Variation of T2R38 Gene Encoding PTC Bitter Taste Receptor in Leaf-Eating Monkey
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
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 G3410012
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
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
Judul Skripsi : Variation of T2R38 Gene Encoding PTC Bitter Taste Receptor in
Leaf-Eating Monkey
Nama
: Laurentia Henrieta Permita Sari Purba
NIM
: G34100121
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:
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 2014.
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
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
DAFTAR TABEL
1 Variation of T2R38 gene of leaf-eating monkey
2 Amino acid of T2R38 receptor of human PTC taster compared to amino
acid T2R38 receptor of leaf-eating monkey
3
4
DAFTAR LAMPIRAN
1 Nucleotide Sequence of T2R38 Gene of Leaf-Eating Monkey Alligned to
T2R38 Gene of Macaca mullata
7
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.
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
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
Trachypithecus cristatus
Trachypithecus auratus
Presbytis melalophos
Trachypithecus auratus
Name
8
25
S1
L3
Site number 25
M
A
C
N
Site number 338
T
C
T
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).
Table 2 Amino acid of T2R38 receptor of human PTC taster compared to
amino acid T2R38 receptor of leaf-eating monkey
Amino acid
49
site number
Homo sapiens
Pro
T. auratus
*
T. cristatus
*
P. melalophos
*
Note: * = same as above
99
100
103
255
259
262
296
Try
*
*
*
Met
*
*
*
Asp
*
*
*
Phe
*
*
*
Ser
*
*
*
Ala
*
*
*
Val
*
*
*
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. 2010. Insights into the binding of
phenyltiocarbamide (PTC) agonist to its target human TAS2R38 bitter
receptor. PLoS ONE 5(8): e12394. doi:10.1371/journal.pone.0012394.
Brandon-Jones D, Eudey AA, Geissmann T, Groves CP, Melnick DJ, Morales JC,
Shekelle M, Stewart CB. 2004. Asian primates classification. Intl. J.
Primatol. 25 (1): 100-164.
Bufe B, Breslin PA, Kuhn C, Reed DR, Tharp CD, Slack JP, Kim UK, Drayna D,
Meyerhof W. 2005. The molecular basis of individual differences in
phenylthiocarbamide and propylthiouracil bitterness perception. Curr.
Biol. 15:322–327.
Chandrashekar J, Mueller KL, Hoon MA, Adler E, Feng L, Guo W, Zuker CS,
Ryba NJ. 2000. T2Rs function as bitter taste receptors. Cell, 100:703–711.
Kim UK, Jorgenson E, Coon H, Leppert M, Risch N, Drayna D. 2003. Positional
cloning of the human quantitative trait locus underlying taste sensitivity to
phenylthiocarbamide. Science 299:1221–1225.
Nei M, Niimura Y, Nozawa M. 2008. The revolution of animal chemosensory
receptor gene repertoires: role of chances and necessity. Nature reviews 9:
951-963.
Suzuki N, Sugawara T, Matsui A, Go Y, Hirai H, Imai H. 2011. Identification of
non-taster Japanese macaques for a specific bitter taste. Primates 51:285–
289.
5
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5:
molecular evolutionary genetics analysis using maximum likelihood,
evolutionary distance, and maximum parsimony methods. Mol. Biol.
Evol. 28: 2731–2739.
Wooding S. 2001. Sequence of Macaca mullata T2R38 [Internet].
http://www.ncbi.nlm.niv.gov/nuccore/384369985?report=fasta.
Wooding S, Bufe B, Grassi C, Howard MT, Stone AC, Vazquez M, Dunn DM,
Meyerhof W, Weiss RB, Bamshad MJ. 2006. Independent evolution of
bitter-taste sensitivity in humans and chimpanzees. Nature 440:930–934.
APPENDIX
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
#Sample-No.8
... ..G ... ... ... ... A..
#Sample-No.25 ... ..G ... ... ... ... A..
#S1
... ..G ... ... ?.. ... A..
