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1842304402
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note
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A PCR-amplified cDNA fragment corresponding either to the first exon (Tat72) or both exons (Tat101) of the HIV-1 Tat gene was amplified with the use of the HIV-1 cDNA clone pCV1 (29) as a template and primers containing an additional Bam HI site for cloning. The amplified fragment was cloned into the unique Bam HI site of the episomal vector pRep9 (Invitrogen, San Diego, CA) under the control of the Rous sarcoma virus promoter. Because pCV1 cDNA encodes a truncated 86-amino acid form of Tat, a mutation (TAG [→] TCG, changing codon 86 from a stop to a serine) was introduced in the 3′ primer used in the PCR reaction to reopen the Tat reading frame to its full length. Recombinant plasmids were transfected into Jurkat cells by electroporation, followed by selection with Geneticin (600 μg/ml, Gibco-BRL) and cloning by limiting dilution. All clones were frozen rapidly after identification, and aliquots were periodically thawed (every 6 weeks) to maintain the identity of the clones. During that interval, all phenomena described in this report (Tat mRNA, Tat activity, and IL-2 hyperresponsiveness) were stable and reproducible. All cell lines used in this study were negative for Mycoplasma (MycoTest, Life Technologies).
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1842400880
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note
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32P-labeled antisense riboprobe for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (30) was hybridized in a separate reaction with 2 μg of total RNA.
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16
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1842383285
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note
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Transient transfection of supercoiled plasmid DNA was performed with the use of DEAE-dextran, and CAT activity was measured with a diffusion assay as previously reported (30).
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unpublished observation
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note
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7 cells). Culture supernatants from cells stimulated with antibodies directed against either CD3 or CD3+CD28 were harvested at different times after infection and analyzed for RT activity.
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33
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1842349472
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note
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Anti-CD3 (454.3.21) was provided by N. Chiorazzi (North Shore University Hospital, Manhasset, NY) and precoated on tissue culture wells with 3 μg of antibody per milliliter of buffer [35 mM bicarbonate and 15 mM carbonate (pH 9.6)] at 4°C overnight. Anti-CD28 (clone 28.2) was provided by D. Olive (INSERM, Marseilles, France) (37).
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34
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1842268727
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note
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4, and 0.01 M tris (pH 8.0)], and 15 ml of buffer C (same composition as buffer B, but at a pH of 6.3). The recombinant protein was eluted with 15 ml of buffer C containing 250 mM imidazole. This eluate was loaded directly on a high-performance liquid chromatography C4 column run in a gradient of acetonitrile (0 to 100%) in 0.1% trifluoroacetic acid. Fractions containing Tat were lyophilized in small aliquots and stored at -70°C under anaerobic conditions to prevent Tat oxidation. Tat was resuspended in degassed phosphate-buffered saline plus 0.1 mM dithiothreitol immediately before use. The biological activity of Tat purified according to this protocol was tested by incubating it with U1 cells, which contain a virus exhibiting a Tat-defective phenotype (32). Tat treatment of these cells caused a 50-fold induction of viral expression as detected by p24 secretion into culture supernatant. Endotoxin contamination of Tat prepared with this protocol was below the detection limit of the assay (<59 EU/ml; Limulus Amebocyte Lysate Assay, Biowhittaker, Walkersville, MD).
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1842278475
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note
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pIL-2luc was constructed by cloning a 390-base pair Kpn I-Hind III fragment containing the human IL-2 promoter (nt -340 to nt +50) (a gift from R. Gaynor) into the corresponding sites of pGL2-BASIC (Promega, Madison, WI). This plasmid was used as a substrate for the mutagenesis of the CD28RE by means of the Transformer site-directed mutagenesis method (Clontech, Palo Alto, CA). The oligonucleotide (5′-GGGTTTAAAGAAGCCTCAAAGAGTCATCA-3′) was used to introduce four mutations within the CD28RE (mutations are highlighted in bold), abolishing binding of the CD28RC to the element (27). The oligonucleotide (5′-CTTATCATGTCTGACGTCGTCGACCGATGC-3′) changes a Bam HI into an Aat II restriction site (highlighted in bold) and was used for selection during mutagenesis. The region corresponding to the IL-2 promoter was fully resequenced to confirm the mutation, and the resequenced fragment was subcloned into pGL2-BASIC as described above.
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37
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0028293290
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C. Van Lint, J. Ghysdael, P. Paras Jr., A. Burny, E. Verdin, J. Virol. 68, 2632 (1994).
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39
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1842269685
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note
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89.6, p89.6 (5) to change codon 72 of the Tat ORF from CAG into TAG. The mutation was introduced with the use of the Transformer kit (Clontech) with the following two oligonucleotides: 5′-CTCTATCAAAGTAGTAAGTAGTAC-3′ (Tat mutation) and 5′-GTGCCACCTGATATCTAAGAAACC-3′ (selection primer). The presence of the mutation was verified by sequencing, and a fully resequenced Sal I-Stu 1 fragment containing the mutation was subcloned back into p89.6. Supernatants from CEM x 174 cells transfected with this DNA were harvested and their RT titer was measured. To confirm that virus stocks had not reverted to wild type, virus stocks were centrifuged and purified RNA was used in RT-PCR to amplify a fragment containing the mutated Tat gene. This PCR fragment was cloned with the TA cloning kit (Invitrogen), 10 individual clones were resequenced, and all contained the original mutation in the Tat gene.
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40
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1842323509
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note
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This paper is dedicated to the memory of Kenneth Warren. We thank R. Colmann, D. Olive, N. Chiorazzi, R. Gaynor, C. Metz, and U. Siebenlist for reagents used in these studies; L. Bonetta and K. Manogue for their helpful comments on the manuscript; D. Olive and Y. Colette for helpful discussions; K.-T. Jeang for pointing out to us the Tat mutation resulting in a truncated Tat protein; and N. Yarlett for testing cell lines for Mycoplasma. C.V.L. is Chargé de Recherches of the Fond National de la Recherche Scientifique, Belgium.
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