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J. Biol. Chem. 282, 14194-14204. Regulation of the interleukin-7 receptor α-promoter by the Ets transcription factors PU.1 and GA-binding protein in developing B cells. 2007

Dekoter, R.P., Schweitzer, B.L., Kamath, M.B., Jones, D., Tagoh, H., Bonifer, C., Hildeman, D.A., and Huang, K.J.

Notes: The interleukin-7 receptor is composed of γ and α subunits, encoded by the genes il7rg and il7r, respectively. The α subunit is expressed in developing B cells and is downregulated upon maturation. These authors investigated the mechanisms of transcriptional regulation of the il7r gene using 5´ RACE, EMSA, RNA interference and chromatin immunoprecipitation analyses. Potential promoter regions identified by 5´ RACE analysis were cloned into the pGL3-Basic luciferase reporter vector for further study. The promoter constructs were transiently transfected into the 38B9 pro-B cell line along with the control pRL-TK Vector, which expresses Renilla luciferase, and the Dual-Luciferase® Reporter Assay System was used to assess luciferase activity from the various promoter constructs. The promoter construct having the highest activity was chosen, and site directed mutagenesis was used to identify specific regions within the promoter fragment that may be important for activity. Sequence analysis was then used to identify a conserved Ets transcription factor binding site within the putative il7r promoter region. To determine whether the ETS transcription factor GABP binds to this Ets region, the authors performed chromatin immunoprecipitation analysis with an anti-GABP antibody. Immunoprecipitated DNA was then PCR-amplified with primers specific for the Ets region or control primers. The Wizard® SV Gel and PCR Clean-Up System was used to purify the amplified fragments prior to semiquantitative PCR analysis. (3626)

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Infect. Immun. 75, 3478–89. Reversal of the antichlamydial activity of putative type III secretion inhibitors by iron. 2007

Slepenkin, A., Enquist, P.A., Hägglund, U., de la Maza, L.M., Elofsson, M. and Peterson, E.M.

Notes: The authors screened members of a class of acylated hydrazones of salicylaldehydes (INPs) to characterize their ability to inhibit growth of Chlamydia by affecting the type III secretion (T3S) system, a potent virulence mechanism. Expression levels of various T3S genes and gene markers of early, middle and late developmental cycles were examined by RT-PCR in Chlamydia trachomatis-infected HeLa 229 cells in the presence and absence of INPs. HeLa229 RNA was isolated at 4, 8, 24 and 36 hours postinfection, treated with RQ1 RNase-Free DNase and amplified using the Access RT-PCR System in the presence of 0.5 units of RNasin RNase Inhibitor. Each set of experiments included a no-reverse transcriptase control reaction to control for DNA contamination and a positive control reaction using the Positive Control RNA with Carrier and control primers supplied with the kit. (3795)

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Genetics 176, 161–180. Role of the mod(mdg4) common region in homolog segregation in Drosophila male meiosis. 2007

Soltani-Bejnood, M., Thomas, S.E., Villeneuve, L., Schwarz, K.T., Hong, C.S. and McKee, B.D.

Notes: In this paper, the authors studied the common region of mod(mdg4) and its role in homolog conjunction during meiosis. Genomic DNA was isolated from adult Drosophila using the Wizard® Genomic DNA Purification Kit and mutations identified by PCR and sequencing of the introns. (3576)

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J. Biol. Chem. 282, 8380–8392. RSK2 Mediates Muscle Cell Differentiation through Regulation of NFAT3. 2007

Cho, Y.Y., Yao, K., Bode, A.M., Bergen, H.R. 3rd, Madden, B.J., Oh, S.M., Ermakova, S., Kang, B.S., Choi, H.S., Shim, J.H. and Dong, Z.

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to screen for protein-binding partners for RSK2, a ribosomal S6 kinase involved in myoblast differentiation. The RSK2 cDNA was cloned into the pBIND Vector as bait while several transcription factors were amplified by PCR and cloned into the pACT Vector. The bait pBIND-RSK2 construct, the pACT-transcription factors and pG5-luc Vector were transfected into 293 cells at a 1:1:1 molar ratio. To assess the protein interaction, the cells were lysed, and the firefly luciferase activity was normalized to Renilla luciferase activity. The strongest interaction was with NFAT3. (3574)

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Genetics 175, 1047-1058. Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes. 2007

Lamb, J.C., Danilova, T., Bauer, M.J., Meyer, J.M., Holland, J.J., Jensen, M.D., and Birchler, J.A.

