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J. Biol. Chem. 279(10), 8761–8768. CzcR-CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa. 2004

Perron, K., Caille, O., Rossier, C., van Delden, C., Dumas, J.L., and Kohler, T.

Notes: Two genes (CzcR and CzcS) encoding the CzcCBA (cobalt/zinc/cadmium) heavy metal efflux pump were cloned using  Pfu DNA Polymerase and genomic DNA from PT5 and PT1105 strains of Pseudomonas aeruginosa. The products were 900 and 1,600 bp, respectively. RNA from 5 different strains of Pseudomonas aeruginosa was isolated and treated with RQ1 RNase-Free DNase.  The RNA was used in reverse transcription reactions with the ImProm-II™ Reverse Transcriptase and random primers.  Copy-DNAs produced from the reaction were stored at -20°C until use in real-time PCR.  (3044)

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Plant Physiol. 135, 1540–1551. Isolation and characterization of a TERMINAL FLOWER homolog and its correlation with juvenility in citrus. 2004

Pillitteri, L.J., Lovatt, C.J. and Walling, L.L.

Notes: The authors identified a TERMINAL FLOWER homolog, CsTFL, in Washington navel oranges (Citrus sinensis) and investigated its role and the role of other genes in juvenility and flower production. The CsTFL gene was amplified from genomic DNA using PCR and degenerate primers, and amplification products were cloned into the pGEM®-T Easy Vector. The resulting clones were sequenced using the fmol® DNA Cycle Sequencing System. The CsTFL cDNA was amplified by RT-PCR, using 4 µg of total RNA from whole flowers and ImProm-II™ Reverse Transcriptase. The amplified cDNA was then cloned into the pGEM®-T Easy Vector. To evaluate CsTFL gene copy number and allele origins, the authors performed a Southern blot with 10 µg of genomic DNA and a CsTFL probe labeled with the Prime-a-Gene® Labeling System. To characterize CsTFL expression in various citrus tissues, RT-PCR was performed with 2 µg of total RNA and ImProm-II™ Reverse Transcriptase. The levels of CsTFL RNA and other RNAs were determined by cDNA synthesis using ImProm-II™ Reverse Transcriptase, followed by real-time PCR. Amplification products were quantitated by SYBR® Green fluorescence. Standard curves for each real-time PCR target were generated using known amounts of in vitro transcribed RNA. Prior to reverse transcription and real-time PCR, total RNA samples were treated with RQ1 RNase-Free DNase to remove DNA contaminants. (3650)

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RNA 10, 277–286. Localization of a promoter in the putative internal ribosome entry site of the Saccharomyces cerevisiae TIF4631 gene. 2004

Verge´, V., VonLanthenm, M., Masson, J.M., Trachsel, H., and Altmann, M.

Notes: Researchers cloned the Photinus and Renilla luciferase ORFs into the pSP64 Poly(A) Vector to create a dual-reporter vector named SP6P. A similar vector, SP6R.4G(-508/-3).P, was created in which a 5´ untranslated region from the Saccharomyces cerevisiae TIF4631 gene was cloned between the two reporter genes. These two vectors were used to transform yeast strains. The resultant transformants were lysed using Passive Lysis Buffer and a modified lysis procedure.   Lysates were analyzed for luciferase activities using the Dual-Luciferase® Reporter Assay System and a TD20/20 luminometer. The researchers also cloned and sequenced the 5´  untranslated region of TIF4631 by using a RACE-PCR technique followed by cloning the PCR amplimers into the pGEM®-T Vector. (2845)

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Am. J. Physiol. Cell Physiol. 287, 1031-1040. Molecular analysis of fiber type-specific expression of murine myostatin promoter. 2004

Salerno, M.S., Thomas, M., Forbes, D., Watson, T., Kambadur, R., and Sharma, M.

Notes: In this article, a 2.5- and a 1.7-kb fragment of the myostatin promoter were amplified by PCR and cloned into the pGEM®-T Easy vector.  These clones were then subcloned into the pGL3-Basic Vector.  Deletion mutants of the constructs were made and used to transient transfect mouse muscle C2C12 cells.  The Luciferase Assay System was then used to analyze transfectants.  The researchers also injected luciferase-reporter constructs into mouse muscle.  Luciferase activity in the mouse muscle was examined by grinding isolated muscles in liquid nitrogen and resuspending them in Cell Culture Lysis Buffer.  Ten micron cryosections of the muscles were also used in immunohistochmical staining experiments.  For these experiments, the researchers used a 1:50 dilution of Promega’s polyclonal anti-luciferase antibody, a secondary antibody and tertiary fluorescein-labeled conjugate.  The slides were counter stained with DAPI. (3147)

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J. Gen. Virol. 85(Pt 7), 2111-21. Molecular characterization of Penicillium chrysogenum virus reconsideration of the taxonomy of the genus Chrysovirus. 2004

Jiang, D. and Ghabrial, S.A.

