Data Link 11

Appendix of Plasmids and Primers for

Leeman JR, MA Weniger, TF Barth and TD Gilmore. 2008. Deletion analysis and alternative splicing define a transactivation inhibitory domain in human oncoprotein REL. Oncogene 27: 6770-6781

pGEM-based plasmids

pGEM4: Cloning vector for in vitro transcription/translation with either SP6 or T7 promoter elements (Promega)

pGEM-Hu-cRel: XbaI-XhoI/Klenow fragment of REL subcloned into XbaI-HincII- digested pGEM4 (Barkett et al, 2001)

pGEM-REL aa 1-322: pGEM-Hu-cRel used as a template for PCR amplification to create deletions in RID; A PCR fragment was generated with a 5’ primer located in the SP6 promoter within pGEM-4 and the REL-DRID-R primer located at REL codon 322 containing a novel SalI digestion site. The PCR fragment was digested with XbaI/SalI and subcloned into pGEM-4 digested with XbaI/SalI

pGEM-RELDRID: pGEM-Hu-cRel used as a template for PCR amplification; a PCR fragment was generated with 5’ REL-DRID-F forward primer overlapping REL codon 322, also containing a novel SalI site, and a 3’ primer within the T7 promoter of pGEM-4. To generate pGEM RELDRID, the 3’ REL fragment was digested with SalI/HindIII and subcloned into SalI/HindIII-digested pGEM-REL aa 1-322

pGEM-REL-RIDD1: pGEM-Hu-cRel was used as a template for PCR amplification; a PCR fragment was generated with a 5’ primer overlapping REL amino acid 356 and containing a novel 5’ SalI site (REL-RIDD1) and a 3’ T7 primer. The fragment was digested with SalI/HindIII and subcloned into pGEM-RELDRID digested SalI/HindIII

pGEM-REL-RIDD1+2: pGEM-Hu-cRel used as a template for PCR amplification; a PCR fragment was generated with a 5’ primer overlapping REL codon 389 and containing a novel 5’ SalI site (REL-RIDD2F) and a 3’ T7 primer. The fragment was digested with SalI/HindIII and subcloned into pGEM-RELDRID digested SalI/HindIII

pGEM-REL-RIDD2+3: pGEM-Hu-cRel used as a template for PCR amplification; a PCR fragment was generated with a 5’ SP6 primer and a 3’ primer overlapping REL codon 355 and containing a novel SalI site (REL-RIDD2R). The fragment was digested with XbaI/SalI and subcloned into pGEM-RELDRID digested XbaI/SalI

pGEM-REL-RIDD3: pGEM-Hu-cRel used as a template for PCR amplification; a PCR fragment was generated with a 5’ SP6 primer and a 3’ primer overlapping REL codon 388 and containing a novel SalI site (REL-RIDD3). The fragment was digested with XbaI/SalI and subcloned into pGEM-RELDRID digested XbaI/SalI

pGEM-RELD164/VP16: REL aa 424-587 replaced with VP16 transactivation sequences. VP16 activation sequences from pSL118 digested with EcoRV/XhoI and subcloned into Swa/XhoI-digested pGEM-Hu-c-Rel (D Starczynowski & TD Gilmore, unpublished)

pGEM-RELD164/VP16DN: VP16 aa 413-453 were removed; pGEM-RELD164/VP16 was digested with 1) BamHI/XbaI (to obtain vector sequences); 2) BamHI/SwaI (to obtain aa 1-423 REL sequences); 3) SmaI/XbaI (to obtain C-terminal VP16 transactivation domain sequences aa 454-490), resulting in a truncated VP16 transactivation domain fused to RELD164. The three fragments were ligated (D Starczynowski & TD Gilmore, unpublished)

pGEM-RELDRID-LacZ-12-106: LacZ-12-106 fragment was subcloned as a SalI fragment from pBluescript-SK(+)-LacZ-12-106 into pGEMRELDRID digested with SalI.  This ligation inserts 95 LacZ codons into REL where RID was removed, along with 4 codons contributed by the flanking SalI sites

