MUTANTS AND THEIR APPLICATION
IN GENOMICS
Methods in genomics and proteomics
(CG980)
Genomics – lesson 6
Mgr. Markéta Žďárská, Ph.D.
Outline
Transgenic plants and functional genomics
Mutagenesis, mutagen types
Forward genetics approaches
EMS mutagenesis
Positional cloning
Sequencing
Reverse genetics approaches
Tilling
Reporter genes
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Transgenic organisms
transgene – a gene (genetic material) that has been
transferred naturally, or by any of a number of genetic
engineering techniques from one organism to another
synthetic, modified or heterogeneous genes
introduced into a different animals and plants
„non-native“ segment of DNA
retains the ability to produce RNA/protein in
the transgenic organism
alters the normal function of the transgenic organism's
genetic code
Transgenic organisms are used for the study of
gene functions 3
Transgenic organisms
GloFish are a type of transgenic zebrafish (Danio rerio) that have been
modified through the insertion of a green fluorescent protein (gfp) gene.
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Functional genomics
 aim: search for genes and determination of their
function in genome
 2 different approaches:
› „forward genetics“
 phenotype → gene
› „reverse genetics“
 Sequence of DNA (gene ) → phenotype
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Mutagenesis
mutants – a tool in both approaches
different type of mutagens
1. chemical
2. physical (radiation)
a huge number of random mutations, affecting all genes
3. biological
• modern
• insertion mutagenesis, lower number of mutations, leave a
molecular marker behind
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classical
Chemical mutagens
 cause point mutations
1. during DNA replication but also in non-replicating DNA
› alkylating agents (transfer an alkyl group to nucleobases; random)
 Sulfur mustard (mustard gas; Ch. Auberbach)
 ENU - N-ethyl-N-nitroso urea
 ethyl group of ENU interacts usually with thymine
 Bill Russell (1951) mouse strain („T-test stock”) used in genetic screens
for testing mutagens such as radiations and chemicals
 Ethyl methane sulfonate (EMS)
 ethyl group of EMS reacts with guanine in DNA, forming the abnormal
base O-6-ethylguanine (original G:C can become A:T)
 Maple J. & Moller SG, 2007 - Mutagenesis in Arabidopsis
› HNO2 (deamination of amino groups) 7
Chemical mutagens
2. only during replication
› base analogs: 5-bromuracil (5-BU),
2-aminopurine
 cause transition mutations
› acridine dyes: proflavine, acridine
orange
 cause addition or deletion of 1or more
bases → change in frame read
→nonfunctional gene products
› hydroxylamine
 specific – transition induced only in
direction G:C → A:T
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5-BU paring with adenine
5-BU paring with guanine
Physical mutagens
 ionizing: X-ray, gamma radiation, radioactive carbon 14C
› Cause DNA molecules break downs
 nonionizing: UV light 254nm is absorbed by bases−> pyrimidine
dimers (distorting sugar phosphate backbone)
 cause extensive insertions and chromosome alterations
 able to delete more genes or insert new regulations sequences
 improper for precise mutagenesis
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Biological mutagens
 insertion mutagenesis
1. T-DNA
› Agrobacterium tumefaciens is able to insert a part of DNA into plant
genome
› Results of T-DNA insertion into genome are various, caused by
insert nature or location of insertion
› effects:
 Gene inactivation
 activation (carrying a promoter, enhancer)
2. Transposons – transposable gene elements (TE or transposon)
› Mobile genetic elements, less stable than T-DNA
› Can jump from original location of insertion −> recovery of normal
phenotype
› The footprint of insertion stays in the location also if the insertion is
no longer in the genome; impropriate for large mutagenesis
(Petersen et al, 2000) 10
T-DNA a transposons
 Insertion mutagenesis
 Insertion into:
› coding region
› noncoding region –
affecting intron splicing,
gene expression
  pros:
› reversible mutation
› Easy to map and the
region is easy for cloning
  cons:
› Insertion is not random 11
usually nonfunctional gene
How to search for mutations?
1. small mutations
 a new single nucleotide polymorphism (SNP) at a specific
position in the genome
 altered transcriptional profile
2. chromosome alterations
 extensive reconstruction → in situ hybridization at metaphase
chromosomes – resolution 5 Mbp
 reconstruction smaller → Comparative Genome Hybridization
(CGN)
 „DNA microarrays“ using DNA probe covering diverse sites of a genome
 hybridization with genomic DNA – resolution 5-10 Mbp
 a full genome analysis of in 1 experiment
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The upper DNA
molecule differs from
the lower DNA
molecule at a single
base-pair location (a
C/A polymorphism).
