P di   f Proceedings of gBiotechnology International ConferenceBiotechnology International Conference

i h l f if“Biotechnology for Better Life”gy

InstitutTeknologi Bandung15‐16 June 2009

PREFACEPREFACE

This CD contains the abstracts of the talks presented in Biotechnology International Conference (BIC) with an This CD contains the abstracts of the talks presented in Biotechnology International Conference (BIC) with an overall theme on “Energy and the Environment: Reinvention for Developing Countries”, especially overall theme on  Energy and the Environment: Reinvention for Developing Countries , especially “Biotechnology: Biotechnology for Better Life”. This is in line with commemorating the 50th anniversary of ITB Biotechnology: Biotechnology for Better Life . This is in line with commemorating the 50 anniversary of ITB (InstitutTeknologi Bandung) as one of the oldest education and science institution in Indonesia. (InstitutTeknologi Bandung) as one of the oldest education and science institution in Indonesia. 

Bi h l  I i l C f      h ld i  W  H ll (A l  B )  d E  H ll (A l  Ti )   Biotechnology International Conference 2009 was held in West Hall (Aula Barat) and East Hall (Aula Timur) at I tit t T k l i B d     th 6th f J    Th  BIC  i  t   t  th  d l t  f InstitutTeknologi Bandung on 15th‐16th of June 2009. The BIC aims to promote the development of bi t h l   d th i   l  i  th  d l t  f  i   d t h l  i  I d i   d R i l  biotechnology and their roles in the development of science and technology in Indonesia and Regional. 

There are three areas of research covered in this conference. They are biotechnology of human health, y gy ,biotechnology of industrial and applications, and biotechnology of agricultural.  The topics of research were gy pp , gy g ppresented in six invited speakers and 34 contributed talks. We would like to thank the invited speakers and p p pthose who have submitted papers to this conference. 

We wish you a enjoy conference and stay in BandungWe wish you a enjoy conference and stay in Bandung.

Bandung, 10th of June 2009g, 9On behalf of the Organizing Committee,g g ,

Adi PancoroAdi PancoroDirector of Biotechnology Research Center ‐ ITBDirector of Biotechnology Research Center  ITB

SCIENTIFIC COMMITTEESCIENTIFIC COMMITTEE

Prof. Dr. Tjandra Setiadi, Bandung Institute of Technology, Indonesia

Prof. Dr. Wisjnuprapto, Bandung Institute of Technology, Indonesia

Dr.Achmad Saifudin Noer, Bandung Institute of Technology, Indonesiag f gy

Dr. Adi Pancoro, Bandung Institute of Technology, Indonesia, g f gy,

Dr. Akhmaloka, Bandung Institute of Technology, Indonesia, g f gy,

Dr. Anggraini Barlian, Bandung Institute of Technology, IndonesiaDr. Anggraini Barlian, Bandung Institute of Technology, Indonesia

Dr  Edwan Kardena  Bandung Institute of Technology  IndonesiaDr. Edwan Kardena, Bandung Institute of Technology, Indonesia

Dr  Enny Ratnaningsih  Bandung Institute of Technology  IndonesiaDr. Enny Ratnaningsih, Bandung Institute of Technology, Indonesia

Dr  Ernawati A  Bandung Institute of Technology  IndonesiaDr. Ernawati A., Bandung Institute of Technology, Indonesia

Dr  Fida MadayantiW  Bandung Institute of Technology  IndonesiaDr. Fida MadayantiW., Bandung Institute of Technology, Indonesia

D  I G  W t  B d  I tit t   f T h l  I d iDr. I.G. Wenten, Bandung Institute of Technology, Indonesia

D  M li R d i M i  B d  I i   f T h l  I d iDr. Maelita Ramdani Moies, Bandung Institute of Technology, Indonesia

l d f h l dDr. Marselina IrasoniaTan, Bandung Institute of Technology, Indonesia

Dr. Debbie S. Retnoningrum, Bandung Institute of Technology, Indonesia

Dr. Dessy Natalia, Bandung Institute of Technology, Indonesia

Dr. Elfahmi, Bandung Institute of Technology, Indonesia

Dr. Ronny Purwadi, Bandung Institute of Technology, Indonesia

Dr. Sony H. Sumarsono, Bandung Institute of Technology, Indonesiay , g f gy,

Dr. Sony Suhandono, Bandung Institute of Technology, Indonesiay , g f gy,

Dr. Sri Nanan B. Widiyanto, Bandung Institute of Technology, IndonesiaS a a d ya to, a du g st tute of ec ology, do es a

