MCO 34-42 Flashcards
Why are bacteria good model organisms
- Haploid
- Asexual reproduction
- Short generation time
- Easy to manipulate
- Easy to store
- Grow of define media
Process of binary fission
- Cell elongates and all content increases
- Septum forms in the middle
- Growth from both sides of the cell
- They are segregated and there are 2 identical daughter cells
What is a prototroph
An organism that doesn’t require special nutritional factors
What is an auxotroph
Organisms that have mutants that impair some metabolic capabilities
What it catabolic auxotrophs
Organisms that have lost the ability to catabolise some carbon sources
What is bio-synthetic auxotrophs
Organisms that have lost the ability to synthesise amino acids, nucleotide or a vitamin
what is a conditional lethal mutants
Mutants affected in housekeeping genes, which are essential for survival
What are temperature sensitive mutants
They grow at permissive temperatures, usually 30 degrees for E.coli
what are cold sensitive mutants
Mutants that grow at high temperatures
Lamarckian evolution
If an organism uses something more it will increase. Change is directed by the environment.
Darwinian evolution
Change is spontaneous and natural selection ensures survival of the fittest. Change is randomly selected.
Luria-Delbruck experiment
- E.coli cultures were grown
- Aliquots plated on plates with T1 phage
- T1 phage kills E.coli
- Muations are already present in culture
Darwinian hypothesis
Random mutations predicts that mutants appear in culture prior to adding selective agent.
Lamarckian hypothesis
Directed change predicts that mutants appear in culture only after adding the selective agent.
Newcombe experiment
- E.coli cultures are grown
- Plated onto 2 plates and let them grow
- 1 plate is left the same and nothing is done. The other is re-spreaded.
- Both sprayed with phage
- Both are grown and colonies are counted
More colonies on the re-spreaded plate as resistant colonies are spread around.
Lederberg and Lederberg replica plating experiment
- Make many replica plates of master plate of E.coli
- Then add phage
- The position of the resistant E.coli colonies on the replica plates are always the same
Therefore the resistance mutation happens before the replica plating.
What are point mutations
A change to one base pair. This can be by substitution, insertions or deletions
Types of substitutions
Transitions and transversions
What is a transition substitution
A point mutation that changes a purine to another purine or a pyrimidine to another pyrimidine
What is a transversion substitution
A point mutation that changes a purine to a pyrimidine or vice versa.
What is a silent mutation
A change in base but no change in phenotype
what is a missense mutation
A substitution where one amino acid is substituted for another
What is a non-sense mutation
Substitution leads to a stop codon being introduced
What are inversions
When kilobases are flipped
What are tandem repeats
When part of the genome is duplicated, it can lead to the overproduction of proteins
What are transposons
Nucleotide sequences that are able to move themselves around
What are reversions
A point mutation resulting in the restoration of the original sequence
What is a tautomere
Isomers of a compound which exists together in equilibrium
What happens in an isomersation switch
The rare (enol) form of a nucleotide is reverted back to the normal (keto) form.
What is a suppressor mutation
A second mutation that results in the original phenotype being restored
What is intergenic suppression
A second mutation in a different gene to the one in which the first mutation occurs. It suppresses the phenotype of the first mutation.
What is a non-sense suppressor
Inhibits the effect of a nonsense mutation. A mutated tRNA binds with the termination codon.
They tend to translate past the normal stop codon, and produce larger proteins. They not also function correctly as they may fold incorrectly.
What is a mutagen
A chemical or physical agent causing damage to DNA
How does spontaneous mutations cause damage to DNA
Replication errors and tautomers
- Base pair slipping
- Repeat nucleotides can lead to frameshift mutations
How does deamination of bases cause damage to DNA
- Removal of am amine group which causes different base pairings
- Can be caused by nitrous acid
- xanthine pairs with C
- U pairs with A
- Hypoxanthine pairs with C
How does a reactive oxygen species cause damage to DNA
-Cause a variety of changes to DNA as they are reactive:
Oxidation and addition to double bonds
-Natural side product of aerobic respiration
-Chemical reactions caused by UV light or ionising radiation .
