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157 Cards in this Set

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Stage 1 Rolling-Circle Replication
The Rep protein binds to DSO; SS break; Rep remains attached to 5' end; DNA-PolIII uses free 3' end as primer; Replication around the circle; Second break by Rep and ligation by Rep; Give replicated DS plasmid and SS plasmid
Theta Replication
Most common form of plasmid replication; Uni- or bidirectional; Gram negative plasmids
Gel Electrophoresis
Purified plasmid from other processes; DNA migrates according to size and shape; supercoiled moves furthest
Separation on CsCl-EtBr gradient
Centrifugation separates based on density; EtBr intercalates into DNA but not plasmid as it is circular; Plasmid is lower in density
Amplification by Cloramphenicol
Chloramphenicol inhibits chromosomal replication by protein synthesis inhibition; plasmid continues to replicate, building up inside the cell
Alkaline lysis
Treat bacteria with lysozyme and alkali which lyses the cell and denatures DNA; Add salt which precipitates DNA and cell wall material; centrifuge; Detect by running supernatant on agarose
4 techniques for plasmid isolation
Alkaline lysis; Amplification with chloramphenicol; separation on ScCl-EtBr gradient; Gel electrophoresis
Curing Experiment
Grow bacteria in prescence of acridine orange or EtBr which causes loss of plasmids; Thest whether trait is lost when plasmid is lost
What do you use to determine whether mutants act in cis or trans?
Complementation Test
Stage 2 Rolling-Circle Replication
RNA polymerase makes a primer at SSO and this RNA primes replication around the circle by DNA Polymerase III;
How is the second stage of rolling-circle replication regulated
the SSO is located immediately before the DSO so the SSO will not appear until the first stage is almost complete
What are relaxed Plasmids?
high copy-number plasmids
What is ColE1-derived Plasmid replication regulated by
How does RNAI regulate ColE1-derived plasmid replication
forms a kissing complex with RNAII. RNAII is then not available as a primer to start replication
What is Rop?
Stabilizes kissing complex of RNAI-RNAII complex
What is Handcuffing or Coupling?
RepA, which regulates plasmid replication, binds to iterons on two plasmids, linking the two and preventing both from replicating
What are the parts of the par-system
parS - cis acting site; plasmids are pulled apart
parA - ATPase, binds to parS/parB complex
parB - binds to parS
How do kill-proteins work in relation to plasmids
production of a protein that kills cells that do not contain the plasmid. The plasmid expresses an antidote. Both the toxin and antitoxin are present in cells with the plasmid. If the plasmid is lost, the toxin is activated by the instability of the antitoxin
What is Inc
An incompatibility group of different plasmids based on identical replication control and identical par system
What is Conjugation?
Plasmid mediated exchange of DNA between bacteria
What is self-transmissible plasmid?
both conjugative and mobilizable
What is a Conjugative plasmid
Can form pairs
What is a Mobilizable plasmid
Can be moved by self transmissible plasmid
What is a donor in conjugation
bacterium containing the conjugative plasmid initially
What is a recipient in conjugation
plasmid free bacterium
What is a transconjugate in conjugation
result of a mating. It is now a potential donor
What is promiscuous in conjugation
conjugative plasmid with a wide host range
Most naturally occuring plasmids are what in relation to conjugation?
transmissable or mobilizable
How are promiscuous plasmids important?
important in evolution and spread of antibiotic resistance
What is the cis-acting site in genes of self-transmissable plasmids
What are trans-acting factors encoded by in self-transmissable plasmids
What is a Dtr component
DNA transfer and replication
What is an Mpf component
mating pair formation
What does an mpf form
large, membrane associated structures between mating pairs, holding them together
How do coupling proteins communicate
through dtr and mpf
Are pili vehicles of DNA transfer?
