Transcript Biophysics 101 Genomics and Computational Biology

Biophysics 101 Genomics and Computational Biology
Schedule:
Tue Sep 19
Tue Sep 26
Tue Oct 03
Tue Oct 10
Tue Oct 17
Tue Oct 24
Tue Oct 31
Tue Nov 07
Tue Nov 14
Tue Nov 21
Tue Nov 28
Tue Dec 05
Tue Dec 12
Tue Dec 19
Tue Jan 02
Tue Jan 09
DNA 1: Life & computers; comparative genomics, databases; model utility
DNA 2: Polymorphisms, populations, statistics, pharmacogenomics
DNA 3: Dynamic programming, Blast, Multi-alignment, HiddenMarkovModels
RNA 1: Microarrays, library sequencing & quantitation concepts
RNA 2: Clustering by gene or condition & other regulon data sources
RNA 3: Nucleic acid motifs; the nature of biological "proofs".
Protein 1: 3D structural genomics, homology, dynamics, function & drug design
Protein 2: Mass spectrometry, post-synthetic modifications,
Protein 3: Quantitation of proteins, metabolites, & interactions
Network 1: Metabolic kinetic & flux balance optimization methods
Network 2: Molecular computing, self-assembly, genetic algorithms, neuralnets
Network 3: Cellular, developmental, social, ecological & commercial models
Team Project presentations
Project Presentations
Project Presentations
Project follow-up & course synthesis
101 Section meetings
Tue
Wed
Thu
3:00 - 4:00
Haley
HMS MEC 342
7:00 - 8:00 pm Jason
HMS MEC 342
12:00 - 1:00
Dan
HMS MEC 342
(Except on 12-Oct & 9-Nov he will use MEC 338)
Thu
12:00 - 1:00
Nick
HMS MEC 340
Tue
7:30 - 9:00 pm Doug
Science Cntr 110
Tue
7:30 - 8:30 pm Allegra
Science Cntr 101B
Tue
7:30 - 8:30 pm Yonatan
Science Cntr 102B
Wed
6:00 - 7:00 pm Peter
Science Cntr 112
Thu
8:00 - 9:00 pm Adnan
Science Cntr 209
Despite recruitment of new TFs, the sections are crowded so there
are no auditor sections. Anyone registered who did not receive
email should check the list at the break.
(Email to schedule another "Biology tutorial" for Math/CS experts)
Last week's take home lessons
Life & computers : Self-assembly
Math: be suspicious of approximations
Catalysis by RNA & proteins
"The Code": treasure (but don't memorize) exceptions
Replication
Differential equation: dx/dt=kx
Mutation & the single molecule: Noise is overcome
Human disease: SNPs <1 ppb & 1.5 fold dosage
Directed graphs & pedigrees
Bell curve statistics: Binomial & Poisson
Selection
Today's story, logic & goals
Types of mutants
Mutation, drift, selection
Binomial & exponential dx/dt = kx
Association studies c2 statistic
Linked and causative alleles
Haplotypes
Computing the first genome,
the second ...
New technologies
Random and systematic errors
Types of Mutants
Null: PKU
Dosage: Trisomy 21
Conditional (e.g. temperature or chemical)
Gain of function: HbS
Altered ligand specificity
Altered specificity mutants
A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity.
A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity.
A mutant of Escherichia coli with altered inducer specificity for the fad regulon.
A mutation in the xanthine dehydrogenase (purine hydroxylase I) of Aspergillus nidulans resulting in altered specificity. Implications for the
A point mutation in the gamma2 subunit of gamma-aminobutyric acid type A receptors results in altered benzodiazepine binding site
A point mutation leads to altered product specificity in beta-lactamase catalysis.
A site-specific endonuclease derived from a mutant Trp repressor with altered DNA-binding specificity.
A spontaneous point mutation in the aac(6')-Ib' gene results in altered substrate specificity of aminoglycoside 6'-N-acetyltransferase of a
A streptavidin mutant with altered ligand-binding specificity.
A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy.
