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BIOL 240 > Module 9 > Flashcards

Module 9 Flashcards

Microbial Genetics

1
Q

define: microbial genetics

A

study of DNA-encoded functions

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2
Q

what forms can bacterial genomes take

A
  • single or multiple chromosomes
  • plasmids
  • bacteriophage DNA
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3
Q

what form do bacteria genomes usually take

A

one circular chromosome, possibly a plasmid as well

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4
Q

how does the plasmid copy number vary

A

plasmid copy number is very carefully regulated

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5
Q

define: prophage

A

bacteriophage DNA integrated within the genome of a bacterium

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6
Q

define: replicon

A

all the chromosomes and plasmids that replicate within a bacterial cell and are passed on to subsequent generations

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7
Q

how do plasmids replicate

A

self-replicating, replicated by host DNA polymerase

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8
Q

what sorts of genes are encoded by plamids

A

those that are not essential housekeeping genes
e.g.
- enabling making nodules on leguminous plants
- introducing tumors
- ability to degrade certain compounds
- ability to confer metal resistance
- ability to be pathogenic
- antibiotic resistance

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9
Q

what is the size of plasmids

A

smaller than most host genomes

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10
Q

is losing a plasmid lethal

A

no, genes are non-essential

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11
Q

define: wild type

A
  • strain most like that found in nature
  • source for deriving mutants
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12
Q

define: mutant

A

strain derived from its wild type that carries a mutation

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13
Q

define: allele

A

gene variant associated with the mutant strain

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14
Q

what are the categories of alleles

A
  • gain-of-function
  • loss-of-function
  • change-of-function
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15
Q

define: auxotroph

A

strain that cannot make an organic compound required for growth

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16
Q

what is the most common example of auxotrophic mutants

A

mutation in amino acid biosynthesis

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17
Q

define: phototroph

A

nutritionally wild-type strain that does not need any additional growth supplement, often the parental strain that gave rise to the auxotrophic mutant

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18
Q

how are genes named

A

three-letter abbreviation in italics, followed by a capital letter to separate genes in the same pathway

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19
Q

how are proteins named

A

same three-letter abbreviation designation as genes but with the first letter capitalized and no italics

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20
Q

define: genotype

A

description of alleles within an organism, generally reflects differences from wild-type

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21
Q

define: phenotype

A

observable properties of a strain

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22
Q

take the hisC gene, what does hisC- indicate

A

a mutation in the hisC gene, such that it cannot make its own histidine

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23
Q

what is the phenotype of a mutant that cannot make histidine

A

His- mutant

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24
Q

how does screening for mutants work

A
  • separating & growing individual cells to look for a mutant phenotype
  • grown on agar place or in liquid medium in multi-welled plates
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25
Q

when is screening for mutants used

A

when there is no growth advantage, nonselectable mutations

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26
Q

why is phenotypic selection preferred over screening

A

screening is enormously laborious and time-consuming, a large number of colonies must be screened

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27
Q

how does phenotypic selection work

A

growth conditions where only desired mutants can grow are provided

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28
Q

when is phenotypic selection used

A

used for selectable mutations, there is a growth advantage

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29
Q

what are the steps of replica plating

A
  • prepare a spread plate of a population of cells and form colonies
  • make a print of those colonies on velvet fabric placed on a wooden block and secured by an elastic band
  • stamp the velvet onto secondary plates of varying mediums
  • compare the growth on the replica plates
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30
Q

describe patching for finding mutants

A
  • pick colonies from master plate with sterile toothpick
  • sequentially inoculate gridded test plates with each picked colony, then incubate
  • compare growth on the test plates
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31
Q

what are the 4 types of mutations

A
  • silent
  • missense
  • nonsense
  • frame shift
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32
Q

define: silent mutation

A

don’t change amino acid sequence and don’t have a phenotypic effect

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33
Q

define: missense mutation

A

involve a codon change, different amino acid gets introduced

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34
Q

define: nonsense mutation

A

stop codon is suddenly introduced

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35
Q

define: frame shift mutations

A

involves an insertion or deletion of a nucleotide, amino acid sequences aren’t related to the protein at all

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36
Q

what is the most common type of mutation in labs

A

missense mutations

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37
Q

define: reversion [mutation]

A

a mutation that “corrects” a metabolic abnormality back to the wild-type form

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38
Q

how are reversions avoided [mutation]

A

mutant strains with multiple mutations are formed, double and triple auxotroph mutant strands are used

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39
Q

who developed the replica plating method

A

Esther Lederberg

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40
Q

what did Esther Lederberg prove with replica plating

A

spontaneous mutation, mutations arise even without selective pressures

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41
Q

what did Luria and Delbruck show

A

variable resistance to phage infection arises in bacteria without selective pressure

