Lecture 1 DNA: The Molecule of Heredity and Biological Information 1/29/2013 8:30:00 PM

Definition of Information: that which reduces uncertainty; not data, since data can be useless and doesnt reduce uncertainty Quantitating Information: Uncertainty = log2(M) o M=# of possible symbols a given spot (=4 in DNA: AGCT) o Uncertainty of DNA = log2(4) = 2 bits o This means if we were to convert information in DNA to binary code, each symbol/letter needs 2 bits; 00, 01,10, 11 (cant use 0, 1, 10, 11 because when jumbled together you dont know which ones are single/double digit ambiguous) A bit = a unit of information for a log base of 2 A digit = a unit of information for a log base of 10 Maximum information content of any sequence = L[log2(M)] o L=length of sequence (e.g. # of bp in a DNA sequence); hence longer sequence = can contain more information o E.g. insulin gene = 1789 bp, contains 1789x[log2(4)] = 3578 bits of information o Formula only holds when each symbol has equal probability of appearing in a sequence (0.25 for each letter in DNA) Math note: log2(4) = log4/log2

Experiments proving DNA as carrying biological information 1. Griffith: inserted streptococcus pneumoniae (2 forms: smooth and rough) into mice. S=lethal, R=non-lethal. Used heat to kill S form and injected, non-lethal, but heat-killed S + R = lethal. R bacteria took up cellular debris from dead S and transformed! Proof of transfer of biological information 2. Avery & Macleod: want to find which part of cell debris conveyed the message its DNA, because only cells treated with DNase (to destroy DNA) didnt transform

3. Chase & Hershey: Proved DNA is heritable. Virus (bacteriophage) inject something into bacteria that produce new virus. Bacteriophages were grown in two different environments: one that contained radioactive phosphorous (32P found only in DNA) and one that contained radioactive sulfur (35S found only in protein). Only phosphorous found in phage progeny, so DNA = heritable = what virus inject into bacteria, not protein.

Chemical Constituents of DNA Nucleoside: A sugar bonded to base

Nucleotide: nucleoside bonded to a phosphate Specific order/sequence of DNA set by phosphodiester linkage between sugar and phosphate of DNA backbone

Chargaffs Rule: amount A = amount T, amount C = amount G

Foundation for Watson and Cricks proposal of DNA structure, and the semi-conservative nature of DNA replication. If you know one strand, also know the other strand with 0 uncertainty.

The Flow of Information in Biological System

1st transformation = central dogma of biology, well understood

2nd and 3rd transformation, not so much. How could 4 letters encode for the entire brain? Mind-blowing!

Lecture 2 - The Eukaryotic Chromosome1/29/2013 8:30:00 PM

Intro: Human genome / cell = 6 billion bp, 2m in length, packed into each nucleus (diameter = 10 microns/m) Double helix = 2nm (20 Angstronms) in width, 3.4nm length/turn The angle b/w glycosidic bonds of minor groove = 120 degrees, while that of major groove = 240 degrees o Major and minor grooves = result of geometry of base pairing

Flexibility in DNA Structure 2 biologically relevant form: B-DNA (right handed thumb up, fingers clockwise) and Z-DNA which forms in vivo transiently (more jagged and not smooth, left handed)

There are also other forms of DNA like A-DNA, but wont form under physiological conditions Z-DNA: may have biological role within cells, but exact role unclear o Form transiently in association with transcription o There are viral proteins that exhibit highly specific Z-DNA

binding activity o Antibodies also bind to Z-DNA in its transcriptionally active regions Flexibility In Helical Structure & Base Flipping: Crucial property of double helix = ability to separate 2 strands without disrupting covalent bonds o Makes it possible to separate and reform the strands under physiological conditions (helix structure = energetically favourable) o Important for DNA replication, transcription and DNA repair Base flipping: enzymes (for DNA repair) can scan DNA for lesions by actually flip out the bases

 Flexibility in DNA Organization & Topology Humans have linear double-stranded DNA (though its double stranded circular in mitochondria), bacteria = single stranded OR double stranded circular Topology = 3D orientation of a molecule in space o Linear DNA molecule = not topologically constrained, can denature and strands separate o Circular DNA or linear DNA with binding proteins = topologically constrained, cant separate the two strands, thus can supercoil!