In the F plasmid tranfer in E. coli, what does F+ make and which cell contains the plasmid (donor or recipient)?
makes sex pilus
What two items are required for transfer in conjugation
relaxase and oriT
What does relaxase do
creates a ss break at the nic site in oriT
What does relaxase interact with during tranfer
coupling protein of mpf-complex
What are male specific phages
bacteriophages that use sex pili as a receptor
What is M13
a filamentous, ss DNA, phage
The expression of tra genes depends on what
TraJ, a transcriptional activator
What regulates traJ expression
finO and finP
FinO stabilizes what
How does F always contain a pili
F lacks finO, a fertility inhibitor, so the pili is always there
What is required for mobilization
self-transmissable plasmids
What do mobilizable plasmids not encode for
mpf gene functions
what region encodes tra genes in mobilizable plasmids
What does the mob region do in terms of host range
expands host range
Agrobacterium tumefaciens has what kind of plasmid
Ti or tumor inducing
Ti plasmid contains two DNA transfer machineries. What are they?
bacterium to plant and bacterium to bacterium
A pilus is not involved in what kind of conjugation
conjugation in gram positives
Pheremone plasmids are found in what bacteria
Enterococcus faecalis
What are pheremone plasmids
plasmids that can carry antibiotic resistances and virulence factors giving E. faecalis a high rate of antibiotic resistance
What activates plasmid transfer in inducible plasmid transfer
Peptides secreted by recipient and used to activate plasmid in donor
Which cell releases the pheremones
What is transformation
uptake of naked DNA molecules
What is compentent in reference to transformation?
capability to take up DNA
What is a transformant in transformation?
Cells that successfully took up DNA
What is natural competence in transformation?
Bacteria that can take up DNA on their own
What is artificial competence in transformation?
chemical or physical manipulation to achieve competence in bacteria
In transformation, do ds DNA or ss DNA enter into the cytoplasm of the cell
One strand is degraded in a ds DNA and a ss enters into the cytoplasm
Integration of naked DNA into the cell's DNA is done by what process
homologous recombination
What are the com genes in Bacillus subtilis
comEFG - operon structural genes
comEA - DNA binding
comF - DNA translocation
comG - channel formation
When does B. subtilis develop competence and sporulates
under nutrient deprivation
comEFG regulated by what
comA and comK
What is meant by induction density dependent in respect to regulation of competence in B. subtilis
accumulation of peptide signals is required to induce competence
comX pheremone and CSF are used in what process
regulation of compentence in B. subtilis
CSF is imported into the cell by what
How do you test DNA uptake efficiency in respect to naked DNA uptake
Label DNA, Add to cells, treat mix with DNase, Collect cells on filter
How do yout test specificity in DNA uptake
DNase treatment of the DNA outside of the cell,
What is the mechanism of DNA uptake in S. pneumoniae
dsDNA binds to receptors on cell surface, broken into smaller pieces by endonucleases, one strand degraded, one transported into cytoplasm, integration into chromosome by recombination
What is the mchanism of DNA uptake in H. influenzae
initial uptake of dsDNA in transformasome
How do we know that ds DNA is broken down and ss DNA enters the cytoplasm
DNase only breaks down dsDNA
Plasmids have to be what in order to be taken up in naturally competent bacteria
has to be dimeric so it can recyclize once inside the cell
What are the possible roles for natural competence
DNA repair although compentence genes not induced in response to DNA damage
Nutrition is unlikely
What is the chemical treatment for artificially induced competence
CaCl2. This is used for plasmid transformation
What is electroporation
an intense electric field used in artificially induced competence
What are bacteriophages
viruses that infect bacteria
What are plaques
holes in bacterial lawn from phage activity
What is the host range of phages
specific to bacterial strains
What is T4
One of the largest known viruses
What are immediate-early and delayed-early genes transcribed from
o70 promoters
Middle genes require what as alternate promoters
delayed-early gene products
What do the true late genes code for
Head, tail, and tail fibers
What is the alternate promoter factor in true late genes
Some true late genes are only expressed if what occurs
DNA replication
What is required for gp55-RNA pol complex to start transcribing
interaction between virus sliding clap and gp33
Is T4 linear or circular
What is a concatemer formation
individual DNA linked end to end
What are the two stages in DNA replication in T4
replication from well-defined origins; ss ends invade daughter strands, form D-loops, prime replication to form branched concatemers. This is recombination dependent replication.