Forward genetics
 based on saturation mutagenesis „saturation screen“
› a mutagen induction to an organism −> analysis of progeny for a
specific phenotype
› identification of mutants −> sorting into complementary groups
› mapping into general chromosomal locations employing known
markers and then cloning, sequencing
 a mutation for every locus exists−> possible to determine
a groups of genes responsible for an exact trait
 aim is to achieve a saturation point −> detect all genes
responsible for a phenotype
 mutagens (RTG, EMS, transposons)
› Examples:
› plants missing the reaction to light
› bacteria unable to growth in the presence of some sugars,.. 13
Identification of a mutated gene in
a mutant line selected by its
phenotype
 using genetic map
1. mapping - (co)segregation analysis
› finding approximate position of a gene in a genetic map, based
on genetic linkage with genetic markers (traits with
polymorphism = they are divergent between parental genotypes)
2. search for an exact sequence carrying a mutation
› chromosome „walking“
› sequencing, matching with WT sequence
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= trait with known (easily checked) position in the
genetic map, featuring polymorphism (divergent
between parental genotypes)
1. Morphologic
2. Molecular
DNA markers – able to detect differences in a sequence
DNA with known and explicit location in genome
easily detected loci with known position at the chromosome
single nucleotide polymorphism (SNP)
ideal – equally localized
Types of genetic markers
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Natural morphologic variability
of Arabidopsis – ecotypes
„accessions“
Fig. 1: Geographical distribution of Arabidopsis thaliana (green
area on the world map) and overall phenotype of the rosette
of a subset of accessions grown under 3 contrasted
environment scenario.
http://www.mpipz.mpg.de/102840/reymond 16
DNA molecular markers
(= a visible band at electrophoretic gel or
blot)
 SSLP (Simple Sequence Length
Polymorphism)
› a genome length (PCR products) amplified
using spec. primers
 RFLP (Restriction fragment length
polymorfism) + Southern
› restriction fragments lengths of a genome
segment, PCR followed by genome DNA
cleavage and adaptors ligation
 RAPD (Random amplified polymorphism
detection)
› a length of randomly amplified genome
segments (short primers 8-10bp)
 AFLP (Amplified fragment length
polymorphism)
› a length of genome fragments, PCR followed
by cleavage of genome DNA a adaptors
ligation
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Other Markers Acronym
Variable Number
Tandem Repeat
VNTR
Oligonucleotide
Polymorphism
OP
Inverse Sequencetagged
Repeats
ISTR
Inter-retrotransposon
Amplified
Polymorphism
IRAP
DNA molecular markers
Arabidopsis thaliana
crossing 2 ecotypes: Columbia a Landsberg erecta (Col X Ler)
recombinant map contained originally 67 markers (Lister & Dean, 1993)
today more than 1300 markers (Hou et al, 2010)
Arabidopsis thaliana physical map with
indication of the positions of the markers.
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Positional cloning,
map-based cloning
gene isolation based on the position on the map
gene function is usually unknown
gene mapping based on genetic linkage with molecular marker followed by
a gene isolation with approximate chromosomal location - this is known as
the candidate region.
need for a standard (WT) line, which is crossed with a mutant line to proceed
with a recombinant analysis using markers (“linkage analysis”)
aim of positional cloning:
find a gene with desired mutation located in the interval of two closest markers
region small enough −> to choose a candidate gene and identify a mutation in it
generally – complicated and time consuming
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Lukowitz et al, 2000
Positional cloning - a scheme
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Lukowitz et al, 2000
Positional cloning - application
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 Arabidopsis
› a map of positional cloned genes contains approx. 620 mutant genes with a
phenotype (Meinke et al, 2003)
› a number of genes gained by positional cloning is increasing every year →
clarification of new gene functions in a model plant
 Wheat (Tritium aestivum)- crops
› molecular markers:
 SSR (Simple Sequence Repeat)
 SCAR (Sequence – Characterized Amplified Region)
› gene isolation with markers is more complicated – hexapodies
› a lot of unknown genes coding crop related traits (up to now approx. 20), i.e.