A technique for the isolation of yeast alcohol dehydrogenase mutants with altered substrate specificity.
A U1 small nuclear ribonucleoprotein particle with altered specificity induces alternative splicing of an adenovirus E1A mRNA precursor.
Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations.
An altered specificity mutation in the lambda repressor induces global reorganization of the protein-DNA interface.
An altered-specificity mutation in a human POU domain demonstrates functional analogy between the POU-specific subdomain and
Analysis of estrogen response element binding by genetically selected steroid receptor DNA binding domain mutants exhibiting altered
Antiprotease targeting: altered specificity of alpha 1-antitrypsin by amino acid replacement at the reactive centre.
AraC proteins with altered DNA sequence specificity which activate a mutant promoter in Escherichia coli.
Assessment of the role of an omega loop of cholesterol oxidase: a truncated loop mutant has altered substrate specificity.
Butyramide-utilizing mutants of Pseudomonas aeruginosa 8602 which produce an amidase with altered substrate specificity.
Carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and DNA binding specificities.
Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities.
Computational method for the design of enzymes with altered substrate specificity.
Crystallographic analysis of trypsin-G226A. A specificity pocket mutant of rat trypsin with altered binding and catalysis.
Designing zinc-finger ADR1 mutants with altered specificity of DNA binding to T in UAS1 sequences.
Dinitrogenase with altered substrate specificity results from the use of homocitrate analogues for in vitro synthesis of the iron-molybdenum
Dissecting Fas signaling with an altered-specificity death-domain mutant: requirement of FADD binding for apoptosis but not Jun
DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.
E461H-beta-galactosidase (Escherichia coli): altered divalent metal specificity and slow but reversible metal inactivation.
EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodeoxynucleotides.
Engineering proteases with altered specificity.
Engrailed (Gln50-->Lys) homeodomain-DNA complex at 1.9 A resolution: structural basis for enhanced affinity and altered specificity.
Enhanced activity and altered specificity of phospholipase A2 by deletion of a surface loop.
Escherichia coli hemolysin mutants with altered target cell specificity.
Evidence for an altered operator specificity: catabolite repression control of the leucine operon in Salmonella typhimurium.
Evidence that HT mutant strains of bacteriophage P22 retain an altered form of substrate specificity in the formation of transducing
Ferrichrome transport in Escherichia coli K-12: altered substrate specificity of mutated periplasmic FhuD and interaction of FhuD with the
Generation of estrogen receptor mutants with altered ligand specificity for use in establishing a regulatable gene expression system.
Altered specificity mutants (continued)
Genetic strategy for analyzing specificity of dimer formation: Escherichia coli cyclic AMP receptor protein mutant altered in dimerization
Immunoglobulin V region variants in hybridoma cells. I. Isolation of a variant with altered idiotypic and antigen binding specificity.
In vitro selection for altered divalent metal specificity in the RNase P RNA.
In vitro selection of zinc fingers with altered DNA-binding specificity.
In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities.
Isolation and properties of Escherichia coli ATPase mutants with altered divalent metal specificity for ATP hydrolysis.
Isolation of altered specificity mutants of the single-chain 434 repressor that recognize asymmetric DNA sequences containing TTAA
Mechanisms of spontaneous mutagenesis: clues from altered mutational specificity in DNA repair-defective strains.
Molecular basis of altered enzyme specificities in a family of mutant amidases from Pseudomonas aeruginosa.
Mutants in position 69 of the Trp repressor of Escherichia coli K12 with altered DNA-binding specificity.
Mutants of eukaryotic initiation factor eIF-4E with altered mRNA cap binding specificity reprogram mRNA selection by ribosomes in
Mutational analysis of the CitA citrate transporter from Salmonella typhimurium: altered substrate specificity.
Na+-coupled transport of melibiose in Escherichia coli: analysis of mutants with altered cation specificity.
Nuclease activities of Moloney murine leukemia virus reverse transcriptase. Mutants with altered substrate specificities.
