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MICR 338 > Midterm 2 Materials > Flashcards

Midterm 2 Materials Flashcards

1
Q

What are the advantages of plasmids?

A

Antibiotic resistance, toxin degradation, virulence and/or symbiosis-related functions

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2
Q

What do Col plasmids do?

A

Synthesize colicin proteins

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3
Q

What are colicin proteins?

A

Type of bacteriocin unique to E.coli strains

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4
Q

What are bacteriocins?

A

Proteins that can kill sensitive bacteria

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5
Q

What are R Plasmids (Resistance)?

A

They carry resistance to antibiotics and other factors

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6
Q

What are characteristics of R Plasmids?

A

Often broad-host-range and self-transmissible

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7
Q

What do F (Fertility) plamids do?

A

Carry the F pilus and other conjugation factors

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8
Q

What is the characteristic of F plasmids?

A

Self-transmissible

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9
Q

What are F’ plasmids?

A

Derivatives of F carrying cell DNA

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10
Q

What can F plasmids be transferred into?

A

F-

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11
Q

What are the genes relevant to F plasmids?

A

oriT and tra

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12
Q

What are Hfr strains?

A

High frequency of recombination

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13
Q

What do Hfr cells have?

A

Replication fusion of F and the chromosome

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14
Q

What happens when the F plasmid is integrated into the chromosome?

A

It can mobilize the entire genome

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15
Q

What does the oriT mark?

A

The beginning of DNA transfer

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16
Q

What is conjugation?

A

Transfer of genetic material between cells via direct cell-to-cell contact

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17
Q

What’s the difference between an F+ and F-cell?

A

F- lacks fertility plasmid

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18
Q

What is an F+ cell?

A

An E.coli cell with its own chromosome

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19
Q

Whats the difference between an F+ cell and Hfr?

A

Hfr cell has the F factor within its chromosome

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20
Q

What is an F’ factor?

A

It’s an F factor that’s carrying some of the bacterial chromosome with it

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21
Q

For Hfr strains, where is the higher recombination frequency?

A

For genes closer to and downstream of Hfr oriT

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22
Q

How are recombinant plasmids made?

A

Combining parts of various plasmids and phages with properties like pRK600, pLAFR1, and pBSKS

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23
Q

What is pRK600?

A

An R plasmid with colE1 replicon

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24
Q

What is pLAFR1?

A

An R plasmid with lambda cos but without tra genes

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25
Q

What is pBSKS?

A

A highly modified col plasmid

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26
Q

What does pBSKS contain?

A

Phage, bacterial & synthetic sequences

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27
Q

What are examples of phage based vectors?

A

Lambda and M13

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28
Q

What are Ti and RI

A

Plasmid derivatives for plant transformation

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29
Q

What do Ti plasmids form?

A

Plant tumours

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30
Q

What is merodiploid?

A

Partial diploid

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31
Q

What do merodiploid may have?

A

Duplicated genes on a plasmid or recombined into the genome

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32
Q

What is complementation?

A

1 gene makes up for the lack of function of another one

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33
Q

What is a conjugative plasmid?

A

Plasmid carrying the genes that determine the effective contact function

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34
Q

What is a mobilizable plasmid?

A

Can prepare its DNA for transfer

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35
Q

What is a self-transmissible plasmid?

A

Like F, is both conjugative and mobilizable

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36
Q

What is an example of self-transmissible plasmids?

A

pRK600

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37
Q

What is an example of a plasmid that’s mobilizable but not self-transmissible?

A

pLAFR1

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38
Q

What is an example of non-mobilizable plasmids?

A

pBSKS

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39
Q

What is a condition that needs to be met for non-mobilizable plasmids?

A

Must be introduced into cells via transformation

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40
Q

What are the functions of Merodiploid analysis?

A

1) Can be used to identify/prove which gene a mutation is in
2) Can be used to isolate wild-type genes from a clone bank
3) Can be used to determine if linked genes are in an operon

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41
Q

How can pRK600 be used?

A

1) As a suicide vector to deliver transposons to cells in which it cannot replicate
2) To mobilize other plasmids

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42
Q

What are the functions of the tra genes

A

1) Are the proteins of the sex pilus
2) Enzymes which nick at the oriT and then mediate transfer of ssDNA into the recipient
3) A. tumefaciens T-DNA export system

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43
Q

Where is the DNA via conjugation transferred from?

A

DNA is transferred from oriT in a ssDNA form and in a unidirectional led by the 5’ end

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44
Q

What is the uptake of DNA into the recipient followed by?

