TABLE 2.
Oligonucleotides used in this study
| Activity and plasmid | Oligonucleotide pair(s)a |
|---|---|
| Deletion mutagenesis | |
| pJEB297 (Δpcr3) | pPcr3a: 5′-GCA TGT GAA TTC GAA CTG AAG CGT CTC TAC CG-3′ (EcoRI); pPcr3b: 5′-GGC ACC GAC CCG GCT CAT-3′ |
| pPcr3c: 5′-AGC CGG GTC GGT GCC CTG TTG CAC AAG GTA TGA ATC-3′; pPcr3d: 5′-ACT TCA AAG CTT TAC ACC GAC ATC ATC AGC AG-3′ (HindIII) | |
| pJEB298 (Δpcr4) | pPcr4a: 5′-GCA TGT GAA TTC GCT GCG TTG CGC CTG GAG C-3′; (EcoRI); pPcr4b: 5′-CGT CGG TTT CAA CGT CAT CG-3′ |
| pPcr4c: 5′-ACG TTG AAA CCG ACG GCG CTG GAG CTG GAG GCG CGG GAA TGA-3′; pPcr4d: 5′-ACT TCA AAG CTT CAT GTC GCC GTC GAT GCT CAT-3′ (HindIII) | |
| pMF535 (ΔyscX) | pyscXa: 5′-ACG TAC CTC GAG ACA CTG ACA TTG TGG T-3′ (XhoI); pyscXb: 5′- TAG CTC TTC CAG GCT AAT CG-3′ |
| pyscXc: 5′-TTA GCC TGG AAG AGC TAG ATG ATC GGT TAC TGC AA-3′; pyscXd: 5′-ACG TAC TCT AGA TCG CCG TTC AGT T-3′ (XbaI) | |
| pMF534 (ΔyscY) | pyscYa: 5′-ACG TAC CTC GAG TCG AAC CAC ATT-3′ (XhoI); pyscYb: 5′-CAA GAA CTC CTG TTG TCG TT-3′ |
| pyscYc: 5′-ACA ACA GGA GTT CTT GAA CGG CGA TCT TGA TA-3′; pyscYd: 5′-ACG TAC TCT AGA GGA GAG TTG ATA TAG-3′ (XbaI) | |
| pJEB342 (ΔyscX yscY) | pyscXa; pyscXb |
| pyscXYc: 5′-ATT AGC CTG GAA GAG CTA AAC GGC GAT CTT GAT AAG G-3′; pyscYd | |
| Site-directed mutagenesis | |
| pJEB85 (PcrH) | PcrH5F: 5′-ACT ACT GAA TTC GAT CTA GAG GTA TCC ATG AAC C-3′ (EcoRI); PcrH6R: 5′-GCA GCA GGA TCC TCA AGC GTT ATC GGA TTC ATA-3′ (BamHI) |
| pJEB87 (PcrHNEISS) | pMutH2: 5′-C GGC GGC ACC CTG GCG ATG CTT AAC GAA ATC AGC TCG GAC ACC CTG GAG CAG CTC TAT-3′ |
| Complementation | |
| pJEB121 (Hyb1-FLAG) | pLcrH3F: 5′-ACT ACT AAG CTT GA CAC GAG GTA ATT ATG CAA C-3′ (HindIII); pHybLcrH1: 5′-AGT GTC ACT TGA AAT TTC GTT GAG-3′ |
| pHybPcrH1: 5′-ATT TCA AGT GAC ACT CTG GAG CAG CTC TAT GCG CT-3′; pPcrHRFlag: 5′-ACT TCA GTC GAC TTA TTT ATC GTC ATC ATC TTT ATA ATC AGC GTT ATC GGA TTC ATA TGT TC-3′ (SalI) | |
| pJEB122 (Hyb2-FLAG) | pLcrH3F; pHybLcrH2: 5′-ATC ATA GTG GTC TAG CAC ACA GA-3′ |
| pHybPcrH2: 5′-CTA GAC CAC TAT GAT GCC CGC TAC TTT CTC GGC CTG-3′; pPcrHRFlag | |
| pJEB123 (Hyb3-FLAG) | pLcrH3F; pHybLcrH3: 5′-TTC TTT TAT ATC CAT TAT GGC GC-3′ |
