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Basic Genetics III Lecture 26: Mutation I – types & consequences See lecture notes Lecture 27: Mutation II – repair & consequences Convention & DNA/RNA sequences o When discussing gene sequences in practice we refer to the non-template strand since it approximates the RNA & allows prediction of protein sequences Spontaneous mutations o Do they even occur? o Spontaneous mutation vs. adaptation Are mutations caused only by environmental changes? Luria-Delbruck fluctuation test (1943) 10 years before Watson & Crick If treatment leads to adaptation, then resistance not developed until phages are added – should see similar levels in all samples. o Low frequency adaptation as opposed to low frequency spontaneous mutation If spontaneous mutations exist, then resistance can arise at any point before/during/after addition of phage so we would expect to see widely varying [fluctuating] resistance between multiple samples. Results consistent with the idea that mutations arise spontaneously and do not follow addition of the phage o Highly fluctuating number of phage-resistant cells in each culture o How do they affect us? Pretty rare, but can result in disease See table. o Types of spontaneous mutation Point mutations caused by the chemical nature of the bases” Replication errors o Tautomeric shifts Adenine Single bond to NH2 is replaced by a double bond to NH o 1 out of a million Hydrogen bonding pairs it to a normal cytosine Similar base pairs between: G and T o Bases usually in keto form o Rare enol & imino forms can mispair Less energetically favourable Depurination o One cell loses about 10,000 purine residues per day o If this happens during DNA replication, DNA polymerase will not know what base to substitute in the non-template strand at the apurinic site Deamination o Can lead to G-C to A-T transitions Cytosine becomes uracil when it is deaminated Mismatches at a replication fork o G-T Wobble-pair In the first generation of progeny, one strand has replicated correctly, one strand has a mismatch (mutant). In the second generation, three strands have replicated correctly, one strand has a mismatch (mutant). Other Replication slippage (DNA polymerase) o Usually associated with repeated DNA sequences o Can lead to additions and deletions Extra base loops out on the synthesized strand and is stabilized by repetitive sequences Addition Extra base loops out on the template strand and is stabilized by repetitive sequences Deletion o Trinucleotide repeat diseases in humans Fragile X syndrome – FMR-1 gene Most common cause of mental retardation Upstream region from gene o Enhancer If >200 copies of CGG gene transcription is silenced Huntington disease Repeat in coding region of CAG (glutamine) Normal = 19-21 copies of CAG repeat Affected = Average 46 copies of CAG repeat o Affects transcription of the gene and stability of the protein Kennedy disease Number of copies reflects severity Similar mechanism to Huntingdon diseases in androgen receptor All three have neural degeneration o Induced mutations and mutagens In the lab or environment Chemicals that affect the structure of DNA in different ways: Base analogs o Chemicals that resemble nucleotides o Can be integrated into DNA in place of nucleotides o Less stable forms than bases, shift base-pairing affinities & lead to changes o 5-bromouracil (5-BU) – causes A-T G-C or G-C A-T transitions Thymine analog Switches tautomeric forms more than adenine o 2-aminopurine (2-AP) – also causes transitions Analog of adenine Switches tautomeric forms more than adenine Base alteration – alkylating agents o React with nucleotide bases and change their base pairing properties Ethylmethane sulfonate (EMS) – adds ethyl groups to nucleotide bases which leads to mismatched base pairing Nitroguanidine (NG) – adds methyl groups to keto oxygen of nucleotide base which leads to mismatched base pairing G-C A-T transitions G-C A-T transitions Base alteration – intercalating agents o Mimic basepairs and slide into double helix o Can cause single insertion/deletions Falls out during replication DNA polymerase deletes or inserts a nucleotide leads to frameshift mutations o Includes DNA dye ethidium bromide Used for visualizing DNA o Three aromatic rings connected together, similar to nucleotide basepairs Base damage – photoproducts o UV light can cause interactions between adjacent pyrimidines Thymine residues crosslink creating a cyclobutyl ring (dimer) Problems for replication Covalent bonds Changes shape of DNA and causes problems for DNA polymerase Base damage – other mutagens o Aflatoxin B1 From infected peanuts Leads to apurinic site One strand remains normal, one strand has a random nucleotide added Problem in developing countries Mutagens in our environment o See table. Mutagens in our environment – radiation o See table. Ames test – detection of mutagenic agents o Developed in the 1970s o Way to detect mutagenic substances developed by Bruce Ames Bacterial strain containing mutation making it deficient in production of histidine Auxotroph o An organism, such as a strain of bacteria, that has lost the ability to synthesize certain substances required for its growth and metabolism as the result of mutational changes. Test if a chemical can revert with second mutation Can happen spontaneously, but if a chemical is mutagenic, the number of reversals will increase in its presence Compare with spontaneous mutation rate Initially only done with a bacterial gene, however, many chemicals in the environment are not mutagens themselves but reactant with chemicals in the body to become mutagenic Solution was to add rat liver enzymes to see if chemicals were metabolically activated Using different His- strains can test for nucleotide changes versus frameshift Mutations happen o At quite a high frequency! o How do organisms deal with it? If they all accumulated, over time, would be lethal. o Very efficient DNA repair systems. See rest of lecture 27 for repair mechanisms. Lecture 28: Transposable Elements I – types & mechanisms Pieces of DNA with capacity to move in genome o Also source of spontaneous mutation Unexpected observations made in different systems o Patchy kernel derived from single cell Some are patchy, cannot be explained by Mendelian genetics Unstable alleles Genetics and the detection of the unexpected: Barbara McClintock and unstable alleles of maize First case: o Found chromosome breakage o Ds mutation in presence of Ac there is chromosome breakage Cells loose downstream traits on chromosome with Ds These include: Pigmented Plump Shiny Second case: o C-Ds reverts to C in the presence of Ac Rare unstable alleles Affect only a single gene distal to usual breakage point Suggest that Ds is mobile, but only in the presence of Ac Couldn’t map Ac so seemed to be mobile itself Mapped in different places in different strains of corn Both explained by mobile elements Genetics and the detection of the unexpected: Hybrid dysgenesis in Drosophila o Crosses of wild isolate males (P) x lab strain females (M) o F1 progeny sterile – chromosome breakage, unstable mutations in germ line o Unstable mutations of white from dysgenic cross reminiscent of what seen before… Genetics and the detection of the unexpected: “Enlarged” mutant alleles in E.coli o Unstable lac- and gal- mutants of E. coli Found to contain extra DNA Inserted into the genes causing the mutations All explained by transposable elements Lecture 29 : Transposable Elements II – transposable elements & genomes See lecture slides Lecture 30 : Genetic Dissection I – reverse genetics & functional dissection See notes Lecture 31 : Genetic Dissection II – forward genetic analysis Lecture 32: Transcription attenuation, bacteriophage lambda, and sigma factors