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111 Cards in this Set

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Why does E.Coli have a lower chance of receiving a mutation?
This is possible because E.Coli has a high fidelty machinery in DNA replication
Besides spontaneous mutation, how else might one receive a mutation?
Induced: Mutagens (alkylating agents, UV radiation or base analogs), including radiation, chemicals, DNA (transposons, viral/phage insertions), other
What are five forms of mutation?
Point mutations – single base change
Deletions – portion of sequence is lost
Insertions – DNA acts as a mutagen
Duplications – makes two or more copies of a sequence
Transpositions – DNA sequence moves from one place to another
Why is it bad when there is base tautomerization ?
The base can go from enol to keto and vice versa, which ultimately changes where the base can hydrogen bond to. For example:
Thymine going to enol means it can bind to guanine
Cytosine and Adenine can tautomerize to an imino form lead to _:_ base pairs
C:A
Tautomerization leads to a ________ mutation
Transition;
Tautomerization don't create transversion
Why are transversion mutations rare?
They are rare due to steric hinderance
What are the three general mutagens?
Radiation
Viral
Chemical
UV radiation causes what?
Thymine dimers
BrdU is a what and describe what BrdU does?
This is a base analog of Thymine and it will compete with Thymine.


BrdU will be incorporated by DNA polymerase. BrdU will compete with thymine for
Incorporation.

BrdU is much more likely to adopt the enol form and improperly base pair than Thymine. This leads to
BrdU:Guanine base pairing, a transition mutation (purine to purine)
Since E.Coli is an haploid this means what?
All traits are dominant
What is the difference between prototroph and auxotroph?
Prototroph: wild type, capable of growing on minimal media
Auxotroph: mutant, must have media supplemented
What are the four enzymes needed to create phenylalanine?
Phosphoenolpyruvate →(1) shikimate →(2) chorismate →(3) prephenate→(4) phenylalanine
What is a conditional mutation?
Mutation only expressed or observed under certain conditions
What is usually the reason for temp sensitive mutations?
Proteins that can't work at elevated temperatures.
What is the meaning of Resistant?
survival of something that kills normal/wild type cells
What is a true reversion?
This when a mutation returns back to its original nucleotide and protein sequence
What are two types of equivalent reversions?
Change in nucleotide sequence, but same protein sequence
e.g: Original mutation mutates serine to proline. Reversion changes proline back to serine, but nucleotide sequence is different from original (two different serine codons)




Conservative amino acid substitution – nucleotide and protein sequence changed, but enzyme still functional (pseudo wild-type)
e.g. original mutation changes aspartate (acidic) to leucine (hydrophobic) and protein loses function. Reversion changes leucine to glutamate (acidic) and protein is functional. Glutamate mutant functions approximately as well as aspartate wild type
Describe what happens in a nonsense suppressor and a physiological suppressors.
Nonsense suppresor-
1st mutation is a nonsense. Creates a stop codon in the ORF
2nd mutation is the suppresor. There is a mutation in the tRNA anticodon that will recognize the stop codon. Instead of recognizing the stop codon, it will bring in another amino acid and continue protein translation. Important concept is that the protein is still made.

Physiological suppresor-
Example-
There is a transporter that brings in lactose and it loses its function. A mutation then happens in another transporter that can then bring in lactose so there is no lose of lactose.

These are extragenic suppresors because mutation happens in another gene.
Describe what happens in an intragenic suppresor?
Possibly different ORF from original, but enzyme functions normally.
e.g. Original mutation changes glutamate to leucine and protein loses function (negative charge to uncharged). Second mutation changes an adjacent amino acid to glutamate. The new glutamate is close enough in position to carry out the function of the original glutamate (negative charge is now found in nearly the same place).

e.g. cysteine residue that participates in disulfide bond is mutated to another aa, so the disulfide bond can’t be formed and the protein doesn’t function. Suppressor mutation changes a nearby aa to cysteine, allowing disulfide bond to form, so protein has nearly identical structure to original.
A minimal media is usually some inorganic salts and a carbon/energy source, with the carbon/energy source usually coming from _________
Glucose
Glucose is usually included as both a carbon and energy source, but other carbon/energy sources may be used.
e.g. NH4Cl, MgSO4, Na2HPO4, KH2PO4, CaCl2, and glucose
What is an anabolic mutant?
This is an mutant that cannot synthesize certain necessary components such as amino acids
Why are mutagens used in screening (replica screening)
This is a way to get your mutant phenotypes without having to simply culture so many bacteria
What occurs in replica screening?
Take a large colony of bacteria and then add a mutant

