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Genomics Book (20 chapters)
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Chapter 7: High throughput genetics
Chapter 8: Proteomics
Chapter 13: Genome Structure
Chapter 14: Human origin
Chapter 15: Genomics and Medicine
Chapter 18: Pharmacogenomics
Chapter 19: Genomics and Agriculture
Chapter 20: Ethical issues of genomics
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Chapter 7
High-Throughput Genetics
Applications of genomics approaches to
genetics
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Contents
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Basics of forward genetics
Genomics approaches to forward genetics
Basics of reverse genetics
Genomics approaches to reverse genetics
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Background
 Genetics is the study of gene function
 Gene function is inferred from the resulting
phenotype when the gene is mutated
 Genomics is changing the way genetics is
performed
 Global, high-throughput approaches
 Genomics approaches are being applied to
both forward and reverse genetics
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Forward and reverse genetics
 Forward genetics starts with identification of
interesting mutant phenotype
 Then aims to discover the function of genes
defective in mutants by chromosome walking
 Reverse genetics starts with a known gene and
alters its function by transgenic technology
 Then aims to determine the role of the gene
from the effects on the organism
 This chapter focuses on applications of
genomics to genetics in model organisms
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Basics of forward genetics
 Forward genetics usually starts with
mutagenesis of organism
 Can use chemicals
 e.g., ethyl methyl sulfonate (EMS)
 Or can use radiation
 e.g., X rays
 Then screen progeny of mutagenized
individuals for phenotypes of interest
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Phenotype
 Empirical definition: features that are different
from those of the wild type (normal)
 Can be something visible
 e.g., change in hair color or anatomy
 Or may require invasive analysis
 e.g., different mobility of enzyme during
electrophoresis
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Mouse hair-color mutant
Beige
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Drosophila anatomical mutant
Mutant with legs
instead of antennae
wild
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Genetic analysis
 First step in analysis of mutants: precisely
describe phenotype
 Basis of predicting gene function: identify
precisely what has malfunctioned (wrong) in
the mutant
 That is what the gene product does in the wild
type
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Phenotype to gene function
 Example: mouse
 Small-eye mutant
 Gene codes for Pax6
transcription factor
 Required for normal
eye development
 Same transcription
factor required for eye
development in
humans and fruit flies
Poor rat
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Genetic screens
 For diploid organisms
 Two chromosomes
 Mutagenesis performed
 Then mutagenized individuals are mated or
self-crossed
 Screen progeny for mutant phenotypes
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Recessive traits
 Recessive trait
 Normally loss-offunction mutation
 When heterozygous
parents are crossed, the
mutant phenotype
appears in 1/4 of the
progeny
homozygous heterozygous homozygous
wildtype
mutant
no
phenotype
no
phenotype
mutant
phenotype
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Dominant traits
 Dominant trait
 Normally gain-offunction mutation
 When heterozygous
parents are crossed, the
mutant phenotype
appears in 3/4 of the
progeny
homozygous heterozygous homozygous
wildtype
mutant
no
phenotype
mutant
phenotype
mutant
phenotype
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Genomics applied to genetics
 Genomics characterized by the following:
 High throughput
 Using automation to speed up a process
 Global approach
 All genes in genome
 Applied to both forward and reverse genetics
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Genomics and forward genetics
 High-throughput genetic screens
 Candidate-gene approach
 To go from phenotype to gene
 Insertional mutagenesis
 Loss-of-function mutation
 Activation tagging
 Enhancer trapping
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
High-throughput genetic screens
 Some genetic screens are relatively
straightforward
 e.g., For a visible phenotype like eye color
 If phenotype is subtle or needs to be measured,
the screen is more time consuming
 Examples
 Seed weight
 Behavioral traits
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Industrial setting for screens
 2002 Para digm Genetics, Inc. All rights reserved. Used with permission.
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
High-throughput genetic screen
 Paradigm Genetics, Inc.
