Data Link 2

Appendix

Presented below is a more detailed version of plasmid constructions and site-directed mutagenesis for recombinant DNA molecules used in

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

Expanded Section 2.1. Plasmid constructions and in vitro mutagenesis

Several site-directed mutations were created using the Kunkel method [see ref. 20 below].For v-rel mutants, an XbaI fragment from a v-rel cDNA was subcloned into the corresponding site of vector M13mp19.The following oligonucleotides were used on single-stranded DNA templates to create the following v-Rel mutants:v-G91D, 5’-GTTGGAAAAGATTGCAGAGATGGC-3’; v- N437D, 5’-CCTGATGAGAAGGATTCC -3’; v-N437I,

5’- CTATAACCCACTGAACTGGCCTGATGAGAAGATTTC-3’; v-DEKDA,

5’-CCTATCCCCACTGATGAAAAGGACGCAGTCAACCCTG-3’.The v-G91D mutation eliminates a PstI site and the v-N437D and v-N437I mutations eliminate an EcoRI site; these sites were used to screen for the respective mutants.For c-Rel mutant c-D82G, an SphI fragment from a c-rel cDNA was subcloned into M13mp18, and for c-Rel mutant c-D432N, an EcoRI fragment was subcloned into M13mp18.The following oligonucleotides were used to create the indicated c-Rel mutants:c-D82G, 5’-GTTGGAAAAGGGTGCAGAGAT-3’; c-D432N,

5’-CCTGATGAGAAGAATTCC-3’.The D82G mutation eliminates a PstI site and the c-D432N mutation creates an EcoRI site, which were used to screen for mutants.

Mutations in the codons for C-terminal Asp residues of chicken c-Rel were made by PCR overlap extension mutagenesis [21] using plasmid pGEM3-c-D82G/D432N as the DNA template for the primary PCR reactions.In each case, the downstream primer was the vector primer T7 and the upstream primer was a chicken c-Rel-specific oligonucleotide (5’ch-c-Rel XbaI,

5’-CAGCTTTCTAGACCAAGAAG-3’).Sense (s) and anti-sense (as) oligonucleotides were designed to create the following mutants:c- D568E,

5’-CTTACAGAGAACCAGTTC-3’ (s) and 5’-GAACTGGTTCTCTGTAAG-3’ (as); c-D573E, 5’-CAGTTCTATGAGACCGATGG-3’ (s) and

5’-ccatcggtctCATAGAACTG-3’ (as); c-D575E,

5’-CAGTTCTATGACACCGAGGGTGTC-3’ (s) and

5’-GACACCCTCGGTGTCATAGAACTG-3’ (as); c-D580E,

5’-GTCCACACTGAAGAGCTCTATCAG-3’ (s) and

5’-CTGATAGAGCTCTTCAGTGTGGAC-3’ (as); c-D575E/D580E,

5’-GACACCGAGGGTGTCCACACTGAAGAGCTCTATCAG-3’ (s) and

5’-CTGATAGAGCTCTTCAGTGTGGACACCCTCGGTGTC-3’ (as).PCR conditions were as follows:94°C, 4 min; 94°C, 1 min; 52°C, 1 min; 72°C, 1 min; repeated for 35 cycles and concluded by a 10 min extension at 72°C.PCR products were digested with XbaI and subcloned into an XbaI-digested pGEM3-c-D82G/D432N vector.All mutants, made by either single-stranded or PCR mutagenesis, were confirmed by DNA sequencing.

pGEM plasmids for the in vitro expression of v-Rel and c-Rel have been described previously [8,22].In vitro expression plasmids for human c-Rel and c-Rel-Nrg were made by subcloning XbaI to XhoI/Klenow-treated cDNA fragments into pGEM4 digested with XbaI and HincII.An EcoRI to HindIII fragment from a mouse c-rel cDNA was subcloned into pGEM4 digested with EcoRI and HindIII to make pGEM4-mu-c-Rel.To create the in vitro expression vector for chicken c-RelD, pGEM3-ch-c-Rel was digested with XbaI and the vector backbone was religated.Similarly, pGEM3-c-D82G/D432ND was created by digesting pGEM3-c-D82G/D432N with XbaI and religating.

