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J. Immunol. 179, 491–504. Evolution of killer cell Ig-like receptor (KIR) genes: definition of an orangutan KIR haplotype reveals expansion of lineage III KIR associated with the emergence of MHC-C. 2007

Guethlein, L.A., Older Aguilar, A.M., Abi-Rached, L. and Parham, P.

Notes: The authors assessed the sequence of the less evolved Orangutan (Pongo pygmaeus) MHC-C, specifically the structure and complexity of the orangutan killer cell Ig-like receptor (KIR) locus. After identifying twelve cosmids from an orangutan library that contain the KIR, the cosmids were cut with EcoRI and BamHI and the digested fragments subcloned into pBluescriptKS+ to determine the haplotype. The medium length subclones (2.5–10kb) were sequenced by unidirectional deletion mutagenesis using the Erase-A-Base® System. (3696)

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Nucl. Acids Res. 35, 2390–2402. Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance. 2007

Wu, J., Apontes, P., Song, L., Liang, P., Yang, L. and Li, F.

Notes: To study how Hoechst33342 upregulates the expression and promoter activity of survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, nested deletions of the survivin promoter driving a firefly luciferase reporter gene (pLuc-1430c ) were created using the Erase-a-Base® System. The vector was digested with SalI, the ends filled in using α-phosphorothioate dNTPs, digested a second time with BamHI and subjected to Exonuclease III digestion at 25°C. Aliquots of the 5’ end deletions were removed every 15–30 seconds, religated, transformed and analyzed by PCR and sequencing. Transient transfection experiments were carried out using HeLa cells seeded in 24-well plates and cotransfected 490ng of a pLuc-survivin construct and 10ng of pRL-TK Vector or in U937 cells using 2µg of survivin promoter constructs. After 24 hours, the HeLa cells were treated with Hoechst33342 and harvested 8–24 hours later. For U937 cells, the medium was changed with or without added drugs and the cells lysed after 36 hours. Reporter expression was assessed using the Dual-Luciferase® Reporter Assay System. (3697)

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J. Biol. Chem. 281, 1137–1144. Identification of the fibroblast growth factor (FGF)-interacting domain in a secreted FGF-binding protein by phage display. 2006

Xie, B., Tassi, E., Swift, M.R., McDonnell, K., Bowden, E.T., Wang, S., Ueda, Y., Tomita, Y., Riegel, A.T. and Wellstein, A.

Notes: Starting with the fibroblast growth factor-binding protein (FGF-BP1) open reading frame cloned into the SP72 Vector, the Erase-a-Base® System was used to generate 5’ and 3’ deletions in the gene to use in phage library display. For the 5’ deletion library, BamHI was used to generate the 5’ overhang, which is susceptible to exonuclease III treatment, while KpnI generated a 3’ overhang to protect the plasmid backbone. To create the 3’ deletion library, NotI and ApaI restriction sites were introduced into pSP72-FGF-BP1 Vector with NotI close to the 3’ end of FGF-BP1. NotI was used to generate the 5' overhang and ApaI generated a 3’ overhang. Exonuclease III was added at 20°C with an estimated erasing rate at around 90bp/minute. Enzymatic digestion of plasmids was stopped at 30-second intervals. Digested plasmids were then pooled, religated and cloned at the EcoRI and HindIII sites into the phage 10-3b vector. In vitro packaging generated the stock library solution, which was then used to localize the interaction interface between FGF-2 and FGF-BP1. (3508)

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J. Bacteriol. 188, 1920–1928. Involvement of the LlaKR2I methylase in expression of the AbiR bacteriophage defense system in Lactococcus lactis subsp. lactis biovar diacetylactis KR2. 2006

Yang, J.M., Deurraza, P.J., Matvienko, N. and O'Sullivan, D.J.

Notes: To avoid additional unwanted mutations introduced by a restriction enzyme deletion strategy, the Erase-a-Base® System was employed to create deletions of the LlaKR2I methylase gene, which may have a role in the abortive infection (Abi) mechanism, AbiR, that negatively affects bacteriophage DNA replication. The plasmid construct pDOU012, which contains part of the bacteriophage defense system genes, was digested with BsgI and BstEII to introduce a 3’ extension and a 5’ overhang, respectively. Exonuclease III reactions were carried out for 12 minutes at 30°C, and aliquots were taken every minute. Erythromycin-resistant transformants were selected, and deletion derivatives were identified by restriction analysis of plasmid DNA as well as by PCR and sequence analysis. Using phenotype analysis, the authors confirmed that the methylase gene was critical for expression of the AbiR phenotype. (3509)

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J. Biol. Chem. 281, 17635–17643. The constitutive expression of anticoagulant protein S is regulated through multiple binding sites for Sp1 and Sp3 transcription factors in the protein S gene promoter. 2006

de Wolf, C.J., Cupers, R.M., Bertina, R.M. and Vos, H.L.

