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Western University
Biology 2581B
Susanne Kohalmi

Biology 2581: Genetics Midterm Lectures Lecture 2 – DNA, The Molecule of Heredity & Biological Information • Information is “that which reduces uncertainty” • Uncertainty = log2(M), where M is the number of possible symbols • Why log 2 The base determines the units (bits) • Information generated depends on the length of the sequence (L) • Maximum information content of any sequence = L[log (M)2 • For DNA: 4 possible symbols A, G, C, or T o Uncertainty = log (4) = 2 bits 2 o For the insulin gene: 1789 [log (2)] = 3578 bits o These calculations assume an equal probability (¼) of seeing each symbol in the sequence • Log b = log a / logab • Why bits? If you want encode a sequence, you would need 2 bits / symbol to encode that unambiguously • A DNA sequence has the capacity to store information, but can this be shown experimentally?  • Expt: Streptococcus Pneumoniae o Smooth / rough phenotype – smooth colony were virulent and rough colony were avirulent o Virulent s form  inject in mouse  pneumonia  death o S form and isolate rough colonies  inject in mouse  alive o Virulent form  heat to kill cell  inject in mouse  alive o Virulent form  heat to kill cell and mix avirulent form  inject in mouse  death (mouse’s tissue has original S form) • Implies molecule of heredity is somewhere in that cellular degree, DNA carries biological information • Expt: S / R Form o Transformation of R to S was independent of the mouse o Heat-killed S mixed with living R  able to isolate living S form from the culture o Took principle and treated with different enzymes to destroy / eliminates components o The only treatment that destroyed the ability to transform R to S was DNAase (destroys DNA) • Expt: Bacteria Phage o Used radioactivity to track where the different molecules are going o 32P – phosphorus is in DNA, take phage and inoculate with E. coli  spin cells  separate phage ghosts and what is being introduced inside the cells  all the radioactivity was present within that cell 35llet (DNA is the genetic material) o S – sulfur is in protein, take phage and inoculate with E.coli  spin cells  separate phage ghosts and what is being introduced inside the cells  all the radioactivity is associated with the phage ghosts o Protein wasn’t getting inside the cell, couldn’t be directing new synthesis of the phage – proved DNA is molecule of heredity • DNA molecule: phosphate group, purine / pyrimidine base, and deoxyribose • If you attach a base to the deoxyribose = nucleoside • If you attach the 5’ phosphate to nucleoside = nucleotide • Uncertainty is the choice of base that appears • Each letter has to be in the proper position to be able to convey information • DNA doesn’t exist as a single helix, it exists as a double helix • Expt: Chargaff’s Rule o Measured amount of ACGT present in the DNA of different organisms o Amount of A always equaled the amount of T, and C equaled the amount G (true for any organism) • Watson and Crick found that you could line up the G and C, three H bonds would form and when you line A and T, two H bonds formed • Based on that, they came up with the double helix which would explain Chargaff’s Rule • Two strands DNA (antiparallel) = pointing inwards are the bases • Assuming the double helix, it also explains how that information is transmitted from generation to generation • All you have to do is replicate the molecule, pull apart the two strands and then each nucleotide in each of the strands could form a template and allow synthesis of a new strand • If you know the sequence of one of the two strands, the uncertainty of the other strand is 0 • Each individual strand can act as a template to produce two new identical double helices • Flow of information in biological systems: 1D DNA  3D protein  4D cells (neurons)  4D human brain Lecture 3 – The Eukaryotic-10romosome • Angstrom (Å) = 10 • Angle between glycosidic bonds of minor groove is 120° while between the major groove is 240° • B-DNA represents only one possible conformation that a DNA double helix can form (right-handed helix) • Another form is Z-DNA (left-handed helix), size of major groove is much bigger and minor groove is much smaller • How the backbone looks generates the name Z-DNA (jagged compared to smooth backbone of B-DNA) • A-form, D form, S, form – other forms of DNA also exist, able to take conformations only in extremes of pH, temperature, and solvents • Only the B and Z are biologically relevant, and for the most part almost all your DNA is in the B-form • Biological role of Z-DNA remains a mystery, does exist in vivo o Formed transiently