VIROLOGY (viruses and non-chromosomal genetic elements) VIRAL GENETICS.
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Transcript of VIROLOGY (viruses and non-chromosomal genetic elements) VIRAL GENETICS.
Mutation types :Biochemical characterization phenotypic expression
MUTATION FREQUENCIES OF VIRUSES Interaction between viruses and
between viruses and cellsphenotypic mixing
ReasortimentsHelper virusesInterference
restriction-modificationCRISP/Cas system
The lytic and lysogenic development cycle, immunityTransduction
VIRAL GENETICS
TYPES OF MUTATION:
single nucleotide replacement : transition or transversion misssense, nonsense or silent
insertion /deletion of nucleotidesrecombination genomic mutations:
translocations inversions deletions duplications
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 sup D, E, F, P tRNS
amber UAG ser, glu, tyr, leuochre UAA (UCG) (CAA) (UAU) (UUG)opal UGA
J.W. Drake, B. Charlesworth, D. Charlesworth, J. F. CrowRates of Spontaneous MutationGenetics, Vol. 148, 1667-1686, 1998
MUTATION RATES
G – size of genome (bp); Ge – size of encoding genome;b – mutation rate per bp in a replication cycleg – mutation rate per genome in a replication cycleeg – mutation rate per genome equivalent encoding replication in a replication cycle
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
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
REASSORTMENTof viruses with segmented genome
Opportunities for the development of vaccines using the reassortment of influenza virus genome
Interference
The defective particles compete for the coat proteins and inhibit the replication
VIRAL GENETICS
From infected cells purified DNA Virion DNA
DNA zonde K DNA zonde S
Membrane Treatment - hybridization with a probe K
Ad12 5’-gala KpnI fragments, 589 b.p.
Virion DNA From infected cells purified DNA
DNA zonde K DNA 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.
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
Mali P. et al. RNA-Guided Human Genome Engineering via Cas9. Science, V339, p. 824, 2013
Novel approaches to genome modification
CRISP-Cas
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
Genetic map of the lambda () phage
VIRAL GENETICS
http://202.204.115.67/jpkch/jpkch/2008/wswx/chapter%209.htm
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
1. 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}
VIRAL GENETICS
THE EARLY STAGE OF INFECTION - A CHOICE
VIRAL GENETICS
THE EARLY STAGE OF INFECTION - A CHOICE
http://biology.bard.edu/ferguson/course/bio404/Lecture_08.pdf
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.
Vīrusu ģenētika
Choice - INTEGRATION
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 - INTEGRATIONVIRAL GENETICS
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.
VIRAL GENETICS
Choice – LYTIC CYCLE
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
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
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