{"id":568,"date":"2011-04-26T15:15:41","date_gmt":"2011-04-26T19:15:41","guid":{"rendered":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link-2\/"},"modified":"2011-04-28T18:23:29","modified_gmt":"2011-04-28T22:23:29","slug":"data-link-2","status":"publish","type":"page","link":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link-2\/","title":{"rendered":"Data Link 2"},"content":{"rendered":"

Appendix<\/h3>\n

Presented below is a more detailed version of plasmid constructions and site-directed mutagenesis for recombinant DNA molecules used in<\/p>\n

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.<\/p>\n

Expanded Section 2.1. Plasmid constructions and in vitro mutagenesis<\/h3>\n

Several site-directed mutations were created using the Kunkel method [see ref. 20 below].For v-rel<\/em> mutants, an Xba<\/em>I fragment from a v-rel<\/em> 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\u2019-GTTGGAAAAGAT<\/span>TGCAGAGATGGC-3\u2019; v- N437D, 5\u2019-CCTGATGAGAAGG<\/span>ATTCC -3\u2019; v-N437I,<\/p>\n

5\u2019- CTATAACCCACTGAACTGGCCTGATGAGAAGAT<\/span>TTC-3\u2019; v-DEKDA,<\/p>\n

5\u2019-CCTATCCCCACTGATGAAAAGGACGCA<\/span>GTCAACCCTG-3\u2019.The v-G91D mutation eliminates a Pst<\/em>I site and the v-N437D and v-N437I mutations eliminate an Eco<\/em>RI site; these sites were used to screen for the respective mutants.For c-Rel mutant c-D82G, an Sph<\/em>I fragment from a c-rel<\/em> cDNA was subcloned into M13mp18, and for c-Rel mutant c-D432N, an Eco<\/em>RI fragment was subcloned into M13mp18.The following oligonucleotides were used to create the indicated c-Rel mutants:c-D82G, 5\u2019-GTTGGAAAAGGG<\/span>TGCAGAGAT-3\u2019; c-D432N,<\/p>\n

5\u2019-CCTGATGAGAAGA<\/span>ATTCC-3\u2019.The D82G mutation eliminates a Pst<\/em>I site and the c-D432N mutation creates an Eco<\/em>RI site, which were used to screen for mutants.<\/p>\n

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\u2019ch-c-Rel Xba<\/em>I,<\/p>\n

5\u2019-CAGCTTTCTAGACCAAGAAG-3\u2019).Sense (s) and anti-sense (as) oligonucleotides were designed to create the following mutants:c- D568E,<\/p>\n

5\u2019-CTTACAGAG<\/span>AACCAGTTC-3\u2019 (s) and 5\u2019-GAACTGGTTC<\/span>TCTGTAAG-3\u2019 (as); c-D573E, 5\u2019-CAGTTCTATGAG<\/span>ACCGATGG-3\u2019 (s) and<\/p>\n

5\u2019-ccatcggtctC<\/span>ATAGAACTG-3\u2019 (as); c-D575E,<\/p>\n

5\u2019-CAGTTCTATGACACCGAG<\/span>GGTGTC-3\u2019 (s) and<\/p>\n

5\u2019-GACACCC<\/span>TCGGTGTCATAGAACTG-3\u2019 (as); c-D580E,<\/p>\n

5\u2019-GTCCACACTGAA<\/span>GAGCTCTATCAG-3\u2019 (s) and<\/p>\n

5\u2019-CTGATAGAGCTCT<\/span>TCAGTGTGGAC-3\u2019 (as); c-D575E\/D580E,<\/p>\n

5\u2019-GACACCGAG<\/span>GGTGTCCACACTGAA<\/span>GAGCTCTATCAG-3\u2019 (s) and<\/p>\n

5\u2019-CTGATAGAGCTCT<\/span>TCAGTGTGGACACCC<\/span>TCGGTGTC-3\u2019 (as).PCR conditions were as follows:94\u00b0C, 4 min; 94\u00b0C, 1 min; 52\u00b0C, 1 min; 72\u00b0C, 1 min; repeated for 35 cycles and concluded by a 10 min extension at 72\u00b0C.PCR products were digested with Xba<\/em>I and subcloned into an Xba<\/em>I-digested pGEM3-c-D82G\/D432N vector.All mutants, made by either single-stranded or PCR mutagenesis, were confirmed by DNA sequencing.<\/p>\n

