Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome...
-
Upload
ann-cora-merritt -
Category
Documents
-
view
218 -
download
3
Transcript of Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome...
![Page 1: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/1.jpg)
Genome biologyGenome biology
![Page 2: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/2.jpg)
TopicsTopics1.1. DefinitionsDefinitions
2.2.The structure of the genomeThe structure of the genome
3.3.The function of the genomeThe function of the genome
4.4.Methods of genomicsMethods of genomics
![Page 3: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/3.jpg)
1.Definitions1.Definitions
![Page 4: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/4.jpg)
Definitions- 1Genome: definition 1. The information coded in the material of inheritence of an organism
definition 2. The haploid DNA (of a cell) of an organism
1. Nuclear genome 2. Mitochondrial and chloroplast genomes
Transcriptome: 1. Full transcriptom: - the total amount of mRNAs of an
organism 2. Cellular transcriptome:- the total amount mRNAs of a cell of an
organism in an experimental situation
Proteome: 1. Full proteome: - the total ammount of proteins of an organism 2. Cellular proteome: - the total ammount of proteins of a cell in an
experimental situation
![Page 5: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/5.jpg)
Definitions- 2Genomics (genome biology)
1. Structural genomics, def: genetic mapping and comparison of individuals
a. determination of the genomic sequence (human, mouse, chimpanzee, etc.)b. genome variability: intraspecific polimorfismc. genome evolution: interspecific polimorfism
2. Functional genomics:def 1: examination of the transcriptomedef 2: examination of the function of the
genes
2/1 Functional genomics-I: transcriptomics 2/2 Functional gemomics-II: proteomics
Scope: - collecting of cDNAs - measuring the differentions in mRNA expression: transcriptomics - measuring the differentions in protein expression : proteomics
![Page 6: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/6.jpg)
Definitions- 3
Alternative grouping:
• Genomics
• Functional genomics (transcriptomics)
• Proteomics
![Page 7: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/7.jpg)
Definitions-4 Other „omics”:
• Phosphorylomics:The interaction between kinases and their substrates
• Metilomics: The methylation markings of the full DNA (3-5% in
mammals)
• Metabolomics: The interactions between enzimes and their substrates
• involved in metabolism
• Interactomics: Interaction between genes
• Lipidomics: The collection of lipids
Omics: system biological approach
![Page 8: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/8.jpg)
The phosphorylome of the yeast
Red dots: kinasesBlue dots: substratesGreen lines: connections
![Page 9: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/9.jpg)
Metabolome
![Page 10: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/10.jpg)
2. The structure 2. The structure of the genomeof the genome
2a. The structure of the DNA
2b. The variation of the DNA
2c. The evolution of the DNA
![Page 11: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/11.jpg)
2a.The structure of the 2a.The structure of the DNADNA
Genome programs
The human genome
![Page 12: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/12.jpg)
Genome programs- history
1990 The genomes of the viruses
1995 The first prokaryotic genome – H. influenzae
1996 The first eukaryotic genome – yeast
1998 The first multicellular genome – C. elegans (string worm)
2000 Drosophila melanogaster, Arabidopsis thaliana (goose-weed)
2001 Human genome: draw version (90%): 30-35,000 gene
2002 Mouse genome: draw version
2004 Human genome: full version (99%): 20-25,000 gene
2005 Chimpanzee genome: draw version
![Page 13: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/13.jpg)
Genome programes- active ( 300)
a. Non mammals: Lot of viruses: small genomeE. coli: model organismOther bacteria: H. influenzae, etcAmoeba: different genomeString worm (C. elegans): model organism Fruit fly: model organismBee: livestock, intelligent insect3 wasp speciesTripanosoma + malaria gnat: health care Triboleum castaneum: pest, modell animal of beetlesSea star: modell animal Goose-weed (arabidopsis) modell, rice + coffee: agriculture
b. Mammalshuman: vanity, self-study, health caremouse, rat: model organismbovine: livestockdog: huge number of genetic variants, homogenic races (in-bred breeds)chimpanzee: relativeorangutan: Rhesus monkeyWallaby (kanguru)Marmoset (monkey)
![Page 14: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/14.jpg)
Genome programs – the competition
director, NIH National Human Genome Research Institute
Craig Venter
Francis Collins
Bill Clinton
Head of the Celera Genomics
President: USA
![Page 15: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/15.jpg)
The set-up of the human genome
–What did they found?I. Not 100,000 - 150,000 genes, but: 20,000 - 25,000 - barely more, than fruit fly and the C. elegans, but the proteome is ~10x
