VARIATION OF T2R38 GENE ENCODING PTC BITTER TASTE RECEPTOR IN LEAF-EATING MONKEY LAURENTIA HENRIETA PERMITA SARI PURBA

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1 VARIATION OF T2R38 GENE ENCODING PTC BITTER TASTE RECEPTOR IN LEAF-EATING MONKEY LAURENTIA HENRIETA PERMITA SARI PURBA DEPARTEMEN BIOLOGI FAKULTAS MATEMATIKA DAN ILMU PENGETAHUAN ALAM INSTITUT PERTANIAN BOGOR 2014

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3 PERNYATAAN MENGENAI SKRIPSI DAN SUMBER INFORMASI SERTA PELIMPAHAN HAK CIPTA Dengan ini saya menyatakan bahwa skripsi saya berjudul Variation of T2R38 Gene Encoding PTC Bitter Taste Receptor adalah benar karya saya dengan arahan dari komisi pembimbing dan belum diajukan dalam bentuk apa pun kepada perguruan tinggi mana pun. Sumber informasi yang berasal atau dikutip dari karya yang diterbitkan maupun tidak diterbitkan dari penulis lain telah disebutkan dalam teks dan dicantumkan dalam Daftar Pustaka di bagian akhir skripsi ini. Dengan ini saya melimpahkan hak cipta dari karya tulis saya kepada Institut Pertanian Bogor. Bogor, Maret 2014 Laurentia Henrieta Permita Sari Purba NIM G

4 ABSTRACT LAURENTIA HENRIETA PERMITA.Variation of T2R38 Gene Encoding PTC Bitter Taste Receptor in Leaf-Eating Monkey. Supervised by KANTHI ARUM WIDAYATI and BAMBANG SURYOBROTO. In mammals, mechanism of taste responding is mediated by G proteincoupled taste receptors in taste bud cells of tongue area. They are encoded by multigene families named taste receptor type 1 (T1R) and type 2 (T2R). T2R38 gene is a member of T2R family that encode the receptor for the bitter phenylthiocarbamide (PTC) compound. T2R38 gene have been identified in human, chimpanzees and Japanese macaques and exhibit within-species polymorphism. The aim of this research is to know the variation of T2R38 gene encoding receptor of PTC bitter taste compound in Trachypithecus cristatus, T. auratus and Presbytis melalophos. DNA were obtained from fecal sampels from captive leaf-eating monkeys in Ragunan Zoo, Jakarta. I found that T2R38 gene from leaf-eating monkey were 1,002 bp and single open reading frame. T2R38 gene of three species of leaf-eating monkey have two nucleotide sites that specific in their group and differ from T2R38 gene of Macaca mullata. In comparison to human, all of these indicate genes is inferred to encode functional protein to taste PTC. Keywords: bitter taste, T2R38, PTC, variation, leaf-eating monkey ABSTRAK LAURENTIA HENRIETA PERMITA. Variasi Gen T2R38 Penyandi Reseptor Rasa Pahit PTC pada Monyet Pemakan Daun. Dibimbing oleh KANTHI ARUM WIDAYATI dan BAMBANG SURYOBROTO. Pengenalan rasa mamalia diperantarai oleh reseptor yang terdapat pada sel kuncup perasa di daerah lidah. Reseptor rasa disandikan oleh kelompok gen reseptor rasa tipe 1 (T1R) dan tipe 2 (T2R). Gen T2R38 merupakan anggota dari kelompok gen T2R yang berfungsi menyandikan reseptor rasa pahit dari komponen phenylthiocarbamide (PTC). Gen T2R38 telah diteliti pada manusia, simpanse dan makaka Jepang. Hasil penelitian tersebut menunjukkan terjadinya polimorfisme intra spesies. Tujuan penelitian ini adalah untuk mengetahui variasi dari gen T2R38 penyandi reseptor rasa pahit PTC pada Trachypithecus cristatus, T. auratus dan Presbytis melalophos. DNA didapatkan dari sampel feses monyet pemakan daun di Kebun Binatang Ragunan, Jakarta. Hasil sekuens menunjukkan gen T2R38 pada monyet pemakan daun memiliki ukuran 1,002 bp dan merupakan single open reading frame. Gen T2R38 pada tiga spesies monyet pemakan daun memiliki dua situs nukleotida yang spesifik dan berbeda dari gen T2R38 pada Macaca mullata. Analisis sekuens gen T2R38 pada monyet pemakan daun menunjukkan bahwa semua gen T2R38 berfungsi menyandikan reseptor rasa pahit untuk PTC, berdasarkan perbandingannya dengan gen T2R38 pada manusia. Keywords: rasa pahit, T2R38, PTC, variasi, monyet pemakan daun

