive marker for lung cancer. Here, the promoter methylation of p16, MGMT, RASSF1A and DAPK genes was investigated in sputum of individuals exposed to smoky coal emissions in Xuan Wei, China, where the lung cancer rate is more than 6 times the Chinese national average. Sputum DNA of 107 noncancer individuals and 58 lung cancer patients was screened for promoter methylation using methylation-specific PCR. Promoter methylation of the p16 gene was detected in about half (51.4% (551107)) of sputum DNA from noncancer individuals, a frequency higher than that observed for the RASSF1A (29.9%), MGMT (17.8%) and DAPK (15.9%) genes. Furthermore, the p16 gene was affected by promoter methylation at a frequency even higher among the lung cancer group, compared with the noncancer group (70.7% (41/58) versus 51.7% (55/107), p=0.017). Individuals exposed to smoky coal emissions in this region harbored frequent promoter methylation of these genes in their sputum and some of such alterations may be involved in lung tumor development.

  6. In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer.

    LENUS (Irish Health Repository)

    Murphy, Therese M


    Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

  7. Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in breast and lung carcinomas. (United States)

    Virmani, A K; Rathi, A; Sathyanarayana, U G; Padar, A; Huang, C X; Cunnigham, H T; Farinas, A J; Milchgrub, S; Euhus, D M; Gilcrease, M; Herman, J; Minna, J D; Gazdar, A F


    The adenomatous polyposis coli (APC) gene is a tumor suppressor gene associated with both familial and sporadic cancer. Despite high rates of allelic loss in lung and breast cancers, point mutations of the APC gene are infrequent in these cancer types. Aberrant methylation of the APC promoter 1A occurs in some colorectal and gastric malignancies, and we investigated whether the same mechanism occurs in lung and breast cancers. The methylation status of the APC gene promoter 1A was analyzed in 77 breast, 50 small cell (SCLC), and 106 non-small cell (NSCLC) lung cancer tumors and cell lines and in 68 nonmalignant tissues by methylation-specific PCR. Expression of the APC promoter 1A transcript was examined in a subset of cell lines by reverse transcription-PCR, and loss of heterozygosity at the gene locus was analyzed by the use of 12 microsatellite and polymorphic markers. Statistical tests were two-sided. Promoter 1A was methylated in 34 of 77 breast cancer tumors and cell lines (44%), in 56 of 106 NSCLC tumors and cell lines (53%), in 13 of 50 SCLC cell lines (26%), and in 3 of 68 nonmalignant samples (4%). Most cell lines tested contained the unmethylated or methylated form exclusively. In 27 cell lines tested, there was complete concordance between promoter methylation and silencing of its transcript. Demethylation with 5-aza-2'-deoxycytidine treatment restored transcript 1A expression in all eight methylated cell lines tested. Loss of heterozygosity at the APC locus was observed in 85% of SCLCs, 83% of NSCLCs, and 63% of breast cancer cell lines. The frequency of methylation in breast cancers increased with tumor stage and size. In summary, aberrant methylation of the 1A promoter of the APC gene and loss of its specific transcript is frequently present in breast and NSCLC cancers and cell lines and, to a lesser extent, in SCLC cell lines. Our findings may be of biological and clinical importance.

  8. Dysfunction of endothelial NO system originated from homocysteine-induced aberrant methylation pattern in promoter region of DDAH2 gene

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jing-ge; LIU Jun-xu; LI Zhu-hua; WANG Li-zhen; JIANG Yi-deng; WANG Shu-ren


    Background Hyperhomocysteinemia (HHcy)-mediated dysfunction of endothelial NO system is an important mechanism for atherosclerotic pathogenesis.Dimethylarginine dimethylaminohydrolase (DDAH) is the key enzyme for degrading asymmetric dimethylarginine (ADMA),which is an endogenous inhibitor of endothelial nitric oxide (NO) synthase (eNOS).This study was designed to investigate whether the dysfunction of endothelial NO system originates from HHcy-mediated aberrant methylation modification in promotor region of DDAH2 gene.Methods Human umbilical vein endothelial cells (HUVECs) were cultured to the third generation and treated with homocysteine (Hcy) at different concentrations (0,10,30,100,and 300 μmol/L) for 72 hours.The methylation pattern in promoter region CpG island of DDAH2 gene was analyzed by nested methylation-specific PCR (nMSP).The mRNA expression of eNOS gene and DDAH2 gene was detected by semi-quantitative RT-PCR.The activity of DDAH2 and eNOS in cells,and the concentrations of ADMA and NO in culture medium were assayed respectively.Results Mild increased concentration of Hcy (10 and 30 μmol/L) induced hypomethylation,while high concentration of Hcy (100 and 300 μmol/L) induced hypermethylation in the promoter CpG island of DDAH2 gene.The mRNA expression of DDAH2 increased in mild enhanced concentration of Hcy,and decreased in high concentration of Hcy correspondingly.The inhibition of DDAH2 activity,the increase of ADMA concentration,the reduction of eNOS activity and the decrease of NO production were all consistently relevant to the alteration of Hcy concentration Conclusion The increased concentration of Hcy induced aberrant methylation pattern in promotor region of DDAH2 gene and the successive alterations in DDAH/ADMA/NOS/NO pathway,which showed highly relevant and dose-effect relationship.The results suggested that the dysfunction of endothelial NO system induced by HHcy could be partially originated from Hcy-mediated aberrant methylation in

  9. Aberrant Methylation of the E-Cadherin Gene Promoter Region in the Endometrium of Women With Uterine Fibroids. (United States)

    Li, Yan; Ran, Ran; Guan, Yingxia; Zhu, Xiaoxiong; Kang, Shan


    A uterine fibroid is a leiomyoma that originates from the smooth muscle layer of the uterus. A variety of endometrial abnormalities are associated with uterine fibroids. This study aims to investigate the methylation status of the E-cadherin gene (CDH1) promoter region in the endometrium of patients with uterine fibroids. The methylation of CDH1 was studied using methylation-specific polymerase chain reaction in the endometrial tissue of 102 patients with uterine fibroids and 50 control patients. The E-cadherin expression was examined by flow cytometry. The methylation rate of CDH1 promoter region was 33.3% in the endometrium of patients with uterine fibroids and 8% in the endometrium of women without fibroids. The frequency of CDH1 promoter methylation in the endometrium of patients with fibroids was significantly higher than that in the endometrium of women without fibroids (P = .001). Furthermore, the E-cadherin expression level in methylation-positive tissues was significantly lower than that in methylation-negative tissues (P = .017). These results suggest that epigenetic aberration of CDH1 may occur in the endometrium of patients with fibroids, which may be associated with E-cadherin protein expression in endometrial tissue.

  10. Aberrant promoter methylation and gene expression of H-cadherin gene is associated with tumor progression and recurrence in epithelial ovarian carcinoma

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    Rahul Bhagat


    Full Text Available Background: Loss of expression of cadherins by promoter hypermethylation has been described in many epithelial cancers, and it may play a role in tumor cell invasion and metastasis. Previously, we reported that E-cadherin gene is frequently methylated in epithelial ovarian cancer. Aim: The aim of this study was to compare the promoter hypermethylation of H-cadherin gene in ovarian epithelial neoplasms to better understand the role of epigenetic silencing in carcinogenesis. Materials and Methods: We examined the promoter methylation of the H-cadherin gene in 134 epithelial ovarian carcinomas (EOC, 23 low malignant potential (LMP tumors, 26 benign cystadenomas and 15 normal ovarian tissues. Methylation was investigated by methylation specific polymerase chain reaction (MSP and the results confirmed by bisulfite DNA sequencing. Relative gene expression of H-cadherin was done using quantitative reverse transcriptase PCR on 51 EOC cases, 9 LMP tumors, 7 benign cystadenomas with 5 normal ovarian tissues. Results: Aberrant methylation of H-cadherin was present in 20 of 134 (15% carcinoma cases, 2 of 23 (09% LMP tumors and 1 of 26 (4% benign cystadenomas. No methylation was observed in any of the normal ovarian tissues. The mRNA expression level of H-cadherin was significantly down-regulated in EOC and LMP tumors than the corresponding normal tissues, whereas the expression level was normal in benign cystadenomas. A significant correlation of H-cadherin promoter methylation was observed with reduced gene expression in EOC. The prevalence of H-cadherin methylation was associated significantly with stage, histopathological grade, and menopausal status of the patient. H-cadherin methylation also had significant association with recurrence and differentiation of tumor. Conclusion: Our findings suggest an association between H-cadherin methylation, tumor progression and recurrence in EOC.


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    Full Text Available PURPOSE: The promoter hypermethylation patterns of Thrombospodin - 1 gene in 50 EOC patients were studied and the methylation pattern was correlated with various clinic pathological parameters. METHODS: The promoter hypermethylation pattern of the TSP - 1 gene was assessed using nested PCR and Methylation specific PCR. STATISTICAL ANALYSIS: All the available data was statistically analyzed using the Chi square test or Fisher Exact Test on the SPSS software version 22.0 and a value <0.0 5 was considered statistically significant. RESULTS: Forty of the fifty ovarian carcinoma samples reported positive for methylation corresponding to a methylation frequency of 80%. A methylation frequency of 89.2%, 83.3% and 42.8% was observed in malignant , Low malignant potential (borderline and benign sample cohorts. CONCLUSION: From the results drawn from this study, it clearly shows that the anti angiogenic protein TSP - 1 is extensively hypermethylated in ovarian carcinoma and that it accumulates over t he progression of the disease from benign to malignant. As previous reports suggest that there is no evidence of mutation of this gene, promoter hypermethylation may be a crucial factor for the down regulation of the gene. Further by clubbing together the promoter hypermethylation pattern of TSP - 1 gene with hypermethylation patterns of other TSG may provide a better insight into the application of using methylation profiles of TSG as a biomarker in the detection of ovarian carcinoma.

  12. Cigarette Smoking, BPDE-DNA Adducts, and Aberrant Promoter Methylations of Tumor Suppressor Genes (TSGs) in NSCLC from Chinese Population. (United States)

    Jin, Yongtang; Xu, Peiwei; Liu, Xinneng; Zhang, Chunye; Tan, Cong; Chen, Chunmei; Sun, Xiaoyu; Xu, Yingchun


    Non-small cell lung cancer (NSCLC) is related to the genetic and epigenetic factors. The goal of this study was to determine association of cigarette smoking and BPDE-DNA adducts with promoter methylations of several genes in NSCLC. Methylation of the promoters of p16, RARβ, DAPK, MGMT, and TIMP-3 genes of tumor tissues from 199 lung cancer patients was analyzed with methylation-specific PCR (MSP), and BPDE-DNA adduct level in lung cancer tissue was obtained by ELISA. Level of BPDE-DNA adduct increased significantly in males, aged people (over 60 years), and smokers; however, no significant difference was found while comparing the BPDE-DNA adduct levels among different tumor types, locations, and stages. Cigarette smoking was also associated with increased BPDE-DNA adducts level (OR = 2.43, p > .05) and increased methylation level in at least 1 gene (OR = 5.22, p smoking also significantly increase the risk of p16 or DAPK methylation (OR = 3.02, p smoking for more than 40 pack-years (OR = 4.21, p smoking is significantly associated with the increase of BPDE-DNA adduct level, promoter hypermethylation of p16 and DAPK genes, while BPDE-DNA adduct was not significantly related to abnormal promoter hypermethylation in TSGs, suggesting that BPDE-DNA adducts and TSGs methylations play independent roles in NSCLC.

  13. Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer. (United States)

    Kuhmann, Christine; Li, Carmen; Kloor, Matthias; Salou, Mariam; Weigel, Christoph; Schmidt, Christopher R; Ng, Linda W C; Tsui, Wendy W Y; Leung, Suet Y; Yuen, Siu T; Becker, Natalia; Weichenhan, Dieter; Plass, Christoph; Schmezer, Peter; Chan, Tsun L; Popanda, Odilia


    Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.

  14. Aberrant promoter hypermethylation in serum DNA from patients with silicosis. (United States)

    Umemura, Shigeki; Fujimoto, Nobukazu; Hiraki, Akio; Gemba, Kenichi; Takigawa, Nagio; Fujiwara, Keiichi; Fujii, Masanori; Umemura, Hiroshi; Satoh, Mamoru; Tabata, Masahiro; Ueoka, Hiroshi; Kiura, Katsuyuki; Kishimoto, Takumi; Tanimoto, Mitsune


    It is well established that patients with silicosis are at high risk for lung cancer; however, it is difficult to detect lung cancer by chest radiography during follow-up treatment of patients with silicosis because of preexisting diffuse pulmonary shadows. The purpose of this study is to evaluate the usefulness of detection of serum DNA methylation for early detection of lung cancer in silicosis. Serum samples from healthy controls (n = 20) and silicosis patients with (n = 11) and without (n = 67) lung cancer were tested for aberrant hypermethylation at the promoters of the DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT), p16(INK4a), ras association domain family 1A (RASSF1A), the apoptosis-related gene death-associated protein kinase (DAPK) and retinoic acid receptor beta (RARbeta) by methylation-specific polymerase chain reaction. Aberrant promoter methylation in at least one of five tumor suppressor genes was detected more frequently in the serum DNA of silicosis patients with lung cancer than in that of patients without it (P = 0.006). Furthermore, the odds ratio of having lung cancer was 9.77 (P = 0.009) for those silicosis patients with methylation of at least one gene. Extended exposure to silica (>30 years) was correlated with an increased methylation frequency (P = 0.017); however, methylation status did not correlate with age, smoking history or radiographic findings of silicosis. These results suggest that testing for aberrant promoter methylation of tumor suppressor genes using serum DNA may facilitate early detection of lung cancer in patients with silicosis.

  15. Aberrant Gene Expression in Acute Myeloid Leukaemia

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    Bagger, Frederik Otzen

    model to investigate the role of telomerase in AML, we were able to translate the observed effect into human AML patients and identify specific genes involved, which also predict survival patterns in AML patients. During these studies we have applied methods for investigating differentially expressed......Summary Acute Myeloid Leukaemia (AML) is an aggressive cancer of the bone marrow, affecting formation of blood cells during haematopoiesis. This thesis presents investigation of AML using mRNA gene expression profiles (GEP) of samples extracted from the bone marrow of healthy and diseased subjects....... Here GEPs from purified healthy haematopoietic populations, with different levels of differentiation, form the basis for comparison with diseased samples. We present a mathematical transformation of mRNA microarray data to make it possible to compare AML samples, carrying expanded aberrant...

  16. Aberrant gene promoter methylation of p16, FHIT, CRBP1, WWOX, and DLC-1 in Epstein-Barr virus-associated gastric carcinomas. (United States)

    He, Dan; Zhang, Yi-wang; Zhang, Na-na; Zhou, Lu; Chen, Jian-ning; Jiang, Ye; Shao, Chun-kui


    Alterations in global DNA methylation and specific regulatory gene methylation are frequently found in cancer, but the significance of these epigenetic changes in EBV-associated gastric carcinoma (EBVaGC) remains unclear. We evaluated global DNA methylation status in 49 EBVaGC and 45 EBV-negative gastric carcinoma (EBVnGC) tissue samples and cell lines by 5-methylcytosine immunohistochemical staining and methylation quantification. We determined promoter methylation status and protein expression for the p16, FHIT, CRBP1, WWOX, and DLC-1 genes in tissues and studied the correlation between CpG island methylator phenotype (CIMP) class and clinicopathological characteristics. Changes in gene methylation and mRNA expression in EBVaGC cell line SNU-719 and in EBVnGC cell lines SGC-7901, BGC-823, and AGS were assessed after treatment with 5-aza-2'-deoxycytidine (5-aza-dC), trichostatin A (TSA), or a combination of both, by methylation-specific PCR and quantitative real-time RT-PCR. Global genomic DNA hypomethylation was more pronounced in EBVnGC than in EBVaGC. Promoter methylation of all five genes was more frequent in EBVaGC than in EBVnGC (p < 0.05). p16 and FHIT methylation was reversely correlated with protein expression in EBVaGC. Most (41/49) EBVaGC exhibited CIMP-high (CIMP-H), and the prognosis of CIMP-H patients was significantly worse than that of CIMP-low (p = 0.027) and CIMP-none (p = 0.003) patients. Treatment with 5-aza-dC and/or TSA induced upregulation of RNA expression of all five genes in SNU-719; meanwhile, individual gene expression increased in EBVnGC cell lines. In summary, EBV-induced hypermethylation of p16, FHIT, CRBP1, WWOX, and DLC-1 may contribute to EBVaGC development. Demethylation therapy may represent a novel therapeutic strategy for EBVaGC.

  17. Aberrant promoter CpG methylation and its translational applications in breast cancer

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    Ting-Xiu Xiang; Ying Yuan; Li-Li Li; Zhao-Hui Wang; Liang-Ying Dan; Yan Chen; Guo-Sheng Ren; Qian Tao


    Breast cancer is a complex disease driven by multiple factors including both genetic and epigenetic alterations.Recent studies revealed that abnormal gene expression induced by epigenetic changes,including aberrant promoter methylation and histone modification,plays a critical role in human breast carcinogenesis.Silencing of tumor suppressor genes (TSGs) by promoter CpG methylation facilitates cells growth and survival advantages and further results in tumor initiation and progression,thus directly contributing to breast tumorigenesis.Usually,aberrant promoter methylation of TSGs,which can be reversed by pharmacological reagents,occurs at the early stage of tumorigenesis and therefore may serve as a potential tumor marker for early diagnosis and therapeutic targeting of breast cancer.In this review,we summarize the epigenetic changes of multiple TSGs involved in breast pathogenesis and their potential clinical applications as tumor markers for early detection and treatment of breast cancer.

  18. Analysis of aberrant methylation on promoter sequences of tumor suppressor genes and total DNA in sputum samples: a promising tool for early detection of COPD and lung cancer in smokers

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    Guzmán Leda


    Full Text Available Abstract Background Chronic obstructive pulmonary disease (COPD is a disorder associated to cigarette smoke and lung cancer (LC. Since epigenetic changes in oncogenes and tumor suppressor genes (TSGs are clearly important in the development of LC. In this study, we hypothesize that tobacco smokers are susceptible for methylation in the promoter region of TSGs in airway epithelial cells when compared with non-smoker subjects. The purpose of this study was to investigate the usefulness of detection of genes promoter methylation in sputum specimens, as a complementary tool to identify LC biomarkers among smokers with early COPD. Methods We determined the amount of DNA in induced sputum from patients with COPD (n = 23, LC (n = 26, as well as in healthy subjects (CTR (n = 33, using a commercial kit for DNA purification, followed by absorbance measurement at 260 nm. The frequency of CDKN2A, CDH1 and MGMT promoter methylation in the same groups was determined by methylation-specific polymerase chain reaction (MSP. The Fisher’s exact test was employed to compare frequency of results between different groups. Results DNA concentration was 7.4 and 5.8 times higher in LC and COPD compared to the (CTR (p  Conclusions We provide evidence that aberrant methylation of TSGs in samples of induced sputum is a useful tool for early diagnostic of lung diseases (LC and COPD in smoker subjects. Virtual slides The abstract MUST finish with the following text: Virtual Slides The virtual slide(s for this article can be found here:


    Institute of Scientific and Technical Information of China (English)

    彭晓春; 耿小平; 朱立新; 孙昀; 李晓明


    To study the relationship between the gene promoter methylation state of DAPK, FHIT and SLIT2 genes and the clinical pronosis of patient in hepatocellular carcinoma ( HCC). Methods The technique of methylation-specific PCR ( MSP) was adopted to investigate the promoter hypermethylalion of DAPK,FHIT 及 SLIT2 genes in 50 HCCs after a curative resection. The relationship between the frequency of hypermethylation of the genes and tumor recrudescence data was analyzed. Results In all patients with HCC, the frequency of hypermethylation in DAPK ,FHIT and SLIT2 were 82. 0% , 68. 0% and 54. 0% , respectively. We divided all those cases into two groups according to the follow-up neoplasm recurrence results( group I ; the group of one year without tumor recrudescence, group Ⅱ : the group of less than one year with tumor recrudescence) . In group I , the frequency of hypermethyla tion in DAPK,FHIT and SLIT2 were 76. 9% , 53. 8% , 50. 0% , respectively; in group II they were 87. 5% , 83. 3% , 58. 3% , re spectively. Those three genes have higer frequency among group D , The frequency hypermethylation of FHIT gene is especially higher in group II (P = 0. 036). In group Ⅱ , there is twenty-two cases which have two or three genes hypermethylation; and in group Ⅰ, the cases are fifteen. There is a statistical prognosis difference between them ( P = 0. 006 ) . Conclusions Hypermethylation of multiple gene promotors are common events in HCC. In patients with HCC, aberrant DNA methylation is significantly associated with poor prog nosis. FHIT maybe can serve as a biomarker for the prognosis, after a curative resection.%目的 了解肝细胞癌(hepatucellular carcinoma,HCC)中,DAPK、FHIT及SLIT2基因的甲基化状况与病人生存预后的关系.方法 应用甲基化特异性PCR(methylation- specific PCR,MSP)技术,检测50例HCC组织中上述基因启动子区域的甲基化状况,并分析每种基因甲基化情况和肿瘤复发之间相关性.结果 50

  20. A DNA methylation signature associated with aberrant promoter DNA hypermethylation of DNMT3B in human colorectal cancer. (United States)

    Huidobro, Covadonga; Urdinguio, Rocío G; Rodríguez, Ramón María; Mangas, Cristina; Calvanese, Vincenzo; Martínez-Camblor, Pablo; Ferrero, Cecilia; Parra-Blanco, Adolfo; Rodrigo, Luis; Obaya, Alvaro J; Suárez-Fernández, Laura; Astudillo, Aurora; Hernando, Henar; Ballestar, Esteban; Fernández, Agustín F; Fraga, Mario F


    Altered promoter DNA methylation, one of the most important molecular alterations in cancer, is proposed to correlate with deregulation of DNA methyltransferases, although the molecular mechanisms implicated are still poorly understood. Here we show that the de novo DNA methyltransferase DNMT3B is frequently repressed in human colorectal cancer cell lines (CCL) and primary tumours by aberrant DNA hypermethylation of its distal promoter. At the epigenome level, DNMT3B promoter hypermethylation was associated with the hypomethylation of gene promoters usually hypermethylated in the healthy colon. Forced DNMT3B overexpression in cancer cells restored the methylation levels of these promoters in the healthy colon. Our results show a new molecular mechanism of aberrant DNMT3B regulation in colon cancer and suggest that its expression is associated with the methylation of constitutively hypermethylated promoters in the healthy colon.

  1. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. (United States)

    Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Gingras, Marie-Claude; Muthuswamy, Lakshmi B; Johns, Amber L; Miller, David K; Wilson, Peter J; Patch, Ann-Marie; Wu, Jianmin; Chang, David K; Cowley, Mark J; Gardiner, Brooke B; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J; Gill, Anthony J; Pinho, Andreia V; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R Scott; Humphris, Jeremy L; Kaplan, Warren; Jones, Marc D; Colvin, Emily K; Nagrial, Adnan M; Humphrey, Emily S; Chou, Angela; Chin, Venessa T; Chantrill, Lorraine A; Mawson, Amanda; Samra, Jaswinder S; Kench, James G; Lovell, Jessica A; Daly, Roger J; Merrett, Neil D; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M; Fisher, William E; Brunicardi, F Charles; Hodges, Sally E; Reid, Jeffrey G; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R; Dinh, Huyen; Buhay, Christian J; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E; Yung, Christina K; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A; Petersen, Gloria M; Gallinger, Steven; Hruban, Ralph H; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Schulick, Richard D; Wolfgang, Christopher L; Morgan, Richard A; Lawlor, Rita T; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A; Mann, Karen M; Jenkins, Nancy A; Perez-Mancera, Pedro A; Adams, David J; Largaespada, David A; Wessels, Lodewyk F A; Rust, Alistair G; Stein, Lincoln D; Tuveson, David A; Copeland, Neal G; Musgrove, Elizabeth A; Scarpa, Aldo; Eshleman, James R; Hudson, Thomas J; Sutherland, Robert L; Wheeler, David A; Pearson, John V; McPherson, John D; Gibbs, Richard A; Grimmond, Sean M


    Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.


    Institute of Scientific and Technical Information of China (English)

    LIN Qing; CHEN Long-bang; TANG Yong-ming; WANG Jing


    Objective: To analyze the aberrant methylation of p16 gene and DAPK gene in sera from primary liver cancer patients ad to evaluate the clinical significance. Methods: A methylation-specific PCR was performed for the detection of promoter hypermethylation of p16 gene and DAPK gene in blood DNA from 64 cases of HCC patients, and to analyze the relation of the aberrant methylation of p16 gene and KAPK gene and the clinical pathological data. Results: 76.6%(49/64) of the sera from 64 cases of HCC patients showed hypermethylation for p16 promoter and 40.6% (26/64) for KAPK promoter, whereas no methylated p16 gene promoter and DAPK gene promoter were found in sera from benign liver diseases patients and normal control. Methylated p16 gene and KAPK gene promoters in sera did not strongly correlated with HBsAg, stage,metastasis and differentiation in HCC; but strongly correlated with AFP. Conclusion: Detection of the aberrant methylation of p16 gene and KAPK gene in blood DNA from HCC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.

  3. Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes. (United States)

    Huang, Yi; Greene, Eriko; Murray Stewart, Tracy; Goodwin, Andrew C; Baylin, Stephen B; Woster, Patrick M; Casero, Robert A


    Epigenetic chromatin modification is a major regulator of eukaryotic gene expression, and aberrant epigenetic silencing of gene expression contributes to tumorigenesis. Histone modifications include acetylation, phosphorylation, and methylation, resulting in a combination of histone marks known collectively as the histone code. The chromatin marks at a given promoter determine, in part, whether specific promoters are in an open/active conformation or closed/repressed conformation. Dimethyl-lysine 4 histone H3 (H3K4me2) is a transcription-activating chromatin mark at gene promoters, and demethylation of this mark by the lysine-specific demethylase 1 (LSD1), a homologue of polyamine oxidases, may broadly repress gene expression. We now report that novel biguanide and bisguanidine polyamine analogues are potent inhibitors of LSD1. These analogues inhibit LSD1 in human colon carcinoma cells and affect a reexpression of multiple, aberrantly silenced genes important in the development of colon cancer, including members of the secreted frizzle-related proteins (SFRPs) and the GATA family of transcription factors. Furthermore, we demonstrate by chromatin immunoprecipitation analysis that the reexpression is concurrent with increased H3K4me2 and acetyl-H3K9 marks, decreased H3K9me1 and H3K9me2 repressive marks. We thus define important new agents for reversing aberrant repression of gene transcription.

  4. Gene expression and epigenetic aberrations in F1-placentas fathered by obese males. (United States)

    Mitchell, Megan; Strick, Reiner; Strissel, Pamela L; Dittrich, Ralf; McPherson, Nicole O; Lane, Michelle; Pliushch, Galyna; Potabattula, Ramya; Haaf, Thomas; El Hajj, Nady


    Gene expression and/or epigenetic deregulation may have consequences for sperm and blastocysts, as well as for the placenta, together potentially contributing to problems observed in offspring. We previously demonstrated specific perturbations of fertilization, blastocyst formation, implantation, as well as aberrant glucose metabolism and adiposity in offspring using a mouse model of paternal obesity. The current investigation analyzed gene expression and methylation of specific CpG residues in F1 placentas of pregnancies fathered by obese and normal-weight male mice, using real-time PCR and bisulfite pyrosequencing. Our aim was to determine if paternal obesity deregulated placental gene expression and DNA methylation when compared to normal-weight males. Gene methylation of sperm DNA was analyzed and compared to placentas to address epigenetic transmission. Of the 10 paternally expressed genes (Pegs), 11 genes important for development and transport of nutrients, and the long-terminal repeat Intracisternal A particle (IAP) elements, derived from a member of the class II endogenous retroviral gene family, we observed a significant effect of paternal diet-induced obesity on deregulated expression of Peg3, Peg9, Peg10, and the nutrient transporter gene Slc38a2, and aberrant DNA methylation of the Peg9 promoter in F1 placental tissue. Epigenetic changes in Peg9 were also found in sperm from obese fathers. We therefore propose that paternal obesity renders changes in gene expression and/or methylation throughout the placental genome, which could contribute to the reproductive problems related to fertility and to the metabolic, long-term health impact on offspring.

  5. Role of aberrant promoter hypermethylation of the CHFR gene in the clinical significance of esophageal cancer%CHFR基因启动子区超甲基化在食管癌临床意义中的研究进展

    Institute of Scientific and Technical Information of China (English)

    吴书胜; 孙玉蓓; 何义富; 季楚舒


    CHFR是一个新的有丝分裂早前期检查点基因,在有丝分裂应激时,延迟染色体凝集和中心体分离,阻止细胞进入有丝分裂期,其表达增强细胞对应激的生存能力.研究显示,CHFR在人正常组织中广泛表达,而在肿瘤组织中表达缺失,如胃癌、子宫内膜癌、宫颈癌等.CHFR基因启动子区超甲基化是其表达沉寂的主要原因,并与肿瘤的发生发展有关,在食管癌中已有相关报道.现对CHFR基因启动子区超甲基化在食管癌的发生、诊断、治疗及判断预后上的意义做一综述.%CHFR,a new mitosis early checkpoint gene, of which the expression enhances the cell survival ability to stress through delaying romosome aggregation and. Centrosome separation, blocking tne ceils into mitotic period, in mitosis stress, riesearcnes snow tnat CHFR was widely expressed in normal tissue,and was lost in tumor tissues such as gastric cancer,endometrial cancer,cervical cancer. Aberrant promoter hyp erm ethyl at ion of the CHFR gene was the main cause of silence, and was associated with the development of tumors, which was corroborated in esophageal cancer. We conducted a review of the relationship between aberrant promoter hyp erm ethyl at ion of the CHFR gene and significance in occurrence,diagnosis,treatment and prognosis of esophageal cancer.

  6. Aberrant DNA methylation of cancer-related genes in giant breast fibroadenoma: a case report

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    Orozco Javier I


    Full Text Available Abstract Introduction Giant fibroadenoma is an uncommon variant of benign breast lesions. Aberrant methylation of CpG islands in promoter regions is known to be involved in the silencing of genes (for example, tumor-suppressor genes and appears to be an early event in the etiology of breast carcinogenesis. Only hypermethylation of p16INK4a has been reported in non-giant breast fibroadenoma. In this particular case, there are no previously published data on epigenetic alterations in giant fibroadenomas. Our previous results, based on the analysis of 49 cancer-related CpG islands have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas. Case presentation A 13-year-old Hispanic girl was referred after she had noted a progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected and axillary lymph nodes were not enlarged. After surgical removal the lump was diagnosed as a giant fibroadenoma. Because of the high growth rate of this benign tumor, we decided to analyze the methylation status of 49 CpG islands related to cell growth control. We have identified the methylation of five cancer-related CpG islands in the giant fibroadenoma tissue: ESR1, MGMT, WT-1, BRCA2 and CD44. Conclusion In this case report we show for the first time the methylation analysis of a giant fibroadenoma. The detection of methylation of these five cancer-related regions indicates substantial epigenomic differences with non-giant fibroadenomas. Epigenetic alterations could explain the higher growth rate of this tumor. Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case, there exist no previous data regarding the role of methylation in giant fibroadenomas, considered by definition as a benign breast lesion.

  7. Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome

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    Chih-Jie Shen


    Full Text Available Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation between OPN mRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcine OPN was frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that the OPN promoter region −2615 to −2239 nucleotides (nt may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation of OPN promoter was found in dose-dependent response of 5-aza-dc treatment and followed the OPN mRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the −2615 to −2239 nt region cause significant downregulation of OPN promoter activity. These data suggested that methylation in the OPN promoter plays a crucial role in the regulation of OPN expression that we found in cloned pigs genome.

  8. Metastatic suppressor genes inactivated by aberrant methylation in gastric cancer

    Institute of Scientific and Technical Information of China (English)


    AIM: To screen out the differentially methylated DNA sequences between gastric primary tumor and metastatic lymph nodes, test the methylation difference of gene PTPRG between primary gastric tumor and metastatic lymph nodes, and test the regulatory function of 5-aza-2-deoxycytidine which is an agent with suppression on methylation and the level of methylation in gastric cancer cell line.METHODS: Methylated DNA sequences in genome were enriched with methylated CpG islands amplification (MCA)to undergo representational difference analysis (RDA),with MCA production of metastatic lymph nodes as tester and that of primary tumor as driver. The obtained differentially methylated fragments were cloned and sequenced to acquire the base sequence, which was analyzed with bioinformatics. With methylation-specific PCR (MSP) and RT-PCR, methylation difference of gene PTPRG was detected between primary tumor and metastatic lymph nodes in 36 cases of gastric cancer.Methylation of gene PTPRG and its regulated expression were observed in gastric cancer cell line before and after being treated with methylation-suppressive agent.RESULTS: Nineteen differentially methylated sequences were obtained and located at 5' end, exons, introns and 3' end, in which KL59 was observed to be located at 9p21 as the first exon of gene p16 and KL22 to be located at promoter region of PRPRG. KL22, aS the probes, was hybridized with driver, tester and 3-round RDA products respectively with all positive signals except with the driver. Significant difference was observed in both methylation rate of gene PTPRG and PTPRG mRNA expression rate between primary tumor and metastatic lymph nodes. Demethylation of gene PTPRG, with recovered expression of PTPRG mRNA, was observed after gastric cancer cell line being treated with methylation-suppressive agent.CONCLUSION: Difference exists in DNA methylation between primary tumor and metastatic lymph nodes of gastric cancer, with MCA-RDA as one of the good analytical

  9. Regulation of MYC gene expression by aberrant Wnt/β-catenin signaling in colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Sherri; Rennoll; Gregory; Yochum


    The Wnt/β-catenin signaling pathway controls intestinal homeostasis and mutations in components of this pathway are prevalent in human colorectal cancers(CRCs).These mutations lead to inappropriate expression of genes controlled by Wnt responsive DNA elements(WREs). T-cell factor/Lymphoid enhancer factor transcription factors bind WREs and recruit the β-catenin transcriptional co-activator to activate target gene expression. Deregulated expression of the c-MYC proto-oncogene(MYC) by aberrant Wnt/β-catenin signaling drives colorectal carcinogenesis. In this review,we discuss the current literature pertaining to the identification and characterization of WREs that control oncogenic MYC expression in CRCs. A common theme has emerged whereby these WREs often map distally to the MYC genomic locus and control MYC gene expression through long-range chromatin loops with the MYC proximal promoter. We propose that by determining which of these WREs is critical for CRC pathogenesis,novel strategies can be developed to treat individuals suffering from this disease.

  10. Gene promoters dictate histone occupancy within genes. (United States)

    Perales, Roberto; Erickson, Benjamin; Zhang, Lian; Kim, Hyunmin; Valiquett, Elan; Bentley, David


    Spt6 is a transcriptional elongation factor and histone chaperone that reassembles transcribed chromatin. Genome-wide H3 mapping showed that Spt6 preferentially maintains nucleosomes within the first 500 bases of genes and helps define nucleosome-depleted regions in 5' and 3' flanking sequences. In Spt6-depleted cells, H3 loss at 5' ends correlates with reduced pol II density suggesting enhanced transcription elongation. Consistent with its 'Suppressor of Ty' (Spt) phenotype, Spt6 inactivation caused localized H3 eviction over 1-2 nucleosomes at 5' ends of Ty elements. H3 displacement differed between genes driven by promoters with 'open'/DPN and 'closed'/OPN chromatin conformations with similar pol II densities. More eviction occurred on genes with 'closed' promoters, associated with 'noisy' transcription. Moreover, swapping of 'open' and 'closed' promoters showed that they can specify distinct downstream patterns of histone eviction/deposition. These observations suggest a novel function for promoters in dictating histone dynamics within genes possibly through effects on transcriptional bursting or elongation rate.

  11. Gene promoter hypermethylation in leukoplakia of the oral mucosa

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    Mingli Liu


    Full Text Available Mingli Liu1, Lei Feng2, Ximing Tang3, Shanchun Guo41Department of Physics, Tufts University School of Medicine, Boston, Massachussetts; 2Department of Thoracic/Head and Neck Medical Oncology, 3Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas; 4Sylvester Cancer Center, University of Miami School of Medicine, Florida, USAAbstract: To examine whether aberrant DNA methylation in the promoter region might occur earlier in tumorigenesis, particularly in premalignant lesions, we examined biopsies from 111 participants in a chemoprevention trial aimed at reversal of oral leukoplakia, using methylation-specific polymerase chain reaction for the promoter regions of the tumor suppressor gene CDKN2A (p16, the putative metastasis suppressor gene for death-associated protein kinase (DAP-K, the DNA repair gene O6-methyguanine-DNA-methyltransferase (MGMT, and the detoxification gene glutathione S-transferase p1(GSTP1. p16 promoter hypermethylation was detected in 21 of 82 (25.6%, DAP-K hypermethylation in 28 of 87 (32.2%, and MGMT hypermethylation in 32 of 106 (30.2% oral leukoplakia lesions analyzed. No aberrant methylation was found at the GSTP1 gene in 110 lesions examined. Among 68 biopsies analyzed for all three genes (p16, DAP-K, MGMT, 17 biopsies were detected with an abnormal methylation pattern at only one gene, 15 at two genes, and 8 at all three genes. Among clinical characteristics and their correlation with methylation, only alcohol consumption was correlated with DAP-K methylation (P = 0.027, while MGMT methylation was more frequent in females (P = 0.003 and nonsmokers (P = 0.0005. A significant correlation was found between p16 and DAP-K hypermethylation; p16 promoter was methylated in 14 (56% of 25 lesions with DAP-K methylation, and only 5 (11.1% of 45 DAP-K methylation-negative lesions (P = 0.0001. DAP-K aberrant methylation was also significantly correlated with MGMT methylation (16 of 31 in MGMT methylation

  12. Deciphering causal and statistical relations of molecular aberrations and gene expressions in NCI-60 cell lines

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    Li Shyh-Dar


    Full Text Available Abstract Background Cancer cells harbor a large number of molecular alterations such as mutations, amplifications and deletions on DNA sequences and epigenetic changes on DNA methylations. These aberrations may dysregulate gene expressions, which in turn drive the malignancy of tumors. Deciphering the causal and statistical relations of molecular aberrations and gene expressions is critical for understanding the molecular mechanisms of clinical phenotypes. Results In this work, we proposed a computational method to reconstruct association modules containing driver aberrations, passenger mRNA or microRNA expressions, and putative regulators that mediate the effects from drivers to passengers. By applying the module-finding algorithm to the integrated datasets of NCI-60 cancer cell lines, we found that gene expressions were driven by diverse molecular aberrations including chromosomal segments' copy number variations, gene mutations and DNA methylations, microRNA expressions, and the expressions of transcription factors. In-silico validation indicated that passenger genes were enriched with the regulator binding motifs, functional categories or pathways where the drivers were involved, and co-citations with the driver/regulator genes. Moreover, 6 of 11 predicted MYB targets were down-regulated in an MYB-siRNA treated leukemia cell line. In addition, microRNA expressions were driven by distinct mechanisms from mRNA expressions. Conclusions The results provide rich mechanistic information regarding molecular aberrations and gene expressions in cancer genomes. This kind of integrative analysis will become an important tool for the diagnosis and treatment of cancer in the era of personalized medicine.

  13. Aberrant Glycosylation of Plasma Proteins in Severe Preeclampsia Promotes Monocyte Adhesion (United States)

    Kazanjian, Avedis A.; Tinnemore, Deborah; Gafken, Philip R.; Ogata, Yuko; Napolitano, Peter G.; Stallings, Jonathan D.; Ippolito, Danielle L.


    Glycosylation of plasma proteins increases during pregnancy. Our objectives were to investigate an anti-inflammatory role of these proteins in normal pregnancies and determine whether aberrant protein glycosylation promotes monocyte adhesion in preeclampsia. Plasma was prospectively collected from nonpregnant controls and nulliparous patients in all 3 trimesters. Patients were divided into cohorts based on the applicable postpartum diagnosis. U937 monocytes were preconditioned with enzymatically deglycosylated plasma, and monocyte adhesion to endothelial cell monolayers was quantified by spectrophotometry. Plasma from nonpregnant controls, first trimester normotensives, and first trimester patients with mild preeclampsia inhibited monocyte–endothelial cell adhesion (P < .05), but plasma from first trimester patients with severe preeclampsia and second and third trimester normotensives did not. Deglycosylating plasma proteins significantly increased adhesion in all the cohorts. These results support a role of plasma glycoprotein interaction in monocyte–endothelial cell adhesion and could suggest a novel therapeutic target for severe preeclampsia. PMID:23757314

  14. From DNA Copy Number to Gene Expression: Local aberrations, Trisomies and Monosomies (United States)

    Shay, Tal

    The goal of my PhD research was to study the effect of DNA copy number changes on gene expression. DNA copy number aberrations may be local, encompassing several genes, or on the level of an entire chromosome, such as trisomy and monosomy. The main dataset I studied was of Glioblastoma, obtained in the framework of a collaboration, but I worked also with public datasets of cancer and Down's Syndrome. The molecular basis of expression changes in Glioblastoma. Glioblastoma is the most common and aggressive type of primary brain tumors in adults. In collaboration with Prof. Hegi (CHUV, Switzerland), we analyzed a rich Glioblastoma dataset including clinical information, DNA copy number (array CGH) and expression profiles. We explored the correlation between DNA copy number and gene expression at the level of chromosomal arms and local genomic aberrations. We detected known amplification and over expression of oncogenes, as well as deletion and down-regulation of tumor suppressor genes. We exploited that information to map alterations of pathways that are known to be disrupted in Glioblastoma, and tried to characterize samples that have no known alteration in any of the studied pathways. Identifying local DNA aberrations of biological significance. Many types of tumors exhibit chromosomal losses or gains and local amplifications and deletions. A region that is aberrant in many tumors, or whose copy number change is stronger, is more likely to be clinically relevant, and not just a by-product of genetic instability. We developed a novel method that defines and prioritizes aberrations by formalizing these intuitions. The method scores each aberration by the fraction of patients harboring it, its length and its amplitude, and assesses the significance of the score by comparing it to a null distribution obtained by permutations. This approach detects genetic locations that are significantly aberrant, generating a 'genomic aberration profile' for each sample. The 'genomic

  15. Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer. (United States)

    Galamb, Orsolya; Kalmár, Alexandra; Péterfia, Bálint; Csabai, István; Bodor, András; Ribli, Dezső; Krenács, Tibor; Patai, Árpád V; Wichmann, Barnabás; Barták, Barbara Kinga; Tóth, Kinga; Valcz, Gábor; Spisák, Sándor; Tulassay, Zsolt; Molnár, Béla


    The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.

  16. Oligoamine analogues in combination with 2-difluoromethylornithine synergistically induce re-expression of aberrantly silenced tumour-suppressor genes. (United States)

    Wu, Yu; Steinbergs, Nora; Murray-Stewart, Tracy; Marton, Laurence J; Casero, Robert A


    Epigenetic gene silencing is an important mechanism in the initiation and progression of cancer. Abnormal DNA CpG island hypermethylation and histone modifications are involved in aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) was the first enzyme identified to specifically demethylate H3K4 (Lys(4) of histone H3). Methylated H3K4 is an important mark associated with transcriptional activation. The flavin adenine dinucleotide-binding amine oxidase domain of LSD1 is homologous with two polyamine oxidases, SMO (spermine oxidase) and APAO (N(1)-acetylpolyamine oxidase). We have demonstrated previously that long-chain polyamine analogues, the oligoamines, are inhibitors of LSD1. In the present paper we report the synergistic effects of specific oligoamines in combination with DFMO (2-difluoromethylornithine), an inhibitor of ornithine decarboxylase, in human colorectal cancer cells. DFMO treatment depletes natural polyamines and increases the uptake of exogenous polyamines. The combination of oligoamines and DFMO results in a synergistic re-expression of aberrantly silenced tumour-suppressor genes, including SFRP2 (secreted frizzled-related protein 2), which encodes a Wnt signalling pathway antagonist and plays an anti-tumorigenic role in colorectal cancer. The treatment-induced re-expression of SFRP2 is associated with increased H3K4me2 (di-methyl H3K4) in the gene promoter. The combination of LSD1-inhibiting oligoamines and DFMO represents a novel approach to epigenetic therapy of cancer.

  17. Computational genes: a tool for molecular diagnosis and therapy of aberrant mutational phenotype

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    Ignatova Zoya


    Full Text Available Abstract Background A finite state machine manipulating information-carrying DNA strands can be used to perform autonomous molecular-scale computations at the cellular level. Results We propose a new finite state machine able to detect and correct aberrant molecular phenotype given by mutated genetic transcripts. The aberrant mutations trigger a cascade reaction: specific molecular markers as input are released and induce a spontaneous self-assembly of a wild type protein or peptide, while the mutational disease phenotype is silenced. We experimentally demostrated in in vitro translation system that a viable protein can be autonomously assembled. Conclusion Our work demostrates the basic principles of computational genes and particularly, their potential to detect mutations, and as a response thereafter administer an output that suppresses the aberrant disease phenotype and/or restores the lost physiological function.

  18. Improved antisense oligonucleotide design to suppress aberrant SMN2 gene transcript processing: towards a treatment for spinal muscular atrophy.

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    Chalermchai Mitrpant

    Full Text Available Spinal muscular atrophy (SMA is caused by loss of the Survival Motor Neuron 1 (SMN1 gene, resulting in reduced SMN protein. Humans possess the additional SMN2 gene (or genes that does produce low level of full length SMN, but cannot adequately compensate for loss of SMN1 due to aberrant splicing. The majority of SMN2 gene transcripts lack exon 7 and the resultant SMNΔ7 mRNA is translated into an unstable and non-functional protein. Splice intervention therapies to promote exon 7 retention and increase amounts of full-length SMN2 transcript offer great potential as a treatment for SMA patients. Several splice silencing motifs in SMN2 have been identified as potential targets for antisense oligonucleotide mediated splice modification. A strong splice silencer is located downstream of exon 7 in SMN2 intron 7. Antisense oligonucleotides targeting this motif promoted SMN2 exon 7 retention in the mature SMN2 transcripts, with increased SMN expression detected in SMA fibroblasts. We report here systematic optimisation of phosphorodiamidate morpholino oligonucleotides (PMO that promote exon 7 retention to levels that rescued the phenotype in a severe mouse model of SMA after intracerebroventricular delivery. Furthermore, the PMO gives the longest survival reported to date after a single dosing by ICV.

  19. Hypermethylation and aberrant expression of secreted fizzled-related protein genes in pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Xian-Min Bu; Cheng-Hai Zhao; Ning Zhang; Feng Gao; Shuai Lin; Xian-Wei Dai


    AIM:To determine the methylation status and aberrant expression of some secreted frizzled-related protein (SFRP) genes in pancreatic cancer and explore their role in pancreatic carcinogenesis. METHODS:Methylation status and expression of SFRP genes were detected by methylation-specific PCR (MSPCR) and reverse-transcription PCR (RT-PCR) respectively. RESULTS:The frequencies of methylation for SFRP genes 1,2,4,5 were 70%, 48.3%,60% and 76.7% in pancreatic cancer samples, and 21.7%, 20%,10% and 36.7% in matched cancer adjacent normal tissue samples,respectively (χ2=28.23,P<0.0001 for SFRP gene 1; χ2=10.71,P=0.001 for SFRP gene 2;χ2=32.97,P<0.0001 for SFRP gene 4;χ2=19.55,P<0.0001 for SFRP gene 5). Expression loss of SFRP genes 1,2,4 and 5 was found in 65%,40%,55% and 71.7% of 60 pancreatic cancer samples, and 25%,15%,18.3% and 31.7% of matched cancer adjacent normal tissue samples,respectively (χ2=19.39,P<0.0001 for SFRP gene 1;χ2=9.40,P=0.002 for SFRP gene 2;χ2=17.37,P<0.0001 for SFRP gene 4;χ2=19.22,P<0.0001 for SFRP gene 5).SFRP gene 1 was methylated but not expressed in PC-3 and PANC-1,SFRP gene 2 was methylated but not expressed in PANC-1 and CFPAC-1,SFRP gene 4 was methylated but not expressed in PC-3,and SFRP gene 5 was methylated but not expressed in CFPAC-1. CONCLUSION:Hypermethylation and aberrant expression of SFRP genes are common in pancreatic cancer,which may be involved in pancreatic carcinogenesis.

  20. Sonic Hedgehog Signaling Affected by Promoter Hypermethylation Induces Aberrant Gli2 Expression in Spina Bifida. (United States)

    Lu, Xiao-Lin; Wang, Li; Chang, Shao-Yan; Shangguan, Shao-Fang; Wang, Zhen; Wu, Li-Hua; Zou, Ji-Zhen; Xiao, Ping; Li, Rui; Bao, Yi-Hua; Qiu, Z-Y; Zhang, Ting


    GLI2 is a key mediator of the sonic hedgehog (Shh) signaling pathway and plays an important role in neural tube development during vertebrate embryogenesis; however, the role of gli2 in human folate-related neural tube defects remains unclear. In this study, we compared methylation status and polymorphisms of gli2 between spina bifida patients and a control group to explore the underlying mechanisms related to folate deficiency in spina bifida. No single nucleotide polymorphism was found to be significantly different between the two groups, although gli2 methylation levels were significantly increased in spina bifida samples, accompanied by aberrant GLI2 expression. Moreover, a prominent negative correlation was found between the folate level in brain tissue and the gli2 methylation status (r = -0.41, P = 0.014), and gli2 hypermethylation increased the risk of spina bifida with an odds ratio of 12.45 (95 % confidence interval: 2.71-57.22, P = 0.001). In addition, we established a cell model to illustrate the effect of gli2 expression and the accessibility of chromatin affected by methylation. High gli2 and gli1 mRNA expression was detected in 5-Aza-treated cells, while gli2 hypermethylation resulted in chromatin inaccessibility and a reduced association with nuclear proteins containing transcriptional factors. More meaningful to the pathway, the effect gene of the Shh pathway, gli1, was found to have a reduced level of expression along with a decreased expression of gli2 in our cell model. Aberrant high methylation resulted in the low expression of gli2 in spina bifida, which was affected by the change in chromatin status and the capacity of transcription factor binding.

  1. Chromosomal Aberrations and DNA Repair Gene Variants in a Radon-exposed Population

    Energy Technology Data Exchange (ETDEWEB)

    Kiuru, A.; Lindholm, C.; Koivistoinen, A.; Salomaa, S.


    Polymorphisms of XRCC1 (X-ray repair cross-complementing group 1), XRCC3 (X-ray repair cross-complementing group 3), and hOGG1 (the human homologue of the yeast OGG1 gene) DNA repair genes have been associated with altered DNA repair capacity and risk of various cancers. In the present study our goal was to clarify the influence of various DNA repair gene variants on the frequency of chromosomal aberrations (CA) in subjects exposed to residential radon. The study group of 84 non-smoking, healthy individuals exposed to domestic radon were analysed using the fluorescence in-situ hybridization (FISH) technique. No association between radon concentration and CA frequencies was observed. However, a significant increase with age was shown as well as a large variability in translocation frequencies between individuals within the same age group. In order to investigate the role of individual susceptibility to this variation genotypes of DNA repair genes XRCC1 (codons 194, 280 and 399), XRCC3 (codon 241) and hOGG1 (codon 326) were determined from leukocyte DNA using methods based on polymerase chain reaction. Multiple regression analysis was applied to evaluate the effect of the polymorphisms and the other confounding factors (age, exposure to randon etc) to the frequency of CA. The preliminary statistical analyses showed that the different gene appeared not to be related to a pronounced increase in chromosome aberration frequencies observed by FISH painting. However, the analysis indicated that the homozygous variant of XRCC3 codon 241 was associated (P<0.05) with two-ways translocations in conjunction with age. Larger studies, both with regard to the cohort and the number of gene variants are needed to elucidate the influence of other DNA repair variants to the yield of chromosomal aberrations. The results indicate that the chromosomal translocations accumulated by age (spontaneous background) may be partly explained by defects in homologous recombination repair. (Author

  2. Aberrant and unstable expression of immunoglobulin genes in persons infected with human immunodeficiency virus. (United States)

    Bessudo, A; Rassenti, L; Havlir, D; Richman, D; Feigal, E; Kipps, T J


    We examined the IgM VH gene subgroup use-distribution in serial blood samples of 37 human immunodeficiency virus (HIV)-infected patients and a group of HIV-seronegative healthy adults. The IgM VH gene repertoires of healthy adults were relatively similar to one another and were stable over time. In contrast, individuals infected with HIV had IgM VH gene repertoires that were significantly more heterogeneous and unstable. Persons at early stages of HIV infection generally had abnormal expression levels of Ig VH3 genes and frequently displayed marked fluctuations in the relative expression levels of this VH gene subgroup over time. In contrast, persons with established acquired immunodeficiency syndrome (AIDS) had a significantly lower incidence of abnormalities in Ig VH3 expression levels, although continued to display abnormalities and instability in the expression levels of the smaller Ig VH gene subgroups. Moreover, the skewing and/or fluctuations in the expressed-IgM VH gene repertoire appeared greatest for persons at earlier stages of HIV infection. These studies show that persons infected with HIV have aberrant and unstable expression of immunoglobulin genes suggestive of a high degree humoral immune dysregulation and ongoing humoral immune responses to HIV-associated antigens and superantigens.

  3. Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

    Institute of Scientific and Technical Information of China (English)


    High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

  4. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal


    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...... knockout and strong overexpression. However, applications such as metabolic optimization and control analysis necessitate a continuous set of expression levels with only slight increments in strength to cover a specific window around the wildtype expression level of the studied gene; this requirement can...... be met by using promoter libraries. This approach generally consists of inserting a library of promoters in front of the gene to be studied, whereby the individual promoters might deviate either in their spacer sequences or bear slight deviations from the consensus sequence of a vegetative promoter. Here...

  5. Epigenomic elements enriched in the promoters of autoimmunity susceptibility genes. (United States)

    Dozmorov, Mikhail G; Wren, Jonathan D; Alarcón-Riquelme, Marta E


    Genome-wide association studies have identified a number of autoimmune disease-susceptibility genes. Whether or not these loci share any regulatory or functional elements, however, is an open question. Finding such common regulators is of considerable research interest in order to define systemic therapeutic targets. The growing amount of experimental genomic annotations, particularly those from the ENCODE project, provide a wealth of opportunities to search for such commonalities. We hypothesized that regulatory commonalities might not only delineate a regulatory landscape predisposing to autoimmune diseases, but also define functional elements distinguishing specific diseases. We further investigated if, and how, disease-specific epigenomic elements can identify novel genes yet to be associated with the diseases. We evaluated transcription factors, histone modifications, and chromatin state data obtained from the ENCODE project for statistically significant over- or under-representation in the promoters of genes associated with Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), and Systemic Sclerosis (SSc). We identified BATF, BCL11A, IRF4, NFkB, PAX5, and PU.1 as transcription factors over-represented in SLE- and RA-susceptibility gene promoters. H3K4me1 and H3K4me2 epigenomic marks were associated with SLE susceptibility genes, and H3K9me3 was common to both SLE and RA. In contrast to a transcriptionally active signature in SLE and RA, SSc-susceptibility genes were depleted in activating epigenomic elements. Using epigenomic elements enriched in SLE and RA, we identified additional immune and B cell signaling-related genes with the same elements in their promoters. Our analysis suggests common and disease-specific epigenomic elements that may define novel therapeutic targets for controlling aberrant activation of autoimmune susceptibility genes.

  6. Aberrant gene expression patterns in extraembryonic tissue from cloned porcine embryos. (United States)

    Park, Mi-Ryung; Im, Gi-Sun; Kim, Sung Woo; Hwang, Seongsoo; Park, Jae-Hong; Kim, Hyun; Do, Yoon Jung; Park, Soo Bon; Yang, Bo-Suck; Song, Young Min; Cho, Jae-Hyeon; Ko, Yeoung-Gyu


    The abnormal development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is considered to be associated with consequent changes in gene expression following errors in epigenetic reprogramming. In this study, we carried out SCNT using donor fibroblast cells derived from 3-way hybrids (Landrace×Duroc×Yorkshire). A total of 655 SCNT embryos were transferred, and 6.97±2.3 cloned fetuses were successfully recovered from three surrogates at gestational day 30. An analysis of the 6.97±2.3 cloned embryos revealed that most had severe extraembryonic defects. The extraembryonic tissue from the SCNT embryos was abnormally small compared with that of the control. To investigate the differentially expressed genes between the SCNT and control extraembryonic tissues, we compared the gene expression profiles of the extraembryonic tissues from gestational day 30 cloned pig embryos with those from the control using an annealing control primer-based GeneFishing polymerase chain reaction. As a result, we found that a total of 50 genes were differentially expressed by utilizing 120 ACPs, 38 genes of which were known. Among them, 26 genes were up-regulated, whereas 12 genes were down-regulated. Real-time RT-PCR showed that apoptosis-related genes were expressed significantly higher in SCNT extraembryonic tissue than in the control, whereas metabolism-related genes were expressed at significantly lower levels in the SCNT extraembryonic tissue. These observations strongly indicate that early gestational death of SCNT embryo is caused, at least in part, by the disruption of developing extraembryonic tissues as a result of aberrant gene expression, which results in abnormal apoptosis and metabolism.

  7. Aberrant DNA methylation of ESR1 and p14ARF genes could be useful as prognostic indicators in osteosarcoma

    Directory of Open Access Journals (Sweden)

    Sonaglio V


    Full Text Available Viviane Sonaglio,1 Ana C de Carvalho,2 Silvia R C Toledo,3,4 Carolina Salinas-Souza,3,4 André L Carvalho,5 Antonio S Petrilli,3 Beatriz de Camargo,6 André L Vettore21Pediatrics Department, A C Camargo Hospital, São Paulo, Brazil; 2Biological Science Department, Federal University of São Paulo, Diadema, Brazil; 3Department of Pediatrics, Pediatric Oncology Institute, GRAACC/Federal University of São Paulo, São Paulo, Brazil; 4Department of Morphology and Genetics, Federal University of São Paulo, São Paulo, Brazil; 5Department of Head and Neck Surgery, PIO XII Foundation, Barretos Cancer Hospital, Barretos, São Paulo, Brazil; 6Research Program Pediatric Oncology Program, CPNq, Instituto Nacional do Cancer, Rio de Janeiro, BrazilAbstract: Osteosarcoma (OS is the eighth most common form of childhood and adolescence cancer. Approximately 10%–20% of patients present metastatic disease at diagnosis and the 5-year overall survival remains around 70% for nonmetastatic patients and around 30% for metastatic patients. Metastatic disease at diagnosis and the necrosis grade induced by preoperative treatment are the only well-established prognostic factors for osteosarcoma. The DNA aberrant methylation is a frequent epigenetic alteration in humans and has been described as a molecular marker in different tumor types. This study evaluated the DNA aberrant methylation status of 18 genes in 34 OS samples without previous chemotherapy treatment and in four normal bone specimens and compared the methylation profile with clinicopathological characteristics of the patients. We were able to define a three-gene panel (AIM1, p14ARF, and ESR1 in which methylation was correlated with OS cases. The hypermethylation of p14ARF showed a significant association with the absence of metastases at diagnoses, while ESR1 hypermethylation was marginally associated with worse overall survival. This study demonstrated that aberrant promoter methylation is a common event

  8. Clinical Omics Analysis of Colorectal Cancer Incorporating Copy Number Aberrations and Gene Expression Data

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Yoshida


    Full Text Available Background: Colorectal cancer (CRC is one of the most frequently occurring cancers in Japan, and thus a wide range of methods have been deployed to study the molecular mechanisms of CRC. In this study, we performed a comprehensive analysis of CRC, incorporating copy number aberration (CRC and gene expression data. For the last four years, we have been collecting data from CRC cases and organizing the information as an “omics” study by integrating many kinds of analysis into a single comprehensive investigation. In our previous studies, we had experienced difficulty in finding genes related to CRC, as we observed higher noise levels in the expression data than in the data for other cancers. Because chromosomal aberrations are often observed in CRC, here, we have performed a combination of CNA analysis and expression analysis in order to identify some new genes responsible for CRC. This study was performed as part of the Clinical Omics Database Project at Tokyo Medical and Dental University. The purpose of this study was to investigate the mechanism of genetic instability in CRC by this combination of expression analysis and CNA, and to establish a new method for the diagnosis and treatment of CRC. Materials and methods: Comprehensive gene expression analysis was performed on 79 CRC cases using an Affymetrix Gene Chip, and comprehensive CNA analysis was performed using an Affymetrix DNA Sty array. To avoid the contamination of cancer tissue with normal cells, laser micro-dissection was performed before DNA/RNA extraction. Data analysis was performed using original software written in the R language. Result: We observed a high percentage of CNA in colorectal cancer, including copy number gains at 7, 8q, 13 and 20q, and copy number losses at 8p, 17p and 18. Gene expression analysis provided many candidates for CRC-related genes, but their association with CRC did not reach the level of statistical significance. The combination of CNA and gene

  9. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes. (United States)

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I


    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations.