#L3
... ..G ... ... ... ... A..
TGC
...
...
...
...
ACT
M..
...
C..
?..
GTG
...
...
...
...
TCC
...
...
...
...
TAT
...
...
...
...
GAA
...
...
...
...
GTC
...
...
...
...
AGG
...
...
...
...
AGC
...
...
...
...
[
[
[
[
[
48]
48]
48]
48]
48]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
ACA
...
...
...
...
TTT
...
...
...
...
CTG
...
...
...
...
TTC
...
...
...
...
ATT
...
...
...
...
TCA
...
...
...
...
GTC
...
...
...
...
CTG
...
...
...
...
GAG
...
...
...
...
TTT
...
...
...
...
GCA
...
...
...
...
GTG
...
...
...
...
GGG
...
...
...
...
TTT
...
...
...
...
CTG
..A
..A
..A
..A
ACC
...
...
...
...
[
[
[
[
[
96]
96]
96]
96]
96]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
AAC
...
...
...
...
GCC
...
...
...
...
TTC
...
...
...
...
ATT
...
...
...
...
TCC
.T.
.T.
.T.
.T.
TTG
...
...
...
...
GTG
...
...
...
...
AAT
...
...
...
...
TTT
...
...
...
...
TGG
...
...
...
...
GAC
...
...
...
...
GTA
A..
A..
A..
A..
GTG
...
...
...
...
AAG
...
...
...
...
AGG
...
...
...
...
CAG
...
...
...
...
[
[
[
[
[
144]
144]
144]
144]
144]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
CCA
...
...
...
...
CTG
...
...
...
...
AGC
...
...
...
...
AAC
...
...
...
...
AGT
...
...
...
...
GAT
...
...
...
...
TGT
...
...
...
...
GTG
...
...
...
...
CTT
...
...
...
...
CTG
...
...
...
...
TGT
...
...
...
...
CTC
...
...
...
...
AGC
...
...
...
...
ATC
...
...
...
...
AGC
...
...
...
...
CGG
...
...
...
...
[
[
[
[
[
192]
192]
192]
192]
192]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
CTT
...
...
...
...
TTC
...
...
...
...
CTG
...
...
...
...
CAT
...
...
...
...
GGA
...
...
...
...
CTG
...
...
...
...
CTC
...
...
...
...
TTC
...
...
...
...
CTG
...
...
...
...
AGT
..Y
...
...
...
GCT
...
...
...
R..
ATC
...
...
...
...
CAG
...
...
...
...
CTT
...
...
...
...
ACC
...
...
...
...
CAC
...
...
...
.?.
[
[
[
[
[
240]
240]
240]
240]
240]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TTC
...
...
...
...
CAG
...
...
...
...
AAG
...
...
...
...
TTG
...
...
...
...
AGT
...
...
...
...
GAA
...
...
...
...
CCA
...
...
...
...
CTG
...
...
...
...
AAC
...
...
.?.
...
CAC
...
...
...
...
AGC
...
...
...
...
TAC
...
...
...
...
CAA
G..
G..
G..
G..
GCC
...
...
...
...
ATC
...
...
...
...
CTC
...
...
...
...
[
[
[
[
[
288]
288]
288]
288]
288]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
ATG
...
...
...
...
CTA
...
...
...
...
TGG
...
...
...
...
ATG
...
...
...
...
ATT
...
...
...
...
GCA
...
...
...
...
AAC
...
...
...
...
CAA
...
...
...
...
GCC
...
...
...
...
AAC
...
...
...
...
CTC
...
...
...
...
TGG
...
...
...
...
CTT
...
...
...
...
GCC
...
...
...
...
GCC
...
...
...
...
TGC
...
...
...
...
[
[
[
[
[
336]
336]
336]
336]
336]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
CTC
...
.C.
...
...
AGC
...
...
...
...
CTG
..A
..A
..A
..A
CTC
...
...
...
...
TAC
...
...
...
