VIROLOGY

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VIROLOGY (viruses and non-chromosomal genetic
elements)
VIRAL GENETICS
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VIRAL GENETICS
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-modificati
on CRISP/Cas system
The lytic and lysogenic development cycle,
immunityTransduction
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TYPES OF MUTATION single nucleotide
replacement transition or transversion
misssense, nonsense or silent insertion
/deletion of nucleotidesrecombination genomic
mutations translocations inversions deletions
duplications
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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
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MUTATION RATES
G size of genome (bp) Ge size of encoding
genomemb 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
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MUTATION RATES
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MUTATION RATES
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MUTATION OUTCOMES
R.Sanjua, et al. (2004)The distribution of
fitness effects caused by single-nucleotide
substitutions in an RNA virus (VSV) PNAS, 101,
83968401
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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
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REASSORTMENT of viruses with segmented genome
Opportunities for the development of vaccines
using the reassortment of influenza virus genome
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VIRAL GENETICS
PHENOTYPIC MIXING
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VIRAL GENETICS
PHENOTYPIC MIXING
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VIRAL GENETICS
PHENOTYPIC MIXING
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VIRAL GENETICS
PHENOTYPIC MIXING
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VIRAL GENETICS
Helper viruses
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CHIMERIC VIRUS-LIKE PARTICLES
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VIRAL GENETICS
Interference
The defective particles compete for the coat
proteins and inhibit the replication
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DNADNA hybridization (Southern blotting)
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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
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DNA zonde K
DNA zonde S
Membrane Treatment - hybridization with a probe S
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
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What makes up the Ad 12 genome 3'-end "excess"
sequence?
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VIRAL GENETICS
Restriction - modification
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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
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Novel approaches to genome modification
CRISP-Cas
Mali P. et al. RNA-Guided Human Genome
Engineering via Cas9. Science, V339, p. 824,
2013
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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)

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VIRAL GENETICS
Lysis / Lysogeny
Strategy Choice of the lphage replication
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VIRAL GENETICS
Lysis / Lysogeny
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VIRAL GENETICS
Genetic map of the lambda (l) phage
http//202.204.115.67/jpkch/jpkch/2008/wswx/chapte
r209.htm
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Virulence / Lysogeny
VIRAL GENETICS
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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 and ligase
• Transcription - immediate early, delayed early,
  late genes
• Replication - Q first, then rolling circle
  mechanism, specific cleavage in cos sequences,
  the separation of the sticky ends, assembling of
  phage
• Lysis of bacterial cell

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cos site nucleotide sequence of the l phage
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Lambda (l) phage replication
teta (Q) mechanism of DNA replication
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VIRAL GENETICS
THE EARLY STAGE OF INFECTION - A CHOICE

• Weak transcription from PL and PR.
• Antitermination protein N that interacts with RNA
  polymerase and promotes transcription in both
  directions is formed. Cro regulatory protein that
  promotes transkription of PR is formed.
• 2. N promotes CIII (CII stabilizer) PL as well
  as CII (CI stimulator) O, P, (DNA synthesis, ?
  mechanism), Q gene transcription PR

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VIRAL GENETICS
THE EARLY STAGE OF INFECTION - A CHOICE
http//biology.bard.edu/ferguson/course/bio404/Lec
ture_08.pdf
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VIRAL GENETICS
THE EARLY STAGE OF INFECTION - A CHOICE
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Virusu genetika
Choice - INTEGRATION
LYSOGENY. CII activates the PRE (CI synthesis
starts) and PI (integrase). Formed CI, which
extorts Cro from PL and PR, activates PRM Int
promotes attP and attB interaction and a fusion
of DNA of phage with the DNA of bacteria.
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Choice - INTEGRATION
VIRAL GENETICS
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Choice - INTEGRATION
VIRAL GENETICS
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VIRAL GENETICS
Choice - INTEGRATION
att site nucleotide sequence of the l phage
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VIRAL GENETICS
Choice - INTEGRATION
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VIRAL GENETICS
Choice - INTEGRATION
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VIRAL GENETICS
Choice - INTEGRATION

• Lysogenic cells
• Contain l 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
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VIRAL GENETICS
Gene expression in prophage
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VIRAL GENETICS
INDUCTION
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Choice LYTIC CYCLE
VIRAL GENETICS
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Lambda (l) phage replication
DNA replication, rolling circle mechanism
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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.
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GENETIC SWITCH
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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
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GENETIC SWITCH
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• OTHER E. coli LYSOGENE PHAGES
• l 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

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VIRAL GENETICS

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

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SPECIFIC TRANSDUCTION
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SPECIFIC TRANSDUCTION
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NON-SPECIFIC (GENERAL) TRANSDUCTION
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NON-SPECIFIC (GENERAL) TRANSDUCTION
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NON-SPECIFIC (GENERAL) TRANSDUCTION
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NON-SPECIFIC (GENERAL) TRANSDUCTION
http//bio.classes.ucsc.edu/bio105l/EXERCISES/P1/m
asters.pdf