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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 page 1 ©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 page 2 ©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 page 3 ©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 page 4 ©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 page 5 ©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 page 6 ©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 page 7 ©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 page 8 ©copyright Bruce Blumberg 2010. All rights reserved Antisense methods to knock out gene function (contd) Oligodeoxyribonucleotide O B O Morpholino Oligonucleotide O B = A, C, T, G O B N O O P N O P O O O O B O N O O P N O P O O O BioSci D145 lecture 9 page 9 ©copyright Bruce Blumberg 2010. All rights reserved B 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 page 10 ©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 page 11 ©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 page 12 ©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 page 13 ©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 page 14 ©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 page 15 ©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 page 16 ©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 page 17 ©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 page 18 ©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 page 19 ©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 page 20 ©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 page 21 ©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 page 22 ©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 page 23 ©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 page 24 ©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 page 25 ©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 page 26 ©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 page 27 ©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 page 28 ©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 page 29 ©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 page 30 ©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 page 31 ©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 page 32 ©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 page 33 ©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 BioSci D145 lecture 10 page 34 ©copyright Bruce Blumberg 2009. All rights reserved