TABLE 2.
Primer sets used to construct plasmids employed in this work
Sequences *,Gln3(1631–1636), 5′-CGTCCG-3′, and **,Gln3(1761–1766), 5′-CTCCGT-3′, were changed to 5′-CGGCCG-3′ to generate a unique EagI restriction site for cloning purposes. These changes did not alter the amino acid sequence of the protein.
| Plasmids | Sequence changes | Primer sets |
|---|---|---|
| pRR536 | Gln3(1–730) (F.L. wild type) | 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ |
| 5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
| pRR849** | Gln3S580D,S582D,S583D,S584D,S589D | 5′-CAATCGGCCgacTTGCAACAACTGCACGAACAGCAACAAGTGGAC-3′ |
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| 5′-CAATCGGCCGCCATAAAgtcatcgtcGTAatcTGCATTGCGCGACATCTTTCTTCTTGG-3′ | ||
| 5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
| pRR876** | Gln3S580A,S582A,S583A,S584A,S589A | 5′-CAATCGGCCGGTAcgcTGCATTGCGCGACATCTTTCTTCTTGG-3′ |
| 5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
| 5′-CAATcggccggcgTTTATGGCTGCGgctTTGCAACAACTGCACGAACAG-3′ | ||
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| pRR924 | Gln3S544D,S545D,S550D,S552D | 5′-GTACGATGCATTGCGCGACATCTTTCTTCTTGGTATATTCATTCCTTGACGTTGGAAAGAATTTGATAATAGATTCTGCTGTAGAGGatcATTgtcGTTAAAGTTTGCgtcgtcTATAATTCTTGGTG-3′ |
| 5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
| pRR926 | Gln3S550D,S552D,S560D,S562D | 5′-GTACGATGCATTGCGCGACATCTTTCTTCTTGGTATATTCATTCCTTGACGTTGGAAatcATTatcTAATAGATTCTGCTGTAGAGGatcATTgtcGTTAAAGTTTGCGGACG-3′ |
| 5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
| pRR992 | Gln3S550A,S552A,S560A,S562A | 5′-GTACGATGCATTGCGCGACATCTTTCTTCTTGGTATATTCATTCCTTGACGTTGGAAagcATTtgcTAATAGATTCTGCTGTAGAGGagcATTtgcGTTAAAG-3′ |
| 5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
| pRR1003 | Gln3S544A,S545A,S550A,S552A | 5′-GTACGATGCATTGCGCGACATCTTTCTTCTTGGTATATTCATTCCTTGACGTTGGAAAGAATTTGATAATAGATTCTGCTGTAGAGGagcATTtgcGTTAAAGTTTGCagctgcTATAATTCTTGGTG-3′ |
| 5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
| pRR1015* | Gln3N518D,N520D,N522D,N525D | 5′-CAATCGGCCGATATAATTCTTGGTGAGGATGCGACACTATTTCCTGCACTTACATTATTTGTTGTgtcCATTAGgtcCATgtcCATgtcGAACTGCTGTGAATTAG-3′ |
| 5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
| 5′-CAATCGGCCGCAAACTTTAACTCAAATAGTCCTCTACAG-3′ | ||
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| pRR1017* | Gln3T526A,T527A,S531A,S535A,S538A,S539A | 5′-CAATCGGCCGATATAATTCTTGGtgcggcTGCGACcgcATTTCCTGCcgcTACATTATTtgctgcGTTCATTAGGTTCATGTTC-3′ |
| 5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
| 5′-CAATCGGCCGCAAACTTTAACTCAAATAGTCCTCTACAG-3′ | ||
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| pRR1034 | Gln3S576D | 5′-GTACGATGCATTGCGgtcCATCTTTCTTCTTGGTATATTC-3′ |
| 5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
| pRR1037 | Gln3S576A | 5′-GTACGATGCATTGCGcgcCATCTTTCTTCTTGGTATATTC-3′ |
| 5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
| pRR1045* | Gln3T526D,T527D,S531D,S535D,S538D,S539D | 5′-CAATCGGCCGCAAACTTTAACTCAAATAGTCCTCTACAG-3′ |
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| 5′-AATCGGCCGATATAATTCTTGGtgctgcTGCGACgtcATTTCCTGCgtcTACATTATTgtcgtcGTTCATTAGGTTCATGTTC-3′ | ||
| 5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
| pRR1065 | Gln3 (1–730) | 5′-CATGCCATGGAGATGCAAGACGACCCCGAAAATTCGAAGCTG-3′ |
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| pRR1109 | Gln3 (512–608) | 5-CATGCCATGGAGATGGCTAATTCACAGCAGTTCAACATGAACATG-3′ |
| 5′-CGCGGATCCGATTCGTGTTTGTGTTGGAATTCACGTCCACTTG-3′ (template, pRR536) | ||
| pRR1112 | Gln3 (512–608) | 5′-CATGCCATGGAGATGGCTAATTCACAGCAGTTCAACATGAACATG-3′ |
| 5′-CGCGGATCCGATTCGTGTTTGTGTTGGAATTCACGTCCACTTG-3′ (template, pRR1045) | ||
| pRR1115 | Gln3N510D,N513D,Q515D,Q516D | 5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACCTTCACCTgatAGCGCTgatTCAgacgacTTCAACATGAAC-3′ |
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| pRR1171 | Gln3N510D,N513D | 5′-TCATCCAGATCTGTTGTTCCGATATTACCAAAACCTTCACCTgatAGCGCTgatTCACAGCAGTTC-3′ |
| 5′-GTACGATGCATTGCGCGACATCTTTCTTCTTGG-3′ | ||
| pRR1190 | Gln3S583D,S584D | 5′-TCGCGCAATGCATCGTACTCCgacgacTTTATGGCTGCGTCTTTG-3′ |
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| pRR1192 | Gln3S583A,S584A | 5′-TCGCGCAATGCATCGTACTCCgcagcgTTTATGGCTGCGTCTTTG-3′ |
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| pRR1194 | Gln3S589D | 5′-TCGCGCAATGCATCGTACTCCTCATCGTTTATGGCTGCGgacTTGCAACAACTGCAC-3′ |
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| pRR1196 | Gln3S589A | 5′-TCGCGCAATGCATCGTACTCCTCATCGTTTATGGCTGCGgctTTGCAACAACTGCAC-3′ |
| 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
| pRR1248 | Gln3 (508–730) | 5′-CATGCCATGGAGTCACCTAATAGCGCTAATTCACAGCAG-3′ |
| 5′-CGCGGATCCGTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ (template, pRR536) | ||
| pRR1250 | Gln3 (508–730) | 5′-CATGCCATGGAGTCACCTAATAGCGCTAATTCACAGCAG-3′ |
| 5′-CGCGGATCCGTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ (template, pRR850) |