2113E McGaugh Hall - office hours Wed 10-11 AM (or by appointment) BioSci 145B Lecture #3 4/19/2005 Bruce Blumberg 2113E McGaugh Hall - office hours Wed 10-11 AM (or by appointment) phone 824-8573 blumberg@uci.edu TA – Suman Verma 2113 McGaugh Hall, 924-6873, 3116 lectures will be posted on web pages http://eee.uci.edu/05s/05705/ - link only here http://blumberg-serv.bio.uci.edu/bio145b-sp2005 http://blumberg.bio.uci.edu/bio145b-sp2005 Many people have not spoken to me about term paper topics By the end of the day today, I need to have topics for everyone Please e-mail me your topic BioSci 145B lecture 3 page 1 ©copyright Bruce Blumberg 2004. All rights reserved

Genome mapping (contd) Radiation hybrid mapping Old but very useful technique Lethally irradiate cells with X-rays Fuse with cells of another species, e.g., blast human cells then fuse with hamster cells Chunks of human DNA will remain in mouse cells Expand colonies of cells to get a collection of cell lines, each containing a single chunk of human cDNA Collection = RH panel Now map markers onto these RH panels Can identify which of any type of markers map together STS, EST (very commonly used), etc Can then map others by linkage to the ones you have mapped Compare RH panel with other maps Utility – great for cloning gaps in other maps BioSci 145B lecture 3 page 2 ©copyright Bruce Blumberg 2004. All rights reserved

Genome mapping (contd) How should maps be made with current knowledge? All methods have strengths and weaknesses – must integrate data for useful map e.g, RH panel, BAC maps, STS, ESTs Size and complexity of genome is important More complex genomes require more markers and time mapping Breakpoints and markers are mapped relative to each other Maps need to be defined by markers (cities, lakes, roads in analogy) Key part of making a finely detailed map is construction of genomic libraries and cell lines for common use Efforts by many groups increase resolution and utility of maps Current strategies BAC end sequencing Whole genome shotgun sequencing EST sequencing Mapping of above to RH panels BioSci 145B lecture 3 page 3 ©copyright Bruce Blumberg 2004. All rights reserved

Sequence stability in E. coli How can we be sure that our genomic and cDNA libraries do not change during growth and screening? What are the sorts of factors that might modulate whether a sequence can be stably propagated in E. coli? 1 2 3 Use the correct bacterial strain Check for errors (look at multiple colonies) toxicity restriction recombination BioSci 145B lecture 3 page 4 ©copyright Bruce Blumberg 2004. All rights reserved

Sequence stability in E. coli toxicity sequence may lead to the production of a toxic product or toxic levels of an otherwise innocuous product more problematic with cDNA than genomic clones restriction - Raleigh 1987 Meth. Enzymol. 152, 130-141 virtually all microorganisms have systems to destroy non-endogenous DNA host range restriction four classes of restriction endonucleases very important for cloning purposes are recently discovered systems that degrade DNA containing 5-methyl cytosine or 6-methyl adenine. If you are cloning genomic DNA, or hemimethylated cDNA these are very important! virtually all eukaryotic DNA contains 5-methyl cytosine and/or 6-methyl adenine mcrA,B,C - methylcytosine mrr - methyl adenine BioSci 145B lecture 3 page 5 ©copyright Bruce Blumberg 2004. All rights reserved

Sequence stability in E. coli (contd) Restriction (contd) foreign DNA escapes restriction 1/105 for EcoK and EcoB, 1/10 for mcrA. one needs to be conscious of the mcr and mrr restriction status of strains and packaging extracts to be used. Recombination - Wyman and Wertman (1987) Meth Enzymol 152, 173-180 genomic DNA contains lots of repeated sequences direct repeats inverted repeats interspersed repeats (e.g. Alu) repeated sequences unstable in recombination proficient E. coli if in: lambda plasmid cosmid seems not to apply to single copy vectors such as BAC, PAC, fosmid What does this imply? ~30% of the human genome is unstable in plasmid or phage clones phages with such sequences don’t grow or get shorter with time Recombination is intermolecular process BioSci 145B lecture 3 page 6 ©copyright Bruce Blumberg 2004. All rights reserved