in association with transcription o Several proteins identified with highly specific Z-DNA binding activities o Antibodies to Z-DNA bind transcriptionally active regions • Crucial property of the double helix is the ability to separate the two strands without disrupting covalent bonds • Makes it possible for the strands to separate and reform under physiological conditions • Base flipping – enzymes involved in DNA repair may scan for DNA lesions by flipping out bases • Great deal of flexibility in overall DNA organization o Humans have linear chromosomes o Bacteria / prokaryotes have tgenome in the form of circular double- stranded molecule o Viruses have either linear or circular single strand / double strand genomes • 3D orientation in space (topology) – classify DNA molecules as being topologically constrained or not constraint • If you’re not constraint, it would be possible to pull apart the two strands • In a topologically constrained molecule, the strands would still be intertwined even if you destroy all H-bonds • Proteins can hold the DNA together to create topologically constrained molecules (pitched together even if H-bonds were gone) • For a constrained double helix, torsional stress will introduce supercoiling o Overwound: (+) writhe  (+) supercoiling  (+) superhelicity, (+) supercoiled DNA o Underwound: (-) writhe  (-) supercoiling  (-) superhelicity, (-) supercoiled DNA • Almost all living things store their DNA with negative superhelical energy • One way to compact molecule is to introduce negative superhelical energy, molecule gets more and more compact • No torsional stress (relaxed state) DNA has about 10.5 bp / turn of the double helix • If you took the ends and attached together covalently, you could get a circle and you would find those two strands would cross each other about 25 times (linking number) • Supercoiling can be induced if the DNA molecule is underwound before the circle is made • This destabilizes the helix, can restabilize the DNA molecule in two ways • Introduced “energy” into the molecule, it can converts the circle into super coils or use that energy to break apart the H-bonds and separate the strands • It’s an advantage for cells to carry DNA with negative energy, stored energy could aid in processes that require strand separation • One exception: organisms that live in hot springs, living at really high temperatures – temperature is so high that it’s trying to pull apart double strands (thus, storing their DNA with + superhelical energy to be overwound to counteract the heat) • Negative supercoiling is also useful in making the DNA molecule more compact in prokaryotes • DNA packaging in eukaryotes: o Average DNA in human chromosome = 3 cm long o 46 chromosomes = almost 2 m of DNA per cell, yet fits in nucleus that is 10 microns in diameter o DNA is very efficiently packed into the chromosome • Chromatin: complex of DNA, chromosomal proteins, and other chromosomal constituents isolated from nuclei o Primarily DNA and protein with some RNA o Proteins fall into two classes: histones and non-histone proteins • Histones: small proteins with basic, positively charged amino acids lysine and arginine o Bind to and neutralize negatively charged DNA o Make up half of all chromatin protein by weight o Five types: H1, H2A, H2B, H3, H4 • Core histones make up nucleosome: H2A, H2B, H3, H4 • Highly conserved, high level of similarity of histones among diverse organisms (serving a very important function) • DNA and histone synthesis regulated so that both are synthesized together during S phase • Nonhistone proteins are a heterogeneous group: o Half proteins in chromatin are nonhistone o Large variety of functions: scaffold (backbone), DNA replication, chromosome segregation, transcriptional regulation, disentangle DNA molecules • Nucleosome: fundamental unit of chromosomal packaging arises from association of DNA with histones • Chromatin fibers with beads having diameter of about 10 nm and strings having diameter of 2 nm o Bead is a nucleosome with about 160 bp of DNA wrapped twice around a core of 8 histones o 40 bp of DNA link individual nucleosomes together o H1 lies outside and helps keep DNA tightly wrapped around • Wrapping of DNA around histone core stores negative superhelicity • Since negatively supercoiled DNA favours DNA unwinding, removal of nucleosomes will: o Increase access to DNA o Promote DNA unwinding of nearby DNA sequences o Important for DNA replication and transcription • If you isolate chromatin very carefully so that H1 is not removed, you’ll see 30 nm fiber • If you isolate chromatin and H1 is removed, you’ll see 10 nm fiber • 30 nm fiber? 