What is the first stage of DNA replication in T4
Primers invade DNA strand, form R-loop which primes the leading strand
What is the second stage of replication in T4
3' end serves as primer, producing long concatemers, which are then ready for packaging
What is required for pairing of invading strand in T4 DNA replication
What is cut from concatemers during packaging
individual genome length DNA
How is terminal redundancy created during packaging
a larger than necessary phage head
Are filamentous phages ss or ds
When is the protein coat removed with filamentous phages
removed on entry into the cell
Do cells lyse with filamentous phages
What is the + strand in phage replication
infecting DNA
What is RF in phage replication
replicative form
What is primed by host RNA-Pol in replication of ssDNA phages in M13
negative strand, ligation, and supercoiling
How is the + strand replicated in phage replication
rolling circle replication
What is used to nick the ori in phage replication during RC replication
endonuclease GpII
What does GpV do in phage replication
binds to ssDNA and prevents the negative strand formation
Why can M13 be used as a cloning vehicle
no fixed length
What are permissive conditions
phages can multiply
What are non-permissive conditions
phage cannot multiply
What is MOI
multiplicity of infection
number of phage/number of bacteria
What does a high MOI signify
phage numbers greatly exceed bacterial numbers
What are crosses in reference to MOI
infection of one host with two genetic distinct phages
How do you find percentage of population infected in a cross
take the percentage of infected population by one phage and square it.
What type of Test uses the permissive host for both phages
Recombination test
What uses a non-permissive host for testing
Complementation test
In T4 analysis of rII how do you make a phenotypic distinction between the mutant and wild type
mutant has hard edges
WT has fuzzy edges
What type of phage has a stable relationship with the host
lysogenic phage
What is the phage called in lysogenic phages
What is the host in lysogenic phages
When are lysogenic phages activated then leave the host
cell damage
Lysogenic phages prevent what from other phages of the same type
What are the first genes expressed in phage lambda
N and cro
What is coded for in late gene expression in phage lambda
head, tail, lytic enzymes
What is cro in phage lambda
inhibitor of repressor synthesis, which is required for lysogeny
What is N in phage lambda
antitermination factor
What does N bind to and interact with
binds to nut, interacts with RNA-Pol
What is the general name for host proteins involved in antitermination
What joins ends of linear lambda DNA and what are the cohesive ends called
cohesive ends called cos sites
DNA ligase joins ends
During packaging in lambda phage replication, where are the head proteins recognized and cut
cos sites
What are the two lambda gene requirements in lambda replication
O and p, a primer at ori
What does gam do in lambda replication
shifts to rolling circle replication
what does gam mutant do in lambda replication
shifts to theta replication
What are cII and cIII transcribed from
respectively, pR and pL
What is the function of cIII in Lysogeny
inhibits host protease that degrades cII
What is the function of cII in Lysogeny
activates transcription of pI which integrates the phage
cI shuts down what in the Lysogeny system
pR and pL
Integrase promotes what in Lambda integrating
site specific recombination between attP(attachment site phage) and attB(bacteria attachment site)
What is meant by the repressor in cI synthesis in Lambda integration acting as a homodimer
repressor not active at low concentrations
What is oR1 and what does it repress in relation to cI synthesis in lambda integration
binding site for cI in low concentrations. represses pR.
What does oR2 activate in relation to lambda integration
in middle concentrations of cI, it binds to oR2 and activates transcription of cI from pRM
What does oR3 repress in relation to lambda integration
at high concentration of cI, it binds to oR3 and represses pRM and no transcription of cI
What two things lead to the degradation of cI
RecA + ssDNA
During induction of lambda, Cro binds to what, preventing what
binds to oR, preventing cI transcription
What is Mu
A mutator phage
What does an Mu consist of
transposon with phage coat
What depends on the prescence of P2 as an example of parasitic growth on a virus
What does Sid do in P2-P4 interaction
causes P2 proteins to make smaller heads, thus only fitting P4 DNA into them
What are pathogenicity islands
large selections of bacterial genomes that are not common to all strains
in packaging of host DNA into phage head in transduction, what is the phage unable to do
generate progeny
Transducing lambda are a defect in either one of two things. What are these
head and tail formation or lysogen formation
Generally, what are transposons
DNA elements that can hop
What is the responsible enzyme in transposons
What are the smallest transposons called
Insertion sequence or IS elements
Most times, what do two IS elements of the same type flanking other genes mean
antibiotic resistance genes
What are suicide vector systems
replication defective phages or plasmids
What is replicative transposition
copy of transposon in donor and target DNA
cut and paste transposition
only one copy remains; donor DNA is destroyed