Notes: These authors generated a set of probes that could be used in fluorescence in situ hybridization (FISH) analyses for karyotyping studies on maize chromosomes. Specific target regions composed of genes or gene clusters and free from repetative elements were identified for each chromosome. Target regions were amplified by PCR, gel purified using the Wizard® SV Gel and PCR Clean-Up System, and tested in a FISH assay. Probes showing low background were selected, subcloned into the pGEM® -T Vector and sequenced to confirm identity. (3627)

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J. Biol. Chem. 282, 19052–19061. SOX6 suppresses cyclin D1 promoter activity by interacting with beta-catenin and histone deacetylase 1, and its down-regulation induces pancreatic beta-cell proliferation. 2007

Iguchi, H., Urashima, Y., Inagaki, Y., Ikeda, Y., Okamura, M., Tanaka, T., Uchida, A., Yamamoto, T.T., Kodama, T. and Sakai, J.

Notes: Sex-determining Y-box (SOX) 6 is a transcription factor downregulated in obesity-related insulin-resistant animals. The authors examined the interaction between SOX 6 and β-catenin, a protein that modulates cyclin D1 promoter activity. To characterize the physical interaction, in vitro binding assays were performed using GST-fused SOX 6 and deletion mutants of β-catenin, which were expressed as 35S-labeled proteins in the TNT® T7 Quick Coupled Transcription/Translation System. The GST-fusion proteins were bound to MagneGST® particles and allowed to interact with the β-catenin mutants. Purified GST was used as a negative control to determine nonspecific protein binding. The authors were able to identify the protein domains necessary for SOX 6/β-catenin interaction. Similar binding assays were performed with GST-β-catenin and 35S-labeled T-cell factor in the presence or absence of SOX 6 to show that SOX 6 does not interfere with the binding of β-catenin to TCF. (3685)

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Mol. Cell. Biol. 26, 8448–8460. Specific isoforms of translation initiation factor 4GI show differences in translational activity. 2007

Coldwell, M.J. and Morley, S.J.

Notes: The authors explored the role of five different eukaryotic initiation factor (eIF) 4GI protein isoforms, which are encoded by alternatively spliced mRNAs, by using short interfering RNAs (siRNAs) to silence the eIF4GI gene. Three eIF4GI siRNA target sequences were evaluated for their ability to reduce eIF4GI mRNA levels in HeLa cells. To quantify the extent of gene silencing, a control plasmid that encodes an eIF4GI/Renilla luciferase fusion mRNA was created using the psiCHECK™-2 Vector. Cotransfection of HeLa cells with the eIF4GI siRNAs and psiCHECK™-2 control plasmid resulted in degradation of the eIF4GI/Renilla luciferase mRNA, leading to reduced Renilla luciferase activity and lower light output. The psiCHECK™-2 Vector encodes the firefly luciferase gene, which allowed normalization of Renilla luciferase expression. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. Quantitative PCR (qPCR) was used to quantify the silencing of endogenous eIF4GI mRNA splice variants. Prior to qPCR, total RNA was isolated from siRNA-expressing HeLa cells, then reverse transcribed using the ImProm-II™ Reverse Transcription System. qPCR was The pGEM®-T Easy Vector was used in the creation of plasmids encoding siRNA-resistant eIF4GI isoforms, which were transfected into siRNA-expressing HeLa cells to restore eIF4GI function. (3778)

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Am. J. Pathol. 171, 1312–1323. Target genes of neuron-restrictive silencer factor are abnormally up-regulated in human myotilinopathy. 2007

Barrachina, M., Moreno, J., Juvés, S., Moreno, D., Olivé, M. and Ferrer, I.

Notes: These authors used chromatin immunoprecipitation to show that neuron-restrictive silencer factor interacts with the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) promoter in U87-MG, DMS53 and HeLa cells. The neuron-restrictive silencing element (NRSE1) of the UCHL1 promoter was amplified using GoTaq® Flexi DNA Polymerase in a 25µl PCR. (3705)

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Mol. Cancer Ther. 6, 856-865. The glycotope-specific RAV12 monoclonal antibody induces oncosis in vitro and has antitumor activity against gastrointestinal adenocarcinoma tumor xenografts in vivo. 2007

Loo, D., Pryer, N., Young, P., Liang, T., Coberly, S., King, K.L., Kang, K., Roberts, P., Tsao, M., Xu, X., Potts, B. and Mather, J.P.