10 nm fiber is still coiling around itself, introduces another level of supercoiling • Models of higher level compaction seek to explain extreme compaction of chromosomes at mitosis • DNA can be compacted even further through radial loop-scaffold • Each loop contains 60 – 100 kb of DNA tethered by nonhistone scaffold proteins, creating a rosette to be stacked on top of each other (compacts DNA even further) • Condenses DNA to rodlike mitotic chromosome that is 10,000x more compact than naked DNA • Experimental support comes from electron micrograph that shows long DNA loops emanating from the protein scaffold • Researchers took chromosome and removed all the histones – “string”, no free ends from scaffold but loops instead • Multineme Model: many DNA molecules that run in parallel through the chromosome • Unineme Model: just one DNA double helix extending from one chromosome end to the other end • There is one giant linear DNA molecule per eukaryotic chromosome • If the multineme was correct, you should’ve seen lots of really small fragments as opposed to this one giant molecule • Recoil time of “stretched” DNA is a function of the size of DNA molecules in the solution being analyzed • Chromosome is actually one big long DNA molecule Lecture 4 – Gene & Genome Structure • DNA stores the biological information to create a diverse range of proteins  cell types  tissues  organisms • Advantages: ease of storage (large quantity of data) and can be copied reliably • DNA stores information “digitally” – ACTG • Central Dogma: DNA (complementary strands)  RNA (single strand complementary to DNA strand)  protein (amino acid subunits) • Exon sequencing occurs when only coding sequences are sequenced • Gene: basic unit of biological information, specific segment of DNA at a specific location in the genome (on a region of chromosome) that serves as a unit of function encoding RNA or protein • Eukaryotic genes have introns • Upstream in 5’ region – consensus regulator sequences that interact with RNA polymerase II and all complexes to be transcribed (not present in all genes) • RNA polymerase starts transcribing at the 5’ UTR (part of RNA transcript but not part of final protein) • Transcription stops at end of 3’ UTR • Translation starts at AUG and stops at a stop codon in 3’ UTR • At ends of exons, there are splice donor and splice acceptor which indicate where splicing will occur • After transcription, product is pre-mRNA – splicing and RNA processing produce final transcript • Single-stranded sequences are unstable and at risk for degradation, cell might think it’s virus • Translation of final mRNA transcript produces protein that starts at AUG and finishes at stop codon • Coding strand – similar 5’  3’ sequence as RNA (sense, non-template, Crick) • Non-Coding Strand: used as template to transcribe RNA (antisense, template, Watson) • Template strand is used to synthesize mRNA primary transcript • Open Reading Frame: in frame sequence of DNA that starts with AUG • Organisms prefer to use one codon for each amino acid rather than all • Due to wobble, single base mutations may not results in amino acid changes • Coding sequence is region of DNA that is translated to form proteins • Amino acid sequence of polypeptide determine 3D shape which determines biological function • Genome: sum total of genetic informational in a particular organism • Genomes differ greatly between species • In yeast, majority of genome is coding but in humans, majority is non- coding (regulatory, functional) and repeats • As complexity increases, correspondingly: o Increase in genome size, number of genes, complexity of gene structure, repetitive elements o Decrease in gene density (% coding) • Each chromosome is a single, giant linear DNA molecule (~5 cm) and average mammalian nucleus is 6 μm in diameter • Packaging must allow DNA to perform biological functions • Biological information is stored in chromosomes which allows for transmission of information via meiosis (Law of Segregation & Independent Assortment) • Replication of chromosomes in S phase allows transmission of genetic info • Once replicated, goes through cell cycle to make a copy of cell or undergo meiosis to form gametes • Chromosomes: self-replicating genetic structures of cells containing DNA, linear array of genes is carried in nucleotide sequence • Chromatids: one of two copies of a chromosome that exist immediately after DNA replication, sister chromatids are joined at centromere • Homolgous chromosomes: pair of chromosomes containing same linear gene sequence derived from one parents (both have same centromeric position, approximate size, order) • Alleles: alternative forms of a single gene • Locus: designated location on a chromosome, can be either coding or non-coding • Wild-Type: form of a gene whose frequency is more than 1% in a population (a+, A, +) • Mutant: form a gene whose frequency is less than 1% in population (a-, a- -) • Many alleles are named after mutant phenotype • Not all genes can be described as having wild type and mutant forms because many genes are polymorphic (many forms exist in nature) • Transmission of genetic info involves segregation of alleles • Information is transmitted during meiosis according to Mendel’s rules of Segregation and Independent Assortment • 4 gametes arise from 1 parent (Mendelian phenotypic ratios 3:1 monohybrid, 9:3:3:1 dihybrid) • Law of Segregation: during meiosis, copies of gene separate so each gamete receives only one copy (one or the other) • Law of Independent Assortment: unlinked genes for different traits will pair independently of others • Genotype: alleles present in an individual • Phenotype: observable characteristic resulting from genotype • Homozygous: genotype in which two copies that determine trait are same allele • Heterozygous: genotype in which two copies of gene are different alleles • Dominant Allele: phenotype is expressed in heterozygote • Recessive Allele: phenotype is not expressed in heterezygote Lecture 5 – From Genotype to Phenotype: Deviations From Mendelian Ratios • Matings of inbred agouti mice to yellow mice always results in a 1:1 ratio of yellow to agouti • Matings of yellow to yellow mice always results in a 2:1 ratio of yellow to agouti?  • Example of lethal allele – cannot generate a homozygous yellow mouse because it dies as an embryo • Pleiotropy: phenomenon in which a single gene determines a number of distinct and seemingly unrelated characteristics Y • A is dominant to A with respect to coat colour, but recessive with respect to viability • Simple extensions of Mendel’s core model can explain relationship between genotype and phenotype in cases where we see deviations • Types of lethal alleles: o Early onset – gene necessary for cellular function, death at embryogenesis o Late onset – gene essential for survival but until maturity o Semi lethal – kills some mutant individuals o Conditional – only lethal under certain environmental conditions • Chi-Square test determines whether or not deviations are due to sampling error o Null hypothesis: observed deviation from expected 3:1 phenotypic ratio is due to sampling error o Degrees of Freedom = # classes – 1 = 1 o Chi-square table has p values – measure of probability of whether or not deviation can be explained by sampling error o 5% chance – deviations are so big the less likely sampling error is a likely explanation o We conclude that observed differences from expected 3:1 are not due to sampling error (null hypothesis rejected) • Extensions to Mendel: single gene inheritance • Complete Dominance: one trait is completely dominant to the other • Incomplete Dominance: blending of the traits, incompletely dominant relative to each other o Heterozygote expresses a phenotype that is different than either of the parents  • Codominance: both traits are seen, codominant relative to each other o Heterozygote expresses a phenotype in which both traits are observed equally • Dominance relationships are only meaningful when considering two specific alleles • There is no limit on the number of alleles you can have on a locus • Dominance Series: what’s dominant to what? o # of alleles = n o Kinds of genotypes = n (n+1) / 2 o Kinds of homozygotes = n o Kinds of heterozygotes = n (n-1) / 2 • Penetrance: proportion of members of a population with a given genotype showing expected phenotype • Expressivity: intensity with which a particular genotype is expressed (everbody shows traits but not to the same degree) • Probability of penetrance and level of expressivity cannot be derived from Mendelian principles and must be determined empirically • Neurofibromatosis: dominant disease that shows 50 – 80% penetrance and variable expressivity o Same mutant allele at the locus involved with this trait but some people have hardly any phenotype (café au lait spots) which in more severe cases neurofibromas are also seen o Has to do with what’s in the background of the individual • Single Gene Inheritance: traits determined by allelic variations of a single gene • Multifactorial Inheritance: traits determined by action of two or more genes • How can you determine whether two distinct genes are affecting a single trait? Complementation cross • Interactions between genes can results in seemingly non-Mendelian rations • Complementation defines a gene – you can infer there must be 2 loci