A

1) Synthesis of the second DNA strand
2) Reformation of the plasmid
3) Homologous recombination into the recipient gene

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45
Q

How is chromosomal DNA be mobilized?

A

When oriT is inserted into the genome

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46
Q

How can the oriT be inserted into the genome?

A

1) An oriT carrying tansposon (Tn5 - mob)
2) Integration of an oriT-carrying plasmid like F (Hfr strains)

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47
Q

What is Recombination frequency?

A

The frequencies at which different donor alleles are recombined into a recipient genome

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48
Q

What does the closer a gene is to the oriT entail?

A

The higher the recombination frequency

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49
Q

What is the objective of triparental matings?

A

Isolate a recipient strain with the target plasmid

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50
Q

What are the strains required in triparental matings?

A

Donor, Mobilizer, and Recipient

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51
Q

How many plasmid transfers via conjugation are required in triparental mobilization?

A

2

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52
Q

What are the two different types of conjugations?

A

1) Conjugation between Mobilizer and Donor strains
2) Conjugation between Donor and Recipient strains

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53
Q

What are genomic clone banks?

A

Contain the complete genome as large fragments on plasmids

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54
Q

What are cosmid vectors?

A

Plasmids that include E.coli phage lambda cos site

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55
Q

What are lambda heads also known as?

A

Capsids

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56
Q

What is the size of the lambda genome?

A

48.5 kb

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57
Q

What does the cos site allow?

A

Packaging into lambda heads of recombinant plasmids close in size (37-52kb) to the lambda genome

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58
Q

How are cosmid clone banks useful?

A

The clones contain large enough stretches of DNA to allow for complementation studies

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59
Q

How are shotgun cloning libraries created?

A

1) Cut a vector
2) Cut genomic DNA
3) Ligate both of these together

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60
Q

What type of clone bank has a higher proportion of small rather than large fragments of cloned DNA?

A

Shotgun clone bank

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61
Q

What can also produce chimeric clones?

A

ligation

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62
Q

How many EcoRI sties does the vector pLAFR1 contain?

A

1

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63
Q

What are the steps needed for the preparation of vector DNA?

A

1) Must be completely digested by EcoRI
2) Linearized

64
Q

What is the goal of preparation of genomic DNA for a cosmid clone bank?

A

To have large (20kb) fragments with intact (functional) genes and operons

65
Q

What is chromosomal DNA subjected to in regards to preparation of genomic DNA for a cosmid clone bank?

A

Partial digestion with a restriction endonuclease

66
Q

How do you prepare genomic DNA for a cosmid clone bank?

A

1) Use low concentrations of enzyme or short digestion times
2) Do several partial digestions

67
Q

What is size fractionation?

A

You can size fraction DNA in a sucrose gradient, these sizes will be separated through a gradient in sucrose

68
Q

What are the steps to Ligation?

A

1) Mix restricted genomic and plasmid DNA
2) add DNA ligase enzyme, plus ATP and Mg2+

69
Q

What are the undesired products of ligation?

A

1) Religation of plasmid itself
2) Ligation of non-contigous stretches of S.meliloti
3) Cloning of small pieces of S.meliloti DNA into pLAFR1

70
Q

What is the desired ligation product?

A

Concatemers

71
Q

What are concatemers?

A

Linear fusion of DNA fragments

72
Q

What does ligation result in?

A

cos sites approximately 48kb apart

73
Q

Why is the result of cos sites being 48kb apart in ligation a good thing?

A

It’s the perfect distance for packaging by bacteriophage lambda

74
Q

Why doesn’t pLAFR1 carry tra genes?

A

1) They are numerous and thus are encoded over many kb
2) Plasmid would be too big to be packaged into lambda gene

75
Q

What does bacteriophage lambda packaging extract not contain?

A

no Lambda DNA

76
Q

What does packaging of a cosmid clone bank entail?

A

Combine ligated DNA with a lambda packaging extract

77
Q

In terms of packaging of a cosmid clone bank, what is the condition for the lambda head to cut DNA?

A

Lambda head will cut DNA at a cos site and will package the DNA only if there is a second two cos sites 40kb from the first

78
Q

What are the ligation products most likely to be produced?

A

1) Concatemers of chromosomal DNA ligated to a complete copy of pLAFR1

79
Q

What is the only thing that is able to replicate in E.coli cells after transfection?

A

Packaged products with a complete copy of pLAFR1

80
Q

What does not occur in shotgun cloning but does in cosmid?