How does an alkylating agent cause damage to DNA
- Chemicals react with DNA adding alkyl groups
- Can lead to point mutations
- Can affect the ability to coil/uncoil
- Can affect how proteins bind to the DNA
How do interchalating agents cause damage to DNA
Flat multiple ring structures which binds between base pairs causing a frameshift.
How pyrimidine dimers cause damage to DNA
Two consecutive bases on one strand bind together, destroying normal base pairing. Affects transcription and replication - DNA is misread or progress is blocked.
How do bacteria minimised damage to DNA
- Bacteria have enzymes that repair their DNA
- Bacteria can produce melanin and other pigments to protect from radiation damage.
What is phenotype lag
Phenotype is not seen for several generations.
- mutation to gene and no more protein is made
- have to wait generations for copies of the gene to disappear
How do we select for mutants
- selectable phenotypes: drug resistance, phage resistance
- Conditional lethal mutants: expressed in certain conditions e.g. temperature
Making histidine auxotroph
- Expose to mutagen
- Grow in complex medium to allow expression of phenotype
- Penicillin enrichment: change medium to minimal medium, add penicillin to kill any growing bacteria. Auxotrophs will not grow,
- Plate on minimal medium + histidine and grow
- Replica plate to double check
What is cross feeding
Where metabolic pathway is blocked and metabolites before the block accumulate. The blocked metabolites diffuse to another bacterial cell and continue the metabolic pathway. Both mutants can grow but are dependent on each other.
What is the Ames test
Used to identify chemicals that are mutagenic and therefore carcinogenic
Steps of Ames test and its limitation
- Grow strain
- Plate same number of bacteria on plates with and without chemical investigated. Incubate overnight.
- If chemical is mutagenic there are lots of reversions,
if not only a few reversions
A limitation is that the chemical may not be mutagenic but a metabolite.
What is the operon
A group of genes under control of the same promoter. This means genes can be regulated together.
What are housekeeping genes
Genes that are always kept active and are involved in replication and transcription. They are constitutively expressed
What is diauxic growth
2 growth phases caused by the presence of two sugars on a culture growth media, one of which is easier for the target bacterium to metabolize. There is a lagging phase where genes are being turned on
What is allolactose
An inducer that disables the repressor turning the lac gene on. Even in a repressed state a small amount of allolactose is produced
What is a catabolite activator protein (CAP)
Enhances transcription by binding to cAMP and to DNA
What Catabolite repression
Glucose is the preferred and metabolism of other sugars are repressed.
Glucose inhibits adenylate cyclase:
-No cAMP
-Lac operon off
Transformation experiment
- R cells, non-pathogenic, live
- S cells, pathogenic, die
- Heated S cells, live
- Heated S cells and R cells, die
what is competence
The ability for a bacterial cell to take up extracellular “naked” DNA from the environment
-some bacteria are naturally competent, for others it has to be induced
Method for artificial transformation
Electroporation - DNA is pushed into the cell through pores created by an electric pulse.
What does gene exchange lead to
- Drug resistance
- Novel virulence characteristics
- Novel metabolic capabilities
When are cells most competent
Just before the stationary phase. This is because of quorum sensing, where genes are regulated based on population density
Mechanisms of competence
- cells secrete comx
- comx concentration increases
- binding of comx to comP set of a reaction of events leading to changes in gene expression
- change means the cell became competent
- It uptakes lysed DNa
Uptake of DNA
- DNA binds to the surface of the protein
- DNA enters the cell and binds to competence specific protein
- RecA mediated integration
Bacteria recognise recognition sequences
potential roles for DNA uptake
- Gain nutrients: nucleotides could provide carbon, nitrogen and phosphorus
- Genetic diversity
- DNA repair
What is transduction
genetic exchange in bacteria mediated by bacteriophages
Phage therapy
- T2 page infects and kills E.coli. It is lytic phage.
