MOLECULAR EVOLUTION STUDY OF VERTEBRATE RPS27 HOMOLOGUES

Patrick Tiong Joon Kiat

Bachelor of Science with Honours (Resource Biotechnology) ~~1 2010T594

2010

Pusal Kbidmat umat Akuem ik: vERS1TI , 1ALAYSIA SAi.,AWAJ{

P.KHICMAT MAKLUMAT AKACEMIK

111111111 IINiiili III1III11 1000212417

MOLECULAR EVOLUTION STUDY OF VERTEBRATE RPS27 HOMOLOGUES

Patrick Tiong Joon Kiat

(19685)

A Thesis subrWtted in partial fulfillment of the requirements for the degree of Bachelor of Science with Honours

(Resource Biotechnology)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK

2010

I

I

Declaration

I declare that this thesis entitled "MOLECULAR EVOLUTION STUDY OF

VERTEBRATE RPS27 HOMOLOGUES" is the result of my own research except as cited

in the references. The thesis has not been accepted for any degree and is not concurrently

submitted in candidature of any other degree .

.' Signature .... .~.... ............. .. .

Name ...~'1..~ ...-p~ ..~.~~.... .....~. 9. ....'!.?~j....~~~....................Date

i

Acknowledgements

First and foremost, I would like to thank to my supervisor, Assc. Prof. Dr. Edmund

Sim who always willing to contribute his valuable time, advice and guidance on this

project. He taught us to work independently and trigger scientific thinking among us in this

research. Besides that, I would like to thank the Committee member and participant from

Basic Phylogenetic Workshop FSTS which was held on 24 February 2010. Dr Ramlah and

Dr Leaw who introduce some basic bioinfonnatics tools that widely used in phylogenetic

research and provides basic guidance on using the PAUP*4 beta software to conduct

phylogenetic analysis. Also, I would like to take this opportunity to thank to UNIMAS,

provides free access to some of the available journal and interschool loan service for

literature required in this project.

Other than supervisor and lecturers, I greatly like to deliver my special thanks and

appreciation to my best friend, Yii Ming Leong, for his spiritual support when I feeling

frustrated during completing this proJht. Without his encouragement, I would end up with

stress and might never able to finish this project.

Finally, the greatest honour I would like to delivers to my families and friends for

their understandings and supports upon completing this project.

11

I

P t hidrnat Maklumal ('0 1 . UNlVERSrTI MALAY ~ 1A.fU...W

Table of Contents

Page

DECLARATION

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS 111

LIST OF ABBREVIATIONS v

LIST OF TABLES Vl

LIST OF FIGURES Vll

ABSTRACT Ix

CHAPTER 1 INTRODUCTION

CHAPTER 2 LITERATURE REVIEW 4 I 2.1 The structure & function of RPS27 ribosomal gene 5

2.2 The homology of the RPS27 across taxa. 5 2.3 RPS27 gene isoform 5 2.4 RPS27L gene homologues 6 2.5 Early gene evolution in vertebrates 6 2.6 . Sequence Databank (Genbank) 7 2.7 Bioinformatics tool 8

2.7.1 Multiple Sequence "tignment 8 2.7.2 Modelling of the evolutionary nucleotides sequence 9 2.7.3 Phylogeny tree reconstruction 9

CHAPTER 3 MATERIALS AND METHODS 11 3.1 Data Mining 11

3.1.1 Entrez Search 11 3.1.2 Blast Search 12 3.1.3 Optimization of sequence search 13 3.1.4 Data confinement and validation 13

3.2 Multiple Sequence analysis 13 3.2.1 Intra-species Multiple Sequence Alignment 13 3.2.2 Inter-species Multiple Sequence Alignment 14

