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EXAM II Study Guide Virus/Phage + DNA + Immunology + Pathogenicity
VIRUS/PHAGE
Define viruses and phages. Differentiate Viroid, Prion, GTA
VirusGenetic element containing either RNA/DNA surrounded by a protein capsid and that replicates
only inside host cells
Phage Virus that infects only bacterial cells
Viroid Naked molecules of RNA important infectious disease agents in plants
PrionAn infectious agent composed only of protein that is responsible for causing a variety of
spongiform encephalopathies (e.g., scrapie).
GTA* Phage-like element produced by several bacteria that mediates horizontal gene transfer
*Gene Transfer Agent
Which of these is not UV sensitive?
Estimate the abundance of viruses in aquatic systems and postulate the significance of these pathogens in
terms of controlling primary productivity and horizontal gene transfer.
A. 106- 10
9ml
-1in seawater
B. Control 1 productivity: Each infection has the potential to introduce new genetic information into an
organism or progeny virus, thereby driving the evolution of both host and viral assemblages
a. Viruses kill ~20% of biomass/day
Contrast viruses with bacteria in terms of genome structure, metabolic activity, life cycle and size (in nm).
Bacteria Virus
Cell wall Peptidoglycan/LPS No cell wall; protein coat
Reproduction Fission; asexual Lysogenic; lyticCellular organization Complex Simple
Ribosome + -
Enzymes + Some
DNA/RNA Both DNA/RNA DNA ORRNA
Life cycle Some obligate IC parasites Obligate intracellular parasites
Size 1-5 m 10-400 nm
UV sensitivity: Bacteriophage > Bacterium > Gene > Prions
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Viral Genome
Structureo Genetic information may be coded as DNA or RNA and in double-stranded or single-stranded form.
o The viral genome may contain exotic bases.
Replication
o Takes place only after successful infection of an appropriate host.
o Proceeds when the injected viral genome subverts normal replicative processes of the host,
producing new virus particles.
Bacteriophage types
o May be discerned by their mode of propagation.
o Lytic phages quickly produce many copies of themselves as they kill the host.
o Temperate phages can lie seemingly dormant in the host (prophage state), timing replication of
prophage genetic material to replication of the host cell. Various activation signals trigger the
prophage to enter a lytic cycle, resulting in host death and the release of new phages.
Sketch these viral morphologies:
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Virus Morphology
TMV (Tobacco Mosaic Virus) Helical
T4 Phage Complex
HIV Enveloped
HSV Enveloped
Polyomavirus Icosahedral
Explain the role of reverse transcriptase, RNA replicase, holin and endolysin in the life cycle of viruses and
phages.
Enzyme Role Type of Phage
Reverse transcriptase Generation of cDNA from RNA; integration of retrovirus into host Retrovirus
RNA Replicase Catalyzes replication of phage RNA in host Lysogenesis
Holin Host lysis; produces lesions in cell membrane T4 phage protein
Endolysin Attacks peptidoglycan T4 phage protein
Contrast the biology of common phages:
Virus Genome Transmission
Cycle
Mode of Transmission Morbidity Mortality
Polio
+ ssRNA
Non-human
host
Fecal - oral
Endemic Pakistan
< 1%
WNV
+ ssRNA
Vector borne
zoonotic
Arthropod vector
Differential
4%
Ebola
- ssRNA
Zoonotic
Contact
Endemic (Africa)
High (5090%)
influenza
- ssRNA
sgmtd
Zoonotic
Aerosols
Humidity
50,000/year; seasonal
Adults: 5-10%
Kids: 20-30%
HIV
retro
Human
STD
---
100% w/o therapy
HSV
dsDNA
Human
STD; mother fetus
5060 million US
Low
Membrane = Peplomeres (spikes)
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smallpox
dsDNA
Zoonotic
Contact
eradicated
High
Discuss how the above examples maintain virulence and evade specific immunity through antigenic drift
and shift.
A. Antigenic drift: natural mutagenesis of viruses as viruses replicate results in altered antigenic profile
B. Antigenic shift: segmented genome of select viruses (influenza) allows reassortment of different
strains; occurs through infection of same host cells and new-intermixed viral genome
Sketch lytic and lysogenic cycles using lamba phage as an example.
Lytic: phage life cycle that culminates with host cell bursting, releasing virions
Virulent phages: phages that lyse their host during the reproductive cycle i.e. T4Phage
(1) Early mRNA synthesis (2) Synthesis of proteins that (3) Enable T4 to take over host cell (4) Phage
DNA replication (5) Late mRNA synthesis (6) encode capsid proteins and other proteins needed for
phage assembly
Lysogenic: Nonlytic relationship b/t phage-host; integration of phage genome into host DNA
Defined terms:
a. Prophageintegrated phage genome (lysogeny)
b. Temperate phagephages able to establish lysogeny
c. InductionAn event in the life cycle of some viruses (e.g., temperate bacteriophage) that results in
the provirus initiating synthesis of mature virions and entering the lytic cycle.