Dr. Zeily Nurachman, Bandung Institute of Technology, IndonesiaDr. Zeily Nurachman, Bandung Institute of Technology, Indonesia

CONTENTSCONTENTS

A.  BIOTECHNOLOGY OF HUMAN HEALTHA.  BIOTECHNOLOGY OF HUMAN HEALTH

Antioxidant and Antibacterial Activities of Extracts  from Hoan Ngoc (Pseuderatherum Platiferum)   a Medicinal PlantAntioxidant and Antibacterial Activities of Extracts  from Hoan Ngoc (Pseuderatherum Platiferum),  a Medicinal PlantChantana KankamolChantana Kankamol

Humanization of MurineAnti CEA antibody and the Selection of High Antibody Producing Cell Lines Using  a Rapid Humanization of MurineAnti‐CEA antibody and the Selection of High Antibody‐Producing Cell Lines Using  a Rapid Automated SystemAutomated SystemHeilly Chong, Ong Sheau Ching, Suba Dharshanan, Siong Jong Hang,  Khairul Ezani Najamudin, Zulkeflie Zamrod

Single Amino Acid Residue Substitution in Two Isolates of Isoniazid‐Resistant Mycobacterium tuberculosis Catalase‐g yPeroxidasePurkan, Ihsanawati, Debby Soffie Retnoningrum, Dessy Natalia, and Achmad Saifuddin Noer

Analysis of Alpha‐Glucosidase Inhibitors Extracted From Several Medicinal HerbsStephanie, Elisa Friska Romasi, Maggy Thenawidjaja

From Science to Farming and the Seek for the Future Policy on Agricultural Biotechnology Product Commercializationl dRagil Yoga Edi

DNA Methylation on Rar Β2 Promotor Is Epigenetic Process on Breast CancerI K t t G dh M li th  Wibi Ri  A h Hid ti d Fit i A iI Ketut Gedhe Muliartha, Wibi Riawan, Asnah Hidayati and Fitri Armania

Identification of a Calcium‐Dependent Protein KinaseGene Involved in Osmoregulation in Chlamydomonas reinhardtiiReinhard PinontoanReinhard Pinontoan

Mi h d i l DNA Di i  i  I di  P l i   f S h  P   d i  i li i    N i  P  Mitochondrial DNA Diversity in Indigenous Populations of Southern Papua and its implications on Native Papuan HaplogroupsHaplogroupsRichardo Ubyaan, Epiphani I. Y. Palit, Hendry Yanuar, Yohanis NgiliRichardo Ubyaan, Epiphani I. Y. Palit, Hendry Yanuar, Yohanis Ngili

The Role  Expression and Production of Brain Receptor Protein of Humpback grouper Cromileptes altivelis to Viral The Role, Expression and Production of Brain Receptor Protein of Humpback grouper Cromileptes altivelis to Viral Nervous Necrotic Infection for Development of Diagnostic MaterialNervous Necrotic Infection for Development of Diagnostic MaterialUun Yanuhar

Nucleotide Polymorphism on D‐Loop Human Mitochondrial DNA from Endoderm, Mesoderm, and Ectodermy p p , ,Yohanis Ngili, A. Saifuddin Noer, Adang Suwandi Ahmad, and Yoni F. Syukriani

B.  BIOTECHNOLOGY FOR INDUSTRIAL AND APPLICATIONB.  BIOTECHNOLOGY FOR INDUSTRIAL AND APPLICATION

Earth to Space: The Adventure of T1 LipaseEarth to Space: The Adventure of T1 LipaseAbu Bakar Salleh  Thean Chor Leow   Raja Noor Zaliha Raja Abdul Rahman  and Mahiran BasriAbu Bakar Salleh, Thean Chor Leow,  Raja Noor Zaliha Raja Abdul Rahman, and Mahiran Basri

Design of an Enzymatic Membrane Microreactor and Its Aplication for Transesterification ReactionDesign of an Enzymatic Membrane Microreactor and Its Aplication for Transesterification ReactionAchmadin Luthfi, Siswa Setyahadi, Misri Gozan, Mohammad Nasikinc ad ut f , S s a Setya ad , s Go a , o a ad as k

