1. BioSci 145B lecture 2 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B Lecture #2 4/13/2004 Bruce Blumberg –2113E McGaugh Hall - office hours Wed 12-1 PM (or by appointment) –phone 824-8573 –blumberg@uci.edu TA – Curtis Daly –2113 McGaugh Hall, 924-6873, 3116 lectures will be posted on web pages after lecture –http://eee.uci.edu/04s/05705/ - link only herehttp://eee.uci.edu/04s/05705/ –http://blumberg-serv.bio.uci.edu/bio145b-sp2004http://blumberg-serv.bio.uci.edu/bio145b-sp2004 –http://blumberg.bio.uci.edu/bio145b-sp2004http://blumberg.bio.uci.edu/bio145b-sp2004 On 3 x 5 card write –On lined side – something that you recall clearly from last week’s class –On other side – something that was unclear to you from last week

2. BioSci 145B lecture 2 page 2 © copyright Bruce Blumberg 2004. All rights reserved Requirements for the oral presentation Goal – again to get you to think more analytically –Exposure to literature (classic and current) –Learn critical reading –Discuss practical applications of what we are learning Powerpoint (“journal club”) presentation – as a presenter –20 minutes with time allowed for discussion (max of 15 – 20 slides) –Frame the problem – what is the big picture question? What was known before they started? What was unknown? Present background (few slides), handouts helpful but not required –What are specific questions or hypotheses to be tested Discuss figures –What is the question being asked in each figure or panel? –What experiments did the authors do to answer questions? –Do the data support the conclusions drawn? »Were controls done? What did they conclude overall? What could have been improved? –Point out a few papers for further reading (reviews, followups, etc)

3. BioSci 145B lecture 2 page 3 © copyright Bruce Blumberg 2004. All rights reserved Requirements for the oral presentation (contd) Powerpoint presentation – as a listener –READ THE PAPERS –Study the figures What points don’t you understand? –Make notations, ask the speaker to clarify these –Listen to the speaker If presentation is unclear, ask the speaker to elaborate Always feel free to ask questions – we want an open discussion Papers are posted on the web sites listed –I have CDs for everyone Logistics –Prepare presentation and either e-mail to me or bring it on a CD-ROM Floppy disk USB flash memory drive –Or bring your own laptop

4. BioSci 145B lecture 2 page 4 © copyright Bruce Blumberg 2004. All rights reserved Presentation schedule Week 1 – Berget et al., 1977; Chow et al., 1977 - Curtis Week 2 – (1) Geisler et al., 1999 (2) Maniatis et al., 1978 (3) Osoegawa et al., 2000 –Erik Week 3 – (4) Adams et al., 1991 (5) Sargent and Dawid, 1983 (6) Wang and Brown, 1991 Week 4 - (7) Innis et al., 1988 (8) Myers et al., 2000 (9) Sanger et al., 1977 Week 5 – midterm, no presentations Week 6 –(10) Hsu et al., 2002 (11) Tyson et al., 2004 (12) Venter et al., 2004 Week 7 – (13) Cawley et al., 2004 (14) Kapranov et al., 2002 (15) Schena et al., 1996 Week 8 – (16) Boutros et al., 2004 (17) Carlson et al., 2003 (18) Golling et al., 2002 Week 9 – (19) Fields and Song, 1989 (20) Ito et al., 2001 (21) Uetz et al., 2000 Week 10 - (22) Gavin et al., 2002 (23) Hamadeh et al., 2002 (24) Hoffmeyer et al., 2000

5. BioSci 145B lecture 2 page 5 © copyright Bruce Blumberg 2004. All rights reserved Lecture Outline 4/13/2004 – Mapping Genomes Today’s topics –Finish up from last time –Construction of genomic libraries –Modern mapping methods The big picture – How does one go about creating a genomic map? –In preparation for sequencing –To locate disease loci This week’s papers – Classic paper on library construction and screening and two recent ones, one about library construction the other about radiation hybrid mapping

6. BioSci 145B lecture 2 page 6 © copyright Bruce Blumberg 2004. All rights reserved Origins of intron/exon organization When did introns arise –Introns early – Walter Gilbert There from the beginning, lost in bacteria and many simpler organisms –Introns late – Cavalier-Smith, Ford Doolittle, Russell Doolittle Introns acquired over time as a result of transposable elements, aberrant splicing, etc If introns benefit protein evolution – why would they be lost? –Which is it? Introns late (at the moment)