  10. Dopamine signaling leads to loss of Polycomb repression and aberrant gene activation in experimental parkinsonism.

    Directory of Open Access Journals (Sweden)

    Erik Södersten


    Full Text Available Polycomb group (PcG proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation. The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP experiments showed that increased H3K27me3S28p was accompanied by reduced PcG binding to regulatory regions of genes. An analysis of the genome wide distribution of L-DOPA-induced H3K27me3S28 phosphorylation by ChIP sequencing (ChIP-seq in combination with expression analysis by RNA-sequencing (RNA-seq showed that the induction of H3K27me3S28p correlated with increased expression of a subset of PcG repressed genes. We found that induction of H3K27me3S28p persisted during chronic L-DOPA administration to parkisonian mice and correlated with aberrant gene expression. We propose that dopaminergic transmission can activate PcG repressed genes in the adult brain and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson's disease.

  11. Aberrations of ERBB2 and TOP2A Genes in Breast Cancer

    DEFF Research Database (Denmark)

    Nielsen, Kirsten Vang; Müller, Sven; Møller, Susanne;


    Copy number changes in TOP2A have frequently been linked to ERBB2 (HER2) amplified breast cancers. To study this relationship, copy number changes of ERBB2 and TOP2A were investigated by fluorescence in situ hybridization (FISH) in two cell lines; one characterized by having amplification of both...... genes and the other by having amplification of ERBB2 and deletion of TOP2A. The characteristics are compared to findings on paired ERBB2 and TOP2A data from 649 patients with invasive breast cancer from a previously published biomarker study. The physical localization of FISH signals in metaphase...... compared to TOP2A. In the majority of breast cancer patients, simultaneous aberration of ERBB2 and TOP2A is not explained by simple co-amplification....

  12. HCV core protein-induced down-regulation of microRNA-152 promoted aberrant proliferation by regulating Wnt1 in HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Shifeng Huang

    Full Text Available BACKGROUND: Hepatitis C virus (HCV has been reported to regulate cellular microRNAs (miRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC, but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152 by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells. METHODS: MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR. Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting. RESULTS: HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT-PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001, miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels. CONCLUSION: These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1

  13. Molecular Network Analysis Suggests Aberrant CREB-Mediated Gene Regulation in the Alzheimer Disease Hippocampus

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    Jun-ichi Satoh


    Full Text Available The pathogenesis of Alzheimer disease (AD involves the complex interaction between genetic and environmental factors affecting multiple cellular pathways. Recent advances in systems biology provide a system-level understanding of AD by elucidating the genome-wide molecular interactions. By using KeyMolnet, a bioinformatics tool for analyzing molecular interactions on the curated knowledgebase, we characterized molecular network of 2,883 all stages of AD-related genes (ADGs and 559 incipient AD-related genes (IADGs identified by global gene expression profiling of the hippocampal CA1 region of AD brains in terms of significant clinical and pathological correlations (Blalock et al., Proc Natl Acad Sci USA 101: 2173-2178, 2004. By the common upstream search, KeyMolnet identified cAMP-response element-binding protein (CREB as the principal transcription factor exhibiting the most significant relevance to molecular networks of both ADGs and IADGs. The CREB-regulated transcriptional network included upregulated and downregulated sets of ADGs and IADGs, suggesting an involvement of generalized deregulation of the CREB signaling pathway in the pathophysiology of AD, beginning at the early stage of the disease. To verify the in silico observations in vivo, we conducted immunohistochemical studies of 11 AD and 13 age-matched control brains by using anti-phoshorylated CREB (pCREB antibody. An abnormal accumulation of pCREB imunoreactivity was identified in granules of granulovacuolar degeneration (GVD in the hippocampal neurons of AD brains. These observations suggest that aberrant CREB-mediated gene regulation serves as a molecular biomarker of AD-related pathological processes, and support the hypothesis that sequestration of pCREB in GVD granules is in part responsible for deregulation of CREB-mediated gene expression in AD hippocampus.

  14. Aberrant trafficking of NSCLC-associated EGFR mutants through the endocytic recycling pathway promotes interaction with Src@

    Directory of Open Access Journals (Sweden)

    Band Vimla


    Full Text Available Abstract Background Epidermal growth factor receptor (EGFR controls a wide range of cellular processes, and altered EGFR signaling contributes to human cancer. EGFR kinase domain mutants found in non-small cell lung cancer (NSCLC are constitutively active, a trait critical for cell transformation through activation of downstream pathways. Endocytic trafficking of EGFR is a major regulatory mechanism as ligand-induced lysosomal degradation results in termination of signaling. While numerous studies have examined mutant EGFR signaling, the endocytic traffic of mutant EGFR within the NSCLC milieu remains less clear. Results This study shows that mutant EGFRs in NSCLC cell lines are constitutively endocytosed as shown by their colocalization with the early/recycling endosomal marker transferrin and the late endosomal/lysosomal marker LAMP1. Notably, mutant EGFRs, but not the wild-type EGFR, show a perinuclear accumulation and colocalization with recycling endosomal markers such as Rab11 and EHD1 upon treatment of cells with endocytic recycling inhibitor monensin, suggesting that mutant EGFRs preferentially traffic through the endocytic recycling compartments. Importantly, monensin treatment enhanced the mutant EGFR association and colocalization with Src, indicating that aberrant transit through the endocytic recycling compartment promotes mutant EGFR-Src association. Conclusion The findings presented in this study show that mutant EGFRs undergo aberrant traffic into the endocytic recycling compartment which allows mutant EGFRs to engage in a preferential interaction with Src, a critical partner for EGFR-mediated oncogenesis.

  15. Prioritizing cancer-related genes with aberrant methylation based on a weighted protein-protein interaction network

    Directory of Open Access Journals (Sweden)

    Lv Jie


    Full Text Available Abstract Background As an important epigenetic modification, DNA methylation plays a crucial role in the development of mammals and in the occurrence of complex diseases. Genes that interact directly or indirectly may have the same or similar functions in the biological processes in which they are involved and together contribute to the related disease phenotypes. The complicated relations between genes can be clearly represented using network theory. A protein-protein interaction (PPI network offers a platform from which to systematically identify disease-related genes from the relations between genes with similar functions. Results We constructed a weighted human PPI network (WHPN using DNA methylation correlations based on human protein-protein interactions. WHPN represents the relationships of DNA methylation levels in gene pairs for four cancer types. A cancer-associated subnetwork (CASN was obtained from WHPN by selecting genes associated with seed genes which were known to be methylated in the four cancers. We found that CASN had a more densely connected network community than WHPN, indicating that the genes in CASN were much closer to seed genes. We prioritized 154 potential cancer-related genes with aberrant methylation in CASN by neighborhood-weighting decision rule. A function enrichment analysis for GO and KEGG indicated that the optimized genes were mainly involved in the biological processes of regulating cell apoptosis and programmed cell death. An analysis of expression profiling data revealed that many of the optimized genes were expressed differentially in the four cancers. By examining the PubMed co-citations, we found 43 optimized genes were related with cancers and aberrant methylation, and 10 genes were validated to be methylated aberrantly in cancers. Of 154 optimized genes, 27 were as diagnostic markers and 20 as prognostic markers previously identified in literature for cancers and other complex diseases by searching Pub

  16. DNA methylation directly silences genes with non-CpG island promoters and establishes a nucleosome occupied promoter. (United States)

    Han, Han; Cortez, Connie C; Yang, Xiaojing; Nichols, Peter W; Jones, Peter A; Liang, Gangning


    Despite the fact that 45% of all human gene promoters do not contain CpG islands, the role of DNA methylation in control of non-CpG island promoters is controversial and its relevance in normal and pathological processes is poorly understood. Among the few studies which investigate the correlation between DNA methylation and expression of genes with non-CpG island promoters, the majority do not support the view that DNA methylation directly leads to transcription silencing of these genes. Our reporter assays and gene reactivation by 5-aza-2'-deoxycytidine, a DNA demethylating agent, show that DNA methylation occurring at CpG poor LAMB3 promoter and RUNX3 promoter 1(RUNX3 P1) can directly lead to transcriptional silencing in cells competent to express these genes in vitro. Using Nucleosome Occupancy Methylome- Sequencing, NOMe-Seq, a single-molecule, high-resolution nucleosome positioning assay, we demonstrate that active, but not inactive, non-CpG island promoters display a nucleosome-depleted region (NDR) immediately upstream of the transcription start site (TSS). Furthermore, using NOMe-Seq and clonal analysis, we show that in RUNX3 expressing 623 melanoma cells, RUNX3 P1 has two distinct chromatin configurations: one is unmethylated with an NDR upstream of the TSS; another is methylated and nucleosome occupied, indicating that RUNX3 P1 is monoallelically methylated. Together, these results demonstrate that the epigenetic signatures comprising DNA methylation, histone marks and nucleosome occupancy of non-CpG island promoters are almost identical to CpG island promoters, suggesting that aberrant methylation patterns of non-CpG island promoters may also contribute to tumorigenesis and should therefore be included in analyses of cancer epigenetics.

  17. Focal chromosomal copy number aberrations identify CMTM8 and GPR177 as new candidate driver genes in osteosarcoma.

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    Joeri Both

    Full Text Available Osteosarcoma is an aggressive bone tumor that preferentially develops in adolescents. The tumor is characterized by an abundance of genomic aberrations, which hampers the identification of the driver genes involved in osteosarcoma tumorigenesis. Our study aims to identify these genes by the investigation of focal copy number aberrations (CNAs, <3 Mb. For this purpose, we subjected 26 primary tumors of osteosarcoma patients to high-resolution single nucleotide polymorphism array analyses and identified 139 somatic focal CNAs. Of these, 72 had at least one gene located within or overlapping the focal CNA, with a total of 94 genes. For 84 of these genes, the expression status in 31 osteosarcoma samples was determined by expression microarray analysis. This enabled us to identify the genes of which the over- or underexpression was in more than 35% of cases in accordance to their copy number status (gain or loss. These candidate genes were subsequently validated in an independent set and furthermore corroborated as driver genes by verifying their role in other tumor types. We identified CMTM8 as a new candidate tumor suppressor gene and GPR177 as a new candidate oncogene in osteosarcoma. In osteosarcoma, CMTM8 has been shown to suppress EGFR signaling. In other tumor types, CMTM8 is known to suppress the activity of the oncogenic protein c-Met and GPR177 is known as an overexpressed upstream regulator of the Wnt-pathway. Further studies are needed to determine whether these proteins also exert the latter functions in osteosarcoma tumorigenesis.

  18. Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation. (United States)

    Katoh, Y; Katoh, M


    Hedgehog signaling is aberrantly activated in glioma, medulloblastoma, basal cell carcinoma, lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and other tumors. Hedgehog signals activate GLI family members via Smoothened. RTK signaling potentiates GLI activity through PI3K-AKT-mediated GSK3 inactivation or RAS-STIL1-mediated SUFU inactivation, while GPCR signaling to Gs represses GLI activity through adenylate cyclase-mediated PKA activation. GLI activators bind to GACCACCCA motif to regulate transcription of GLI1, PTCH1, PTCH2, HHIP1, MYCN, CCND1, CCND2, BCL2, CFLAR, FOXF1, FOXL1, PRDM1 (BLIMP1), JAG2, GREM1, and Follistatin. Hedgehog signals are fine-tuned based on positive feedback loop via GLI1 and negative feedback loop via PTCH1, PTCH2, and HHIP1. Excessive positive feedback or collapsed negative feedback of Hedgehog signaling due to epigenetic or genetic alterations leads to carcinogenesis. Hedgehog signals induce cellular proliferation through upregulation of N-Myc, Cyclin D/E, and FOXM1. Hedgehog signals directly upregulate JAG2, indirectly upregulate mesenchymal BMP4 via FOXF1 or FOXL1, and also upregulate WNT2B and WNT5A. Hedgehog signals induce stem cell markers BMI1, LGR5, CD44 and CD133 based on cross-talk with WNT and/or other signals. Hedgehog signals upregulate BCL2 and CFLAR to promote cellular survival, SNAI1 (Snail), SNAI2 (Slug), ZEB1, ZEB2 (SIP1), TWIST2, and FOXC2 to promote epithelial-to-mesenchymal transition, and PTHLH (PTHrP) to promote osteolytic bone metastasis. KAAD-cyclopamine, Mu-SSKYQ-cyclopamine, IPI-269609, SANT1, SANT2, CUR61414 and HhAntag are small-molecule inhibitors targeted to Smoothened, GANT58, GANT61 to GLI1 and GLI2, and Robot-nikinin to SHH. Hedgehog signaling inhibitors should be used in combination with RTK inhibitors, GPCR modulators, and/or irradiation for cancer therapy.

  19. Focal chromosomal copy number aberrations identify CMTM8 and GPR177 as new candidate driver genes in osteosarcoma. (United States)

    Both, Joeri; Krijgsman, Oscar; Bras, Johannes; Schaap, Gerard R; Baas, Frank; Ylstra, Bauke; Hulsebos, Theo J M


    Osteosarcoma is an aggressive bone tumor that preferentially develops in adolescents. The tumor is characterized by an abundance of genomic aberrations, which hampers the identification of the driver genes involved in osteosarcoma tumorigenesis. Our study aims to identify these genes by the investigation of focal copy number aberrations (CNAs, GPR177 as a new candidate oncogene in osteosarcoma. In osteosarcoma, CMTM8 has been shown to suppress EGFR signaling. In other tumor types, CMTM8 is known to suppress the activity of the oncogenic protein c-Met and GPR177 is known as an overexpressed upstream regulator of the Wnt-pathway. Further studies are needed to determine whether these proteins also exert the latter functions in osteosarcoma tumorigenesis.

  20. Aberrant hypomethylation-mediated CD147 overexpression promotes aggressive tumor progression in human prostate cancer. (United States)

    Liang, Yu-Xiang; Mo, Ru-Jun; He, Hui-Chan; Chen, Jia-Hong; Zou, Jun; Han, Zhao-Dong; Lu, Jian-Ming; Cai, Chao; Zeng, Yan-Ru; Zhong, Wei-De; Wu, Chin-Lee


    Our previous study revealed the potential role of CD147 in human prostate cancer (PCa). Here, we investigated the CD147 promoter methylation status and the correlation with tumorigenicity in human PCa. CD147 mRNA and protein expression levels were both significantly higher in the 4 PCa cell lines, than in the 2 non-tumorigenic benign human prostatic epithelial cell lines (all PCD147 in PCa cell lines with significant CD147 expression as compared to non-tumorigenic benign human prostatic epithelial cell lines slowly expressing CD147. Additionally, the treatment of methylated cell lines with 5-aza-2'-deoxycytidine increased CD147 expression significantly in low-expressing cell lines and also activated the expression of matrix metalloproteinase (MMP)-2, which may be one of the most important downstream targets of CD147. Furthermore, PCa tissues displayed decreased DNA methylation in the promoter region of CD147 compared to the corresponding non-cancerous prostate tissues, and methylation intensity correlated inversely with the CD147 mRNA levels. There was a significant negative correlation between CD147 mRNA levels and the number of methylated sites in PCa tissues (r=-0.467, PCD147 may be one of the regulatory mechanisms involved in the cancer-related overexpression of CD147 and may play a crucial role in the tumorigenesis of PCa.

  1. Novel Genomic Aberrations in Testicular Germ Cell Tumors by Array-CGH, and Associated Gene Expression Changes

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    Rolf I. Skotheim


    Full Text Available Introduction: Testicular germ cell tumors of adolescent and young adult men (TGCTs generally have near triploid and complex karyotypes. The actual genes driving the tumorigenesis remain essentially to be identified. Materials and Methods: To determine the detailed DNA copy number changes, and investigate their impact on gene expression levels, we performed an integrated microarray profiling of TGCT genomes and transcriptomes. We analyzed 17 TGCTs, three precursor lesions, and the embryonal carcinoma cell lines, NTERA2 and 2102Ep, by comparative genomic hybridization microarrays (array-CGH, and integrated the data with transcriptome profiles of the same samples. Results: The gain of chromosome arm 12p was, as expected, the most common aberration, and we found CCND2, CD9, GAPD, GDF3, NANOG, and TEAD4 to be the therein most highly over-expressed genes. Additional frequent genomic aberrations revealed some shorter chromosomal segments, which are novel to TGCT, as well as known aberrations for which we here refined boundaries. These include gains from 7p15.2 and 21q22.2, and losses of 4p16.3 and 22q13.3. Integration of DNA copy number information to gene expression profiles identified that BRCC3, FOS, MLLT11, NES, and RAC1 may act as novel oncogenes in TGCT. Similarly, DDX26, ERCC5, FZD4, NME4, OPTN, and RB1 were both lost and under-expressed genes, and are thus putative TGCT suppressor genes. Conclusion: This first genome-wide integrated array-CGH and gene expression profiling of TGCT provides novel insights into the genome biology underlying testicular tumorigenesis.

  2. Prognostic significance of numeric aberrations of genes for thymidylate synthase, thymidine phosphorylase and dihydrofolate reductase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Søren Astrup; Vainer, B.; Witton, C.J.


    ) in colorectal cancer, and to evaluate its prognostic significance following adjuvant chemotherapy, since these enzymes are closely related to efficacy of 5-fluorouracil (5FU). PATIENTS AND METHODS: Consecutive patients (n = 314), who were completely resected for colorectal cancer stages II-IV and adjuvantly...... treated with 5-FU were retrospectively evaluated. Paraffin embedded tumor specimens were assessed for gene copies per nucleus of TYMS, TP and DHFR by fluorescence in situ hybridisation (FISH) using specific peptide nucleic acid probes. Outcome according to gene copies per nucleus above and below...... 1.1-2.2; p = 0.02) and death (HR = 1.6; 95%CI 1.1-2.3; p = 0.01). No significant differences in outcome appeared according to TP and DHFR gene ratios. CONCLUSION: Aberration of TYMS gene is of significance to expression of TYMS, which may influence the biology and 5-FU sensitivity of colorectal...

  3. Aberrant expression of Notch1, HES1, and DTX1 genes in glioblastoma formalin-fixed paraffin-embedded tissues. (United States)

    Narayanappa, Rajeswari; Rout, Pritilata; Aithal, Madhuri G S; Chand, Ashis Kumar


    Glioblastoma is the most common malignant brain tumor accounting for more than 54 % of all gliomas. Despite aggressive treatments, median survival remains less than 1 year. This might be due to the unavailability of effective molecular diagnostic markers and targeted therapy. Thus, it is essential to discover molecular mechanisms underlying disease by identifying dysregulated pathways involved in tumorigenesis. Notch signaling is one such pathway which plays an important role in determining cell fates. Since it is found to play a critical role in many cancers, we investigated the role of Notch genes in glioblastoma with an aim to identify biomarkers that can improve diagnosis. Using real-time PCR, we assessed the expression of Notch genes including receptors (Notch1, Notch2, Notch3, and Notch4), ligands (JAG1, JAG2, and DLL3), downstream targets (HES1 and HEY2), regulator Deltex1 (DTX1), inhibitor NUMB along with transcriptional co-activator MAML1, and a component of gamma-secretase complex APH1A in 15 formalin-fixed paraffin-embedded (FFPE) patient samples. Relative quantification was done by the 2(-ΔΔCt) method; the data are presented as fold change in gene expression normalized to an internal control gene and relative to the calibrator. The data revealed aberrant expression of Notch genes in glioblastoma compared to normal brain. More than 85 % of samples showed high Notch1 (P = 0.0397) gene expression and low HES1 (P = 0.011) and DTX1 (P = 0.0001) gene expression. Our results clearly show aberrant expression of Notch genes in glioblastoma which can be used as putative biomarkers together with histopathological observation to improve diagnosis, therapeutic strategies, and patient prognosis.

  4. Association between promoter methylation of DAPK gene and HNSCC: A meta-analysis (United States)

    Cai, Fucheng; Xiao, Xiyue; Niu, Xun; Zhong, Yi


    Background The death-associated protein kinase (DAPK) is a tumor suppressor gene, which is a mediator of cell death of INF-γ–induced apoptosis. Aberrant methylation of DAPK promoter has been reported in patients with head and neck squamous cell carcinoma (HNSCC). However, the results of these studies are inconsistent. Hence, the present study aimed to evaluate the association between the promoter methylation of DAPK gene and HNSCC. Methods Relevant studies were systematically searched in PubMed, Web of Science, Ovid, and Embase. The association between DAPK promoter methylation and HNSCC was assessed by odds ratio (ORs) and 95% confidence intervals (CI). To evaluate the potential sources of heterogeneity, we conducted the meta-regression analysis and subgroup analysis. Results Eighteen studies were finally included in the meta-analysis. The frequency of DAPK promoter methylation in patients with HNSCC was 4.09-fold higher than the non-cancerous controls (OR = 3.96, 95%CI = 2.26–6.95). A significant association between DAPK promoter methylation and HNSCC was found among the Asian region and the Non-Asia region (Asian region, OR = 4.43, 95% CI = 2.29–8.58; Non-Asia region, OR = 3.39, 95% CI = 1.18–9.78). In the control source, the significant association between DAPK promoter methylation and HNSCC was seen among the autologous group and the heterogeneous group (autologous group, OR = 2.71, 95% CI = 1.49–4.93; heterogeneous group, OR = 9.50, 95% CI = 2.98–30.27). DAPK promoter methylation was significantly correlated with alcohol status (OR = 1.85, 95% CI = 1.07–3.21). Conclusion The results of this meta-analysis suggested that aberrant methylation of DAPK promoter was associated with HNSCC. PMID:28249042

  5. Tumoral Environment Triggers Transcript Anomalies in Established Tumors: Induction of Altered Gene Expression and of Aberrant, Truncated and B2 Repeat-Containing Gene Transcripts

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    Pieter Rottiers


    Full Text Available In addition to eugenetic changes, cancerous cells exhibit extensive modifications in the expression levels of a variety of genes. The phenotypic switch observed after inoculation of T lymphoma cells into syngenic mice illustrates the active participation of tumoral environment in the induction of an aberrant gene expression pattern. To further substantiate this contribution, we performed polymerase chain reaction (PCR-based subtraction suppression hybridization (SSH to identify genes that are differentially expressed in tumor-derived EL4/13.3 cells compared to the same cells isolated from cultures. Besides a number of unknown genes, the subtracted library contained several known genes that have been reported to be expressed at increased levels in tumors and/or to contribute to carcinogenesis. Apart from clones representing translated transcripts, the subtracted library also contained a high number of clones representing B2 repeat elements, viz. short interspersed repetitive elements that are transcribed by RNA polymerase III. Northern blotting confirmed the induction of B2 transcripts in tumor tissue and also revealed induction of chimeric, B2 repeat-containing mRNA. The appearance of chimeric transcripts was accompanied by aberrant, shorter-than-full-length transcripts, specifically from upregulated genes. Accordingly, in addition to altered gene expression, tumoral environmental triggers constitute a potent mechanism to create an epigenetic diversity in cancers by inducing extensive transcript anomalies.

  6. Gene expression profile of brain regions reflecting aberrations in nervous system development targeting the process of neurite extension of rat offspring exposed developmentally to glycidol. (United States)

    Akane, Hirotoshi; Saito, Fumiyo; Shiraki, Ayako; Imatanaka, Nobuya; Akahori, Yumi; Itahashi, Megu; Wang, Liyun; Shibutani, Makoto


    We previously found that exposure to glycidol at 1000 ppm in drinking water caused axonopathy in maternal rats and aberrations in late-stage hippocampal neurogenesis, targeting the process of neurite extension in offspring. To identify the profile of developmental neurotoxicity of glycidol, pregnant Sprague-Dawley rats were given drinking water containing glycidol from gestational day 6 until weaning on day 21 after delivery, and offspring at 0, 300 and 1000 ppm were subjected to region-specific global gene expression profiling. Four brain regions were selected to represent both cerebral and cerebellar tissues, i.e., the cingulate cortex, corpus callosum, hippocampal dentate gyrus and cerebellar vermis. Downregulated genes in the dentate gyrus were related to axonogenesis (Nfasc), myelination (Mal, Mrf and Ugt8), and cell proliferation (Aurkb and Ndc80) at ≥ 300 ppm, and upregulated genes were related to neural development (Frzb and Fzd6) at 1000 ppm. Upregulation was observed for genes related to myelination (Kl, Igf2 and Igfbp2) in the corpus callosum and axonogenesis and neuritogenesis (Efnb3, Tnc and Cd44) in the cingulate cortex, whereas downregulation was observed for genes related to synaptic transmission (Thbs2 and Ccl2) in the cerebellar vermis; all of these changes were mostly observed at 1000 ppm. Altered gene expression of Cntn3, which functions on neurite outgrowth-promotion, was observed in all four brain regions at 1000 ppm. Gene expression profiles suggest that developmental exposure to glycidol affected plasticity of neuronal networks in the broad brain areas, and dentate gyrus neurogenesis may be the sensitive target of this type of toxicity.

  7. Aberrant methylation of the M-type phospholipase A2 receptor gene in leukemic cells

    Directory of Open Access Journals (Sweden)

    Menschikowski Mario


    Full Text Available Abstract Background The M-type phospholipase A2 receptor (PLA2R1 plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS or acute leukemia. Methods Sites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM analysis was then carried out to quantify PLA2R1 methylation at 5`-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA. Results Expression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2´-deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% ± 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia. Conclusions The study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis.

  8. Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

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    Oliveira Jorge


    Full Text Available Abstract Background Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. Methods A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC, 13 papillary (pRCC, 10 chromophobe (chRCC, and 10 oncocytomas and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. Results Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007, PTGS2 (p = 0.002, and RASSF1A (p = 0.0001. CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively, whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004. RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035. In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031 and nuclear grade (p = 0.022, respectively. Conclusion The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.

  9. An integrative multi-dimensional genetic and epigenetic strategy to identify aberrant genes and pathways in cancer

    Directory of Open Access Journals (Sweden)

    Lockwood William W


    Full Text Available Abstract Background Genomics has substantially changed our approach to cancer research. Gene expression profiling, for example, has been utilized to delineate subtypes of cancer, and facilitated derivation of predictive and prognostic signatures. The emergence of technologies for the high resolution and genome-wide description of genetic and epigenetic features has enabled the identification of a multitude of causal DNA events in tumors. This has afforded the potential for large scale integration of genome and transcriptome data generated from a variety of technology platforms to acquire a better understanding of cancer. Results Here we show how multi-dimensional genomics data analysis would enable the deciphering of mechanisms that disrupt regulatory/signaling cascades and downstream effects. Since not all gene expression changes observed in a tumor are causal to cancer development, we demonstrate an approach based on multiple concerted disruption (MCD analysis of genes that facilitates the rational deduction of aberrant genes and pathways, which otherwise would be overlooked in single genomic dimension investigations. Conclusions Notably, this is the first comprehensive study of breast cancer cells by parallel integrative genome wide analyses of DNA copy number, LOH, and DNA methylation status to interpret changes in gene expression pattern. Our findings demonstrate the power of a multi-dimensional approach to elucidate events which would escape conventional single dimensional analysis and as such, reduce the cohort sample size for cancer gene discovery.

  10. Influence of DNA repair gene polymorphisms of hOGG1, XRCC1, XRCC3, ERCC2 and the folate metabolism gene MTHFR on chromosomal aberration frequencies. (United States)

    Skjelbred, Camilla Furu; Svendsen, Marit; Haugan, Vera; Eek, Anette Kildal; Clausen, Kjell Oskar; Svendsen, Martin Veel; Hansteen, Inger-Lise


    We have studied the effect of genetic polymorphisms in the DNA repair genes hOGG1, XRCC1, XRCC3, ERCC2 and the MTHFR gene in the folate metabolism on the frequencies of cells with chromosomal aberrations (CA), chromosome-type aberrations (CSA), chromatid-type aberrations (CTA), chromatid breaks (CTB) and chromatid gaps (CTG) scored in peripheral blood lymphocytes from 651 Norwegian subjects of Caucasian descendant. DNA was extracted from fixed cell suspensions. The log-linear Poisson regression model was used for the combined data which included age, smoking, occupational exposure and genotype for 449 subjects. Our results suggest that individuals carrying the hOGG1 326Cys or the XRCC1 399Gln allele have an increased risk of chromosomal damage, while individuals carrying the XRCC1 194Trp or the ERCC2 751Gln allele have a reduced risk regardless of smoking habits and age. Individuals carrying the XRCC1 280His allele had an increased risk of CSA which was only apparent in non-smokers. This was independent of age. A protective effect of the XRCC3 241Met allele was only found in the older age group in non-smokers for CA, CSA and CTA, and in smokers for CSA. In the youngest age group, the opposite effect was found, with an increased risk for CA, CTA and CTG in smokers. Carrying the MTHFR 222Val allele gave an increased risk for chromosome and chromatid-type aberrations for both non-smokers and smokers, especially for individuals in the older age group, and with variable results in the youngest age group. The variables included in the different regression models accounted, however, for only 4-10% of the variation. The frequency ratio for CTG was significantly higher than for CTA and CTB for only 7 of the 43 comparisons performed. Some of the gap frequencies diverge from the trend in the CA, CSA, CTA and CTB results.

  11. Cancer specificity of promoters of the genes controlling cell proliferation. (United States)

    Kashkin, Kirill; Chernov, Igor; Stukacheva, Elena; Monastyrskaya, Galina; Uspenskaya, Natalya; Kopantzev, Eugene; Sverdlov, Eugene


    Violation of proliferation control is a common feature of cancer cells. We put forward the hypothesis that promoters of genes involved in the control of cell proliferation should possess intrinsic cancer specific activity. We cloned promoter regions of CDC6, POLD1, CKS1B, MCM2, and PLK1 genes into pGL3 reporter vector and studied their ability to drive heterologous gene expression in transfected cancer cells of different origin and in normal human fibroblasts. Each promoter was cloned in short (335-800 bp) and long (up to 2.3 kb) variants to cover probable location of core and whole promoter regulatory elements. Cloned promoters were significantly more active in cancer cells than in normal fibroblasts that may indicate their cancer specificity. Both versions of CDC6 promoters were shown to be most active while the activities of others were close to that of BIRC5 gene (survivin) gene promoter. Long and short variants of each cloned promoter demonstrated very similar cancer specificity with the exception of PLK1-long promoter that was substantially more specific than its short variant and other promoters under study. The data indicate that most of the important cis-regulatory transcription elements responsible for intrinsic cancer specificity are located in short variants of the promoters under study. CDC6 short promoter may serve as a promising candidate for transcription targeted cancer gene therapy.

  12. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

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    V Shilpa


    Full Text Available Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O 6 -methyguanine-DNA methyltransferase (MGMT is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 -position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC tissue samples, 14 low malignant potential (LMP tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression.

  13. Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA hypermethylation.

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    Kevin Pruitt


    Full Text Available The class III histone deactylase (HDAC, SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs in which 5' CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively, had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA-mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.

  14. Physiological characterization and genetic modifiers of aberrant root thigmomorphogenesis in mutants of Arabidopsis thaliana MILDEW LOCUS O genes. (United States)

    Bidzinski, Przemyslaw; Noir, Sandra; Shahi, Shermineh; Reinstädler, Anja; Gratkowska, Dominika Marta; Panstruga, Ralph


    Root architecture and growth patterns are plant features that are still poorly understood. When grown under in vitro conditions, seedlings with mutations in Arabidopsis thaliana genes MLO4 or MLO11 exhibit aberrant root growth patterns upon contact with hard surfaces, exemplified as tight root spirals. We used a set of physiological assays and genetic tools to characterize this thigmomorphogenic defect in detail. We observed that the mlo4/mlo11-associated root curling phenotype is not recapitulated in a set of mutants with altered root growth patterns or architecture. We further found that mlo4/mlo11-conditioned root curling is not dependent upon light and endogenous flavonoids, but is pH-sensitive and affected by exogenous calcium levels. Based upon the latter two characteristics, mlo4-associated root coiling appears to be mechanistically different from the natural strong root curvature of the Arabidopsis ecotype Landsberg erecta. Gravistimulation reversibly overrides the aberrant thigmomorphogenesis of mlo4 seedlings. Mutants with dominant negative defects in α-tubulin modulate the extent and directionality of mlo4/mlo11-conditioned root coils, whereas mutants defective in polar auxin transport (axr4, aux1) or gravitropism (pgm1) completely suppress the mlo4 root curling phenotype. Our data implicate a joint contribution of calcium signalling, pH regulation, microtubular function, polar auxin transport and gravitropism in root thigmomorphogenesis.

  15. Promoter methylation confers kidney-specific expression of the Klotho gene. (United States)

    Azuma, Masahiro; Koyama, Daisuke; Kikuchi, Jiro; Yoshizawa, Hiromichi; Thasinas, Dissayabutra; Shiizaki, Kazuhiro; Kuro-o, Makoto; Furukawa, Yusuke; Kusano, Eiji


    The aging suppressor geneKlotho is predominantly expressed in the kidney irrespective of species. Because Klotho protein is an essential component of an endocrine axis that regulates renal phosphate handling, the kidney-specific expression is biologically relevant; however, little is known about its underlying mechanisms. Here we provide in vitro and in vivo evidence indicating that promoter methylation restricts the expression of the Klotho gene in the kidney. Based on evolutionary conservation and histone methylation patterns, the region up to -1200 bp was defined as a major promoter element of the human Klotho gene. This region displayed promoter activity equally in Klotho-expressing and -nonexpressing cells in transient reporter assays, but the activity was reduced to ∼20% when the constructs were integrated into the chromatin in the latter. Both endogenous and transfected Klotho promoters were 30-40% methylated in Klotho-nonexpressing cells, but unmethylated in Klotho-expressing renal tubular cells. DNA demethylating agents increased Klotho expression 1.5- to 3.0-fold in nonexpressing cells and restored the activity of silenced reporter constructs. Finally, we demonstrated that a severe hypomorphic allele of Klotho had aberrant CpG methylation in kl/kl mice. These findings might be useful in therapeutic intervention for accelerated aging and several complications caused by Klotho down-regulation.

  16. Two types of chloroplast gene promoters in Chlamydomonas reinhardtii. (United States)

    Klein, U; De Camp, J D; Bogorad, L


    Structures of the promoters of Chlamydomonas reinhardtii plastid atpB and 16S rRNA-encoding genes were analyzed in vivo. Chimeric constructs, containing the Chlamydomonas chloroplast atpB or 16S rRNA-encoding gene promoter coupled to the Escherichia coli uidA (beta-glucuronidase, GUS) reporter gene and bordered by C. reinhardtii chloroplast sequences, were stably introduced into the chloroplast of Chlamydomonas by microprojectile bombardment. Activity of the promoters in the chloroplast of GUS gene-positive transformants was assayed by measuring the abundance of GUS transcripts and determining the relative rates of GUS transcription in vivo. Deletion analyses of the 16S rRNA gene and atpB promoter fragments showed that the two promoters differ structurally. The 16S rRNA gene promoter resembles the bacterial sigma 70 type with typical -10 and -35 elements. The atpB promoter, on the other hand, lacks a conserved motif in the -35 region but contains, in the -10 region, a characteristic octameric palindrome (TATAATAT) that is conserved in the promoter sequences of some other C. reinhardtii chloroplast genes. For maximum activity, the atpB promoter requires sequences of approximately 22 base pairs upstream and approximately 60 base pairs downstream of the transcription start site.

  17. Precise regulation of gene expression dynamics favors complex promoter architectures.

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    Dirk Müller


    Full Text Available Promoters process signals through recruitment of transcription factors and RNA polymerase, and dynamic changes in promoter activity constitute a major noise source in gene expression. However, it is barely understood how complex promoter architectures determine key features of promoter dynamics. Here, we employ prototypical promoters of yeast ribosomal protein genes as well as simplified versions thereof to analyze the relations among promoter design, complexity, and function. These promoters combine the action of a general regulatory factor with that of specific transcription factors, a common motif of many eukaryotic promoters. By comprehensively analyzing stationary and dynamic promoter properties, this model-based approach enables us to pinpoint the structural characteristics underlying the observed behavior. Functional tradeoffs impose constraints on the promoter architecture of ribosomal protein genes. We find that a stable scaffold in the natural design results in low transcriptional noise and strong co-regulation of target genes in the presence of gene silencing. This configuration also exhibits superior shut-off properties, and it can serve as a tunable switch in living cells. Model validation with independent experimental data suggests that the models are sufficiently realistic. When combined, our results offer a mechanistic explanation for why specific factors are associated with low protein noise in vivo. Many of these findings hold for a broad range of model parameters and likely apply to other eukaryotic promoters of similar structure.

  18. The core promoter: At the heart of gene expression. (United States)

    Danino, Yehuda M; Even, Dan; Ideses, Diana; Juven-Gershon, Tamar


    The identities of different cells and tissues in multicellular organisms are determined by tightly controlled transcriptional programs that enable accurate gene expression. The mechanisms that regulate gene expression comprise diverse multiplayer molecular circuits of multiple dedicated components. The RNA polymerase II (Pol II) core promoter establishes the center of this spatiotemporally orchestrated molecular machine. Here, we discuss transcription initiation, diversity in core promoter composition, interactions of the basal transcription machinery with the core promoter, enhancer-promoter specificity, core promoter-preferential activation, enhancer RNAs, Pol II pausing, transcription termination, Pol II recycling and translation. We further discuss recent findings indicating that promoters and enhancers share similar features and may not substantially differ from each other, as previously assumed. Taken together, we review a broad spectrum of studies that highlight the importance of the core promoter and its pivotal role in the regulation of metazoan gene expression and suggest future research directions and challenges.

  19. Splicing aberrations caused by constitutional RB1 gene mutations in retinoblastoma

    Indian Academy of Sciences (India)

    Vidya Latha Parsam; Mohammed Javed Ali; Santosh G Honavar; Geeta K Vemuganti; Chitra Kannabiran


    Analysis of RB1 mRNA from blood leukocytes of patients with retinoblastoma identified the effects of mutations involving consensus splice site, exonic substitution and whole-exon deletions identified in genomic DNA of these patients. In addition, this study identified mutations in cases in which no mutations were detectable in the genomic DNA. One proband had mutation at the canonical splice site at +5 position of IVS22, and analysis of the transcripts in this family revealed skipping of exon 22 in three members of this family. In one proband, a missense substitution of c.652T > G (g.56897T > G; Leu218Val) in exon 7 led to splicing aberrations involving deletions of exons 7 and 8, suggesting the formation of a cryptic splice site. In two probands with no detectable changes in the genomic DNA upon screening of RB1 exons and flanking intronic sequences, transcripts were found to have deletions of exon 6 in one, and exons 21 and 22 in another family. In two probands, RNA analysis confirmed genomic deletions involving one or more exons. This study reveals novel effects of RB1 mutations on splicing and suggests the utility of RNA analysis as an adjunct to mutational screening of genomic DNA in retinoblastoma.

  20. Integrating chromosomal aberrations and gene expression profiles to dissect rectal tumorigenesis

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    Eilers Paul HC


    Full Text Available Abstract Background Accurate staging of rectal tumors is essential for making the correct treatment choice. In a previous study, we found that loss of 17p, 18q and gain of 8q, 13q and 20q could distinguish adenoma from carcinoma tissue and that gain of 1q was related to lymph node metastasis. In order to find markers for tumor staging, we searched for candidate genes on these specific chromosomes. Methods We performed gene expression microarray analysis on 79 rectal tumors and integrated these data with genomic data from the same sample series. We performed supervised analysis to find candidate genes on affected chromosomes and validated the results with qRT-PCR and immunohistochemistry. Results Integration of gene expression and chromosomal instability data revealed similarity between these two data types. Supervised analysis identified up-regulation of EFNA1 in cases with 1q gain, and EFNA1 expression was correlated with the expression of a target gene (VEGF. The BOP1 gene, involved in ribosome biogenesis and related to chromosomal instability, was over-expressed in cases with 8q gain. SMAD2 was the most down-regulated gene on 18q, and on 20q, STMN3 and TGIF2 were highly up-regulated. Immunohistochemistry for SMAD4 correlated with SMAD2 gene expression and 18q loss. Conclusion On basis of integrative analysis this study identified one well known CRC gene (SMAD2 and several other genes (EFNA1, BOP1, TGIF2 and STMN3 that possibly could be used for rectal cancer characterization.

  1. Aberrant DNA methylation of cancer-associated genes in gastric cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC-EURGAST)

    NARCIS (Netherlands)

    Balassiano, Karen; Lima, Sheila; Jenab, Mazda; Overvad, Kim; Tjonneland, Anne; Boutron-Ruault, Marie Christine; Clavel-Chapelon, Francoise; Canzian, Federico; Kaaks, Rudolf; Boeing, Heiner; Meidtner, Karina; Trichopoulou, Antonia; Laglou, Pagona; Vineis, Paolo; Panico, Salvatore; Palli, Domenico; Grioni, Sara; Tumino, Rosario; Lund, Eiliv; Bueno-de-Mesquita, H. Bas; Numans, Mattjis E.; Peeters, Petra H. M.; Ramon Quiros, J.; Sanchez, Maria-Jose; Navarro, Carmen; Ardanaz, Eva; Dorronsoro, Miren; Hallmans, Goran; Stenling, Roger; Ehrnstrom, Roy; Regner, Sara; Allen, Naomi E.; Travis, Ruth C.; Khaw, Kay-Tee; Offerhaus, G. Johan A.; Sala, Nuria; Riboli, Elio; Hainaut, Pierre; Scoazec, Jean-Yves; Sylla, Bakary S.; Gonzalez, Carlos A.; Herceg, Zdenko


    Epigenetic events have emerged as key mechanisms in the regulation of critical biological processes and in the development of a wide variety of human malignancies, including gastric cancer (GC), however precise gene targets of aberrant DNA methylation in GC remain largely unknown. Here, we have comb

  2. Dopamine signaling leads to loss of Polycomb repression and aberrant gene activation in experimental parkinsonism

    DEFF Research Database (Denmark)

    Södersten, Erik; Feyder, Michael; Lerdrup, Mads


    Polycomb group (PcG) proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. ...... and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson's disease....

  3. APC and K-ras gene mutation in aberrant crypt foci of human colon

    Institute of Scientific and Technical Information of China (English)

    Ping Yuan; Meng Hong Sun; Jin Sheng Zhang; Xiong Zeng Zhu; Da Ren Shi


    AIM To study the genetic alteration in ACF andto define the possibility that ACF may be a veryearly morphological lesion with molecularchanges, and to explore the relationshipbetween ACF and colorectal adenoma evencarcinoma.METHODS DNA from 35 CRC, 15 adenomas, 34ACF and 10 normal mucus was isolated by meansof microdissection. Direct gene sequencing of K-ras gene including codon 12, 13 and 61 as well asthe mutation cluster region (MCR) of APC genewas performed.RESULTS K-ras gene mutation frequency inACF, adenoma and carcinoma was 17.6% (6/34), 13.3% (2/ 15), and 14.3% (5/ 35)respectively, showing no difference ( P > 0.05)in K-fas gene mutation among three pathologicprocedures. The K-ras gene mutation inadenoma, carcinoma and 4 ACF restricted incodon 12 (GGT→GAT), but the other 2 mutationsfrom ACF located in codon 13 (GGC→GAC). K-res gene mutation was found more frequently inolder patients and patients with polypoidcancer. No mutation in codon 61 was found in thethree tissue types. Mutation rate of APO gene inadenoma and carcinoma was 22.9% (8/35) and26.7% (4/ 15), which was higher than ACF(2.9%) (P < 0.05). APC gene mutation incarcinoma was not correlated with age ofpatients, location, size and differentiation oftumor.CONCLUSION ACF might be a very earlymorphological lesion in the tumorogenesis ofcolorectal tumor. The morphological feature andgene mutation status was different in ACF andadenoma. ACF is possibly putative"microadenoma" that might be the precursor ofadenoma. In addition, the development of asubgroup of colorectal carcinomas mightundergo a way of "normal epithelium→ ACF→carcinomas".

  4. Histone Modifications Depict an Aberrantly Heterochromatinized FMR1 Gene in Fragile X Syndrome


    Coffee, Bradford; Zhang, Fuping; Ceman, Stephanie; Warren, Stephen T.; Reines, Daniel


    Fragile X syndrome is caused by an expansion of a polymorphic CGG triplet repeat that results in silencing of FMR1 expression. This expansion triggers methylation of FMR1's CpG island, hypoacetylation of associated histones, and chromatin condensation, all characteristics of a transcriptionally inactive gene. Here, we show that there is a graded spectrum of histone H4 acetylation that is proportional to CGG repeat length and that correlates with responsiveness of the gene to DNA demethylation...

  5. DNA repair gene polymorphisms in relation to chromosome aberration frequencies in retired radiation workers

    Energy Technology Data Exchange (ETDEWEB)

    Wilding, Craig S. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom)]. E-mail:; Relton, Caroline L. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Paediatric and Lifecourse Epidemiology Research Group, School of Clinical Medical Sciences (Child Health), Newcastle University, Sir James Spence Institute, Royal Victoria Infirmary, Newcastle-upon-Tyne NE1 4LP (United Kingdom); Rees, Gwen S. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Tarone, Robert E. [International Epidemiology Institute, 1455 Research Boulevard, Suite 550, Rockville, MD 20850 (United States); Whitehouse, Caroline A. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Tawn, E. Janet [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom)


    Polymorphic variation in DNA repair genes was examined in a group of retired workers from the British Nuclear Fuels plc facility at Sellafield in relation to previously determined translocation frequencies in peripheral blood lymphocytes. Variation at seven polymorphisms in four genes involved in the base excision repair (XRCC1 R194W, R399Q and a [AC]{sub n} microsatellite in the 3' UTR) and double strand break repair (XRCC3 T241M and a [AC]{sub n} microsatellite in intron 3 of XRCC3, XRCC4 I134T, and a GACTAn microsatellite located 120kb 5' of XRCC5) pathways was determined for 291 retired radiation workers who had received cumulative occupational external radiation doses of between 0 and 1873mSv. When the interaction between radiation dose and each DNA repair gene polymorphism was examined in relation to translocation frequency there was no evidence for any of the polymorphisms studied influencing the response to occupational exposure. A positive interaction observed between genotype (individuals with at least one allele >=20 repeat units) at a microsatellite locus in the XRCC3 gene and smoking status should be interpreted cautiously because interactions were investigated for seven polymorphisms and two exposures. Nonetheless, further research is warranted to examine whether this DNA repair gene variant might be associated with a sub-optimal repair response to smoking-induced DNA damage and hence an increased frequency of translocations.

  6. Archaeal promoter architecture and mechanism of gene activation

    DEFF Research Database (Denmark)

    Peng, Nan; Ao, Xiang; Liang, Yun Xiang;


    Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory...... element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked...

  7. Topoisomerase-1 gene copy aberrations are frequent in patients with breast cancer

    DEFF Research Database (Denmark)

    Kümler, Iben; Balslev, Eva; Poulsen, Tim S.


    Topoisomerase-1 (Top1) targeting drugs have shown promising efficacy in patients with metastatic breast cancer (BC). However, these drugs are rather toxic calling for development and validation of predictive biomarkers to increase the therapeutic index. As these drugs are targeting the Top1 protein...... of TOP1 gene copy gain in BC. The prevalence of TOP1 gene copy gain was investigated by fluorescence in situ hybridization with a TOP1/CEN-20 probemix in normal breast tissue (N=100) and in tissue from patients with metastatic BC in a discovery (N=100) and a validation cohort (N=205). As amplification...... of 20q including CEN-20 is common in BC a TOP1/CEN-2 probemix was applied to the validation cohort. More than 30% of the patients had gene copy numbers of ≥ 4 and approximately 20% of the patients had TOP1/CEN-20 ratios ≥ 1.5. The CEN-2 probe did not add any information. Gain of the TOP1 gene appears...

  8. Functional annotation of rare gene aberration drivers of pancreatic cancer | Office of Cancer Genomics (United States)

    As we enter the era of precision medicine, characterization of cancer genomes will directly influence therapeutic decisions in the clinic. Here we describe a platform enabling functionalization of rare gene mutations through their high-throughput construction, molecular barcoding and delivery to cancer models for in vivo tumour driver screens. We apply these technologies to identify oncogenic drivers of pancreatic ductal adenocarcinoma (PDAC).

  9. Epigenetic regulation of transposable element derived human gene promoters. (United States)

    Huda, Ahsan; Bowen, Nathan J; Conley, Andrew B; Jordan, I King


    It was previously thought that epigenetic histone modifications of mammalian transposable elements (TEs) serve primarily to defend the genome against deleterious effects associated with their activity. However, we recently showed that, genome-wide, human TEs can also be epigenetically modified in a manner consistent with their ability to regulate host genes. Here, we explore the ability of TE sequences to epigenetically regulate individual human genes by focusing on the histone modifications of promoter sequences derived from TEs. We found 1520 human genes that initiate transcription from within TE-derived promoter sequences. We evaluated the distributions of eight histone modifications across these TE-promoters, within and between the GM12878 and K562 cell lines, and related their modification status with the cell-type specific expression patterns of the genes that they regulate. TE-derived promoters are significantly enriched for active histone modifications, and depleted for repressive modifications, relative to the genomic background. Active histone modifications of TE-promoters peak at transcription start sites and are positively correlated with increasing expression within cell lines. Furthermore, differential modification of TE-derived promoters between cell lines is significantly correlated with differential gene expression. LTR-retrotransposon derived promoters in particular play a prominent role in mediating cell-type specific gene regulation, and a number of these LTR-promoter genes are implicated in lineage-specific cellular functions. The regulation of human genes mediated by histone modifications targeted to TE-derived promoters is consistent with the ability of TEs to contribute to the epigenomic landscape in a way that provides functional utility to the host genome.

  10. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter (United States)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  11. Gene transfer to promote cardiac regeneration. (United States)

    Collesi, Chiara; Giacca, Mauro


    There is an impelling need to develop new therapeutic strategies for patients with myocardial infarction and heart failure. Leading from the large quantity of new information gathered over the last few years on the mechanisms controlling cardiomyocyte proliferation during embryonic and fetal life, it is now possible to devise innovative therapies based on cardiac gene transfer. Different protein-coding genes controlling cell cycle progression or cardiomyocyte specification and differentiation, along with microRNA mimics and inhibitors regulating pre-natal and early post-natal cell proliferation, are amenable to transformation in potential therapeutics for cardiac regeneration. These gene therapy approaches are conceptually revolutionary, since they are aimed at stimulating the intrinsic potential of differentiated cardiac cells to proliferate, rather than relying on the implantation of exogenously expanded cells to achieve tissue regeneration. For efficient and prolonged cardiac gene transfer, vectors based on the Adeno-Associated Virus stand as safe, efficient and reliable tools for cardiac gene therapy applications.

  12. Gene Targets in Prostate Tumor Cells that Mediate Aberrant Growth and Invasiveness (United States)


    named EIAF ( Higashino et al. 1993). Finally, PEA3/EI AF was found to be the fourth Ets factor involved in chromosomal fusions with EWS (Kaneko et al. transcriptional repression mediated by the t(8;21), t(16;21), t(12;21), and inv(16) fusion proteins. Curr Opin Hematol 8:197-200 Higashino F...Yoshida K, Handa M, Toyoda Y, Nishihira H, Tanaka Y, Sasaki Y, Ishida S, Higashino F, Fuj inaga K (1996) Fusion of an ETS-family gene, EIAF, to EWS by t

  13. Aberrations in the Iron Regulatory Gene Signature Are Associated with Decreased Survival in Diffuse Infiltrating Gliomas. (United States)

    Weston, Cody; Klobusicky, Joe; Weston, Jennifer; Connor, James; Toms, Steven A; Marko, Nicholas F


    Iron is a tightly regulated micronutrient with no physiologic means of elimination and is necessary for cell division in normal tissue. Recent evidence suggests that dysregulation of iron regulatory proteins may play a role in cancer pathophysiology. We use public data from The Cancer Genome Atlas (TCGA) to study the association between survival and expression levels of 61 genes coding for iron regulatory proteins in patients with World Health Organization Grade II-III gliomas. Using a feature selection algorithm we identified a novel, optimized subset of eight iron regulatory genes (STEAP3, HFE, TMPRSS6, SFXN1, TFRC, UROS, SLC11A2, and STEAP4) whose differential expression defines two phenotypic groups with median survival differences of 52.3 months for patients with grade II gliomas (25.9 vs. 78.2 months, p< 10-3), 43.5 months for patients with grade III gliomas (43.9 vs. 87.4 months, p = 0.025), and 54.0 months when considering both grade II and III gliomas (79.9 vs. 25.9 months, p < 10-5).

  14. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis

    Directory of Open Access Journals (Sweden)

    Zhang Lisheng


    Full Text Available Abstract Background Hepatocellular carcinoma (HCC presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes. Methods We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors. Results While the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARβ, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a , RASSF1a, CASP8 and CDH13 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6% and less significantly methylated in non-cancerous tissue (4/29. 13.79%. The RASSF1a was fully methylated in all tumor tissues (29/29, 100%, and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%. Conclusions Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the CDH13 genes. For the

  15. Differential regulation of genes by retrotransposons in rice promoters. (United States)

    Dhadi, Surendar Reddy; Xu, Zijun; Shaik, Rafi; Driscoll, Kyle; Ramakrishna, Wusirika


    Rice genome harbors genes and promoters with retrotransposon insertions. There is very little information about their function. The effect of retrotransposon insertions in four rice promoter regions on gene regulation, was investigated using promoter-reporter gene constructs with and without retrotransposons. Differences in expression levels of gus and egfp reporter genes in forward orientation and rfp in reverse orientation were evaluated in rice plants with transient expression employing quantitative RT-PCR analysis, histochemical GUS staining, and eGFP and RFP fluorescent microscopy. The presence of SINE in the promoter 1 (P1) resulted in higher expression levels of the reporter genes, whereas the presence of LINE in P2 or gypsy LTR retrotransposon in P3 reduced expression of the reporter genes. Furthermore, the SINE in P1 acts as an enhancer in contrast with the LINE in P2 and the gypsy LTR retrotransposon in P3 which act as silencers. CTAA and CGG motifs in these retrotransposons are the likely candidates for the downregulation compared to TCTT motif (SINE) which is a candidate for the upregulation of gene expression. The effect of retrotransposons on gene regulation correlated with the earlier investigation of conservation patterns of these four retrotransposon insertions in several rice accessions implying their evolutionary significance.

  16. Potassium Channel Ether à go-go1 Is Aberrantly Expressed in Human Liposarcoma and Promotes Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Jin Wu


    Full Text Available The ether à go-go1 (Eag1 channel is overexpressed in a variety of cancers. However, the expression and function of Eag1 in liposarcoma are poorly understood. In the present study, the mRNA expression of Eag1 in different adipose tissue samples was examined by real-time PCR. Then, the protein expression of Eag1 in 131 different adipose tissues from 109 patients was detected by immunohistochemistry. Next, the associations between Eag1 expression and clinicopathological features of liposarcoma were analyzed. In addition, the effects of Eag1 on liposarcoma cell proliferation and cycle were evaluated by CCK-8, colony formation, xenograft mouse model, and flow cytometry, respectively. Finally, the activation of p38 mitogen-activated protein kinase (MAPK was detected by Western blot analysis to explain the detailed mechanisms of oncogenic potential of Eag1 in liposarcoma. It was found that Eag1 was aberrantly expressed in over 67% liposarcomas, with a higher frequency than in lipoma, hyperplasia, inflammation, and normal adipose tissues. However, Eag1 expression was not correlated with clinicopathological features of liposarcoma. Eag1 inhibitor imipramine or Eag1-shRNA significantly suppressed the proliferation of liposarcoma cells in vitro and in vivo, accompanying with accumulation of cells in the G1 phase. These results suggest that Eag1 plays an important role in regulating the proliferation and cell cycle of liposarcoma cells and might be a potential therapeutic target for liposarcoma.

  17. Lactic acid induces aberrant amyloid precursor protein processing by promoting its interaction with endoplasmic reticulum chaperone proteins.

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    Yiwen Xiang

    Full Text Available BACKGROUND: Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of β-amyloid peptide (Aβ of Alzheimer's disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP. METHODOLOGY/PRINCIPAL FINDINGS: Lactic acid increased levels of Aβ40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y, whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes. CONCLUSIONS/SIGNIFICANCE: These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer's disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.

  18. Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization

    Institute of Scientific and Technical Information of China (English)

    Ti Liu; Tianhong Wang; Xian Li; Xuan Liu


    To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene (cbh1) promoters was obtained. The region from-677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from-620 to-820 of the modified cbh1 promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbh1 promoter, obtaining promoters with copy numbers 2, 4,and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase (gus) reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.

  19. Mechanosensitive promoter region in the human HB-GAM gene

    DEFF Research Database (Denmark)

    Liedert, Astrid; Kassem, Moustapha; Claes, Lutz;


    expression through specific transcription factor binding sites in the promoter region of mechanosensitive genes. In the present study, we demonstrate that the expression of HB-GAM, which is known to have stimulating effects on osteogenic differentiation, is rapidly induced by mechanical loading in hMSC-TERT4...... cells. Analysis of the human HB-GAM gene upstream regulatory region with luciferase reporter gene assays revealed that the upregulation of HB-GAM expression occurred at the transcriptional level and was mainly dependent on the HB-GAM promoter region most upstream containing three potential AP-1 binding...

  20. Gene Expression Meta-Analysis identifies Cytokine Pathways and 5q Aberrations involved in Metastasis of ERBB2 Amplified and Basal Breast Cancer

    DEFF Research Database (Denmark)

    Thomassen, Mads; Tan, Qihua; Burton, Mark


    Background: Breast tumors have been described by molecular subtypes characterized by pervasively different gene expression profiles. The subtypes are associated with different clinical parameters and origin of precursor cells. However, the biological pathways and chromosomal aberrations that differ...... the subgroups impact metastasis. Results: We have scrutinized publicly available gene expression datasets and identified molecular subtypes in 1,394 breast tumors with outcome data. By analysis of chromosomal regions and pathways using “Gene set enrichment analysis” followed by a meta-analysis, we identified...... show that high expression of 5q14 genes and low levels of TNFR2 pathway genes were associated with poor survival in basal-like cancers. Furthermore, low expression of 5q33 genes and interleukin-12 pathway genes were associated with poor outcome exclusively in ERBB2-like tumors. Conclusion...

