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GENET 270 > Transformation > Flashcards

Transformation Flashcards

1
Q

summarize transformation

A

when a cell is competent and can take up free naked DNA which recombines with homologous regions to produce recombinant transformants

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2
Q

what are recombinant transformants

A

homologous recombination between chromosomal DNA and and free naked DNA that was taken up by the cell

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3
Q

what is competence

A

ability for cell to take up free naked DNA

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4
Q

what experiment was used in the discovery of transformation OR transforming principles

A

Griffiths experiment with the mice, R cells, and S cells
- note: r cells avirulent mixed with dead S cells virulent produced virulent effect on mice.

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5
Q

when is streptococcus pneumoniae competent

A

only in early exponential growth phase

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6
Q

when is bacillus subtilis competent

A

competent when lacking nutrients

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7
Q

when is haemophilus influenzae competent

A

competent when lacking nutrients

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8
Q

when is Neisseria gonorrhoea

A

always competent

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9
Q

what is two component system in bacillus subtilis
(hint: explain competence in reference to gm+ bacteria)

A

include comP sensor proteins which detect competence stimulating factor (pheromones)

ComP signals comA response regulator which turns on gene expression for DNA uptake (competence)

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10
Q

what is ComP

A

sensor protein that detects competence stimulating factor (comX- pheromones) and auto-phosphorylates, giving P to comA

IN BACILLUS SUBTILIS

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11
Q

what is comA

A

protein that receives P from comP, recruits comK (transcription) to turn on competence gene expression

IN BACILLUS SUBTILIS

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12
Q

what is comK

A

transcription factor that stimulates expression of DNA uptake and machinery (RNAP)

IN BACILLUS SUBTILIS

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13
Q

what is comS

A

protects comK from proteolytic degradation

IN BACILLUS SUBTILIS

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14
Q

what is proteolytic degradation

A

proteases (enzymes) that break down proteins

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15
Q

what is two component system in streptococcus pneumonia

A

peptide pheromone increase in density signaling comD sensor kinase which signals comE response regulator

  • phosphorylated comE can signal for competence gene expression
    OR
  • can signal alternative sigma factor (comX), region on RNAP which tells it what promotor to bind to for competence gene expression
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16
Q

whats comD

A

sensor kinase that is signaled by density of peptide pheromone and signals comE

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17
Q

Whats comE

A

response regulator that turns on gene expression in streptococcus pneumonia

18
Q

what is comG operon genes

A

gene involved in building of pseudopilus which grabs foreign DNA

19
Q

what is comGC

A

the major pilus protein with which the pilus is comprised of

20
Q

what is comGB and comGA

A

proteins involved in pilus assembly

21
Q

what is comC processing endopeptidase

A

cleaves pilus proteins involved in pilus retraction (?)

22
Q

explain Gm+ DNA uptake machinery
(bringing DNA into the cell)

A

DNA binds to pilus, comEA holds dna through electrostatic interactions
- dsDNA is cleaved by nucA
- Pilus retraction brings DNA into cell pulling one strand in with the other out -> directed by comFA
- EndA nuclease degrades outside strand while other goes through comEC channel and is held by SSB to prevent degradation
- enters 3’ to 5’

23
Q

what is comEA

A

holds DNA through electrostatic interaction

24
Q

what is NucA

A

nucleases that cleaves DNA

25
Q

what is comEC

A

channel with which ssDNA goes through

26
Q

what is comFA ATP-dependent DNA translocator

A

uses energy to direct ssDNA through comEC from 3’ to 5’

27
Q

what is endA nuclease

A

nuclease that degrades other strand of ssDNA (outside cell)

28
Q

explain Gm- uptake machinery

A

uses type II secretion system
- DNA goes through pilQ channel
- pilE is pseudopilus that grabs DNA
- comE degrades int ssDNA
- comA is second channel that ssDNA goes through
- pilT is motor that directs ssDNA into cell

29
Q

what is role of SSB

A

ssDNA in cell is coated with SSB to prevent ssDNA degradation
- its also a substrate for recA so that it does homologous recombination

30
Q

what experiment is used to observe the efficacy of DNA uptake

A
  • donor DNA is labelled with radioactivity
  • added to competent recipients and treated with DNase (cleaves dna) at various times
  • cells are precipitated and radioactivity is counted
31
Q

what type of DNA is favored for uptake

A

competence favors free DNA that is similar or the same as bacterial DNA/ plasmid DNA because foreign DNA could be lethal

32
Q

what are specific uptake sequences (USSs)

A

signals that allow for4 the uptake of bacteria/plasmids own DNA more efficiently or rapidly than other DNA

33
Q

what is the eclipse phase of transformation

A

The time elapsed between successful cell infection and the start of virus production
ssDNA is in the cell but is not yet integrated into bacterial chromosome to become dsDNA

34
Q

what are arguments against competence for nutrition

A

1) some bacterial are only specific in taking up their own kind of DNA

2) competence develops in only a portion of a population

35
Q

what are arguments for competence for nutrition

A

1) uptake of DNA may keep from species uptaking harmful DNA (B.subtilis)

2) feeding on cells that are incompetent may be normal process

36
Q

what is B. Subtilis cell killing mechanism

A

process by which bacteria are programmed to kill themselves and donate their contents so that other bacterial cells can thrive (improve fitness)

37
Q

what are arguments for competence for DNA repair

A

damage in a bacterial chromosome can be replaced by foreign DNA that was taken up

38
Q

what are arguments against competence for DNA repair

A

1) recA gene is not induced during competence in all species, thus no recombination of new genes occurs in some bacterial cells

2) competence genes are not induced in response to DNA damage

39
Q

what is the evolutionary hypothesis for transformation and recombination
use neisseria spp. as an example

A

hypothesis:
transformation provides exchange in genetic material and increases genetic diversity

an example is Neisseria spp. as it has type 2/4 secretion systems for DNA import and type 4 for DNA export
- DNA export undergoes mutations and modifications continuously which lead to diversity

40
Q

what is calcium ion induction artificial transformation

A

calcium+ heat shock induces cell surface to open–> any proteins is sucked inside and induces competence

41
Q

what is electroporation artificial transformation

A

cells exposed to electric field which induce small forms which make cells permeable to molecules like DNA for competence
- more efficient than calcium induction

GENET 270 (6 decks)

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