Sequence stability in E. coli (contd) Recombination (contd) E. coli has a variety of recombination pathways. These are the major players in causing sequence underrepresentation recA required for all pathways recBCD - major recombination pathway sbcB,C - suppressor of B,C minor pathways recE recF recJ rule of thumb - the more recombination pathways mutated, the sicker the cells and the slower they grow major players for inverted repeats are recBCD and sbc recA is most important for stabilizing direct repeats and preventing plasmid concatamerization BioSci 145B lecture 3 page 7 ©copyright Bruce Blumberg 2004. All rights reserved

Sequence stability in E. coli (contd) Plating a genomic library whenever possible, select a cell type that is recA, recD, sbcB and deficient in all restriction systems. Conveniently, EcoK, mcrB,C and mrr are all linked and often deleted together in strains can get more than 100 fold difference in numbers of phage between wild type and recombination deficient recD is preferred over recB,C because recD promotes rolling circle replication in lambda which improves yields BioSci 145B lecture 3 page 8 ©copyright Bruce Blumberg 2004. All rights reserved

What do I need to know about E. coli genetics? You look in a supplier’s catalog and see lots of E. coli with different genotypes of the following general form: F’{lacIq Tn10 (TetR)} mcrA, Δ(mrr-hsdRMS-mcrBC), Φ80lacZΔM15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(StrR), endA1, nupG Does this make any difference for your experiments? Or should you simply follow the supplier’s instructions? Or just use whatever people in the next lab are using without thinking about it? BioSci 145B lecture 3 page 9 ©copyright Bruce Blumberg 2004. All rights reserved

What do I need to know about E. coli genetics? F’{lacIq Tn10 (TetR)} mcrA, Δ(mrr-hsdRMS-mcrBC), Φ80lacZΔM15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(StrR), endA1, nupG restriction systems mcrA - cuts Cm5CGG mcrB,C - complex cuts at Gm5C mrr - restricts 6-methyl adenine containing DNA Why are these important? hsdRMS - EcoK restriction system R cuts 5'-AAC(N)6 GTGC-3’ M/S methylates A residues in this sequence Why methylate the DNA? for stability of long repeated sequences recA1 - deficient in general recombination recD - deficiency in Exonuclease V sbcB,C - Exonuclease I deoR - allows uptake of large DNA Most eukaryotic DNA is methylated! Protects own DNA from digestion BioSci 145B lecture 3 page 10 ©copyright Bruce Blumberg 2004. All rights reserved

What do I need to know about E. coli genetics? (contd) for lac color selection lacZ ΔM15 either on F’ or on Φ80 prophage lacIq - constitutive expression of lac repressor. Prevents leaky expression of promoters containing lac operator for high quality DNA preps recA1 - deficient in general recombination endA1 - deficient in endonuclease I if you buy ESTs from Research Genetics (InVitrogen) or OpenBiosystems tonA - resistant to bacteriophage T1 for recombinant protein expression lon - protease deficiency OmpT - protease found in periplasmic space most important protease inhibitor for E. coli protein preps is pepstatin A BioSci 145B lecture 3 page 11 ©copyright Bruce Blumberg 2004. All rights reserved

What do I need to know about E. coli genetics? (contd) suppressors supE - inserts glutamine at UAG (amber) codons supF - inserts tyrosine at UAG (amber) codons many older phages have S100am which can only be suppressed by supF λZAP, λgt11, λZipLOX, BioSci 145B lecture 3 page 12 ©copyright Bruce Blumberg 2004. All rights reserved

Construction of cDNA libraries What is a cDNA library? What are they good for? Collection of DNA copies representing the expressed mRNA population of a cell, tissue, organ or embryo Identifying and isolating expressed mRNAs functional identification of gene products cataloging expression patterns for a particular tissue EST sequencing and microarray analysis Mapping gene boundaries Promoters Alternative splicing BioSci 145B lecture 3 page 13 ©copyright Bruce Blumberg 2004. All rights reserved

Determinants of library quality What constitutes a full-length cDNA? Strictly, it is an exact copy of the mRNA full-length protein coding sequence considered acceptable for most purposes mRNA full-length, capped mRNAs are critical to making full-length libraries cytoplasmic mRNAs are best – WHY? 1st strand synthesis complete first strand needs to be synthesized issues about enzymes 2nd strand synthesis thought to be less difficult than 1st strand (probably not) choice of vector plasmids are best for EST sequencing phages are best for manual screening how will library quality be evaluated test with 2, 4, 6, 8 kb probes to ensure that these are well represented They are processed, i.e., introns removed and pA+ added BioSci 145B lecture 3 page 14 ©copyright Bruce Blumberg 2004. All rights reserved

