Supporting Information

Transcription

1 upporting Information hiota et al..73/pnas I Materials and Methods Yeast trains. Yeast strains used in this study are described in Table 1. TOM22FLAG, a yeast haploid strain for expression of C-terminally FLAG-tagged Tom22 instead of corresponding T Tom22 was constructed as follows. A DNA fragment for the triple-flag epitope tag and that containing the Candida glabrata I3 (CgI3) marker were amplified from ptye247 by PCR using primers Tom22 FLAG F (5 -ATT GGC CCA AGG TGA AAA AGA TGC TGC AGC AAC AGC CAA TGG CGA ATT GGG TAC CGG G-3 ) and Tom22-tag-R (5 -CCT TTT CTA AAA CCC TCT CTT TTC TTT TAC ATC ATTAAA ACA GGA AAC AGC TAT GAC-3 ). The amplified DNA fragments were integrated into the 3 -end of the corresponding T gene of the haploid strain 33-1A, and his transformants were selected. GAL7-TOM22, a yeast strain for expression of Tom22 from the GAL7 promoter, was constructed as follows. A DNA fragment containing the GAL7 promoter was amplified from plasmid pcgi3 GAL7 by PCR using primer pairs Tom22-off-F (5 - TCA AAA CGG AAC TAT ATA CCC CAA AAT AAG CAT CAT TCA AAA GCT TGG GTC TTC TGG AGC-3 ) and Tom22-off-R (5 -ATT GAA CGA CAT CGT CTT TAA TTT CAG TTA ATT CGA CCA TTT TTG AGG GAA TAT TCA ACT-3 ). The amplified DNA fragment was integrated into the chromosome upstream of the TOM22 gene of the T strain 33-1A and his transformants were selected. Plasmids. Plasmids and primers used in this study are described in Tables 2 4. The pt1 plasmid was constructed as follows. The TOM22 I gene was amplified from pu22 (see below) by PCR using primer pairs of Tom22C (5 -CGC GAA TTC ATG GTC GAA TTA ACT GAA-3 ) and Tom22E (5 -GCG AAG CTT TTA ATG ATG ATG ATG ATG GTG ATG ATG GTG GTG CCC GGG GGA TCC ATT GGC TGT TGC TGC AGC-3 ). The amplified DNA fragment was digested with EcoRI and indiii, and inserted into the EcoRI/indIII site between the GAL1 promoter and CMK1 terminator of the CEN-URA3 plasmid, YCpUG578T, to generate pu22. A pei/xhoi fragment of pu22 containing the GAL1 promoter, TOM22 I gene and CMK1 terminator was introduced into the pei/xhoi site of the CEN LEU2 plasmid, pr315, to generate pt1. For in vivo photocross-linking, pt1-x plasmids were constructed as follows. The codon for residue x of Tom22is in pt1 was replaced with the amber codon by Quick-change using a primer pair of Tom22(x)-amber-Fw and Tom22(x)-amber-Rv (Table 3) to generate pt1-x. For in vivo and in organello photocross-linking, pt2-x plasmids were constructed as follows. The TOM22 promoter and terminator were amplified from the yeast genomic DNA by PCR using primer