For supercoiling, think of twist telephone cord.

Positive supercoiling = same direction as helix negative supercoiling = whats found in cells, because its against natural curve of DNA, make the shape more accessible for transcription/replication etc.)

DNA Supercoiling Relaxed DNA (most energetically-favourable state) has 10.5 bp for every turn (a turn = when DNA strand completely circles the axis of the double helix once; usually = 2 loops shown in the diagram) Linking number = number of times 2 strands cross each other o =number of bp / bp per turn (10.5) E.g. If you unwind DNA before it becomes circular through covalently joining of ends, decrease number of turns, so bp per turn increases, you destabilized the double helix which isnt energetically favourable. To stabilize: 1) some part of the 2 strands come apart (not E.g. favourable) 2) supercoils introduced! the energetically favourable state again

1)

2)

Living cells store DNA with negative superhelicity; stores energy and aid in processes that require strand separation (replication & transcription), can also make DNA molecules compact in prokaryotes (just like positive can) DNA Packaging In Eukaryotes, Chromatin & Its Protein Components: Chromatin: complex of DNA, chromosomal proteins, and other chromosomal constituents isolated from nuclei

Chemical analysis reveals chromatin = primarily DNA, proteins (50% histones & 50% non-histone proteins by weight), with some RNA (to hold up DNA, transiently part of transcription) Histones: small proteins with basic, positively charged amino acids lysine and arginine allowing them to bind to and neutralize negatively-charged DNA backbone; there are five types of histones Core histones: H2A, H2B, H3, and H4 make up the nucleosome Non-core histone: H1 1 per linker region Histones have been carefully conserved throughout evolution among seemingly diverse species thus important function Non-histone proteins: a heterogeneous group with large variety of functions: creating the scaffold (backbone of the chromosome) DNA replication (e.g. DNA polymerases) chromosome segregation (motor proteins of kinetochores), transcriptional regulation Unknotting/disentangling DNA (topoisomerases). The Nucleosome: the fundamental unit of chromosomal packaging that arises from the association of DNA with histones (7 fold compaction) form chromatin fibres (shows when H1 is removed) with beads (nucleosome core particles, d=10nm) on a string (linker regions in between nucleosome core particles, d=2nm) nucleosome core particle: 160bp of DNA wraps twice around a core of 8 histones (two of each) linker region: 40bp of DNA link individual nucleosome (core particles) together total: each nucleosome = 200 bp

 H1 non-core histone is on the outside of DNA and helps keep the DNA tightly-bound to the histone core The wrapping of DNA around the histone core stores negative superhelicity, which favours DNA unwinding, and the removal of nucleosomes will: increase access to DNA promote DNA unwinding of nearby DNA sequences (important for transcription and replication of DNA). Higher Order Chromosome Structures & The Radial Loop-Scaffold Model: 1. 30nm fibre: a higher order of compaction, forms with the help of H1 the chromatin fiber coils coil around themselves (total of 40-50 fold)

2. radial loop-scaffold model: 30nm fibre form loops around a central scaffold (of non-histone scaffold proteins) to form a rossett, and rossets pack together into a single bundle (total 10,000 fold compaction compared to naked DNA) each loop = 60-100kb of DNA

Chromosome Compaction Summary:

Naked DNA molecule strings wrapped around nucleosome core particle chromatin fibre (beads on a string) + H1 = 30nm fibre (coiled coils) + non-histone scaffolding proteins = radial loop-scaffolding chromosome. The Unineme & Multineme Theory Of Chromosome Structure: (incorrect) The Multineme model: many DNA molecules run in parallel throughout the chromosome (correct) The Unineme model: there is just one DNA double helix extending from one chromosome end to other end Proof experiment 1: a chromosome was carefully unwound to a final length that corresponded to a single DNA molecule. Proof experiment 2: viscoelastometry - take relaxed (coiled) DNA, stretch it and let go amount of time taken to recoil to relaxed state tells you the size recoil time of stretched DNA is a function of DNA molecule size Results also support for the Unineme theory (long duration, a very long DNA molecule)