Xylanase Production from Winter Mushroom Flamulina velutipes Grown on Optimized Cultural ConditionsXylanase Production from Winter Mushroom Flamulina velutipes Grown on Optimized Cultural ConditionsAltaf Simairf

The Prospective of Raw Material and Pathway Modification in Polylactic Acid Production as Ecofriendly The Prospective of Raw Material and Pathway Modification in Polylactic Acid Production as Ecofriendly Biodegradable PlasticgDiana Lo

Ethanol Production from Glycerol of Biodiesel Plant using Batch and Semi‐continuous Fermentation by Enterobacter gaerogenes TISTR 1468kb k k k hAkbar Ciptanto, Poonsuk Prasertsan, Aran Hanpongkittikun, Sompong O‐Thong

Biodegradable Plastic Made from Hidrocoloid Alginate Extracted from Sargasum filifendulaM di hMurdinah

Solid State Fermentation for Improvement of  Fatty Acids in By ProductsMohammad Faseleh JahromiMohammad Faseleh Jahromi

C i   f W  f  Bi di l I d  i  V l bl  P d    I i i l S    Bi fiConversion of Waste from Biodiesel Industry into Valuable Products: an Initial Step to BiorefineryAndi  Ronny Purwadi  M T A P  Kresnowati  and Tjandra SetiadiAndi, Ronny Purwadi, M.T.A.P. Kresnowati, and Tjandra Setiadi

……

Extraction and Characterization of Protease from  The Viscera of Skipjack Tuna Fish  (Katsuwonus pelamis Linnaeus)Extraction and Characterization of Protease from  The Viscera of Skipjack Tuna Fish  (Katsuwonus pelamis Linnaeus)Mery Tambaria Damanik Ambarita  Jeni Anggrek  Maggy ThenawidjajaMery Tambaria Damanik Ambarita, Jeni Anggrek, Maggy Thenawidjaja

Mechanism of Interaction between  Prefoldin and Group II ChaperoninMechanism of Interaction between  Prefoldin and Group II ChaperoninMuhamad Sahlan  Taro Kanzaki  Phan The Tai  Tomatsu Zako   Mizuo Maeda and Masafumi YohdaMuhamad Sahlan, Taro Kanzaki, Phan The Tai, Tomatsu Zako,  Mizuo Maeda and Masafumi Yohda

Cloning of a Soft Coral Associated Bacillus aquimaris Α Amylase GeneCloning of a Soft Coral Associated Bacillus aquimaris Α‐Amylase GeneF  Puspasari, D  Natalia, Z  Nurachman, O  K  Radjasa, A  S  NoerF. Puspasari, D. Natalia, Z. Nurachman, O. K. Radjasa, A. S. Noer

Effects of Temperature and pH on the Capability of Enterobacter aerogenes as Hg remediatorsEffects of Temperature and pH on the Capability of Enterobacter aerogenes as Hg remediatorsLanny Hartanti, Oktavina Maharani and Dien A. Limyatiy , y

Lignin recovery from pulp and paper mill wastewaterLignin recovery from pulp and paper mill wastewaterSandhya Babel, Massalin Suksai and Phattaraporn Sarikkha

Isolation, purification and characterization of cellulase from giant snail (Achatina fulica) and application to hydrolyze , p g ( ) pp y ycellulolytic materialsSendy Adhika Noor and Aran H‐Kittikun

The prospect of gadung (Dioscorea hispida Dennst.) tuber as carbon source for Aspergillus niger in amylase productionS  Ri  R t ih  W d  Nil bSuraya, Rina Ratnasih, Wardono. Niloperbowo

i id l i i filNitrous Oxide Gas Removal Using Compost BiofilterTania Surya Utami  Josia Simanjuntak  Heri Hermansyah  Slamet  Mohammad NasikinTania Surya Utami, Josia Simanjuntak, Heri Hermansyah, Slamet, Mohammad Nasikin

C.  BIOTECHNOLOGY FOR AGRICULTURAL PRODUCTIONC.  BIOTECHNOLOGY FOR AGRICULTURAL PRODUCTION

Expression of Nucleoprotein Gene of Newcastle Disease Virus and Its Utility in Seroprofiling of the Disease in Expression of Nucleoprotein Gene of Newcastle Disease Virus and Its Utility in Seroprofiling of the Disease in ChickenChickenVinod Kumar

