CG920 Genomics
Finishing Lesson 2
Genes Identification
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
 Identification of genes ab initio
 Constructing gene-enriched libraries using
methylation filtration technology
 EST libraries
 Forward and reverse genetics
 Experimental identification of genes
 Genomic colinearity and genomic homology
 Structure of genes and searching for them
 Forward and reverse genetics approaches
 Differences between the approaches used
for identification of genes and their function
Outline
(finishing Lesson 02)
2
 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 revers genetics
Forward and reverse
genetics
3
 Alteration of phenotype after mutagenesis
 Forward genetics
 Principles of experimental identification of
genes using forward and revers genetics
Forward and reverse
genetics – summary
4
Hormonal regulations of plant development
Cloning of CKI1
 CKI1 was identified via activation mutagenesis in Arabidopsis
 Overexpresion of CKI1 leads to
CK-like response in the
hypocotyl explants
 CKI1 encodes a protein with
similarity to bacterial histidine
klinases
Kakimoto, Science (1996)
NO hormones
t-zeatin
ctrl1 ctrl2
plasmid
rescue Pro35S::CK1
5
Hormonal regulations of plant development
Signal Transduction via MSP
NUCLEUS
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
Recent Model of the CK Signaling via Multistep
Phosphorelay (MSP) Pathway
6
Hormonal regulations of plant development
 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
Forward and reverse
genetics
7
Hormonal regulations of plant development
Identification of insertional
cki1 mutant allele
8
Hormonal regulations of plant development
CKI1 Regulates Female
Gametophyte Development
 CKI1 is necessary for proper megagametogenesis in Arabidopsis
CKI1/CKI1CKI1/cki1-i
Hejátko et al., Mol Genet Genomics (2003)
9
Hormonal regulations of plant development
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
10
Hormonal regulations of plant development
FG 0FG 1FG 2FG 3FG 4
CKI1 and
megagametogenesis
11
Hormonal regulations of plant development
cki1-iCKI1
late FG5FG6FG7
24 HAE48 HAE
CKI1 and
megagametogenesis
Hejátko et al., Mol Genet Genomics (2003)
12
Hormonal regulations of plant development
 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 revers genetics
Forward and reverse
genetics
13
Hormonal regulations of plant development
CKI1 is Expressed During
Megagametogenesis
FG0-FG1
FG3-FG4
FG4-FG5
FG7
14
Hormonal regulations of plant development
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)
15
Hormonal regulations of plant development
CG020 Genomics
Bi7201 Genomics – a basic course
Lesson 3
Reverse genetics
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
16
 Literature sources for Chapter 03:
 Bioinformatics and Functional Genomics, 2009, Jonathan Pevsner, WilleyBlackwell,
Hobocken, New Jersey
http://www.bioinfbook.org/index.php
 Plant Functional Genomics, ed. Erich Grotewold, 2003, Humana Press,
Totowa, New Jersey
 Mello, C.C. and Conte Jr., D. (2004) Revealing the world of RNA interference.
Nature, 431, 338-342.
 Klinakis et al.. (2000) Genome-wide insertional mutagenesis in human cells by
the Drosophila mobile element Minos. EMBO Rep, 1, 416.
 Hansen et al.. (2003) A large-scale, gene-driven mutagenesis approach for the
functional analysis of the mouse genome. PNAS, 100, 9918.
