Transcript Mosaic Analysis

Contents
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Definition of proteomics
Protein profiling
Protein-protein interactions
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Yeast two-hybrid method
Protein chips
TAP tagging/Mass spectrometry
Biochemical genomics
Pregenomics biochemical assays
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Methods used to find genes responsible
for specific biochemical activity before the
inception of genomics
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Laboriously purify responsible protein
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Often expensive and time consuming
Expression cloning
Introduce cDNA pools into cells
 Look for biochemical activity in those cells
 Caveat: Often difficult to detect biochemical
activity in cell’s biochemical “background”
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Biochemical genomics
( “Enzomics”? )
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Genome of an organism is already known
Approach
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Construct plasmids for all ORFs
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Attach ORFs to sequence that will facilitate
purification
Transform cells
Isolate ORF products
Test for biochemical activity
Biochemical genomics in yeast
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6,144 ORF yeast strains made
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ORFs fused to glutathione S-transferase (GST) for
purification purposes
Biochemical assay revealed three new
biochemical reactions associated with yeast
ORFs
pre-tRNA
Ligated tRNA
tRNA halves
Abc1
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Microfluidics
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Proteomics requires
greater automation
Microfluidics: a “lab
on a chip”
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Microvalves and
pumps allow control of
nanoliter amounts
Can control
biochemical reactions
A microfluidics chip
Microfluidics in action
loading
mixing
500 mm
compartmentalization
purging
Summary I
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Goals of proteomics
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Identify and ascribe function to proteins under
all biologically plausible conditions
Proteomics methods
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2-D gel electrophoresis for separating proteins on the
basis of charge and molecular weight
Mass spectrometry for identifying proteins by
measuring the mass-to-charge ratio of their ionized
peptide fragments
Protein chips to identify proteins, to detect protein–
protein interactions, to perform biochemical assays,
and to study drug–target interactions
Summary II
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Proteomics methods (continued)
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Yeast two-hybrid method for studying protein–protein
interactions
Biochemical genomics for high-throughput assays
Some accomplishments of proteomics
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Example: yeast
Yeast two-hybrid method reveals interactome
 Transcriptional regulatory networks deduced
 Biochemical genomics uncovers new ORF functions
 Subcellular localization of proteins
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Genomics IV:
“Phenomics”
High-Throughput Genetics
Applications of genomics
approaches to genetics
Background
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Genetics is the study of gene function
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Genomics is changing the way genetics is
performed
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Gene function is inferred from the resulting
phenotype when the gene is mutated
Global, high-throughput approaches
Genomics approaches are being applied to
both forward and reverse genetics
Forward and reverse genetics
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Forward genetics starts with identification
of interesting mutants
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Reverse genetics starts with a known gene
and alters its function
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Then aims to discover the function of genes
defective in mutants
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
Basics of forward genetics
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Forward genetics usually starts with
mutagenesis of organism
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Can use chemicals
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Or can use radiation
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e.g., ethyl methyl sulfonate (EMS)
e.g., X rays
Then screen progeny of mutagenized
individuals for phenotypes of interest
Genomics applied to genetics
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Genomics characterized by the following:
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High throughput
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Global approach
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Using automation to speed up a process
All genes in genome
Applied to both forward and reverse
genetics
Genomics and forward genetics
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High-throughput genetic screens
Candidate-gene approach
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Insertional mutagenesis
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To go from phenotype to gene
Loss-of-function mutation
Activation tagging
Enhancer trapping
High-throughput genetic
screens
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Some genetic screens are relatively
straightforward
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e.g., For a visible phenotype like eye color
If phenotype is subtle or needs to be
measured, the screen is more time
consuming
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Examples
Seed weight
 Behavioral traits
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Industrial setting for screens
 2002 Para digm Genetics, Inc. All rights reserved. Used with permission.
High-throughput genetic screen
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Paradigm Genetics,
Inc. performs
“phenotypic profiling”
Take measurements
of mutants’ physical
and chemical
parameters
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e.g., plant height, leaf
size, root density, and
nutrient utilization
Different
developmental times: 2002 Para digm Genetics, Inc. All rights reserved. Used with permission.
