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The GENOME structure, function & evolution

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Page 1: The GENOME structure, function & evolution structure, function & evolution

The GENOMEThe GENOME

structure, function &

evolution

structure, function &

evolution

Page 2: The GENOME structure, function & evolution structure, function & evolution

DefinitionsDefinitions

Page 3: The GENOME structure, function & evolution structure, function & evolution

From genome to cell biochemistry From genome to cell biochemistry

GENOMEGENOME

TRANSCRIPTOMETRANSCRIPTOME

PROTEOMEPROTEOME

Biochemistry of the cellBiochemistry of the cell

Full genome: Total amount of DNA of an organism

Cellular genome: Haploid DNA content of a cell of an organism

Transcription

Translation

Proteome activity

Full transccriptome: Total amount of RNA of an organism

Cellular transcriptome: Total amount of RNA of a cell of an organism

Full proteome: Total amount of proteins of an organsim

Cellular proteom: Total amount of proteins of a cell of an organsim

Metabolome, lipome, phosphorylome, methylome, etc.

Page 4: The GENOME structure, function & evolution structure, function & evolution

Disciplines of genome biology Disciplines of genome biology

Metabolome, lipome, methylomephosphorylome, interactome, etc

Proteome activity

GENOMICSGENOMICS

TRANSCRIPTOMICSTRANSCRIPTOMICS

PROTEOMICSPROTEOMICS

Metabolomics, lipomics, Metabolomics, lipomics, methylomics, phosphorylomics…methylomics, phosphorylomics…

Structural genomics

Functional genomics

(Functional genomics)

(Functional genomics)

GENOMEGENOME

TRANSCRIPTOMETRANSCRIPTOME

PROTEOMEPROTEOME

Transcription

Translation

Page 5: The GENOME structure, function & evolution structure, function & evolution

Remarks Remarks

- Structural genomics collects elements of the DNA, the full transcriptome and proteome, but does not deal with their functions

- The scope of functional genomics:

(A) change of transcriptome and proteome

(1) in different cell types (2) healthy/diseased tissues and cells (3) treated/untreated tissues and cells

(B) Interaction between the elements of transcriptome and proteome - interaction maps

Page 6: The GENOME structure, function & evolution structure, function & evolution

Yeast protein interaction mapYeast protein interaction map

Each dots represents a protein, with connecting lines indicating interactions between pairs of proteins.

Red dots: essential proteins – an inactivating mutation is lethalGreen dots: non-essential proteins - mutation is nonlethalOrange dots: non-essential proteins - mutation leads to slow growth

Page 7: The GENOME structure, function & evolution structure, function & evolution

Yeast protein interaction map Yeast protein interaction map

Red: cell cycle Dark green: signalingDark blue: transcription, chromatin structurePink: protein and RNA transportOrange: RNA metabolism Light green: protein synthesis and turnoverBrown: cell polarityViolet: intermediate or energy metabolismLight blue: membrane biogenesis and/or traffic

Each oval represents a protein complex, with connections shown between complexes that share at least 1 protein.

Page 8: The GENOME structure, function & evolution structure, function & evolution

Yeast protein interaction map

- the complete network

Yeast protein interaction map

- the complete network

Hubs: proteins with many interactions. A much larger number of protein has only few individual connections-This architecture is thought to minimize the effect on the proteome of mutations which might inactivate individual proteins

Page 9: The GENOME structure, function & evolution structure, function & evolution

Yeast protein interaction map

- removal of party hubs

Yeast protein interaction map

- removal of party hubs

Party hubs: interact with all their partners simultaneously- their removal has little effect on the overall structure of the network

Page 10: The GENOME structure, function & evolution structure, function & evolution

Yeast protein interaction map

- removal of date hubs

Yeast protein interaction map

- removal of date hubs

Date hubs: interact with different partners at different times- their removal breaks the network into a small subnetworks

Page 11: The GENOME structure, function & evolution structure, function & evolution

Red dots: kinasesBlue dots: substratesGreen lines: connections

Phosphorylome of the yeastPhosphorylome of the yeast

Page 12: The GENOME structure, function & evolution structure, function & evolution

Gene networkGene network

nucleus

mitochondrion

ER

cytoplasm

extracellular space

In a strict sense: only transcription factors are the gene network components

Page 13: The GENOME structure, function & evolution structure, function & evolution

