Transcript No Slide Title

BioSci D145 Lecture #9
• Bruce Blumberg ([email protected])
– 4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment)
– phone 824-8573
• TA – Bassem Shoucri ([email protected])
– 4351 Nat Sci 2, 824-6873, 3116 – office hours M 2-4
• lectures will be posted on web pages after lecture
– http://blumberg.bio.uci.edu/biod145-w2015
– http://blumberg-lab.bio.uci.edu/biod145-w2015
– Term papers due Friday March 6 by 12
midnight (23:59.59) (-1 point/day late)
– Last year’s final exam is posted
– No office hours Thursday 3/5
BioSci D145 lecture 1
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©copyright
Bruce Blumberg 2010. All rights reserved
Genome wide analysis of gene function (contd)
• Main method for gene targeting in more complex organisms is random
insertional mutagenesis
– Transposon mutagenesis
• Bacteria – Tn transposons
• Yeast - Ty transposons
• Drosophila - P- elements
• Vertebrates - Sleeping Beauty transposons
– Viral infection
• Typically retroviruses – host range selectivity is obstacle
– Gene or enhancer trapping
– modified viruses
or transposons
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2009 All rights reserved
Insertional mutagenesis - Gene trapping – enhancer trip
• viruses and transposable elements
can deliver DNA to random
locations
– can disrupt gene function
– put inserted gene under the
control of adjacent regulatory
sequences
– BOTH
• enhancer trap is designed to bring inserted reporter gene under the control
of local regulatory sequences
– put a reporter gene adjacent to a weak promoter (enhancer-less), e.g. a
retrovirus with enhancers removed from the LTRs
– may or may not disrupt expression
– Hopkins zebrafish group used unmodified virus
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2009 All rights reserved
Insertional mutagenesis - Gene trapping –enhancer trap (contd)
Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in
two developmental genes: tcf7 and synembryn-like. Nagayoshi S, Hayashi E, Abe G, Osato N, Asakawa
K, Urasaki A, Horikawa K, Ikeo K, Takeda H, Kawakami K. Development 2008 Jan;135(1):159-69.
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2009 All rights reserved
Insertional mutagenesis - Gene trapping –enhancer trap (contd) stopped here
2015
• enhancer trap (contd)
– expression only when integrate into an active transcription unit
• reporter expression duplicates the temporal and spatial pattern of
the endogenous gene
– reporters used
• -galalactosidase was the most widely used reporter
• GFP is now popular
• -lactamase is seeing increasing use
– advantages
• relatively simple to perform
• active promoters frequently targeted, perhaps due to open chromatin
– Disadvantages
• Inactive promoters probably not targeted
• insertional mutagenesis not the goal, and not frequent
– overall frequency is not that high
• relies on transposon or retroviruses to get insertion
– may not be available for all systems, requires transgenesis or
good viral vectors
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2009 All rights reserved
Insertional mutagenesis – Gene trapping (contd)
• expressed gene trap (many variations possible)
– goal -> ablate expression of endogenous gene, replace with transgene
– Make insertion construct – reporter, selection, polyA sites
• No promoter but has a splice-acceptor sequence 5’ of reporter
• Can only be expressed if spliced into an endogenous mRNA
– Transfer into embryonic cells, generate a library of insertional mutagens
• Mouse, Drospophila, zebrafish, frog
– reporter expression duplicates the temporal and spatial pattern of the
endogenous gene
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2009 All rights reserved
Insertional mutagenesis - Gene trapping (contd)
• Expressed gene trapping (contd)
– advantages
• insertional mutagen
– gives information about expression patterns
– can be made homozygous to generate phenotypes
• higher efficiency than original trapping methods
• selectable markers allow identification of mutants
– many fewer to screen
– dual selection strategies possible
– disadvantages
• overall frequency is still not that high
• frequency of integration into transcription unit is not high either
• relies on transposon or retroviruses to get insertion
– may not be available in your favorite system.