  1. Functional analysis of the Myostatin gene promoter in sheep

    Institute of Scientific and Technical Information of China (English)

    DU; Rong; AN; XiaoRong; CHEN; YongFu; QIN; Jian


    Compared with the understanding for the functional mechanism of the myostatin gene, little is known about the regulatory mechanism of the myostatin gene transcription and expression. To better understand the function of the myostatin gene promoter (MSTNpro) in the transcriptional regulation of the myostatin gene and to further investigate the transcriptional regulation mechanism of the myostatin gene, the promoter region of the myostatin gene in sheep has been cloned in our recent study (AY918121). In this study, the wild (W) type MSTNProW-EGFP vectors and E-box (E) (CANNTG) mutant (M) type MSTNProE(3+5+7)M-EGFP vectors were constructed and the transcriptional regulation activities were compared by detecting the fluorescent strength of EGFP (enhanced green fluorescent protein) in C2C12 myoblasts (or myotubes) and sheep fibroblasts transfected with the vectors. Results showed that the 0.3―1.2 kb sheep myostatin promoter could activate the transcription and expression of EGFP gene in C2C12 myoblasts to different extent and the 1.2 kb promoter was the strongest. However, fluorescence was not observed in the sheep fibroblasts transfected with the 1.2 kb sheep myostatin promoter. These results suggested that the specific nature of the myostatin gene expression in skeletal muscle was attributed to the specific nature of the myostatin promoter activity. The increasing growth density of C2C12 myoblasts inhibited the transcriptional regulation activity of the wild type sheep myostatin promoter by a mechanism of feedback. The transcriptional regulation activity of the 1.2 kb wild type sheep myostatin promoter increased significantly after C2C12 myoblasts were differentiated, while the activity of 1.2 kb E(3+5+7)-mutant type myostatin promoter had no obvious change. This result suggested that MyoD may be responsible for the difference of the myostatin gene transcription and expression between growing and differentiating conditions by binding to E-box of the myostatin

  2. Aberrant Promoter Methylation of the Tumour Suppressor RASSF10 and Its Growth Inhibitory Function in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Antje M. Richter


    Full Text Available Breast cancer is the most common cancer in women, with 1.7 million new cases each year. As early diagnosis and prognosis are crucial factors in cancer treatment, we investigated potential DNA methylation biomarkers of the tumour suppressor family Ras-association domain family (RASSF. Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby promotes cancer development and progression. In this study we analysed the tumour suppressors RASSF1A and RASSF10. Our study shows that RASSF10 is expressed in normal breast but inactivated by methylation in breast cancer. We observed a significant inactivating promoter methylation of RASSF10 in primary breast tumours. RASSF10 is inactivated in 63% of primary breast cancer samples but only 4% of normal control breast tissue is methylated (p < 0.005. RASSF1A also shows high promoter methylation levels in breast cancer of 56% vs. 8% of normal tissue (p < 0.005. Interestingly more than 80% of breast cancer samples harboured a hypermethylation of RASSF10 and/or RASSF1A promoter. Matching samples exhibited a strong tumour specific promoter methylation of RASSF10 in comparison to the normal control breast tissue. Demethylation treatment of breast cancer cell lines MCF7 and T47D reversed RASSF10 promoter hypermethylation and re-established RASSF10 expression. In addition, we could show the growth inhibitory potential of RASSF10 in breast cancer cell lines MCF7 and T47D upon exogenous expression of RASSF10 by colony formation. We could further show, that RASSF10 induced apoptotic changes in MCF7 and T47D cells, which was verified by a significant increase in the apoptotic sub G1 fraction by 50% using flow cytometry for MCF7 cells. In summary, our study shows the breast tumour specific inactivation of RASSF10 and RASSF1A due to DNA methylation of their CpG island promoters. Furthermore RASSF10 was characterised by the ability to block growth of breast cancer cell lines by apoptosis

  3. SINE retrotransposons cause epigenetic reprogramming of adjacent gene promoters. (United States)

    Estécio, Marcos R H; Gallegos, Juan; Dekmezian, Mhair; Lu, Yue; Liang, Shoudan; Issa, Jean-Pierre J


    Almost half of the human genome and as much as 40% of the mouse genome is composed of repetitive DNA sequences. The majority of these repeats are retrotransposons of the SINE and LINE families, and such repeats are generally repressed by epigenetic mechanisms. It has been proposed that these elements can act as methylation centers from which DNA methylation spreads into gene promoters in cancer. Contradictory to a methylation center function, we have found that retrotransposons are enriched near promoter CpG islands that stay methylation-free in cancer. Clearly, it is important to determine which influence, if any, these repetitive elements have on nearby gene promoters. Using an in vitro system, we confirm here that SINE B1 elements can influence the activity of downstream gene promoters, with acquisition of DNA methylation and loss of activating histone marks, thus resulting in a repressed state. SINE sequences themselves did not immediately acquire DNA methylation but were marked by H3K9me2 and H3K27me3. Moreover, our bisulfite sequencing data did not support that gain of DNA methylation in gene promoters occurred by methylation spreading from SINE B1 repeats. Genome-wide analysis of SINE repeats distribution showed that their enrichment is directly correlated with the presence of USF1, USF2, and CTCF binding, proteins with insulator function. In summary, our work supports the concept that SINE repeats interfere negatively with gene expression and that their presence near gene promoters is counter-selected, except when the promoter is protected by an insulator element.

  4. Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jun-Xiong Wang; Yuan-Long He; Sheng-Tao Zhu; Shuo Yang; Shu-Tian Zhang


    AIM: To identify the novel methylation-silenced gene pentraxin 3 (PTX3) in esophageal squamous cell carcinoma (ESCC). METHODS: PTX3 mRNA expression was examined in six human ESCC cell lines, one human immortalized normal esophageal epithelial cell line, primary ESCC tumor tissue, and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction (RT-PCR). Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels. Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene. RESULTS: In the majority of ESCC cell lines, we found that PTX3 expression was down-regulated due to gene promoter hypermethylation, which was further confirmed by bisulphite genomic sequencing. Demethyl-ation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines. Methylation was more common in tumor tissues (85%) than in adjacent nontumor tissues (25%) (P < 0 .01). CONCLUSION: PTX3 is down-regulated through promoter hypermethylation in ESCC, and could potentially serve as a biomarker of ESCC.

  5. Conditional gene expression and promoter replacement in Zymoseptoria tritici using fungal nitrate reductase promoters. (United States)

    Marchegiani, Elisabetta; Sidhu, Yaadwinder; Haynes, Ken; Lebrun, Marc-Henri


    Studying essential genes in haploid fungi requires specific tools. Conditional promoter replacement (CPR) is an efficient method for testing gene essentiality. However, this tool requires promoters that can be strongly down-regulated. To this end, we tested the nitrate reductase promoters of Magnaporthe oryzae (pMoNIA1) and Zymoseptoria tritici (pZtNIA1) for their conditional expression in Z. tritici. Expression of EGFP driven by pMoNIA1 or pZtNIA1 was induced on nitrate and down-regulated on glutamate (10-fold less than nitrate). Levels of differential expression were similar for both promoters, demonstrating that the Z. tritici nitrogen regulatory network functions with a heterologous promoter similarly to a native promoter. To establish CPR, the promoter of Z. tritici BGS1, encoding a β-1,3-glucan synthase, was replaced by pZtNIA1 using targeted sequence replacement. Growth of pZtNIA1::BGS1 CPR transformants was strongly reduced in conditions repressing pZtNIA1, while their growth was similar to wild type in conditions inducing pZtNIA1. This differential phenotype demonstrates that BGS1 is important for growth in Z. tritici. In addition, in inducing conditions, pZtNIA1::BGS1 CPR transformants were hyper-sensitive to Calcofluor white, a cell wall disorganizing agent. Nitrate reductase promoters are therefore suitable for conditional promoter replacement in Z. tritici. This tool is a major step toward identifying novel fungicide targets.

  6. A novel PTEN gene promoter mutation and untypical Cowden syndrome

    Institute of Scientific and Technical Information of China (English)

    Chen Liu; Guangbing Li; Rongrong Chen; Xiaobo Yang; Xue Zhao; Haitao Zhao


    Cowden syndrome (CS),an autosomal dominant disorder,is one of a spectrum of clinical disorders that have been linked to germline mutations in the phosphatase and tensin homolog (PTEN) gene.Although 70-80% of patients with CS have an identifiable germline PTEN mutation,the clinical diagnosis presents many challenges because of the phenotypic and genotypic variations.In the present study,we sequenced the exons and the promoter of PTEN gene,mutations and variations in the promoter and exons were identified,and a PTEN protein expression negative region was determined by immunohistochemistry (IHC).In conclusion,a novel promoter mutation we found in PTEN gene may turn off PTEN protein expression occasionally,leading to the disorder of PTEN and untypical CS manifestations.

  7. Cloning and Characterization of Gene Promoters from Bacillus pumilus

    Institute of Scientific and Technical Information of China (English)

    Pan Jiao(潘皎); Zhang Yizheng


    DNA fragments obtained from Sau3AI partially digested total DNA of Bacillus pumilus UN31-C-42 are first inserted into BamHI site of pSUPV4, a promoter-probe vector. The recombinant DNA molecules are transformed into Escherichia coli cells and eight-three Kanr clones (named pSUBp1- pSUBp83) are obtained. The inserted fragments in pSUBp53, pSUBp57, pSUBp21, which showed high level of kanamycin - resistance, are sequenced and analyzed, respectively. These fragments contain some conserved sequences of prokaryotic gene promoters, such as TATAAT and TTGACA box. The promoter fragment Bp53 could efficiently promote the alkaline protease gene of B.pumilus expression not only in E.coli but also in B.subtilis cells.

  8. Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome.

    Directory of Open Access Journals (Sweden)

    Steven D Sheridan

    Full Text Available Fragile X syndrome (FXS is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation (FMR1 gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP. Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid

  9. Molecular cloning and analysis of the Catsper1 gene promoter. (United States)

    Mata-Rocha, Minerva; Alvarado-Cuevas, Edith; Hernández-Sánchez, Javier; Cerecedo, Doris; Felix, Ricardo; Hernández-Reyes, Adriana; Tesoro-Cruz, Emiliano; Oviedo, Norma


    CatSper channels are essential for hyperactivity of sperm flagellum, progesterone-mediated chemotaxis and oocyte fertilization. Catsper genes are exclusively expressed in the testis during spermatogenesis, but the function and regulation of the corresponding promoter regions are unknown. Here, we report the cloning and characterization of the promoter regions in the human and murine Catsper1 genes. These promoter regions were identified and isolated from genomic DNA, and transcriptional activities were tested in vitro after transfection into human embryonic kidney 293, mouse Sertoli cells 1 and GC-1spg cell lines as well as by injecting plasmids directly into mouse testes. Although the human and murine Catsper1 promoters lacked a TATA box, a well-conserved CRE site was identified. Both sequences may be considered as TATAless promoters because their transcriptional activity was not affected after deletion of TATA box-like sites. Several transcription initiation sites were revealed by RNA ligase-mediated rapid amplification of the cDNA 5'-ends. We also found that the immediate upstream region and the first exon in the human CATSPER1 gene negatively regulate transcriptional activity. In the murine Catsper1 promoter, binding sites for transcription factors SRY, SOX9 and CREB were protected by the presence of nuclear testis proteins in DNAse degradation assays. Likewise, the mouse Catsper1 promoter exhibited transcriptional activity in both orientations and displayed significant expression levels in mouse testis in vivo, whereas the suppression of transcription signals in the promoter resulted in low expression levels. This study, thus, represents the first identification of the transcriptional control regions in the genes encoding the human and murine CatSper channels.

  10. A novel BDNF gene promoter directs expression to skeletal muscle

    Directory of Open Access Journals (Sweden)

    Heinrich Gerhard


    Full Text Available Abstract Background Cell-specific expression of the gene that encodes brain-derived neurotrophic factor (BDNF is required for the normal development of peripheral sensory neurons and efficient synaptic transmission in the mature central and peripheral nervous system. The control of BDNF gene expression involves multiple tissue and cell-specific promoters that are differentially regulated. The molecular mechanisms that are responsible for tissue and cell-specific expression of these promoters are still incompletely understood. Results The cloning and analysis of three additional zebrafish (Danio rerio BDNF gene exons and two associated promoters, is reported. Among them are two exons that generate a novel tripartite mature transcript. The exons were located on the transcription unit, whose overall organization was determined by cloning, Southern blot hybridization and sequence analysis, and compared with the pufferfish (Fugu rubripes and mammalian BDNF loci, revealing a conserved but more compact organization. Structural and functional analysis of the exons, their adjacent promoters and 5' flanks, showed that they are expressed cell-specifically. The promoter associated with the 5' exon of the tripartite transcript is GC-rich, TATA-less and the 5' flank adjacent to it contains multiple Sp1, Mef2, and AP1 elements. A fusion gene containing the promoter and 1.5 KB of 5' flank is directed exclusively to skeletal muscle of transiently transfected embryos. The second promoter, whose associated 5' exon contains a 25-nucleotide segment of identity with a mammalian BDNF gene exon, was transiently expressed in yolk of the early embryo. RT-PCR analysis of total RNA from whole juvenile fish and adult female skeletal muscle revealed tissue-specific expression of the 5' exons but the novel exon could not be detected even after two rounds of nested PCR. Conclusion The zebrafish BDNF gene is as complex as the mammalian gene yet much more compact. Its exons are

  11. Stimulation of the mouse rRNA gene promoter by a distal spacer promoter.


    Paalman, M H; Henderson, S L; Sollner-Webb, B


    We show that the mouse ribosomal DNA (rDNA) spacer promoter acts in vivo to stimulate transcription from a downstream rRNA gene promoter. This augmentation of mammalian RNA polymerase I transcription is observed in transient-transfection experiments with three different rodent cell lines, under noncompetitive as well as competitive transcription conditions, over a wide range of template concentrations, whether or not the enhancer repeats alone stimulate or repress expression from the downstre...

  12. Control of Glycosylation-Related Genes by DNA Methylation: the Intriguing Case of the B3GALT5 Gene and Its Distinct Promoters. (United States)

    Trinchera, Marco; Zulueta, Aida; Caretti, Anna; Dall'Olio, Fabio


    Glycosylation is a metabolic pathway consisting of the enzymatic modification of proteins and lipids through the stepwise addition of sugars that gives rise to glycoconjugates. To determine the full complement of glycoconjugates that cells produce (the glycome), a variety of genes are involved, many of which are regulated by DNA methylation. The aim of the present review is to briefly describe some relevant examples of glycosylation-related genes whose DNA methylation has been implicated in their regulation and to focus on the intriguing case of a glycosyltransferase gene (B3GALT5). Aberrant promoter methylation is frequently at the basis of their modulation in cancer, but in the case of B3GALT5, at least two promoters are involved in regulation, and a complex interplay is reported to occur between transcription factors, chromatin remodelling and DNA methylation of typical CpG islands or even of other CpG dinucleotides. Transcription of the B3GALT5 gene underwent a particular evolutionary fate, so that promoter hypermethylation, acting on one transcript, and hypomethylation of other sequences, acting on the other, cooperate on one gene to obtain full cancer-associated silencing. The findings may also help in unravelling the complex origin of serum CA19.9 antigen circulating in some patients.

  13. Characterization of the promoter region of the mouse Xist gene. (United States)

    Pillet, N; Bonny, C; Schorderet, D F


    The mouse Xist gene is expressed exclusively from the inactive X chromosome and may be implicated in initiating X inactivation. To better understand the mechanisms underlying the control of Xist expression, we investigated the upstream regulatory region of the mouse Xist promoter. A 1.2-kb upstream region of the Xist gene was sequenced and promoter activity was studied by chloramphenicol acetyltransferase (CAT) assays after transfection in murine XX and XY cell lines. The region analyzed (-1157 to +917 showed no in vitro sex-specific promoter activity. However, a minimal constitutional promoter was assigned to a region from -81 to +1, and a cis element from -41 to -15 regulates promoter activity. We showed that a nuclear factor binds to an element located at -30 to -25 (TTAAAG). A second sequence at -41 to -15 does not act as an enhancer and is unable to confer transcriptional activity to the Xist gene on its own. A third region from -82 to -41 is needed for correct expression. Deletion of the segment -441 to -231 is associated with an increase in CAT activity and may represent a silencer element. Images Fig. 3 PMID:8618932

  14. In vitro mapping of Myotonic Dystrophy (DM) gene promoter

    Energy Technology Data Exchange (ETDEWEB)

    Storbeck, C.J.; Sabourin, L. [Univ. of Ottawa (Canada); Baird, S. [Children`s Hospital of Eastern Ontario, Ottawa (Canada)] [and others


    The Myotonic Dystrophy Kinase (DMK) gene has been cloned and shared homology to serine/threonine protein kinases. Overexpression of this gene in stably transfected mouse myoblasts has been shown to inhibit fusion into myotubes while myoblasts stably transfected with an antisense construct show increased fusion potential. These experiments, along with data showing that the DM gene is highly expressed in muscle have highlighted the possibility of DMK being involved in myogenesis. The promoter region of the DM gene lacks a consensus TATA box and CAAT box, but harbours numerous transcription binding sites. Clones containing extended 5{prime} upstream sequences (UPS) of DMK only weakly drive the reporter gene chloramphenicol acetyl transferase (CAT) when transfected into C2C12 mouse myoblasts. However, four E-boxes are present in the first intron of the DM gene and transient assays show increased expression of the CAT gene when the first intron is present downstream of these 5{prime} UPS in an orientation dependent manner. Comparison between mouse and human sequence reveals that the regions in the first intron where the E-boxes are located are highly conserved. The mapping of the promoter and the importance of the first intron in the control of DMK expression will be presented.

  15. Comparative analysis of ADS gene promoter in seven Artemisia species

    Indian Academy of Sciences (India)

    Mojtaba Ranjbar; Mohammad Reza Naghavi; Hoshang Alizadeh


    Artemisinin is the most effective antimalarial drug that is derived from Artemisia annua. Amorpha-4,11-diene synthase (ADS) controls the first committed step in artemisinin biosynthesis. The ADS gene expression is regulated by transcription factors which bind to the cis-acting elements on the ADS promoter and are probably responsible for the ADS gene expression difference in the Artemisia species. To identify the elements that are significantly involved in ADS gene expression, the ADS gene promoter of the seven Artemisia species was isolated and comparative analysis was performed on the ADS promoter sequences of these species. Results revealed that some of the cis-elements were unique or in terms of number were more in the high artemisinin producer species, A. annua, than the other species. We have reported that the light-responsive elements, W-box, CAAT-box, 5′-UTR py-rich stretch, TATA-box sequence and tandem repeat sequences have been identified as important factors in the increased expression of ADS gene.

  16. Conditional promoters for analysis of essential genes in Zymoseptoria tritici. (United States)

    Kilaru, S; Ma, W; Schuster, M; Courbot, M; Steinberg, G


    Development of new fungicides, needed for sustainable control of fungal plant pathogens, requires identification of novel anti-fungal targets. Essential fungal-specific proteins are good candidates, but due to their importance, gene deletion mutants are not viable. Consequently, their cellular role often remains elusive. This hindrance can be overcome by the use of conditional mutants, where expression is controlled by an inducible/repressible promoter. Here, we introduce 5 inducible/repressible promoter systems to study essential genes in the wheat pathogen Zymoseptoria tritici. We fused the gene for enhanced green-fluorescent protein (egfp) to the promoter region of Z. tritici nitrate reductase (Pnar1; induced by nitrogen and repressed by ammonium), 1,4-β-endoxylanase A (Pex1A; induced by xylose and repressed by maltodextrin), l-arabinofuranosidase B (PlaraB; induced by arabinose and repressed by glucose), galactose-1-phosphate uridylyltransferase 7 (Pgal7; induced by galactose and repressed by glucose) and isocitrate lyase (Picl1; induced by sodium acetate and repressed by glucose). This was followed by quantitative analysis of cytoplasmic reporter fluorescence under induced and repressed conditions. We show that Pnar1, PlaraB and Pex1A drive very little or no egfp expression when repressed, but induce moderate protein production when induced. In contrast, Pgal7 and Picl1 show considerable egfp expression when repressed, and were strongly induced in the presence of their inducers. Normalising the expression levels of all promoters to that of the α-tubulin promoter Ptub2 revealed that PlaraB was the weakest promoter (∼20% of Ptub2), whereas Picl1 strongly expressed the reporter (∼250% of Ptub2). The use of these tools promises a better understanding of essential genes, which will help developing novel control strategies that protect wheat from Z. tritici.

  17. Optical Aberrations and Wavefront

    Directory of Open Access Journals (Sweden)

    Nihat Polat


    Full Text Available The deviation of light to create normal retinal image in the optical system is called aberration. Aberrations are divided two subgroup: low-order aberrations (defocus: spherical and cylindrical refractive errors and high-order aberrations (coma, spherical, trefoil, tetrafoil, quadrifoil, pentafoil, secondary astigmatism. Aberrations increase with aging. Spherical aberrations are compensated by positive corneal and negative lenticular spherical aberrations in youth. Total aberrations are elevated by positive corneal and positive lenticular spherical aberrations in elderly. In this study, we aimed to analyze the basic terms regarding optic aberrations which have gained significance recently. (Turk J Ophthalmol 2014; 44: 306-11

  18. Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration. (United States)

    Im, Gun-Il


    Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes.

  19. Food-grade TiO2 impairs intestinal and systemic immune homeostasis, initiates preneoplastic lesions and promotes aberrant crypt development in the rat colon (United States)

    Bettini, Sarah; Boutet-Robinet, Elisa; Cartier, Christel; Coméra, Christine; Gaultier, Eric; Dupuy, Jacques; Naud, Nathalie; Taché, Sylviane; Grysan, Patrick; Reguer, Solenn; Thieriet, Nathalie; Réfrégiers, Matthieu; Thiaudière, Dominique; Cravedi, Jean-Pierre; Carrière, Marie; Audinot, Jean-Nicolas; Pierre, Fabrice H.; Guzylack-Piriou, Laurence; Houdeau, Eric


    Food-grade titanium dioxide (TiO2) containing a nanoscale particle fraction (TiO2-NPs) is approved as a white pigment (E171 in Europe) in common foodstuffs, including confectionary. There are growing concerns that daily oral TiO2-NP intake is associated with an increased risk of chronic intestinal inflammation and carcinogenesis. In rats orally exposed for one week to E171 at human relevant levels, titanium was detected in the immune cells of Peyer’s patches (PP) as observed with the TiO2-NP model NM-105. Dendritic cell frequency increased in PP regardless of the TiO2 treatment, while regulatory T cells involved in dampening inflammatory responses decreased with E171 only, an effect still observed after 100 days of treatment. In all TiO2-treated rats, stimulation of immune cells isolated from PP showed a decrease in Thelper (Th)-1 IFN-γ secretion, while splenic Th1/Th17 inflammatory responses sharply increased. E171 or NM-105 for one week did not initiate intestinal inflammation, while a 100-day E171 treatment promoted colon microinflammation and initiated preneoplastic lesions while also fostering the growth of aberrant crypt foci in a chemically induced carcinogenesis model. These data should be considered for risk assessments of the susceptibility to Th17-driven autoimmune diseases and to colorectal cancer in humans exposed to TiO2 from dietary sources. PMID:28106049

  20. Aberrant Behaviours of Reaction Diffusion Self-organisation Models on Growing Domains in the Presence of Gene Expression Time Delays

    KAUST Repository

    Seirin Lee, S.


    Turing\\'s pattern formation mechanism exhibits sensitivity to the details of the initial conditions suggesting that, in isolation, it cannot robustly generate pattern within noisy biological environments. Nonetheless, secondary aspects of developmental self-organisation, such as a growing domain, have been shown to ameliorate this aberrant model behaviour. Furthermore, while in-situ hybridisation reveals the presence of gene expression in developmental processes, the influence of such dynamics on Turing\\'s model has received limited attention. Here, we novelly focus on the Gierer-Meinhardt reaction diffusion system considering delays due the time taken for gene expression, while incorporating a number of different domain growth profiles to further explore the influence and interplay of domain growth and gene expression on Turing\\'s mechanism. We find extensive pathological model behaviour, exhibiting one or more of the following: temporal oscillations with no spatial structure, a failure of the Turing instability and an extreme sensitivity to the initial conditions, the growth profile and the duration of gene expression. This deviant behaviour is even more severe than observed in previous studies of Schnakenberg kinetics on exponentially growing domains in the presence of gene expression (Gaffney and Monk in Bull. Math. Biol. 68:99-130, 2006). Our results emphasise that gene expression dynamics induce unrealistic behaviour in Turing\\'s model for multiple choices of kinetics and thus such aberrant modelling predictions are likely to be generic. They also highlight that domain growth can no longer ameliorate the excessive sensitivity of Turing\\'s mechanism in the presence of gene expression time delays. The above, extensive, pathologies suggest that, in the presence of gene expression, Turing\\'s mechanism would generally require a novel and extensive secondary mechanism to control reaction diffusion patterning. © 2010 Society for Mathematical Biology.

  1. Germline promoter hypermethylation of tumor suppressor genes in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Pu-Yuan Wu; Zheng Zhang; Jing-Mei Wang; Wen-Wen Guo; Nong Xiao; Qiong He; Ya-Ping Wang; Yi-Mei Fan


    AIM: To explore germline hypermethylation of the tumor suppressor genes MLH1 , CDH1 and P16INK4a in suspected cases of hereditary gastric cancer (GC). METHODS: A group of 140 Chinese GC patients in whom the primary cancer had developed before the age of 60 or who had a familial history of cancer were screened for germline hypermethylation of the MLH1 , CDH1 and P16INK4a tumor suppressor genes. Genomic DNA was extracted from peripheral blood leukocytes and modified by sodium bisulfite. The treated DNA was then subjected to bisulfite DNA sequencing for a specific region of the MLH1 promoter. The methylation status of CDH1 or P16INK4a was assayed using methylation- specific PCR. Clonal bisulfite allelic sequencing in positive samples was performed to obtain a comprehensive analysis of the CpG island methylation status of these promoter regions. RESULTS: Methylation of the MLH1 gene promoter was detected in the peripheral blood DNA of only 1/140 (0.7%) of the GC patient group. However, this methylation pattern was mosaic rather than the allelic pattern which has previously been reported for MLH1 in hereditary non-polyposis colorectal cancer (HNPCC) patients. We found that 10% of the MLH1 alleles in the peripheral blood DNA of this patient were methylated, consistent with 20% of cells having one methylated allele. No germline promoter methylation of the CDH1 or P16INK4a genes was detected. CONCLUSION: Mosaic germline epimutation of the MLH1 gene is present in suspected hereditary GC patients in China but at a very low level. Germline epimutation of the CDH1 or P16INK4a gene is not a frequent event.

  2. Isolation and characterization of the murine Nanog gene promoter

    Institute of Scientific and Technical Information of China (English)

    Da Yong WU; Zhen YAO


    Nanog protein is expressed in the interior cells of compacted morulae and maintained till epiblasts but downregulated by implantation stage. It is also expressed in embryonic stem cells, embryonic carcinoma cells and embryonic germ cells but disappeared in differentiated ES cells. In this study, we have isolated, sequenced, and performed the first characterization of the Nanog promoter. The transcription start sites were mapped by primer extension analysis. Two promoter regions were found upstream the transcription start sites and the expression of major Nanog promoter/reporter gene construct is abolished in differentiated F9 EC cells as compared to the undifferentiated counterpart. We also showed that a putative octamer motif (ATGCAAAA) is necessary for the major promoter activity. Gel shift and supershift assays showed that Oct-1, Oct-4 and Oct-6 protein selectively bind to the octamer motif.

  3. Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island.

    Directory of Open Access Journals (Sweden)

    Heather M O'Hagan


    Full Text Available Chronic exposure to inducers of DNA base oxidation and single and double strand breaks contribute to tumorigenesis. In addition to the genetic changes caused by this DNA damage, such tumors often contain epigenetically silenced genes with aberrant promoter region CpG island DNA hypermethylation. We herein explore the relationships between such DNA damage and epigenetic gene silencing using an experimental model in which we induce a defined double strand break in an exogenous promoter construct of the E-cadherin CpG island, which is frequently aberrantly DNA hypermethylated in epithelial cancers. Following the onset of repair of the break, we observe recruitment to the site of damage of key proteins involved in establishing and maintaining transcriptional repression, namely SIRT1, EZH2, DNMT1, and DNMT3B, and the appearance of the silencing histone modifications, hypoacetyl H4K16, H3K9me2 and me3, and H3K27me3. Although in most cells selected after the break, DNA repair occurs faithfully with preservation of activity of the promoter, a small percentage of the plated cells demonstrate induction of heritable silencing. The chromatin around the break site in such a silent clone is enriched for most of the above silent chromatin proteins and histone marks, and the region harbors the appearance of increasing DNA methylation in the CpG island of the promoter. During the acute break, SIRT1 appears to be required for the transient recruitment of DNMT3B and subsequent methylation of the promoter in the silent clones. Taken together, our data suggest that normal repair of a DNA break can occasionally cause heritable silencing of a CpG island-containing promoter by recruitment of proteins involved in silencing. Furthermore, with contribution of the stress-related protein SIRT1, the break can lead to the onset of aberrant CpG island DNA methylation, which is frequently associated with tight gene silencing in cancer.

  4. Hypermethylation of E-Cadherin and Estrogen Receptor-a Gene Promoter and Its Association with Clinicopathological Features of Breast Cancer in Iranian Patients

    Directory of Open Access Journals (Sweden)

    Mozhgan Rasti


    Full Text Available Background: Aberrant methylation of cytosine-guanine dinucleotideislands leads to inactivation of tumor suppressorgenes in breast cancer. Tumor suppressor genes are unmethylatedin normal tissue and often become hypermethylatedduring tumor formation, leading to gene silencing. We investigatedthe association between E-cadherin (CDH1 and estrogenreceptor-α (ESRα gene promoter methylation andmajor clinical and pathological features of breast cancer inIranian women.Methods: DNA was extracted from 67 primary breast tumorsand gene promoter methylation was analyzed by methylationspecificpolymerase chain reaction method.Results: Fifty percent of the samples showed aberrant methylationin at least one of the two tested loci. We detectedCDH1 hypermethylation in 41% of invasive tumors and receptor-α gene hypermethylation in 18% of invasive tumorsamples. We found no association between CDH1 and receptor-α gene hypermethylation (P=0.45. There was a correlationbetween hypermethylation of CDH1 locus and tumorsize ≥5 cm (P=0.019.Conclusion: Our data suggest that the malignant progressionof human ductal and lobular breast carcinoma in Iranianwomen involves a heterogeneous pattern of cytosine-guaninedinucleotide island hypermethylation of the CDH1 gene.

  5. Methylation of Gene CHFR Promoter in Acute Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    GONG Hui; LIU Wengli; ZHOU Jianfeng; XU Huizhen


    Summary: In order to explore whether gene CHFR was inactivated by methylation in leukemia cells, the expression of CHFR was examined before and after treatment with demethylation agent in Molt-4, Jurkat and U937 leukemia cell lines by means of RT-PCR. The methylation of promoter in Molt-4, Jurkat and U937 cells as well as 41 acute leukemia patients was analyzed by MS-PCR. The results showed that methylation of CHFR promoter was inactivated and could be reversed by treatment with a demethylating agent in Molt-4, Jurkat and U937. CHFR promoter methylation was detected in 39 % of acute leukemia patients. There was no difference in incidence of CHFR promoter methylation between acute myelocytic leukemia and acute lymphocytic leukemia. In conclusion, CHFR is frequently inactivated in acute leukemia and is a good candidate for the leukemia supper gene. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in acute leukemia. Moreover, the methylation of gene CHFR appears to be a good index with which to predict the sensitivity of acute leukemia to microtubule inhibitors.

  6. Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

    Directory of Open Access Journals (Sweden)

    María Berdasco

    Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

  7. Detection of aberrant methylation of a six-gene panel in serum DNA for diagnosis of breast cancer. (United States)

    Shan, Ming; Yin, Huizi; Li, Junnan; Li, Xiaobo; Wang, Dong; Su, Yonghui; Niu, Ming; Zhong, Zhenbin; Wang, Ji; Zhang, Xianyu; Kang, Wenli; Pang, Da


    Detection of breast cancer at an early stage is the key for successful treatment and improvement of outcome. However the limitations of mammography are well recognized, especially for those women with premenopausal breast cancer. Novel approaches to breast cancer screening are necessary, especially in the developing world where mammography is not feasible. In this study, we examined the promoter methylation of six genes (SFN, P16, hMLH1, HOXD13, PCDHGB7 and RASSF1a) in circulating free DNA (cfDNA) extracted from serum. We used a high-throughput DNA methylation assay (MethyLight) to examine serum from 749 cases including breast cancer patients, patients with benign breast diseases and healthy women. The six-gene methylation panel test achieved 79.6% and 82.4% sensitivity with a specificity of 72.4% and 78.1% in diagnosis of breast cancer when compared with healthy and benign disease controls, respectively. Moreover, the methylation panel positive group showed significant differences in the following independent variables: (a) involvement of family history of tumors; (b) a low proliferative index, ki-67; (c) high ratios in luminal subtypes. Additionally the panel also complemented some breast cancer cases which were neglected by mammography or ultrasound. These data suggest that epigenetic markers in serum have potential for diagnosis of breast cancer.

  8. Gene promoter analysis and molecular diagnostics – Application to erythroid-specific gene expression

    NARCIS (Netherlands)

    de Vooght, K.M.K.


    Gene expression is regulated at many levels. Most regulation, however, is believed to occur at the level of transcription initiation. Transcription factors, chromatin-modifying enzymes, and basal transcription factors unite to activate genes and are recruited in a precise order to promoters. This th

  9. Antipsychotic drugs attenuate aberrant DNA methylation of DTNBP1 (dysbindin) promoter in saliva and post-mortem brain of patients with schizophrenia and Psychotic bipolar disorder. (United States)

    Abdolmaleky, Hamid M; Pajouhanfar, Sara; Faghankhani, Masoomeh; Joghataei, Mohammad Taghi; Mostafavi, Ashraf; Thiagalingam, Sam


    Due to the lack of genetic association between individual genes and schizophrenia (SCZ) pathogenesis, the current consensus is to consider both genetic and epigenetic alterations. Here, we report the examination of DNA methylation status of DTNBP1 promoter region, one of the most credible candidate genes affected in SCZ, assayed in saliva and post-mortem brain samples. The Illumina DNA methylation profiling and bisulfite sequencing of representative samples were used to identify methylation status of the DTNBP1 promoter region. Quantitative methylation specific PCR (qMSP) was employed to assess methylation of DTNBP1 promoter CpGs flanking a SP1 binding site in the saliva of SCZ patients, their first-degree relatives and control subjects (30, 15, and 30/group, respectively) as well as in post-mortem brains of patients with SCZ and bipolar disorder (BD) versus controls (35/group). qRT-PCR was used to assess DTNBP1 expression. We found DNA hypermethylation of DTNBP1 promoter in the saliva of SCZ patients (∼12.5%, P = 0.036), particularly in drug-naïve patients (∼20%, P = 0.011), and a trend toward hypermethylation in their first-degree relatives (P = 0.085) versus controls. Analysis of post-mortem brain samples revealed an inverse correlation between DTNBP1 methylation and expression, and normalization of this epigenetic change by classic antipsychotic drugs. Additionally, BD patients with psychotic depression exhibited higher degree of methylation versus other BD patients (∼80%, P = 0.025). DTNBP1 promoter DNA methylation may become a key element in a panel of biomarkers for diagnosis, prevention, or therapy in SCZ and at risk individuals pending confirmatory studies with larger sample sizes to attain a higher degree of significance.

  10. Gene activation regresses atherosclerosis, promotes health, and enhances longevity

    Directory of Open Access Journals (Sweden)

    Luoma Pauli V


    Full Text Available Abstract Background Lifestyle factors and pharmacological compounds activate genetic mechanisms that influence the development of atherosclerotic and other diseases. This article reviews studies on natural and pharmacological gene activation that promotes health and enhances longevity. Results Living habits including healthy diet and regular physical activity, and pharmacotherapy, upregulate genes encoding enzymes and apolipoprotein and ATP-binding cassette transporters, acting in metabolic processes that promote health and increase survival. Cytochrome P450-enzymes, physiological factors in maintaining cholesterol homeostasis, generate oxysterols for the elimination of surplus cholesterol. Hepatic CTP:phosphocholine cytidylyltransferase-α is an important regulator of plasma HDL-C level. Gene-activators produce plasma lipoprotein profile, high HDL-C, HDL2-C and HDL-C/cholesterol ratio, which is typical of low risk of atherosclerotic disease, and also of exceptional longevity together with reduced prevalence of cardiovascular, metabolic and other diseases. High HDL contributes to protection against inflammation, oxidation and thrombosis, and associates with good cognitive function in very old people. Avoiding unhealthy stress and managing it properly promotes health and increases life expectancy. Conclusions Healthy living habits and gene-activating xenobiotics upregulate mechanisms that produce lipoprotein pattern typical of very old people and enhance longevity. Lipoprotein metabolism and large HDL2 associate with the process of living a very long life. Major future goals for health promotion are the improving of commitment to both wise lifestyle choices and drug therapy, and further the developing of new and more effective and well tolerated drugs and treatments.

  11. BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

    NARCIS (Netherlands)

    E.H.J. van Roon (Eddy); A. Boot (Arnoud); A.A. Dihal (Ashwin); R.F. Ernst (Robert); T. van Wezel (Tom); H. Morreau (Hans); J.M. Boer (Judith)


    textabstractBackground: Cancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have

  12. Promoter Hypermethylation and Its Impact on Expression of MGMT Gene in the GIT Malignant Patients of Kashmiri Origin. (United States)

    Bhat, Arif Akbar; Wani, Hilal Ahmad; Ishaq, Shiekh; Waza, Ajaz Ahmad; Malik, Rawoof Ahmad; Shabir, Iram; Jeelani, Showkat; Kadla, Showkat; Qureshie, Waseem; Masood, Akbar; Majid, Sabhiya


    Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Gastrointestinal tract (GIT) cancer is a major medical and economic burden worldwide. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumor suppressor genes. Although a number of cancer-associated genes have been found to be hypermethylated in GIT cancer, valuable methylation markers for early diagnosis and prognostic evaluation of this cancer remain largely unknown. O6-methyguanine DNA methyltransferase (MGMT) is a DNA-repair gene that removes mutagenic and cytotoxic adducts from the O6 position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression have been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human GIT carcinomas. A total of 210 GIT tumor samples and 90 adjacent normal tissues were analyzed for MGMT promoter methylation by methylation-specific polymerase chain reaction after bisulfite modification of DNA and same samples were analyzed for MGMT protein expression by Western blotting. The methylation frequencies of MGMT gene promoter were 41.4%, 34.2%, and 44.2% in stomach, esophageal, and colorectal cancer cases while as 16.6, 13.3, and 13.3 in respective controls. MGMT protein was found downregulated in controls of all GIT. The results suggest that methylation at CpG islands of MGMT may be responsible for the downregulation of MGMT protein expression in GIT cancers.

  13. Correlation between ECT2 gene expression and methylation change of ECT2 promoter region in pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Mang-Li Zhang; Sen Lu; Lin Zhou; Shu-Sen Zheng


    BACKGROUND: Pancreatic cancer is closely related to epigenetic abnormality. The epithelial cell transforming sequence 2 gene (ECT2) plays a critical role in Rho activation during cytokinesis, and thus may play a role in the pathogenesis of pancreatic cancer. In this study, we investigated the relationships between aberrant expression and epigenetic changes of the ECT2 gene in pancreatic cancer. METHODS: Four cell lines (PANC-1, Colo357, T3M-4 and PancTuⅠ) and pancreatic ductal adenocarcinoma (PDAC) tissues were used for mRNA detection. After restriction isoschizomer endonucleases (MspⅠ/HpaⅡ) were used to digest the DNA sequence (5'-CCGG-3'), PCR was made to amplify the product. And RT-PCR was applied to determine the expression of the gene. RESULTS: The mRNA expression of the ECT2 gene was higher in pancreatic tumor tissue than in normal tissue. The gene was also expressed in the 4 PDAC cell lines. The methylation states of the upstream regions of the ECT2 gene were almost identical in normal, tumor pancreatic tissues, and the 4 PDAC cell lines. Some of the 5'-CCGG-3' areas in the upstream region of ECT2 were methylated, while others were unmethylated. CONCLUSIONS: The oncogene ECT2 is overexpressed in pancreatic tumor tissues as veriifed by RT-PCR detection. The methylation status of DNA in promoter areas is involved in the gene expression, along with other factors, in pancreatic cancer.

  14. Analysis of promoter activity in transgenic plants by normalizing expression with a reference gene: anomalies due to the influence of the test promoter on the reference promoter

    Indian Academy of Sciences (India)

    Simran Bhullar; Suma Chakravarthy; Deepak Pental; Pradeep Kumar Burma


    Variations in transgene expression due to position effect and copy number are normalized when analysing and comparing the strengths of different promoters. In such experiments, the promoter to be tested is placed upstream to a reporter gene and a second expression cassette is introduced in a linked fashion in the same transfer DNA (T-DNA). Normalization in the activity of the test promoter is carried out by calculating the ratio of activities of the test and reference promoters. When an appropriate number of independent transgenic events are analysed, normalization facilitates assessment of the relative strengths of the test promoters being compared. In this study, using different modified versions of the Cauliflower Mosaic Virus (CaMV) 35S promoter expressing the reporter gene -glucuronidase (gus) (test cassette) linked to a chloramphenicol acetyl transferase (cat) gene under the wild-type 35S promoter (reference cassette) in transgenic tobacco lines, we observed that cat gene expression varied depending upon the strength of the modified 35S promoter expressing the gus gene. The 35S promoter in the reference cassette was found to have been upregulated in cases where the modified 35S promoter was weaker than the wild-type 35S promoter. Many studies have been carried out in different organisms to study the phenomenon of transcriptional interference, which refers to the reduced expression of the downstream promoter by a closely linked upstream promoter. However, we observed a positive interaction wherein the weakened activity of a promoter led to upregulation of a contiguous promoter. These observations suggest that, in situations where the promoters of the test and reference gene share the same transcription factors, the activity of the test promoter can influence the activity of the reference promoter in a way that the test promoter’s strength is underestimated when normalized by the reference promoter.

  15. Silencing of CHD5 gene by promoter methylation in leukemia.

    Directory of Open Access Journals (Sweden)

    Rui Zhao

    Full Text Available Chromodomain helicase DNA binding protein 5 (CHD5 was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network. CHD5 is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of CHD5 down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that CHD5 was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the CHD5 promoter was hypermethylated in human leukemia cells and samples. Thus, CHD5 expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC activates CHD5 expression in human leukemia cell lines. In vitro luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2 as a potential transcription factor involved in CHD5 expression and indicated that treatment with DAC increases the recruitment of AP2 to the CHD5 promoter. In vitro transcription-factor activity studies showed that AP2 over-expression was able to activate CHD5 promoter activity. Our findings indicate that repression of CHD5 gene expression in human leukemia is mediated in part by DNA methylation of its promoter.

  16. The Histone Demethylase Jarid1b Ensures Faithful Mouse Development by Protecting Developmental Genes from Aberrant H3K4me3

    DEFF Research Database (Denmark)

    Albert, Mareike; Schmitz, Sandra U; Kooistra, Susanne M


    of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line...... with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis...... of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like...

  17. Promoter methylation analysis of IDH genes in human gliomas

    Directory of Open Access Journals (Sweden)

    Simon eFlanagan


    Full Text Available Mutations in isocitrate dehydrogenase (IDH -1 or -2 are found in the majority of WHO grade II and III astrocytomas and oligodendrogliomas, and secondary glioblastomas. Almost all described mutations are heterozygous missense mutations affecting a conserved arginine residue in the substrate binding site of IDH1 (R132 or IDH2 (R172. But the exact mechanism of IDH mutations in neoplasia is not understood. It has been proposed that IDH mutations impart a ‘toxic gain of function’ to the mutant protein, however a dominant-negative effect of mutant IDH has also been described, implying that IDH may function as a tumour suppressor gene. As most, if not all, tumour suppressor genes are inactivated by epigenetic silencing, in a wide variety of tumours, we asked if IDH1 or IDH2 carry the epigenetic signature of a tumour suppressor by assessing cytosine methylation at their promoters. Methylation was quantified in 68 human brain tumours, including both IDH-mutant and IDH wildtype, by bisulfite pyrosequencing. In all tumours examined, CpG methylation levels were less than 8%. Our data demonstrate that inactivation of IDH function through promoter hypermethylation is not common in human gliomas and other brain tumours. These findings do not support a tumour suppressor role for IDH genes in human gliomas.

  18. Exclusion of APC and VHL gene deletions by array-based comparative hybridization in two patients with microscopically visible chromosomal aberrations. (United States)

    Wallerstein, Robert J; Brooks, Susan Sklower; Streck, Deanna L; Kurvathi, Rohini; Toruner, Gokce A


    Karyotyping is a major component of the genetic work-up of patients with dysmorphism. Cytogenetic aberrations close to a known tumor suppressor gene raise important clinical issues because deletion of that tumor suppressor gene can cause genetic predisposition to cancer. We present two cancer-free dysmorphic patients with karyotypes of 46,XX,del(5)(q15q22.3) and 46,XX,del(3)(p25.2~pter). These deletions are close to the APC and VHL genes that confer susceptibility to familial Adenomatous polyposis (OMIM #17510) and von-Hippel-Lindau syndrome (OMIM #193300), respectively. The array-based comparative genomic hybridization (array-CGH) analysis using a custom Agilent 44K oligonucleotide array demonstrated an interstitial 20.7-megabase (Mb) deletion on 5q (chr5: 89,725,638-110,491,345) and a terminal 9.45-Mb deletion on 3p (chr3:pter-9,450,984). According to the March 2006 human reference sequence, the APC gene is located at chr5: 112,101,483-112,209,835 and the VHL gene is located at chr3: 10,158,319-10,168,746. These results indicate that the APC gene is 2,300 kilobases (kb) and the VHL gene is 700 kb away from deleted regions. Southern blot analysis for APC and VHL genes were negative, consistent with array-CGH findings. These results demonstrate the power of array-CCH to assess potential tumor suppressor gene involvement and cancer risk in patients with microscopically visible deletions in areas near tumor suppressors.

  19. The tumor-selective over-expression of the human Hsp 70 gene is attributed to the aberrant controls at both initiation and elongation levels of transcription

    Institute of Scientific and Technical Information of China (English)


    The tumor selective over-expression of the human Hsp70 gene has been well documented in human tumors, linked to the poor prognosis, being refractory to chemo- and radio-therapies as well as the advanced stage of tumorous lesions in particular. However, both the nature and details of aberrations in the control of the Hsp70 expression in tumor remain enigmatic. By comparing various upstream segments of the Hsp70gene for each's ability to drive the luciferase reporter genes in the context of the tumor cell lines varying in their p53 status and an immortal normal liver cell line, we demonstrated in a great detail the defects in the control mechanisms at the both initiation and elongation levels of transcription being instrumental to the tumor selective profile of its expression. Our data should not only offer new insights into our understanding of the tumor specific over-expression of the human Hsp70 gene, but also paved the way for the rational utilization of the tumor selective mechanism with the Hsp70 at the central stage for targeting the therapeutic gene expression to human tumors.

  20. Downregulation of anti-oncomirs miR-143/145 cluster occurs before APC gene aberration in the development of colorectal tumors. (United States)

    Kamatani, Akemi; Nakagawa, Yoshihito; Akao, Yukihiro; Maruyama, Naoko; Nagasaka, Mitsuo; Shibata, Tomoyuki; Tahara, Tomomitsu; Hirata, Ichiro


    Accumulating data indicate that some microRNAs (miRNAs or miRs) can function as tumor suppressors or oncogenes and as such are important in cancer development. We previously reported that miR-143 and -145 are frequently downregulated in colon adenomas and cancers, acting as tumor suppressors. In this present study, we investigated the relationship between the downregulation of the miR-143/145 cluster and genetic aberrations of adenomatous polyposis coli (APC), which are early genetic events in the development of colorectal tumors. The expression levels of both miRs were determined by performing real-time PCR on tissue samples of familial adenomatous polyposis (FAP), colorectal adenoma, colorectal cancer, and paired non-tumorous tissues. Also, the expression of C- or N-terminus of the APC protein and that of the p53 protein in these tissues were examined immunohistochemically. Our data clearly indicated that the decreased expression of miR-143 and -145 frequently occurred before APC gene aberrations. The downregulation of miR-143 and -145 is thus an important genetic event for the initiation step in colorectal tumor development.

  1. Construction of chimeric antibodies: cloning of immunoglobulin genes including their promoter regions by PCR. (United States)

    Mocikat, R; Kütemeier, G; Harloff, C


    In the production of recombinant antibodies, it is necessary to have an immunoglobulin gene promoter for driving the expression of the antibody genes. Here we describe a simple PCR method that allows cloning of the immunoglobulin genes together with their own promoters despite the fact that the sequence of the upstream part of the gene is unknown.

  2. Inferring a role for methylation of intergenic DNA in the regulation of genes aberrantly expressed in precursor B-cell acute lymphoblastic leukemia. (United States)

    Almamun, Md; Kholod, Olha; Stuckel, Alexei J; Levinson, Benjamin T; Johnson, Nathan T; Arthur, Gerald L; Davis, J Wade; Taylor, Kristen H


    A complete understanding of the mechanisms involved in the development of pre-B ALL is lacking. In this study, we integrated DNA methylation data and gene expression data to elucidate the impact of aberrant intergenic DNA methylation on gene expression in pre-B ALL. We found a subset of differentially methylated intergenic loci that were associated with altered gene expression in pre-B ALL patients. Notably, 84% of these regions were also bound by transcription factors (TF) known to play roles in differentiation and B-cell development in a lymphoblastoid cell line. Further, an overall downregulation of eRNA transcripts was observed in pre-B ALL patients and these transcripts were associated with the downregulation of putative target genes involved in B-cell migration, proliferation, and apoptosis. The identification of novel putative regulatory regions highlights the significance of intergenic DNA sequences and may contribute to the identification of new therapeutic targets for the treatment of pre-B ALL.

  3. COX-2 gene promoter haplotypes and prostate cancer risk. (United States)

    Panguluri, Ramesh C K; Long, Layron O; Chen, Weidong; Wang, Songping; Coulibaly, Aoua; Ukoli, Flora; Jackson, Aaron; Weinrich, Sally; Ahaghotu, Chiledum; Isaacs, William; Kittles, Rick A


    Cyclooxygenase-2 (COX-2) is a key rate-limiting enzyme that converts arachidonic acid into pro-inflammatory prostaglandins. COX-2 expression is strongly correlated with increased tumor microvasculature density and plays an important role in inhibiting apoptosis, stimulating angiogenesis and promoting tumor cell metastasis and invasion. However, little is known about the role that sequence variation of the COX-2 gene contributes to prostate cancer. Thus, we searched for polymorphisms in the promoter region of the COX-2 gene using denaturing high-performance liquid chromatography. Four single nucleotide polymorphisms (SNPs), -1285A/G, -1265G/A, -899G/C and -297C/G, were detected and confirmed by direct sequencing. Three of the SNPs in the promoter region of COX-2 gene create at least three putative transcription factor binding sites and eliminate CCAAT/enhancer binding protein alpha (C/EBP alpha) and NF-kappa B binding sites. A case-control study of the four SNPs in African American (n = 288), Bini Nigerian (n = 264) and European American (n = 184) prostate cancer cases and age-matched controls revealed that SNP -297G was associated with a decreased risk for prostate cancer [odds ratio (OR) = 0.49; CI = 0.2-0.9; P = 0.01]. The effect on risk was observed in both African Americans (OR = 0.51; CI = 0.2-0.9; P = 0.01) and European Americans (OR = 0.33; CI = 0.1-0.9; P = 0.02). In addition, SNPs -1265A and -899C were associated with increased prostate cancer risk in African Americans (OR = 2.72; CI = 1.3-5.8; P = 0.007 and OR = 3.67; CI = 1.4-9.9; P = 0.007, respectively). Haplotype analyses revealed modest effects on susceptibility to prostate cancer across populations. Haplotype GGCC conferred increased risk in the African American and Nigerian populations. Conversely, haplotype AGGG exhibited a negative association with prostate cancer risk in African Americans (OR = 0.4; CI = 0.1-0.9; P = 0.02) and European Americans (OR = 0.2; CI = 0.1-0.9; P = 0.03). These data

  4. Aberrantly methylated DNA as a biomarker in breast cancer

    DEFF Research Database (Denmark)

    Kristiansen, Søren; Jørgensen, Lars Mønster; Guldberg, Per;


    hypermethylation events, their use as tumor biomarkers is usually not hampered by analytical signals from normal cells, which is a general problem for existing protein tumor markers used for clinical assessment of breast cancer. There is accumulating evidence that DNA-methylation changes in breast cancer patients......Aberrant DNA hypermethylation at gene promoters is a frequent event in human breast cancer. Recent genome-wide studies have identified hundreds of genes that exhibit differential methylation between breast cancer cells and normal breast tissue. Due to the tumor-specific nature of DNA...... into subgroups based on DNA biomarkers may improve prognosis. Serial monitoring of DNA-methylation markers in blood during treatment may be useful, particularly when the cancer burden is below the detection level for standard imaging techniques. Overall, aberrant DNA methylation has a great potential...

  5. Detection of gene copy number aberrations in mantle cell lymphoma by a single quantitative multiplex PCR assay: clinicopathological relevance and prognosis value. (United States)

    Jardin, Fabrice; Picquenot, Jean-Michel; Parmentier, Françoise; Ruminy, Philippe; Cornic, Marie; Penther, Dominique; Bertrand, Philippe; Lanic, Hélène; Cassuto, Ophélie; Humbrecht, Catherine; Lemasle, Emilie; Wautier, Agathe; Bastard, Christian; Tilly, Hervé


    The t(11;14)(q13;q32) is the hallmark of mantle cell lymphoma (MCL). Additional genetic alterations occur in the majority of cases. This study aimed to design a polymerase chain reaction (PCR) assay to determine the incidence and relevance of recurrent gene copy number aberrations in this disease. Forty-two MCL cases with frozen- or paraffin-embedded (FFPE) tissues were selected. Three different quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) assays were designed to simultaneously analyse eight genes (CDKN2A, RB1, ATM, CDK2, TP53, MYC, CDKN1B, MDM2), to analyse the 9p21 locus (CDKN2A/CDKN2B) and FFPE tissues. Gains of MYC, CDK2, CDKN1B, and MDM2 were observed in 10% of cases. Losses of RB1, CDKN2A, ATM or TP53 were observed in 38%, 31%, 24% and 10% of cases, respectively. Analysis of the 9p21 locus indicated that, in most cases, tumours displayed a complete inactivation of p14(ARF)/p15I(NK4B)/p16I(NK4A). CDKN2A and MYC aberrations were associated with a high MCL international prognostic index (MIPI). CDK2/MDM2 gains and CDKN2A/TP53 losses correlated with an unfavourable outcome. PCR experiments with frozen and FFPE-tissues indicated that our approach is valid in a routine diagnostic setting, providing a powerful tool that could be used for patient stratification in combination with MIPI in future clinical trials.

  6. Epigenetic aberrations and therapeutic implications in gliomas. (United States)

    Natsume, Atsushi; Kondo, Yutaka; Ito, Motokazu; Motomura, Kazuya; Wakabayashi, Toshihiko; Yoshida, Jun


    Almost all cancer cells have multiple epigenetic abnormalities, which combine with genetic changes to affect many cellular processes, including cell proliferation and invasion, by silencing tumor-suppressor genes. In this review, we focus on the epigenetic mechanisms of DNA hypomethylation and CpG island hypermethylation in gliomas. Aberrant hypermethylation in promoter CpG islands has been recognized as a key mechanism involved in the silencing of cancer-associated genes and occurs at genes with diverse functions related to tumorigenesis and tumor progression. Such promoter hypermethylation can modulate the sensitivity of glioblastomas to drugs and radiotherapy. As an example, the methylation of the O6-methylguanine DNA methyltransferase (MGMT) promoter is a specific predictive biomarker of tumor responsiveness to chemotherapy with alkylating agents. Further, we reviewed reports on pyrosequencing - a simple technique for the accurate and quantitative analysis of DNA methylation. We believe that the quantification of MGMT methylation by pyrosequencing might enable the selection of patients who are most likely to benefit from chemotherapy. Finally, we also evaluated the potential of de novo NY-ESO-1, the most immunogenic cancer/testis antigen (CTA) discovered thus far, as an immunotherapy target. The use of potent epigenetics-based therapy for cancer cells might restore the abnormally regulated epigenomes to a more normal state through epigenetic reprogramming. Thus, epigenetic therapy may be a promising and potent treatment for human neoplasia.

  7. Chromosome aberrations induced by zebularine in triticale. (United States)

    Ma, Xuhui; Wang, Qing; Wang, Yanzhi; Ma, Jieyun; Wu, Nan; Ni, Shuang; Luo, Tengxiao; Zhuang, Lifang; Chu, Chenggen; Cho, Seong-Woo; Tsujimoto, Hisashi; Qi, Zengjun


    Chromosome engineering is an important approach for generating wheat germplasm. Efficient development of chromosome aberrations will facilitate the introgression and application of alien genes in wheat. In this study, zebularine, a DNA methylation transferase inhibitor, was successfully used to induce chromosome aberrations in the octoploid triticale cultivar Jinghui#1. Dry seeds were soaked in zebularine solutions (250, 500, and 750 μmol/L) for 24 h, and the 500 μmol/L treatment was tested in three additional treatment times, i.e., 12, 36, and 48 h. All treatments induced aberrations involving wheat and rye chromosomes. Of the 920 cells observed in 67 M1 plants, 340 (37.0%) carried 817 aberrations with an average of 0.89 aberrations per cell (range: 0-12). The aberrations included probable deletions, telosomes and acentric fragments (49.0%), large segmental translocations (28.9%), small segmental translocations (17.1%), intercalary translocations (2.6%), long chromosomes that could carry more than one centromere (2.0%), and ring chromosomes (0.5%). Of 510 M2 plants analyzed, 110 (21.6%) were found to carry stable aberrations. Such aberrations included 79 with varied rye chromosome numbers, 7 with wheat and rye chromosome translocations, 15 with possible rye telosomes/deletions, and 9 with complex aberrations involving variation in rye chromosome number and wheat-rye translocations. These indicated that aberrations induced by zebularine can be steadily transmitted, suggesting that zebularine is a new efficient agent for chromosome manipulation.

  8. Integrated genome-wide genotyping and gene expression profiling reveals BCL11B as a putative oncogene in acute myeloid leukemia with 14q32 aberrations. (United States)

    Abbas, Saman; Sanders, Mathijs A; Zeilemaker, Annelieke; Geertsma-Kleinekoort, Wendy M C; Koenders, Jasper E; Kavelaars, Francois G; Abbas, Zabiollah G; Mahamoud, Souad; Chu, Isabel W T; Hoogenboezem, Remco; Peeters, Justine K; van Drunen, Ellen; van Galen, Janneke; Beverloo, H Berna; Löwenberg, Bob; Valk, Peter J M


    Acute myeloid leukemia is a neoplasm characterized by recurrent molecular aberrations traditionally demonstrated by cytogenetic analyses. We used high density genome-wide genotyping and gene expression profiling to reveal acquired cryptic abnormalities in acute myeloid leukemia. By genome-wide genotyping of 137 cases of primary acute myeloid leukemia, we disclosed a recurrent focal amplification on chromosome 14q32, which included the genes BCL11B, CCNK, C14orf177 and SETD3, in two cases. In the affected cases, the BCL11B gene showed consistently high mRNA expression, whereas the expression of the other genes was unperturbed. Fluorescence in situ hybridization on 40 cases of acute myeloid leukemia with high BCL11B mRNA expression [2.5-fold above median; 40 out of 530 cases (7.5%)] revealed 14q32 abnormalities in two additional cases. In the four BCL11B-rearranged cases the 14q32 locus was fused to different partner chromosomes. In fact, in two cases, we demonstrated that the focal 14q32 amplifications were integrated into transcriptionally active loci. The translocations involving BCL11B result in increased expression of full-length BCL11B protein. The BCL11B-rearranged acute myeloid leukemias expressed both myeloid and T-cell markers. These biphenotypic acute leukemias all carried FLT3 internal tandem duplications, a characteristic marker of acute myeloid leukemia. BCL11B mRNA expression in acute myeloid leukemia appeared to be strongly associated with expression of other T-cell-specific genes. Myeloid 32D(GCSF-R) cells ectopically expressing Bcl11b showed decreased proliferation rate and less maturation. In conclusion, by an integrated approach involving high-throughput genome-wide genotyping and gene expression profiling we identified BCL11B as a candidate oncogene in acute myeloid leukemia.

  9. Mutation of the gene promoter synthase nitric oxide eNOS T786С in patients with pseudarthrosis of long bones: relationship with lipid levels and endothelial function

    Directory of Open Access Journals (Sweden)

    Bezsmertnyi Yu.O.