pairs of Tom22P-f (5 -CGG GAT CCC AAA AAG AGC TAA TCA ACT CCT TG-3 ) with Tom22P-r (5 -CGG AAT TCG ATG CTT ATT TTG GGG TAT ATA GT-3 ) and Tom22T-f (5 -CCC CAA GCT TTG ATG TAA AAG AAA AGA GAG GGT T-3 ) with Tom22T-r (5 -CCA TCG ATT GAA ATA TGT ACA AGT TTA TAC AT-3 ), respectively. The amplified DNA fragment containing the TOM22 promoter was digested with BamI and EcoRI, and inserted into the BamI/EcoRI site of the CEN-URA3 plasmid, YCpUG578T, to generate YCpUG578T-Tom22ownP. The amplified DNA fragment containing the TOM22 terminator was digested with indiii and ClaI, and inserted into the indiii/clai site of the YCpUG578T- Tom22ownP plasmid to generate YCpUG578T-Tom22ownPT. The EcoRI/indIII fragment of pt1-x containing the TOM22 I gene with the amber codon for position X was introduced into the EcoRI/indIII site of YCpUG578T-Tom22ownPT to generate pt2-x. The pt3 plasmid was constructed as follows. The TOM22 gene was amplified from the genomic DNA by PCR using a primer pair of Tom22C (5 -CGC GAA TTC ATG GTC GAA TTA ACT GAA-3 ) and Tom22B (5 -GCG AAG CTT TTA ATT GGC TGT TGC TGC-3 ). The amplified DNA fragment was digested with EcoRI and indiii, and inserted into the EcoRI/indIII site of the CEN-URA3 plasmid, YCpUG578T, to generate pu22. A BamI/XhoI fragment of pu22 containing the GAL1 promoter, TOM22 gene, and CMK1 terminator was introduced into the BamI/XhoI site of the CEN-LEU2 plasmid, pr315, to generate pt3. For in vivo photocross-linking, pt3-x plasmids were constructed as follows. The codon for residue x of Tom22 in pt3 was replaced with the amber codon by Quickchange using a primer pair of Tom22(x)-amber-Fw and Tom22 (x)-amber-rv (Table 3) to generate pt3-x. The pt4 plasmid was constructed as follows. The TOM2 gene was amplified from the genomic DNA by PCR using a primer pair of Tom2-is-N (5 -CGC AGA TCT ATG TCC CAG TCG AAC CCT AT-3 ) and Tom2-is-C (5 -CGC AAG CTT TCA ATG ATG ATG ATG ATG GTG ATG ATG GTG GTG GTC ATC GAT ATC GTT AGC TT-3 ). The amplified DNA fragment was digested with BglII and indiii, and inserted into the BglII/indIII site of the CEN-URA3 plasmid, YCpUG578T, to generate pt4. For in vivo and in organello photocross-linking, pt4-x plasmids were constructed as follows. The codon for residue x of Tom2is in pt4 was replaced with the amber codon by Quick-change using Tom2(x)-amber-Fw and Tom2 (x)-amber-rv (Table 4) to generate pt4-x. The pt5 plasmid was constructed as follows. The gene for pb 2 ð167þδ19 DFR was amplified from pgem4z pb 2 ð167þδ19 DFR (7) by PCR using a primer pair of pb2(bglii)-n (5 -CCC AGATCTATGCTAAAATACAAACCTTT-3 ) anddfr (indiii)-c (5 -GGG AAG CTT TTA GTC TTT CTC CTC GTA GA-3 ). The amplified DNA fragment was digested with BglII and indiii, and inserted into the BglII/indIII site of CEN-URA3 plasmid, YCpUG578T, to generate YCpUG578T pb 2 ð167þδ19 DFR. A ali/acii fragment of YCpUG578T pb 2 ð167þδ19 DFR containing the GAL1 promoter, pb 2 ð167þδ19 DFR gene, and CMK1 terminator gene was introduced into the ali/acii site of a 2μ-URA3 plasmid, pyo326, to generate pt5. Growth Conditions. Cells were grown in YPD (1% yeast extract, 2% polypeptone, and 2% glucose), D (.67% yeast nitrogen base without amino acids and 2% glucose), CD (.67% yeast nitrogen base without amino acids,.5% casamino acid, and 2% glucose), Gal (.67% yeast nitrogen base without amino acids and 2% galactose), or CGal (.67% yeast nitrogen base without amino acids,.5% casamino acid, and 2% galactose) media with appropriate supplements. In Vivo Photocross-Linking. For overexpression of Tom22 is containing DL-2-amino-3-(p-benzoylphenyl)pentanoic acid (BPA), saturated cultures of TY1-X and TY3-X grown in D ( Trp and Leu) were transferred to Gal ( Trp, Leu, and 1 mm BPA) and grown for 16 h. To express Tom22 is containing BPA and suppress expression of endogenous Tom22, a saturated culture of TY2-X grown in CGal ( Trp and Ura) was transferred to CD ( Trp, Ura, and 1 mm BPA) and grown for 24 h. For overexpression of Tom2-is containing BPA, a saturated 1of6

2 culture of TY-4 grown in CD ( Trp and Ura) was transferred to CGal ( Trp, Ura, and 1 mm BPA) and grown for 16 h. BPA (BACEM) was added to appropriate media from 1 M of stock solution in 1 N NaO. During cultivations, cells were kept in the dark to prevent BPA from cross-linkng reactions. Yeast cells were harvested and divided into halves. One half was UV-irradiated for min at room temperature at a distance 5 cm from a 365-nm UV lamp (22; μ cm 2 ; B-AP; UVP) and the other half was kept on ice. The yeast cells of 6 OD 66 of yeast culture were resuspended in 1 ml of TE buffer ( mm Tris-Cl, p 7.5 and 1 mm EDTA) containing 63 μl of5nnaoand 7 ml of β-mercaoptoethanol, and incubated for 4 min on ice. Then proteins were precipitated with 1 μl of % (v v) TCA and washed with ice-cold acetone. In Organello Photocross-Linking. Mitochondria were isolated from TY2-X grown in CD ( Trp, Ura, and 1 mm BPA) as described for in vivo photocross-linking. The pgem4z vector carrying the gene for pu9 DFR was used for in vitro transcription with P6 RNA polymerase. Radiolabeled pu9 DFR was synthesized with rabbit reticulocyte lysate in the presence of 35 -methionine. Isolated mitochondria