| pHybPcrH3: 5′-ATG GAT ATA AAA GAA CCG CGC TTT CCC TTC CAT GCC-3′; pPcrHRFlag | |
| pJEB124 (Hyb4-FLAG) | pLcrH3F; pHybLcrH4: 5′-TTT GTC TGC GAT AAG CTC TTG A-3′ |
| pHybPcrH4: 5′-CTT ATC GCA GAC AAA CCG GCG CAC GAG GCC CTG GC-3′; pPcrHRFlag | |
| pJEB125 (Hyb5-FLAG) | pPcrHF: 5′-GCA TGT AAG CTT GAT CTA GAG GTA TCC ATG-3′ (HindIII); pHybPcrH5: 5-CTG TGC CGC GGC CAG GGC-3′ |
| pHybLcrH5: 5′-CTG GCC GCG GCA CAG CCT GAG TTT AAG GAG CTT TCC-3′; pLcrHRFlag: 5′-ACT TCA GTC GAC TTA TTT ATC GTC ATC ATC TTT ATA ATC TGG GTT ATC AAC GCA CTC ATG TTC-3′ (SalI) | |
| pJEB126 (Hyb6-FLAG) | pPcrHF; pHybPcrH6: 5′-CTC GTT GAT GTC CAT CAG CG-3′ |
| pHybLcrH6: 5′-ATG GAC ATC AAC GAG CCT CGT TTT CCG TTT CAT GCG-3′; pLcrHRFlag | |
| pJEB127 (Hyb7-FLAG) | pPcrHF; pHybPcrH7: 5′-GTC GTA GTG GTC GAG CAT GCA-3′ |
| pHybLcrH7: 5′-CTC GAC CAC TAC GAC TCA CGT TTC TTT TTA GGG CTA G-3′; pLcrHRFlag | |
| pJEB128 (Hyb8-FLAG) | pPcrHF; pHybPcrH8: 5′-GGT GTC CTC GCT GAG TCC G-3′ |
| pHybLcrH8: 5′-CTC AGC GAG GAC ACC TTA GAG CAA CTC TAC TCT CTT G-3′; pLcrHRFlag | |
| pJEB129 (Hyb9-FLAG) | pLcrH3F; pHybLcrH9: 5′-GGA TTC CAT TGC CAG CTG GTA TTC T-3′ |
| pHybPcrH9: 5′-CTG GCA ATG GAA TCC TTC CTG CGC GAC GGC GGC ACC CTG-3′; pPcrHRFlag | |
| pJEB130 (PcrH-FLAG) | pPcrHF; pPcrHRFlag |
| pJEB132 (PcrHNEISS-FLAG) | pPcrHF; pPcrHRFlag |
| pJEB133 (LcrH-FLAG) | pLcrH3F; pLcrHRFlag |
| pJEB199 (Hyb10-FLAG) | pLcrH3F; pHybLcrH10: 5′-GAG CAT GGC GAT AGT TCC CCC TCC-3′ |
| pHybPcrH10: 5′-GAA CTA TCG CCA TGC TC C GCG GAC TCA GCG AGG ACA CCC TG-3′; pPcrHRFlag | |
| pKEC005 (PcrH) | pPcrH5F: 5′-ACT ACT GAA TTC GAT CTA GAG GTA TCC ATG AAC C-3′ (EcoRI); pPcrH6R: 5′-GCA GCA GGA TCC TCA AGC GTT ATC GGA TTC ATA-3′ (BamHI) |
| pPJE020 (LcrH) | plcrH1: 5′-CTG GTG AAT TCC ACG AGG TAA TTA TGC-3′ (EcoRI); plcrH2: 5′-GTT ACT GCA GTT GAG CGG TCA T-3′ (PstI) |
| pJEB291 (YscX) | pYscXF: 5′-ACT ACT GAA TTC AAG AGG TGC TTG CGC CGT GAG-3′ (EcoRI); pYscXR: 5′-ACT ACT AAG CTT TCA TAC TTT GTG CAA CAG GTT-3′ (HindIII) |
| pJEB292 (YscY) | pYscY(20) for: 5′-GAA TTC AAC CTG TTG CAC AAA GTA TGA-3′ (EcoRI); pYscY(20)rev: 5′-CTG CAG TCA TGG GGA TTC ATT ATG ATC-3′ (PstI) |