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42
Q

how does the growth of mutations compare to their parental strains

A

mutations continually confer growth advantages

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43
Q

what is the purpose of restriction enzymes

A

cut DNA at a specific recognition

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44
Q

what is a special feature of restriction enzyme recognition sites

A

they are usually palindromic

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45
Q

what is the most common recognition site length

A

six bases

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46
Q

what is the use of staggered cuts produced by restriction enzymes

A

allows for the ligation of DNA

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47
Q

what are restriction enzymes always paired with

A

corresponding modification system

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48
Q

what is the purpose of methyltransferase in the context of modification enzymes

A

protects the DNA from restriction enzyme activity

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49
Q

what is the purpose of modification enzymes

A

they restrict incoming bacteriophage DNA that may be harmful to the host

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50
Q

where are methyltransferase and the restriction enzyme often located

A

close together, often in a single operon

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51
Q

what enzyme is responsible for ligating DNA fragments

A

DNA ligase - reattaches phosphate backbone

52
Q

define: vectors

A

mobile genetic elements that can be used to get DNA shuttled into a cell for cloning experiments

53
Q

what are the types of vectors

A
  • plasmid vectors
  • phage vectors
  • cosmids
54
Q

what is the purpose of recombinant molecules

A

used to clone a bacterial gene of interest

55
Q

how are plasmid vectors usually cloned

A

the DNA and plasmid are cut with the same restriction enzyme and then are ligated together

56
Q

what are the traits of plasmid cloning vectors for easier gene cloning

A
  • origin of replication
  • selectable marker gene
  • multiple cloning site
  • small size
  • high copy number
57
Q

what is the purpose of the origin of replication

A

allows the plasmid to replicate within a host

58
Q

what is the purpose of the selectable marker gene

A

ensure recipient cells have the plasmid of interest
e.g. antibiotic resistance

59
Q

what is a multiple cloning site

A

a site that contains many enzyme recognition sites side-by-side

60
Q

what does it mean for plasmids to be in the same Inc group

A

plasmids that are incompatible with one another and are closely related, replication is controlled by the same genes

61
Q

which gene gets disrupted in blue-white screening

A

lacZ’ gene - codes for one-half of β-galactosidase

62
Q

what is used to test for β-galactosidase activity

A

X-gal

63
Q

where is the lacZ omega subunit encoded (blue-white screening)

A

E. coli genome

64
Q

why are alternate hosts sometimes needed for cloning

A

toxicity issues or codon biases

65
Q

what are shuttle-vector plasmids

A

vectors with multiple types of origins or replication, expanding the range of hosts they can be inserted into

66
Q

what else is needed in addition to multiple origins or replication in shuttle-vector plasmids

A

multiple selectable markers

67
Q

what is plasmid replication rate governed by

A

the origin of replication (oriV) sequence

68
Q

what colour are colonies with an insert successfully added in blue-white screening

A

white, β-galactosidase is disrupted

69
Q

how do phage vectors work

A

mix viral DNA with a fragment of interest

70
Q

around how large of a fragment can lysogenic lambda phages carry

A

~20-kb fragments

71
Q

why do lysogenic lambda phages become lytic after DNA of interest is added

A

the part that codes for the lysogenic pathway gets replaced

72
Q

what is a drawback to using phage vectors

A

the host cells lyse and die in the process

73
Q

define: cosmids

A

phage genomes that omit nearly all the phage DNA, leaving more room for the fragment

74
Q

what portion of the phage genome is left in cosmids

A

the cos site

75
Q

how big does the cosmid vector + insert have to be

A

~50 kb

76
Q

what elements are present in cosmids

A
  • cos site
  • oriV
  • multiple cloning site
  • antibiotic selection marker
77
Q

how large can the fragments be in cosmids

A

35-45 kb

78
Q

what are 3 methods of DNA transfer

A
  • transformation
  • conjugation
  • transposition
79
Q

describe: transformation [DNA transfer]

A

introduction of naked extracellular DNA directly into an organism

80
Q

does transformation require cell-to-cell contact [DNA transfer]

A

it does not

81
Q

define: natural competence

A

the ability for bacteria to take up DNA directly from the environment

82
Q

what is free DNA used for in bacteria

A
  • source of nutrients
  • repairing DNA
  • recombination to provide new traits
83
Q

what is a risk of recombination with free DNA

A

deleterious traits can also be acquired

84
Q

what happens to a DNA strand brought into a naturally competent cell

A
  • it’s introduced single-stranded
  • other strand of DNA is digested
  • single-stranded DNA is coated with the RecA protein
85
Q

why is free DNA introduced as a single strand

A

it’s protected from restriction enzymes

86
Q

why is the single-stranded DNA coated with the RecA protein [transformation, DNA transfer]