...
TGC
...
...
...
...
TCC
...
...
...
...
AAG
...
...
...
...
CTC
...
...
...
...
ATC
...
...
...
...
CGT
...
...
...
...
TTC
...
...
...
...
TCT
...
...
...
...
CAC
...
...
...
...
ACC
...
...
...
...
TTC
...
...
...
...
[
[
[
[
[
384]
384]
384]
384]
384]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
CTG
...
...
...
...
ATC
...
...
...
...
TGC
...
...
...
...
TTG
...
...
...
...
GCA
...
...
...
...
AGC
...
...
...
...
TGG
...
...
...
...
GTC
...
...
...
...
TCC
...
...
...
...
AGG
...
...
...
...
AAG
...
...
...
...
ATA
..C
..C
..C
..C
TCC
...
...
...
...
CAG
...
...
...
...
ATG
...
...
...
...
CTC
...
...
...
...
[
[
[
[
[
432]
432]
432]
432]
432]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
CTG
...
...
...
...
GGT
...
...
...
...
ATT
...
...
...
...
ATT
T..
T..
T..
T..
CTT
...
...
...
...
TGC
...
...
...
...
TCC
...
...
...
...
TGC
...
...
...
...
ATC
...
...
...
...
TGC
...
...
...
...
ACT
...
...
...
...
GTC
...
...
...
...
CTC
...
...
...
...
TGT
...
...
...
...
GTT
...
...
...
...
TGG
...
...
...
...
[
[
[
[
[
480]
480]
480]
480]
480]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TGC
...
...
...
...
TTT
...
...
...
Y..
TTT
...
...
...
...
GGC
A..
A..
A..
A..
AGA
...
...
...
...
CTT
...
...
...
...
CAC
.C.
.C.
.C.
.C.
TTC
...
...
...
...
ACA
...
...
...
...
GTC
...
...
...
...
ACA
...
...
...
...
ACT
...
...
...
...
GTG
...
...
...
...
CTA
...
...
...
...
TTC
...
...
...
...
ATG
...
...
...
...
[
[
[
[
[
528]
528]
528]
528]
528]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
AAT
...
...
...
...
AAC
...
...
...
...
AAT
...
...
...
...
ACA
...
...
..R
...
AGG
...
...
...
...
CTC
...
...
...
...
AAC
...
...
...
...
TGG
...
...
...
...
CAG
...
...
...
...
ATT
...
...
...
...
AAA
...
...
...
...
GAT
..C
..C
..C
..C
CTC
...
...
...
...
AAC
...
...
...
...
TTA
...
...
...
...
TTT
...
...
...
...
[
[
[
[
[
576]
576]
576]
576]
576]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TAT
...
...
...
...
TCC
...
...
...
...
TTT
...
...
...
...
CTC
...
...
...
...
TTC
...
...
...
...
TGC
...
...
...
...
TAT
...
...
...
M..
CTG
...
...
.Y.
...
TGG
...
...
...
...
TCT
...
...
...
...
GTC
...
...
...
...
CCT
...
...
...
...
CCT
...
...
...
...
TTC
...
...
...
...
CTA
...
...
...
...
TTG
...
...
...
...
[
[
[
[
[
624]
624]
624]
624]
624]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TTT
...
...
...
...
CTG
..A
..A
..A
..A
GTT
...
...
...
...
TCT
...
...
...
...
TCT
...
...
...
...
GGG
...
...
...
...
ATG
...
...
...
...
CTG
...
...
...
...
ACT
...
...
...
...
GTC
...
...
...
...
TCC
...
...
...
...
CTG
..T
..T
..T
..T
GGA
...
...
...
...
AGG
...
...
...
...
CAC
...
...
...
...
ATG
...
...
...
...
[
[
[
[
[
672]
672]
672]
672]
672]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
AGG
.A.
.A.
.R.
.A.
ACA
...
...
...
...
ATG
...
...
...
...
AAG
...
...
...
...