- Lambda phage can be lytic or lysogenic
Lytic cycle
DNA is inserted into cell, replication, transcription and translation. New virons assembled. Lysis and release of new virons.
Lysogenic cycle
DNA is inserted and integrated into the genome. DNA stays there and is transmitted to daughter cells.
what is a lysogen
strain of bacteria carrying lysogenic phage. In lysogens the lytic cycle is suppressed by expression of phage protein.
what is a prophage
phage in lysogenic state
what is generalised transduction
Transfer of any DNA to the recipient by lytic or lysogenic cycle
what is specialised transduction
Transfer of specific genes via lysogenic phage
Conjugation
the process of moving gentic information via direct cell-to-cell contact
Lederberg and tatum experiment
- 2 strains of bacteria require certain nutrients
- Individually they dont grow on minimal media but grow together when mixed together
Davies experiment
- 2 strains of bacteria are put in a u bend glass tube seperated by a semi-permeable filter. allowing media through but not cells.
- Then apply a vacuum to mix media
- plate both bacteria and no growth
- when filter is removed there is growth
Why are some plasmids incompatible
Because related plasmids sharing common mechanisms for replication often cant co-exist
What is curing
when a plasmid is lost from the host spontaneously
What are episomes
special hosts that can integrate into host genome
How does the f pilus work
pilus makes contact, pulls the cell closer and the f plasmid is transferred
Rolling circle replication
- One of the ds is nicked
- nicked strand is transferred to the other cell
- Single starnd nicked acts as a template for the lagging strand
- Joined by DNA ligase and both cells have F plasmid
What is a Hfr strain
A bacterium with a conjugative plasmid integrated into its chromosomal DNA
How does the Hfr plasmind integrate into the genome
- integrated f plasmid is nicked and unravels
- DNA polymerase synthesises starnd being pulled off.
- Replaced strand is transferred to the other cell. chromosomal DNA is broken off as well
This DNA is linear and cannot circularise as not all of chromosomal DNA is transferred.
This DNA is degraded most of the time but occasionally recombination takes place.
How does an integrated F plasmid, become an F plasmid
It can excise itself.
occasionally some chromosomal genes end up in the plasmid. F’ plasmid.
what is recombination and what are it roles
Breaking down and rejoining DNA into new combinations.
- allows rapid evolution
- genetic diversity
- switch from auxotroph to prototroph
What is homologous recombination
switching DNA that is similar. requires extensive homology
What is Non-homologous recombination
repair of double stranded DNA break by simply joining with another piece of DNA.
Mechanisms of homologous recombination
- Alignment
- Breakage - one strand nicked by RecBCD
- Invasion - DNA stabilised by ssb protein, catalysed by RecA
- Cross strand exchange
- Branch migration
Holliday junction
- Bend both duplexes 90 degrees from crossover
- Rotate lower half 180, to uncross the crossover
2 outcomes from the holliday junction
- non recombinant products, horizontal cleavage
- Recombinant products, vertical cleavage, products are hybrid
RecA
- all organisms have there own version of RecA
- essential for DNA repair
- Binds single stranded DNA
RecBCD
- Has nuclease activity and catalyses initial nick in DNA needed for recombination, requires a specific sequence
- Has helicase activity, unwinds DNA for single stranded DNA binding protein and RecA to bind
Non-homologous recombination
- No homology need
- Transposons events
what are insertion sequences
small pieces of DNA that can hop from one position to another.
- Hop is called transpostion catalysed by transpotase
- It distrupts genes but there is a high degree of reversion
- distrupting a housekeeping gene is lethal
What are transposons
same features as insertion sequences but carries additional genes.
Mechanism of transposons
- Transpotases bind to each end
- transposon is cut out
- chromosome is repaired but there may be change in the original sequence
- New target sequence is found somewhere else
- Transposons inserts
2 types of transposition and what are they
- Conservative: Transposon moves from place to another
- Replicative: The original copy is maintained and a new copy is made and inserted elsewhere