5 VARIATION OF T2R38 GENE ENCODING PTC BITTER TASTE RECEPTOR IN LEAF-EATING MONKEY LAURENTIA HENRIETA PERMITA SARI PURBA Skripsi sebagai salah satu syarat untuk memperoleh gelar Sarjana Sains pada Departemen Biologi DEPARTEMEN BIOLOGI FAKULTAS MATEMATIKA DAN ILMU PENGETAHUAN ALAM INSTITUT PERTANIAN BOGOR BOGOR 2014

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7 Judul Skripsi : Variation of T2R38 Gene Encoding PTC Bitter Taste Receptor in Leaf-Eating Monkey Nama : Laurentia Henrieta Permita Sari Purba NIM : G Disetujui oleh Dr Kanthi Arum Widayati Pembimbing I Dr Bambang Suryobroto Pembimbing II Diketahui oleh Dr Ir Iman Rusmana, M.Si Ketua Departemen Tanggal lulus:

8 PRAKATA Puji syukur kepada Bapa, Putra dan Roh Kudus untuk berkat dan penyertaan-nya. Skripsi ini disusun berdasarkan hasil penelitian berjudul Variation of T2R38 Gene Encoding PTC Bitter Taste Receptor in Leaf-Eating Monkey yang berlangsung dari November 2013 sampai Februari Terima kasih kepada Dr Kanthi Arum Widayati dan Dr Bambang Suryobroto selaku dosen pembimbing atas bimbingan dan saran yang diberikan. Terima kasih juga saya sampaikan kepada Hiroo Imai, Ph.D dan Mrs. Nami Suzuki untuk bimbingan dan pendampingannya saat melakukan penelitian di laboratorium dan saran selama proses penulisan. Terima kasih banyak kepada Ibu, Indhra, Patrick, Nico, bude, pakde dan Kreszens cinta dan dukungannya. Terima kasih juga untuk Mbak Puji, Kak Ari, Kak Ziah, Kak Andi untuk bantuan dan sarannya selama penulisan dan untuk Kak Sarah, Kak Okta, Kak Arvin, Elly, Tya, Ismi, Sabeth, Nisa, Indri, Amel, Christyne, Ega, Agnes, Iren, Ibeth, Ian, semua teman-teman Biologi 47 dan Zoo Corner untuk kebersamaan dan keceriannya selama ini. Semoga karya ilmiah ini bermanfaat. Bogor, Maret 2014 Laurentia Henrieta Permita Sari Purba

9 DAFTAR ISI DAFTAR TABEL viii DAFTAR LAMPIRAN viii INTRODUCTION 1 Background 1 Aim 1 MATERIALS AND METHOD 2 Time and Place 2 Sample collection 2 DNA extraction 2 Specific amplification of T2R38 gene 2 Verification of T2R38 gene 2 Sequencing of T2R38 gene 2 RESULTS 2 DISCUSSION 3 CONCLUSION 4 REFERENCES 4 APPENDIX 6 RIWAYAT HIDUP 9

10 DAFTAR TABEL 1 Variation of T2R38 gene of leaf-eating monkey 3 2 Amino acid of T2R38 receptor of human PTC taster compared to amino acid T2R38 receptor of leaf-eating monkey 4 DAFTAR LAMPIRAN 1 Nucleotide Sequence of T2R38 Gene of Leaf-Eating Monkey Alligned to T2R38 Gene of Macaca mullata 7

