1. Last year’s final exam is posted
BioSci D145 Lecture #9
Bruce Blumberg
4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment)
phone
TA – Riann Egusquiza
4351 Nat Sci 2– office hours M 1-3
Phone
Updated lectures will be posted on web pages after lecture
Term papers due Friday, March 10 by 12 midnight (23:59.59) (-1 point for each day late)
Last year’s final exam is posted
BioSci D145 lecture 9 page 1 ©copyright Bruce Blumberg All rights reserved

2. Phenotypic rescue by RNAi – synthetic lethal and related approaches
How can we find other members of pathways we already know something about?
Or, how can we find drugs that act on a pathway to kill cells (e.g., cancer cells?)
Synthetic lethal is one relatively new and promising approach
2 mutations are synthetic lethal if either single mutation is viable but the double mutant is lethal
BioSci D145 lecture 9 page 2 ©copyright Bruce Blumberg All rights reserved

3. Phenotypic rescue by RNAi – synthetic lethal and related approaches
How can we find other members of pathways we already know something about?
In cancer screening, what if we combine a mutation and a drug to find a combination that increases the kill rate, or reveals a phenotype that is similar to the total loss of function?
Could find novel drug targets (pathways that kill cells in presence of siRNA+drug (usually sublethal amount of drug)
Can find genes that are targeted by drugs – pathway analysis
Could find new biomarkers that are required for cell viability following drug treatment
BioSci D145 lecture 9 page 3 ©copyright Bruce Blumberg All rights reserved

4. Mohr et al., 2010, Genomic Screening with RNAi: Results and Challenges. Annual Review of Biochemistry, 29: 37-64
BioSci D145 lecture 9 page 4 ©copyright Bruce Blumberg All rights reserved

5. Genome wide analysis of gene function
How to mutate all genes (we know about) in a given genome?
Easy with microbial genomes – can mutate all yeast genes by homologous recombination
Recombine in selectable marker
Propagate strain and analyze phenotypes
Homology region
Unique oligonucleotide
“barcodes” for PCR
Selectable marker (antibiotic resistance)
Target gene
BioSci D145 lecture 9 page 5 ©copyright Bruce Blumberg All rights reserved

6. Genome wide analysis of gene function (contd)
How about gene targeting in other organisms
With more complex genomes and more genes?
Huge undertaking to specifically target 30K+ genes in mammalian cells
Difficulty
Expense
Inability to target all possible loci
Some efforts to make mouse collection
Lexicon Genetics has a collection of ES cells
Drosophila collection as well
Driving force behind these efforts is
Genome annotation
Drug target discovery (Lexicon)
Functional analysis
BioSci D145 lecture 9 page 6 ©copyright Bruce Blumberg All rights reserved

7. Figure 5.18 The three major types of mutagen
BioSci D145 lecture 9 page 7 ©copyright Bruce Blumberg All rights reserved

8. Genome wide analysis of gene function (contd)
A 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 9 page 8 ©copyright Bruce Blumberg All rights reserved

9. Insertional mutagenesis - enhancer trap
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 9 page 9 ©copyright Bruce Blumberg All rights reserved

10. Insertional mutagenesis - 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):
BioSci D145 lecture 9 page 10 ©copyright Bruce Blumberg All rights reserved

11. Insertional mutagenesis - enhancer trap
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 9 page 11 ©copyright Bruce Blumberg All rights reserved

12. Insertional mutagenesis – Gene trapping
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 9 page 12 ©copyright Bruce Blumberg All rights reserved

13. 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
BioSci D145 lecture 9 page 13 ©copyright Bruce Blumberg All rights reserved

14. Antisense methods to inhibit 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
BioSci D145 lecture 9 page 14 ©copyright Bruce Blumberg All rights reserved

15. Antisense methods to inhibit gene function (contd)
Oligodeoxyribonucleotide
Morpholino Oligonucleotide
B = A, C, T, G
BioSci D145 lecture 9 page 15 ©copyright Bruce Blumberg All rights reserved

16. Antisense methods to inhibit 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 page 16 ©copyright Bruce Blumberg All rights reserved

17. Antisense methods to inhibit 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 page 17 ©copyright Bruce Blumberg All rights reserved

18. Antisense methods to inhibit 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 page 18 ©copyright Bruce Blumberg All rights reserved

19. 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 page 19 ©copyright Bruce Blumberg All rights reserved

20. Each protein interacts with average of 3 others
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 page 20 ©copyright Bruce Blumberg All rights reserved

21. How to identify protein-protein interactions on a genome wide scale?
You have one protein and want to identify proteins that interact with it
You want to identify all proteins that interact with all other proteins
straight biochemistry
Co-immunoprecipitation
GST-pulldown
Library based methods
phage display
Yeast two hybrid
in vitro expression cloning
Proteomic analysis
Protein microarrays
Large scale two-hybrid
BioSci D145 lecture 9 page 21 ©copyright Bruce Blumberg All rights reserved

22. Mapping protein-protein interactions
biochemical approach –
what are some ways to purify cellular proteins that interact with your protein
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
co-immunoprecipitation
GST-pulldown
affinity chromatography
biochemical fractionation
BioSci D145 lecture 9 page 22 ©copyright Bruce Blumberg All rights reserved

23. Mapping protein-protein interactions (contd)
GST (glutathione-S-transferase) pulldown assay
Versatile and general
Fuse protein of interest to GST
Incubate with cell or tissue extracts
Mix with glutathione-sepharose 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 page 23 ©copyright Bruce Blumberg All rights reserved

24. 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 page 24 ©copyright Bruce Blumberg All rights reserved

25. 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 page 25 ©copyright Bruce Blumberg All rights reserved

26. 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 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 page 26 ©copyright Bruce Blumberg All rights reserved

27. 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 page 27 ©copyright Bruce Blumberg All rights reserved

28. 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 page 28 ©copyright Bruce Blumberg All rights reserved

29. 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?
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
Lambda and M13 phages don’t have legs…
BioSci D145 lecture 9 page 29 ©copyright Bruce Blumberg All rights reserved

30. 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 page 30 ©copyright Bruce Blumberg All rights reserved

31. 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?
approach
transfect library into cells and either select for survival or activation of reporter gene
purify and characterize positive clones
no activation domain in bait!
BioSci D145 lecture 9 page 31 ©copyright Bruce Blumberg All rights reserved

32. 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 page 32 ©copyright Bruce Blumberg All rights reserved

33. 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 mass-spectrometry
Iteratively build up a map of protein interactions from such complexes
BioSci D145 lecture 9 page 33 ©copyright Bruce Blumberg All rights reserved

34. Global profiling of protein expression
Proteomics is name given to study of the proteome (what is proteome)?
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
Proteome -> a cell or organism’s complement of expressed proteins
Not necessarily identical to transcriptome
BioSci D145 lecture 9 page 34 ©copyright Bruce Blumberg All rights reserved

35. 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 page 35 ©copyright Bruce Blumberg All rights reserved

36. 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 page 36 ©copyright Bruce Blumberg All rights reserved

37. 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 page 37 ©copyright Bruce Blumberg All rights reserved

38. 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
BioSci D145 lecture 10 page 38 ©copyright Bruce Blumberg All rights reserved

39. 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 page 39 ©copyright Bruce Blumberg All rights reserved

40. Global profiling of protein expression (contd)
Protein arrays now available from many vendors
Immobilized proteins
Spot proteins on slides and ask what interacts with particular ones
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
BioSci D145 lecture 9 page 40 ©copyright Bruce Blumberg All rights reserved