111

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I

3.3 Protein secondary structure prediction 15 3.4 Model Selection 15 3.5 Phylogeny reconstruction 15

CHAPTER 4 RESULTS AND DISCUSSION 17 4.1 Homology relationship on RPS27 and its homologues sequences 17 4.2 The substitution patterns among RPS27 homologues 21 4.3 Evolution relationship with RPS27 protein functional properties 24 4.4 Conservation of RPS27 sequence at different regions in respects to protein 25

functionalities 4.5 Evolutionary history of RPS27 homologues inferred from phylogenetic tree 20

CHAPTER 5 CONCLUSION AND RECOMMENDATION 36

REFERENCES 38

APPENDIX A- F 41

IV

I

AIC

BIC

CI

dLRT

DNA

DOS

DT

GUI

HI

hLRT

MCMC

ML J MP

MPS-I

NCBI

NIH

NJ

PAUP*

RC

RI

RPS27

List of Abbreviations

Akaike information criteria

Bayesian information criteria

Consistency index

Dynamic likelihood ratio test

Deoxyribonucleic Acid

Disk Operating System

Decision theory method

Graphic User Interface

Homoplasy Index

Hierarchical likelihood ratio test

Markov chain Monte Carlo analyses

Maximum Likelihood

Maximum Parsimony

Metallopanstimulin-l

National Center for Biotechnology Information .~

National Institutes of Health

Neighbour-Joining

Phylogenetic Analysis Using Paximony

Rescaled Consistency Index

Retention Index

Ribosomal Protein Small Subunit 27

v

List of Tables

Table Page

Table 1 The finalised set of sequences and respective assigned annotation 18 after confinement of raw data sequence obtained from Entrez and Blastn.

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Table 2 The transition and transversion rate generated from MEGA4. 22

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VI

l. I

List of Figures

Figure

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Page

Ribbon illustration of paralogous sequence, RPS27 (left) and 20 RPS27L (right) constructed through 1-TASSER with C-score value of -0.82 and -0.92, respectively.

The conservation scores plotted against the nucleotides position 21 shows 51 sites are highly conserved (conservation score = 100) and 204 sites are variable (conservation score < 100) from a total of255 sites.

The conservation scores plotted against the amino acids 22 position shows 53 sites are highly conserved (conservation score = 100) and 31 sites are variable (conservation score < 100) from a total of 84 sites.

RPS27 homologues nucleotides alignment with single 26 translational amino acid shows highly conserved Cysteine amino acids residues (in black box) in Zinc Finger like structural domain.

RPS27 homolo:~s nuc1eotides alignment with single 28 translational amino acid shows well conserved basic clustered amino acid residues (blue box) and rather well conserved acidic (red box) and aliphatic (green box) amino acid residues.

Space filled and ribbon diagram representing the conserved 29

clusters of aliphatic and acidic amino acid residue that flanking basic amino acids residue which potentially function as nuclear localisation signal.

VII

Figure 6a Neighbour-Joining (NJ) tree analysis generated by using coding region of RPS27 mRNA homologue sequences with Branchiostoma be/cheri tsingtaunese as outgroup. The branch lengths indicate the numbers of change along each branch. The values indicate the bootstrapping with 1000 replicates.

32

Figure 6b Maximum Parsimony tree analysis (first tree) generated by using coding region of RPS27 mRNA homologue sequences with Branchiostoma be/cheri tsingtaunese as outgroup. The branch lengths indicate the numbers of change along each branch. The values indicate the bootstrapping with 1000 replicates.

33

Figure 6c Maximum Likelihood (ML) tree analysis generated from coding region of RPS27 mRNA homologue sequences with Branchiostoma be/cheri tsingtaunese as outgroup. The branch lengths indicate the number of changes along each branch. The branching values indicate the bootstrapping with 100 replicates.

34

Figure 6d Bayesian tree analysis generated by using coding region of RPS27 mRNA homologue sequences with Branchiostoma be/cheri tsingtaunese as outgroup. The branch lengths indicate the numbers of change along each branch. The branching values indicate the posterior probability.