7. BioSci 145B lecture 2 page 7 © copyright Bruce Blumberg 2004. All rights reserved Evolution of gene clusters Many genes occur as multigene families (e.g., actin, tubulin, globins, Hox) –Inference is that they evolved from a common ancestor –Families can be clustered - nearby on chromosomes (α-globins, HoxA) Dispersed – on various chromosomes (actin, tubulin) Both – related clusters on different chromosomes (α,β-globins, HoxA,B,C,D) –Members of clusters may show stage or tissue-specific expression Implies means for coregulation as well as individual regulation

8. BioSci 145B lecture 2 page 8 © copyright Bruce Blumberg 2004. All rights reserved Evolution of gene clusters (contd) multigene families (contd) –Gene number tends to increase with evolutionary complexity Globin genes increase in number from primitive fish to humans –Clusters evolve by duplication and divergence

9. BioSci 145B lecture 2 page 9 © copyright Bruce Blumberg 2004. All rights reserved Evolution of gene clusters (contd) History of gene families can be traced by comparing sequences –Molecular clock model holds that rate of change within a group is relatively constant Not totally accurate – check rat genome sequence paper –Distance between related sequences combined with clock leads to inference about when duplication took place

10. BioSci 145B lecture 2 page 10 © copyright Bruce Blumberg 2004. All rights reserved Types and origin of repetitive elements DNA sequences are not random –genes, restriction sites, methylation sites Repeated sequences are not random either –Some occur as tandemly repeated sequences –Usually generated by unequal crossing over during meiosis –These resolve in ultracentrifuge into satellite bands because GC content differs from majority of DNA –This “satellite” DNA is highly variable Between species And among individuals within a population Can be useful for mapping genotyping, etc –Much highly repetitive DNA is in heterochromatin (highly condensed regions) Centromeres, telomeres, others

11. BioSci 145B lecture 2 page 11 © copyright Bruce Blumberg 2004. All rights reserved Types and origin of repetitive elements (contd) Dispersed tandem repeats are “minisatellites” 14-500 bp in length –First forensic DNA typing used satellite DNA – Sir Alec Jeffreys –Minisatellite DNA is highly variable and perfect for fingerprinting

12. BioSci 145B lecture 2 page 12 © copyright Bruce Blumberg 2004. All rights reserved Types and origin of repetitive elements – dispersed repeated sequences

13. BioSci 145B lecture 2 page 13 © copyright Bruce Blumberg 2004. All rights reserved Types and origin of repetitive elements – dispersed repeated sequences Book discusses types of elements, similarities, differences at great length –Main point is to understand how such elements can affect evolution of genes and genomes –Gene transduction has long been known in bacteria (transposons, P1, etc) –LINE (long interspersed nuclear elements) can mediate movement of exons between genes Pick up exons due to weak poly- adenylation signals The new exon becomes part of LINE by reverse transcription and is inserted into a new gene along with LINE –Voila – gene has a new exon –Experiments in cell culture proved this model and suggested it is unexpectedly efficient –Likely to be a very important mechanism for generating new genes

14. BioSci 145B lecture 2 page 14 © copyright Bruce Blumberg 2004. All rights reserved Genome Structure The big picture –Chromosomes consist of coding (euchromatin) and noncoding (heterochromatin) regions Various physical methods can distinguish these regions –Staining –Buoyant density –Restriction digestion Heterochromatin is primarily tandemly repeated sequences Euchromatin is everything else –Genes including promoters, introns, exons –LINES, SINES micro and minisatellite DNA Patterns of euchromatin and heterochromatin can be useful for constructing genetic maps –Heterochromatin is trouble for large scale physical mapping and sequencing May be hard to cross gaps

15. BioSci 145B lecture 2 page 15 © copyright Bruce Blumberg 2004. All rights reserved Genome evolution Genomes evolve increasing complexity in various ways –Whole genome duplications Particularly important in plants –Recombination and duplication mediated by SINEs, LINEs, etc. Expands repeats, exon shuffling, creates new genes –Meiotic crossing over Expands repeats, duplicates genes –Segmental duplication – frequent in genetic diseases Interchromosomal – duplications among non-homologous chromosomes Intrachromosomal – within or across homologous chromosomes