Production and Application of Inoculant Acid‐Alumunium Tolerant Bradyrhizobium japonicum Strains with SoybeanProduction and Application of Inoculant Acid Alumunium Tolerant Bradyrhizobium japonicum Strains with SoybeanNisa Rachmania Mubarik

Detection and Cloning of A Gene Involved in Zwitermicin A Biosynthesis from Plant Growth Promoting g y gRhizobacterium Bacillus sp CR64Rika Indri Astuti, Aris Tri Wahyudi, Sarah Asih Faulina, and Nisa Rachmania

Analysis Polymorphism Gene in Cattle by DNA SequencingTety Hartatik, Tri Satya Mastuti Widi and Mohammad Cahyadi

Neuroethics: The Pros and ConshPriya Chatteree

Proceedings ofgBiotechnology International Conference“Biotechnology for Better Life”ISBN : 978-979-19994-0-3

Additional copies of this publication are available from:ITB Research Center on Biotechnology (PP Bioteknologi ITB)nd fl I t U i it C t (PAU B ildi )2nd floor, Inter University Center (PAU Building),

Institut Teknologi BandungInstitut Teknologi BandungJl. Ganesha no. 10, Bandung, IndonesiaPhone : +62‐22‐253 4115F 6Fax : +62‐22‐253 4115

The ITB’s Golden Anniversary

International Conference on

Energy and the Environment: Reinvention for Developing Countries

Bandung, 15th-20

th of June 2009

Biotechnology for a Better Life

Day 1: June 15 / Monday

Venue: West Hall

08:00-08:30 Registration of Participants

08:30-09:30 Opening Ceremony Rector of ITB Keynote: Economic Challenging in the Next Five Year

Tentative Keynote:

09.00-09.30 Keynote Speech National Research Council and its Roles in Solving Problems and Needs in Developing

World by Prof. Dr. Ir. Andrianto Handojo (DRN) (*) ** Ibu Tusi Adibroto

09.30-10.00 Invited Speaker Earthquake by Prof. Sri Widiyantoro (ITB)

10.00-10.30 C O F F E E B R E A K

Session PLENARY (Chairman : Dr. Achmad Saifudin Noer)

10.30-11.00 Invited Speaker Current and Future Trends of Therapeutic Proteins by Dr. Debbie S Retnoningrum (School of Pharmacy-ITB)

11.00-11.30 Invited Speaker Current Status of Biotechnology in Agriculture by Tumpal Silalahi (PT Monsanto-Indonesia).

11.30-12.00 Invited Speaker Value Added Products from Palm Oil mill by enzyme technology by Prof. Aran H-Kittikun (Prince of Songkla University Thailand).

12.00-13.00 L U N C H B R E A K

13.00-15.00 Technical Session 1 by Chairman: - (in detailed agenda)

15.00-15.30 C O F F E E B R E A K

15.30-17.00 Technical Session 5 by Chairman: - (in detailed agenda)

17.00-19.00 PREPARATION TO DINNER

Venue: East Hall 13.00-15.00 Technical Session 2 by Chairman: -

(in detailed agenda)

Technical Session 3 by Chairman: -

(in detailed agenda)

Technical Session 4 by Chairman:

- (in detailed agenda)

15.00-15.30 C O F F E E B R E A K

15.30-17.00 Technical Session 6 by Chairman: - (in detailed agenda)

Technical Session 7 by Chairman: - (in detailed agenda)

Technical Session 8 by Chairman: -(in detailed agenda)

17.00-19.00 PREPARATION TO DINNER

Day 2: June 16 / Tuesday

Venue: East Hall

08.30-10.00 Technical Session 9 by Chairman:- (in detailed agenda)

Technical Session 10 by Chairman: - (in detailed agenda)

Technical Session 11 by Chairman: - (in detailed agenda)

10.00-10.30 C O F F E E B R E A K

Session PLENARY (Chairman : Dr.Ronny Purwadi )

10.30-11.30 Invited Speaker Toward Self Sufficient in Food and Biopharmaceutical Through Biotechnology by Dr. Sony Suhandono (School of Life Sciences and Technology – ITB).

11.00-11.30 Invited Speaker Marine Biotechnology in Indonesia : Present status and future Development by Prof. Dr. Hari Eko Irianto (Director, Research Center for Marine and Fisheries

product Processing and Biotechnology, Ministry of Marine Affairs and Fisheries).