were incubated with radiolabeled pu9-dfr in binding buffer ( mm sucrose, mm MOP-KO (potassium 3-[N-morpholino] propanesulfonate), p 7.2, 15 mm KCl, 5 mm MgCl 2, 2.5 mm KPi, 5 mm dithiothreitol, 1% (w v) BA, μg ml valinomycin) with 2 mm methionine at 4 or 3 C for min, or incubated with the purified rat-pald peptide in binding buffer for min on ice. The reaction mixtures were divided into halves, and the one half was UV-irradiated for min at room temperature at a distance 5 cm from a 365-nm UV lamp and the other half kept on ice. For preparation of the rat-pald peptide, a fusion protein consisting of gene protein plus one glutamate followed by pald was expressed in Escherichia coli cells by using an expression vector pet-17xb (Novagen). The fusion protein was recovered as inclusion bodies, solubilized with 4 M urea, and subjected V8 protease treatment (1 5 w w) to yield pald, which was further purified by reversed-phase PLC. A Galactose BPA p6 trna Tom22(136BPA)-is Tom22 Tom22(1-136aa) B Log (cell number) T -BPA T BPA 75amber BPA 48amber BPA 75amber -BPA 48amber -BPA vector -BPA vector BPA Time (h) Fig. 1. (A) trains TY1/pT1-136 containing both plasmids p6 trna and pt-136 (lanes 2, 4, and 6) and its parental strain 33-1A containing the plasmid pt-136, but not p6 trna, (lanes 1, 3, and 5) were grown on D medium (lanes 1 and 2) or Gal medium (lanes 3 6) in the presence (lanes 5 and 6) or absence (lanes 1 4) of BPA. Proteins in total lysates were analyzed by D-PAGE and immunoblotting with anti-tom22 antibodies. Tom22(136BPA) is, Tom22 with BPA at position 136 and the C-terminal is tag; Tom22, endogenous Tom22; Tom22(1 136aa), Tom22 fragment with translation termination at residue 136. (B) trains TY2/pT2-48 and TY2/pT2-75 for expression of Tom22 with BPA at residue 48 (48amber) or 75 (75amber), respectively, from its own promoter, pty2/pr316 (vector), and TY2/pT2 for expression of Tom22 from its own promoter were grown in CD ( Trp, Ura)with BPA (BPA)or without BPA ( BPA) for indicated time. Cell numbers were calculated from OD 66. 2of6

3 A αtom7 αtom4 C Tom4 Tom4 Tom7 Tom22T Tom22(136BPA)-is Tom22 α Tom4 α Tom4 αtom4 B T T Tom4 Tom22 T 48BPA 6BPA 118BPA 118BPA αtom4 Fig. 2. (A) The yeast strain (TY1/pT1-136) for overexpression of Tom22 with BPA at residue 136 from the GAL1 promoter was cultured in D medium ( Trp, Leu), then shifted to Gal medium ( Trp, Leu) with BPA and grown for 16 h. Mitochondria were isolated from the cells, solubilized with 1% digitonin, and analyzed by 4% glycerol density gradient centrifugation at 2; g for h at 4 C. After