Midterm 2 Materials Flashcards
What are the advantages of plasmids?
Antibiotic resistance, toxin degradation, virulence and/or symbiosis-related functions
What do Col plasmids do?
Synthesize colicin proteins
What are colicin proteins?
Type of bacteriocin unique to E.coli strains
What are bacteriocins?
Proteins that can kill sensitive bacteria
What are R Plasmids (Resistance)?
They carry resistance to antibiotics and other factors
What are characteristics of R Plasmids?
Often broad-host-range and self-transmissible
What do F (Fertility) plamids do?
Carry the F pilus and other conjugation factors
What is the characteristic of F plasmids?
Self-transmissible
What are F’ plasmids?
Derivatives of F carrying cell DNA
What can F plasmids be transferred into?
F-
What are the genes relevant to F plasmids?
oriT and tra
What are Hfr strains?
High frequency of recombination
What do Hfr cells have?
Replication fusion of F and the chromosome
What happens when the F plasmid is integrated into the chromosome?
It can mobilize the entire genome
What does the oriT mark?
The beginning of DNA transfer
What is conjugation?
Transfer of genetic material between cells via direct cell-to-cell contact
What’s the difference between an F+ and F-cell?
F- lacks fertility plasmid
What is an F+ cell?
An E.coli cell with its own chromosome
Whats the difference between an F+ cell and Hfr?
Hfr cell has the F factor within its chromosome
What is an F’ factor?
It’s an F factor that’s carrying some of the bacterial chromosome with it
For Hfr strains, where is the higher recombination frequency?
For genes closer to and downstream of Hfr oriT
How are recombinant plasmids made?
Combining parts of various plasmids and phages with properties like pRK600, pLAFR1, and pBSKS
What is pRK600?
An R plasmid with colE1 replicon
What is pLAFR1?
An R plasmid with lambda cos but without tra genes
What is pBSKS?
A highly modified col plasmid
What does pBSKS contain?
Phage, bacterial & synthetic sequences
What are examples of phage based vectors?
Lambda and M13
What are Ti and RI
Plasmid derivatives for plant transformation
What do Ti plasmids form?
Plant tumours
What is merodiploid?
Partial diploid
What do merodiploid may have?
Duplicated genes on a plasmid or recombined into the genome
What is complementation?
1 gene makes up for the lack of function of another one
What is a conjugative plasmid?
Plasmid carrying the genes that determine the effective contact function
What is a mobilizable plasmid?
Can prepare its DNA for transfer
What is a self-transmissible plasmid?
Like F, is both conjugative and mobilizable
What is an example of self-transmissible plasmids?
pRK600
What is an example of a plasmid that’s mobilizable but not self-transmissible?
pLAFR1
What is an example of non-mobilizable plasmids?
pBSKS
What is a condition that needs to be met for non-mobilizable plasmids?
Must be introduced into cells via transformation
What are the functions of Merodiploid analysis?
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
How can pRK600 be used?
1) As a suicide vector to deliver transposons to cells in which it cannot replicate
2) To mobilize other plasmids
What are the functions of the tra genes
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
Where is the DNA via conjugation transferred from?
DNA is transferred from oriT in a ssDNA form and in a unidirectional led by the 5’ end
What is the uptake of DNA into the recipient followed by?
1) Synthesis of the second DNA strand
2) Reformation of the plasmid
3) Homologous recombination into the recipient gene
How is chromosomal DNA be mobilized?
When oriT is inserted into the genome
How can the oriT be inserted into the genome?
1) An oriT carrying tansposon (Tn5 - mob)
2) Integration of an oriT-carrying plasmid like F (Hfr strains)
What is Recombination frequency?
The frequencies at which different donor alleles are recombined into a recipient genome
What does the closer a gene is to the oriT entail?
The higher the recombination frequency
What is the objective of triparental matings?
Isolate a recipient strain with the target plasmid
What are the strains required in triparental matings?
Donor, Mobilizer, and Recipient
How many plasmid transfers via conjugation are required in triparental mobilization?
2
What are the two different types of conjugations?
1) Conjugation between Mobilizer and Donor strains
2) Conjugation between Donor and Recipient strains
What are genomic clone banks?
Contain the complete genome as large fragments on plasmids
What are cosmid vectors?
Plasmids that include E.coli phage lambda cos site
What are lambda heads also known as?
Capsids
What is the size of the lambda genome?
48.5 kb
What does the cos site allow?
