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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BC102-Spe/Sna: BC102 with linker containing SpeI and SnaBI sites at the unique EcoRI site (Dave & Gilmore, unpublished)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the N-terminal Rel Homology domain and lacking C-terminal transactivation domains; from aa 1-322 (Harkness & Gilmore, unpublished)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up to the ClaI restriction site (aa 148) and C-terminal 5’c-Rel sequences to the Hinc II site (Harkness & Gilmore, unpublished)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sequences up to the ClaI restriction site and C-terminal 5’v-Rel sequences to the HincII site (Harkness & Gilmore, unpublished)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GB 5’v-Rel: 5’v-Rel from aa 2-331 fused to GAL4 DNA-binding domain (Harkness & Gilmore, unpublished)
GB 5’c-Rel: 5’c-Rel from aa 1-322 fused to GAL4 DNA-binding domain (Harkness & Gilmore, unpublished)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v-Rel sequences (aa 332-503) starting from the Hinc II site, includes v-Rel transactivation domains (Koedood & Gilmore, unpublished)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