GTC
...
...
...
...
TAT
...
...
...
...
ACC
...
...
...
...
AGA
...
...
...
...
GAC
...
.W.
...
...
TCT
...
...
...
...
CGT
...
...
...
...
GAC
...
...
...
...
CCC
...
...
...
...
AGC
...
...
...
..Y
CTG
...
...
...
...
GAG
...
...
...
...
[
[
[
[
[
720]
720]
720]
720]
720]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
GCC
...
...
...
...
CAC
...
...
...
...
ATT
...
...
...
...
AAA
...
...
...
...
GCC
...
...
...
...
CTC
...
...
...
...
AAG
...
...
...
...
TCT
...
...
...
...
CTT
...
...
...
...
ATC
...
...
...
...
TCC
..A
..A
..A
..A
TTT
...
...
...
...
TTC
...
...
...
...
TGC
...
...
...
...
TTC
...
...
...
...
TTT
...
...
...
...
[
[
[
[
[
768]
768]
768]
768]
768]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
GTG
...
...
...
...
ATA
...
...
...
...
TCA
...
...
...
...
TCC
...
...
...
...
TGT
...
...
...
...
GCT
...
...
...
...
GCC
...
...
...
...
TTC
...
...
...
...
ATC
...
...
...
...
TCA
...
...
...
...
GTG
...
...
...
...
CCC
...
...
...
...
CTA
...
...
...
...
CTT
...
...
...
...
ATT
...
...
...
...
CTG
...
...
...
...
[
[
[
[
[
816]
816]
816]
816]
816]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TGG
...
...
...
...
CAT
...
...
...
...
GAC
...
...
...
...
AAA
...
...
...
...
ATA
...
...
...
...
GGG
...
...
...
...
GTG
...
...
...
...
ATG
...
...
...
...
GTT
...
...
...
...
TGT
...
...
...
...
GTT
...
...
...
...
GGG
...
...
...
...
ATA
...
...
...
...
ATG
...
...
...
...
GCA
...
...
...
...
GCT
...
...
...
...
[
[
[
[
[
864]
864]
864]
864]
864]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TGT
...
...
...
...
CCC
...
...
...
...
TCT
...
...
...
...
GGG
...
...
...
...
CAT
...
...
...
...
GCA
...
...
...
...
GCC
...
...
...
...
GTC
...
...
...
...
CTG
...
...
...
...
ATC
...
...
...
...
TCA
...
...
...
...
GGC
...
...
...
...
AAT
...
...
...
...
GCC
...
...
...
...
AAG
...
...
...
...
TTG
...
...
...
...
[
[
[
[
[
912]
912]
912]
912]
912]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
AGG
...
...
...
...
AGA
...
...
...
...
GCT
...
...
...
...
GTG
...
...
...
...
ACA
...
...
...
...
ACC
...
...
...
...
ATT
...
...
...
...
CTG
...
...
...
...
CTC
...
...
...
...
TGG
...
...
...
...
GCT
...
...
...
...
CAG
...
...
...
...
AGC
...
...
...
...
AGC
...
...
...
...
CTG
...
...
...
...
AAG
...
...
...
...
[
[
[
[
[
960]
960]
960]
960]
960]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
GTA
...
...
...
...
AGA
...
...
...
...
GCC
...
...
...
...
GAT
..C
..C
..C
..C
CAC
...
...
...
...
ATG
...
...
...
...
GCA
...
...
...
...
GAT
...
...
...
...
TCC
...
...
...
...
AGG
.A.
.A.
.A.
.A.
ACA
...
...
...
...
CTG
...
...
...
...
TGC
...
...
...
...
TGA
...
...
...
...
[1002]
[1002]
[1002]
[1002]
[1002]
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 2012-2013 dan Struktur Hewan pada tahun
2013-2014.