Spleen necrosis virus vectors pGM282BS+, pJD214BS+ and pJD-chc-RelD have been described previously [5,23].To create JD-c D82G/D432ND, a BsmI to XbaI fragment was used to replace the corresponding wild-type sequences in JD-chc-RelD.For v-N437D mutant, a BstXI to SalI fragment replaced the wild-type fragments in GM282BS+ and CG129, creating JD-v-N437D and pGEM2-v-N437D, respectively.An XbaI fragment of v-GD/ND was subcloned into JD214BS+ to create JD-v-G91D/N437D.Plasmid JD-v-G91D was made by replacing a BstXI to SalI fragment of JD-v-G91D/N437D with the wild-type fragment from pGM282BS+.pGEM2-v-G91D/N437D was created by subcloning a BsmI to ClaI fragment containing the v-G91D mutation into the corresponding sites of pGEM2-v-N437D.To create the in vitro expression vector for v-Rel mutant v-DEKDA, an XbaI to NruI fragment containing wild-type sequences of v-rel was subcloned into pGEM4 digested with XbaI and HincII.pGEM4-v-DEKDA was then made by subcloning a StuI to HincII fragment containing the insertion mutation into the corresponding sites.To create retroviral vector JD-v-DEKDA, a StuI to BstXI fragment from pGEM4-v-DEKDA was used to replace the corresponding wild-type sequences in pGM282BS+.

A CMV promoter expression vector for chicken c-RelD was made by subcloning an XbaI to HindIII fragment from pGEM3-c-RelD into pcDNA3.1+ digested with XbaI and HindIII.Similarly, pcDNA3.1 ch c-D82G/D432ND was made by subcloning an XbaI to HindIII fragment from pGEM3-c-D82G/D432ND, into pcDNA3.1+ digested with XbaI and HindIII.

GAL4 site reporter plasmid G5BCAT and GAL4-v-Rel expression plasmid SG-3’v-Rel have been described previously [24].Plasmid SG3’-v-N437D was made by replacing a BstXI to XbaI wild-type fragment in SG-3’v-Rel with the corresponding mutant fragment of v-N437D.Plasmid SG-3’c-Rel was made by subcloning a HindIII to BamHI fragment containing the GAL4-3’c-Rel coding sequence into pSG424 [25] digested with HindIII and BamHI.SG-3’D432N was created by using a BstXI to KpnI fragment of c-D432N to replace the corresponding wild-type sequences in SG-3’c-Rel.

References

5.J. Kamens, P. Richardson, G. Mosialos, R. Brent, T. Gilmore, Mol. Cell.

Biol. 10 (1990) 2840-2847.

8.G. Mosialos, T.D. Gilmore, Oncogene 8 (1993) 721-730.

20. T.A. Kunkel, Proc. Natl. Acad. Sci. USA 82 (1985) 488-492.

21. R.M. Horton, H.D. Hunt, S.N. Ho, J.K. Pullen, L.R. Pease, Gene 77

(1989) 61-68.

22. A.J. Capobianco, T.D. Gilmore, Oncogene 6 (1991) 2203-2210.

23. S. Sif, A.J. Capobianco, T.D. Gilmore, Oncogene 8 (1993) 2501-2509.

24. S. Sarkar, T.D. Gilmore, Oncogene 8 (1993) 2245-2252.

25. I. Sadowski, M. Ptashne, Nucleic Acids Res. 17 (1989) 7539.

Table 1. Oligonucleotides used to create v-rel and c-rel mutants

Mutant Oligonucleotide^a

Kunkel single-stranded mutagenesis

v-G91D                  5’-GTTGGAAAAGATTGCAGAGATGGC-3’

v-N437D                5’-CCTGATGAGAAGGATTCC -3’

v-N437I                 5’- CTATAACCCACTGAACTGGCCTGATGAGAAGATTTC-3’

v-DEKDA              5’-CCTATCCCCACTGATGAAAAGGACGCAGTCAACCCTG-3’

c-D82G                  5’-GTTGGAAAAGGGTGCAGAGAT-3’;

c-D432N                5’-CCTGATGAGAAGAATTCC-3’

PCR overlap extension mutagenesis

c-D568E                5’-CTTACAGAGAACCAGTTC-3’ (s)

5’-GAACTGGTTCTCTGTAAG-3’ (as)

c-D573E                5’-CAGTTCTATGAGACCGATGG-3’ (s)

5’-ccatcggtctCATAGAACTG-3’ (as)

c-D575E                5’-CAGTTCTATGACACCGAGGGTGTC-3’ (s)

5’-GACACCCTCGGTGTCATAGAACTG-3’ (as)

c-D580E                 5’-GTCCACACTGAAGAGCTCTATCAG-3’ (s)

5’-CTGATAGAGCTCTTCAGTGTGGAC-3’ (as)

c-D575E/D580E        5’-GACACCGAGGGTGTCCACACTGAAGAGCTCTATCAG-3’ (s)

5’-CTGATAGAGCTCTTCAGTGTGGACACCCTCGGTGTC-3’ (as)

^aunderlined residues designate mutant residues; s, sense-strand oligonucleotide; as, antisense- strand oligonucleotide.See text for further details.