1. IN THE NAME OF GOD Islamic Azad University Falavarjan Branch Falavarjan Branch School of Biological Sciences Department of Microbiology

2. Microbial Genetics By: Keivan Beheshti Maal

3. Genetic Mutations

4. Mutations: Changes in DNA Why are mutations in DNA important to humans? 2 types of mutations: Spontaneous Mutations: –occur in the natural environment without the addition of mutagens (agents that cause mutations) –Occur randomly and spontaneously Induced Mutations: –Mutations that are created by the addition of mutagens

5. Spontaneous Mutations Two types: 1. Base substitutions 2. Frameshift mutations

6. Spontaneous Mutations: Base substitutions Most common type of substitution Mistake during DNA replication, incorrect base incorporated into DNA Three types: 1. Silent mutation: no effect on protein (remember- several codons code for the same amino acid) 2. Missense mutation: codon has changed and different amino acid is incorporated 3. Nonsense mutation: codon has changed to a stop codon

7. Figure 8.15 - Overview

8. Spontaneous Mutation: Base-pair deletion or insertion Insert or delete a nucleotide- very disastrous Shifts codons of DNA when transcribed into RNA (also called frameshift mutation) All nucleotides downstream of mutation will be grouped into improper codons, and wrong amino acids will be added Protein will be non-functional

9. Mutation Mutation –Change in the base sequence of DNA may cause change in the product coded by the gene –Beneficial –Lethal –Neutral Occur commonly Degeneracy

10. Mutations Types of mutations –Base substitution (point mutation) AT substituted for CG mRNA carries incorrect base Translation –Insertion of incorrect amino acid into protein –Missense mutation, nonsense mutation, frame shift mutation, and spontaneous mutations

11. Base substitution

12. Mutations Normal –No mutations –DNA strand properly transcribed by mRNA –Correct sequence of amino acids for protein

13. Mutations Mis sense mutation –Base substitution results in an amino acid substitution in protein –Sickle cell anemia A to T Glutamic acid to valine Hb shape changed during low oxygen

14. Mutations Non sense mutation –Base substitution creates a nonsense or stop codon –Protein is not produced –Only a fragment of protein is produced

15. Mutations Frame shift mutation –One or a few nucleotide pairs are deleted or inserted in the DNA –Shifts the translation reading frame –Almost always result in a long stretch of altered amino acids –Inactive protein

16. Mutations Insertion of extra bases into a gene –Huntington's disease Spontaneous mutations –Occur occasionally in DNA replication Mutagens –Chemically of physically alters DNA and effects a change is called a mutagen Radiation, ultraviolet light

17. Mutagens Chemical Mutagens –Nitrous acid Converts adenine (A) to a form that doesn’t bind with thymine (T), but instead binds with cytosine (C) Alters base pair on DNA, works on random locations

18. Mutagens Chemical mutagens (cont) –Nucleoside analogs Structurally similar to normal nitrogenous bases 2 - aminopurine –Adenine 5 – bromouracil –Thymine analog –Will bind with guanine

19. Mutagens Chemical mutagens (cont) –During replication analogs cause base pairing mistakes –Antiviral and antitumor drugs AZT (azidothymi dine)

20. Mutagens Chemical mutagens (cont) –Other chemicals cause deletions, frameshifts, or insertions Benzyprene – present in smoke and soot –Frameshift Aflatoxin – Aspergillus flavus –Frameshift

21. Mutagens Radiation mutagens –X – rays –Gamma rays –Ultraviolet Forms covalent bond between certain bases Thymine dimers –Death of damage to cell Light repair enzymes –Photolyases Use visible light energy to separate dimer

22. Mutagens Ultraviolet damage –Nucleotide excision repair Enzymes cut out distorted thymines Creates gap Gap is filled with newly synthesized DNA DNA ligase joins strand to surrounding backbone

23. Mutation frequency Mutation rate –Probability that a gene will mutate when a cell divides –Expressed in power of 10 10 -4 mutation rate (1 in 10,000 chance of mutation) 10 -6 ( 1 in 1,000,000) –Mutagens Increase spontaneous mutation by 10 – 10,000 times 10 -6 becomes 10 -3 to 10 -5

24. Identifying Mutants Positive (direct) selection –Detection of mutant cells by rejection of unmutated parent cells Penicillin in agar Unmutated parental cell will not grow Only mutated cells grow

25. Identifying Mutants Negative (indirect) selection –Replica plating technique

26. Replica Plating

27. Replica plating Auxotroph –A mutant microorganism having a nutritional requirement that is absent in the parent.

28. Identifying Chemical Carcinogens Carcinogen –A substance found to cause cancer in animals –Often mutagens are carcinogens as well –Previously used animal testing Time consuming Expensive

29. Ames test Ames test utilizes bacteria to act as carcinogen indicator Based on observation that exposure to mutant bacteria to mutagenic substance may reverse effect of the original mutation

30. Ames test These are called reversions –Back mutations Measures the reversion of Salmonella –Auxotrophs Have lost there ability to synthesize histidine (his - ) (his + ) bacteria have ability to synthesize histidine 90% of substances that cause reversion have been shown to be carcinogens