Notes: The Protein S promoter (PROS1) fragment –5948/–1 was cloned directly 5’ to the firefly luciferase reporter gene in the pGL3-Basic Vector using the KpnI and XhoI enzyme sites. This construct, PS5948-luc, was linearized with KpnI and NdeI and subsequently subjected to progressive deletion using the Erase-a-Base® System. The size of the resulting 5’-deletion was determined by sequence analysis, and the deletion constructs were used for transient transfection assays. HepG2, HuH7, HeLa and HUVEC cells were transfected at 60–80% confluency in 12-well plates using 3µl of Tfx™-20 per microgram DNA. In each transfection, an equimolar concentration of construct was used and supplemented with an additional plasmid to keep the amount of transfected DNA constant. pRL-SV40 Vector was co-transfected as a transfection control using a 1:500 ratio to the total transfected amount of DNA in HepG2, HuH7 and HeLa cell lines, and a 1:100 ratio in transfections with HUVEC and 1 × 106 Meg01 suspension cells. Transcription factor expression vector (250ng) was co-transfected, and expression vector without the transcription factor cDNA was used as a negative control. Cell extracts were harvested at either 24 (HepG2 and HuH7) or 48 hours (Meg01, HUVEC, and HeLa) post-transfection using 250µl of Passive Lysis Buffer per well. Luciferase activity was determined using 20–100µl of lysate with the Dual-Luciferase® Reporter Assay System. (3510)

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Infect. Immun. 73, 2611-2620. Molecular cloning and characterization of three beta-defensins from canine testes. 2005

Sang, Y., Ortega, M.T., Blecha, F., Prakash, O. and Melgarejo, T.

Notes: The investigators cloned three β-defensins, which are antimicrobial peptides, from canine testes. A canine expressed sequence tag (EST) was identified based on similarity to human β-defensins. Full-length cDNAs were obtained using 5´- and 3´RACE, then amplified by PCR and cloned into the pGEM®-T Easy Vector. cDNA sequences were confirmed using the SP6 and T7 Promoter Primers. The tissue-specific expression of canine β-defensins (cBDs) was characterized using the AccessQuick™ RT-PCR System to amplify β-defensin RNA from a variety of tissues. RNA was treated with RQ1 RNase-Free DNase prior to RT-PCR. The identity of the RT-PCR products was confirmed by electrophoresis, transfer to nylon membranes and hybridization to probes derived from sequence-confirmed β-defensin clones; the probes were synthesized using the Prime-a-Gene® Labeling System. To localize expression of the three β-defensin isoforms in canine testes, in situ hybridization (ISH) was performed. The 3´-RACE products were cloned into the pGEM®-T Vector, which was then linearized and treated with exonuclease III (Erase-a-Base® System) to delete an approximately 80-bp region shared by the three cBD isoforms. The resulting product was used to synthesize sense and antisense ISH probes. (3453)

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J. Virol. 78(6), 3072-3082. Cis and trans requirements for rolling circle replication of a satellite RNA. 2004

Song, S.I. and Miller, W.A.

Notes: In this report, satellite RNA from the full-length RPV serotype (satRPV RNA) of Cereal yellow dwarf virus was transcribed in vitro using the RiboMAX™ Large Scale RNA Production System. Self-cleavage of the synthesized RNA was then induced by incubation with a cleavage buffer. Serial deletions were created in the wild-type satRPV cloned in pGEM®-3Zf(–) Vector using the Erase-a-Base® System. The mutants generated were tested to see how well they replicated with internal sections of satRPV sequence removed.  (3080)

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Eur. J. Biochem. 267, 7224–7229. Identification and characterization of a novel transcriptional regulator, MatR, for malonate metabolism in Rhizobium leguminosarum bv. trifolii 2000

Lee, H.Y., An, J.H. and Kim, Y.S.

Notes: Total RNA was isolated from Rhizobium leguminosarum bv. trifolii using the SV Total RNA Isolation System. This RNA was used in a primer extension assay using Promega's AMV Reverse Transcriptase to determine the transcriptional start site of the mat operon. Nested deletions during the sequencing of the mat promoter were prepared using the Erase-a-Base® System. The firefly luciferase gene from pGL3 Basic was used to construct a reporter construct containing the mat promoter to study transcriptional effects of MatR. The interaction of the MatR protein with the mat operator was mapped using the Core Footprinting System. (2308)

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J. Biol. Chem. 274, 11220-11228. Cloning of the cyclin A1 genomic structure and characterization of the promoter region. GC boxes are essential for cell cycle-regulated transcription of the cyclin A1 gene. 1999

Muller, C., Yang, R., Beck-von-Peccoz, L., Idos, G., Verbeek, W. and Koeffler, H.P.

