{"id":586,"date":"2011-04-26T15:27:14","date_gmt":"2011-04-26T19:27:14","guid":{"rendered":"https:\/\/www.bu.edu\/nf-kb\/?page_id=586"},"modified":"2011-04-30T13:58:58","modified_gmt":"2011-04-30T17:58:58","slug":"data-link-6","status":"publish","type":"page","link":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link-6\/","title":{"rendered":"Data Link 6"},"content":{"rendered":"

Appendix of plasmids and plasmid constructions used in<\/strong><\/p>\n

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<\/p>\n

Vectors for in vitro transcription\/translation <\/strong><\/p>\n

<\/span><\/p>\n

pGEM4: <\/span> Vector for in vitro transcription\/translation (Promega)<\/p>\n

<\/span><\/p>\n

pGEM-Hu-cRel:<\/span> 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)<\/p>\n

<\/span><\/p>\n

pGEM-RELD29:<\/span> 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<\/p>\n

pGEM- RELD58:<\/span> 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<\/p>\n

pGEM- RELD90:<\/span> 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<\/p>\n

pGEM-RELD110:<\/span> 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<\/p>\n

pGEM-RELD132:<\/span> 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<\/p>\n

pGEM-RELD150:<\/span> 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<\/p>\n

pGEM-RELD164:<\/span> 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<\/p>\n

and religating<\/p>\n

pGEM-RELD282:<\/span> 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<\/p>\n

<\/span><\/p>\n

pGEM-RELD424-490:<\/span> 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<\/p>\n

<\/span><\/p>\n

pGEM-RELD164-VP16:<\/span> REL aa 424-587 replaced with VP16 transactivation sequences; VP16 activation sequences cloned from pSL1180 (EcoRV-XhoI) into SwaI-XhoI sites of<\/p>\n

pGEM-Hu-c-Rel<\/p>\n

pGEM-RELD164-VP16DN:<\/span> 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\u2206164, and the three fragments were ligated<\/p>\n

Spleen necrosis virus-based avian r<\/strong>etroviral vectors <\/strong><\/p>\n

pMH105:<\/span> Bi-cistronic spleen necrosis retroviral vector containing v-rel<\/em>-IRES-neo<\/em> (White & Gilmore, 1996)<\/p>\n

pMH-Bcl2:<\/span> Contains only chicken bcl-2<\/em> as the 3\u2019 gene (White & Gilmore, 1996)<\/p>\n

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

pRELD29\/Bcl-2:<\/span> The REL<\/em> 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<\/em><\/p>\n

pRELD58\/Bcl-2:<\/span> The REL<\/em> 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<\/em><\/p>\n

pRELD90\/Bcl-2:<\/span> The REL<\/em> 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<\/em><\/p>\n

pRELD110\/Bcl-2:<\/span> The REL<\/em> 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<\/em><\/p>\n

pRELD132\/Bcl-2:<\/span> The REL<\/em> 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<\/em><\/p>\n

pRELD150\/Bcl-2:<\/span> The REL<\/em> fragment from pGEM-RELD150 digested with XbaI-XhoI was subcloned into\u00a0pc-Rel\/Bcl2 digested with XbaI-XhoI and used to replace wild-type REL<\/em><\/p>\n

pRELD164\/Bcl-2<\/span> The REL<\/em> 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<\/em><\/p>\n

pRELD282\/Bcl-2:<\/span> The REL<\/em> 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<\/em><\/p>\n

pRELD424-490\/Bcl-2:<\/span> The REL<\/em> 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<\/em><\/p>\n

pRELD164-VP16\/Bcl-2: <\/span>The REL<\/em> 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 <\/em><\/p>\n

<\/em><\/p>\n

JD214 BS+:<\/span> Spleen necrosis virus vector (Sif et al, 1993)<\/p>\n

GM282BS+:<\/span> JD214BS+ containing v-rel subcloned into the XbaI site (Sif et al, 1993)<\/p>\n