pCRII-based Plasmids

pCRII-RELD9: cDNA isolated from the Karpas1106 mediastinal B-lymphoma cell line was used as a template for REL PCR amplification. A PCR fragment was generated using the 5’ REL-cDNA-F forward primer and the 3’ REL-cDNA-R reverse primer. PCR products were A-tailed and ligated into pCRII

pCRII-REL+Alu: cDNA isolated from the Karpas1106 mediastinal B-lymphoma cell line was used as a template for REL PCR amplification. A PCR fragment was generated using the 5’ REL-cDNA-F forward primer and the 3’ REL-cDNA-R reverse primer. PCR products were A-tailed and ligated into pCRII

pBluescript Intermediate Plasmids

pBluescript SK(+): Cloning Vector (Stratagene)

pBluscript SK(+)-REL: a wild-type REL EcoRV/XhoI fragment was subcloned into pBluescript SK(+) digested EcoRV/XhoI (D Starczynowski & TD Gilmore)

pBluscript SK(+)-RELD150: RELD150 EcoRV/XhoI fragment subcloned into EcoRV/XhoI digested pBluescript SK(+) (D Starczynowski & TD Gilmore)

pBluescript SK(+)-RELD424-490: RELD424-490 fragment digested EcoRV/XhoI was subcloned into pBluescript SK(+) digested with EcoRV/XhoI (D Starczynowski & TD Gilmore)

pBluescript SK(+)-RELDRID: RELDRID subcloned as an EcoRV/HindIII fragment into pBluescript SK(+) digested with EcoRV/HindIII

pBluescript SK(+)-REL-RIDD1: RIDD1 was  subcloned as an EcoRV/HindIII fragment into pBluescript SK(+) digested with EcoRV/HindIII

pBluescript SK(+)-REL-RIDD1+2: RIDD1+2 subcloned as an EcoRV/HindIII fragment into pBluescript SK(+) digested with EcoRV/HindIII

pBluescript SK(+)-REL-RIDD2+3: RIDD2+3 subcloned as an EcoRV/HindIII fragment into pBluescript SK(+) digested with EcoRV/HindIII

pBluescript SK(+)-REL-RIDD3: RIDD3 subcloned as an EcoRV/HindIII fragment into pBluescript SK(+) digested with EcoRV/HindIII

pBluescript SK(+)-RELD164/VP16DN: REL aa 427-587 replaced with VP16 activation sequences; pGEM-RELD164/VP16 digested with EcoRV/XhoI and subcloned into EcoRV/XhoI sites in pBluescript SK(+) (D Starczynowski & TD Gilmore)

pBluescript SK(+)-LacZ-12-106: pRSV-bGAL was used as a template for PCR amplification; the 5’ LacZ-R1/Sal1-F forward primer contained both PCR-generated EcoRI and SalI sites and overlapped LacZ codon 12. The 3’ LacZ-SalI-R reverse primer contained a PCR-generated SalI site and overlapped LacZ codon 106. The final PCR fragment was subcloned as an EcoRI-partial SalI fragment into pBS-SK+

pSL1180: Cloning vector

pSL1180-VP16: VP16 subcloned from CRF3 KS+ (EcoRV/XbaI) into EcoRV/XbaI sites of pSL1180 (D Starczynowski & TD Gilmore)

Avian Retroviral Vectors

JD214BS+: Spleen necrosis virus vector (Sif et al, 1993)

JD-REL: JD214BS+ containing human REL subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI (Gilmore et al, 2001)

JD-RELDRID: pGEM-RELDRID was digested with XbaI/HindIII and subcloned into JD-REL plasmid digested with XbaI/HindIII

JD-RELD424-490: JD214BS+ containing human RELD424-490 subcloned as an XbaI/XhoI fragment into JD214BS+ digested with XbaI/SalI (Starczynowski et al, 2003)

JD-RELD9: RELD9 XhoI/HindIII fragment subcloned into JD214 BS+ digested with SalI/HindIII

JD-REL+Alu: REL+Alu XhoI/HindIII fragment subcloned into JD214BS+ digested with SalI/HindIII