11.30-12.00 Invited Speaker Overview of Current Status and Development Strategy of Biotechnology in Indonesia by Dr. Bambang Prasetya (Head of Biotechnology Consortium Indonesia).

12.00-13.00 L U N C H B R E A K

Venue: West Hall 13.00-15.00 MRA FORUM for Biotechnology. Chairman: Dr. Adi Pancoro

15.00-15.30 C O F F E E B R E A K

15.30-16.30 MRA FORUM for Biotechnology. Chairman: Dr. Adi Pancoro

16.30-17.00 Closing

17.00-19.00 PREPARATION TO DINNER

The ITB’s Golden Anniversary

International Conference on

Energy and the Environment: Reinvention for Developing Countries

Bandung, 15th-20

th of June 2009

Day 1 : MONDAY, JUNE 15th 2009

TIME PARALLEL SECTION

VENUE : WEST HALL VENUE : EAST HALL - 1 VENUE : EAST HALL - 2 VENUE : EAST HALL - 3

Chairman: Dr. Sony H.

Sumarsono

Chairman: Dr. Zeily

Nurachman

Chairman: Dr. Fida

Madayanti W

Chairwoman: Dr. Sri Nanan

B. Widiyanto

Biotechnology of Human

Health

Biotechnology of Industrial

and Application

Biotechnology of Industrial

and Application

Biotechnology of Agricultural

Productions

13.00 :13.30 A.1 Chantana Kankamol

Antioxidant and antibacterial

activities of extracts from

Hoan Ngoc (Pseuderatherum

platiferum), a medicinal plant

B.1 Abu Bakar Salleh

Earth To Space: the

Adventure of Ti Lipase

B.8 Tjandra Setiadi

Conversion of waste from

Biodiesel Industry into

Valuable: an Initial step to

Biorefinery

C. 1 Vinod Kumar Sahu

Expression of nucleoprotein

gene of Newcastle disease virus

and its utility in seroprofiling of

the disease in chicken

13.30 :14.00 A.2 Zulkeflie Zamrod

Humanization of Murine

Anti-CEA and Selection of

High Antibody Producing

Cell Lines Using A Rapid

Automated Clonal Selection

Method

B.2 Achmadin Luthfi

Design Of An Enzymatic

Membrane Microreactor And

ITS Application for

Transesterification Reaction

B.9 Mery Tambaria

Damanik Ambarita

Extraction and

Characterization of Potease

from the Viscera of Skipjack

Tuna Fish(Katsuwonus

pelamis Linnaeus)

C.2 Nisa Rachmania

Mubarik Production and

Application of Inoculant Acid-

Aluminium Tolerant

Bradyrhizobium japonicum

Strains with Soybean

14.00 :14.30 A.3 Purkan

Single Amino Acid Residue

Substitution in two Isolates of

Isoniazid- resistant

Mycobacterium Tuberculosis

Catalase Peroxidase

B.3 Altaf Simiar

Xylanase Production from

Winter Mushroom Flamulina

velutipes grown on optimized

cultural conditions

B.10 Mohammad Sahlan

Mechanism of Interaction

between and Group II

Chaperonin

C.3 Rika Indri Astuti

Detection and Cloning of a

Gene Involved Zwetermicin A

Biosynthesis from Plant

Growth Promoting

Rhizobacterium Bacillus sp

CR64

14.30: 15.00 A.4 Elisa Friska Romasi

Analysis of Alpha -

Glucosidase Inhibitors

Extracted From Several

Medical Herbs

B.4 Diana Lo

The Prospective of Raw

Material and Pathway

Modification in Polylactic

Acid Production as

Ecofriendly Biodegradable

Plastic

B.11 Fernita Puspasari

Cloning of A soft Coral

Associated Bacillus

aquimaris α-Amylase Gene

C.4 Tety Hartatik

Analysis Polymorphism Gene

in Cattle by DNA Sequencing

15.00:15.30 C O F F E E B R E A K

Chairwoman : Dr. Elfahmi Chairwoman : Dr. Ernawati

A.

Chairwoman : Dr.

Maelita Ramdani M.

