1. VIROLOGY (viruses and non-chromosomal genetic elements) VIRAL GENETICS

2. Mutation types : Biochemical characterization phenotypic expression MUTATION FREQUENCIES OF VIRUSES Interaction between viruses and between viruses and cells phenotypic mixing Reasortiments Helper viruses Interference restriction-modification CRISP/Cas system The lytic and lysogenic development cycle, immunity Transduction VIRAL GENETICS

3. TYPES OF MUTATION: single nucleotide replacement : transition or transversion misssense, nonsense or silent insertion /deletion of nucleotides recombination genomic mutations: translocations inversions deletions duplications

4. VIRAL GENETICS Zero (silent) mutations: inactivating of the gene (nonsense, missense) nonsense suppression Temperature sensitivity (ts) mutation: conditionally lethal (missense) Host range mutations Plaque morphology, enzyme resistance mutations; “hot" mutants, attenuated mutants E.coli supD, E, F, P tRNS amberUAG ser, glu, tyr, leu ochreUAA (UCG) (CAA) (UAU) (UUG) opalUGA

5. J.W. Drake, B. Charlesworth, D. Charlesworth, J. F. Crow Rates of Spontaneous Mutation Genetics, Vol. 148, 1667-1686, 1998 MUTATION RATES G – size of genome (bp); G e – size of encoding genome;  b – mutation rate per bp in a replication cycle  g – mutation rate per genome in a replication cycle  eg – mutation rate per genome equivalent encoding replication in a replication cycle

6. MUTATION RATES

8. R.Sanjua, et al. (2004)The distribution of fitness effects caused by single- nucleotide substitutions in an RNA virus (VSV) PNAS, 101, 8396–8401 MUTATION OUTCOMES

9. HOMOLOGOUS RECOMBINATION The mechanism of copy choice in the replication of viruses The mechanism of strand exchange in replication of eucariot cells Mapping genomes, Marker rescue, Inclusion of host cell genome fragments into virus

10. REASSORTMENT of viruses with segmented genome Opportunities for the development of vaccines using the reassortment of influenza virus genome

11. PHENOTYPIC MIXING VIRAL GENETICS

12. PHENOTYPIC MIXING

13. VIRAL GENETICS PHENOTYPIC MIXING

14. VIRAL GENETICS

15. Helper viruses VIRAL GENETICS

16. CHIMERIC VIRUS-LIKE PARTICLES

17. Interference The defective particles compete for the coat proteins and inhibit the replication VIRAL GENETICS

18. DNA–DNA hybridization (Southern blotting)

19. From infected cells purified DNA Virion DNA DNA zonde KDNA zonde S Membrane Treatment - hybridization with a probe K Ad12 5’-gala KpnI fragments, 589 b.p.

20. Virion DNA From infected cells purified DNA DNA zonde KDNA zonde S Membrane Treatment - hybridization with a probe S + 273 b.p. no Ad12 33845 - 34118 2x (+ 273 b.p. no Ad12 33845 – 34118) 3x (+ 273 b.p. no Ad12 33845 – 34118) Ad12 3’-gala SacI fragments, 615 b.p.

21. What makes up the Ad 12 genome 3'-end "excess" sequence?

22. Restriction - modification VIRAL GENETICS

23. CRISPR (clustered regularly interspaced short palindromic repeat) Cas (CRISPR-associated) genes, CRISPR-based adaptive immune systems Terns and Terns, 2011 Bacterial defence against viral infections CRISP-Cas

24. Mali P. et al. RNA-Guided Human Genome Engineering via Cas9. Science, V339, p. 824, 2013 Novel approaches to genome modification CRISP-Cas

25. Transfection Protein unprotected viral delivery of genetic material in the cell (electroporation, liposomes, hydroxyapatite) Transduction Gene transfer with the help of virus Specialized ( phage, gal, bio operons) Non-specific (P1,P22 phage, 40-50 kbp. genomic fragments) VIRAL GENETICS

