BioSci 203 blumberg lecture 4 page 1 © copyright Bruce Blumberg All rights reserved Bio Sci 203 BB-lecture 4 - cDNA library screening Bruce Blumberg –office – 2113E McGaugh Hall – –lab (x46873,x43116) –NO office hours today - sorry. Link is also main class web site Today –wrap up cDNA library screening –characterization of clones obtained from screening –Protein protein binding assays

BioSci 203 blumberg lecture 4 page 2 © copyright Bruce Blumberg All rights reserved Full-length mRNA isolation and cDNA synthesis (contd) Cloning of cDNAs –most methods require linker or adapter addition followed by restriction digestion –relies on methylation to protect internal sites or use of rare cutters –A new alternative is ExoIII-mediated subcloning no methylation no restriction digestion no ligation no multimerization of vector or inserts 100% oriented

BioSci 203 blumberg lecture 4 page 3 © copyright Bruce Blumberg All rights reserved Vectors for cDNA cloning Plasmids vs phage –phage preferred for high density manual screening –plasmids are better for functional screening microinjection transfection panning –phage packaging and infection more efficient than electroporation x better than best transformation frequency what will the library be used for ? –Consider the intended use as well as other contemplated uses will the library go to an EST project? –Plasmid will it be screened manually –phage or arrayed and screened on high density filters –plasmid will we normalize it? –Probably plasmid

BioSci 203 blumberg lecture 4 page 4 © copyright Bruce Blumberg All rights reserved Vectors for cDNA cloning (contd) Analysis of cDNAs obtained –rate limiting step in clone analysis is getting them into a usable form usually a plasmid –cloning is tedious, particularly if one has many positives some tricks can be used but this is still the bottleneck in about 1985 or so, Stratagene introduced lambda ZAP –phage with an embedded plasmid and M13 packaging signals –plasmid can be automatically excised by adding a helper phage gene II protein replicates plasmid into ss phagemid which is secreted –this was a major advance and many phage libraries today are made in ZAP or its derivatives –early protocols had problems with helper phage but this has been overcome later, others developed a Cre-lox based system –instead of M13 used loxP sites. –When Cre recombinase is added, recombination between the loxP sites excises a plasmid both methods work very well and make analysis of many clones very straightforward

BioSci 203 blumberg lecture 4 page 5 © copyright Bruce Blumberg All rights reserved Vectors for cDNA cloning (contd)

BioSci 203 blumberg lecture 4 page 6 © copyright Bruce Blumberg All rights reserved Vectors for cDNA cloning (contd)

BioSci 203 blumberg lecture 4 page 7 © copyright Bruce Blumberg All rights reserved mRNA frequency and cloning mRNA frequency classes –classic references Bishop et al., 1974 Nature 250, Davidson and Britten, 1979 Science 204, –abundant mRNAs that together represent 10-20% of the total RNA mass > 0.2% –intermediate 1,000-2,000 mRNAs together comprising 40-45% of the total % abundance –rare 15,000-20,000 mRNAs comprising 40-45% of the total abundance of each is less than 0.05% of the total some of these might only occur at a few copies per cell How does one go about identifying genes that might only occur at a few copies per cell?

BioSci 203 blumberg lecture 4 page 8 © copyright Bruce Blumberg All rights reserved Normalization and subtraction How to identify genes that might only occur at a few copies per cell? –Improve your chances - alter the representation of the cDNAs Normalization - reducing the frequency of abundant and increasing the frequency of rare mRNAs - Bonaldo et al., 1996 Genome Research 6, –Theoretically, normalization brings all cDNAs into the same order of magnitude abundance, i.e., within 10 fold of each other rarely works this well. Typically, abundant genes reduced 10x, rare ones increased 3-10x Intermediate class genes do not change much at all –Approach make a population of cDNAs single stranded –tester hybridize with a large excess of cDNA or mRNA to C o t =5.5 –driver C o t value is critical for success of normalization –5-10 is optimal –higher values are not better

BioSci 203 blumberg lecture 4 page 9 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) –Approach (contd) various approaches to make driver –use mRNA - may not be easy to get –make ssRNA by transcribing library –ssDNA from gene II/ExoIII treating inserts from plasmid library –PCR amplification of library best approach is to use driver derived from the same library by PCR –rapid, simple and effective –other approaches each have various technical difficulties –see the Bonaldo review for details.

