Transcript Genes & Development

Genes & Development
Part 2: Molecular Methodology
Proves Differential Gene Expression
Differential Gene Expression
• Demonstration of differential gene expression
has been done through a variety of methods
• Cytogenetics in Drosophila
– Polytene chromosomes
• RNA hybridization competition & Rot curves
• RNA localization
• Biochemical & Immunological techniques
Differential Gene Expression
• Polytene chromosomes
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–
–
–
10 rounds of replication without cell division
1024 side-by-side chromatids
Stained chromosomes seen during interphase
Puffs –
• Regions where polytene chromosome appears much wider
• Locations of puffs change during development and in
different cell types
• Location of transcription
• Implies that transcription occurs in different places on
chromosomes in different cells
Polytene Chromosomes
Fluorescence microscopy
of propidium I stained
Drosophila polytene
chromosomes (red)
overlayed with immunostained chromatin scaffold
protein (yellow).
Chromocenter
(centromere) indicated
with arrowhead. Puffs
indicated by arrows
Methods: Microscopy
• Low power stereo microscopy (dissecting scope; 4-100X)
• High-power light microscope (20-400X)
– Nemarski interference optics
• Fluorescence microscopy
– Confocal laser microscopy (stereoimage; 3D/optical sectioning)
• Electron microscopy
– Scanning (SEM)
• Photomicroscopy
Differential Gene Expression
• RNA hybridization
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–
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Isolate RNA from different cell types
Make radiolabeled cDNA from one mRNA
Hybridize labeled cDNA to mRNA
Compete mRNA-cDNA hybridization by adding
excess mRNA from other cell type
– Amount of competition indicates number of same
mRNA species in both cells.
– The presence of mRNA-cDNA hybrids remaining after
competition with unlabeled mRNA indicates unique
transcripts
Methods: RNA hybridization
mRNA from cell type B
mRNA from cell type A
1st
All B mRNAs
hybridize to cDNAs
Only some type A mRNAs
hybridize
Synthesize
strand cDNA
RNA Localization
• Northern analysis
• Isolate total or pA+ RNA
• Separate by size with electrophoresis (special gel
to prevent secondary structure)
• Transfer electrophoresed RNA to
nitrocellulose/nylon membrane (blot)
• Probe blot with radiolabeled cDNA probe
• Northern analysis requires that cDNA be cloned
Northern Analysis: Gel
Electrophoresis
Formaldehyde gels
Methyl-mercury-OH gels
Northern Analysis: ElectroBlotting
Anode (-)
RNA gel
Support pad
Nylon or nitrocellulose filter
Cathode (+)
Support pad
Northern Analysis: ElectroBlotting
100V
Northern Analysis: Probing
RNA Localization
• Developmental Northerns
– Temporal expression information
– Spatial expression information (limited to
microdissected embryo regions and tissues)
– Requires that RNA species being detected be
fairly abundant (large amounts of RNA must be
isolated/purified)
RNA
Localization
Developmental
Stages
RNA
Localization
Developmental
Stages
RNA Localization in Tissues
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
RNA Expression
Following
Experimental
Treatments
RNA Expression
Following
Experimental
Treatments
RNA Localization
• RT-PCR
– Isolate RNA (total or pA+)
– Reverse transcribe using oligo dT primer
– Amplify cDNA by PCR using gene specific
primers and using only 10-15 rounds
– Run PCR product on gel (regular agarose)
– Southern blot and probe with cDNA probe
– Much more sensitive than northern blot
RNA Localization
• Whole mount in situ hybridization allows us
to see which cells express a given mRNA.
• Hybridize antisense RNA probe to mRNA
in embryo
• Information: temporal and spatial
expression pattern
• Putative function of gene product based on
expression pattern
Methods: Whole-mount in situ
hybridization
Antisense RNA
Methods: Whole mount in situ
hybridization
m
FGF8 expression in
3-day chick.
