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BIOL 240 Microbiology > Topic 9 - Genetics > Flashcards

Topic 9 - Genetics Flashcards

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Biology 101 flashcards
1
Q

microbial genetics grew from ____, led to ___ ____

A

microbiology
molecular biology

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2
Q

when did microbial genetics start

A

1940s-1950s

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3
Q

required development of 2 model systems for genetic investigations:

A
  • Escherichia coli and Salmonella typhimurium
  • E.coli K-12 = not pathogenic, studied
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4
Q

organization of bacterial genomes

A

single chromosome and plasmids

bacteriophage DNA can also be present

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5
Q

what is replicon?

A

chromosomes + plasmids in cell

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6
Q

plasmid copy # in cell is ___ regulated

A

CLOSELY
- diff plasmids can be copied diff times

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7
Q

plasmids typically ____ than genomes

A

smaller

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8
Q

T/F plasmids usually do not encode housekeeping codes

A

T

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9
Q

T/F antibiotic resistance genes uncommon in plasmids?

A

F

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10
Q

what is plasmid copy number governed by?

A

plasmid-encoded genes

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11
Q

plasmids with similar replication controls =?

A

incompatible (Inc)

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12
Q

why are bacteria ideal genetic research candidates

A
  • one chromosome for easy detection of mutations
  • early studies, nutritional mutants were used
    – allowed study of one gene based on its inability to use or produce a particular nutrient
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13
Q

wild type meaning

A
  • strain most like that found in nature
  • original isolate
  • source for deriving mutants
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14
Q

mutant meaning

A
  • strain carrying a mutation, relative to wild type
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15
Q

mutation meaning

A
  • change in a gene that disrupts/alters functions
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16
Q

allele meaning

A

variant of a gene
- may be gain of function
- may be loss of function
- may be change of function

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17
Q

auxotroph meaning

A
  • mutant that is unable to make a specific compound
  • often a mutation in AA biosynthesis
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18
Q

prototroph meaning

A
  • strain capable of making all required organic compounds
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19
Q

how are genes/proteins named generally?

A

genes
- three-letter abbr. in italics, followed by a capital letter to separate genes in same pathway

proteins
- given same three-letter designation, first letter capitalized, no italics

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20
Q

why do microbial geneticists compare wild-type strains and mutant strains?

A

goal: to identify differing alleles of genes

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21
Q

mutant selection

A
  • isolation of cells with a particular genotype on basis of growth
  • can select for His+ on basis of growth
  • canNOT select directly for His-
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22
Q

mutant screening

A
  • identification of cells with a phenotype
  • colour, morphology, “no growth”
  • CAN identify His- by screening
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23
Q

difference between selection vs screening

A

selection: cannot directly identify auxotroph on basis of growth

screening: can directly identify auxotroph through phenotype

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24
Q

what makes a mutation selectable or non-selectable?

A

selectable mutations generally give a growth advantage under specific conditions
- e.g., conditions that kill wild-type
- useful in genetic research

non-selectable mutations confer NO advantage or confers a DISadvantage
- detection requires screening of a large # of colonies

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25
Q

phenotypic selection

A
  • use a growth medium that inhibits microbes lacking desired gene
  • antibiotic selection commonly used
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26
Q

screening - replica plating

A
  • more tedious than selection
  • can facilitate screening with replica plating
  • duplicate plates are created (one lacks particular nutrient)
  • a mutation has occurred where a colony grows on full support plate but doesn’t grow on partial support plate
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27
Q

screening - patching

A
  • transferring colonies (w/ toothpick) to a gridded plate
  • usu more accurate and reproducible than standard velvet replica plating
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28
Q

types of mutations (4)

A

silent - no change in AA seq of protein; usu third position of codon

missense - change in codon that results in a diff AA

nonsense - early STOP

frameshift - shifts reading frame (insertion or deletion of nucleotides)

29
Q

reversion mutation meaning (why problematic? how to avoid?)

