MCB 3020, Spring 2005

1. MCB 3020, Spring 2005 Chapter 10: Microbial Genetics I Mutations

2. RNA protein DNA phenotype Genetics : the study of the mechanisms of heredity and variation in organisms DNA Central dogma

3. A. The genotype determines the possible phenotypes of an organism. Genotype the exact genetic composition (DNA sequence) of an organism Phenotype the observable characteristics of an organism

4. B. Why study prokaryotic genetics? 1. Prokaryotes are relatively simple: haploid, easy to grow 2. Many principles of genetics are the same in prokaryotes and eukaryotes. 3. Molecular cloning and biotechnology 4. Control of pathogenic microorganisms TB

5. extant species mutation, DNA transfer and natural selection (evolutionary time) ancestral cell Life evolves. This leads to diversity. It is likely that all organisms are related to a single ancestral cell or group of cells. TB

6. D. Genetic diversity can result from 1. Mutations 2. DNA transfer

7. Molecular Genetics I: Mutations I. Mutations II. Effects of mutations on protein structure III. Effects of mutations on protein function IV. Effects of mutations on phenotype V. Mutagens

8. Mutations can play an important role in genetic diversity and evolution. I. Mutations inheritable changes in the genotype (DNA sequence) of an organism

9. A. A base pair change is an example of a mutation. ...GATCGGATC... ...CTAGCCTAG... mutation ...GATAGGATC... ...CTATCCTAG... TB

10. B. Mutations can lead to biological variation Most mutations are harmful or neutral. Rare beneficial mutations and natural selection lead to new species. TB

11. C. Most mutations result from DNA replication errors. DNA polymerases sometimes make mistakes that are not repaired. DNA damage increases the likelihood of such mistakes. TB

12. DNA damage can lead to mutation, but is not a mutation per se because it is not heritable. ...GATCGGATC... ...CTAGCCTAG... methyl-G CH3 ...GATCGGATC... ...CTAGCCTAG... DNA damage (alkylation) TB

13. D. Mutation frequencies are thought to be roughly similar in all organisms: ~10-9 to 10-10 / base pair / generation Thus, in general, mutations are rare. TB

14. mutation E. Mutants can be derived from wild-type strains (or from other mutant strains). Wild-type: the original strain of an organism isolated from nature mutation Mutant: an organism with a genome that carries a mutation

15. Mutations in genes that encode proteins can affect • protein structure • protein function • the phenotype of the organisms

16. II. Effects of mutations on protein structure A. base pair changes 1. silent mutations 2. missense mutations 3. nonsense mutations B. deletions C. insertions D. frameshift mutations E. inversions F. duplications

17. Overview: Effect of mutations on protein structure transcription mRNA translation OR mutation gene Translation has two possible outcomes: (1) a change in the amino acid sequence, or (2) no change. TB

18. A. Base-pair changes (point mutations) A heritable change in a single base pair of DNA 1. silent mutations 2. missense mutations 3. nonsense mutations

19. mutation ...TAT... ...ATA... ...UAC... ...UAU... tyrosine tyrosine 1.Silent mutations ...TAC... ...ATG... DNA RNA Polypeptide No change in the polypeptide TB

20. mutation ...AAC... ...TTG... ...UAC... ...AAC... asparagine tyrosine 2. Missense mutations ...TAC... ...ATG... DNA RNA Polypeptide One amino acid is changed in the polypeptide. TB

21. 3. Nonsense mutations (mutation results in a stop codon) mutation ...TAG... ...ATC... ...UAC... ...UAG... stop codon tyrosine ...TAC... ...ATG... DNA RNA Polypeptide A truncated polypeptide is made. TB

22. B. Deletions One or more base pairs are lost ATGAAAGAG.... ATGGAG.... Possible results a. amino acids or polypeptides can be lost b. frameshifts can occur (see below) TB

23. C. Insertions One or more base pairs are gained ATGGAG.... ATGAAAGAG.... Possible results a. amino acids or polypeptides can be gained. b. frameshifts can occur (see below) TB

24. D. Frameshift mutations ATGAAGTTG.... Insertions or deletions that change the translational frame ATGCAAGTTG.... one base pair deletion Two changes in polypeptides are possible: (1) every amino acid downstream of the mutation is changed, (2) a truncated (shortened) protein is produced. TB

25. A T G C A A G T T G A #1 met gln val A T G C A A G T T G A #2 cys lys leu A T G C A A G T T G A #3 ala ser STOP DNA can have 3 reading frames: A T G C A A G T T G A

26. met gln val ile lys leu Frameshift mutations change the translational reading frame. ATGCAAGTTGA…. one base pair deletion (Frameshifts occur only if insertion or deletion is in the reading frame section of a protein-encoding gene.) ATCAAGTTGA

27. E. Inversions chromosomal segment is inverted ...ATGGAAGAG.... ...TACCTTCTC.... ...ATTTCCGAG.... ...TAAAGGCTC.... A number of changes in polypeptides are possible. TB

28. F. Duplications chromosomal segment is duplicated ...ATGGAAGAG.... ...TACCTTCTC.... ...ATGGAAGGAAGAG.... ...TACCTTCCTTCTC.... A number of changes in polypeptides are possible. TB