BioSci 203 blumberg lecture 4 page 10 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) –What are normalized libraries good for? EST sequencing gene identification –biggest use is to reduce the number of cDNAs that must be screened –good general purpose target to screen »subtracted libraries are useful but limited in utility –Drawbacks Not trivial to make Size distribution of library changes –Longer cDNAs lost

BioSci 203 blumberg lecture 4 page 11 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) Subtraction - removing cDNAs (mRNAs) expressed in two populations leaving only differentially expressed –Sagerström et al. (1997) Ann Rev. Biochem 66, /- screening St. John and Davis (1979) Cell 16, –Hybridize the same library with probes prepared from two different sources and compare the results example - hybridize normal liver cDNA library with probes from normal and cancerous liver –Colonies or plaques that are expressed in target tissue (tumor) compared with control are picked –Why aren’t all colonies labeled in normal tissue?

BioSci 203 blumberg lecture 4 page 12 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) What about rare mRNAs? These might be differential but not abundant enough to detect –Do reverse experiment -> select for absence of a signal –example - hybridize a tumor cDNA library with probe prepared from normal liver –select for genes absent in tumor »Get genes lost from normal tissue »or upregulated in tumor by this approach

BioSci 203 blumberg lecture 4 page 13 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) +/- screening (contd) –Advantages Relatively simple approach Doesn’t require difficult manipulations on probes –Disadvantages Housekeeping genes often appear to be differential Sensitivity less than subtracted screening –+/- screening typically requires >10 fold difference in expression levels using standard methods not widely used any longer BUT –Multicolor microarray analysis is really just a refined version of +/- screening fluorescence ratios give good internal standards –more precise quantitation –increased sensitivity

BioSci 203 blumberg lecture 4 page 14 © copyright Bruce Blumberg All rights reserved DNA microarray

BioSci 203 blumberg lecture 4 page 15 © copyright Bruce Blumberg All rights reserved DNA microarray

BioSci 203 blumberg lecture 4 page 16 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) Subtractive screening - Sargent and Dawid (1983) Science 222, –Make 1st strand cDNA from a tissue and then hybridize it to excess mRNA from another larger C o t is best >20 at least –remove double stranded materials -> common seqs –make a probe or library from the remaining single stranded cDNA – fold more sensitive than +/- screening

BioSci 203 blumberg lecture 4 page 17 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) Subtractive screening (contd) –benefits sensitive can simultaneously identify all cDNAs that are differentially present in a population good choice for identifying unknown, tissue specific genes –drawbacks easy to have abundant housekeeping genes slip through –multistage subtraction is best –in effect normalize first, then subtract libraries have limited applications –may not be useful for multiple purposes

BioSci 203 blumberg lecture 4 page 18 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) –rule of thumb make a high quality representative library from a tissue of interest save subtraction and other fancy manipulations for making probes to screen such libraries with –unlimited screening –easy to use libraries for different purposes, e.g. the liver library »hepatocarcinoma »cirrhosis »regeneration specific genes

BioSci 203 blumberg lecture 4 page 19 © 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 4 page 20 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) What is the most important piece of information you need to clone a cDNA? First step in any hybridization based method (high or low stringency) is to get information on expression –high stringency homologous screening - Northern analysis –cross species screening requires more care perform a genomic Southern to identify hybridization and washing conditions that identify a small number of hybridizing fragments –standard conditions - 1 M Na +, 43% formamide, 37° C –begin washing at RT in 2 x SSC and expose –increase stringency until signal/noise ratio is acceptable –use these conditions for Northern. If Northern is unsuccessful - obtain a genomic clone and repeat the screening at high stringency –this approach will never fail to identify a homologous gene –Information on where the mRNA is expressed either what tissue or what time during development –such information is indispensable!!

BioSci 203 blumberg lecture 4 page 21 © 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 4 page 22 © 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 such Make genomic library from mutant –What type of library will you make? Why? Screen with transposon –Recover positives, sequence flanking region Use flanking sequence to screen normal genomic library –What type of library will you screen? –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 4 page 23 © 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 4 page 24 © 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 4 page 25 © 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 4 page 26 © 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 -> melting temperature 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 4 page 27 © 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 4 page 28 © 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 –

BioSci 203 blumberg lecture 4 page 29 © 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 4 page 30 © 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 4 page 31 © 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