PCR-SSP primer mixes for KIR3DL3 non-synonymous polymorphism, and SNP linkage (L) reactions. Reaction KIR3DL3 Polymorphism Sense primers Antisense primers Amplicon Control no size (bp) Product 1 b Exon 3, 155A GACTCTTCAGTGTCGCTCTCA 0.6 GAGTGTGGGTGTGAACTGCA 0.6 171 HLA-DRB1 2 b Exon 3, 155G CTCTTCAGTGTCGCTCTCG 1.2 GAGTGTGGGTGTGAACTGCA 1.2 169 HLA-DRB1 3 b Exon 3, 168T/C a TCGCTCTCGTCTTGGGTTTAAT TCGCTCTCGTCTTGGGTTTAACG TCGCTCTCATCTTGGGTTTAACG 0.6 0.6 0.6 GAGTGTGGGTGTGAACTGCA 1.2 159 HLA-DRB1 4 b Exon 3, 168A TCGCTCTCGTCTTGGGTTTAAA 0.6 GAGTGTGGGTGTGAACTGCA 0.6 159 HLA-DRB1 5 b Exon 4, 447T GCAATGTTGGTCAGATGTCAGT 1.2 GACAACTCATAGGGTAAGTGAGTG 1.2 194 HLA-DRB1 6 b Exon 4, 447G CAATGTTGGTCAGATGTCAGG 1.2 GACAACTCATAGGGTAAGTGAGTG 1.2 193 HLA-DRB1 7 b Exon 4, 455A GTCAGATGTCAGGTTTGAGCA 1.2 GACAACTCATAGGGTAAGTGAGTG 1.2 106 HLA-DRB1 8 b Exon 4, 455G a TCAGATGTCAGTTTTGAGCG TCAGATGTCAGGTTTGAGCG 1.8 1.8 GACAACTCATAGGGTAAGTGAGTG 1.8 105 HLA-DRB1 9 b Exon 4, 502A AGGACCCCTTGCGCCTCA 1.2 GACAACTCATAGGGTAAGTGAGTG 1.2 136 HLA-DRB1 10 b Exon 4, 502G GGACCCCTTGCGCCTCG 1.2 GACAACTCATAGGGTAAGTGAGTG 1.2 135 HLA-DRB1 11 b Exon 5, 869C TGCAGCTCCCGGAGCTTG 0.6 AGAGCCGAAGCATCTGTAGG 0.6 159 HLA-DRB1 12 b Exon 5, 869A TGCAGCTCCCGGAGCTTG 1.2 AAGAGCCGAAGCATCTGTAGT 1.2 160 HLA-DRB1 13 b Exon 6, 961A a TTCCTCCAGGTAACTCCAGAA 1.2 CAGGCTCTTGGTCCATTACAA CAGGCTCCTGGTCCATTACAA 1.2 1.2 603 APC 14 b Exon 6, 961C TTCCTCCAGGTAACTCCAGAC 1.2 CAGGCTCTTGGTCCATTACAA 1.2 603 APC 15 b Exon 6, 961T TTCCTCCAGGTAACTCCAGAT 2.4 CAGGCTCTTGGTCCATTACAA 2.4 603 APC 16 b Exon 6, 971C ACTCCAGATACCTGCACGC 1.2 CAGGCTCTTGGTCCATTACAA 1.2 590 APC L1 Exon 2, 69G Exon 4, 621A GGCCCTGGCCACATGTG 1.2 CAGAGGGTCACTGGGAGCT 1.2 2630 APC L2 Exon 2, 69A Exon 4, 621G GGCCCTGGCCACATGTA 1.2 AGAGGGTCACTGGGAGCC 1.2 2629 APC L3 b Exon 3, 155A Exon 4, 502G GACTCTTCAGTGTCGCTCTCA 0.3 CATCGTGGAGCTGTCCAAC 0.3 1659 APC L4 b Exon 3, 155G Exon 4, 502A CTCTTCAGTGTCGCTCTCG 0.12 GCATCGTGGAGCTGTCCAAT 0.12 1658 APC L5 b Exon 3, 155G Exon 4, 502G CTCTTCAGTGTCGCTCTCG 0.3 CATCGTGGAGCTGTCCAAC 