29. III. Effects of mutations on protein function 1. No effect (common) 2. Loss of function (common) 3. Partial loss of function (leaky) 4. Conditional loss of function 5. Change of function (rare) 6. Restoration of function (reversion) TB

30. 1. No effect (common) wild type (normal protein) mutant protein Depending on the protein, up to 80% of the amino acids may only function as spacers. TB

31. 2. Loss of function (common) mutant protein Examples: a. A change in an amino acid that participates directly in catalysis (change in the active site) wild type (normal protein) TB

32. 2. Loss of function (contd.) mutant protein misfolded degradation amino acids b. A change in an amino acid that causes the protein to misfold. wild type protein TB

33. 3. Partial loss of function, "leaky" (common) Reduction in the catalytic activity of an enzyme due to a change in 3-D shape, and / or stability. wild type (normal) protein mutant protein TB

34. 4. Conditional loss of function (common) mutant protein misfolded degradation amino acids e.g. Temperature-sensitive (heat- sensitive) mutations. 42°C 30°C properly folded TB

35. 5. Change of function (rare) mutant protein e.g. change in specificity wild type protein converts lactose to glucose and galactose converts maltose to 2 glucose TB

36. 6. Restoration of function, reversion ("back mutation") (rare) functional mutant protein nonfunctional a second mutation TB

37. a. Same site revertants ii. others A second change at the same site results in a less harmful amino acid change, or the original amino acid. i. true revertants A second mutation restores the original DNA sequence. TB

38. b. Second site revertants (suppressors) ii. intergenic A second mutation in a different gene restores function. i. intragenic A second mutation at a different site within the same gene restores function. TB

39. Example of an intragenic suppressor a salt bridge between Lys 121 and Asp 44 is essential to protein folding. _ + Asp 44 Lys 121 A mutation that converts Lys 121 to Glu destroys protein activity. A second mutation that converts Asp 44 to His restores protein activity. Note that Asp and Glu are negatively charged and that Lys and His are positively charged. TB

40. IV. Effects of mutations on phenotype Phenotype The observable characteristics of an organism Mutations can have many different effects on phenotype. TB

41. A. Loss of enzyme activity Examples: 1. If a mutations destroys an enzyme needed for pigment formation, an albino can result. 2. If a mutation inactivates an enzyme for lactose catabolism, a microbe unable to grow on lactose will result. TB

42. B. Loss of regulatory proteins 1. inability to induce enzymes 2. inability to differentiate 3. inability to tax toward nutrients etc. etc. etc. C. Loss of structural proteins D. Mutations in tRNA or rRNA TB

43. V. Mutagens Substances that increase mutation frequency. In the lab, mutagens can be used to create mutations for genetic analysis. TB

44. Mutagens are used to increase mutation frequencies to ~10-6 to 10-7 / base pair / generation A. Mutation frequencies Spontaneous mutations occur with a frequency of about 10-9 / base pair / generation (~10-3 to 10-4 / gene / generation). TB

45. B. Types of mutagens and the mutations they cause. 1. Base analogs Compounds structurally similar to the normal DNA bases TB

46. O H CH3 N N O H Thymine Bromouracil O H Br N N O H • Bromouracil will be incorporated into DNA in place of thymine. • During DNA replication, bromouracil can mispair with guanine and cause point mutations. TB

47. 2. Alkylating agents Compounds that chemically modify DNA bases via alkylation During DNA replication modified bases mispair causing single base pair change (point) mutations. Example: dimethyl sulfate TB

48. 3. Intercalating agents ... ... intercalating agent (ethidium bromide) ... ... ... ... Chemicals that insert between DNA base pairs. DNA bases H-bonds backbone Intercalating agents lead to small deletions and insertions during DNA replication. TB

49. 4. Radiation Ultraviolet light (UV) O O CH3 CH3 H H N N N N O O Thymine dimer: two "T"s on the same strand become covalently bonded. Thymine dimers lead to various replication errors. TB

50. 1. Understand how genotype affects phenotype. 2. Define mutation. Understand the role of mutations in genetic diversity and evolution. Is chemical modification of a DNA base considered a mutation? why? 3. What is the most common cause of spontaneous mutations? What is the typical mutation frequency in most organisms? Define wildtype and mutant. 4. What is a point mutation? Understand the effects of silent, missense and nonsense mutations on protein primary structure. 5. Define deletions, insertions, frameshift mutations, inversions, and duplications. Understand how these mutations influence protein structure. 6. Be able to distinguish between the different effects of mutations on protein function. What are most common effects that mutations have on protein function? Which are rare? Understand the terms leaky mutant, conditional loss of function, temperature-sensitive mutations, back mutation, reversion, revertants (know the different types), intragenic and intergenic suppression. 7. Describe how a mutation might change the substrate specificity of an enzyme. 8. In general how do mutations affect phenotype? 9. In genetics, what is main use of mutagens? How do they affect mutation freq? Describe how base pair analogs, alkylating agents, intercalating agents and UV radiation lead to mutations. Know the examples! What is a thymine dimer?