Probing the altered specificity and catalytic properties of mutant subtilisin chemically modified at position S156C and S166C in the S1
Products of alternatively spliced transcripts of the Wilms' tumor suppressor gene, wt1, have altered DNA binding specificity and regulate
Proline transport in Salmonella typhimurium: putP permease mutants with altered substrate specificity.
Random mutagenesis of the substrate-binding site of a serine protease can generate enzymes with increased activities and altered
Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position.
Selection and characterization of amino acid substitutions at residues 237-240 of TEM-1 beta-lactamase with altered substrate specificity
Selection strategy for site-directed mutagenesis based on altered beta-lactamase specificity.
Site-directed mutagenesis of yeast eEF1A. Viable mutants with altered nucleotide specificity.
Structure and dynamics of the glucocorticoid receptor DNA-binding domain: comparison of wild type and a mutant with altered specificity.
Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions.
Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity & altered
T7 RNA polymerase mutants with altered promoter specificities.
The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket.
The structural basis for the altered substrate specificity of the R292D active site mutant of aspartate aminotransferase from E. coli.
Thymidine kinase with altered substrate specificity of acyclovir resistant varicella-zoster virus.
U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in
Use of altered specificity mutants to probe a specific protein-protein interaction in differentiation: the GATA-1:FOG complex.
Use of Chinese hamster ovary cells with altered glycosylation patterns to define the carbohydrate specificity of Entamoeba histolytica
Using altered specificity Oct-1 and Oct-2 mutants to analyze the regulation of immunoglobulin gene transcription.
Variants of subtilisin BPN' with altered specificity profiles.
Yeast and human TFIID with altered DNA-binding specificity for TATA elements.
From genomics to public health
Vaccines, drugs, lifestyle, public health measures
Pharmacogenomics
Targets (proteins or phenotypes)
Chemical diversity
Gene therapy, DNA vaccines, ribozymes, nutrition
High-throughput screening of compounds
Animal testing
Clinical trials phase 1,2,3
Formulation: Bioavailability
Toxicity
Delivery: time release ,feedback
Marketing and societal priorities
Pharmacogenomics
Gene/Enzyme
Examples of
clinically
relevant genetic
polymorphisms
influencing drug
metabolism and
effects.
Additional data
Drug
Quantitative effect
CYP2C9
Tolbutamide, warfarin, phenytoin, nonsteroidal antiinflammatories
Anticoagulant effect of warfarin
CYP2D6
Beta blockers, antidepressants, antipsychotics,
codeine, debrisoquin, dextromethorphan, encainide,
flecainide, guanoxan, methoxyamphetamine, N propylajmaline, perhexiline, phenacetin, phenformin,
propafenone, sparteine
Tardive dyskinesia from
antipsychotics; narcotic side
effects, efficacy, and dependence;
imipramine dose requirement; betablocker effect
Dihydropyrimidine dehydrogenase
Fluorouracil
Thiopurine methyltransferase
Mercaptopurine, thioguanine, azathioprine
ACE
Enalapril, lisinopril, captopril
Fluorouracil neurotoxicity
Thiopurine toxicity and efficacy; risk
of second cancers
Renoprotective effects, cardiac
indices, blood pressure,
immunoglobulin A nephropathy
Potassium channels
HERG
Quinidine
Drug-induced long QT syndrome
KvLQT1
Cisapride
Terfenadine, disopyramide, meflaquine
Drug-induced torsade de pointes
Drug-induced long QT syndrome
hKCNE2
Clarithromycin
Drug-induced arrhythmia
Diversity Databases
45 genomes completed & 324 started 216.190.101.28/GOLD
(DBCat & NAR) 513 bio-databases plus
List of SNP databasesariel.ucs.unimelb.edu.au:80/~cotton/mdi.htm
803557 human SNPs www.ncbi.nlm.nih.gov/SNP
296990 mapped snp.cshl.org
21591 SNPs in genes
http://www.uwcm.ac.uk/uwcm/mg/hgmd0.html
A significant basepair
aggtcatctgagGtcaggagttca
ANALYSIS:
ALU repeat found upstream of
Iodothyronine deiodinase, Myeloperoxidase,
Keratin K18, HoxA1,etc.