– Uses
• Insertional mutagenesis
• Marking genes to identify interesting ones
• Gene cloning
• http://www.genetrap.org/
BioSci D145 lecture 8
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©copyright
Bruce Blumberg 2009 All rights reserved
Antisense methods to knock out gene function
• Antisense oligonucleotides can transiently target endogenous RNAs
– For destruction
• Many methods and oligo chemistries available
• Most are very sensitive to level of antisense oligo, these are degraded
and rapidly muck up cellular nucleotide pools leading to toxicity
– For translational inhibition
• Morpholino oligos appear to work the best
– Morpholine sugar is substituted for deoxyribose
– Is not a substrate for cellular DNAses or RNAse H
– Base-pairs with RNA or DNA more avidly than standard DNA
– The oligo binds to the area near the AUG in the transcript and
inhibits translation of the protein
–
Deoxyribose
morpholine
O
O
N
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Antisense methods to knock out gene function (contd)
Oligodeoxyribonucleotide
Morpholino Oligonucleotide
O
O
B
O
B = A, C, T, G
O
B
N
O
O
P
O
O
P
N
O
O
O
B
O
B
N
O
O
O
P
O
O
O
BioSci D145 lecture 9
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P
©copyright
Bruce Blumberg 2010. All rights reserved
N
Antisense methods to knock out gene function
• Morpholinos (contd)
– For translational inhibition
• AUG morpholinos – make within about 50 bp of AUG
– Inhibits translation of the mRNA but mRNA is still present
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Antisense methods to knock out gene function
• Morpholinos (contd)
– For translational inhibition
• Splice morpholinos are very effective
– Target intron exon borders with the morpholino
– Morpholino prevents splicing
– No splicing -> no mature mRNA -> no transport out of nucleus
– Or mis-splicing to get nonsense proteins
» Or to get some unexpected product….
– Mature mRNA is depleted from cells leading to loss of protein
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Antisense methods to knock out gene function
• Morpholinos (contd)
– AUG morpholinos – make within about 50 bp of AUG
• Inhibits translation of the mRNA but mRNA is still present
– Splice morpholinos are very effective
• Block or alter splicing to make no, or non-functional
• How do we verify that morpholinos worked as expected?
– AUG morpholinos
• Western blot to verify loss of protein – requires an antibody
• Rescue with mRNA to which MO doesn’t bind
– Most frequently used method in Xenopus
• Obtain same phenotype with a different MO
− Good but gets EXPENSIVE
– splice morpholinos
• RT-PCR to test for mature mRNA
• QPCR to quantitate
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Most Molecules Function in Complexes
• Given a target, how can we
identify interacting proteins?
• Complex members may be
important new targets
– pharmacology
– toxicology
– Endocrine disrupter action
• High throughput, genome wide
screen is preferred
– 20 years is too long
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
How can we approach whole genome analysis of protein complex formation?
• Each protein interacts
with average of 3
others
• Many are much more
complex
• Papers this Thursday
describe two different
approaches to this
problem.
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
How to identify protein-protein interactions on a genome wide scale?
• You have one protein and want to identify proteins that interact with it
– straight biochemistry
• Co-immunoprecipitation
• GST-pulldown
– Library based methods
• phage display
• Yeast two hybrid
• in vitro expression cloning
• You want to identify all proteins that interact with all other proteins
– Proteomic analysis
– Protein microarrays
– Large scale two-hybrid
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Mapping protein-protein interactions
• biochemical approach –
– what are some ways to purify cellular proteins that interact with your
protein
•
•
•
•
co-immunoprecipitation
GST-pulldown
affinity chromatography
biochemical fractionation
– pure protein(s) are microsequenced
– advantage
• functional approach
• stringency can be manipulated
• can identify multimeric proteins or complexes
• will work if you can purify proteins
– disadvantages
• much skill required
• low throughput
• considerable optimization required
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Mapping protein-protein interactions (contd)
• GST (glutathione-Stransferase) pulldown assay
– Versatile and general
– Fuse protein of interest
to GST
– Incubate with cell or
tissue extracts
– Mix with glutathionesepharose beads
• Binds GST-fusion
protein and anything
bound to it
– Run SDS-PAGE
– Identify bands
• Co-IP (immunoprecipitation) is identical except that antibody is used to pull
down protein X
• Many sorts of tags can be used
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Mapping protein-protein interactions (contd)
• scintillation proximity assay
– Target is bound to solid phase –
bead or plate
– radioactive protein or ligand is added
and allowed to reach equilibrium
• 35S, 125I, 3H work best
– radioactive decay is quenched in
solution, only detected when in
“proximity” of the solid phase, e.g.
