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APPENDIX

Epinat J-C, Dvorin EL and Gilmore TD. (2000) Envelope-dependent transactivation by the retroviral oncoprotein is required for transformation of chicken spleen cells. Oncogene 19 :3131-3137

A.  Descriptions of plasmids used in this paper. (The source of the plasmid is indicated in parentheses.)

1.  Plasmids for the expression of Rel proteins in yeast.

BC102:            Vector alone with Adh promoter, Trp-selectable (Kamens & Brent, 1991)

BC102-Spe/Sna:  BC102 with linker containing SpeI and SnaBI sites at the unique EcoRI site
(Dave & Gilmore, unpublished; see Epinat et al., 2000a)

BC 5’v-Rel:    Contains sequences up to v-rel’s unique Hinc II restriction site, includes

mainly the N-terminal Rel Homology domain and lacking the C-

terminal transactivation domains; from amino acids (aa) 1-331 (Mosialos &

Gilmore, 1993; see Epinat et al., 2000a)

BC5′-RevA-vRel:  Identical to BC-5′v-Rel, but contains the Envelope sequences of the Rev-A helper virus Env (this study)

BC5′v-N6D:  Identical to BC-5′v-Rel but contains an Asn to Asp mutation at amino acid 6 of the Env (this study)

BC5’3,9L-RevA-vRel:  Identical to BC-RevA-v-Rel but has Cys->Leu and Ser->Leu mutations at aa 3 and 9, respectively (this study)

BC5’3,9Y-RevA-Rel:  Identical to BC-RevA-v-Rel but has Cys->Tyr and Ser->Tyr mutations at aa 3 and 9, respectively (this study)

BC 5′DEnv-v-Rel :  A 5′v-Rel protein that has a deletion of aa 1-11 (Envelope sequence) and the addition of an AUG start codon (Epinat et al., 2000a)

IgK6:  LacZ reporter plasmid with 6 upstream kB sites, Ura-selectable (Epinat et al., 2000a)

2.  Plasmids used to express the GAL4-Rel proteins in yeast.

GBT9: GAL4 DNA-binding domain alone, aa 1-147 (Clontech), Trp-selectable

GB 5’v-Rel: 5’v-Rel from aa 2-331 fused to GAL4 DNA-binding domain (Epinat et al., 2000a)

GB-5′RevA-vRel:  identical to GB5′v-Rel except has aa 1-331 of v-Rel fused to the GAL4-DNA binding domain, and has the Rev-A Env aa substitutions at aa 3, 6, and 9

GB-F3C-vRel, GB-F9S-vRel, GB-F3C/F9S-vRel, GB-N6D-v-Rel, GB-F3C/N6D-vRel, GB-N6D/F9S-vRel, GB-3,9L-RevA-vRel, GB-3,9Y-RevA-vRel, GB-C3L/S9F-RevA-vRel, GB-C3F/S9L-RevA-vRel, GB-C3Y/S9F-RevA-vRel, GB-C3F/S9Y-RevA-v-Rel, GB-F10-v-Rel, GB-S10-vRel:  All are identical to GB-5′RevA-vRel except with the substitutions as described in Figures 2a and 3a of the manuscript

GB 5’-Ch-cRel: 5’c-Rel from aa 1-322 fused to GAL4 DNA-binding domain (Epinat et al., 2000a)

GB-5′-Env-Ch-cRel: Chicken c-Rel from aa 2-322 with 11 N-terminal Env aa of v-Rel and fused to GAL4 DNA-binding domain (Epinat et al., 2000a)

GB-5′-Hu-cRel:  Human c-Rel from aa 1-322 fused to GAL4 DNA-binding domain (this study)

GB5′-Env-Hu-cRel:  Human c-Rel from aa 2-322 with 11 N-terminal Env aa of v-Rel and fused to GAL4 DNA-binding domain (this study)

GB 5′DEnv-v-Rel : A GAL4-5′v-Rel protein which consists of a deletion of aa 1-11, v-Rel Envelope sequence (Epinat et al., 2000a)

SD5-Leu:  GAL4 site lacZ reporter plasmid, Leu-selectable (Epinat et al., 2000a)

3.  Plasmids used to express aa 1-331 of v-Rel mutants in vitro.

Plasmids for the expression of aa 1-331 of wild-type v-Rel and RevA-vRel are described in Epinat et al. (2000a).  These plasmids have a stop codon after aa 331 of v-Rel.