as big
II. The bigger part of the genome is non-coding: waste - or selfish DNA? –maybe functional?
III. Nearly all insect and string-wormal genes are inside of us as well.
IV. This is not true conversely: immunity genes: antibodies, MHC, cytokinines; apoptotic genes, etc.
V. Numerous proteins from one gene: a human gene codes for an avarige of 2,6 protein: alternative splicing
VI. More transcription factors
VII. Huge enhancer region
VIII. More complex domain structure
![Page 16: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/16.jpg)
The human genome
Total genome45% Transposable elements 25% introns + UTR20,7% other intergenic sequences5%
Simple repeats (microsatelites; VNTR-s)
Protein coding sequences
large duplications
3%
1,2%
21% LINE 13% SINE 8% 3%
Non-LTR retrotransposonsLTR - retrotransposons
DNA transposons
53% repetitive sequences
LTR:long terminal repeat (regulatory role)LINE: long interspersed elements;SINE: short interspersed elements:
![Page 17: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/17.jpg)
Total genome45% Transposable elements 25% introns + UTR5%
21% LINE 13% SINE 8% 3%
Retrotransposons DNS transposons
LTR retrotransposones(Retroviruses, and other functioning retroposons) (450,000 copies)
Non-LTR retrotransposons(degenerated viral genes)(850,000 LINE, 1500,000 SINE)
Protein coding sequences
1,2%
„Copy and paste”
„cut and paste”
The human genomeSimple repeats
3%
LINE: long interspesed elements; SINE: short interspersed elements:
20,7% other inter-genic sequences
large duplications
![Page 18: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/18.jpg)
The human genome -transposable elements
CP NC Pr RT RNáz H Int
gag pol env
LTR capsid nucleocapsid proteinase ribonuclease H envelope LTR
Reverse transcriptase integrase
CP NC Pr RT RNase HInt
gag pol
RT RNase H
gag? pol
A B
ORF
transposase
I. class
II. class
Retroviruses (1%)
SINE-s (Alu)
Retrogenes
DNA transposons
LTR retro-transposones
polyA
polyA
polyA
LINE-s (pl. L1)
I. Class: retotransposons I/1. LTR transposons I/2. Non-LTR transposons II/21. LINE-s II/22. SINE-s II/23. Retrogenes
II. Class: DNA transposons
3%
33%
IR IR
LTR: long terminal repeat
LTR LTR
7%
![Page 19: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/19.jpg)
The human genome-retroviruses
CP NC Pr RT RNase HInt
gag pol env
LTR capsid nucleocapsid proteinase ribonuclease H envelope LTR
Reverse transcriptase integrase
LTR: long terminal repeat
gag: capsid (structural elements)pol: polimerase: reverse transcriptase, integrase, proteinase, RNase H env: envelope (structural elements)
Low copy number (10-1000 copies) human endogene retroviruses are presentin 1% of the genome
![Page 20: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/20.jpg)
The human genome- Retroviral infection
![Page 21: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/21.jpg)
The human genome - retrotransposons
CP NC Pr RT RNaseHInt
gag pol
RTRNase H
gag? pol
A B
ORF I. class
SINE-s (Alu)
Retrogenes
LTR retro-transposons
polyA
polyA
polyA
LINE-s (pl. L1)
LTR retrotransposones: from human endogene retroviruses, 10 – 1000 copies
LINE-s: in human L1 is the most common; present in 100,000 copies, but Lots of them are degenerated pseudogenes (non perfect reverse transcription).The 3,500 full length (6,1 kb) L1 –s 1% have promoter and two intact ORFs. LINE mobilisation in germ line and somatic cells as well.
SINE-s: 500,000 – 900,000 Alu copies (the most succesful transposone in human). All Alu element was created from a 280 bp + polIII promoter containing 7SL RNS gene. AluI restriction enzyme recognition sites are present in them.