    Full Text Available Objective: The prevalence of gene promoter polymorphism eNOS T786С and its relationship with the level of lipids and vascular endothelial function in patients with pseudoarthrosis of long bones was investigated. Materials and methods. In 118 patients with pseudoarthrosis of long bones frequency of mutation of gene promoter eNOS T786C was studied and its relationship to lipid levels and vascular endothelial function was determined. The control group consisted of 48 patients with consolidated diaphyseal fractures, representative by age, gender and location of the damage. Results and discussion. Pathological homozygotes eNOS 786-CC accumulate predominantly in patients with hypoplastic and atrophic type of pseudoarthrosis. The frequency of genotypes distribution in patients with normoplastic type of pseudoarthrosis corresponded to the same in people with consolidated fractures. In patients with genotype eNOS 786-CC more often individuals with aberrant lipid levels and structural and functional changes in the common carotid, brachial and femoral arteries are identified. Conclusions. In patients with pseudarthrosis of long bones tendency to decrease in the proportion of normal homozygotes (786-CT, increase in heterozygotes (786-CT and the proportion of individuals with abnormal genotype 786-CC is observed. The mutation in gene T786C eNOS was associated with the development of hypoplastic and aplastic types of pseudarthrosis, increasing the proportion of persons with aberrant lipid levels, endothelial dysfunction of the central and peripheral vessels.

  10. IL6 gene promoter polymorphisms and type 2 diabetes

    DEFF Research Database (Denmark)

    Huth, Cornelia; Heid, Iris M; Vollmert, Caren;


    Several lines of evidence indicate a causal role of the cytokine interleukin (IL)-6 in the development of type 2 diabetes in humans. Two common polymorphisms in the promoter of the IL-6 encoding gene IL6, -174G>C (rs1800795) and -573G>C (rs1800796), have been investigated for association with type...... 2 diabetes in numerous studies but with results that have been largely equivocal. To clarify the relationship between the two IL6 variants and type 2 diabetes, we analyzed individual data on >20,000 participants from 21 published and unpublished studies. Collected data represent eight different...... countries, making this the largest association analysis for type 2 diabetes reported to date. The GC and CC genotypes of IL6 -174G>C were associated with a decreased risk of type 2 diabetes (odds ratio 0.91, P = 0.037), corresponding to a risk modification of nearly 9%. No evidence for association was found...

  11. Characterization of gene rearrangements resulted from genomic structural aberrations in human esophageal squamous cell carcinoma KYSE150 cells. (United States)

    Hao, Jia-Jie; Gong, Ting; Zhang, Yu; Shi, Zhi-Zhou; Xu, Xin; Dong, Jin-Tang; Zhan, Qi-Min; Fu, Song-Bin; Wang, Ming-Rong


    Chromosomal rearrangements and involved genes have been reported to play important roles in the development and progression of human malignancies. But the gene rearrangements in esophageal squamous cell carcinoma (ESCC) remain to be identified. In the present study, array-based comparative genomic hybridization (array-CGH) was performed on the ESCC cell line KYSE150. Eight disrupted genes were detected according to the obviously distinct unbalanced breakpoints. The splitting of these genes was validated by dual-color fluorescence in-situ hybridization (FISH). By using rapid amplification of cDNA ends (RACE), genome walking and sequencing analysis, we further identified gene disruptions and rearrangements. A fusion transcript DTL-1q42.2 was derived from an intrachromosomal rearrangement of chromosome 1. Highly amplified segments of DTL and PTPRD were self-rearranged. The sequences on either side of the junctions possess micro-homology with each other. FISH results indicated that the split DTL and PTPRD were also involved in comprising parts of the derivative chromosomes resulted from t(1q;9p;12p) and t(9;1;9). Further, we found that regions harboring DTL (1q32.3) and PTPRD (9p23) were also splitting in ESCC tumors. The data supplement significant information on the existing genetic background of KYSE150, which may be used as a model for studying these gene rearrangements.

  12. Functional Analysis of Promoters in the Nisin Gene Cluster of Lactococcus lactis

    NARCIS (Netherlands)

    Ruyter, Pascalle G.G.A. de; Kuipers, Oscar P.; Beerthuyzen, Marke M.; Alen-Boerrigter, Ingrid van; Vos, Willem M. de


    The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless β-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expr

  13. Common Genetic Variation In Cellular Transport Genes and Epithelial Ovarian Cancer (EOC) Risk

    NARCIS (Netherlands)

    Chornokur, G.; Lin, H.Y.; Tyrer, J.P.; Lawrenson, K.; Dennis, J.; Amankwah, E.K.; Qu, X.; Tsai, Y.Y.; Jim, H.S.; Chen, Z.; Chen, A.Y.; Permuth-Wey, J.; Aben, K.; Anton-Culver, H.; Antonenkova, N.; Bruinsma, F.; Bandera, E.V.; Bean, Y.T.; Beckmann, M.W.; Bisogna, M.; Bjorge, L.; Bogdanova, N.; Brinton, L.A.; Brooks-Wilson, A.; Bunker, C.H.; Butzow, R.; Campbell, I.G.; Carty, K.; Chang-Claude, J.; Cook, L.S.; Cramer, D.W; Cunningham, J.M.; Cybulski, C.; Dansonka-Mieszkowska, A.; Bois, A. du; Despierre, E.; Dicks, E.; Doherty, J.A.; Dork, T.; Durst, M.; Easton, D.F.; Eccles, D.M.; Edwards, R.P.; Ekici, A.B.; Fasching, P.A.; Fridley, B.L.; Gao, Y.T.; Gentry-Maharaj, A.; Giles, G.G.; Glasspool, R.; Goodman, M.T.; Gronwald, J.; Harrington, P.; Harter, P.; Hein, A.; Heitz, F.; Hildebrandt, M.A.T.; Hillemanns, P.; Hogdall, C.K.; Hogdall, E.; Hosono, S.; Jakubowska, A.; Jensen, A.; Ji, B.T.; Karlan, B.Y.; Kelemen, L.E.; Kellar, M.; Kiemeney, L.A.L.M.; Krakstad, C.; Kjaer, S.K.; Kupryjanczyk, J.; Lambrechts, D.; Lambrechts, S.; Le, N.D.; Lee, A.W.; Lele, S.; Leminen, A.; Lester, J.; Levine, D.A.; Liang, D.; Lim, B.K.; Lissowska, J.; Lu, K.; Lubinski, J.; Lundvall, L.; Massuger, L.F.A.G.; Matsuo, K.; McGuire, V.; McLaughlin, J.R.; McNeish, I.; Menon, U.; Milne, R.L.; Modugno, F.; Moysich, K.B.; Ness, R.B.; Nevanlinna, H.; Eilber, U.; Odunsi, K.; Olson, S.H.; Orlow, I., et al.


    BACKGROUND: Defective cellular transport processes can lead to aberrant accumulation of trace elements, iron, small molecules and hormones in the cell, which in turn may promote the formation of reactive oxygen species, promoting DNA damage and aberrant expression of key regulatory cancer genes. As

  14. Common Genetic Variation In Cellular Transport Genes and Epithelial Ovarian Cancer (EOC) Risk

    DEFF Research Database (Denmark)

    Chornokur, Ganna; Lin, Hui-Yi; Tyrer, Jonathan P


    BACKGROUND: Defective cellular transport processes can lead to aberrant accumulation of trace elements, iron, small molecules and hormones in the cell, which in turn may promote the formation of reactive oxygen species, promoting DNA damage and aberrant expression of key regulatory cancer genes. ...

  15. Genome-wide analysis of regions similar to promoters of histone genes

    KAUST Repository

    Chowdhary, Rajesh


    Background: The purpose of this study is to: i) develop a computational model of promoters of human histone-encoding genes (shortly histone genes), an important class of genes that participate in various critical cellular processes, ii) use the model so developed to identify regions across the human genome that have similar structure as promoters of histone genes; such regions could represent potential genomic regulatory regions, e.g. promoters, of genes that may be coregulated with histone genes, and iii/ identify in this way genes that have high likelihood of being coregulated with the histone genes.Results: We successfully developed a histone promoter model using a comprehensive collection of histone genes. Based on leave-one-out cross-validation test, the model produced good prediction accuracy (94.1% sensitivity, 92.6% specificity, and 92.8% positive predictive value). We used this model to predict across the genome a number of genes that shared similar promoter structures with the histone gene promoters. We thus hypothesize that these predicted genes could be coregulated with histone genes. This hypothesis matches well with the available gene expression, gene ontology, and pathways data. Jointly with promoters of the above-mentioned genes, we found a large number of intergenic regions with similar structure as histone promoters.Conclusions: This study represents one of the most comprehensive computational analyses conducted thus far on a genome-wide scale of promoters of human histone genes. Our analysis suggests a number of other human genes that share a high similarity of promoter structure with the histone genes and thus are highly likely to be coregulated, and consequently coexpressed, with the histone genes. We also found that there are a large number of intergenic regions across the genome with their structures similar to promoters of histone genes. These regions may be promoters of yet unidentified genes, or may represent remote control regions that

  16. Cooperative binding of transcription factors promotes bimodal gene expression response.

    Directory of Open Access Journals (Sweden)

    Pablo S Gutierrez

    Full Text Available In the present work we extend and analyze the scope of our recently proposed stochastic model for transcriptional regulation, which considers an arbitrarily complex cis-regulatory system using only elementary reactions. Previously, we determined the role of cooperativity on the intrinsic fluctuations of gene expression for activating transcriptional switches, by means of master equation formalism and computer simulation. This model allowed us to distinguish between two cooperative binding mechanisms and, even though the mean expression levels were not affected differently by the acting mechanism, we showed that the associated fluctuations were different. In the present generalized model we include other regulatory functions in addition to those associated to an activator switch. Namely, we introduce repressive regulatory functions and two theoretical mechanisms that account for the biphasic response that some cis-regulatory systems show to the transcription factor concentration. We have also extended our previous master equation formalism in order to include protein production by stochastic translation of mRNA. Furthermore, we examine the graded/binary scenarios in the context of the interaction energy between transcription factors. In this sense, this is the first report to show that the cooperative binding of transcription factors to DNA promotes the "all-or-none" phenomenon observed in eukaryotic systems. In addition, we confirm that gene expression fluctuation levels associated with one of two cooperative binding mechanism never exceed the fluctuation levels of the other.

  17. Association of NDRG1 gene promoter methylation with reduced NDRG1 expression in gastric cancer cells and tissue specimens. (United States)

    Chang, Xiaojing; Zhang, Shuanglong; Ma, Jinguo; Li, Zhenhua; Zhi, Yu; Chen, Jing; Lu, Yao; Dai, Dongqiu


    NDRG1 (N-myc downstream-regulated gene 1) plays a role in cell differentiation and suppression of tumor metastasis. This study aims to determine the expression of NDRG1 mRNA and protein in gastric cancer cell lines and tissue specimens and then assess the possible cause of its aberrant expression. Six gastric cancer cell lines and 20 pairs of normal and gastric cancer tissue samples were used to assess NDRG1 expression using Real-time PCR and Western blot. High-resolution melting analysis (HRM) and methylation-specific PCR (MSP) were performed to detect gene mutation and methylation, respectively, in cell lines and tissues samples. Expression of NDRG1 mRNA and protein was downregulated in gastric cancer cell lines and tissues. Specifically, expression of NDRG1 mRNA and protein was lower in all six gastric cancer cell lines than that of normal gastric cells, while 15 out of 20 cases of gastric cancer tissues had the reduced levels of NDRG1 mRNA and protein. HRM data showed that there was no mutation in NDRG1 gene, but MSP data showed high levels of NDRG1 gene promoter methylation in the CpG islands in both cell lines and tissue samples. Moreover, treatment with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine upregulated NDRG1 expression in gastric cancer HGC27 cells, but not in the histone deacetylase inhibitor trichostatin A-treated HGC27 cells. In conclusion, this study has shown that expression of NDRG1 mRNA and protein was reduced in gastric cancer cell lines and tissues, which is due to methylation of NDRG1 gene promoter. Further study will unearth the clinical significance of the reduced NDRG1 protein in gastric cancer.

  18. Aberrant Expression of Critical Genes during Secondary Cell Wall Biogenesis in a Cotton Mutant, Ligon Lintless-1 (Li-1

    Directory of Open Access Journals (Sweden)

    James J. Bolton


    Full Text Available Over ninety percent of the value of cotton comes from its fiber; however, the genetic mechanisms governing fiber development are poorly understood. Due to their biochemical and morphological diversity in fiber cells cotton fiber mutants have been useful in examining fiber development; therefore, using the Ligon Lintless (Li-1 mutant, a monogenic dominant cotton mutant with very short fibers, we employed the high throughput approaches of microarray technology and real time PCR to gain insights into what genes were critical during the secondary cell wall synthesis stage. Comparative transcriptome analysis of the normal TM-1 genotype and the near isogenic Li-1 revealed that over 100 transcripts were differentially expressed at least 2-fold during secondary wall biogenesis, although the genetic profile of the expansion phase showed no significant differences in the isolines. Of particular note, we identified three candidate gene families-expansin, sucrose synthase, and tubulin—whose expression in Li-1 deviates from normal expression patterns of its parent, TM-1. These genes may contribute to retarded growth of fibers in Li-1 since they are fiber-expressed structural and metabolic genes. This work provides more details into the mechanisms of fiber development, and suggests the Li gene is active during the later stages of fiber development.

  19. Aberrant gene methylation in the peritoneal fluid is a risk factor predicting peritoneal recurrence in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Masatsugu; Hiraki; Yoshihiko; Kitajima; Seiji; Sato; Jun; Nakamura; Kazuyoshi; Hashiguchi; Hirokazu; Noshiro; Kohji; Miyazaki


    AIM:To investigate whether gene methylation in the peritoneal fluid (PF) predicts peritoneal recurrence in gastric cancer patients.METHODS: The gene methylation of CHFR (checkpoint with forkhead and ring finger domains), p16, RUNX3 (runt-related transcription factor 3), E-cadherin, hMLH1 (mutL homolog 1), ABCG2 (ATP-binding cassette, sub-family G, member 2) and BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3) were analyzed in 80 specimens of PF by quantitative methylation-specific polymerase chain r...

  20. Aberrant expression of shared master-key genes contributes to the immunopathogenesis in patients with juvenile spondyloarthritis.

    Directory of Open Access Journals (Sweden)

    Lovro Lamot

    Full Text Available Association of juvenile spondyloarthritis (jSpA with the HLA-B27 genotype is well established, but there is little knowledge of other genetic factors with a role in the development of the disease. To date, only a few studies have tried to find those associated genes by obtaining expression profiles, but with inconsistent results due to various patient selection criteria and methodology. The aim of the present study was to identify and confirm gene signatures and novel biomarkers in highly homogeneous cohorts of untreated and treated patients diagnosed with jSpA and other forms of juvenile idiopathic arthritis (JIA according to ILAR criteria. For the purposes of the research, total RNA was isolated from whole blood of 45 children with jSpA and known HLA genotype, 11 children with oligo- and polyarticular forms of JIA, as well as 12 age and sex matched control participants without diagnosis of inflammatory disease. DNA microarray gene expression was performed in 11 patients with jSpA and in four healthy controls, along with bioinformatical analysis of retrieved data. Carefully selected differentially expressed genes where analyzed by qRT-PCR in all participants of the study. Microarray results and bioinformatical analysis revealed 745 differentially expressed genes involved in various inflammatory processes, while qRT-PCR analysis of selected genes confirmed data universality and specificity of expression profiles in jSpA patients. The present study indicates that jSpA could be a polygenic disease with a possible malfunction in antigen recognition and activation of immunological response, migration of inflammatory cells and regulation of the immune system. Among genes involved in these processes TLR4, NLRP3, CXCR4 and PTPN12 showed almost consistent expression in study patients diagnosed with jSpA. Those genes and their products could therefore potentially be used as novel biomarkers, possibly predictive of disease prognosis and response to

  1. Chicago aberration correction work. (United States)

    Beck, V D


    The author describes from his personal involvement the many improvements to electron microscopy Albert Crewe and his group brought by minimizing the effects of aberrations. The Butler gun was developed to minimize aperture aberrations in a field emission electron gun. In the 1960s, Crewe anticipated using a spherical aberration corrector based on Scherzer's design. Since the tolerances could not be met mechanically, a method of moving the center of the octopoles electrically was developed by adding lower order multipole fields. Because the corrector was located about 15 cm ahead of the objective lens, combination aberrations would arise with the objective lens. This fifth order aberration would then limit the aperture of the microscope. The transformation of the off axis aberration coefficients of a round lens was developed and a means to cancel anisotropic coma was developed. A new method of generating negative spherical aberration was invented using the combination aberrations of hexapoles. Extensions of this technique to higher order aberrations were developed. An electrostatic electron mirror was invented, which allows the cancellation of primary spherical aberration and first order chromatic aberration. A reduction of chromatic aberration by two orders of magnitude was demonstrated using such a system.

  2. Homeotic-like modification of stamens to petals is associated with aberrant mitochondrial gene expression in cytoplasmic male sterile Ogura Brassica juncea

    Indian Academy of Sciences (India)

    Gargi Meur; K. Gaikwad; S. R. Bhat; S. Prakash; P. B. Kirti


    We have previously reported correction of severe leaf chlorosis in the cytoplasmic male sterile Ogura (also called Ogu) Brassica juncea line carrying Ogura cytoplasm by plastid substitution via protoplast fusion. Two cybrids obtained from the fusion experiment, Og1 and Og2, were green and carried the plastid genome of B. juncea cv. RLM198. While Og1 displayed normal flower morphology comparable to that of its euplasmic B. juncea counterpart except for sterile anthers, Og2 retained homeotic-like floral modification of stamens to petal-like structures and several other floral deformities observed in the chlorotic (Ogu) B. juncea cv. RLM198 (or OgRLM). With respect to the mitochondrial genome, Og1 showed 81% genetic similarity to the fertile cultivar RLM while Og2 showed 93% similarity to OgRLM. In spite of recombination and rearrangements in the mitochondrial genomes in the cybrids, expression patterns of 10 out of 11 mitochondrial genes were similar in all the three CMS lines; the only exception was atp6, whose expression was altered. While Og1 showed normal atp6 transcript similar to that in RLM, in Og2 and OgRLM weak expression of a longer transcript was detected. These results suggest that the homeotic-like changes in floral patterning leading to petaloid stamens in Og2 and OgRLM may be associated with aberrant mitochondrial gene expression.

  3. Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii


    Muh. Alias L. Rajamuddin; Alimuddin A; Utut Widyastuti; Irvan Faizal


    Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select asuitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Greenfl uorescent protein (GFP) gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycusalvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP), caulifl ower mosaic virus (pCaMV-GFP),medaka β-actin (pmBA-GFP) and Japanese fl...

  4. High-throughput mapping of the promoters of the mouse olfactory receptor genes reveals a new type of mammalian promoter and provides insight into olfactory receptor gene regulation. (United States)

    Clowney, E Josephine; Magklara, Angeliki; Colquitt, Bradley M; Pathak, Nidhi; Lane, Robert P; Lomvardas, Stavros


    The olfactory receptor (OR) genes are the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion in olfactory neurons. Using a high-throughput approach, we mapped the transcription start sites of 1085 of the 1400 murine OR genes and performed computational analysis that revealed potential transcription factor binding sites shared by the majority of these promoters. Our analysis produced a hierarchical model for OR promoter recognition in which unusually high AT content, a unique epigenetic signature, and a stereotypically positioned O/E site distinguish OR promoters from the rest of the murine promoters. Our computations revealed an intriguing correlation between promoter AT content and evolutionary plasticity, as the most AT-rich promoters regulate rapidly evolving gene families. Within the AT-rich promoter category the position of the TATA-box does not correlate with the transcription start site. Instead, a spike in GC composition might define the exact location of the TSS, introducing the concept of "genomic contrast" in transcriptional regulation. Finally, our experiments show that genomic neighborhood rather than promoter sequence correlates with the probability of different OR genes to be expressed in the same olfactory cell.

  5. Aberrant nuclear localization of β-catenin without genetic alterations in β-catenin or Axin genes in esophageal cancer

    Directory of Open Access Journals (Sweden)

    Shinoda Noriyuki


    Full Text Available Abstract Background β-catenin is a multifunctional protein involved in two apparently independent processes: cell-cell adhesion and signal transduction. β-catenin is involved in Wnt signaling pathway that regulates cellular differentiation and proliferation. In this study, we investigated the expression pattern of β-catenin and cyclin D1 using immunohistochemistry and searched for mutations in exon 3 of the β-catenin gene and Axin gene in esophageal squamous cell carcinoma. Materials and methods Samples were obtained from 50 esophageal cancer patients. Immunohistochemical staining for β-catenin and cyclin D1 was done. Mutational analyses of the exon3 of the β-catenin gene and Axin gene were performed on tumors with nuclear β-catenin expression. Results Four (8% esophageal cancer tissues showed high nuclear β-catenin staining. Overexpression of cyclin D1 was observed in 27 out of 50 (54% patients. All four cases that showed nuclear β-catenin staining overexpressed cyclin D1. No relationship was observed between the expression pattern of β-catenin and cyclin D1 and age, sex, tumor size, stage, differentiation grade, lymph node metastasis, response to chemotherapy, or survival. No mutational change was found in β-catenin exon 3 in the four cases with nuclear β-catenin staining. Sequencing analysis of the Axin cDNA revealed only a splicing variant (108 bp deletion, position 2302–2409 which was present in the paired normal mucosa. Conclusion A fraction of esophageal squamous cell carcinomas have abnormal nuclear accumulation of β-catenin accompanied with increased cyclin D1 expression. Mutations in β-catenin or axin genes are not responsible for this abnormal localization of β-catenin.

  6. A preliminary study of the relationship between promoter methylation of the ABCG1, GALNT2 and HMGCR genes and coronary heart disease.

    Directory of Open Access Journals (Sweden)

    Ping Peng

    Full Text Available AIMS: To investigate the association of ABCG1, GALNT2 and HMGCR genes promoter DNA methylation with coronary heart disease (CHD and explore the interaction between their methylation status and the CHD patients' clinical characteristics in Han Chinese population. METHODS AND RESULTS: Methylation-specific polymerase chain reaction (MSP technology was used to examine the role of the aberrant gene promoter methylation in CHD in Han Chinese population. A total of 85 CHD patients and 54 participants without CHD confirmed by angiography were recruited. 82.8% of the participants with ABCG1 gene promoter hypermethylation have CHD, while only 17.4% of the participants without hypermethylation have it. The average age of the participants with GALNT2 gene promoter hypermethylation is 62.10 ± 8.21, while that of the participants without hypermethylation is 57.28 ± 9.87; in the former group, 75.4% of the participants have CHD, compared to only 50% in the latter group. As for the HMGCR gene, the average age of the participants with promoter hypermethylation is 63.24 ± 8.10 and that of the participants without hypermethylation is 57.79 ± 9.55; its promoter hypermethylation is likely to be related to smoking. Our results indicated a significant statistical association of promoter methylation of the ABCG1 gene with increased risk of CHD (OR = 19.966; 95% CI, 7.319-54.468; P*<0.001; P*: adjusted for age, gender, smoking, lipid level, hypertension, and diabetes. Similar results were obtained for that of the GALNT2 gene (OR = 2.978; 95% CI, 1.335-6.646; P* = 0.008, but not of HMGCR gene (OR = 1.388; 95% CI, 0.572-3.371; P*  = 0.469. CONCLUSIONS: The present work provides evidence to support the association of promoter DNA methylation status with the risk profile of CHD. Our data indicates that promoter DNA hypermethylation of the ABCG1 and GALNT2 genes, but not the HMGCR gene, is associated with an increased risk of CHD. CHD, smoking and aging are likely to

  7. Comprehensive annotation of bidirectional promoters identifies co-regulation among breast and ovarian cancer genes.

    Directory of Open Access Journals (Sweden)

    Mary Q Yang


    Full Text Available A "bidirectional gene pair" comprises two adjacent genes whose transcription start sites are neighboring and directed away from each other. The intervening regulatory region is called a "bidirectional promoter." These promoters are often associated with genes that function in DNA repair, with the potential to participate in the development of cancer. No connection between these gene pairs and cancer has been previously investigated. Using the database of spliced-expressed sequence tags (ESTs, we identified the most complete collection of human transcripts under the control of bidirectional promoters. A rigorous screen of the spliced EST data identified new bidirectional promoters, many of which functioned as alternative promoters or regulated novel transcripts. Additionally, we show a highly significant enrichment of bidirectional promoters in genes implicated in somatic cancer, including a substantial number of genes implicated in breast and ovarian cancers. The repeated use of this promoter structure in the human genome suggests it could regulate co-expression patterns among groups of genes. Using microarray expression data from 79 human tissues, we verify regulatory networks among genes controlled by bidirectional promoters. Subsets of these promoters contain similar combinations of transcription factor binding sites, including evolutionarily conserved ETS factor binding sites in ERBB2, FANCD2, and BRCA2. Interpreting the regulation of genes involved in co-expression networks, especially those involved in cancer, will be an important step toward defining molecular events that may contribute to disease.

  8. Different Effects of Homocysteine and Oxidized Low Density Lipoprotein on Methylation Status in the Promoter Region of the Estrogen Receptor α Gene

    Institute of Scientific and Technical Information of China (English)

    Yushan HUANG; Kejun PENG; Juan SU; Yuping HUANG; Yizhou XU; Shuren WANG


    We investigated the effects of homocysteine (Hcy) and oxidized low density lipoprotein (oxLDL) on DNA methylation in the promoter region of the estrogen receptor α (ERα) gene, and its potential mechanism in the pathogenesis of atherosclerosis. Cultured smooth muscle cells (SMCs) of humans were treated by Hcy and ox-LDL with different concentrations for different periods of time. The DNA methylation status was assayed by nested methylation-specific polymerase chain reaction, the lipids that accumulated in the SMCs and foam cell formations were examined with Oil red O staining. The proliferation of SMCs was assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The results showed that ox-LDL in moderate concentrations (10-40 mg/L) induced de novo methylation in the promoter region of the ERα gene of SMCs. However, high concentrations (50 mg/L) of ox-LDL, resulted in demethylation of ERα. The Hcy treatment resulted in de novo methylation in the promoter region of the ERα gene with a concentration- and treating time-dependent manner, and a dose-dependent promoting effect on SMC proliferation. These data indicated that the two risk factors for atherosclerosis had the function of inducing de novo methylation in the promoter region of the ERα gene of SMCs. However, high concentrations (50mg/L) of ox-LDL induced demethylation, indicating that different risk factors of atherosclerosis with different potency might cause different aberrant methylation patterns in the promoter region of the ERα gene. The atherogenic mechanism of Hcy might involve the hypermethylation of the ERα gene, leading to the proliferation of SMCs in atherosclerotic lesions.

  9. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice. (United States)

    Li, Jing; Jiang, Dagang; Zhou, Hai; Li, Feng; Yang, Jiawei; Hong, Laifa; Fu, Xiao; Li, Zhibin; Liu, Zhenlan; Li, Jianming; Zhuang, Chuxiong


    Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  10. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Directory of Open Access Journals (Sweden)

    Jing Li

    Full Text Available Antisense and RNA interference (RNAi-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  11. Chicago aberration correction work

    Energy Technology Data Exchange (ETDEWEB)

    Beck, V.D., E-mail: [1 Hobby Drive, Ridgefield, CT 06877-01922 (United States)


    The author describes from his personal involvement the many improvements to electron microscopy Albert Crewe and his group brought by minimizing the effects of aberrations. The Butler gun was developed to minimize aperture aberrations in a field emission electron gun. In the 1960s, Crewe anticipated using a spherical aberration corrector based on Scherzer's design. Since the tolerances could not be met mechanically, a method of moving the center of the octopoles electrically was developed by adding lower order multipole fields. Because the corrector was located about 15 cm ahead of the objective lens, combination aberrations would arise with the objective lens. This fifth order aberration would then limit the aperture of the microscope. The transformation of the off axis aberration coefficients of a round lens was developed and a means to cancel anisotropic coma was developed. A new method of generating negative spherical aberration was invented using the combination aberrations of hexapoles. Extensions of this technique to higher order aberrations were developed. An electrostatic electron mirror was invented, which allows the cancellation of primary spherical aberration and first order chromatic aberration. A reduction of chromatic aberration by two orders of magnitude was demonstrated using such a system. -- Highlights: Black-Right-Pointing-Pointer Crewe and his group made significant advances in aberration correction and reduction. Black-Right-Pointing-Pointer A deeper understanding of the quadrupole octopole corrector was developed. Black-Right-Pointing-Pointer A scheme to correct spherical aberration using hexapoles was developed. Black-Right-Pointing-Pointer Chromatic aberration was corrected using a uniform field mirror.

  12. [Functional interactions between promoters of neighboring yellow and CG3777 genes in Drosophila melanogaster]. (United States)

    Leman, D V; Parshikov, A F; Georgiev, P G; Maksimenko, O G


    It was shown by us previously that the transcription of the yellow gene can be affected by the promoter of the neighboring gene CG3777, which has a similar expression profile. In the present work, we continued studying the functional interactions between the promoters of the yellow and CG3777 genes in transgenic Drosophila strains. In this work, we used the failure of the yeast activator GAL4 to stimulate transcription from the promoter of the yellow gene for the case when GAL4-binding sites are localized at the 3'-end of the gene. It has been found that, if the 983-bp CG3777 gene promoter is inserted in transgenic strains in the same orientation with the yellow gene promoter, downstream from the sites of the GAL4 activator, the CG3777 promoter provides a strong stimulation of the yellow gene by the GAL4 activator. When the promoters of the yellow and CG3777 genes are inserted in opposite orientations relative to one another, no stimulation of the yellow gene by GAL4 is observed. Additional results obtained in the work demonstrate that the functional interacton between the CG3777 and yellow promoters depends on their mutual orientation and position relative to the GAL4-binding sites.

  13. Molecular gate keepers succumb to gene aberrations in colorectal cancer in Kashmiri population, revealing a high incidence area

    Directory of Open Access Journals (Sweden)

    Sameer A


    Full Text Available Background/Aim: Colorectal cancer (CRC is one of the leading malignancies worldwide and has been reported to show geographical variation in its incidence, even within areas of ethnic homogeneity. The aim of this study was to identify p53 and K-ras gene mutations in CRC patients in a Kashmiri population, and to assess whether these mutations are linked with clinicopathological parameters. Materials and Methods: Paired tumor and normal tissue samples from a consecutive series of 53 patients undergoing resective surgery for CRC were prospectively studied for p53 and K-ras gene mutations by PCR/single strand conformation polymorphism (SSCP. Results: Less than half (45%, 19/42 of the patients presented mutations in the p53 gene. Twenty eight mutations were found in the p53 gene, which comprised of 23 substitutions (17 transitions + 6 transversions, and five insertions. The 23 substitutions constituted 18 missense mutations, two nonsense mutations, and three silent mutations. Of the 28 mutations (7.14% observed in this study, 2 were not previously reported for CRC samples and were identified as novel p53 mutations. A few patients (22.64%, 12/53 presented with mutations in K-ras, constituting 13 missense mutations, out of which 11 were G→A transitions, one was a G→C transversion, and one a G→T transversion. More than half (61.5% of the mutations occurred in codon 12 whereas a few (38.5% occurred in codon 13. One tumor contained missense mutations in both codons. Comparison of the mutation profiles of our patients with those of other ethnic populations and regions reflected both differences and similarities, indicating co-exposure to a unique set of risk factors. Conclusion: Mutations of the p53 and K-ras genes are some of the most common genetic changes in the development of human CRC. The high frequency of p53 gene mutations implicates p53 as a predominant factor for CRC in the high-risk ethnic Kashmiri population.

  14. Genome-wide prediction of transcriptional regulatory elements of human promoters using gene expression and promoter analysis data

    Directory of Open Access Journals (Sweden)

    Kim Seon-Young


    Full Text Available Abstract Background A complete understanding of the regulatory mechanisms of gene expression is the next important issue of genomics. Many bioinformaticians have developed methods and algorithms for predicting transcriptional regulatory mechanisms from sequence, gene expression, and binding data. However, most of these studies involved the use of yeast which has much simpler regulatory networks than human and has many genome wide binding data and gene expression data under diverse conditions. Studies of genome wide transcriptional networks of human genomes currently lag behind those of yeast. Results We report herein a new method that combines gene expression data analysis with promoter analysis to infer transcriptional regulatory elements of human genes. The Z scores from the application of gene set analysis with gene sets of transcription factor binding sites (TFBSs were successfully used to represent the activity of TFBSs in a given microarray data set. A significant correlation between the Z scores of gene sets of TFBSs and individual genes across multiple conditions permitted successful identification of many known human transcriptional regulatory elements of genes as well as the prediction of numerous putative TFBSs of many genes which will constitute a good starting point for further experiments. Using Z scores of gene sets of TFBSs produced better predictions than the use of mRNA levels of a transcription factor itself, suggesting that the Z scores of gene sets of TFBSs better represent diverse mechanisms for changing the activity of transcription factors in the cell. In addition, cis-regulatory modules, combinations of co-acting TFBSs, were readily identified by our analysis. Conclusion By a strategic combination of gene set level analysis of gene expression data sets and promoter analysis, we were able to identify and predict many transcriptional regulatory elements of human genes. We conclude that this approach will aid in decoding

  15. MBD3 localizes at promoters, gene bodies and enhancers of active genes. (United States)

    Shimbo, Takashi; Du, Ying; Grimm, Sara A; Dhasarathy, Archana; Mav, Deepak; Shah, Ruchir R; Shi, Huidong; Wade, Paul A


    The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status.

  16. Tacrolimus increases Nox4 expression in human renal fibroblasts and induces fibrosis-related genes by aberrant TGF-beta receptor signalling.

    Directory of Open Access Journals (Sweden)

    Georg Kern

    Full Text Available Chronic nephrotoxicity of immunosuppressives is one of the main limiting factors in the long-term outcome of kidney transplants, leading to tissue fibrosis and ultimate organ failure. The cytokine TGF-β is considered a key factor in this process. In the human renal fibroblast cell line TK-173, the macrolide calcineurin inhibitor tacrolimus (FK-506 induced TGF-β-like effects, manifested by increased expression of NAD(PH-oxidase 4 (Nox4, transgelin, tropomyosin 1, and procollagen α1(V mRNA after three days. The macrolide mTOR inhibitor rapamycin had similar effects, while cyclosporine A did not induce fibrose-related genes. Concentration dependence curves were sigmoid, where mRNA expression was induced already at low nanomolar levels of tacrolimus, and reached saturation at 100-300 nM. The effects were independent of extracellular TGF-β as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-β receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a "leaky" TGF-β receptor. The myofibroblast marker α-smooth muscle actin was neither induced by tacrolimus nor by TGF-β1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus- and TGF-β1-induced Nox4 protein upregulation was confirmed by Western blotting, and was accompanied by a rise in intracellular H2O2 concentration. Si-RNA mediated knock-down of Nox4 expression prevented up-regulation of procollagen α1(V mRNA in tacrolimus-treated cells, but induced procollagen α1(V expression in control cells. Nox4 knock-down had no significant effect on the other genes tested. TGF-β is a key molecule in fibrosis, and the constant activation of aberrant receptor signaling by tacrolimus might contribute to the long-term development of interstitial kidney fibrosis in immunosuppressed patients. Nox4 levels possibly play a regulatory role in these processes.

  17. Genetic Determinants for Promoter Hypermethylation in the Lungs of Smokers: A Candidate Gene-Based Study


    Leng, Shuguang; Stidley, Christine A.; Liu, Yushi; Edlund, Christopher K.; Willink, Randall P.; Han, Younghun; Landi, Maria Teresa; Thun, Michael; Picchi, Maria A.; Bruse, Shannon E.; Crowell, Richard E.; Van Den Berg, David; Neil E Caporaso; Amos, Christopher I.; Siegfried, Jill M.


    The detection of tumor suppressor gene promoter methylation in sputum-derived exfoliated cells predicts early lung cancer. Here we identified genetic determinants for this epigenetic process and examined their biological effects on gene regulation. A two-stage approach involving discovery and replication was employed to assess the association between promoter hypermethylation of a 12-gene panel and common variation in 40 genes involved in carcinogen metabolism, regulation of methylation, and ...

  18. Identification of the transcriptional promoters in the proximal regions of human microRNA genes. (United States)

    Long, Yue-Sheng; Deng, Guang-Fei; Sun, Xun-Sha; Yi, Yong-Hong; Su, Tao; Zhao, Qi-Hua; Liao, Wei-Ping


    To identify the transcriptional promoters in the proximal regions of human microRNA (miRNA) genes, we analyzed the 5' flanking regions of intergenic miRNAs and intronic miRNAs. With the TSSG program prediction, we found that the ratio of intronic-s miRNA genes with a least one promoter was significantly lower than those of intergenic miRNA genes and intronic-a miRNA genes. More than half of the miRNA genes have only one promoter and less than 20% of the miRNA genes have more than three promoters in the 5-kb upstream regions. All potential promoters are randomly distributed within these regions. Approximately 60% of the miRNA promoters have a TATA-like box, being significantly higher than that of all human promoters. Luciferase reporter assays showed that 22 of the 30 promoters drove gene expression in HEK-293 cells, indicating a high accuracy of the promoter prediction. This study lays a foundation for future investigation into the transcriptional regulatory mechanisms of human miRNA genes.

  19. Cancer specificity of promoters of the genes involved in cell proliferation control. (United States)

    Kashkin, K N; Chernov, I P; Stukacheva, E A; Kopantzev, E P; Monastyrskaya, G S; Uspenskaya, N Ya; Sverdlov, E D


    Core promoters with adjacent regions of the human genes CDC6, POLD1, CKS1B, MCM2, and PLK1 were cloned into a pGL3 vector in front of the Photinus pyrails gene Luc in order to study the tumor specificity of the promoters. The cloned promoters were compared in their ability to direct luciferase expression in different human cancer cells and in normal fibroblasts. The cancer-specific promoter BIRC5 and non-specific CMV immediately early gene promoter were used for comparison. All cloned promoters were shown to be substantially more active in cancer cells than in fibroblasts, while the PLK1 promoter was the most cancer-specific and promising one. The specificity of the promoters to cancer cells descended in the series PLK1, CKS1B, POLD1, MCM2, and CDC6. The bidirectional activity of the cloned CKS1B promoter was demonstrated. It apparently directs the expression of the SHC1 gene, which is located in a "head-to-head" position to the CKS1B gene in the human genome. This feature should be taken into account in future use of the CKS1B promoter. The cloned promoters may be used in artificial genetic constructions for cancer gene therapy.

  20. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jacob Daniela


    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  1. Quantitative Detection of ID4 Gene Aberrant Methylation in the Differentiation of Myelodysplastic Syndrome from Aplastic Anemia

    Institute of Scientific and Technical Information of China (English)

    Mian-Yang Li; Yuan-Yuan Xu; Hui-Yuan Kang; Xin-Rong Wang; Li Gao; Jian Cen; Wei Wang


    Background:The diagnosis of myelodysplastic syndrome (MDS),especially hypoplastic MDS,and MDS with low blast counts or normal karyotype may be problematic.This study characterized ID4 gene methylation in patients with MDS and aplastic anemia (AA).Methods:The methylation status ofID4 was analyzed by bisulfite sequencing polymerase chain reaction (PCR) and quantitative real-time methylation-specific PCR (MethyLight PCR) in 100 patients with MDS and 31 patients with AA.Results:The MDS group had a higher ID4 gene methylation positivity rate (22.22%) and higher methylation levels (0.21 [0-3.79]) than the AA group (P < 0.05).Furthermore,there were significant differences between the hypoplastic MDS and AA groups,the MDS with low blast count and the AA groups,and the MDS with normal karyotype and the AA groups.The combination of genetic and epigenetic markers was used in much more patients with MDS (62.5% [35/56]) than the use of genetic markers only (51.79% [29/56]).Conclusions:These results showed that the detection ofID4 methylation positivity rates and levels could be a useful biomarker for MDS diagnosis.

  2. Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

    Institute of Scientific and Technical Information of China (English)

    CHAI Yurong; LU Yumin; WANG Tianyun; HOU Weihong; XUE Lexun


    Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficientpromoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase (CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics.Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

  3. A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease.

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    Kaisa Kyöstilä


    Full Text Available Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136 in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

  4. NFAT targets signaling molecules to gene promoters in pancreatic β-cells. (United States)

    Lawrence, Michael C; Borenstein-Auerbach, Nofit; McGlynn, Kathleen; Kunnathodi, Faisal; Shahbazov, Rauf; Syed, Ilham; Kanak, Mazhar; Takita, Morihito; Levy, Marlon F; Naziruddin, Bashoo


    Nuclear factor of activated T cells (NFAT) is activated by calcineurin in response to calcium signals derived by metabolic and inflammatory stress to regulate genes in pancreatic islets. Here, we show that NFAT targets MAPKs, histone acetyltransferase p300, and histone deacetylases (HDACs) to gene promoters to differentially regulate insulin and TNF-α genes. NFAT and ERK associated with the insulin gene promoter in response to glucagon-like peptide 1, whereas NFAT formed complexes with p38 MAPK (p38) and Jun N-terminal kinase (JNK) upon promoters of the TNF-α gene in response to IL-1β. Translocation of NFAT and MAPKs to gene promoters was calcineurin/NFAT dependent, and complex stability required MAPK activity. Knocking down NFATc2 expression, eliminating NFAT DNA binding sites, or interfering with NFAT nuclear import prevented association of MAPKs with gene promoters. Inhibiting p38 and JNK activity increased NFAT-ERK association with promoters, which repressed TNF-α and enhanced insulin gene expression. Moreover, inhibiting p38 and JNK induced a switch from NFAT-p38/JNK-histone acetyltransferase p300 to NFAT-ERK-HDAC3 complex formation upon the TNF-α promoter, which resulted in gene repression. Histone acetyltransferase/HDAC exchange was reversed on the insulin gene by p38/JNK inhibition in the presence of glucagon-like peptide 1, which enhanced gene expression. Overall, these data indicate that NFAT directs signaling enzymes to gene promoters in islets, which contribute to protein-DNA complex stability and promoter regulation. Furthermore, the data suggest that TNF-α can be repressed and insulin production can be enhanced by selectively targeting signaling components of NFAT-MAPK transcriptional/signaling complex formation in pancreatic β-cells. These findings have therapeutic potential for suppressing islet inflammation while preserving islet function in diabetes and islet transplantation.

  5. Promoter methylation of E-cadherin, p16, and RAR-beta(2) genes in breast tumors and dietary intake of nutrients important in one-carbon metabolism (United States)

    Aberrant DNA methylation plays a critical role in carcinogenesis, and the availability of dietary factors involved in 1-carbon metabolism may contribute to aberrant DNA methylation. We investigated the association of intake of folate, vitamins B(2), B(6), B(12), and methionine with promoter methylat...

  6. Relationship between polymorphism of class Ⅱ transactivator gene promoters and chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    Ying-Ren Zhao; Ling Gong; Ying-Li He; Fang Liu; Chang Lu


    AIM: To investigate the relationship between the polymorphism of class Ⅱ transactivator (CⅡTA) gene promoters and chronic hepatitis B (CHB).METHODS: Genomic DNA was prepared from peripheral blood leukocytes. Promoters Ⅰ, Ⅲ and Ⅳ of gene were analyzed respectively with polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) in 65 patients with CHB, 26 patients with acute hepatitis B (AHB) and 85 normal controls.RESULTS: No abnormal migration was found in PCR-SSCP analysis of the three promoters in the three groups. Also,no sequential difference was observed at the three promoters among the CHB patients, AHB patients and normal controls.CONCLUSION: No polymorphism in promoters Ⅰ, Ⅲ and Ⅳ of CⅡTA gene exists in CHB patients, ABH patients and normal controls, suggesting that the promoter of CⅡTA gene might be a conserved domain.

  7. Positional bias of general and tissue-specific regulatory motifs in mouse gene promoters

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    Farré Domènec


    Full Text Available Abstract Background The arrangement of regulatory motifs in gene promoters, or promoter architecture, is the result of mutation and selection processes that have operated over many millions of years. In mammals, tissue-specific transcriptional regulation is related to the presence of specific protein-interacting DNA motifs in gene promoters. However, little is known about the relative location and spacing of these motifs. To fill this gap, we have performed a systematic search for motifs that show significant bias at specific promoter locations in a large collection of housekeeping and tissue-specific genes. Results We observe that promoters driving housekeeping gene expression are enriched in particular motifs with strong positional bias, such as YY1, which are of little relevance in promoters driving tissue-specific expression. We also identify a large number of motifs that show positional bias in genes expressed in a highly tissue-specific manner. They include well-known tissue-specific motifs, such as HNF1 and HNF4 motifs in liver, kidney and small intestine, or RFX motifs in testis, as well as many potentially novel regulatory motifs. Based on this analysis, we provide predictions for 559 tissue-specific motifs in mouse gene promoters. Conclusion The study shows that motif positional bias is an important feature of mammalian proximal promoters and that it affects both general and tissue-specific motifs. Motif positional constraints define very distinct promoter architectures depending on breadth of expression and type of tissue.

  8. HOX Gene Promoter Prediction and Inter-genomic Comparison: An Evo-Devo Study

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    Marla A. Endriga


    Full Text Available Homeobox genes direct the anterior-posterior axis of the body plan in eukaryotic organisms. Promoter regions upstream of the Hox genes jumpstart the transcription process. CpG islands found within the promoter regions can cause silencing of these promoters. The locations of the promoter regions and the CpG islands of Homeo sapiens sapiens (human, Pan troglodytes (chimpanzee, Mus musculus (mouse, and Rattus norvegicus (brown rat are compared and related to the possible influence on the specification of the mammalian body plan. The sequence of each gene in Hox clusters A-D of the mammals considered were retrieved from Ensembl and locations of promoter regions and CpG islands predicted using Exon Finder. The predicted promoter sequences were confirmed via BLAST and verified against the Eukaryotic Promoter Database. The significance of the locations was determined using the Kruskal-Wallis test. Among the four clusters, only promoter locations in cluster B showed significant difference. HOX B genes have been linked with the control of genes that direct the development of axial morphology, particularly of the vertebral column bones. The magnitude of variation among the body plans of closely-related species can thus be partially attributed to the promoter kind, location and number, and gene inactivation via CpG methylation.

  9. Tumor Restrictive Suicide Gene Therapy for Glioma Controlled by the FOS Promoter.

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    Jianqing Pan

    Full Text Available Effective suicide gene delivery and expression are crucial to achieving successful effects in gene therapy. An ideal tumor-specific promoter expresses therapeutic genes in tumor cells with minimal normal tissue expression. We compared the activity of the FOS (FBJ murine osteosarcoma viral oncogene homolog promoter with five alternative tumor-specific promoters in glioma cells and non-malignant astrocytes. The FOS promoter caused significantly higher transcriptional activity in glioma cell lines than all alternative promoters with the exception of CMV. The FOS promoter showed 13.9%, 32.4%, and 70.8% of the transcriptional activity of CMV in three glioma cell lines (U87, U251, and U373. Importantly, however, the FOS promoter showed only 1.6% of the transcriptional activity of CMV in normal astrocytes. We also tested the biologic activity of recombinant adenovirus containing the suicide gene herpes simplex virus thymidine kinase (HSV-tk driven by the FOS promoter, including selective killing efficacy in vitro and tumor inhibition rate in vivo. Adenoviral-mediated delivery of the HSV-tk gene controlled by the FOS promoter conferred a cytotoxic effect on human glioma cells in vitro and in vivo. This study suggests that use of the FOS-tk adenovirus system is a promising strategy for glioma-specific gene therapy but still much left for improvement.

  10. Bidirectional promoters of insects: genome-wide comparison, evolutionary implication and influence on gene expression. (United States)

    Behura, Susanta K; Severson, David W


    Bidirectional promoters are widespread in insect genomes. By analyzing 23 insect genomes we show that the frequency of bidirectional gene pairs varies according to genome compactness and density of genes among the species. The density of bidirectional genes expected based on number of genes per megabase of genome explains the observed density suggesting that bidirectional pairing of genes may be due to random event. We identified specific transcription factor binding motifs that are enriched in bidirectional promoters across insect species. Furthermore, we observed that bidirectional promoters may act as transcriptional hotspots in insect genomes where protein coding genes tend to aggregate in significantly biased (p promoters. Natural selection seems to have an association with the extent of bidirectionality of genes among the species. The rate of non-synonymous-to-synonymous changes (dN/dS) shows a second-order polynomial distribution with bidirectionality between species indicating that bidirectionality is dependent upon evolutionary pressure acting on the genomes. Analysis of genome-wide microarray expression data of multiple insect species suggested that bidirectionality has a similar association with transcriptome variation across species. Furthermore, bidirectional promoters show significant association with correlated expression of the divergent gene pairs depending upon their motif composition. Analysis of gene ontology showed that bidirectional genes tend to have a common association with functions related to "binding" (including ion binding, nucleotide binding and protein binding) across genomes. Such functional constraint of bidirectional genes may explain their widespread persistence in genome of diverse insect species.

  11. Engineering of Promoter Replacement Cassettes for Fine-Tuning of Gene Expression in Saccharomyces cerevisiae



    The strong overexpression or complete deletion of a gene gives only limited information about its control over a certain phenotype or pathway. Gene function studies based on these methods are therefore incomplete. To effect facile manipulation of gene expression across a full continuum of possible expression levels, we recently created a library of mutant promoters. Here, we provide the detailed characterization of our yeast promoter collection comprising 11 mutants of the strong constitutive...

  12. A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters. (United States)

    Zhang, Ning; McHale, Leah K; Finer, John J


    Introns, especially the first intron in the 5' untranslated region (5'UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.

  13. Nonviral gene transfer strategies to promote bone regeneration. (United States)

    Im, Gun-Il


    Despite the inherent ability of bone to regenerate itself, there are a number of clinical situations in which complete bone regeneration fails to occur. In view of shortcomings of conventional treatment, gene therapy may have a place in cases of critical-size bone loss that cannot be properly treated with current medical or surgical treatment. The purpose of this review is to provide an overview of gene therapy in general, nonviral techniques of gene transfer including physical and chemical methods, RNA-based therapy, therapeutic genes to be transferred for bone regeneration, route of application including ex vivo application, and direct gene therapy approaches to regenerate bone.

  14. Hypermethylation of Syk gene in promoter region associated with oncogenesis and metastasis of gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Shui Wang; Yong-Bin Ding; Guo-Yu Chen; Jian-Guo Xia; Zhen-Yan Wu


    AIM: To investigate the rrelationship between methylation of Syk (spleen tyrosine kinase) gene in promoter region and oncogenesis, metastasis of gastric carcinoma. The relation between silencing of the Syk gene and methylation of Syk promoter region was also studied.METHODS: By using methylation-specific PCR (MSP)technique, the methylation of Syk promoter region in specimens from 61 gastric cancer patients (tumor tissues and adjacent normal tissues) was detected. Meanwhile, RTPCR was used to analyse syk expression exclusively.RESULTS: The expression of the Syk gene was detected in all normal gastric tissues. Syk expression in gastric carcinoma was lower in 14 out of 61 gastric cancer samples than in adjacent normal tissues (x2=72.3, P<0.05). No methylation of Syk promoter was found in adjacent normal tissues, hypermethylation of Syk gene in promoter was detected 21 cases in 61 gastric carcinoma patients. The rate of methylation of Syk promoter in gastric carcinoma was higher than that in adjacent normal tissues (x2=25.1,P<0.05). In 31 patients with lymph node metastasis, 17 were found with Syk promoter methylation. A significant difference was noted between two groups (x2=11.4, P<0.05).CONCLUSION: Hypermethylation leads to silencing of the Syk gene in human gastric carcinoma. Methylation of Syk promoter is correlated to oncogenesis and metastasis of gastric carcinoma. Syk is considered to be a potential tumor suppressor and anti-metastasis gene in human gastric cancer.

  15. The human desmin promoter drives robust gene expression for skeletal muscle stem cell-mediated gene therapy. (United States)

    Jonuschies, Jacqueline; Antoniou, Michael; Waddington, Simon; Boldrin, Luisa; Muntoni, Francesco; Thrasher, Adrian; Morgan, Jennifer


    Lentiviral vectors (LVs) represent suitable candidates to mediate gene therapy for muscular dystrophies as they infect dividing and non-dividing cells and integrate their genetic material into the host genome, thereby theoretically mediating longterm expression. We evaluated the ability of LVs where a GFP reporter gene was under the control of five different promoters, to transduce and mediate expression in myogenic and non-myogenic cells in vitro and in skeletal muscle fibres and stem (satellite) cells in vivo. We further analysed lentivirally-transduced satellite cell-derived myoblasts following their transplantation into dystrophic, immunodeficient mouse muscles. The spleen focus-forming virus promoter mediated the highest gene expression in all cell types; the CBX3-HNRPA2B1 ubiquitously-acting chromatin opening element (UCOE) promoter was also active in all cells, whereas the human desmin promoter in isolation or fused with UCOE had lower activity in non-muscle cells. Surprisingly, the human skeletal muscle actin promoter was also active in immune cells. The human desmin promoter mediated robust, persistent reporter gene expression in myogenic cells in vitro, and satellite cells and muscle fibres in vivo. The human desmin promoter combined with UCOE did not significantly increase transgene expression. Therefore, our data indicate that the desmin promoter is suitable for the development of therapeutic purposes.

  16. A novel method for the determination of basal gene expression of tissue-specific promoters: an analysis of prostate-specific promoters.

    NARCIS (Netherlands)

    Poel, H.G. van der; McCadden, J.; Verhaegh, G.W.C.T.; Kruszewski, M.; Ferrer, F.; Schalken, J.A.; Carducci, M.; Rodriguez, R.


    Because the toxicity of suicide gene therapeutics is directly related to basal promoter activity, we developed an assay to test for promoter "leakiness" using a diphtheria toxin mutant. Sequences of 15 prostate-specific gene promoter constructs were cloned in an expression plasmid (pBK; Stratagene,

  17. [Comparative analysis of activity of different promoters for NIS gene expression in melanoma cells]. (United States)

    Kuz'mich, A I; Kopantsev, E P; Vinogradova, T V; Sverdlov, E D


    Development of targeted drug delivery system is key problem of cancer gene therapy. To ensure specific delivery of these therapeutic compounds to the tumor it is preferable for therapeutic gene expression to occur predominantly in cancer cells. Therefore, when testing drug in vivo, it is necessary to study distribution of therapeutic gene expression products in different tissues of the organism. Sodium iodide symporter (NIS) is attractive reporter because its tissue level is easily quantitatively detected by noninvasive imaging methods. Different promoters are used to direct expression of therapeutic genes in tumor cells: strong nonspecific, moderate tissue-specific and tumor-specific. Tumor-specific promoters function in wide range of tumor cells, however they are relatively weak. Relationship between promoter and sodium iodide symporter activity is unclear to date. In this report we examined activity of different promoters in two melanoma cell lines, functional activity of NIS driven by these promoters, also we compared promoter strength and NIS activity. We demonstrated that in spite of strong differences in promoter activity functional activity of NIS directed by these promoters varies weakly. Relatively weak melanoma-specific promoter directs high NIS activity in melanoma cell, however weaker cancer-specific promoters drive high NIS activity only in certain melanoma cell line.

  18. Aberration Corrected Emittance Exchange

    CERN Document Server

    Nanni, Emilio A


    Full exploitation of emittance exchange (EEX) requires aberration-free performance of a complex imaging system including active radio-frequency (RF) elements which can add temporal distortions. We investigate the performance of an EEX line where the exchange occurs between two dimensions with normalized emittances which differ by orders of magnitude. The transverse emittance is exchanged into the longitudinal dimension using a double dog-leg emittance exchange setup with a 5 cell RF deflector cavity. Aberration correction is performed on the four most dominant aberrations. These include temporal aberrations that are corrected with higher order magnetic optical elements located where longitudinal and transverse emittance are coupled. We demonstrate aberration-free performance of emittances differing by 4 orders of magnitude, i.e. an initial transverse emittance of $\\epsilon_x=1$ pm-rad is exchanged with a longitudinal emittance of $\\epsilon_z=10$ nm-rad.

  19. Multiobjective H2/H∞ synthetic gene network design based on promoter libraries. (United States)

    Wu, Chih-Hung; Zhang, Weihei; Chen, Bor-Sen


    Some current promoter libraries have been developed for synthetic gene networks. But an efficient method to engineer a synthetic gene network with some desired behaviors by selecting adequate promoters from these promoter libraries has not been presented. Thus developing a systematic method to efficiently employ promoter libraries to improve the engineering of synthetic gene networks with desired behaviors is appealing for synthetic biologists. In this study, a synthetic gene network with intrinsic parameter fluctuations and environmental disturbances in vivo is modeled by a nonlinear stochastic system. In order to engineer a synthetic gene network with a desired behavior despite intrinsic parameter fluctuations and environmental disturbances in vivo, a multiobjective H(2)/H(∞) reference tracking (H(2) optimal tracking and H(∞) noise filtering) design is introduced. The H(2) optimal tracking can make the tracking errors between the behaviors of a synthetic gene network and the desired behaviors as small as possible from the minimum mean square error point of view, and the H(∞) noise filtering can attenuate all possible noises, from the worst-case noise effect point of view, to achieve a desired noise filtering ability. If the multiobjective H(2)/H(∞) reference tracking design is satisfied, the synthetic gene network can robustly and optimally track the desired behaviors, simultaneously. First, based on the dynamic gene regulation, the existing promoter libraries are redefined by their promoter activities so that they can be efficiently selected in the design procedure. Then a systematic method is developed to select an adequate promoter set from the redefined promoter libraries to synthesize a gene network satisfying these two design objectives. But the multiobjective H(2)/H(∞) reference tracking design problem needs to solve a difficult Hamilton-Jacobi Inequality (HJI)-constrained optimization problem. Therefore, the fuzzy approximation method is

  20. Effect of glucocorticoid on promoter of 11β-hydroxysteroid dehydrogenase I gene

    Institute of Scientific and Technical Information of China (English)

    何平; 孙刚


    Objective: To study the effect of glucocorticoid on the promoter of the pre-receptor glucocorticoid metabolizing enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) gene. Methods: The 1.2 kb length sequence upstream to the transcription start site of the 11β-HSD1 gene was amplified with polymerase chain reaction (PCR) and then was cloned into pBLCAT6 plasmid carrying chloramphenicol acetyltransferase (CAT) reporter gene. The plasmid pBLCAT6 carrying the promoter and reporter gene was used to transfect HeLa cells to study the regulation of 11β-HSD1 gene expression by glucocorticoids in terms of reporter gene expression. Results: PCR showed that there was a complete alignment of the amplified sequence with the sequence 1.2 kb upstream to the transcription start site of 11β-HSD1 gene. When cloned into pBLCAT6 plasmid carrying the reporter gene, this part of the promoter is functional in terms of regulation of reporter gene expression upon transfection into HeLa cells. The synthetic glucocorticoid-dexamethasone induced the reporter gene expression in the system described above, which was blocked by glucocorticoid receptor antagonist RU486. Conclusion: Glucocorticoids can modulate the expression of 11β-HSD1 through a mechanism involving activation of GR and interaction of the promoter of 11β-HSD1 gene.

  1. Identification and refinement of two strong constitutive promoters for gene expression system of Schizosaccharomyces pombe. (United States)

    Wang, Hongcheng; Wang, Haiyang; Wang, Meng; Zhang, Lei; Wang, Ren; Mei, Yanzhen; Shao, Weilan


    Fission yeast Schizosaccharomyces pombe shares various important properties with higher eukaryotes and is now considered a useful host for elevated production of mammalian proteins for medicinal applications. The full-length nmt1 promoter has been widely used as a strong promoter in S. pombe expression system. In the present study, the promoters of the eno101 and gpd3 genes in S. pombe were identified as strong constitutive promoters. For convenient applications in the plasmids of S. pombe, these promoters were refined to 276-bp eno and 273-bp gpd promoters by deleting undesired sequences and examining the expression of reporter genes including lacZ and xynA. Both the refined eno and gpd promoters provided approximately 1.5-fold higher expression of LacZ than nmt1 promoter. Furthermore, gene expression under the control of the eno or gpd promoter was not repressed by the components of YES medium while nmt1 promoter was inhibited by thiamine in yeast extract. Therefore, both eno and gpd promoters offer opportunities for efficient production of recombinant proteins by S. pombe in high cell-density fermentation.

