BioSci 203 blumberg lecture 5 page 1 © copyright Bruce Blumberg All rights reserved Bio Sci 203 bb-lecture 5 - cDNA library screening & sequence characterization Bruce Blumberg –office – 2113E McGaugh Hall – –lab (x46873,x43116) –office hours MWF This week –cDNA identification –Protein protein binding assays –Characterization of Selected DNA Sequences DNA sequence analysis –mRNA Analysis –Transcript mapping

BioSci 203 blumberg lecture 5 page 2 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest Screening methods depend on what type of information you have in hand. –Related gene from another species? –A piece of genomic DNA? –A mutant –A functional assay? –An antibody? –A partial amino acid sequence? –A DNA element required for expression of an interesting gene? –An interacting protein? –A specific tissue or embryonic stage? Low stringency hybridization Hybridization Complementation Positional cloning Expression screening Expression library screening Oligonucleotide screening Various binding protein strategies Interaction screening Subtracted or +/- screening

BioSci 203 blumberg lecture 5 page 3 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Cloning by complementation –generally only useful with manipulable genetic systems yeast Drosophila C. elegans zebrafish –presumes that complemented mutant is readily observable –Approach transfer pooled cDNA libraries in expression vectors into the mutant –or mRNA pools derived from libraries assay for rescue subdivide positive pools and repeat –advantages direct functional test rapid compared with chromosome walking –disadvantages fairly tedious dependent on library quality requires easily observable rescue

BioSci 203 blumberg lecture 5 page 4 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Positional cloning –If your mutant results from a transposon insertion then this can be recovered –If insertion is a P-element or gene trap Make genomic library from mutant –What type of library will you make (λ, BAC, etc)? Why? Screen with transposon –Recover positives, sequence flanking region Use flanking sequence to screen normal genomic library –What type of library will you screen (λ, BAC, etc)? Why? –If insertion is a gene trap or related You can digest mutant DNA with an enzyme that linearizes the vector Ligate and transform Colonies that form should have flanking region –sequence Use this to screen normal library –OR Use inverse PCR to get flanking sequence from plasmid and use this to probe library

BioSci 203 blumberg lecture 5 page 5 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Functional screening (expression cloning) –if you have a functional assay, expression cloning is reasonable –strategy: Large pools (~10,000) of cDNAs tested for function –microinjection –transfection –receptor binding (panning) positive pools are subdivided and retested to obtain pure cDNAs cycle is repeated until single clones obtained –Advantages functional approach in vivo testing is possible can identify secreted proteins and receptors –Disadvantages Slow and tedious sensitivity issue due to pool size extensive retesting of pools is required –applications: many receptors and transporters cloned this way

BioSci 203 blumberg lecture 5 page 6 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Antibody screening of cDNA expression libraries –requirements antibody must recognize denatured epitope (western blot) –many monoclonals recognize 3-D or sugar epitopes affinity purified antibodies work best cDNA expression library, e.g., λgt11 series –approach plate library and induce replicate filters incubate with antibody, wash and develop the filters repeat until a pure clone is obtained –verification affinity purify antibody with phage fusion protein – western with original protein –advantages best choice if only antibody is available –disadvantages λgt11 and relatives are painful to work with your antibody may not be suitable –sugar directed –structural epitope

BioSci 203 blumberg lecture 5 page 7 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) A partial amino acid sequence? –Purified protein and have one or more partial amino acid sequences make a peptide antibody and screen (slow) Oligonucleotide screening based on aa sequence –multiple codons for most aa PCR between multiple primers –three types of oligos in use long guess-mers - pick the wobble base –relies on low stringency hybridization inosine - use inosine for multiple bases –I:C >> others degenerate oligos (mixtures of all possible seqs) –mixtures of < 1024 virtually always work

BioSci 203 blumberg lecture 5 page 8 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) A partial amino acid sequence (contd)? –approach pick an aa sequence that predicts a reasonable probe complexity (~1024 fold)(avoid ser, leu, arg) WHY? synthesize fully degenerate mixture of probes and label hybridize at low stringency (T m -25 for the most AT rich sequence) wash at high stringency in 3M tetramethylammonium chloride –TMAC stabilizes AT base pairs A-T = G-C –T m is a strict function of length –works best for mers –degenerate oligo and TMAC advantages –degenerate oligos always work –fast –only requires a single sequence disadvantages –TMAC method requires strict adherence to technique –aa sequence may not predict a good oligo »e.g., too many leu, ser or arg

