C6215 Advanced Biochemistry
and its Methods
Lesson 1
Introduction into Genomics
Jan Hejátko
Functional Genomics and Proteomics of Plants,
Mendel Centre for Plant Genomics and Proteomics,
Central European Institute of Technology (CEITEC), Masaryk University, Brno
hejatko@sci.muni.cz, www.ceitec.muni.cz
 Definition Of Genomics
 Forward vs Reverse Genetics
 Genes Structure and Identification
 Nucleic Acid Sequencing
 Analysis of Gene Expression
Outline
 Definition Of Genomics
Outline
 Sensu lato (in the broad sense) – it is interested in STRUCTURE
and FUNCTION of genomes
 Sensu stricto (in the narrow sense) – it is interested in
FUNCTION of INDIVIDUAL GENES – FUNCTIONAL
GENOMICS
 It uses mainly the reverse genetics approaches
 Necessary prerequisite: knowledge of the genome
(sequence) – work with databases
GENOMICS – What is it?
 Definition Of Genomics
 Forward vs Reverse Genetics
Outline
3 : 1
Forward („classical“) Genetics Approaches Reverse Genetics Approaches
?
Insertional mutagenesis
5‘TTATATATATATATTAAAAAATAAAATAAAA
GAACAAAAAAGAAAATAAAATA….3‘
GENOMICS – What is it?
The role of BIOINFORMATICS in FUNCTIONAL GENOMICS
BIOINFORMATICS
FUNCTIONAL GENOMICS
 Definition Of Genomics
 Forward vs Reverse Genetics
 Genes Structure and Identification
Outline
 Promoter
 Transcriptional
start
 5´UTR
 Translational
start
 Splicing sites
 Stop codon
 3´UTR
 Polyadenylation
signal
TATA ATG….ATTCATCAT
ATTATCTGATATA
5´UTR
3´UTR
….ATAAATAAATGCGA
Genes Structure
 Omitting 5‘ and 3‘ UTR
 Identification of translation start (ATG) and stop
codon (TAG, TAA, TGA)
 Finding donor (typically GT) and acceptor (AG)
splicing sites
 Many ORFs are not real coding sequences – in
Arabidopsis, there are on average approximately
350 milion ORFs in every 900 bp of sequence(!)
 Using various statistic models (e.g. Hidden
Markov Model – HMM, see recommended
literature, Majoros et al., 2003) to evaluate and
score the weight of identified donor and acceptor
sites
Identification of Genes Ab
Initio
 Alteration of phenotype after mutagenesis
 Forward genetics
 Identification of sequence-specific mutant
and analysis of its phenotype
 Reverse genetics
 Analysis of expression of a particular gene
and its spatiotemporal specifity
 Principles of experimental identification of
genes using forward and revers genetics
Experimental Gene
Identification
 Alteration of phenotype after mutagenesis
 Forward genetics
 Principles of experimental identification of
genes using forward and reverse genetics
Forward Genetics
Identification of CKI1 via
Activation Mutagenesis
 CKI1 overexpression mimics cytokinin response
Kakimoto, Science, 1996
NO
hormones
tZ
ctrl1 ctrl2Plasmid Rescue Pro35S::CK1
Signal Transduction via MSP
NUCLEUS
CYTOKININ
PM
AHK sensor histidine kinases
• AHK2
• AHK3
• CRE1/AHK4/WOL
REGULATION OF TRANSCRIPTION
INTERACTION WITH EFFECTOR PROTEINS
HPt Proteins
• AHP1-6
Response Regulators
• ARR1-24
 Alteration of phenotype after mutagenesis
 Forward genetics
 Identification of insertional mutant and
analysis of its phenotype
 Reverse genetics
 Principles of experimental identification of
genes using forward and revers genetics
Reverse Genetics
Identification of insertional
cki1 mutant allele
CKI1 Regulates Female
Gametophyte Development
 CKI1 is necessary for proper megagametogenesis in Arabidopsis
CKI1/CKI1CKI1/cki1-i
Hejátko et al., Mol Genet Genomics (2003)
A. ♂ wt x ♀ CKI1/cki1-i
B. ♂ CKI1/cki1-i x ♀ wt
C. ♂ wt x ♀ CKI1/cki1-i
D. ♂ CKI1/cki1-i x ♀ wt
CKI1 specific primers (PCR positive control)
cki1-i specific primers
CKI1 and
Megagametogenesis
 cki1-i is not transmitted through the female gametophyte
FG 0FG 1FG 2FG 3FG 4
CKI1 and
Megagametogenesis
cki1-iCKI1
late FG5FG6FG7
24 HAE48 HAE
CKI1 and
Megagametogenesis
Hejátko et al., Mol Genet Genomics (2003)
 Alteration of phenotype after mutagenesis
 Forward genetics
 Identification of insertional mutant and
analysis of its phenotype
 Reverse genetics
 Analysis of expression of a particular gene
and its spatiotemporal specifity
 Principles of experimental identification of
genes using forward and reverse genetics
Forward and Reverse
Genetics
CKI1 is Expressed During
Megagametogenesis
FG0-FG1
FG3-FG4
FG4-FG5
FG7
12 HAP
(hours
after
pollination)