0.3 1657 APC L6 Exon 3, 155A Exon 4, 621G GACTCTTCAGTGTCGCTCTCA 1.2 AGAGGGTCACTGGGAGCC 1.2 1777 APC L7 Exon 3, 155G Exon 4, 621G CTCTTCAGTGTCGCTCTCG 1.2 AGAGGGTCACTGGGAGCC 1.2 1775 APC L8 Exon 3, 168T Exon 4, 408A TCGCTCTCGTCTTGGGTTTAAT 1.2 CAGGATGACCGTCTCTCCT 1.2 1553 APC L9 Exon 3, 168A Exon 4, 621A TCGCTCTCGTCTTGGGTTTAAA 1.2 CAGAGGGTCACTGGGAGCT 1.2 1766 APC L10 b Exon 4, 408G Exon 5, 777A CAGGTCCCCTGGTGAAATCG 1.2 CCTAAGTTCACCGGCCTCT 1.2 1985 APC L11 b Exon 4, 408A Exon 5, 777G CAGGTCCCCTGGTGAAATCA 1.2 TAAGTTCACCGGCCTCCG 1.2 1983 APC L12 Exon 4, 447T Exon 5, 869C GCAATGTTGGTCAGATGTCAGT 1.2 AGAGCCGAAGCATCTGTAGG 1.2 2041 APC L13 Exon 4, 447G Exon 5, 869A CAATGTTGGTCAGATGTCAGG 1.2 AAGAGCCGAAGCATCTGTAGT 1.2 2041 APC L14 Exon 4, 502A Exon 5, 869A AGGACCCCTTGCGCCTCA 1.2 AAGAGCCGAAGCATCTGTAGT 1.2 1983 APC L15 Exon 4, 502G Exon 5, 869A GGACCCCTTGCGCCTCG 1.2 AAGAGCCGAAGCATCTGTAGT 1.2 1982 APC All mixes contained control primer pairs designed to amplify a 796 bp region of HLA-DRB1 (Bunce et al. 1995; Olerup et al. 1993) or a 256 bp amplicon of the APC gene (Sadler et al. 1994). PCR conditions are as described in the text. a Degenerate primers used in these reactions. b Reactions used to type all samples used in this study
Long-range PCR-SSP primer mixes for KIR3DL3 Reaction KIR3DL3 Polymorphism Sense primers Antisense primers Amplicon Control no size (bp) Product 1 Exon 5, 777G Exon 6, 961T TTACCATCTATCCAGGGAGGCG 2.4 TCCCAATCAGAGCGTGCAGGTA 2.4 5694 APC 2 Exon 5, 777G Exon 6, 961A TTACCATCTATCCAGGGAGGCG 2.4 TCCCAATCAGAACGTGCAGGTT 2.4 5694 APC 3 Exon 5, 777A Exon 6, 961A TTTACCATCTATCCAGGGAGGCA 2.4 TCCCAATCAGAACGTGCAGGTT 2.4 5695 APC 4 Exon 5, 869A Exon 6, 961T CTGTGACCCACGGAGGGAA 2.4 CCAATCAGAGCGTGCAGGTA 2.4 5598 APC 5 Exon 5, 869A Exon 6, 961C CCTGTGACCCACGGAGGGAA 2.4 TCCCAATCAGAACGTGCAGGTG 2.4 5601 APC 6 Exon 5, 869C Exon 6, 961T CCTGTGACCCACGGAGGGAC 2.4 TCCCAATCAGAGCGTGCAGGTA 2.4 5601 APC All primer mixes contained control primer pairs to amplify a 256 bp region of the APC gene (Sadler et al. 1994).