11 1 INTRODUCTION Background In mammals, mechanism of taste responding is mediated by G proteincoupled taste receptors in taste bud cells of tongue area (Chandrasekar et al 2000). They are encoded by multigene families named taste receptor type 1 (TAS1R or T1R) and type 2 (T2R). The T1R genes encode receptors that function to detect sweet and umami tastants and T2R genes encode receptors to detect bitter tastants. The number of T2R genes is larger than the number of T1R genes. This difference may be caused by survival function of the receptors (Nei et al. 2008). Bitter taste receptors help mammals to avoid ingesting poisonous foods that usually associated with bitter taste. T2R38 gene is a member of T2R family that encode receptor for the bitter phenylthiocarbamide (PTC) compound. T2R38 gene have been identified in human, chimpanzees and Japanese macaques and exhibit within-species polymorphism (Kim et al. 2003; Suzuki et al. 2011; Wooding et al. 2006). Polymorphism of T2R38 gene in human (ht2r38) leads to various receptors with difference in amino acid numbers 49, 262 and 296 of the encoded protein. The change of amino acid at position 49 from proline to alanine and at position 262 from alanine to valin diminish the receptor function (phenotypic PTC non-taster (Bufe et al. 2005)). Variation in position 296 from valin to isoleucine only had little effect on sensitivity to PTC (Bufe et al. 2005). The phenomena of non-taster individual was also reported in Japanese macaques from Kii region. The start codon of this haplotype change from the common ATG to ACG and make T2R38 protein defective (Suzuki et al. 2011). The non-taster individuals in chimpanzees have the same mutation (Wooding et al. 2006). Present research was focused to study the variation of gene encoding PTC receptor in leaf-eating monkeys. Those monkeys are members of subfamily Colobinae which is unique among other primates because they are predominantly leaf-eater. Colobinae are divided into seven genus (Brandon-Jones et al. 2004). These include Semnopithecus, Trachypithecus, Presbytis, Rhinopithecus Pygathrix, Nasalis and Simias. Recently, Widayati KA (3 Oktober 2013, personal communication) conducted behavioral study to test the responses of three species of leaf-eating monkeys (Trachypithecus auratus, T. cristatus, Presbytis melalophos) to PTC. It followed classical genetic and anthropological experiments where human that taste PTC tend to spit out the PTC-containing food. Her result showed that all of leaf-eating individuals eat the PTC-containing food without spitting out, thus they may be thought of as showing PTC non-tasting behavior. Aim The aim of this research is to know the variation of gene encoding receptor of PTC bitter taste compound in leaf-eating monkeys.

12 MATERIALS AND METHOD Time and Place This research was held on November 2013 until February 2014 in Molecular Biology Section, Department of Cellular and Molecular Biology Section, Primate Research Institute, Kyoto University. Data was analyzed in Biosystematics and Ecology of Animals, Department of Biology, IPB. Sample collection Fecal samples were obtained from 22 captive leaf-eating monkeys in Ragunan Zoo, Jakarta in tubes containing 1 ml lysis buffer. Those samples were stored at room temperature. DNA extraction Genomic DNA was exctracted using QIAamp DNA Stool Mini Kit (QIAGEN). Specific amplification of T2R38 gene T2R38 gene was amplified using polymerase chain reaction with primers constructed from Macaca mullata genome (Suzuki 2010). Verification of T2R38 gene The PCR products of supposed T2R38 gene was visualized in agarose gel using electrophoresis. Sequencing of T2R38 gene Sequencing was conducted using standard Big Dye Terminator chemistry (Applied Biosystem, California, USA). Sequence data were alligned to Macaca mullata T2R38 gene as the reference (Wooding 2001; Access number: JQ272208). In verification step, I found that out of 22 samples only four samples, those are 8, 25, S1 and L3 that showed clear single band of DNA with size around 1 kb. Those four samples were sequenced, analyzed and assemblied using ATGC sequence assembly software (Genetyx Corporation, Tokyo, Japan) and MEGA version 5 (Tamura et al. 2011). RESULTS In the four samples, I found that all of T2R38 gene from leaf-eating monkeys were 1,002 bp (Appendix 1). I found that in several sites nucleotides