Notes: The promoter of the human cyclin A1 gene was studied by the authors of this work. To define promoter activity, various genomic fragments from upstream of the cyclin A1 gene were analyzed in the pGL3-Basic luciferase vector. The fragments were generated by the 5´ deletion technique using the Erase-a-Base® System. These constructs were expressed in HeLa cells and in the Drosophila cell line S2. For studies in S2 cells, an expression vector encoding the transcription factor Sp1 was also used. The data for HeLa cells is displayed as fold activation over that of the pGL3-Basic Vector without insert normalized against a CMV driven β-galactosidase control.  No β-galactosidase control was used in S2 cells because the viral promoter control vectors all cross-reacted with the Sp1 expression vector. The data in S2 cells is expressed as fold activation over similar transfections without Sp1. (3002)

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Genetics 151, 1027-1039. Evolution of the RECQ family of helicases. A drosophila homolog, dmblm, is similar to the human bloom syndrome gene. 1999

Kusano, K., Berres, M.E., Engels, W.R.

Notes: The Erase-a-Base® System was used for preparing plasmid DNA with nested deletions in the insert region for DNA sequencing. (0867)

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J. Biol. Chem. 274, 19699-19706. Identification and characterization of a ran gene promoter in the protozoan pathogen Giardia lamblia 1999

Sun, C.-H., Tai, J.-H.

Notes: The pSP-luc+ Vector was used as a source of luciferase cDNA for construction of a expression vector in G. lamblia. The promoter of interest was combined with luc+ in a standard cloning vector for study. Deletion mutants of the promoter were constructed with the Erase-a-Base® System. The authors note that the pGL3 Control Vector produced some luciferase activity in the G. lamblia but only 0.5% of the RAN promoter-luc+ construct. (0317)

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Eur. J. Biochem. 261, 137-147. Identification of chURP, a nuclear calmodulin-binding protein related to hnRNP-U. 1999

Lodge, A.P., Walsh, A., McNamee, C.J., Moss, D.J.

Notes: The chURP protein was expressed from the PinPoint™ Xa-3 Vector in a 2-liter culture of JM109. Much detail is provided for purification of the protein to make an immunogen for antibody production. The Wizard® Lambda Preps System was used to purify cDNA clones of the chURP and the Erase-a-Base® System was used to make nested deletions for sequencing. (0741)

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J. Biol. Chem. 273, 8376-8381. A rare 920-kilobase chromosomal inversion mediated by IS1 transposition causes constitutive expression of the yiaK-S operon for carbohydrate utilization in Eschericia coli. 1998

Badía, J., Ibáñez, E., Sabaté, M., Baldomà, L. and Aguilar, J.

Notes: Chromosomal DNA was obtained from E. coli using the Wizard® Genomic DNA Purification Kit. The DNA was used for EMBL4 library construction (with the aid of the Packagene® Lambda DNA Packaging System) as well as Southern analysis. The Erase-a-Base® System was produce nested deletions prior to sequencing. (1490)

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Genetics 150, 1625-1637. Extraordinary ribosomal spacer length heterogeneity in a neotyphodium endophyte hybrid: implications for concerted evolution. 1998

Ganley, A. R. , Scott, B.

Notes: Serial deletions of a 4.1kb insert were made by using the Erase-a-Base® System for DNA sequencing. (1138)

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Genetics 150, 119-128. Genetic and molecular characterization of the Caenorhabditis elegans gene, mel-26, a postmeiotic negative regulator of mei-1, a meiotic- specific spindle component. 1998

Dow, M. R. , Mains, P. E.

Notes: Nested deletions of cDNA and genomic clones were generated using the Erase-a-Base® System. (1247)

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Biochem. J. 332, 773-780. Transcript heterogeneity of the human reduced folate carrier results from the use of multiple promoters and variable splicing of alternative upstream exons. 1998

Zhang, L. , Wong, S. C. , Matherly, L. H.

Notes: The authors used the PolyATtract® mRNA Isolation System to isolate mRNA from total RNA. They used this mRNA-rich fraction for primer extension analysis using Promega's AMV Reverse Transcriptase. The Wizard® Plus Midiprep DNA Purification System was used for various plasmid isolations and the Erase-a-Base®  System was used to generate deletion series. The Dual-Luciferase® Reporter Assay System was used to study promoters cloned into the pGL3-Basic Vector. The pRL-SV40 was used as a transfection control plasmid. (0095)

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Immunity 6, 119-129. cDNA cloning and primary structure analysis of C1qR(P), the human C1q/MBL/SPA receptor that mediates enhanced phagocytosis in vitro. 1997

Nepomuceno, R.R., Henschen Edman, A.H., Burgess, W.H., Tenner, A.J.