Literature
17
„Classical genetics“ approach
1. IDENTIFICATION OF PHENOTYPE
2. GENE MAPPING
3. GENE IDENTIFICATION
- position cloning
„Reverse genetics“ approach
1. ISOLATION OF SEQUENCE-SPECIFIC
MUTANT
2. IDENTIFICATION OF
PHENOTYPE
3. PROOF OF CAUSAL RELATIONSHIP
BETWEEN INSERTION AND
PHENOTYPE
RANDOM MUTAGENESIS
„Classical“ genetics versus „reverse genetics“
approaches in functional genomics
EMS
T-DNA
(retro)transposons
18
 Analysis of phenotype and confirmation of causality between
phenotype and insertional mutation
 Mechanism of RNA interference
 Gene silencing using RNA interference
 Using unstable insertional mutagens and isolation
of revertant lines
 Co-segregation analysis
 Methods of identification of sequence-specific mutants
 Preparation of mutants collection
 Searching for sequence-specific mutants using
PCR
 Searching for sequence-specific mutants in
electronic databases
 Identification of independent insertional allele
Outline
19
 Methods of identification of sequence-specific mutants
 Preparation of mutants collection
Outline
20
• Mobile elements
• Stable elements
 Autonomous transposons (En-1)
 Non-autonomous transposons (dSpm)
 T-DNA
 They contain a gene for transponase, enabling
excision and reintegration into the genome
 At both ends they contain short inverted repeat,
which are recognized by transponase
 mutant of En/Spm transposon, which has lost
autonomy because of mutation in a gene for
transponase
 It can be activated by crossing with a line carrying
the En/Spm transposon
 completely stable, however, its insertion can lead
to chromosome rearrangements (inversions,
deletions, transpositions)
Types of insertional mutagens
21
Searching for sequence-specific mutants
by PCR
Libraries of insertional
mutants (plants)
Preparation of transgenic plants
Creating the population of mutants
22
Libraries of insertional
mutants (animals)
Transfection into human cell cultures (HeLa) or
mouse embryonic stem (ES) cells
Generating a population of mutant cell lines and
frequence-analysis of insertions
in vitro analysis or preparation of library of
insertional mutants by reintroingression ES into
mouse embryos
23
 Methods of identification of sequence-specific mutants
 Preparation of mutants collection
 Searching for sequence-specific mutants using
PCR
 PCR-based three-dimensional screening
Outline
24
Isolation of sequence-specific
mutants
1. Library of En-1 insertional mutants
• 3000 independent lines
• 5 copies per line on average
• PCR-based three-dimensional screening
• autonomous En/Spm, without selection
25
Základy genomiky III, Přístupy reverzní a přímé genetiky
 PCR-based three-dimensional screening
 Isolation of genomic DNA from the individual plants of mutant population and
creating sets of DNA („triads“, rows and columns of triads and individual trays)
p o o l 1 ......p o o l 2 8
28x3-tray pools
90
R
28 x 7 row pools
28 x 5 column pools
35
B
35
B
28 x 3 1-tray pools
3.000 mutant lines of A. thaliana (5 copies of En-1/line)
Isolation of sequence-specific
mutants
26
Základy genomiky III, Přístupy reverzní a přímé genetiky
 PCR-based three-dimensional screening
 Isolation of genomic DNA from the individual plants of mutant
population and creating sets of DNA („triads“, rows and columns of
triads and individual trays)
 Identification of positive „triad“ with PCR, blotting of PCR products
and hybridization of the PCR products with gene-specific probe
Isolation of sequence-specific
mutants
27
Základy genomiky III, Přístupy reverzní a přímé genetiky
p o o l 1 ......p o o l 2 8
28x3-tray pools
Identification of the PCR product by
hybridization with a gene-specific probe
1. 3-tray pools screen
En-1
En-8130
En-205
Xho67
1262
CKI1 CKI1
En-1
En-8130
En-205Xho67
1262
CKI1 CKI1
(2x2x28=112 PCR reactions)
3.000 mutant lines of A. thaliana (5 copies of En-1/line)
Isolation of sequence-specific
mutants
28
Základy genomiky III, Přístupy reverzní a přímé genetiky
 PCR-based three-dimensional screening
 Isolation of genomic DNA from the individual plants of mutant
population and creating sets of DNA („triads“, rows and columns of
triads and individual trays)
 Identification of positive „triad“ with PCR, blotting of PCR products and
hybridization of the PCR products with gene-specific probe
 Identification of the positive line through identification of positive tray,
row and column