performs “phenotypic
profiling”
 Take measurements of
mutants’ physical and
chemical parameters
 e.g., plant height, leaf
size, root density, and
nutrient utilization
 Different developmental
times: compare to wild
type
 2002 Para digm Genetics, Inc. All rights reserved. Used with permission.
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
From phenotype to gene
 Once an interesting
mutant is found and
characterized, we want chromosome
to find the gene in
which the mutant
occurred
 Positional cloning
contig
candidate genes mutation
 First use genetic
mapping
 Then use chromosome
walking
 Needle in hay stack
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Candidate-gene approach
 If the mutated gene is
localized to a sequenced
region of the
chromosome, then look
for genes that could be
involved in the process
under study
 Last step: confirm gene
identification
 Rescue of phenotype
 Mutations in same
gene in different alleles Tau mutation in circadian rhythm
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Insertional mutagenesis
 Alternative to chromosome walking
 To reduce time and effort required to identify
mutant gene
 Insert piece of DNA that disrupts genes
 Inserts randomly in chromosomes
 Make collection of individuals
 Each with insertion in different place
 Screen collection for phenotypes
 Use inserted DNA to identify mutated gene
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Insertional mutagens
 Transposable elements
 Mobile elements jump from introduced DNA
 e.g., P elements in Drosophila
 Or start with a small number of nonautonomous
elements
 Mobilize by introducing active element
 e.g., AC/DS elements in plants
 Single-insertion elements
 e.g., T-DNA in plants
 Once insert, can’t move again
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Insertional mutagenesis in Arabidopsis
 T-DNA inserts into plant
chromosome
 Screen for mutations
that affect flower
formation
 Make genomic library
from mutant DNA
 Probe with T-DNA
 Identify mutant gene
T-DNA
AGAMOUS
T-DNA
inserts
into
gene
T-DNA
probe
probe library
made from
T-DNA tagged
mutant with
T-DNA
T-DNA
sequence
DNA flanking
T-DNA to
identify gene
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Activation tagging
 A variation on insertional mutagenesis
 Makes gain-of-function mutations instead of
loss-of-function mutations
 Potential to identify gene function not
detectable through loss-of-function screens
 Useful for the following cases:
 Functionally redundant genes
 Genes required for viability
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Activation tagging in Arabidopsis I
 Strong constitutive viral
promoter
35S enhancer
T-DNA vector
 CaMV 35S
 Inserted randomly
 With T-DNA
 When inserts are near a
gene, the following
results occur:
 Activation
 Constitutive expression
 Can result in abnormal
phenotype
35S enhancer
gene X
constitutive expression of gene X
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Activation tagging in Arabidopsis II
 Examples of mutant
phenotypes found in
activation-tagging screen
 In an activation-tagging
experiment carried out by
Detlef Weigel’s laboratory at
the Salk Institute, many
different abnormal
phenotypes were observed
for Arabidopsis. Among the
genes that were activated
were Flowering Locus T
(FT), which controls
flowering time, and genes
that control plant growth and
leaf shape.
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Enhancer trapping
 Type of insertional mutagenesis
 Used to find genes with interesting expression
patterns
 Insert carries a reporter gene
 Expresses foreign protein
 No effect on organism
 Enhancer trap
 Has minimal promoter in front of reporter gene
 Enhancer near point of insertion acts on it
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
By chance it will go to right place
Enhancer
Need very large population to increase your chances
Not good for all genes! Randon=m event.
Need targeted approach!
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Enhancer trapping in Drosophila I
 Use transposon P element
 Carries reporter gene
 b-galactosidase
 Hops into genome
 When lands near
enhancer, activates gene
expression
 Expression similar to that
of neighboring gene
enhancer
P element recognition sites
b-galactosidase
P element vector
enhancer
gene Y
b-galactosidase
gene Y
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Enhancer trapping in Drosophila II
 Reporter gene: Green
Fluorescent Protein
(GFP)
 The enhancer trap has
inserted into a gene
expressed in part of the
fly eye
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Gene trapping
 Similar to enhancer
trapping
 Instead of minimal
promoter has splice
acceptor site (AG)
before reporter gene
 Expressed only when
correctly spliced
 Usually causes gene
disruption as well
GT
AG
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Basics of reverse genetics
To find out the function of a known gene
 Reverse genetics starts with known genes
 e.g., from genomic sequencing
 Goal: to determine function through targeted
modulation of gene activity
 Decrease
 Increase
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Ways to modulate gene activity
 Delete gene
 Homologous recombination
 Works well in yeast
 Can be done in mouse and flies not plants
 Interfere with transcription
 Antisense RNA
 Interfering RNA (RNAi)
 Identify gene affected by mutagenesis
 Insertional or chemical
