Bruce Blumberg (blumberg@uci.edu) BioSci D145 Lecture #2 Bruce Blumberg (blumberg@uci.edu) 4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment) phone 824-8573 TA – Bassem Shoucri (bshoucri@uci.edu) 4351 Nat Sci 2, 824-6873 – office hours TBA check e-mail and noteboard daily for announcements, etc.. Please use the course noteboard for discussions of the material lectures will be posted on web pages after lecture http://blumberg.bio.uci.edu/biod145-w2014 http://blumberg-lab.bio.uci.edu/biod145-w2014 BioSci D145 lecture 1 page 1 ©copyright Bruce Blumberg 2014. All rights reserved

How about term paper topics? Example term papers are posted on web site Specific aims Background and significance Research plan References Please e-mail me (or stop by and discuss) your topic as soon as possible Remember that your goal is to propose a study of something you find interesting that has not already been done Exercise your imagination Indulge your intellectual curiosity Expand your BioSci 199 research interests. ~2 pages ~3 pages No limit BioSci D145 lecture 2 page 2 ©copyright Bruce Blumberg 2007. All rights reserved

Organization and Structure of Genomes (contd) Gene content is proportional to single copy DNA Amount of non-repetitive DNA has a maximum,total genome size does not What is all the extra DNA, i.e., what is it good for? Where did all this junk come from and why is it still around? BioSci D145 lecture 1 page 3 ©copyright Bruce Blumberg 2010. All rights reserved

Organization and Structure of Genomes (contd) What is this highly repetitive DNA? Selfish DNA? Parasitic sequences that exist solely to replicate themselves? Or evolutionary relics? Produced by recombination, duplication, unequal crossing over BioSci D145 lecture 1 page 4 ©copyright Bruce Blumberg 2010. All rights reserved

Transcription of Prokaryotic vs Eukaryotic genomes Prokaryotic genes are expressed in linear order on chromosome mRNA corresponds directly to gDNA Most eukaryotic genes are interrupted by non-coding sequences Introns (Gilbert 1978) These are spliced out after transcription and prior to transport out of nucleus Post-transcriptional processing in an important feature of eukaryotic gene regulation Why do eukaryotes have introns, i.e., what are they good for? BioSci D145 lecture 1 page 5 ©copyright Bruce Blumberg 2010. All rights reserved

Alternative splicing can generate protein diversity Introns and splicing Alternative splicing can generate protein diversity Many forms of alternative splicing seen Some genes have numerous alternatively spliced forms Dozens are not uncommon, e.g., cytochrome P450s BioSci D145 lecture 1 page 6 ©copyright Bruce Blumberg 2010. All rights reserved

Alternative splicing can generate protein diversity (contd) Introns and splicing Alternative splicing can generate protein diversity (contd) Others show sexual dimorphisms Sex-determining genes Classic chicken/egg paradox how do you determine sex if sex determines which splicing occurs and spliced form determines sex? BioSci D145 lecture 1 page 7 ©copyright Bruce Blumberg 2010. All rights reserved

Origins of intron/exon organization Introns and exons tend to be short but can vary considerably “Higher” organisms tend to have longer lengths in both First introns tend to be much larger than others – WHY? BioSci D145 lecture 1 page 8 ©copyright Bruce Blumberg 2010. All rights reserved

Origins of intron/exon organization Exon number tends to increase with increasing organismal complexity Possible reasons? BioSci D145 lecture 1 page 9 ©copyright Bruce Blumberg 2010. 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? What is common factor among animals that share intron locations? BioSci D145 lecture 1 page 10 ©copyright Bruce Blumberg 2010. 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 BioSci D145 lecture 1 page 11 ©copyright Bruce Blumberg 2010. 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 BioSci D145 lecture 1 page 12 ©copyright Bruce Blumberg 2010. 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 BioSci D145 lecture 1 page 13 ©copyright Bruce Blumberg 2010. 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 are one such place BioSci D145 lecture 2 page 14 ©copyright Bruce Blumberg 2007. 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 BioSci D145 lecture 2 page 15 ©copyright Bruce Blumberg 2007. All rights reserved

Types and origin of repetitive elements – dispersed repeated sequences BioSci D145 lecture 2 page 16 ©copyright Bruce Blumberg 2007. All rights reserved

Types and origin of repetitive elements – dispersed repeated sequences 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 BioSci D145 lecture 2 page 17 ©copyright Bruce Blumberg 2007. All rights reserved

Various physical methods can distinguish these regions Staining 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 BioSci D145 lecture 2 page 18 ©copyright Bruce Blumberg 2007. All rights reserved

Genomes evolve increasing complexity in various ways 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 BioSci D145 lecture 2 page 19 ©copyright Bruce Blumberg 2007. 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. cerevisiae vs two other species of yeast Saw genome duplications and evolution or loss of one duplicated member but never both 90 mb->3Gb 115 mb 12 mb 4.6 mb 4.2 mb 1.44 mb 1.66 mb BioSci D145 lecture 2 page 20 ©copyright Bruce Blumberg 2007. All rights reserved