Notes: The authors examined the effectiveness of a monoclonal antibody treatment to human tumor-derived cells implanted under the kidney capsule of male athymic mice. These tumors were recovered from mouse kidney and the total genomic DNA isolated using the Wizard® SV Genomic DNA Purification System. The human tumor DNA was quantified using a TaqMan® qPCR method. (3748)

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Mol. Endocrinol. Mar. 13, Epub (ahead of print). The micro-RNA miR-206 targets the human estrogen receptor-α, and represses ERα mRNA and protein expression in breast cancer cells. 2007

Adams, B.D., Furneaux, H. and White, B.

Notes: This study investigated the mechanism of silencing of the estrogen receptor α mRNA in the human breast cancer cell line, MCF-7. The authors initially used software for miRNA target prediction to analyze the 3´ UTR of the human ERα gene for potential miR-206 target sites. Two potential targets, designated hERα1 and hERα2, were identified. ERα levels were repressed in a dose-dependent manner in MCF-7 cells transfected with a synthetic pre-miR-206 duplex, and transfection of an miR-206 expression construct into MCF-7 cells also resulted in specific inhibition of ERα expression, as measured by real-time PCR and Northern blot assays. A luciferase reporter assay was then used to determine whether miR-206 interacted directly with the hERα1 and hERα2 sites in the ERα 3´UTR. Luciferase reporter constructs containing either the hERα1 or hERα2 cloned 3´ of the firefly luciferase gene showed miR-206-medisted repression of luciferase expression in HeLa cells. Mutation of the hERα1 or hERα2 sites to disrupt hybridization with the 5´ region of miR-206 restored luciferase activity, as did co-transfection with an miRNA antagonist of miR-206. Transformation of the luciferase constructs into the breast cancer cell lines, MCF-7 and MDA-MB-231, both of which expressed high levels of miR-206 as measured by real-time PCR, resulted in repression of luciferase activity. Treatment with estrogen was then shown to reduce miR-206 levels in MCF-7 cells. Luciferase assays were used to confirm this result, and levels of luciferase activity from the reporter constructs were increased upon exposure to estrogen, indicating that ERα agonists were able to decrease miR-206 levels in MCF- cells. (3603)

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Appl. Environ. Microbiol. 73, 2860-2870. The microbial community structure in petroleum-contaminated sediments corresponds to geophysical signatures. 2007

Allen, J.P., Atekwana, E.A., Atekwana, E.A., Duris, J.W., Werkema, D.D., and Rossbach, S.

Notes: These authors studied microbial community structure at various locations in an aged underground petroleum plume. DNA was purified from soil samples collected from different sites within a contaminated area. 16S rRNA genes were then amplified from the isolated DNA, and the PCR products were run on a gel and purified using the Wizard® SV Gel and PCR Clean-Up System. After subcloning into a TA vector, the 16S RNA genes were sequenced and used to identify the various Phyla represented and characterize the microbial populations present throughout the site. (3625)

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Forensic Sci. Int. 152, 89–94. Y-chromosomal STR haplotypes in a Belgian population sample and identification of a micro-variant with a flanking site mutation at DYS19. 2007

De Maesschalck, K,. Vanhoutte, E., Knaepen, K., Vanderheyden, N., Cassiman, J.J., and Decorte, R.

Notes: The authors collected DNA samples from 113 unrelated Belgian males. The PowerPlex® Y System and a GeneAmp® 9700 PCR system were used to amplify 12 Y-chromosome STR loci (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439). Amplification products were detected using an ABI PRISM® 3100 genetic analyzer and POP-6™ polymer. Allele and haplotype frequencies and haplotype diversity were calculated. A total of 99 different haplotypes were observed. (3655)

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Exp. Parasitol. 112, 63-65. Babesia canis vogeli: A novel PCR for its detection in dogs in Australia. 2006

Martin, A.R., Dunstan, R.H., Roberts, T.K., and Brown, G.K.

Notes: GoTaq® DNA Polymerase was used in PCR to test dog blood for the presence of Babesia canis. Genomic DNA isolated from dog blood was analyzed with primers to the variable 5’ region of the Babesia canis 18S rRNA gene. PCR was performed in 50µl reactions containing 1.25 units of GoTaq® DNA Polymerase and 10µl of GoTaq® Reaction Buffer. (3367)

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J. Biol. Chem. 281, 9963–9970. A novel hematopoietic granulin induces proliferation of goldfish (Carassius auratus L.) macrophages. 2006

Hanington, P.C., Barreda, D.R. and Belosevic, M.

