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Ch2. Genome Organization and Evolution (continue). 阮雪芬 Jan02, 2003 NTUST. Pick out Genes in Genomes. Open reading frames (ORFs) Start codon ------------------  stop codon A potential protein-coding region Approaches to identify protein-coding regions

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Ch2. Genome Organization and Evolution (continue)

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Pick out genes in genomes
Pick out Genes in Genomes

  • Open reading frames (ORFs)

    • Startcodon------------------stop codon

    • A potential protein-coding region

  • Approaches to identify protein-coding regions

    • Detection of regions similar to known coding regions from other organisms

    • Ab inition methods

      • It is more complete and accurate for bacteria than eukaryotes


Pick out genes in genomes1
Pick out Genes in Genomes

  • A framework for ab initio gene identification in eukaryotic genomes



Genomes of prokaryotes
Genomes 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 e coli
The Genome of the Bacterium E. coli

大腸桿菌

  • 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


The genome of the bacterium e coli1
The Genome of the Bacterium E. coli

  • 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.


The genome of the bacterium e coli2
The Genome of the Bacterium E. coli

  • 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 methanococcus jannnaschii
The genome of the archaeon Methanococcus jannnaschii

古甲烷球菌

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


The genome of the archaeon methanococcus jannnaschii1
The genome of the archaeon Methanococcus 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 non-coding DNA.

  • In archaea, protein involved in transcription, translation, 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 organisms mycoplasma genitalium
The genome of one of the simplest organisms: Mycoplasma genitalium

黴漿菌

  • An infectious bacterium.

  • Its genome was sequenced in 1995 by TIGR, The 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 immune system.

    • Transport proteins


Genomes of eukaryotes
Genomes of Eukaryotes

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

  • Each chromosome contains a singledouble-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.


Genomes of eukaryotes1
Genomes of Eukaryotes

  • Saccaromyces cerevisiae (Ibaker’s yeast)

    • Protein-protein interaction

      • Yeast two-hybrid system


Yeast two hybrid system
Yeast 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).


Yeast two hybrid system1
Yeast Two-hybrid System

GAL4 DNA-activation domain

GAL4 DNA-binding domain

Nature, 2000


Yeast two hybrid system2
Yeast Two-hybrid System

  • Library-based yeast two-hybrid screening method

Nature, 2000


Protein protein interactions on the web
Protein-protein Interactions on the 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 data
Yeast Protein Linkage Map Data

  • New protein-protein interactions in yeast

List of interactions with links to YPD

Stanley Fields Lab

http://depts.washington.edu/sfields/yplm/data



Genomes of eukaryotes2
Genomes of Eukaryotes

  • Caenorhabditis elegans

    • The genome was completed in 1998

    • The first full DNA sequence of a multicellular organism

    • XX genotype: a self-fertilizing hermaphrodite.

    • XO genotype: a male.


Genomes of eukaryotes3
Genomes of Eukaryotes

  • Drosophila melanogaster

    • Its genome sequence was announced in 1999 by a collaboration 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.


Genomes of eukaryotes4
Genomes of Eukaryotes

  • Arabidopsis thaliana

    • A flowering plant

    • ~125 Mbp DNA


Genomes of eukaryotes human
Genomes of Eukaryotes-Human

  • 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


Genomes of eukaryotes human1

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

Genomes of Eukaryotes-Human

  • Human protein coding gene


Genomes of eukaryotes human2
Genomes of Eukaryotes-Human

  • Repeat sequences

    • 50% of the genome

    • Contain

      • Transposable elements

      • Retroposed pseudogenes

      • Simple “sutters”

      • Segmental duplications

      • Blocks of tandem repeats


Genomes of eukaryotes human3
Genomes of Eukaryotes-Human

  • RNA

    • 497 transfer RNA genes

    • Genes for 28S and 5.8S ribosomal RNAs

    • Small nucleolar RNAs

    • Spliceosomal snRNAs


Ch2 genome organization and evolution continue
SNPs

  • Single-nucleotide polymorphisms (SNPs)

    • A genetic variation between individuals, limited 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

      • AT mutation in the beta-globin gene changes a GluVal


Ch2 genome organization and evolution continue
SNPs

  • Single-nucleotide polymorphisms (SNPs)

    • Nearly 1.8 million SNPs

    • Occurring on the average every 2000 base pairs.

    • Not all SNPs are linked to disease

    • The A, B, and O alleles of genes for blood groups illustrate these possibilities.

      • A and B alleles differ by four SNP substitutions.


Abo blood groups
ABO Blood Groups

N-acetylgalactosamine

Galactose

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


Evolution of genomes
Evolution 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



Example the effect of rgd mimetic peptide in breast cancer cell line mcf7
Example- The Effect of RGD Mimetic Peptide in Breast Cancer Cell Line MCF7


Introduction
Introduction

  • RGD has been used as inhibitor of integrin-ligand interaction.

  • Loss of integrin-mediated signaling will induce apoptosis.


Introduction1
Introduction

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.

Aggregation

Cell Death

Control


Ch2 genome organization and evolution continue

Our Study

Human breast cancer cell MCF-7

Genomic Study

RGD mimetic peptides

Proteomics

Bioinformatics

Cell Apotosis


The structures of rgd mimetic peptides

Gly

Asp

Arg

Trp

Asp

Tpa

Pro

Arg

Gly

Cys

Cyclic-RGD

The Structures of RGD Mimetic Peptides


Ch2 genome organization and evolution continue

RGD

cRGD

control

control

1mM

0.5mM

5mM

1mM


Cdna microarray
cDNA Microarray

C-RGD, 24hr

C-RGD, 6hr

C-RGD, 48hr

C-RGD, 72hr


Apoptosis
Apoptosis

  • Total 34 genes, but after filtering there are only 19 genes

  • Total 11genes have expression fold >2 (up or down changes)




Caspase pathway in c rgd treated mcf7 cell
Caspase Pathway in CRGD-treated MCF7 Cell

Caspase 10

Caspase 3

Caspase 9

Caspase 8 and FADD

Caspase 4

Caspase 7


Searching and clustering of rgd containing protein in swiss prot database
Searching and Clustering of RGD-containing Protein in Swiss-Prot Database

  • In Swiss-Prot database, there are541 human RGD-containing protein containing 5 caspase proteins.

  • Caspase 8 was clustered with integrin beta4

  • Caspase 1, caspase 2, caspase 3 and caspase7 are clustered.


Please pass the genes horizontal gene transfer
Please pass the genes: horizontal gene transfer Swiss-Prot Database

  • Horizontal gene transfer is the acquisition of genetic material by one organism from the other.

    • Direct uptake

    • Via a viral carrier


Genome databases
Genome Databases Swiss-Prot Database

  • PIR


Genome databases1
Genome Databases Swiss-Prot Database

  • Entrez Genomes


Exercises
Exercises Swiss-Prot Database

  • Weblem 2.1

  • Weblem 2.9

  • Weblem 3.1

Deadline: Jan 16


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