Grow the colonies then in a minimal plate w/(for example) histidine

Then grow the colonies in minimal media (w/o) histidine and then evaluate
How do you select a Phe+ revertant from a Phe- population?
Starting with Phe- (auxotrophic) culture, plate on media without Phenylalanine, any growth is due to reversion mutants

i.e. do experiment from previous slide, to isolate Phe- mutant strain. Grow this strain to large numbers, then mutagenize and plate on minimal media.
How do you select for resistance (Amp+) mutants
i.e. take strain known to be sensitive, mutate, grow large amounts, plate on media with Ampicillin – look for growth
Describe how to find a colony that cannot metabolize lactose?
Grow a colony of bacteria that in minimal media with glucose, then use that replica plate and grow on minimal media with lactose as the sugar source and evaluate
What are the enzymes used to synthesize phenylalanine?
Shikimate dehydrogenase
Chorismate synthetase
Chorismate mutase
Phenylalanine transaminase
Give the pathway of phenylalanine synthesis?
Phosphoenolpyruvate(Shikimate synthetase)---------Shikimate (Chrosmiate dehydrogenase)-------Chromosate (Chorismate mutase)------prephenate

At prephenate you can then go to either tyrosine (tyr transaminase) or phenylalanine (phe transaminase)
How would you find a strain with a mutation in Chorismate synthetase?
Simply grow the mutated strains in a minimal media with Chorismate and then replicate the strain in a minimal media w/o Chorismate and evaluate.
What is the Ames test?
This test for carcinogenes
Describe what happens in the Ames test?....just in general
Basic idea: Grow His- mutants (auxotrophic strain of Salmonella typhimurim) on minimal media: only revertants will grow.

Add a small amount of test chemical. If it’s a mutagen, then the number of colonies will increase in comparison to a plate with no added chemical.

A small amount of histidine is also included on the plate. Histidine is “limiting” in this test. The his- mutant cells can undergo a few rounds of replication and then the histidine is used up. This allows for some DNA replication (and hence mutation) but not enough growth for visible colonies.

Another feature of these strains is they have reduced ability to exclude chemicals from their cytoplasm. This increases the potency of the chemicals.

Plates include liver extract (i.e. a collection of liver enzymes) to simulate physiological processing of test chemical

Growth of revertants (as compared to control with no putative carcinogen/mutagen) indicates the chemical is mutagenic (in presence of liver extract) and therefore perhaps carcinogenic
Following ingestion, inhalation, or injection, most organic chemicals are chemically modified/processed in the liver by a family of enzymes known as cytochrome ________.
these enzymes give the liver its distinctive _____color
P450's
Brown
Metabolic processing of chemicals can reduce or increase toxicity?
This often reduces toxicity and/or enhances the excretion rate of the chemical. It might also increase the toxicity… Then decrease it or increase it again… it’s impossible to predict.
Base analogs usually cause what kind of mutations?
Point mutations (transversion and transition)
Alkylating agents usually cause what kind of mutations?
Alkylating agents will cause deletions resulting in frameshifts
Why must you use (at least) two strains in the Ames test?
In case your suspected mutagen causes one specific kind of mutation
e.g. a mutagen that causes deletions is unlikely to revert a transition mutation. A mutagen that causes transition mutations is likely to revert a transition mutation, but unlikely to revert a frameshift mutation.
Thus, there is a strain of bacteria that will show a large amount of revertants only if the mutagen causes frameshift mutations, while another strain(s) will show large amounts of revertants if the mutagen causes transitions and/or transversions
Transition mutations are most likely to revert a previous _______ ______
transition mutation
Give a quick definition of what a bacteriophage is?
It is an obligate intracellular parasite....its a virus for a bacteria
The nucleic acid in a bacteriophage can be what?
DNA or RNA
Single or Double Stranded
And can contain modified bases
The protein in a bacteriophage is supposed to do what?
Protein forms a capsule and protects nucleic acid from degradation.
The capsule can also contain carbohydrates, internal membrane
Describe the lytic pathway?
Attachment begins
Insert the nucleic acid
Translation or transcription occurs and creates the components
Forms the bacteriophage
Lysis of the components
The early promoters of the bacteriophage are extremely strong, what in the host directs transcription of these promoters?
Host sigma70
What sigma factor in the bacteriophage is seen to contribute for the DNase activity the degrades the host DNA?
Phage sigma factor gp55
Besides the early mRNA giving the DNase activity, what else does the early mRNA do?
ADP ribosylation of alpha subunits
(i.e. ADP is covalently attached to host RNA polymerase alpha subunits, these seems to favor txn of T4 genes over E. coli genes)