Chairman : Dr. Adi Pancoro

Biotechnology of Human Health Biotechnology of Industrial and

Application

Biotechnology of Industrial

and Application

Biotechnology of Agricultral

Productions

15.30:16.00 A.5 Ragil Yoga Edi

From Science to farming and

the Seek for the Future Policy

on agricultural Biotechnology

Products Commercializattion

B.5 Akbar Ciptanto

Ethanol Production from

Glycerol of Biodiesel Plant

using Batch and Semi-

continuous Fermentation by

Enterobacter aerogenes

TISTR 1468

B.12 Lanny Hartanti

Effects of Temperature and

pH on the Capability of

Enterobacter aerogenes as

Hg remediator

C.5 Indi Hendraswari

Assessing Growth And

Microsatellite Diversity Of

Timor Mountain Gum

(Eucalyptus Urophylla St.

Blake) Provenances For Tree

Improvement Programs And

The ITB’s Golden Anniversary

International Conference on

Energy and the Environment: Reinvention for Developing Countries

Bandung, 15th-20

th of June 2009

Breeding Assistance

16.00:16.30 A.6 Ketut Gede Muliartha

DNA Methylation of Rar β2

Promotor is Epigenetic

Process on Breast Cancer

B.6 Murdinah

Biodegradable Plastic Made

Hidrocoloid Alginate Extracted

from Sargasum filifend

B.13 Sandhya Babel

Lignin recovery from pulp

and paper mill wastewater

C.6 Priya Chatterjee

Neuroethics: The Pros and Co

16.30:17.00 A.7 Reinhard Pinontoan

Identification of a Calcium-

Dependent Protein Kinase

Gene Involved in

Osmoregulation in

Chlamydomonas reinhardtii

B.7 Mohammad Faseleh

Jahromi

Solid state fermentation for

improvement of fatty acids in

byproducts

B.14 Sendy Adhika Noor

Isolation, purification and

characterization of cellulase

from giant snail (Achatina

fulica) and application to

hydrolyze cellulosic

materials

-

17.00 : 19.00 PREPARATION TO DINNER

The ITB’s Golden Anniversary

International Conference on

Energy and the Environment: Reinvention for Developing Countries

Bandung, 15th-20

th of June 2009

Day 2 : TUESDAY, JUNE 16th 2009

TIME PARALLEL SECTION

VENUE : WEST HALL VENUE : EAST HALL - 1 VENUE : EAST HALL – 2 VENUE : EAST HALL - 3

- Chairwoman : Dr. Marselina

Irasonia Tan

Chairman : Dr. Zeily

Nurachman

Chairman : -

- Biotechnology of Human Health Biotechnology of Industrial and

Application

Biotechnology of Agricultral

Productions

08.30 - 09.00 - A.8 Ricardo Ubyaan

Mitochondrial DNA Variations in

Indigenous Populations of Southern

Papua and its implications on

Native Papuan Haplogroups

B.15 Suraya

The Prospect of Gadung

(Dioscorea hispida Dennst).

Tuber as Carbon Source for

Aspergillus Niger to Produce

Amylase

-

09.00 - 09.30 - A. 9 Uun Yanuhar

The Role, Expression and

Production of Brian Receptor

Protien Of Humpback grouper

Cromileptes altivelis to Viral

Nervous Necrotic Infection for

Development of Diagnostic

Material

B.16 Tania Surya Utami

Nitrous Oxide Gas Removal

Using Compost Biofilter

-

09.30 – 10.00 - A.10 Yohanis Ngili

Nucleotide Polymorphism on D-

Loop Human Mitochondrial DNA

from Endoderm, Mesoderm, and

Ectoderm

- -

10.00 - 10.30 C O F F E E B R E A K

VENUE : EAST HALL

Session PLENARY (Chairman : Dr.Ronny Purwadi )

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Dr. Bambang Prasetya

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The ITB’s Golden Anniversary

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15.30 - 16.00 Millennium Research Agenda

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Dr. Bambang Prasetya

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16.30 - 17.00

17.00 - 19.00 PREPARATION TO DINNER

SEAN

138

Analysis Polymorphism Gene in Cattle by DNA

Sequencing

Tety Hartatik, Tri Satya Mastuti Widi and Muhammad Cahyadi

Department of Animal Production, Faculty of Animal Science,

Gadjah Mada University, Yogyakarta

Jl. Fauna No. 3 Bulaksumur Yogyakarta 55281

ABSTRACT

Studying research technologies on molecular biology is useful to improve the understanding on

molecular genetics in animal. The study of genetic variation of animal population involves the change

of allele size, genotype and phenotype. One of the molecular-based types of genetic variation is the

polymorphism of DNA sequences. Polymorphism can be identified by investigating the changes in

nucleotides which could be transition or tranversion and insertion or deletion. Mitochondrial DNA

(mtDNA) restriction fragment length polymorphism (RFLP) are useful tools for studying genetic

variation and in elaborating relationships between and within species and populations. Here we

examined the polymorphism genetics using Polymerase Chain Reaction (PCR)-base methods, PCR-

RFLP and DNA sequencing for partial mtDNA cyt b gene. We have analyzed the 464 base PCR

product of mtDNA cytochrome b gene from domestic cattle with HaeIII and HinfI restriction enzyme.

