Data Link

Appendix

Descriptions of plasmids used to express the Rel proteins studied. The source of the plasmid is indicated in parentheses.

BC102:  Vector alone (Kamens & Brent, 1991)

BC102-Spe/Sna:  BC102 with linker containing SpeI and SnaBI sites at the unique EcoRI site (Dave & Gilmore, unpublished)

JK v-Rel: Wild-type full-length v-Rel in BC102 (Kamens & Brent, 1991)

JK c-Rel:  Wild-type full-length c-Rel in BC102 (Kamens & Brent, 1991)

BC 5’v-Rel:    Contains sequences up to v-rel’s unique HincII 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)

JK 5’c-Rel: Contains sequences up to c-rel’s unique HincII restriction site, including mainly the N-terminal Rel Homology domain and lacking C-terminal transactivation domains; from aa 1-322 (Harkness & Gilmore, unpublished)

BC 5’V/C: A 5’Rel protein that consists of N-terminal 5’v-Rel sequences up to the ClaI restriction site (aa 148) and C-terminal 5’c-Rel sequences to the Hinc II site (Harkness & Gilmore, unpublished)

BC 5’C/V: A 5’Rel protein that consists of N-terminal 5’c-Rel sequences up to the ClaI restriction site and C-terminal 5’v-Rel sequences to the HincII site (Harkness & Gilmore, unpublished)

BC 5′DEnv-v-Rel : A 5′v-Rel protein that consists of a deletion of aa 1-11 (Envelope sequence) and the addition of an AUG start codon (this publication) BC 5′Env-c-Rel: A 5′c-Rel protein that consists of an addition of the 11 aa Envelope sequence (this publication)

BC-p100:   Human NF-kB1-p100 subcloned into BC102

BC-p52:  Human NF-kB-p52 (aa 1-399, up to an internal XmaI site) subcloned into BC102

BC-p85:   Human NF-kB1-p85 cDNA from Hut-78 cells subcloned into BC102


Descriptions of Plasmids used to express the GAL4-Rel proteins.

The source of the plasmid is indicated in parentheses.

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

GB 5’v-Rel: 5’v-Rel from aa 2-331 fused to GAL4 DNA-binding domain (Harkness & Gilmore, unpublished)

GB 5’c-Rel: 5’c-Rel from aa 1-322 fused to GAL4 DNA-binding domain (Harkness & Gilmore, unpublished)

GB 3’v-Rel: GAL4 DNA-binding domain fused to carboxy-terminal v-Rel sequences (aa 332-503) starting from the Hinc II site, includes v-Rel transactivation domains (Koedood & Gilmore, unpublished)

GB 3’c-Rel: GAL4 DNA-binding domain fused to C-terminal c-Rel sequences (aa 324-598) starting from Hinc II site, includes c-Rel transactivation domains (Gilmore, unpublished)

GB 5’v-Rel SPW: GB 5’v-Rel with a proline-tryptophan insertion mutation in the Rel Homology domain at aa 273 that inhibits the formation of homodimers (Harkness & Gilmore, unpublished)
GB 5′V/C (Cla): A GAL4-5’Rel protein which consists of N-terminal 5’v-Rel sequences up to Cla I restriction site and C-terminal 5’c-Rel sequences to the Hinc II site (this publication)

GB 5′C/V (Cla): A GAL4-5’Rel protein which consists of N-terminal 5’c-Rel sequences up to Cla I restriction site and C-terminal 5’v-Rel sequences to the Hinc II site (this publication)
GB 5′V/C (Sca): A GAL4-5’Rel protein which consists of N-terminal 5’v-Rel sequences up to Sca I restriction site and C-terminal 5’c-Rel sequences to the Hinc II site (this publication)

GB 5′C/V (Sca): A GAL4-5’Rel protein which consists of N-terminal 5’c-Rel sequences up to Sca I restriction site and C-terminal 5’v-Rel sequences to the Hinc II site (this publication)

GB 5′DEnv-v-Rel : A GAL4-5′v-Rel protein which consists of a deletion of aa 1-11, v-Rel Envelope sequence (this publication)