Reverse Transcription!
LTR LTR
I. Class: retotransposons I/1. LTR transposons I/2. Non-LTR transposons II/21. LINE-s II/22. SINE-s II/23. Retrogenes
II. Class: DNA transposons
![Page 22: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/22.jpg)
The human genome - DNA transposons
transposase II. class
DNA transposons
DNA transposons:
- The transposase responsible for the flip: how does it multiply?- More than 60 families: Charlie, mariner, Tigger, THE1, etc -The mariner family is similar to the the transposones present in insects: horizontal gene transfer?
IR IR
IR: inverted repeat
21% LINE 13% SINE 8% 3%
Retrotransposons DNA transposons
I. Class: retotransposons I/1. LTR transposons I/2. Non-LTR transposons II/21. LINE-s II/22. SINE-s II/23. Retrogenes
II. Class: DNA transposons
![Page 23: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/23.jpg)
Total genome45% Transposable elements 25% introns+ UTR5%
Simple repeats (microsatellites; VNTR-s)
Large duplications: minisatellites and macrosatellites
3%
The human genome - microsatellites, minisatellites, macrosatellites
Microsatellites: small 4 base pair long or shorter repeats: 1 – 15 kilobasepairs long-CA/TG repeats in the 0.5% of the genom – yet their function is not known, „replication slippage” - AAAAs and TTTTTs- trinucleotid repeats CAA (Glu), ACA (ala): neuronal disorders; transcription factors in dogs
Minisatellites: 1 – 15 kbs repeats: like telomer: 15 kb: TTAGGG hexamer -the telomerse forges to the end of the chromosomes
Macrosatellites: several hundred kbs repeats
Exons
1,2%
Satellites: highly repetitve sequencesDuplications: importance in evolution
DNAsatellites
20,7% other inter-genic sequences
![Page 24: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/24.jpg)
Total genome45% Transposable elements 25% introns + UTR5%
Simple repeats Protein coding sequences
Large duplications 3% 1.3%
The human genome - exons and introns
Exons: - protein coding DNA sequences + UTRs
Introns: - cut out- alternative splicing, other alternative processes.- are they functional?
leaderleader
E1E1
I1I1 E2E2 I2I2 trailertrailer
E3E3
Coding sequenceAUG Stop
pre-mRNS
polyA signal
5’-UTR 3’-UTR
20,7% other inter-genic sequences
![Page 25: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/25.jpg)
Total genome45% Transposable elements 25% introns + UTR20,7% other inter-genic sequences5%
Simple repeats
Protein coding DNA sequences
Large duplications
3%
1.3%
The human genome- other intergenic sequences
1. Unidentifyable degenerated transposones2. Pseudogenes: 2 types (reverse transcripted RNA, duplicated DNA)3. Regulatory elements: promoters, enhancers, silencers4. others
![Page 26: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/26.jpg)
2b. The variability of 2b. The variability of the DNAthe DNA
- intraspecific variability- intraspecific variability
Human genom diversity programes
The genetic code of the phenotypic variability – coding vs regulatory sequences
![Page 27: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/27.jpg)
Human genome diversity programs
-From 1990 programs to map the polimorfism of the human genome. Importance: genealogic, medical
-From 2005 Genographic Project (National Geographic)
-mtDNA-Y chromosome
Genetic markers
The practicability of the data:
- Two theories on the origin of Homo sapiens (From Homo erectus):
multiregional theory – African origin (mitochondrial Éva)
- The wanderings of modern man.
![Page 28: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/28.jpg)
Inheritence
somatic chromosomes
![Page 29: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/29.jpg)
Inheritance
somatic chromosomesY chromosomeMitochondrial DNA
![Page 30: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/30.jpg)
Inheritance
somatic chromosomesY chromosomeMitochondrial DNA
![Page 31: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/31.jpg)
Genes STR*-s
Genetic markers on the Y chromosome
STR: short tandem repeats
![Page 32: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/32.jpg)
16,569 nukleotide
Genes on the mitochondrial DNA
Hiper variable region
![Page 33: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/33.jpg)
„Common” origin„Common” origin
100.000 years ago
Homo sapiens Homo sapiens Homo neanderthalensis
Homo erectus African Homo erectus
Asia
n H
omo
erec
tus
European Homo erectus
1.8 million years ago
Hypotheses: Multiregional Origin Out of Africa------------------
√
![Page 34: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/34.jpg)
67,000 yr
African originAfrican origin
13,000 yr40-60,000 yr
20,000 yr
130,000 yr
40,000 yr
Comparison of mitochondrial DNAs: winning of „Out of Africa” hypothesis over „Multiregional Origin” hypothesis.