SW253: Replication-competent Rev-A avian helper virus (Watanabe & Temin, 1983)

pcDNA-based Expression Vectors

pcDNA 3.1(-): CMV promoter-driven expression vector (Invitrogen)

pcDNA-REL: Full-length wild=type REL XbaI/HindIII fragment subcloned into pcDNA3.1(-) digested with XbaI/HindIII

pcDNA-RELDRID: RELDRID XbaI/HindIII fragment subcloned into pcDNA-REL plasmid digested with XbaI/HindIII

pcDNA-RELD424-490: RELD424-490. RELD424-490 XbaI/HindIII fragment subcloned into pcDNA3.1(-) digested with XbaI/HindIII (D Starczynowski & TD Gilmore)

pcDNA-RELD9: A RELD9 XhoI/BamHI cDNA fragment subcloned into pcDNA3.1(-) digested with XhoI/BamHI

pcDNA-REL+Alu: A REL+Alu XhoI/BamHI cDNA fragment subcloned into pcDNA3.1(-) digested with XhoI/BamHI

pcDNA-FLAG: From Bakary Sylla (World Health Organization, Lyon, France)

pcDNA-FLAG-RHD-RID: pGEM-Hu-cRel was used as a template for the following PCR steps. A PCR fragment was generated using a 5’ primer overlapping REL aa 1 with a novel 5’ EcoRI site and a 3’ primer overlapping REL codon 423 with a novel 3’ BamHI site. The fragment was cloned into pcDNA-FLAG digested with EcoRI/BamHI (Michael Garbati, Gilmore Lab)

pcDNA-FLAG-REL: pGEM-Hu-cRel was digested EcoRV/XhoI generating a C-terminal REL fragment that was subcloned into EcoRV/XhoI-digested pcDNA-FLAG-RHD-RID (Michael Garbati, Gilmore Lab)

pcDNA-FLAG-RELDRID: pcDNA-RELDRID was digested EcoRV/XhoI generating a C-terminal REL fragment lacking codons 323-422 that was subcloned into EcoRV/XhoI-digested pcDNA-FLAG-REL

Expression Vectors for GAL4 Fusion Proteins in A293 Cells

pSG424: Expression vector with the SV40 early promoter controlling the GAL4 DNA-binding domain (aa 1-147) upstream of multi-cloning site (Sadowski & Ptashne, 1989)

pSG-REL: Wild-type human c-Rel (aa 278-587) fused to GAL4-DBD; pBluescript SK+-REL cut with BamHI-KpnI was subcloned into BamHI/KpnI-digested pSG424 (Starczynowski et al, 2003)

pSG-RELD424-490: REL (aa 278-423, 491-587) fused to GAL4 DBD; pBluescript SK+RELD424-490 cut with BamHI-KpnI was subcloned into BamHI-KpnI-digested pSG424 (Starczynowski et al., 2003)

pSG-RELDRID: pBluescript SK(+)-RELDRID digested with BamHI-KpnI was subcloned into BamHI-KpnI-digested pSG-REL

pSG-REL-RIDD1: pBluescript SK(+)-REL-RIDD1 digested with BamHI-KpnI was subcloned into BamHI/KpnI digested pSG424-REL

pSG-REL-RIDD1+2: pBluescript SK(+)-REL RIDD1+2 digested with BamHI-KpnI was subcloned into BamHI/KpnI-digested pSG-REL

pSG-REL-RIDD2+3: REL RIDD2+3 BamHI-KpnI fragment subcloned into BamHI/KpnI-digested pSG-REL

pSG-REL-RIDD3: REL RIDD3 BamHI-KpnI fragment subcloned into BamHI/KpnI- digested pSG-REL

pSG-RELD150: RELD150 (aa 278-436) BamHI-KpnI fragment subcloned into BamHI/KpnI-digested pSG424 (Starczynowski et al, 2003)

pSG-VP16DN: VP16 transactivation domain sequences were subcloned as an XmaI/XbaI fragment into the XmaI/XbaI sites in pSG424

pSG-REL-RID-VP16DN: REL amino acids 424-587 replaced with VP16 activation sequences; pBluescript SK+- REL-RID-VP16DN cut with BamHI-KpnI was subcloned into BamHI/KpnI-digested pSG424 (also referred to as pSG-3’ RELD164/VP16; Starczynowski et al, 2003)