Notes: The authors cloned and sequenced the dsRNA from Penicillium chrysogenum virus (PcV) and analyzed the gene products of the segmented virus. After reverse transcription followed by PCR to confirm the 5’ and 3’ ends of the dsRNA segments, RT-PCR was used to amplify the complete cDNAs for the four viral segments. The products were cloned into pGEM®-T Vector after A-tailing. To confirm the ORFs predicted for each of the four segments, the cDNAs were added to TNT® T7 Quick Coupled Transcription/Translation System reactions that included radiolabeled methionine. The resulting proteins ranged in size from 94-128 kDa and were separated on an 8% SDS-PAGE gel and analyzed by autoradiography. Gradient-purified PcV virions were digested with Sequencing Grade Modified Trypsin at 37°C for 18 hours and the digestion products were separated by reverse-phase HPLC on a C18 column. Two highly resolved peptides were selected for amino acid sequencing by automated Edman degradation. (3089)

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J. Cell Biol. 166, 61–71. Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac. 2004

D’Avino, P.P., Savoian, M.S., and Glover, D.M.

Notes: The entire sti gene (~7kb) of Drosophila was PCR cloned into the pGEM®-T Vector. The cloned sequences were then sequenced and analyzed for mutations. The researchers also used the T7 RiboMAX™ Large Scale RNA Production System to create dsRNAs from PCR-amplified regions of the sti and GFP coding regions. TransFast™ Transfection Reagent was used to transfect 2 x 106 Schneider S2 cells with 10 μg of dsRNA in a 35mm Petri dish. The cells were fixed after transfection and examined for multinucleate cells, or immunocytochemically stained for actin and anillin. (3259)

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J. Cell Biol. 279, 50069–50077. p51/p63 controls subunit 3 of the major epidermis integrin anchoring the stem cells to the niche. 2004

Kurata, S., Okuyama, T., Osada, M., Watanabe, T., Tomimori, Y., Sato, S., Iwai, A., Tsuji, T., Ikawa, Y. and Katoh, I.

Notes: HeLa and Saos-2 were transiently transfected with luciferase reporter vectors made from the pGL3-Promoter vector containing various intron sequences from the human integrinα3 gene ITGA3.  The first intron sequence was originally cloned by PCR cloning into the pGEM®-T Easy vector. Cell cultures were assessed for luciferase activity by making lysates with the Glo Lysis Buffer and assaying with the Steady-Glo® Assay kit.    (3225)

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Reproduction 127, 201–205. Quantitative analysis of mitochondrial DNAs in macaque embryos reprogrammed by rabbit oocytes. 2004

Yang, C.X., Kou, Z.H., Wang, K., Jiang, Y., Mao, W.W., Sun, Q.Y., Sheng, H.Z. and Chen, D.Y.

Notes: Nuclear transfer (NT) embryos (macaque fibroblasts introduced into matured metaphase II stage rabbit oocytes) were placed into PCR tubes containing 10µl of lysis solution, ReadyAmp™ Genomic DNA Purification System resin and 200µg/ml proteinase K. To break open the cells and access the genomic DNA, the embryos were incubated for 30 minutes at 55°C, boiled for 10 minutes, centrifuged for 1 minute then used for PCR. The amplified DNA was then cloned into the pGEM®-T Easy Vector and sequenced to determine if the product was macaque or rabbit. (3423)

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Biochimie 86, 849-856. Targeted disruption of the perxisomal thiolase B gene in mouse: A new model to study disorders related to peroxisomal lipid metabolism. 2004

Chevillard, G., Clémencet, M.C., Latruffe, N., and Nicolas-Francès, V.

Notes: GoTaq® DNA Polymerase was used in a multiplexed genotyping reaction with three primers to differentiate wild-type, heterozygous, and mutant transgenic mouse alleles simultaneously. The wild-type target was 670bp and the mutant or knockout target was 1,030bp. Various other amplimers (1.4kb, 471bp, & 311bp) were subcloned with the aid of the pGEM®-T Easy Vector System. (3360)

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RNA 10, 469-481. Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. 2004

van Eden, M.E., Byrd, M.P., Sherrill, K.W. and Lloyd, R.E.

