Supplementary information. Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus

Transcription

1 Supplementary information Lysine and arginine iosyntheses mediated y a common carrier protein in Sulfolous Takuya Ouchi 1, Takeo Tomita 1, Akira Horie 1, Ayako Yoshida 1, Kento Takahashi 1, Hiromi Nishida 2, Kerstin Lassak 3, Hikari Taka 4, Reiko Mineki 4, Tsutomu Fujimura 4, Saori Kosono 1, Chiharu Nishiyama 5, Ryoji Masui 6, 7, Seiki Kuramitsu 6, 7, Sonja-Verena Alers 3, Tomohisa Kuzuyama 1, and Makoto 1, 7* Nishiyama 1 Biotechnology Research Center, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo , Japan. 2 Agricultural Bioinformatics Research Unit, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo , Japan. 3 Max Planck Institute for terrestrial Microiology, Karl von Frisch Strasse 10 D Marurg, Germany. 4 Division of Biochemical Analysis, Central Laoratory of Medical Sciences, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo , Japan. 5 Atopy (Allergy) Research Center, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo , Japan. 6 Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka , Japan. 7 RIKEN SPring-8 Center, Kouto, Sayo-cho, Sayo-gun, Hyogo , Japan. Present address: Nihon Unisys, Ltd., Tokyo, Japan. Central Laoratories for Frontier Technology, Kirin Holdings Co., Yokohama, Japan. *To whom correspondence should e addressed: umanis@mail.ecc.u-tokyo.ac.jp

2 SUPPLEMENTARY RESULTS a Optical Density at 600 nm 0.1 Optical Density at 600 nm Time (h) Time (h) Supplementary Figure 1. Effects of various concentrations of lysine on growth. (a) Parental MW001 strain. () Δ0754 strain. Brock s asal salt medium supplemented with 0.2% D-xylose and μg/ml uracil was used as the asal culture medium. Additives are: white diamonds, 0.1 mm lysine plus 0.1 mm arginine; lue diamonds, none; red squares, 0.01 mm lysine; purple crosses, 0.05 mm lysine; light green triangles, 0.1 mm lysine; orange circles, 0.5 mm lysine. Each strain was cultured in triplicate. Bars indicate standard deviations.

3 M.W. (kda) Supplementary Figure 2. Tricine SDS-PAGE (full size). Purified LysW-AAA and LysW- were incuated with or without Saci_0751 in the presence of 1 mm MgSO4, 160 mm KOH, 160 mm NH2OH-HCl, 10 mm ATP, 0 mm HEPES-NaOH, ph8.0, for 1 h at 60 C. Lanes 1 and 6, molecular size marker; lanes 2 and 3, LysW-AAA was used as the sustrate; Lanes 4 and 5, LysW- was used as the sustrate. Lanes 2 and 4, with Saci_0751; Lanes 3 and 5, without Saci_0751.

4 a y 11 y 10 y 9 y 8 y 7 y 6 y 5 y 4 y 3 y 2 y 1 L A E Q V G W E G D E X NH OH y y y y 6 y 10 %Intensity m/z y 11 y 10 y 9 y 8 y 7 y 6 y 5 y 4 y 3 y 2 y 1 L A E Q V G W E G D E X OH y y y y %Intensity m/z c y 11 y 10 y 9 y 8 y 7 y 6 y 5 y 4 y 3 y 2 y 1 L A E Q V G W E G D E E NH OH m/z: y y y y y y y %Intensity m/z d y 11 y 10 y 9 y 8 y 7 y 6 y 5 y 4 y 3 y 2 y 1 L A E Q V G W E G D E E OH y y y y y %Intensity m/z m/z: Supplementary Figure 3. LC-MS/MS of C-terminal tryptic fragments of LysW derivatives. (a) C-terminal tryptic fragments of the and shown in an asterisk in lane 2 in Figure 3. () C-terminal tryptic fragments of the and shown in an asterisk in lane 3 in Figure 3. (c) C-terminal tryptic fragments of the and shown in an asterisk in lane 4 in Figure 3. (d) C-terminal tryptic fragments of the and shown in an asterisk in lane 5 in Figure 3. X denotes AAA residue. 8 m/z: m/z:

5 a Asorance at 280 nm Radius [cm] 1 1 c(s) (S -1 ) c Sedimentation coefficient [S] 0 d Asorance at 280 nm Radius [cm] - c(s) (S -1 ) Sedimentation coefficient [S] Supplementary Figure 4. Analytical ultra centrifugation of StArgX and StLysW. (a) Data plot for StArgX. () Distriution plot of sedimentation coefficent of StArgX. (c) Data plot for StLysW. (d) Distriution plot of sedimentation coefficent of StLysW.