Viral sigma factor (gp55) confers specificity for phage promoters

T4 DNA polymerase
T4 genomic DNA isn't made with regular cysitine it is made with what?
hydroxymethycytosine (HMC)
Once a T4 genomic DNA is created it is then further modified with what?
Glucose is added and this gives further resistance from nucleases
What are the functions of late mRNA?
Capsid structural proteins
Assembly proteins
Lysis enzymes (peptidoglycan degradation, cell membrane disruption)
How can one find phages?
Phages will be seen as circular holes on a lawn of bacteria that are called plaques.
Finding plaques are usually done on what kind of agar?
Top agar
When determining the lifecycle of plaque what chemical is used to lyse the bacterial cell, without doing damage to the cell?
Chloroform
What is happening in the eclipse period of the phage lifecycle?
No detectable infectious particle in or out of cell
What happens during intracellular growth of the phage life cycle?
Infectious particles can be found inside but not outside cells
What happens during the latent of the phage lifecycle?
Time while no phage particles are outside cell (Eclipse + Intracellular)
What is the burst size of a phage lifecycle?
Number of particles released per infected cell
The capsid of a bacteriophage may have an internal membrane and may be composed of a single subunit. It is also in the ______ shape.
icosahedral (20 triangular sides),
Some tails of bacteriophages are hollow and can be used for what?
The tail can be used to put the nucleic acid into the bacteria cell
What is the function of a tail fiber on a bacteriophage?
The tail fiber is used to recognize Receptors on bacteria. Receptors may be many things – LPS, flagella, pili, techoic acid, etc
The bacteriophage commitment step for binding is when the _____ ____ binds to the bacteria.
Base plate
The ______ of the bacteriophage will contract and cause further attachment, allowing nucleic acid to go through a hollow tail.
Sheath
When classifying genes of phages they fall into early, mid and late. What are typically the functions of these genes?
Early – takeover host machinery
Mid – synthesis of phage components (chromosome)
Late – capsids, assembly and lysis
Why might a bacteriophage go into the lysogenic phase as opposed to the lytic phase?
A bacteriophage might enter the lysogenic phase because bacteria is in the stationary phase. The lytic phase because the bacteria is in the log phase.
The lambda bacteriophage infects E. Coli by binding to the _____, this acts as a receptor?
Porin
The lambda genome is a _______ DNA molecule, approximately 50kb long. While in a phage particle, it’s maintained in a ___ state, with cohesive single stranded ends, known as ____ sites. When the DNA enters the host cell, the _____sites hybridize to each other, so the DNA assumes a _____structure. The remaining gaps or nicks are sealed by DNA ligase, resulting in a closed, _____DNA genome.
single;
linear ;
cos
cos
circular
circular
This is long.....but describe the lambda lifecycle?
1. Binding of particle and DNA entry

2. Linear genome has ss overhangs
(cos sites) at each end

3. The ss overhangs at each end
are complimentary to each other.

When they hybridize, the genome becomes
circular, with two ss nicks (one one each
strand) where the cos sites are

4. Host DNA ligase
seals the nicks

5. Bidirectional “theta” replication
makes copies of lambda genome.

6. Circular genomes undergo
Rolling circle replication,
Creating a ss concatemer of
Lambda genomes

7. Second strand replication

8. Concatemers cleaved at cos sites

9. Packaging into phage particles, lysis
Describe the lambda lifecycle during lysogeny?
The linear DNA will enter the cell
The cos sites will anneal
Nicks sealed by DNA ligase.
Transcription and translation of the lambda repressor protein and integrase
What is the function of of the lambda repressor protein and integrase?
Lambda repressor forms a dimer, binds DNA and represses transcription of all lambda genes except the repressor gene itself
Will also repress transcription of newly introduced lambda (interference)
Repressor constantly made at low levels through lysogenic phase

Integrase of course integrates Circular DNA into specific site in E coli chromosome
What is the name of a DNA copy of virus in host genome?
Prophage
The att region of phage DNA and the small region of chromosomal DNA are both recognized by DNA binding ________
Proteins
What happens if the cro gene product wins in lambda phages?
cro will cause expression of numerous other phage genes, including those necessary for phage DNA synthesis, particle assembly, etc. Lysis is usually favored in log phase cultures
What happens if the repressor gene product wins in lambda phages?
Repressor binds phage DNA causes expression of more repressor. Phage DNA will integrate into host genome and continue to make repressor. Synthesis of cro is inhibited! So, prophage DNA makes repressor protein only. Lysogeny is usually favored in stationary phase cells.
Repressor is also responsible for ______ in lambda
interference
DNA damage can cause the shift from _______ to ______ via the production of RecA
DNA damage (UV irradiation etc) and some other events will cause induction (switch from lysogeny to lysis).
UV radiation activates the protease activity of RecA
How does Rec A switch phages from lysogeny to lysis?
Cellular proteins (RecA in particular) will proteolyze lambda repressor.