yr
![Page 35: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/35.jpg)
The genetic base of the phenotypic variability: coding vs regulatory sequences
1. Genes and proteins functional variance
2. The theory of neutrality
3. Intragenic variability in the regulatory regions
4. Variance in the coding region of the regulatory genes
![Page 36: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/36.jpg)
Genes and proteins - functional variance
Traditional concept
The different gene-products are responsible for the phenotypic variance in efficiency and function
![Page 37: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/37.jpg)
The theory of neutrality
The gene variants (alleles) are functionally the same !
Motoo Kimura
- The majority of gene substitutions are not responsible for amino-acid changes (sinonim changes)
- The vast majority of aminoacid substitutions do not changes the function of the protein (chemically similar aminoacid substitutions: conservative change)
- A The function of the genes did not changed through evolution, Gene variability do not cause phenotypic variability. These are true for the most genes.- except for defective genes
![Page 38: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/38.jpg)
Of Mice and ManSignificant polimorfism in the regulatory sequences- variability: expression level and tissue-specifity
Intraspecfic variability in the regulatory regions
![Page 39: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/39.jpg)
Intraspecfic variability in the regulatory regions
P gene A
P gene A
P gene A
P gene A
enhancers promotersindividuals
1
2
3
4
The gene regulation theory: variability in gene expression
Variants of gene „A” with identical function but differing controlling regions
![Page 40: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/40.jpg)
Coding variance: number of triplet repeats (number of glutamine and alanine repetitions)
1931
1976
Q19A14
Q19A13
. . . CAACAAGCACAAGCAGCA . . .
Q Q QA A AQ: glutamineA: alanine
bull terrier
Harold Garner and John W. Fondon Revival of gene function theory:
Variability in the sequence of transcription factors
runx-2 gene
Intraspecfic variability in the regulatory regions
![Page 41: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/41.jpg)
2c. The evolution of the 2c. The evolution of the DNADNA
- interspecific variability- interspecific variability
Differences between genomes
The chimpanzee in us
![Page 42: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/42.jpg)
Exaples Size (bp) Length (m)
HIV-1 9.8x103 10-6
fage 4.8x104 10-5
T4 fage 1.7x105 10-4
E. Coli 4.6x106 10-3
Drosophila 1.8x108 10-1
Mouse 3.5x109 1Dog 3.4x109 1Horse 3.3x109 1Human 3.4x109 1Corn 5.0x109 1Lilie 3.6x1010 10Amoeba 2.9x1011 100
The genome size differs between species and is not in relation with phenotypic complexity
The differences between genomes - Genome size
![Page 43: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/43.jpg)
The differences between genomes - Number of the genes compared with the total number of the cells
Number of the genes: number of the cells:
Human: 20 – 25,000 1014
Fruit fly: 13,500 -C. elegans: 19,100 959
![Page 44: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/44.jpg)
Differences between genomes Sctructural and functional differences
DNA similarity:
Human - chiken: 60%Human – mouse: 88%Human – chimpanzee: >98%