The result showed two cutting site of Hae III and one cutting site of HinfI. HaeIII produced three

fragments of 126 bp, 159 bp and 179 bp, but HinfI produced two fragments at 305 bp and 159 bp. In

this study, we also examined partial mtDNA cyt b sequence of local cattle in Indonesia such as

Madura cattle, Ongole hybrid cattle (PO), local cattle of Pacitan and crossing bred of those cattle with

exotic breed (Limousin). Madura cattle was considered as a result of cross breeding between Bos

Javanicus (Bali cattle), and Bos indicus (Ongole cattle). Local cattle of Pacitan have a distinct

phenotype with both Madura cattle and Ongole hybrid cattle, but genetically local cattle of Pacitan

have the same branch with Madura cattle. Thus, based on analysis polymorphism of cytochrome b

gene in cattle shows the diverge branch between species.

Keywords: PCR-RFLP, mtDNA cyt b, Madura cattle, local cattle of Pacitan

INTRODUCTION

Cattle are one of the most economically important domestic animals in the world. Their origin,

genetic diversity, conservation and sustainable utilization have received close attention for long time.

Bali cattle are the original cattle in Indonesia. Bali cattle were domesticated from the Banteng (Bos

Javanicus), a wild cattle species. The other local cattle breed in Indonesia is the Madura cattle. It was

considered as a result of cross breeding between Bos Javanicus, and Bos indicus. Bos indicus (Ongole

cattle) was imported to Indonesia in 1930s. Ongole were used in cross breeding programs to generate

Ongole hybrid (PO) cattle as they can adapt to hot and humid climate and a good type of working.

Madura cattle are similar to Javanese cattle, indigenous hybrids with genes from the Bos indicus, Bos

taurus and Bos (bibos) species (Namikawa,1981 cited by Payne and Hodges, 1997). The local cattle

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of Pacitan are expected as Javanese cattle which lived in East Java have the phenotype of various

kinds coat color.

To study the genetic variation of domestic cattles in Indonesia, we examined the sequence of

mt-DNA cytochrome b. Mitochondrial DNA evolves much faster than nuclear DNA and thus contains

more sequence diversity compared to nuclear DNA, facilitating the identification of closely related

species (Brown et al., 1996). In addition, maternal inheritance of the mt-DNA generally results in lack

of heterocygosity. Mitochondrial DNA of several vertebrates, including mammals, was mainly

investigated for evolutionary studies (Wolf et al., 1999). Comparisons of nucleotide sequences of the

complete mt-DNA or of single genes, e.g. cytochrome b, were used to study inter- and intraspecies

relationships of animals in order to establish the molecular phylogeny (Birstein and DeSalle, 1998;

Birstein et al., 1998).

MATERIALS AND METHODS

Blood and ear tissue samples of cattle were collected from the smallholder of cattle in Yogyakarta,

Madura and Pacitan. As a control sample, the semen of Limousin were obtained from the inseminator

in those region. DNA was isolated from the whole blood and ear tissue using standard SDS/proteinase

K extraction (Sambrook et al, 1989) and DNA KIT high pure PCR template preparation (ROCHE). A

fragment of the mtDNA cyt b was amplified by PCR methods from 50 ng templates DNA with the

primers L14735 and H15149 (Wolf et al, 1999). PCR product were purified with the QIAquick

isolation system (Qiagen, USA) and were sequenced using BigDye Terminator v3.1 Cycle sequencing

Kit and an ABI PrismTM

310 sequencer Systems (Applied Biosystems). The prediction of restriction

enzyme sites was performed base on sequence results. A phylogenetic tree using partial mtDNA cyt b

sequence was constructed using ClustalW 1.81.