Structure and operation of the genome

Structure and operation of the genome

Page 14: The GENOME structure, function & evolution structure, function & evolution

Nuclear genome Mitochondrial genome

Two genomes in a cellTwo genomes in a cell

Page 15: The GENOME structure, function & evolution structure, function & evolution

16,569 nukleotid

The mitochondrial genomeThe mitochondrial genome

Mitochondrial

DNA strands

Page 16: The GENOME structure, function & evolution structure, function & evolution

Mitochondrion: arose1.5x109 years ago from a purple bacterium sp.: endosymbiosis

Mammalian mitochondrion:

- most of genes has been lost or got to the chromosome - 13 polypeptide (all of the are the enzymes for oxydative phosphorylation) - 12S and 16S rRNA genes - 22 tRNA gene

Mitochondrial DNA: several thousands copies/cell

Deviations from the universal code:

codon amino acid normally amino acid in mitochondrion

UGA stop Trp (mammals, insects, yeast, fungi) AGA Arg stop (mammals, insects)

ACG Arg stop (mammals)

AUA Ile Met (mammals, insects, yeast)

CUN Leu Thr (yeast)

CGG Arg Trp (maize)

Remark: there are alterations in the genomial DNAs in some species, too (prokaryotes and eukariotes)

The mitochondrial genomeThe mitochondrial genome

Page 17: The GENOME structure, function & evolution structure, function & evolution

The nuclear genomeThe nuclear genome

chromatin

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Genome sizeGenome size

Page 19: The GENOME structure, function & evolution structure, function & evolution

The ratio of noncoding sequences

The ratio of noncoding sequences

Prokaryotes unicellular plants/ protochordata human organisms fungi invertebrates vertebrates

%

Page 20: The GENOME structure, function & evolution structure, function & evolution

The human genome projectThe human genome project

The human variom projectThe human variom project

Human genome: 2001: raw version (90%)2004: full version (99%)

The missing 1%: repetitive sequences near the centromere

Collection of variable sequences from different individuals - primary focus on medical application

Richard Cotton

Craig Venter

Bill Clinton

Francis Collins

Page 21: The GENOME structure, function & evolution structure, function & evolution

Genome programsGenome programs

Relatives of human: chimp, orang utan

Model organisms of science: E. coli, yeast, C. elegans, fruit fly, arabidopsis, mouse

Pathogens and their vectors: viruses, bacteria, plasm. malariae + malaria mosquito

Agric. animals and plants: wheat, chick, cow, pig

Pets: dog

Others: archaebacteria, amoeba, wallabi kangaroo, etc.

Ascertaining the sequence of DNA is not enough to understand its operation!

Ascertaining the sequence of DNA is not enough to understand its operation!

Page 22: The GENOME structure, function & evolution structure, function & evolution

Initiation of DNA synthesis „A” dideoxynucleotide

Template DNABase (A)

The dATP –OH group was changed to –H ddATP

For the synthesis dATP/ddATP mixture is added (less ddATP), therefore the synthesis stops at „T”s

DNA sequencing

DNA sequencing

Frederick Sanger

1.

2.

3.

1.

2.

3.

Synthesis is terminated upon incorporation

Page 23: The GENOME structure, function & evolution structure, function & evolution

The different ddNTPs are labeled with distinct colors

detector

50 nucleotide

synthesis, then gel electrophoresis 10 nucleotide

DNA sequencing

DNA sequencing

Page 24: The GENOME structure, function & evolution structure, function & evolution

Human genome Human genome

exons

LINEs

SINEs

LTR retrotransposonsDNA transposonsSimple repeats

Large duplications

miscellaneous heterochromatin

Miscellaneous unique sequences

introns

retrotransposons

– 3,2 GB (3,2 billion base pair)– 3,2 GB (3,2 billion base pair)

LINE: long interspersed nuclear elementsSINE: short interspersed nuclear elements

Page 25: The GENOME structure, function & evolution structure, function & evolution

Coding sequencesGene-related sequences

Intergenic sequences

Non-coding RNA coding „genes”

48 MB 1152 MB 2000 MB

Gene-related sequenc. Intergenic sequences

1,5% 36% 62,5%

Coding sequences

Human genome Human genome

Page 26: The GENOME structure, function & evolution structure, function & evolution