Isolation of sequence-specific
mutants
29
Základy genomiky III, Přístupy reverzní a přímé genetiky
p o o l 1 ......p o o l 2 8
28x3-tray pools
Identification of the PCR product by
hybridization with a gene-specific probe
1. 3-tray pools screen
2. Identification of line carrying
the insertion
En-1
En-8130
En-205
Xho67
1262
CKI1 CKI1
En-1
En-8130
En-205Xho67
1262
CKI1 CKI1
(2x2x28=112 PCR reactions)
(another 5+7+3=15 PCR reactions)
In total: 112+15=127 PCR reactions
9 0
R
7 row pools
5 column pools
3 5
B
3 5
B
3 x 1-tray pools
3.000 mutant lines of A. thaliana (5 copies of En-1/line)
Isolation of sequence-specific
mutants
30
 Methods of identification of sequence-specific mutants
 Preparation of mutants collection
 Searching for sequence-specific mutants using
PCR
 PCR-based three-dimensional screening
 Hybridization with iPCR products on filters
Outline
31
Insertion library of dSpm mutants
 48.000 lines
 non-autonomous transposon
 SINS (sequenced insertion sites) database
 PCR searching or hybridization with iPCR filters
 The Sainsbury Laboratory (SLAT-lines),
John Innes Centre, Norwich Research Park
 DNA and seeds in Nottingham Seed Stock Centre
http://nasc.nott.ac.uk
 1.2 insertion per line on average
Isolation of sequence-specific
mutants
32
T-DNA
 Hybridization with products of iPCR on filters
 Isolation of genomic DNA from the
individoul plants of mutant
population
 Restriction endonuclease cleavage
 Ligation, formation of circular DNA
 Inverse PCR (iPCR) using the TDNA
specific primers
 Preparation of nylon filters with PCR
products in the exact position using
a robot
 Hybridization with a gene-specific
probe
Isolation of sequence-specific
mutants
33
 Methods of identification of sequence-specific mutants
 Preparation of mutants collection
 Searching for sequence-specific mutants using
PCR
 Searching for sequence-specific mutants in
electronic databases
Outline
34
Preparation of librares from population of A. thaliana
mutated by T-DNA
GABI-Kat (MPIZ, Köln)
Isolation of sequence-specific
mutants
35
Searching in electronic libraries of insertional
mutants
36
Searching in electronic libraries of insertional mutants
| | | | | | | | | | | | | | | | | | | | | | | |
gtgactaaagtgtaattaataagtga………
1923
13
37
 Analysis of phenotype and confirmation of causality between
phenotype and insertional mutation
 Using unstable insertional mutagens and isolation
of revertant lines
 Co-segregation analysis
 Methods of identification of sequence-specific mutants
 Preparation of mutants collection
 Searching for sequence-specific mutants using
PCR
 Searching for sequence-specific mutants in
electronic databases
 Identification of independent insertional allele
Outline
38
 Presence of multiple insertions in one line
Why is it necessary to analyze the causality
between the insertion and the observed
phenotype?
 Posibility of independent point mutation occurrence
 Insertions of T-DNA are often associated with chromosomal
aberrations (duplications, inversions, deletions)
39
 Co-segregation analysis
 Co-segregation of specific fragment, e.g. after insertion of T-DNA (or
exposure to EMS etc.) into the genome of the observed phenotype
cki1::En-1
+ ++ + + + + + ++
Causality between insertion
and phenotype
40
 However, excision of transposons is not always entirely accurate
– point mutations occurr – isolation of revertant lines with silent
mutation, or even isolation of the stable mutants
Use of autonomous transposons for the isolation
of new stable mutations and of revertant lines
 Transposons are often characterized by excision and reinsertion
into a nearby region – use for the isolation of new mutant alleles
41
Phenotype of silicles cki1::En-1/CKI1
cki1::En-1/CKI1 CKI1/CKI1
42
1. Isolation of revertant lines
• PCR-searching in 246 plants of segregating population
• from 90 cki1::En-1 positive plants, 9 plants had both mutant and
standard silicles
Offspring analysis
• confirmation of absention of insertion using PCR
• PCR amplification and cloning the part of the
genomic DNA at the insertion site
• sequencing
Confirmation of phenotype cki1::En-1/CKI1
43
Use of autonomous transposons for the
isolation of new stable mutations and
revertant lines
44
2. Isolation of a stable mutant line
• analysis of the phenotype of the segregating population
(CKI1/CKI1 CKI1/cki1::En-1)