6 a ,532 monomer Intensity x 10 6 Intensity x dimer 63,064 trimer 94,636 tetramer 126, Mass (kda) m/z Supplementary Figure 5. MS analysis of StArgX. (a) Spectrum deconvoluted from mass spectrum for StArgX in ().

7 a StArgX (D-chain) StArgX (C-chain) 90 StArgX (A-chain) AMP -PNP AMP -PNP StArgX (B-chain) TtLysX (D-chain) TtlysX (C-chain) 90 AMP-PNP TtLysX (A-chain) AMP-PNP TtlysX (B-chain) Supplementary Figure 6. Overall structures of StArgX/ and TtLysX/AMP-PNP complex. (a) StArgX/ complex. () TtLysX/AMP-PNP complex. A-, B-, C-, and D-chains of these tetramer are shown as green, cyan, magenta, and yellow figures, respectively. The ound ligands are shown as stick models.

8 a N-terminal gloular domain C9 C29 Zn 2+ C6 H27 N-ter N158 A S159 B E52 E E48 E D53 A R59 A W54 E S56 A R135 A N158 A S159 B E52 E E48 E D53 A R59 A W54 E S56 A R135 A M57 A V55 A G55 E V254 A I9 A W133 A M57 A V55 A G55 E V254 A I9 A W133 A S132 A S132 A E256 A E256 A C-terminal extension E52 D53 W54 G55 C-ter E56 D253 A E56 E S235 A R178A D253 A E56 E S235 A R178A Supplementary Figure 7. Structure of StLysW and the recognition y StArgX. (a) Domain architecture of StLysW. N-terminal gloular domain and C-terminal extension are shown as pink and purple models, respectively. The Zn 2+ ion ound to N-terminal gloular domain and the coordinating residues are shown as a sphere model and stick models, respectively. The residues of strictly conserved C-terminal EDWGE motif are shown as stick models. () Stereo view of interaction etween StArgX and C-terminal extension of StLysW. Another sustrate glutmate molecule, the α-amino group of which is to e ligated with the γ -caroxyl group of 56 residue of StLysW, is also shown y orange stick model. The residues involved in the interactions are shown as stick models and hydrogen onds and electrostatic interactions are shown y dotted lines.

9 a K87A N2 A Mg R192A R178 A 2+ E250A Zn2+ SO42- N252A K87A N2 A R192A Mg R178 A 2+ E250A Zn2+ SO42- E56 E N252A K125 R225 N235 Mg 2+ E281 Mg2+ SO42- R210 K125 R225 N235 E281 Mg2+ SO42- R210 N283 GSH Mg 2+ N283 GSH E56 E d c V3A R192A Y190A A4A G259A GSH E56E F260A GSH E56E A4A G259A V3A R192A Y190A E256A SO42E56E F260A E256A SO42E56E Supplementary Figure 8. Active site of StArgX. (a) Stereoview of the active site structure of StArgX inding StLysW,,, SO42-, and metal ions. Bound Mg2+ and Zn2+ are shown y green and gray sphere models. The Fo-Fc maps of ligands at the active site of StArgX contoured at 3.0 σ are shown as lack mesh. () Stereoview of the active site structure of the pseudo-michaelis complex of glutathione synthetase (GSHase) inding GSH,, SO42-, and metal ions (PDB ID: 1gsa). (c) Superposition of (a) and (). Colors used are the same as in (a) and (). (d) Recognition of ound glutamate y StArgX.