A

reversion: a mutation that “corrects” a metabolic abnormality back to wild-type form
- problematic when trying to determine mutation rates of a chemical or DNA exchange rates between two microbes
- to avoid this problem, double and triple auxotroph mutant strains were studied (decreases possibility of a spontaneous reversion mutation)

30
Q

spontaneous mutations (2 experiments - Lederberg + Luria-Delbruck)

A
  • experiments by Esther Lederberg used replica plating to illustrate spontaneous mutation without selective pressure
    (mutations did not adapt in real-time, were always there)
  • Luria and Delbruck showed variable resistance to phage infection arises in bacteria without selective pressure (random)
31
Q

Richard Lenski E. coli experiment

A

cultures given an extended generational time w/ selective pressures -> compared to original cultures w/o selective pressures

  • culture grown under selective pressure displayed higher fitness
32
Q

restriction enzymes

A
  • name explains what organism they came from
  • cut DNA at a specific recognition site
  • usu palindromic (sticky/blunt ends)
  • similar ends of cut DNA can be paired together and ligated
33
Q

modification enzymes

A
  • REs ALWAYS paired with modification enzymes
    – often in a single operon
    – referred to as “R/M systems”
  • recognize the same site as the paired RE
  • methyltransferase activity protects DNA from endonuclease activity (e.g., EcoRI doesn’t cut E.coli)
34
Q

cloning vectors

A
  • REs allow researchers to stitch together DNA fragments into recombinant molecules
  • recombinant molecules can be used to clone a bacterial gene of interest
  • vectors are used to insert a recombinant DNA molecule into a recipient host bacterial cell (plasmids, phages, cosmids)
35
Q

plasmid cloning vectors

A
  • first used in 1970s by Cohen
  • cut fragments from 2 plasmids carrying antibiotic resistance genes w/ same RE
  • transformed strain exhibited traits from both plasmids
36
Q

oriV = ?

A

origin of replication
(v = vegetative growth/replication)

37
Q

what does the ori control? (2)

A

(1) what types of organisms the plasmid can be in
(2) # of copies of plasmid that will be made

38
Q

desirable plasmid traits for easier gene cloning (5)

A
  • origin of replication (ori)
  • selectable marker gene
  • multiple cloning site
  • small size
  • high copy #
39
Q

X-gal screening for transformed cells

A
  • no beta-galactosidase = white, meaning insert successful!
  • X-gal = analog of lactose (cleaved by beta-galactosidase)

X-gal = white (yes insert)
X-gal = blue (no insert)

40
Q

shuttle-vector plasmids

A
  • shuttle-vector plasmids have MULTIPLE types of ori
  • expands range of host cell types that the plasmids can be inserted into
    (replication in certain hosts is restricted by ori)
41
Q

phage vectors

A
  • mix viral DNA with fragment of interest
  • lysogenic lambda phage can carry ~20kb fragments -> take this genetic material out, add gene of interest (needs to be same length)
  • Cos (abbrv of “cohesive end site”

phages infect cells, replicates and lyses cells, then we can recover recombinant phage from plaques

42
Q

Cos =?

A

cohesive end site

43
Q

cosmids

A
  • phage genome that omit nearly all the phage DNA, leaving more room for the fragment
  • only the critical phage cos packaging recognition sites remain
  • other elements incl a multiple cloning site and an antibiotic selection marker
  • cosmids can typically carry 35-45kb fragments
  • NO LYSIS, viruses just added to inject genomic data
44
Q

list cosmid vector components

A
  • cos site
  • cloning site
  • oriV
  • antibiotic resistance
45
Q

transformation

A
  • introduction of extracellular DNA into organism (from env)
  • doesn’t require cell-to-cell contact
  • a part of SOS reaction to genetic dmg***
  • some bacteria naturally competent for transformation
  • other bacteria can be artificially induced to become competent
    – treatment w/ Ca2+ cations (double charged, attracts DNA ss and cell membrane to get the physically close)
    – electroporation
46
Q

during transformation, the foreign DNA comes into the cell in ss/ds?

A

single strand!!! (activates SOS response)

47
Q

transformation of what response? AKA?

