CG920 Genomics
Lesson 4
Forward Genetics
Jan Hejátko
Functional Genomics and Proteomics of Plants,
CEITEC - Central European Institute of Technology
And
National Centre for Bimolecular Research,
Faculty of Science,
Masaryk University, Brno
hejatko@sci.muni.cz, www.ceitec.eu
2
2
 Forward vs. Reverse Genetics
 Use of Libraries of Insertional Mutants in Forward Genetics
 Searching in Libraries of Insertional Mutants According to:
 anatomically or morphologically detectable phenotype
 metabolic profile
 expression of genes of interest
 Identification of the Mutated Locus
 plasmid rescue
 iPCR
 Use of Libraries of Point Mutants in Forward Genetics
 Positional Cloning
Outline
3
3
 Forward vs. Reverse Genetics
Outline
4
„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
4
5
5
 Forward vs. Reverse Genetics
 Use of Libraries of Insertional Mutants in Forward Genetics
 Searching in Libraries of Insertional Mutants According to:
 anatomically or morphologically detectable phenotype
Outline
6
6
Insertional mutagenesis in
forward genetics approaches
 Use of insertional mutagenesis for study of
carcinogenesis
 Infection of EμMyc mice by MoMuLV retrovirus leads to lymphomas
formation, which arose due to activation of Pim kinases (40 % activation
of Pim1, 15 % activation of Pim2), molecular targets of these kinases
were unknown
Mikkers et al., Nature Gen (2002)
?
7
7
 Infection of EμMyc pim1 mutants by MoMuLV retrovirus leads to
lymphomas formation, which in 90 % contain insertion nearby
(activation) Pim2
 Use of insertional mutagenesis for study of
carcinogenesis
Mikkers et al., Nature Gen (2002)
?
Insertional mutagenesis in
forward genetics approaches
8
8
 Use of insertional mutagenesis for study of
carcinogenesis
 Infection of EμMyc double mutants pim1, pim2 by MoMuLV retrovirus leads to
lymphomas formation, which can be expected to activate either one of the
signalling partner of Pim proteins (Y), one of the downsteram proteins of Pim
signalling pathway (X) or to activate some of the related pathways leading to
lymphomagenesis (Z).
Mikkers et al., Nature Gen (2002)
?
Insertional mutagenesis in
forward genetics approaches
9
9
 Cleavage of genomic DNA and ligation
of special linkers, so-called splincerettes
(increasing the specifity of amplification)
 Isolation of genomic regions adjacent to the insertion site
of the provirus
Mikkers et al., Nature Gen (2002)
Devon et al., Nucl Acid Res (1994)
Insertional mutagenesis in
forward genetics approaches
10
10
 First amplification using specific primers
 Isolation of genomic regions adjacent to the insertion site
of the provirus
Mikkers et al., Nature Gen (2002)
Devon et al., Nucl Acid Res (1994)
Insertional mutagenesis in
forward genetics approaches
11
11
 Second amplification using nested
primers (increasing the specifity)
 Isolation of genomic regions adjacent to the insertion site
of the provirus
Mikkers et al., Nature Gen (2002)
Devon et al., Nucl Acid Res (1994)
Insertional mutagenesis in
forward genetics approaches
12
12
 Sequencing and localization of regions
adjacent to provirus by searching in
annotated databases of mouse genome
 Isolation of genomic regions adjacent to the insertion site
of the provirus
Mikkers et al., Nature Gen (2002)
Devon et al., Nucl Acid Res (1994)
Insertional mutagenesis in
forward genetics approaches
In case of splincerette, the primer is of the same sequence as the top
strand and therefore it is unable to act as a primer until the complement of
this strand has been synthesized (from the insert-specific primer at the
right-hand side).
13
13
 Forward vs. Reverse Genetics
 Use of Libraries of Insertional Mutants in Forward Genetics
 Searching in Libraries of Insertional Mutants According to:
 anatomically or morphologically detectable phenotype
 metabolic profile
Outline
14
 Automated analysis of metabolites (up to
25.000) by GC-MS techniques in libraries
of T-DNA mutants
 Metabolic profiling of plants
Metabolic profiling
14
15
 Automated analysis of metabolites (up to
25.000) by GC-MS techniques in libraries
of T-DNA mutants
 Identification of interesting (even
comercially interesting) mutants
 Metabolic profiling of plants
Metabolic profiling
15
16
Metabolic profiling
 Automated analysis of metabolites (up to
25.000) by GC-MS techniques in libraries
of T-DNA mutants
 Identification of interesting (even
comercially interesting) mutants
 Fast and easy isolation of genes through
identification of sequences adjacent to T-
DNA
 Metabolic profiling of plants
16
17
 Possibility to use special techniques, e.g. microdissection
 Metabolic profiling of plants
Metabolic profiling
17
18
18
 Forward vs. Reverse Genetics
 Use of Libraries of Insertional Mutants in Forward Genetics
 Searching in Libraries of Insertional Mutants According to:
 anatomically or morphologically detectable phenotype
 metabolic profile
 expression of genes of interest
Outline
19
 Analysis of expression profile (pattern) of the gene and
identification of mutants with altered expression pattern
 Identification of mutants with a change in the expression profile
Expression profile
19
20
 Analysis of expression profile (pattern) of the gene and
identification of mutants with altered expression pattern
 Identification of mutants with a change in the
expression profile
 Possibility of partial automation (virtual digital microscopy)
Expression profile
20
21
Automated Microscopy Screening
Tady by mohla být reference zpět na CEITEC, jaké skvělé vybavení v něm je a
jak dobře se vám s tím pracuje
Pokud tam nezůstane video, ikonu bych dala pryč
22
WT
Expression profile
22
23
LPH controls expression of CKI1
Dobisova et al., Plant Physiol, 2017
Dobré použití otázky „so what did we do“?