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 Xba<\/em>I to Xho<\/em>I\/Klenow-treated cDNA fragments into pGEM4 digested with Xba<\/em>I and Hin<\/em>cII.An Eco<\/em>RI to Hin<\/em>dIII fragment from a mouse c-rel<\/em> cDNA was subcloned into pGEM4 digested with Eco<\/em>RI and Hin<\/em>dIII to make pGEM4-mu-c-Rel.To create the in vitro expression vector for chicken c-RelD, pGEM3-ch-c-Rel was digested with Xba<\/em>I and the vector backbone was religated.Similarly, pGEM3-c-D82G\/D432ND was created by digesting pGEM3-c-D82G\/D432N with Xba<\/em>I and religating.<\/p>\n

Spleen necrosis virus vectors pGM282BS+, pJD214BS+ and pJD-chc-RelD have been described previously [5,23].To create JD-c D82G\/D432ND, a Bsm<\/em>I to Xba<\/em>I fragment was used to replace the corresponding wild-type sequences in JD-chc-RelD.For v-N437D mutant, a Bst<\/em>XI to Sal<\/em>I fragment replaced the wild-type fragments in GM282BS+ and CG129, creating JD-v-N437D and pGEM2-v-N437D, respectively.An Xba<\/em>I fragment of v-GD\/ND was subcloned into JD214BS+ to create JD-v-G91D\/N437D.Plasmid JD-v-G91D was made by replacing a Bst<\/em>XI to Sal<\/em>I fragment of JD-v-G91D\/N437D with the wild-type fragment from pGM282BS+.pGEM2-v-G91D\/N437D was created by subcloning a Bsm<\/em>I to Cla<\/em>I 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 Xba<\/em>I to Nru<\/em>I fragment containing wild-type sequences of v-rel<\/em> was subcloned into pGEM4 digested with Xba<\/em>I and Hin<\/em>cII.pGEM4-v-DEKDA was then made by subcloning a Stu<\/em>I to Hin<\/em>cII fragment containing the insertion mutation into the corresponding sites.To create retroviral vector JD-v-DEKDA, a Stu<\/em>I to Bst<\/em>XI fragment from pGEM4-v-DEKDA was used to replace the corresponding wild-type sequences in pGM282BS+.<\/p>\n

A CMV promoter expression vector for chicken c-RelD was made by subcloning an Xba<\/em>I to Hin<\/em>dIII fragment from pGEM3-c-RelD into pcDNA3.1+ digested with Xba<\/em>I and Hin<\/em>dIII.Similarly, pcDNA3.1 ch c-D82G\/D432ND was made by subcloning an Xba<\/em>I to Hin<\/em>dIII fragment from pGEM3-c-D82G\/D432ND, into pcDNA3.1+ digested with Xba<\/em>I and Hin<\/em>dIII.<\/p>\n

GAL4 site reporter plasmid G5BCAT and GAL4-v-Rel expression plasmid SG-3\u2019v-Rel have been described previously [24].Plasmid SG3\u2019-v-N437D was made by replacing a Bst<\/em>XI to Xba<\/em>I wild-type fragment in SG-3\u2019v-Rel with the corresponding mutant fragment of v-N437D.Plasmid SG-3\u2019c-Rel was made by subcloning a Hin<\/em>dIII to Bam<\/em>HI fragment containing the GAL4-3\u2019c-Rel coding sequence into pSG424 [25] digested with Hin<\/em>dIII and Bam<\/em>HI.SG-3\u2019D432N was created by using a Bst<\/em>XI to Kpn<\/em>I fragment of c-D432N to replace the corresponding wild-type sequences in SG-3\u2019c-Rel.<\/p>\n

