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Microbio > lecture exam 3 > Flashcards

lecture exam 3 Flashcards

1
Q

what do restriction endonucleases do?

A

cut DNA 5’ to 3’

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2
Q

cloning vector

A

engineered plasmid to put gene of interest

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3
Q

insert

A

piece of DNA you are cloning into a vector

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4
Q

PCR

A
  • polymerase chain reaction
  • genomic NDA libraries
  • ex: ddNTP
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5
Q

agarose gels

A
  • to maximize efficiency of separating
  • the larger the bands, the longer it takes to get through (smaller goes through first)
  • want less dense gel if you have big bands because will get stuck there
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6
Q

what is loading dye for?

A

increase density

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7
Q

plasmids

A
  • DNA addition to chromosome DNA
  • have multiple cloning sites
  • selectable markers encoded in the plasmid
  • used for PCR products
  • negatively supercoiled
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8
Q

Metagenomic sequencing

A
  1. illumina sequencing- fluorescent nucleotides that glow when added to chain (get lots of short reads)
  2. pyrosequencing- detection of pyrophosphate release and generation of light on n.t. (instead of chain termination w ddNTP) !!! 2 phosphates released (stuck together) !!!
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9
Q

viruses
viroids
virusoids
prions

composition

A

viruses: protein and nucleic acid
viroids: RNA
virusoids: RNA
prions: protein

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10
Q

PrP

A

normal protein in all cells that can exist as PC, PSC, or Press

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11
Q

PrPC vs PrPSC vs PrPress

A

PC: non-infected
PSC: infectious form
Press: non-infectious misfolded PrP (resistant to proteases –> cant turn over)

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12
Q

Creutzfeldt-Jakob disease (CJD)

A

inherited form of scrapie for humans

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13
Q

prions

A
  • infectious agent made up of protein
  • causes a cascade of misfolding events
  • cause Bovine spongiform encephalopahty (BSE) aka mad cow
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14
Q

Gerstmann-Straussler-Scheinker syndrome

A
  • strictly familiar
  • change in codon 102 proline –> leucine in PRNP
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15
Q

fatal familial insomnia
&
sFI

A

insomnia, dementia
sFI: sporadic fatal insomnia

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16
Q

Kuru

A
  • due to eating human flesh
  • Papua New Guinea
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17
Q

viruses importance

A
  1. major cause of disease and new source of therapy
  2. important members of aquatic world- move organic matter from particulate to dissolved
  3. important in evolution -> transfer genes between bacteria, etc
  4. important model systems in molecular bio
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18
Q

virion

A
  • complete virus particle with DNA or RNA in coat of protein
  • cannot reproduce independently nor carry out cell division
  • can exist extracellulary (have strange envelope structures)
  • may have enzymes
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19
Q

capsids

A
  • protein coat to protect genetic material
  • aids in transfer between host cells
  • made of protomers
  • helical, icosahedral, or complex
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20
Q

general structure of viruses

A
  • size range ~10 to 400 nm in diameter
  • nucleocapsids (DNA/RNA + capsid)
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21
Q

enveloped vs nonenveloped viruses

A

enveloped: has protein coat and lipid membrane, as well as
spikes (to target and penetrate cells)

22
Q

animal virus envelope

A
  • made up of lipids and carbs
  • usually arise from host cell plasma or nuclear membranes
23
Q

viral envelope proteins

A
  • viral encoded as spikes (peplomers) –> host-specific
  • for attachment to host cell (ex: hemaglutin of influenza)
  • for identification of virus
  • may have enzymatic activity (ex: neuraminidase of influenza)
  • may be involved in nucleic acid replication
24
Q

why are viral envelope proteins host-specific?