Plasmids for the expression of aa 1-331 of N6D-vRel, 3,9L-RevA-vRel, 3,9Y-RevA-vRel, and DEnv-vRel are identical to the pGEM-v-Rel and pGEM-RevA-vRel plasmids, except with mutations in the N-terminal Env sequences.

4.  Plasmids used to express the GAL4-Rel proteins in CEF.

SG424: SV40 promoter plasmid controlling expression of GAL4 (1-147) (see Epinat et al., 2000a).

SG-5-vRel:  GAL4 fused to a portion of v-Rel encoding aa 2-331 (see Epinat et al., 2000a).

All other SG-mutant Env-v-Rel plasmids are as described above for the GBT9-based plasmids, and they were constructed by subcloning XhoI-BamHI fragments from the GBT9-based plasmids into SG-5′RevA-Rel (from Epinat et al., 2000a).

5.  Plasmids used to express full-length v-Rel mutants in spleen cell transformation assays.

JD-v-Rel (GM282BS+):  Spleen necrosis virus vector for the expression of wild-type v-rel (Sif et al., 1993)

JD-RevA-v-Rel:  Spleen necrosis virus vector for the expression of RevA-vRel (v-Rel with Rev-A Env aa at its N terminus) (this study)

JD-vN6D, JD-3,9L-RevA-v-Rel, JD-3,9Y-RevA-vRel, JD-3,9P-RevA-v-Rel:  Spleen necrosis virus vectors for the expression of the indicated v-Rel mutants (this study)

B. Construction of plasmids used in this paper.

1.  Plasmids with citations in parenthesis above were constructed previously.

2.  Mutant Rel plasmids used in this study were all constructed in the following order:

1) PCR mutagenesis was used to create the desired Env mutants.  For the Env mutants, PCR was performed with an upstream mutagenic primer (see below) and a downstream Rev-Rel primer (5′-CTA TAC GTA CTA GTT GAC AGT GGG GAT TGG AGC TGC TTT TGG-3′; see also below) on GB 5’v-Rel: 5’v-Rel or GB-5′RevA-vRel;

2) The PCR product was digested with XmaI and ClaI and the 450 bp fragment was used to replace the corresponding fragment of GB-5′v-Rel (which was also digested with XmaI and ClaI);

3)  The GB-derived vector was sequenced with a GAL4-annealing primer (5′-ACAGCATAGAATAAGTG-3′) to confirm the identity of the mutation, to confirm the precision of the GAL4-Rel fusion point, and to screen for other spurious mutations; and

4)   The correct GB-mutant Rel plasmid was then digested with XmaI-StuI and these fragments were used to replace the corresponding fragments in a pGEM5′v-Rel fragment or in JD-v-Rel.  Correct clones were identified by size and by digestion with XbaI (since a correct subcloning would remove a 5′ XbaI site in these clones).  In addition, the GBT9-based subclone was digested with XhoI-BamHI and the fragment was used to replace the corresponding fragment in SG-5′RevA-vRel (described in Epinat et al., 2000a).  Finally, for expression of aa 1-331 of certain Env Rel mutant proteins (5′Rev-Rel; 5′v-N6D; 5’3,9L-RevA-vRel; 5’3,9Y-RevA-v-Rel; 5′DEnv-vRel) in yeast, the corresponding GB-mutant Rel plasmids were digested with SmaI and HincII and the fragment was subcloned into the SnaBI site of BC102-Spe/SnaBI.

3.  Primers used for Env mutagenesis of v-Rel:

a.  Upstream Env primers for mutagenesis:

RevA2 (RevA-vRel)
XmaI                        Cys                  Asp                    Ser
CCCCCCGGGG ATG GAC TGT CTC ACC GAC CTC CGA TCC ACT GAG

RevA3 (F3C-vRel)
Cys                   Asn                   Phe
CCCCCCGGGG ATG GAC TGT CTC ACC AAC CTC CGA TTC ACT GAG

RevA4 (N6D-vRel)
Phe                  Asp                   Phe
CCCCCCGGGG ATG GAC TTT CTC ACC GAC CTC CGA TTC ACT GAG

RevA5 (F9S-vRel)
Phe                  Asn                    Ser
CCCCCCGGGG ATG GAC TTT CTC ACC AAC CTC CGA TCC ACT GAG

RevA6 (S9F-RevA-vRel)
Cys                   Asp                   Phe
CCCCCCGGGG ATG GAC TGT CTC ACC GAC CTC CGA TTC ACT GAG

