Data Link 8
APPENDIX
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 can enhance its in vitro transforming ability. Oncogene 24: 7355-7368
Vectors for In Vitro Transcription/Translation
pGEM4: Vector for in vitro transcription/translation (Promega)
pGEM-Hu-cRel: Vector for in vitro transcription/translation of wild-type human REL using SP6 polymerase; XbaI-XhoI/Klenow fragment of REL from CM216 subcloned into XbaI-HincII-digested pGEM4 (Barkett et al., 2001)
pGEM-RELD424-490: aa 424-490 deleted from within the C terminal half of REL; an EcoRV-XhoI fragment from a PCR-generated deletion (ask for details for primers) was used to replace an EcoRV-XhoI fragment in pGEM-Hu-c-Rel
pGEM-RELD424-490/bgal: aa 424-490 from RELwere replaced with 66 aa of b-galactosidase sequences from pBluescript SK+ (ask for details for primers)
pGEM-REL-S454A: pBluescriptSK+-REL-S454A digested with SwaI was subcloned into pGEM-RELD164 (SwaI) (Starczynowski et al, 2003)
pGEM-REL-S460A: pBluescriptSK+-REL-S460A digested with SwaI-XhoI was subcloned into pGEM-RELD150 (SwaI-XhoI) (Starczynowski et al, 2003)
pGEM-REL-S471A: PCR generated fragment (see Table 2.1 for primers) using pGEM-Hu-c-Rel as a template was subcloned EcoRV-XhoI into pGEM-RELD424-490 (EcoRV-XhoI)
pGEM-REL-S491,494A: pBluescriptSK+-REL-S491,494A digested with SwaI was subcloned into pGEM-RELD164 (SwaI) (Starczynowski et al, 2003)
Retroviral vectors
JD-REL: JD214BS+ containing human REL subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI (Gilmore et al, 2001)
JD-RELD424-490: Spleen necrosis virus vector for expressing RELD424-490; JD214BS+ containing human RELD424-490 subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI
JD-RELD424-490/bgal: Spleen necrosis virus vector for expressing RELD424-490/bgal; JD214BS+ containing RELD424-490/bgal, subcloned as an XbaI-NdeI fragment into JD-RELD424-490 digested with XbaI-NdeI (Starczynowski et al, 2003)
JD-REL-S443,444,447A: JD214BS+ containing REL-S443,444,447A, subcloned as an ApaI-NdeI fragment from pUC18-REL-S443,444,447A into JD-RELD424-490 digested with ApaI-NdeI
JD-REL-S454A: JD214BS+ containing REL-S454A, subcloned as an Xba-XhoI fragment from pGEM-REL-S454A into JD214BS+ digested with XbaI-SalI
JD-REL-S460A: JD214BS+ containing REL-S460A, subcloned as an Xba-XhoI fragment from pGEM-REL-S460A into JD214BS+ digested with XbaI-SalI
JD-REL-S471N: JD214BS+ containing REL-S471N, subcloned as an ApaI-HindIII fragment from pGEM-REL-S471N into JD-RELD424-490 digested with ApaI-HindIII
JD-REL-S471A: JD214BS+ containing REL-S471D, subcloned as an ApaI-HindIII fragment from a PCR-generated fragment into JD-RELD424-490 digested with ApaI-HindIII
JD-REL-S471D: JD214BS+ containing REL-S471D, subcloned as an Xba-HindIII fragment from pGEM-REL-S471D into JD214 BS+ digested with XbaI-HindIII
JD-REL-S491,494A: JD214BS+ containing REL-S491,494A, subcloned as an Xba-XhoI fragment from pGEM-REL-S494,494A into JD214 BS+ digested with XbaI-SalI
JD-REL-S533,536A: JD214BS+ containing REL-S533,536A, subcloned as an ApaI-NdeI fragment from pUC18-REL-S533,536A into JD-REL-D424-490 digested with ApaI-NdeI
JD-REL-S460F: JD214BS+ containing REL-S460F, subcloned as an Xba-XhoI fragment from pGEM-REL-S460F into JD214BS+ digested with XbaI-SalI
Cloning Vectors
pBluescript SK+: Plasmid containing the MCS within lacZ (Stratagene)
pBluescript SK+-REL: Wild-type REL fragment (EcoRV-XhoI) subcloned into the corresponding sites (EcoRV-XhoI) of pBluescript SK+
pBluescript SK+-RELD424-490: RELD424-490 EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript SK+
pBluescriptSK+-3’REL-S454A: Plasmid containing REL fragment from SwaI to 5’ end; REL-S454A fragment (SacI-BglII) from pUC18-3’REL-S454A subcloned into pBluescript SK+ (SacI-BamHI)
