TABLE 2.
Oligonucleotides used in this study
| Oligonucleotidea | Oligonucleotide sequenceb | Presence, loss, or gain of restriction site |
|---|---|---|
| Oligonucleotides for modification of the Shine-Dalgarno region | ||
| KPIfc | 5′ CTCGGTAC(CCAAAAAGT)GAGAAGACAATG 3′ | Gain of KpnI |
| BEIIrc | 5′ CAAGTAGCTGGTGACCTTCGGCCC 3′ | BstEII |
| Mutant oligonucleotidesd | ||
| L285W | 5′ CCAGCTACTGGACCGAAGGCCCGGCG 3′ | Loss of DraII |
| A291S | 5′ GAAGGCCCCGCGTCGGAAAAGGCGG 3′ | Loss of SacII |
| G404D | 5′ GCTGAGATGTCCATGGACCAAACCGTTG 3′ | Gain of NcoI |
| I301V/T305S/I307L/L309V | 5′ GCTCGACCATGAGTTTGGAGCCGCGCTCCACACTAC 3′ | Gain of BstXI |
| V324I/I327V | 5′ CCCAGGTATCAATACGGTCCGAACATGG 3′ | Gain of AvaII |
| I412V | 5′ CGACCCGGTATACCCTGGTCG 3′ | Gain of Bst1107I |
| K436R | 5′ GCACATTGGCTCCGGATGATGACC 3′ | Gain of BspEI |
| E444D | 5′ GACTGGGACGCATTAAAGGCGACG 3′ | Gain of BsmFI |
| Nonmutagenic oligonucleotides for PCR-based site-directed mutagenesis and/or cloning of bod2C1, bod3C1, todC1-bedC2, and bodC1-bedC2 genes | ||
| SBIfc | 5′ CGTCAGACACTACGTACCTTCTCTGCC 3′ | SnaBI |
| MUIrc | 5′ TTAGGTTTAACGCGTCGCCTTTAATGC 3′ | MluI |
| BEIIfc | 5′ GGGCCGAAGGTCACCAGCTACTTG 3′ | BstEII |
| SBIrc | 5′ GGCAGAGAAGGTACGTAGTGTCTGACG 3′ | SnaBI |
| HDIIIrc | 5′ GGCTGAAAATCTTCTCTCATCCGC 3′ | HindIII |
| PKKc | 5′ GCGGATAACAATTTCACACAGG 3′ | EcoRI |
| AGIr | 5′ CGGGCGCTACCGGTGCCGGC 3′ | AgeIe |
Most oligonucleotides were synthesized by Phil Marsh (King's College London); the exceptions were KPIf and BEIIr, which were synthesized by Pharmacia (UK) and Perkin-Elmer Ltd., respectively.
The base changes are indicated by boldface type. The underlined bases indicate loss or addition of the restriction site. Additional bases are in parentheses.
For the locations of nonmutagenic oligonucleotides see the positions of restriction sites in Fig. 1, sequences are within or close to the primer sequence.
In the mutant oligonucleotide designations the first letter is the amino acid in the ISPBEDα sequence, the number corresponds to the position in the amino acid sequence, and the second letter is the replacing amino acid in the ISPTODα sequence.
The AgeI restriction site is located between the bedC1 and bedC2 genes in the pJRM502 plasmid.