involved • Dihybrid Cross: two heterozygotes produce a 9:3:3:1 ratio • Dihybrid cross involving complementary gene action gives you a 9:7 ratio instead – dominant allele of both genes must be present to produce a phenotype • Dihybrid cross showing recessive epistasis produces a 9:3:4 ratio • Recessive Epistasis: homozygousity for a recessive allele is required to hide effects of another gene o Lab coat colours: Yellow  E  Brown  B  Black o Genotype at E locus has the potential to hide the effects of B locus o Little e will only give you yellow, doesn’t matter what allele you have at the B locus (irrelevant) • Ex: Blood type is determines by sugars presented, for the sugars to be seen you have to have substance H (connects sugar to membrane) o No sugars can be presented if you have hh (epistatic – masks what you have the A / B locus)  Bombay phenotype (recessive allele masking) • Dominant Epistasis: dominant allele of one gene hides the effects of another gene o I – (12:3:1) o II – (13:3) • Continuous Variation: quantitative traits Lecture 6 – Linkage, Recombination & Mapping • Recombination: sorting of alleles into new combinations • Is genetic linkage a violation of Mendel’s Law of Independent Assortment? • When you cross a dihybrid parent with a homozygous recessive at both loci, you would expect equal gametes of each genotype but this is not the case • Conduct chi-square test to see if deviations from expected 1:1:1:1 phenotypic ratio due to sample error o Degrees of freedom = # of classes – 1 = 3 o Compare stat, p < 0.005 o We conclude that observed differences from expected 1:1:1:1 ratio isn’t sampling error (null hypothesis is rejected) • Repeat experiment – second trial with phenotypically identical parents gives converse phenotypic ratios in the progeny • Observed results are different from the first trial • What is genetic basis for the deviation from expected 1:1:1:1 ratio? • Why is there an excess of AaBb and aabb flies (deficiency of Aabb and aaBb) in some trials, while in others this pattern is reversed? • New combinations of alleles are recovered for genes on the same chromosome through crossing over chiasma • In addition to independent assortment of homologous chromosomes during meiosis, crossing over between two nearby loci on the same chromosome provides an alternate mechanism of deriving recombinants • Chiasma: point of crossing over • Recombination results from reciprocal exchanges between homologous chromosomes • Synaptonemal complex aligns homologous chromosomes o Two transverse elements with lateral elements overlap o Literally able to “zip up” chromosomes o Nodules corresponds with subsequent crossovers, when you see a recombination nodule you’ll see a crossover later on (1:1 correspondence) • Experiment: isolated chromosomes with physical abnormalities that they can see under microscope o On the chromosomes, there are two markers (both affect eye phenotype) o Whenever there is a cross over, you’d see gametes with no abnormalities or both abnormalities • Crossing over between two loci in close physical proximity on the same chromosome during meiosis I is the genetic basis of the deviation from expected 1:1:1:1 phenotypic ratio • Arrangement (cis / trans) of alleles on chromosome determines recombinant or parental classes • Recombination frequency is related to distance between loci • As distance between loci increase, probability of cross-over occurring between them increases • % Recombination = # of recombinant / total # of gametes • 1% recombination = 1 map unit = 1 centimorgan (cM) • Why are map data not internally consistent? • What is genetic basis behind apparent underestimation of map distance? • Probability of multiple crossovers increases the greater the distance between markers • Thus the farther apart two loci are, the greater the disparity between “detectable” cross-over events (recombinant gametes) and “actual” number of cross-over events • The farther apart loci are, the greater the underestimation of map distance • Three point test-cross: “tri-hybrid parent” x homozygous recessive at all loci o Least likely event is a case where you get a double crossover o Most likely event is a case where you have no crossovers • Coefficient of Coincidence (c) = actual # of DCOs / expected # of DCOs • Interference = 1 – c • Mathematical analysis demonstrates that crossing over does not occur randomly along a chromosome • Crossover decrease likelihood of another crossover in an adjacent region • Recombination at molecular level: o 1) Double-strand break formation (in one of the chromatids) o 2) Resection – 5’ – 3’ exonuclease chews back the strands and gives you single-stranded tails o 3) First