Bio Sci 145 B Lecture 9 612004 Bruce

Bio. Sci 145 B Lecture #9 6/1/2004 • Bruce Blumberg – 2113 E Mc. Gaugh Hall - office hours Wed 12 -1 PM (or by appointment) – phone 824 -8573 – blumberg@uci. edu • TA – Curtis Daly cdaly@uci. edu – 2113 Mc. Gaugh Hall, 924 -6873, 3116 – Office hours Tuesday 11 -12 • lectures will be posted on web pages after lecture – http: //eee. uci. edu/04 s/05705/ - link only here – http: //blumberg-serv. bio. uci. edu/bio 145 b-sp 2004 – http: //blumberg. bio. uci. edu/bio 145 b-sp 2004 • DON’T FORGET – TERM PAPERS ARE DUE BY 5 PM on FRIDAY JUNE 4 copyright 2000 Bruce Blumberg, all rights reserved

Requirements for the term paper • Goals – Analytical thinking – Improved writing • Select a topic related to genomic or proteomic analysis of an interesting problem – Talk with me about your topic • Write a short paper (~5 pages) in the style of a research grant describing how you will attack this problem (example is posted). – Specific aims – questions, hypotheses – Background and significance • What is known, what remains to be learned • why should someone give you money to study this problem? – Research plan – specific experiments to answer the questions posed in specific aims • Expected vs unexpected results Bio. Sci 145 B lecture 9 page 2 ©copyright Bruce Blumberg 2004. All rights reserved

Conditional gene targeting - contd • Approach – recombinases perform site-specific excision between recognition sites – FLP system from yeast • doesn’t work well – Cre/lox system from bacteriophage P 1 • P 1 is a temperate phage that hops into and out of the bacterial genome • recombination requires – 34 bp recognition sites locus of crossover x in P 1 (lox. P) – Cre recombinase • if lox. P sites are directly repeated then deletions • if inverted repeats then inversions result Bio. Sci 145 B lecture 9 page 3 ©copyright Bruce Blumberg 2004. All rights reserved

Conditional gene targeting (contd) • Strategy – Make targeting construct (minimum needed for grant) – homologous recombination, – transfect CRE, select for loss of tk – Southern to select correct event • Result called “floxed allele” – inject into blastocysts, select chimeras – establish lines – cross with Cre expressing line and analyze function Bio. Sci 145 B lecture 9 page 4 ©copyright Bruce Blumberg 2004. All rights reserved

Conditional gene targeting (contd) – Tissue- or stage-specific knockouts from crossing floxed mouse with specific Cre-expressing line – requirement for Cre lines • must be well characterized – promoters can’t be leaky • Andras Nagy’s database of Cre lines and other knockout resources http: //www. mshri. on. ca/nagy/cre. htm Bio. Sci 145 B lecture 9 page 5 ©copyright Bruce Blumberg 2004. All rights reserved

Conditional gene targeting (contd) • advantages – can target recombination to specific tissues and times – can study genes that are embryonic lethal when disrupted – can use for marker eviction – can study the role of a single gene in many different tissues with a single mouse line – can use for engineering translocations and inversions on chromosomes • disadvantages – not trivial to set up, more difficult than std ko but more information possible – requirement for Cre lines • must be well characterized regarding site and time of expression • promoters can’t be leaky (expressed when not intended) Bio. Sci 145 B lecture 9 page 6 ©copyright Bruce Blumberg 2004. All rights reserved

Genome wide analysis of gene function • How to mutate all genes 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 Bio. Sci 145 B lecture 9 page 7 ©copyright Bruce Blumberg 2004. All rights reserved

Genome wide analysis of gene function (contd) • How about gene targeting in other organisms – With more complex genomes and more genes? – Not really feasible to specifically target 20 -30 K genes • Difficulty • Expense • Inability to target all possibile loci – Some efforts to make mouse collection • Lexicon Genetics has a collection – Drosophila collection as well – Driving force behind these efforts is • Genome annotation • Drug target discovery (Lexicon) • Functional analysis Bio. Sci 145 B lecture 9 page 8 ©copyright Bruce Blumberg 2004. 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 Bio. Sci 145 B lecture 9 page 9 ©copyright Bruce Blumberg 2004. All rights reserved