Notes: The authors investigate a potential internal ribosome entry site (IRES) in the 5´ untranslated region (UTR) of cellular inhibitor of apoptosis protein 1 (c-IAP1). The c-IAP1 5´ UTR was amplified, cloned into pGEM®-T Vector, sequenced, then inserted into a dicistronic reporter vector between Renilla and firefly luciferase sequences. Using the Dual-Luciferase® Reporter Assay System, IRES activity was evaluated in Rabbit Reticulocyte Lysate and transiently transfected cells. The pSV-β-Galactosidase Control Vector was used as a control for transfection efficiency. Because splicing events were removing part of the Renilla luciferase coding region, the authors chose to use RNA transfection of cells. The ImProm-II™ Reverse Transcription System was used for the reverse transcription step of RT-PCRs to amplify intercistronic regions of the dicistronic RNA to examine mRNA splicing. (3429)

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J. Biol. Chem. 278, 1784-1793. Alternate activation of two divergently transcribed mouse genes from a bidirectional promoter is linked to changes in histone modification. 2003

Schuettengruber, B., Doetzlhofer, A., Kroboth, K., Wintersberger, E. and Seiser, C.

Notes: The acetylation status of histones was explored as an indicator of functional gene expression. The acetylation status of core histones H3 and H4 were assessed in treated and untreated cells by crosslinking chromatin, immunoprecipitating the chromatin with acetyl-H3 and acetyl-H4 specific antibodies. The crosslinking was reversed and the immunoprecipitated were phenol chloroform extracted then amplified for the mouse histone H4 gene. Amplifications were also set up with untreated genomic DNA to insure the amplification was within the linear range and to supply standards for gel-based quantitation.  All amplifications were performed with the PCR Master Mix. (2611)

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Infect. Immun. 71, 3914–3919. Detection of a luxS-signaling molecule in Bacillus anthracis. 2003

Jones M.B. and Blaser, M.J.

Notes: The Wizard® Genomic DNA Purification Kit was used to purify Bacillus anthracis chromosomal DNA, which was then used in PCR to amplify a luxN ortholog (named open reading frame BA5047). Amplified DNA containing the luxN ortholog region was then cloned into the pGEM®-T Easy Vector. This construct, when transformed into Vibrio harveyi (AI-1-, and AI-2+), allowed the detection of an upregulated signaling system by a bioluminescent assay. (3092)

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Infect. Immun. 71, 3971-3978. Development of DNA vaccines against hemolytic-uremic syndrome in a murine model. 2003

Capozzo, A.V.E., Creydt, V.P., Dran, G., Fernández, G., Gómez, S., Bentancor, L.V., Rubel, C., Ibarra, C., Isturiz, M. and Palermo, M.S.

Notes: Researchers used the pGEM®-T Vector System to clone the entire 1.4kb Shiga toxin type 2 gene (Stx2) from E. coli O157-H7 C600 (933W). The resultant construct, named pGEMTStx2, was used as a template in PCR to amplify each region of the gene corresponding to Shiga toxin type 2 subunits A and B. Each PCR product was digested with BamHI and EcoR I before ligation into pCDNA 3.1+ (Invitrogen) to create pStx2ΔA and pStx2B. Mice were then immunized with either one or both of these constructs and another construct expressing murine granulocyte-macrophage colony-stimulating factor. Expression of each subunit in mouse tissue was verified by RT-PCR with specific primers and the AccessQuick™ RT-PCR System. (2701)

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Clin. Can. Res. 9, 3052-3057. Expression and functional analyses of breast cancer resistance protein in lung cancer 2003

Kawabata, S., Oka, M., Soda, H., Shiozawa, K., Nakatomi, K., Tsurutani, J., Nakamura, Y., Doi, S., Kitazaki, T., Sugahara, K., Yamada, Y., Kamihira, S., Kohno, S.

Notes: Total RNA was obtained from non-small cell lung cancer cell lines and the breast cancer resistance protein message was amplified using real-time quantitative RT-PCR. RT-PCR products were subcloned into the pGEM®-T Easy Vector. (2714)

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J. Virol. 77, 1992-2002. Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio. 2003

Altmann, S.M., Mellon, M.T., Distel, D.L. and Kim, C.H.