24 HAP48 HAP72 HAP
♀ wt x ♂ ProCKI1:GUS
Paternal CKI1 is Expressed in the
Arabidopsis Sporophyte Early after
Fertilization
24 HAP
Hejátko et al., Mol Genet Genomics (2003)
 Definition Of Genomics
 Forward vs Reverse Genetics
 Genes Structure and Identification
 Nucleic Acid Sequencing
Outline
Frederick Sanger
1958 – Nobel prize – insuline structure
1975 - Dideoxy sequencing method
1980 – second Nobel prize for NA
sequencing
Sanger Sequencing
Sanger Sequencing
https://youtu.be/3M0PyxFPwkQ
NGS Sequencing
https://youtu.be/‐7GK1HXwCtE
 Definition Of Genomics
 Forward vs Reverse Genetics
 Genes Structure and Identification
 Nucleic Acid Sequencing
 Analysis of Gene Expression
Outline
 Methods of gene expression analysis
 Quantitative analysis of gene expression
 DNA chips
 Next generation transcriptional profiling
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of
analysed gene with a reporter gene
 Preparation of translational fusion of the coding
region of the analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
Gene Expression Assays
 Methods of gene expression analysis
 Quantitative analysis of gene expression
 DNA chips
Gene Expression Assays
 Method, which provides quick comparison of a large
number of genes/proteins between the test sample and
control
 Oligo DNA chips are used the most
 There are commercialy available kits for the whole genome
 company Operon (Qiagen), 29.110 of 70-mer oligonucleotides representing
26.173 genes coding proteins, 28.964 transcripts and 87 microRNA genes
of Arabidopsis thaliana
 Possibility of use for the preparation of photolithography chips – facilitation
of oligonucletide synthesis e.g. for the whole human genome (about 3,1 x
109 bp) jit is possible to prepare 25-mers in only 100 steps, by this
technique
Affymetrix ATH1 Arabidopsis genome array
 Chips not only for the analysis of
gene expression, but also for e.g.
Genotyping (SNPs, sequencing
with chips, …)
DNA Chips
 For the correct interpretation of the results, good knowledge of
advanced statistical methods is required
 Control of accuracy of the measurement (repeated
measurements on several chips with the same
sample, comparing the same samples analysed on
different chips with each other)
 It is necessary to include a sufficient number of controls and
repeats
 Control of reproducibility of measurements
(repeated measurements with different samples
isolated under the same conditions on the same
chip – comparing with each other)
Che et al., 2002
 Identification of reliable measurement treshold
nespolehlivé
spolehlivé
 Finally comparing the experiment with the control or
comparing different conditions with each other ->
the result
 Currently there‘s been a great number of results of various
experiments in publicly accessible databases
DNA Chips
 Methods of gene expression analysis
 Quantitative analysis of gene expression
 DNA chips
 Next generation transcriptional profiling
Gene Expression Assays
WT hormonal mutant
Next Gen Transcriptional
Profiling
□ Transcriptional profiling via RNA sequencing
mRNA
Sequencing by Illumina and
number of transcripts determination
mRNA
cDNA cDNA
Results of –omics Studies vs
Biologically Relevant Conclusions
□ Transcriptional profiling yielded more then 7K differentially
regulated genes…
gene locus sample_1 sample_2 status value_1 value_2 log2(fold_change) test_stat p_value q_value significant
AT1G07795 1:2414285-2414967 WT MT OK 0 1,1804 1.79769e+308
1.79769e+
308 6.88885e-05
0,00039180
1 yes
HRS1 1:4556891-4558708 WT MT OK 0 0,696583 1.79769e+308
1.79769e+
308 6.61994e-06
4.67708e-
05 yes
ATMLO14 1:9227472-9232296 WT MT OK 0 0,514609 1.79769e+308
1.79769e+
308 9.74219e-05
0,00053505
5 yes
NRT1.6 1:9400663-9403789 WT MT OK 0 0,877865 1.79769e+308
1.79769e+
308 3.2692e-08
3.50131e-
07 yes
AT1G27570 1:9575425-9582376 WT MT OK 0 2,0829 1.79769e+308
1.79769e+
308 9.76039e-06 6.647e-05 yes
AT1G60095
1:22159735-
22162419 WT MT OK 0 0,688588 1.79769e+308
1.79769e+
308 9.95901e-08
9.84992e-
07 yes
AT1G03020 1:698206-698515 WT MT OK 0 1,78859 1.79769e+308
1.79769e+
308 0,00913915 0,0277958 yes
AT1G13609 1:4662720-4663471 WT MT OK 0 3,55814 1.79769e+308
1.79769e+
308 0,00021683 0,00108079 yes