Explain why stress triggers induction events
Triggered by drop in levels of lambda repressor caused by exposure to UV light and chemicals that
cause DNA damage
a.
Catalyzed by excisionase: binds integrase which enables integrase to reverse integration process
DNA/RNA
Describe these key experiments that elucidated the central dogma of molecular biology in terms of
objective, methods, results & conclusions:
a. Gliffith (1928), Avery (1944)
i. Griffith: Transformation; Dead S (infectious) + Live R strain injected Mouse dies (live S strain
isolated); conclusion: hereditary element can be transformed from dead to live cells
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ii. Avery: Protein not transforming agent; R & S culture PRO/lipid removed transforming
element had DNA chemistry (destroyed by DNAse)
b. Heshey & Chase (1952)
i. DNA injected by viruses; bacteriophage infected cells had radioisotope labeled DNA (phosphorus) +
protein (sulfur) agitation/centrifugation revealed only P isotope still present in cells
c. Fraenkel-Conrat (1957)
i.
NA (RNA), not protein coat is infective agent; Tobacco infected with hybrid: TMV protein coat andHRV RNA observed lesions characteristic of HRV
d. Watson & Crick and Meselson & Stahl (1958)
i. Double helix 2 strands (parental & complementary)
ii. Meselson & Stahl radioisotope labeled DNA revealed semi-conservative replication in DNA
Identify the sugar in nucleic acids and features associated with I ', 2', 3' and 5' positions
Explain difference In strength of G-C pairing versus A-T.
A. GC capable of 3 H bonding
Contrast the structure(s) and function(s) of DNA & RNA including mRNA, tRNA & rRNA
Describe how these enzymes: gyrase, DNA polymerase, helicase and ligase, contribute to DNA replication
A. Gyrase: Remove supercoil
B.
DNA Polymerase: polymerizes dNTP in 5 3C. Helicase: Unwinds double-helix
D. Ligase: Fuses okazaki fragments
Describe polypetide synthesis using the terms transcription and translation.
RNA polymerase (a large multi-subunit enzyme) is responsible for the synthesis of RNA
The core enzyme (2, , ' subunits) catalyzes RNA synthesis
The sigma subunit () helps the core enzyme bind DNA at the appropriate site
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sequences centered at -35 and -10 BP before the transcription starting point are important in directing
RNA polymerase to the promoter
Prokaryotic mRNA can code for one polypeptide (monogenic) or many polypeptides (polygenic)
Leader sequences consist of 25 to 150 bases at the 5 end of the mRNA, and precede the initiation
codon
Spacer regions separate the segments that code for individual polypeptides in polygenic mRNAs
Trailer regions are found at the 3 end of the mRNA after the last termination codon
Identify where sigma factors bind to activate transcription and explain their role stress response and
antibiotic resistance.
A. Binds to RNA polymerase @ specific promoter sequence; each factor binds to unique sequence
Sketch a biphasic growth curve using the example of diauxic growth observed when E. coli is grown on
glucose and lactose as sole carbon sources.
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Discuss transcription regulation in this lac operon example using these terms: cAMP, promoter,
repressor, polygenic and inducer.
Discuss how eukaryotes and bacteria generate genotype and phenotype diversity.
Explain degeneracy and wobble in the genetic code and how point mutations and frame-shift mutations in
DNA alter polypeptide sequences or, in the case of silent mutations, don't.
Define wild type, prototrophs and auxotrophs to explain how the Ames test works and how horizontal gene
transfer (MGT) was quantified Lederberg and Zinder (1951).
a. Auxotrophrequires an organic growth factor
b.
Prototrophcan synthesize all necessary growth factors
Using the terms pili, cell-to-cell contact, prophage, lytic phase and phage- like particles, contrast
conjugation, transformation, specialized transduction, generalized transduction and gene transfer agents.
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Conjugation
Define plasmids, episomes and conjugative plasmids.
PlasmidSmall replicons, double-stranded, usually circular DNA molecules; have their own origin of
replication; can exist as single copies or as multiple copies
Episome Plasmids that can exist either with or without integrating into chromosome
onjugationThe form of gene transfer and recombination in procaryotes that requires direct cell-to-cell
contact
Conjugative
Plasmids
Have genes for pili (ex F factor in E. coli); can transfer copies of themselves to other bacteria during
conjugation
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CuringElimination of plasmid; spontaneous or induced by treatments that inhibit plasmid replication but no
host cell reproduction
F FactorF plasmid; the fertility factor; a plasmid that carries genes for bacterial conjugation and makes its E.