Notes: RT-PCR was performed with degenerate primers and the resulting 110bp product was subcloned with the pGEM®-T Vector System. The 110bp fragment was used to screen a cDNA library and four positive plaques were identified. The lambda clones were sequenced with the fmol® DNA Cycle Sequencing System. To sequence across the entire lambda clone nested deletions were made with the Erase-a-Base® System. (0650)

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J. Neurosci. 17, 4159-4169. Cloning and functional characterization of Roaz, a zinc finger protein that interacts with O/E-1 to regulate gene expression: implications for olfactory neuronal development. 1997

Tsai, R. Y. , Reed, R. R.

Notes: Reporter studies were performed in human embryonic kidney 293 cells using constructs prepared in the pGL2-Basic Vector. The Erase-A-Base® System was used to create unidirectional deletion mutants of promoter elements. (0236)

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J. Biol. Chem. 272, 7464-7472. Differential effects of protein kinase C, Ras, and Raf-1 kinase on the induction of the cardiac B-type natriuretic peptide gene through a critical promoter-proximal M-CAT element. 1997

Thuerauf, D.J., Glembotski, C.C.

Notes: Luciferase studies were performed in primary neonatal rat ventricular myocardiocytes and constructs were prepared in the pGL2-Basic Vector. The promoter mutations of the reporter constructs were made by Altered Sites® II in vitro Mutagenesis System. (0264)

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Proc. Natl. Acad. Sci. USA 94, 3837-3841. Gypsy retrotransposon as a tool for the in vivo analysis of the regulatory region of the optomotor-blind gene in Drosophila. 1997

Tsai, S.F., Jang, C.C., Prikhod'ko, G.G., Bessarab, D.A., Tang, C.Y., Pflugfelder, G.O , Sun, Y.H.

Notes: The authors cloned a 5.7 kb PCR product into pGEM®-T Vector (0237)

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Proc. Natl. Acad. Sci. USA 94(9), 4354-4359. Purification and characterization of a human RNA adenosine deaminase for glutamate receptor B pre-mRNA editing. 1997

Yang, J.H., Sklar, P., Axel, R. and Maniatis, T.

Notes: Poly A+ RNA was isolated from HeLa cell total RNA using the PolyATtract® mRNA Isolation System and used for RT-PCR. The Erase-A-Base® System was used in this study. (1699)

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J. Biol. Chem. 272, 5899-5908. Regulation of murine cytochrome oxidase Vb gene expression in different tissues and during myogenesis. Role of a YY-1 factor-binding negative enhancer. 1997

Basu, A., Lenka, N., Mullick, J. and Avadhani, N.G.

Notes: Reporter studies were performed in 3T3, COS, Hep3B and C2C12 cells using the pCAT® Basic Vector. The Erase-A-Base® System was used to make deletion mutants of the promoter being studied. (An improved version of the pCAT®  Basic Vector, the pCAT® 3 Basic Vector is now available) (1466)

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J. Biol. Chem. 272, 17112-17117. Structure of the m1 muscarinic acetylcholine receptor gene and its promoter 1997

Pepitoni, S., Wood, I.C. Buckley, N.J.

Notes: The Dual-Luciferase® Reporter Assay System was used to study transfections of IMR32 and NIH3T3 cells with firefly luciferase vector (pGL3 Basic) constructs. Transfections were controlled with co-transfected pRL-CMV Vector. The promoter under study was truncated with the Erase-A-Base® System. Tth DNA Polymerase was used as a high temperature reverse transcriptase. SP6 RNA Polymerase was used to produce probes for RNase protection assays. (0558)

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J. Biol. Chem. 272, 26285-26294. TGT3, thyroid transcription factor I, and Sp1 elements regulate transcriptional activity of the 1.3-kilobase pair promoter of t1alpha, a lung alveolar type I cell gene 1997

Ramirez, M.I., Rishi, A.K., Cao, Y.X., Williams, M.C.

Notes: Luciferase studies were performed in SV40 T type II cells and IMR90 fibroblasts using constructs prepared in the pGL3 Basic Vector. Luciferase activity was measured with the Luciferase Assay System. The Erase-A-Base® System was used to generate deletion mutants of the promoter. The hepatocyte nuclear factor 3beta was produced in vitro with the TNT® Coupled Reticulocyte Lysate System and used for gel shift analysis. (0495)

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J. Neurosci. 16(21), 6839-6852. Characterization of Densin-180, a new brain-specific synaptic protein of the O-sialoglycoprotein family. 1996

Apperson, M.L., Moon, I.S. and Kennedy, M.B.

Notes: The PolyATtract® mRNA Isolation System was used to isolate Poly(A+) RNA from the total RNA of various rat tissues ranging from the forebrain to the testes. The isolated RNA was used for Northern analysis. The Erase-A-Base System was used to produce nested deletions to simplify sequencing of the cDNA clones. (2141)

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