TABLE 2.
LcrV mutanta | Oligonucleotide pair (sequence [5′-3′)b |
---|---|
Deletion mutants | |
V1 (2-20) | pLcrVdelA (GCA TGT CTC GAG CAG AAC TGG CAA TAG CCG AC) and pLcrVdel2-20b (CAT ATT AAA TAA TTT GCC CTC GCA TC) |
pLcrVdel2-20C (GCA AAT TAT TTA ATA TGG TGG AAC AAC TTA CTG GTC ATG G) and pLcrVdelD1 (GCA TGT GAG CTC ACT CGC TTG ATG CCA TTT TGC AG) | |
V2 (3-20) | pLcrVdelA and pLcrVdel3-20b (AAT CAT ATT AAA TAA TTT GCC CTC GC) |
pLcrVdel3-20c (ATT ATT TAA TAT GAT TGT GGA ACA ACT TAC TGG TCA TGG) and pLcrVdelD1 | |
V3 (5-20) | pLcrVdelA and pLcrVdel5-20b (GGC TCT AAT CAT ATT AAA TAA T) |
pLcrVdel5-20c (TAA TAT GAT TAG AGC CGT GGA ACA ACT TAC TGG TCA TGG) and pLcrVdelD1 | |
V4 (7-20) | pLcrVdelA and pLcrVdel7-20b (TTC GTA GGC TCT AAT CAT ATT) |
pLcrVdel7-20c (ATT AGA GCC TAC GAA GTG GAA CAA CTT ACT GGT CAT GG) and pLcrVdelD1 | |
V5 (9-20) | pLcrVdelA and pLcrVdel9-20b (GTT TTG TTC GTA GGC TCT AAT) |
pLcrVdel9-20c (GCC TAC GAA CAA AAC GTG GAA CAA CTT ACT GGT CAT GG) and pLcrVdelD1 | |
V6 (11-20) | pLcrVdelA and pLcrVdel11-20b (TTG TGG GTT TTG TTC GTA GGC T) |
pLcrVdel11-20c (CGA ACA AAA CCC ACA AGT GGA ACA ACT TAC TGG TCA TGG) and pLcrVdelD1 | |
V7 (13-20) | pLcrVdelA and pLcrVdel13-20b (AAA TGT TGT GGG TTT TGT TCG) |
pLcrVdel13-20c (AAC CCA CAA CAT TTT GTG GAA CAA CTT ACT GGT CAT GG) and pLcrVdelD1 | |
V8 (15-20) | pLcrVdelA and pLcrVdel15-20b (CTC AAT AAA ATG TTG TGG GTT) |
pLcrVdel15-20c (CAA CAT TTT ATT GAG GTG GAA CAA CTT ACT GGT CAT GG) and pLcrVdelD1 | |
V9 (17-20) | pLcrVdelA and pLcrVdel17-20b (TAG ATC CTC AAT AAA ATG TTG TG) |
pLcrVdel17-20c (TTT ATT GAG GAT CTA GTG GAA CAA CTT ACT GGT CAT GG) and pLcrVdelD1 | |
V10 (19-20) | pLcrVdelA and pLcrVdel19-20b (TTT TTC TAG ATC CTC AAT AAA ATG) |
pLcrVdel19-20c (GAG GAT CTA GAA AAA GTG GAA CAA CTT ACT GGT CAT GG) and pLcrVdelD1 | |
V11 (25-40) | pLcrVdelA and pLcrVdel25-40b (AAG TTG TTC CAC CCT AAC TT) |
pLcrVdel25-40c (AGG GTG GAA CAA CTT GAT AAA AAT ATA GAT ATT TCC ATT A) and pLcrVdelD2 (GCA TGT GAG CTC CTA CGG GCA TCA CCA TGA TGA T) | |
V12 (2-4) | pLcrVdelA and pLcrVdel2-20b |
pLcrVdel2-4c (GCA AAT TAT TTA ATA TGT ACG AAC AAA ACC CAC AAC ATT) and pLcrVdelD1 | |
V13 (5-7) | pLcrVdelA and pLcrVdel5-20b |
pLcrVdel5-7c (TAA TAT GAT TAG AGC CAA CCC ACA ACA TTT TAT TGA GG) and pLcrVdelD1 | |
V14 (8-10) | pLcrVdelA and pLcrVdel8-10b (TTG TTC GTA GGC TCT AAT CAT) |
pLcrVdel8-10C (TTA GAG CCT ACG AAC AAC ATT TTA TTG AGG ATC TAG AA) and pLcrVdelD1 | |
V15 (11-13) | pLcrVdelA and pLcrVdel11-20b |
pLcrVdel11-13c (CGA ACA AAA CCC ACA AGA GGA TCT AGA AAA AGT TAG G) and pLcrVdelD1 | |
V16 (14-16) | pLcrVdelA and pLcrVdel14-16b (AAT AAA ATG TTG TGG GTT TTG TTC G) |
pLcrVdel14-16c (CCC ACA ACA TTT TAT TGA AAA AGT TAG GGT GGA ACA ACT T) and pLcrVdelD1 | |
V17 (17-18) | pLcrVdelA and pLcrVdel17-20b |
pLcrVdel17-18c (TTT ATT GAG GAT CTA GTT AGG GTG GAA CAA CTT ACT GGT) and pLcrVdelD1 | |
Frameshift and wobble base mutants | |
FS +1 (4-13) | pLcrVdelA and pLcrV + 1(4-13)b (TTG TGG GTT TTG TTC GTA GGC TTC TAA TCA TAT TAA ATA A) |
pLcrV + 1(4-13)c (CTA CGA ACA AAA CCC ACA ACA TTT TAT GAG GAT CTA GAA AAA GTT AGG GT) and pLcrVdelD1 | |
FS −1 (2-15) | pLcrVdelA and pLcrV-1(2-15)b (TTG TGG GTT TTG TTC GTA GGC TCT AAC ATA TTA AAT AAT TTG CCC TCG CA) |
pLcrV-1(2-15)c (CGA ACA AAA CCC ACA ACA TTT TAT TGA GGA TTC TAG AAA AAG TTA GGG TG) and pLcrVdelD1 | |
Scramble (2-15) | pLcrVdelA and pLcrVmRNAmutb (CTG CGG ATT CTG CTC ATACGC CCT GAT CAT ATT AAA TAA TTT GCC CTC GCA T) |
pLcrVmRNAmutc (TAT GAG CAG AAT CCG CAG CAC TTC ATC GAA GAC CTA GAA AAA GTT AGG GTG GAA CA) and pLcrVdelD1 |
Numbers in parentheses indicate the internal amino acids deleted or altered in LcrV (316 amino acids).
Italic type indicates the incorporated XhoI and SacI restriction sites used for cloning of the PCR-amplified DNA fragments. Underlining indicates the complementary overlap between the corresponding B and C primers. Bold type indicates altered or inserted oligonucleotides.