Mutations I. TERMINOLOGY A. Mutant 1. Genetic mutant a) An organisms whose base sequence of DNA differs from the wild type 2. Phenotypic mutant a) An organisms whose phenotype differs from the wild type 3. A genetic mutant does not necessarily need to be a phenotypic mutant, while a phenotypic mutant must be a genotypic mutant B. Wild type 1. The "normal" standard against which a mutant is compared to a) Originally referred to organisms in nature (wild) C. Mutation 1. A stable and heritable change in the sequence or number of nucleotides in a nucleic acid molecule a) Common types of point mutations (1) Substitutions (2) Additions (3) Deletions 2. Chromosomal aberrations are covered in a different section D. Mutagen 1. An agent that causes mutations E. Mutagenesis 1. Process of producing mutations 2. Categories a) Spontaneous mutagenesis (1) If it occurs without the addition of a known mutagen b) Induced mutagenesis (1) Mutation produced by a known mutagen (a) These include chemicals and radiation II.TYPES OF MUTATIONS A. Nature of mutation 1. Point mutation a) Definition (1) One base pair difference from wild type b) Base substitution (1) Transition (a) A purine (A,G) replaced by another purine, or a pyrimidine (T,C) replaced by another pyrimidine (2) Transversion (a) A purine (A,G) replace by a pyrimidine (T,C), or a pyrimidine (T,C) replaced by a purine (A,G) (3) Example (a) Sickle cell hemoglobin (i) Contains a valine in a position that normally contains glutamate c) Single base addition (1) Will throw off reading frame (2) Usually causes missense and nonsense mutations downstream d) Single base deletion (1) Will throw off reading frame (2) Usually causes missense and nonsense mutations downstream 2. Changes in two or more bases a) Deletions (1) Causes (a) Breaks in chromosomes (b) Mistakes in replication b) Additions (1) Lysogenic virus (2) Transposon (3) Mistakes in replication c) Frame shift (1) Additions or deletions of bases (as long as not in multiples of three) d) Inversions (1) A section of DNA is rotated 180 e) Rearrangements B. Effect of mutation 1. Non-lethal mutation a) A mutation that can be overcome with nutritional supplements 2. Lethal mutation a) A mutation that interferes with an essential function that cannot be overcome with a nutritional supplement b) e.g., inactive ribosomes, defective polymerases 3. Missense mutation a) Results in substitution of one amino acid for another b) Base substitution only 4. Temperature sensitive mutation a) Ts mutation b) Protein is active at one temperature, but inactivate at higher temperatures (1) Usually a result of a missense mutation, in which interactions between particular amino acids are weakened, thus broken as temperature is raised (2) These mutations are valuable in studying lethal mutations as the organism can grow at the permissive temperature 5. Nonsense mutation a) Chain termination mutation (1) Results in a shorter, or truncated, protein b) A codon for a particular amino acid is changed to a stop codon 6. Silent mutation a) A mutation that does not result in any phenotypic change b) Degeneracy of the genetic code (1) Many amino acids are encoded for my more than one codon (a) Many codons varying only in the third base code for the same amino acid (2) Therefore, a base substitution in DNA might not result in any amino acid change in a protein c) Missense mutation resulting in the replacement of an amino acid with a similar amino acid (1) A substitution of one hydrophobic amino acid for another hydrophobic amino acid might not disrupt the structure of the protein 7. Leaky mutation a) Usually a missense mutation, resulting in a change of one amino acid with a similar one, resulting in an enzyme that is less effective than the wild-type (1) For example, the new amino acid might be more bulky than the wild type, slightly disrupting protein structure, not enough to completely deactivate enzymatic activity, but enough so that it does not work as efficiently b) The following amino acid changes rarely result in a leaky mutation (1) polar to non-polar (2) non-polar to polar (3) change in sign of charge (4) sulfhydryl group to anything else (5) small side chains to bulky side chains 8. Conditional mutants a) Mutant phenotype is observed only under certain conditions b) Example (1) A mutation which prevented a cell from synthesizing an amino acid would not be detected if that amino acid was present in the growth media (a) This mutation would only be observed under conditions when that particular amino acid was absent (2) An organism that grows only at lower temperatures compared to the wild-type strain 9. Advantageous mutation a) A mutation which makes the organism more suited to its environment (1) For example, a mutation leading to an altered ribosome that no longer binds the antibiotic rifamicin would be advantageous when that antibiotic is around 10. Suppressor mutations a) A secondary mutation that restores a mutant organism's phenotype to that of the wild type III.NOMENCLATURE A. Phenotype versus genotype 1. Phenotype a) Usually designated by three letters (1) Only first letter is capitalized (2) Not italicized b) Example (1) Trp+ (a) Has the ability to produce tryptophan (2) Trp- (a) Lacks the ability to produce tryptophan c) No need to give type of mutation, the mutant number, or the