BioSci 203 blumberg lecture 5 page 9 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) A partial amino acid sequence (contd) –PCR – design primers to two conserved sequences, amplify, clone advantages –very fast –almost anyone can manage disadvantages –requires 2 good sequences –PCR errors may give incorrect sequence

BioSci 203 blumberg lecture 5 page 10 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) A DNA element required for expression of an interesting gene? –How to identify what factors bind to putative elements? examine the sequence –does it contain known binding sites? –Check TRANSFAC database » –if yes, do such proteins bind to the isolated element in gel-shift experiments? do the elements bind proteins from nuclear extracts? –gel shift (EMSA) experiments clone the elements into reporters with minimal promoters. –do these constructs recapitulate activity? –What does the sequence tell you about the binding protein? AGGTCATGACCT Dyad symmetry always means multimeric protein No symmetry usually means monomeric protein

BioSci 203 blumberg lecture 5 page 11 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Biochemical purification of binding proteins –tedious, considerable biochemical skill required –two basic approaches fractionate nuclear extracts chromatographically and test fractions for ability to bind the element DNA-affinity chromatography –multimerize the element and bind to a resin –pass nuclear extracts across column and purify specific binding proteins –protein microsequencing –predict DNA sequence from amino acid sequence look in the database prepare oligonucleotides and screen library

BioSci 203 blumberg lecture 5 page 12 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Biochemical purification of binding proteins (contd) –advantages gold standard if you can purify proteins, this will always work –not so many good protein biochemists works for dimeric proteins and complexes –disadvantages slow, tedious need good protein sequencing facility biochemical expertise required expense of preparing preparative quantities of nuclear extracts

BioSci 203 blumberg lecture 5 page 13 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Molecular biological approaches to identifying binding proteins –oligonucleotide screening of expression libraries (Singh screening) multimerize oligonucleotide and label with 32 P screen expression library to identify binding proteins advantages –straightforward –much less biochemical expertise required than biochemical purification –relatively fast disadvantages –can’t detect binding if multiple partners are required –fair amount of “touch” required

BioSci 203 blumberg lecture 5 page 14 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd)) Molecular biological approaches to identifying binding proteins –expression cloning (sib screening) clone element of interest (or promoter) into a suitable reporter construct (e.g. luciferase) transfect (or inject, or infect, etc) pools (~10,000 cDNAs each) of cDNA expression libraries and assay for reporter gene retest positive pools in smaller aliquots (~1000) repeat until a pure cDNA is found –advantages functional approach presumably using the appropriate cell type so modifications occur possibility to detect dimers with endogenous proteins –disadvantages VERY TEDIOUS very slow, much duplication in pools, extensive rescreening is required could be expensive

BioSci 203 blumberg lecture 5 page 15 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Molecular biological approaches to identifying binding proteins –in vitro expression cloning (IVEC) transcribe and translate cDNA libraries in vitro into small pools of proteins (~100) EMSA to test protein pools for element binding unpool cDNAs and retest advantages –functional approach –smaller pools increase sensitivity disadvantages –can’t detect dimers –very expensive (TNT lysate) –considerable rescreening still required –tedious, countless DNA minipreps required

BioSci 203 blumberg lecture 5 page 16 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Molecular approaches (contd) –yeast one-hybrid assay clone element into a reporter construct and make stable yeast strain transfect aliquots of cDNA expression libraries that have fragments of DNA fused to yeast activator if the fusion protein binds to your element then the reporter gene will be activated advantages –somewhat of a functional approach –eukaryotic milieu allows some protein modification disadvantages –slow, tedious purification of positives –can’t readily detect heterodimeric proteins –sensitivity is not so great –usual yeast false positives

BioSci 203 blumberg lecture 5 page 17 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) You have one protein and want to identify proteins that interact with it –some sort of interaction screen is indicated straight biochemistry phage display two hybrid in vitro expression cloning

BioSci 203 blumberg lecture 5 page 18 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) biochemical approach –purify cellular proteins that interact with your protein co-immunoprecipitation affinity chromatography biochemical fractionation –pure protein(s) are microsequenced if not in database then make oligonucleotides and screen cDNA library from appropriate tissues –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