pBluescriptSK+-3’REL-S460A: Plasmid containing REL fragment from SwaI to 5’ end; REL-S460A fragment (SacI-BglII) from pUC18-3’REL-S454A subcloned into pBluescript SK+ (SacI-BamHI)
pBluescriptSK+-3’REL-S491,494A: Plasmid containing REL fragment from SwaI to 5’end; REL-S491,494A fragment (SacI-BglII) from pUC18-3’REL-S454A subcloned into pBluescript SK+ (SacI-BamHI)
pBluescriptSK+-3’REL-S454A: Plasmid containing REL fragment from EcoRV to 5’ end; REL-S454A fragment (EcoRV-HindIII) from JD-REL-S454A subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+
pBluescriptSK+-3’REL-S460A: Plasmid containing REL fragment from EcoRV to 5’ end; REL-S460A fragment (EcoRV-HindIII) from JD-REL-S460A subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+
pBluescriptSK+-3’REL-S471N: Plasmid containing REL fragment from EcoRV to 5’ end; REL-S471N fragment (EcoRV-HindIII) from JD-REL-S471N subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript KS+
pBluescriptSK+-3’REL-S471A: Plasmid containing REL fragment from EcoRV to 5’ end; REL-S471A fragment (EcoRV-HindIII) from JD-REL-S471A subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+
pBluescriptSK+-3’REL-S471D: Plasmid containing REL fragment from EcoRV to 5’ end; REL-S471D fragment (EcoRV-HindIII) from JD-REL-S471D subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+
pBluescriptSK+-3’REL-S491,494A: Plasmid containing REL fragment from EcoRV to 5’ end; REL-S491,494A fragment (EcoRV-HindIII) from JD-REL-S491,494A subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+
pBluescriptSK+-3’REL-S460F: Plasmid containing REL fragment from EcoRV to 5’end; REL-S460F fragment (EcoRV-HindIII) from JD-REL-S460F subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+
Expression Vectors
SG424: Expression plasmid containing GAL4 DNA-binding domain (DBD) upstream of MCS and downstream of SV40 promoter (Sadowski and Ptashne, 1989)
SG-3’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 sites in SG424
SG-3’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 sites in SG424
SG-3’REL-S443,444,447A: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S443,444,447A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424
SG-3’REL-S454A: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-454A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424
SG-3’REL-S460A: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S460A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424
SG-3’REL-S471A: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S471A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424
SG-3’REL-S471N: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S471N cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424
SG-3’REL-S471D: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S471D cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424
SG-3’REL-S491,494A: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S491,494A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424
SG-3’REL-S533,536A: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S533,536A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424
SG-3’REL-S460F: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S460F cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424
CM216: CMV promoter expression vector for full-length REL (Inder Verma)
pcDNA3.1(-): CMV promoter expression vector (Promega)
pcREL-S443,444,447A: pcDNA3.1(-) containing REL-S443,444,447A, subcloned as an XbaI-HindIII fragment from JD-REL-S443,444,447A into pcDNA3.1(-) digested with XbaI-HindIII
pcREL-S454A: pcDNA3.1(-) containing REL-S454A. REL-S454A subcloned as an XbaI-HindIII fragment from JD-REL-S454A into pcDNA3.1(-) digested with XbaI-HindIII