This allows production of cro, which leads to the lytic cycle, including excision of the prophage from the genome.

----The prophage leaves the bacterial genome as a circle. From there it may undergo bidirectional and/or rolling circle replication, and proceed with the lytic cycle.
The lambda repressor and cro gene product compete for binding at the what component of the phage promoter?
OL and OR are operator/promoter sites found in the lambda genome
How can one tell the difference between a non-virulent and virulent S.pneumoniae?
Virulent is shiny, and has a capsule

Non-virulent is rough and has no capsule.Rough, non-virulent strain is a mutant unable to make capsule
If one heats up the virulent S.pneumoniae and combines it with the non-virulent S.pneumoniae the mouse will then get infected. And isolation of bacteria from mouse shows both smooth and rough colony How is this possible?
Rough colony was “transformed” into smooth virulent colony
Why is transformation not very efficient?
Because DNA is very large and hydrophilic making it very hard to cross cell membrane
What DNA is not naturally competent?
E.Coli
In a lab many methods are used to make the cell membrane competent, such as?
using agents which will partially disrupt the cell membrane, microinjection, coating the DNA with more hydrophobic molecules, and many other methods
What bacteria species has a receptor for DNA uptake?
Naturally competent Heamophilus species express a specific receptor for DNA binding and uptake.
E.Coli is not naturally competent but is used in the lab to uptake plasmid DNA. How is this accomplished?
Ballistic-metal beads coated with DNA fired at cells

Electroporation-put bacteria in low ionic strength buffer, shock with brief electric current (very high voltage)

CaCl2-will lead to water influx and some swelling
Bacteria sometimes has naturally occurring transformation, how does this happen in the real world?
Bacteria dies and DNA is lyses and released to the environment

Bacteria will then uptake this DNA, species like Haemoophilus species
What is homologous recombination?
Breaking and rejoining of DNA over regions of identical or highly similar sequences
--Promoted by base pairing between the two double strands – more homology = more likely recombination
What is site specific recombination? What bacteriophage does this happen?
Enzyme mediated breaking and rejoining of DNA at specific enzyme-recognized sites
Lambda
What is illegitimate recombination?
Breaking and rejoining by non-homologous DNA strands
What highly multifunctional 352 amino acid protein is required for most E.Coli recombination pathways?
RecA;
Acts as ATPase, catalyzes strand exchange and also acts as a coprotease.
What has cooperative binding of ssDNA?
recA
What are two primary functions of RecBCD?
They act as ATP dependent DNA helicase and an exonuclease
What is the chi site and its function?
GCTGGTGG
and it inhibits the exonuclease activity of RecBCD
Describe the steps of RecBCD and RecA activity?
1.RecBCD recognizes and binds free end of DNA

2.RecBCD unwinds the DNA and acts as a 3’-5’ exonuclease

3.When the RecBCD complex encounters a Χ (chi) site, it pauses and undergoes a conformational shift.
The RecD subunit is released and the complex resumes unwinding the dsDNA. Exonuclease activity ceases, however.

4.DNA Pol I fills in DNA behind RecBCD or BC

5.RecA and SSB bind the displaced ssDNA

-----
RecA mediates a strand invasion of nearby
homologous dsDNA. The dsDNA is unwound, allowing base
pairing of ssDNA strand to homologous sequences. This is
an ATP-dependent process (RecA is an ATPase).

RecA also stimulates branch migration, further
unwinding the dsDNA and increasing base pairing with
invading ssDNA strand

6.Displaced DNA in D loop is degraded

7.Cleavage of invading strand, or RecBC reaches end of invading DNA

8.DNA ligase seals gaps
How would you use a wild type strain of E. coli to correct an auxotrophic mutant (assume it’s Phe-)?

Using:
Sample of each E. coli strain
Minimal media plates
A bunsen burner and some glassware


Assuming you are successful and get growth on minimal media plates (Phe+ phenotype):
----How do you know you’ve transformed the mutant strain and not simply contaminated your plate with the wild type E. coli?