  2. Activity of heat shock genes' promoters in thermally contrasting animal species. (United States)

    Astakhova, Lyubov N; Zatsepina, Olga G; Funikov, Sergei Yu; Zelentsova, Elena S; Schostak, Natalia G; Orishchenko, Konstantin E; Evgen'ev, Michael B; Garbuz, David G


    Heat shock gene promoters represent a highly conserved and universal system for the rapid induction of transcription after various stressful stimuli. We chose pairs of mammalian and insect species that significantly differ in their thermoresistance and constitutive levels of Hsp70 to compare hsp promoter strength under normal conditions and after heat shock (HS). The first pair includes the HSPA1 gene promoter of camel (Camelus dromedarius) and humans. It was demonstrated that the camel HSPA1A and HSPA1L promoters function normally in vitro in human cell cultures and exceed the strength of orthologous human promoters under basal conditions. We used the same in vitro assay for Drosophila melanogaster Schneider-2 (S2) cells to compare the activity of the hsp70 and hsp83 promoters of the second species pair represented by Diptera, i.e., Stratiomys singularior and D. melanogaster, which dramatically differ in thermoresistance and the pattern of Hsp70 accumulation. Promoter strength was also monitored in vivo in D. melanogaster strains transformed with constructs containing the S. singularior hsp70 ORF driven either by its own promoter or an orthologous promoter from the D. melanogaster hsp70Aa gene. Analysis revealed low S. singularior hsp70 promoter activity in vitro and in vivo under basal conditions and after HS in comparison with the endogenous promoter in D. melanogaster cells, which correlates with the absence of canonical GAGA elements in the promoters of the former species. Indeed, the insertion of GAGA elements into the S. singularior hsp70 regulatory region resulted in a dramatic increase in promoter activity in vitro but only modestly enhanced the promoter strength in the larvae of the transformed strains. In contrast with hsp70 promoters, hsp83 promoters from both of the studied Diptera species demonstrated high conservation and universality.

  3. Alternative splicing, promoter methylation, and functional SNPs of sperm flagella 2 gene in testis and mature spermatozoa of Holstein bulls. (United States)

    Guo, F; Yang, B; Ju, Z H; Wang, X G; Qi, C; Zhang, Y; Wang, C F; Liu, H D; Feng, M Y; Chen, Y; Xu, Y X; Zhong, J F; Huang, J M


    The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

  4. Endogenous retroviral LTRs as promoters for human genes: a critical assessment. (United States)

    Cohen, Carla J; Lock, Wynne M; Mager, Dixie L


    Gene regulatory changes are thought to be major factors driving species evolution, with creation of new regulatory regions likely being instrumental in contributing to diversity among vertebrates. There is growing appreciation for the role of transposable elements (TEs) in gene regulation and, indeed, laboratory investigations have confirmed many specific examples of mammalian genes regulated by promoters donated by endogenous retroviruses (ERVs) or other TEs. Bioinformatics studies have revealed hundreds of additional instances where this is likely to be the case. Since the long terminal repeats (LTRs) of retroviruses naturally contain abundant transcriptional regulatory signals, roles for ERV LTRs in regulating mammalian genes are eminently plausible. Moreover, it seems reasonable that exaptation of an LTR regulatory module provides opportunities for evolution of new gene regulatory patterns. In this Review we summarize known examples of LTRs that function as human gene alternative promoters, as well as the evidence that LTR exaptation has resulted in a pattern of novel gene expression significantly different from the pattern before LTR insertion or from that of gene orthologs lacking the LTR. Available data suggest that, while new expression patterns can arise as a result of LTR usage, this situation is relatively rare and is largely restricted to the placenta. In many cases, the LTR appears to be a minor, alternative promoter with an expression pattern similar to that of the native promoter(s) and hence likely exerts a subtle overall effect on gene expression. We discuss these findings and offer evolutionary models to explain these trends.

  5. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene

    NARCIS (Netherlands)

    Kop, D.A.M. van der; Schuyer, M.; Pinas, J.E.; Zaal, B.J. van der; Hooykaas, P.J.J.


    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the β-glucuronidase (gusA) reporter gene. Subsequently, seeds were tr

  6. Cloning and characterization of the human USP22 gene promoter.

    Directory of Open Access Journals (Sweden)

    Jianjun Xiong

    Full Text Available Ubiquitin-specific processing enzyme 22 (USP22 plays a direct role in regulating cell cycle, and its overexpression has been reported to be involved in tumor progression. However, little is known about the regulation of USP22 transcription. In this study, we cloned and characterized the human USP22 promoter. Using 5' RACE (rapid amplification of cDNA ends analysis, the transcriptional initiation site was identified. Promoter deletion analysis showed that the sequence between -210 and -7 contains the basal promoter for USP22 in human fibroblast and tumor cells. Surprisingly, mutations in a putative Sp1 binding site immediately upstream of the USP22 transcriptional start site (-13 to -7 resulted in a significant induction of promoter activity. Further study revealed that Sp1 binds to this site in human normal fibroblast cells, and treatment with the Sp1 inhibitor mithramycin A led to a marked increase in USP22 transcript levels. Forced expression of exogenous Sp1 repressed the USP22 promoter activity in HeLa cells. In contrast, knockdown of Sp1 enhanced USP22 promoter activity and mRNA levels. These data suggest that Sp1 is a crucial regulator of USP22 transcription.

  7. Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA (United States)

    Bredel, Markus; Bredel, Claudia; Juric, Dejan; Kim, Young; Vogel, Hannes; Harsh, Griffith R.; Recht, Lawrence D.; Pollack, Jonathan R.; Sikic, Branimir I.


    Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalin-fixed/paraffin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fine-needle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens. PMID:15858140

  8. Optimization of reporter gene assay: several factors influencing detection of promoter activity

    Institute of Scientific and Technical Information of China (English)

    XUE Li-xiang; WENG Mo; ZHANG Zong-yu; TONG Tan-jun


    Background Promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay.Methods Human embryonic lung fibroblast cells (2BS), HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined various factors that affect promoter activity determination,such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments.Results The sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein (EGFP). Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA.Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity.Conclusions To detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.

  9. Structure, variation and expression analysis of glutenin gene promoters from Triticum aestivum cultivar Chinese Spring shows the distal region of promoter 1Bx7 is key regulatory sequence. (United States)

    Wang, Kai; Zhang, Xue; Zhao, Ying; Chen, Fanguo; Xia, Guangmin


    In this study, ten glutenin gene promoters were isolated from model wheat (Triticum aestivum L. cv. Chinese Spring) using a genomic PCR strategy with gene-specific primers. Six belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoters, and four to low-molecular-weight glutenin subunit (LMW-GS). Sequence lengths varied from 1361 to 2,554 bp. We show that the glutenin gene promoter motifs are conserved in diverse sequences in this study, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show that HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (> -700 bp) compared with LMW-GS. The y-type HMW-GS gene promoters possess unique motifs including RY repeat and as-2 box compared to the x-type. We also identified important motifs in the distal region of HMW-GS gene promoters including the 5'-UTR Py-rich stretch motif and the as-2 box motif. We found that cis-acting elements in the distal region of promoter 1Bx7 enhanced the expression of HMW-GS gene 1Bx7. Taken together, these data support efforts in designing molecular breeding strategies aiming to improve wheat quality. Our results offer insight into the regulatory mechanisms of glutenin gene expression.

  10. Biological Activity of the Alternative Promoters of the Dictyostelium discoideum Adenylyl Cyclase A Gene. (United States)

    Rodriguez-Centeno, Javier; Sastre, Leandro


    Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation.

  11. A novel binary T-vector with the GFP reporter gene for promoter characterization.

    Directory of Open Access Journals (Sweden)

    Shu-Ye Jiang

    Full Text Available Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens.

  12. Evaluation of a novel promoter from Populus trichocarpa for mature xylem tissue specific gene delivery. (United States)

    Nguyen, Van Phap; Cho, Jin-Seong; Choi, Young-Im; Lee, Sang-Won; Han, Kyung-Hwan; Ko, Jae-Heung


    Wood (i.e., secondary xylem) is an important raw material for many industrial applications. Mature xylem (MX) tissue-specific genetic modification offers an effective means to improve the chemical and physical properties of the wood. Here, we describe a promoter that drives strong gene expression in a MX tissue-specific manner. Using whole-transcriptome genechip analyses of different tissue types of poplar, we identified five candidate genes that had strong expression in the MX tissue. The putative promoter sequences of the five MX-specific genes were evaluated for their promoter activity in both transgenic Arabidopsis and poplar. Among them, we found the promoter of Potri.013G007900.1 (called the PtrMX3 promoter) had the strongest activity in MX and thus was further characterized. In the stem and root tissues of transgenic Arabidopsis plants, the PtrMX3 promoter activity was found exclusively in MX tissue. MX-specific activity of the promoter was reproduced in the stem tissue of transgenic poplar plants. The PtrMX3 promoter activity was not influenced by abiotic stresses or exogenously applied growth regulators, indicating the PtrMX3 promoter is bona fide MX tissue-specific. Our study provides a strong MX-specific promoter for MX-specific modifications of woody biomass.

  13. Molecular assays in detecting EGFR gene aberrations: an updated HER2-dependent algorithm for interpreting gene signals; a short technical report. (United States)

    Tsiambas, Evangelos; Ragos, Vasileios; Lefas, Alicia Y; Georgiannos, Stavros N; Rigopoulos, Dimitrios N; Georgakopoulos, Georgios; Stamatelopoulos, Athanasios; Grapsa, Dimitra; Syrigos, Konstantinos


    Purpose: Among oncogenes that have already been identified and cloned, Epidermal Growth Factor Receptor (EGFR) remains one of the most significant. Understanding its deregulation mechanisms improves critically patients' selection for personalized therapies based on modern molecular biology and oncology guidelines. Anti-EGFR targeted therapeutic strategies have been developed based on specific genetic profiles and applied in subgroups of patients suffering by solid cancers of different histogenetic origin. Detection of specific EGFR somatic mutations leads to tyrosine kinase inhibitors (TKIs) application in subsets of them. Concerning EGFR gene numerical imbalances, identification of pure gene amplification is critical for targeting the molecule via monoclonal antibodies (mAbs). In the current technical paper we demonstrate the main molecular methods applied in EGFR analyses focused also on new data in interpreting numerical imbalances based on ASCO/ACAP guidelines for HER2 in situ hybridization (ISH) clarifications.

  14. From amplification to gene in thyroid cancer: A high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, X.N.; Gonsky, R.; Korenberg, J.R. [UCLA School of Medicine, Los Angeles, CA (United States). Cedars-Sinai Research Inst.; Knauf, J.A.; Fagin, J.A. [Univ. of Cincinnati, OH (United States). Div. of Endocrinology/Metabolism; Wang, M.; Lai, E.H. [Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Pharmacology; Chissoe, S. [Washington Univ. School of Medicine, St. Louis, MO (United States). Genome Sequencing


    Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. The authors now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. They used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3--6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKC{epsilon}), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKC{epsilon} as a previously unmapped candidate gene involved in thyroid tumorigenesis.

  15. Discovery of inhibitors of aberrant gene transcription from Libraries of DNA binding molecules: inhibition of LEF-1-mediated gene transcription and oncogenic transformation. (United States)

    Stover, James S; Shi, Jin; Jin, Wei; Vogt, Peter K; Boger, Dale L


    The screening of a >9000 compound library of synthetic DNA binding molecules for selective binding to the consensus sequence of the transcription factor LEF-1 followed by assessment of the candidate compounds in a series of assays that characterized functional activity (disruption of DNA-LEF-1 binding) at the intended target and site (inhibition of intracellular LEF-1-mediated gene transcription) resulting in a desired phenotypic cellular change (inhibit LEF-1-driven cell transformation) provided two lead compounds: lefmycin-1 and lefmycin-2. The sequence of screens defining the approach assures that activity in the final functional assay may be directly related to the inhibition of gene transcription and DNA binding properties of the identified molecules. Central to the implementation of this generalized approach to the discovery of DNA binding small molecule inhibitors of gene transcription was (1) the use of a technically nondemanding fluorescent intercalator displacement (FID) assay for initial assessment of the DNA binding affinity and selectivity of a library of compounds for any sequence of interest, and (2) the technology used to prepare a sufficiently large library of DNA binding compounds.

  16. Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

    Directory of Open Access Journals (Sweden)

    Fauteux François


    Full Text Available Abstract Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP gene promoters from three plant families, namely Brassicaceae (mustards, Fabaceae (legumes and Poaceae (grasses using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L. Heynh., soybean (Glycine max (L. Merr. and rice (Oryza sativa L. respectively. We have identified three conserved motifs (two RY-like and one ACGT-like in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination

  17. Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

    Directory of Open Access Journals (Sweden)

    Daniel Meier

    Full Text Available The Fanconi anemia (FA gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS. In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs, and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

  18. Establishment of a cell-based assay to screen regulators for Klotho gene promoter

    Institute of Scientific and Technical Information of China (English)

    Zhi-liang XU; Hong GAO; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU


    AIM: To discover compounds which can regulate Klotho promoter activity. Klotho is an aging suppressor gene. A defect in Klotho gene expression in the mouse results in the phenotype similar to human aging. Recombinant Klotho protein improves age-associated diseases in animal models. It has been proposed that up-regulation of Klotho gene expression may have anti-aging effects. METHODS: Klotho promoter was cloned into a vector containing luciferase gene, and the reporter gene vector was transfected into HEK293 cells to make a stable cell line (HEK293/KL). A model for cellular aging was established by treating HEK293/KL cells with H2O2. These cells were treated with extracts from Traditional Chinese Medicines (TCMs). The luciferase activity was detected to identify compounds that can regulate Klotho promoter. RESULTS:The expression of luciferase in these cells was under control of Klotho promoter and down-regulated after H2O2 treatment The down-regulation of luciferase expression was H2O2 concentration-dependent with an IC50 at approximately 0.006 %. This result demonstrated that the Klotho gene promoter was regulated by oxidative stress. Using the cell-based reporter gene assay, we screened natural product extracts for regulation of Klotho gene promoter. Several extracts were identified that could rescue the H2O2effects and up-regulated Klotho promoter activity. CONCLUSION: A cell -based assay for high-throughput drug screening was established to identify compounds that regulate Klotho promoter activity, and several hits were discovered from natural products. Further characterization of these active extracts could help to investigate Klotho function and aging mechanisms.

  19. Structural Properties of Gene Promoters Highlight More than Two Phenotypes of Diabetes. (United States)

    Ionescu-Tîrgovişte, Constantin; Gagniuc, Paul Aurelian; Guja, Cristian


    Genome-wide association studies (GWAS) published in the last decade raised the number of loci associated with type 1 (T1D) and type 2 diabetes (T2D) to more than 50 for each of these diabetes phenotypes. The environmental factors seem to play an important role in the expression of these genes, acting through transcription factors that bind to promoters. Using the available databases we examined the promoters of various genes classically associated with the two main diabetes phenotypes. Our comparative analyses have revealed significant architectural differences between promoters of genes classically associated with T1D and T2D. Nevertheless, five gene promoters (about 16%) belonging to T1D and six gene promoters (over 19%) belonging to T2D have shown some intermediary structural properties, suggesting a direct relationship to either LADA (Latent Autoimmune Diabetes in Adults) phenotype or to non-autoimmune type 1 phenotype. The distribution of these promoters in at least three separate classes seems to indicate specific pathogenic pathways. The image-based patterns (DNA patterns) generated by promoters of genes associated with these three phenotypes support the clinical observation of a smooth link between specific cases of typical T1D and T2D. In addition, a global distribution of these DNA patterns suggests that promoters of genes associated with T1D appear to be evolutionary more conserved than those associated with T2D. Though, the image based patterns obtained by our method might be a new useful parameter for understanding the pathogenetic mechanism and the diabetogenic gene networks.

  20. Selective AR Modulators that Distinguish Proliferative from Differentiative Gene Promoters (United States)


    dependent compound screen, aided by the University of Michigan Center for Chemical Genomics . Differential AR activation in transfected cells was assessed...WR, Parker JS, Lee MX, Kass EM, Spratt DE, Iaquinta PJ, Arora VK, Yen WF, Cai L, Zheng D, Carver BS, Chen Y, Watson PA, Shah NP, Fujisawa S, Goglia...for known genes and genome -wide by ChIP-seq. Results will strengthen our overall hypothesis that genes with similar function (i.e

  1. Anthracyclines induce double-strand DNA breaks at active gene promoters. (United States)

    Yang, Fan; Kemp, Christopher J; Henikoff, Steven


    Doxorubicin is a widely used chemotherapeutic drug that intercalates between DNA base-pairs and poisons Topoisomerase II, although the mechanistic basis for cell killing remains speculative. Doxorubicin and related anthracycline compounds have been shown to increase nucleosome turnover and/or eviction around promoters, which suggests that the resulting enhanced exposure of DNA might underlie cell killing. Previously, we showed that low doses of anthracyclines increase nucleosome turnover around active gene promoters, which suggests that loss of nucleosomes might contribute to cancer cell killing. Here we apply a genome-wide method to precisely map DNA double-strand breaks (DSBs) in cancer cells. We find that spontaneous DSBs occur preferentially around promoters of active genes, and that both anthracyclines and etoposide, a Topoisomerase II poison, increase DSBs around promoters, although CpG islands are conspicuously protected from DSBs. We propose that torsion-based enhancement of nucleosome turnover by anthracyclines exposes promoter DNA, ultimately causing DSBs around promoters.

  2. Identification of epigenetically downregulated Tmem70 and Ube2e2 in rat liver after 28-day treatment with hepatocarcinogenic thioacetamide showing gene product downregulation in hepatocellular preneoplastic and neoplastic lesions produced by tumor promotion. (United States)

    Mizukami, Sayaka; Yafune, Atsunori; Watanabe, Yousuke; Nakajima, Kota; Jin, Meilan; Yoshida, Toshinori; Shibutani, Makoto


    The present study identified genes showing promoter region hypermethylation by CpG island microarrays in the liver of rats treated with hepatocarcinogen thioacetamide (TAA) for 28days. Among 47 hypermethylated genes, Hist1h2aa, Tmem70, Ube2e2, and Slk were confirmed to show hypermethylation by methylation-specific quantitative polymerase chain reaction (PCR) and pyrosequencing analyses as well as downregulation of transcript levels by real-time reverse transcription-PCR analysis in the livers of rats treated with TAA. All gene products of the 4 selected genes showed decreased immunoreactivity forming negative liver cell foci in a subpopulation of glutathione S-transferase placental form (GST-P)(+) foci in TAA-promoted rat livers in a two-stage hepatocarcinogenesis model. Among them, TMEM70 and UBE2E2 showed increased incidences of negative foci in GST-P(+) foci by promotion of all examined TAA, β-naphthoflavone, piperonyl butoxide, fenbendazole and phenobarbital, while HIST1H2AA and SLK did not respond to all promotive treatments. In the late stage of tumor promotion by TAA, the incidence of GST-P(+) proliferative lesions with downregulation of TMEM70 or UBE2E2 was higher in adenomas and carcinomas than liver cell foci. TMEM70 plays a role in mitochondrial oxidative phosphorylation, and UBE2E2 participates in the stabilization of cell cycle regulatory proteins. Therefore, our results indicate that aberrant epigenetic gene downregulation suggestive of a metabolic shift of cellular respiration from oxidative phosphorylation to glycolysis and aberrant cell cycle regulation facilitating cell proliferation from as early as 28days after hepatocarcinogen treatment contribute to tumor development.

  3. Structure of the BoLA-DRB3 gene and promoter. (United States)

    Russell, G C; Smith, J A; Oliver, R A


    The cattle major histocompatibility complex (MHC) class II DR gene product is a heterodimer encoded by the BoLA-DRA and -DRB3 genes. Several groups have isolated cDNA and genomic clones for these genes, but their full genomic organization has not been described. We used a combination of long-range polymerase chain reaction (PCR), cloning and sequencing to define the organization of the DRB3 gene on existing genomic clones and in genomic DNA. We estimate the size of the coding region to be 11.4 kbp. Sequencing of full-length PCR clones from two different haplotypes confirmed that they carried complete DRB3 genes and allowed the design of probes and primers to isolate and characterize the DRB3 promoter and 3' end. Fragments carrying the 5' end of the DRB3 gene and its promoter were identified on bacterial artificial chromosome (BAC) clones carrying the BoLA-DR genes. A 10-kbp promoter fragment was subcloned from one clone and a 1.7-kbp region including exon 1 and the promoter was sequenced. A 3-kbp fragment encoding exons 4-6 and the entire 3' untranslated region of the DRB3 gene was isolated from lambda clone A1 and sequenced. This provides us with improved characterization of the DRB3*0101 and DRB3*2002 alleles, and also subcloned 5' and 3' flanking regions of the polymorphic DRB3 gene for use in functional studies.

  4. Rational promoter selection for gene transfer into cardiac cells

    NARCIS (Netherlands)

    Maass, A; Langer, SJ; Oberdorf-Maass, S; Bauer, S; Neyses, L; Leinwand, LA


    Cardiomyocytes (CMCs) are extremely difficult to transfect with non-viral techniques, but they are efficiently infected by adenoviruses. The most commonly used promoters to drive protein expression in cardiac myocytes are of viral origin, since they are believed to be constitutively active and minim

  5. ATRX promotes gene expression by facilitating transcriptional elongation through guanine-rich coding regions. (United States)

    Levy, Michael A; Kernohan, Kristin D; Jiang, Yan; Bérubé, Nathalie G


    ATRX is a chromatin remodeling protein involved in deposition of the histone variant H3.3 at telomeres and pericentromeric heterochromatin. It also influences the expression level of specific genes; however, deposition of H3.3 at transcribed genes is currently thought to occur independently of ATRX. We focused on a set of genes, including the autism susceptibility gene Neuroligin 4 (Nlgn4), that exhibit decreased expression in ATRX-null cells to investigate the mechanisms used by ATRX to promote gene transcription. Overall TERRA levels, as well as DNA methylation and histone modifications at ATRX target genes are not altered and thus cannot explain transcriptional dysregulation. We found that ATRX does not associate with the promoter of these genes, but rather binds within regions of the gene body corresponding to high H3.3 occupancy. These intragenic regions consist of guanine-rich DNA sequences predicted to form non-B DNA structures called G-quadruplexes during transcriptional elongation. We demonstrate that ATRX deficiency corresponds to reduced H3.3 incorporation and stalling of RNA polymerase II at these G-rich intragenic sites. These findings suggest that ATRX promotes the incorporation of histone H3.3 at particular transcribed genes and facilitates transcriptional elongation through G-rich sequences. The inability to transcribe genes such as Nlgn4 could cause deficits in neuronal connectivity and cognition associated with ATRX mutations in humans.

  6. Interleukin 10 gene promoter polymorphism and risk of diffuse large B cell lymphoma (DLBCL

    Directory of Open Access Journals (Sweden)

    Roba M. Talaat


    Conclusions: Taken together, our findings demonstrated that IL-10 promoter gene polymorphism (−1082 and −819 may not have an influence on the clinical outcome of DLBCL, especially in terms of overall secretion level. Further investigations of other cytokine gene polymorphisms will lead to a better understanding of the disease’s biological background.

  7. Spermatogenesis-related ring finger gene ZNF230 promoter: identification and functional analysis

    DEFF Research Database (Denmark)

    Xu, Wenming; Zhang, Sizhong; Qiu, Weimin


    The ZNF230 gene is a recently cloned gene which is transcribed only in fertile male testes and may be related to human spermatogenesis. To characterize the multiple stage-specific transcription elements necessary for ZNF230 expression, we cloned ZNF230 promoter and constructed chimeric luciferase...

  8. CMP Promoters Database: A systematic study on site-specific transcription factors in CMP genes

    Directory of Open Access Journals (Sweden)

    Meera A


    motifs as NKX and AP2 making up the structural feature of the non coding genes are absent from few genes. Keywords: Non-coding sequence, Phylogeny, TCA, Glycolysis, TRANSFAC, Promoter, Database, Central Metabolic Pathway. Received: 17 March 2008 / Received in revised form: 5 February 2009, Accepted: 31 April 2009 Published online: 14 May 2009

  9. GUS Gene Expression Driven by A Citrus Promoter in Transgenic Tobacco and 'Valencia' Sweet Orange (United States)

    The objective of this work was the transformation of tobacco and ‘Valencia’ sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL) gene promoter (CsPP). Transformation was accomplished by co-cultivation of tobacco and ‘Valencia’ sweet orange explants with Agrobacteriu...

  10. A cII-dependent promoter is located within the Q gene of bacteriophage lambda.


    Hoopes, B C; McClure, W R


    We have found a cII-dependent promoter, PaQ, within the Q gene of bacteriophage lambda. Transcription experiments and abortive initiation assays performed in vitro showed that the promoter strength and the cII affinity of PaQ were comparable to the other cII-dependent lambda promoters, PE and PI. The location and leftward direction of PaQ suggests a possible role in the delay of lambda late-gene expression by cII protein, a phenomenon that has been called cII-dependent inhibition. We have con...

  11. Decreased expression of BRCA1 in SK-BR-3 cells is the result of aberrant activation of the GABP Beta promoter by an NRF-1-containing complex

    Directory of Open Access Journals (Sweden)

    MacDonald Gwen


    Full Text Available Abstract Background BRCA1 has recently been identified as a potential regulator of mammary stem/progenitor cell differentiation, and this function may explain the high prevalence of breast cancer in BRCA1 mutation carriers, as well as the downregulation of BRCA1 in a large proportion of sporadic breast cancers. That is, loss of BRCA1 function results in blocked differentiation with expansion of the mammary stem/progenitor cells. Because BRCA1 also maintains genomic integrity, its loss could produce a pool of genetically unstable stem/progenitor cells that are prime targets for further transforming events. Thus, elucidating the regulatory mechanisms of BRCA1 expression is important to our understanding of normal and malignant breast differentiation. Results Loss of BRCA1 expression in the ErbB2-amplified SK-BR-3 cell line was found to be the result of loss of activity of the ets transcription factor GABP, a previously characterized regulator of BRCA1 transcription. The expression of the non-DNA binding GABPβ subunit was shown to be deficient, while the DNA binding subunit, GABPα was rendered unstable by the absence of GABPβ. Deletion analysis of the GABPβ proximal promoter identified a potential NRF-1 binding site as being critical for expression. Supershift analysis, the binding of recombinant protein and chromatin immunoprecipitation confirmed the role of NRF-1 in regulating the expression of GABPβ. The siRNA knockdown of NRF-1 resulted in decreased GABPβ and BRCA1 expression in MCF-7 cells indicating that they form a transcriptional network. NRF-1 levels and activity did not differ between SK-BR-3 and MCF-7 cells, however the NRF-1 containing complex on the GABPβ promoter differed between the two lines and appears to be the result of altered coactivator binding. Conclusions Both NRF-1 and GABP have been linked to the regulation of nuclear-encoded mitochondrial proteins, and the results of this study suggest their expression is

  12. Assessment of gene promoter hypermethylation for detection of cervical neoplasia

    NARCIS (Netherlands)

    Wisman, G. Bea A.; Nijhuis, Esther R.; Hoque, Mohammad O.; Reesink-Peters, Nathalie; Koning, Alice J.; Volders, Haukeline H.; Buikema, Henk J.; Boezen, H. Marike; Hollema, Harry; Schuuring, Ed; Sidransky, David; van der Zee, Ate G. J.


    Current cervical cancer screening is based on morphological assessment of Pap smears and associated with significant false negative and false positive results. Previously, we have shown that detection of hypermethylated genes in cervical scrapings using quantitative methylation-specific PCR (QMSP) i

  13. Gene promoter methylation patterns throughout the process of cervical carcinogenesis

    NARCIS (Netherlands)

    Yang, Nan; Nijhuis, Esther R.; Volders, Haukeline H.; Eijsink, Jasper J. H.; Lendvai, Agnes; Zhang, Bo; Hollema, Harry; Schuuring, Ed; Wisman, G. Bea A.; van der Zee, Ate G. J.


    Objectives: To determine methylation status of nine genes, previously described to be frequently methylated in cervical cancer, in squamous intraepithelial lesions (SIL). Methods: QMSP was performed in normal cervix, low-grade ( L) SIL, high-grade (H) SIL, adenocarcinomas and squamous cell cervical

  14. Plant defense gene promoter enhances the reliability of shiva-1 gene-induced resistance to soft rot disease in potato. (United States)

    Yi, Jung Yoon; Seo, Hyo Won; Yang, Moon Sik; Robb, E Jane; Nazar, Ross N; Lee, Shin Woo


    PAL5, a tomato (Lycopersicon esculentum Mill.) plant defense gene that encodes phenylalanine ammonia-lyase, is known to respond to a variety of environmental stresses including pathogen infection and wounding. A shiva-1 gene recombinant that encodes a small synthetic antibacterial peptide under the PAL5 gene promoter was transformed into potato (Solanum tuberosum L.) and its ability to induce resistance to Erwinia carotovora was compared with a construct under the control of the constitutive and widely used cauliflower mosaic virus (CaMV) 35S promoter. The shiva-1 peptide, an analog of natural cecropin B, was shown previously to have high bactericidal activity in vitro, but when expressed in vivo under the control of the CaMV 35S promoter, the effects were very inconsistent. As observed previously, in the present studies a few transformants with the CaMV 35S promoter were highly resistant when assayed for susceptibility to soft rot disease. In marked contrast the majority of transformants with the PAL5 gene promoter were highly resistant. More-detailed analyses of the incorporated DNA indicated that most of the transformants with the CaMV 35S promoter contained multiple copies of the transforming DNA while all of the PAL5 recombinants contained single copies. The highly resistant CaMV 35S recombinant also was present as a single copy. The results indicate that, at least in this instance, a constitutive promoter may not be ideal for the effective expression of a foreign gene and suggest that multiple insertions may have negative consequences.

  15. The Mouse Solitary Odorant Receptor Gene Promoters as Models for the Study of Odorant Receptor Gene Choice.

    Directory of Open Access Journals (Sweden)

    Andrea Degl'Innocenti

    Full Text Available In vertebrates, several anatomical regions located within the nasal cavity mediate olfaction. Among these, the main olfactory epithelium detects most conventional odorants. Olfactory sensory neurons, provided with cilia exposed to the air, detect volatile chemicals via an extremely large family of seven-transmembrane chemoreceptors named odorant receptors. Their genes are expressed in a monogenic and monoallelic fashion: a single allele of a single odorant receptor gene is transcribed in a given mature neuron, through a still uncharacterized molecular mechanism known as odorant receptor gene choice.Odorant receptor genes are typically arranged in genomic clusters, but a few are isolated (we call them solitary from the others within a region broader than 1 Mb upstream and downstream with respect to their transcript's coordinates. The study of clustered genes is problematic, because of redundancy and ambiguities in their regulatory elements: we propose to use the solitary genes as simplified models to understand odorant receptor gene choice.Here we define number and identity of the solitary genes in the mouse genome (C57BL/6J, and assess the conservation of the solitary status in some mammalian orthologs. Furthermore, we locate their putative promoters, predict their homeodomain binding sites (commonly present in the promoters of odorant receptor genes and compare candidate promoter sequences with those of wild-caught mice. We also provide expression data from histological sections.In the mouse genome there are eight intact solitary genes: Olfr19 (M12, Olfr49, Olfr266, Olfr267, Olfr370, Olfr371, Olfr466, Olfr1402; five are conserved as solitary in rat. These genes are all expressed in the main olfactory epithelium of three-day-old mice. The C57BL/6J candidate promoter of Olfr370 has considerably varied compared to its wild-type counterpart. Within the putative promoter for Olfr266 a homeodomain binding site is predicted. As a whole, our findings

  16. Rice Mitochondrial Genes Are Transcribed by Multiple Promoters That Are Highly Diverged

    Institute of Scientific and Technical Information of China (English)

    Qun-Yu Zhang; Yao-Guang Liu


    Plant mitochondrial genes are often transcribed into complex sets of mRNA. To characterize the transcription initiation and promoter structure, the transcript termini of four mitochondrial genes, atp1, atp6, cob,rps7, in rice (Oryza sativa L.), were determined by using a modified circularized RNA reverse transcriptionpolymerase chain reaction method. The results revealed that three genes (atp1, atp6, rps7) were transcribed from multiple initiation sites, indicating the presence of multiple promoters. Two transcription termination sites were detected in three genes (atp6, cob, rps7), respectively. Analysis on the promoter architecture showed that the YRTA (Y=T or C, R=A or G) motifs that are widely present in the mitochondrial promoters of other monocot and dicot plant species were detected only in two of the 12 analyzed promoters.Our data suggest that the promoter sequences in the rice mitochondrial genome are highly diverged in comparison to those in other plants, and the YRTA motif is not an essential element for the promoter activity.

  17. Cloning and functional analysis of SEL1L promoter region, a pancreas-specific gene. (United States)

    Cattaneo, M; Sorio, C; Malferrari, G; Rogozin, I B; Bernard, L; Scarpa, A; Zollo, M; Biunno, I


    We examined the promoter activity of SEL1L, the human ortholog of the C. elegans gene sel-1, a negative regulator of LIN-12/NOTCH receptor proteins. To understand the relation in SEL1L transcription pattern observed in different epithelial cells, we determined the transcription start site and sequenced the 5' flanking region. Sequence analysis revealed the presence of consensus promoter elements--GC boxes and a CAAT box--but the absence of a TATA motif. Potential binding sites for transcription factors that are involved in tissue-specific gene expression were identified, including: activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF3 beta), homeobox Nkx2-5 and GATA-1. Transcription activity of the TATA-less SEL1L promoter was analyzed by transient transfection using luciferase reporter gene constructs. A core basal promoter of 302 bp was sufficient for constitutive promoter activity in all the cell types studied. This genomic fragment contains a CAAT and several GC boxes. The activity of the SEL1L promoter was considerably higher in mouse pancreatic beta cells (beta TC3) than in several human pancreatic neoplastic cell lines; an even greater reduction of its activity was observed in cells of nonpancreatic origin. These results suggest that SEL1L promoter may be a useful tool in gene therapy applications for pancreatic pathologies.

  18. Characteristic differences between the promoters of intron-containing and intronless ribosomal protein genes in yeast

    Directory of Open Access Journals (Sweden)

    Vingron Martin


    Full Text Available Abstract Background More than two thirds of the highly expressed ribosomal protein (RP genes in Saccharomyces cerevisiae contain introns, which is in sharp contrast to the genome-wide five percent intron-containing genes. It is well established that introns carry regulatory sequences and that the transcription of RP genes is extensively and coordinately regulated. Here we test the hypotheses that introns are innately associated with heavily transcribed genes and that introns of RP genes contribute regulatory TF binding sequences. Moreover, we investigate whether promoter features are significantly different between intron-containing and intronless RP genes. Results We find that directly measured transcription rates tend to be lower for intron-containing compared to intronless RP genes. We do not observe any specifically enriched sequence motifs in the introns of RP genes other than those of the branch point and the two splice sites. Comparing the promoters of intron-containing and intronless RP genes, we detect differences in number and position of Rap1-binding and IFHL motifs. Moreover, the analysis of the length distribution and the folding free energies suggest that, at least in a sub-population of RP genes, the 5' untranslated sequences are optimized for regulatory function. Conclusion Our results argue against the direct involvement of introns in the regulation of transcription of highly expressed genes. Moreover, systematic differences in motif distributions suggest that RP transcription factors may act differently on intron-containing and intronless gene promoters. Thus, our findings contribute to the decoding of the RP promoter architecture and may fuel the discussion on the evolution of introns.

  19. Taproot promoters cause tissue specific gene expression within the storage root of sugar beet. (United States)

    Oltmanns, Heiko; Kloos, Dorothee U; Briess, Waltraud; Pflugmacher, Maike; Stahl, Dietmar J; Hehl, Reinhard


    The storage root (taproot) of sugar beet (Beta vulgaris L.) originates from hypocotyl and primary root and contains many different tissues such as central xylem, primary and secondary cambium, secondary xylem and phloem, and parenchyma. It was the aim of this work to characterize the promoters of three taproot-expressed genes with respect to their tissue specificity. To investigate this, promoters for the genes Tlp, His1-r, and Mll were cloned from sugar beet, linked to reporter genes and transformed into sugar beet and tobacco. Reporter gene expression analysis in transgenic sugar beet plants revealed that all three promoters are active in the storage root. Expression in storage root tissues is either restricted to the vascular zone (Tlp, His1-r) or is observed in the whole organ (Mll). The Mll gene is highly organ specific throughout different developmental stages of the sugar beet. In tobacco, the Tlp and Mll promoters drive reporter gene expression preferentially in hypocotyl and roots. The properties of the Mll promoter may be advantageous for the modification of sucrose metabolism in storage roots.

  20. Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression

    Directory of Open Access Journals (Sweden)

    Mansukhani Mahesh


    Full Text Available Abstract Background Cervical Cancer (CC exhibits highly complex genomic alterations. These include hemizygous deletions at 4p15.3, 10q24, 5q35, 3p12.3, and 11q24, the chromosomal sites of Slit-Robo pathway genes. However, no candidate tumor suppressor genes at these regions have been identified so far. Slit family of secreted proteins modulates chemokine-induced cell migration of distinct somatic cell types. Slit genes mediate their effect by binding to its receptor Roundabout (Robo. These genes have shown to be inactivated by promoter hypermethylation in a number of human cancers. Results To test whether Slit-Robo pathway genes are targets of inactivation at these sites of deletion, we examined promoter hypermethylation of SLIT1, SLIT2, SLIT3, ROBO1, and ROBO3 genes in invasive CC and its precursor lesions. We identified a high frequency of promoter hypermethylation in all the Slit-Robo genes resulting in down regulated gene expression in invasive CC, but the inhibitors of DNA methylation and histone deacetylases (HDACs in CC cell lines failed to effectively reactivate the down-regulated expression. These results suggest a complex mechanism of inactivation in the Slit-Robo pathway in CC. By analysis of cervical precancerous lesions, we further show that promoter hypermethylation of Slit-Robo pathway occurs early in tumor progression. Conclusion Taken together, these findings suggest that epigenetic alterations of Slit-Robo pathway genes (i play a role in CC development, (ii further delineation of molecular basis of promoter methylation-mediated gene regulation provides a potential basis for epigenetic-based therapy in advanced stage CC, and (iii form epigenetic signatures to identify precancerous lesions at risk to progression.

  1. Predicting Polymerase Ⅱ Core Promoters by Cooperating Transcription Factor Binding Sites in Eukaryotic Genes

    Institute of Scientific and Technical Information of China (English)

    Xiao-Tu MA; Min-Ping QIAN; Hai-Xu TANG


    Several discriminate functions for predicting core promoters that based on the potential cooperation between transcription factor binding sites (TFBSs) are discussed. It is demonstrated that the promoter predicting accuracy is improved when the cooperation among TFBSs is taken into consideration.The core promoter region of a newly discovered gene CKLFSF1 is predicted to locate more than 1.5 kb far away from the 5′ end of the transcript and in the last intron of its upstream gene, which is experimentally confirmed later. The core promoters of 3402 human RefSeq sequences, obtained by extending the mRNAs in human genome sequences, are predicted by our algorithm, and there are about 60% of the predicted core promoters locating within the ± 500 bp region relative to the annotated transcription start site.

  2. Optimization of TaDREB3 gene expression in transgenic barley using cold-inducible promoters. (United States)

    Kovalchuk, Nataliya; Jia, Wei; Eini, Omid; Morran, Sarah; Pyvovarenko, Tatiana; Fletcher, Stephen; Bazanova, Natalia; Harris, John; Beck-Oldach, Kontanze; Shavrukov, Yuri; Langridge, Peter; Lopato, Sergiy


    Constitutive over-expression of the TaDREB3 gene in barley improved frost tolerance of transgenic plants at the vegetative stage of plant development, but leads to stunted phenotypes and 3- to 6-week delays in flowering compared to control plants. In this work, two cold-inducible promoters with contrasting properties, the WRKY71 gene promoter from rice and the Cor39 gene promoter from durum wheat, were applied to optimize expression of TaDREB3. The aim of the work was to increase plant frost tolerance and to decrease or prevent negative developmental phenotypes observed during constitutive expression of TaDREB3. The OsWRKY71 and TdCor39 promoters had low-to-moderate basal activity and were activated by cold treatment in leaves, stems and developing spikes of transgenic barley and rice. Expression of the TaDREB3 gene, driven by either of the tested promoters, led to a significant improvement in frost tolerance. The presence of the functional TaDREB3 protein in transgenic plants was confirmed by the detection of strong up-regulation of cold-responsive target genes. The OsWRKY71 promoter-driven TaDREB3 provides stronger activation of the same target genes than the TdCor39 promoter. Analysis of the development of transgenic plants in the absence of stress revealed small or no differences in plant characteristics and grain yield compared with wild-type plants. The WRKY71-TaDREB3 promoter-transgene combination appears to be a promising tool for the enhancement of cold and frost tolerance in crop plants but field evaluation will be needed to confirm that negative development phenotypes have been controlled.

  3. Promoter polymorphism of transforming growth factor-β1 gene and ulcerative colitis

    Institute of Scientific and Technical Information of China (English)

    B Tamizifar; KB Lankarani; S Naeimi; M Rismankar Zadeh; A Taghavi; A Ghaderi


    AIM: To elucidate the possible difference in two promoter polymorphisms of the transforming growth factor-β1 (TGF-β1) gene (-800G > A, -509C > T)between ulcerative colitis (UC) patients and normal subjects.METHODS: A total of 155 patients with established ulcerative colitis and 139 normal subjects were selected as controls. Two single nucleotide polymorphisms within the promoter region of TGF-β1 gene (-509C > T and -800G > A) were genotyped using PCR-RFLP.RESULTS: There was a statistically significant difference in genotype and allele frequency distributions between UC patients and controls for the -800G > A polymorphism of the TGF-β1 gene (P A of TGF-β1 gene promoter between Iranian patients with UC and normal subjects.

  4. Inhibition of nodule development by multicopy promoters of Rhizobium meliloti nif/fix genes

    Institute of Scientific and Technical Information of China (English)

    吴桐; 朱家璧; 俞冠翘; 沈善炯


    Using luc gene as a reporter to study the activation of Rhizobium meliloti nif/fix genes in thedevelopment of symbiosis,the authors observed that nodule development and nitrogen fixation were inhibitedby both multicopy promoters of nifHDK and fixABCX.The phenotype of R.meliloti containing multicopynif/fix promoters appeared exactly like that of nifA mutant.Using lacZ as a reporter,the authors got the same re-sults.By contrast,the rhizobia containing low-copy promoters of nif/fix genes were normal fornodule development and nitrogen fixation.These results substantiate the evidence that the product of nifAgene not only acts as a transcriptional activator of nif/fix genes,but also plays an important role in thedevelopment of root nodules.

  5. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

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    Miyuki Uno


    Full Text Available OBJECTIVES: 1 To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT promoter to its gene and protein expression levels in glioblastoma and 2 to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001. However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297. The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing, and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue.

  6. Methylation Status of Vitamin D Receptor Gene Promoter in Benign and Malignant Adrenal Tumors

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    Catia Pilon


    Full Text Available We previously showed a decreased expression of vitamin D receptor (VDR mRNA/protein in a small group of adrenocortical carcinoma (ACC tissues, suggesting the loss of a protective role of VDR against malignant cell growth in this cancer type. Downregulation of VDR gene expression may result from epigenetics events, that is, methylation of cytosine nucleotide of CpG islands in VDR gene promoter. We analyzed methylation of CpG sites in the VDR gene promoter in normal adrenals and adrenocortical tumor samples. Methylation of CpG-rich 5′ regions was assessed by bisulfite sequencing PCR using bisulfite-treated DNA from archival microdissected paraffin-embedded adrenocortical tissues. Three normal adrenals and 23 various adrenocortical tumor samples (15 adenomas and 8 carcinomas were studied. Methylation in the promoter region of VDR gene was found in 3/8 ACCs, while no VDR gene methylation was observed in normal adrenals and adrenocortical adenomas. VDR mRNA and protein levels were lower in ACCs than in benign tumors, and VDR immunostaining was weak or negative in ACCs, including all 3 methylated tissue samples. The association between VDR gene promoter methylation and reduced VDR gene expression is not a rare event in ACC, suggesting that VDR epigenetic inactivation may have a role in adrenocortical carcinogenesis.

  7. NADPH Oxidase 4-Derived H2O2 Promotes Aberrant Retinal Neovascularization via Activation of VEGF Receptor 2 Pathway in Oxygen-Induced Retinopathy

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    Jingming Li


    Full Text Available NADPH oxidase 4 (Nox4 is a major isoform of NADPH oxidase in retinal endothelial cells. Our previous study suggests that upregulation of Nox4 in retinal endothelial cells contributes to retinal vascular leakage in diabetes. In the current study, we investigated the role and mechanism of Nox4 in regulation of retinal neovascularization (NV, a hallmark of proliferative diabetic retinopathy (PDR, using a mouse model of oxygen-induced retinopathy (OIR. Our results confirmed that Nox4 was expressed predominantly in retinal vasculature of mouse retina. Retinal expression of Nox4 was markedly increased in OIR, in parallel with enhanced phosphorylation of ERK. In human retinal microvascular endothelial cells (HRECs, overexpression of Nox4 by adenovirus significantly increased extracellular H2O2 generation, resulting in intensified VEGFR2 activation and exacerbated angiogenesis upon VEGF stimulation. In contrast, silencing Nox4 expression or scavenging H2O2 by polyethylene glycol- (PEG- conjugated catalase inhibited endothelial migration, tube formation, and VEGF-induced activation of VEGFR2 signaling. Importantly, knockdown of retinal Nox4 by adenovirus-delivered siRNA significantly reduced ERK activation and attenuated retinal NV formation in OIR. Taken together, our data indicate that Nox4 promotes retinal NV formation through H2O2/VEGFR2/ERK signaling pathway. Reducing retinal Nox4 expression may represent a promising therapeutic approach for neovascular retinal diseases such as PDR.

  8. Identification of learning and memory genes in canine; promoter investigation and determining the selective pressure. (United States)

    Seifi Moroudi, Reihane; Masoudi, Ali Akbar; Vaez Torshizi, Rasoul; Zandi, Mohammad


    One of the important behaviors of dogs is trainability which is affected by learning and memory genes. These kinds of the genes have not yet been identified in dogs. In the current research, these genes were found in animal models by mining the biological data and scientific literatures. The proteins of these genes were obtained from the UniProt database in dogs and humans. Not all homologous proteins perform similar functions, thus comparison of these proteins was studied in terms of protein families, domains, biological processes, molecular functions, and cellular location of metabolic pathways in Interpro, KEGG, Quick Go and Psort databases. The results showed that some of these proteins have the same performance in the rat or mouse, dog, and human. It is anticipated that the protein of these genes may be effective in learning and memory in dogs. Then, the expression pattern of the recognized genes was investigated in the dog hippocampus using the existing information in the GEO profile. The results showed that BDNF, TAC1 and CCK genes are expressed in the dog hippocampus, therefore, these genes could be strong candidates associated with learning and memory in dogs. Subsequently, due to the importance of the promoter regions in gene function, this region was investigated in the above genes. Analysis of the promoter indicated that the HNF-4 site of BDNF gene and the transcription start site of CCK gene is exposed to methylation. Phylogenetic analysis of protein sequences of these genes showed high similarity in each of these three genes among the studied species. The dN/dS ratio for BDNF, TAC1 and CCK genes indicates a purifying selection during the evolution of the genes.

  9. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    Energy Technology Data Exchange (ETDEWEB)

    Kurayoshi, Kenta [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan); Ozono, Eiko [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ (United Kingdom); Iwanaga, Ritsuko; Bradford, Andrew P. [Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, CO 80045 (United States); Komori, Hideyuki [Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 (United States); Ohtani, Kiyoshi, E-mail: [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan)


    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  10. Regional differences in gene expression and promoter usage in aged human brains

    KAUST Repository

    Pardo, Luba M.


    To characterize the promoterome of caudate and putamen regions (striatum), frontal and temporal cortices, and hippocampi from aged human brains, we used high-throughput cap analysis of gene expression to profile the transcription start sites and to quantify the differences in gene expression across the 5 brain regions. We also analyzed the extent to which methylation influenced the observed expression profiles. We sequenced more than 71 million cap analysis of gene expression tags corresponding to 70,202 promoter regions and 16,888 genes. More than 7000 transcripts were differentially expressed, mainly because of differential alternative promoter usage. Unexpectedly, 7% of differentially expressed genes were neurodevelopmental transcription factors. Functional pathway analysis on the differentially expressed genes revealed an overrepresentation of several signaling pathways (e.g., fibroblast growth factor and wnt signaling) in hippocampus and striatum. We also found that although 73% of methylation signals mapped within genes, the influence of methylation on the expression profile was small. Our study underscores alternative promoter usage as an important mechanism for determining the regional differences in gene expression at old age.

  11. Nucleosome Stability Distinguishes Two Different Promoter Types at All Protein-Coding Genes in Yeast. (United States)

    Kubik, Slawomir; Bruzzone, Maria Jessica; Jacquet, Philippe; Falcone, Jean-Luc; Rougemont, Jacques; Shore, David


    Previous studies indicate that eukaryotic promoters display a stereotypical chromatin landscape characterized by a well-positioned +1 nucleosome near the transcription start site and an upstream -1 nucleosome that together demarcate a nucleosome-free (or -depleted) region. Here we present evidence that there are two distinct types of promoters distinguished by the resistance of the -1 nucleosome to micrococcal nuclease digestion. These different architectures are characterized by two sequence motifs that are broadly deployed at one set of promoters where a nuclease-sensitive ("fragile") nucleosome forms, but concentrated in a narrower, nucleosome-free region at all other promoters. The RSC nucleosome remodeler acts through the motifs to establish stable +1 and -1 nucleosome positions, while binding of a small set of general regulatory (pioneer) factors at fragile nucleosome promoters plays a key role in their destabilization. We propose that the fragile nucleosome promoter architecture is adapted for regulation of highly expressed, growth-related genes.

  12. Characterization of promoter sequence of toll-like receptor genes in Vechur cattle

    Directory of Open Access Journals (Sweden)

    R. Lakshmi


    Full Text Available Aim: To analyze the promoter sequence of toll-like receptor (TLR genes in Vechur cattle, an indigenous breed of Kerala with the sequence of Bos taurus and access the differences that could be attributed to innate immune responses against bovine mastitis. Materials and Methods: Blood samples were collected from Jugular vein of Vechur cattle, maintained at Vechur cattle conservation center of Kerala Veterinary and Animal Sciences University, using an acid-citrate-dextrose anticoagulant. The genomic DNA was extracted, and polymerase chain reaction was carried out to amplify the promoter region of TLRs. The amplified product of TLR2, 4, and 9 promoter regions was sequenced by Sanger enzymatic DNA sequencing technique. Results: The sequence of promoter region of TLR2 of Vechur cattle with the B. taurus sequence present in GenBank showed 98% similarity and revealed variants for four sequence motifs. The sequence of the promoter region of TLR4 of Vechur cattle revealed 99% similarity with that of B. taurus sequence but not reveals significant variant in motifregions. However, two heterozygous loci were observed from the chromatogram. Promoter sequence of TLR9 gene also showed 99% similarity to B. taurus sequence and revealed variants for four sequence motifs. Conclusion: The results of this study indicate that significant variation in the promoter of TLR2 and 9 genes in Vechur cattle breed and may potentially link the influence the innate immunity response against mastitis diseases.

  13. Enhancer activity of Helitron in sericin-1 gene promoter from Bombyx mori. (United States)

    Huang, Ke; Li, Chun-Feng; Wu, Jie; Wei, Jun-Hong; Zou, Yong; Han, Min-Jin; Zhou, Ze-Yang


    Sericin is a kind of water-soluble protein expressed specifically in the middle silk gland of Bombyx mori. When the sericin-1 gene promoter was cloned and a transgenic vector was constructed to express a foreign protein, a specific Helitron, Bmhel-8, was identified in the sericin-1 gene promoter sequence in some genotypes of Bombyx mori and Bombyx mandarina. Given that the Bmhel-8 Helitron transposon was present only in some genotypes, it could be the source of allelic variation in the sericin-1 promoter. The length of the sericin-1 promoter sequence is approximately 1063 or 643 bp. The larger size of the sequence or allele is ascribed to the presence of Bmhel-8. Silkworm genotypes can be homozygous for either the shorter or larger promoter sequence or heterozygous, containing both alleles. Bmhel-8 in the sericin-1 promoter exhibits enhancer activity, as demonstrated by a dual-luciferase reporter system in BmE cell lines. Furthermore, Bmhel-8 displays enhancer activity in a sericin-1 promoter-driven gene expression system but does not regulate the tissue-specific expression of sericin-1.

  14. Cloning and characterizing of the murine IRF-3 gene promoter region. (United States)

    Xu, Hua-Guo; Liu, Lifei; Gao, Shan; Jin, Rui; Ren, Wei; Zhou, Guo-Ping


    The interferon regulatory factor 3 (IRF-3) plays essential roles in inflammation and immune response. Here, we cloned the nucleotide sequence of the 5'-flanking region of the murine IRF-3 gene (mIRF-3) and characterized the molecular mechanisms controlling the mIRF-3 transcriptional activity in NIH3T3 cells. Analyses of a series of 5' deletion constructs demonstrated that a 301 bp region (-255/+46) of the mIRF-3 gene is sufficient for full promoter activity. This region contains IK1, Egr2, Cmyb, E2F1 and YY1 putative transcription factor binding sites. Mutation of Egr2 or YY1 site led to 52-68 % decrease of the mIRF-3 promoter activity, and double Egr2 and YY1 mutation reduced the promoter activity to 20 % of the wild-type promoter activity. Furthermore, knockingdown of endogenous Egr2 or YY1 by a siRNA strategy markedly inhibited the mIRF-3 promoter activity. Chromatin immunoprecipitation assays showed that Egr2 and YY1 interact with the mIRF-3 promoter in vivo. These results suggested that the basal promoter activity of the mIRF-3 gene is regulated by transcription factors Egr2 and YY1 in NIH3T3 cells.

  15. Fruit preferential activity of the tomato RIP1 gene promoter in transgenic tomato and Arabidopsis. (United States)

    Agarwal, Priyanka; Kumar, Rahul; Pareek, Amit; Sharma, Arun K


    Isolation and functional characterization of tissue- and stage-specific gene promoters is beneficial for genetic improvement of economically important crops. Here, we have characterized a putative promoter of a ripening-induced gene RIP1 (Ripening induced protein 1) in tomato. Quantification of the transcript level of RIP1 showed that its expression is fruit preferential, with maximum accumulation in red ripe fruits. To test the promoter activity, we made a reporter construct by cloning 1450 bp putative RIP1 promoter driving the GUS (ß-glucuronidase) gene expression and generated stable transgenic lines in tomato and Arabidopsis. Histochemical and fluorometric assays validated the fruit-specific expression of RIP1 as the highest GUS activity was found in red ripe tomatoes. Similarly, we detected high levels of GUS activity in the siliques of Arabidopsis. On the contrary, weak GUS activity was found in the flower buds in both tomato and Arabidopsis. To characterize the specific regions of the RIP1 promoter that might be essential for its maximum activity and specificity in fruits, we made stable transgenic lines of tomato and Arabidopsis with 5'-deletion constructs. Characterization of these transgenic plants showed that the full length promoter is essential for its function. Overall, we report the identification and characterization of a ripening-induced promoter of tomato, which would be useful for the controlled manipulation of the ripening-related agronomic traits in genetic manipulation studies in future.

  16. Culex tarsalis vitellogenin gene promoters investigated in silico and in vivo using transgenic Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Song Chen

    Full Text Available INTRODUCTION: Genetic modification, or transgenesis, is a powerful technique to investigate the molecular interactions between vector-borne pathogens and their arthropod hosts, as well as a potential novel approach for vector-borne disease control. Transgenesis requires the use of specific regulatory regions, or promoters, to drive expression of genes of interest in desired target tissues. In mosquitoes, the vast majority of described promoters are from Anopheles and Aedes mosquitoes. RESULTS: Culex tarsalis is one of the most important vectors of arboviruses (including West Nile virus in North America, yet it has not been the subject of molecular genetic study. In order to facilitate molecular genetic work in this important vector species, we isolated four fat body-specific promoter sequences located upstream of the Cx. tarsalis vitellogenin genes (Vg1a, Vg1b, Vg2a and Vg2b. Sequences were analyzed in silico to identify requisite cis-acting elements. The ability for promoter sequences to drive expression of green fluorescent protein (GFP in vivo was investigated using transgenic Drosophila melanogaster. All four promoters were able to drive GFP expression but there was dramatic variation between promoters and between individual Drosophila lines, indicating significant position effects. The highest expression was observed in line Vg2bL3, which was >300-fold higher than the lowest line Vg1aL2. CONCLUSIONS: These new promoters will be useful for driving expression of genes of interest in transgenic Cx. tarsalis and perhaps other insects.

  17. Mutations in the promoter region of the aldolase B gene that cause hereditary fructose intolerance. (United States)

    Coffee, Erin M; Tolan, Dean R


    Hereditary fructose intolerance (HFI) is a potentially fatal inherited metabolic disease caused by a deficiency of aldolase B activity in the liver and kidney. Over 40 disease-causing mutations are known in the protein-coding region of ALDOB. Mutations upstream of the protein-coding portion of ALDOB are reported here for the first time. DNA sequence analysis of 61 HFI patients revealed single base mutations in the promoter, intronic enhancer, and the first exon, which is entirely untranslated. One mutation, g.-132G>A, is located within the promoter at an evolutionarily conserved nucleotide within a transcription factor-binding site. A second mutation, IVS1+1G>C, is at the donor splice site of the first exon. In vitro electrophoretic mobility shift assays show a decrease in nuclear extract-protein binding at the g.-132G>A mutant site. The promoter mutation results in decreased transcription using luciferase reporter plasmids. Analysis of cDNA from cells transfected with plasmids harboring the IVS1+1G>C mutation results in aberrant splicing leading to complete retention of the first intron (~5 kb). The IVS1+1G>C splicing mutation results in loss of luciferase activity from a reporter plasmid. These novel mutations in ALDOB represent 2% of alleles in American HFI patients, with IVS1+1G>C representing a significantly higher allele frequency (6%) among HFI patients of Hispanic and African-American ethnicity.

  18. Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species (United States)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR


    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  19. Promoter sequence of 3-phosphoglycerate kinase gene 2 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR


    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  20. Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster. (United States)

    Magoulas, C; Loverre-Chyurlia, A; Abukashawa, S; Bally-Cuif, L; Hickey, D A


    Previous studies have demonstrated that the expression of the alpha-amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the alpha-amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.

  1. The expression of foreign gene under the control of cauliflower mosaic virus 35s RNA promoter

    Institute of Scientific and Technical Information of China (English)

    WangHao; BaiYongyan


    The promoter region of cauliflower mosaic virus (CaMV) 35s RNA was employed to construct an intermediate expression vector which can be used in Ti plasmid system of Agrobacterium iumefaciens.The original plasmid,which contains a polylinker between CaMV 35s RNA and its 3' termination signal in pUC18 was modified to have another antibiotic resistance marker (kanamycin resistance gene Kmr) to facilitate the selection of recombinant with Ti plasmid.Octopine synthase (ocs) structural gene was inserted into this vector downstream of CaMV 35s RNA promoter.This chimaeric gene was introduced into integrative Ti plasmid vector pGV 3850,and then transformed into Nicotiana tobaccum the chimaeric gene into tobacco cells.In both cases,the expression of ocs gene was demonstrated.The amount of octopine was much more than the nopaline synthesized by nopaline synthase (nos) gene transferred at the same time with Ti plasmid vector.This demonstrated that CaMV 35s RNA promoter is stronger in transcriptional function than the promoter of nos in tobacco cells.