Insertional mutagenesis - Gene trapping • 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 Bio. Sci 145 B lecture 9 page 10 ©copyright Bruce Blumberg 2004. All rights reserved

Insertional mutagenesis - Gene trapping (contd) • 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 • -gal 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 Bio. Sci 145 B lecture 9 page 11 ©copyright Bruce Blumberg 2004. 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, poly. A sites • No promoter but has a splie-acceptor sequence 5’ of reporter • Can only be expressed if spliced into an endogenous m. RNA – 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 • As in Golling paper we heard about on Thursday Bio. Sci 145 B lecture 9 page 12 ©copyright Bruce Blumberg 2004. All rights reserved

Insertional mutagenesis - Gene trapping (contd) • Expressed gene trapping (contd) – advantages • insertional mutagen – gives information about expression patterns – can be homozygosed 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 id interesting ones • Gene cloning Bio. Sci 145 B lecture 9 page 13 ©copyright Bruce Blumberg 2004. All rights reserved

Generating phenocopies of mutant alleles • How to inactivate endogenous genes in a targeted but general way? – Important new development is RNAi – RNA interference – Observation is that introduction of doublestranded RNAs into cells lead to destruction of corresponding m. RNA (if there is one) – Principle is si. RNA – small interfering RNAs – These generate small single stranded RNAs that target m. RNAs for destruction by – RISC – RNA interference silencing complex – First applied in C. elegans where it works extremely well • Can introduce si. RNA into cells even by feeding to the worms! • Works very well in Drosophila • poorly in mammalian cells • Poorly in Xenopus Bio. Sci 145 B lecture 9 page 14 ©copyright Bruce Blumberg 2004. All rights reserved

RNAi (contd) • Dicer complex generates short duplexes from ds. RNA in the cell – Important to have 2 -nt overhangs • si. RNAs are generated from these fragments – Antisense strand binds to m. RNA and this recruits the RISC - RNAi silencing complex – Complex leads to m. RNA cleavage and destruction • Two important reviews to read – Mc. Manus and Sharp (2002) Nature Reviews Genetics 3, 737 -747 – Dykxhoorn et al. (2003) Nature Reviews, Molecular Cellular Biology 4, 457 -467 Bio. Sci 145 B lecture 9 page 15 ©copyright Bruce Blumberg 2004. All rights reserved

RNAi (contd) • Micro RNAs are small cellular RNAs that previously lacked any known function – Always form a hairpin structure with mismatches in stem • Turn out that micro RNAS direct gene silencing via translational repression – (mi. RNAs) are mismatched duplexes that dicer processes into st. RNAs – Use the same cellular complex as si. RNAs – Perfect matches -> target cleavage – Imperfect matches -> translational repression of target Bio. Sci 145 B lecture 9 page 16 ©copyright Bruce Blumberg 2004. All rights reserved

RNAi (contd) • Parallels between si. RNA and mi. RNA-directed RNAi Bio. Sci 145 B lecture 9 page 17 ©copyright Bruce Blumberg 2004. All rights reserved

RNAi (contd) • Ways to generate short RNAs that silence gene expression in vitro – a) chemical synthesis of si. RNA, introduce into cell – b) synthesize long ds. RNA, use dicer to chop into si. RNA – c) introduce perfect duplex hairpin, dicer generates si. RNA – d) make mi. RNA based hairpin, dicer generates silencing RNA • Introduce into cells or organism by microinjection, transfection, etc. – Expression is transient – can only generate phenotypes for a short time after introduction Bio. Sci 145 B lecture 9 page 18 ©copyright Bruce Blumberg 2004. All rights reserved

RNAi (contd) • Ways to generate short silencing RNAs in vivo – Continuing expression to generate stable phenotype – a) produce long hairpin from pol II promoter, let dicer make si. RNA – b) produce two transcripts from pol III promoter, let anneal in cells – c) produce a short hairpin from pol III promoter, let dicer generate si. RNAs – d) produce imperfect hairpin from pol II promoter, let dicer generate mi. RNAs that direct gene silencing Bio. Sci 145 B lecture 9 page 19 ©copyright Bruce Blumberg 2004. All rights reserved