How do we go about mapping whole genomes? Mapping Genomes Why map genomes? How do we go about mapping whole genomes? BioSci D145 lecture 2 page 21 ©copyright Bruce Blumberg 2007. All rights reserved

Mapping Genomes – comparison of maps BioSci D145 lecture 2 page 22 ©copyright Bruce Blumberg 2007. All rights reserved

Construction of genomic libraries What do we commonly use genomic libraries for? Genome sequencing (most approaches use genomic libraries) 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 BioSci D145 lecture 2 page 23 ©copyright Bruce Blumberg 2007. 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 BioSci D145 lecture 2 page 24 ©copyright Bruce Blumberg 2007. All rights reserved

Genomic libraries (contd.) 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 BioSci D145 lecture 2 page 25 ©copyright Bruce Blumberg 2007. 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 BioSci D145 lecture 2 page 26 ©copyright Bruce Blumberg 2007. All rights reserved

Construction of genomic libraries (contd) stopped here 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 BioSci D145 lecture 2 page 27 ©copyright Bruce Blumberg 2007. 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 BioSci D145 lecture 2 page 28 ©copyright Bruce Blumberg 2007. All rights reserved

Bacteriophage library cloning systems All are relatively similar to each other Lambda, cosmid, fosmid, P1 We will hear about some cloning systems on next 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 BioSci D145 lecture 2 page 29 ©copyright Bruce Blumberg 2007. 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) BioSci D145 lecture 2 page 30 ©copyright Bruce Blumberg 2007. All rights reserved

Screening a bacteriophage library BioSci D145 lecture 2 page 31 ©copyright Bruce Blumberg 2007. All rights reserved

Large insert vectors - 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 BioSci D145 lecture 2 page 32 ©copyright Bruce Blumberg 2007. All rights reserved

can propagate extremely large fragments 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 BioSci D145 lecture 2 page 33 ©copyright Bruce Blumberg 2007. All rights reserved

partial digests are cloned into dephosphorylated vector BAC cloning BAC – Bacterial artificial chromosome (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 BioSci D145 lecture 2 page 34 ©copyright Bruce Blumberg 2007. All rights reserved

PAC - P1 artificial chromosome 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 BioSci D145 lecture 2 page 35 ©copyright Bruce Blumberg 2007. All rights reserved

all the advantages of BACS stability replication as plasmids 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 BioSci D145 lecture 2 page 36 ©copyright Bruce Blumberg 2007. All rights reserved

Comparison of cloning systems BioSci D145 lecture 2 page 37 ©copyright Bruce Blumberg 2007. 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.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 BioSci D145 lecture 2 page 38 ©copyright Bruce Blumberg 2007. All rights reserved

The problem – genomes are large, workable fragments are small 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? 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? BioSci D145 lecture 2 page 39 ©copyright Bruce Blumberg 2007. All rights reserved

Genome mapping (contd) How are maps made? What do we map these days? BioSci D145 lecture 2 page 40 ©copyright Bruce Blumberg 2007. 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 BioSci D145 lecture 2 page 41 ©copyright Bruce Blumberg 2007. All rights reserved

Genome mapping (contd) Useful markers (contd) ESTs – expressed sequence tags randomly acquired cDNA sequences Lots of value in ESTs 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 BioSci D145 lecture 2 page 42 ©copyright Bruce Blumberg 2007. 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 method for ligation mediated SNP detection that they use for evolutionary analyses BioSci D145 lecture 2 page 43 ©copyright Bruce Blumberg 2007. 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. BioSci D145 lecture 2 page 44 ©copyright Bruce Blumberg 2007. 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 BioSci D145 lecture 2 page 45 ©copyright Bruce Blumberg 2007. 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 BioSci D145 lecture 2 page 46 ©copyright Bruce Blumberg 2007. All rights reserved

Genome mapping (contd) Mapping by hybridization Array library – pick a “seed clone” See where it hybridizes, pick new seed and repeat Product BioSci D145 lecture 2 page 47 ©copyright Bruce Blumberg 2007. All rights reserved

Genome mapping (contd) Restriction mapping of large insert clones Mapping by restriction digest fingerprinting Order clones by comparing patterns from restriction enzyme digestion BioSci D145 lecture 2 page 48 ©copyright Bruce Blumberg 2007. 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 painting marker detection BioSci D145 lecture 2 page 49 ©copyright Bruce Blumberg 2007. 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 HAPPY Mapping – PCR-based method – see Amanda’s presentation BioSci D145 lecture 3 page 50 ©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 HAPPY mapping Mapping of above to RH panels BioSci D145 lecture 3 page 51 ©copyright Bruce Blumberg 2004. All rights reserved