  2. Cloning and Analysis of the Promoter Region of Rat uPA Gene

    Institute of Scientific and Technical Information of China (English)

    Yan LIU; Jin-wen XIONG; Li-gang CHEN; Yong-hong TIAN; Cheng-liang XIONG


    Objective To clone and analyze the promoter sequence of rat urokinase plasminogen activator protein gene.Methods The genomic DNA was extracted from rat testicular tissue. According to urokinase plasminogen activator, the gene sense primer and antisense primer of uPA gene were designed and synthesized, then Touch-Down PCR were performed. After proper purification, the PCR product was sequenced, analyzed with the promoter prediction software and compared with the DNA sequence of rattuas urokinase plasminogen activator.Results The cloned uPA gene was about 1 572 bp in length, which contained a full open-reading frame with 21 bp in length exons, and the upper region of transcriptional start was 1 551 bp in length which was eucaryon transcriptional control area.The 5' UTR had a promoter region including a non-responsive TATA-box. Not only the GC-box binding region was found in this gene, but also active protein 1 (AP1) and SP1 were seen in other regions.Conclusion A 1 572 bp uPA gene fragment (GenBank accession No. X65651) was obtained from rat genomic DNA library, containing eucaryon transcriptional control area with a promoter region, non-conspicuous TATA-box, GC-box and an extron. A non-responsive TATA-box is located at the upper -30 region.

  3. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters. (United States)

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F


    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

  4. Gene controlled by promoter--PTH4 depending on whiG of Streptomyces coelicolor

    Institute of Scientific and Technical Information of China (English)

    谭华荣; 杨海花; 田宇清; 吴畏; 董可宁; K.F.Chater


    The downstream gene controlled by promoter--PTH4 which is related to Streptomycesdifferentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.

  5. Probing the effect of promoters on noise in gene expression using thousands of designed sequences. (United States)

    Sharon, Eilon; van Dijk, David; Kalma, Yael; Keren, Leeat; Manor, Ohad; Yakhini, Zohar; Segal, Eran


    Genetically identical cells exhibit large variability (noise) in gene expression, with important consequences for cellular function. Although the amount of noise decreases with and is thus partly determined by the mean expression level, the extent to which different promoter sequences can deviate away from this trend is not fully known. Here, we present a high-throughput method for measuring promoter-driven noise for thousands of designed synthetic promoters in parallel. We use it to investigate how promoters encode different noise levels and find that the noise levels of promoters with similar mean expression levels can vary more than one order of magnitude, with nucleosome-disfavoring sequences resulting in lower noise and more transcription factor binding sites resulting in higher noise. We propose a kinetic model of gene expression that takes into account the nonspecific DNA binding and one-dimensional sliding along the DNA, which occurs when transcription factors search for their target sites. We show that this assumption can improve the prediction of the mean-independent component of expression noise for our designed promoter sequences, suggesting that a transcription factor target search may affect gene expression noise. Consistent with our findings in designed promoters, we find that binding-site multiplicity in native promoters is associated with higher expression noise. Overall, our results demonstrate that small changes in promoter DNA sequence can tune noise levels in a manner that is predictable and partly decoupled from effects on the mean expression levels. These insights may assist in designing promoters with desired noise levels.

  6. Functional analysis of the human somatic angiotensin I-converting enzyme gene promoter. (United States)

    Testut, P; Soubrier, F; Corvol, P; Hubert, C


    Angiotensin I-converting enzyme (ACE) is a key enzyme in the regulation of systemic blood pressure and plays a major role in the renin-angiotensin and bradykinin-kinin systems, at the luminal surface of the vascular endothelia. To identify the promoter region, the transcription regulatory elements and the cell specificity of the ACE gene, five successive DNA deletions of the 5' upstream region (-1214, -754, -472, -343, -132 bp relative to the start site of transcription) were isolated and fused in sense and antisense orientations to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene in the promoterless plasmid pBLCAT3. Promoter activities were measured in transient transfection assays using three different cell lines from rabbit endothelium (RE), human embryocarcinoma (Tera-1) and hepatocarcinoma cells (HepG2). All five fragments of the ACE promoter region directed expression of the CAT gene when transfected into the endothelial and the embryocarcinoma cells, which contain endogenous ACE mRNA and express ACE activity. In contrast only minimal levels of promoter activity were obtained on transfection into hepatocarcinoma cells in which endogenous ACE mRNA and ACE activity were not detected. Transfection of RE and Tera-1 cells demonstrated that promoter activity was defined by the length of the ACE promoter sequence inserted into the construct. The 132 bases located upstream from the transcription start site were sufficient to confer ACE promoter activity, whereas the sequences upstream from -472 bp and between -343 bp and -132 bp were responsible for a decrease of promoter activity. Furthermore, the minimal 132 bp of the ACE promoter contains elements which direct cell-specific CAT expression. In addition, the DNA transfection study in the presence of dexamethasone suggested that the potential glucocorticoid regulatory elements, located in the sequence of the ACE promoter, are not functional.

  7. An investigation of MYC gene aberration in diffuse large B-cell lymphomas%弥漫性大B细胞淋巴瘤C-MYC基因异常分析

    Institute of Scientific and Technical Information of China (English)

    梁艳; 潘毅; 房爱菊; 管冰心; 霍颖颖; 王妍; 孙保存; 付凯; 孟斌


    Objective:This study aims to investigate MYC gene aberration and analyze the correlation of gene aberrations among MYC, BCL-2, and BCL-6 in diffuse large B-cell lymphomas (DLBCL). Methods:Aberrations of MYC, BCL-2, and BCL-6 genes were detected using interphase fluorescence in situ hybridization (FISH), and the protein markers (CD10, BCL-6, MUM1, and Ki67) were stained using immunohistochemistry in the tissue microarrays of 194 DLBCL cases. The correlations among them were analyzed using statistical methods. Results:In 164 of the 194 cases that obtained FISH results of MYC, 38 cases revealed MYC gene aberration (38/164;23.17%). Of the 38 cases, 9 (9/164;5.49%) were MYC translocation, and the other 29 (29/164;17.68%) were MYC gene amplification. No significant difference was observed in the distribution of the aberrations between the cases with germinal central B-cell (GCB) (5/49;10.20%) and the non-GCB (24/115; 20.87%) subtypes (P=0.187). Of the 159 cases with complete FISH test data, coexistent MYC and BCL-6 gene rear-rangements were found in only two"double hit"cases. Aberrations of MYC, BCL-2, and BCL-6 genes or a coexistent rearrangement of the three was not found in the cases. A significantly positive correlation was observed between MYC (28/159, 17.61%) and BCL-2 gene amplification (38/159, 23.90%) (r=0.2916, P=0.000 4). The expression rate of Ki67 (≥90%) was apparently higher in the cases with MYC translocation (5/8, 62.50%) than those without (33/149, 22.15%) (P=0.027 7). High Ki67 expression was found in both"double hit"cases. No significant correlation was found between MYC gene amplification and high Ki67 expression. Conclusion: In addition to gene translocation, gene amplification and other activation pathways of the MYC gene were found in DLBCL. Further studies are needed to elucidate the role of MYC gene aberration in DLBCL.%  目的:探讨弥漫性大B细胞淋巴瘤(DLBCL)MYC基因异常情况及其与BCL-2、BCL-6基因异常的关系

  8. Genomic organisation of the channel catfish Mx1 gene and characterisation of multiple channel catfish Mx gene promoters. (United States)

    Plant, Karen P; Thune, Ronald L


    In order to further characterise channel catfish (Ictalurus punctatus) Mx1, studies were initiated to amplify and clone the Mx1 promoter into a reporter vector, pGL3basic. Initially the Mx1 gene was amplified from genomic DNA and was found to have 12 exons and 11 introns, spanning a region over 6 kilobases (kb) in length. The Mx1 promoter was amplified using genome walking and during this process four additional Mx promoters were identified, suggesting the presence of five Mx genes in the channel catfish. All five promoters possess an interferon stimulated response element (ISRE) and the Mx1 promoter possessed two potential NF-kappabeta transcription sites. Following cloning each construct was transiently transfected into COS-7 and EPC cells for 24h and treated with 5 microg/ml poly I:C for 24h. An increase in expression of the reporter gene in response to poly I:C was noted in both cell lines in the pGL3Mx1 construct only. However, the reporter gene was also constitutively expressed in these cells. Constitutive expression was also observed in channel catfish ovary cells transiently transfected with pGL3Mx1 only. Treatment with 5 microg/ml poly I:C did not increase this expression, which may be due to high levels of cell death in this difficult to transfect cell line. The constitutive expression observed implies that a repressor element is missing in the 390 base pair sequence of the Mx1 promoter used in this study. These results suggest that only channel catfish Mx1 is involved in the type I interferon pathway and that the presence of an ISRE in a regulatory region is not necessarily indicative of a role in the type I interferon response.

  9. Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos. (United States)

    Edman, R M; Linger, R J; Belikoff, E J; Li, F; Sze, S-H; Tarone, A M; Scott, M J


    The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. hominivorax from North and Central America. This involved area-wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making 'male-only' strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro-apoptotic gene by the tetracycline-dependent transactivator. Sex-specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro-apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro-apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro-apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests.

  10. Regulation of a Mammalian Gene Bearing a CpG Island Promoter and a Distal Enhancer

    Directory of Open Access Journals (Sweden)

    Georgina Berrozpe


    Full Text Available A quantitative nucleosome occupancy assay revealed rules for nucleosome disposition in yeast and showed how disposition affects regulation of the GAL genes. Here, we show how those findings apply to the control of Kit, a mammalian gene. The Kit promoter lies in a CpG island, and its enhancer (active in mast cells lies some 150 kb upstream. Nucleosomes form with especially high avidities at the Kit promoter, a reaction that, we surmise, ensures extremely low basal expression. In mast cells, transcriptional activators displace nucleosomes that are less tightly formed at the Kit enhancer. In turn, the active enhancer replaces a single Kit promoter nucleosome with the transcriptional machinery, thereby inducing transcription over 1,000-fold. As at the yeast GAL genes, the inhibitory effects of nucleosomes facilitate high factors of induction by mammalian activators working in the absence of specific repressors.

  11. GAL promoter-driven heterologous gene expression in Saccharomyces cerevisiae Δ strain at anaerobic alcoholic fermentation. (United States)

    Ahn, Jungoh; Park, Kyung-Min; Lee, Hongweon; Son, Yeo-Jin; Choi, Eui-Sung


    The removal of Gal80 protein by gene disruption turned into efficient GAL promoter-driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae. Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter (P(GAL10) ) in Δgal80 mutant that does not require galactose as an inducer was compared to those of ADH1, PDC1, and PGK promoters, which have been known to work well anaerobically in actively fermenting yeast cells under high glucose concentration. P(GAL10) in the Δgal80 mutant showed 0.8-fold (ADH1), fourfold (PDC1), and 50-fold (PGK) in promoter strength.

  12. The promoter of the glucoamylase-encoding gene of Aspergillus niger functions in Ustilago maydis

    Energy Technology Data Exchange (ETDEWEB)

    Smith, T.L. (Dept. of Agriculture, Madison, WI (United States) Univ. of Wisconsin, Madison (United States)); Gaskell, J.; Cullen, D. (Dept. of Agriculture, Madison, WI (United States)); Berka, R.M.; Yang, M.; Henner, D.J. (Genentech Inc., San Francisco, CA (United States))


    Promoter sequences from the Aspergillus niger glucoamylase-encoding gene (glaA) were linked to the bacterial hygromycin (Hy) phosphotransferase-encoding gene (hph) and this chimeric marker was used to select Hy-resistant (Hy[sup R]) Ustilago maydis transformants. This is an example of an Ascomycete promoter functioning in a Basidiomycete. Hy[sup R] transformants varied with respect to copy number of integrated vector, mitotic stability, and tolerance to Hy. Only 216 bp of glaA promoter sequence is required for expression in U. maydis but this promoter is not induced by starch as it is in Aspergillus spp. The transcription start points are the same in U. maydis and A. niger.

  13. Diverse flavonoids stimulate NodD1 binding to nod gene promoters in Sinorhizobium meliloti. (United States)

    Peck, Melicent C; Fisher, Robert F; Long, Sharon R


    NodD1 is a member of the NodD family of LysR-type transcriptional regulators that mediates the expression of nodulation (nod) genes in the soil bacterium Sinorhizobium meliloti. Each species of rhizobia establishes a symbiosis with a limited set of leguminous plants. This host specificity results in part from a NodD-dependent upregulation of nod genes in response to a cocktail of flavonoids in the host plant's root exudates. To demonstrate that NodD is a key determinant of host specificity, we expressed nodD genes from different species of rhizobia in a strain of S. meliloti lacking endogenous NodD activity. We observed that nod gene expression was initiated in response to distinct sets of flavonoid inducers depending on the source of NodD. To better understand the effects of flavonoids on NodD, we assayed the DNA binding activity of S. meliloti NodD1 treated with the flavonoid inducer luteolin. In the presence of luteolin, NodD1 exhibited increased binding to nod gene promoters compared to binding in the absence of luteolin. Surprisingly, although they do not stimulate nod gene expression in S. meliloti, the flavonoids naringenin, eriodictyol, and daidzein also stimulated an increase in the DNA binding affinity of NodD1 to nod gene promoters. In vivo competition assays demonstrate that noninducing flavonoids act as competitive inhibitors of luteolin, suggesting that both inducing and noninducing flavonoids are able to directly bind to NodD1 and mediate conformational changes at nod gene promoters but that only luteolin is capable of promoting the downstream changes necessary for nod gene induction.

  14. Structural analysis and promoter characterization of the human collagenase-3 gene (MMP13)

    Energy Technology Data Exchange (ETDEWEB)

    Pendas, A.M.; Balbin, M.; Llano, E. [Universidad de Oviedo (Spain)] [and others


    Human collagenase-3 (MMP13) is a recently identified member of the matrix metalloproteinase (MMP) family that is expressed in breast carcinomas and in articular cartilage from arthritic patients. In this work we have isolated and characterized genomic clones coding for human collagenase-3. This gene is composed of 10 exons and 9 introns and spans over 12.5 kb. The overall organization of the collagenase-3 gene is similar to that of other MMP genes clustered at chromosome 11q22, including fibroblast collagenase (MMP-1), matrilysin (MMP-7), and macrophage metalloelastase (MMP-12), but is more distantly related to genes coding for stromelysin-3 (MMP-11), gelatinase-A (MMP-2), and gelatinase-B (MMP-9), which map outside of this gene cluster. Nucleotide sequence analysis of about 1 kb of the 5{prime}-flanking region of the collagenase-3 gene revealed the presence of a TATA box, an AP-1 motif, a PEA-3 consensus sequence, an osteoblast specific element (OSE-2), and a TGF-{beta} inhibitory element. Transient transfection experiments in HeLa and COS-1 cells with chloramphenicol acetyltransferase (CAT)-containing constructs showed that the AP-1 site is functional and responsible for the observed inducibility of the reporter gene by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). However, and in contrast to other MMP genes, no significative synergistic effect on CAT activity between the AP-1 and PEA-3 elements found in the collagenase-3 gene promoter was found. DNA binding analysis with nuclear extracts from HeLa cells revealed the formation of specific complexes between collagenase-3 promoter sequences containing the AP-1 site and nuclear proteins. The presence of this AP-1 functional site, which is able to confer responsiveness to a variety of tumor promoters and oncogene products, may contribute to explaining the high-level expression of collagenase-3 in breast carcinomas and degenerative joint diseases. 48 refs., 5 figs., 2 tabs.

  15. Identification of MGMT promoter methylation sites correlating with gene expression and IDH1 mutation in gliomas. (United States)

    Zhang, Jie; Yang, Jian-Hui; Quan, Jia; Kang, Xing; Wang, Hui-Juan; Dai, Peng-Gao


    O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation was reported to be an independent prognostic and predictive factor in glioma patients who received temozolomide treatment. However, the predictive value of MGMT methylation was recently questioned by several large clinical studies. The purpose of this study is to identify MGMT gene promoter CpG sites or region whose methylation were closely correlated with its gene expression to elucidate this contradictory clinical observations. The methylation status for all CpG dinucleotides in MGMT promoter and first exon region were determined in 42 Chinese glioma patients, which were then correlated with MGMT gene expression, IDH1 mutation, and tumor grade. In whole 87 CpG dinucleotides analyzed, three distinct CpG regions covering 28 CpG dinucleotides were significantly correlated with MGMT gene expression; 10 CpG dinucleotides were significantly correlated with glioma classification (p MGMT gene hypermethylation significantly co-existed, but not for MGMT gene expression. The validation cohort of gliomas treated with standard of care and comparison of the CpGs we identified with the current CpGs used in clinical setting will be very important for gliomas individual medicine in the future.

  16. Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene. (United States)

    Xia, Mengna; Malkaram, Sridhar A; Zempleni, Janos


    Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2 and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon and kidney cell lines; activities consistently followed the pattern P1>P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene.

  17. Characterization of the Promoter of a Homolog of Maize MADS-Box Gene m18

    Institute of Scientific and Technical Information of China (English)

    QIN Hui-juan; PAN Hong; FAN Xian-wei; WU Qiao; LI You-zhi


    Maize (Zea mays L.) is one of the world’s major food crops, and often suffers from tremendous yield loss caused by abiotic stresses. The MADS-box genes are known to play versatile roles in plants, controlling plant responses to multiple abiotic stresses. However, understanding of regulation of their expressions by the conventional loss-of-function approach is very dififcult. So far, regulation of MADS-box gene expression is little known. The best approach to retrieve expression regulation of this category of genes is to characterize expression of their promoters. In this study, the promoter of a homolog (GenBank accession no. EC864166) of maize MADS-box gene m18 was cloned by way of genome-walking PCR, named Pro66. Predicative analysis indicated that Pro66 contains more than one TATA box and multiple cis-acting environmental conditions-responsive elements (ECREs). Pro66 could drive expression of theβ-glucuronidase (GUS)-encoding gene in maize, and heterologous expression of GUS in red pepper stressed by water deifcit, salt, copper, iron deifciency, heat, cold, and grown under short and long photoperiods, echoing predicative ECREs. Conclusively, maize MADS-box gene m18 likely plays versatile functions in maize response to multiple abiotic stresses due to the promoter with multiple cis-acting elements. The complex arrangement of multiple cis-acting elements in the promoter features meticulously regulated expression of m18. The results give informative clues for heterologous utilisation of the promoters in monocot and dicot species. The copy of the ECREs and heterologous expression of the promoter in dicot species are also discussed.

  18. Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination. (United States)

    Reynolds, David L; Hofmeister, Brigitte T; Cliffe, Laura; Siegel, T Nicolai; Anderson, Britta A; Beverley, Stephen M; Schmitz, Robert J; Sabatini, Robert


    The genomes of kinetoplastids are organized into polycistronic gene clusters that are flanked by the modified DNA base J. Previous work has established a role of base J in promoting RNA polymerase II termination in Leishmania spp. where the loss of J leads to termination defects and transcription into adjacent gene clusters. It remains unclear whether these termination defects affect gene expression and whether read through transcription is detrimental to cell growth, thus explaining the essential nature of J. We now demonstrate that reduction of base J at specific sites within polycistronic gene clusters in L. major leads to read through transcription and increased expression of downstream genes in the cluster. Interestingly, subsequent transcription into the opposing polycistronic gene cluster does not lead to downregulation of sense mRNAs. These findings indicate a conserved role for J regulating transcription termination and expression of genes within polycistronic gene clusters in trypanosomatids. In contrast to the expectations often attributed to opposing transcription, the essential nature of J in Leishmania spp. is related to its role in gene repression rather than preventing transcriptional interference resulting from read through and dual strand transcription.

  19. Methylation status of the interferon-gamma gene promoter in chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)


    Objective To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-γ) gene promoter and its effect on IFN-γ expression in chronic hepatitis B. Method The authors recruited 30 patients with HBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequencing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood mononuclear cells (PBMCs). The expres...

  20. A cII-dependent promoter is located within the Q gene of bacteriophage lambda. (United States)

    Hoopes, B C; McClure, W R


    We have found a cII-dependent promoter, PaQ, within the Q gene of bacteriophage lambda. Transcription experiments and abortive initiation assays performed in vitro showed that the promoter strength and the cII affinity of PaQ were comparable to the other cII-dependent lambda promoters, PE and PI. The location and leftward direction of PaQ suggests a possible role in the delay of lambda late-gene expression by cII protein, a phenomenon that has been called cII-dependent inhibition. We have constructed a promoter down mutation, paq-1, by changing a single base pair in the putative cII binding site of the promoter by oligonucleotide site-directed mutagenesis. The paq-1 mutant promoter required about 4-fold higher cII concentrations for maximal activation compared to the wild-type PaQ. We tested the hypothesis that PaQ is responsible in part for the delay of lambda late-gene expression by recombining the paq-1 mutation into a phage showing severe cII-dependent inhibition. We found that the paq-1 mutation relieved the cII-dependent growth defect of this phage. The paq-1 mutation (in combination with lambda cI857) resulted in a clear-plaque phenotype at the permissive temperature of 32 degrees C. The role of the PaQ-initiated antisense transcript in the control of lambda development is discussed.

  1. JAK2V617F/STAT5 signaling pathway promotes cell proliferation through activation of Pituitary Tumor Transforming Gene 1 expression

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Xu-Liang [Department of Hematology, Heping Hospital of Changzhi Medical College, Changzhi 046000 (China); Department of Hematology, Xiangya Hospital of Centre-South University, Changsha 410008 (China); Wei, Wu; Xu, Hong-Liang; Zhang, Mei-Xiang; Qin, Xiao-Qi; Shi, Wen-Zhi; Jiang, Zhi-Ping [Department of Hematology, Heping Hospital of Changzhi Medical College, Changzhi 046000 (China); Chen, Yi-Jian [Department of Hematology, The First Affiliated Hospital, GanNan Medical University, GanZhou 341000 (China); Chen, Fang-Ping, E-mail: [Department of Hematology, Xiangya Hospital of Centre-South University, Changsha 410008 (China)


    Research highlights: {yields} AG490, a member of tyrosine kinase inhibitors, could inhibit the JAK2V617F/STAT5 signaling pathway in HEL cell which harbor JAK2V617F mutation. {yields} Inhibition of the JAK2V617F/STAT5 signaling pathway inhibited the growth of HEL cells. {yields} JAK2V617F mutation promotes cell proliferation through activation of PTTG1 expression. {yields} JAK2V617F/STAT5 signaling pathway regulate PTTG1 expression at transcriptional level. -- Abstract: Gain-of-function mutations of JAK2 play crucial roles in the development of myeloproliferative neoplasms; however, the underlying downstream events of this activated signaling pathway are not fully understood. Our experiment was designed and performed to address one aspect of this issue. Here we report that AG490, a potent JAK2V617F kinase inhibitor, effectively inhibits the proliferation of HEL cells. Interestingly, AG490 also decreases the expression of PTTG1, a possible target gene of the aberrant signaling pathway, in a dose- and time-dependent manner. Furthermore, the promoter activity analyses reveal that the inhibition of the PTTG1 expression is affected at the transcriptional level. Thus, our results suggest that the JAK2V617F/STAT5 signaling pathway promotes cell proliferation through the transcriptional activation of PTTG1.

  2. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells. (United States)

    Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y


    Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality.

  3. Quantitative assessment of the relationship between RASSF1A gene promoter methylation and bladder cancer (PRISMA) (United States)

    Zhan, Leyun; Zhang, Bingyi; Tan, Yaojun; Yang, Chengliang; Huang, Chenhong; Wu, Qiongya; Zhang, Yulin; Chen, Xiaobo; Zhou, Mi; Shu, Aihua


    Abstract Background: Methylation of the Ras-association domain family 1 isoform A (RASSF1A) gene promoter region is thought to participate in the initiation and development of many different cancers. However, in bladder cancer the role of RASSF1A methylation was unclear. To evaluate the relationship between RASSF1A methylation and bladder cancer, a quantitative assessment of an independent meta-analysis was performed. In addition, a DNA methylation microarray database from the cancer genome atlas (TCGA) project was used to validate the results of the meta-analysis. Methods: We searched published articles from computerized databases, and DNA methylation data were extracted from TCGA project. All data were analyzed by R software. Results: The results of the meta-analysis indicated that the frequency of RASSF1A gene methylation in bladder cancer patients is significantly higher than in healthy controls. The hazard ratio (HR) was 2.24 (95% CI = [1.45; 3.48], P = 0.0003) for overall survival (OS), and the RASSF1A gene promoter methylation status was strongly associated with the TNM stage and differentiation grade of the tumor. The similar results were also found by the data from TCGA project. Conclusion: There was a significant relationship between the methylation of the RASSF1A gene promoter and bladder cancer. Therefore, RASSF1A gene promoter methylation will be a potential biomarker for the clinical diagnosis of bladder cancer. PMID:28207521

  4. Association of polymorphisms of interleukin-18 gene promoter region with polycystic ovary syndrome in chinese population

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    Li Mei-zhi


    Full Text Available Abstract Background Recent research shows that polycystic ovary syndrome (PCOS may have an association with low-grade chronic inflammation, and that PCOS may induce an increase in serum interleukin-18 (IL-18 levels. Methods To investigate the polymorphisms of the IL-18 gene promoters with PCOS, two single nucleotide polymorphisms (SNPs in the promoter of the IL-18 gene (at positions -607C/A and -137G/C in 118 Chinese women with PCOS and 79 controls were evaluated using polymerase chain reaction (PCR. Results No significant differences were found in the genotype distribution, allele frequency and haplotype frequency between the PCOS and control groups. Further analysis demonstrated a relationship between IL-18 gene promoter polymorphisms and PCOS insulin resistance (IR. Regarding the -137 allele frequency, G and C allele frequencies were 93.5% and 6.5%, respectively, in the PCOS with IR patients; G and C allele frequencies were 85.4% and 14.6%, respectively, in PCOS patients without IR (chi2 = 3.601, P = 0.048. Conclusions The presence of a polymorphism in the IL-18 gene was found to have no correlation with the occurrence of PCOS. Carriage of the C allele at position -137 in the promoter of the IL-18 gene may play a protective role from the development of PCOS IR.

  5. Rice bZIP protein, REB, interacts with GCN4 motif in promoter of Waxy gene

    Institute of Scientific and Technical Information of China (English)

    程世军; 王宗阳; 洪孟民


    A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5′ upstream region of many seed storage protein genes accounting for these genes expression exclusive in endosperm among various cereals. Many reports demonstrated that the bZIP transcription activators isolated from wheat, barley and maize, etc. regulate the gene expression through binding to the GCN4 motif. In this research, we showed that GCN4 sequence could be recognized by nuclear proteins extracted from immature rice seeds. Furthermore, a rice bZIP protein, REB was isolated by using PCR method and REB fusion protein was expressed in E. coli. The results of gel shift analysis showed that REB could recognize and bind to the GCN4 motif in the Wx gene in addition to binding to the target sequence in the promoter of α-globulin.

  6. The early gene product EUO is a transcriptional repressor that selectively regulates promoters of Chlamydia late genes. (United States)

    Rosario, Christopher J; Tan, Ming


    The obligate intracellular bacterium Chlamydia has an unusual developmental cycle in which there is conversion between two forms that are specialized for either intracellular replication or propagation of the infection to a new host cell. Expression of late chlamydial genes is upregulated during conversion from the replicating to the infectious form, but the mechanism for this temporal regulation is unknown. We found that EUO, which is expressed from an early gene, binds to two sites upstream of the late operon omcAB, but only the downstream site was necessary for transcriptional repression. Using gel shift and in vitro transcription assays we showed that EUO specifically bound and repressed promoters of Chlamydia trachomatis late genes, but not early or mid genes. These findings support a role for EUO as a temporal repressor that negatively regulates late chlamydial genes and prevents their premature expression. The basis of this specificity is the ability of EUO to selectively bind promoter regions of late genes, which would prevent their transcription by RNA polymerase. Thus, we propose that EUO is a master regulator that prevents the terminal differentiation of the replicating form of chlamydiae into the infectious form until sufficient rounds of replication have occurred.

  7. Promoter hypermethylation and loss of CD133 gene expression in colorectal cancers

    Institute of Scientific and Technical Information of China (English)

    You-Kyung; Jeon; Sung-Hee; Kim; Seung-Ho; Choi; Kyung-Hee; Kim; Byong-Chul; Yoo; Ja-Lok; Ku; Jae-Gahb; Park


    AIM: To understand CD133 promoter hypermethyl-ation and expression in 32 colorectal cancer cell lines. METHODS: Nucleic acid was isolated from 32 colorectal cancer cell lines and CD133 expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Promoter methylation status of the CD133 gene was analyzed with a methylation-specific PCR after sodium-bisulfi te modification and by clonal sequencing analysis. The correlation between expression and promoter methy...

  8. Cloning and characterizing of the ovine MX1 gene promoter/enhancer region. (United States)

    Assiri, A M; Ott, T L


    Ovine MX1 (MX1) is expressed in the uterus during the estrous cycle and is strongly up-regulated during early pregnancy in the uterus and peripheral blood leukocytes. In this study we cloned the MX1 gene promoter/enhancer, and tested its response to interferon tau (IFN-tau). To address the role of IFN tau in regulating MX1 expression, serial deletion mutants were prepared along with a clone that contained a full-length promoter including the two proximal ISREs but lacking an intronic ISRE site. Promoter deletions showed the two proximal ISRE sites, but not the intronic ISRE site, were required for maximal response to IFN tau. Interestingly, MX1 promoter deletion mutants revealed the presence of distal positive (-920 to -715) and negative (-715 to -437) regulatory regions. Identifying positive and negative regulatory regions in MX1 promoter will help define the complex regulation of MX1 during early pregnancy in ruminants.

  9. Gene targeting in melanoma therapy: exploiting of surface markers and specific promoters

    Directory of Open Access Journals (Sweden)

    Sverdlov E. D.


    Full Text Available One of the problems of gene therapy of melanoma is effective expression of therapeutic gene in tumor cells and their metastases but not in normal cells. In this review, we will consider a two-step approach to a highly specific gene therapy. At the first step, therapeutic genes are delivered specifically to tumor cells using cell surface markers of melanoma cells as targets. At the second step, a specific expression of the therapeutic genes in tumor cells is ensured. Surface markers of melanoma cells were analyzed as potential targets for therapeutic treatment. Criteria for choosing the most promising targets are proposed. The use of specific melanoma promoters allows to further increase the specificity of treatment via transcriptional control of therapeutic gene expression in melanoma cells.

  10. An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters.

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    Tianqiao Song


    Full Text Available Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters.

  11. RNA polymerase V targets transcriptional silencing components to promoters of protein-coding genes. (United States)

    Zheng, Qi; Rowley, M Jordan; Böhmdorfer, Gudrun; Sandhu, Davinder; Gregory, Brian D; Wierzbicki, Andrzej T


    Transcriptional gene silencing controls transposons and other repetitive elements through RNA-directed DNA methylation (RdDM) and heterochromatin formation. A key component of the Arabidopsis RdDM pathway is ARGONAUTE4 (AGO4), which associates with siRNAs to mediate DNA methylation. Here, we show that AGO4 preferentially targets transposable elements embedded within promoters of protein-coding genes. This pattern of AGO4 binding cannot be simply explained by the sequences of AGO4-bound siRNAs; instead, AGO4 binding to specific gene promoters is also mediated by long non-coding RNAs (lncRNAs) produced by RNA polymerase V. lncRNA-mediated AGO4 binding to gene promoters directs asymmetric DNA methylation to these genomic regions and is involved in regulating the expression of targeted genes. Finally, AGO4 binding overlaps sites of DNA methylation affected by the biotic stress response. Based on these findings, we propose that the targets of AGO4-directed RdDM are regulatory units responsible for controlling gene expression under specific environmental conditions.

  12. Promoter Hypermethylation of DNA Repair Gene MGMT in Laryngeal Squamous Cell Carcinoma

    Institute of Scientific and Technical Information of China (English)


    The relationship between hypermethylation of CpG islands in the promoter regions of O6methylguanine DNA methyltransferase (MGMT)genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR and semi-quantitative RT-PCR were used to study the promoter methylation and mRNA expression of the MGMT gene in laryngeal carcinoma tissues, t issues adjacent to the tumor and normal laryngeal tissues. Hypermethylation of MGMT gene was detected in 16 samples of 46 (34.8 %) laryngeal squamous cell carcinoma samples. However, the MGMT hypermethylation was not detected in all tissues adjacent to the tumors and normal tissues. No significant difference in MGMT gene hypermethylation was found in samples with different histological grades (x2= 3. 130, P=0. 077) or in samples from patients with different TNM status (x2=3. 957, P=0. 138). No expression of MGMT mRNA was detected in all hypermethylated laryngeal carcinoma tissues. The expression of MGMT mRNA was detected in all unmethylated laryngeal carcinoma tissues, tissues adjacent to the tumors and normal tissues. It suggests that MGMT gene promoter hypermethylation is associated with MGMT gene transcription loss in laryngeal carcinoma tissues and possibly plays an important role in carcinogenesis of laryngeal tissues.

  13. An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters. (United States)

    Song, Tianqiao; Ma, Zhenchuan; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong


    Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters.

  14. Core histone genes of Giardia intestinalis: genomic organization, promoter structure, and expression

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    Adam Rodney D


    Full Text Available Abstract Background Giardia intestinalis is a protist found in freshwaters worldwide, and is the most common cause of parasitic diarrhea in humans. The phylogenetic position of this parasite is still much debated. Histones are small, highly conserved proteins that associate tightly with DNA to form chromatin within the nucleus. There are two classes of core histone genes in higher eukaryotes: DNA replication-independent histones and DNA replication-dependent ones. Results We identified two copies each of the core histone H2a, H2b and H3 genes, and three copies of the H4 gene, at separate locations on chromosomes 3, 4 and 5 within the genome of Giardia intestinalis, but no gene encoding a H1 linker histone could be recognized. The copies of each gene share extensive DNA sequence identities throughout their coding and 5' noncoding regions, which suggests these copies have arisen from relatively recent gene duplications or gene conversions. The transcription start sites are at triplet A sequences 1–27 nucleotides upstream of the translation start codon for each gene. We determined that a 50 bp region upstream from the start of the histone H4 coding region is the minimal promoter, and a highly conserved 15 bp sequence called the histone motif (him is essential for its activity. The Giardia core histone genes are constitutively expressed at approximately equivalent levels and their mRNAs are polyadenylated. Competition gel-shift experiments suggest that a factor within the protein complex that binds him may also be a part of the protein complexes that bind other promoter elements described previously in Giardia. Conclusion In contrast to other eukaryotes, the Giardia genome has only a single class of core histone genes that encode replication-independent histones. Our inability to locate a gene encoding the linker histone H1 leads us to speculate that the H1 protein may not be required for the compaction of Giardia's small and gene-rich genome.

  15. Novel strong tissue specific promoter for gene expression in human germ cells

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    Kuzmin Denis


    Full Text Available Abstract Background Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene NDUFV1 containing an endogenous retroviral sequence. Results Among seven established human cell lines and five primary cultures, this modified NDUFV1 upstream sequence (mNUS was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102, where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter. To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1, whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293. In further experiments, we tested mNUS-driven expression of a hyperactive Sleeping Beauty transposase (SB100X. The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter. Conclusions We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream NDUFV1 transcriptional start site plays a crucial role in the activity of this gene promoter in vitro in the majority of tested cell types (10/12, and an important role - in the rest two cell lines.

  16. Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii

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    Muh. Alias L. Rajamuddin


    Full Text Available Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select asuitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Greenfl uorescent protein (GFP gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycusalvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP, caulifl ower mosaic virus (pCaMV-GFP,medaka β-actin (pmBA-GFP and Japanese fl ounder keratin (pJfKer-GFP promoters were introduced by electroporationmethod. Electroporation was performed using a gene pulser (BIORAD with voltage of 300 V, pulse length of 0.5 ms,pulse numbers of 4, and pulse interval of 0.1 s. Promoter activity was determined by analyzing GFP gene expressionlevel using a fl uorescent microscope. The results showed that CMV regulated highest number of fi lament callus(34.10%±1.49 expressing GFP at medium to strong fl uorescence levels. CaMV promoter had relatively similar activitywith CMV, but lower number of fi lament callus expressing GFP (10.48%±0.25. mBA promoter drove GFP expressionat medium level and similar number of fi lament callus (8.85%±2.31 expressing GFP with CaMV, while JfKer promoterhad lowest activity by means in number of fi lament callus expressing GFP (4.79%±0.26 and GFP expression level. PCRanalysis for transgenic confi rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressingfi lament callus in the same size (about 0.6 kb with positive control of plasmid. Thus, CMV and CaMV promoters wasan appropriate promoter and foreign gene could be transferred to fi lament callus by electroporation method. Combiningthis achievement with developing a culture method of fi lament callus to be thallus, stable transgenic breeding in K.alvarezii can be feasible.

  17. Characterization of the promoter region of biosynthetic enzyme genes involved in berberine biosynthesis in Coptis japonica

    Directory of Open Access Journals (Sweden)

    Yasuyuki Yamada


    Full Text Available The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs, a plant-specific WRKY-type transcription factor, CjWRKY1, and a basic helix-loop-helix (bHLH transcription factor, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4’OMT and CYP719A1 were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay (EMSA and by a chromatin immunoprecipitation (ChIP assay. In addition, CjbHLH1 also activated transcription from truncated 4’OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. japonica are discussed.

  18. Potential transcriptional regulatory regions exist upstream of the human ezrin gene promoter in esophageal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Shuying Gao; Yanpeng Dai; Meijun Yin; Jing Ye; Gang Li; Jie Yu


    We previously demonstrated that the region -87/+ 134 of the human ezrin gene (VIL2) exhibited promoter activity in human esophageal carcinoma EC109 cells, and a further upstream region -1324/-890 positively regulated transcription.In this study, to identify the transcriptional regulatory regions upstream of the VIL2 promoter, we cloned VIL2 - 1541/- 706 segment containing the -1324/-890, and investigated its transcriptional regulatory properties via luciferase assays in transiently transfected cells.In EC109 cells, it was found that VIL2 -1541/-706 possessed promoter and enhancer activities.We also localized transcriptional regulatory regions by fusing 5′- or 3′-deletion segments of VIL2 -1541/-706 to a luciferase reporter.We found that there were three positive and one negative transcriptional regulatory regions ithin VIL2 -1541/-706 in EC109 cells.When these regions were separately located upstream of the luciferase gene without promoter, or located upstream of the VIL2 promoter or SV40 promoter directing the luciferase gene, only VIL2 -1297/-1186 exhibited considerable promoter and enhancer activities, which were lower than those of -1541/-706.In addition, transient expression of Sp1 increased ezrin expression and the transcriptional activation of VIL2 -1297/-1186.Other three regions,although exhibiting significantly positive or negative transcriptional regulation in deletion experiments, showed a weaker or absent regulation.These data suggested that more than one region upstream of the VIL2 promoter participated in VIL2 transcription, and the VIL2 -1297/-1186, probably as a key transcriptional regulatory region, regulated VIL2 transcription in company with other potential regulatory regions.

  19. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene. (United States)

    van der Kop, D A; Schuyer, M; Pinas, J E; van der Zaal, B J; Hooykaas, P J


    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the beta-glucuronidase (gusA) reporter gene. Subsequently, seeds were treated with EMS to obtain mutants in which both reporter gene fusions were up-regulated. Northern analysis showed that the mRNA level of a related, endogenous auxin-inducible GST gene of Arabidopsis was increased in some of these mutants as well. Two of the gup (GST up-regulated) mutants were characterized in more detail and roughly mapped. Both had epinastic cotyledons and leaves, a phenotype that turned out to be linked to the gup mutation.

  20. ParaHox genes in pancreatic cell cultures: effects on the insulin promoter regulation

    Directory of Open Access Journals (Sweden)

    Anna Rosanas-Urgell, Jordi Garcia-Fernàndez, Gemma Marfany


    Full Text Available The gene encoding PDX1 (pancreatic duodenum homeobox 1, the main transcription factor regulating the glucose-dependent transactivation of the insulin promoter in pancreatic β-cells, clusters with two closely related homeobox genes (Gsh1 and Cdx2/3, all of them belonging to the ParaHox gene family. The ParaHox gene evolutionary history in the vertebrate lineage involved duplications of the cluster and subsequent loss of some members, so that eventually, the human and murine genomes contain only 6 ParaHox genes. The crucial role of PDX1 in pancreas development, beta-cell formation and insulin transcription regulation has long been established. There is some data on CDX2/3 function in α-cells, but remarkably, nothing is known on the role of the other ParaHox genes, which are also expressed in the endocrine pancreas. Homeobox transcription factors that belong to the same family show high conservation of the homeodomain and share similar target sites and oligomeric partners, and thus may act redundantly, synergistically or antagonistically on the same promoters. Therefore, we explored the effects of the Parahox proteins (GSH1, GSH2, CDX1, CDX2/3 and CDX4 on the regulation of the insulin promoter in transfected α- and β- cultured cell lines at different glucose concentrations and compared them to those of PDX1. Noticeably, several ParaHox transcription factors are able to transactivate or inhibit the insulin promoter, depending on the cell type and glucose concentration, thus suggesting their possible participation in the regulation of similar target genes, such as insulin, either by silencing or activating them, in the absence of PDX1.

  1. Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Piotr Szymczyk


    Full Text Available The promoter, 5' UTR, and 34-nt 5' fragments of protein encoding region of the Salvia miltiorrhiza copalyl diphosphate synthase gene were cloned and characterized. No tandem repeats, miRNA binding sites, or CpNpG islands were observed in the promoter, 5' UTR, or protein encoding fragments. The entire isolated promoter and 5' UTR is 2235 bp long and contains repetitions of many cis-active elements, recognized by homologous transcription factors, found in Arabidopsis thaliana and other plant species. A pyrimidine-rich fragment with only 6 non-pyrimidine bases was localized in the 33-nt stretch from nt 2185 to 2217 in the 5' UTR. The observed cis-active sequences are potential binding sites for trans-factors that could regulate spatio-temporal CPS gene expression in response to biotic and abiotic stress conditions. Obtained results are initially verified by in silico and co-expression studies based on A. thaliana microarray data. The quantitative RT-PCR analysis confirmed that the entire 2269-bp copalyl diphosphate synthase gene fragment has the promoter activity. Quantitative RT-PCR analysis was used to study changes in CPS promoter activity occurring in response to the application of four selected biotic and abiotic regulatory factors; auxin, gibberellin, salicylic acid, and high-salt concentration.

  2. Monoamine Oxidase a Promoter Gene Associated with Problem Behavior in Adults with Intellectual/Developmental Disabilities (United States)

    May, Michael E.; Srour, Ali; Hedges, Lora K.; Lightfoot, David A.; Phillips, John A., III; Blakely, Randy D.; Kennedy, Craig H.


    A functional polymorphism in the promoter of the gene encoding monoamine oxidase A has been associated with problem behavior in various populations. We examined the association of MAOA alleles in adult males with intellectual/developmental disabilities with and without established histories of problem behavior. These data were compared with a…

  3. Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

    DEFF Research Database (Denmark)

    Mortensen, O V; Thomassen, M; Larsen, M B;


    To investigate the structural basis for genetic regulation of the human serotonin transporter gene, a 1.8 kb fragment upstream to the cap site was cloned and sequenced. The promoter possesses a polymorphic repeat region with 16 and 14 repeats, respectively. Both were cloned and characterized. The...

  4. The promoter analysis of the human C17orf25 gene, a novel chromosome 17p13.3 gene

    Institute of Scientific and Technical Information of China (English)


    The human C17orf25 gene (Accession No. AF177342) is one of thirteen genes cloned from a regiondisplaying a high score of loss of heterozygosity within chromosome 17p13.3 in human hepatocellular car-cinoma in China[1]. To unveil the underlying mechanisms for the transcription regulation of this gene andunderstand its implication to the hepatocellular carcinogenesis, we looked into the relevant aspects by bothbioinformatic and experimental executions. We found: 1, The abundant expression of the C17orf25 genewas evident in all the cell lines and tissue samples tested, showing little hepatoma-selectivity; 2, Its tran-scription starts at a single site, locating at -60 from the translation initiation codon; 3, A 58 bp fragmentcontaining the transcription start, extending from -112 to -55, represents the minimal promoter; 4, Theconsensus sequence within this fragment recognized by SP1 contributes predominantly to the activity of theminimal promoter; 5, The bioinformatic analysis suggests that the C17orf25 gene may encode a protein inthe family of the glyoxalase. Our data has provided some deep insight into both function and regulation ofthe C17orf25 gene in the context of the normal liver and hepatocellular carcinoma.

  5. Examination of the phosphoenolpyruvate carboxykinase gene promoter in patients with noninsulin-dependent diabetes mellitus

    Energy Technology Data Exchange (ETDEWEB)

    Ludwig, D.S. [Beth Israel Hospital, Boston, MA (United States)]|[Children`s Hospital, Boston, MA (United States); Vidal-Puig, A.; Moller, D.E. [Beth Israel Hospital, Boston, MA (United States)] [and others


    Expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis, is under dominant negative regulation by insulin. In this study, we sought to test the hypothesis that mutations in the PEPCK gene promoter may impair the ability of insulin to suppress hepatic glucose production, thereby contributing to both the insulin resistance and increased rate of gluconeogenesis characteristic of NIDDM. The proximal PEPCK promoter region in 117 patients with noninsulin-dependent diabetes mellitus and 20 obese Pima Indians was amplified by PCR and analyzed with single strand conformation of polymorphism techniques. In addition, limited direct DNA sequencing was performed on the insulin response sequence and flanking regions. No DNA sequence polymorphisms were found in any patient. This result suggests that mutations in cis-acting PEPCK gene regulatory elements do not constitute a common cause of noninsulin-dependent diabetes mellitus. The significance of genetic variation in promoter regions to human disease is discussed. 40 refs., 1 figs., 1 tab.

  6. Gene trap-based identification of a guard cell promoter in Arabidopsis. (United States)

    Francia, Priscilla; Simoni, Laura; Cominelli, Eleonora; Tonelli, Chiara; Galbiati, Massimo


    Preserving crop yield under drought stress is a major challenge for modern agriculture. To cope with the detrimental effects of water scarcity on crop productivity it is important to develop new plants with a more sustainable use of water and capable of higher performance under stress conditions. Transpiration through stomatal pores accounts for over 90% of water loss in land plants. Recent studies have increased our understanding of the networks that control stomatal activity and have led to practical approaches for enhancing drought tolerance. Genetic engineering of target genes in stomata requires effective expression systems, including suitable promoters, because constitutive promoters (i.e., CaMV35S) are not always functional or can have negative effects on plant growth and productivity. Here we describe the identification of the CYP86A2 guard cell promoter and discuss its potential for gene expression in stomata.

  7. Functional analysis of the larval serum protein gene promoter from silkworm,Bombyx mori.

    Institute of Scientific and Technical Information of China (English)

    TANG Shunming; YI Yongzhu; SHEN Xingjia; ZHANG Zhifang; LI Yiren; HE Jialu


    The regulation region of larval serum protein gene, Bombyx mori. (BmLSP), consisting of the first intron, the first exon, the central promoter region and 5′-upstream region, is cloned from genomic DNA from the silkworm variety of Suju×Minghu. Using PCR and restriction endonuclease methods, a series of luciferase reporter plasmids, driven by different length of BmLSP promoters, are constructed. Via the transient expression system in BmN cells, the effects of the regulation elements and foreign insect hormones on the BmLSP promoter activity are investigated. The results demonstrate that the promoter activity of BmLSP is 5.8- or 4.4-fold higher than that of BmLSPs whose first intron or the element in 5′-upstream region harboring the homologous sequence with the first intron of light-chain fibroin gene (EHIF) is deleted, respectively, suggesting that both the first intron and EHIF contain the main positive cis-acting elements. However, the inactive mariner transposable element (MTE) in 5′-upstream region presents a negative effect. Furthermore, the effects of juvenile hormone analogue (JHA) on the BmLSP promoter activity show a typical dose-dependent manner, that is, low concentration treatments increase the BmLSP promoter activity and high concentration treatments decrease it. Meanwhile, insect ecdysone (MH) treatments present no significant effect.

  8. Isolation and Functional Characterization of a Lycopene β-cyclase Gene Promoter from Citrus. (United States)

    Lu, Suwen; Zhang, Yin; Zheng, Xiongjie; Zhu, Kaijie; Xu, Qiang; Deng, Xiuxin


    Lycopene β-cyclases are key enzymes located at the branch point of the carotenoid biosynthesis pathway. However, the transcriptional regulatory mechanisms of LCYb1 in citrus with abundant carotenoid accumulation are still unclear. To understand the molecular basis of CsLCYb1 expression, we isolated and functionally characterized the 5' upstream sequences of CsLCYb1 from citrus. The full-length CsLCYb1 promoter and a series of its 5' deletions were fused to the β-glucuronidase (GUS) reporter gene and transferred into different plants (tomato, Arabidopsis and citrus callus) to test the promoter activities. The results of all transgenic species showed that the 1584 bp upstream region from the translational start site displayed maximal promoter activity, and the minimal promoter containing 746 bp upstream sequences was sufficient for strong basal promoter activity. Furthermore, the CsLCYb1 promoter activity was developmentally and tissue-specially regulated in transgenic Arabidopsis, and it was affected by multiple hormones and environmental cues in transgenic citrus callus under various treatments. Finer deletion analysis identified an enhancer element existing as a tandem repeat in the promoter region between -574 to -513 bp and conferring strong promoter activity. The copy numbers of the enhancer element differed among various citrus species, leading to the development of a derived simple sequence repeat marker to distinguish different species. In conclusion, this study elucidates the expression characteristics of the LCYb1 promoter from citrus and further identifies a novel enhancer element required for the promoter activity. The characterized promoter fragment would be an ideal candidate for genetic engineering and seeking of upstream trans-acting elements.

  9. Isolation and functional characterization of a lycopene β-cyclase gene promoter from citrus

    Directory of Open Access Journals (Sweden)

    Suwen Lu


    Full Text Available Lycopene β-cyclases are key enzymes located at the branch point of the carotenoid biosynthesis pathway. However, the transcriptional regulatory mechanisms of LCYb1 in citrus with abundant carotenoid accumulation are still unclear. To understand the molecular basis of CsLCYb1 expression, we isolated and functionally characterized the 5’ upstream sequences of CsLCYb1 from citrus. The full-length CsLCYb1 promoter and a series of its 5’ deletions were fused to the β-glucuronidase (GUS reporter gene and transferred into different plants (tomato, Arabidopsis and citrus callus to test the promoter activities. The results of all transgenic species showed that the 1584 bp upstream region from the translational start site displayed maximal promoter activity, and the minimal promoter containing 746 bp upstream sequences was sufficient for strong basal promoter activity. Furthermore, the CsLCYb1 promoter activity was developmentally and tissue-specially regulated in transgenic Arabidopsis, and it was affected by multiple hormones and environmental cues in transgenic citrus callus under various treatments. Finer deletion analysis identified an enhancer element existing as a tandem repeat in the promoter region between -574 to -513 bp and conferring strong promoter activity. The copy numbers of the enhancer element differed among various citrus species, leading to the development of a derived simple sequence repeat (SSR marker to distinguish different species. In conclusion, this study elucidates the expression characteristics of the LCYb1 promoter from citrus and further identifies a novel enhancer element required for the promoter activity. The characterized promoter fragment would be an ideal candidate for genetic engineering and seeking of upstream trans-acting elements.

  10. The expression profile and promoter analysis of ultraspiracle gene in the silkworm Bombyx mori. (United States)

    Huang, Ming-xia; Du, Jie; Su, Bao-jin; Zhao, Guo-dong; Shen, Wei-de; Wei, Zheng-guo


    The nuclear receptor, ultraspiracle protein (USP), is a transcription factor and an essential component of a heterodimeric receptor complex with ecdysone receptor. However, the mechanisms underlying the transcriptional regulation of USP in silkworm are unknown. In this study, using dual-spike-in qPCR method, we examined the expression of Bombyx ultraspiracle gene (BmUSP) in various tissues of silkworm as well as expression changes after stimulation with ecdysone. The results showed that the expression levels of BmUSP gene varied in different tissues and were increased 2 h after exposure to ecdysone. To identify the molecular mechanism underlying the regulation of USP gene expression in silkworm Bombyx mori, promoter truncation analyses were performed using the luciferase reporter assay and Bac-to-Bac expression system in several tissues of B. mori. BmUSP gene promoter with 5' end serial deletions showed different levels of activity in various tissues, higher in fat body and Malpighian tubule. Deletion of the region from -485 to -445 and -307 to -281 upstream of BmUSP gene abolished and increased its promoter activity, respectively. This region contains AP-1, Dfd transcription factor binding sites. These results indicate that BmUSP are expressed at different levels in different tissues of the silkworm, but all are subjected to the regulation by ecdysone. This study would provide an important foundation for investigating the mechanism underlying the transcriptional regulation of BmUSP in the silkworm.

  11. Lentiviral gene therapy using cellular promoters cures type 1 Gaucher disease in mice. (United States)

    Dahl, Maria; Doyle, Alexander; Olsson, Karin; Månsson, Jan-Eric; Marques, André R A; Mirzaian, Mina; Aerts, Johannes M; Ehinger, Mats; Rothe, Michael; Modlich, Ute; Schambach, Axel; Karlsson, Stefan


    Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease.

  12. The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription. (United States)

    Corbi, N; Libri, V; Fanciulli, M; Tinsley, J M; Davies, K E; Passananti, C


    Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

  13. Novel and functional DNA sequence variants within the GATA5 gene promoter in ventricular septal defects

    Institute of Scientific and Technical Information of China (English)

    Ji-Ping Shan; Xiao-Li Wang; Yuan-Gang Qiao; Hong-Xin Wan Yan; Wen-Hui Huang; Shu-Chao Pang; Bo Yan


    Background: Congenital heart disease (CHD) is the most common human birth defect. Genetic causes for CHD remain largely unknown. GATA transcription factor 5 (GATA 5) is an essential regulator for the heart development. Mutations in the GATA5 gene have been reported in patients with a variety of CHD. Since misregulation of gene expression have been associated with human diseases, we speculated that changed levels of cardiac transcription factors, GATA5, may mediate the development of CHD. Methods: In this study, GATA5 gene promoter was genetically and functionally analyzed in large cohorts of patients with ventricular septal defect (VSD) (n=343) and ethnic-matched healthy controls (n=348). Results: Two novel and heterozygous DNA sequence variants (DSVs), g.61051165A>G and g.61051463delC, were identified in three VSD patients, but not in the controls. In cultured cardiomyocytes, GATA5 gene promoter activities were significantly decreased by DSV g.61051165A>G and increased by DSV g.61051463delC. Moreover, fathers of the VSD patients carrying the same DSVs had reduced diastolic function of left ventricles. Three SNPs, g.61051279C>T (rs77067995), g.61051327A>C (rs145936691) and g.61051373G>A (rs80197101), and one novel heterozygous DSV, g.61051227C>T, were found in both VSD patients and controls with similar frequencies. Conclusion: Our data suggested that the DSVs in the GATA5 gene promoter may increase the susceptibility to the development of VSD as a risk factor.

  14. Effect of promoter architecture on the cell-to-cell variability in gene expression.

    Directory of Open Access Journals (Sweden)

    Alvaro Sanchez


    Full Text Available According to recent experimental evidence, promoter architecture, defined by the number, strength and regulatory role of the operators that control transcription, plays a major role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effort that addresses the question of how changes in promoter architecture affect variability in gene expression in a systematic rather than case-by-case fashion. In this article we make such a systematic investigation, based on a microscopic model of gene regulation that incorporates stochastic effects. In particular, we show how operator strength and operator multiplicity affect this variability. We examine different modes of transcription factor binding to complex promoters (cooperative, independent, simultaneous and how each of these affects the level of variability in transcriptional output from cell-to-cell. We propose that direct comparison between in vivo single-cell experiments and theoretical predictions for the moments of the probability distribution of mRNA number per cell can be used to test kinetic models of gene regulation. The emphasis of the discussion is on prokaryotic gene regulation, but our analysis can be extended to eukaryotic cells as well.

  15. Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum. (United States)

    James, S J; Shpyleva, S; Melnyk, S; Pavliv, O; Pogribny, I P


    Epigenetic mechanisms regulate programmed gene expression during prenatal neurogenesis and serve as a mediator between genetics and environment in postnatal life. The recent discovery of 5-hydroxymethylcytosine (5-hmC), with highest concentration in the brain, has added a new dimension to epigenetic regulation of neurogenesis and the development of complex behavior disorders. Here, we take a candidate gene approach to define the role 5-hmC in Engrailed-2 (EN-2) gene expression in the autism cerebellum. The EN-2 homeobox transcription factor, previously implicated in autism, is essential for normal cerebellar patterning and development. We previously reported EN-2 overexpression associated with promoter DNA hypermethylation in the autism cerebellum but because traditional DNA methylation methodology cannot distinguish 5-methylcytosine (5-mC) from 5-hmC, we now extend our investigation by quantifying global and gene-specific 5-mC and 5-hmC. Globally, 5-hmC was significantly increased in the autism cerebellum and accompanied by increases in the expression of de novo methyltransferases DNMT3A and DNMT3B, ten-eleven translocase genes TET1 and TET3, and in 8-oxo-deoxyguanosine (8-oxo-dG) content, a marker of oxidative DNA damage. Within the EN-2 promoter, there was a significant positive correlation between 5-hmC content and EN-2 gene expression. Based on reports of reduced MeCP2 affinity for 5-hmC, MeCP2 binding studies in the EN-2 promoter revealed a significant decrease in repressive MeCP2 binding that may contribute to the aberrant overexpression of EN-2. Because normal cerebellar development depends on perinatal EN-2 downregulation, the sustained postnatal overexpression suggests that a critical window of cerebellar development may have been missed in some individuals with autism with downstream developmental consequences. Epigenetic regulation of the programmed on-off switches in gene expression that occur at birth and during early brain development warrants

  16. Computational promoter analysis of mouse, rat and human antimicrobial peptide-coding genes

    Directory of Open Access Journals (Sweden)

    Kai Chikatoshi


    Full Text Available Abstract Background Mammalian antimicrobial peptides (AMPs are effectors of the innate immune response. A multitude of signals coming from pathways of mammalian pathogen/pattern recognition receptors and other proteins affect the expression of AMP-coding genes (AMPcgs. For many AMPcgs the promoter elements and transcription factors that control their tissue cell-specific expression have yet to be fully identified and characterized. Results Based upon the RIKEN full-length cDNA and public sequence data derived from human, mouse and rat, we identified 178 candidate AMP transcripts derived from 61 genes belonging to 29 AMP families. However, only for 31 mouse genes belonging to 22 AMP families we were able to determine true orthologous relationships with 30 human and 15 rat sequences. We screened the promoter regions of AMPcgs in the three species for motifs by an ab initio motif finding method and analyzed the derived promoter characteristics. Promoter models were developed for alpha-defensins, penk and zap AMP families. The results suggest a core set of transcription factors (TFs that regulate the transcription of AMPcg families in mouse, rat and human. The three most frequent core TFs groups include liver-, nervous system-specific and nuclear hormone receptors (NHRs. Out of 440 motifs analyzed, we found that three represent potentially novel TF-binding motifs enriched in promoters of AMPcgs, while the other four motifs appear to be species-specific. Conclusion Our large-scale computational analysis of promoters of 22 families of AMPcgs across three mammalian species suggests that their key transcriptional regulators are likely to be TFs of the liver-, nervous system-specific and NHR groups. The computationally inferred promoter elements and potential TF binding motifs provide a rich resource for targeted experimental validation of TF binding and signaling studies that aim at the regulation of mouse, rat or human AMPcgs.

  17. Aberrant gene expression profiles, during in vitro osteoblast differentiation, of telomerase deficient mouse bone marrow stromal stem cells (mBMSCs)

    DEFF Research Database (Denmark)

    Saeed, H.; Iqtedar, M.


    Background: Telomerase deficiency has been associated with inadequate differentiation of mesenchymal stem cells. However, the effect of telomerase deficiency on differential regulation of osteoblast specific genes, based on functional gene grouping, during in vitro osteoblast differentiation has ...