compare to wild type
From phenotype to gene
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Once an interesting
mutant is found and chromosome
characterized, we
want to find the gene
in which the mutant
occurred
Positional cloning
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First use genetic
mapping
Then use chromosome
walking
contig
candidate genes mutation
Candidate-gene approach
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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
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Rescue of phenotype
Mutations in same
gene in different
alleles
Insertional mutagenesis
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Alternative to chromosome walking
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Insert piece of DNA that disrupts genes
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Inserts randomly in chromosomes
Make collection of individuals
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To reduce time and effort required to identify
mutant gene
Each with insertion in different place
Screen collection for phenotypes
Use inserted DNA to identify mutated
gene
Insertional mutagens
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Transposable elements
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Mobile elements jump from introduced DNA
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Or start with a small number of
nonautonomous elements
Mobilize by introducing active element
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e.g., P elements in Drosophila
e.g., AC/DS elements in plants
Single-insertion elements
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e.g., T-DNA in plants
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Once insert, can’t move again
Basics of reverse genetics
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Reverse genetics starts with known genes
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E.g., from genomic sequencing
Goal: to determine function through
targeted modulation of gene activity
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Decrease
Increase
Ways to modulate gene activity
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Delete gene
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Homologous recombination
Works well in yeast
 Can be done in mouse and flies
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Interfere with transcription
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Antisense RNA
Interfering RNA (RNAi)
Identify gene affected by mutagenesis
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Insertional or chemical
Reverse-genetics example
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Gene that encodes
muscle-specific
transcription factor in
mouse
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neo
genome locus
myogenin
selection
Myogenin
Homologous
recombination used to
delete gene
Mice born, but can’t
make muscle
targeting vector
Tk
neo
product of homologous recombination
selectable marker disrupts
myogenin gene
RNAi and antisense RNA
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Double-stranded RNA able to disrupt gene
expression
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Cells have machinery that destroy doublestranded RNA
Appears to be basis for the following:
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Interfering RNA (RNAi)
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Double-stranded RNA introduced into cells
Antisense RNA
Introduce complementary RNA
 Forms double-stranded RNA in cells
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Finding random mutations in your
gene of interest (or every gene in
the genome)
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Random insertion of transposons
Random point mutations/indels
Screening an insertion library
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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
P element
piggyBac
Summary of P element Gene
Disruption Project
Insect transposon vectors
Host range of transposons
Mosquito
Silkmoth
Flour Beetle
Transformation of Planaria
(roundworm)
Potential for broad host range
transposons in mutagenesis
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Insertional mutagenesis (random)
Transgenic RNAi
Homologous recombination? (a la Drosophila)
TILLING
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Method for finding mutations produced by
chemical mutagens in specific genes
Chemical mutagenesis
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Usually produces point mutations
Very high mutagenic efficiency
Generally gives more subtle phenotypes than
insertions
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e.g., hypomorphs, temperature sensitive mutants
TILLING in Arabidopsis I
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EMS
mutagenize
seed
EMS used to
mutagenize
Arabidopsis
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Grow individual
mutagenized lines
Make primers flanking
gene of interest
Amplify using PCR
gene Z
WT
gene Z
mutant
PCR amplification
from wild type
and mutant
WT
mutant
TILLING in Arabidopsis II
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Denature DNA from
pools of mutant lines
Allow to hybridize to
wild-type DNA
Detect mismatches in
hybridized DNA
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Denaturing HPLC
Cel I enzyme cuts at
mismatches
Sequence to identify
site of mutation
ATGCGGACTG
|||||| ||| +
TACGCCGGAC
ATGCGG
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TACGCC
CTG
|||
GAC
Cel 1
Arabidopsis TILLING Project
Mapping mutations by DHPLC
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Prepare heteroduplexes as
for Cel I method
Rather than digest sample,
separate on denaturing HPLC
Cleaner result, but much
slower analysis than Cel I
digestion
Mapping mutations by TGCE
Summary I
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Forward genetics
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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
Summary II
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Reverse genetics
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From gene to function
Genomics approaches to reverse genetics
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RNAi screens
Identifying mutations in insertional libraries
TILLING