Packaging into lambda heads of recombinant plasmids close in size (37-52kb) to the lambda genome
How are cosmid clone banks useful?
The clones contain large enough stretches of DNA to allow for complementation studies
How are shotgun cloning libraries created?
1) Cut a vector
2) Cut genomic DNA
3) Ligate both of these together
What type of clone bank has a higher proportion of small rather than large fragments of cloned DNA?
Shotgun clone bank
What can also produce chimeric clones?
ligation
How many EcoRI sties does the vector pLAFR1 contain?
1
What are the steps needed for the preparation of vector DNA?
1) Must be completely digested by EcoRI
2) Linearized
What is the goal of preparation of genomic DNA for a cosmid clone bank?
To have large (20kb) fragments with intact (functional) genes and operons
What is chromosomal DNA subjected to in regards to preparation of genomic DNA for a cosmid clone bank?
Partial digestion with a restriction endonuclease
How do you prepare genomic DNA for a cosmid clone bank?
1) Use low concentrations of enzyme or short digestion times
2) Do several partial digestions
What is size fractionation?
You can size fraction DNA in a sucrose gradient, these sizes will be separated through a gradient in sucrose
What are the steps to Ligation?
1) Mix restricted genomic and plasmid DNA
2) add DNA ligase enzyme, plus ATP and Mg2+
What are the undesired products of ligation?
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
What is the desired ligation product?
Concatemers
What are concatemers?
Linear fusion of DNA fragments
What does ligation result in?
cos sites approximately 48kb apart
Why is the result of cos sites being 48kb apart in ligation a good thing?
It’s the perfect distance for packaging by bacteriophage lambda
Why doesn’t pLAFR1 carry tra genes?
1) They are numerous and thus are encoded over many kb
2) Plasmid would be too big to be packaged into lambda gene
What does bacteriophage lambda packaging extract not contain?
no Lambda DNA
What does packaging of a cosmid clone bank entail?
Combine ligated DNA with a lambda packaging extract
In terms of packaging of a cosmid clone bank, what is the condition for the lambda head to cut DNA?
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
What are the ligation products most likely to be produced?
1) Concatemers of chromosomal DNA ligated to a complete copy of pLAFR1
What is the only thing that is able to replicate in E.coli cells after transfection?
Packaged products with a complete copy of pLAFR1
What does not occur in shotgun cloning but does in cosmid?
Size selection as the critical step as it eliminates the undesired ligation products
What is transfection?
The use of viruses or viral particles to transfer DNA into a cell
In transfection, what happens upon infection of E.coli cells?
The DNA will be injected and recombination between the cos sites will circulize the plasmid?
Why will plaques not be formed in transfection?
None of the phage heads contain a virus genome
Why are overlapping clones desired?
Ensures all genes and operons are present
How can clones be isolated?
Complementation studies
What does complementation select for?
Genes that encode functional gene products
What can lambda be used for?
specialized transduction in E.coli
What can phiM12 be used for?
Generalized transduction in S. meliloti
What are factors influencing plasmid stability?
1) copy number
2) Partitioning
3) Incompatibility
What are the two ways in which complementation groups can be defined?
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
What type of copy number plasmids should complementation experiments be done with? (high or low) and what is an example of one?
Low copy number plasmids
ex. pLAFR1
What is the issue with high copy number plasmids?
Over-expression and thus may result in pleiotropic effects
In terms of Merodiploid analysis experiment 1, what are the three genes E.coli requires?
galK, galT, and galE
In terms of Merodiploid analysis experiment 1, what is the plasmid that carries all three genes?
pGAL101
In terms of Merodiploid analysis experiment 1, which mutants does pGal101 complement?
galK, galT, and galE
How many genes are involved in arabinose metabolism in S.meliloti?
4! (Ara1,ara2,ara3,ara4)
What are the two ways in which complementation groups be defined?
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
What was the objective of merodiploid experiment #3 which a MSc student did at McGill?
To understand acetate metabolism in S. meliloti
What are the four steps of Merodiploid experiment 3?
1) Isolate mutants
2) Eliminate non-acetate specific mutants
3) Isolate complementing clones
4) Merodiploid analysis
What is transduction?
The transfer of DNA from one cell to another via phage particle
What are transducing particles?
Phage particles carrying bacterial DNA
What are virulent particles?
Phage particles carrying viral DNA only
What are the two types of bacteriophages?
1) Lytic Phages
2) Temperate phages
What is the lytic cycle?
Infection by virulent particles lead to cell lysis
How is phage DNA packaged by?