mRNA is isolated from source of interest cDNA synthesis Scheme mRNA is isolated from source of interest 1-10 μg are denatured and annealed to primer containing d(T)nV To minimize length of poly A tail in libraries for sequencing reverse transcriptase copies mRNA into cDNA DNA polymerase I and Rnase H convert remaining mRNA into DNA cDNA is rendered blunt ended linkers or adapters are added for cloning cDNA is ligated into a suitable vector vector is introduced into bacteria Caveats there is lots of bad information out there much is derived from vendors who want to increase sales of their enzymes or kits all manufacturers do not make equal quality enzymes most kits are optimized for speed at the expense of quality small points can make a big difference in the final outcome BioSci 145B lecture 3 page 15 ©copyright Bruce Blumberg 2004. All rights reserved

cDNA synthesis (contd) Preparation of mRNA want minimum of non poly A+ mRNAs affinity chromatography on oligo d(T) or (U) Oligo d(T)30 latex (Nippon Roche) works best overall (a.k.a. OligoTex Qiagen) 2 successive runs gives ~90% pure A+ mRNA denaturation of mRNA critical step most protocols use heat denaturation Heat RNA in the presence of metal ions = chemical cleavage! CH3HgOH is method of choice for best libraries Potent, reversible denaturant But VERY TOXIC! BioSci 145B lecture 3 page 16 ©copyright Bruce Blumberg 2004. All rights reserved

cDNA synthesis (contd) First strand synthesis - lots of misinformation about enzymes reverse transcriptase contains 2 subunits polymerase RNase H - critical for processivity of the enzyme! What is processivity? Manufacturers prefer to sell MMLV RNase H- RT – cloned and cheap best enzyme for 1st strand synthesis is AMV RT from Seikagaku America But not best overall thought that 1st strand is main failure point in cDNA synthesis - NOT addition of 0.6M trehalose to AMV reactions increases yield allows rxns to run at ~60° C Betaine is very big help for MMLV RT A recent publication claimed that betaine plus trehalose was the way to go for library making Is it? How many nt are polymerized before falling off BioSci 145B lecture 3 page 17 ©copyright Bruce Blumberg 2004. All rights reserved

cDNA synthesis (contd) R B T both R B T both 9 6 4 2 1 AMV Sigma AMV Superscript Example of comparisons between enzymes and buffers Mfg supplied buffers NOT optimal Literature references not optimal either Enzymes vary a lot Between AMV and MMLV And AMV between suppliers BioSci 145B lecture 3 page 18 ©copyright Bruce Blumberg 2004. All rights reserved

cDNA synthesis (contd) 2nd strand must remove mRNA best way is with RNAse H so that fragments serve as primers for DNA pol I Gubler and Hoffman (1983) Gene 25, 263 in my experience, 2nd strand synthesis is the point of failure in cDNA virtually all kits shortcut this step (1-2 hrs) should be overnight recent improvement is to use thermostable RNAse H, DNA ligase and DNA polymerase to maximize production of 2nd strand. BioSci 145B lecture 3 page 19 ©copyright Bruce Blumberg 2004. All rights reserved

cDNA synthesis (contd) BioSci 145B lecture 3 page 20 ©copyright Bruce Blumberg 2004. All rights reserved

cDNA synthesis (contd) Cloning after 2nd strand is made, the ends must be blunted and linkers or adapters added usually T4 DNA polymerase WHY? perfect cDNAs will retain 2-20 bp of RNA at the 5’ end. Linkers can not be added to this by any DNA ligase! T4 RNA ligase ligates DNA-RNA and stimulates blunt end ligation 10x no commercial products use T4 RNA ligase so it is no wonder that full-length cDNAs are lost if internal restriction sites have not been protected, they need to be methylated now before linkers are added. Most methylase preps are not clean Very strong proofreading activity m R N A c D N A BioSci 145B lecture 3 page 21 ©copyright Bruce Blumberg 2004. All rights reserved

Full-length mRNA isolation and cDNA synthesis Ways to capture cap structures and presumably full-length mRNAs affinity chromatography with eIF-4E (cap binding protein a.k.a. Capture selection with antibody to cap structure oligo capping biotinylated cap trapper 5’ oligo capping - Maruyama, K., and Sugano, S. (1994). Gene 138, 171-4. uncapped mRNAs are dephosphorylated so that they cannot be ligated cap structure is removed, only previously capped mRNAs have 5’ PO4 RNA ligase can ligate a 5’-OH oligo to the 5’ end of the mRNA This can be used to prime 2nd strand synthesis BioSci 145B lecture 3 page 22 ©copyright Bruce Blumberg 2004. All rights reserved