fungi and their resistance
 Genetic disorders (a genetic problem caused by one or more
abnormalities in the genome, especially a condition that is present
from birth (congenital)
› Huntington's Disease (no signs until adulthood; autosomal dominant)
› Down syndrome (DS or DNS), also known as trisomy 21
› Cystic fibrosis (CF) (affects mostly the lungs but also the pancreas, liver, kidneys, and
intestine; autosomal recessive)
Identification of mutant gene –
an exact sequence carrying a mutation
 allelic test - crossing a desired mutant line with a „knock-out“
candidate gene – check if the mutant phenotype is maintained
 molecular complementation (recessive mutations)
› transformation of mutant plant with sequences of WT DNA in
restricted space in order to detect the one recovering the WT
phenotype
 analysis of entire DNA sequence in restricted genetic
interval −> search for alterations causing the mutation
› SSCP(Single Stranded Conformational Polymorphism)
› HMA (Heteroduplex Mobility Assay)
› DGGE (Denaturing Gradient Gel Electrophoresis)
› dHPLC (denaturing High Performance Liquid Chromatography)
› a DNA chip hybridization
› “pyrosequencing”
› „chromosome walking“
 sequencing
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„Chromosome walking“
search for 2 optimal markers surrounding
mutated gene
libraries of huge genome DNA fragments
(redundant; random):
YACs, BACs
= yeast (bacterial) artificial chromosome, ~ 300
(100) kbp
search for overlaps based on hybridization
Mutated
gene X
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Genomic DNA is shown in blue. Selected clones from a library
of cloned genomic DNA fragments are shown in red. The initial
probe, probe a, is specific to gene A or exon A and allows
identification of clones 1 and 2. A new probe, probe b, is
prepared from one end of clone 2 and used to isolate new
clones 3 and 4 from the genomic library. Probe c, prepared from
clone 4 is used to identify clone 5, etc. The orientation of the
clones is determined by restriction mapping of the clones. Clone
6 contains the desired gene B or exon B.
Sequencing - classic - Sanger
 based on the selective incorporation of chain-terminating
dideoxynucleotides by DNA polymerase during in vitro DNA
replication
 Developed by Frederick Sanger and colleagues in 1977
 requires a single-stranded DNA template, a DNA primer, a
DNA polymerase, normal dNTPs, and modified ddNTPs), the
latter of which terminate DNA strand
 The DNA sample is divided into four separate sequencing
reactions, containing all four of the standard deoxynucleotides
(dATP, dGTP, dCTP and dTTP) and the DNA polymerase
 due to length of reading frames (700 to 800 bp) – still
commonly used and precise
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Sequencing - classic - Sanger
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Pyrosequencing –
„Next-generation sequencing“
DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by
synthesis" principle
relies on the detection of pyrophosphate release on nucleotide incorporation, rather than
chain termination with dideoxynucleotides; no need for electrophoresis
developed by Mostafa Ronaghi and Pål Nyrén at the Royal Institute of Technology in
Stockholm in 1996
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„Next-generation sequencing“
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„Next-generation sequencing“
„Next-generation sequencing“
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„Next-generation sequencing“
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„Next-generation sequencing“
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Application of next-gen
sequencing
 whole genomes
› resequencing −> search for variability in human population
› de novo sequencing −> non-model organisms
 transcriptome (RNA-seq)
› identification of unknown transcripts
› more precise than „microarrays“
 targeted −> to a specific part of a genome or focused
on selected group of genes
› random genome regions −> based on length after
restriction cleavage of genome DNA (RAD-seq)
› hybridization to approx.100bp probes with DNA fragments
that are sequenced (Hyb-seq)
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Large-scale random
mutagenesis and „screening“
systematic mutagenesis – gradual
EMS mutagenesis
search for a phenotype (forward), gene of interest with
alterations in nucleotides
common „screen“ of 1000 or 10000 individuals
use of PCR gene of interest −> search for mild changes in PCR
product migration on a gel or a column
be aware that all changes are not covered by a „knockout“
gene, some of them can be “silenced”- present at non-essential
AK positions
methods commonly used:
DHPLC –“Denaturing High Performance Liquid Chromatography“
DGGE – „Denaturing Gradient Gel Electrophoresis“
SSCP – „Single-Stranded Conformation Polymorphism“
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A scientific example of EMS
mutagenesis (Feraru et al, 2010)
A fluorescence imaging-based forward genetic
screen“
As a tool for the screening to identify novel components of plant intracellular
trafficking, they used a well characterized plant cargo, the auxin efflux carrier
PIN1 (Petrasek et al., 2006).
With this strategy,they aimed to identify novel regulators at different stages of
subcellular protein trafficking.
EMS-mutagenized PIN1pro:PIN1-GFP (for green fluorescent protein)
population using epifluorescent microscopy for seedlings displaying aberrant
PIN1-GFP distribution in the root.