"-463 G creates a stronger SP1 binding site &
retinoic acid response element (RARE) in the allele...
overrepresented in acute promyelocytic leukemia"
Piedrafita FJ, et al. 1996 JBC 271: 14412
Critique of a basepair
1. 97% of the genome is noncoding.
2. Even repeats have regulatory & health relevance.
3. H. sapiens as a model system: Saturation
mutagenesis screen of 6x109 heterozygotes;
many hits per basepair on average.
4. One key basepair may be too reductionistic.
Whole genome, whole population, whole network
analyses are becoming increasingly feasible.
Today's story, logic & goals
Types of mutants
Mutation, drift, selection
Binomial & exponential dx/dt = kx
Association studies c2 statistic
Linked and causative alleles
Haplotypes
Computing the first genome,
the second ...
New technologies
Random and systematic errors
Where do allele frequencies come from?
Mutation (T), Migration(M), Drift (D), Selection(S), …
Tj=Sj+S(SiFj-i - SjRj-i) + S(SiRi-j - SjF i-j)
i=0,j-1
i=j+1,N
Mj= Tj + analogous to above
Dj= S Mi*B(N,j,i/N)
i=0,N
Sj= Dj * w (w=relative fitness of i mutants to N-i original).
__________________________________
T,M,D,Si = frequency of i mutants in a pop. size N
Fi= forward rate = B(N,i,PF), Ri=reverse
B(N,i,p)= Binomial = C(N,i) pi (1-p)N-i
(ref)
Random Genetic Drift
very dependent upon population size
Directional & Stabilizing Selection
• codominant mode of selection
(genic selection)
– fitness of heterozygote is the mean
of the fitness of the two
homozygotes
AA = 1; Aa = 1 + s; aa = 1 + 2s
– always increase frequency of one
allele at expense of the other
• overdominant mode
– heterozygote has highest fitness
AA = 1, Aa = 1 + s; aa = 1 + t
where 0 < s > t
– reach equilibrium where two
alleles coexist
Fixation Times
• for neutral mutations, K = µ
• for advantageous mutations, K = 4Nsµ
Role of Genetic Exchange
• Effect on distribution of fitness in the whole population
• Can accelerate rate of evolution at high cost (50%)
Network genomics
Environment
Metabolites
DNA
RNA
Interactions
Protein
Growth rate
stem cells
cancer cells
viruses
organisms
Expression
Multiplex Competitive Growth Experiments
t=0
64 Conditions
48 (to 600)
Strains
Intensity calibrated
to strain abundance
(selection coeficient)
Ratio of strains over environments, e ,
times, te , selection coefficients, se,
R = Ro exp[-Ssete]
80% of 34 random yeast insertions have s<0.3% or s>0.3%
t=160 generations, e=1 (rich media); ~50% for t=15, e=7.
Should allow comparisons with population allele models.
Other multiplex competitive growth experiments:
Thatcher, et al. (1998) PNAS 95:253.
Link AJ (1994) thesis; (1997) J Bacteriol 179:6228.
Smith V, et al. (1995) PNAS 92:6479.
Shoemaker D, et al. (1996) Nat Genet 14:450.
Today's story, logic & goals
Types of mutants
Mutation, drift, selection
Binomial & exponential dx/dt = kx
Association studies c2 statistic
Linked and causative alleles
Haplotypes
Computing the first genome,
the second ...