10 Mg 2+ Mg 2+ R178 R178 Zn 2+ Zn 2+ E56 E E56 E Supplementary Figure 9. Structural model of the phosphoryl intermediate in the StArgX reaction. Sustrate glutamate,, Arg178 and the C-terminal residue (phosphorylated) of StLysW are shown. Based on the crystal structure of the StArgX/StLysW complex, a model of reaction intermediate of StArgX was generated. In the determined structure, the γ-caroxyl group of 56 of StLysW was not present at the position suitale for acyltransfer due to charge repulsion against the ound sulfate ion that mimics the γ -phosphate group from ATP. Therefore, the sulfate ion was replaced with the phosphate moiety of acylphosphate intermediate of StLysW and the conformation of the 56 side chain of StLysW was manually adjusted to form the acylphosphate linkage with the phosphate group. In the intermediate state, Arg178 completely conserved among LysX-ArgX proteins may form ionic interactions with the side-chain caroxyl group of 56 and the phosphoryl group of the intermediate.

11 β1 α1 β2 β3 η1 β4 α2 β5 α3 StArgX StArgX MRVVLIVDIVRQEEKLIAKALEENKVQYDIINVAQEPLPFN...KALGRYDVAIIRPVSMYRALYSSAVLEAAGVHTINSSDVINVCGD SaArgX MRVALVVDIVRQEEKLIAKALEKFQLQYDVINVAQEPLPFN...KALGRYDVAIIRPISMYRALYASAVLESAGVHTINSSDTISLCGD StLysX MILGVIYDLLRWEEKNLIQEARKLGHTVIPIYTKDFYYFYNNDSNETLGDLDVVIQRNTSHARAVITSTIFENLSYKTINDSSTLIKCEN SaLysX...MRWEEKDIITEAKKSGFKAIPIFTKDFYSAIGVGENYSELEADVIIQRNTSHARALTTSLIFEGWNYNVVNDATSLFKCGN TtLysX.MLAILYDRIRPDERMLFERAEALGLPYKKVYVPALPMVLG.ERPEALEGVTVALERCVSQSRGLAAARYLTALGIPVVNRPEVIEACGD TtLysX β1 α1 β2 η1 TT β3 η2 β4 α2 β5 α3 α4 β6 α5 β7 β8 α6 η2 β9 StArgX StArgX KILTYSKLYREGIPIPDSIIALSAEAALKAYEQRGFPLIDKPPIGSWGRLVSLIRDVFEGKTIIEHRELMGNSALKAHIVQEYIQYKGRD SaArgX KILTYSKLYREGIPIPDSIIAMSSDAALKAYEQKGFPLIDKPPIGSWGRLVSLIRDIFEGKTIIEHRELMGNSALKVHIVQEYINYKSRD StLysX KLYTLSLLSKHGIRVPKTIVAFSKEKALELANKLSYPVVIKPVEGSWGRMVARAIDEDTLRNFLEYQEYTTLQFRYIYLIQEFVKKPDRD SaLysX KLYTLSLLAKHNIKTPRTIVTFSKDKAVDLAKKIGFPAVIKPIEGSWGRMVAKAVDEDILYSFLEYQEYTTSQFRQIYLVQEFVKKPNRD TtLysX KWATSVALAKAGLPQPKTALATDREEALRLMEAFGYPVVLKPVIGSWGRLLAKVTDRAAAEALLEHKEVLGGFQHQLFYIQEYVEKPGRD TtLysX TT α4 β6 α5 β7 β8 α6 η3 β9 StArgX β10 β11 β12 α7 β13 β14 α8 TT TT TTT StArgX IRCIAIGEELLGCYARNIPPNEWRANVALGGTPSNIEVDEKLKETVVKAVSIVHGEFVSIDILEHPNKGYVVNELNDVPEFKGFMVATNI SaArgX IRCIVIGSELLGCYARNIPSNEWRANIALGGYPSQIEVDHKLKETVLKATSIIGGEFVSIDVMEHQSKNYVINEFNDVPEFKGFMLATNI StLysX IRIFTIGDEAPVGIYRVN.SRNWKTNTALGAKAEPLKIDEELQDLALKVKDIIGGFFLGIDVFEDPERGYIINEVNGVPEYKNTVRVNNF SaLysX IRIFVMGDEAPVGIYRVN.ERNWKTNTALGARALPLKIDDELRDLALKVRDIMGGFFLGIDIFEDPERGYLVNEVNGVPEYKNTVRVNNF TtLysX IRVFVVGERAIAAIYRR..SAHWITNTARGGQAENCPLTEEIARLSVGAAEAVGGGVVAVDLFES.ERGLLVNEVNHTMEFKNSVHTTGV TtLysX β10 TT β11 η4 β12 α7 β13 TT β14 α8 α9 StArgX StArgX NVAQKLVEYIKENYSK SaArgX DVAEELVSYVKNNYLR StLysX NVSEYLIRKIEEWIKK SaLysX NVSSYLLNKLREWIKK TtLysX DIPGEILRYAWEVARG TtLysX α9 Signature motif Supplementary Figure 10. Amino acid sequence alignment of LysX/ArgX family proteins. Structure-ased alignment was performed y ClustalW and ESPript. Areviations are: StArgX, ArgX from S. tokodaii; SaArgX, ArgX from S. acidocaldarius; StLysX, LysX from S. tokodaii; SaLysX, LysX from S. acidocaldarius; TtLysX, LysX from T. thermophilus. Signature motif that determines the sustrate specificity of this protein family is oxed.