coli host the gene donor during conjugation
eneralized
ransduction
The transfer of any part of a prokaryotic genome when the DNA fragment is packaged within a virus
capsid by mistake
HGT The process by which genes are transferred from one mature, independent organ- ism to another
Prototroph A microorganism that requires the same nutrients as most of the members of its species
Specialized
ransduction
A transduction process in which only a specific set of bacterial or archaeal genes is carried to a
recipient cell by a temperate virus
ransduction The transfer of genes between bacterial or archaeal cells by viruses.
ansformationMode of gene transfer in prokaryotes in which a piece of free DNA is taken up by a cell and stably
maintained
ranspositionThe movement of a piece of DNA around a cell's genome. transposon A mobile genetic element that
carries the genes required for transposition
Explain relationship between methylation of cytosine in viral DNA replication and restriction enzymes.
Contains hydroxymethyl-cytosine (HMC) instead of cytosine
o Synthesized by two phage encoded enzymes
Protects phage DNA from host restriction endonucleases
Restriction defends against viral infection
Contrast restriction enzymes and CRISPR as bacterial defenses against phages. Insertion Sequences
a. Short DNA sequences (6003,000 bp)
b. Carry transposease
Transposons
a. Known for carrying antibiotic resistance
b. Include conjugative
CRISPR (clustered regularly interspaced shortpalindromic repeats)
a. Bacteria andArchaea not only produce restriction endonucleases (Sections 8.6 and 11.1) that
function to destroy incoming foreign DNA, they also have an RNA-based defense program to
destroy invading DNA from viral infection and sometimes conjugation. This type of prokaryotic
immune system helps preservegenome stability and is called the CRISPR system, which stands
for clustered regularly interspaced shortpalindromic repeats
b. DNA repeats spacing between DNA complementary to foreign DNA
c. Processed to generate crRNAs
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The CRISPR region is transcribed as a whole into a long RNA molecule that is then cleaved in
the middle of each of the repeated sequences by the nuclease activity of Cas proteins. This
converts the long RNA molecule into spacer segments of small RNAs called CRISPR RNAs
(crRNAs)
2 Methods of Microbial Genetic Diversity
Mutationalteration in existing DNA sequence
DNA transfer (horizontal gene transfer)acquisition of DNA from another source
Discuss, citing Corvaglia et al. (2010), the importance of type Ill-like restriction enzymes in the virulence of S.
aureus through acquisition of multiple antibiotic resistance.
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Explain how the roles of RecA and LexA, uvrD in the SOS response.
LexA:repressor; in absence of DNA damage LexA binds to operator
RecA: inducer; DNA damage activates; cleave LexA
UvrD: helicase; removes thymine dimer
Compare structure and organization of chromosomes and genes in archaea, bacteria and eukaryotes usingthe terms intron, start amino acids, size (bp), circular/linear, haploid/diploid.
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Explain the function of 5' cap and poly A tail in eukaryotic primary transcripts.
Protect from exonuclease activity in Eukaryotes
Capping is the addition of a methylated guanine nucleotide at the 5-phosphate end of the mRNAa. The cap nucleotide is added in reverse orientation relative to the rest of the mRNA molecule and is
needed to initiate translation.
Poly A tail
a. The tail recognition sequence, AAUAAA, is located close to the 3 end of the primary transcript.
b. The poly(A) tail stabilizes mRNA and must be removed before the mRNA can be degraded.
Discuss how alternative splicing, insertion sequences, methylation and RNA interference contribute to
complexity of organisms.
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Alternative splicing: # of introns; increases w/ complexity of organism
Insertion sequences: Carry transposease (transposons); most bacteria have few; increase variability of
organisms (antibiotic resistance)
RNA interference
Explain why obligate mutualist bacteria generally have small genomes and few insertion sequences.
No selective pressure to undergo evolutionary beneficial adaptations
Describe in general terms nonspecific responses to infection.
Innate Immune System (PAMPs)
a. Cells: Macrophages + Neutrophils + NK cells
b. Proteins: Complement (opsonization; c3b) + Interferons
c. Systemic: Inflammatory + Temperature
i.
Cytokines: Proinflammatory (IL-1 + TNF- + IL-6)
Discuss why these PAMPs illicit a general immune response by binding PRRs and identify the class of
microbes associated with them: LPS, teichoic acid, peptidoglycan, chitosan, CpG DNA (methylationpatterns), SS DNA and DS DNA.
PAMPs:
Class of
MicrobesPAMP
PRR (Patern
Recognition
Receptor)
Result
Gram - LPS TLR-2/4 Phagocyte activation + inflam
Gram + Teichoic Acid CD14TLR-4 Phagocyte + inflam
Gram -/+ Peptidoglycan TLR-2; NOD1/2
Phagocyte activation + inflam; Antimicrobial
peptide production + proinflamm cytokinesFungal cell
wallChitosan TLR-6 Phagocyte activation + inflam
Bacteria
CpG DNA
(methylation
patterns
TLR-9 Phagocyte activation + inflam
Virus ss DNA TLR-7 Phagocyte activation + inflam
Virus ds DNA TLR-3 Phagocyte activation + inflam
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Describe how these PAMPs stimulate systemic and local responses by stimulating the release of
endogenous pyrogens, particularly IL-I and TNF-a, or, for viral associated PAMPs, interferon.
