Transcript PPT - Bruce Blumberg

Bio Sci 203 bb-lecture 5 - cDNA library screening & sequence characterization
• Bruce Blumberg ([email protected])
– office – 2113E McGaugh Hall
– 824-8573
– lab (x46873,x43116)
– office hours MWF 11-12.
•
•
http://blumberg-serv.bio.uci.edu/bio203-w2002/index.htm
http://blumberg.bio.uci.edu/bio203-w2002/index.htm
• 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 1
©copyright
Bruce Blumberg 2001-2005. 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?
• Low stringency hybridization
– A piece of genomic DNA?
• Hybridization
– A mutant
• Complementation
• Positional cloning
– A functional assay?
• Expression screening
– An antibody?
• Expression library screening
– A partial amino acid sequence?
• Oligonucleotide screening
– A DNA element required for expression of an interesting gene?
• Various binding protein strategies
– An interacting protein?
• Interaction screening
– A specific tissue or embryonic stage?
• Subtracted or +/- screening
BioSci 203 blumberg lecture 5
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©copyright
Bruce Blumberg 2001-2005. 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
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©copyright
Bruce Blumberg 2001-2005. 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 4
©copyright
Bruce Blumberg 2001-2005. 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 5
©copyright
Bruce Blumberg 2001-2005. 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
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©copyright
Bruce Blumberg 2001-2005. 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 7
©copyright
Bruce Blumberg 2001-2005. 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 (Tm-25 for the most AT rich sequence)
• wash at high stringency in 3M tetramethylammonium chloride
– TMAC stabilizes AT base pairs A-T = G-C
– Tm is a strict function of length
– works best for 21-23 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 8
©copyright
Bruce Blumberg 2001-2005. 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 9
©copyright
Bruce Blumberg 2001-2005. 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
» http://www.gene-regulation.com/
– 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
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©copyright
Bruce Blumberg 2001-2005. 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
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©copyright
Bruce Blumberg 2001-2005. 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 12
©copyright
Bruce Blumberg 2001-2005. 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 32P
• 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 13
©copyright
Bruce Blumberg 2001-2005. 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
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©copyright
Bruce Blumberg 2001-2005. 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 15
©copyright
Bruce Blumberg 2001-2005. All rights reserved
How to identify your gene of interest (contd)
• Molecular approaches (contd)
AD
– yeast one-hybrid assay
• clone element into a
His
lacZ
reporter construct and
Bait elements
Reporter(s)
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 16
©copyright
Bruce Blumberg 2001-2005. 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 17
©copyright
Bruce Blumberg 2001-2005. 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
BioSci 203 blumberg lecture 5
page 18
©copyright
Bruce Blumberg 2001-2005. All rights reserved