Exam 4 Flashcards
Homologous recombination in Euk.
vertical gene transfer during meiosis
Homologous recombination in Prok.
horizontal gene transfer by transformation, transduction, conjugation
Genetic recombination
the physical exchange of genes between genetic elements
RecA
promotes strand invasion
Transformation
involves the uptake of naked DNA from environment
Transformation experiment
Streptococcus pneumoniae
Streptococcus pneumoniae smooth
Capsule and Pathogenic
Streptococcus pneumoniae rough
No Capsule and Nonpathogenic
Mechanism of transformation
DNA binding proteins expressed on the cell’s surface; stand cleaved by endonucleases (5-15 kilobases); while being taken up, one strand is completely degraded by exonucleases
Competency
the ability to take up exogenous DNA; Bacillus, Streptococcus, Haemophilus, Neisseria; Gram +, -, Archaea
Poorly transforming bacteria
Electrical shock; exposure to Calcium chloride
Electroporation
usually used with Gram +, and then chill them after
Streptococcus pneumoniae competency
can take up any DNA; cells must be in exp. Phase of growth; 10^7-8 bact/mL; competence factor secreted; triggers upregulation of genes involved in translation
Bacillus competency
only 20% competent after competence factors secreted
Haemophilus competency
will only take up its own DNA; an 11 base sequence repeated 1400 times must be recognized
Transduction
DNA transfer that is mediated by bacteriophage
lytic
generalized transduction
lysogenic
specialized transduction
Conjugative plasmids
relies on formation of a pili; mediates direct exchange between bacterial cells
tra region
a plasmid must have this to transfer DNA
R (resistance) plasmids
confer resistance to antibiotics and other growth inhibitors by having genes that encode for proteins that inactivate it
Virulence plasmids
carry genes that allow attachment to specific cells and/or the formation of substances that cause damge to host
Conjugation
the transfer of DNA by direct cell contact
Pilus
thin, fragile, and hard to maintain; retracts for more stability, but still hard to keep in place
Biofilm
on the rocks in a stream; a large community of cells create an EXOPOLYMER SUBSTANCE; aids in conjugation
Hfr conjugation
chromosomal genes take longer to transfer than plasmids
Transposition
recombination due to themovement of DNA sequences form one part of the genome to another; can occur in Euk.; diversifies genome
transposons
jumping genes
transposable elements
insertion sequences: contain only the genes for those enzymes required for its movement; transposons: carry other genes
Discovery of Viruses
A. Mayer was trying to find the cause of Tobacco mosaic: hypothesis- either the organism was ultra-small or toxin mediated
Viruses
average size: 10-400nm
Enveloped virus
nucleocapsid; main component from host
nucleocapsid
main component from host; any proteins within it will be viral
Naked virus
lacks a envelope
Glycoprotein spikes
used for attachment
capsomeres
usually one expressed; MAX 4
Viral capsid structure
copmosed of protein subunits called capsomers that spontaneously self assemble
2 Arrangements of capsid structure
Helical and Icosahedron
Helical structure
rigid or flexible; hollow inside
Icosahedron
aka polyhedral capsule; much more common; 20 triangular faces and 12 vertices
Vertices
glycoprotein spikes protrude from these in naked viruses
Atypical/complex viruses
Pox virus: coating made up of glyco and fibral protein; Bacteriophage
Viral nucleic acid
quite small; usually 4-100 genes; can contain either DNA or RNA, NOT BOTH
Viral DNA
can be double stranded or single stranded; double more common
Viral RNA
can be double stranded or single stranded; single more common
Positive sense RNA
can act as mRNA
Negative sense RNA
cannot act as mRNA
Segmented viral genome
multiple pieces of genome; one gene per piece
Viral Order
-virales
Viral Family
-viridae
Viral Genus
-virus
Viral Species
_virus
Viral Taxonomy
based on the International Committee for the Taxonomy of Viruses
International Committee for the Taxonomy of Viruses
genome structure; life cycle; morphology; genetic relatedness
The Baltimore classifcation system of viruses
genome and replication strategy only; 7 classes
Adsorption
a specific glycoprotein spike binds to a specific host cell receptor protein
Host cell receptors
tend to deal with the immune system; typically cytokine receptors
lipid rafts
lipid microdomains that allow only certain proteins to interact; usually have more cholesterol; GPI-anchored protein found here
Penetration and Uncoating: Endocytosis
NAKED or ENVELOPED; endocytic entrance; endosomal compartment and a decreease in pH triggers uncoating event
Penetration and Uncoating: Fusion
ENVELOPED ONLY; viral envelope directly fused with host cell membrane; uncoating events hard to define
Synthesis: DNA Viruses
enter host’s nucleus and exert control over host’s machinery
Why can’t RNA viruses use Euk’s machinery?