A
  • codon usage- so genomes are compatible
  • similar promoter sequences in transcription
25
Q

virion genome

A
  • can be single or double stranded DNA or RNA
  • size varies
  • segmented or circular
26
Q

viral multiplication 6 steps

A
  1. attachment to host cell (adsorption)
  2. entry
  3. uncoating of genome
  4. synthesis of genome
  5. assembly
  6. release
27
Q

virulent phage

A
  • 1 reproductive choice: lytic
  • multiplies immediately upon entry
28
Q

temperate phages

A

2 reproductive options
1. reproduce lytically and kill host cell
2. remain within host cell without destroying it (integrate genome- lysogeny)

change genotype of its host
bacteria gain immunity

29
Q

prophage

A
  • genetic material of a bacteriophage
  • in lysogenic state
30
Q

lysogens

A
  • bacteria carrying a prophage
  • lysogenic bacteria
  • appear normal and can switch from lysogenic to lytic cycle
31
Q

2 advantages of lysogenic conversion for virus

A
  1. phage stays viable but may not replicate
  2. multiplicity of infection ensures survival of host cell

(every time bacterium divides, makes copies, doesnt want to kill cell)
(phage doesnt want to compete with similar phages)

32
Q

induction

A
  • conditions in cell cause prophage to inititate synthesis of new phage particles
  • bacteria is struggling, phage activates lytic cycle
  • triggered by SOS response (single stranded DNA accumulation)

(going back)

33
Q

lysogenic conversion

A
  • temeperate phage changes phenotype of its host
  • bacteria gain immunity
  • phage may express pathogenic toxin or enzyme (ex: virulance factors)
34
Q

bacteriophages

A
  • bacterial viruses
  • most abundant “biological entities” on earth
35
Q

plaques

A

clearing on plates that contain a bacterial lawn (resulting from lytic cycle)

36
Q

what is the viral tail for?

A

binding and injecting phage genome

37
Q

tiles

A

groups of genes in phage for similar function (ex: DNA rep or capsid formation)

38
Q

host range

A

phage makes plaques on only certain bacteria

39
Q

early genes

A
  • involved in synthesis of the genome
  • transcription of these genes begins as DNA is being injected into bacteria
  • share same promoter/ mimic promoters of the host cell
40
Q

late genes

A
  • involved in assembly of head and tail, as well as lytic cycle
  • different promoters from host cell –> unrecognized by host RNA pol. or require specific sigma factors
  • degrade outside cell wall (ex: tail fibers)
41
Q

lytic cycle

A
  • process for breaking the host bacterium and release of new phage
  • has to be timed (inducible operator system)
42
Q

transcriptional regulation

A

responsible for most of the regulation of phage particles ( shows activation and repression of gene expression)

43
Q

what is the primer problem?

A
  • lagging strand replication gets to the end of a linear template molecule and there is no DNA upstream to synthesize a primer to inititate replication
  • basically, you end up with segments of DNA you cant replicate)
  • bacteria don’t run into this problem because circular DNA
44
Q

how do phages overcome primer problem?

A
  1. synthesize single stranded circles
  2. make protein primers to complete replication
  3. use terminal redundancies
  4. hair pin end to replicate around teh ends and then are resolved by a protelomerase (a recombinase-like rxn)
45
Q

telomeres

A
  • sequences synthesized by telomerase at the end of DNA without needed a template
  • in euk cells
46
Q

concatemers

A

continuous DNA molecule with multiple copies of the same DNA sequences linked together

47
Q

Pac sites

A
  • phage packaging site
  • monomers cut out of concatemer and packed into phage head
  • only in lytic life cycle
48
Q

3 proteins involved in phage lysis

A
  1. lysozymes- degrade cell wall
  2. holins- form pore for lysozyme to exit
  3. antiholins- prevent lysozyme from leaving
49
Q

defective phage

A
  • can’t replicate
  • can contribute useful genes
50
Q

Phage T7

A
  • responsible for turning on late genes
  • infects E. Coli bacteria
  • double-stranded DNA genome
  • has icosahedral head (aka capsid)
  • lytic life cycle only. NOT lysogenic
  • rapid life cycle ~17 mins
51
Q

pET plasmid

A
  • target gene cloned downstream of T7 promoter
  • lac repressor binding site engineered into T7 promoter —> IPTG —> T7 RNA polymerase
52
Q

pLysS or E

A

plasmid-encoded lysozyme that will bind and inactive residual production of T7 RNA polymerase

Microbio (5 decks)

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