12 Signature motif Archaea Bacteria TTS NP Sul Py Pyc NS Chloroflexus sp. Y400fl Chy NS Chloroflexus aurantiacus Caur 0334 NS Chloroflexus aggregans Cagg 3597 NS Herpetosiphon aurantiacus Haur 3306 NS Roseiflexus castenholzii DSM13941 Rcas 3469 NS Roseiflexus sp. RS1 RoseRS 1059 NS Thermoaculum terrenum Tter 0316 NS Thermomicroium roseum trd A0165 NS Sphaeroacter thermophilus Sthe 2943 NS Deinococcus deserti Deide NS Deinococcus radiodurans DR 2194 NS Deinococcus geothermalis Dgeo 1151 NS Truepera radiovictrix Trad 1392 NS Meiothermus ruer Mru 2724 NS Meiothermus silvanus Mesil 0438 NS Thermanaerovirio acidaminovorans Taci 0444 NS Thermus thermophilus HB8 TTHA1907 NS Thermus thermophilus HB27 TTC1543 GL Thermoaculum terrenum Tter 0318 GL Thermomicroium roseum trd 1519 GL Sphaeroacter thermophilus Sthe 0215 NA Candidatus Korarchaeum cryptofilum Kcr 0812 LA Pyrococcus furiosus PF09 NA Pyrococcus ayssi PAB0290 NA Thermococcus kodakarensis TK0278 NA Pyrococcus horikoshii PH1721 NA Pyrococcus furiosus PF1682 NT Nitrosopumilus maritimus Nmar 1295 NT Picrophilus torridus PTO1469 GL Nitrosopumilus maritimus Nmar 1288 AL Natronomonas pharaonis NP5258A AL Halalkalicoccus jeotgali HacjB3 000 AL Halorurum lacusprofundi Hlac 2616 AL Haloarcula marismortui rrnac2679 AL Halomicroium mukohataei Hmuk 0772 AL Haloferax volcanii HVO 0046 AL Natriala magadii Nmag 1761 AL Haloterrigena turkmenica Htur 0328 AL Halorhadus utahensis Huta 1502 AL Haloquadratum walsyi HQ3714A GA Picrophilus torridus PTO1467 NT Ignicoccus hospitalis Igni 0057 NT Ignisphaera aggregans Igag 1752 NT Sulfolous tokodaii ST0192 NT Sulfolous acidocaldarius Saci 0754 NT Metallosphaera sedula Msed 0168 NT Sulfolous solfataricus SSO0159 NT Sulfolous islandicus M M NT Sulfolous islandicus Y.N YN NT Sulfolous islandicus M M NT Sulfolous islandicus L.D.8.5 LD NT Sulfolous islandicus Y.G YG NT Sulfolous islandicus M.16.4 M NT Sulfolous islandicus L.S.2.15 LS NT Aeropyrum pernix APE 1463 NV Thermoproteus neutrophilus Tneu 0259 NV Pyroaculum calidifontis Pcal 1379 NV Pyroaculum islandicum Pisl 1291 NV Pyroaculum arsenaticum Pars 0291 NV Pyroaculum aerophilum PAE1240 NV Caldivirga maquilingensis Cmaq 1298 NV Vulcanisaeta distriuta Vdis 00 GL Vulcanisaeta distriuta Vdis 0196 AI Caldivirga maquilingensis Cmaq 1297 AL Pyroaculum calidifontis Pcal 0115 AL Pyroaculum aerophilum PAE0948 AL Pyroaculum arsenaticum Pars 0145 AL Pyroaculum islandicum Pisl 1185 AL Thermoproteus neutrophilus Tneu 0136 GF Ignicoccus hospitalis Igni 1403 GF Sulfolous tokodaii ST1505 GF Sulfolous acidocaldarius Saci 1621 GF Metallosphaera sedula Msed 11 GF Sulfolous solfataricus SSO0645 GF Sulfolous islandicus L.D.8.5 LD85 16 GF Sulfolous islandicus Y.N YN GF Sulfolous islandicus Y.G YG GF Sulfolous islandicus L.S.2.15 LS GF Sulfolous islandicus M.16.4 M GF Sulfolous islandicus M M GF Sulfolous islandicus M M GI Dickeya dadantii Ech586 Dd GV Serratia proteamaculans Spro 1643 GM Ferrimonas alearica Fal 1405 GF Planctomyces limnophilus Plim 2585 NL Oceanoacillus iheyensis OB59 GM Methanococcus vannielii Mevan 0912 AV Streptomyces griseus SGR 3478 HS Lactoacillus plantarum WCFS1 lp 0484 GF Catenulispora acidiphila Caci 6289 LysX ArgX LysX/ArgX ArgX LysX/ArgX LysX ArgX LysX LysX ArgX ArgX DAP pathway lacking LysW Supplementary Figure % ootstrap consensus tree of 90 argx/lysx-like gene products ased on maximum likelihood analysis (full version).