13 3 could not be decided because when analyzed in chromatrogram, these nucleotides have two peaks. Using ORF finder (NCBI) it was verified that all of the sequences were single open reading frame (Kim et al. 2003). T2R38 gene of three species of leaf-eating monkey have two nucleotide sites (number 25 and 338) variation that specific in their group and differ from T2R38 gene of M. mullata. The intragroup difference of T2R38 gene of leaf-eating monkey is shown in Table 1. Furthermore, T2R38 gene of T. cristatus has nine nucleotide sites that differ from T2R38 of M. mullata. The nine nucleotide differences in those sites implicated amino acid differences because the the differences occured in first or second nucleotide on triplets codon (Appendix 1). T2R38 gene of T. auratus which is obtained from sample 25 has ten sites that differ from T2R38 gene of M. mullata. T2R38 gene of T. auratus which is obtained from sample L3 had nine sites that differ from T2R38 gene of M. mullata. T2R38 gene of P. melalophos has nine nucleotide sites that differ from T2R38 of M. mullata. Nucleotide differences in those sites also caused amino acid differences because the differences occured in first or second nucleotide on triplets codon (Appendix 1). Table 1 Variation of T2R38 gene of leaf-eating monkey Species Name Site number 25 Site number 338 Trachypithecus cristatus 8 M T Trachypithecus auratus 25 A C Presbytis melalophos S1 C T Trachypithecus auratus L3 N T DISCUSSION I checked the translated amino acid of T2R38 gene of leaf-eating monkey. The translated amino acid were obtained from converting DNA sequence to protein sequence using MEGA version 5 (Table 2). In human, changes in amino acid site 49 and 262 could cause PTC nontaster phenotype (Bufe et al. 2005). Human with PTC taster phenotype have proline on site 49 and alanine on site 262 (Table 2) while non-taster phenotype have alanine and valine in those site. In the translated amino acid of T23R8 gene of T. auratus, T. cristatus and P. melalophos, I found that site number 49 is proline and site number 262 is alanine. I also checked amino acid site numbers 99,100, 103, 255, 259, and 296 (Table 2). The amino acid changes in those sites would alter PTC binding by the receptor and disturbed receptor activation that caused non-taster phenotype (Biarnes et al. 2010). In translated amino acid of T2R38 gene of T. auratus, T. cristatus and P. melalophos, I found that there are no amino acid changes in those sites. In comparison to human, these indicated that all gene will encode functional protein to taste PTC. However, these result is contrary with the result of behavioral study conducted by Widayati KA (3 Oktober 2013, personal communication).

14 Table 2 Amino acid of T2R38 receptor of human PTC taster compared to amino acid T2R38 receptor of leaf-eating monkey Amino acid site number Homo sapiens Pro Try Met Asp Phe Ser Ala Val T. auratus * * * * * * * * T. cristatus * * * * * * * * P. melalophos * * * * * * * * Note: * = same as above CONCLUSION In the four samples, I found that all of T2R38 gene from leaf-eating monkey were 1,002 bp and single open reading frame. T2R38 gene of three species of leaf-eating monkey have two nucleotide sites (number 25 and 338) that vary in their group and nine to ten sites that differ from T2R38 gene of M. mullata. In comparison to human, all of these indicate genes is inferred to encode functional protein to taste PTC. REFERENCES Biarnes X, Marchiori A, Giorgetti A, Lanzara C, Gasparini P, Carloni P, Born S, Brockhoff A, Behrens M, Meyerhof W Insights into the binding of phenyltiocarbamide (PTC) agonist to its target human TAS2R38 bitter receptor. PLoS ONE 5(8): e doi: /journal.pone Brandon-Jones D, Eudey AA, Geissmann T, Groves CP, Melnick DJ, Morales JC, Shekelle M, Stewart CB Asian primates classification. Intl. J. Primatol. 25 (1): Bufe B, Breslin PA, Kuhn C, Reed DR, Tharp CD, Slack JP, Kim UK, Drayna D, Meyerhof W The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception. Curr. Biol. 15: Chandrashekar J, Mueller KL, Hoon MA, Adler E, Feng L, Guo W, Zuker CS, Ryba NJ T2Rs function as bitter taste receptors. Cell, 100: Kim UK, Jorgenson E, Coon H, Leppert M, Risch N, Drayna D Positional cloning of the human quantitative trait locus underlying taste sensitivity to phenylthiocarbamide. Science 299: Nei M, Niimura Y, Nozawa M The revolution of animal chemosensory receptor gene repertoires: role of chances and necessity. Nature reviews 9: Suzuki N, Sugawara T, Matsui A, Go Y, Hirai H, Imai H Identification of non-taster Japanese macaques for a specific bitter taste. Primates 51:

15 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28: Wooding S Sequence of Macaca mullata T2R38 [Internet]. Wooding S, Bufe B, Grassi C, Howard MT, Stone AC, Vazquez M, Dunn DM, Meyerhof W, Weiss RB, Bamshad MJ Independent evolution of bitter-taste sensitivity in humans and chimpanzees. Nature 440:

16 APPENDIX

17 7 Appendix 1 Nucleotide Sequence of T2R38 Gene of Leaf-Eating Monkey Alligned to T2R38 Gene of Macaca mullata DataType=Nucleotide CodeTable=Standard NSeqs=5 NSites=1002 Identical=. Missing=? Indel=-;!Domain=Data property=coding CodonStart=1; #MamumuTAS2R38 ATG TTA ACT CTA ACT CAC GTC TGC ACT GTG TCC TAT GAA GTC AGG AGC [ 48] #Sample-No G A..... M [ 48] #Sample-No G A [ 48] #S1.....G......?..... A..... C [ 48] #L3.....G A.....? [ 48] #MamumuTAS2R38 ACA TTT CTG TTC ATT TCA GTC CTG GAG TTT GCA GTG GGG TTT CTG ACC [ 96] #Sample-No A... [ 96] #Sample-No A... [ 96] #S A... [ 96] #L A... [ 96] #MamumuTAS2R38 AAC GCC TTC ATT TCC TTG GTG AAT TTT TGG GAC GTA GTG AAG AGG CAG [ 144] #Sample-No T A [ 144] #Sample-No T A [ 144] #S T A [ 144] #L T A [ 144] #MamumuTAS2R38 CCA CTG AGC AAC AGT GAT TGT GTG CTT CTG TGT CTC AGC ATC AGC CGG [ 192] #Sample-No [ 192] #Sample-No [ 192] #S [ 192] #L [ 192] #MamumuTAS2R38 CTT TTC CTG CAT GGA CTG CTC TTC CTG AGT GCT ATC CAG CTT ACC CAC [ 240] #Sample-No Y [ 240] #Sample-No [ 240] #S [ 240] #L R ?. [ 240] #MamumuTAS2R38 TTC CAG AAG TTG AGT GAA CCA CTG AAC CAC AGC TAC CAA GCC ATC CTC [ 288] #Sample-No G [ 288] #Sample-No G [ 288] #S ? G [ 288] #L G [ 288] #MamumuTAS2R38 ATG CTA TGG ATG ATT GCA AAC CAA GCC AAC CTC TGG CTT GCC GCC TGC [ 336] #Sample-No [ 336] #Sample-No [ 336] #S [ 336] #L [ 336] #MamumuTAS2R38 CTC AGC CTG CTC TAC TGC TCC AAG CTC ATC CGT TTC TCT CAC ACC TTC [ 384] #Sample-No A [ 384] #Sample-No.25.C......A [ 384] #S A [ 384] #L A [ 384] #MamumuTAS2R38 CTG ATC TGC TTG GCA AGC TGG GTC TCC AGG AAG ATA TCC CAG ATG CTC [ 432] #Sample-No C [ 432] #Sample-No C [ 432] #S C [ 432] #L C [ 432] #MamumuTAS2R38 CTG GGT ATT ATT CTT TGC TCC TGC ATC TGC ACT GTC CTC TGT GTT TGG [ 480] #Sample-No T [ 480] #Sample-No T [ 480] #S T [ 480] #L T [ 480] #MamumuTAS2R38 TGC TTT TTT GGC AGA CTT CAC TTC ACA GTC ACA ACT GTG CTA TTC ATG [ 528] #Sample-No A C [ 528] #Sample-No A C [ 528] #S A C [ 528] #L3... Y..... A C [ 528] #MamumuTAS2R38 AAT AAC AAT ACA AGG CTC AAC TGG CAG ATT AAA GAT CTC AAC TTA TTT [ 576] #Sample-No C [ 576] #Sample-No C [ 576] #S R C [ 576] #L C [ 576]