New technologies
Random and systematic errors
Caution: phases of human genetics
Monogenic vs. Polygenic dichotomy
Method
Problems
Mendelian Linkage
Common direct (causative)
Common indirect (LD)
All alleles (causative)
need large families
3% coding + ?non-coding
recombination & new alleles
expensive
LD= linkage disequilibrium = non-random association of k alleles
Electron magnetic moment to Bohr magneton ratio
me/mB = 1.0011596521869 (41) Ur= 4.1x 10-12
"99.5%…to accept unambiguously that the Higgs
has been spotted, the chances …
have to be reduced to one in ten million"
Number of genes in the human
genome
34,000 to 120,000
Peter J. Mohr and Barry N. Taylor, CODATA &
Reviews of Modern Physics, Vol. 72, No. 2, 2000.
physics.nist.gov/cuu/Constants
Nature 407: 118
Nature Genetics July 2000
False negatives & positive rates
One form of HIV-1 Resistance
An association test for CCR-5 & HIV
resistance
Alleles
CCR-5+
D ccr-5
total
SeroNeg SeroPos
total
ExpecNeg EXpecPos
1278
1368
2646
1305
1341
130
78
208
103
105
1408
1446
2854
dof=(r-1)(c-1)=1
ChiSq=sum[(o-e)^2/e]=
12.047374
15.122772
P
0.00052
0.00010
0.00008
But what if we test more than one locus?
Y= Number of Sib Pairs (Assocation)
X= Number of Alleles (Hypotheses) Tested
Y= Number of Sib Pairs (Association)
X= Population frequency (p)
GRR=1.5, p= 0.5
1,600
GRR=1.5, #alleles=1E6
1,400
1E+10
1,200
1E+9
1,000
1E+8
800
600
1E+7
[based on Risch & Merikangas
(1996) Science 273: 1516]
|
400
1E+6
200
1E+5
0
[based on Risch & Merikangas
(1996) Science 273: 1516]
1E+4
1E+4
1E+6
1E+8
1E+10
1E+12
1E+14
1E+16
1E+18
1E+20
1E+22
1E+3
|
Y= Number of Sib Pairs (Association)
X= Genotypic Relative Risk (GRR)
1E+2
1
0.1
0.01
0.001
0.0001 0.00001
1E-06
1E-07
1E-08
1E-09
#alleles=1E6, p=0.5
1E+8
The future of genetic studies
of complex human diseases.
ref
1E+7
1E+6
1E+5
[based on Risch & Merikangas
| (1996) Science 273: 1516]
1E+4
1E+3
1E+2
|
1E+1
0.001
0.01
1.001
0.1
1.01
1
1.1
2
10
11
100
101
1000
1,001
10,001
10000
1-GRR
GRR
How many "new" polymorphisms?
G= generations of exponential population growth = 5000
N'= population size = 6 x 109 now; N= 104 pre-G
m= mutation rate per bp per generation = 10-8 to 10-9 (ref)
L= diploid genome = 6 x 109 bp
ekG = N'/N; so k= 0.0028
Av # new mutations < S Lektm = 4 x 103 to 4 x 104
t=1 to 5000
per genome
Take home: "High genomic deleterious mutation rates in hominids"
accumulate over 5000 generations & confound LD.
How well linked?
G= generations of exponential population growth = 5000
N= population size = 6 x 109 now; N= 104 pre-G
for each haplotype H,
frequency of H on the variant gametes = nvH/nv
frequency of H on the + gametes= n+H/n+
linkage disequilibrium: d2 = (nvH/nv - n+H/n+)2 = 0 to 1
q= marker separation 1% recomb = 1 Mbp
If S= sample size needed to detect variant & disease assoc.
then approx. S/d2 is required for the LD marker.
(Kruglyak ref)
LD as a function of marker spacing &
population expansion times
Variant at 50%
Variant at 10%
LD as a function of recombination and
population size
Finding & Creating mutants
Isogenic
Proof of causality:
Find > Create a copy > Revert
Caution:
Effects on nearby genes
Aneuploidy (ref)
Pharmacogenomics
Example
5-hydroxytryptamine transporter
Lesch KP, et al Science 1996 274:1527-31
Association of anxiety-related traits with
a polymorphism in the serotonin transporter
gene regulatory region. Pubmed
Caution: phases of human genetics
Monogenic vs. Polygenic dichotomy
Method
Problems
Mendelian Linkage (300bp)
Common indirect/LD (106bp)
Common direct (causative)
All alleles (109)
need large families
recombination & new alleles
3% coding + ?non-coding
expensive ($0.20 per SNP)
Today's story, logic & goals
Types of mutants
Mutation, drift, selection
Binomial & exponential dx/dt = kx
Association studies c2 statistic
Linked and causative alleles
Haplotypes
Computing the first genome,
the second ...