pcREL-S460A: pcDNA3.1(-) containing REL-S460A. REL-S460A subcloned as an XbaI-HindIII fragment from JD-REL-S460A into pcDNA3.1(-) digested with XbaI-HindIII
pcREL-S471A: pcDNA3.1(-) containing REL-S471A. REL-S471A subcloned as an XbaI-HindIII fragment from JD-REL-S471A into pcDNA3.1(-) digested with XbaI-HindIII
pcREL-S471N: pcDNA3.1(-) containing REL-S471N. REL-S471N subcloned as an XbaI-HindIII fragment from JD-REL-S471N into pcDNA3.1(-) digested with XbaI-HindIII
pcREL-S471D: pcDNA3.1(-) containing REL-S471D. REL-S471D subcloned as an XbaI-HindIII fragment from JD-REL-S471D into pcDNA3.1(-) digested with XbaI-HindIII
pcREL-S491,494A: pcDNA3.1(-) containing REL-S491,494A. REL-S491,494A subcloned as an XbaI- HindIII fragment from JD-REL-S491,494A into pcDNA3.1(-) digested with XbaI-HindIII
pcREL-S533,536A: pcDNA3.1(-) containing REL-S533,536A. REL-S533,536A subcloned as an XbaI-HindIII fragment from JD-REL-S533,536A into pcDNA3.1(-) digested with XbaI-HindIII
pcREL-S460F: pcDNA3.1(-) containing REL-S460F. REL-S460F subcloned as an XbaI-HindIII fragment from JD-REL-S460F into pcDNA3.1(-) digested with XbaI-HindIII
pcRELD424-490: pcDNA3.1(-) containing RELD424-490. RELD424-490 subcloned as an XbaI-HindIII fragment from JD-RELD424-490 into pcDNA3.1(-) digested with XbaI-HindIII
pUC18-3’REL-S443,444,447A: REL-S443,444,447A (aa 309-587) fused to GAL4 DBD (Martin et al, 2001)
pUC18-3’REL-S454A: REL-S454A (aa 309-587) fused to GAL4 DBD (Martin et al, 2001)
pUC18-3’REL-S460A: REL-S460A (aa 309-587) fused to GAL4 DBD (Martin et al, 2001)
pUC18-3’REL-S491,494A: REL-S491,494A (aa 309-587) fused to GAL4 DBD (Martin et al, 2001)
pUC18-3’REL-S533,536A: REL-S533,536A (aa 309-587) fused to GAL4 DBD (Martin et al, 2001)
pRc/CMV-REL-S471N: CMV-containing plasmid for expression of REL-S471N (Martin et al, 2000)
Reporter Plasmids
3x-kB-pGL2: 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 Luciferase (Mitchell and Sugden, 1995) .
IkB-pGL2: IkBa-Luciferase-pGL2 reporter plasmid contains a 1.3 kb HindIII-EcoRI fragment of the chicken IkBa genomic clone, containing the transcriptional start site and 900 bp of upstream sequence upstream of Luciferase (Schatzle et al, 1995) .
SOD2-pGL3: The SOD2-Luciferase-pGL3 reporter plasmid contains 3.3 kb of the human SOD2 gene promoter and 0.4 kb of the SOD2 intronic enhancer upstream of Luciferase (Abid et al, 2004) .
CMV-bgal: CMV promoter–driven expression plasmid for b-galactosidase; used for normalization of transfection efficiency
pGK-bgal: SV40 promoter-driven expression plasmid for b-galactosidase; used for normalization of transfection efficiency
Primers used for PCR
REL Forward: 5’-GAAGTTAGTGAATCTATCGATTTT-3’
REL Reverse: 5’-CAATTGACTGACTCCTTTCCATATGAA-3’
REL-S471A Forward: 5’-ATGACAACCAGCGCTGACAGCATGGGAGAG-3’
REL-S471A Reverse: 5’-CTCTCCCATGCTGTCAGCGCTGGTTGTCAT-3’
REL-S471D Forward: 5’-ATGACAACCAGCGATGACAGCATGGGAGAG-3’
REL-S471D Reverse: 5’-CTCTCCCATGCTGTCATCGCTGGTTGTCAT-3’
Oligonucleotide used for kB probe (annealed to be double-stranded)
kB-WT: 5’-TCGAGAGGTCGGGAAATTCCCCCCCG-3’kB site underlined
References
Abid MR, IG Schoots, KC Spokes, SQ Wu, Mawhinney C & 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
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
Martin AG, B San-Antonio & M Fresno (2001) Regulation of NF-kB transactivation: implication of phosphatiylinositol 3-kinase and protein kinase C zeta in c-Rel activation by tumor necrosis factor alpha. Journal of Biological Chemistry 276: 15840-15849
Mitchell T & Sugden B (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 & Ptashne M (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
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
