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Schedule:Tue Sep 19 DNA 1: Life & computers; comparative genomics, databases; model utilityTue Sep 26 DNA 2: Polymorphisms, populations, statistics, pharmacogenomicsTue Oct 03 DNA 3: Dynamic programming, Blast, Multi-alignment, HiddenMarkovModelsTue Oct 10 RNA 1: Microarrays, library sequencing & quantitation concepts Tue Oct 17 RNA 2: Clustering by gene or condition & other regulon data sourcesTue Oct 24 RNA 3: Nucleic acid motifs; the nature of biological "proofs".Tue Oct 31 Protein 1: 3D structural genomics, homology, dynamics, function & drug designTue Nov 07 Protein 2: Mass spectrometry, post-synthetic modifications, Tue Nov 14 Protein 3: Quantitation of proteins, metabolites, & interactionsTue Nov 21 Network 1: Metabolic kinetic & flux balance optimization methodsTue Nov 28 Network 2: Molecular computing, self-assembly, genetic algorithms, neuralnetsTue Dec 05 Network 3: Cellular, developmental, social, ecological & commercial modelsTue Dec 12 Team Project presentationsTue Dec 19 Project PresentationsTue Jan 02 Project PresentationsTue Jan 09 Project follow-up & course synthesis
Biophysics 101 Genomics and Computational Biology
101 Section meetingsTue 3:00 - 4:00 Haley HMS MEC 342Wed 7:00 - 8:00 pm Jason HMS MEC 342Thu 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 340Tue 7:30 - 9:00 pm Doug Science Cntr 110Tue 7:30 - 8:30 pm Allegra Science Cntr 101BTue 7:30 - 8:30 pm Yonatan Science Cntr 102BWed 6:00 - 7:00 pm Peter Science Cntr 112Thu 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 approximationsCatalysis by RNA & proteins"The Code": treasure (but don't memorize) exceptionsReplication Differential equation: dx/dt=kxMutation & the single molecule: Noise is overcome Human disease: SNPs <1 ppb & 1.5 fold dosage Directed graphs & pedigrees Bell curve statistics: Binomial & PoissonSelection
Today's story, logic & goals
Types of mutantsMutation, drift, selection Binomial & exponential dx/dt = kxAssociation studies 2 statisticLinked and causative allelesHaplotypesComputing the first genome, the second ... New technologiesRandom and systematic errors
Types of Mutants
Null: PKUDosage: Trisomy 21Conditional (e.g. temperature or chemical)Gain of function: HbSAltered ligand specificity
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
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.
Altered specificity mutants (continued)
From genomics to public healthVaccines, 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 Drug Quantitative
effect
Cisapride Drug-induced torsade de pointesKvLQT1 Terfenadine, disopyramide, meflaquine Drug-induced long QT syndrome
"-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.
4. One key basepair may be too reductionistic. Whole genome, whole population, whole network analyses are becoming increasingly feasible.
3. H. sapiens as a model system: Saturation mutagenesis screen of 6x109 heterozygotes; many hits per basepair on average.
Today's story, logic & goals
Types of mutantsMutation, drift, selection Binomial & exponential dx/dt = kxAssociation studies 2 statisticLinked and causative allelesHaplotypesComputing the first genome, the second ... New technologiesRandom and systematic errors
Where do allele frequencies come from?Mutation (T), Migration(M), Drift (D), Selection(S), …
Tj=Sj+(SiFj-i - SjRj-i) + (SiRi-j - SjF i-j)
i=0,j-1 i=j+1,N
Mj= Tj + analogous to above
Dj= 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%)
Ratio of strains over environments, e ,times, te , selection coefficients, se,R = Ro exp[-sete]
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 mutantsMutation, drift, selection Binomial & exponential dx/dt = kxAssociation studies 2 statisticLinked and causative allelesHaplotypesComputing the first genome, the second ... New technologiesRandom and systematic errors
Caution: phases of human genetics
Monogenic vs. Polygenic dichotomy
Method ProblemsMendelian Linkage need large familiesCommon direct (causative) 3% coding + ?non-codingCommon indirect (LD) recombination & new allelesAll alleles (causative) expensive
LD= linkage disequilibrium = non-random association of k alleles
Electron magnetic moment to Bohr magneton ratio e/B = 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"
Peter J. Mohr and Barry N. Taylor, CODATA & Reviews of Modern Physics, Vol. 72, No. 2, 2000.
[based on Risch & Merikangas (1996) Science 273: 1516]|
How many "new" polymorphisms?
G= generations of exponential population growth = 5000N'= population size = 6 x 109 now; N= 104 pre-Gm= 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 < Lektm = 4 x 103 to 4 x 104
per genome t=1 to 5000
Take home: "High genomic deleterious mutation rates in hominids"accumulate over 5000 generations & confound LD.
How well linked?
G= generations of exponential population growth = 5000N= population size = 6 x 109 now; N= 104 pre-Gfor each haplotype H,frequency of H on the variant gametes = nvH/nv
Lesch KP, et al Science 1996 274:1527-31 Association of anxiety-related traits with a polymorphism in the serotonin transportergene regulatory region. Pubmed
Method ProblemsMendelian Linkage (300bp) need large familiesCommon indirect/LD (106bp) recombination & new allelesCommon direct (causative) 3% coding + ?non-codingAll alleles (109) expensive ($0.20 per SNP)
Today's story, logic & goals
Types of mutantsMutation, drift, selection Binomial & exponential dx/dt = kxAssociation studies 2 statisticLinked and causative allelesHaplotypesComputing the first genome, the second ... New technologiesRandom and systematic errors
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)*
Single molecule methods (not production)• Fluorescent exonuclease (Davis91)• Patch clamp current during ss-DNA pore transit (Kasianowicz96)• Electron, STM, optical microscopy (Lagutina96, Lin99)
New Genotyping
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
Fluorecent primers or ddNTPs
Ewing, Hillier, Wendl, & Green
1998
Indel=I+DTotal= I+D+N+S
For (clone) template isolation?
For sequencing?
For assembly?
What are examples of random & systematic errors?
For (clone) template isolation:restriction sites, repeats
For sequencing:Hairpins, tandem repeats
For assembly:repeats, errors, polymorphisms, chimeric clones, read mistracking
Examples of systematic errors
Project completion % vs coverage redundancy
(see Roach 1995)
0%
20%
40%
60%
80%
100%
120%
140%
160%
0 1 2 3 4 5 6 7 8 9 10 11 12
Closure Probab. 1939
Av Island length 1995
Island Length 1988
Whole-genome shotgun
X= mean coverage
Weber & Myers 1997
Sequential dNTP addition (pyrosequencing)> 30 base reads; no hairpin artefacts
A T A T A
Conventionaldideoxy gelwith 2 hairpin
B B’
3’ 5’
CTA
GA
Systematic errors
Use of DNA Chips for SNP ID & Scoring
Wang et al., Science 280 (1998): 1077
• 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
Use of Mass Spec for Analysis and Scoring
A single nucleotide primer extension assay
Haff and Smirnov, Genome Research 7 (1997): 378
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.)
Types of mutantsMutation, drift, selection Binomial & exponential dx/dt = kxAssociation studies 2 statisticLinked and causative allelesHaplotypesComputing the first genome, the second ... New technologiesRandom and systematic errors
END Sep 26, 2000
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Please fill out your questionaires and hand them to a teaching fellow now.