New technologies
Random and systematic errors
New Genotyping
& haplotyping technologies
de novo sequencing > scanning > selected sequencing > diagnostic methods
Sequencing by synthesis
• 1-base Fluorescent, isotopic or Mass-spec* primer extension (Pastinen97)
• 30-base extension Pyrosequencing (Ronaghi99)*
• 700-base extension, capillary arrays dideoxy* (Tabor95, Nickerson97, Heiner98)
SNP & mapping methods
• Sequencing by hybridization on arrays (Hacia98, Gentalen99)*
• Chemical & enzymatic cleavage: (Cotton98)
• SSCP, D-HPLC (Gross 99)*
Femtoliter scale reactions (105 molecules)
• 20-base restriction/ligation MPSS (Gross 99)
• 30-base fluorescent in situ amplification sequencing (Mitra 1999)
Single molecule methods (not production)
• Fluorescent exonuclease (Davis91)
• Patch clamp current during ss-DNA pore transit (Kasianowicz96)
• Electron, STM, optical microscopy (Lagutina96, Lin99)
Fluorecent primers
or ddNTPs
Anal Biochem 1997 Oct 1;252(1):78-88
Optimization of spectroscopic and electrophoretic
properties of energy transfer primers.
Hung SC, Mathies RA, Glazer AN
http://www.pebio.com/ab/apply/dr/dra3b1b.html
Ewing, Hillier,
Wendl, & Green
1998
Indel=I+D
Total= I+D+N+S
What are examples of random &
systematic errors?
For (clone) template isolation?
For sequencing?
For assembly?
Examples of systematic errors
For (clone) template isolation:
restriction sites, repeats
For sequencing:
Hairpins, tandem repeats
For assembly:
repeats, errors, polymorphisms,
chimeric clones, read mistracking
Whole-genome shotgun
Project completion % vs coverage redundancy
160%
140%
120%
100%
80%
60%
Closure Probab. 1939
40%
Av Island length 1995
20%
Island Length 1988
0%
0
1
2
3
4
(see Roach 1995)
5
6
7
8
X= mean coverage
9
10
11
12
Weber & Myers 1997
Conventional
dideoxy gel
with 2 hairpin
Systematic errors
3’ 5’
B B’
CG
TA
A
A
T A
TA
Sequential dNTP addition (pyrosequencing)
> 30 base reads; no hairpin artefacts
Use of DNA Chips for SNP ID & Scoring
• already used for mutation
detection with HIV-1,
BRCA1, mitochondria
• higher detection rate than
gel-based assays
• higher throughput and
potential for automation
• ID of > 2000 SNPs in 2 Mb
of human DNA
• can multiplex reactions
Wang et al., Science 280 (1998): 1077
Use of Mass Spec for Analysis and Scoring
Haff and Smirnov, Genome Research 7 (1997): 378
A single nucleotide primer extension assay
Mass Spectrometry
for Analysis and
Scoring
Use mass spec to score
which base was
added
Can also multiplex as
long as primer
masses are known
Haff and Smirnov,
Genome Res. 7 (1997):
378
Searching for Perls
(If only finding mutations were as easy as finding words.)
#!/usr/local/bin/perl
undef $/;
$dnatext = <>;
$dnatext =~ s/\>.+?\n//g;
$mutation = $text =~ s/mutation/mutation/gi;
print " found: $mutation\n";
Today's story, logic & goals
Types of mutants
Mutation, drift, selection
Binomial & exponential dx/dt = kx
Association studies c2 statistic
Linked and causative alleles
Haplotypes
Computing the first genome,
the second ...
New technologies
Random and systematic errors
1 2000
END Sep
26,
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Please fill out your questionaires and hand them to a teaching fellow now.
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