AT1G21550 1:7553100-7553876 WT MT OK 0 0,562868 1.79769e+308
1.79769e+
308 0,00115582 0,00471497 yes
AT1G22120 1:7806308-7809632 WT MT OK 0 0,617354 1.79769e+308
1.79769e+
308 2.48392e-06
1.91089e-
05 yes
AT1G31370
1:11238297-
11239363 WT MT OK 0 1,46254 1.79769e+308
1.79769e+
308 4.83523e-05
0,00028514
3 yes
APUM10
1:13253397-
13255570 WT MT OK 0 0,581031 1.79769e+308
1.79769e+
308 7.87855e-06
5.46603e-
05 yes
AT1G48700
1:18010728-
18012871 WT MT OK 0 0,556525 1.79769e+308
1.79769e+
308 6.53917e-05
0,00037473
6 yes
AT1G59077
1:21746209-
21833195 WT MT OK 0 138,886 1.79769e+308
1.79769e+
308 0,00122789 0,00496816 yes
AT1G60050
1:22121549-
22123702 WT MT OK 0 0,370087 1.79769e+308
1.79769e+
308 0,00117953 0,0048001 yes
Ddii et al., unpublished
AT4G15242 4:8705786-8706997 WT MT OK 0,00930712 17,9056 10,9098 -4,40523 1.05673e-05 7.13983e-05 yes
AT5G33251
5:12499071-
12500433 WT MT OK 0,0498375 52,2837 10,0349 -9,8119 0 0 yes
AT4G12520 4:7421055-7421738 WT MT OK 0,0195111 15,8516 9,66612 -3,90043 9.60217e-05 0,000528904 yes
AT1G60020
1:22100651-
22105276 WT MT OK 0,0118377 7,18823 9,24611 -7,50382 6.19504e-14 1.4988e-12 yes
AT5G15360 5:4987235-4989182 WT MT OK 0,0988273 56,4834 9,1587 -10,4392 0 0 yes
 Methods of gene expression analysis
 Quantitative analysis of gene expression
 DNA chips
 Next generation transcriptional profiling
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of
analysed gene with a reporter gene
Gene Expression Assays
 Identification and cloning of the promoter region of the gene
 Preparation of recombinant DNA carrying the promoter and
the reporter gene (uidA, GFP)
TATA box
Iniciation of transcription
promoter
5’ UTR
ATG…ORF of reporter gene
Transcriptional Fusion
 Identification and cloning of the promoter region of the gene
 Preparation of recombinant DNA carrying the promoter and
the reporter gene (uidA, GFP)
 Preparation of transgenic organisms carrying this
recombinant DNA and their histological analysis
Transcriptional Fusion
GUS Reporter in Mouse
Embryos
 Methods of gene expression analysis
 Quantitative analysis of gene expression
 DNA chips
 Next generation transcriptional profiling
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of
analysed gene with a reporter gene
 Preparation of translational fusion of the coding
region of the analysed gene with reporter gene
Gene Expression Assays
 Identification and cloning of the promoter and coding region of
the analyzed gene
 Preparation of a recombinant DNA carrying the promoter and
the coding sequence of the studied gene in a fusion with the
reporter gene (uidA, GFP)
TATA box
promoter
5’ UTR
ATG…ORF of analysed gene…..….ATG…ORF of reporter gene….….....STOP
Translational Fusion
 Preparation of transgenic organisms carrying the
recombinant DNA and their histological analysis
 Compared to transcriptional fusion, translation fusion allows
analysis of intercellular localization of gene product (protein)
or its dynamics
Histone 2A-GFP in Drosophila embryo by PAMPIN1-GFP in Arabidopsis
Translational Fusion
Translational Fusion
 Methods of gene expression analysis
 Quantitative analysis of gene expression
 DNA chips
 Next generation transcriptional profiling
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of
analysed gene with a reporter gene
 Preparation of translational fusion of the coding
region of the analysed gene with reporter gene
 Tissue- and cell-specific gene expression analysis
Gene Expression Assays
Fluorescence-Activated Cell Sorting (FACS)
□ High-Resolution Expression Map in Arabidopsis Root
Expression Maps - RNA
Brady et al., Science, 2007
BAR ePlant
https://bar.utoronto.ca/eplant/
□ High-Resolution Expression Map in Drosophilla
Expression Maps - RNA
Nikos Karaiskos et al. Science 2017;science.aan3235
Drosophila Virtual Expression
eXplorer
https://shiny.mdc‐berlin.de/DVEX/
Expression Maps - Proteins
Ponten et al., J Int Med, 2011
□ Human Protein Atlas
□ Human Protein Atlas
(http://www.proteinatlas.org/)
Expression Maps - Proteins
□ Human Protein Atlas
(http://www.proteinatlas.org/)
Expression Maps - Proteins
 Definition Of Genomics
 Forward vs Reverse Genetics
 Genes Structure and Identification
 Nucleic Acid Sequencing
 Analysis of Gene Expression
Summary
Discussion