35

V1ll

MOLECULAR EVOLUTION STUDY OF VERTEBRATE RPS27 HOMOLOGUES

Patrick Tiong Joon Kiat

Resource Biotechnology Faculty of Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

RPS17 is a protein coding gene that constitutes one of the components in small ribosomal subunit and its housekeeping fun'i tion makes RPS27 gene likely to be expressed in every cells types of all living organisms. It has unique Zinc finger like structure that have been deduced potentially function to bind and interact with nucleic acids. In this study, multiple sequence aligrunent of protein coding sequence and phylogenetic analysis were carried out to delineate the substitution pattern and the evolutionary relationship of the RPS27 homologues sequence among vertebrates. High similarity from mUltiple sequence aligrunent and protein secondary structure of the RPS27 and RPS27L genes which located in different genome location have lead to the possibility that RPS27 have undergo gene duplication during divergence of the vertebrates species and both sharing orthologs and paralogs relationships. However, the exact divergence time of RPS27 homologue are not investigated in this study. Besides that, this research also revealed that RPS27 homologous are highly conserved in the amino acids sequence and therefore reflected strict selective pressure acting on the homologues sequences to maintain indigenous functionality of the protein. Further evolutionary functionality of the protein also have been investigated, suggesting that acidic and aliphatic amino acids residue flanking the clustered basic amino acid residues have evolved in the vertebrates RPS27 proteins may facilitate the appropriate orientation of the nucleic acids from interaction with the nuclear localisation signal towards Zinc finger structure ofRPS2 7 protein.

Key words: RPS27, MPS-I , molecular evolution, gene duplication, vertebrates, bioinfonnatics

ABSTRAK

RPS27 merupakan gen pengekod protein dan merupakan salah satu komponen membentuk ribosom subunit kecil. Fungsi penting ribosom menjadikan RPS27 gen mungkin disajikan dalam semua jenis sel-sel daripada setiap organisma hidup. RPS27 protein mempunyai struktur jari Zink yang unik membolehkan ia berpotensi berfungsi un/uk mengikat dan berinteraksi dengan asid nukleik. Dalam kajian ini, penjajaran (alignment) dan analisis filogenetik dilakukan pada wutan nukleotida pengekod protein untuk menggambarkan pola subsitusi dan hubungan evolusi homolo!!{JflPS27 pada vertebrata. Hasil daripada kajian ini telah membuktikan RPS27 dan RPS27L mempunyai identiti yang tinggi dalam segi penjajaran urutan protein dan juga struktur 3D protein sedangkan gen-gen ini didapati pada lokasi genom yang berbeza. Ini telah menimbulkan kemungkinan RPS27dan RPS27L gen adalah berasal daripada gen yang sama dan merupakan hasil duplikasi sejak dari pemesonganlperbezaan spesies vertebrata. Namun, masa perbezaan RPS27 homolog pada vertebrate tidak ditelitikan dalam analisis ini. Selain itu, kajian ini juga telah mendedahkan bahawa dalam urutan asid amino RPS27 homolog dikekalkan pada tahap yang tinggi dan ini juga mencerminkan wujudnya tekanan selektif yang ketat bertindak ke atas RPS27 homolog untuk mempertahankan fungsi protein. Evolusi pada fungsi protein homologs juga telah ditelitikan, menunjukkan bahawa asid amino berasid dan alifatik residu yang mengapit antara amino asid residu berbasic dijllmpai pada vertebrata RPS27 protein memlldahkan orientasi yang sesllai oleh asid nukleik dengan interaksi penyetempalon nuklear isyarat terhadap struktllr jari Zink pada RPS2 7protein.

ta Kunci: RPS27, MPS-I, evolusi molekul, duplikasi gene, vertebrata, bioinfonnatik

IX

1.0 INTRODUCTION

Recently, ribosomal protein small subunit 27, RPS27 gene has been intensively

sequenced among species and its usefulness has been proposed to be used as the molecular

I

markers for phylogenetic analysis (Manchado, Infante, Asensio, Canavate & Douglas,

2007). Besides that, the molecular functions of RPS27 genes such its physiological roles in

association with the established isoforms (Revenkova, Masson, Koncz, Asfar, lakoveleva

& Paszkowski 1999) or differential expression in respect with cancer development (Sim,

Toh & Tiong, 2008) have been widely studied. However, the evolutionary relationships of

RPS27 homologues among vertebrate species are yet to be well established.