24
lph is novel allele of HEME
OXYGENASE 1
Dobisova et al., Plant Physiol, 2017
25
lph is novel allele of HEME
OXYGENASE 1
Terry et al., Biochem Soc Trans, 2002
26
Light controls spatiotemporal
specificity of CKI1 expression
Dobisova et al., Plant Phys, 2017
WT
Short Day
Darkness
Red
Far-Red
lph/hy1-7
27
27
 Forward vs. Reverse Genetics
 Use of Libraries of Insertional Mutants in Forward Genetics
 Searching in Libraries of Insertional Mutants According to:
 anatomically or morphologically detectable phenotype
 metabolic profile
 expression of genes of interest
 Identification of the Mutated Locus
 plasmid rescue
 iPCR
Outline
28
 Identification of chromosomal rearrangements responsible
for bushy phenotype of Arabidopsis
 Description of phenotype
Identification of mutated locus
28
29
Identification of mutant
 Crinkled leaves
 No trichomes on leaves and
stems
 Late senescence
 Bushy phenotype (branching
defective)
29
30
 Male sterility, defects in stamen
filament elongation (A,B)
(compare with wild type C)
Identification of mutant
30
31
 Identification of chromosomal rearrangements responsible
for bushy phenotype of Arabidopsis
 Description of phenotype
 Identification of T-DNA mutated region
Identification of mutated locus
31
32
1. Identification of region of genomic DNA adjacent to the left
border using plasmid rescue
 Restriction digestion (EcoRI)
of mutant genomic DNA
 Religation and transformation of
E. coli
 Isolation of plasmid DNA from
positively selected clones
 Identified sequence was
identical to gene for NAD7
coded by mtDNA
Identification of mutated locus
32
33
2. Identification of region of genomic DNA adjacent to the right
border using inversion PCR (iPCR)
 Restriction digestion
(EcoRI) of mutant genomic
DNA
 Purification, religation and
PCR using T-DNA specific
primers
 Cloning and sequencing
 Identified sequence was
not homologous to any
sequences with known
function
Identification of mutated locus
33
34
 Identification of chromosomal rearrangements responsible
for bushy phenotype of Arabidopsis
 Description of phenotype
 Identification of T-DNA mutated region
 Localization of T-DNA insertion site in Arabidopsis genome
Identification of mutated locus
34
35
Searching in library IGF-BAC
 Genome library containing 10.752 clones with an average size of an
insert of 100 kb
 Bacterial clones arranged in the microtiter plates
 Library loaded onto nylon filters for hybridization with the
radiolabeled probe
35
36
I. Sequences adjacent to the left border of T-DNA
 28 positively hybridizing clones in total
 19 of them located on chromosome 2
 18 of them similar with mtDNA
II. Sequences adjacent to the right border of T-DNA
 6 positively hybridizing clones in total
 all of them located on chromosome 2
Mapping with IGF-BAC database
36
37
left border of T-DNASequences adjacent to right and
Localization of genomic T-DNA adjacent to both left
and right T-DNA borders on chromosome 2
 There was probably an inversion of almost entire
chromosome 2
37
38
38
 Forward vs. Reverse Genetics
 Use of Libraries of Insertional Mutants in Forward Genetics
 Searching in Libraries of Insertional Mutants According to:
 anatomically or morphologically detectable phenotype
 metabolic profile
 expression of genes of interest
 Identification of the Mutated Locus
 plasmid rescue
 iPCR
 Use of Libraries of Point Mutants in Forward Genetics
 Positional Cloning
Outline
39
 Positional cloning
 Principle: co-segregation analysis of segregating population (mostly of
offspring of backcrosses) with molecular markers
 RFLP (Restriction Fragment Length Polymorphism)
 RAPD (Randomly Amplified Polymorphic DNA)
 SSLP (Simple Sequence Length Polymorphism)
 Polymorphism of genome (PCR products) length, amplified using
specific primers
 Detection by Southern blot (PCR after digestion of the genomic DNA
and ligation of adapters)
 Polymorphism of length of randomly amplified genome segments,
using short 8-10bp primers
 CAPS (Cleaved Amplified Polymorphic Sequence)
 Restriction fragment length polymorphism, genome segments
amplified by PCR
Identification of mutated locus
39
40
m m
M M
Col Col
+
M
Col Ler
m
M
+
M
+
M
Ler Ler
M M
m m
Col Ler
M M
m m
Ler Ler
m +
M M
Col Ler
m
M
+
M
Ler Ler
+ +
M M
Col Col Col Ler
+
M
+
M
M M
m +
Col Ler
+ +
M M
♂
Ler Ler
♀
Col Col
mm
M M
Preparation of mapping population
M
m+
M M
Positional cloning
40
41
Recombinant analysis – determining the percentage of
recombination between mutation and molecular marker
r [%] = number of chromosomes of Col /
number of all the chromosomes × 100
marker I – linked
5 mutants
1/10×100 = 10%
marker II - no linkage
6 mutants
7/12×100 = 58%
• Analysis of approximately 2000 mutant plants
• Determining the closest (still segregating) marker
• Identification of mutation by sequencing
41
42
Map of DNA molecular markers
42
43
Markers for fine mapping
43
44
44
 Forward genetics allows targeted screening for interesting
phenotypes, whose association with a given gene/locus is
unknown
 Employs both insertional mutagenes as well as point
mutations
 Inserional mutation
 (mostly) loss-of-function mutation
 Identification via
 iPCR
 plasmid rescue
 Point mutation
 Both loss-of-function as well as
 gain-of-function mutations
 Identification via
 map-based cloning
 GWAS
Key Concepts
45
45
Discussion