31. Ames Test

32. Induced Mutation Mutations are induced by either certain chemical mutagens or physical mutagens Sometimes scientists intentionally mutate DNA to study it

33. Physical Mutagens: Radiation

34. Repair of thymine dimers

35. Effects of Mutation

37. Gene Transfer

38. Three methods of horizontal gene transfer: 1. Transformation 2. Transduction 3. Conjugation

39. Gene Transfer Vertical Gene Transfer= When genes are passed from an organism to it’s offspring Horizontal Gene Transfer= Occurs between bacteria

41. Horizontal Gene Transfer Two types of cells: 1. Donor: transfers DNA to recipient 2. Recipient: receives the DNA

42. Genetic transfer and recombination Eukaryotes –Meiosis Prophase I Prokaryotes –Numerous different ways

43. Genetic Transfer and Recombination Vertical gene transfer –Genetic information passed from an organism to its offspring Plants and animals Horizontal gene transfer –Bacteria transfer genetic information form one organism to another in the same generation –Genetic information passed laterally

44. Horizontal Gene Transfer Horizontal gene transfer –Donor cell Organism gives up its entire DNA Part goes to recipient cell Part is degraded by cellular enzymes –Recipient cell Receives portion of donor cells DNA Incorporates donor DNA into its own DNA –Recombinant DNA –Less than 1 % of population

45. Transformation Genes transferred from one bacterium to another in solution –Naked DNA –Discovered by Griffith –Used Streptococcus pneumoniae Two strains –Virulent (pathologic) strain Had a polysaccharide capsule resists phagocytosis –Avirulent (non- pathogenic) strain Lacked a capsule

46. Griffith’s Experiment

47. Transformation Bacteria after cell death and lysis could release DNA into environment Recipient cell can take up DNA fragments and incorporate into their own DNA –Resulting in a hybrid (recombinant cell) –Recombinant cell must be competent Able to alter cell wall to allow DNA (large molecule) to enter Bacillus, Haemophilus, Neisseria, Acinetobacter, and some Staph and Strep

48. Genetic Transformation

49. Conjugation Conjugation –Involves plasmid Circular piece of DNA Replicates independent of chromosome Non essential for growth genes –Requires cell to cell contact –Opposite mating type Donor cell carries plasmid Recipient cell lacks plasmid

50. Conjugation Gram positive –Sticky surfaces cause bacteria to come in contact with one another Gram negative –Utilize sex pili

51. Conjugation E coli model –F factor plasmid Fertility factor Donors (F + ) Recipients (F - ) –Converted to (F + ) –F + factor integrated into chromosome Becomes Hfr (high frequency of recombination) cell

52. Conjugation Hfr conjugates with F - cell Chromosomal strand replicates and transferred to recipient Incomplete transfer of donor DNA Recipient integrates new DNA –Acquires new versions of chromosome –Remains F - cell

53. Conjugation Minutes and conjugation –Identify locations of various genes –Hfr His, pro, thr, leu, and F (+) –F( - ) His, pro, thr, leu, and F( - )

55. Conjugation In some cells carrying F factors, the F factor integrates into the host chromosome Now called Hfr cell Conjugation between Hfr and F - –Chromosome replicates –Transferred to F - cell –Usually chromosome breaks off before completely transferred –Generally remains F - because does not receive F factor

57. Transduction in Bacteria Transfer of bacterial DNA transferred via bacteriophage Bacteriophage –Virus that infects bacteria

58. Transduction Steps of transduction –1- bacteriophage infects donor bacterial cell –2- Phage DNA and proteins, and bacterial chromosome is broken into pieces

59. Transduction Steps of transduction –3- during phage reassembly, bacterial DNA incorporated in capsid of bacteriophage –4 – donor cell lysis releasing new bacteriophage particles

60. Transduction Steps in transduction –5- phage carrying donor DNA infects new recipient cell –6- recombination can occur Producing bacteria with genotype different than donor and recipient

61. Transduction Generalized transduction –Previously explained Specialized transduction –Only certain genes are transferred –i.e. phage codes for toxins to be produced Cornybacterium diphtheriae – diphtheria toxin Streptococcus pyogenes – erythrogenic toxin Escherichia coli – Shiga toxin (hemorrhagic diarrhea)

62. Plasmids Plasmids –Self replicating rings of DNA –1-5% size of chromosomal DNA –Non – essential genes –Conjugative plasmid F factor –Dissimilation plasmids Code for enzymes to breakdown unusual sugars and hydrocarbons Help in survival of unusual environments

63. Plasmids Other plasmids –Toxins (Anthrax, tetanus, Staph) –Bacterial attachment –Bacteriocins Toxic proteins that kill other bacteria –Resistance factors (R factors) Resistance to antibiotics, heavy metals, cellular toxins

64. Plasmids Resistance factors –Two groups RTF – resistance transfer factor –Includes genes for plasmid replication and conjugation r-determinant –Resistance genes –Codes for production of enzymes that inactivate drugs or toxic substances Bacteria can conjugate and transfer plasmids between species –Neisseria Penicillinase resists penicillin

65. R factor Plasmids

66. R plasmid Resistance plasmid- confer antibiotic resistance Two parts: 1. Resistance genes (R genes) 2. Resistance transfer factor (RTF)

67. Figure 8.22