pSG-RELDRID+LacZ: pSG-RELDRID+LacZ was generated by digestion of pGEM-RELDRID+LacZ with EcoRV/NdeI and subcloned into pSG-RELD424-490 that had been digested with EcoRV/NdeI

pSG-RELD9: JD-RELD9 digested with EcoRV/NdeI was subcloned into EcoRV/NdeI-digested pSG-REL

pSG-REL+Alu: JD-REL+Alu digested with EcoRV/NdeI was subcloned into EcoRV/NdeI-digested pSG-REL

Yeast GAL4 expression vectors

pGBT9:  yeast expression vector for the GAL4 DNA-binding domain (aa 1-147) (Epinat et al, 2000)

pGB-REL: REL (aa 278-587) fused to GAL4-DBD for expression in yeast; pSG424-REL cut with BamHI/XhoI was subcloned into BamHI/SalI-digested pGBT9

pGB-RELDRID: RELDRID (from aa 278) BamHI/XhoI fragment subcloned into BamHI/SalI-digested pGBT9

Vertebrate Reporter Plasmids

CMV-bgal: CMV promoter-driven expression plasmid for b-galactosidase (Starczynowski et al, 2003, 2005)

pRSV-bgal: Contains the RSV LTR upstream of the b-galactosidase gene (gift of U. Hansen, Boston University)

pGL2-3x-kB-LMP1-luciferase: 3x-kB-luciferase-pGL2 reporter plasmid has a minimal c-fos promoter element and three copies of the major histocompatibility complex (MHC) class I kB element (TGGGGATTCCCCA) upstream of  the luciferase gene (Mitchell & Sugden, 1995; gift of G. Mosialos, Fleming Institute)

IkB-pGL2 luciferase: 1.3 kb HindIII-EcoRI fragment of chicken IkBa, containing the transcriptional start site and 900 bp of upstream sequence all upstream of the luciferase gene (Schatzle et al, 1995; Starczynowski et al, 2007)

PolyA-GAL4E1b GAL4-luc: Contains GAL4 DNA-binding sites upstream of the luciferase gene (Starczynowski et al, 2003). Gift of Joseph Lipsick (Stanford Medical School)

Primers used for PCR

SP6 Promoter: 5’-GATTTAGGTGACACTATAG-3’

T7 Promoter: 5’-GTAATACGACTCACTATAGGGC-3’

REL-423-R: 5’-CGCAGGATCCCAATCATTCCCAACAGG-3’

PCR generated BamHI site (GGATCC): underlined

RELDRID-F: 5’-GGGGTCGACTTAAATGCTTCTAATGC-3’

RELDRID-R: 5’-TCCGTCGACAATTGAACCGAGGA-3’

REL-RIDD1: 5’-AATCGTCGACTATCCCTCACCTGGGCCCATC-3’

REL-RIDD3: 5’-AATAGTCGACTGGGGTGGGGTGGGCCAC-3’

REL-RIDD2R: 5’-ATACGTCGACGTAGGATTCTGCTTGACTTGAAACC-3’

REL-RIDD2F: 5’-ACGCGTCGACCGCTCAGGCAATACAAACCCACTG-3’

PCR generated SalI sites (GTCGAC): underlined

LacZ-R1/Sal1-F: 5’-AAGAATTCGTCGACTTACAACGTCGTGACTGGGAAAAC-3’

PCR generated EcoRI site (GAATTC) and SalI site (GTCGAC): underlined

LacZ-Sal1-R: 5’-TAATGTCGACATAGGTTACGTTGGTGTAGATGGG-3’

PCR generated SalI site (GTCGAC): underlined

REL-cDNA-F: 5′-CACCATGGCCTCCGGTGCGTATA-3′

REL-cDNA-R: 5′-TTATACTTGAAAAAATTCATATGGAAAGGAGTC-3′

Primers used for Real-time PCR

GAPDH-For: 5’-TGGTATCGTGGAAGGACTCATGAC-3’