Notes: The pGEM®-T Easy Vector was used to subclone products of a 5´ RACE reaction. A promoter construct, assembled in the pGL3 Basic Vector, was co-transfected with a zebrafish interferon expression vector in the ZF4 zebrafish embryo fibroblast cell line using the TransFast™ Reagent (details provided). Luciferase levels were examined with the BrightGlo™ Luciferase Assay Reagent. Induction of zebrafish mRNA was also examined in zebrafish liver cells (ZFL) following treatment with the known interferon inducer, poly(I)-poly(C). RNA was extracted and reverse transcribed using ImProm-II™ Reverse Transcriptase. The resulting cDNA was used for quantitative, real-time RT-PCR with a SYBR green-based assay. (2627)

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Clin. Can. Res. 9, 1906-1916. Novel kidney cancer immunotherapy based on the granulocyte-macrophage colony-stimulating factor and carbonic anhydrase IX fusion gene 2003

Hernández, J.M., Bui, M.H.T., Han, K-r., Mukouyama, H., Freitas, D.G., Nguyen, D., Caliliw, R., Shintaku, P.I., Paik, S.H., Tso, C-L., Figlin, R.A., Belldegrun, A.S.

Notes: pGEM®-T Easy Vector was used to clone PCR products.  The CytoTox 96® Non-Radioactive Cytotoxicity Assay  was used to determine specific cytotoxicity of human dendritic cells that were transduced with recombinant adenoviruses containing the gene encoding a fusion protein of granulocyte-macrophage colony stimulating factor and carbonic anhydrase IX. (2674)

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J. Cell Sci. 116, 2421-2430. PTHrP [67–86] regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinase-plasminogen activator and MMP-1 expression. 2003

Luparello, C., Sirchia, R. and Pupello, D.

Notes: Total and poly(A)+ RNA from treated and untreated 8701-BC breast cancer tumor cells were treated with RQ1 RNase-free DNase. The RNA samples were reverse transcribed using the M-MLV RNase H– point mutant reverse transcriptase and random primers to create cDNA used in downstream analyses. The cDNA from the reactions were used in PCR, differential display PCR, and semi-quantitative multiplex PCR reactions. In differential display PCR studies, the authors analyzed differentially displayed bands by staining 6% polyacrylamide gels with the SILVER SEQUENCE™ Staining Reagents. Differentially displayed bands were re-amplified and cloned using the pGEM®-T Easy Vector and high efficiency JM109 competent cells. (3020)

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Proc. Natl. Acad. Sci. USA 99, 13723-13728. A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity. 2002

Bufler, P., Azam, T., Gamboni-Robertson, F., Reznikov, L.L., Kumar, S., Dinarello, C.A., and Kim, S.-H.

Notes: The cDNA for the IL-1F7b protein was amplified from a human spleen cDNA library and directly subcloned in the pGEM®- T Easy Vector and sequenced. The clone was transferred to a bacterial expression vector with a purification tag. The expressed protein was used to make a polyclonal rabbit antibody and antibody purification column. The cDNA for the IL-1F7b was reamplified and directly subcloned into the pTARGET™ Mammalian Expression Vector. The cDNA was stably expressed in RAW264.7 mouse monocyte/macrophage cell line.  Lysates from the cells stably expressing IL-1F7b were used to test the antibodies by western blotting. Expressing cells were also tested by immunocytochemistry. (2598)

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Plant Physiol. 130, 58–67. Differential expression of a metallothionein gene during the presymbiotic versus the symbiotic phase of an arbuscular mycorrhizal fungus. 2002

Lanfranco, L., Bolchi, A., Ros, E.C., Ottonello, S. and Bonfante, P.

Notes: In this paper, the SV Total RNA Isolation System was used to isolate total RNA from Gigaspora margarita. The authors reported isolating total RNA from 100 spores or 100mg of mycorrhizal roots. One microliter of the isolated RNA was used in RT-PCR to amplify Gigaspora margarita metallothionein (MT)-like polypeptide (GmarMT1) RNA. The researchers also cloned the GmarMT1 coding region using the pGEM®-T Vector. (3077)

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J. Biol. Chem. 277, 42280–42288. GAGA factor down-regulates its own promoter. 2002

Kosoy, A., Pagans, S., Espina´s, M.L., Azorýn, F. and Bernue´s, J.