Same proteins
Ascidian –human: 80%Human – fruit fly: 40%Fruit fly – human: 61%C. elegans – human: 43%Yeast – human: 46%
Same domains:
Human – fruit fly, C. elegans > 90%,Exon shuffling in human , 2x as much gene
Rather „copy and paste”, than „cut and paste” mechanism
![Page 45: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/45.jpg)
4 theories about the differences:
1. Evolution of regulatory proteinsa. FOXP2 gene: mutation: disorder in speech and articulation; 2 amino acid differences between human and chimp b. ASPM and MCPH1 genes mutation: microcephaly; their expression levels are higher in neural precursor cells
2. Evolution of regulatory sequences A general increase in the expression level of genes in brain; it is difficult to detect it at the level of DNA
3. Retainment of juvenile characters Lack of body fur, higher brain/body weigh ratio, the form of out skull is similar to that of chimp kid
4. Neotenia theoryCompare to the rest of the body, the development of head accelerated
Human genome - chimp genome
1. Chromosome number: 23 vs. 24
2. Genetic alterations:
3. More Alu and L1 sequences in human (short repetitive sequences)
- Point mutations (complete genome)… 1.23%- Point mutations (coding sequences).... 1% - Duplications: ……………………….. 2.7%- Insertions, deletions:………………… 3.0%- Several Chromosomal rearrangements
The chimp in usThe chimp in us
![Page 46: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/46.jpg)
3. The function of the 3. The function of the genomegenome
3a. Gene expression
3b. Genome expression
3c. An astonishing RNA world
![Page 47: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/47.jpg)
cytoplasmcytoplasmnucleusnucleus
ERER
GolgiGolgi
pol-II
pre-mRNA
mRNApolyA
cap
DNADNA
protein
ribosome
RNA polimerase-II
Gene expression
![Page 48: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/48.jpg)
Alternative gene usage - instrument of complexity
Alternative……
- …promoter-usage- …splicing- …polyadenylation- …gene expression
![Page 49: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/49.jpg)
P1P1 Ex1Ex1
Coding region
pre-mRNA
mRNA
protein
TT
Alternative promoter usage
Ex2Ex2 Ex3Ex3I2I2I1I1Promoter 1 terminator
ribosome
Ex: exonI: intron
P2P2Promoter 2
Epidermal cells
pol
![Page 50: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/50.jpg)
Coding region
pre-mRNA
mRNA
protein
TTEx1Ex1 I2I2I1I1 Ex3Ex3Ex2Ex2terminator
Ex: exonI: intron
Alternative promoter usage
P1P1Promoter 2
P2P2
Neuronal cellsPromoter 1
pol
![Page 51: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/51.jpg)
Alternative RNA splicing
E2E2PP E1E1 E4E4
E1E1 E2E2 E4E4
E3E3E1E1 E3E3 E4E4
E1E1P1P1 E3E3 E4E4E2E2P2P2
E1E1 E3E3 E4E4
E2E2 E3E3 E4E4
E2E2PP E1E1 E4E4
E1E1 E2E2 E4E4
E3E3E1E1 E3E3 E3E3
PA2PA2PA1PA1
DNADNA
DNADNA
DNADNA
Alternative splicingAlternative splicing
Alternative splicing + promoter usage Alternative splicing + promoter usage
Alternative splicing + polyadenylationAlternative splicing + polyadenylation
![Page 52: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/52.jpg)
gene 2
Nerve cells
Neuron-specific enhancer
P2 TEx2 Ex3 Ex4I3I2
Cell-specific gene expression
gene 1
P1 TEx1 Ex2 Ex3I2I1
Ex1 I1
Skin cell-specific enhancer
TF
gene 2
P2 TEx2 Ex3 Ex4I3I2
gene 1
P1 TEx1 Ex2 Ex3I2I1
Ex1 I1
Skin cell-specific enhancer
S
Neuron-specific enhancer
TF
Skin cells
Cell-specific gene expression: histone pattern!