centrifugation, fractions were collected from the top and analyzed by D-PAGE and immunoblotting with the indicated antibodies. (B) The yeast strains (TY2/pT2-48, TY2/pT2-6, TY2/pT2-118, and TY2/pT2-136) for expression of Tom22 with BPA at residue 48, 6, 118, or 136 from its own promoter were cultured in Gal medium ( Trp, Ura), then shifted to D medium ( Trp, Ura) with BPA and grown for 24 h. Mitochondria were isolated from the cells and analyzed as in A. (C) The yeast strain (TY2/pT2-118) for expression of Tom22 with BPA at residue 118 from its own promoter was cultured as in B. Mitochondria were isolated from the cells and, after incubation with indicated antibodies, were analyzed as in A., Tom22 with BPA and the C-terminal is tag;, endogenous Tom22;, Tom22 fragment with translation termination specified by the amber codon. 3of6

4 A Prec Time (h) 37 (kda) p m αmdj1p p m αdfr f B BPA UV pald (mg/ml) (kda) 22 αtom4 C 136 UV (kda) αtim5 Fig. 3. (A) The yeast strains (TY1/pT1-48) for overexpression of Tom22 with BPA at residue 48 from the GAL1 promoter with (lanes 5 8) or without (lanes 1 4) a plasmid for overexpression of pb 2 ð167þδ19 DFR from the GAL1 promoter were cultured in D medium, then shifted to Gal medium with BPA and grown for indicated time. Proteins were analyzed by D-PAGE and immunoblotting with the indicated antibodies. Prec, pb 2 ð167þδ19-dfr; p, precursor form; m, mature form; f, degraded fragment. (B) Mitochondria containing Tom22 with BPA at residue 5 were purified and subjected to in vitro binding of indicated amounts of pald peptides in the absence of ΔΨ as in Fig. 2C. After UV-irradiation, proteins were analyzed as in Fig. 1A. The 22, Tom22; 22 4, Tom22 crosslinked with Tom4. (C) The yeast strain (TY3/pT3 136) with overexpressed Tom22 containing BPA at residue 136 without the C-terminal is tag was UV-irradiated and subjected to affinity purification of Tim5 with the FLAG tag. Proteins were analyzed as in Fig. 1A. These results rule out the possibility that the cross-linking between Tom22 and Tim5 is due to the artificially attached C-terminal is tag in Tom22. 2, Tom2; 2 4 Tom2 cross-linked with Tom4; 5, Tim5; 22 5 Tom22 cross-linked with Tim5. Table 1. Yeast strains train Genotype ource 33-1A MATa ade2-1 his3-11,15 ura3-1 leu2-3,112 trp1-1 can1- R. Rothstein (Columbia University, NY) GAL7-TOM22 MATa ade2-1 his3-11,15 ura3-1 leu2-3,112 trp1-1 can1- GAL7-Tom22-CgI3 This study TOM22FLAG MATa ade2-1 his3-11,15 ura3-1 leu2-3,112 trp1-1 can1- TOM22-FLAG CgI3 This study TY1 MATa ade2-1 his3-11,15 ura3-1 leu2-3,112 trp1-1 can1- [p6xtrna] This study