Ch2. Genome Organization and Evolution (continue)

Ch2. Genome Organization Ch2. Genome Organization and Evolution (continue)and Evolution (continue)

阮雪芬阮雪芬Jan02, 2003Jan02, 2003

NTUSTNTUST

Pick out Genes in GenomesPick out Genes in Genomes

• Open reading frames (ORFs)– Start codon------------------stop codon– A potential protein-coding region

• Approaches to identify protein-coding regions– Detection of regions similar to known coding regions f

rom other organisms– Ab inition methods

• It is more complete and accurate for bacteria than eukaryotes


• A framework for ab initio gene identification in eukaryotic genomes

Genomes of ProkaryotesGenomes of Prokaryotes

• Most prokaryotic cells contain – A large single circular piece of double-

stranded DNA (< 5 Mb)– Plasmids

• E. coli only ~11% of the DNA is non-coding.

The Genome of the Bacterium The Genome of the Bacterium E. E. colicoli

• Strain K-12 contains 4639221 bp in a single circular DNA molecules, with no plastids.

• An inventory reveals– 4285 protein-coding genes– 122 structural RNA genes– Non-coding repeat sequences– Regulatory elements– Transcription/translation guides– Transposase– Prophage remnants– Insertion sequence elements– Patches of unusual composition

大腸桿菌


• The average size of an ORF is 317 amino acids.

• 630-700 operons, operons vary in size, although few contain more than five genes. Genes within operons vary to have related functions.


• Several features of E. coli– It can synthesize all components of proteins

and nucleic acids, and cofactors.– It has metabolic flexibility– A wide range of transporters– Even for specific metabolic reactions there

are many cases of multiple enzymes.– Does not posses a complete range of

enzymatic capacity.

The genome of the archaeon The genome of the archaeon MethMethanococcus jannnaschiianococcus jannnaschii

• Methanococcus jannnaschii was collected from a hydrothermal vent 2600m deep off the coast of Baja California, Mexico, in 1983.

• Thermophilic organism• The genome was sequenced in 1996 by T

he Institute for Genomic Research (TIGR). It was the first archaeal genome sequenced.

古甲烷球菌

The genome of the archaeon The genome of the archaeon MethMethanococcus jannnaschiianococcus jannnaschii

• It contains a large chromosome containing a circular double-stranded DNA molecule 1664976 bp long.

• 1743 predicted coding regions.• Some RNA genes contain introns.• As in other prokaryotic genomes there is a little n

on-coding DNA.• In archaea, protein involved in transcription, tran

slation, and regulation are more similar to those of eukaryotes.

• Archaeal proteins involved in metabolism are more similar to those of bacteria.

The genome of one of the simplest The genome of one of the simplest organisms: organisms: Mycoplasma genitaliumMycoplasma genitalium• An infectious bacterium.• Its genome was sequenced in 1995 by TIGR, Th

e Johns Hopkins University and The University of North Carolina.

• The gene repertoire includes some that encode proteins– DNA replication– Transcription– Translation– Adhesions– Other molecules for defence against the host’s immun

e system.– Transport proteins

黴漿菌

Genomes of EukaryotesGenomes of Eukaryotes

• In eukaryotic cells, the majority of DNA is in the nucleus, separated into bundles of nucleoproteins, the chromosomes.

• Each chromosome contains a single double-stranded DNA molecule.

• Nuclear genomes of different species vary widely in size.

• Eukaryotic species vary in the number of chromosomes and distribution of genes among them.– Human chromosome 2~~a fusion of chimpanzee

chromosomes 12 and 13.


• Saccaromyces cerevisiae (Ibaker’s yeast)– Protein-protein interaction

• Yeast two-hybrid system

Yeast Two-hybrid SystemYeast Two-hybrid System

• Useful in the study of various interactions• The technology was originally developed during

the late 1980's in the laboratory Dr. Stanley Fields (see Fields and Song, 1989, Nature).