Viscoelastometry
100000

D. melanogaster

Log M (Daltons x10-6)

10000

1000

B. subtilis

E. coli

100

Phage T2 Phage T7
0.01 0.1 1 10 100 1000 10000

10

Log Retardation Time (seconds)

Lecture 3 Gene and Genome Structure1/29/2013 8:30:00 PM

Flow Of Information In Biological Systems: Just recapping the central dogma of biology; note: N-terminus to C-terminus of polypeptide correspond to the 5 end and 3 end of mRNA respectively (add new aa to C, read the next code on 3)

proteomics: study of proteins and their genetics makeup Genes: basic unit of biological information A specific segment of DNA at a specific location in the genome/chromosome that serves as a unit of function (must code for something functional) Encodes RNA or protein Anatomy Of A Eukaryotic Gene:

Note: picture below shows the non-template strand (coding strand); the actual strand being transcribed is the template strand

DNA is read 3 to 5, mRNA is made and read 5 to 3, protein made N to C Regulatory flanking region: usually contain GC/CAAT/TATA box (repeats of these letters, but these boxes not always present) which is/enhances the promoter

o usually on 5 end, but can be anywhere along the gene (upstream, downstream, or in introns) 5UTR (Untranslated Region; 5 of the mRNA not template DNA strand): where transcription begins, contains 5 cap 3UTR: contained AATAA in the middle that later signals the recruitment of poly-A tail to be added onto end of 3UTR (where transcription stops) GT(splice donor site)/AG(splice acceptor site): introns on coding strand always begin and end with these (GU and AG in mRNA) Newly-transcribed mRNA has intron removed through splicing 5UTR & 3UTR and 5cap and 3tail are all important for stability and regulation

Genes Between Species: Simpler eukaryotic organisms = fewer exons and introns in pre-mRNA, humans have much more and 4-5 different tissue-specific promoters that can give rise to different combos of mRNA transcripts. Sequence To Function:

Open Reading Frame (ORF): an in-frame (divisible by 3) sequence of DNA that starts with the start codon (ATG) and ends with a stop codon (stop) codons (TAA, TAG, TGA); can potentially code for proteins, but we dont know for sure Coding Sequence (CDS): a region of DNA that we know is translated to form proteins Amino acid (each with different properties) sequence determines 3D shape of protein, which determines function Genetic Code Codes for every organism on this planet (shows aa for a given 5mRNA3). There are 4^3 = 64 possible codons, lots of redundancy, 3rd nucleotide doesnt matter too much due to wobble safe mechanisms to minimize effects of mutation in 3rd codon

Anatomy Of A Genome: Genome: sum total of genetic information in a particular cell or organism As organisms increase in complexity, so does their gene structure Larger genome more # of genes decreasing gene density (more % non-coding sequence; which doesnt include introns because they are still coded, just spliced out) increasing complexity of gene structure (more & larger introns, more complex gene regulation)

 More repetitive elements Genome Storage: Chromosome = a single giant linear DNA molecule in its most condensed form (after S-phas) Chromatin: physiologically form of DNA: most DNA packed as chromatin =30nm chromatin fibre/solenoid fibre Replication allows transmission of genetic information Genetic Terms To Know:

Wild Type, Mutant Alleles & Natural Allelic Variation: Wild-type allele: form of a gene-frequency > 1% in a population (a+ / A / +) Mutant allele: form of a gene-frequency < 1% in a population (a-, a, or -) Many genes are named after their mutant allele phenotype e.g. tubby gene which can be tub+ (normal) or tub- (tubby) note: mutant allele = always smaller frequency, but can be both dominant (rare) or recessive (most common).