KIR3DL3 Sequencing primer mixes Reaction KIR3DL3 Region Sense primers Antisense primers Amplicon no size (bp) 1 Exons 1 2 GAACTGAGCTGGGGCGCA 1.2 CATGGTCAGCCCATCAGTCA 1.2 894/933 b 2 Exon 3 TCCACATCCTCCTCTCTAAGGT 1.2 TCTTCCCACCACAGCAACT 1.2 483 3 Exon 3 (168T linked Intron 2 SNP) a TCCACATCCTCCTCTCTAAGGTA 1.2 TCTTCCCACCACAGCAACT 1.2 483 4 Exon 4 CACTTATTTCAGGTCCCATGA 1.2 CCAGTCCTAGATCATTCACTCCA 1.2 434 5 Exon 4, 621G a CACTTATTTCAGGTCCCATGA 1.2 AGAGGGTCACTGGGAGCC 1.2 354 6 Exon 5 GAACCTCCCTGAGGAAACCA 1.2 TTCTCACCTGTGACAGAAACG 1.2 337 7 Exon 5, 777G a CCATCTATCCAGGGAGGCG 1.2 TTCTCACCTGTGACAGAAACG 1.2 198 8 Exon 6 TGATTAGCTTCTTATTGGTGTCTTG 1.2 GCCCTGAGCTCTCTGGC 1.2 178 9 Exon 6 (961T & 971C linked Intron 6 SNP) a TGATTAGCTTCTTATTGGTGTCTTG 1.2 GCAGGGGATGTGAGGATAT 1.2 356 10 Exons 6-8 TGATTAGCTTCTTATTGGTGTCTTG 1.2 CCGGTTTTGAGACAGGGCTA 1.2 993 a Reactions designed to amplify a single allelic sequence: The forward primer of reaction 3 is positioned on a SNP 20 bp upstream of exon 3 found to be in linkage with the 168T polymorphism; the reverse primer of reaction 5 is specific to the 621G polymorphism found within exon 4; Forward primer used in reaction 7 is specific to the 777G polymorphism within exon 5; the reverse primer of reaction 9 is positioned on a SNP located at nucleotide 197 of intron 6, found to be in linkage with the exon 6 polymorphisms 961T and 971C b Amplicon size variable due to an insertion within Intron 1 in certain alleles.
Interspecies dn and ds values for KIR3DL3 and four other conserved KIR loci in higher primates D0 a D1 D2 dn dn Covariance ds ds Covariance dn/ds dn dn Covariance ds ds Covariance dn/ds dn dn Covariance ds ds Covariance dn/ds KIR3DL3 Hs v. Gg 0.0046 0.0046 0.0000 0.0000 N/A 0.0000 0.0000 0.0290 0.0208 N/A - - - - - Hs v. Pt 0.0046 0.0048 0.0309 0.0222 0.1501 0.0047 0.0048 0.0144 0.0146 0.3291 0.0188 0.0096 0.0409 0.0238 0.4589 Pt v. Gg 0.0093 0.0067 0.0311 0.0223 0.2992 0.0047 0.0050 0.0440 0.0261 0.1076 b - - - - - KIR2DL4 Hs v. Gg 0.0187 0.0090 0.0000 0.0000 N/A - - - - - 0.0573 0.0164 0.0715 0.0329 0.8010 Hs v. Pt 0.0000 0.0000 0.0000 0.0000 N/A - - - - - 0.0376 0.0140 0.0873 0.0371 0.4308 Pt v. Gg 0.0187 0.0090 0.0000 0.0000 N/A - - - - - 0.0378 0.0132 0.0418 0.0245 0.9042 KIR2DL5 Hs v. Gg 0.0242 0.0112 0.0577 0.0298 0.4192 - - - - - 0.0328 0.0119 0.0139 0.0139 2.3663 Hs v. Pt 0.0192 0.0093 0.0000 0.0000 N/A - - - - - 0.0327 0.0124 0.0426 0.0247 0.7692 Pt v. Gg 0.0340 0.0131 0.0581 0.0300 0.5850 - - - - - 0.0280 0.0113 0.0280 0.0199 0.9985 KIR2DS4 Hs v. Pt 0.0189 0.0095 0.0313 0.0224 0.6046 0.0313 0.0110 0.0451 0.0262 0.6931 0.0092 0.0069 0.0580 0.0302 0.1582 b KIR3DL1 Hs v. Pt 0.0289 0.0142 0.0651 0.0337 0.4437 0.0270 0.0105 0.0000 0.0000 N/A 0.0420 0.0146 0.0899 0.0390 0.4676 Hs= Homo sapiens; Gg= Gorilla gorilla; Pt= Pan troglodytes (common chimp) ; N/A: Not applicable (due to a dn or ds value equalling 0); Dashes (-) indicate the absence of that Ig domain within a loci. a Pseudoexon 3 in the case of KIR2DS4 b p<0.05 using Fisher s exact test for purifying selection (Zhang et al. 1997)
Interspecial values for dn, ds, covariance and dn/ds were calculated using SNAP (http://www.hiv.lanl.gov/content/hivdb/snap/websnap/snap.html ). For polymorphic loci, consensus representative sequences were produced using (Cons, Emboss package). dn/ds values greater than 1 suggest positive evolutionary pressure, those less than 1 suggest purifying pressure. Low dn values obtained for KIR3DL3 reflect its conserved nature at the protein level. The relatively low values obtained for dn/ds across all the Ig domains of KIR3DL3 suggest it has undergone purifying evolutionary pressure.