TASTE RECEPTOR IN LEAF-EATING MONKEY
LAURENTIA HENRIETA PERMITA SARI PURBA
DEPARTEMEN BIOLOGI
FAKULTAS MATEMATIKA DAN ILMU PENGETAHUAN ALAM
INSTITUT PERTANIAN BOGOR
2014
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 G3410012
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
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
Judul Skripsi : Variation of T2R38 Gene Encoding PTC Bitter Taste Receptor in
Leaf-Eating Monkey
Nama
: Laurentia Henrieta Permita Sari Purba
NIM
: G34100121
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:
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 2014.
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
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
DAFTAR TABEL
1 Variation of T2R38 gene of leaf-eating monkey
2 Amino acid of T2R38 receptor of human PTC taster compared to amino
acid T2R38 receptor of leaf-eating monkey
3
4
DAFTAR LAMPIRAN
1 Nucleotide Sequence of T2R38 Gene of Leaf-Eating Monkey Alligned to
T2R38 Gene of Macaca mullata
7
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.
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
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
Trachypithecus cristatus
Trachypithecus auratus
Presbytis melalophos
Trachypithecus auratus
Name
8
25
S1
L3
Site number 25
M
A
C
N
Site number 338
T
C
T
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).
Table 2 Amino acid of T2R38 receptor of human PTC taster compared to
amino acid T2R38 receptor of leaf-eating monkey
Amino acid
49
site number
Homo sapiens
Pro
T. auratus
*
T. cristatus
*
P. melalophos
*
Note: * = same as above
99
100
103
255
259
262
296
Try
*
*
*
Met
*
*
*
Asp
*
*
*
Phe
*
*
*
Ser
*
*
*
Ala
*
*
*
Val
*
*
*
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. 2010. Insights into the binding of
phenyltiocarbamide (PTC) agonist to its target human TAS2R38 bitter
receptor. PLoS ONE 5(8): e12394. doi:10.1371/journal.pone.0012394.
Brandon-Jones D, Eudey AA, Geissmann T, Groves CP, Melnick DJ, Morales JC,
Shekelle M, Stewart CB. 2004. Asian primates classification. Intl. J.
Primatol. 25 (1): 100-164.
Bufe B, Breslin PA, Kuhn C, Reed DR, Tharp CD, Slack JP, Kim UK, Drayna D,
Meyerhof W. 2005. The molecular basis of individual differences in
phenylthiocarbamide and propylthiouracil bitterness perception. Curr.
Biol. 15:322–327.
Chandrashekar J, Mueller KL, Hoon MA, Adler E, Feng L, Guo W, Zuker CS,
Ryba NJ. 2000. T2Rs function as bitter taste receptors. Cell, 100:703–711.
Kim UK, Jorgenson E, Coon H, Leppert M, Risch N, Drayna D. 2003. Positional
cloning of the human quantitative trait locus underlying taste sensitivity to
phenylthiocarbamide. Science 299:1221–1225.
Nei M, Niimura Y, Nozawa M. 2008. The revolution of animal chemosensory
receptor gene repertoires: role of chances and necessity. Nature reviews 9:
951-963.
Suzuki N, Sugawara T, Matsui A, Go Y, Hirai H, Imai H. 2011. Identification of
non-taster Japanese macaques for a specific bitter taste. Primates 51:285–
289.
5
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5:
molecular evolutionary genetics analysis using maximum likelihood,
evolutionary distance, and maximum parsimony methods. Mol. Biol.
Evol. 28: 2731–2739.
Wooding S. 2001. Sequence of Macaca mullata T2R38 [Internet].
http://www.ncbi.nlm.niv.gov/nuccore/384369985?report=fasta.
Wooding S, Bufe B, Grassi C, Howard MT, Stone AC, Vazquez M, Dunn DM,
Meyerhof W, Weiss RB, Bamshad MJ. 2006. Independent evolution of
bitter-taste sensitivity in humans and chimpanzees. Nature 440:930–934.
APPENDIX
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
#Sample-No.8
... ..G ... ... ... ... A..
#Sample-No.25 ... ..G ... ... ... ... A..