TY2 MATa ade2-1 his3-11,15 ura3-1 leu2-3,112 trp1-1 can1- GAL7-Tom22-CgI3 [p6xtrna] This study TY3 MATa ade2-1 his3-11,15 ura3-1 leu2-3,112 trp1-1 can1- TIM5-FLAG CgI3 [p6xtrna] This study TY4 MATa ade2-1 his3-11,15 ura3-1 leu2-3,112 trp1-1 can1- TOM22-FLAG CgI3 [p6xtrna] This study Table 2. Expression plasmids for yeast Plasmid name Expressed protein (trna) Promoter Vector pt1 Tom22 is GAL1 pr315 pt1-x Tom22 is with BPA at position X GAL1 pr315 pt2 Tom22 is TOM22 YCpUG578T Tom22ownPT pt2-x Tom22 is with BPA at position X TOM22 YCpUG578T Tom22ownPT pt3 Tom22 GAL1 pr315 pt3-x Tom22 with BPA at position X GAL1 pr315 pt4 Tom2 is GAL1 YCpUG578T pt4-x Tom2 is with BPA at position X GAL1 YCpUG578T pt5 pb 2 ð167þδ19 DFR GAL1 pyo326 p6xtrna BpaR and amber supressor trna N/A N/A 4of6

5 Table 3. Primers for amber mutations in the TOM22 gene X Tom22 (x) amber Fw Tom22 (x) amber Rv 5 TGGTCGAATTATAGGAAATTAAAGA TCTTTAATTTCCTATAATTCGACCA AAATTAAAGACTAGGTCGTTCAATT AATTGAACGACCTAGTCTTTAATTT 15 TCGTTCAATTATAGGAACCACAATT AATTGTGGTTCCTATAATTGAACGA 2 AACCACAATTTTAGAGAAATCAGGC GCCTGATTTCTCTAAAATTGTGGTT GAAATCAGGCCTAGGTGGAAGAAAA TTTTCTTCCACCTAGGCCTGATTTC 3 TGGAAGAAAAGTAGTCTGCAACAAA TTTGTTGCAGACTACTTTTCTTCCA 35 CTGCAACAAACTAGGACGTTGTCGA TCGACAACGTCCTAGTTTGTTGCAG 4 ACGTTGTCGATTAGGAAGATGACTC GAGTCATCTTCCTAATCGACAACGT 41 TTGTCGATGATTAGGATGACTCTGA TCAGAGTCATCCTAATCATCGACAA 42 TCGATGATGAATAGGACTCTGATAG CTATCAGAGTCCTATTCATCATCGA 44 ATGAAGATGACTAGGATAGTGATTT AAATCACTATCCTAGTCATCTTCAT 45 AAGATGACTCTTAGAGTGATTTTGA TCAAAATCACTCTAAGAGTCATCTT 46 ATGACTCTGATTAGGATTTTGAAGA TCTTCAAAATCCTAATCAGAGTCAT 47 ACTCTGATAGTTAGTTTGAAGATGA TCATCTTCAAACTAACTATCAGAGT 48 CTGATAGTGATTAGGAAGATGAATT AATTCATCTTCCTAATCACTATCAG 49 ATAGTGATTTTTAGGATGAATTTGA TCAAATTCATCCTAAAAATCACTAT 5 GTGATTTTGAATAGGAATTTGATGA TCATCAAATTCCTATTCAAAATCAC 51 ATTTTGAAGATTAGTTTGATGAAAA TTTTCATCAAACTAATCTTCAAAAT 52 TTGAAGATGAATAGGATGAAAATGA TCATTTTCATCCTATTCATCTTCAA 55 AATTTGATGAATAGGAAACATTGTT AACAATGTTTCCTATTCATCAAATT 56 TTGATGAAAATTAGACATTGTTGGA TCCAACAATGTCTAATTTTCATCAA 6 AAACATTGTTGTAGAGAATCGTTGC GCAACGATTCTCTACAACAATGTTT 61 CATTGTTGGACTAGATCGTTGCTTT AAAGCAACGATCTAGTCCAACAATG 62 TGTTGGACAGATAGGTTGCTTTAAA TTTAAAGCAACCTATCTGTCCAACA 63 TGGACAGAATCTAGGCTTTAAAAGA TCTTTTAAAGCCTAGATTCTGTCCA 64 ACAGAATCGTTTAGTTAAAAGACAT ATGTCTTTTAACTAAACGATTCTGT 65 GAATCGTTGCTTAGAAAGACATTGT ACAATGTCTTTCTAAGCAACGATTC 7 AAGACATTGTCTAGCCAGGTAAGAG CTCTTACCTGGCTAGACAATGTCTT 75 CAGGTAAGAGATAGACAATTTCTAA TTAGAAATTGTCTATCTCTTACCTG 8 CAATTTCTAATTAGTTTGGTTTTAC GTAAAACCAAACTAATTAGAAATTG 85 TTGGTTTTACTTAGTCTTTTGTGAG CTCACAAAAGACTAAGTAAAACCAA 9 CTTTTGTGAGATAGGCTTTCACAAA