GB 5′Env-c-Rel: A GAL4-5′c-Rel protein which consists of an addition of the 11 aa
v-Rel Envelope sequence (this publication)

GB 5′Env C/V (Cla): A GAL4-5′CV (Cla) protein with the addition of the 11 aa
v-Rel Envelope sequence at its N terminus (this publication)

GB 5′RevA-v-Rel: A GAL4-5′v-Rel protein with a substitution of its Envelope sequence for Rev-A Envelope sequence (this publication)

GB 5′RevA-c-Rel: A GAL4-5′c-Rel protein which consists of an addition of the 11 aa Rev-A Envelope sequence (this publication)

GB 5′Env: A fusion that only consists of the GAL4 DNA-binding domain fused to the 11 Envelope-derived aa (this publication)

GB-3′p100:  A fusion that contains aa 399-899 of human p100 (from XmaI site at codon 399)

GB-3′p85:  A fusion that contains aa 399-666 from human p85 (from XmaI site at codon 399)


Construction of BC102 and GBT9 (GAL4) subclones.
Subclone Original Plasmid containing rel fragment and restriction site (insert) Vector and restriction site cloned into
BC 5′v-Rel DEnv GB 5′v-Rel DEnv (Sma I-Hinc II) BC102 (SnaB I)
BC 5′Env-c-Rel PCR fragment (Spe I-SnaB I) BC102 (Spe I-SnaB I)
GB 5′V/C (Cla) GB 5′v-Rel (Cla I-Sal I) GB 5′c-Rel (Cla I-Sal I)
GB 5′C/V (Cla) GB 5′c-Rel (Cla I-Sal I) GB 5′v-Rel (Cla I-Sal I)
GB 5′V/C (Sca) GB 5′v-Rel (Sca I-partial Sal I) GB 5′c-Rel (Sca I-Sal I)
GB 5′C/V (Sca) GB 5′c-Rel (Sca I-partial Sal I) GB 5′v-Rel (Sca I-Sal I)
GB 5′DEnv-v-Rel PCR fragment (Sal I-Ava I) GBT9 (SalI)
GB 5′Env-c-Rel GB 5′V/C (Sca I-HincII) GBT9 (Sca I/HincII)
GB Env Env oligo (EcoR I-Sma I) GBT9 (EcoR I-Sma I)
GB 5′Env C/V(Cla) GB 5′Env c-Rel-(Cla I-Sal I) GB 5′v-Rel (Cla I-Sal I)
GB 5′RevA Env-v-Rel PCR fragment (Xma I-Hinc II) GBT9 (Xma I/Pst I + T4 polmerase)
GB 5′RevA Env-c-Rel PCR fragment (Xma I-Hinc II) GBT9 (Xma I/Pst I + T4 polmerase)

Oligonucleotide sequences used as primers in PCR and subclone construction

Sequence of Oligonucleotides Used to Create Plasmids by PCR

BC 5′Env-c-Rel

Primer 1: GA CTA GTC ATG GAC TTT CTC ACC AAC CTC CGA TTC

Primer 2: CTA TAC GTA CTA GTT GAC AGT GGG GAT TGG AGC TGC TTT TGG

GB 5′DEnv-v-Rel

Primer 1: C CCT CGA GGG ATG GGT ATC TCA GAG CCC TAC ATT

Primer 2: T7 primer

GB 5′RevA-v-Rel & 5′RevA-c-Rel

Primer 1: C CCC CCG GGG ATG GAC TGT CTC ACC GAC CTC CGA TCC ACT GAG

Primer 2: CTA TAC GTA CTA GTT GAC AGT GGG GAT TGG AGC TGC TTT TGG

GB Env

Oligo 1: AA TTC AGA TCT ATG GAC TTT CTC ACC AAC CTC CGA TTC ACT GAG GGC CC

Oligo 2: GG GCC CTC AGT GAA TCG GAG GTT GGT GAG AAA GTC CAT AGA TCT G