#S1
... ..G ... ... ?.. ... A..
#L3
... ..G ... ... ... ... A..
TGC
...
...
...
...
ACT
M..
...
C..
?..
GTG
...
...
...
...
TCC
...
...
...
...
TAT
...
...
...
...
GAA
...
...
...
...
GTC
...
...
...
...
AGG
...
...
...
...
AGC
...
...
...
...
[
[
[
[
[
48]
48]
48]
48]
48]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
ACA
...
...
...
...
TTT
...
...
...
...
CTG
...
...
...
...
TTC
...
...
...
...
ATT
...
...
...
...
TCA
...
...
...
...
GTC
...
...
...
...
CTG
...
...
...
...
GAG
...
...
...
...
TTT
...
...
...
...
GCA
...
...
...
...
GTG
...
...
...
...
GGG
...
...
...
...
TTT
...
...
...
...
CTG
..A
..A
..A
..A
ACC
...
...
...
...
[
[
[
[
[
96]
96]
96]
96]
96]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
AAC
...
...
...
...
GCC
...
...
...
...
TTC
...
...
...
...
ATT
...
...
...
...
TCC
.T.
.T.
.T.
.T.
TTG
...
...
...
...
GTG
...
...
...
...
AAT
...
...
...
...
TTT
...
...
...
...
TGG
...
...
...
...
GAC
...
...
...
...
GTA
A..
A..
A..
A..
GTG
...
...
...
...
AAG
...
...
...
...
AGG
...
...
...
...
CAG
...
...
...
...
[
[
[
[
[
144]
144]
144]
144]
144]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
CCA
...
...
...
...
CTG
...
...
...
...
AGC
...
...
...
...
AAC
...
...
...
...
AGT
...
...
...
...
GAT
...
...
...
...
TGT
...
...
...
...
GTG
...
...
...
...
CTT
...
...
...
...
CTG
...
...
...
...
TGT
...
...
...
...
CTC
...
...
...
...
AGC
...
...
...
...
ATC
...
...
...
...
AGC
...
...
...
...
CGG
...
...
...
...
[
[
[
[
[
192]
192]
192]
192]
192]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
CTT
...
...
...
...
TTC
...
...
...
...
CTG
...
...
...
...
CAT
...
...
...
...
GGA
...
...
...
...
CTG
...
...
...
...
CTC
...
...
...
...
TTC
...
...
...
...
CTG
...
...
...
...
AGT
..Y
...
...
...
GCT
...
...
...
R..
ATC
...
...
...
...
CAG
...
...
...
...
CTT
...
...
...
...
ACC
...
...
...
...
CAC
...
...
...
.?.
[
[
[
[
[
240]
240]
240]
240]
240]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TTC
...
...
...
...
CAG
...
...
...
...
AAG
...
...
...
...
TTG
...
...
...
...
AGT
...
...
...
...
GAA
...
...
...
...
CCA
...
...
...
...
CTG
...
...
...
...
AAC
...
...
.?.
...
CAC
...
...
...
...
AGC
...
...
...
...
TAC
...
...
...
...
CAA
G..
G..
G..
G..
GCC
...
...
...
...
ATC
...
...
...
...
CTC
...
...
...
...
[
[
[
[
[
288]
288]
288]
288]
288]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
ATG
...
...
...
...
CTA
...
...
...
...
TGG
...
...
...
...
ATG
...
...
...
...
ATT
...
...
...
...
GCA
...
...
...
...
AAC
...
...
...
...
CAA
...
...
...
...
GCC
...
...
...
...
AAC
...
...
...
...
CTC
...
...
...
...
TGG
...
...
...
...
CTT
...
...
...
...
GCC
...
...
...
...
GCC
...
...
...
...
TGC
...
...
...
...
[
[
[
[
[
336]
336]
336]
336]
336]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
CTC
...
.C.
...
...
AGC
...
...
...
...
CTG
..A
..A
..A
..A
CTC
...