16. BioSci 145B lecture 2 page 16 © copyright Bruce Blumberg 2004. All rights reserved Genome evolution (contd) Several recent papers discuss details of genome evolution as studied in closely related species –Dietrich et al. (2004) Science 304, 304-7 –Kellis et al. (2004) Nature 428, 617-24. –S. cerevvisiae vs two other species of yeast Saw genome duplications and evolution or loss of one duplicated member but never both

17. BioSci 145B lecture 2 page 17 © copyright Bruce Blumberg 2004. All rights reserved Mapping Genomes Why map genomes? –Locate genes causing mutations or diseases Figure out where identified genes are –Prepare to sequence –Discern evolutionary relationships How do we go about mapping whole genomes? –Book describes restriction endonuclease digestion Impossible for all but the tiniest genomes Requires ability to precisely resolve very large fragments of DNA –Must be able to separate chromosomes or huge fragments thereof Then map various types of markers onto these fragments STS, ESTs, RFLPs –Modern approach Construct large insert genomic libraries –Map relationship to each other –map markers onto large insert library members Map to chromosomes

18. BioSci 145B lecture 2 page 18 © copyright Bruce Blumberg 2004. All rights reserved Construction of genomic libraries What do we commonly use genomic libraries for? –Genome sequencing –gene cloning prior to targeted disruption or promoter analysis –positional cloning genetic mapping –Radiation hybrid, STS (sequence tagged sites), ESTs, RFLPs chromosome walking gene identification from large insert clones disease locus isolation and characterization Considerations before making a genomic library –what will you use it for what size inserts are required? –Are high quality validated libraries available? Caveat emptor –Research Genetics X. tropicalis BAC library is really Xenopus laevis apply stringent standards, your time is valuable

19. BioSci 145B lecture 2 page 19 © copyright Bruce Blumberg 2004. All rights reserved Genomic libraries (contd.) Considerations before making a genomic library (contd) –availability of equipment? PFGE laboratory automation if not available locally it may be better to use a commercial library or contract out the construction

20. BioSci 145B lecture 2 page 20 © copyright Bruce Blumberg 2004. All rights reserved Goals for a genomic library –Faithful representation of genome clonability and stability of fragments essential >5 fold coverage i.e., library should have a complexity of five times the genome size for a 99% probability of a clone being present. –easy to screen plaques much easier to deal with colonies UNLESS you are dealing with libraries spotted in high density on filter supports –easy to produce quantities of DNA for further analysis Genomic libraries (contd.)

21. BioSci 145B lecture 2 page 21 © copyright Bruce Blumberg 2004. All rights reserved Construction of genomic libraries Prepare HMW DNA –bacteriophage λ, cosmids or fosmids partial digest with frequent (4) cutter followed by sucrose gradient fractionation or gel electrophoresis –Sau3A (^GATC) most frequently used, compatible with BamHI (G^GATCC) why can’t we use rare cutters? Ligate to phage or cosmid arms then package in vitro –Stratagene >>> better than competition –Vectors that accept larger inserts prepare DNA by enzyme digestion in agarose blocks –why? Partial digest with frequent cutter Separate size range of interest by PFGE (pulsed field gel electrophoresis) ligate to vector and transform by electroporation

22. BioSci 145B lecture 2 page 22 © copyright Bruce Blumberg 2004. All rights reserved Construction of genomic libraries (contd) What is the potential flaw for all these methods? Solution? –Shear DNA or cut with several 4 cutters, then methylate and attach linkers for cloning –benefits should get accurate representation of genome can select restriction sites for particular vector (i.e., not limited to BamHI) –pitfalls quality of methylases more steps potential for artefactual ligation of fragments –molar excess of linkers –Unequal representation of restriction sites, even 4 cutters in genome –large regions may exist devoid of any restriction sites tend not to be in genes

23. BioSci 145B lecture 2 page 23 © copyright Bruce Blumberg 2004. All rights reserved Construction of genomic libraries (contd) What sorts of vectors are useful for genomic libraries? –Plasmids? –Bacteriophages? –Others? Standard plasmids nearly useless Bacteriophage lamba once most useful and popular –Size limited to 20 kb Lambda variants allow larger inserts – 40 kb –Cosmids –Fosmids Bacteriophage P1 – 90 kb YACs – yeast artificial chromosomes - megabases New vectors BACs and PACs - 300 kb