1. Type of transcription factor expressed in a cell
2. The accesability of the regulatory region of a gene
![Page 53: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/53.jpg)
The expression of the genome
Expression changes for a lot of genes:
1. Different tissues2. Between humans3. Diseased - healthy
Transcriptome
![Page 54: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/54.jpg)
Astounding RNA world
1. The larger half of the genome is transcribed
- The ncRNA coding part of the genome is 50 times longer, than the part which encodes coding RNA (genome tiling arrays, cDNA cloning)
2. There are regulatory antisense RNAs everywhere
- a significant portion of the genes are under the control of trans-asRNAs (miRNAs):
1 miRNA – more gene; 1 gene – more miRNA (transcriptome analysis)
- a significant portion of the genes are under the control of trans-asRNAs (miRNAs): (EST analysis)
- Two discoveries
![Page 55: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/55.jpg)
1. New functions of RNAs
- Traditional function: information transmission from DNA to proteins (mRNA), and conribution in this process (tRNA, rRNA)
- New functions:●RNAs are independent information carriers●RNAs regulate the expression of genetic information
2. Genetic regulation
- Traditional theory: the genetic regulation is achieved by theinteraction between transcription factors and the cis regulatoryelements (promoter, enhancer, silencer)
– Present theory: essential rule of regulatory ncRNAs
An astonishing RNA world - Two surprises
![Page 56: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/56.jpg)
coding sequences non-coding sequences non-coding/coding Species protein-coding gene genome size MB % MB % sequence ratio (db) (MB)Complete genome Human 20-25 000 2851 34 1,2 1619 57 47,5 : 1 Mouse 20-25 000 2490 31 1,3 1339 54 42,5 : 1 Fruit fly 13 500 120 22 18 53 44 2,4 : 1 C. elegans 19 000 100 26 26 33 33 1,3 : 1
Non-repetitive sequences Human 1455 33 2,3 867 60 26,1 : 1 Mouse 1422 29 2,0 811 57 28,5 : 1 Fruit fly 109 21 20 48 44 2,2 : 1 C. elegans 86 25 29 26 33 1,1 : 1
Coding – non coding
![Page 57: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/57.jpg)
Types of RNAs
Types of RNAs
RNAs
Coding RNA Non-coding RNA
mRNA tRNA rRNA aoRNA miRNA snRNA snoRNA
Transcription RNA
siRNA
Regulatory RNA
messenger transfer ribosomal small interfering micro small nuclear
antisense overlapping small nucleolar
33.
![Page 58: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/58.jpg)
a. trans-antisense RNAs: imperfect homology
b. cis-antisense RNAs: perfect homology
gene
antisense RNA
cis trans
DNA
Regulating antisense RNAs
![Page 59: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/59.jpg)
Micro RNAs
Micro RNAs
nucleus
Droshatranscription
pri-miRNA
pre-miRNA
DICER
exportin-5
RISC
mature miRNA
blocked mRNA
1 23
4
5
cytoplasm
(trans-antisense RNAs)
Discovered in 2000
34.
![Page 60: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/60.jpg)
degradation translation block
pre-miRNA
miRNA
The mechanisms of miRNA action
The mechanisms of miRNA action
The function of miRNAs: - Ontogenesis (timing), apoptosis, cell proliferation, oncogenesis
35.
![Page 61: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/61.jpg)
cis-antisense RNAs - overlapping antisense RNAs
5’3’DNA
mRNA
5’ 3’
5’ 3’
coding strand
non-coding (antisense) strand
over-lapping
RNAs
5’3’5’ 3’
5’3’5’ 3’
5’3’5’ 3’
Complete overlap
Convergent
Divergent
![Page 62: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/62.jpg)
5’3’5’ 3’
5’3’5’ 3’
1. Blocking of tranlation elongation
3. RNA interferencie (?)
RISC
RNáz RNáz
ribosome
FUNCTION
5’3’5’ 3’
FactorFactor: translation, regulation of half-life
2. Blocking of translation initiation
Overlapping RNAsOverlapping RNAs ((ciscis-antisense -antisense
RNAs)RNAs)
![Page 63: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/63.jpg)
4.Tools of Genomics4.Tools of Genomics4a. The structure of the DNA 1. cloning – genome library
construction 2. sequencing
4b. The expression of the DNA
1. cDNA library, EST library 2. DNA mikroarrays 3. Protein arrays
4c. Bioinformatics
![Page 64: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/64.jpg)
Gene libraries
Genom library: collection of clones, in wich every pieces of the genome of a particular organism can be found.
Usage: sequencing (genome projects), isolation of genes.
cDNA library: (cDNA: copy DNA) The cDNA library contains a cDNA copy of each mRNA of an organism (tissue or cell type). It represents the transcriptome.
Usage: gene structure determination, isolation of cDNSs (intronless gene).
EST library: ‘Expressed Sequence Tag’: either the 5’ or the 3’ end of a cDNA. It also represents the transcriptome of an organism (tissue or cell type), however, because of the smaller clone sizes, it can be handled, sequenced easier and faster.