Not all genes = either wild-type (+) or mutant (m) forms; many genes are naturally polymorphic (locus with two or more distinct alleles in a population). E.g. snail shells, human blood types. Mendels Law Of Segregation: During meiosis, the 2 copies of a gene separate so that each gamete cell receives only one allele

Mendels Law Of Independent Assortment: (Unlinked) genes for different biological traits will pair independently of other traits. Homologous chromosomes will choose how they line up at the metaphase plate of the cell independently of other genes (metaphase I)

Lecture 4 - Deviations from Mendelian Ratios

Lucien

1/29/2013 8:30:00 PM

Cunots Odd Yellow Mice & Pleiotropy: Agouti=recessive allele, Yellow=dominant allele Heterozygous = yellow Mated hetero yellow with hetero yellow, should be 3:1 yellow:agouti, but got 2:1 yellow:agouti, and all yellow=hetero

turns out AyAy is lethal Example of pleiotropy: single gene determines a number of distinct and seemingly unrelated characteristics In this case, Ay (yellow allele) was shown to be dominant to A with respect to coat colour, but recessive with respect to viability (wont kill when hetero because agouti dominant in viability) Types Of Recessive Lethal Alleles:

Genes that are essential, and the recessive of which is lethal Early onset alleles: the gene is necessary for cellular function as well as death at embryogenesis or early on in life (e.g. Cunots mice). Late onset alleles: the gene is essential for survival, but not until the individual has matured (e.g. 30-40s) Semi lethal alleles: the gene kills some mutant individuals in the population, but not all. Conditional alleles: the mutation is only lethal under certain environmental conditions (e.g. fruit fly mutants and temperature). The Chi Square Test: Chi Square - Null hypothesis: observed = expected, and deviation from expected is only due to sampling error.

Red=wild type dominant, white=recessive; expected = 3:1 ratio

Critical value = 0.05 (95% confidence), look for chi square value (99.1) and its corresponding p-value; p-value smaller than 0.005 so must be smaller than critical value, therefore reject null hypothesis and the observed differences from expected can be attributed to genetic phenomenon rather than chance.

Note: table below = for single gene (monohybrid ratios)

Incomplete Dominance & Codominance Incomplete dominance: heterozygote = blend of traits, different than either of the parents (incomplete b/c neither can show itself) e.g. white and red flower pink flower Codominance: heterozygote = both traits are observed equally (both show) Human glycolipid blood types (type A and type B give type AB, both glycolipids present)

Dominance Series Mendel focused on genes with only 2 alleles, but there are genes with multiple (>2) alleles and thus phenotypes (e.g. lentils appearance) still only 2 allele per offspring/zygote though, just more options to choose from Dominant/recessive = only describes relationship b/w 2 alleles, so it is necessary to set up a dominance series (rank the dominance of all alleles) through series of 2-generation crosses and deduce ranking (if 2 alleles = incomplete/co dominant, rank the same) o Cross 2 phenotypes, then cross offsprings again to verify 3:1 ratio which tells you which one is dominant (the initial parents are pure bred (homozygous), so F1 = must be heterozygote) If n=number of alleles o Kinds of homozygotes = n o kinds of genotypes = n(n+1) / 2 o kinds of heterozygotes = n(n-1) / 2.

o Penetrance & Expressivity: Variable penetrance: same genotype at a given locus, but some show the expected phenotype and others do not o Penetrance: proportion of members showing phenotype(exp.) Variable expressivity: same genotype at a given locus, but phenotype expressed to varying o Expressivity: intensity with which genotype is expressed

Variable penetrance and expressivity combination: same genotype at a given locus, some doesnt show trait at all, while others express it to varying degrees o E.g. neurofibromatosis The probability of penetrance and expressivity level must be determined empirically; not Mendelian-able.

Above = extension to mendel for single gene inheritance: lethal alleles, incomplete dominance, co-dominance, dominance series, and penetrance/expressivity ***** Multifactorial Inheritance Traits determined by the expression of two or more genes; usually involved in a biochemical pathway with intermediates (compare to pleiotropy: gene that determine two or more traits dominance series: traits determined by two or more alleles of the same gene) Below = extensions to multifactorial inheritance

Complementation: interactions between genes that can result in seemingly non-Mendelian ratios. a and b = the 2 genes. +=dominant, number=recessive (non-functional)

Example of red (dominant) & white (recessive) flower: biochemical pathway with 2 enzymes/genes required to attain red pigment. White flower = homo recessive allele for one of the enzyme. If 2 whites have homo recessive (non-functional) allele at different genes (but 1 has a+ and other has b+), their progeny=red due to complementation (since a+ and b+ are dominant). o Note: if complementation occur (recessive+recessive = dominant), must have 2 (or more) genes!