• PCR analysis of plants with the mutant phenotype – identification of plants
without insertion
• PCR amplification and cloning the part of the genomic DNA at the
insertion site
• sequencing
Confirmation of phenotype cki1::En-1/CKI1
45
Use of autonomous transposons for the
isolation of new stable mutations and
revertant lines
46
 Analysis of phenotype and confirmation of causality between
phenotype and insertional mutation
 Mechanism of RNA interference
 Gene silencing using RNA interference
 Using unstable insertional mutagens and isolation
of revertant lines
 Co-segregation analysis
 Methods of identification of sequence-specific mutants
 Preparation of mutants collection
 Searching for sequence-specific mutants using
PCR
 Searching for sequence-specific mutants in
electronic databases
 Identification of independent insertional allele
Outline
47
 Molecular basis of posttranscriptional gene silencing (PTGS)
 RNAi found in plants and in Coenorhabditis elegans
 Silencing was induced by both sense and antisense RNA
(probably contamination by both during in vitro transcription)
 dsRNA induced silencing about 10-100 times more effectively
RNA interference
Waterhaus et al., PNAS (1998)
48
 Molecular basis of posttranscriptional gene silencing (PTGS)
 dsRNA induction is dependent on its own genes – gene
searching
RNA interference
RNAi rnai
Mello and Conte, Nature (2004)
49
 Molecular basis of posttranscriptional gene silencing (PTGS)
 RNAi found in Coenorhabditis elegans and in plants
 It is a natural mechanism of regulation of gene expression
in all eukaryotes
 The principle is creating dsRNA, which can be triggered in
several ways:
 By presence of foreign „aberrant“ DNA
 Specific transgenes containing inverted repeats of the cDNA parts
 Transcription of own genes for shRNA (short hairpin RNA) or miRNA
(micro RNA, endogenous hairpin RNA)
 dsRNA is processed by enzyme complex (DICER), which
leads to the formation of siRNA (short interference RNA),
which is then bound to enzyme complex RITS (RNAinduced
transcriptional silencing complex) or RISC (RNAinduced
silencing komplex)
 RISC mediates either degradation of mRNA (in case of full
similarity of siRNA and the target mRNA) or leads only to
termination of translation (in case of incomplete
homology, e.g. as in the case of miRNA)
 RITS mediates reorganization of genomic DNA
(heterochromatin formation and inhibition of transcription)
RNA interference
50
RNA-dependent RNA polymerase
short hairpin RNA
micro RNA
Mechanism of RNA interference
+ tasiRNAs
21-25 bp
Mello and Conte, Nature (2004)
51
It has been found that dsRNA might be either an intermediate or a trigger in PTGS.
In the first case, dsRNA is formed by the action of RNA-dependent RNA polymerases
(RdRPs), which use specific transcripts as a template. It is still not clear, how these
transcripts are recognized, but it might be e.g. abundant RNA that is a result of viral
amplification or transcription of foreign DNA.
It is not clear, how the foreign DNA might be recognized, possibly, lack of bound proteins on
the foreign “naked” DNA and its subsequent “signature” (e.g. by specific methylation pattern)
during packing of the foreign DNA into the chromatin structure might be involved.
The highly abundant transcripts might be recruited to the RdRPs by the defects in the RNA
processing, e.g. lack of polyadenylation.
In the case when dsRNA is a direct trigger, there are two major RNA molecules involved in
the process: Short interference RNA (siRNA) and micro RNA (miRNA), both encoded by the
endogenous DNA.
52
These two functionally similar molecules differ in their origin:
siRNAs are dominantly product of the cleavage of the long dsRNA that are produced by the
action of cellular or viral RdRPs. However, there are also endogenous genes, e.g. short
hairpin RNAs (shRNAs) allowing production of the siRNA (see the figure).
miRNAs are involved in the developmental-specific regulations and are product of
transcription of endogenous genes encoding for small dsRNAs with specific structure (see the
figure).
In addition to siRNAs, there are trans-acting siRNAs (tasiRNAs) that are a special class of
siRNAs that appear to function in development (much like miRNAs) but have a unique mode
of origin involving components of both miRNA and siRNA pathways.
Developmental regulations via miRNAs are more often used in animals then in plants.