Full-length mRNA isolation and cDNA synthesis (contd) 5’ oligo capping (contd) advantages very simple no homopolymeric regions to worry about can put arbitrary sequence at 5’ end. Enables custom vector construction also enables PCR to make driver for normalization disadvantages cap trapper paper claims this method only gives 70% full-length cDNAs high quality TAP is not easy to find original paper used PCR between 5’ and 3’ primer to make cDNAs PCR => bias! BioSci 145B lecture 3 page 23 ©copyright Bruce Blumberg 2004. All rights reserved

Full-length mRNA isolation and cDNA synthesis (contd) Cap trapping Carninci, P. et al. (1996) Genomics 37: 327- 336. biotin residue is chemically added to the cap structure approach 1st strand cDNA is synthesized treatment with RNAse I cuts any cDNA:mRNA duplexes which are not absolutely complete complete cDNAs are isolated by streptavidin chromatography RNA is hydrolyzed cDNA is tailed with dG What are pitfalls of this? 2nd strand synthesis is primed with dC adapter added cloned Homopolymers troublesome BioSci 145B lecture 3 page 24 ©copyright Bruce Blumberg 2004. All rights reserved

Full-length mRNA isolation and cDNA synthesis (contd) Cap trapping (contd) advantages claimed to give 90% recovery of full-length cDNAs lots of history at RIKEN disadvantages homopolymeric region many steps -> points of failure BioSci 145B lecture 3 page 25 ©copyright Bruce Blumberg 2004. 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 145B lecture 3 page 26 ©copyright Bruce Blumberg 2004. 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 10-100x 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 145B lecture 3 page 27 ©copyright Bruce Blumberg 2004. 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 helper phage problems - solved 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 145B lecture 3 page 28 ©copyright Bruce Blumberg 2004. All rights reserved

Vectors for cDNA cloning (contd) BioSci 145B lecture 3 page 29 ©copyright Bruce Blumberg 2004. All rights reserved

Vectors for cDNA cloning (contd) BioSci 145B lecture 3 page 30 ©copyright Bruce Blumberg 2004. All rights reserved

mRNA frequency and cloning mRNA frequency classes classic references Bishop et al., 1974 Nature 250, 199-204 Davidson and Britten, 1979 Science 204, 1052-1059 abundant 10-15 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 0.05-0.2% 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 145B lecture 3 page 31 ©copyright Bruce Blumberg 2004. All rights reserved

Normalization and subtraction How to identify genes that might only occur at a few copies per cell? alter the representation of the cDNAs in a library or probe Normalization - process of reducing the frequency of abundant and increasing the frequency of rare mRNAs Bonaldo et al., 1996 Genome Research 6, 791-806 Subtraction - removing cDNAs (mRNAs) expressed in two populations leaving only differentially expressed Sagerström et al. (1997) Ann Rev. Biochem 66, 751-783 BioSci 145B lecture 3 page 32 ©copyright Bruce Blumberg 2004. All rights reserved

Normalization and subtraction Normalization - reducing abundant, increase rare mRNAs - normalization should bring cDNA abundunce to within 10x 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 Cot =5.5 driver Cot value is critical for success of normalization 5-10 optimal, higher values NOT better BioSci 145B lecture 3 page 33 ©copyright Bruce Blumberg 2004. 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 145B lecture 3 page 34 ©copyright Bruce Blumberg 2004. 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 145B lecture 3 page 35 ©copyright Bruce Blumberg 2004. All rights reserved

Normalization and subtraction (contd) Subtractive screening - Sargent and Dawid (1983) Science 222, 135-139. Make 1st strand cDNA from a tissue and then hybridize it to excess mRNA from another larger Cot is best >20 at least remove double stranded materials -> common seqs make a probe or library from the remaining single stranded cDNA BioSci 145B lecture 3 page 36 ©copyright Bruce Blumberg 2004. 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 145B lecture 3 page 37 ©copyright Bruce Blumberg 2004. 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 145B lecture 3 page 38 ©copyright Bruce Blumberg 2004. 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 screening BioSci 145B lecture 3 page 39 ©copyright Bruce Blumberg 2004. 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 145B lecture 3 page 40 ©copyright Bruce Blumberg 2004. All rights reserved