----How do you know any colonies you isolate are a result of transformation and not a reversion mutation?
Explain
Conjugation is the transfer of DNA from one bacteria to another, is unidirectional and requires ___ to ___ contact.
Cell to cell
In can't reversion be considered the reason the auxotrophic mutants are then able to grow like the wild types via using the process of conjugation.
Simultaneous complementation of 3 mutations – virtually no chance of simple reversion accounting for this
Describe how the process of conjugation works
A pili will extend from a bacteria and attach to a proximal bacteria.

The pili will then contract and bring the bacteria closer

The bacteria's DNA will then be transported from the bacteria to another foreign bacteria.

The donors bacteria will stay keep a copy of the DNA
What are the three different types of f factors?
F'-Plasmid exists extrachromosomally, but contains fragments of host genome.

Hfr-Plasmid integrated into host genome

F+-Plasmid exists extrachromosomally, by itself
The F factor contains tra genes which create proteins involved in...?
Involved in the transfer of DNA from one bacteria to another
-DNA nicking and unwinding
-Pili structural genes
oriT is...?
oriT: origin of transfer – site (a specific sequence) where plasmid gets nicked to initiate transfer of DNA
The relaxosome is complex of proteins which bind to oriT and cause a single stranded nick. The relaxosome involves TraI (bifunctional-__________ and _________)TraY and IHF (Integration Host Factor, derived from the _____ not the _____).

The first step involves the binding of TraY and IHF to sequences near ____ nick site.

This binding facilitates the binding of _____ and formation of the _____, leading to nicking of one strand.

The nick on the DNA strand will leave the ____ ___ and _____ _____.

The TraI will then covalently attach to the 5'phosphate.

This complex will wait for signal indicating formation of a stable mating pair.

Once the mating pair is formed, the mating pair signal activates the helicase activity of ____.

TraI drags___ end of DNA through some pore structure to other cell.
Nuclease and Helicase
Host plasmid
oriT
TraI
Relaxosome
3'OH and the 5'Phosphate
TraI
5'
Nicking of the single strand DNA by the relaxosome is both _____ and _______ specific.
Site and strand
Can F+ plasmids receive genetic info from F+?
Nope
Why is complementation of an auxotrophic mutations rare with F only as a basic plasmid?
Because usually only plasmid (not chromosomal genes needed for complementation) gets transferred.

--Transfer moves only one strand of ds plasmid to recipient.
--Recipient and host must synthesize complementary strand for their plasmids.
Which of the three f factor plasmids can result with the recipient having the most of the host gene?
Hfr because it can transfer the entire genome....if given enough time

F+ will give the least with Little transfer of host genes

F' is in the middle with specific genes (those incorporated in plasmid) transferred frequently. Other genes not so frequently.
-F' cell is partially diploid
Which of the three f factor plasmids results with the recipient not always becoming F+?
Hfr
Why doesn't the Hfr usually transfer its entire genome?
Not enough time usually interrupted during the process
When performing mapping of Hfr to a recipient what is needed for the donor to be...in comparison to the recipient.
The recipient must have some form of resistance and the donor must not.

For example,
Recipient is usually resistant to streptomycin (strR), while donor is sensitive (strS). Adding streptomycin to the media prevents growth of the donor strain.
i.e. it’s important that the donor strain not be able to grow
How might a F' plasmid be created?
Formed due to improper excision, similar to lambda. . But in this case, it can take large or small parts of the E. coli genome, and doesn’t leave parts of F behind.
After the recipient cell has received genetic information from the F' plasmid, the cell is _____for all genes incorporated into plasmid - one copy in plasmid, one in genome.
diploid
If a cell is phe-phe+, what is the dominant trait?
Phe+
Just Know
Radiation response/DNA repair:
Treat with mutagen
Plate colonies on enriched (complex) media
Irradiate with near-lethal radiation
Near lethal to WT. Should be lethal to mutants in DNA repair, or any other resistance mechanism
Replica plate to another enriched media plate
Mutants will be dead from radiation and won’t grow.

Temperature sensitive mutants:
Treat with mutagen
Grow on enriched (complex) media at 37C.
Replica plate, grow at 42C
Cell wall mutants:

Treat with mutagen
Grow in isotonic media
Replica plate to non-isotonic media
Describe the steps needed to determine which is closer to F' with two closely linked genes>
First, isolate mutants in one gene, grow these up, mutate again and select for mutants in second gene.

Conjugate and look for single reversions (revertants in one of the two genes, not both). The mutation that is reverted in the single reversions is likely closer to F