Data Link 6

Appendix of plasmids and plasmid constructions used in

Starczynowski D, 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

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-RELD29: aa 559-587 deleted from the C terminus of REL; an EcoRV-XhoI fragment from a PCR-generated deletion (see primers below) was used to replace an EcoRV-XhoI fragment in pGEM-Hu-c-Rel

pGEM- RELD58: aa 530-587 deleted from the C terminus of REL; an EcoRV-XhoI fragment from a PCR-generated deletion (see primers below) was used to replace an EcoRV-XhoI fragment in pGEM-Hu-c-Rel

pGEM- RELD90: aa 498-587 deleted from the C terminus of REL; an EcoRV-XhoI fragment from a PCR-generated deletion (see primers below) was used to replace an EcoRV-XhoI fragment in pGEM-Hu-c-Rel

pGEM-RELD110: a 478-587 deleted from the C terminus of REL; an EcoRV-XhoI fragment from a PCR-generated deletion (see primers below) was used to replace an EcoRV-XhoI fragment in pGEM-Hu-c-Rel

pGEM-RELD132: aa 456-587 deleted from the C terminus of REL; an EcoRV-XhoI fragment from a PCR-generated deletion (see primers below) was used to replace an EcoRV-XhoI fragment in pGEM-Hu-c-Rel

pGEM-RELD150: aa 438-587 deleted from the C terminus of REL; an EcoRV-XhoI fragment from a PCR-generated deletion (see primers below) was used to replace an EcoRV-XhoI fragment in pGEM-Hu-c-Rel

pGEM-RELD164: aa 424-587 deleted from the C terminus of REL and contains 4 additional vector-encoded aa; created by digesting wild-type pGEM4-Hu-c-Rel with SwaI

and religating

pGEM-RELD282: aa 306-587 deleted from the C terminus of REL; an EcoRV-XhoI fragment from a PCR-generated deletion (see primers below) was used to replace an EcoRV-XhoI fragment in pGEM-Hu-c-Rel

pGEM-RELD424-490: aa 424-490 deleted from within the C-terminal half of REL; an EcoRV-XhoI fragment from a PCR-generated deletion (see primers below) was used to replace an EcoRV-XhoI fragment in pGEM-Hu-c-Rel

pGEM-RELD164-VP16: REL aa 424-587 replaced with VP16 transactivation sequences; VP16 activation sequences cloned from pSL1180 (EcoRV-XhoI) into SwaI-XhoI sites of

pGEM-Hu-c-Rel

pGEM-RELD164-VP16DN: VP16 aa 413-453 were removed; pGEM-RELD164-VP16 was digested with 1) BamH1/XbaI (to obtain vector sequences); 2) BamH1/XmaI (to obtain C-terminal REL sequences); 3) XmaI/XbaI (to obtain C-terminal VP16 transactivation domain sequences 454-490), resulting in a truncated VP16 transactivation domain fused to REL∆164, and the three fragments were ligated

Spleen necrosis virus-based avian retroviral vectors

pMH105: Bi-cistronic spleen necrosis retroviral vector containing v-rel-IRES-neo (White & Gilmore, 1996)

pMH-Bcl2: Contains only chicken bcl-2 as the 3’ gene (White & Gilmore, 1996)

pc-Rel/Bcl-2: Wild-type human REL was digested with XbaI-SpeI and subcloned intoXbaI-digested pMH-Bcl2 (Gilmore et al, 2001)

pRELD29/Bcl-2: The REL fragment from pGEM-RELD29 digested with XbaI-XhoI was subcloned into pc-Rel/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL

pRELD58/Bcl-2: The REL fragment from pGEM-RELD58 digested with XbaI-XhoI was subcloned into pc-Rel/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL

pRELD90/Bcl-2: The REL fragment from pGEM-RELD90 digested with XbaI-XhoI was subcloned into pc-Rel/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL

pRELD110/Bcl-2: The REL fragment from pGEM-RELD110 digested with XbaI-XhoI was subcloned into pc-Rel/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL

pRELD132/Bcl-2: The REL fragment from pGEM-RELD132 digested with XbaI-XhoI was subcloned into pc-Rel/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL

pRELD150/Bcl-2: The REL fragment from pGEM-RELD150 digested with XbaI-XhoI was subcloned into pc-Rel/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL

pRELD164/Bcl-2 The REL fragment from pGEM-RELD164 digested with XbaI-XhoI was subcloned into pc-Rel/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL

pRELD282/Bcl-2: The REL fragment from pGEM-RELD282 digested with XbaI-XhoI was subcloned into pc-Rel/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL

pRELD424-490/Bcl-2: The REL fragment from pGEM-RELD424-490 digested with XbaI-XhoI was subcloned into pc-Rel/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL

pRELD164-VP16/Bcl-2: The REL fragment from pGEM-RELD164-VP16 digested with XbaI- XhoI was subcloned into pc-Rel/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL

JD214 BS+: Spleen necrosis virus vector (Sif et al, 1993)

GM282BS+: JD214BS+ containing v-rel subcloned into the XbaI site (Sif et al, 1993)

JD-Hu-c-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-RELD58: Spleen necrosis virus vector for expressing RELD58; JD214BS+ containing human RELD58 subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI

JD-RELD164: Spleen necrosis virus vector for expressing RELD164; JD214BS+ containing human RELD164 subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI

JD-RELD164-VP16: Spleen necrosis virus vector for expressing RELD164-VP16; JD214BS+ containing human RELD164-VP16 subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI

JD-RELD164-VP16DN: Spleen necrosis virus vector for expressing RELD164-VP16DN; JD214BS+ containing human RELD164-VP16DN subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI

Intermediate Subcloning Plasmids

CRF3 KS+ VP16: VP16 activation sequences (aa 413-490) were isolated as a BglII-BamHI fragment and ligated into the BamHI site in pBluescript KS+ (Gilmore, unpublished)

pSL1180-VP16: VP16 sequences subcloned from CRF3 KS+ VP16 (EcoRV-XbaI) into EcoRV-XbaI sites in pSL1180

pBluescript KS+: Plasmid containing the MCS within lacZ (Stratagene)

pBluescript KS+-REL: Wild-type REL fragment (EcoRV-XhoI) subcloned into the corresponding sites (EcoRV-XhoI) of pBluescript KS+

pBluescript KS+-RELD29: RELD29 EcoRV-XhoI fragment subcloned into the EcoRV-XhoI sites of pBluescript KS+

pBluescript KS+-RELD58: RELD58 EcoRV-XhoI fragment subcloned into the EcoRV-XhoI sites of pBluescript KS+

pBluescript KS+-RELD90: RELD90 EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+

pBluescript KS+-RELD110: RELD110 EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+

pBluescript KS+-RELD132: RELD132 EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+

pBluescript KS+-RELD150: RELD150 EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+

pBluescript KS+-RELD164: RELD164 EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+

pBluescript KS+-RELD282: RELD282 EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+

pBluescript KS+-RELD424-490: RELD424-490 EcoRV-XhoI fragment subcloned into EcoRV- XhoI sites of pBluescript KS+

Expression vectors (primarily for GAL4-fusion proteins)

CM216: CMV promoter expression vector for full-length REL

CMV-bgal: CMV promoter-driven expression plasmid for b-galactosidase; used for normalization of transfection efficiency

SG424: Expression plasmid containing GAL4 DNA-binding domain (DBD; aa 1-147) upstream of MCS and controlled by SV40 promoter (Sadowski & Ptashne, 1989)

SG-3’REL: Wild-type REL (aa 278-587) fused to GAL4 DBD; pBluescript KS+-REL cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in SG424 (Epinat et al., 2000;

Wang & Gilmore, 2001)

SG-3’RELD29: RELD29 (aa 278-557) fused to GAL4 DBD; pBluescript KS+-RELD29 cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424

SG-3’RELD58: RELD58 (aa 278-528) fused to GAL4 DBD; pBluescript KS+-RELD58  cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424

SG-3’RELD90: RELD90 (aa 278-496) fused to GAL4 DBD; pBluescript KS+-RELD90  cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424

SG-3’RELD110: RELD110 (aa 278-476) fused to GAL4 DBD; pBluescript KS+-RELD110 cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424

SG-3’RELD132: RELD132 (aa 278-454) fused to GAL4 DBD; pBluescript KS+-RELD132  cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in SG424

SG-3’RELD150: RELD150 (aa 278-436) fused to GAL4 DBD; pBluescript KS+-RELD150 cut with

BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424

SG-3’RELD164: RELD164 (aa 278-423) fused to GAL4 DBD; pBluescript KS+-REL cut with SwaI and religated

SG-3’RELD164/VP16: REL amino acids 424-587 replaced with VP16 activation sequences; pGEM4-RELD164/VP16 cut with ApaI-XbaI was subcloned into ApaI-XbaI sites in

pSG-3’REL

SG-3’RELD282: RELD282 (aa 278-304) fused to GAL4 DBD; pBluescript KS+-RELD282 cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424

SG-3’RELD424-490: RELD424-490 (aa 278-423,491-587) fused to GAL4 DBD; pBluescriptKS+-RELD424-490 digested with BamHI-KpnI was subcloned into BamHI-KpnI

sites in pSG424

Primers for PCR-based creation of REL deletion mutants

REL upstream primer: 5′- GAAGTTAGTGAATCTATGGATTTT-3′

RELD29: 5’-TATCCTCGAGCTAACCATGACTGTTTG G-3’

RELD58: 5’-CAGACTCGAGCTATGCATCTGATTGTGAAAC-3’

RELD90: 5’-TATCCTCGAGCTAGTCTAACACTGAATTACATGAGG-3’

RELD110: 5’-TATCCTCGAGCTAAGTCTCTCCCATGCTGTCACTGC-3’

RELD132: 5’-TATCCTCGAGCTAATCAGAAATACCATATAAATCTGC-3’

RELD150: 5’-CAGACTCGAGCTAGACTATGTCATCGGCATT-3’

RELD282: 5’-CAGACTCGAGCTAATCCTGGCACAGTTTCAG-3’

RELD424-490: 5’-CCGGATTTAAATTCATGTAATTCAGTGTTAGAC-3’

(PCR generated SwaI site at amino acid 489)

References

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

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 sequencs 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

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

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

Wang Y & TD Gilmore (2001) LIM domain protein Trip6 has a conserved nuclear export signal, nuclear targeting sequences, and multiple transactivation domains. Biochimica et Biophysica Acta 1538: 260-272

White DW & TD Gilmore (1996) Bcl-2 and CrmA have different effects on transformation, apoptosis, and the stability of IkB-a in chicken spleen cells transformed by temperature-sensitive v-Rel oncoproteins. Oncogene 13: 891-899