1. Mutation

2. Learning Outcomes To define Mutation To define Mutation To classify mutation by type To classify mutation by type To define Mutagens To define Mutagens To outline the method of identifying mutants To outline the method of identifying mutants

3. Mutation any inheritable change in the base sequence of DNA a strain carrying such a change-- mutant mutant differ from parental strain (wild-type stain) in genotype (precise sequence of nucleotides) and phenotype (observable properties) silent-cause no change in activity beneficial- new and enhanced activity disadvantageous- inactive or less active

4. Wild-type Mutant

5. Can be spontaneous or induced Can be spontaneous or induced spontaneous mutation -a mutation that occurs without a mutagen spontaneous mutation -a mutation that occurs without a mutagen can occur as a result of a natural radiation- alters the structure of bases in the DNA can occur as a result of a natural radiation- alters the structure of bases in the DNA occur during replication- errors in the pairing of bases, leading to changes in replicated DNA occur during replication- errors in the pairing of bases, leading to changes in replicated DNA Induced mutation- chemical and physical agents to induced the mutation Induced mutation- chemical and physical agents to induced the mutation

6. Types of Mutations base substitutions (point mutation)- a single base at one point in the DNA sequence is replace with a different base base substitutions (point mutation)- a single base at one point in the DNA sequence is replace with a different base silent silent missense missense nonsense nonsense frameshift mutation frameshift mutation insertion or deletion insertion or deletion shift in the reading frame shift in the reading frame

8. Missense mutations Changes in first or second base lead to significant changes in protein Instead of the original amino acid in the polypeptide chain, a different one is substituted. E.g mRNAUACAAC amino acidtyrosine asparagine Phenotypic effect can range from non-existent to devastating changes occurs at critical point- protein could be inactive/reduced activity

10. Silent mutation Genetic code is degenerate--not all mutation in protein-encoding genes result in changes in protein Genetic code is degenerate--not all mutation in protein-encoding genes result in changes in protein Silent mutation --Almost always occur in the third base of a codon (except, arg and leu- at first base) Silent mutation --Almost always occur in the third base of a codon (except, arg and leu- at first base) no apparent effect no apparent effect e.g mRNAUACUAU e.g mRNAUACUAU amino acidtyrosine tyrosine

11. Nonsense mutations results from stop codon (UAG, UAA, UGA) formation change from codon for an amino acid (sense codon) to a stop codon ( nonsense codon) termination of translation production of a truncated protein may or may not be functional depending on degree of shortening

14. Frameshift mutation Genetic code is read from one end in conservative blocks of 3 bases mutations that add or remove a base shift the reading frame of the genetic message results in a totally different protein from the point of mutation most likely to get termination of protein synthesis soon after the mutation

17. Mutation Rate the probability that a gene will be mutated in a single generation Mutation Frequency the frequency at which a specific kind of mutation (or mutant) is found in a population of individuals

18. Spontaneous mutations no mutagens involved approx. 10 -5 to 10 -6 per locus per generation 2 mechanisms: –errors occurring during replication –spontaneous alteration of a base eg. spontaneous deamination of 5-methylcytosine –GMeC AT

19. Types of mutagens base analogues chemical mutagen radiation intercalating agents

20. Base analogues compounds sufficiently similar to one of four bases substitutes a standard base during replication which appear in daughter cells in a later generation eg.5-Bromouracil 2-Aminopurine

22. Chemical Mutagens substances that can alter a base that is already incorporated in DNA and change its hydrogen bonding specificity chemically alters a base so that a new base pair appears in daughter cells in a later generation nitrous acid- Actiondeaminates A and C ResultAT GC GC AT hydroxylamine- Action react with C (GC AT) alter DNA at random location