Ribosomal protein small subunit 27, RPS27 gene is also known as metallopanstimulin-

I, MPS-l is a protein coding gene that encodes a component of eukaryotic 40S ribosome

which function as machinery of mRNA-directed protein biosynthesis. This gene may

function as mediator of cellular proliferation (Fernandez-Pol, Dennis, & Paul, 1993) and

may exhibit extra-ribosomal functions in certain species (Revenkova et aI., 1999). The

important role in housekeeping and additional extra-ribosomal function of RPS27 genes

indicate that this gene should be present and expressed continually in most of the cells in .. vertebrates. Thus, it should exist among the eukaryotes (Ma et aI., 2005).

Along with the determination of cDNA libraries and protein sequences have

implied increasing importance on information in molecular evolutionary relationship of

RPS27 gene. In years 2005, deduced RPS27 protein from amphioxus Branchiotoma

belcheri tsingtauense which has been claimed to be extant invertebrate of the most closely

ated to proximate ancestor of vertebrates was compared with its homologues among

vertebrate species (Ma, Zhang, Liu, Li & Xia, 2005). However, the great similarities in

I

RPS27 protein sequences exist among its homologues have proven this to be highly

conserved throughout evolution.

The investigation of RPS27 gene has further carried out by Manchado et al. (2007),

RPS27 cDNA gene sequences is compared between Solea senegalensis and Hippoglossus

hippoglossus, two commercially important flatfish species with a numbers of RPS27

homologues from marines species and the phylogenetic tree has been constructed for

species identification. However, the molecular evolution of RPS27 homologues has not

explained throughout the research.

Thus, in order to discern complete evolutionary relationship among vertebrates

RPS27 homologues, a proper comparative analysis which incorporates wider taxonomic

families are required. Even so, neither proper research is conducted with the aim to access

the molecular evolution nor genetic relationship of RPS27 gene phylogenetic tree is

available. Therefore, in this study, the genetic relationship of RPS27 homologues from

broader taxonomic families of vertebrate species was assessed.

2

1.1 OBJECTIVES

The objectives of this study are:

1. To compare genetic relationship, in terms of nucleotides similarities and

differences among RPS27 gene homologues.

2. To estimate the rate and investigate pattern of the nucleotides substitution

contribute to the evolutionary of RPS27 genes among vertebrates RPS27

sequences.

3. To deduce the orthology and paralogy of RPS27 homologues from the

phylogenetic tree constructed.

3

2.0 LITERATURE REVIEW

2.1 The structure & function of RPS27 ribosomal gene

RPS27 gene is ribosomal protein coding gene which is also known as

metallopanstimulin-I (MPS-I). The product of RPS27 gene constitutes as a component of

the small ribosomal subunit (40S) of the eukaryotic ribosome. Interestingly, this ribosomal

protein has an unusual structural domain of C4-type Zinc finger like motif which is

associates with zinc ion and potentially coordinates the interaction of zinc ions with

nucleic acids such as DNA and RNA. In the secondary and tertiary structure of the C4 type

zinc finger motif found in the ribosomal protein S27 shows that presentation of the tandem

repeats of the a-helix is able to bind with DNA to major groove. These Zinc finger-like

motif structure have shared similarities with those proteins found in the DNA binding such

as the transcription factor and protein involve in response to DNA injury, however the

specific roles of this structure in the ribosome are currently unavailable. Throughout the

studies, some of researchers have also suggested that this structure might highly associate

with the function such as nuclear localization signal (Ma et al., 2005), recruitment of ..' DNA-binding protein (Revenkova et al., 1999) and binding of mRNA during translation.