Mean dn and ds values for individual immunoglobulin domains of human KIR3DL3 alleles and four other polymorphic KIR loci D0 D1 D2 dn dn Covariance ds ds Covariance dn/ds dn dn Covariance ds ds Covariance dn/ds dn dn Covariance ds ds Covariance dn/ds KIR3DL3 0.0025 0.0018 0.0066 0.0049 0.3792 0.0028 0.0017 0.0084 0.0061 0.3292 0.0023 0.0023 0.0144 0.0086 0.1569 KIR2DL4 0.0027 0.0014 0.0015 0.0015 1.7665 - - - - - 0.0059 0.0030 0.0069 0.0058 0.8468 KIR2DL5 0.0027 0.0020 0.0247 0.0143 0.1104 - - - - - 0.0065 0.0037 0.0000 0.0000 N/A KIR3DL1/S1 0.0132 0.0049 0.0138 0.0099 0.9558 0.0067 0.0031 0.0027 0.0014 2.4550 0.0048 0.0026 0.0085 0.0055 0.5667 KIR3DL2 0.0060 0.0030 0.0025 0.0025 2.4103 0.0031 0.0019 0.0021 0.0022 1.4377 0.0021 0.0012 0.0021 0.0021 0.9965 N/A: Not applicable (due to a ds or dn value equalling 0); dashes (-) indicate the absence of that Ig domain within a loci. Values for dn, ds and covariance were calculated using SNAP (http://www.hiv.lanl.gov/content/hiv-db/snap/websnap/snap.html) for all KIR3DL3 alleles listed in Table 4. Additional values were calculated for several other KIR loci using allele sequences present on the IPD-KIR sequence database (http://www.ebi.ac.uk/ipd/kir/index.html). The dn/ds ratios were calculated by dividing the mean dn average with that of ds. The relatively low dn/ds values suggest KIR3DL3 is undergoing purifying pressure over all Ig domains, favouring the appearance of synonymous polymorphism.
References Bunce, M., O'Neill, C. M., Barnardo, M. C., Krausa, P., Browning, M. J., Morris, P. J., and Welsh, K. I.: Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilising sequence-specific primers (PCR-SSP). Tissue Antigens 46: 355-67., 1995 Olerup, O., Aldener, A., and Fogdell, A.: HLA-DQB1 and -DQA1 typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours. Tissue Antigens 41: 119-34., 1993 Sadler, A. M., Petronzelli, F., Krausa, P., Marsh, S. G., Guttridge, M. G., Browning, M. J., and Bodmer, J. G.: Low-resolution DNA typing for HLA-B using sequence-specific primers in allele- or group-specific ARMS/PCR. Tissue Antigens 44: 148-54, 1994 Zhang, J., Kumar, S., and Nei, M.: Small-sample tests of episodic adaptive evolution: a case study of primate lysozymes. Mol Biol Evol 14: 1335-8, 1997