when bound to target
– applications
• ligand-receptor binding with 3H small molecules
• protein:protein interaction
• protein:DNA
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Mapping protein-protein interactions (contd)
• FRET - fluorescent resonance energy transfer
– transfer of energy from one fluor
to another not normally excited at that
wavelength
– Many types of fluorescent
moieties possible
• rare earth metals
– europium cryptate
• fluorescent proteins
– GFP and variants
– allophycocyanin
• Tryptophan residues
in proteins
• Use in protein:protein
interactions?
− If proteins are close AND
if emission of A matches
excitation of B
FRET occurs
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Mapping protein-protein interactions (contd)
• FRET (contd)
– application
• commonly used for protein:protein interaction screening in industry
• FRET microscopy can be used to prove interactions between proteins
within single cells
– Roger Tsien at UCSD is expert
– advantages
• can be very sensitive
• may be inexpensive or not depending on materials
• non-radioactive
• equilibrium assay
• single cell protein:protein interactions possible
• time resolved assays possible
– disadvantage
• poor dynamic range - 2-3 fold difference full scale
• must prepare labeled proteins or ligands
– Difficult to do whole genome analysis this way
• multiwavelength capable fluorometer required (we have one here)
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Mapping protein-protein interactions (contd)
• Biacore (surface plasmon resonance)
– surface plasmon waves are excited at a metal/liquid interface
– Target bound to a thin metal foil and test sample flowed across it
– Foil is blasted by a laser from behind
• SPR alters reflected light intensity at a specific angle and wavelength
• Binding to target alters refractive index which is detected as change
in SPR
• Change is proportional to change in mass and independent of
composition of binding agent
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Mapping protein-protein interactions (contd)
• Biacore (contd)
– Advantages
• Can use any target
• Biological extracts possible
• Can measure kinetics
– Generate Kd directly
• Small changes detectable with correct instrument
– 360 d ligand binding to 150 kd antibody
• Can use as purification and identification system
– Disadvantages
• Machine is expensive (we have three)
• “high throughput” very expensive
• Not trivial to optimize
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Library-based methods to map protein-protein interactions (contd)
• Phage display screening (a.k.a. panning)
– requires a library that expresses
inserts as fusion proteins with a
phage capsid protein
• most are M13 based
• some lambda phages used
• What is wrong with this picture?
Lambda and M13 phages don’t
have legs…
– prepare target protein
• as affinity matrix
• or as radiolabeled probe
– test for interaction with library members
• if using affinity matrix you purify phages from a mixture
• if labeling protein one plates fusion protein library and probes with
the protein
– called receptor panning based on similarity with panning for
gold
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Library-based methods to map protein-protein interactions (contd)
• Phage display screening (a.k.a. panning) (contd)
– advantages
• stringency can be manipulated
• if the affinity matrix approach works the cloning could go rapidly
– Disadvantages
• Multiple attempts required to optimize binding
• Limited targets possible
• may not work for heterodimers
• unlikely to work for complexes
• panning can take many months for each screen
– Greg Weiss in Chemistry is local expert
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Mapping protein-protein interactions (contd)
• Two hybrid screening
– originally used in yeast, now
other systems possible
– prepare bait - target protein
fused to DBD (GAL4) usual
• stable cell line is commonly
used
– prepare fusion protein library
with an activation domain - prey
– What is the key factor required for success?
• no activation domain in bait!