A

it initiates the SOS pathway regulon, DNA introduces itself into the genome through homologous recombination

87
Q

what is required in the host genome for transformation [DNA transfer]

A

a region of homology

88
Q

what are two ways to artificially induce competence in bacteria

A
  • treatment with calcium cations
  • electroporation
89
Q

define: conjugation

A

the transfer of DNA from cell to cell via direct contact/sex pilus formation

90
Q

what is the F plasmid

A
  • “fertility plasmid”
  • best-studied plasmid that encodes for conjugation
91
Q

what genes are critical for mediating the conjugation process

A

tra region

92
Q

what does the tra gene encode for

A

production of a mating bridge for transferring plasmid DNA in a specific direction

93
Q

what gets transferred first in conjugation

A

oriT, the origin of transfer

94
Q

what are F+ cells

A

E. coli with the F plasmid

95
Q

what cells do F+ cells conjugate with

A

F- cells

96
Q

what ensures that F+ cells don’t conjugate with other F+ cells

A

tra genes

97
Q

what happens to an F- cell one it has been conjugated with

A

it becomes a F+ cell

98
Q

how does the F+ cell retain the F plasmid

A

it replicates itself while mating, and threads one strand into the recipient cell

99
Q

how does F plasmid occasionally integrate into the genome of E. coli

A

by homologous recombination

100
Q

define: episome

A

DNA that can integrate into the chromosome but also exist autonomously

101
Q

what are some examples of episomes

A
  • F plasmid
  • lysogenic phages
  • insertion sequences
  • transposons
102
Q

how does the transfer of DNA work with F plasmid genomes

A
  • begins with a portion of the plasmid because of the origin of transfer
  • genome itself starts passing through the mating bridge after
  • very last bit of DNA passing through would be the rest of the plasmid
103
Q

what is the name of a bacteria cell in which the F plasmid is integrated into the genome

A

Hfr strain - “High-Frequency of Recombination”

104
Q

how can Hfr stains be used to map genomes

A

Hfr stains were allowed to mate with recipients for extended periods of time before being disrupted by a blender

105
Q

who discovered using Hfr stains for genome mapping

A

Joshua Lederberg

106
Q

how can E.coli go from being Hfr to F+

A

the integrated F factor can excise itself from the genome by homologous recombination

107
Q

how is it possible for F’ to contain chromosomal DNA for E. coli

A

by homologous recombination, when the integrated F factor gets excised, some genomic DNA is excised as well

108
Q

how does triparental conjugation work

A
  • helper plasmid contains tra genes
  • helper plasmid conjugates into donor strain
  • helper plasmid encodes proteins needed for transfer of recombinant plasmid from donor strain
109
Q

what does transposition involve

A

movement of insertion sequences and transposons

110
Q

what are some moves DNA can make through transposition

A
  • one plasmid to another
  • within and between genomes
  • between genomic DNA and plasmids
  • from a plasmid back to the genome
111
Q

define: insertion sequence

A

mobile genetic element that only encodes the proteins needed for transposition

112
Q

define: transposons

A

mobile genetic elements that contains genes in addition to those needed for transposition

113
Q

does transposition depend on homologous recombination

A

no, sequence homology is not required

114
Q

what is required in insertion sequences

A

transposase gene with inverted repeat (IR) regions

115
Q

how long are IR regions

A

~20 bases

116
Q

what does the resolvase gene do

A

it recognizes the res site in the transposon

117
Q

describe: replicative transposition

A

copies the genetic element and moves the copy to another location

118
Q

describe: non-replicative transposition

A

cuts the genetic element and moves the excised element to another location

119
Q

which form of transposition is resolvase responsible for

A

replicative transposition

120
Q

what can be used to generate mutants (aside from UV light)

A

transposons

121
Q

what is the purpose of suicide vectors

A

delivering transposons to recipient cells

122
Q

define: transposon mutagenesis

A

the creation of mutants by randomly embedding a transposon in the genome

123
Q

define: transducing particles

A

viral particles that contain host DNA instead of, or in addition to, viral DNA

124
Q

describe: generalized transduction

A
  • virus accidentally packages a fragment of host cell DNA
  • virus delivers random host genome fragment instead of viral DNA to the next cell
  • virus is unable to replicate
  • few transducing phages
  • homologous recombination must occur to integrate into a recipient genome
125
Q

describe: specialized transduction

A
  • lysogenic bacteriophage integrates at specific site
  • virus excises incorrectly, includes some host genome
  • all virus particles are transducing phages following lysis
  • homologous recombination must occur to integrate into a recipient genome
126
Q

what was used to map bacterial genomes prior to DNA sequencing

A

co-transduction frequency

127
Q

is there phage DNA in transducing particles for generalized transduction

A

no, it only contains the host DNA

BIOL 240 (12 decks)

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