A

Size selection as the critical step as it eliminates the undesired ligation products

81
Q

What is transfection?

A

The use of viruses or viral particles to transfer DNA into a cell

82
Q

In transfection, what happens upon infection of E.coli cells?

A

The DNA will be injected and recombination between the cos sites will circulize the plasmid?

83
Q

Why will plaques not be formed in transfection?

A

None of the phage heads contain a virus genome

84
Q

Why are overlapping clones desired?

A

Ensures all genes and operons are present

85
Q

How can clones be isolated?

A

Complementation studies

86
Q

What does complementation select for?

A

Genes that encode functional gene products

87
Q

What can lambda be used for?

A

specialized transduction in E.coli

88
Q

What can phiM12 be used for?

A

Generalized transduction in S. meliloti

89
Q

What are factors influencing plasmid stability?

A

1) copy number
2) Partitioning
3) Incompatibility

90
Q

What are the two ways in which complementation groups can be defined?

A

1) A set of mutant alleles that are complemented by a plasmid carrying the same WT genes
2) A set of mutant alleles that fail to complement each other

91
Q

What type of copy number plasmids should complementation experiments be done with? (high or low) and what is an example of one?

A

Low copy number plasmids
ex. pLAFR1

92
Q

What is the issue with high copy number plasmids?

A

Over-expression and thus may result in pleiotropic effects

93
Q

In terms of Merodiploid analysis experiment 1, what are the three genes E.coli requires?

A

galK, galT, and galE

94
Q

In terms of Merodiploid analysis experiment 1, what is the plasmid that carries all three genes?

A

pGAL101

95
Q

In terms of Merodiploid analysis experiment 1, which mutants does pGal101 complement?

A

galK, galT, and galE

96
Q

How many genes are involved in arabinose metabolism in S.meliloti?

A

4! (Ara1,ara2,ara3,ara4)

97
Q

What are the two ways in which complementation groups be defined?

A

1) A set of mutant alleles that are complemented by the same WT gene
2) A set of mutant alleles that fail to complement each other

98
Q

What was the objective of merodiploid experiment #3 which a MSc student did at McGill?

A

To understand acetate metabolism in S. meliloti

99
Q

What are the four steps of Merodiploid experiment 3?

A

1) Isolate mutants
2) Eliminate non-acetate specific mutants
3) Isolate complementing clones
4) Merodiploid analysis

100
Q

What is transduction?

A

The transfer of DNA from one cell to another via phage particle

101
Q

What are transducing particles?

A

Phage particles carrying bacterial DNA

102
Q

What are virulent particles?

A

Phage particles carrying viral DNA only

103
Q

What are the two types of bacteriophages?

A

1) Lytic Phages
2) Temperate phages

104
Q

What is the lytic cycle?

A

Infection by virulent particles lead to cell lysis

105
Q

How is phage DNA packaged by?

A

Headful mechanism

106
Q

How does phage DNA replicate?

A

Rolling circle mechanism

107
Q

How does packaging by headful mechanism work?

A

1) Cut on replicated phage genome concatemer occurs at pac site
2) All the following cuts on the concatemer occur at ‘headful distance’ from the initial pac site

108
Q

What is the result of packaging by headful mechanism?

A

Virulent phage particles in which the terminal sequences of the genome are redundant

109
Q

What are the conditions that need to be met for generalized transduction to be possible?

A

1) Sequences in the bacterial genome similar to pac sites
2) Phage pac nucleases incorrectly recognizes these sites and begin cleavage

110
Q

What are the two things infection by virulent particles leads to?

A

1) Cell lysis
2) Integration of phage DNA into the host genome

111
Q

What does the lambda genome do following infection?

A

circularize

112
Q

How does the lambda genome when infected?

A

Annealing of the sticky ends of the cos sites

113
Q

What are the two steps required in phage DNA replication?

A

1) Theta replication
2) rolling circle replication

114
Q

What does rolling circle replication produce?

A

Linear concatemers of the phage genome

115
Q

When does the lysogenic cycle occur?

A

Only if the lytic cycle is not induced

116
Q

What are lysogens?

A

Bacterial cells carrying prophage

117
Q

What are the three different ways prophage can be maintained in the host genome?

A

1) Integrated into the hot genome via site-specific recombination
2) Replicated in a circular plasmid like form
3) Integrated into non-specific (random) sites in the host genome in a transposon-like manner

118
Q

What is lambda (vector)?