JD-Hu-c-Rel:<\/span> JD214BS+ containing human REL<\/em> subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI (Gilmore et al, 2001)<\/p>\n

JD-RELD424-490:<\/span> Spleen necrosis virus vector for expressing RELD424-490; JD214BS+ containing human RELD424-490<\/em> subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI<\/p>\n

JD-RELD58:<\/span> Spleen necrosis virus vector for expressing RELD58; JD214BS+ containing human RELD58<\/em> subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI<\/p>\n

JD-RELD164:<\/span> Spleen necrosis virus vector for expressing RELD164; JD214BS+ containing human RELD164<\/em> subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI<\/p>\n

JD-RELD164-VP16:<\/span> Spleen necrosis virus vector for expressing RELD164-VP16; JD214BS+ containing human RELD164-VP16<\/em> subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI<\/p>\n

JD-RELD164-VP16DN: <\/span>Spleen necrosis virus vector for expressing RELD164-VP16DN; JD214BS+ containing human RELD164-VP16DN<\/em> subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI<\/p>\n

Intermediate Subcloning\u00a0Plasmids<\/h3>\n

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

pSL1180-VP16:<\/span> VP16<\/em> sequences subcloned from CRF3 KS+ VP16 (EcoRV-XbaI) into EcoRV-XbaI sites in pSL1180 <\/span><\/p>\n

pBluescript KS+:<\/span> Plasmid containing the MCS within lacZ<\/em> (Stratagene)<\/p>\n

pBluescript KS+-REL:<\/span> Wild-type REL<\/em> fragment (EcoRV-XhoI) subcloned into the corresponding sites (EcoRV-XhoI) of pBluescript KS+<\/p>\n

pBluescript KS+-RELD29:<\/span> RELD29<\/em> EcoRV-XhoI fragment subcloned into the EcoRV-XhoI sites of pBluescript KS+<\/p>\n

pBluescript KS+-RELD58:<\/span> RELD58<\/em> EcoRV-XhoI fragment subcloned into the EcoRV-XhoI sites of pBluescript KS+<\/p>\n

pBluescript KS+-RELD90:<\/span> RELD90<\/em> EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+<\/p>\n

pBluescript KS+-RELD110:<\/span> RELD110<\/em> EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+<\/p>\n

pBluescript <\/span>KS+-RELD132:<\/span> RELD132<\/em> EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+<\/p>\n

<\/span><\/p>\n

pBluescript <\/span>KS+-RELD150:<\/span> RELD150<\/em> EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+<\/p>\n

pBluescript <\/span>KS+-RELD164:<\/span> RELD164<\/em> EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+<\/p>\n

pBluescript <\/span>KS+-RELD282:<\/span> RELD282<\/em> EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript KS+<\/p>\n

pBluescript <\/span>KS+-RELD424-490:<\/span> RELD424-490<\/em> EcoRV-XhoI fragment subcloned into EcoRV- XhoI sites of pBluescript KS+<\/p>\n

Expression vectors (primarily for GAL4-fusion proteins)<\/h3>\n

CM216:<\/span> CMV promoter expression vector for full-length REL<\/p>\n

CMV-bgal:<\/span> CMV promoter-driven expression plasmid for b-galactosidase; used for normalization of transfection efficiency<\/p>\n

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

SG-3\u2019REL:<\/span> 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.,<\/em> 2000;<\/p>\n

Wang & Gilmore, 2001)<\/p>\n

SG-3\u2019RELD29:<\/span> RELD29 (aa 278-557) fused to GAL4 DBD; pBluescript KS+-RELD29 cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424<\/p>\n

SG-3\u2019RELD58: <\/span> RELD58 (aa 278-528) fused to GAL4 DBD; pBluescript KS+-RELD58\u00a0 cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424<\/p>\n