RNAi (contd) • RNAi for whole genome functional analysis – First generate library of constructs that generates si. RNA or st. RNA – Introduce these into cells, embyos (fly, frog, mouse) or animals (C. elegans, plants) • For C. elegans, make the library in E. coli and simply feed bacteria to worms • Must microinject or transfect with other animals – Evaluate phenotypes Bio. Sci 145 B lecture 9 page 20 ©copyright Bruce Blumberg 2004. 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 ATG in the transcript and inhibits translation of the protein – Bio. Sci 145 B lecture 9 Deoxyribose page 21 ©copyright morpholine Bruce Blumberg 2004. All rights reserved

Antisense methods to knock out gene function (contd) Oligodeoxyribonucleotide Morpholino Oligonucleotide B = A, C, T, G Bio. Sci 145 B lecture 9 page 22 ©copyright Bruce Blumberg 2004. 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 Bio. Sci 145 B lecture 9 page 23 ©copyright Bruce Blumberg 2004. 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 • Two papers this Thursday and one next Thursday describe two different approaches to this problem. Bio. Sci 145 B lecture 9 page 24 ©copyright Bruce Blumberg 2004. 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 Bio. Sci 145 B lecture 9 page 25 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions • biochemical approach – 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 Bio. Sci 145 B lecture 9 page 26 ©copyright Bruce Blumberg 2004. 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 Bio. Sci 145 B lecture 9 page 27 ©copyright Bruce Blumberg 2004. 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 • 35 S, 125 I, 3 H 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 3 H small molecules • protein: protein interaction • protein: DNA Bio. Sci 145 B lecture 9 page 28 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) • FRET - fluorescent resonance energy transfer – based on the transfer of energy from one fluor to another that is 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 – application • very commonly used for protein: protein interaction screening in industry • FRET microscopy can be used to prove interactions between proteins within single cells – Roger Tsien Bio. Sci 145 B lecture 9 page 29 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) • FRET (contd) – 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 – Not suitable for whole genome analysis • tunable (or multiwavelength capable) fluorometer required (we have one here) Bio. Sci 145 B lecture 9 page 30 ©copyright Bruce Blumberg 2004. 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 Bio. Sci 145 B lecture 9 page 31 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) • Biacore (contd) – Advantages • Can use any target • Biological extracts possible • Measure kinetics • 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 two) • “high throughput” very expensive • Not trivial to optimize Bio. Sci 145 B lecture 9 page 32 ©copyright Bruce Blumberg 2004. 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 M 13 based • some lambda phages used – 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 Bio. Sci 145 B lecture 9 page 33 ©copyright Bruce Blumberg 2004. 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 • Fusion proteins bias the screen against full-length c. DNAs • 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 Bio. Sci 145 B lecture 9 page 34 ©copyright Bruce Blumberg 2004. 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 (GAL 4) usual • stable cell line is commonly used – prepare fusion protein library with an activation domain - prey – Key factor required for success is 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 Bio. Sci 145 B lecture 9 page 35 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) • 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 Bio. Sci 145 B lecture 9 page 36 ©copyright Bruce Blumberg 2004. All rights reserved

Mapping protein-protein interactions (contd) • In vitro interaction screening - based on in vitro expression cloning (IVEC) – transcribe and translate c. DNAs in vitro into small pools of proteins (~100) – test for their ability to interact with your protein of interest • EMSA • co-ip • FRET • SPA – advantages • functional approach • smaller pools increase sensitivity • diversity of targets – proteins, complexes, nucleic acids, protein/nucleic acid complexes, small molecule drugs – very fast – disadvantages • can’t detect heterodimers unless 1 partner known • expensive consumables (but cheap salaries) – Typical screen may cost $10 -15 K • expense of automation Bio. Sci 145 B lecture 9 page 37 ©copyright Bruce Blumberg 2004. All rights reserved