Headful mechanism
How does phage DNA replicate?
Rolling circle mechanism
How does packaging by headful mechanism work?
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
What is the result of packaging by headful mechanism?
Virulent phage particles in which the terminal sequences of the genome are redundant
What are the conditions that need to be met for generalized transduction to be possible?
1) Sequences in the bacterial genome similar to pac sites
2) Phage pac nucleases incorrectly recognizes these sites and begin cleavage
What are the two things infection by virulent particles leads to?
1) Cell lysis
2) Integration of phage DNA into the host genome
What does the lambda genome do following infection?
circularize
How does the lambda genome when infected?
Annealing of the sticky ends of the cos sites
What are the two steps required in phage DNA replication?
1) Theta replication
2) rolling circle replication
What does rolling circle replication produce?
Linear concatemers of the phage genome
When does the lysogenic cycle occur?
Only if the lytic cycle is not induced
What are lysogens?
Bacterial cells carrying prophage
What are the three different ways prophage can be maintained in the host genome?
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
What is lambda (vector)?
A virus that infects E.coli
What is included in the integration of lambda into the E.coli genome?
1) Recombination between homologous attachment sites on the phage (attP) and host (attB) genomes
2) Involves site-specific recombination enzymes
What are the site-specific recombination enzymes involved in lambda integration into E.coli genomes?
1) lambda integrase (Int)
2) Lambda excisionase (Xis)
What is the protein CI?
A gene regulatory protein (repressor) that prevents expression of the lytic cycle genes
What is the function of CI when it comes to lambda?
CI holds lambda in the lysogenic cycle
How is artificial lysogenic induction mediated?
Exposure of lysogens to UV light (via SOS response by cell) or heat
What are single stranded breaks called in the DNA?
nicks
What are the 5 DNA damage and causative agents?
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)
What does UV cause the formation of?
Pyrimidine dimers
What is catalyzed by UV?
Covalent bonding of adjacent T bases
Which polymerase does DNA damage block?
Polymerase 3
What are the two general classes of repair mechanisms for thymine dimers?
1) Non-mutagenic (“faithful”) repair
2) Error-prone (“SOS”) repair (transdimer synthesis)
What are the 3 major mechanisms of non-mutagenic DNA repair?
1) Photo reactivation
2) Nucleotide Excision Repair (NER)
3) Recombinant Repair
What is referred to as light repair?
Photo-reactivation
What is referred to as dark repair and does not require visible light?
2) Nucleotide excision repair
3) Recombination Repair
What is photoreactivation?
If cells were flashed with UV light before plating
What is the mechanism of photoreactivation?
Photolyase enzyme cleaves T-T dimers
What is a liquid holding recovery?
If cells were incubated in a dark, rich liquid medium
phr mutants survive as well as a WT strain if they’re subjected to what?
Liquid holding recovery prior to plating
What are the components NER requires?
1) UvrABC excision endonuclease
2) UvrD helicase
3) DNA polymerase 1
4) DNA ligase
5) ATP
What is RecA
Protein that’s essential for the repair and maintenance of DNA
What are the reasons why Nucleotide Excision Repair could fail to correct a mutation?
It takes time!
What happens if NER fails to correct a mutation?
POL 3 can eventually bypass the damage
What cuts the parental strand opposite of the gap?
RecBCD
What facilitated the migration of the 3’ end of cut strand?
RecA
What is the gap on parental strand resulting from strand exchange filled by?
Pol1
How does SOS system repair DNA?
Blindly inserting bases when template strand cannot be read
What did Weigle observe?
1) Reactivation of UV-damaged phage
2) High mutation frequency in “surviving” page
What are the two proteins which regulates the SOS operon?
LexA and RecA
What is LexA?
Repressor of SOS-operon
What is RecA?
Inducer of SOS-operon
What are the four treatments of the Weigle Reactivation Experiments?
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
In terms of the Weigle reactivation, what can mutations cause lambda to form?
Clear plaques
What do plaques made by lytic phage tend to be (clear or opaque)?
Clear
For the Weigle reactivation, what does lethal DNA damage result in?
Reduction of number of plaques
For the Weigle reactivation, how does the number of plaques decrease?
As UV dose increases
What are the results observed for the first Weigle experiment?
1) High yield of phage
2) Low frequency of mutation in phage
What are the 3 conclusions of SOS system?
1) Able to repair potentially-lethal DNA damage
2) Error-prone
3) Inducible