Expression detected
in somites (s), limb
buds (l), brachial
arches (b) &
midbrain-hindbrain
boundary (m)
b
s
l
l
Whole Mount In Situs
Whole Mount In Situs
Biochemical & Immunological
Techniques
• Biochemical purification & characterization
• Generation of antibodies that specifically
recognize proteins
– pAb – polyclonal antibodies
• Many Abs varieties generated against a protein
• Each Ab may recognize a unique epitope
– mAb – moloclonal antibodies
• One Ig producing cell (B-cell) reacts to protein
• One variety of Ab produced which recognizes one
specific epitope
Biochemical Techniques
• Classical protein biochemistry
– Column chromatography
– Preparative electrophoresis
• Amino acid sequencing
• Structural Biochemistry
– X-ray crystallography, CD-ORD, MS-AS,
NMR
– Computer generated structure predictions
Immunological Techniques
• Immunohistochemistry (whole mount & section)
• Immunolocalization
• Western analysis
• Immunoprecipitation IP
• Co-immunoprecipitation coIP
• cDNA expression library screening
Western Analysis
• Separate proteins by electrophoresis (SDSPAGE)
• Incubate with antibody to specific protein
and detect presence/absence
• IP protein using Ab to first protein
• Run gel and probe with 2nd Ab to second
protein to determine if two proteins interact
Western Analysis
Identifying Developmentally
Significant Genes
• Differential Screens
• Homology Screens
• Functional Screens
Require generation of cDNA libraries
followed by screening for desired clones
• Mutagenic Screens
Requires an assayable phenotype and viable
heterozygote (doesn’t work well for
dominant lethal mutations)
Differential Screening
• Use of a subtracted probe to screen a tissue
specific cDNA library
• Subtracted probe is not gene-specific rather it is
differentiation-state-specific probe
• cDNA made from one tissues mRNA
• cDNA is hybridized to mRNA from another
related yet different cell type to remove all
transcripts in common (housekeeping genes)
• Probe has then has common RNA transcripts
subtracted from it
Differential Screening:
Subtracted Library
Epidermal cells
Neuroblasts & Neuroectoderm
pA+ RNA
pA+ RNA
1st strand cDNA
Anneal epi RNA + neuro cDNA (2X)
recover ss cDNA from hydroxyapatite column
(neuro + epi specific cDNAs)
Anneal ss cDNAs from HA column to neuro mrna
recover ds cDNA/mRNA hybrids from HA column
(neuro specific cDNAs)
Use neuro specific cDNAs to make library
Differential Screen: Subtracted
Probe & +/- Screen
Epidermal cells
Neuroblasts & Neuroectoderm
pA+ RNA
pA+ RNA
1st strand cDNA (32P-label)
Anneal epi RNA + neuro cDNA
recover ss cDNA (HA column)
(neuro + epi specific cDNAs)
Anneal ss cDNAs from HA column to unlabeled,neuro mRNA
recover ds cDNA/mRNA hybrids from HA column
(neuro specific cDNAs)
Use labeled cDNA as - probe
to screen library
Use labeled cDNA as + probe
to screen library
Differential Screen: Subtracted
Probe & +/- Screen
Plus-Probe
Minus-Probe
Homology Screens
• Screen a cDNA library for a related gene from a
different species
• Reduce stringency of hybridization so that to
compensate for slight differences in the gene
sequences between the two species
• Find a gene in Drosophila
– antennapedia
• Is there a related gene in mammals?
– Hox genes
Functional Screens
• Add back mRNAs to rescue a mutant
phenotype
– Drosophila
• Cloning of bicoid by adding back fractions of mRNAs
from wt eggs to bicoid mutant eggs
– Xenopus
• Cloning of noggin by adding back mRNAs to
ventralized embryos
• Screening of expression libraries
• Look for functional interactions – two hybrid
Functional Screen: Yeast 2Hybrid
• Fuse DNA binding domain to bait
– cDNA coding DNA binding domain of yeast txn factor
– Ligated in-frame with cDNA coding protein of interest
– bait
• Make cDNA library in vector where cDNAs will
be in-frame with txn activation domain of yeast
txn factor
– This is the prey
• If bait and prey proteins interact, txn-activation &
DNA-binding domains of yeast txn factor will be
next to each other and will drive expression of
gene in yeast that allows yeast to live
Functional Screen: Yeast 2Hybrid
Prey
UnkP
UnkP
Bait
UnkP
LexA-DBD POI
UnkP
Transformed into yeast
Gal4-AD
UnkP
Gal4-AD
Gal4-AD
Gal4-AD
Gal4-AD
UnkP
Gal4-AD
Transform yeast cells expression bait
with cDNA prey library. Each yeast
cell will make 1 of the prey fusion
proteins
Functional Screen: Yeast 2Hybrid
When bait & prey interact, functional txn factor is