GAL4 DNA-binding

domain

GAL4 DNA-activation domain

Nature, 2000


• Library-based yeast two-hybrid screening method

Nature, 2000

Protein-protein Interactions on Protein-protein Interactions on the Webthe Web

• Yeast http://depts.washington.edu/sfields/yplm/data/index.html

http://portal.curagen.com

http://mips.gsf.de/proj/yeast/CYGD/interaction/

http://www.pnas.org/cgi/content/full/97/3/1143/DC1

http://dip.doe-mbi.ucla.edu/

http://genome.c.kanazawa-u.ac.jp/Y2H

• C. Elegans http://cancerbiology.dfci.harvard.edu/cancerbiology/ResLabs/Vidal/

• H. Pylori

http://pim/hybrigenics.com

• Drosophila

http://gifts.univ-mrs.fr/FlyNets/Flynets_home_page.html

Yeast Protein Linkage Map Yeast Protein Linkage Map DataData

• New protein-protein interactions in yeast

Stanley Fields Lab http://depts.washington.edu/sfields/yplm/data

List of interactions with links to YPD

Yeast Protein Linkage Map Yeast Protein Linkage Map DataData


• Caenorhabditis elegans– The genome was completed in 1998– The first full DNA sequence of a multicellular o

rganism– XX genotype: a self-fertilizing hermaphrodite.– XO genotype: a male.


• Drosophila melanogaster– Its genome sequence was announced in 1999 by a co

llaboration between Celera Genomics and the Berkeley Drosophila Genome Project.

– Despite the fact that insects are not very closely related to mammals, the fly genome is useful in the study of human disease.

– It contains homolgues of 289 human genes implicated in various disease:

• Cancer• Cardiovascular disease….etc.


• Arabidopsis thaliana– A flowering plant– ~125 Mbp DNA

Genomes of Eukaryotes-Genomes of Eukaryotes-HumanHuman

– In Feb 2001, the International Human Genome Sequencing Consortium and Celera Genomics published, separately, drafts of the human genome.

– 22 chromosome pairs +X, Y – Protein coding gene

• ~32000 genes in all


– Nucleic acid binding– Transcription factor binding– Cell cycle regulator– Chaperone– Motor– Actin binding– Defense/immunity protein– Enzyme– Enzyme activator– Enzyme inhibitor

– Apoptosis– Signal transduction– Storage protein– Cell adhesion– Structural protein– Transporter– Ligand binding or carrier– Tumour suppressor– Unclassified

•Human protein coding gene


• Repeat sequences– 50% of the genome– Contain

• Transposable elements• Retroposed pseudogenes• Simple “sutters”• Segmental duplications• Blocks of tandem repeats


• RNA– 497 transfer RNA genes– Genes for 28S and 5.8S ribosomal RNAs– Small nucleolar RNAs– Spliceosomal snRNAs

SNPsSNPs

• Single-nucleotide polymorphisms (SNPs)– A genetic variation between individuals, limite

d to a single base pair which can be substituted, inserted or deleted.

– Sickle-cell anaemia is an example of a disease caused by a specific SNP

• AT mutation in the beta-globin gene changes a GluVal

SNPsSNPs

• Single-nucleotide polymorphisms (SNPs)– Nearly 1.8 million SNPs – Occurring on the average every 2000 base pa

irs.– Not all SNPs are linked to disease– The A, B, and O alleles of genes for blood gro

ups illustrate these possibilities.• A and B alleles differ by four SNP substitutions.

ABO Blood GroupsABO Blood Groups

The human ABO blood groups illustrate the effect of glycosyl-transferases.

N-acetylgalactosamine Galactose

Evolution of GenomesEvolution of Genomes

• Synonymous nucleotide substitution

• Non- synonymous nucleotide substitution Ka = the number of non- synonymous

nucleotide substitution

Ks = the number of synonymous nucleotide substitution

Ka/ Ks : high ratio

possibly functional changes

Databases of Aligned Gene Databases of Aligned Gene FamiliesFamilies

ExampleExample- The Effect of RGD Mimetic - The Effect of RGD Mimetic Peptide in Breast Cancer Cell Line Peptide in Breast Cancer Cell Line

MCF7MCF7

IntroductionIntroduction

•RGD has been used as inhibitor of integrin-ligand interaction.•Loss of integrin-mediated signaling will induce apoptosis.