13 a c kda kda kda kda 1 d e kda kda 1 f Supplementary Figure 12. Purification of proteins involved in lysine and arginine iosynthesis. (a) Saci_0754 (lanes 2-10) and Saci_1621 (lanes 11-19). Lanes 2 and 11, precipitate of cell lysate y centrifugation; lanes 3 and 12, supernatant of cell lysate y centrifugation; lanes 4 and 13, heat-stale franction of lanes 3 and 12; lanes 5 and 14, passing fraction through Ni 2+ -column; lanes 6-9 and 15-18, washing fraction; lanes 10 and 19, eluate y 500 mm imidazole (purified proteins) () Saci_0751. Lane 2, precipitate of cell lysate y centrifugation; lane 3, supernatant of cell lysate y centrifugation; lane 4, heat-stale franction of lane 3; lane 5, passing fraction through Ni 2+ -column; lane 6, washing fraction; lanes 7 and 8, eluate y 0 mm and 500 mm imidazole (purified protein). (c) Saci_0753. (d) TtLysX. (e) St1505 (StArgX). (f) STs023 (StLysW). Pane (c)-(f), gel filtration profiles. Purified fractins are shown y divergent arrows. In every panel, lane 1 contains molecular size markers.

14 Supplementary Tale 1 Data collection and refinement statistics (molecular replacement) StArgX/ TtLysX/AMP-PNP StArgX/StLysW Data collection Space group P1 P P2 1 Cell dimensions a,, c (Å) 63.3, 67.8, , 130.3, , 114.0, 78.6,, ( ) 87.8, 75.3, , 90.0, , 102.4, 90.0 Resolution (Å) 1.87 ( ) * 1.95 ( ) * 1.80 ( ) * R sym or R merge 7.8 (30.1) 7.3 (47.9) 7.3 (58.1) I / I 14.7 (1.8) 24.2 (2.7) 12.3 (2.0) Completeness (%) 95.8 (87.3).7 (.1).7 (100.0) Redundancy 1.3 (1.3) 4.9 (4.4) 1.8 (1.8) Refinement Resolution (Å) No. reflections R work / R free 19.8 / / / 23.5 No. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å) Bond angles ( ) Each data set was collected from a single crystal. *Highest-resolution shell is shown in parentheses.