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Reverse-genetics example
 Gene that encodes
muscle-specific
transcription factor in
mouse
 Myogenin required
 Homologous
recombination used to
delete gene
 Mice born, but can’t
make muscle
neo
targeting vector
Tk
genome locus
myogenin
selection
neo
product of homologous recombination
selectable marker disrupts
myogenin gene
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RNAi and antisense RNA
 Double-stranded RNA able to disrupt gene
expression
 Cells have machinery that destroy doublestranded RNA: viruses/ cDNA
 Appears to be basis for the following:
 Interfering RNA (RNAi)
 Double-stranded RNA introduced into cells
 Antisense RNA
 Introduce complementary RNA
 Forms double-stranded RNA in cells
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
RNAi mechanism I
 RNAi probably depends on a system used to
detect the following:
 Double-stranded (ds) RNA viruses
 Other abnormal gene expression
 Initially characterized in the following:
 Fungus
 Quelling in Neurospora
 Plants
 Resistance to spread of virus
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
RNAi mechanism II
 Cellular RNase
recognizes dsRNA
 Cleaves to small (23 bp)
fragments
 Fragments hybridize to
transcripts
 RNA-dependent RNA
polymerase forms
dsRNA
 RISC nuclease chews up
dsRNA
RNase
RNApol
RISC
(RNA induced silencing complex)
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Systematic RNAi screens
 In the roundworm, C. elegans, RNAi is easy to do
 Soak in solution of dsRNA
 Feed bacteria expressing dsRNA
 Or inject dsRNA
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Makes systematic RNAi screens possible
~ 75% of time, RNAi gives a reduction in RNA levels
(Not 100% silencing but leaky)
Has been used to reduce expression of genes
 On particular chromosomes, or
 Expressed at a particular developmental time
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
RNAi on all genes on chromosome
 Goal: In C. elegans,
determine function of all
2,300 genes on
chromosome III
 RNAi constructs made
for each gene
 Worms microinjected
with double-stranded
RNA
predicted ORF
T7
T3
construct with
promoter sequence
add T3 + T7
polymerase
dsRNA
Dr. A. Hyman
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Phenotypes found in RNAi screen
Wild type
RNAi embryos
Pronuclear/nuclear
appearance
(phenotypic class B1)
Pronuclear migration
(phenotypic class C1)
Cytokinesis
(phenotypic class C6)
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RNAi screen of embryonic genes
 Microarray analysis identified genes active in
early embryogenesis
 Used RNAi to target each gene
 Worms injected with RNAi construct
 Progeny tested for phenotypes
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Wild-type C. elegans embryogenesis
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are needed to see this picture.
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Effects of RNAi on actin
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are needed to see this picture.
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Finding genes in libraries of mutants
cDNA
 Goal: Find known genes disrupted in
collections of mutants
 Screening of functional-genomics libraries
 Can be used for mutations that affect lethality
or fertility
 Screen heterozygotes
 Can either use PCR to identify mutations in a
particular gene or sequence flanking sequences
of all inserts
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Screening an insertion library
(Insertion in promoter region)
 PCR used to find
insertion
 One primer
complementary to insert
 Other primer
complementary to gene
 If get an amplification
product then you have
insertion
 Sequence product for
exact location
PCR primers
insert
gene Z
PCR amplification
insert
gene Z
+
–
amplification product
on gel indicates
presence of insert
near gene
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TILLING
 Method for finding mutations produced by
chemical mutagens in specific genes
 Chemical mutagenesis
 Usually produces point mutations
 Generally gives more subtle phenotypes than
insertions
 e.g., hypomorphs, temperature sensitive mutants
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
TILLING in Arabidopsis I
 EMS used to
mutagenize Arabidopsis
 Grow individual
mutagenized lines
 Make primers flanking
gene of interest
 Amplify using PCR
EMS
mutagenize
seed
gene Z
WT
gene Z
mutant
PCR amplification
from wild type
and mutant
WT
mutant
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TILLING in Arabidopsis II
 Denature DNA from
pools of mutant lines
 Allow to hybridize to
wild-type DNA
 Detect mismatches in
hybridized DNA
 Denaturing HPLC
 Cel I enzyme cuts at
mismatches
 Sequence to identify site
of mutation
ATGCGGACTG
|||||| ||| +
TACGCCGGAC
ATGCGG
||||||
TACGCC
Cel 1
CTG
|||
GAC
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Summary I
 Forward genetics
 Mutation to gene function
 Genetic screens
 Cloning genes identified in screens
 Genomics approaches to forward genetics
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High-throughput genetic screens
Insertional mutagenesis
Activation tagging
Enhancer trapping and gene trapping
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Summary II
 Reverse genetics
 From gene to function
 Genomics approaches to reverse genetics
 RNAi screens
 Identifying mutations in insertional libraries
 TILLING
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458