References<\/h3>\n

5.J. Kamens, P. Richardson, G. Mosialos, R. Brent, T. Gilmore, Mol. Cell.<\/p>\n

Biol. 10 (1990) 2840-2847.<\/p>\n

8.G. Mosialos, T.D. Gilmore, Oncogene 8 (1993) 721-730.<\/p>\n

20. T.A. Kunkel, Proc. Natl. Acad. Sci. USA 82 (1985) 488-492.<\/p>\n

21. R.M. Horton, H.D. Hunt, S.N. Ho, J.K. Pullen, L.R. Pease, Gene 77<\/p>\n

(1989) 61-68.<\/p>\n

22. A.J. Capobianco, T.D. Gilmore, Oncogene 6 (1991) 2203-2210.<\/p>\n

23. S. Sif, A.J. Capobianco, T.D. Gilmore, Oncogene 8 (1993) 2501-2509.<\/p>\n

24. S. Sarkar, T.D. Gilmore, Oncogene 8 (1993) 2245-2252.<\/p>\n

25. I. Sadowski, M. Ptashne, Nucleic Acids Res. 17 (1989) 7539.<\/p>\n

Table 1.<\/strong> Oligonucleotides used to create v-rel<\/em> and c-rel<\/em> mutants<\/h3>\n

Mutant<\/span><\/strong> Oligonucleotide<\/strong><\/span>a<\/sup> <\/span><\/h3>\n

Kunkel single-stranded mutagenesis<\/h3>\n

v-G91D\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019-GTTGGAAAAGAT<\/span>TGCAGAGATGGC-3\u2019<\/p>\n

v-N437D\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019-CCTGATGAGAAGG<\/span>ATTCC -3\u2019<\/p>\n

v-N437I\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019- CTATAACCCACTGAACTGGCCTGATGAGAAGAT<\/span>TTC-3\u2019<\/p>\n

v-DEKDA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019-CCTATCCCCACTGATGAAAAGGACGCA<\/span>GTCAACCCTG-3\u2019<\/p>\n

c-D82G\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019-GTTGGAAAAGGG<\/span>TGCAGAGAT-3\u2019;<\/p>\n

c-D432N\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019-CCTGATGAGAAGA<\/span>ATTCC-3\u2019<\/p>\n

PCR overlap extension mutagenesis<\/h3>\n

c-D568E\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019-CTTACAGAG<\/span>AACCAGTTC-3\u2019 (s)<\/p>\n

5\u2019-GAACTGGTTC<\/span>TCTGTAAG-3\u2019 (as)<\/p>\n

c-D573E\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019-CAGTTCTATGAG<\/span>ACCGATGG-3\u2019 (s)<\/p>\n

5\u2019-ccatcggtctC<\/span>ATAGAACTG-3\u2019 (as)<\/p>\n

c-D575E\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019-CAGTTCTATGACACCGAG<\/span>GGTGTC-3\u2019 (s)<\/p>\n

5\u2019-GACACCC<\/span>TCGGTGTCATAGAACTG-3\u2019 (as)<\/p>\n

c-D580E\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019-GTCCACACTGAA<\/span>GAGCTCTATCAG-3\u2019 (s)<\/p>\n

5\u2019-CTGATAGAGCTCT<\/span>TCAGTGTGGAC-3\u2019 (as)<\/p>\n

c-D575E\/D580E\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u2019-GACACCGAG<\/span>GGTGTCCACACTGAA<\/span>GAGCTCTATCAG-3\u2019 (s)<\/p>\n

5\u2019-CTGATAGAGCTCT<\/span>TCAGTGTGGACACCC<\/span>TCGGTGTC-3\u2019 (as)<\/p>\n

a<\/sup>underlined residues designate mutant residues; s, sense-strand oligonucleotide; as, antisense- strand oligonucleotide.See text for further details.<\/p>\n","protected":false},"excerpt":{"rendered":"

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 […]<\/p>\n","protected":false},"author":4258,"featured_media":0,"parent":337,"menu_order":8,"comment_status":"closed","ping_status":"open","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/568"}],"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=568"}],"version-history":[{"count":3,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/568\/revisions"}],"predecessor-version":[{"id":597,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/568\/revisions\/597"}],"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=568"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}