Gene-related sequences

Intergenic sequences

pseudo-genes

gene frag-ments

introns UTRs

1,5% 36% 62,5%

1,5% 24%

10,5%

Coding sequences

Human genome Human genome

Page 27: The GENOME structure, function & evolution structure, function & evolution

othersRepeated

sequences

retroposonsDNA trans-

posonsSimple repeats

Large repeats

11%51,5%

2,8%41% 2,8% 5%

transposons

Human genome Human genome

Gene-related sequences

Intergenic sequences

pseudo-genes

gene frag-ments

introns UTRs

1,5% 36% 62,5%

1,5% 24%

10,5%

Coding sequences

Page 28: The GENOME structure, function & evolution structure, function & evolution

leaderleader

E1E1

I1I1 E2E2 I2I2 trailertrailer

E3E3

AUG Stop

pre-mRNA

polyA signal5’-UTR 3’-UTR

Amino acid coding parts of exons

leaderleader

E1E1

E2E2 trailertrailer

E3E3

Coding sequencesAUG Stop

mRNA

polyA signal5’-UTR 3’-UTR

Coding sequencesCoding sequences

Page 29: The GENOME structure, function & evolution structure, function & evolution

10-12,000 genes; the functions of the rest 10,000 genes are unknown!!

Page 30: The GENOME structure, function & evolution structure, function & evolution

UTR: regulation of translation and half-life of mRNAs

Intron: 1. genetic junks2. it can contain regulatory elements3. in case of alternative splicing it can serve as an exon

Introns and UTRsIntrons and UTRs

Page 31: The GENOME structure, function & evolution structure, function & evolution

Pseudogenes & gene fragmentsPseudogenes & gene fragments

Fossils in the genetic cemetery

2 types: 1. intron-containing: chromosomal segment duplication

2. intronless: reverse transcription, then reinsertion

Function: 1. In some cases regulation of the original gene by means of antisense interaction

2. Genetic junk

Genetic junksGene fragmentsGene fragments

PseudogenesPseudogenes

Page 32: The GENOME structure, function & evolution structure, function & evolution

Gene-related sequences

Intergenic sequences

1,5% 36% 62,5%

OthersRepetative sequences

retroposonsDNA trans-

posonsSimplerepeats

Large repeats

11%51,5%

2,8%41% 2,8% 5%transposons

Coding sequences

Page 33: The GENOME structure, function & evolution structure, function & evolution

Transposable elements in the human genomeTransposable elements in the human genome

class family copy number occurrance %

retr

otr

ansp

oso

ns

Page 34: The GENOME structure, function & evolution structure, function & evolution

Transposable elementsTransposable elements

CP NC Pr RT RNaseHInt

gag pol env

LTR capsid nucleocapsid protease ribonuclease H envelope LTR

reverse transcriptase integrase

CP NC Pr RT RNaseHInt

gag pol

RT RNaseH

gag? pol

A B

OR

transposase

I. class

II. class

Endogenous retroviruses: all inactive

1%

SINEs

DNA transposons

LTR retro-transposons8%

polyA

polyA

LINEs

I. class: retotransposons

I/1. LTR transposons I/2. Non-LTR transposons II/21. LINEs II/22. SINEs

II. class: DNA transposons

3%

33%

IR IR

LTR LTR

IR: inverted repeat

7%

1%

Page 35: The GENOME structure, function & evolution structure, function & evolution

retroposonsDNA trans-

posonsSimple repeats

Large repeats

2,8%41% 2,8% 5%

20% LINE 13% SINE 8%

LTR retrotransposons

Endogenous retroviruses (more than 20 families;

450,000 copies)

Non-LTR retrotransposons

(850,000 LINE, 1500,000 SINE)

„Copy and paste” „cut and paste”

TransposonsTransposons

degenerated virus genes

Derived from7S RNA „gene”

DNA transposons

Colonized the genome by horizontal gene transfer

Vector is unknown

LTR: long terminal regionLINE: long interspersed nuclear elementsSINE: short interspersed nuclear elements

Page 36: The GENOME structure, function & evolution structure, function & evolution

Retrovirus infection

Retrovirus infectionenvelope

capsid

Virus RNA

Page 37: The GENOME structure, function & evolution structure, function & evolution

Human endogenous retroviruses (HERVs)

& LTR-transposons

Human endogenous retroviruses (HERVs)

& LTR-transposons

gag: capsid (structural element)pol: polymerase: reverse transcriptase, integrase, protease, RNase H env: envelope (structural element)

LTR (long terminal repeat): promoter

LTR retrotransposons are compose of 8% of the genome, but only 1% of them has a structure similar to those of retroviruses, the others are degenerated. All of them are mutant: they are not able to form infective virions but, de some of them can move by the enzymes of other elements.