Notes: Researchers PCR amplified and cloned the Trl promoter into the pGEM®-T vector. The Trl promoter sequence, as well as the eve promoter sequence and/or a CMV promoter, were used to make luciferase reporter constructs using the pGL3-Basic Vector. Transient transfection experiments were performed with the promoter constructs and GAGA factor expression constructs. Relative luciferase activity from each experiment was measured and compared to that of a control.  (2775)

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J. Am. Soc. Nephrol. 13, 1992–1998. Human CLC-KB gene promoter drives the EGFP expression in the specific distal nephron segments and inner ear. 2002

Kobayashi, K., Uchida, S., Okamura, H.O., Marumo, F. and Sasaki, S.

Notes: The pGEM®-T Easy Vector was used to clone a 12.5 kb fragment containing human kidney specific chloride channel (CLC-KB) gene fused to an enhanced green fluorescence protein (EGFP) created in a long and accurate PCR (LA-PCR) reaction.  The resultant construct was further manipulated and used to make transgenic mice.  (3131)

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J. Biol. Chem. 277, 17349-17358. Structural Determinants of BRCA1 Translational Regulation 2002

Sobczak, K. and Krzyzosiak, W.J.

Notes: The authors investigated expression of BRCA1 from alternative transcripts possessing different 5' UTRs. RT-PCR using Promega's AMV reverse transcriptase was performed to generate cDNAs from total RNA isolated from human tissue. Promega's Taq DNA Polymerase was used for the PCR reaction. The authors also prepared two mRNAs that contained one or the other of the 2 alternative 5' UTRs (ex1a or ex1b) fused with the luciferase coding region. To generate the corresponding DNA for these constructs, the luciferase coding region was amplified from Promega's pGEM vector and ligated to PCR amplified ex1a or exlb. These ligation products served as the template amplifying two cDNA constructs (exla-luc or ex1b-luc).Ten additional cDNAs were made containing a variety of changes to the 5'UTR region of BRCA1. In vitro translation experiments were performed to determine how the composition of the 5'UTR affected protein expression levels (using Promega's RNasin to protect transcribed mRNA; T7 polymerase buffer; and rabbit reticulocyte and wheat germ extracts for translation). The authors conclude that secondary structures associated with elements in the longer 5'UTR reduced translation rates and could be responsible for the reduced expression of BRCA1 often associated with spontaneous ovarian and breast cancers. (2441)

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Oncogene 20, 1041-1051. Isolation and characterization of the TERE1 gene, a gene down-regulated in transitional cell carcinoma of the bladder 2001

McGarvey, T.W., Nguyen, T., Tomaszewski, J.E., Monson, F.C. and Malkowicz, S.B.

Notes: The cDNA for the TERE1 gene was amplified by PCR and directly subcloned into the pTARGET™ Mammalian Expression Vector. The 338-amino acid (36.8kDa) TERE1 protein was stably expressed in three human bladder transitional cell carcinomas (J82, T24 and 1376 TCC) and the human embryonic kidney cell line, HEK 293. Stable transfectants were selected with the antibiotic G-418. Overexpression of the protein caused 80-90% inhibition of cellular proliferation in two of the transitional cell carcinoma cell lines and inhibition of aneuploidy in the third. Transfections were accomplished in 24-well plates with the Tfx™-20 Transfection Reagent. Excellent details of the 1 hour transfection protocol are presented. (2596)

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Nucl. Acids Res. 29, 4502-4508. Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection. 2001

Cao, Z., Wanagat, J., McKiernan, S.H. and Aiken J.M.

Notes: Taq Polymerase was used to amplify mitochondrial sequences from various laser-capture microdissected rat DNA samples. A 15.7 kilobase PCR fragment was cloned into the pGEM®-T Easy Vector. The resultant clone was used to identify sequence breakpoints. (3234)

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Oncogene 20, 260-269. Modulation of apoptosis by procaspase-2 short isoform: selective inhibition of chromatin condensation, apoptotic body formation and phosphatidylserine externalization 2001

Droin, N., Rebe, C., Bichat, F., Hammann, A., Bertrand, R., and Solary, E.

Notes: The cDNA for the short isoform of the caspase-2 mRNA was amplified and cloned directly into the pTARGET™ Mammalian Expression Vector. The protein was stably expressed in U937 human leukemic cells by selection with the antibiotic G-418.  Stable clones were identified by PCR of the genomic DNA of selected colonies with primers to the T7 promoter of the pTARGET™ Vector and the downstream primer of the caspase-2 message. Overexpression of the protein interfered with etoposide-mediated apoptosis. (2597)

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