RESULTS AND DISCUSSIONS

The mtDNA cytb gene has been widely considered as one of the preferential DNA targets. In this

study, we analyzed the 464 bp PCR product of mtDNA cytochrome b gene from domestic cattle with

HaeIII and HinfI restriction enzyme. The result of restriction enzyme site analysis showed mostly the

domestic cattle of Indonesia have two cutting site of Hae III and one cutting site of HinfI. Fifty two

samples of 62 analyzed (84%) showed a digested PCR product with HaeIII produce the fragments of

126 bp, 159 bp and 179 bp. Eighty nine persen of the samples was cut with HinfI and produce two

fragments at 305 bp and 159 bp

Base on the mtDNA cyt b sequence alignment analysis, local cattle of Pacitan (Pc13) have similar

sequence similarity to Madura cattle (M1) with score 99%. We found polymorphism of mtDNA

cytochrome b gene. Only four single nucleotides were detected in 400 bp nucleotides sequence. The

four points of difference nucleotide were identified as transversiton (AC→AG), deletion (TT→T- )

and transition (AG→GA) (Figure 1). On the other hand, PO cattle of Pacitan (PO1P) and PO cattle of

DIY (PO68) showed higher number of point’s mutation. There are 34 different nucleotides (Figure 2)

which consist of two tranversions (T→G, T→A), one deletion (TC→T-) and 31 transition

((T→C(13), C→T(11), A→G(4), G→A(3)).

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Figure 1. Multiple Sequence Alignments of partial mtDNA cyt b in Madura Cattle (M1) and Local cattle of

Pacitan (Pc13)

Figure 2. Multiple Sequence Alignments of partial mtDNA cyt b in PO Cattle of DIY (PO68) and PO cattle of

Pacitan (PO1P).

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The exterior phenotype of PO1P and PO68 was similar, however we found 34 nucleotides

differences. On the contrary, Madura cattle (M1) has different coat color with Pc13, but the similarity

of DNA sequences were higher than those of PO1P and PO68. Limpo cattle of Pacitan were derived

from crossing of three breeds, Limousin, PO and local cattle itself. We compared mtDNA cyt b in

Limousin and Limpo cattle of Pacitan at Figure 3. There are 31 different nucleotides which consist of

two tranversions (T→G, C→A), one transition-deletion (TT→C-) and 28 transition ((T→C(11),

C→T(11), A→G(4), G→A(2)).

Sequence analyses indicated that mtDNA cyt b gene for Local cattle of Pacitan were closely

related to Madura cattle. Local cattle of Pacitan has distinct sequences significantly from the Ongole

hybrid (Bos indicus) and Limousin (Bos Taurus) sequences with several point mutation, tranversion,

transition, and deletion/insertion.

mtDNA is a marker of maternal lineage, which in cattle corresponds to the history of the herd

(Nijman et al, 2003). Neighbor-Joining tree using of mtDNA cytochrome b was constructed using

ClustalW 1.81.

Figure 3. Multiple Sequence Alignments of partial mtDNA cyt b in Limpo Cattle of Pacitan and Limmousine

(Lim).

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Figure 4. Neighbor-Joining tree was constructed using ClustalW 1.81. The individual sample represented local

cattle from Pacitan (1,2,3), Madura cattle (4), Limousin as exotic breed (5) and Ongole hybrid (6).

The existence of three groups ((Bos Taurus(5), Bos Indicus (6) and Bos Javanicus (1-4)) was

confirmed (Figure 4). Madura cattle and local cattle of Pacitan shows the diverge branch from Bos

Taurus and Bos Indicus. This branch indicate that local cattle of Pacitan and the new hybrids of these

cattle are originated from Bos Javanicus.

CONCLUSION

Local cattle of Pacitan indicate the spesific characteristic exterior and specific mtDNA cyt b. For

preventing genetics erosion of local cattle by cross breeding, further investigation on local cattle in

Indonesia is necessary. It is needed to study the molecular genetics of local cattle and to conserve the

local genetics resources.

ACKNOWLEDGMENTS

This work was partly supported by Research Grant from Gadjah Mada University through Hibah

Bersaing XVI year 2008. We thank Prof. Masahiko Hatano and Prof. Takeshi Tokuhisa for laboratory

facilities and valuable things related to sequencing DNA. We thank Mrs. Hanasono for technical

guidance in Biomedical Research Center, Chiba University. We also thank the staff of veterinary

services in Pacitan Regency and Madura island, and the students who spent the time for sample

collection.

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