strand invasion – one strand complementary binds to another strand in another chromatid (DMC1 promotes the invasion) o 4) Formation of double holiday junction – the other strand can also attack the first strand o 5) Branch migration – these junctions can actually migrate if the strands unzip (length of migration is completely at random) – chromatids are intertwined o 6) Holiday intermediate – all four arms push out, two chromatid arms rotate o 7) Alternative resolutions – you can cut vertically (exchange of the chromosome arms) or horizontally (makes the original structure) to resolve the junction o 8) Resolution of two intermediates – what happens in each junction determines if there was a crossover or not o Only get crossovers when you make one horizontal resolution and one vertical Lecture 7 – Understanding Mutations • Mutation: change in DNA sequence and this change can be inherited • In yeast colonies in a normal medium, all are growing • In a medium that is lacking, some are growing (able to make what is lacking in the medium) • Mutant: organism which experiences a change in DNA sequence • Wildtype is considered to be norm, more frequent and usually the first • Mutant displays a “negative effect” • Most mutations do not have any effect, can cause neutral changes o Very few are beneficial o Some cause problems, some are lethal (depends on conditions) • Mutation  change in phenotype o Altered appearance, growth conditions, behaviour, molecules… something one can track • Small changes – based on nucleotides, base pair substitutions o Insertions, deletions, inversions (insert reverse complement), translocation • Transitions – replacing purine to purine, pyrimidine to pyrimidine o GC to AT o One nucleotide to one that has similar molecular structure • Purine to pyrimidine / pyrimidine to purine are transversions o GC to TA o GC to CG o AT to CG o AT to TA • Large changes – affect chromosomes / parts of chromosomes o Chromosome rearrangements (duplication, deletion, insertion, inversion, translocation, reciprocal translocation, genome duplication) • Mutations are rare events (2 – 12 x 10 ) and gene dependent • Can happen all the time, random, and have many different reasons (errors during replication, radicals, UV damage) • Spontaneous mutation – are they a response to an environmental stimulus or do they happen randomly at some time? o Treating bacteria with penicillin – mutation frequency should be the same in all test tubes if penicillin (environmental) is the cause of mutation o If you assume mutations happen all the time randomly, they will happen one test tube earlier on (can propagate) and in another they will happen later on / no mutation o Take different cultures and plate them, look for number of colonies o When they actually performed the experiment, the plates did not have equal number of colonies o Mutations happen in the different cultures at different times o Using replica plating, grow strain with no penicillin and then put it on velvet (creates mirror image) o Take fresh plates (all contain penicillin) and put it on velvet and get an exact replica of the colonies from first plate o If penicillin is a cause of mutation, then you would have different colonies being to grow on different plates o Always the same colonies on all the plates able to resist penicillin (ability to grow penicillin was always there) o Spontaneous mutations are not caused by something • Change in genome allows organisms to adjust to changes in environment, it was always there not because of the environmental stimulus • Spontaneous (randomly occur over time) VS induced (take a mutagen and expose organism to it  cause DNA changes) • Endogenous (things in your body – from within) VS exogenous (from outside) • How do spontaneous mutations occur? o Depurination o Deamination o Breaking of DNA backbone (X-ray) o Formation of pyrimidine dimers (UV light) o Mistakes during replication o Unequal crossovers o Slippage / unstable trinucleotide repeats • Deamination: changes cytosine to uracil o Loses amino group  also changes base pairing at that particular site o C-G (C loses amino group to become U) – mismatch o C-G  U-A o Wherever the G is, you get C on the otherside o Wherever the U is, you will get A o The next round of replication, you get WT (CG) and mismatch (UA) and mutant (TA)  • Nucleotides mistake happen during replication, polymerase makes a mistake 6 • Typically polymerase have an error rate of 10 bases • DNA repair mechanisms for mutations, proofreading ability (3’ to 5’ exonuclease) and remove the base to do it again • Wrong base gets excised by proofreading a
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