particular gene mutated, since this refers only to the phenotype 2. Genotype a) All letter lowercase and italicized b) Example (1) trp+ (a) Has the wild type sequence of the tryptophan operon (2) trp- (a) Has a mutation in the tryptophan operon B. Genotype mutant nomenclature 1. geneZisolate#(Type) a) The operon in which the mutation occurs is written first b) The particular gene in the operon that is mutated (1) Omitted in monocistronic operons and in unknown cases c) The number states the order in which this mutation was isolated d) The type of mutation (if known) is given in parenthesis e) Example (1) galY223(Ts) (a) A mutation in the galactose operon (b) The Y gene contains the mutation (c) It was the 223 isolated galactose mutant (d) It is a temperature sensitive mutant IV.SPONTANEOUS MUTATIONS A. Errors in replication 1. DNA polymerase is an extremely accurate molecule a) Has proof-reading ability (1) If newly incorporated bases do not properly hydrogen bonded to the template strand, DNA polymerase exhibits 3' to 5' exonuclease activity, removing the improperly H-bonded base, then replacing it with the correct base using 5' to 3' polymerase activity (2) It inserts the wrong base only about 10-7 to 10-11 times (a) Makes on mistake in 10 million to 100 billion bases added (b) 99.999999999% accuracy b) If the wrong base is incorporated, mismatch repair mechanism can repair (1) DNA adenine methylase (DAM) acts at the sequence GATC, adding a methyl group to the A (a) The parent strand should be more methylated than the newly synthesized daughter strand (2) In mismatch repair systems, the base to be removed is taken from the under methylated strand 2. If the wrong base is added and is not corrected (or is corrected improperly) before the next replicative cycle, the mutation will be permanent in one of the daughter molecules a) The other molecule will have the wild type sequence B. Spontaneous conversion of a base incorporated in the DNA 1. Tautomer a) Each nucleotide base can exist in two forms, the normal one, and a rare tautomeric isomer (1) Tautomeric isomers have different base pairing properties than the normal isomer (a) Imino form of A binds with C (i) AT - AC - GC (b) Imino form of C binds with A (i) CG - CA - TA (c) Enol form of T binds with G (i) TA - TA - CG (d) Enol form of G binds with T (i) GC - GT - AT C. Frequency 1. Definition a) Ratio of the number of organisms bearing a mutation for a given character to the total viable population in the sample 2. Usually phenotype is used a) Of course, an altered phenotype could occur by mutations in several genes 3. Usually a large population is sampled a) That population should have reached an equilibrium of forward and reverse mutations 4. Spontaneous mutation frequencies a) 10-9 to 10-5 b) Though these numbers seem low, remember that a person may have 1013 body cells and bacteria can reach a concentration of 109 per ml D. Mutation rate 1. Definition a) Proportion of mutants, for a given characteristic, appearing per generation V. INDUCED MUTATIONS A. Base analog mutagens 1. A compound that resembles one of the four bases enough to be incorporated into DNA a) Must properly hydrogen bound to template DNA or editing activity of DNA polymerase will remove it 2. If tautormerization occurs, so that it could H-bond with other nucleotides, it will be a mutagen 3. Example a) 5-bromouracil (1) An analogue to thymine (2) Results in AT to GC switch (a) 5-bromouracil is incorporated instead of T (b) It tautorizes to the enol form, so that it can H-bond to G (i) Just like thymine, but more often (3) Results in GC to AT switch (a) It many be incorporated in its rare enol form, H-bonding to C (b) It tautorizes to the keto form, resulting in H-bonding to A during replication 4. Transition mutation a) In both cases, the purine-pyrimidine orientation remains the same b) All base analogue mutations are transition mutations B. Altering proportions of nucleotides 1. If the concentration of a purine or pyrimidine decreases in large proportion to the other purine or pyrimidine, the opposite may be incorporated in its place a) There are repair mechanisms to remove the incorrect base, but rate of incorporation increases as the differences in amounts increase (1) The incorporation rate can exceed the repair rate 2. 5-bromouracil a) Excess TTP inhibits NTP synthesis (1) This insures that the appropriate ratio between the NTP is maintains (2) 5-bromouracil also inhibits the synthesis of C, but not T, resulting in a large proportion of T to C (3) Hence T will replace C, resulting in a CG to a TA mutation (a) A transition mutation C. Intercalating mutagens 1. Examples a) Acridine orange, ethidium bromide, acriflavine 2. Characteristics a) Planar, three ring compounds (1) Three rings about the same size as a purine (2 rings) H-bonded to a pyrimidine (1 ring) (a) About the thickness of a base pair 3. Mechanism of mutagenesis a) Intercalates between base pairs in DNA b) During replication, extra bases are added (1) Both strands give rise to mutant daughter strands c) This results in a frame shift addition mutation D. Chemical mutagens 1. Definition a) A chemical that alters a base already incorporated into DNA 2. Oxidative deamination a) Nitrous oxide b) Converts amino groups to keto groups (1) C to U (H-bonds to A) (a) CG - UG - UA - TA (2) A to hypoxanthine (H-bonds to C) (a) AT - HT - HC - GC (3) G to xanthine (H-bonds to C) (a) GC - XC - GC c) Results in (1) CG to TA (2) AT to GC (3) GC to GC 3. Alkylating agents a) Example (1) Ethylmethane sulfonate (a) Widely used mutagen in eukaryotes b) Addition of alkyl groups to oxygen of G and T results in G H-bonding to T; and T H-bonding to G (1) Results in GC to AT, and TA to CG mutations E. Activation of mistake prone repair systems 1. The SOS repair system is a system to allow DNA replication to occur even with much damage a) If DNA polymerase cannot recognize a damage base, it adds any nucleotide, so that it can continue replicating (1) This, of course, results in the incorporation of many incorrect bases b) Less damage is done by alkylating agents in cells deficient in the SOS repair mechanism F. Depurination 1. Alkylation of guanine causes the breakage of the n-glycosidic bond of this purine from deoxyribose of the DNA backbone a) Can be repaired by the n-glycosidic endonuclease repair mechanism (1) Apurinic glycosylase breaks phosphodiester bond of DNA backbone when sugar is lacking its purine base (2) DNA polymerase and DNA ligase then restore the correct structure b) If encountered during DNA replication before repaired, DNA polymerase will stall (1) It will most likely install an A opposite the apurinic acid (a) G-C, to _-C, to _-A, to T-A (2) Transversion (a) A purine-pyrimidine orientation to a pyrimidine-purine orientation 2. Depurination also occurs with low pH or high temperatures VI.GENETICALLY INDUCED MUTATIONS A. Mutator genes 1. Function is to limit mutations a) When nonfunctional, mutations increase 2. DNA polymerase a) Lacking 3’-5’exonuclease activity 3. DAM a) Mismatch repair mechanism has to guess B. Transposon-mediated mutagenesis 1. Replicated copies of the transposon inserts itself into the genome VII.SITE-SPECIFIC MUTAGENESIS A. Plasmids 1. The following experiments are carried out on DNA sequences of plasmids, which are then returned to the host cell B. Deletions 1. Restriction endonuclease restriction near bases to be deleted 2. Remove small DNA segment 3. Ligate C. Base substitution 1. S1 nuclease restriction to obtain about 1 ss nick per plasmid 2. ss intact circular DNA is isolated after denaturation 3. Add short, chemically synthesized oligonucleotide with substituted base 4. Renature 5. Add DNA polymerase lacking 3’-5’ exonuclease ability (and ligase) a) Serves as a primer 6. Transform cell a) Mismatch repair system has a fifty-fifty chance VIII.REVERSION MUTATIONS A. Regaining wild type phenotype B. Second site mutations 1. Often called suppressor mutations 2. Intragenic or intergenic a) Intragenic (1) Distant amino acid that used to bind to changed one (2) Mutated amino acid switches to a similar one (3) Frame shift (a) Appropriate addition or deletion b) Intergenic (1) tRNA (2) Regulatory protein (3) Mutation in protein it interacts with (a) Antibiotic resistance C. Ames test 1. Theory a) Various chemical compounds increase the rate of mutations (1) It is important to identify such mutagens because they present a potential health risk (2) Additionally, the carcinogenic (cancer-causing) potential of many compounds to which we are exposed in our environment is highly correlated with their ability to induce mutation b) The mutagenic potential of various chemicals can be demonstrated most easily by using mutants of Salmonella typhimurium (1) The system usually involve measuring the rate of revertant mutations in mutant strains that are auxotrophic (mutants requiring specific nutrients for growth) (a) The Ames test relies on the use of a nutritional mutants of Salmonella typhimurium (i) In addition to nutritional deficiencies they also lack mechanisms for DNA repair (2) Because many compounds must be activated before they demonstrate carcinogenicity, the reliability of the Ames Salmonella test is enhanced by the use of a liver enzyme system, which may convert inactive substances to a carcinogenic form as sometimes occurs in animal metabolism. 2. Procedure a) Microorganisms are exposed to a suspect chemical incorporated into a solid growth medium that lack histidine (1) Normally, the auxotrophic Salmonella test strains cannot grow in this medium, and in the absence of a mutagen no colonies will develop (2) If the chemical is a mutagen, however, some of the cells will revert by mutation to the prototrophic state, that is, they can now synthesize their own histidine (a) These reverse-mutant cells are able to grow and produce visible colonies on the histidine-deficient agar medium (b) The appearance of bacterial colonies thus indicates that the chemical has mutagenic properties IX.USE OF MUTANTS A. To define a function of a protein 1. A mutant lacking a particular protein should then not be able to perform a particular task B. Elucidation of metabolic pathways 1. For example, glycolysis take glucose to two pyruvate molecules a) Isolate many mutants and grow with radioactive glucose b) Look for various radioactive products (glucose, glucose-6-phosphate, fructose-6-phosphate, etc.) each representing a substrate in glycolysis where the mutant is lacking an enzyme C. Correlate in vivo with in vitro events 1. D. Relationships between different systems 1.
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