The genome of chimp and other monkeys contains infective retroviruses.

CP NC Pr RT RNaseHInt

gag pol env

LTR capsid nucleocapsid protease ribonuclease H envelope LTR

reverse transcriptase integrase

Page 38: The GENOME structure, function & evolution structure, function & evolution

Retroviruses and their fossilsRetroviruses and their fossils

Solitary LTR

Wild type retroviruses

Human endogenous retroviruses

....... and their fossils

Page 39: The GENOME structure, function & evolution structure, function & evolution

The effect of endogenous retroviruses on gene

expression

The effect of endogenous retroviruses on gene

expression

HERV: human endogenous retroviruses

a cellular gene

1. No effect

2. Transcription from the LTRl(HERV splice donor site can also be active)

3. The activity of LTR can be modulated

methylation

polymorphism

Cell-specific activation/inhibition

Page 40: The GENOME structure, function & evolution structure, function & evolution

Non-LTR retrotrasposonsNon-LTR retrotrasposons

CP NC Pr RNaseHInt

gag pol

RTRNaseH

gag? pol

A B SINEs

LTR retrotransposons

polyA

polyA

LINEs: autonomous transposons

LTR LTR

Non-autonomous transposons

RT

Page 41: The GENOME structure, function & evolution structure, function & evolution

LINE-okLINE-ok RTRNaseH

gag? polpolyA

- 21% of the human genome (850,000), 17% L1 (500,000), 10,000 full-length (6,1 kb), however, only 50-100 functional

- Some part of the rest can jump with the help of the enzymes of the intact ones.

- LINE mobilization both in germ line and somatic cells

LINE: long interspersed nuclear elementsSINE: short interspersed nuclear elementsIRES: internal ribosome entry site

ORF1 ORF2

IRESpromoter

RTRNaseHpolyA

DNA

RNAribosome

protease

Page 42: The GENOME structure, function & evolution structure, function & evolution

A LINE-1 „propagation”

A LINE-1 „propagation”

copying

perfect 5’-deleted 5’-deletes

+ inverted

Page 43: The GENOME structure, function & evolution structure, function & evolution

The effect of LINE-1 on the genome – formation of pseudogenes

The effect of LINE-1 on the genome – formation of pseudogenes

gene

Intronless pseudogene

Page 44: The GENOME structure, function & evolution structure, function & evolution

insertion

to the exon

to the intron

*: stop codon

L1 mRNA

The effect of LINE-1 on the genome – gene inactivation

The effect of LINE-1 on the genome – gene inactivation

to the intron

Page 45: The GENOME structure, function & evolution structure, function & evolution

The poly A signal of LINE is weak readthrough of adjacent gene exon

Gene „B”

An exon of gene „A”

Insertion of a piece of LINE and the exon of gene „A” to gene „B”

Or only the exon of gene „A”

mRNA

The effect of LINE-1 on the genome – transduction

The effect of LINE-1 on the genome – transduction

Page 46: The GENOME structure, function & evolution structure, function & evolution

SINEsSINEs A BpolyA

- 13% of the genome, 11% Alu sequences; non-protein coding

- AluI restriction enzyme recognition site

- An average SINE repeat unit 100 - 400 bp (Alu: 300 bp: 280 bp + pol III promoter)

-More than 1 million copies, the most successful transposon in human

-Ancestor: SRP (signal recognition particle; ribonucleoprotein) RNA component (7SL RNA)

Alu domain S domain

Page 47: The GENOME structure, function & evolution structure, function & evolution

The hyperparasite Alu sequences

The hyperparasite Alu sequences

Page 48: The GENOME structure, function & evolution structure, function & evolution

DNA transposonsDNA transposons

transzposase

- Infection mechanism is not known, what could be the vector?

- transposase executes the jumping: „cut and space” mechanism – how do they multiply?

- More than 60 families: Charlie, mariner, Tigger, THE1, etc

- The mariner family resembles to the those of insects transposons: horizontal gene transfer?