GAPDH-Rev: 5’-ATGCCAGTGAGCTTCCCGTTCAGC-3’

REL-558-Rev: 5’-CCATGACTGTTTGGATTAGTACTGTTTG-3’

REL-AltSplic-d9-For: 5’-AAACTGTGCCAGGATCACGAACC-3’

REL-AltSplice-For: 5’-GCTATCACAGAACCCGTAACAG-3’

REL-AltSplice-Rev: 5’-ACCCCTGTAGGCATTTCTCTCACA-3’

Oligonucleotides used for kB probe

IFN-b kB-site: 5’-TCGAGAGGTCGGGAAATTCCCCCCCG-3’

MHC kB-site: 5’-TCGAGAGGTTGGGGATTCCCCACCCG-3’

SOD2 kB-site:  5’-TCGAGAGGTCGGGAATACCCCCCCCG-3’ (Bernard et al, 2001)

kB site underlined

References

Abid MR, IG Schoots, KC Spokes, SQ Wu, C Mawhinney & WC Aird (2004) Vascular endothelial growth factor-mediated induction of manganese superoxide dismutase occurs through redox-dependent regulation of forkhead and IkB/NF-kB. Journal of Biological Chemistry 279: 44030-44038

Barkett M, JE Dooher, L Lemonnier, L Simmons, JN Scarpati, Y Wang & TD Gilmore (2001) Three mutations in the retroviral oncoprotein v-Rel render it resistant to cleavage by caspase-3. Biochimica et Biophysica Acta 1526: 25-36

Bernard D, B Quatannens, A Begue, B Vandenbunder & C Abbadie (2001) Antiproliferative and antiapoptotic effects of cRel may occur witin the same cells via the up-regulation of manganese superoxide dismutase. Cancer Research 61: 2656-2664

Epinat J-C, D Kazandjian, DD Harkness, S Petros, J Dave, DW White & TD Gilmore (2000) Mutant envelope residues confer a transactivation function onto N-terminal sequences of the v-Rel oncoprotein. Oncogene 19: 599-607

Gilmore TD, C Cormier, J Jean-Jacques & M-E Gapuzan (2001) Malignant transformation of primary chicken spleen cells by human transcription factor c-Rel. Oncogene 20: 7098-7103

Kalaitzidis D, RE Davis, A Rosenwald, LM Staudt & TD Gilmore (2002) The human B-cell lymphoma cell line RC-K8 has multiple genetic alterations that dysregulate the Rel/NF-kB signal transduction pathway. Oncogene 21: 8759-8768

Mitchell T & B Sugden  (1995) Stimulation of NF-kB-mediated transcription by mutant derivatives of the latent membrane protein of Epstein-Barr virus. Journal of Virology 69: 2968-2976

Sadowski I & M Ptashne (1989) A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Research 17: 7539

Schatzle JD, J Kralova & HR Bose Jr (1995) Avian IkBa is transcriptionally induced by c-Rel and v-Rel with different kinetics. Journal of Virology 69: 5383-5390

Sif S, AJ Capobianco & TD Gilmore (1993) The v-Rel oncoprotein increases expression from Sp1 site-containing promoters in chicken embryo fibroblasts. Oncogene 8: 2501-2509

Starczynowski DT, JG Reynolds & TD Gilmore. (2003) Deletion of either C-terminal transactivation subdomain enhances the in vitro transforming activity of human transcription factor REL in chicken spleen cells. Oncogene 22: 6928-6936

Starczynowski DT, JG Reynolds & TD Gilmore (2005) Mutations of tumor necrosis factor alpha-responsive serine residues within the C-terminal transactivation domain of human transcription factor REL enhance its in vitro transforming ability. Oncogene 24: 7355-7368

Starczynowski DT, H Trautmann, C Pott, L Harder, N Arnold, JA Africa, JR Leeman, R Siebert & TD Gilmore (2007) Mutation of an IKK phosphorylation site within the transactivation domain of REL in two patients with B-cell lymphoma enhances REL’s in vitro transforming activity. Oncogene 26: 2685-2694

Watanabe S & HM Temin (1983) Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors. Molecular and Cellular Biology 3: 2241-2249