TABLE 1.
Construction of A14 mutantsa
| Primer | Sequence |
|---|---|
| O1 | 5′ CCATCGATGGACATGATGCTTAT 3′ |
| O4 | 5′ CGGGATCCTTAGTTCATGGAAATAT 3′ |
| S12A-B | 5′ CACGCCGGCAAAATAATTTCCAATCA 3′ |
| S12A-C | 5′ ATTATTTTGCCGGCGTGCTAATCGCT 3′ |
| S25A-B | 5′ GATACACGCAAGAATCAAAAGAATGA 3′ |
| S25A-C | 5′ TGATTCTTGCGTGTATCTTCGCCTTT 3′ |
| S34A-B | 5′ GTAGACTTAGCAAAGTCAATAAAGGCGA 3′ |
| S34A-C | 5′ TTGACTTTGCTAAGTCTACCAGTCCCA 3′ |
| S36A-B | 5′ ACTGGTAGCCTTACTAAAGTCAATAA 3′ |
| S36A-C | 5′ TTAGTAAGGCTACCAGTCCCACTCGT 3′ |
| S38A-B | 5′ GAGTGGGAGCGGTAGACTTACTAAAGT 3′ |
| S38A-C | 5′ AGTCTACCGCTCCCACTCGTACATGGA 3′ |
| S34,36,38A-B | 5′ GAGTGGGAGCGGTAGCCTTAGCCAAAGTCAATAAAGGCG 3′ |
| S34,36,38A-C | 5′ TTGACTTTGCTAAGGCTACCGCTCCCACTCGTACATGGA 3′ |
| S47A-B | 5′ GCCATAATAGCCAATACTTTCCATGTA 3′ |
| S47A-C | 5′ AAGTATTGGCTATTATGGCGTTTATAC 3′ |
| S65A-B | 5′ CCACATAGCATAAATTAGCATTCCG 3′ |
| S65A-C | 5′ TAATTTATGCTATGTGGGGAAAGCA 3′ |
| S77A-B | 5′ AATGACTCCGGCAACTCTGTGGGGTGCG 3′ |
| S77A-C | 5′ CCCACAGAGTTGCCGGAGTCATTCATACC 3′ |
| S88A | 5′ GGGATCCTTAGTTCATGGCAATATCGCTATGATTG 3′ |
| N′ deletion | 5′ CCATCGATGATTGGAAATTATTTTTCCGGC 3′ |
| C′ deletion | 5′ CGGGATCCTTACATAGAATAAATTAG 3′ |
| PTRTWK-B | 5′ TGCCGCTGCAGCAGCGGCACTGGTAGACTTACTA 3′ |
| PTRTWK-C | 5′ GCCGCTGCTGCAGCGGCAGTATTGAGTATTATG 3′ |
| P39A-B | 5′ ACGAGTGGCACTGGTAGACTTACTA 3′ |
| P39A-C | 5′ CTACCAGTGCCACTCGTACATGGAAAG 3′ |
| R41K44-B | 5′ TACTGCCCATGTAGCAGTGGGACTGGTAGA 3′ |
| R41K44-C | 5′ ACTGCTACATGGGCAGTATTGAGTATTATG 3′ |
| W43A-B | 5′ ATACTTTCGCTGTACGAGTGGGACTG 3′ |
| W43A-C | 5′ CTCGTACAGCGAAAGTATTGAGTATT 3′ |
| W43K44-B | 5′ TCAATACTGCCGCTGTACGAGTGGGACTG 3′ |
| W43K44-C | 5′ CTCGTACAGCGGCAGTATTGAGTATTATG 3′ |
| NYF-B | 5′ AGCAGCAGCTCCAATCATAAGCATC 3′ |
| NYF-C | 5′ GCTGCTGCTTCCGGCGTGCTAATC 3′ |
| CIFAF-B | 5′ AGCGGCGGCGGCAGCCGAAAGAATCAAAAG 3′ |
| CIFAF-C | 5′ GCTGCCGCCGCCGCTATTGACTTTAGTAAG 3′ |
| DFSK-B | 5′ CGCAGCAGCGGCAATAAAGGCGAAGATA 3′ |
| DFSK-C | 5′ GCCGCTGCTGCGTCTACCAGTCCCACT 3′ |
| MWG-B | 5′ TGCCGCCGCAGAATAAATTAGCATT 3′ |
| MWG-C | 5′ GCGGCGGCAAAGCACTGCGCACCC 3′ |
| C71S-B | 5′ TGGGGTGCGCTGTGCTTTCCCCACAT 3′ |
| C71S-C | 5′ GAAAGCACAGCGCACCCCACAGAGTT 3′ |
| N83Q | 5′ CGGGATCCTTAGTTTACGGAAATATCGCTATGCAGGGTATGAATGACTCCA 3′ |
| S85A | 5′ CGGGATCCTTAGTTCAGTGAAATATCGGCATGATTGGTATGAATG 3′ |
| vS85A-B | 5′ GAAATATCCCGATGATTGGTATGAAT 3′ |
| vS85A-C | 5′ CCAATCATGCCGATATTTCCATGAAC 3′ |
Primer O1 inserts a ClaI site (in bold) at the 5′ terminus, overlapping the translational start site (underlined) of A14. Primer O4 inserts a BamHI site (in bold) at the 3′ terminus, immediately downstream of the translational stop site (underlined) of the A14 open reading frame. The B and C primers serve to mutate the various residues indicated in the table. Construction of most of these mutants involved two sequential rounds of PCR. In the first round, two reactions utilizing primers O1 plus B or O4 plus C were performed to generate subgenic fragments of the A14 gene containing the desired mutations within a 23-bp region of overlap. These products were purified on glass beads (39), with aliquots of each serving as the template for a second round of PCR using primers O1 and O4. Mutants of A14 not generated by overlap PCR include N′ deletion, C′ deletion, S85A, S88A, and N83Q. These mutants were generated in one round of PCR by substituting the N′ deletion primer for O1 or the C′ deletion S85A, S88A, or N83Q primer for O4. In all cases, the final products were purified on glass beads and were then subjected to digestion with BamHI and ClaI. These inserts were then ligated into pUC1246 plasmid DNA that had been previously digested with BamHI and ClaI and treated with calf intestinal alkaline phosphatase. pUC1246 places the A14 alleles under the regulation of a strong, late promoter derived from the cowpox ATI gene (23, 36). E. coli transformants carrying the pUC1246-A14 plasmids were generated, and the plasmids were purified by using Qiagen Endofree Maxi kits (Valencia, Calif.). All A14 alleles were verified by restriction enzyme digestion and automated DNA sequencing.