Usage: determination of the transcription of a cell or tissue type (What kind of genes are expressed in this tissue?).
EST libraries were used for the rapid sequencing of the „active” genome.
![Page 65: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/65.jpg)
VectorsSeveral different vectors can be used for theconstruction of gene libraries, like:
PlasmidBacteriophage (lambda)CosmidBAC (Bacterial Artificial Chromosome)YAC (Yeast Artificial Chromosome)
Vector Maximal clone size Numb. of clones requiredfor a complete library
Plasmid 10 Kb 107
Bacteriophage 20 Kb 5 x 105
Cosmid 45 Kb 2 x 105
BAC 500 Kb 5 x 104
YAC 1 Mb 104
![Page 66: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/66.jpg)
Structure of vectors
Plasmid: replication origin, antibiotic resistance gene, multi cloning site with many unique restriction sites.
BAC: Bacterial Arteficial Chromosome. In fact, it is a plasmid, with a replication origin, that can be found on native giant plasmids (like F-factor).
![Page 67: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/67.jpg)
Construction of genome librariesFragmenting the DNAThe aim is to produce overlapping DNA fragments, that have an appropriate lenght, according to the vector content. We can use restriction endonuclease with 4pbs long recognition site, or we can physically shear the DNA (e.g. pressing through a thin capillary).The animation shows, how the overlapping DNA fragments are generated by partial digestion with an endonuclease.
![Page 68: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/68.jpg)
1 2 3 4 5 6
Constructing genome libraries2.: ligation into plasmid vector1.: partial digestion with restriction endonuclease
1
2 53 4 6
1
2 5
3
4
6
![Page 69: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/69.jpg)
3.: transforming into E. coli
3
1
2
4
5
6
3
1
2
4
5
6
Constructing genome libraries2.: ligation into plasmid vector
![Page 70: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/70.jpg)
Screening of gene librariesHybridization
5’ 3’
5’3’G T G C A C
5’3’G T G C A C
C A C G T G
probetarget
![Page 71: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/71.jpg)
Southern blotAgaroze gel electrophoresis
transfer
Nitrocellulose or plasticmembrane
Hybridization with a labeledDNA probe
![Page 72: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/72.jpg)
Screening of gene librariesColony hybridization
lysis
transfer onto membrane
hybridizationwith a labeledprobe
![Page 73: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/73.jpg)
cDNA-librariesHow can we produce cDNA?
AAAAAAAAA 3’mRNA 5’
TTTTTTTTT 5’3’cDNSfirst strand
3’ CCCCC
5’ GGGGGcDNASecondstrand
3’
1. RNA (mRNA) purification: we can use total RNA or mRNA extract
2. Reverse transcription: by using of oligo dT primers and reverse transcriptase (RNA-dependent DNA polimerase) the first strand of cDNA is synthesized
3. RNase treatment
4. Linker synthesis: the terminal deoxinucleotidil transferase (DNA polimerase, which doesn’t require any template) adds the C linker to the 3’ end
5. Second strand synthesis: oligo dG primers are added and the DNA polimerasesynthesizes the second strand of cDNA.
![Page 74: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/74.jpg)
The DNA chip (microarray)
It is for measuring the expression pattern of a large number of genes at the same time. In fact, the DNA chip is an inverted Southern blot: the known probes are covalently bound onto the glass surface. One chip contains 6-10000 gene specific probes. There are:
-cDNA-oligonucleotide and-sequencing chips.
![Page 75: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/75.jpg)
1. Preparing the chip:- printing- in situ synthesis
2. Collection of tissue samples
control treated
3. RNA purification
4. Reverse transcription (fluorescent labeling)
5. Hybridization 6. Reading
![Page 76: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/76.jpg)
The DNA chipReading and evaluation of data
![Page 77: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/77.jpg)
Protein chipHow does it work?
control treated
Protein purification
labeling
Immune reaction
detection
![Page 78: Genome biology. Topics 1. Definitions 2.The structure of the genome 3.The function of the genome 4.Methods of genomics.](https://reader038.fdocuments.net/reader038/viewer/2022110402/56649e535503460f94b4962e/html5/thumbnails/78.jpg)
Protein chipLabeling strategies
1. Direct labeling
2. Sandwitch method