 If they have the non-functional alleles at the same loci(e.g. a1 in one flower and a2 in the other), non-complementation and progeny = still white.

 Complementary Gene Action: A typical Mendelian dihybrid crosses (heterozygous x heterozygous) have expected result in a typical 9:3:3:1 ratio (see below: lentil coat colours)

An extension to complementary gene action: genes working in a consecutive chain to achieve a particular trait. Basically instead of each genotypic combination = a different phenotype, each gene now corresponds to an enzyme, and only when the entire pathway is carried through can you reach the purple phenotype.

 So instead of 9:3:3:1, anything not A-B-=white and you get 9:(3+3+1)=9:7 Epistasis=one gene (dominant or recessive, determines the name) mask the effect of another gene Recessive Epistasis Where homozygosity of recessive allele for one gene can hide the effects of another gene.

Labrador retriever coat colour: whenever there is ee, phenotype=yellow no matter what b is

 Another

Because only E- can convert yellow to brown. So if ee, stuck at yellow. Again, sequential process example = Bombay blood phenotype Need substance H to attach blood sugar for determining blood type If hh genotype (recessive masks other gene), no H produced, and so it doesnt matter what blood type you are, you get Bombay phenotype (no sugar surface marker) ii H (results in O phenotype) and IAIB H (results in AB phenotype).

Dominant Epistasis Dominant allele of one gene hides the effects of another gene.

As long as theres a B-, will be white. In a molecular context: B can convert both yellow and green to white, so it doesnt matter what A is.

Continuous Variation: Ronald Fisher provided the foundation of modern quantitative genetics by fusing Mendelian theory with the study of continuous (or quantitative) traits. Continuous variation is the combined effect of many genes (>2) and is often significantly affected by environmental influences. Many examples of continuous traits in human populations include height, weight and skin colour; these traits are not as clear-cut as the discontinuous traits that Mendel exclusively studied. Continuous variation can however, be explained by Mendelian genetics. The more genes or alleles there are responsible for the trait, the more possible phenotypic classes and the greater the similarity to continuous variation. In these examples, several pairs of incompletely dominant alleles have additive effects. The percentages shown at the bottom denote the frequencies of each genotype expressed as fractions of the total population.

Lecture 5 Linkage, Recombination & Mapping

1/29/2013 8:30:00 P

Recombination, Genetic Linkage, & The Chi Square Test: Recombination is the sorting of alleles into new combinations when making gametes, during independent assortment of meiosis (thus not necessarily just crossing over, but meiosis metaphase I too)

Genetic linkage: tendency of genes that are located proximal to each other on a chromosome to be inherited together during meiosis; also a violation of Mendels Law of Independent Assortment as the resultant gametes deviate from the expected 1:1:1:1 ratio (validated using chi square) so in the above picture, if A and B are linked, AB and ab gametes will be produced more often (which can be seen from test cross F1)

Crossing Over, Synapsis & Reciprocal Changes: Crossing over: the physical exchange of nearby loci in homologous chromosome pair allele flipping to form hybrid chromatids Another mechanism for deriving recombinants, in addition to independent assortment (which also gives recombinants diff from either parents) occurs during prophase I of meiosis subsequent to synapsis Synapsis: when the synaptonemal complex zip the homologous chromosomes together (actually like a zipper) such that they are aligned (?)Chiasma/recombination nodules: site of exchange of parts between non-sister chromatids; number and location of recombination nodules correlates with the number and location of crossover events.

-Curt Stern: demonstrated in experiments that recombination results from reciprocal changes between homologous chromosomes (observed as physical change of shape in DNA) using Drosophila Genetic Crosses & Linkage Notation:

 cis: 2 alleles of the same type (AB or ab) on same chromosome trans: Ab or aB on same chromosome. The cis/trans arrangement of the alleles on the chromosome determines the recombinant or parental classes. The ratio between the 2 classes determined by gene linkage. More linked, more parental, less recombinant. Recombination Frequency: Recombination frequency is a measure of the physical distance between the loci (the less the recomb, the closer/more linked they are) % Recombination = Number of recombinant gametes / Number of total gametes 1% recombination = 1 map unit b/w gene = 1 centiMorgan (cM) Note: recombination is an underestimate of the physical distance between loci, because it counts multiple crossovers (DCO) as no recombination The farther apart loci are, the greater the underestimation of map distance, the greater the disparity b/w detectable & actual crossover