From 1500 M1 families, they identified several protein affected trafficking
(pat) mutants defining three independent loci (mapping with simple sequence
length polymorphism (SSLP) and cleaved amplified polymorphic sequence
(CAPS and dCAPS) markers
The At3G55480 candidate gene was sequenced and a point mutation that
caused a stop codon was found at the position 705 downstream of ATG)
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A scientific example of EMS
mutagenesis (Feraru et al, 2010)
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Figure 1. The pat2 Mutant Displays Ectopic
Intracellular Protein Accumulation.
(A) to (D) Both PIN1-GFP ([A] and [B]) and
aleurain-GFP ([C] and [D]) accumulate
intracellularly in pat2-1 (B) or pat2-2 (D) root
cells compared with
control ([A] and [C]).
•pat2 mutant lytic vacuoles display altered morphology and
accumulation of proteins
• unlike other mutants affecting the vacuole, pat2 is
specifically defective in the biogenesis, identity, and
function of lytic vacuoles but shows normal sorting of
proteins to storage vacuoles
• PAT2 encodes a putative b-subunit of adaptor protein
complex 3 (AP-3)
•AP-3 b functions in mediating lytic vacuole performance
and transition of storage into the lytic vacuoles
independently of the main prevacuolar compartment-based
trafficking route
„Reverse“ genetics
today −> post-genomic era
genes are known (sequences)
unknown
functions of genes
usually >50% predicted genes in eukaryotes
phenotypes causing mutations in theses genes
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TILLING
(Targeting induced local lesions in
genomes)
 to create libraries of mutagenized
individuals that are later subjected
to high-throughput screens for the
discovery of mutations
› potential changes in regulation,
interaction, …
 introduced on Arabidopsis
thaliana (McCallum et al, 2000)
 Principle
› random induction of point
mutations(EMS)
› followed by search of lines
with mutations in targeted
gene using PCR and
heteroduplex analysis
(McCallum et al, 2000)
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TILLING – detection of
mutations, strategy
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ATP http://tilling.fhcrc.org
TILLING: A five step process
1. You decide whether your gene is worth TILLING.
”I have an insertion in my gene but the knockout phenotype is
lethal.” ....TILLING can provide the sub-lethal phenotypes you want.
“The knockout phenotype is interesting.”.....TILLING can provide an
allelic series that may help you better ascertain the function of your gene.
“I have an insertion in my gene that knocks out gene function but
my plants have no phenotype.”.....TILLING is not for you. In this scenario,
a gain-of-function mutation is needed to investigate the potential in vivo role of
this gene.The large majority of phenotypes arising from our populations will
cause full or partial loss of function.
”I have a candidate gene and I want to know the knockout
phenotype.”....There are very good reasons why you should start by insertional
mutagenesis rather than by TILLING. First, only a small percentage of EMS-induced
mutations will yield a change likely to truncate the protein (~5%). Second, the Arabidopsis
community has access to excellent insertional mutagenesis resources. Third, if a knockout
mutation causes no phenotype, then the TILLING allelic series is not expected to either.
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ATP http://tilling.fhcrc.org/
TILLING: A five step process
2. You find the best the region to be targeted and
place your order.
3. ATP screens the region for mutations.
4. ATP sequences the mutation and enters it in
our public database.
5. ATP sends you a mutant report and you order
seed.
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TILLING centres
Several TILLING centers exists over the world that focus on agriculturally
important species:
Rice – UC Davis (USA)
Maize – Purdue University (USA)
Brassica napus – University of British Columbia (CA)
Brassica rapa – John Innes Centre (UK)
Arabidopsis – Fred Hutchinson Cancer Research
Soybean – Southern Illinois University (USA)
Lotus and Medicago – John Innes Centre (UK)
Wheat – UC Davis (USA)
Pea, Tomato - INRA (France)
Tomato - University of Hyderabad (India)
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Plants with insertion in an exact
gene
public collections of available mutants in diverse regions of
genome
insertions in almost all genes in Arabidopsis thaliana
selection in silico, ordering seeds with insertion in your gene of
interest
Gen1 Gen2 Gen3
= T-DNA insertion points in individual mutant lines
1 2 3 4 5 6 7 8 num. of line
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Reporter genes
a gene attached to a regulatory sequence of another gene of
interest in bacteria, cell culture, animals or plants
reporters – used as an indication of whether a certain gene has
been taken up by or expressed in the cell or organism
population
fluorescent (GFP)
β-galactosidase (GUS)
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TCS::GFP
45(Zurcher et al, 2013)
GUS reporter system
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(Mason et al, 2004)
Thank you for your attention 
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c/o Masaryk University
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601 77 Brno, Czech Republic
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