  18. Gene aberrations of RRM1 and RRM2B and outcome of advanced breast cancer after treatment with docetaxel with or without gemcitabine

    DEFF Research Database (Denmark)

    Jørgensen, Charlotte Lt; Ejlertsen, Bent; Bjerre, Karsten D


    of the hypothesis that aberrations of RRM1 or RRM2B, neither individually nor in combination, are associated with an altered clinical outcome following chemotherapy with gemcitabine in combination with docetaxel compared to docetaxel alone in advanced breast cancer patients....... agent docetaxel in advanced breast cancer patients. Methods Primary tumor samples from patients randomly assigned to gemcitabine plus docetaxel or docetaxel alone were analyzed for RRM1 and RRM2B copy number changes using Fluorescence In Situ Hybridization (FISH) technology with probes covering...... endpoint. Overall survival (OS) and response rate (RR) were secondary endpoints. Associations between RRM1/CEN-11 and/or RRM2B/CEN-8 ratios and time-to-event endpoints were analyzed by unadjusted and adjusted Cox proportional hazards regression models. Heterogeneity of treatment effects on TTP and OS...

  19. DNA methylation profiling of sorted cells from myelofibrosis patients reveals aberrant epigenetic regulation of immune pathways and identifies early MPN driver genes

    DEFF Research Database (Denmark)

    Nielsen, H. M.; Andersen, C. L.; Kristensen, L. S.;


    Methylation 450K BeadChip. Candidate genes were validated by pyrosequencing in a second cohort of 30 MF patients where DNA was extracted from full blood (PB). To identify potential driver genes, the DNA methylation status of candidate genes was likewise analyzed in PB from a larger cohort consisting of 60 ET...

  20. Effect of folic acid in preventing aberrant methylation of fetal endometriosis susceptibility gene HOXA10%叶酸在预防胎源性子宫内膜异位症易感基因HOXA10异常甲基化中的作用

    Institute of Scientific and Technical Information of China (English)

    刘木彪; 黄雪梅; 许苏容; 李蕾


    目的 通过检测和比较子宫内膜异位症(EMS)孕妇有无补充叶酸其女性胎儿脐血EMS易感基因HOXA10启动区CpG岛甲基化状态,探讨叶酸优化宫内环境的作用.方法 收集标本2010年1月~2012年12月21例患有EMS孕妇服用和15例未服用叶酸的女性胎儿脐血标本,利用甲基化特异性聚合酶链反应(MSP)及亚硫酸氢盐修饰后测序(BSP)检测和比较各组HOXA10的甲基化率.结果 补充叶酸组女性胎儿EMS易感基因HOXA10甲基化率显著降低,两组差异有统计学意义(P<0.05).结论 提示叶酸在预防胎源性EMS易感基因HOXA10异常甲基化中可能起一定作用.%Objective To detect aberrant methylation in the promoter region of fetal endometriosis susceptibility gene homeobox-10 (HOXA10) in women with and without folic acid supplementation and explore the effect of folic acid in optimizing intrauterine environment.Methods Thirty-six cord blood specimens were collected between January,2010 and December,2012 from pregnant women with endometriosis,including 22 with folic acid treatment and 15 without.Methylation-specific polymerase chain reaction (MSP) and bisulfite salt modified sequencing (BSP) were employed to detect aberrant methylation of HOXA10 gene in these specimens.Results The methylation rate of HOXA10 gene differed significantly between pregnant women with endometriosis taking folic acid and those who did (P<0.05).Conclusion Folic acid treatment can significantly reduce the methylation rate of fetal endometriosis susceptibility gene HOXA10.

  1. Expression of the promoter for the maltogenic amylase gene in Bacillus subtilis 168. (United States)

    Kim, Do-Yeon; Cha, Choon-Hwan; Oh, Wan-Seok; Yoon, Young-Jun; Kim, Jung-Wan


    An additional amylase, besides the typical alpha-amylase, was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a maltogenic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the beta-galactosidase activity produced from the bbmA promoter fused to the amino terminus of the lacZ structural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The promoter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing beta-cyclodextrin (beta-CD), maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the beta-CD hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regulatory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the beta-CD hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing beta-CD in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and beta-CD utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.

  2. A database of annotated promoters of genes associated with common respiratory and related diseases

    KAUST Repository

    Chowdhary, Rajesh


    Many genes have been implicated in the pathogenesis of common respiratory and related diseases (RRDs), yet the underlying mechanisms are largely unknown. Differential gene expression patterns in diseased and healthy individuals suggest that RRDs affect or are affected by modified transcription regulation programs. It is thus crucial to characterize implicated genes in terms of transcriptional regulation. For this purpose, we conducted a promoter analysis of genes associated with 11 common RRDs including allergic rhinitis, asthma, bronchiectasis, bronchiolitis, bronchitis, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, eczema, psoriasis, and urticaria, many of which are thought to be genetically related. The objective of the present study was to obtain deeper insight into the transcriptional regulation of these disease-associated genes by annotating their promoter regions with transcription factors (TFs) and TF binding sites (TFBSs). We discovered many TFs that are significantly enriched in the target disease groups including associations that have been documented in the literature. We also identified a number of putative TFs/TFBSs that appear to be novel. The results of our analysis are provided in an online database that is freely accessible to researchers at Promoter-associated TFBS information and related genomic features, such as histone modification sites, microsatellites, CpG islands, and SNPs, are graphically summarized in the database. Users can compare and contrast underlying mechanisms of specific RRDs relative to candidate genes, TFs, gene ontology terms, micro-RNAs, and biological pathways for the conduct of metaanalyses. This database represents a novel, useful resource for RRD researchers. Copyright © 2012 by the American Thoracic Society.

  3. Functional Characterization of a Putative Nitrate Transporter Gene Promoter from Rice

    Institute of Scientific and Technical Information of China (English)

    Ting-Zhang HU; Kai-Ming CAO; Mian XIA; Xi-Ping WANG


    Drought is one of the most significant abiotic stresses that influence plant growth and development. Expression analysis revealed that OsNRT1.3, a putative nitrate transporter gene in rice, was induced by drought. To confirm if the OsNRT1.3 promoter can respond to drought stress, a 2019 bp upstream sequence of OsNRT1.3 was cloned. Three OsNRT1.3 promoter fragments were generated by 5'-deletion, and fused to the β-glucuronidase (GUS) gene. The chimeric genes were introduced into rice plants. NRT2019::GUS, NRT1196: :GUS and NRT719::GUS showed similar expression patterns in seeds,roots, leaves and flowers in all transgenic rice, and GUS activity conferred by different OsNRT1.3 promoter fragments was significantly upregulated by drought stress, indicating that OsNRT1.3 promoter responds to drought stress and the 719 bp upstream sequence of OsNRT1.3 contains the drought response elements.

  4. RNAi-based conditional gene knockdown in mice using a U6 promoter driven vector

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    Vivek Shukla, Xavier Coumoul, Chu-Xia Deng


    Full Text Available RNA interference (RNAi is a powerful tool widely used for studying gene function in a number of species. We have previously developed an approach that allows conditional expression of a polymerase III promoter based small hairpin RNA (shRNA in mice using the Cre-LoxP system. This approach uses a U6 promoter, which is inactive due to the presence of a ploxPneo cassette in the promoter; this promoter can be activated after excision of the neo gene in transgenic mice that express a Cre recombinase transgene. As a proof of principle, we have previously knocked down over 95% of Fgfr2 transcripts in mouse germlines, leading to embryonic lethality, while restricting the knockdown to the progress zone of the limb results in live animals with malformation of digits of both the forelimbs and hindlimbs. We now provide a detailed protocol, including a simplified single-step cloning procedure for vector construction. This method provides a fast yet efficient way to decipher gene functions in vivo in a tissue specific manner.

  5. Cloning and characterization of the human integrin β6 gene promoter.

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    Mingyan Xu

    Full Text Available The integrin β6 (ITGB6 gene, which encodes the limiting subunit of the integrin αvβ6 heterodimer, plays an important role in wound healing and carcinogenesis. The mechanism underlying ITGB6 regulation, including the identification of DNA elements and cognate transcription factors responsible for basic transcription of human ITGB6 gene, remains unknown. This report describes the cloning and characterization of the human ITGB6 promoter. Using 5'-RACE (rapid amplification of cDNA ends analysis, the transcriptional initiation site was identified. Promoter deletion analysis identified and functionally validated a TATA box located in the region -24 to -18 base pairs upstream of the ITGB6 promoter. The regulatory elements for transcription of the ITGB6 gene were predominantly located -289 to -150 from the ITGB6 promoter and contained putative binding sites for transcription factors such as STAT3 and C/EBPα. Using chromatin immunoprecipitation assays, this study has demonstrated, for the first time, that transcription factors STAT3 and C/EBPα are involved in the positive regulation of ITGB6 transcription in oral squamous cell carcinoma cells. These findings have important implications for unraveling the mechanism of abnormal ITGB6 activation in tissue remodeling and tumorigenesis.

  6. Identification of SNPs in the promoter of β-lactoglobulin gene in three Sicilian goat breeds

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    Baldassare Portolano


    Full Text Available The aim of this work was to sequence the full-length promoter region of the caprine β-lactoglobulin (β-lg gene in three Sicilian goat breeds (Girgentana, Maltese, and Derivata di Siria, in order to identify polymorphisms, to search for transcription factors (TFs sites, and to check if polymorphisms found lay within TFs binding sites. The promoter region of β-lg gene in Sicilian goat breeds showed high level of polymorphism due to the presence of 31 SNPs. Binding sites for several TFs were found within the goat β-lg promoter and within regions conserved between ovine and caprine species. Two SNPs were detected within TFs binding sites, such as MPBF and NF-I. Further studies are in progress to confirm polymorphic sites, to evaluate the possible effect of these mutations on binding affinity of TFs, their relationship with β-lg gene expression, and the functional role of SNPs within the TFs sites of the promoter region on milk traits.

  7. Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase. (United States)

    Weir, J P; Moss, B


    Nine recombinant vaccinia viruses that contain overlapping segments of the putative promoter region of the vaccinia virus thymidine kinase (TK) gene linked to DNA coding for the prokaryotic enzyme chloramphenicol acetyltransferase (CAT) were constructed. In each case, the RNA start site and 5 bp of DNA downstream were retained. No significant difference in CAT expression occurred as the deletion was extended from 352 to 32 bp before the RNA start site. Deletion of a further 10 bp, however, led to complete cessation of early promoter activity. Primer extension analysis of the 5' ends of the transcripts verified that the natural TK RNA start site was still used when only 32 bp of upstream DNA remained. Loss of early promoter activity was previously found when deletions were extended from 31 to 24 bp before the RNA start site of another vaccinia gene that is expressed constitutively throughout infection (M.A. Cochran, C. Puckett, and B. Moss, 1985, Proc. Natl. Acad. Sci. USA 82, 19-23). Sequence similarities in the promoter regions of these two genes were noted.

  8. Prolonged treatment with DNMT inhibitors induces distinct effects in promoters and gene-bodies.

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    Yan-Fung Wong

    Full Text Available Treatment with the demethylating drugs 5-azacytidine (AZA and decitabine (DAC is now recognised as an effective therapy for patients with Myelodysplastic Syndromes (MDS, a range of disorders arising in clones of hematopoietic progenitor cells. A variety of cell models have been used to study the effect of these drugs on the methylation of promoter regions of tumour suppressor genes, with recent efforts focusing on the ability of these drugs to inhibit DNA methylation at low doses. However, it is still not clear how nano-molar drug treatment exerts its effects on the methylome. In this study, we have characterised changes in DNA methylation caused by prolonged low-dose treatment in a leukemic cell model (SKM-1, and present a genome-wide analysis of the effects of AZA and DAC. At nano-molar dosages, a one-month continuous treatment halved the total number of hypermethylated probes in leukemic cells and our analysis identified 803 candidate regions with significant demethylation after treatment. Demethylated regions were enriched in promoter sequences whereas gene-body CGIs were more resistant to the demethylation process. CGI methylation in promoters was strongly correlated with gene expression but this correlation was lost after treatment. Our results indicate that CGI demethylation occurs preferentially at promoters, but that it is not generally sufficient to modify expression patterns, and emphasises the roles of other means of maintaining cell state.

  9. Isolation and characterization of "GmScream" promoters that regulate highly expressing soybean (Glycine max Merr.) genes. (United States)

    Zhang, Ning; McHale, Leah K; Finer, John J


    To increase our understanding of the regulatory components that control gene expression, it is important to identify, isolate and characterize new promoters. In this study, a group of highly expressed soybean (Glycine max Merr.) genes, which we have named "GmScream", were first identified from RNA-Seq data. The promoter regions were then identified, cloned and fused with the coding region of the green fluorescent protein (gfp) gene, for introduction and analysis in different tissues using 3 tools for validation. Approximately half of the GmScream promoters identified showed levels of GFP expression comparable to or higher than the Cauliflower Mosaic Virus 35S (35S) promoter. Using transient expression in lima bean cotyledonary tissues, the strongest GmScream promoters gave over 6-fold higher expression than the 35S promoter while several other GmScream promoters showed 2- to 3-fold higher expression. The two highest expressing promoters, GmScreamM4 and GmScreamM8, regulated two different elongation factor 1A genes in soybean. In stably transformed soybean tissues, GFP driven by the GmScreamM4 or GmScreamM8 promoter exhibited constitutive high expression in most tissues with preferentially higher expression in proliferative embryogenic tissues, procambium, vascular tissues, root tips and young embryos. Using deletion analysis of the promoter, two proximal regions of the GmScreamM8 promoter were identified as contributing significantly to high levels of gene expression.

  10. Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits. (United States)

    Ravel, Catherine; Fiquet, Samuel; Boudet, Julie; Dardevet, Mireille; Vincent, Jonathan; Merlino, Marielle; Michard, Robin; Martre, Pierre


    The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression.

  11. Tissue-specific regulation of the mouse Pkhd1 (ARPKD) gene promoter (United States)

    Williams, Scott S.; Cobo-Stark, Patricia; Hajarnis, Sachin; Aboudehen, Karam; Shao, Xinli; Richardson, James A.; Patel, Vishal


    Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-1β, which is required for activity in transfected cells. Mutation of the HNF-1β-binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7 kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1β. We conclude that the proximal 2.0-kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo and that HNF-1β is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located from exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues. PMID:24899057

  12. Study on RIZ1 gene promoter methylation status in human esophageal squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Shang-Wen Dong; Peng Zhang; Yi-Mei Liu; Yuan-Tao Cui; Shuo Wang; Shao-Jie Liang; Zhun He; Pei Sun; Yuan-Guo Wang


    AIM: To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) in the human esophageal squamous cell carcinoma (ESCC) cell lines and tissues and verify the relationship between methylation of RIZ1 and oncogen-esis, tumor progression and metastasis etc of ESCC.METHODS: Methylation-specific polymerase chain reaction (MSP) was used to investigate the promoter region methylation status of RIZ1 in 6 ESCC cell lines. One cell line where RIZ1 promoter region methylation was detected was selected for the next study, where the cell line was treated with 5-aza-CdR. Real-time polymerase chain reaction was used to investigate its influence on the transcription of RIZ1. Experiments using frozen pathological specimens from 47 ESCC patients were performed using the same MSP methodology.RESULTS: Promoter methylation of RIZ1 gene was detected in TE13, CaEs17 and EC109 cell lines and the cell line TE13 was chosen for further study. The expression of RIZ1 mRNA in TE-13 was up-regulated after treatment with 5-aza-CdR. The rate of methylation in carcinomas tissues was significantly higher than those in matched neighboring normal and distal ending normal tissue, and the deviation of data was statistically significant (x2 = 24.136, P < 0.01). Analysis of the gender, age familial history, tumour deviation, tumour saturation, lymph gland displacement and clinical staging of 47 samples from ESCC patients showed that the fluctuation of data was not statistically significant.CONCLUSION: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression in human ESCC. RIZ1 is considered to be a potential tumor suppressor gene and may be a biological parameter for testing early stage human ESCC.

  13. Myeloid translocation gene-16 co-repressor promotes degradation of hypoxia-inducible factor 1.

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    Parveen Kumar

    Full Text Available The myeloid translocation gene 16 (MTG16 co-repressor down regulates expression of multiple glycolytic genes, which are targets of the hypoxia-inducible factor 1 (HIF1 heterodimer transcription factor that is composed of oxygen-regulated labile HIF1α and stable HIF1β subunits. For this reason, we investigated whether MTG16 might regulate HIF1 negatively contributing to inhibition of glycolysis and stimulation of mitochondrial respiration. A doxycycline Tet-On system was used to control levels of MTG16 in B-lymphoblastic Raji cells. Results from co-association studies revealed MTG16 to interact with HIF1α. The co-association required intact N-terminal MTG16 residues including Nervy Homology Region 1 (NHR1. Furthermore, electrophoretic mobility shift assays demonstrated an association of MTG16 with hypoxia response elements (HREs in PFKFB3, PFKFB4 and PDK1 promoters in-vitro. Results from chromatin immunoprecipitation assays revealed co-occupancy of these and other glycolytic gene promoters by HIF1α, HIF1β and MTG16 in agreement with possible involvement of these proteins in regulation of glycolytic target genes. In addition, MTG16 interacted with prolyl hydroxylase D2 and promoted ubiquitination and proteasomal degradation of HIF1α. Our findings broaden the area of MTG co-repressor functions and reveal MTG16 to be part of a protein complex that controls the levels of HIF1α.

  14. Methylation status of the interferon-gamma gene promoter in chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)


    Objective To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-7) gene promoter and its effect on IFN-7 expression in chronic hepatitis B. Method The authors recruited 30 patients with UBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequeneing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood mononuclear cells (PBMCs). The expression of IFN-γ was analyzed by real-time RT-PCR and ELISA. HBV DNA in PBMCs was detected by nested PCR. Results The methylation level at CpG site -55 in the IFN-γ gene promoter was significantly increased, resulting in subsequent down-regulation of the expression of this cytoldne in CHB. The methylation level at CpG site -55 was significantly higher in HBeAg-positive patients than in HBeAg-negative ones (P<0.01) and was also significantly higher in PBMCs from HBV DNA-positive patients than from HBV DNA-negative ones (P<0.01) ; the methylation level at CpG site -55 was positively correlated with the amount of HBV DNA in serum (P<0.01). Oonclusion IFN-γ gene expression appears to be regulated by methylation of the IFN-γ gene promoter in CHB; the methylation level at CpG site -55 is associated with HBV infection.

  15. Chicken ovalbumin upstream promoter transcription factor II regulates renin gene expression. (United States)

    Mayer, Sandra; Roeser, Marc; Lachmann, Peter; Ishii, Sumiyashi; Suh, Jae Mi; Harlander, Sabine; Desch, Michael; Brunssen, Coy; Morawietz, Henning; Tsai, Sophia Y; Tsai, Ming-Jer; Hohenstein, Bernd; Hugo, Christian; Todorov, Vladimir T


    This study aimed to investigate the possible involvement of the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in the regulation of renin gene expression. COUP-TFII colocalized with renin in the juxtaglomerular cells of the kidney, which are the main source of renin in vivo. Protein-DNA binding studies demonstrated that COUP-TFII binds to an imperfect direct repeat COUP-TFII recognition sequence (termed hereafter proxDR) in the proximal renin promoter. Because cAMP signaling plays a central role in the control of the renin gene expression, we suggested that COUP-TFII may modulate this cAMP effect. Accordingly, knockdown of COUP-TFII in the clonal renin-producing cell lines As4.1 and Calu-6 diminished the stimulation of the renin mRNA expression by cAMP agonists. In addition, the mutation of the proxDR element in renin promoter reporter gene constructs abrogated the inducibility by cAMP. The proxDR sequence was found to be necessary for the function of a proximal renin promoter cAMP-response element (CRE). Knockdown of COUP-TFII or cAMP-binding protein (CREB), which is the archetypal transcription factor binding to CRE, decreased the basal renin gene expression. However, the deficiency of COUP-TFII did not further diminish the renin expression when CREB was knocked down. In agreement with the cell culture studies, mutant mice deficient in COUP-TFII have lower renin expression than their control strain. Altogether our data show that COUP-TFII is involved in the control of renin gene expression.

  16. Functional analysis of bifidobacterial promoters in Bifidobacterium longum and Escherichia coli using the α-galactosidase gene as a reporter. (United States)

    Sakanaka, Mikiyasu; Tamai, Saki; Hirayama, Yosuke; Onodera, Ai; Koguchi, Hiroka; Kano, Yasunobu; Yokota, Atsushi; Fukiya, Satoru


    Heterologous gene expression in bifidobacteria requires weak, strong, and inducible promoters depending on the objectives of different expression studies. Weak promoters in Escherichia coli can also be desirable for stable heterologous gene cloning. Here, we developed a reporter system using the Bifidobacterium longum α-galactosidase gene and investigated the activity and inducibility of seven bifidobacterial promoters in B. longum and their activities in E. coli. These studies revealed diverse promoter activities. Three promoters were highly active in B. longum, but only slightly active in E. coli. Among these, two phosphoketolase gene (xfp) promoters exhibited strong activity in B. longum cells grown on glucose. In contrast, the promoter activity of the fructose transporter operon (fruEKFG) was strongly induced by carbohydrates other than glucose, including fructose, xylose, and ribose. These promoters will allow strong or highly inducible expression in bifidobacteria and stable gene cloning in E. coli. In contrast to the functions of these promoters, the promoter of sucrose-utilization operon cscBA showed very high activity in E. coli but low activity in B. longum. Other three promoters were functional in both B. longum and E. coli. In particular, two sucrose phosphorylase gene (scrP) promoters showed inducible activity by sucrose and raffinose in B. longum, indicating their applicability for regulated expression studies. The diverse promoter functions revealed in this study will contribute to enabling the regulated expression of heterologous genes in bifidobacteria research.

  17. Promoters for the human beta-hexosaminidase genes, HEXA and HEXB. (United States)

    Norflus, F; Yamanaka, S; Proia, R L


    Human lysosomal beta-hexosaminidases are encoded by two genes, HEXA and HEXB, specifying an alpha- and a beta-subunit, respectively. The subunits dimerize to form beta-hexosaminidase A (alpha beta), beta-hexosaminidase B (beta beta), and beta-hexosaminidase S (alpha alpha). This enzyme system has the capacity to degrade a variety of cellular substrates: oligosaccharides, glycosaminoglycans, and glycolipids containing beta-linked N-acetylglucosaminyl or N-galactosaminyl residues. Mutations in either the HEXA gene or HEXB gene lead to an accumulation of GM2 ganglioside in neurons, resulting in the severe neurodegenerative disorders termed the GM2 gangliosidoses. To identify the DNA elements responsible for hexosaminidase expression, we ligated the 5'-flanking sequences of both the human and mouse hexosaminidase genes to a chloramphenicol acetyltransferase (CAT) gene. The resulting plasmids were transfected into NIH-3T3 cells and CAT activity was determined as a measure of promoter strength. By 5' deletion analysis, it was found that essential sequences for HEXA expression resided within a 40-bp region between 100 bp and 60 bp upstream of the ATG initiation codon. This area contained two potential estrogen response element half-sites as well as potential binding sites for transcription factors NF-E1 and AP-2. Similarly, important HEXB promoter sequences were localized to a 60-bp region between 150 bp and 90 bp upstream of the ATG codon. By performing scanning mutagenesis on a 60-bp region within the 150-bp HEXB construct, we defined an essential promoter element of 12 bp that contained two potential AP-1 sites. The mouse Hexa and Hexb 5'-flanking sequences were found to contain regions similar in sequence, location, and activity to the essential promoter elements defined in the cognate human genes. No sequence similarity was found, however, between 5'-flanking regions of the HEXA and HEXB genes. These essential promoter elements represent potential sites for HEXA and

  18. Structural and functional analysis of the Entamoeba histolytica EhrabB gene promoter

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    Rodríguez Mario A


    Full Text Available Abstract Background The Entamoeba histolytica EhrabB gene encodes for a Rab GTPase involved in phagocytosis. It is located at a virulence locus where the Ehcp112 gene is in the complementary strand at 332 bp of EhrabB start codon, suggesting a finely regulated transcription of both genes. However, the transcription regulation in this parasite is poorly understood. Results To initiate the knowledge of EhrabB gene expression regulation, here we studied the structural characteristics of its gene promoter and its control transcription elements. In silico searches of the EhrabB 5'-flanking region revealed that it contains a motif similar to the upstream regulatory element 1 (URE1 of the E. histolytica hgl5 gene. It also has sequences with homology to C/EBP and GATA1 binding sites, and heat shock elements (HSE. Primer extension experiments revealed that EhrabB has at least four transcription initiation sites. The elements at the 5'-flanking region that drive EhrabB gene expression were detected and characterized using transitory transfected trophozoites with a plasmid carrying the CAT reporter gene. EhrabB transcription is negatively regulated by a sequence located between positions -491 to -428 with respect to the first transcription initiation site. We also showed that the URE1-like motif activates EhrabB transcription. In addition, heat shock activated the EhrabB promoter in episomal constructs and lead to an increase in de novo EhrabB transcription. Conclusion The data suggest that EhrabB transcription is controlled negatively by an unidentified sequence, but it is activated by an URE1-like motif. Our analyses also revealed the presence of activator HSE that function under stress.

  19. MGMT, GATA6, CD81, DR4, and CASP8 gene promoter methylation in glioblastoma

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    Skiriute Daina


    Full Text Available Abstract Background Methylation of promoter region is the major mechanism affecting gene expression in tumors. Recent methylome studies of brain tumors revealed a list of new epigenetically modified genes. Our aim was to study promoter methylation of newly identified epigenetically silenced genes together with already known epigenetic markers and evaluate its separate and concomitant role in glioblastoma genesis and patient outcome. Methods The methylation status of MGMT, CD81, GATA6, DR4, and CASP8 in 76 patients with primary glioblastomas was investigated. Methylation-specific PCR reaction was performed using bisulfite treated DNA. Evaluating glioblastoma patient survival time after operation, patient data and gene methylation effect on survival was estimated using survival analysis. Results The overwhelming majority (97.3% of tumors were methylated in at least one of five genes tested. In glioblastoma specimens gene methylation was observed as follows: MGMT in 51.3%, GATA6 in 68.4%, CD81 in 46.1%, DR4 in 41.3% and CASP8 in 56.8% of tumors. Methylation of MGMT was associated with younger patient age (p CASP8 with older (p MGMT methylation was significantly more frequent event in patient group who survived longer than 36 months after operation (p CASP8 was more frequent in patients who survived shorter than 36 months (p MGMT, GATA6 and CASP8 as independent predictors for glioblastoma patient outcome (p MGMT and GATA6 were independent predictors for patient survival in younger patients’ group, while there were no significant associations observed in older patients’ group when adjusted for therapy. Conclusions High methylation frequency of tested genes shows heterogeneity of glioblastoma epigenome and the importance of MGMT, GATA6 and CASP8 genes methylation in glioblastoma patient outcome.

  20. Transcription factors interacting with herpes simplex virus alpha gene promoters in sensory neurons. (United States)

    Hagmann, M; Georgiev, O; Schaffner, W; Douville, P


    Interference with VP16-mediated activation of herpes virus immediate-early (or alpha) genes is thought to be the major cause of establishing viral latency in sensory neurons. This could be brought about by lack of a key activating transcription factor(s) or active repression. In this study we find that sensory neurons express all important components for VP16-mediated alpha gene induction, such as the POU transcription factor Oct-1, host cell factor (HCF) and GABP alpha/beta. However, Oct-1 and GABP alpha/beta are only present at low levels and the VP16-induced complex (VIC) appears different. We do not find protein expression of the transcription factor Oct-2, implicated by others as an alpha gene repressor. The POU factor N-Oct3 (Brn 2 or POU3F2) is also present in sensory neurons and binds viral TAATGARAT motifs with higher affinity than Oct-1, indicating that it may be a candidate repressor for competitive binding to TAATGARAT motifs. When transfected into HeLa cells, where Oct-1 and GABP alpha/beta are highly abundant, N-Oct3 represses model promoters with multimerized TAATGARAT motifs, but fails to repress complete alpha gene promoters. Taken together our findings suggest that modulation of alpha gene promoters could contribute to viral latency when low concentrations of the activating transcription factors Oct-1 and GABP alpha/beta prevail. Our data, however, refute the notion that competing Oct factors are able to block alpha gene transcription to achieve viral latency. Images PMID:8559654

  1. Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis. (United States)

    Zhang, Zhichun; Tian, Hua; Lv, Ping; Wang, Weiping; Jia, Zhuqing; Wang, Sainan; Zhou, Chunyan; Gao, Xuejun


    Mutation of distal-less homeobox 3 (DLX3) is responsible for human tricho-dento-osseous syndrome (TDO) with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP) genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO) inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.

  2. Characterization of chicken riboflavin carrier protein gene structure and promoter regulation by estrogen

    Indian Academy of Sciences (India)

    Nandini Vasudevan; Urvashi Bahadur; Paturu Kondaiah


    The chicken riboflavin carrier protein (RCP) is an estrogen induced egg yolk and white protein. Eggs from hens which have a splice mutation in RCP gene fail to hatch, indicating an absolute requirement of RCP for the transport of riboflavin to the oocyte. In order to understand the mechanism of regulation of this gene by estrogen, the chicken RCP gene including 1 kb of the 5′ flanking region has been isolated. Characterization of the gene structure shows that it contains six exons and five introns, including an intron in the 5′ untranslated region. Sequence analysis of the 5′ flanking region does not show the presence of any classical, palindromic estrogen response element (ERE). However, there are six half site ERE consensus elements. Four deletion constructs of the 5′ flanking region with varying number of ERE half sites were made in pGL3 basic vector upstream of the luciferase-coding region. Transient transfection of these RCP promoter deletion constructs into a chicken hepatoma cell line (LMH2A) showed 6-12-fold transcriptional induction by a stable estrogen analogue, moxesterol. This suggests that the RCP gene is induced by estrogen even in the absence of a classical ERE and the half sites of ERE in this promoter may be important for estrogen induction.

  3. Study on the Polymorphisms of Porcine Myostatin Gene in Promoter Region by PCR-RFLPS

    Institute of Scientific and Technical Information of China (English)

    YANG Xiu-qin; LIU Di


    In order to further study functions of the porcine myostatin gene, we analyzed the polymorphisms of porcine myostatin gene in promoter region among different breeds including Yorkshire, Landrace, Duroc, Junmu, Min pig and Sanjiang white pig by PCR-RFLPs. The allele T dominated in the imported lean-type pig breeds such as Yorkshire, Landrace and Duroc. No allele A was detected in Junmu and Sanjiang white pig, and the frequencies of three genotypes were about equal in Min pig. The result using X2 analysis showed that the distribution of three genotypes was related to pig breeds.

  4. Interferon gamma response region in the promoter of the human DPA gene.



    The interferon gamma (IFN-gamma) response region of the human class II major histocompatibility complex gene, DPA, has been localized to a 52-base-pair (bp) DNA fragment in the proximal promotor at -107 to -55 bp after transfection into HeLa cells of a series of 5', 3', and gap deletion mutants linked to a reporter gene, human growth hormone, as well as of synthetic oligonucleotides fused to the heterologous promoter thymidine kinase. The 52-mer sequence contains the X and Y box elements cons...

  5. Repressive BMP2 gene regulatory elements near the BMP2 promoter

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Shan [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States); Chandler, Ronald L. [Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, TN (United States); Fritz, David T. [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States); Mortlock, Douglas P. [Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, TN (United States); Rogers, Melissa B., E-mail: [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States)


    The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

  6. Maximal Expression of the Evolutionarily Conserved Slit2 Gene Promoter Requires Sp1. (United States)

    Saunders, Jacquelyn; Wisidagama, D Roonalika; Morford, Travis; Malone, Cindy S


    Slit2 is a neural axon guidance and chemorepellent protein that stimulates motility in a variety of cell types. The role of Slit2 in neural development and neoplastic growth and migration has been well established, while the genetic mechanisms underlying regulation of the Slit2 gene have not. We identified the core and proximal promoter of Slit2 by mapping multiple transcriptional start sites, analyzing transcriptional activity, and confirming sequence homology for the Slit2 proximal promoter among a number of species. Deletion series and transient transfection identified the Slit2 proximal promoter as within 399 base pairs upstream of the start of transcription. A crucial region for full expression of the Slit2 proximal promoter lies between 399 base pairs and 296 base pairs upstream of the start of transcription. Computer modeling identified three transcription factor-binding consensus sites within this region, of which only site-directed mutagenesis of one of the two identified Sp1 consensus sites inhibited transcriptional activity of the Slit2 proximal promoter (-399 to +253). Bioinformatics analysis of the Slit2 proximal promoter -399 base pair to -296 base pair region shows high sequence conservation over twenty-two species, and that this region follows an expected pattern of sequence divergence through evolution.

  7. Absence of mutation at the 5'-upstream promoter region of the TPM4 gene from cardiac mutant axolotl (Ambystoma mexicanum). (United States)

    Denz, Christopher R; Zhang, Chi; Jia, Pingping; Du, Jianfeng; Huang, Xupei; Dube, Syamalima; Thomas, Anish; Poiesz, Bernard J; Dube, Dipak K


    Tropomyosins are a family of actin-binding proteins that show cell-specific diversity by a combination of multiple genes and alternative RNA splicing. Of the 4 different tropomyosin genes, TPM4 plays a pivotal role in myofibrillogenesis as well as cardiac contractility in amphibians. In this study, we amplified and sequenced the upstream regulatory region of the TPM4 gene from both normal and mutant axolotl hearts. To identify the cis-elements that are essential for the expression of the TPM4, we created various deletion mutants of the TPM4 promoter DNA, inserted the deleted segments into PGL3 vector, and performed promoter-reporter assay using luciferase as the reporter gene. Comparison of sequences of the promoter region of the TPM4 gene from normal and mutant axolotl revealed no mutations in the promoter sequence of the mutant TPM4 gene. CArG box elements that are generally involved in controlling the expression of several other muscle-specific gene promoters were not found in the upstream regulatory region of the TPM4 gene. In deletion experiments, loss of activity of the reporter gene was noted upon deletion which was then restored upon further deletion suggesting the presence of both positive and negative cis-elements in the upstream regulatory region of the TPM4 gene. We believe that this is the first axolotl promoter that has ever been cloned and studied with clear evidence that it functions in mammalian cell lines. Although striated muscle-specific cis-acting elements are absent from the promoter region of TPM4 gene, our results suggest the presence of positive and negative cis-elements in the promoter region, which in conjunction with positive and negative trans-elements may be involved in regulating the expression of TPM4 gene in a tissue-specific manner.

  8. Promoter competition assay for analyzing gene regulation in joint tissue engineering. (United States)

    Sun, Hui Bin; Malacinski, George M; Yokota, Hiroki


    We describe a new biochemical technique, "promoter competition assay," for examining the role of cis-acting DNA elements in tissue cultures. Recent advances in tissue engineering permit the culture of a variety of cells. Many tissues are engineered, however, without an appropriate understanding of molecular machinery that regulates gene expression and cellular growth. For elucidating the role of cis-acting regulatory elements in cellular differentiation and growth, we developed the promoter competition assay. This assay uses a transient transfer into cells of double-stranded DNA fragments consisting of cis-acting regulatory elements. The transferred DNA fragments act as a competitor and titrate the function of their genomic counterparts. Using synovial cells derived from a rheumatoid arthritis patient, we examined a role of NF-kappa B binding sites in the regulation of the expression of matrix metalloproteinase (MMP) genes. The results support a stimulatory role of NF-kappa B in transcriptional regulation of MMP-1 and MMP-13.

  9. Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Rachel M. Woodfint


    Full Text Available Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2 expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2, GATA binding protein 4 (GATA4, hepatocyte nuclear factor 4 α (HNF4A, and transcription factor 4 (TCF4 that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

  10. Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells (United States)

    Woodfint, Rachel M.; Chen, Paula R.; Ahn, Jinsoo; Suh, Yeunsu; Hwang, Seongsoo; Lee, Sang Suk; Lee, Kichoon


    Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2) expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR) analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2), GATA binding protein 4 (GATA4), hepatocyte nuclear factor 4 α (HNF4A), and transcription factor 4 (TCF4) that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP) reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine. PMID:28106824

  11. A new PKLR gene mutation in the R-type promoter region affects the gene transcription causing pyruvate kinase deficiency. (United States)

    Manco, L; Ribeiro, M L; Máximo, V; Almeida, H; Costa, A; Freitas, O; Barbot, J; Abade, A; Tamagnini, G


    Mutations in the PKLR gene responsible for pyruvate kinase (PK)-deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A-->G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK-deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA-A element) in the R-type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R-PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the -72A-->G point mutation disables the binding of the erythroid transcription factor GATA-1 to the GATA-A element. Supporting these data, the two patients homozygous for the -72A-->G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C-->T had a mild condition.

  12. Molecular Cloning and Characterization of Human Homeobox Gene Nkx3.1 Promoter

    Institute of Scientific and Technical Information of China (English)

    An-LiJIANG; Jian-YeZHANG; CharlesYOUNG; Xiao-YanHU; Yong-MeiWANG; Zhi-FangLIU; Mei-LanHAO


    Nkx3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. To study its regulation of transcription, 1.06 kb 5′ flanking region of Nkx3.1 gene and its 5′ deletion mutants (861,617,417 and 238 bp) were obtained by PCR and cloned into pGL3-basic, a promoter-less luciferase reporter vector, to examine their promoter activities driving the reporter gene transcription, pRL-TK, a Renilla luciferase reporter vector was used as internal control, and pGL3-control and pGL3-basic were used as positive and negative control respectively. The promoter activities were determined by dual-luciferase reporter assay 48h after pGL3 constructs were cotransfected with pRL-TK into prostate cancer cell LNCaP. The results showed that dual-luciferase reporter assay (M/M2) of pGL3-1.06kb cotransfection with pRL-TK was 2.7, which was about 1.5-fold higher than that of pGL3-control cotransfection with pRL-TK and 50-fold higher than that of pGL3-basic cotransfection with pRL-TK. The results also showed that the relative activities (M1/M2) were 0.71, 0.84, 0.44 and 2.07 respectively for pGL3-861bp, pGL3-617bp, pGL3-417bp, pGL3-238bp, the last one still had 80% promoter activity compared with pGL3-1.06kb, which showed that deletion from 1.06kb to 238 bp had small effects on promoter activity. The conclusion was that the 238bp fragment containing a TATA box and two CAAT boxes had strong promoter activity. However, the deletion from 1.06kb to 861bp reduced activity 3.8-fold while the deletion from 417bp to 238bp enhanced activity 4.7-fold, which indicated that these deleted sequences might contain some important positive or negative regulatory elements. It will be important to identify the elements within the Nkx3.1 promoter that contribute to regulation of the gene transcription in the future studies.

  13. Association between Osteopontin Promoter Gene Polymorphisms and Haplotypes with Risk of Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Balneek Singh Cheema


    Full Text Available Background: Osteopontin (OPN C-443T promoter polymorphism has been shown as a genetic risk factor for diabetic nephropathy (DN in type 2 diabetic patients (T2D. Methods: In the present study we investigated the association of three functional promoter gene polymorphisms C-443T, delG-156G, and G-66T and their haplotypes with the risk of DN and estimated Glomerular Filtration Rate (eGFR in Asian Indians T2D patients using Real time PCR based Taqman assay. A total of 1165 T2D patients, belonging to two independently ascertained Indian Asian cohorts, were genotyped for three OPN promoter polymorphisms C-443T (rs11730582, delG-156G (rs17524488 and G-66T (rs28357094. Results: -156G allele and GG genotypes (delG-156G and haplotypes G-C-G and T-C-G (G-66T, C-443T, delG-156G were associated with decreased risk of DN and higher eGFR. Haplotype G-T-delG and T-T-delG (G-66T, C-443T, delG-156G were identified as risk haplotypes, as shown by lower eGFR. Conclusion: This is the first study to report an association of OPN promoter gene polymorphisms; G-66T and delG-156G and their haplotypes with DN in T2D. Our results suggest an association between OPN promoter gene polymorphisms and their haplotypes with DN.

  14. Single-nucleotide polymorphisms and activity analysis of the promoter and enhancer of the pig lactase gene. (United States)

    Du, Hai-Ting; Zhu, Hong-Yan; Wang, Jia-Mei; Zhao, Wei; Tao, Xiao-Li; Ba, Cai-Feng; Tian, Yu-Min; Su, Yu-Hong


    Lactose intolerance in northern Europeans is strongly associated with a single-nucleotide polymorphism (SNP) located 14 kb upstream of the human lactase gene: -13,910 C/T. We examined whether SNPs in the 5' flanking region of the pig lactase gene are similar to those in the human gene and whether these polymorphisms play a functional role in regulating pig lactase gene expression. The 5' flanking region of the lactase gene from several different breeds of pigs was cloned and analyzed for gene regulatory activity of a luciferase reporter gene. One SNP was found in the enhancer region (-797 G/A) and two were found in the promoter region (-308G/C and -301 A/G). The promoter C-308,G-301(Pro-CG) strongly promotes the expression of the lactase gene, but the promoter G-308,A-301(Pro-GA) does not. The enhancer A-797(Enh-A) genotype for Pro-GA can significantly enhance promoter activity, but has an inhibitory effect on Pro-CG. The Enhancer G-797(Enh-G) has a significant inhibitory effect on both promoters. In conclusion, the order of effectiveness on the pig lactase gene is Enh-A+Pro-GA>Enh-A/G+Pro-CG>Enh-G+Pro-GA.

  15. Novel screening assay for in vivo selection of Klebsiella pneumoniae genes promoting gastrointestinal colonisation

    DEFF Research Database (Denmark)

    Boll, Erik J.; Nielsen, Lene N; Krogfelt, Karen A.;


    Klebsiella pneumoniae is an important opportunistic pathogen causing pneumonia, sepsis and urinary tract infections. Colonisation of the gastrointestinal (GI) tract is a key step in the development of infections; yet the specific factors important for K. pneumoniae to colonize and reside in the GI...... tract of the host are largely unknown. To identify K. pneumoniae genes promoting GI colonisation, a novel genomic-library-based approach was employed....

  16. Finding Combination of Features from Promoter Regions for Ovarian Cancer-related Gene Group Classification

    KAUST Repository

    Olayan, Rawan S.


    In classification problems, it is always important to use the suitable combination of features that will be employed by classifiers. Generating the right combination of features usually results in good classifiers. In the situation when the problem is not well understood, data items are usually described by many features in the hope that some of these may be the relevant or most relevant ones. In this study, we focus on one such problem related to genes implicated in ovarian cancer (OC). We try to recognize two important OC-related gene groups: oncogenes, which support the development and progression of OC, and oncosuppressors, which oppose such tendencies. For this, we use the properties of promoters of these genes. We identified potential “regulatory features” that characterize OC-related oncogenes and oncosuppressors promoters. In our study, we used 211 oncogenes and 39 oncosuppressors. For these, we identified 538 characteristic sequence motifs from their promoters. Promoters are annotated by these motifs and derived feature vectors used to develop classification models. We made a comparison of a number of classification models in their ability to distinguish oncogenes from oncosuppressors. Based on 10-fold cross-validation, the resultant model was able to separate the two classes with sensitivity of 96% and specificity of 100% with the complete set of features. Moreover, we developed another recognition model where we attempted to distinguish oncogenes and oncosuppressors as one group from other OC-related genes. That model achieved accuracy of 82%. We believe that the results of this study will help in discovering other OC-related oncogenes and oncosuppressors not identified as yet.

  17. Division genes in Escherichia coli are expressed coordinately to cell septum requirements by gearbox promoters. (United States)

    Aldea, M; Garrido, T; Pla, J; Vicente, M


    The cell division ftsQAZ cluster and the ftsZ-dependent bolA morphogene of Escherichia coli are found to be driven by gearboxes, a distinct class of promoters characterized by showing an activity that is inversely dependent on growth rate. These promoters contain specific sequences upstream from the mRNA start point, and their -10 region is essential for the inverse growth rate dependence. Gearbox promoters are essential for driving ftsQAZ and bolA gene expression so that the encoded products are synthesized at constant amounts per cell independently of cell size. This mode of regulation would be expected for the expression of proteins that either play a regulatory role in cell division or form a stoichiometric component of the septum, a structure that, independently of cell size and growth rate, is produced once per cell cycle.

  18. Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus. (United States)

    Lee, Ki-Sung; Kim, Jun-Seob; Heo, Paul; Yang, Tae-Jun; Sung, Young-Je; Cheon, Yuna; Koo, Hyun Min; Yu, Byung Jo; Seo, Jin-Ho; Jin, Yong-Su; Park, Jae Chan; Kweon, Dae-Hyuk


    Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P(CYC), P(TEF), P(GPD), and P(ADH)) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters--the gene for the green fluorescence protein (GFP)--CYC1 terminator (T(CYC)) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P(GPD) > P(ADH) ∼ P(TEF) > P(CYC). All promoters except for the P(CYC) exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the

  19. Transcriptional infidelity promotes heritable phenotypic change in a bistable gene network.

    Directory of Open Access Journals (Sweden)

    Alasdair J E Gordon


    Full Text Available Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS. Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.

  20. Brain molecular aging, promotion of neurological disease and modulation by sirtuin 5 longevity gene polymorphism. (United States)

    Glorioso, Christin; Oh, Sunghee; Douillard, Gaelle Guilloux; Sibille, Etienne


    Mechanisms determining characteristic age-of-onset for neurological diseases are largely unknown. Normal brain aging associates with robust and progressive transcriptome changes ("molecular aging"), but the intersection with disease pathways is mostly uncharacterized. Here, using cross-cohort microarray analysis of four human brain areas, we show that neurological disease pathways largely overlap with molecular aging and that subjects carrying a newly-characterized low-expressing polymorphism in a putative longevity gene (Sirtuin5; SIRT5(prom2)) have older brain molecular ages. Specifically, molecular aging was remarkably conserved across cohorts and brain areas, and included numerous developmental and transcription-regulator genes. Neurological disease-associated genes were highly overrepresented within age-related genes and changed almost unanimously in pro-disease directions, together suggesting an underlying genetic "program" of aging that progressively promotes disease. To begin testing this putative pathway, we developed and used an age-biosignature to assess five candidate longevity gene polymorphisms' association with molecular aging rates. Most robustly, aging was accelerated in cingulate, but not amygdala, of subjects carrying a SIRT5 promoter polymorphism (+9 years, p=0.004), in concordance with cingulate-specific decreased SIRT5 expression. This effect was driven by a set of core transcripts (+24 years, p=0.0004), many of which were mitochondrial, including Parkinson's disease genes, PINK-1 and DJ-1/PARK7, hence suggesting that SIRT5(prom2) may represent a risk factor for mitochondrial dysfunction-related diseases, including Parkinson's, through accelerated molecular aging of disease-related genes. Based on these results we speculate that a "common mechanism" may underlie age-of-onset across several neurological diseases. Confirming this pathway and its regulation by common genetic variants would provide new strategies for predicting, delaying, and

  1. Quantification of Atlantic salmon type-I interferon using an Mx1 promoter reporter gene assay. (United States)

    Johansen, Audny; Collet, Bertrand; Sandaker, Elin; Secombes, Christopher J; Jørgensen, Jorunn B


    We here describe an assay for the detection of interferon-like activity in Atlantic salmon based on the transient transfection of chinook salmon embryo cells (CHSE-214 cells) with a rainbow trout Mx1 promoter linked to a luciferase reporter. A beta-galactosidase gene under the control of a constitutively expressed beta-actin promoter was used as a transfection standard, and luciferase and beta gal expression were measured by a commercially available kit. Interferon containing supernatants from poly I:C- or CpG-stimulated leucocytes added to transfected CHSE-cells induced high luciferase expression (>60-fold induction compared to supernatants from non-stimulated cells). There was no response to supernatants from LPS- and ConA/PMA-stimulated leucocytes, demonstrating the specificity for type I interferon-like activity. Duplicate samples analysed using a cell protection assay for detection of antiviral activity correlated well with levels obtained by the Mx1 promoter reporter gene assay (R2=0.97), confirming the reporter assay as a reliable substitute for the standard antiviral assay. The Mx reporter gene assay also has advantages in terms of sensitivity, high dynamic range and reliability over the conventional cell protection assay.

  2. Gene expression of axon growth promoting factors in the deer antler.

    Directory of Open Access Journals (Sweden)

    Wolfgang Pita-Thomas

    Full Text Available The annual regeneration cycle of deer (Cervidae, Artiodactyla antlers represents a unique model of epimorphic regeneration and rapid growth in adult mammals. Regenerating antlers are innervated by trigeminal sensory axons growing through the velvet, the modified form of skin that envelopes the antler, at elongation velocities that reach one centimetre per day in the common deer (Cervus elaphus. Several axon growth promoters like NT-3, NGF or IGF-1 have been described in the antler. To increase the knowledge on the axon growth environment, we have combined different gene-expression techniques to identify and characterize the expression of promoting molecules not previously described in the antler velvet. Cross-species microarray analyses of deer samples on human arrays allowed us to build up a list of 90 extracellular or membrane molecules involved in axon growth that were potentially being expressed in the antler. Fifteen of these genes were analysed using PCR and sequencing techniques to confirm their expression in the velvet and to compare it with the expression in other antler and skin samples. Expression of 8 axon growth promoters was confirmed in the velvet, 5 of them not previously described in the antler. In conclusion, our work shows that antler velvet provides growing axons with a variety of promoters of axon growth, sharing many of them with deer's normal and pedicle skin.

  3. Promoter Hypermethylation of the EMP3 Gene in a Series of 229 Human Gliomas

    Directory of Open Access Journals (Sweden)

    Marta Mellai


    Full Text Available The epithelial membrane protein 3 (EMP3 is a candidate tumor suppressor gene in the critical region 19q13.3 for several solid tumors, including tumors of the nervous systems. The aim of this study was to investigate the EMP3 promoter hypermethylation status in a series of 229 astrocytic and oligodendroglial tumors and in 16 GBM cell lines. The analysis was performed by methylation-specific PCR and capillary electrophoresis. Furthermore, the EMP3 expression at protein level was evaluated by immunohistochemistry and Western blotting analysis. Associations of EMP3 hypermethylation with total 1p/19q codeletion, MGMT promoter hypermethylation, IDH1/IDH2 and TP53 mutations, and EGFR amplification were studied, as well as its prognostic significance. The EMP3 promoter hypermethylation has been found in 39.5% of gliomas. It prevailed in low-grade tumors, especially in gliomas with an oligodendroglial component, and in sGBMs upon pGBMs. In oligodendroglial tumors, it was strongly associated with both IDH1/IDH2 mutations and total 1p/19q codeletion and inversely with EGFR gene amplification. No association was found with MGMT hypermethylation and TP53 mutations. In the whole series, the EMP3 hypermethylation status correlated with 19q13.3 loss and lack of EMP3 expression at protein level. A favorable prognostic significance on overall survival of the EMP3 promoter hypermethylation was found in patients with oligodendroglial tumors.

  4. Association study of promoter polymorphisms at the dopamine transporter gene in Attention Deficit Hyperactivity Disorder

    Directory of Open Access Journals (Sweden)

    Huang Yu-Shu


    Full Text Available Abstract Background Attention deficit hyperactivity disorder (ADHD is a complex neurobehavioral disorder. The dopamine transporter gene (DAT1/SLC6A3 has been considered a good candidate for ADHD. Most association studies with ADHD have investigated the 40-base-pair variable number of tandem repeat (VNTR polymorphism in the 3'-untranslated region of DAT1. Only few studies have reported association between promoter polymorphisms of the gene and ADHD. Methods To investigate the association between the polymorphisms -67A/T (rs2975226 and -839C/T (rs2652511 in promoter region of DAT1 in ADHD, two samples of ADHD patients from the UK (n = 197 and Taiwan (n = 212 were genotyped, and analysed using within-family transmission disequilibrium test (TDT. Results A significant association was found between the T allele of promoter polymorphism -67A/T and ADHD in the Taiwanese population (P = 0.001. There was also evidence of preferential transmission of the T allele of -67A/T polymorphism in combined samples from the UK and Taiwan (P = 0.003. No association was detected between the -839C/T polymorphism and ADHD in either of the two populations. Conclusion The finding suggests that genetic variation in the promoter region of DAT1 may be a risk factor in the development of ADHD.

  5. Cloning and Characterization of β-Carotene Ketolase Gene Promoter in Haematococcus pluvialis

    Institute of Scientific and Technical Information of China (English)

    Chun-Xiao MENG; Chang-Ying TENG; Peng JIANG; Song QIN; Cheng-Kui TSENG


    The unicellular green alga Haematococcuspluvialis accumulates a highly valuable ketocarotenoid,astaxanthin, under various environmental stresses. β-carotene ketolase (BKT) plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, an approximate 700 bp 5'-flanking region of the bkt gene containing a putative promoter was cloned through walking upstream. The results of the sequence analysis showed that this bkt 5'-flanking region might have cis-acting elements such as sterol regulatory element (SRE-1)-like motifs, the C-repeat/dehydration responsive element (DRE) and al-3 proximal element (APE)-like motifs,except for typical TATA and CCAAT boxes. The results of the β-galactosidase assay and the transient expression of lacZ driven by a series of sequential deletions revealed that a minimal promoter-like region might exist from -630 to -408 bp, and the highest promoter activity was observed to span the positions from -630 to -308 bp. The results of the site-directed mutagenesis of a C-repeat/DRE and two APE-like motifs in a promoter-like region (-630 to -308 bp) suggested that two APE-like motifs might be essential for transcriptional control of the bkt gene.

  6. The de novo methyltransferases DNMT3a and DNMT3b target the murine gammaherpesvirus immediate-early gene 50 promoter during establishment of latency. (United States)

    Gray, Kathleen S; Forrest, J Craig; Speck, Samuel H


    The role of epigenetic modifications in the regulation of gammaherpesvirus latency has been a subject of active study for more than 20 years. DNA methylation, associated with transcriptional silencing in mammalian genomes, has been shown to be an important mechanism in the transcriptional control of several key gammaherpesvirus genes. In particular, DNA methylation of the functionally conserved immediate-early replication and transcription activator (RTA) has been shown to regulate Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus Rta expression. Here we demonstrate that the murine gammaherpesvirus (MHV68) homolog, encoded by gene 50, is also subject to direct repression by DNA methylation, both in vitro and in vivo. We observed that the treatment of latently MHV68-infected B-cell lines with a methyltransferase inhibitor induced virus reactivation. In addition, we show that the methylation of the recently characterized distal gene 50 promoter represses activity in a murine macrophage cell line. To evaluate the role of de novo methyltransferases (DNMTs) in the establishment of these methylation marks, we infected mice in which conditional DNMT3a and DNMT3b alleles were selectively deleted in B lymphocytes. DNMT3a/DNMT3b-deficient B cells were phenotypically normal, displaying no obvious compromise in cell surface marker expression or antibody production either in naïve mice or in the context of nonviral and viral immunogens. However, mice lacking functional DNMT3a and DNMT3b in B cells exhibited hallmarks of deregulated MHV68 lytic replication, including increased splenomegaly and the presence of infectious virus in the spleen at day 18 following infection. In addition, total gene 50 transcript levels were elevated in the spleens of these mice at day 18, which correlated with the hypomethylation of the distal gene 50 promoter. However, by day 42 postinfection, aberrant virus replication was resolved, and we observed wild-type frequencies of viral

  7. Genomic organization and promoter cloning of the human X11α gene APBA1.

    LENUS (Irish Health Repository)

    Chai, Ka-Ho


    X11α is a brain specific multi-modular protein that interacts with the Alzheimer\\'s disease amyloid precursor protein (APP). Aggregation of amyloid-β peptide (Aβ), an APP cleavage product, is believed to be central to the pathogenesis of Alzheimer\\'s disease. Recently, overexpression of X11α has been shown to reduce Aβ generation and to ameliorate memory deficit in a transgenic mouse model of Alzheimer\\'s disease. Therefore, manipulating the expression level of X11α may provide a novel route for the treatment of Alzheimer\\'s disease. Human X11α is encoded by the gene APBA1. As evidence suggests that X11α expression can be regulated at transcription level, we have determined the gene structure and cloned the promoter of APBA1. APBA1 spans over 244 kb on chromosome 9 and is composed of 13 exons and has multiple transcription start sites. A putative APBA1 promoter has been identified upstream of exon 1 and functional analysis revealed that this is highly active in neurons. By deletion analysis, the minimal promoter was found to be located between -224 and +14, a GC-rich region that contains a functional Sp3 binding site. In neurons, overexpression of Sp3 stimulates the APBA1 promoter while an Sp3 inhibitor suppresses the promoter activity. Moreover, inhibition of Sp3 reduces endogenous X11α expression and promotes the generation of Aβ. Our findings reveal that Sp3 play an essential role in APBA1 transcription.

  8. Analysis of Hypothetical Promoter Domains of DKFZp564A1164, NPHS1 and HSPOX1 Genes

    Energy Technology Data Exchange (ETDEWEB)

    Hammond, S S


    For this study, a high throughput method for identifying and testing regulatory elements was examined. In addition, the validity of promoters predicted by FirstEF was tested. It was found that by combining computer based promoter and first exon predictions from FirstEF (Davuluri et al., 2001) with PCR-based cloning to generate luciferase reporter constructs, and by testing reporter activity in cultured mammalian cells plated in a 96 well format one could identify promoter activity in a relatively high throughput manner. The data generated in this study suggest that FirstEF predictions are sometimes incorrect. Therefore, having a strategy for defining which FirstEF predicted promoters to test first may accelerate the process. Initially testing promoters that are at a confirmed transcription start site for a gene, at a possible alternate transcription start site or in a region of conserved sequence would be the best candidates, while promoters predicted in gene desert regions may not be as easy to confirm. The luciferase assay lent itself very well to the high throughput search, however the subcloning did not always go smoothly. The numerous steps that this traditional subcloning method requires were time consuming and increased the opportunities for errors. A faster method that skips many of the traditional subcloning steps, such as the Creator{trademark} system by Clontech is currently being investigated by our lab. The development and testing of substantially larger enhancer/silencer regulatory elements may not be possible at this time using these high throughput methods. These regulatory elements are generally GC rich making them more difficult to PCR and subclone. Additionally, confirming upstream untranslated first exons was not possible within this time scale using the SMART RACE protocol. It will be necessary to further explore the limitations within these procedures in order to confirm these and future regulatory elements. Alterations and modifications to

  9. Study on the polymorphisms and promoter methylation and expression of the glutathione Stransferases P1 gene in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)



    Objective To study the relationship between hepatocellular carcinoma (HCC) and the polymorphisms, promoter methylation, and expression of glutathione S-transferases P1 gene (GST)P1 gene. Methods Using methylation -special PCR (MSP), the methylated status of CpG islands of GSTP1 gene in tumor tissues of 53 HCC and its adjacent nontumor tissues were studied. The en-

  10. Analysis of APC and IGFBP7 promoter gene methylation in Swedish and Vietnamese colorectal cancer patients. (United States)

    Dimberg, Jan; Hong, Thai Trinh; Skarstedt, Marita; Löfgren, Sture; Zar, Niklas; Matussek, Andreas


    The tumour suppressor gene adenomatous polyposis coli (APC) is a key component that drives colorectal carcinogenesis. The reported DNA methylation in the promoter of APC varies greatly among studies of colorectal cancer (CRC) in different populations. Insulin-like growth factor binding protein 7 (IGFBP7), also known as IGFBP-related protein 1 (IGFBP-rP1), is expressed in various tissue types, including the lung, brain, prostate and gastrointestinal tract, and has been suggested to play a tumour suppressor role against colorectal carcinogenesis. Studies have indicated that IGFBP7 is inactivated by DNA methylation in human colon, lung and breast cancer. In the present study, we used the methylation-specific polymerase chain reaction to study the methylation status of the APC and IGFBP7 gene promoters in cancerous and paired normal tissue to evaluate its impact on clinical factors and association with ethnicity, represented by Swedish and Vietnamese CRC patients. We also investigated the distribution of CpG islands and the CpG dinucleotide density of each CpG island in the regions which were the subject of our investigation. Overall, normal tissue from Swedish patients exhibited a significantly higher frequency of IGFBP7 gene methylation in comparison with that of Vietnamese patients. Moreover, a significantly higher number of cancer tissues from Vietnamese individuals showed higher levels of methylation versus the paired normal tissue compared with that of Swedish patients. When we studied the methylation in cancer compared with the matched normal tissue in individuals, we found that a significantly higher number of Vietnamese patients had a higher degree of IGFBP7 gene methylation in cancer versus matched normal tissue in comparison with Swedish patients. Taken together, our results suggest that the methylation of the APC and IGFBP7 gene promoter region in cancerous tissue, in combination with the predominance of methylation in normal tissue, may serve as a

  11. Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter

    Energy Technology Data Exchange (ETDEWEB)

    Yanagawa, H.; Nishio, H.; Takeshima, Y. [Kobe Univ. School of Medicine (Japan)] [and others


    The dystrophin gene, which is muted in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously-unidentified alternative promoter. The case study presented is that of patient with Duchenne muscular dystrophy who had a deletion extending from 5{prime} end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5{prime} part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5{prime} end of the new transcript indicated that the 5{prime} end of exon 3 was extended by 9 codons, only the last (most 3{prime}) of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5{prime} of the previously-identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

  12. DNA sequence and structure properties analysis reveals similarities and differences to promoters of stress responsive genes in Arabidopsis thaliana. (United States)

    Zhu, Pan; Zhou, Yanhong; Zhang, Libin; Ma, Chuang


    Understanding regulatory mechanisms of stress response in plants has important biological and agricultural significances. In this study, we firstly compiled a set of genes responsive to different stresses in Arabidopsis thaliana and then comparatively analysed their promoters at both the DNA sequence and three-dimensional structure levels. Amazingly, the comparison revealed that the profiles of several sequence and structure properties vary distinctly in different regions of promoters. Moreover, the content of nucleotide T and the profile of B-DNA twist are distinct in promoters from different stress groups, suggesting Arabidopsis genes might exploit different regulatory mechanisms in response to various stresses. Finally, we evaluated the performance of two representative promoter predictors including EP3 and PromPred. The evaluation results revealed their strengths and weakness for identifying stress-related promoters, providing valuable guidelines to accelerate the discovery of novel stress-related promoters and genes in plants.