24. BioSci 145B lecture 2 page 24 © copyright Bruce Blumberg 2004. All rights reserved Bacteriophage library cloning systems All are relatively similar to each other –Lambda, cosmid, fosmid, P1 –Erik will tell us about lambda on Thursday P1 cloning systems –derived from bacteriophage P1 one of the primary tools of E. coli geneticists for many years –infect cells with packaged DNA then recover as a plasmid. –useful, but size limited to 95 kb by “headfull” packaging mechanism

25. BioSci 145B lecture 2 page 25 © copyright Bruce Blumberg 2004. All rights reserved Cosmid/fosmid cloning P1, cosmids and fosmids replicated as plasmids after infection –Cosmids have ColE1 origin (25- 50 copies/cell_ –Fosmids have F1 origin (1 copy/cell)

26. BioSci 145B lecture 2 page 26 © copyright Bruce Blumberg 2004. All rights reserved Screening a bacteriophage library

27. BioSci 145B lecture 2 page 27 © copyright Bruce Blumberg 2004. All rights reserved YACs, BACs and PACs Three complementary approaches, each with its own strengths and weaknesses YACs - Yeast artificial chromosomes –requires two vector arms, one with an ARS one with a centromere both fragments have selective markers –trp and ura are commonly used background reduction is by dephosphorylation ligation is transformed into spheroplasts colonies picked into microtiter dishes containing media with cryoprotectant

28. BioSci 145B lecture 2 page 28 © copyright Bruce Blumberg 2004. All rights reserved YAC cloning YAC cloning (contd) –advantages can propagate extremely large fragments may propagate sequences unclonable in E. coli –disadvantages tedious to purify away from yeast chromosomes by PFGE grow slowly insert instability generally difficult to handle

29. BioSci 145B lecture 2 page 29 © copyright Bruce Blumberg 2004. All rights reserved BAC cloning Based on the E. coli F’ plasmid –partial digests are cloned into dephosphorylated vector –ligation is transformed into E. coli by electroporation –advantages large plasmids - handle with usual methods Stable - stringently controlled at 1 copy/cell Vectors are small ~7 kb –– good for shotgun cloning strategies –disadvantages low yield no selection against nonrecombinant clones (blue/white only) apparent size limitation

30. BioSci 145B lecture 2 page 30 © copyright Bruce Blumberg 2004. All rights reserved PAC cloning PAC - P1 artificial chromosome –combines best features of P1 and BAC cloning –size selected partial digests are ligated to dephosphorylated vector and electrotransformed into E. coli. Stored as colonies in microtiter plates –Selection against non-recombinants via SacBII selection (nonrecombinant cells convert sucrose into a toxic product) –inducible P1 lytic replicon allows amplification of plasmid copy number

31. BioSci 145B lecture 2 page 31 © copyright Bruce Blumberg 2004. All rights reserved PAC cloning (contd) PAC –advantages all the advantages of BACS –stability –replication as plasmids –stringent copy control selection against nonrecombinant clones inducible P1 lytic replicon –addition of IPTG causes loss of copy control and larger yields –disadvantages effective size limitation (~300 kb) Vector is large – lots of vector fragments from shotgun cloning PACs

32. BioSci 145B lecture 2 page 32 © copyright Bruce Blumberg 2004. All rights reserved Comparison of cloning systems

33. BioSci 145B lecture 2 page 33 © copyright Bruce Blumberg 2004. All rights reserved Which type of library to make Do I need to make a new library at all? –Is the library I need available? http://bacpac.chori.org/home.htmhttp://bacpac.chori.org/home.htm PAC libraries are suitable for most purposes BAC libraries are most widely available If your organism only has YAC libraries available you may wish to make PAC or BACs Much easier to buy pools or gridded libraries for screening –doesn’t always work –What is the intended use? Will this library be used many times? –e.g. for isolation of clones for knockouts –if so, it pays to do it right –who should make the library? Going rate for custom PAC or BAC library is 50K. Most labs do not have these resources if care is taken, construction is not so difficult

34. BioSci 145B lecture 2 page 34 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping The problem – genomes are large, workable fragments are small –How to figure out where everything is? –How to track mutations in families or lineages? Book makes a good analogy to roadmaps –The most useful maps do not have too much detail but have major features and landmarks that everything can be related to Allows genetic markers to be related to physical markers What sorts of maps are useful for genomes? –Restriction maps of various sorts RFLPs, fingerprints –Recombination maps, how often to traits segregate together –Physical maps – which genes occur on same chunks of DNA

35. BioSci 145B lecture 2 page 35 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping (contd) How are maps made? –Restriction digestion and ordering of fragments to build contigs Fingerprinting –Location of marker sequences onto larger chunks –Hybridization of markers to larger chunks –Calculation of recombination frequencies between loci What do we map these days? –BACs are most common target for mapping of new genomes –Radiation hybrid panels still in wide use –Goal is always to map markers onto ordered large fragments and infer location of genes relative to each other.