Notes: These authors expressed a recombinant form of a novel hemapoietic granulin from goldfish. This recombinant granulin was expressed with a His6 tag in a 1-liter culture of BL21 Star™ (DE3) cells and purified from the culture supernatant using the MagneHis™ Protein Purification System. The purified protein was used to immunize rabbits and produce an affinity-purified rabbit anti-goldfish granulin IgG for immunodetection. The purified protein was also added to goldfish primary kidney macrophage cultures to determine if granulin stimulates macrophage proliferation. (3567)

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J. Forensic Sci. 51, 274–281. A proposal for standardization in forensic canine DNA typing: allele nomenclature of six canine-specific STR loci. 2006

Hellmann, A.P., Rohleder, U., Eichmann, C., Pfeiffer, I., Parson, W. and Schleenbecker, U.

Notes: Saliva samples from a total of 142 dogs (36 different pure breeds and several cross breeds) were collected on cotton swabs. Genomic DNA was isolated using the ReadyAmp™ Genomic DNA Purification System. Single amplifications of six canine STR loci were performed using 1–5µl of saliva DNA extract. (3424)

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Clin. Can. Res. 12, 4515-22. Accumulation of promoter methylation suggests epigenetic progression in squamous cell carcinoma of the esophagus 2006

Guo, M., Ren, J., House, M.G., Qi, Y., Brock, M.V., Herman, J.G.

Notes: The authors studied multiple genes for aberrant DNA methylation in esophogeal cancer, to determine timing of methylation and epigenetic changes in early lesions. In addition the authors sought a role for preinvasive lesions in tumor initiation and progression. Tissues from esophageal cancers or various grades of esophogeal dysplasia were collected. A bisulfite modification was performed on genomic DNA to convert unmethylated cytosine to uracil in denatured DNA; methylated cytosines are resistant. The difference in reactivity to bisulfite treatment was used to provide unique sequences for primers to detect methylation differences. The resulting DNA samples were purified using the Wizard® DNA Clean-Up System. (4110)

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Forensic Sci. Int. 164, 75–78. Allele distribution of 15 STR loci used for human identity purposes in the Greek Cypriot population of the island of Cyprus. 2006

Cariolou, M.A., Manoli, P., Demetriou, N., Bashiardes, E., Karagrigoriou, A. and Budowle, B.

Notes: The authors generated population data for 15 autosomal STRs using the PowerPlex® 16 System and DNA samples collected from 1,475 unrelated Greek Cypriot individuals. DNA was extracted from the blood samples using organic extraction, and 0.5ng was amplified per reaction using the GeneAmp® PCR System 9700. Amplified products were analyzed using an ABI PRISM® 3100 Genetic Analyzer. (3815)

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Hum. Mol. Genet. 15, 999–1013. An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements. 2006

Disset, A., Bourgeois, C.F., Benmalek, N., Claustres, M., Stevenin, J. and Tuffery-Giraud, S.

Notes: To construct dystrophin minigenes, genomic DNA containing a mutation in dystrophin was amplified for exons 30, 31 and 32. The three PCR fragments were combined and amplified into one product. This overlap-extension PCR generated two minigenes which were then cloned into the pGEM®-T Vector and sequenced. After EcoR I digestion, the minigenes were ligated into the pSI Mammalian Expression Vector and transiently transfected into C2C12 cells for expression analysis. (3499)

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Appl. Environ. Microbiol. 72, 6070-6078. An oxidoreductase is involved in cercosporin degradation by the bacterium Xanthomonas campestris pv. zinniae. 2006

Taylor, T.V., Mitchell, T.K. and Daub, M.E.