24. Radiation 2 types; ionizing and nonionizing 2 types; ionizing and nonionizing ionizing ionizing more powerful form, short wavelength rays (X-ray) more powerful form, short wavelength rays (X-ray) mutagenic effect indirect (ionization of water/other substance) mutagenic effect indirect (ionization of water/other substance) higher dose- killed the cell higher dose- killed the cell high penetrating power, less used with microbes high penetrating power, less used with microbes nonionizing nonionizing UV radiation at 260 nm is most effective as a lethal agent UV radiation at 260 nm is most effective as a lethal agent induce pyrimidine (thymine) dimers induce pyrimidine (thymine) dimers UV radiation can kill about 90-95% of cell UV radiation can kill about 90-95% of cell survivors are normally mutant survivors are normally mutant

25. Mutation arising from repair of damaged DNA DNA damage can induce DNA repair system SOS regulatory system: initiates DNA repair processes Light-repair enzyme -repair ultraviolet induced damage –Excision repair original base-pair sequence restored repair process can result in error hence mutation

27. Intercalating agents planar, 3-ringed molecules whose dimensions are equivalent to purine (A, G) -pyrimidine (C, T, U) bp no deoxyribose phosphate may insert (between two base pair) during replication, pushing them apart addition or deletion of one or more base pairs; therefore frameshifts acridines, ethidium bromide

28. Identifying mutants Detection by selecting or testing for an altered phenotype Detection by selecting or testing for an altered phenotype 2 kinds, selectable or nonselectable 2 kinds, selectable or nonselectable nonselectable - loss of colour in pigmented organism nonselectable - loss of colour in pigmented organism selectable- drug resistance; an antibiotic-resistant mutant can grow in the presence of antibiotic concentrations that inhibit or killed the parent selectable- drug resistance; an antibiotic-resistant mutant can grow in the presence of antibiotic concentrations that inhibit or killed the parent Positive (direct) selection--isolate mutants that require amino acids or other growth factors (parent cannot grow) Positive (direct) selection--isolate mutants that require amino acids or other growth factors (parent cannot grow) Negative(indirect selection)-- nutritional mutant, inability to grow in agar plate lacking the nutrient (parent grow) Negative(indirect selection)-- nutritional mutant, inability to grow in agar plate lacking the nutrient (parent grow)

29. Wild-type Mutants

30. Petunias moss rose

31. Positive selection Positive selection detection is done by rejecting unwanted parents detection is done by rejecting unwanted parents Growing cells in plates containing antibiotics- those that grow are resistant and form colonies Growing cells in plates containing antibiotics- those that grow are resistant and form colonies Negative selection Negative selection selects cells that are unable to carry out certain function selects cells that are unable to carry out certain function replica-plating technique replica-plating technique for example can identify colonies require histidine or arginine because it cannot synthesized histidine/arginine (auxotroph) for example can identify colonies require histidine or arginine because it cannot synthesized histidine/arginine (auxotroph) auxotroph- any mutant microorganism having a nutritional requirement that is absent in the parent auxotroph- any mutant microorganism having a nutritional requirement that is absent in the parent needs a lots of screening as mutants are rare needs a lots of screening as mutants are rare

32. uv irradiation Plate and incubate Ampicillin-containing agar Amp R colonies grow Positive selection

34. Treatment of cells with mutagen replica plate rich medium/all cell grow rich medium all cell minimal medium mutant cannot grow minimal medium + arginine-mutant that needs arginine grow Negative selection

36. Identifying chemical carcinogens- Ames test relatively inexpensive and rapid relatively inexpensive and rapid assumes that a mutant can revert to wild type in the presence of a mutagen - back mutation assumes that a mutant can revert to wild type in the presence of a mutagen - back mutation many mutagens are carcinogens many mutagens are carcinogens His auxotrophs of Salmonella are exposed to an enzymatically treated potential carcinogen, and reversions to the nonmutant state are selected His auxotrophs of Salmonella are exposed to an enzymatically treated potential carcinogen, and reversions to the nonmutant state are selected the higher the concentration of mutagen used- the more revertant colonies will result the higher the concentration of mutagen used- the more revertant colonies will result