...
...
...
TAC
...
...
...
...
TGC
...
...
...
...
TCC
...
...
...
...
AAG
...
...
...
...
CTC
...
...
...
...
ATC
...
...
...
...
CGT
...
...
...
...
TTC
...
...
...
...
TCT
...
...
...
...
CAC
...
...
...
...
ACC
...
...
...
...
TTC
...
...
...
...
[
[
[
[
[
384]
384]
384]
384]
384]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
CTG
...
...
...
...
ATC
...
...
...
...
TGC
...
...
...
...
TTG
...
...
...
...
GCA
...
...
...
...
AGC
...
...
...
...
TGG
...
...
...
...
GTC
...
...
...
...
TCC
...
...
...
...
AGG
...
...
...
...
AAG
...
...
...
...
ATA
..C
..C
..C
..C
TCC
...
...
...
...
CAG
...
...
...
...
ATG
...
...
...
...
CTC
...
...
...
...
[
[
[
[
[
432]
432]
432]
432]
432]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
CTG
...
...
...
...
GGT
...
...
...
...
ATT
...
...
...
...
ATT
T..
T..
T..
T..
CTT
...
...
...
...
TGC
...
...
...
...
TCC
...
...
...
...
TGC
...
...
...
...
ATC
...
...
...
...
TGC
...
...
...
...
ACT
...
...
...
...
GTC
...
...
...
...
CTC
...
...
...
...
TGT
...
...
...
...
GTT
...
...
...
...
TGG
...
...
...
...
[
[
[
[
[
480]
480]
480]
480]
480]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TGC
...
...
...
...
TTT
...
...
...
Y..
TTT
...
...
...
...
GGC
A..
A..
A..
A..
AGA
...
...
...
...
CTT
...
...
...
...
CAC
.C.
.C.
.C.
.C.
TTC
...
...
...
...
ACA
...
...
...
...
GTC
...
...
...
...
ACA
...
...
...
...
ACT
...
...
...
...
GTG
...
...
...
...
CTA
...
...
...
...
TTC
...
...
...
...
ATG
...
...
...
...
[
[
[
[
[
528]
528]
528]
528]
528]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
AAT
...
...
...
...
AAC
...
...
...
...
AAT
...
...
...
...
ACA
...
...
..R
...
AGG
...
...
...
...
CTC
...
...
...
...
AAC
...
...
...
...
TGG
...
...
...
...
CAG
...
...
...
...
ATT
...
...
...
...
AAA
...
...
...
...
GAT
..C
..C
..C
..C
CTC
...
...
...
...
AAC
...
...
...
...
TTA
...
...
...
...
TTT
...
...
...
...
[
[
[
[
[
576]
576]
576]
576]
576]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TAT
...
...
...
...
TCC
...
...
...
...
TTT
...
...
...
...
CTC
...
...
...
...
TTC
...
...
...
...
TGC
...
...
...
...
TAT
...
...
...
M..
CTG
...
...
.Y.
...
TGG
...
...
...
...
TCT
...
...
...
...
GTC
...
...
...
...
CCT
...
...
...
...
CCT
...
...
...
...
TTC
...
...
...
...
CTA
...
...
...
...
TTG
...
...
...
...
[
[
[
[
[
624]
624]
624]
624]
624]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TTT
...
...
...
...
CTG
..A
..A
..A
..A
GTT
...
...
...
...
TCT
...
...
...
...
TCT
...
...
...
...
GGG
...
...
...
...
ATG
...
...
...
...
CTG
...
...
...
...
ACT
...
...
...
...
GTC
...
...
...
...
TCC
...
...
...
...
CTG
..T
..T
..T
..T
GGA
...
...
...
...
AGG
...
...
...
...
CAC
...
...
...
...
ATG
...
...
...
...
[
[
[
[
[
672]
672]
672]
672]
672]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
AGG
.A.
.A.
.R.
.A.
ACA
...
...
...
...