4

· ",

UNJVE r '

2.2 The homology of the RPS27 across taxa

The RPS27 gene is highly conserved throughout the evolutionary process. It has

been claimed that the zinc finger structure of the RPS27 protein are vestiges of the

evolution (Revenkova et al., 1999). This is consistent with the study by Ma et al. (2005)

revealing that the cDNA coded RPS27 protein from the cephalochordate amphioxus that

recognized as the extant invertebrates which is mostly related to the ancestors of

vertebrates has higher similarities among the higher eukaryotes across the taxa, The finding

of the comparative analysis on 12 of known S27 protein sequence has been shown that, the

S27 protein from the amphioxus (AmphiS27) shares it's homology with homologues in

vertebrates such as humans, Xenopus and fish ranging from 94-99%, whereas 84-94%

among the invertebrate homologues such as annelids, mollusks and crustaceans and 69­

72% homology with other eukaryotes counterparts such as plant and yeasts (Ma, et al.,

2005). This have strengthen the fact that amphioxus as extant invertebrate that most closely

relates with common ancestor of the vertebrates and agreed by Holland and Shimeld

(2005). Therefore, the RPS27 gene sequence from the amphioxus implicates more reliable

outgroup during phylogenetic analysis.

2.3 RPS27 gene isoform

During the evolutionary process, accumulated mutation, transposition, duplication

and gene conversion are the major contribution which leads to genetic variation (Russell,

2006). In higher eukaryotes, there are a numbers of different copies of the RPS27 gene that

actively transcribed and translated into a functional protein known as isoforms

(Revenk.ova, et al. , 1999; Chan, Katsuyuki Suzuki, Olvera, & Wool, 1993) has been

5

detected located at the different location in the genome. These isofonn might derive from a

single gene. Some of the research has come out with the finding deduced that different

isofonn of this gene carries its own specific function beyond the protein synthesis. For

example, ARS27A gene is one of three RPS27 isofonn gene located in the genome of the

Arabidopsis thaliana which has been found that involves in the degradation of the mRNA

trigger by the genotoxic stress (Revenkova et ai., 1999).

2.4 RPS27L gene homologs

RPS27L also known as ribosomal RPS27 like protein is a novel protein from RPS27

gene family that yet to be well characterised (He & Sun, 2007). According to He and Sun

(2007), the RPS27L gene have shows great similarity with 96.3% identity with RPS27

protein in human although the RNA sequence are quite diversified. Meanwhile, Li and his

colleagues (2007) also have justified that both RPS27 and RPS27L are expressed in both

cancerous and normal cells. However, experimental evidences have shown that RPS27L

protein is a direct p53 inducible modulator which promotes cell apoptosis in the respond

toward genotoxic stress (He & Sun, 2007; Li et ai, 2007) .

• 2.5 Early gene evolution in vertebrates

Vertebrates are all organisms characterised with availability of backbones (Benton,

2007) and classified into different class namely Class Agnatha, Placodenni,

Chondrichthyes, Osteichthyes, Amphibia, Reptilia, Aves and Mammalia (Klappenbach,

). The evolutions of vertebrates from palaeontology data are likely to trance back that

oldest organism in ancient period as possible common ancestors. However, most of the

6

fossil evidences from many fossils specles are probably mlssmg and render it to be

impossible to unveil the entire vertebrate evolution (Benton, 2007). Meanwhile continuous

evolution in the molecular data also has been used as useful tool for the phylogenic

reconstruction. Two round genome wide duplication (2R) hypothesis is among the most

popular hypothesis which has been proposed as early vertebrates evolutionary occurred in

vertebrates species (Ohta, 1970).