A

SOS response (to genetic damage)
ALA homologous recombination

48
Q

conjugation (what is it, mechanism)

A
  • transfer of DNA from cell to cell via DIRECT CONTACT/SEX PILUS formation

mechanism:
- F plasmid carries gene to form sex pilus bridge” between 2 cells
- F plasmid can be copied and sent across the bridge (origin of transfer (oriT) first) into recipient cell
- turns an F- cell into an F+ cell capable of conjugating with another F- cell
- note: tra genes

49
Q

big difference between transformation and conjugation

A

transformation: no direct cell-to-cell contact
conjugation: requires direct cell-to-cell contact

50
Q

F plasmid =?

A

fertility plasmid

51
Q

F-plasmid conjugation steps

A
  • sex pili pull cells together, forms mating bridge, ss nick made at oriT
  • one strand of F plasmid is transferred into recipient (one strand kept behind), replication occurs in both donor/recipient -> makes ds F plasmids
  • results in two F+ bacteria
52
Q

how does the F plasmid integrate into host chromosome?

A

homologous recombination

53
Q

what does oriT need?

A

origin of transfer needs tra (tra operon codes for essential proteins needed in conjugation)

54
Q

what does “F plasmid is an episome” mean?

A

F plasmid is DNA that can integrate into chromosome or exist autonomously

55
Q

Hfr =? explanation

A

high frequency of recombination strain DNA transfer
- incorporated F plasmid sends host cell DNA next to its incorporation site across mating bridge over time
- can be used to “map” location of genes in host chromosome

56
Q

Hfr cell vs F+ cell

A

Hfr cell = integrated F plasmid (big host DNA w/ F plasmid combined)

F+ cell = F plasmid separate from chromosome, attached through homologous DNA seq (SITE-SPECIFIC recombination)

57
Q

Hfr gene transfer is like F plasmid but…?

A

they both use sex pili but Hfr uses the integrated F factor to insert genes into F- cell.

by stopping conjugation at diff time intervals, gene location can be mapped (genes closer to site of integration are transferred first)

58
Q

generation process of F’ plasmids

A
  • an incorporated F plasmid excises itself
  • excision is inaccurate, some host DNA is excised too
  • when F’ plasmid conjugates, it sends the HOST cell DNA to recipient
59
Q

triparental conjugation

A
  • conjugation can still occur using recombinant plasmid lacking the required tra genes and a helper plasmid w/ tra genes
  • more room in the recombinant plasmid for a desired DNA fragment

helper plasmid conjugates into donor strain, encodes proteins for transfer of recombinant plasmid from donor strain, conjugates into recipient

60
Q

transposition (what is it, discovered by who)

A
  • movement of DNA via mobile genetic elements
  • transposable elements can move within and between genomes
  • first detected in corn by Barbara McClintock
61
Q

transposition can be subdivided into:

A
  • insertion sequences: encode only proteins needed for transposition
  • transposons: contain other genes in addition to those needed for transposition
62
Q

transposons can be non-replicative or replicative, meaning?

A

non-replicative - cut and paste
replicative - copy and paste

63
Q

mechanisms of transposition:

A
  • requires transposase and resolvase genes
  • replicative transposition: copies element and moves copy to another location
  • non-replicative transposition: cuts and pastes element into new location
64
Q

transpositions can be used to ___ functional genes and observe phenotypic changes

A

disrupt

65
Q

____ vector plasmid carrying the transposon
- details

A

suicide
- recipient cell gets the plasmid, but plasmid can’t replication
- transposition can still occur at random
- screening and/or selection for desired disruption follows

66
Q

transduction

A
  • virus accidentally packages a fragment of host cell DNA (=transducing particle)
  • virus delivers that fragment instead of viral DNA to next cell
  • virus unable to replicate bc lacking viral genome
67
Q

historically, co-transduction frequency was used to?

A

map bacterial genomes
- genomes that were closer to a known marker gene would be transduced with that marker more frequently than ones farther

68
Q

co-transduction can also be used to modify bacteria (example?)

A

ex. specialized transduction: Shigella dysenteriae DNA in E.coli causing romaine lettuce poisoning (Shiga toxins)

BIOL 240 Microbiology (10 decks)

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