SG-3\u2019RELD90:<\/span> RELD90 (aa 278-496) fused to GAL4 DBD; pBluescript KS+-RELD90\u00a0 cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424<\/p>\n

SG-3\u2019RELD110:<\/span> RELD110 (aa 278-476) fused to GAL4 DBD; pBluescript KS+-RELD110 cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424<\/p>\n

SG-3\u2019RELD132:<\/span> RELD132 (aa 278-454) fused to GAL4 DBD; pBluescript KS+-RELD132\u00a0 cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in SG424<\/p>\n

SG-3\u2019RELD150:<\/span> RELD150 (aa 278-436) fused to GAL4 DBD; pBluescript KS+-RELD150 cut with<\/p>\n

BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424<\/p>\n

SG-3\u2019RELD164:<\/span> RELD164 (aa 278-423) fused to GAL4 DBD; pBluescript KS+-REL cut with SwaI and religated<\/p>\n

SG-3\u2019RELD164\/VP16:<\/span> REL amino acids 424-587 replaced with VP16 activation sequences; pGEM4-RELD164\/VP16 cut with ApaI-XbaI was subcloned into ApaI-XbaI sites in<\/p>\n

pSG-3’REL<\/p>\n

SG-3\u2019RELD282:<\/span> RELD282 (aa 278-304) fused to GAL4 DBD; pBluescript KS+-RELD282 cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in pSG424<\/p>\n

SG-3\u2019RELD424-490:<\/span> RELD424-490 (aa 278-423,491-587) fused to GAL4 DBD; pBluescriptKS+-RELD424-490 digested with BamHI-KpnI was subcloned into BamHI-KpnI<\/p>\n

sites in\u00a0pSG424<\/p>\n

Primers <\/strong>for PCR-based creation of REL deletion mutants<\/strong> <\/strong><\/h3>\n

REL upstream primer:<\/span> 5′- GAAGTTAGTGAATCTATGGATTTT-3′<\/p>\n

RELD29:<\/span> 5\u2019-TATCCTCGAGCTAACCATGACTGTTTG G-3\u2019<\/p>\n

RELD58:<\/span> 5\u2019-CAGACTCGAGCTATGCATCTGATTGTGAAAC-3\u2019<\/p>\n

RELD90:<\/span> 5\u2019-TATCCTCGAGCTAGTCTAACACTGAATTACATGAGG-3\u2019<\/p>\n

RELD110:<\/span> 5\u2019-TATCCTCGAGCTAAGTCTCTCCCATGCTGTCACTGC-3\u2019<\/p>\n

RELD132:<\/span> 5\u2019-TATCCTCGAGCTAATCAGAAATACCATATAAATCTGC-3\u2019<\/p>\n

RELD150:<\/span> 5\u2019-CAGACTCGAGCTAGACTATGTCATCGGCATT-3\u2019<\/p>\n

RELD282:<\/span> 5\u2019-CAGACTCGAGCTAATCCTGGCACAGTTTCAG-3\u2019<\/p>\n

RELD424-490:<\/span> 5\u2019-CCGGATTTAAATTCATGTAATTCAGTGTTAGAC-3\u2019<\/p>\n

(PCR generated SwaI site at amino acid 489)<\/p>\n

References<\/strong><\/h3>\n

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<\/p>\n

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<\/p>\n

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<\/p>\n

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<\/p>\n

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

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<\/p>\n

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<\/p>\n","protected":false},"excerpt":{"rendered":"

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 […]<\/p>\n","protected":false},"author":4258,"featured_media":0,"parent":337,"menu_order":18,"comment_status":"closed","ping_status":"open","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/586"}],"collection":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/users\/4258"}],"replies":[{"embeddable":true,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/comments?post=586"}],"version-history":[{"count":4,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/586\/revisions"}],"predecessor-version":[{"id":614,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/586\/revisions\/614"}],"up":[{"embeddable":true,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/337"}],"wp:attachment":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/media?parent=586"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}