RF=0%
Three-Point Test Cross, Interference & The Coefficient Of Coincidence: 1. Given gamete genotype and frequency (may be progeny frequency too) 2 most frequent = parental class (no cross over) 2 least frequent = double cross over (what are the chances of that!) rest = single cross over 2. Gene order / relative location figure out which one is in the middle Compare parental with DCO; the only gene that switched = in the middle o the ones on the outside doesnt matter Or list all possible parietals out like this and find the one that matches DCO

 o cross over follow how those 2 Xs link the gene. Just assume cross over always between genes (?) 3. Determine Map Distance list out how the parental look like For each of the 2 distances, add up number of progeny that had the two genes of interest crossed over (different from parental) and divide over total gametes o Note how if you did y and m directly, you would have underestimated the distance because DCO will be counted as parental (y and m remained same relative to each other) o So you should do yw, then wm, then add them up (or double count DCO?)

 4. Interference & Coefficient of Coincidence chance of crossover is not completely evenly distributed along a chromosome; a given crossover decreases the likelihood of another crossover in an adjacent region (not independent events) o (we knew genes close together will be more likely to end up on same gene, but that was still with the assumption that crossovers occur completely randomly along a chromosome)

 note: expected number of DCO = recombinant frequency @ 1st crossover x recombinant frequency @ 2nd crossover x total number o expected number of DCO o actual double cross overs are less than that (which is calculated using single cross over values) because one crossover interferes with the chance of next one occurring near it. The closer to 1, the more interference, the smaller the c, the much less actual than expected DCOs. Recombination @ The Molecular Level: 1. a double-stranded break in one chromatid of one chromosome 2. crucial and involves the broken strand invading its non-sister chromatid neighbour (they are held together by synapsis)

3. first strand invasion

4. Double Holiday Junction Forms

The dotted strands = to be synthesized by DNA replication 5. Branch Migration (just slides through, expanding cross over area)

6. Holliday Intermediate (imagine twisting bottom 180 degrees w/ arrow)

7. The 2 alternative resolutions

8. Resolution of the 2 intermediates; whether or not there is a crossover depends on how each of the junctions is resolved (vertical: cross, hor.: stay) If both are resolved in the same way (horizontal or vertical), then an aborted crossover results and the loci return to their original state If both are resolved in different ways (horizontal and vertical), then a crossover between loci occurs.

Lecture 6 Mutations & Mutants

1/29/2013 8:30:00 PM

Mutations & Mutants Mutation: an inheritable change in DNA sequence can have positive (rare), negative/lethal, or no effect (most) on the organism o Effect depended on conditions, can be induced by enviro. Mutant: an organism which experiences a change in DNA sequence o Wildtype: the norm, more frequent, there first o Mutant: typically a negative effect on wild type Phenotypes & Mutations: Phenotype: important for tracking & observing mutations in an organisms E.g.: altered appearance, growth conditions, behaviour, even molecules; something one can track Types Of Mutations (Small Changes): based on changes in nucleotides Base pair substitution: substitute one nucleotide for another, always alter the base on the complementary strand too through replication 2 Transition: the base substituted is the same type (purine to purine, or pyrimidine to pyrimidine) 4 Transversion: purine for pyrimidine, and vice versa

Insertion/deletion mutations: addition/removal of base-pairs

Inversions: double-stranded flipping of base-pairs

translocation: movement of base-pairs along the double-helix

Types Of Mutations (Large Changes): chromosome rearrangements affecting chromosomes or parts of chromosomes

For a single chromosome: Chromosome duplication (two copies) deletion (shorter) insertion (longer) translocation (moved) inversion (flipped) Between two or more chromosomes: Translocation (from one to another; one way) reciprocal translocation (exchange of genetic material between 2 chromosomes) Genome duplication of all chromosomes - not necessarily lethal in plants Spontaneous Mutations vs. Induced Mutations: Spontaneous mutations happens naturally by itself (note: she considers anything that we normally do as happens naturally, like UV light exposure) gene-dependent, rare events (2-12 x 10-6 per gene per gamete) can occur all the time throughout the course of life many different natural reasons: replication error, radicals