| pJEB340 (YscX, YscY) | pYscXF; pYscY(20)rev |
| pJEB295 (Pcr3) | pPcr3F: 5′-ACT ACT GAA TTC AGC GAA GTG CTC GCC GCA TG-3′ (EcoRI); pPcr3R: 5′-ACT TCA AAG CTT TCA TAC CTT GTG CAA CAG GTT CAG-3′ (HindIII) |
| pJEB296 (Pcr4) | pPcr4F: 5′-ACT ACT GAA TTC CAC AAG GTA TGA ATC GAT GAC GTT-3′ (EcoRI); pPcr4R: 5′-ACT TCA AAG CTT CGT TCA TTC CCG CGC CTC CAG C-3′ (HindIII) |
| pJEB335 (Pcr3, Pcr4) | pPcr3F and pPcr4R |
| Yeast two-hybrid interaction studies | |
| pJEB211 (Hyb1) | pLcrH1: 5′-CTG GTG AAT TCC ACG AGG TAA TTA TGC-3′ (EcoRI); pHybLcrH1 |
| pHybPcrH1; pPcrH6R | |
| pJEB212 (Hyb2) | pLcrH1; pHybLcrH2 |
| pHybPcrH2; pPcrH6R | |
| pJEB213 (Hyb3) | pLcrH1; pHybLcrH3 |
| pHybPcrH3; pPcrH6R | |
| pJEB214 (Hyb4) | pLcrH1; pHybLcrH4 |
| pHybPcrH4; pPcrH6R | |
| pJEB215 (Hyb5) | pPcrH5F; pHybPcrH5 |
| pHybLcrH5; LcrH1R: 5′-GCA GCA GGA TCC TCA TCA TGG GTT ATC AAC GCA CT-3′ (BamHI) | |
| pJEB216 (Hyb6) | pPcrH5F; pHybPcrH6 |
| pHybLcrH6; LcrH1R | |
| pJEB217 (Hyb7) | pPcrH5F; pHybPcrH7 |
| pHybLcrH7; LcrH1R | |
| pJEB218 (Hyb8) | pPcrH5F; pHybPcrH8 |
| pHybLcrH8; LcrH1R | |
| pJEB219 (Hyb9) | pLcrH1; pHybLcrH9 |
| pHybPcrH9; pPcrH6R | |
| pJEB220 (Hyb10) | pLcrH1; pHybLcrH10 |
| pHybPcrH10; pPcrH6R | |
| pJEB221 (PcrH) | pPcrH5F; pPcrH6R |
| pJEB222 (PcrHNEISS) | pPcrH5F; pPcrH6R |
| pJEB338 (LcrH1-35) | pLcrH1; plcrH-Nterm(Pst): 5′-CTG CAG TCA AGT GTC ACT TGA AAT TTC GTT GAG-3′ (PstI) |
| pPJE025 (Pcr3) | pcr3-A: 5′-CCG GAA TTC ATG AGC CGG GTC GGT G-3′ (EcoRI); pcr3-C: 5′-CCG CTC GAG TCA TAC CTT GTG CAA CAG-3′ (XhoI) |
| pPJE024 (Pcr4) | pcr4-A: 5′-CGC CGC GGA TCC CAA TCG ATG ACG TTG AA-3′ (BamHI); pcr4-B: 5′-AAA ACT GCA GTC ATT CCC GCG CCT CCA-3′ (PstI) |
a
The nucleotide sequences in italics represent the incorporated HindIII, SalI, EcoRI, XhoI, PstI or BamHI restriction sites used for cloning of the PCR amplified DNA fragments. The underlined sequence indicates the complementary overlap between respective primers in the overlap PCR reactions. In the primer pMutH2, for generation of PcrHNEISS by site-directed mutagenesis, the codons substituted are indicated in boldface.