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Discuss evidence that interferon production interferes with viral replication.
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Contrast the three pathways of complement activation and explain the central role of C3.
Explain these effector roles of complement:
Inflammation (C3a & C5a)
Lysis (membrane attack complex
Opsonization.
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Memory B and T cells generated, which confer long-term immunity
Describe the role of macrophages, neutrophils, dendritic cells and in the immunes system.
Macrophage: phagocytosis
Neutophils: phagocytosis
Dendritic: antigen presentation
Describe two mechanisms of activating natural killer cells.
Nonspecific activation signal on target cell
Absence of MHC I (endogenous) marker on target cell for NK cells
Cell-mediated CD16 binds to Fc region of Ig (+) NK cells
Differentiate MHC class I and Il in terms of endogenous'exogenous peptides and CDS & CD4 positive cells.
Rule of 8
MHC I = CD 8+ = endogenous = T killer
MHC II = CD4+ = exogenous = T-helper
Differentiate the two arms of acquired immunity, humoral and cellular, in tenns of cells (B and helper and
cytotoxic T) involved.
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Define antigens, epitopes and haptens. Sketch the basic structure of antibodies including variable and
conserved regions and light and heavy chains.
Identify the structure and function of the five isotypes of antibodies: lgG, IgA, IgM, IgD and lgE.
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Identify those found on mature B cells, where IgM functions as the B cell receptor, and produced following
activation.
IgD
Differentiate active and passive imtiation of specific Immune response, with natural and artificial examples.
Identify the isotypes (IgG and lgA) involved in natural, passive immunity acquis'tion by the fetus and
newborns. Describe how HIV evades the immune system.
List Koch's Postulates.
Pathogen presentin every case of disease
Isolatepathogen and growin pure culture
Isolated pathogen causes diseasewhen inoculatedinto lab animal
Pathogen isolated from inoculatedanimal identical to original organism
Differentiate the terms disease and pathogen.
Contrast mutualism, commensualism and parasitism.
Macro
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Define virulence or pathogen and lethal dose 50
Differentiate opportunistic and nosocomial infections.
Differentiate vectors and fomites.
Vectorsliving organism transmits pathogen
Fomitesinanimate object that transmits pathogen
Explain the relationship between virulence, mode of transmission and reservoirs.
Virulenceextent of pathogenicity
Discuss role of probiotics in prevention of disease including the role of attachment and competition forspace in establishment of infection.
Define these pathogens in these genera as either facultative intracellular, obligate intracellular, or
extracellular: Yersinia, Chlamydia, Vibrio and Pseudomonas.
YersiniaFacultative IC
ChlamydiaObligate IC
Vibrio - EC
Psuedomonas - EC
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Compare exotoxins and endotoxins in terms of chemical nature, host responses, gram positive/negative,
heat stability and toxicity.
Contrast these exotoxin types: superantigens, type AB and cytotoxins.
Superantigens
Cause T cells to release cytokines
e.g., toxic shock syndrome
AB
diptheria
neurotoxins
target nerve tissue
e.g., botulinum toxin
enterotoxins
target intestinal mucosa
e.g., cholera toxin
cytotoxins
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target general tissues
Include hemolysin and membrane disruptors
Identify MHC and TCR bound by superantigens.
Contrast A and B proteins in AB-types.
A = active; effector
B = mediates attachment
Explain how pore-forming and phospholipases cause cytotoxicity.
Pore-forming
a. bind cholesterol
b. e.g., hemolysins, leukocidins
Phospholipases
a. destroy membrane integrity
b. e.g., gangrene toxin
Explain how siderophores and lgA proteases are virulence factors and help bacteria evade the immune
system.
Collagenase
a. Hydrolyzes collagen
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IgA protease
a. Destroys secreted antibodies
sidephores
a. Take essential nutrient, iron, from host enzymes
Capsules
a. not antigenic
b. e.g., Streptococcus pyogenes
N. gonorrheaapproaches
a. genetic variation of surface antigens
b. production of IgA proteases
Interfere with antibody-mediated opsonization
a. produce proteins that bind Fc portions of immunoglobins
b. Can bind macrophages as well as normal antibody response
c.
e.g., Streptococcus pyogenes
Describe how type Ill secretion systems are acquired as pathogenicity islands and contribute to virulence.
Present evidence that capsules and fimbriae are virulence factors for S. pneumonia and E. coli respectively.
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