The dsRNAs of all origins and pre miRNAs are cleaved by DICER or DICER-like (DCL)
enzyme complexes with RNAse activity, leading to production of siRNAs and miRNA,
respectively.
These small RNAs are of 21-24 bp long and bind either to RNA-induced transcriptional
silencing complex (RITS) or RNA-induced silencing complex (RISC).
53
From MacRae, I.J., Zhou, K., Li, F., Repic, A., Brooks, A.N., Cande, W.., Adams, P.D., and Doudna, J.A. (2006)
Structural basis for double-stranded RNA processing by Dicer. Science 311: 195 -198. Reprinted with permission from
AAAS. Photo credit: Heidi
Dicer and Dicer-like proteins
54
In siRNA and miRNA biogenesis, DICER or DICER-like (DCL) proteins cleave long
dsRNA or foldback (hairpin) RNA into ~ 21 – 25 nt fragments.
Dicer’s structure allows it to measure the RNA it is cleaving. Like a cook who
“dices” a carrot, DICER chops RNA into uniformly-sized pieces.
Note the two strands of the RNA molecule. The cleavage sites are indicated by
yellow arrows.
55
Reprinted by permission from Macmillan Publishers Ltd: EMBO J. Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., and Benning, C. (1998)
AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17: 170–180. Copyright 1998; Reprinted from Song, J.-J., Smith,
S.K., Hannon, G.J., and Joshua-Tor, L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305: 1434 – 1437.
with permission of AAAS.
Argonauta argoago1
Argonaute proteins
56
ARGONAUTE proteins bind small RNAs and their targets and it is an important part of both RITS
and RISC complexes.
ARGONAUTE proteins are named after the argonaute1 mutant of Arabidopsis; ago1 has thin
radial leaves and was named for the octopus Argonauta which it resembles (see the figure).
ARGONAUTE proteins were originally described as being important for plant development and for
germline stem-cell division in Drosophila melanogaster.
ARGONAUTE proteins are classified into three paralogous groups: Argonaute-like proteins, which
are similar to Arabidopsis thaliana AGO1; Piwi-like proteins, which are closely related to D.
melanogaster PIWI (P-element induced wimpy testis); and the recently identified Caenorhabditis
elegans-specific group 3 Argonautes.
Members of a new family of proteins that are involved in RNA silencing mediated by Argonautelike
and Piwi-like proteins are present in bacteria, archaea and eukaryotes, which implies that
both groups of proteins have an ancient origin.
The number of Argonaute genes that are present in different species varies. There are 8
Argonaute genes in humans (4 Argonaute-like and 4 Piwi-like), 5 in the D. melanogaster genome
(2 Argonaute-like and 3 Piwi-like), 10 Argonaute-like in A. thaliana, only 1 Argonaute-like in
Schizosaccharomyces pombe and at least 26 Argonaute genes in C. elegans (5 Argonaute-like, 3
Piwi-like and 18 group 3 Argonautes).
http://youdpreferanargonaute.com/2009/06/
57
MIR gene
RNA Pol
AAAn
AGO AAAn
RNA Pol
mRNA
AGO
AGO
RNA Pol
AGO
AGO
AAAn
siRNA
miRNA
post-transcriptional gene silencingtranscriptional gene silencing
transcriptional slicing
translational repression
binding to DNA
binding to specific
transcripts
58
MicroRNAs are encoded by MIR genes, fold into hairpin structures that are
recognized and cleaved by DCL (Dicer-like) proteins.
In summary, siRNAs-mediates silencing via post-transcriptional and transcriptional
gene silencing, while miRNAs -mediate slicing of mRNA and translational
repression.
59
The Nobel Prize in Physiology or Medicine 2006
Andrew Z. Fire Craig C. Mello
USA USA
Stanford University
School of Medicine
Stanford, CA, USA
University of
Massachusetts Medical
School
Worcester, MA, USA
b. 1959 b. 1960
“for their discovery of RNA interference - gene silencing by double-stranded RNA“
60
Mechanism of posttranscriptional gene silencing by RNA
interference (iRNA)
uidA
dsRNA
sense
antisense
61
RNAi approach using regulated
expression system
62