TTTGTGAAAGCCTATCTCACAAAAG 95 CTTTCACAAAATAGGGAAACCTTGC GCAAGGTTTCCCTATTTTGTGAAAG 98 AATCCGGAAACTAGGCTTGGACTTT AAAGTCCAAGCCTAGTTTCCGGATT 99 CCGGAAACCTTTAGTGGACTTTGAC GTCAAAGTCCACTAAAGGTTTCCGG GAAACCTTGCTTAGACTTTGACCAC GTGGTCAAAGTCTAAGCAAGGTTTC 1 ACCTTGCTTGGTAGTTGACCACCAC GTGGTGGTCAACTACCAAGCAAGGT 2 TTGCTTGGACTTAGACCACCACTGC GCAGTGGTGGTCTAAGTCCAAGCAA 3 CTTGGACTTTGTAGACCACTGCTTT AAAGCAGTGGTCTACAAAGTCCAAG 4 GGACTTTGACCTAGACTGCTTTGTT AACAAAGCAGTCTAGGTCAAAGTCC 5 CTTTGACCACCTAGGCTTTGTTACT AGTAACAAAGCCTAGGTGGTCAAAG 6 TGACCACCACTTAGTTGTTACTCGG CCGAGTAACAACTAAGTGGTGGTCA 7 CCACCACTGCTTAGTTACTCGGTGT ACACCGAGTAACTAAGCAGTGGTGG 8 CCACTGCTTTGTAGCTCGGTGTGCC GGCACACCGAGCTACAAAGCAGTGG 9 CTGCTTTGTTATAGGGTGTGCCACT AGTGGCACACCCTATAACAAAGCAG 1 CTTTGTTACTCTAGGTGCCACTATC GATAGTGGCACCTAGAGTAACAAAG 111 TGTTACTCGGTTAGCCACTATCCTT AAGGATAGTGGCTAACCGAGTAACA 112 TACTCGGTGTGTAGCTATCCTTATC GATAAGGATAGCTACACACCGAGTA 113 TCGGTGTGCCATAGTCCTTATCTAT ATAGATAAGGACTATGGCACACCGA 114 GTGTGCCACTATAGTTATCTATACT AGTATAGATAACTATAGTGGCACAC 115 TGCCACTATCCTAGTCTATACTTGC GCAAGTATAGACTAGGATAGTGGCA 116 CACTATCCTTATAGATACTTGCCGA TCGGCAAGTATCTATAAGGATAGTG 117 TATCCTTATCTTAGCTTGCCGAACA TGTTCGGCAAGCTAAGATAAGGATA 118 CCTTATCTATATAGGCCGAACAACA TGTTGTTCGGCCTATATAGATAAGG 119 TATCTATACTTTAGGAACAACAGCT AGCTGTTGTTCCTAAAGTATAGATA 12 CTATACTTGCCTAGCAACAGCTAAT ATTAGCTGTTGCTAGGCAAGTATAG 121 TACTTGCCGAATAGCAGCTAATCGA TCGATTAGCTGCTATTCGGCAAGTA 122 TTGCCGAACAATAGCTAATCGAAAT ATTTCGATTAGCTATTGTTCGGCAA 123 CCGAACAACAGTAGATCGAAATGGA TCCATTTCGATCTACTGTTGTTCGG 124 AACAACAGCTATAGGAAATGGAAAA TTTTCCATTTCCTATAGCTGTTGTT 1 AACAGCTAATCTAGATGGAAAAGAC GTCTTTTCCATCTAGATTAGCTGTT 126 AGCTAATCGAATAGGAAAAGACATT AATGTCTTTTCCTATTCGATTAGCT 127 TAATCGAAATGTAGAAGACATTTGA TCAAATGTCTTCTACATTTCGATTA 128 TCGAAATGGAATAGACATTTGATTT AAATCAAATGTCTATTCCATTTCGA 13 TGGAAAAGACATAGGATTTACAAAG CTTTGTAAATCCTATGTCTTTTCCA 131 AAAAGACATTTTAGTTACAAAGTGA TCACTTTGTAACTAAAATGTCTTTT 132 AGACATTTGATTAGCAAAGTGATGC GCATCACTTTGCTAATCAAATGTCT 133 CATTTGATTTATAGAGTGATGCTAA TTAGCATCACTCTATAAATCAAATG 134 TTGATTTACAATAGGATGCTAATAA TTATTAGCATCCTATTGTAAATCAA 5of6

6 X Tom22 (x) amber Fw Tom22 (x) amber Rv 135 ATTTACAAAGTTAGGCTAATAACAT ATGTTATTAGCCTAACTTTGTAAAT 136 TACAAAGTGATTAGAATAACATATT AATATGTTATTCTAATCACTTTGTA 137 AAAGTGATGCTTAGAACATATTGGC GCCAATATGTTCTAAGCATCACTTT 138 GTGATGCTAATTAGATATTGGCCCA TGGGCCAATATCTAATTAGCATCAC 139 ATGCTAATAACTAGTTGGCCCAAGG CCTTGGGCCAACTAGTTATTAGCAT 14 CTAATAACATATAGGCCCAAGGTGA TCACCTTGGGCCTATATGTTATTAG 141 ATAACATATTGTAGCAAGGTGAAAA TTTTCACCTTGCTACAATATGTTAT 142 ACATATTGGCCTAGGGTGAAAAAGA TCTTTTTCACCCTAGGCCAATATGT 143 TATTGGCCCAATAGGAAAAAGATGC