RevA7 (D6N-RevA-vRel)
Cys                  Asn                    Ser
CCCCCCGGGG ATG GAC TGT CTC ACC AAC CTC CGA TCC ACT GAG

RevA8 (C3F-RevA-vRel)
Phe                   Asp                   Ser
CCCCCCGGGG ATG GAC TTT CTC ACC GAC CTC CGA TCC ACT GAG

RevA9 (3,9L-RevA-vRel)
Leu                   Asp                   Leu
CCCCCCGGGG ATG GAC TTG CTC ACC GAC CTC CGA TTG ACT GAG

RevA10 (3,9Y-RevA-vRel)
Tyr                   Asp                   Tyr
CCCCCCGGGG ATG GAC TAT CTC ACC GAC CTC CGA TAC ACT GAG

RevA11 (F10-vRel)
Met———————-10 Phe———————————-  Gly    Ile
CCCCCCGGGG ATG TTC TTT TTC TTC TTC TTC TTC TTC TTT TTC GGT ATC TC

RevA12 (S10-vRel)
Met———————-10 Ser—————————————  Gly   Ile
CCCCCCGGGG ATG TCC AGT AGC AGC TCC TCC AGT TCC TCT TCG GGT ATC TC

RevA14 (C3F/S9L-RevA-vRel)
Phe                   Asp                  Leu
CCCCCCGGGG ATG GAC TTT CTC ACC GAC CTC CGA TTG ACT GAG

RevA15 (C3Y/S9F-RevA-vRel)
Tyr                   Asp                   Phe
CCCCCCGGGG ATG GAC TAT CTC ACC GAC CTC CGA TTC ACT GAG

RevA16 (C3F/S9Y-RevA-vRel)
Phe                   Asp                  Tyr
CCCCCCGGGG ATG GAC TTT CTC ACC GAC CTC CGA TAC ACT GAG

RevA26 (3,9P-RevA-vRel)
Pro                    Asp                  Pro
CCCCCCGGGG ATG GAC CCT CTC ACC GAC CTC CGA CCC ACT GAG

b.  Downstream primter

Rev-Rel (reverse oligo, around the Hinc2 site of v-Rel, used for all constructs above):

|  SnaB1  |  STOP |   Hinc2            ——————-vRel———————|
CTA TAC GTA CTA GTT GAC AGT GGG GAT TGG AGC TGC TTT TGG

4.  Primers used for PCR amplification and mutagenesis of human c-Rel:

a.  Upstream primer for Env-Hu-c-Rel (c-Rel starts at 3rd codon):

Xma1        Met   Asp  Phe   Leu  Thr    Asn   Leu  Arg  Phe   Thr  Glu   —human c-Rel—————-
5′CCCCCCGGGG ATG GAC TTT CTG ACG AAC CTA CGC TTC ACC GAG ggt gcg tat aac ccg tat ata gag3′

b.  Upstream primer for Rev-hu-c-Rel (c-Rel starts at 3rd codon)

Xma1        Met   Asp  Cys   Leu  Thr    Asp   Leu  Arg  Ser   Thr  Glu   —human c-Rel—————-
5′CCCCCCGGGG ATG GAC TGT CTG ACG GAC CTA CGC TCC ACC GAG ggt gcg tat aac ccg tat ata gag3′

c.  Upstream primer for human c-Rel fusion into GBT9:

Xma1     Met—hc-Rel——————–
CCCCCCGGGG atg gcc tcc ggt gcg tat aac ccg

d.  Downstream primer for Env-Hu-cRel and Hu-c-Rel PCR amplification (reverse oligo)

BamH1|STOP|———human c-Rel
5′CGGGATCCTCAtccaattgaaccgaggagaccagg3′

References

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

2.  Kamens J and Brent R. 1991. A yeast transcription assay defines distinct Rel and Dorsal DNA recognition sequences. New Biologist 3:1005-1013

3.  Mosialos G and Gilmore TD. 1993. v-Rel and c-Rel are differentially affected by mutations at a consensus protein kinase recognition sequence. Oncogene 8:721-730

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