18 #MamumuTAS2R38 TAT TCC TTT CTC TTC TGC TAT CTG TGG TCT GTC CCT CCT TTC CTA TTG [ 624] #Sample-No [ 624] #Sample-No [ 624] #S Y [ 624] #L M [ 624] #MamumuTAS2R38 TTT CTG GTT TCT TCT GGG ATG CTG ACT GTC TCC CTG GGA AGG CAC ATG [ 672] #Sample-No A T [ 672] #Sample-No A T [ 672] #S1.....A T [ 672] #L3.....A T [ 672] #MamumuTAS2R38 AGG ACA ATG AAG GTC TAT ACC AGA GAC TCT CGT GAC CCC AGC CTG GAG [ 720] #Sample-No.8.A [ 720] #Sample-No.25.A W [ 720] #S1.R [ 720] #L3.A Y [ 720] #MamumuTAS2R38 GCC CAC ATT AAA GCC CTC AAG TCT CTT ATC TCC TTT TTC TGC TTC TTT [ 768] #Sample-No A [ 768] #Sample-No A [ 768] #S A [ 768] #L A [ 768] #MamumuTAS2R38 GTG ATA TCA TCC TGT GCT GCC TTC ATC TCA GTG CCC CTA CTT ATT CTG [ 816] #Sample-No [ 816] #Sample-No [ 816] #S [ 816] #L [ 816] #MamumuTAS2R38 TGG CAT GAC AAA ATA GGG GTG ATG GTT TGT GTT GGG ATA ATG GCA GCT [ 864] #Sample-No [ 864] #Sample-No [ 864] #S [ 864] #L [ 864] #MamumuTAS2R38 TGT CCC TCT GGG CAT GCA GCC GTC CTG ATC TCA GGC AAT GCC AAG TTG [ 912] #Sample-No [ 912] #Sample-No [ 912] #S [ 912] #L [ 912] #MamumuTAS2R38 AGG AGA GCT GTG ACA ACC ATT CTG CTC TGG GCT CAG AGC AGC CTG AAG [ 960] #Sample-No [ 960] #Sample-No [ 960] #S [ 960] #L [ 960] #MamumuTAS2R38 GTA AGA GCC GAT CAC ATG GCA GAT TCC AGG ACA CTG TGC TGA [1002] #Sample-No C A [1002] #Sample-No C A [1002] #S C A [1002] #L C A [1002]

19 9 RIWAYAT HIDUP Penulis lahir di Bantul pada tanggal 16 Juni 1992 sebagai anak pertama dari pasangan Agustinus Gasumta Purba dan Andrea Ismargyaning Utami. Penulis menyelesaikan pendidikan di SMA Pangudi Luhur Van Lith pada tahun 2010 dan melanjutkan studi pada Jurusan Biologi, Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor melalui jalur SNMPTN. Penulis melakukan Studi Lapangan pada tahun 2012 dengan judul penelitian Keragaman Semut di sekitar Pohon Pinus di Taman Nasional Gunung Gede Pangrango (TNGGP). Penulis melakukan Praktik Lapangan pada tahun 2013 dengan judul Menejemen dan Kendali Mutu Mikrobiologis pada Produksi Pasta di PT Indofood Sukses Makmur Tbk. Divisi Bogasari. Penulis menjadi asisten praktikum Biologi Dasar pada tahun dan Struktur Hewan pada tahun