GCATCTTTTTCCTATTGGGCCAATA 145 CCCAAGGTGAATAGGATGCTGCAGC GCTGCAGCATCCTATTCACCTTGGG 146 AAGGTGAAAAATAGGCTGCAGCAAC GTTGCTGCAGCCTATTTTTCACCTT Table 4. Primers for amber mutations in the TOM2 gene X Tom2(x)amber Fw Tom2(x)amber Rv 7 AGTCGAACCCTTAGTTACGTGGCCT AGGCCACGTAACTAAGGGTTCGACT CTATCTTACGTTAGCTCGCTATTAC GTAATAGCGAGCTAACGTAAGATAG 13 GTGGCCTCGCTTAGACAACAGCCAT ATGGCTGTTGTCTAAGCGAGGCCAC 19 CAGCCATAGCATAGCTATCAGCCAC GTGGCTGATAGCTATGCTATGGCTG 22 CAGCTCTATCATAGACCGGTTATGC GCATAACCGGTCTATGATAGAGCTG CAGCCACCGGTTAGGCTATCTACTT AAGTAGATAGCCTAACCGGTGGCTG 33 TTGACTATCAATAGAGAAATAGCCC GGGCTATTTCTCTATTGATAGTCAA 41 CGCAATTCAGGTAGGTGTTGAGACA TGTCTCAACACCTACCTGAATTGCG 49 AAAGGGCCAAATAGCAGGCCAAGAT ATCTTGGCCTGCTATTTGGCCCTTT 57 TGGAAGAACAATAGAAAACTCATGC GCATGAGTTTTCTATTGTTCTTCCA 65 CTAAGGAAGTGTAGCTGCAAAAGGT ACCTTTTGCAGCTACACTTCCTTAG 73 TTACCGAATTCTAGTCCATGGAATT AATTCCATGGACTAGAATTCGGTAA 79 TGGAATTAGCCTAGGACCCCATCCC GGGATGGGGTCCTAGGCTAATTCCA 81 TAGCCAAGGACTAGATCCCTAGTGA TCACTAGGGATCTAGTCCTTGGCTA 87 CTAGTGATCCCTAGGAAAGAGAAGC GCTTCTCTTTCCTAGGGATCACTAG 91 CCGAAAGAGAATAGACATTTACCAC GTGGTAAATGTCTATTCTCTTTCGG 93 GAGAAGCTACATAGACCACCAACGT ACGTTGGTGGTCTATGTAGCTTCTC 95 CTACATTTACCTAGAACGTAGAAAA TTTTCTACGTTCTAGGTAAATGTAG 97 TTACCACCAACTAGGAAAATGGTGA TCACCATTTTCCTAGTTGGTGGTAA 98 CCACCAACGTATAGAATGGTGAAAG CTTTCACCATTCTATACGTTGGTGG 99 CCAACGTAGAATAGGGTGAAAGATT AATCTTTCACCCTATTCTACGTTGG 2 AAAATGGTGAATAGTTATCCATGCA TGCATGGATAACTATTCACCATTTT 5 AAAGATTATCCTAGCAACAAGGTAA TTACCTTGTTGCTAGGATAATCTTT 8 CCATGCAACAATAGAAGGAACTGGA TCCAGTTCCTTCTATTGTTGCATGG 111 AAGGTAAGGAATAGGAAGCAGCCTC GAGGCTGCTTCCTATTCCTTACCTT 112 GTAAGGAACTGTAGGCAGCCTCTAA TTAGAGGCTGCCTACAGTTCCTTAC 115 TGGAAGCAGCCTAGAAGTTTTATAA TTATAAAACTTCTAGGCTGCTTCCA 116 AAGCAGCCTCTTAGTTTTATAAAGC GCTTTATAAAACTAAGAGGCTGCTT 118 CCTCTAAGTTTTAGAAAGCATTGAC GTCAATGCTTTCTAAAACTTAGAGG 121 TTTATAAAGCATAGACTGTATACCC GGGTATACAGTCTATGCTTTATAAA 123 AAGCATTGACTTAGTACCCTCAGCC GGCTGAGGGTACTAAGTCAATGCTT 126 CTGTATACCCTTAGCCAGCCGATTT AAATCGGCTGGCTAAGGGTATACAG 128 ACCCTCAGCCATAGGATTTATTGGG CCCAATAAATCCTATGGCTGAGGGT 131 CAGCCGATTTATAGGGAATTTACCA TGGTAAATTCCCTATAAATCGGCTG 134 TATTGGGAATTTAGCAAAGATCCAT ATGGATCTTTGCTAAATTCCCAATA 138 ACCAAAGATCCTAGCCTGAAGCCAT ATGGCTTCAGGCTAGGATCTTTGGT 143 CTGAAGCCATTTAGGAATATATTAT ATAATATATTCCTAAATGGCTTCAG 147 ACGAATATATTTAGTTAATGATTGC GCAATCATTAACTAAATATATTCGT 152 TAATGATTGCCTAGTTGCCTCCTGC GCAGGAGGCAACTAGGCAATCATTA 156 TCTTGCCTCCTTAGAATGTGGCTTC GAAGCCACATTCTAAGGAGGCAAGA 161 ATGTGGCTTCTTAGGTTAAAGGAGT ACTCCTTTAACCTAAGAAGCCACAT 166 TTAAAGGAGTTTAGGGAAGCAAGGC GCCTTGCTTCCCTAAACTCCTTTAA 176 CTGATGCGGTTTAGGAAGCTAACGA TCGTTAGCTTCCTAAACCGCATCAG 6of6