Happens randomly without external stimuli; randomness of its occurrence verified by fluctuation test Proof of spontaneity - Fluctuation test: bacterial resistance experiment Resistance to penicillin = a mutation. If its an induced one, when bacteria put into tubes of penincillin, each tube should have around the same survival rate. But nope, drastic difference, meaning some culture developed resistance way before penincillin was introduced (so the bottom pic).

Therefore, resistance was spontaneous and occurred before environmental signals (only became apparent after exposure)

Induced mutations = DNA response to mutagen (UV=spontaneous when normal exposure, and induced if in high quantity)

Types of Spontaneous Mutations

Deamination: when U replaces C (loss of an amino group from C)

G is now paired with U, after replication, polymerase pair U with A, gradually half of the replicated cells will be mutants o Note: picture is wrong, the top right UA is not a mutant, just a mismatch

Slippage: DNA polymerase forgetting to count bases among tandem repeats New strand slips: insertion Template strand slips: deletion

Spontaneous mistakes during replication: DNA polymerase error rate: 1 mistake per 1 million (10^6) bases. can be fixed by DNA polymerases self exonuclease ability

Other spontaneous mutations: Depurination: A or G removed, results in apurinic site no base there X-rays: can break the sugar-phosphate backbone, split DNA molecule into smaller pieces, which may be spliced back together improperly Ultraviolet (UV): radiation causes adjacent thymines to form dimers, which inhibits DNA replication disrupts reading of DNA Unequal crossing over: recombination takes places between mismatched sequences of homologous chromosome pairs; one chromosome has insertion, other deletion. Repair Mechanisms 1. Proofreading: done by DNA polymerase during synthesis 2. Base excision repair: fixes deamination! Not mismatch. glycosylase recognizes and removes uracil, leaving an AP site (backbone intact though) AP endonuclease then cuts backbone, make nick at AP site

DNA exonuclease remove nucleotides near nick, expand gap DNA polymerase fills gap with new bases and ligase seals nick

3. Nucleotide excision repair: fixes thymine dimer UvrB and UvrC endocnucleases recognize and create nicks surrounding the dimer to remove damaged fragment. DNA polymerase then fill the gap and ligase to seal the nick.

endonuclease: make cut in the backbone exonuclease: chews up

4. Mismatch Repair (Methyl-Directed): fixes mismatches made by DNA polymerase that escaped its self proofreading. But how do you know which base of the mismatch is the error? Want to fix newly synthesized strand assess degree of methylation: o template strand = very methylated o new strand = non-methylated fix this one! again, just remove old up to site of mismatch, fill in new and seal

something recognize it, make a cut, removes a segment, DNA polymerase add back, and ligase seals backbone. Enzymes may be different, but these = common.

Lecture 7 Tetrad Analysis

Introduction:

1/29/2013 8:30:00 PM

Problem: we want to find the gene mutation responsible for a certain phenotype. Sequencing genomes is unhelpful since the gene of interest is among 20,000 other genes. Gene linkage becomes useful as it is the process of finding genes proximal to the gene of interest. o Example: males who have haemophilia A are often also colorblind as these sex-linked mutations are inherited together. In contrast, individuals with haemophilia B do not necessarily inherited colour blindness (the responsible genes

are far away from each other or on different chromsomes). Meiosis In Budding Yeast: Saccharomyces cerevisiae = bakers yeast = budding yeast; weird cell cycle normal individuals = haploid (2 gender, a and ), fuse to form ONE diploid cell, goes through meiosis and form 4 gametes in an asco spore what we take out and are interested in; = yeast gamete

 These asco spores are isolated and cultured in a given medium In rich medium, both wild type URA and mutant ura (lacks enzyme to make uracil) spores will grow In a medium lacking uracil, only wild type will survive. It is impossible to know which spores are mutants just by mere observation. A tetrad = the 4 gametes in the asco spore tetra study makes it possible to study individual meiosis (cant isolate the 4 gametes in humans) Two Unlinked Genes On Different Chromosomes In Budding Yeast: HIS4 & TRP1 = wild types; his4 & trp2 = mutants. 2 different unlinked genes