  13. Analysis of molecular aberrations of Wnt pathway gladiators in colorectal cancer in the Kashmiri population

    Directory of Open Access Journals (Sweden)

    Sameer A


    Full Text Available Abstract The development and progression of colorectal cancer (CRC is a multi-step process, and the Wnt pathways with its two molecular gladiators adenomatous polyposis coli (APC and β-catenin plays an important role in transforming a normal tissue into a malignant one. In this study, we aimed to investigate the role of aberrations in the APC and β-catenin genes in the pathogenesis of CRC in the Kashmir valley, and to correlate it with various clinicopathological variables. We examined the paired tumour and normal-tissue specimens of 86 CRC patients for the occurrence of aberrations in the mutation cluster region (MCR of the APC gene and exon 3 of the β-catenin gene by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP and/or PCR-direct sequencing. Analysis of promoter hypermethylation of the APC gene was also carried out using methylation-specific PCR (MS-PCR. The overall mutation rate of the MCR of the APC gene among 86 CRC cases was 12.8 per cent (11 of 86. Promoter hypermethylation of APC was observed in 54.65 per cent (47 of 86 of cases. Furthermore, we found a significant association between tumour location, tumour grade and node status and the methylation status of the APC gene (p ≤ 0.05. Although the number of mutations in the APC and β-catenin genes in our CRC cases was very low, the study confirms the role of epigenetic gene silencing of the pivotal molecular gladiator, APC, of the Wnt pathway in the development of CRC in the Kashmiri population.

  14. Promoting lumbar spinal fusion by adenovirus-mediated bone morphogenetic protein-4 gene therapy

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jian; ZHAO Dun-yan; SHEN Ai-guo; LIU Fan; ZHANG Feng; SUN Yu; WU Hong-fu; LU Chun-feng; SHI Hong-guang


    Objective: To determine whether an adenoviral construct containing bone morphogenetic protein-4 (BMP-4) gene can be used for lumbar spinal fusion. Methods: Twelve New Zealand white rabbits were randomly divided into two groups, 8 in the experimental group and 4 in the control group. Recombinant, replication-defective type 5 adenovirus with the cytomegalovirus (CMV) promoter and BMP-4 gene (Ad-BMP-4) was used. Another adenovirus constructed with the CMV promoter and β-galactosidase gene (Ad-β-gal) was used as control. Using collagen sponge as a carrier, Ad-BMP-4 (2.9×108 pfu/ml ) was directly implanted on the surface of L5-L6 lamina in the experimental group, while Ad-β-gal was implanted simultaneously in the control group. X-ray was obtained at 3, 6, and 12 weeks postoperatively to observe new bone formation. When new bone formation was identified, CT scans and three-dimensional reconstruction were obtained. After that, the animals were killed and underwent histological inspection.Results: In 12 weeks after operation, new bone formation and fusion were observed on CT scans in the experimental group, without the evidence of ectopic calcification in the canal. Negative results were found in the control group. Histological analysis demonstrated endochondral bone formation at the operative site and fusion at early stage was testified.Conclusions: In vivo gene therapy using Ad-BMP-4 for lumbar posterolateral spinal fusion is practicable and effective.

  15. Structure of the gene for human. beta. /sub 2/-adrenergic receptor: expression and promoter characterization

    Energy Technology Data Exchange (ETDEWEB)

    Emorine, L.J.; Marullo, S.; Delavier-Klutchko, C.; Kaveri, S.V.; Durieu-Trautmann, O.; Strosberg, A.D.


    The genomic gene coding for the human ..beta../sub 2/-adrenergic receptor (..beta../sub 2/AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with ..beta../sub 2/AR properties. Southern blot analyses with ..beta../sub 2/AR-specific probes show that a single ..beta../sub 2/AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two TATA boxes, a CAAT box, and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the ..beta../sub 2/AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins.

  16. Grainyhead and Zelda compete for binding to the promoters of the earliest-expressed Drosophila genes. (United States)

    Harrison, Melissa M; Botchan, Michael R; Cline, Thomas W


    Maternally contributed mRNAs and proteins control the initial stages of development following fertilization. During this time, most of the zygotic genome remains transcriptionally silent. The initiation of widespread zygotic transcription is coordinated with the degradation of maternally provided mRNAs at the maternal-to-zygotic transition (MZT). While most of the genome is silenced prior to the MZT, a small subset of zygotic genes essential for the future development of the organism is transcribed. Previous work in our laboratory and others identified the TAGteam element, a set of related heptameric DNA-sequences in the promoters of many early-expressed Drosophila genes required to drive their unusually early transcription. To understand how this unique subset of genes is regulated, we identified a TAGteam-binding factor Grainyhead (Grh). We demonstrated that Grh and the previously characterized transcriptional activator Zelda (Zld) bind to different TAGteam sequences with varying affinities, and that Grh competes with Zld for TAGteam occupancy. Moreover, overexpression of Grh in the early embryo causes defects in cell division, phenocopying Zld depletion. Our findings indicate that during early embryonic development the precise timing of gene expression is regulated by both the sequence of the TAGteam elements in the promoter and the relative levels of the transcription factors Grh and Zld.

  17. Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters. (United States)

    Thomson, Joshua J; Withey, Jeffrey H


    The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription.

  18. Cloning and Characterization of Largemouth Bass (Micropterus salmoides) Myostatin Encoding Gene and Its Promoter

    Institute of Scientific and Technical Information of China (English)

    LI Shengjie; BAI Junjie; WANG Lin


    Myostatin or GDF-8, a member of the transforming growth factor-β (TGF-β) superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64kb sequence of myostatin encoding gene and its promoter from largemouth bass (Micropterus salmoides). The myostatin encoding gene consisted of three exons (488bp, 371 bp and 1779bp, respectively) and two introns (390bp and 855 bp, respectively). The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Se- quence analysis of 1.569kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 (MEF2), serum response factor (SRF), activator protein 1 (API), etc.. and muscle-specific Mt binding site (MTBF) were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This information will be useful for studying the tran- scriptional regulation of myostatin in fish.

  19. Promotion of growth by Coenzyme Q10 is linked to gene expression in C. elegans. (United States)

    Fischer, Alexandra; Niklowitz, Petra; Menke, Thomas; Döring, Frank


    Coenzyme Q (CoQ, ubiquinone) is an essential component of the respiratory chain, a cofactor of pyrimidine biosynthesis and acts as an antioxidant in extra mitochondrial membranes. More recently CoQ has been identified as a modulator of apoptosis, inflammation and gene expression. CoQ deficient Caenorhabditis elegans clk-1 mutants show several phenotypes including a delayed postembryonic growth. Using wild type and two clk-1 mutants, here we established an experimental set-up to study the consequences of endogenous CoQ deficiency or exogenous CoQ supply on gene expression and growth. We found that a deficiency of endogenous CoQ synthesis down-regulates a cluster of genes that are important for growth (i.e., RNA polymerase II, eukaryotic initiation factor) and up-regulates oxidation reactions (i.e., cytochrome P450, superoxide dismutase) and protein interactions (i.e., F-Box proteins). Exogenous CoQ supply partially restores the expression of these genes as well as the growth retardation of CoQ deficient clk-1 mutants. On the other hand exogenous CoQ supply does not alter the expression of a further sub-set of genes. These genes are involved in metabolism (i.e., succinate dehydrogenase complex), cell signalling or synthesis of lectins. Thus, our work provides a comprehensive overview of genes which can be modulated in their expression by endogenous or exogenous CoQ. As growth retardation in CoQ deficiency is linked to the gene expression profile we suggest that CoQ promotes growth via gene expression.

  20. Effect of TNF{alpha} on activities of different promoters of human apolipoprotein A-I gene

    Energy Technology Data Exchange (ETDEWEB)

    Orlov, Sergey V., E-mail: [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Mogilenko, Denis A. [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Shavva, Vladimir S. [Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Dizhe, Ella B.; Ignatovich, Irina A. [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Perevozchikov, Andrej P., E-mail: [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation)


    Research highlights: {yields} TNF{alpha} stimulates the distal alternative promoter of human apoA-I gene. {yields} TNF{alpha} acts by weakening of promoter competition within apoA-I gene (promoter switching). {yields} MEK1/2 and nuclear receptors PPAR{alpha} and LXRs take part in apoA-I promoter switching. -- Abstract: Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1{beta} and TNF{alpha}. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNF{alpha}-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNF{alpha} on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNF{alpha} leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5'-regulatory region (apoA-I promoter switching) in the cells treated by TNF{alpha}. The MEK1/2-ERK1/2 cascade and nuclear receptors PPAR{alpha} and LXRs are important for TNF{alpha}-mediated apoA-I promoter switching.

  1. Association of Interleukin-10 Gene Promoter Polymorphisms in Saudi Patients with Vitiligo

    Directory of Open Access Journals (Sweden)

    Abdullah Abanmi


    Full Text Available The promoter region of human Interleukin −10 gene is highly polymorphic and has been associated with numerous autoimmune diseases. Recent studies have linked vitiligo with defective autoimmune system. This study is aimed to explore a possible association between IL-10 gene polymorphism and vitiligo in Saudi population. This case control study consisted of 184 Saudi subjects including 83 vitiligo patients (40 males, 43 females mean age 27.85 ± 12.43 years and 101 matched controls. Genomic DNA was extracted from the blood samples of healthy controls and Vitiligo patients visiting out patient clinic of Department of Dermatology, Riyadh Armed Forces Hospital, using QIA ampR DNA mini kit (Qiagen CA, USA. Interleukin-10 gene was amplified by polymerase chain reaction (PCR using Arms primers to detect any polymorphism involved at positions −592, −819 and −1082.

  2. Promoter characterization and genomic organization of the human X11β gene APBA2.

    LENUS (Irish Health Repository)

    Hao, Yan


    Overexpression of neuronal adaptor protein X11β has been shown to decrease the production of amyloid-β, a toxic peptide deposited in Alzheimer\\'s disease brains. Therefore, manipulation of the X11β level may represent a potential therapeutic strategy for Alzheimer\\'s disease. As X11β expression can be regulated at the transcription level, we determined the genomic organization and the promoter of the human X11β gene, amyloid β A4 precursor protein-binding family A member 2 (APBA2). By RNA ligase-mediated rapid amplification of cDNA ends, a single APBA2 transcription start site and the complete sequence of exon 1 were identified. The APBA2 promoter was located upstream of exon 1 and was more active in neurons. The core promoter contains several CpG dinucleotides, and was strongly suppressed by DNA methylation. In addition, mutagenesis analysis revealed a putative Pax5-binding site within the promoter. Together, APBA2 contains a potent neuronal promoter whose activity may be regulated by DNA methylation and Pax5.

  3. Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription. (United States)

    Knight, Britta; Kubik, Slawomir; Ghosh, Bhaswar; Bruzzone, Maria Jessica; Geertz, Marcel; Martin, Victoria; Dénervaud, Nicolas; Jacquet, Philippe; Ozkan, Burak; Rougemont, Jacques; Maerkl, Sebastian J; Naef, Félix; Shore, David


    In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output.

  4. Methylation of Promoter Regions of Genes of the Human Intrauterine Renin Angiotensin System and Their Expression

    Directory of Open Access Journals (Sweden)

    Shane D. Sykes


    Full Text Available The intrauterine renin angiotensin system (RAS is implicated in placentation and labour onset. Here we investigate whether promoter methylation of RAS genes changes with gestation or labour and if it affects gene expression. Early gestation amnion and placenta were studied, as were term amnion, decidua, and placenta collected before labour (at elective caesarean section or after spontaneous labour and delivery. The expression and degree of methylation of the prorenin receptor (ATP6AP2, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AGTR1, and two proteases that can activate prorenin (kallikrein, KLK1, and cathepsin D, CTSD were measured by qPCR and a DNA methylation array. There was no effect of gestation or labour on the methylation of RAS genes and CTSD. Amnion and decidua displayed strong correlations between the percent hypermethylation of RAS genes and CTSD, suggestive of global methylation. There were no correlations between the degree of methylation and mRNA abundance of any genes studied. KLK1 was the most methylated gene and the proportion of hypermethylated KLK1 alleles was lower in placenta than decidua. The presence of intermediate methylated alleles of KLK1 in early gestation placenta and in amnion after labour suggests that KLK1 methylation is uniquely dynamic in these tissues.

  5. Differential control of Bradyrhizobium japonicum iron stimulon genes through variable affinity of the iron response regulator (Irr) for target gene promoters and selective loss of activator function. (United States)

    Jaggavarapu, Siddharth; O'Brian, Mark R


    Bradyrhizobium japonicum Irr is a conditionally stable transcriptional activator and repressor that accumulates in cells under iron-limited, manganese-replete conditions, but degrades in a haem-dependent manner under high iron conditions, manganese limitation or upon exposure to H2 O2 . Here, we identified Irr-regulated genes that were relatively unresponsive to factors that promote Irr degradation. The promoters of those genes bound Irr with at least 200-fold greater affinity than promoters of the responsive genes, resulting in maintenance of promoter occupancy over a wide cellular Irr concentration range. For Irr-repressible genes, promoter occupancy correlated with transcriptional repression, resulting in differential levels of expression based on Irr affinity for target promoters. However, inactivation of positively controlled genes required neither promoter vacancy nor loss of DNA-binding activity by Irr. Thus, activation and repression functions of Irr may be uncoupled from each other under certain conditions. Abrogation of Irr activation function was haem-dependent, thus haem has two functionally separable roles in modulating Irr activity. The findings imply a greater complexity of control by Irr than can be achieved by conditional stability alone. We suggest that these regulatory mechanisms accommodate the differing needs for Irr regulon genes in response to the prevailing metabolic state of the cell.

  6. A novel interferon-inducible domain: structural and functional analysis of the human interferon regulatory factor 1 gene promoter.



    We have cloned and functionally characterized the human interferon regulatory factor 1 (IRF-1) gene promoter. The promoter contains a CpG island, with several GC boxes, a CAAT box, but no TATA box. IRF-1 mRNA is strongly induced by gamma interferon (IFN-gamma) but more weakly and transiently by IFN-alpha. There are several putative kappa B motifs and numerous AA(G/A)G(G/T)A and GAAANN motifs throughout the promoter. The IRF-1 promoter is not autoregulated by the IRF-1 gene product. IFN induci...

  7. The bovine Mx1 gene: characterization of the gene structure, alternative splicing, and promoter region. (United States)

    Kojima, Takatoshi; Oshima, Kazunaga; Watanabe, Hiroko; Komatsu, Masanori


    The Mx gene encodes an antiviral protein and is induced by type 1 interferons (IFNs). In this study, a new bovine Mx gene (designated Mx1B) was isolated from the endometrial cDNA library of the early pregnant cow. Although the Mx1B cDNA contained a single open reading frame (ORF) the same as the known Mx1, the 5' untranslated region (UTR) and 5' coding region of Mx1B were rather different from the corresponding regions of Mx1. Genomic structure analysis revealed that bovine Mx1B was an alternative splicing variant of Mx1 and had transcription regulatory sequences in the upstream region. RT-PCR and its sequencing identified another Mx1 splicing variant and demonstrated that these bovine Mx1 splicing variants were ubiquitously expressed in various tissues. Furthermore, it was found that all the bovine breeds investigated had identical splice sites of Mx1 and Mx1B. It is speculated that cattle have at least two functional Mx isoforms that might provide strong natural resistance to specific viruses.

  8. Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene. (United States)

    Dolferus, R; Jacobs, M; Peacock, W J; Dennis, E S


    The Adh (alcohol dehydrogenase, EC gene from Arabidopsis thaliana (L.) Heynh. can be induced by dehydration and cold, as well as by hypoxia. A 1-kb promoter fragment (CADH: -964 to +53) is sufficient to confer the stress induction and tissue-specific developmental expression characteristics of the Adh gene to a beta-glucuronidase reporter gene. Deletion mapping of the 5' end and site-specific mutagenesis identified four regions of the promoter essential for expression under the three stress conditions. Some sequence elements are important for response to all three stress treatments, whereas others are stress specific. The most critical region essential for expression of the Arabidopsis Adh promoter under all three environmental stresses (region IV: -172 to -141) contains sequences homologous to the GT motif (-160 to -152) and the GC motif (-147 to -144) of the maize Adh1 anaerobic responsive element. Region III (-235 to -172) contains two regions shown by R.J. Ferl and B.H. Laughner ([1989] Plant Mol Biol 12: 357-366) to bind regulatory proteins; mutation of the G-box-1 region (5'-CCACGTGG-3', -216 to -209) does not affect expression under uninduced or hypoxic conditions, but significantly reduces induction by cold stress and, to a lesser extent, by dehydration stress. Mutation of the other G-box-like sequence (G-box-2: 5'-CCAAGTGG-3', -193 to -182) does not change hypoxic response and affects cold and dehydration stress only slightly. G-box-2 mutations also promote high levels of expression under uninduced conditions. Deletion of region I (-964 to -510) results in increased expression under uninduced and all stress conditions, suggesting that this region contains a repressor binding site. Region II (-510 to -384) contains a positive regulatory element and is necessary for high expression levels under all treatments. PMID:7972489

  9. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Vu Hong; Tae, Seong Ho; Le, Nguyen Uyen Chi; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)


    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study.

  10. Characterization of a proximal Sp1 response element in the mouse Dlk2 gene promoter

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    Rivero Samuel


    Full Text Available Abstract Background DLK2 is an EGF-like membrane protein, closely related to DLK1, which is involved in adipogenesis. Both proteins interact with the NOTCH1 receptor and are able to modulate its activation. The expression of the gene Dlk2 is coordinated with that of Dlk1 in several tissues and cell lines. Unlike Dlk1, the mouse Dlk2 gene and its locus at chromosome 17 are not fully characterized. Results The goal of this work was the characterization of Dlk2 mRNA, as well as the analysis of the mechanisms that control its basal transcription. First, we analyzed the Dlk2 transcripts expressed by several mouse cells lines and tissues, and mapped the transcription start site by 5' Rapid Amplification of cDNA Ends. In silico analysis revealed that Dlk2 possesses a TATA-less promoter containing minimal promoter elements associated with a CpG island, and sequences for Inr and DPE elements. Besides, it possesses six GC-boxes, considered as consensus sites for the transcription factor Sp1. Indeed, we report that Sp1 directly binds to the Dlk2 promoter, activates its transcription, and regulates its level of expression. Conclusions Our results provide the first characterization of Dlk2 transcripts, map the location of the Dlk2 core promoter, and show the role of Sp1 as a key regulator of Dlk2 transcription, providing new insights into the molecular mechanisms that contribute to the expression of the Dlk2 gene.

  11. Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels.

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    Xian-E Peng

    Full Text Available Liver fatty acid-binding protein (L-FABP, also known as fatty acid-binding protein 1 (FABP1, is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182 from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032.Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05. The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01. In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = -0.320, P = 0.003, while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014. Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans.

  12. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

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    Lagerstedt Kristina


    Full Text Available Abstract Background Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Method Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Results Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4 showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3 were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Conclusions Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue.

  13. PARP1 Differentially Interacts with Promoter region of DUX4 Gene in FSHD Myoblasts (United States)

    Sharma, Vishakha; Pandey, Sachchida Nand; Khawaja, Hunain; Brown, Kristy J; Hathout, Yetrib; Chen, Yi-Wen


    Objective The goal of the study is to identity proteins, which interact with the promoter region of double homeobox protein 4 (DUX4) gene known to be causative for the autosomal dominant disorder Facioscapulohumeral Muscular Dystrophy (FSHD). Methods We performed a DNA pull down assay coupled with mass spectrometry analysis to identify proteins that interact with a DUX4 promoter probe in Rhabdomyosarcomca (RD) cells. We selected the top ranked protein poly (ADP-ribose) polymerase 1 (PARP1) from our mass spectrometry data for further ChIP-qPCR validation using patients' myoblasts. We then treated FSHD myoblasts with PARP1 inhibitors to investigate the role of PARP1 in the FSHD myoblasts. Results In our mass spectrometry analysis, PARP1 was found to be the top ranked protein interacting preferentially with the DUX4 promoter probe in RD cells. We further validated this interaction by immunoblotting in RD cells (2-fold enrichment compared to proteins pulled down by a control probe, pfisetin (0.5 mM), a polyphenol compound with PARP1 inhibitory property, for 24 h also suppressed the expression of DUX4 (44.8 fold, p<0.01) and ZSCAN4 (2.2 fold, p<0.05) in the FSHD myoblasts. We further showed that DNA methyltransferase 1 (DNMT1), a gene regulated by PARP1 was also enriched at the DUX4 promoter in RD cells through immunoblotting (2-fold, p<0.01) and immortalized FSHD myoblasts (42-fold, p<0.01) but not control myoblasts through ChIP qPCR. Conclusion Our results showed that PARP1 and DNMT1 interacted with DUX4 promoter and may be involved in modulating DUX4 expression in FSHD. PMID:27722032

  14. Functional analysis of promoter variants in the microsomal triglyceride transfer protein (MTTP) gene. (United States)

    Rubin, Diana; Schneider-Muntau, Alexandra; Klapper, Maja; Nitz, Inke; Helwig, Ulf; Fölsch, Ulrich R; Schrezenmeir, Jürgen; Döring, Frank


    The microsomal triglyceride transfer protein (MTTP) is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins from the intestine and liver. According to this function, polymorphic sites in the MTTP gene showed associations to low-density lipoprotein (LDL) cholesterol and related traits of the metabolic syndrome. Here we studied the functional impact of common MTTP promoter polymorphisms rs1800804:T>C (-164T>C), rs1800803:A>T (-400A>T), and rs1800591:G>T (-493G>T) using gene-reporter assays in intestinal Caco-2 and liver Huh-7 cells. Significant results were obtained in Huh-7 cells. The common MTTP promoter haplotype -164T/-400A/-493G showed about two-fold lower activity than the rare haplotype -164C/-400T/-493T. MTTP promoter mutant constructs -164T/-400A/-493T and -164T/-400T/-493T exhibited similar activity than the common haplotype. Activities of mutants -164C/-400A/-493G and -164C/-400A/-493T resembled the rare MTTP promoter haplotype. Electrophoretic mobility shift assays (EMSAs) revealed higher binding capacity of the transcriptional factor Sterol regulatory element binding protein1a (SREBP1a) to the -164T probe in comparison to the -164C probe. In conclusion, our study indicates that the polymorphism -164T>C mediates different activities of common MTTP promoter haplotypes via SREBP1a. This suggested that the already described SREBP-dependent modulation of MTTP expression by diet is more effective in -164T than in -164C carriers.

  15. Genomic structure, organisation, and promoter analysis of the bovine (Bos taurus) Mx1 gene. (United States)

    Gérardin, Joël A; Baise, Etienne A; Pire, Grégory A; Leroy, Michaël P-P; Desmecht, Daniel J-M


    Some MX proteins are known to confer a specific resistance against a panel of single-stranded RNA viruses. Many diseases due to such viruses are known to affect cattle worldwide, raising the possibility that the identification of an antiviral isoform of a bovine MX protein would allow the implementation of genetic selection programs aimed at improving innate resistance of cattle. With this potential application in mind, the present study was designed to isolate the bovine Mx1 gene including its promoter region and to investigate its genomic organisation and promoter reactivity. The bovine Mx1 gene is made up of 15 exons. All exon-intron boundaries conformed to the consensus sequences. A PCR product that contained a approximately 1-kb, 5'-flanking region upstream from the putative transcription start site was sequenced. Unexpectedly, this DNA region did not contain TATA or CCAAT motifs. A computer scan of the region disclosed a series of putative binding sites for known cytokines and transcription factors. There was a GAAAN(1-2)GAAA(C/G) motif, typical of an interferon-sensitive responsive element, between -118 and -107 from the putative transcription start site. There were also a NF-kappaB, two interleukin-6 binding sites, two Sp1 sites and five GC-rich boxes. The region also contained 12 stretches of the GAAA type, as described in all IFN-inducible genes. Bovine Mx1 expression was assessed by Northern blotting and immunofluorescence in the Madin Darby bovine kidney cells (MDBK) cell line treated with several stimuli. In conclusion, the bovine Mx1 gene and promoter region share the major structural and functional characteristics displayed by their homologs described in the rainbow trout, chicken, mouse and man.

  16. Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

    Institute of Scientific and Technical Information of China (English)

    CHEN XiaoGuang; ZHANG YaJing; ZHENG XueLi; WANG ChunMei


    The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles stephensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I digestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively conserved in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.

  17. Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes

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    Rowe J


    Full Text Available Abstract Background Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes. The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusion This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.

  18. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans. (United States)

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S


    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

  19. Coactivator CBP/p300 increased the activity of C/EBP-mediated human interleukin-5 gene promoter

    Institute of Scientific and Technical Information of China (English)

    LIU Chunyan; LU Jun; LI Lin; TAN Jiang; SHAO Yangguang; HUANG Baiqu


    Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an essential role in the development of allergic diseases, such as allergic asthma. Histone acetyltransferase CBP/p300 remodels chromatin by acetylating histones, resulting in open structure of chromatin and active transcription. Adenovirus protein E1A inhibits the activity of CBP/p300. In this study, we analyzed the effects of E1A on IL-5 gene promoter/luciferase reporter activity. The results showed that E1A protein inhibited the activity of PMA/ionomycin-stimulated IL-5 gene promoter/luciferase reporter construct. In contrast, overexpression of the CBP/p300-binding defective E1A A2-36 protein did not inhibit IL-5 gene promoter activity. These data demonstrated for the first time that transcriptional coactivator CBP/p300was involved in the activation of IL-5 gene promoter. E1A protein can modulate CBP/p300 function to activate the transcription of IL-5 gene promoter/luciferase reporter plasmid. Furthermore, in collaboration with transcription factor C/EBP, CBP/p300 activated IL-5 gene promoter/luciferase reporter expression. This study provides further insight into the mechanisms of transcriptional regulation of IL-5 gene.

  20. Isolation and Analysis of the Cppsy Gene and Promoter from Chlorella protothecoides CS-41. (United States)

    Li, Meiya; Cui, Yan; Gan, Zhibing; Shi, Chunlei; Shi, Xianming


    Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg(2+) binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.

  1. Rarity of DNA sequence alterations in the promoter region of the human androgen receptor gene

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    D.F. Cabral


    Full Text Available The human androgen receptor (AR gene promoter lies in a GC-rich region containing two principal sites of transcription initiation and a putative Sp1 protein-binding site, without typical "TATA" and "CAAT" boxes. It has been suggested that mutations within the 5'untranslated region (5'UTR may contribute to the development of prostate cancer by changing the rates of gene transcription and/or translation. In order to investigate this question, the aim of the present study was to search for the presence of mutations or polymorphisms at the AR-5'UTR in 92 prostate cancer patients, where histological diagnosis of adenocarcinoma was established in specimens obtained from transurethral resection or after prostatectomy. The AR-5'UTR was amplified by PCR from genomic DNA samples of the patients and of 100 healthy male blood donors, included as controls. Conformation-sensitive gel electrophoresis was used for DNA sequence alteration screening. Only one band shift was detected in one individual from the blood donor group. Sequencing revealed a new single nucleotide deletion (T in the most conserved portion of the promoter region at position +36 downstream from the transcription initiation site I. Although the effect of this specific mutation remains unknown, its rarity reveals the high degree of sequence conservation of the human androgen promoter region. Moreover, the absence of detectable variation within the critical 5'UTR in prostate cancer patients indicates a low probability of its involvement in prostate cancer etiology.

  2. Transgenic Studies with a Keratin Promoter-Driven Growth Hormone Transgene: Prospects for Gene Therapy (United States)

    Wang, Xiaoming; Zinkel, Sandra; Polonsky, Kenneth; Fuchs, Elaine


    Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at ≈ 1/10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.

  3. Integrin α9 gene promoter is hypermethylated and downregulated in nasopharyngeal carcinoma (United States)

    Hu, Li-Fu; Moumad, Khalid; Pavlova, Tatiana V.; Kashuba, Vladimir; Almgren, Malin; Zabarovsky, Eugene R.; Ernberg, Ingemar


    Epigenetic silencing of tumor suppressor genes (TSGs) by promoter methylation can be an early event in the multi-step process of carcinogenesis. Human chromosome 3 contains clusters of TSGs involved in many cancer types including nasopharyngeal carcinoma (NPC), the most common cancer in Southern China. Among ten candidate TSGs identified in chromosome 3 using NotI microarray, ITGA9 and WNT7A could be validated. 5′-aza-2′ deoxycytidine treatment restored the expression of ITGA9 and WNT7A in two NPC cell lines. Immunostaining showed strong expression of these genes in the membrane and cytoplasm of adjacent control nasopharyngeal epithelium cells, while they were weakly expressed in NPC tumor cells. The ITGA9 promoter showed marked differentially methylation between tumor and control tissue, whereas no differentially methylation could be detected for the WNT7A promoter. The expression level of ITGA9 in NPC tumors was downregulated 4.9-fold, compared to the expression in control. ITGA9 methylation was detected by methylation specific PCR (MSP) in 56% of EBV positive NPC- cases with 100% specificity. Taken together, this suggests that ITGA9 might be a TSG in NPC that is involved in tumor cell biology. The possibility of using ITGA9 methylation as a marker for early detection of NPC should further be explored. PMID:26372814

  4. Isolation and Analysis of the Cppsy Gene and Promoter from Chlorella protothecoides CS-41

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    Meiya Li


    Full Text Available Phytoene synthase (PSY catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg2+ binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.

  5. Association of the Resistin Gene Promoter Region Polymorphism with Kawasaki Disease in Chinese Children

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    Ruixi Liu


    Full Text Available Objectives. The −420C>G polymorphism located in the resistin gene (RETN promoter has recently been suggested to play a potential role in proinflammatory conditions and cardiovascular disease. This study investigated the association of the RETN promoter polymorphism with Kawasaki disease (KD and its clinical parameters in Chinese children. Methods. We compared patients with complete KD to incomplete KD children. Genotyping of the RETN promoter polymorphism was performed using MassARRAY system, and serum resistin levels were estimated using the sandwich enzyme immunoassay method. Results. There was no significant difference in RETN (−420C>G genotypes between KD and control groups. However, the frequency of the G allele was higher in iKD patients than in cKD children due to a significantly increased frequency of the GG genotypes. Serum levels of resistin were significantly higher in KD patients than in controls regardless of the presence of coronary artery lesions (CALs. Conclusion. The present findings suggest that while resistin may play a role in the pathogenesis of KD, there is no apparent association between CAL and the RETN (−420C>G gene polymorphism in KD children. However, the diagnosis of iKD is challenging but can be supported by the presence of the G allele and the GG genotypes.

  6. Baculovirus-mediated promoter assay and transcriptional analysis of white spot syndrome virus orf427 gene

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    Yu Li


    Full Text Available Abstract Background White spot syndrome virus (WSSV is an important pathogen of the penaeid shrimp with high mortalities. In previous reports, Orf427 of WSSV is characterized as one of the three major latency-associated genes of WSSV. Here, we were interested to analyze the promoter of orf427 and its expression during viral pathogenesis. Results in situ hybridization revealed that orf427 was transcribed in all the infected tissues during viral lytic infection and the translational product can be detected from the infected shrimp. A time-course RT-PCR analysis indicated that transcriptional products of orf427 could only be detected after 6 h post virus inoculation. Furthermore, a baculovirus-mediated promoter analysis indicated that the promoter of orf427 failed to express the EGFP reporter gene in both insect SF9 cells and primary shrimp cells. Conclusion Our data suggested that latency-related orf427 might not play an important role in activating virus replication from latent phase due to its late transcription during the lytic infection.

  7. Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey

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    Tung Shu-Yun


    Full Text Available Abstract Background Orchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as Arabidopsis thaliana do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied. Results Several molecular biology tools were used to isolate flower-specific gene promoters from Oncidium 'Gower Ramsey' (Onc. GR. A cDNA library of reproductive tissues was used to construct a microarray in order to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues, and the subcellular locations of the corresponding proteins were identified using lip transient transformation with fluorescent protein-fusion constructs. BAC clones of the 5 genes, together with 7 previously published flower- and reproductive growth-specific genes in Onc. GR, were identified for cloning of their promoter regions. Interestingly, 3 of the 5 novel flower-abundant genes were putative trypsin inhibitor (TI genes (OnTI1, OnTI2 and OnTI3, which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable A. thaliana transformation analyses. Conclusions By combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.

  8. Novel characterisation of the gene encoding conglutinin reveals that previously characterised promoter corresponds to the CL-43 promoter

    DEFF Research Database (Denmark)

    Hansen, Soren; Moeller, Vivi; Holm, Dorte;


    '-flanking region shows 95.8% identity with the sequence previously published, which on the other hand shows 99.7% identity with the CL-43 promoter. We conclude that the previously published promoter corresponds to the CL-43 promoter and that the functional studies performed on it apply to transcription...

  9. Cloning and molecular characterisation of resuscitation promoting factor-like gene from Mycobacterium avium subspecies avium

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    R Kavitha


    Full Text Available Purpose: Resuscitation promoting factor (Rpf-like gene of Mycobacterium avium subspecies paratuberculosis has been known to stimulate the growth of mycobacteria and enhances the recovery of replicating cells from non-replicating phases. The objective of the study was to produce recombinant rpf-like protein of M. avium subspecies avium protein for purification and physico-chemical characterisation. Materials and Methods: The identified rpf gene of M. avium subspecies avium was cloned, subcloned, sequenced and expressed in Escherichia coli expression system for the production of the recombinant protein. The expressed recombinant Rpf protein was confirmed by Western blot and the extract was purified to yield a pure recombinant protein. Results: An rpf-like gene of 675 bp size in the M. avium subspecies avium was identified. This gene was expressed and the recombinant Rpf weighed 65 kDa as confirmed by Western blot. The M. avium recombinant Rpf protein was extracted under denatured conditions and purified yielding a recombinant protein with >90% purity. Conclusions: Identification, cloning, sequencing and expression of a rpf-like gene from M. avium suggest that RpfA is present in this species also, which might be involved in reactivation phenomenon in this high-risk pathogen.

  10. Distinguishing the Transcription Regulation Patterns in Promoters of Human Genes with Different Function or Evolutionary Age

    KAUST Repository

    Alam, Tanvir


    Distinguishing transcription regulatory patterns of different gene groups is a common problem in various bioinformatics studies. In this work we developed a methodology to deal with such a problem based on machine learning techniques. We applied our method to two biologically important problems related to detecting a difference in transcription regulation of: a/ protein-coding and long non-coding RNAs (lncRNAs) in human, as well as b/ a difference between primate-specific and non-primate-specific long non-coding RNAs. Our method is capable to classify RNAs using various regulatory features of genes that transcribe into these RNAs, such as nucleotide frequencies, transcription factor binding sites, de novo sequence motifs, CpG islands, repetitive elements, histone modification marks, and others. Ten-fold cross-validation tests suggest that our model can distinguish protein-coding and non-coding RNAs with accuracy above 80%. Twenty-fold cross-validation tests suggest that our model can distinguish primate-specific from non-primate-specific promoters of lncRNAs with accuracy above 80%. Consequently, we can hypothesize that transcription of the groups of genes mentioned above are regulated by different mechanisms. Feature selection techniques allowed us to reduce the number of features significantly while keeping the accuracy around 80%. Consequently, we can conclude that selected features play significant role in transcription regulation of coding and non-coding genes, as well as primate-specific and non-primate-specific lncRNA genes.

  11. Monoallelic loss of the imprinted gene Grb10 promotes tumor formation in irradiated Nf1+/- mice.

    Directory of Open Access Journals (Sweden)

    Rana Mroue


    Full Text Available Imprinted genes are expressed from only one parental allele and heterozygous loss involving the expressed allele is sufficient to produce complete loss of protein expression. Genetic alterations are common in tumorigenesis but the role of imprinted genes in this process is not well understood. In earlier work we mutagenized mice heterozygous for the Neurofibromatosis I tumor suppressor gene (NF1 to model radiotherapy-associated second malignant neoplasms that arise in irradiated NF1 patients. Expression analysis of tumor cell lines established from our mouse models identified Grb10 expression as widely absent. Grb10 is an imprinted gene and polymorphism analysis of cell lines and primary tumors demonstrates that the expressed allele is commonly lost in diverse Nf1 mutant tumors arising in our mouse models. We performed functional studies to test whether Grb10 restoration or loss alter fundamental features of the tumor growth. Restoring Grb10 in Nf1 mutant tumors decreases proliferation, decreases soft agar colony formation and downregulates Ras signaling. Conversely, Grb10 silencing in untransformed mouse embryo fibroblasts significantly increased cell proliferation and increased Ras-GTP levels. Expression of a constitutively activated MEK rescued tumor cells from Grb10-mediated reduction in colony formation. These studies reveal that Grb10 loss can occur during in vivo tumorigenesis, with a functional consequence in untransformed primary cells. In tumors, Grb10 loss independently promotes Ras pathway hyperactivation, which promotes hyperproliferation, an early feature of tumor development. In the context of a robust Nf1 mutant mouse model of cancer this work identifies a novel role for an imprinted gene in tumorigenesis.

  12. Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis.

    Directory of Open Access Journals (Sweden)

    Zhichun Zhang

    Full Text Available Mutation of distal-less homeobox 3 (DLX3 is responsible for human tricho-dento-osseous syndrome (TDO with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.

  13. Reactivating aberrantly hypermethylated p15 gene in leukemic T cells by a phenylhexyl isothiocyanate mediated inter-active mechanism on DNA and chromatin

    Directory of Open Access Journals (Sweden)

    Jiang Shaohong


    Full Text Available Abstract Background We have previously demonstrated that phenylhexyl isothiocyanate (PHI, a synthetic isothiocyanate, inhibits histone deacetylases and remodels chromatins to induce growth arrest in HL-60 myeloid leukemia cells in a concentration-dependent manner. Methods To investigate the effect of PHI, a novel histone deacetylases inhibitor (HDACi, on demethylation and activation of transcription of p15 in acute lymphoid leukemia cell line Molt-4, and to further decipher the potential mechanism of demethylation, DNA sequencing and modified methylation specific PCR (MSP were used to screen p15-M and p15-U mRNA after Molt-4 cells were treated with PHI, 5-Aza and TSA. DNA methyltransferase 1 (DNMT1, 3A (DNMT3A, 3B (DNMT3B and p15 mRNA were measured by RT-PCR. P15 protein, acetylated histone H3 and histone H4 were detected by Western Blot. Results The gene p15 in Molt-4 cells was hypermethylated and inactive. Hypermethylation of gene p15 was attenuated and p15 gene was activated de novo after 5 days exposure to PHI in a concentration-dependent manner. DNMT1 and DNMT3B were inhibited by PHI (P Conclusion PHI could induce both DNA demethylation and acetylated H3 and H4 accumulation in Molt-4 cells. Hypermethylation of gene p15 was reversed and p15 transcription could be reactivated de novo by PHI.

  14. Epigenetic Perturbations by Arg882-Mutated DNMT3A Potentiate Aberrant Stem Cell Gene-Expression Program and Acute Leukemia Development. (United States)

    Lu, Rui; Wang, Ping; Parton, Trevor; Zhou, Yang; Chrysovergis, Kaliopi; Rockowitz, Shira; Chen, Wei-Yi; Abdel-Wahab, Omar; Wade, Paul A; Zheng, Deyou; Wang, Gang Greg


    DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, we report that the DNMT3A mutational hotspot at Arg882 (DNMT3A(R882H)) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3A(R882H) directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1, and Hoxa gene cluster. DNMT3A(R882H) induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3A(R882H)-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3A(R882H)-induced gene-expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.

  15. Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli beta-glucuronidase gene and analysis of its expression in Aspergillus oryzae. (United States)

    Tada, S; Gomi, K; Kitamoto, K; Takahashi, K; Tamura, G; Hara, S


    Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) down-stream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

  16. Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter. (United States)

    Liu, Dan; Liu, Xin; Wu, Ye; Wang, Wen; Ma, Xinliang; Liu, Huirong


    HtrA serine peptidase 2 (HtrA2), also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at -1205~-838 bp and -146~+93 bp, with the -838~-649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at -709~+37 bp, and eight heat shock transcription factor 1 (HSF1) sites, two Sp1 transcription factor (SP1)sites, one activator protein (AP) site, seven p53 sites, and four YY1 transcription factor(YY1) sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases.

  17. Defining the cutoff value of MGMT gene promoter methylation and its predictive capacity in glioblastoma. (United States)

    Brigliadori, Giovanni; Foca, Flavia; Dall'Agata, Monia; Rengucci, Claudia; Melegari, Elisabetta; Cerasoli, Serenella; Amadori, Dino; Calistri, Daniele; Faedi, Marina


    Despite advances in the treatment of glioblastoma (GBM), median survival is 12-15 months. O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status is acknowledged as a predictive marker for temozolomide (TMZ) treatment. When MGMT promoter values fall into a "methylated" range, a better response to chemotherapy is expected. However, a cutoff that discriminates between "methylated" and "unmethylated" status has yet to be defined. We aimed to identify the best cutoff value and to find out whether variability in methylation profiles influences the predictive capacity of MGMT promoter methylation. Data from 105 GBM patients treated between 2008 and 2013 were analyzed. MGMT promoter methylation status was determined by analyzing 10 CpG islands by pyrosequencing. Patients were treated with radiotherapy followed by TMZ. MGMT promoter methylation status was classified into unmethylated 0-9 %, methylated 10-29 % and methylated 30-100 %. Statistical analysis showed that an assumed methylation cutoff of 9 % led to an overestimation of responders. All patients in the 10-29 % methylation group relapsed before the 18-month evaluation. Patients with a methylation status ≥30 % showed a median overall survival of 25.2 months compared to 15.2 months in all other patients, confirming this value as the best methylation cutoff. Despite wide variability among individual profiles, single CpG island analysis did not reveal any correlation between single CpG island methylation values and relapse or death. Specific CpG island methylation status did not influence the predictive value of MGMT. The predictive role of MGMT promoter methylation was maintained only with a cutoff value ≥30 %.

  18. Genomic structure, promoter analysis, and expression of the porcine (Sus scrofa) Mx1 gene. (United States)

    Thomas, Anne V; Palm, Melanie; Broers, Aurore D; Zezafoun, Hussein; Desmecht, Daniel J-M


    Allelic polymorphisms at the mouse Mx1 locus affect the probability of survival after experimental influenzal disease, raising the possibility that marker-assisted selection using the homologous locus could improve the innate resistance of pigs to natural influenza infections. Several issues need to be resolved before efficient large scale screening of the allelic polymorphism at the porcine (Sus scrofa) Mx1 locus can be implemented. First, the Mx1 genomic structure has to be established and sufficient flanking intronic sequences have to be gathered to enable simple PCR amplification of the coding portions of the gene. Then, a basic knowledge of the promoter region needs to be obtained as an allelic variation there can significantly alter absolute levels and/or tissue-specificity of MX protein expression. The results gathered here show that the porcine Mx1 gene and promoter share the major structural and functional characteristics displayed by their homologs described in cattle, mouse, chicken, and man. The crucial function of the proximal interferon-sensitive response elements motif for gene expression is also demonstrated. The sequence data compiled here will allow an extensive analysis of the polymorphisms present among the widest spectrum possible of porcine breeds with the aim to identify an Mx1 allele providing antiviral resistance.

  19. Identification of the core promoter of STK11 gene and its transcriptional regulation by p53

    Institute of Scientific and Technical Information of China (English)

    Maojin Yao; Chenjie Li; Yi Chu; Fei Wang; Xiaoliu Shi; Yongjun Wang; Hongwei Shen; Wenfeng Ning; Jianguang Tang; Xiangping Wang; Jie Li; Shiquang Zhou; Xin Yi


    Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by mucocutaneous pigmentation and hamartomatous polyps. Most cases of PJS involve the inactivation of germline mutations in the serine/threonine kinase gene STK11 which is also known as LKB1. The function of STK11 was previously linked to the tumor suppressor p53 and was shown to activate the p53 target p21/ WAF1. Recently, STK11 was reported to be interacting with p53 physically in the nucleus and it can directly or indirectly phosphorylate p53. Here we characterized the 5'-flanking region of human STK11 gene and identified a 161-bp fragment with promoter activity. Sequence analysis, mutagenesis and gel shift studies revealed a binding site of Spl and p53, which affects the promoter activity. Mutation analyses showed that this fragment was required for p53-mediated transcriptional activation. This transcriptional activation was further confirmed by real-time quantitative RT-PCR and Western blot analysis. Transient transfection of p53 expression plasmid into fetal liver cell lines increased STK11 mRNA and protein levels. In conclusion, our results reveal a new role for p53 in elevating STK11 gene expression via a positive feedback pattern.

  20. Variants in the dopamine-4-receptor gene promoter are not associated with sensation seeking in skiers. (United States)

    Thomson, Cynthia J; Rajala, Amelia K; Carlson, Scott R; Rupert, Jim L


    Sensation seeking is a personality trait that has been associated with disinhibited behaviours including substance use and gambling, but also with high-risk sport practices including skydiving, paragliding, and downhill skiing. Twin studies have shown that sensation seeking is moderately heritable, and candidate genes encoding components involved in dopaminergic transmission have been investigated as contributing to this type of behaviour. To determine whether variants in the regulatory regions of the dopamine-4-receptor gene (DRD4) influenced sport-specific sensation seeking, we analyzed five polymorphisms (-1106T/C, -906T/C, -809G/A, -291C/T, 120-bp duplication) in the promoter region of the gene in a cohort of skiers and snowboarders (n = 599) that represented a broad range of sensation seeking behaviours. We grouped subjects by genotype at each of the five loci and compared impulsive sensation seeking and domain-specific (skiing) sensation seeking between groups. There were no significant associations between genotype(s) and general or domain-specific sensation seeking in the skiers and snowboarders, suggesting that while DRD4 has previously been implicated in sensation seeking, the promoter variants investigated in this study do not contribute to sensation seeking in this athlete population.

  1. Variants in the dopamine-4-receptor gene promoter are not associated with sensation seeking in skiers.

    Directory of Open Access Journals (Sweden)

    Cynthia J Thomson

    Full Text Available Sensation seeking is a personality trait that has been associated with disinhibited behaviours including substance use and gambling, but also with high-risk sport practices including skydiving, paragliding, and downhill skiing. Twin studies have shown that sensation seeking is moderately heritable, and candidate genes encoding components involved in dopaminergic transmission have been investigated as contributing to this type of behaviour. To determine whether variants in the regulatory regions of the dopamine-4-receptor gene (DRD4 influenced sport-specific sensation seeking, we analyzed five polymorphisms (-1106T/C, -906T/C, -809G/A, -291C/T, 120-bp duplication in the promoter region of the gene in a cohort of skiers and snowboarders (n = 599 that represented a broad range of sensation seeking behaviours. We grouped subjects by genotype at each of the five loci and compared impulsive sensation seeking and domain-specific (skiing sensation seeking between groups. There were no significant associations between genotype(s and general or domain-specific sensation seeking in the skiers and snowboarders, suggesting that while DRD4 has previously been implicated in sensation seeking, the promoter variants investigated in this study do not contribute to sensation seeking in this athlete population.

  2. Dissection of cis-regulatory elements in the C. elegans Hox gene egl-5 promoter. (United States)

    Teng, Yingqi; Girard, Lisa; Ferreira, Henrique B; Sternberg, Paul W; Emmons, Scott W


    Hox genes are highly conserved segmental identity genes well known for their complex expression patterns and divergent targets. Here we present an analysis of cis-regulatory elements in the Caenorhabditis elegans Hox gene egl-5, which is expressed in multiple tissues in the posterior region of the nematode. We have utilized phylogenetic footprinting to efficiently identify cis-regulatory elements and have characterized these with gfp reporters and tissue-specific rescue experiments. We have found that the complex expression pattern of egl-5 is the cumulative result of the activities of multiple tissue or local region-specific activator sequences that are conserved both in sequence and near-perfect order in the related nematode Caenorhabditis briggsae. Two conserved regulatory blocks analyzed in detail contain multiple sites for both positively and negatively acting factors. One of these regions may promote activation of egl-5 in certain cells via the Wnt pathway. Positively acting regions are repressed in inappropriate tissues by additional negative pathways acting at other sites within the promoter. Our analysis has allowed us to implicate several new regulatory factors significant to the control of egl-5 expression.

  3. Aberration Correction in Electron Microscopy

    CERN Document Server

    Rose, Harald H


    The resolution of conventional electron microscopes is limited by spherical and chromatic aberrations. Both defects are unavoidable in the case of static rotationally symmetric electromagnetic fields (Scherzer theorem). Multipole correctors and electron mirrros have been designed and built, which compensate for these aberrations. The principles of correction will be demonstrated for the tetrode mirror, the quadrupole-octopole corrector and the hexapole corrector. Electron mirrors require a magnetic beam separator free of second-order aberrations. The multipole correctors are highly symmetric telescopic systems compensating for the defects of the objective lens. The hexapole corrector has the most simple structure yet eliminates only the spherical aberration, whereas the mirror and the quadrupole-octopole corrector are able to correct for both aberrations. Chromatic correction is achieved in the latter corrector by cossed electric and magnetic quadrupoles acting as first-order Wien filters. Micrographs obtaine...

  4. Camera processing with chromatic aberration. (United States)

    Korneliussen, Jan Tore; Hirakawa, Keigo


    Since the refractive index of materials commonly used for lens depends on the wavelengths of light, practical camera optics fail to converge light to a single point on an image plane. Known as chromatic aberration, this phenomenon distorts image details by introducing magnification error, defocus blur, and color fringes. Though achromatic and apochromatic lens designs reduce chromatic aberration to a degree, they are complex and expensive and they do not offer a perfect correction. In this paper, we propose a new postcapture processing scheme designed to overcome these problems computationally. Specifically, the proposed solution is comprised of chromatic aberration-tolerant demosaicking algorithm and post-demosaicking chromatic aberration correction. Experiments with simulated and real sensor data verify that the chromatic aberration is effectively corrected.

  5. Cloning of Promoter of Chinese Bean GRP 1.8 Gene and Characterization of Its Function in Transgenic Tobacco Plants

    Institute of Scientific and Technical Information of China (English)


    In order to learn the expression pattern of GRP1.8(glycine-rich-protein) gene promoter in transgenic plants and to explore its potential application in plant genetic engineering for vascular-specific expression of interested genes, GRP 1.8 promoter was amplified by PCR from Chinese bean genomic DNA. The intermediate vector was constructed by inserting vascular-specific expression promoter of GRP 1.8 gene in vector pBI 101. The regenerated tobacco plants obtained were analyzed by PCR to select the putative transgenic plants. The histochemical localization of GUS(β-D-glucosidase) activity indicates that as for that of GRP 1.8 promoter we can confer the vascular-specific expression of GUS gene.

  6. Relationship between promoter methylation of the Runx3 and Rassf1a genes and Dnmt1 expression in gastric cancer

    Institute of Scientific and Technical Information of China (English)



    Objective To analyze the promoter methylation of the human runt-related transcription factor3(Runx3) and ras-association domain family1a(Rassf1a) genes and Dnmt1protein expression in gastric cancer and to

  7. Cancer genes hypermethylated in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Vincenzo Calvanese

    Full Text Available Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

  8. Detection of differentially methylated gene promoters in failing and nonfailing human left ventricle myocardium using computation analysis. (United States)

    Koczor, Christopher A; Lee, Eva K; Torres, Rebecca A; Boyd, Amy; Vega, J David; Uppal, Karan; Yuan, Fan; Fields, Earl J; Samarel, Allen M; Lewis, William


    Human dilated cardiomyopathy (DCM) is characterized by congestive heart failure and altered myocardial gene expression. Epigenetic changes, including DNA methylation, are implicated in the development of DCM but have not been studied extensively. Clinical human DCM and nonfailing control left ventricle samples were individually analyzed for DNA methylation and expressional changes. Expression microarrays were used to identify 393 overexpressed and 349 underexpressed genes in DCM (GEO accession number: GSE43435). Gene promoter microarrays were utilized for DNA methylation analysis, and the resulting data were analyzed by two different computational methods. In the first method, we utilized subtractive analysis of DNA methylation peak data to identify 158 gene promoters exhibiting DNA methylation changes that correlated with expression changes. In the second method, a two-stage approach combined a particle swarm optimization feature selection algorithm and a discriminant analysis via mixed integer programming classifier to identify differentially methylated gene promoters. This analysis identified 51 hypermethylated promoters and six hypomethylated promoters in DCM with 100% cross-validation accuracy in the group assignment. Generation of a composite list of genes identified by subtractive analysis and two-stage computation analysis revealed four genes that exhibited differential DNA methylation by both methods in addition to altered gene expression. Computationally identified genes (AURKB, BTNL9, CLDN5, and TK1) define a central set of differentially methylated gene promoters that are important in classifying DCM. These genes have no previously reported role in DCM. This study documents that rigorous computational analysis applied to microarray analysis of healthy and diseased human heart samples helps to define clinically relevant DNA methylation and expressional changes in DCM.

  9. Scattered regulatory regions of the chicken immunoglobulin-β gene and two adjacent promoters of ubiquitously expressed genes interact with the immunoglobulin-β promoter in DT40 cells. (United States)

    Minbuta, Tomohiro; Ono, Masao


    Recent studies indicate that several transcription units assemble to form a 'transcription factory' where active transcription occurs in the nuclei. Previously, we generated chicken B-lymphocyte-derived DT40 cells lacking six transcriptional regulatory regions scattered in and around the immunoglobulin (Ig)-β gene. The deletions caused a complete shut down of transcription and epigenetic regulation of the Ig-β gene, demonstrating that the scattered regulatory regions cooperated in the transcriptional and epigenetic regulation of the gene. However, the in vivo 3-dimensional spatial relationships between the Ig-β promoter and these six regulatory regions were not investigated. In this study, we used chromosome conformation capture (3C) technology and demonstrated that the Ig-β promoter physically interacted with the scattered regulatory regions. We found that the Ig-β promoter also interacted with two downstream promoters of ubiquitously expressed genes, rad motif 1 (RDM1) and Plekhm1, to form a transcription factory, but not with three ubiquitously expressed genes, BAF60b, p45/SUG, and RRMJ3, located upstream of the Ig-β gene. In this factory, the chromatin from the three promoters and the scattered regulatory regions of the Ig-β gene formed a complex structure with many chromatin loops.

  10. Distinct promoter activation mechanisms modulate noise-driven HIV gene expression (United States)

    Chavali, Arvind K.; Wong, Victor C.; Miller-Jensen, Kathryn


    Latent human immunodeficiency virus (HIV) infections occur when the virus occupies a transcriptionally silent but reversible state, presenting a major obstacle to cure. There is experimental evidence that random fluctuations in gene expression, when coupled to the strong positive feedback encoded by the HIV genetic circuit, act as a ‘molecular switch’ controlling cell fate, i.e., viral replication versus latency. Here, we implemented a stochastic computational modeling approach to explore how different promoter activation mechanisms in the presence of positive feedback would affect noise-driven activation from latency. We modeled the HIV promoter as existing in one, two, or three states that are representative of increasingly complex mechanisms of promoter repression underlying latency. We demonstrate that two-state and three-state models are associated with greater variability in noisy activation behaviors, and we find that Fano factor (defined as variance over mean) proves to be a useful noise metric to compare variability across model structures and parameter values. Finally, we show how three-state promoter models can be used to qualitatively describe complex reactivation phenotypes in response to therapeutic perturbations that we observe experimentally. Ultimately, our analysis suggests that multi-state models more accurately reflect observed heterogeneous reactivation and may be better suited to evaluate how noise affects viral clearance.

  11. LuxCDE-luxAB-based promoter reporter system to monitor the Yersinia enterocolitica O:3 gene expression in vivo (United States)

    Bozcal, Elif; Dagdeviren, Melih; Uzel, Atac


    It is crucial to understand the in vitro and in vivo regulation of the virulence factor genes of bacterial pathogens. In this study, we describe the construction of a versatile reporter system for Yersinia enterocolitica serotype O:3 (YeO3) based on the luxCDABE operon. In strain YeO3-luxCDE we integrated the luciferase substrate biosynthetic genes, luxCDE, into the genome of the bacterium so that the substrate is constitutively produced. The luxAB genes that encode the luciferase enzyme were cloned into a suicide vector to allow cloning of any promoter-containing fragment upstream the genes. When the obtained suicide-construct is mobilized into YeO3-luxCDE bacteria, it integrates into the recipient genome via homologous recombination between the cloned promoter fragment and the genomic promoter sequence and thereby generates a single-copy and stable promoter reporter. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core hexasaccharide (OC) of YeO3 are virulence factors necessary to colonization of the intestine and establishment of infection. To monitor the activities of the OC and O-ag gene cluster promoters we constructed the reporter strains YeO3-Poc::luxAB and YeO3-Pop1::luxAB, respectively. In vitro, at 37°C both promoter activities were highest during logarithmic growth and decreased when the bacteria entered stationary growth phase. At 22°C the OC gene cluster promoter activity increased during the late logarithmic phase. Both promoters were more active in late stationary phase. To monitor the promoter activities in vivo, mice were infected intragastrically and the reporter activities monitored by the IVIS technology. The mouse experiments revealed that both LPS promoters were well expressed in vivo and could be detected by IVIS, mainly from the intestinal region of orally infected mice. PMID:28235077

  12. A highly specific pathogen-responsive promoter element from the immediate-early activated CMPG1 gene in Petroselinum crispum. (United States)

    Kirsch, C; Logemann, E; Lippok, B; Schmelzer, E; Hahlbrock, K


    Within the complex signalling network from pathogen-derived elicitor perception to defense-related gene activation, some immediate-early responding genes may have pivotal roles in downstream transcriptional regulation. We have identified the parsley (Petroselinum crispum) ELI17 gene as a particularly fast-responding gene possessing a new type of W box-containing, elicitor-responsive promoter element, E17. Highly selective E17-mediated reporter gene expression at pathogen infection sites in transgenic Arabidopsis thaliana plants demonstrated the potential of this promoter element for designing new strategies in resistance breeding as well as for further analysis of the early components of defense-related gene activation mechanisms. The protein encoded by the ELI17 gene exhibits various structural characteristics of established transcription factors and is designated as a CMPG protein according to the first four strictly conserved amino acids defining a newly emerging class of plant-specific proteins.

  13. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus. (United States)

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin


    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment bet