36. BioSci 145B lecture 2 page 36 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping (contd) Useful markers –STS – sequence tagged sites Short randomly acquired sequences PCRing sequences, then prove by hybridization that only a single sequence is amplified/genome –VERY tedious and slow validated ones mapped back to RH panels Orders sequences on large chunks of DNA –STC – sequence tagged connectors Array BAC libraries to 15x coverage of genome Sequence BAC ends Combine with genomic maps and fingerprints to link clones –Average about 1 tag/5 kb Most useful preparatory to sequencing

37. BioSci 145B lecture 2 page 37 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping (contd) Useful markers (contd) –ESTs – expressed sequence tags randomly acquired cDNA sequences Lots of value in ESTs (paper next week) –Info about diversity of genes expressed –Quick way to get expressed genes Better than STS because ESTs are expressed genes Can be mapped to –chromosomes by FISH –RH panels –BAC contigs –Polymorphic STS – STS with variable lengths Often due to microsatellite differences Useful for determining relationships Also widely used for forensic analysis –OJ, Kobe, etc

38. BioSci 145B lecture 2 page 38 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping (contd) Useful markers (contd) –SNPs – single nucleotide polymorphisms Extraordinarily useful - ~1/1000 bp in humans Much of the differences among us are in SNPs SNPs that change restriction sites cause RFLPs (restriction fragment length polymorphisms Detected in various ways –Hybridization to high density arrays (Affymetrix) –Sequencing –Denaturing electrophoresis or HPLC –Invasive cleavage Tony Long in E&E Biology has new method for ligation mediated SNP detection that they use for evolutionary analyses

39. BioSci 145B lecture 2 page 39 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping (contd) Useful markers (contd) –RAPDs – randomly amplified polymorphic DNA Amplify genomic DNA with short, arbitrary primers Some fraction will amplify fragments that differ among individuals These can be mapped like STS Issues with PCR amplification Benefit – no sequence information required for target –AFLPs – amplified fragment length polymorphisms Cut with enzymes (6 and 4 cutter) that yield a variety of small fragments ( < 1 kb) Ligate sequences to ends and amplify by PCR Generates a fingerprint –Controlled by how frequently enzymes cut Often correspond to unique regions of genome –Can be mapped Benefit – no sequence required.

40. BioSci 145B lecture 2 page 40 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping (contd) Mapping by walking/hybridization –Start with a seed clone then walk along the chromosome –Takes a LOOONNNNGGG time –Benefit – can easily jump repetitive sequences

41. BioSci 145B lecture 2 page 41 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping (contd) Fingerprinting –Array and spot ibraries –Probe with short oligos (10-mers) Repeat –Build up a “fingerprint” for each clone –Can tell which ones share sequences tedious

42. BioSci 145B lecture 2 page 42 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping (contd) Mapping by restriction digest fingerprinting –Order clones by comparing patterns from restriction enzyme digestion Mapping by hybridization –Array library – pick a “seed clone” –See where it hybridizes, pick new seed and repeat –Product

43. BioSci 145B lecture 2 page 43 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping (contd) FISH - Fluorescent in situ hybridization – can detect chromosomes or genes –Can localize probes to chromosomes and –Relationship of markers to each other –Requires much knowledge of genome being mapped –Chromosome paintingmarker detection

44. BioSci 145B lecture 2 page 44 © copyright Bruce Blumberg 2004. All rights reserved Genome mapping (contd) Radiation hybrid mapping –Old but very useful technique Blast cells with x-rays Fuse with cells of another species, e.g., blast human cells then fuse with mouse cells –Chunks of human DNA will remain in mouse cells Can build up a library of different cell lines – 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 –We will discuss a paper on Thursday

45. BioSci 145B lecture 2 page 45 © 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