2.6 Sequence Databank

The GenBank is one of the most popular sequence databank which is developing

National Institutes of Health (NIH) sequence database, where large amount of the DNA

sequence are deposited, annotated and readily retrievable at the National Center for

Biotechnology Information, NCBI website. (http://www.ncbi.nlm.nih.gov/GenbankJ). One

of the easiest ways to access this homologues gene sequence is through Homologene.

According to NCBI, the system such as Homologene which allows the automated detection

of homologues gene among the several completely sequenced eukaryotic genome. Other

than that, Entrez tools able is to provides a broad search to retrieval of DNA or protein

sequence or Medline reference relates to the molecular biology sequence database from a ..~

series of interconnected database throughout the Genbank (Mount, 2005). Therefore,

Entrez tool is able to fulfil the complete search throughout the database.

7

,

2.7 Bioinformatics tool

There are a numbers of the computation tools available online by which the

analysis can be done through the web server or downloadable from the website that aid the

various sequence and phylogeny analysis.

2.7.1 Multiple Sequence Alignment

Multiple sequence alignment is routinely carried out as a part of the phylogenetic

analysis. There are several package of computational tools widely used in performing

multiple sequence alignment which includes Clustal W & Clustal X (Larkin et al., 1997),

T-Coffee (Notredame, Higgins & Heringa, 2000), MAFFT (Katoh, Kuma, Toh & Miyata,

2005), and MUSCLE (Edgar, 2004). ClustalW and ClustalX is the oldest and widely used

program involves in the sequence analysis as it able to work in most types of PC platforms

(Larkin et ai, 2007). Clustal W program able to alignment the nucleotides or amino acids

sequence through global alignment method and this program calculate the best match each

of the sequence so that the identities, similarities and differences among the sequence can

be easily seen (http://www.ebi~.uk/2canJtutorials/nucleotide/clustalw2.html). Both

Clustal W and Clu tal X utilize pairwise progressive alignment method by which the

alignment begins with the most closely related groups of the sequence and build upon the

alignment using the rest of the sequence (Phillips, 2006).

8

1.7.1. Modelling of the evolutionary nucleotides sequence

The nucleotides substitutions of the gene are vary considerably. According to Li

and Graur (1991), the variation of the substitution rates among the gene are due to the rate

of the mutation and the probability of fixation of a mutation. Therefore, it is important to

estimates the rates of nucleotides substitution before the construction of the phylogenetic

analysis. The jModelTest is a tool by which enable researcher to carry out the statistical

selection of best-fit model of the nucleotides substitution. It was released in year 2008,

which superceded the previous version Modeltest by which implement with larger set of

the models (up to 88 models) through five model selection strategies including hierarchical

and dynamical likelihood ratio tests (hLRT and dLRT), Akaike and Bayesian infonnation

criteria (AIC and BIC), and a decision theory method (DT) (Guindon & Gascuel, 2003).

'1..7.3 Phylogenetic tree reconstruction

Phylogenetics analysis is a research field of study involves in the reconstruction of

the evolutionary process of organism or genes. In the evolutionary studies, the systematic

i ultimately based on the notion oihomology. The concept of the homology defined to be

different from the similarity by which the homologues are similar sequence in two

different organisms which has been derived from a common ancestor (Phillips, 2006).

Therefore, the gene homologues has to be detennined prior to the phylogenetic analysis

since during the construction of gene phylogeny the criteria of similarity is used. The

evolutionary relationships are presented in the fonn of bifurcating networks called

ogram (Phillips, 2006) or trees (David, 2005). The phylogenetics analysis orders of

the sequences into interested sets based on the pattern of the similarity of among the gene

9

ences family. In others words, the sequences that has highest similarities will be

clustered as neighbour sequence under the common branch in front them. During the

n:eonstruction of gene phylogenies, in some instance it will not follows the track of the

species phylogeny. This may attributes to several other genetic mechanisms such as the

horizontal gene transfer, introgression, and ancestral polymorphism (Phillips, 2006).