Parents are like this:

Outcome 1. Parental ditype (PD): during metaphase I, lined up just parents; all gametes are like parents, 2 of each (its like parents just duplicated themselves) note: di- here refers to both parents genotype are in progeny

Outcome 2. Non-parental ditypes (NPD): shows that chromosomes aligned differently during metaphase, and all gametes dont look like parents

Outcome 3. Tetratype (T): a cross over occurs, now all gametes are different (2 parental ditype, 2 non-parental ditypes)

Compare results: if #PD ~= #NPD, genes are unlinked (homologous chromosomes can randomly and independently assort/line up)

Two Linked Genes On The Same Chromosome In Budding Yeast: ARG3 & URA2 = wild types; arg3 & ura2 = mutants, 2 linked genes on the same chromosome Outcome 1: no crossover (NCO) parental ditype (no independent assortment since same gene, so if NCO, must be parental ditype)

Outcome 2: single cross over (SCO) tetratype: 2 PD, 2 NPD, all 4 different

Outcome 3: double cross over (DCO) TWO strands parental ditype: since only 2 genes, just go back to original place, like NCO

Outcome 4: double cross over (DCO) THREE strands tetratype Note: DCO means 2 of those X thingies Top: first 2 inner, then top inner and bottom outer Bottom: first 2 inner, then top outer and bottom inner

Outcome 5: double cross over (DCO) FOUR strands: all non-parental ditypes Inner switch, then outer switch

Compare results: PD far outnumber NPD since they are linked genes Summary Of Linked & Unlinked Genes: Parental alleles align together Parental alleles switch Crossover in homologous pair No crossovers Single crossover Double crossover (2 strands) Unlinked Genes Unlinked Genes Unlinked Genes Linked Genes Linked Genes Linked Genes Parental Ditype (2 types) Non-parental Ditype (2 types) Tetratype (2 PD, 2 ND) Parental Ditype (2 types) Tetratype (2 PD, 2 ND) Parental Ditype (2 types)

Double crossover (3 strands) Double crossover (4 strands)

Linked Genes Linked Genes

Tetratype (2 PD, 2 ND) Non-parental Ditype (2 types)

*Remember that linked genes are those that are close together on the same chromosome whereas unlinked genes are those that are either on two different chromosomes or far apart on the same one. Determining Distance Between Loci & The Centromere: Distance between loci: same as calculating recombination frequency, except instead of giving you all the progeny genotypes directly, now grouped into PD (no recomb), NPD(all recomb), and T(1/2 recomb). New formula: Note: tetra in denominator = number of asco spore/group of 4 gametes, not tetratypes

Distance between loci and the centromere: Model: fungus Neurospora , also produce asco spores Colour = only 1 gene involved, either black WS+ or white WS ONLY 2 types of segregation patterns: first/second division First-division segregation pattern: no cross over, the 2 variations arise solely from the law of independent assortment

Second-division segregation pattern: tells you there was a single cross over; but since only 1 loci, the recombination frequency is determined by distance between loci and centromere the shorter their distance, the less likely cross over occurred somewhere between loci and centromere

So now, you can calculate RF, which = distance between loci and centromere Only half of 2nd division gametes actually undergone recombination

Gene Conversion: -In a medium, the amount of surviving spores : dead spores usually exhibit 0:4, 4:0, or 2:2 ratios, but 3:1 ratios have been observed and are rare.

This phenomenon is attributed to gene conversion: recombination of DNA seen as error and fixed such that no recombination on that chromatid At some point during recombination, a heteroduplex region (1 strand from one homologue and 1 strand from another) is formed. A mismatch occurs in this region because a DNA strand of the wild type allele and the mutant allele are bound (not perfectly complementary).

Gene conversion can then occur and the mismatch is corrected in one homologue (but not in other). This rare process accounts for

the resulting tetrad ratio of 3:1 since one chromatid still contains the mismatch, while the others do not.