Control Aggregation Cell Death

RGD(Arg-Gly-Asp) is the smallest motif that bind with the integrin receptor on the cell surface and Play important role in cell cycle.

IntroductionIntroduction

Human breast cancer cell MCF-7

Cell Apotosis

Genomic Study

Proteomics

Bioinformatics

Our Study

The Structures of RGD Mimetic The Structures of RGD Mimetic PeptidesPeptides

Asp

GlyArg

NH

H2N O

O

N

O

HN

NH

O

O

OH

HN

O

HN

O

S

S

HN

O

NH

NH

H2N

ArgGly Asp

Trp

Pro

Cys

Tpa

Cyclic-RGD

RGD cRGDcontrol

1mM

5mM

0.5mM

1mM

control

cDNA MicroarraycDNA Microarray

C-RGD, 6hr C-RGD, 24hr

C-RGD, 48hr C-RGD, 72hr

Apoptosis Apoptosis

• Total 34 genes, but after filtering there are only 19 genes• Total 11 genes have expression fold >2 (up or down

changes)

Apoptosis RegulatorApoptosis Regulator

U60519

U97075

AF051941

U13738

AF005775

U60521

Z48810

AAF19819

U67319

U28976

AF015450

DescriptionGenebankaccession

No.

6 hFold Change

24 hFold Change

48 hFold Change

72 hFold Change

Group 1

caspase 10, apoptosis-related cysteine protease U60519 - - - 0.471

CASP8 and FADD-like apoptosis regulator U97075 - - - 0.355

nucleoside diphosphate kinase type 6 (inhibitorof p53-induced apoptosis-alpha) AF051941 - - - 0.376

Group 2

caspase 3, apoptosis-related cysteine protease U13738 - 2.301 - -

CASP8 and FADD-like apoptosis regulator AF005775 - 2.272 - -

Group 3

caspase 9, apoptosis-related cysteine protease U60521 - - 2.519 -

Group 4

caspase 4, apoptosis-related cysteine protease Z48810 2.615 - 2.796 2.819

Group 5

inhibitor of apoptosis protein AAF19819 - - - 5.249



Group 6

CASP8 and FADD-like apoptosis regulator AF015450 - - - 6.912

Apoptosis RegulatorApoptosis Regulator

6 7224 48

time (hour)0.01

0.1

1

10

Normalized Intensity(log scale)

p1

6 7224 48

time (hour)0.01

0.1

1

10


p1

6 7224 48

time (hour)0.01

0.1

1

10


p1

6 7224 48

time (hour)0.01

0.1

1

10


p1

6 7224 48

time (hour)0.01

0.1

1

10


p1

6 7224 48

time (hour)0.01

0.1

1

10


p1

Caspase Pathway in Caspase Pathway in CCRGD-treRGD-treated MCF7 Cellated MCF7 Cell

Caspase 10

Caspase 9 Caspase 8 and FADD Caspase 4

Caspase 7

Caspase 3

Searching and Clustering of Searching and Clustering of RGD-containing Protein in RGD-containing Protein in

Swiss-Prot DatabaseSwiss-Prot Database• In Swiss-Prot database, there are 541 human

RGD-containing protein containing 5 caspase proteins.

• Caspase 8 was clustered with integrin beta4• Caspase 1, caspase 2, caspase 3 and caspase

7 are clustered.

Please pass the genes: horizontal Please pass the genes: horizontal gene transfer gene transfer

• Horizontal gene transfer is the acquisition of genetic material by one organism from the other.– Direct uptake– Via a viral carrier

Genome DatabasesGenome Databases

• PIR

Genome DatabasesGenome Databases

• Entrez Genomes

ExercisesExercises

• Weblem 2.1

• Weblem 2.9

• Weblem 3.1

Deadline: Jan 16

Ch2. Genome Organization and Evolution (continue)

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