formed and expression of His3 occurs
Prey
Bait
RP
LexA Site
His3
Mutagenic Screens:
Chemical/Radiation
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Mutagenize one sex (usually male)
Mate to wt female
All progeny (F1) are heterozygotes for any mutations
Outcross all F1 females to wt males or backcross FI
males to mother
– 50% of F2 will be heterozygotes
• Testcross F2 sibs to produce
– 25% with mutant phenotype
• Outcrossed F1 becomes founder for further generations
to examine and clone mutated gene
Mutagenic Screens: Insertional
Mutagenesis
• Create transgenic animals with heterologous
marker gene inserted randomly into genome
– LacZ or GFP most common marker gene
• Screen for animals with mutant phenotype
• Identify location of insertion and clone
surrounding sequences
– Use probe corresponding to marker gene
sequences
Functional Analysis of
Developmental Genes
• Mutant phenotype associated with gene
• Generation of mutant phenotype when not
already known
– Targeted disruption (transgenic analysis)
– Mis-expression
• Ectopic expression
• Over expression
– Biochemical analysis
• Subcellular location
• Protein-protein interactions
• Enzymology
Transgenic Analysis
• Random insertion of transgenes (for mutagenesis)
• Targeted insertion of transgenes
– Knockout
– Knockin
• Requires special vectors
– contains flanking sequences to permit homologous
recombination between construct and chromosome
– Contains selectable marker to permit survival only of
homologous recombination and not non-homologous
Transgenic Analysis
Vector for homologous
recombination
Transgenic Analysis
Gene of interest
Transgenic Analysis
neor
Homologous recombination replaces region of gene
with neomycin resistance gene and disrupts generation of
functional protein. Neor allows for cells to be selected for
using antibiotic neomycin.
Transgenic Analysis
HSV-tk
neor
Non-homologous recombination inserts thymidine
kinase. The presence of gene allows cells containing it to be
killed by the thymidine analog gancyclovir or FIAU. Only
HSV (viral) tk will phosphorylate the nucleotide analog so only
the cells with HSV-tk will be killed. The phosphorylated
analog stops DNA synthesis when it is incorporated by DNA
polymerase.
Transgenic Analysis
iodo
arabinose
fluoro
Transgenic Analysis
Transgenic Analysis
& FIAU
insensitivity
Transgenic
Analysis
Transgenic Analysis
Functional Analysis by
Misexpression
• Ectopic expression
– Express protein in cells which would not
normally have the protein present
• Mis-expression at different stages which
could be in normal or ectopic locations
• Drosophila, Xenopus, zebrafish, C. elegans,
cultured cells (ES cells  mouse)
– Systems conducive to DNA/RNA
injection/transfection/injfection
Misexpression
• Allows expression of wt or mutant forms of
protein
• Allows knockout using antisense RNA,
antisense oligos, or dominant-negative
proteins
• Allows increase in activity of protein by
expressing constitutively active proteins
Misexpression
• Injection of either synthetic mRNAs or DNA
expression vectors
• Use of DNA expression vector requires specific
regulatory elements
• Judicious use of regulatory elements allows one to
define the expression pattern
– Use homologous elements – expressed in wt pattern
– Use heterologous elements – expression in pattern of
gene from which element came
– Use of constitutive heterologous elements (viral LTR or
housekeeping gene – drive expression everywhere in
embryo
Biochemical
Analysis:Functional Cloning
• Cloning via DNA interaction or protein
interaction
– Screen cDNA expression library with DNA
probe to identify DNA binding proteins
– Screen by yeast two hybrid to identify proteinprotein interactions
Biochemical Analysis
• Function of secreted morphogens by exposing
whole embryo, explants or cultured cells to
purified morphogen
• Determining differentially expressed genes by
detecting differences in protein expression
– 2D SDS-PAGE
• Separate proteins based on isoelectric point then by size
• Compare proteins from two cell types to identify unique
proteins
• Purify protein, sequence, reverse transcribe
oligonucleotide, screen cDNA library
Biochemical Analysis:
Bioactivity
Biochemical Analysis: 2D Gel
IEF Gel
B
Acidic
-
Basic
+
SDS-PAGE
A
Biochemical Analysis: 2D Gel
Compare 2D Gels of proteins form two cell types
Cell type A
Cell type B
Biochemical Analysis:2D Gel
Overlay 2D gels to identify cell-type-specific proteins