1. Bruce Blumberg (blumberg@uci.edu)
BioSci D145 Lecture #2
Bruce Blumberg
4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment)
phone
TA – Bassem Shoucri
4351 Nat Sci 2, – office hours TBA
check 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
BioSci D145 lecture 1 page 1 ©copyright Bruce Blumberg All rights reserved

2. How about term paper topics?
Example term papers are posted on web site
Specific aims
Background and significance
Research plan
References
Please 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 All rights reserved

3. 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 All rights reserved

4. 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 All rights reserved

5. 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 All rights reserved

6. 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 All rights reserved

7. 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 All rights reserved

8. 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 All rights reserved

9. 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 All rights reserved

10. 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 All rights reserved

11. 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 All rights reserved

12. 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 All rights reserved

13. 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 All rights reserved

14. 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 All rights reserved

15. Types and origin of repetitive elements (contd)
Dispersed tandem repeats are “minisatellites” 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 All rights reserved

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

17. 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 All rights reserved

18. 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 All rights reserved

19. 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 All rights reserved

20. 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,
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 All rights reserved

21. 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 All rights reserved

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

23. 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 All rights reserved

24. 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 All rights reserved

25. 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 All rights reserved

26. 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 All rights reserved

27. 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 All rights reserved

28. 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 kb
BioSci D145 lecture 2 page 28 ©copyright Bruce Blumberg All rights reserved

29. 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 All rights reserved

30. 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 All rights reserved

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

32. 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 All rights reserved

33. 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 All rights reserved

34. 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 All rights reserved

35. 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 All rights reserved

36. 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 All rights reserved

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

38. Which type of library to make
Do I need to make a new library at all?
Is the library I need available?
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 All rights reserved

39. 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 All rights reserved

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

41. 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 All rights reserved

42. 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 All rights reserved

43. 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 All rights reserved

44. 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 All rights reserved

45. 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 All rights reserved

46. 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 All rights reserved

47. 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 All rights reserved

48. 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 All rights reserved

49. 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 All rights reserved

50. 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 All rights reserved

51. 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 All rights reserved