there is no RNA-dependent RNA polymerase to read the RNA
Synthesis: (-) RNA
a viral RNA-dependent RNA polymerase is packaged within the viral capsid
Synthesis: (+) RNA
translation of RNA-dependent RNA polymerase is translated by host’s ribosomes and enzymes
Retroviruses
RNA is transcribed to DNA using reverse transcriptase; the DS DNA enters the nucleus where it is incorporated into host’s genome using integrase
reverse transcriptase
RNA=> DNA
integrase
integrates viral DNA into host’s genome
Early viral proteins
for genome replicationand taking over host cell’s machinery
Late viral proteins
for capsid
matrix proteins
line up underneath lipid domains and grab onto nucleocapsid proteins
budding
reverse endocytosis
Cytopathic effects of viruses
inhibition of trascription, translation, and host’s replication; endosomes damaged; plasma membrane alterations (syncytia); inclusion bodies formed; build up of of viral products; chromosomal incorporation; malignant cells
Syncytia
large multi-nucleated cells where the plasma membrane fused together (50-100 nuclei); syncytium (pl)
Oncogenes
cancer causing genes
Proto-oncogenes
regulate cell growth and division cycle
dsDNA viruses
making a viral protein to inhibit a tumor suppresssor gene (pRb, p53)
Retroviruses and cancer
either bring in their own oncogene protein or insert oncogene into genome
Bacteriophage found…?
in both bact. and arch.; RNA, ss DNA (not common), dsDNA (common and naked)
virulent/lytic
kill host immediately (25 minutes)
temperent/ lysogenic
“hide out” in bacteria
Step 1 of Lytic Attachment
tail fibers mediate interaction
Step 2 of Lytic Attachment
retraction of tail fibers pulls base plate clos; lysozyme-like enzyme on base plate breaks peptidoglycan
Step 3 of Lytic Attachment
tail enters cell wall and contracts
Step 4 of Lytic Attachment
DNA is released into cytoplasm
Early phage PROTEINS
involved in DNA replication and transcription
Middle phage PROTEINS
involved in DNA replication and transcription
Late phage PROTEINS
involved in the making of head and tail fibers and anything else needed for release
Early phage GENES
transcribed using host’s RNA polymerase; promoters identical to host
Last Early Proteins
include an anti-sigma factor; make proteins that modify alpha-subunit to make host RNA polymerase bind to phage promoters; some phage proteins will bind directly to RNA polymerase
Anti-sigma factor
interacts with sigma-factor and disables it
Late phage GENES
make a phage sigma-factor that recognizes late gene promoters
Phage DNA
linear not circular; must overcome th eabsence of telomerase
concatomer
individual genes spliced together; leaving extra ensures that all genes included
Phage endonuclease
cuts a “headful” of DNA
Phage assembly
all parts come from different biosynthetic events and then are assembled
Phage release
two enzymes are used to release; VERY RARE to see budding or envelopment
Holin
puts holes in cell membrane for release
T4 lysozyme
degrades peptidoglycan
Characteristics of lysogenic phage
can’t have late gene synthesis AND phage genome integrates into host genome
Virulent phage
can only be lytic
Temperent phage
can either be lytic or lysogenic
Cohesive ends
linear genome, but the 5’ ends of each strand has a 12 nucleotide sequence that are complementary
cos sites
where the single stranded tails bind to make circular genome
MOI
multiplicity of infection; how many other phage are in the area
Host growing rapidly under nutrient rich conditions and has a low MOI
Lytic favored
Host growing slowly under metabolic stress and high MOI
Lysogenic favored
If cI wins
lysogenic cycle; aka lambda repressor
If Cro wins
lytic cycle
PL
makes N protein
PR
makes Cro
N Protein
an anti-terminator which allows RNA polymerase to go past specific termination sites to make cII and cIII
cII
considered unstable, so targeted by bacterial protein hflA; activates transcription of cI
hflA
degrades cII; available based on how well the cell is doing; cyclic AMP involved
cIII
stabilizing factor for cII; 2 mechanisms: binds directly to cII and the other is unknown
hflA activity when cAMP levels are low in glucose rich environments
hflA activity is high
hflA activity when cAMP levels are high in glucose lacking environments
hflA activity is low
Integrase enzyme
cleaves phage and host genome at specific sites
Can prophage remain integrated indefinitely?
