BioSci 145B lecture 2 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B Lecture #2 4/12/2005 Bruce Blumberg –2113E McGaugh Hall -

BioSci 145B lecture 2 page 1 ©copyright Bruce Blumberg 2004. All rights reserved

BioSci 145B Lecture #2 4/12/2005

• Bruce Blumberg– 2113E McGaugh Hall - office hours Wed 10-11 AM (or by

appointment)– phone 824-8573– [email protected]

• TA – Suman Verma– 2113 McGaugh Hall, 924-6873, 3116

• check e-mail and noteboard daily for announcements, etc..– If you do not have ready access to e-mail or the web speak with me

ASAP– Please use the course noteboard for discussions of the material

• I will post all questions received via e-mail on the course noteboard

• If you object to your question being posted please indicate this clearly in the message..

• 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

http://eee.uci.edu/05s/05705/

http://blumberg-serv.bio.uci.edu/bio145b-sp2005

http://blumberg.bio.uci.edu/bio145b-sp2005


How about term paper topics?

• Example term paper (grant) is posted on web site– A bit longer than necessary for this class but the style is

appropriate– Specific aims– Background and significance– Research plan – References

• Please e-mail me (or stop by and discuss) with your topic by the end of the day on Thursday 4/14

~2 pages

~3 pages

No limit


Types and origin of repetitive elements – dispersed repeated sequences


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 betweengenes

• Pick up exons due to weak poly-adenylation signals

• The new exon becomes part of LINEby reverse transcription and isinserted into a new gene alongwith 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 mechanismfor generating new genes


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


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


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

4.6 mb

4.2 mb

1.44 mb 1.66 mb

12 mb

115 mb90 mb->3Gb


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


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


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 usea commercial libraryor contract out the construction


• 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.)


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

Unequal representation of restriction sites in genome

So DNA does not get mechanically sheared


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


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


Bacteriophage library cloning systems• All are relatively similar to each other

– Lambda, cosmid, fosmid, P1– We will hear about cloning

systems 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


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)


Screening a bacteriophage library


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


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


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


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


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


Comparison of cloning systems

YAC BAC PAC

Host cells S. cerevisiae AB1380, J57D

E. coli DH10B E. coli DH10B

Transformation method

Spheroplast transformation

Electroporation Electroporation

DNA topology of recombinants Linear Circular supercoiled Circular supercoiled

Maximum insert size >>1 Mb ~300 kb ~300 kb

Selection for recombinants

Ade2 supF red-white color selection

Lacz blue-white SacIIb selective growth

Selection for vector Dropout medium (lacking trp and ura)

Chloramphenicol Kanamycin

Enzyme for partial digests EcoRI HindIII MboI or Sau3AI

Stability Variable but can be very unstable

Very stable Very stable

Degree of chimerism

Varies but can be >50%

Very low Very low

Degree of co-cloning Occasional Undetectable Undetectable

Purification of intact inserts Difficult Easy Easy

Direct sequencing of insert Difficult Relatively easy Relatively easy

Clone mating Yes No No’


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

http://bacpac.chori.org/home.htm

http://bacpac.chori.org/home.htm


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


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.



• 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



• 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



• 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



• 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.



• Mapping by walking/hybridization– Start with a seed clone then walk along the chromosome– Takes a LOOONNNNGGG time– Benefit – can easily jump repetitive sequences



• 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



• Mapping by restriction digest fingerprinting– Order clones by comparing

patterns from restriction enzymedigestion

• Mapping by hybridization– Array library – pick a “seed clone” – See where it hybridizes, pick new seed and repeat– Product


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



• 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



• 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

Documents

BioSci 145B lecture 2 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B Lecture #2 4/12/2005 Bruce Blumberg –2113E McGaugh Hall -