Notes: Fungi of the genus Cercospora are plant pathogens that cause leaf spot and blight diseases, and produce the polyketide toxin cercosporin. The bacterium Xanthomonas campestris is able to rapidly degrade cercosporin. In this study, X. campestris mutants unable to degrade cercosporin were created by chemical mutagenesis. Complementation studies with a plasmid-based library of X. campestris DNA showed that the ability to degrade cercosporin was restored upon transformation with plasmids containing an oxidoreductase gene and a putative transcriptional regulator. These genes were then amplified from the mutant strains by high-fidelity PCR. The PCR products were separated by agarose gel electrophoresis, purified using the Wizard® SV Gel and PCR Clean-Up System, and subcloned into the pGEM®-T Easy Vector. The mutant genes were then sequenced to identify the nature of the mutations. (3531)

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J. Biol. Chem. 281, 17410-17419. ATP binding to a unique site in the type-1 S2- inositol 1,4,5-triphosphate receptor defines susceptibility to phosphorylation by protein kinase A. 2006

Wagner, L.E., Betzenhauser, M.J. and Yule, D.I.

Notes: N-terminal GST fusion proteins of portions of the type-1 S2- Ionsitol 1,4,5-triphosphate receptor were created using the pFN2A (GST) Flexi® Vector. The constructs were expresed in BL21 (DE3) pLysS cells and used for ATP-binding assays. (3392)

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Nucl. Acids Res. 34, 6215-6224. Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51 2006

Desfarages, S., San Filippo, J., Fournier, M. Calmels, C., Caumont-Sarcos, A., Litvak, S., Sung, P., Parissi, V.

Notes: In the process of demonstrating the role of IN in HIV-1 integration in yeast, the authors purified all DNA vectors and PCR products with the Wizard® Plus SV Miniprep System and Wizard® SV Gel System. PCR products were generated using Taq DNA Polymerase. The pGEM®-T Vector was used to clone amplification products. Sequencing was performed using BamHI, religated with T4 DNA Ligase. (3704)

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Behav. Ecol. 17, 419–429. Cichlids do not adjust reproductive skew to the availability of independent breeding options. 2006

Heg, D., Bergmuller, R., Bonfils, D., Otti, O., Bachar, A., Burri, R., Heckel, G. and Taborskya, M.

Notes: These authors sought to determine whether helpers in cooperatively breeding species reproduced or if all offspring were from the breeders. They created 32 breeding groups and tested the possibilities of reproductive skew under various conditions. Genomic DNA from breeders and helpers was isolated from ethanol-preserved 1–2 mm2 finclip samples or from whole offspring using the Wizard® Genomic DNA Purification Kit. Purified DNA was resuspended in 50µl of DNA Rehydration Solution and analyzed by PCR using seven microsatellite primer pairs. (3679)

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Appl. Environ. Microbiol. 72, 2539-2546. Cloning and sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula. 2006

Mohan Nair, M.K. and Venkitanarayanan, K.S.

Notes: The outer membrane protein A (ompA) gene of Enterobacter sakazakii was amplified using PCR primers based on E. coli ompA sequences. The resulting PCR product was ligated into the pGEM®-T Easy Vector, and the sequence was confirmed. The ompA sequence was used to develop a PCR for detection of Enterobacter sakazakii in reconsituted infant formula. (3464)

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Appl. Environ. Microbiol. 72, 5463–5468. Comparative, collaborative, and on-site validation of a TaqMan® PCR method as a tool for certified production of fresh, campylobacter-free chickens. 2006

Krause, M., Josefsen, M.H., Lund, M., Jacobsen, N.R., Brorsen, L., Moos, M., Stockmarr, A. and Hoorfar, J.

Notes: To test if a real-time PCR assay would be able to detect Campylobacter spp. in various chicken samples, several laboratories were involved in a collaborative trial. Each lab was given 18 1ml samples, including 6 chicken neck skin samples, 6 shoe cover fecal samples and 6 cloacal swab samples. DNA was extracted from these samples using the MagneSil® KF, Genomic System, first tested using 20µl of paramagnetic particles (PMPs) and then using 75µl PMPs. Five microliters of purified DNA was used in real-time PCR analysis. (3558)

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J. Biol. Chem. 281, 9901–9908. CpG sites preferentially methylated by Dnmt3a in vivo. 2006

Oka, M., Rodic, N., Graddy, J., Chang, L.J. and Terada, N.

Notes: The Wizard® Genomic DNA Purification Kit was used to isolate genomic DNA from mouse embryonic stem (ES) cells, embryonic bodies (EB) and adult tissues. The purified genomic DNA was subjected to methylation-sensitive restriction fingerprinting (MSRF; digestion with methylation-sensitive restriction enzymes followed by amplification of 100ng of digested DNA), or 2µg DNA treated with bisulfite. (3419)

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