Yes
Agents that revert phage back to a lytic cycle
UV radiation, X-rays, chemicals
SOS Response for lytic cycle
activates a protease that destroys lambda repressor; lytic genes can now be transcribed
Cultivating bacteriophage
broth culture containing young, actively growing cells
Cultivating animal viruses in whole animals
hard to observe effects at cellular level; many viruses only infect one host
Cultivating animal viruses in cell culture
animal cells can be free from surrounding tissue and grown on plastic to produce a monolayer
Primary Cell Lines
Straight from the tissue with no passages
Continuous Cell Lines
Have at least one passage
Quantifying viruses
differential centrifugation or gradient centrifugation; mixed with liquid agar and a culture of host cells
Why are Plaque Forming Units used?
cannot be sure if it was one virus or phage that started the colony of death
Who discovered Prions?
Stanley Prusiner
Prion definition
Proteinaceous infectious particles found composed of normal protein PrPC
Normal PrPC
more alpha-helices
PrPSc
more B-sheets
If PrPSc is transmitted by animals
infectious ex) Kuru (cannibalism)
If PrPSc is sporadic
spontaneous random misfolding of PrPC ex) Creutzfeldt-Jacob disease
If PrPSc is inherited
mutation within the PrP gene (20 mutations linked) ex) Gerstmann-Straussler-Scheinker Disease and Fatal familial insomnia
What do prions cause?
Transmissable spongiform encephalopathies
Amyloids
proteinaceous fibers that are resistant to proteases and are an elongation of PrPSc
Highest resistance of microbes
bacterial endospores
Moderate resistance of microbes
naked viruses and bacterial cells with more resistant vegetative cells
Least resistance of microbes
most bacterial vegetative cells, enveloped viruses
Moist Heat
operates at lower temperates and short exposures; causes coagulation and denaturing of proteins; permeates better using moisture
Dry Heat
operates at higher temperatures with long exposures; dehydrates cells
Autoclave
121 degrees Celsius achieved by pressurizing steam by 15psi
Traditional pasteurization
63 degrees C for 30 mins. BATCH
Flash pasteurization
71 degrees C for 15 sec. THIN AMOUNT
Ultrapasteurization
140 degrees C for 1-2 sec. THIN AMOUNT; DOES NOT NEED REFRIGERATION
Ionizing radiation
causes DNA breakages; Gamm rays, X-rays, Electron beams
Nonionizing radiation
causes Thymine dimers; Germicidal lamps at 254nm wavelength UV disinfection
Advantages of ionization
prevent food born illnesses; preservation; control of insects; delay sprouting and ripening; sterilizes food
How many KiloGrays of ionization required depending on DNA?
the more DNA it has, the less KiloGrays necessary
Depth filter
liquid and air; 0.3 micrometers = 99.97% of contaminants
Membrane filter
porous; to be sterilizing 0.2 micrometers
Disinfectant
for a nonliving surface
Antiseptic
for a living surface
Microbicidal
kills organism
Microbiostatic
inhibits growth
What are antimicrobials geared to target?
cell wall, cell membrane, genetic material, proteins
Which chemical agents come closest to fulfilling all the requirements?
Hydrogen Peroxide and Glutaraldehyde
Chlorine as a disinfectant
hypochlorous acid
Iodine as a disinfectant
first used as a tincture of 2% iodine and water, ethanol, mix of Potassium idodide; STAINS, ALLERGENIC
Iodophores
common in use; water soluble; release iodine slowly
Phenol
Joseph Lister; not widely used because of irritating effects
Isopropyl
used at 70% isopropyl and 30% water for better permeability
Hydrogen Peroxide
3% Hyd. Perox. Used; can be vaporized to sterilize enclosed areas
Detergents
break down cell membrane by interacting with lipids
Silver
effective in low concentrations
Glutaraldehyde
used more than formaldehyde; yellow acidic liquid with a mild odor
Why do dsDNA phage have to form a concatemer?
To overcome the absence of telomerase