Phylogenetic Analysis Using Parsimony, PAUP is the most widely used program in

the inference of evolutionary trees. The Latest version of PAUP is PAUP* version 4.0

which has been upgraded and able to works with several common platforms such as

Macintosh, Windows UNIX/VMS and even in DOS based format.

10

.1.1

procedure

3.1.2

MllJ:Iank

MATERIALS AND METHODS

Data Mining

Data mining of RPS27 homologous were carried out by manipulating two major

sequence search engine provided by GenBank (http://www.ncbi.nlm.nih.govl) that is

Bntrez and Blast alignment tool.

Entrez Search

Firstly, the nucleotide database was selected from the search scroll down menu of

the Entrez search engine. The words "RPS27" was then typed into Entrez query box and

click Go button. Further constraint towards the Entrez targeted database search was

introduced by manipulation of Boolean "AND "vertebrate"[porgn:_txid7742]" which was

entered into the Entrez query box after the word "RPS27". The results of the Entrez search

were saved as collection in the GenBank and named as "Entrez RPS27 nuc1eotides". The

were repeated by replacing the search database to protein database for

nucleotide search and again the nu!& tide search database were then saved as collection

named "Entrez RPS27 proteins".

Blast Search

On the other hand, Blast search was executed from an RPS27 nuc1eotides sequence

accession number: AY168455) from Amphioxus Branchiostoma belcheri

lSingtauense which is the most approximate to the ancestor of vertebrates (Holland et al.,

11

..

200S' Ma et ai., 2005) as Blastn query sequence. Before the Blastn alignment executed, the

Blutn default searching preferences were used except the "Choose search set" and "Max

sequences" sections were altered according to parameter listed below.

Choose search set: Non redundant nuc1eotides sequence in vertebrates (taxid: 7742)

excluding Annelida (taxid:6340), Plants (taxid:3193), Fungi (taxid:4751), Synthetic

Construct (taxid:32630) and Crustaceans (taxid:6657) .

Max target sequences: 20000

A rough sequences confinement were first carried out manually based on the four

criteria, Max identity (>70%), Coverage (>80%), Expected value «r40) and Max scores

(>150). Highly similar or related sequences listed in Blast alignment output were later

saved into collection in NCBI database and named as "Blast nuc1eotides sequence".

imilar search method was also applied to protein database by using Blastp algorithm .

•1.3 Optimization of sequence search

From the Entrez and Blast search, four sets of data collections were obtained. These

• include two nucleotide and two protein collections generated from each search method

(Entrez and Blastn, respectively). These sequence collections were then merged into two

major combined collections as nucleotides and proteins collections. The respective

nucleotides sequences which hyperlinked from each proteins sequences were collected and

ubsequently combined with the nucleotides collections. The individual redundant

sequences which obtained from both Blast and Entrez that having same accession number

eliminated automatically by the NCBI database.

12

eliminate unsuitable

8IId inter-species multiple sequence alignments.

I

sets of each species

_

Data confmement and validation

In the final stage of data mining process, all the collected sequences were confined

or less meaningful sequences such as genomic sequence and

,.,adogene sequences. The redundant sequences with identical nucleotides sequence were

eel based on intra-species multiple sequence alignment stated in the next section

(_am 3.2.1). The selection of sequence used in the analysis is based on the nucleotides

Multiple Sequence Alignment

Multiple sequence alignment was carried out in two distinct stages that is the intra­

Intra-species Multiple Sequence Alignment

Intra-species Multiple Seq\\!nce Alignment was carried out to eliminate the

redundant nucleotides sequences and selection of representative sequences for species. The

sequences obtained from data mining were clustered into groups based on types of species.

Subsequently, multiple sequence alignment of RPS27 sequences (does not include RPS27L)

was carried out by using ClustalW 1.4 (Thompson, 1994)

implemented as accessory application in the BioEdit 7.0.5.3 (Hall, 1999). The nucleotides

PCilllces that consists identical sequences in coding region of the gene despite of different

eBank accession number are chosen as representative sequence for RPS27 homologues

13