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VIR OLOGY ( viruses and non-chromosomal genetic elements)

VIR OLOGY ( viruses and non-chromosomal genetic elements). VIRAL GENETICS. VIRAL GENETICS. Mutation types : Biochemical characterization phenotypic expression MUTATION FREQUENCIES OF VIRUSES Interaction between viruses and between viruses and cells phenotypic mixing Reasortiments

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VIR OLOGY ( viruses and non-chromosomal genetic elements)

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

  2. VIRAL GENETICS • Mutation types : • Biochemical characterization • phenotypic expression • MUTATION FREQUENCIES OF VIRUSES Interactionbetweenvirusesand • betweenvirusesandcells • phenotypicmixing • Reasortiments • Helperviruses • Interference • restriction-modification • CRISP/Cassystem The lytic and lysogenic development cycle, immunityTransduction

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

  4. VIRAL GENETICS Zero (silent) mutations: inactivating of the gene (nonsense, missense) nonsense suppression E.coli sup D, E, F, P tRNS amber UAG ser, glu, tyr, leuochre UAA (UCG) (CAA) (UAU) (UUG)opal UGA Temperature sensitivity (ts) mutation: conditionally lethal (missense) Host range mutations Plaque morphology, enzyme resistance mutations; “hot" mutants, attenuated mutants

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

  6. MUTATION RATES

  7. MUTATION RATES

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

  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. VIRAL GENETICS PHENOTYPIC MIXING

  12. VIRAL GENETICS PHENOTYPIC MIXING

  13. VIRAL GENETICS PHENOTYPIC MIXING

  14. VIRAL GENETICS PHENOTYPIC MIXING

  15. VIRAL GENETICS Helper viruses

  16. CHIMERIC VIRUS-LIKE PARTICLES

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

  18. DNA–DNA hybridization (Southern blotting)

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

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

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

  22. VIRAL GENETICS Restriction - modification

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

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

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

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

  27. VIRAL GENETICS Lysis / Lysogeny

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

  29. Virulence / Lysogeny VIRAL GENETICS

  30. Lysis / Lysogeny VIRAL GENETICS • Early stages of the l infection: • Adsorption to the cell receptor (maltose transport protein) • DNA injection, cos sequence –the union of the sticky ends andligase • Transcription - immediate early, delayed early, late genes • Replication - Q first, then rolling circle mechanism, specific cleavagein cos sequences, the separationof thesticky ends, assembling of phage • Lysis of bacterial cell

  31. cos site nucleotide sequence of the lphage

  32. Lambda (l) phage replication teta (Q) mechanism of DNAreplication

  33. VIRAL GENETICS THE EARLY STAGE OF INFECTION - A CHOICE • Weaktranscriptionfrom PLandPR. • AntiterminationproteinN thatinteractswith RNA polymeraseandpromotestranscriptioninbothdirectionsisformed.Croregulatoryproteinthatpromotestranskriptionof PRisformed. • 2. N promotes CIII (CII stabilizer) {PL}; aswellas CII (CI stimulator) O, P, (DNA synthesis, mechanism), Q genetranscription {PR}

  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. Vīrusu ģenētika Choice - INTEGRATION LYSOGENY. CII activates the PRE (CI synthesis starts) and PI (integrase). Formed CI, which extorts Cro from PL and PR, activates PRMInt promotes attP and attB interaction and a fusion of DNA of phage with the DNA of bacteria.

  37. Choice - INTEGRATION VIRAL GENETICS

  38. Choice - INTEGRATION VIRAL GENETICS

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

  40. VIRAL GENETICS Choice - INTEGRATION

  41. VIRAL GENETICS Choice - INTEGRATION

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

  43. VIRAL GENETICS Gene expression in prophage

  44. VIRAL GENETICS INDUCTION

  45. Choice – LYTIC CYCLE VIRAL GENETICS

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

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

  48. GENETIC SWITCH

  49. GENETIC SWITCH 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

  50. GENETIC SWITCH

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