A

A virus that infects E.coli

119
Q

What is included in the integration of lambda into the E.coli genome?

A

1) Recombination between homologous attachment sites on the phage (attP) and host (attB) genomes
2) Involves site-specific recombination enzymes

120
Q

What are the site-specific recombination enzymes involved in lambda integration into E.coli genomes?

A

1) lambda integrase (Int)
2) Lambda excisionase (Xis)

121
Q

What is the protein CI?

A

A gene regulatory protein (repressor) that prevents expression of the lytic cycle genes

122
Q

What is the function of CI when it comes to lambda?

A

CI holds lambda in the lysogenic cycle

123
Q

How is artificial lysogenic induction mediated?

A

Exposure of lysogens to UV light (via SOS response by cell) or heat

124
Q

What are single stranded breaks called in the DNA?

A

nicks

125
Q

What are the 5 DNA damage and causative agents?

A

1) Pyrimidine Dimers (UV light)
2) Single-strand nicks and double-strand breaks (ionizing radiation)
3) Mismatched base pairs (spontaneous deamination)
4) Covalent cross-links (UV light)
5) Apurinic and apyrimidinic sites (spontaneous hydrolysis)

126
Q

What does UV cause the formation of?

A

Pyrimidine dimers

127
Q

What is catalyzed by UV?

A

Covalent bonding of adjacent T bases

128
Q

Which polymerase does DNA damage block?

A

Polymerase 3

129
Q

What are the two general classes of repair mechanisms for thymine dimers?

A

1) Non-mutagenic (“faithful”) repair
2) Error-prone (“SOS”) repair (transdimer synthesis)

130
Q

What are the 3 major mechanisms of non-mutagenic DNA repair?

A

1) Photo reactivation
2) Nucleotide Excision Repair (NER)
3) Recombinant Repair

131
Q

What is referred to as light repair?

A

Photo-reactivation

132
Q

What is referred to as dark repair and does not require visible light?

A

2) Nucleotide excision repair
3) Recombination Repair

133
Q

What is photoreactivation?

A

If cells were flashed with UV light before plating

134
Q

What is the mechanism of photoreactivation?

A

Photolyase enzyme cleaves T-T dimers

135
Q

What is a liquid holding recovery?

A

If cells were incubated in a dark, rich liquid medium

136
Q

phr mutants survive as well as a WT strain if they’re subjected to what?

A

Liquid holding recovery prior to plating

137
Q

What are the components NER requires?

A

1) UvrABC excision endonuclease
2) UvrD helicase
3) DNA polymerase 1
4) DNA ligase
5) ATP

138
Q

What is RecA

A

Protein that’s essential for the repair and maintenance of DNA

139
Q

What are the reasons why Nucleotide Excision Repair could fail to correct a mutation?

A

It takes time!

140
Q

What happens if NER fails to correct a mutation?

A

POL 3 can eventually bypass the damage

141
Q

What cuts the parental strand opposite of the gap?

A

RecBCD

142
Q

What facilitated the migration of the 3’ end of cut strand?

A

RecA

143
Q

What is the gap on parental strand resulting from strand exchange filled by?

A

Pol1

144
Q

How does SOS system repair DNA?

A

Blindly inserting bases when template strand cannot be read

145
Q

What did Weigle observe?

A

1) Reactivation of UV-damaged phage
2) High mutation frequency in “surviving” page

146
Q

What are the two proteins which regulates the SOS operon?

A

LexA and RecA

147
Q

What is LexA?

A

Repressor of SOS-operon

148
Q

What is RecA?

A

Inducer of SOS-operon

149
Q

What are the four treatments of the Weigle Reactivation Experiments?

A

1) Lambda phage treated with high UV dose
2) lambda phage not treated with UV
3) E.coli cells exposed to a low UV dose
4) E.coli cells not treated with UV

150
Q

In terms of the Weigle reactivation, what can mutations cause lambda to form?

A

Clear plaques

151
Q

What do plaques made by lytic phage tend to be (clear or opaque)?

A

Clear

152
Q

For the Weigle reactivation, what does lethal DNA damage result in?

A

Reduction of number of plaques

153
Q

For the Weigle reactivation, how does the number of plaques decrease?

A

As UV dose increases

154
Q

What are the results observed for the first Weigle experiment?

A

1) High yield of phage
2) Low frequency of mutation in phage

155
Q

What are the 3 conclusions of SOS system?

A

1) Able to repair potentially-lethal DNA damage
2) Error-prone
3) Inducible

MICR 338 (3 decks)

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