ATG
...
...
...
...
AAG
...
...
...
...
GTC
...
...
...
...
TAT
...
...
...
...
ACC
...
...
...
...
AGA
...
...
...
...
GAC
...
.W.
...
...
TCT
...
...
...
...
CGT
...
...
...
...
GAC
...
...
...
...
CCC
...
...
...
...
AGC
...
...
...
..Y
CTG
...
...
...
...
GAG
...
...
...
...
[
[
[
[
[
720]
720]
720]
720]
720]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
GCC
...
...
...
...
CAC
...
...
...
...
ATT
...
...
...
...
AAA
...
...
...
...
GCC
...
...
...
...
CTC
...
...
...
...
AAG
...
...
...
...
TCT
...
...
...
...
CTT
...
...
...
...
ATC
...
...
...
...
TCC
..A
..A
..A
..A
TTT
...
...
...
...
TTC
...
...
...
...
TGC
...
...
...
...
TTC
...
...
...
...
TTT
...
...
...
...
[
[
[
[
[
768]
768]
768]
768]
768]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
GTG
...
...
...
...
ATA
...
...
...
...
TCA
...
...
...
...
TCC
...
...
...
...
TGT
...
...
...
...
GCT
...
...
...
...
GCC
...
...
...
...
TTC
...
...
...
...
ATC
...
...
...
...
TCA
...
...
...
...
GTG
...
...
...
...
CCC
...
...
...
...
CTA
...
...
...
...
CTT
...
...
...
...
ATT
...
...
...
...
CTG
...
...
...
...
[
[
[
[
[
816]
816]
816]
816]
816]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TGG
...
...
...
...
CAT
...
...
...
...
GAC
...
...
...
...
AAA
...
...
...
...
ATA
...
...
...
...
GGG
...
...
...
...
GTG
...
...
...
...
ATG
...
...
...
...
GTT
...
...
...
...
TGT
...
...
...
...
GTT
...
...
...
...
GGG
...
...
...
...
ATA
...
...
...
...
ATG
...
...
...
...
GCA
...
...
...
...
GCT
...
...
...
...
[
[
[
[
[
864]
864]
864]
864]
864]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
TGT
...
...
...
...
CCC
...
...
...
...
TCT
...
...
...
...
GGG
...
...
...
...
CAT
...
...
...
...
GCA
...
...
...
...
GCC
...
...
...
...
GTC
...
...
...
...
CTG
...
...
...
...
ATC
...
...
...
...
TCA
...
...
...
...
GGC
...
...
...
...
AAT
...
...
...
...
GCC
...
...
...
...
AAG
...
...
...
...
TTG
...
...
...
...
[
[
[
[
[
912]
912]
912]
912]
912]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
AGG
...
...
...
...
AGA
...
...
...
...
GCT
...
...
...
...
GTG
...
...
...
...
ACA
...
...
...
...
ACC
...
...
...
...
ATT
...
...
...
...
CTG
...
...
...
...
CTC
...
...
...
...
TGG
...
...
...
...
GCT
...
...
...
...
CAG
...
...
...
...
AGC
...
...
...
...
AGC
...
...
...
...
CTG
...
...
...
...
AAG
...
...
...
...
[
[
[
[
[
960]
960]
960]
960]
960]
#MamumuTAS2R38
#Sample-No.8
#Sample-No.25
#S1
#L3
GTA
...
...
...
...
AGA
...
...
...
...
GCC
...
...
...
...
GAT
..C
..C
..C
..C
CAC
...
...
...
...
ATG
...
...
...
...
GCA
...
...
...
...
GAT
...
...
...
...
TCC
...
...
...
...
AGG
.A.
.A.
.A.
.A.
ACA
...
...
...
...
CTG
...
...
...
...
TGC
...
...
...
...
TGA
...
...
...
...
[1002]
[1002]
[1002]
[1002]
[1002]
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 2012-2013 dan Struktur Hewan pada tahun
2013-2014.