– approach
• transfect library into cells and either
select for survival or activation of
reporter gene
• purify and characterize positive clones
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Mapping protein-protein interactions (contd) (stopped here)
• Two-hybrid screening (contd)
– Can be easily converted to
genome wide searching by
making haploid strains, each
containing one candidate
interactor
– Mate these and check for
growth or expression of reporter
gene
Bait plasmid
Prey plasmid
If interact, reporter expressed
and/or
Yeast survive
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Large scale mapping of protein-protein interactions
• GST (glutathione-S-transferase)
pulldown assay
– Or other purification wherein one
protein is tagged and complex of
proteins binding to it is recovered
– Purify complexes from cells
– Characterize complexes by massspectrometry
– Iteratively build up a map of
protein interactions from such
complexes
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Global profiling of protein expression
• Proteomics is name given to study of the proteome (what is proteome)?
– Proteome -> a cell or organism’s complement of expressed proteins
• Not necessarily identical to transcriptome
• Methods
– 2-D gel electrophoresis
– Mass spectrometry of various sorts
• All mass spectrometry requires that molecules “fly” and measures
mass/charge (m/z) ratio
• MALDI-TOF
– Matrix assisted laser desorption ionization – time of flight
– Laser causes matrix to vaporize and molecules to fly, charge is
applied and time molecule takes to fly to detector measured
along with m/z
• ESI
– electrospray ionization – molecules are sprayed, ionized and
detected
• MS-MS
– Tandem mass spec – has two mass analyzers - first detector
shunts selected molecule to second – used for sequencing and
structure analysis
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Global profiling of protein expression (contd)
• 2-D electrophoresis
– Ironically, this is the oldest method for
“proteomics”
– First dimension is isoelectric focusing
• Set up a pH gradient in tube, apply proteins and electrophorese
• each protein migrates to its isoelectric point and stops
– Second dimension is SDS-PAGE – proteins migrate according to size
• Run at 90º to first dimension
BioSci D145 lecture 9
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©copyright
Bruce Blumberg 2010. All rights reserved
Global profiling of protein expression (contd)
• 2-D electrophoresis
– Current technology is to cut out spots and id by mass spec
• Mass spec resurrected 2-D electrophoresis
–
Steep pH gradient
shallow pH gradient
BioSci D145 lecture 10
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©copyright
Bruce Blumberg 2009. All rights reserved
Global profiling of protein expression (contd)
• 2-D electrophoresis (contd)
– Good points
• Straightforward separation
• Can get good resolution with good isoelectric focusing gels
– Downside
• Protein may not be detectable as well-resolved spots that can be
excised and characterized
– Co-migrate
– Abundance
• Variation from experiment to experiment
– Spot position on gel is very sensitive to small changes in pH
BioSci D145 lecture 10
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©copyright
Bruce Blumberg 2009. All rights reserved
Global profiling of protein expression (contd)
• Mass spectrometric methods
– MudPIT is most useful for large scale protein profiling
• Multidimensional protein identification technology
– Separate proteins by microcapillary liquid chromatography
– Characterize and identify proteins by ms-ms
– Prof. Lan Huang is local expert on protein profiling by mass spectrometry
• https://webfiles.uci.edu/lanhuang/www/
BioSci D145 lecture 10
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©copyright
Bruce Blumberg 2009. All rights reserved
Global profiling of protein expression (contd)
• Strategies for high-throughput,
high-resolution protein
identification and analysis
– Equipment is very
expensive but possibilities
are limitless
– Can match proteins with
database sequences OR
– Can sequence proteins de
novo
• Computationally
intensive
BioSci D145 lecture 10
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©copyright
Bruce Blumberg 2009. All rights reserved
Global profiling of protein expression (contd)
• Protein arrays now available
– Immobilized proteins
• Spot proteins on slides and ask what interacts with particular ones
• Luis Villareal runs a facility here that intends to produce all possible
proteins for array generation (from Franciscella tularensis)
– Antibody arrays
• Antibodies spotted on arrays – test for presence of particular proteins
in probe
• Micro-ELISA or RIA
– Antigen arrays
• Known antigens spotted – tests for presence of antibodies in sample
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©copyright
Bruce Blumberg 2009. All rights reserved