15 Supplementary Tale 2 Interactions etween StArgX and StLysW residue on StLysW atom atom residue on StArgX distance (Å) Gloular domain Asp18 O 1 N Lys O Gly22 O N Val O 2 N Lys O 1 O Asp8 3.2 Arg45 N 2 O 1 Gln O * 2.8 C-terminal extension 48 O 1 N 1 Arg O 2 N O 1 O Ser159 * 2.7 N 2.8 N Asn O N Arg Asp53 O O Ser N Met N Tyr O N Met Trp54 O N 1 Arg N Gly55 N O Val O 1 N 1 Arg N N 2.5 O3 2- SO O 2 N 1 Arg O Ser O3 2- SO O N Asp N Val N O Ser * indicates residues from another suunit.

16 Supplementary Tale 3 Oligonucleotides used in this study Primers Sequence Restriction site 0753-Fw 5 - GGGGTACCCATATGGTACTATTAAAATGT -3 KpnI / NdeI 0753-Rv 5 - GGAATTCTTACTCTCCCAGTCCTC -3 EcoRI 0754-Fw 5 - GGGGTACCCCATGGCATGATAATAGGGGTATCG -3 KpnI / NcoI 0754-Rv 5 - GGAATTCTTATTTCTTAATCCACTC -3 EcoRI 1621-Fw 5 - GGGGTACCCCATGGCATGAGAGTAGCACTTGTC -3 KpnI / NcoI 1621-Rv 5 - GGAATTCTTACCTGAGGTAATTATT -3 EcoRI 0751-Fw 5 - GGGGTACCCCATGGCATGATAGTAGTAAAAAC -3 KpnI / NcoI 0751-Rv 5 - GGAATTCTTATTCAATCACCGTGCC -3 EcoRI TtLysX-Fw 5 - GGGGTACCCATATGCTGGCCATCCTCTAC -3 KpnI / NdeI TtLysX-Rv 5 - GGAATTCTAATCCACGGGCCACCT -3 EcoRI 0753up-Fw 5 - GCTTCATATGGGCAGACGTTGATGATAGTGATATTA -3 NdeI 0753up-Rv 5 - CTCTCCCCAGTCACATTTTAATAGTACCATGATGGATC ds-Fw 5 - ACTATTAAAATGTGACTGGGGAGAGTGATAATAGG ds-Rv 5 - GTGGGCCCCCTAAATTGACTTGTTGTATATTCCTG -3 ApaI 0754up-Fw 5 - GCTTCATATGGGTTAAAACTAATCCTCAAATTCCTA -3 NdeI 0754up-Rv 5 -GATCAATTTCATTTCTCTTCCCATCTCAACAGGTC ds -Fw 5 - GATGGGAAGAGAAATGAAATTGATCCAACTGTATGGA ds-Rv 5 - GTGGGCCCGCAGCCGTCTCATTATCTATC -3 ApaI 1621up-Fw 5 - TCCCCGCGGTAATGTCCAATTCGTAGT -3 SacII 1621up-Rv 5 - CGGGATCCTCATATACCTGCCCCATA -3 BamHI 1621ds-Fw 5 - GGAATTCTTACCTCAGGTAAGCCAA -3 EcoRI 1621ds-Rv 5 - CCGCTCGAGCTACATTTACTTGTCTCA -3 XhoI pyref-fw 5 - CGGGATCCTTTGAGCAGTTCTAGTAC -3 BamHI pyref-ev 5 - GGAATTCGACCGGCTATTTTTTCAC -3 EcoRI GF_NT-Fw 5 - GACGTTCCAGAGTTCAAAAATACTATGTTGGCTACCAATATT -3 GF_NT-Rv 5 - AATATTGGTAGCCAACATAGTATTTTTGAACTCTGGAACGTC -3 Y_I-Fw 5 - AGTGAGCTTCTAGGTTGTATCGCTAGGAATATACCTTCT -3 Y_I-Rv 5 - AGAAGGTATATTCCTAGCGATACAACCTAGAAGCTCACT -3 NT_GF-Fw 5 - GGTGTACCGGAATATAAAGGTTTTGTAAGAGTTAATAATTTC -3 NT_GF-Rv 5 - GAAATTATTAACTCTTACAAAACCTTTATATTCCGGTACACC -3 I_Y-Fw 5 - GATGAGGCACCAGTTGGATATTATAGAGTTAATGAACGT -3 I_Y-Rv 5 -ACGTTCATTAACTCTATAATATCCAACTGGTGCCTCATC -3