26. Lysis / Lysogeny Strategy Choice of the –phage replication VIRAL GENETICS

27. Lysis / Lysogeny VIRAL GENETICS

28. Genetic map of the lambda ( ) phage VIRAL GENETICS http://202.204.115.67/jpkch/jpkch/2008/wswx/chapter%209.htm

29. Virulence / Lysogeny VIRAL GENETICS

30. Lysis / Lysogeny Early stages of the infection: 1.Adsorption to the cell receptor (maltose transport protein) 2.DNA injection, cos sequence – the union of the sticky ends and ligase 3.Transcription - immediate early, delayed early, late genes 4.Replication -  first, then rolling circle mechanism, specific cleavage in cos sequences, the separation of the sticky ends, assembling of phage 5.Lysis of bacterial cell VIRAL GENETICS

31. cos site nucleotide sequence of the phage

32. teta (  ) mechanism of DNA replication Lambda ( ) phage replication

33. 1.Weak transcription from P L and P R. Antitermination protein N that interacts with RNA polymerase and promotes transcription in both directions is formed. Cro regulatory protein that promotes transkription of P R is formed. 2. N promotes CIII (CII stabilizer) {PL}; as well as CII (CI stimulator) O, P, (DNA synthesis,  mechanism), Q gene transcription {PR} VIRAL GENETICS THE EARLY STAGE OF INFECTION - A CHOICE

34. VIRAL GENETICS THE EARLY STAGE OF INFECTION - A CHOICE http://biology.bard.edu/ferguson/course/bio404/Lecture_08.pdf

35. VIRAL GENETICS THE EARLY STAGE OF INFECTION - A CHOICE

36. LYSOGENY. CII activates the P RE (CI synthesis starts) and P I (integrase). Formed CI, which extorts Cro from P L and P R, activates P RM Int promotes attP and attB interaction and a fusion of DNA of phage with the DNA of bacteria. Vīrusu ģenētika Choice - INTEGRATION

37. VIRAL GENETICS

38. Choice - INTEGRATION VIRAL GENETICS

39. att site nucleotide sequence of the  phage Choice - INTEGRATION VIRAL GENETICS

40. Choice - INTEGRATION VIRAL GENETICS

41. Choice - INTEGRATION VIRAL GENETICS

42. Lysogenic cells: Contain phage genome integrated in the chromosome, the inactive state Immune to infection with the closely related phages Prophages can be activated by a variety of factors (UV, mutagenic, adverse environmental conditions) PROPHAGES Choice - INTEGRATION VIRAL GENETICS

43. Gene expression in prophage VIRAL GENETICS

44. INDUCTION VIRAL GENETICS

45. Choice – LYTIC CYCLE

46. DNA replication, rolling circle mechanism Lambda ( ) phage replication

47. LYSE. If there is enough Cro, CI synthesis is blocked (first), but later the P L and P R in general. Decisive role is played by P R’ in context with Q antitermination, that runs a phage capcid protein and lysis protein synthesis. DNA synthesis moves from  to the rolling circle mechanism. VIRAL GENETICS Choice – LYTIC CYCLE

48. GENETIC SWITCH

49. O1, 2, 3 sequences are similar but not identical; CI has the best affinity to O1, the weakest – to O3. Cro - best to the O3. In average, CI binds to the operator sites approx. 5 times more efficient than the Cro GENETIC SWITCH

51. OTHER E. coli LYSOGENE PHAGES phage-like – phages 21 f80, 82, 424, 434, crossimmunity; P1, the largest lysogene phage, 97 kbp. DNA rarely integrates - more present in plasmid form of Cre protein and loxP recombination site, 40% of the DNA filling required for aggregation, non-specific transduction; Mu, 42 kbp. DNA, at the ends of phage genome – bacteria sequence, effective transposon, mutation induction; P2, 33,2 kbp. DNA, approx. 10 integration sites in the genome of bacteria, lysis is rare. P2 encoded capsid proteins can be used for P4 (11 kpb. DNA) incapsidation, which in P2 free cells are in multicopy plasmid form

52. TRANSDUCTION Gene transfer with the help of LYSOGENE virus Specialized ( phage, gal, bio operons) Non-specific (P2 phage, 40-50 KBP. genomic fragments) VIRAL GENETICS

53. SPECIFIC TRANSDUCTION

55. NON-SPECIFIC (GENERAL) TRANSDUCTION

58. http://bio.classes.ucsc.e du/bio105l/EXERCISES/ P1/masters.pdf NON-SPECIFIC (GENERAL) TRANSDUCTION