IR IR

IR: inverted repeat

Page 49: The GENOME structure, function & evolution structure, function & evolution

Defense by the hostDefense by the host

1. Heterochromatinization (methylation): inhibition of transcription

2. RNA interference: inhibition of transcription & translation

3. Local raise of mutation rate: inactivation

Page 50: The GENOME structure, function & evolution structure, function & evolution

Benefits of the host from the transposons

Benefits of the host from the transposons

1. Variability of genes encoding antibodies and T cell receptors

2. Genome plasticity

Page 51: The GENOME structure, function & evolution structure, function & evolution

Sleeping Beauty & Frog PrinceSleeping Beauty & Frog Prince

transgene

IR IR

transposasepromoter

Binary system

transgene

Reparated fish and. frog DNA transposons: gene therapy

Page 52: The GENOME structure, function & evolution structure, function & evolution

Tandem repeatsTandem repeats

Microsatellites : small 1-5 base pairs repetitions: up to several hundreds repetitions

- CA/TG repeats 0.5% of the genome – no known function

- Trinucleotid repeats: CAA (Gln), ACA (ala): neurodegenerative diseases; dog transcription factors

genomial DNA

satellites

(Macro)satellites: 1 – several hundred kb repetitions: centromer and constitutive heterochromatin

- Telomer: 15 kb: TTAGGG hexamer – telomerase attaches to the ends of chromosomes

- Satellite 2 and 3: GGAAT

- Alpha satellite: 171 bp units

STR: short tandem repeats; VNTR: variable number of tandem repeat

Minisatellites (VNTR*, STR*): shorter than macrosatellites.

Genetic markers (paternal test, descent); related to several diseases, e.g. diabetes

Consecutive identical or close o identical (degenerated) repeat units

Variability in the length of: (1) repeat unit and (2) the whole repeat

Page 53: The GENOME structure, function & evolution structure, function & evolution

1. Unrecognizably degraded transposons, pseudogenes

2. Regulatory regions: promoters, enhancers, silencers

3. others

Other intergenic sequences

Other intergenic sequences

Page 54: The GENOME structure, function & evolution structure, function & evolution

A new RNA worldA new RNA world

1. The major part of the genome is transcriptionally active

- ncRNAs are encompassing 50x longer genomic region than genes

2. Antisense regulation

- trans-antisense RNAs (miRNAs) 1 miRNA – more gene; 1 gene – more miRNAs

- cis-atiszense RNAs: a huge amount of gene is overlapped by antisense transcripts

Page 55: The GENOME structure, function & evolution structure, function & evolution

Novel functions of RNAs

- Traditional functions: transmission of information between DNA and proteins and other contributions to these processes

- New functions:● Independent carriers of informations (?)●Regulation of the manifestation of genetic information

A new RNA worldA new RNA world

Page 56: The GENOME structure, function & evolution structure, function & evolution

1. Preparation of the chip

- printing

2. Collection of tissue samples

control treated

3. RNA purification

4. Reverse transcription (fluorescent labeling)

5. Hybridization 6. Detection

DNA chipDNA chip

Page 57: The GENOME structure, function & evolution structure, function & evolution

Protein chipProtein chipcontrol treated

Protein purification

labeling

immunoreaction

detection

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The Evolution of genomeThe Evolution of genome

Page 59: The GENOME structure, function & evolution structure, function & evolution

Gene function does not change even across large evolutionary distancesGene function does not change even across large evolutionary distances

- many homologous genes of mouse and fruit fly are interchangable- many homologous genes of mouse and fruit fly are interchangable

Evolution alters gene expression and not gene functionEvolution alters gene expression and not gene function - in different species, the same genes are turned on at different time in different tissues and - in different species, the same genes are turned on at different time in different tissues and are expressed in different amountare expressed in different amount

Evolution of genetic regulationEvolution of genetic regulation

Expression of Expression of 11056 gene in the liver056 gene in the liver:

-------------------------

Expression of 12,000 gene in brainExpression of 12,000 gene in brain: in human 5,6x times higher expression level(human chimp)

rhesus macaco

orangutan

chimp

humanThe same gene expression

Expression of transcription factors differs

Page 60: The GENOME structure, function & evolution structure, function & evolution

Duplication of „B” gene segment

A domain B domain C domain

A domain B domain B domain C domain

Exon/domain duplicationExon/domain duplication

Page 61: The GENOME structure, function & evolution structure, function & evolution

Exon/domain shuffleExon/domain shuffle

A domain B domain C domain X domain Y domain

A domain B domain Y domain

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Exon/domain shuffleExon/domain shuffle

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