JG Microbial Genetics Flashcards
(34 cards)
1
Q
What are the advantages of using microbes for genetics?
A
- They reproduce rapidly
- Are simple to maintain and cultivate
- Large numbers of individual cells can be produced in a short time
- Populations are large enough to contain spontaneous mutants
- Selection techniques can allow the detection of one mutant within a large population
2
Q
What are the advantages of using bacteria for genetics?
A
- Bacteria are haploid so the phenotype of mutations is seen immediately
- Relatively small genome
- Genetic manipulation is straightforward
- Strains carrying desired combinations of mutations can be made with relative ease
3
Q
What is the method for forward genetics studies?
A
- Random genome wide mutagenesis
- Phenotypic screening for desired mutants
- Biochemical/physiological characterisation of the mutants
- Genetic analysis
- Gene isolation
- Gene sequence determination
4
Q
What is the method for reverse genetics studies?
A
- Focus on gene of interest
- Ask what the role of each gene is
- Mutate gene in vitro
- Substitute the mutated allele for the wild-type
- Determine phenotype of resulting mutant
5
Q
What are the uses of mutants?
A
- Mutants define genes involved in particular functions
- Mutant phenotypes can be informative (e.g. if a TF is mutated and 4 genes are affected, we know the TF regulates 4 genes)
- Permit matching a protein to its biological function
- Conditional lethal mutants
- Having a mutant can help us to clone the gene (complementation)
6
Q
How is slip strand mis-pairing used in some pathogenic bacteria?
A
To switch expression of surface exposed proteins on or off for immune evasion (phase variation)
7
Q
What are the different types of DNA repair?
A
- Methyl mis-match repair
- Repair of thymine dimers
- Base excision repair
- Recombinational repair
- Error-prone repair (SOS repair)
8
Q
What is mutation rate?
A
The number of mutations per cell division
9
Q
What is mutation frequency?
A
The ratio of number of mutant cells to total cells in the population
10
Q
What are the key features of pKD46 (Lambda Red)?
A
- PBAD promoter: lambda red genes only expressed in presence of arabinose
- Bla: beta-lactam resistance gene
- OriR: temperature sensitive (at 42 degrees) origin (repA dependent, repA is temperature sensitive
- Lambda Red genes:
- Exo: a 5’-3’ exonuclease that degrades 5’ ends of linear DNA
- Beta: binds to the ss 3’ ends generated by exo and promotes annealing to complementary DNA
- Gam: binds to host the RecBCD complex to inhibit exonuclease activity
11
Q
What are transposons?
A
DNA sequences that can move from one genetic element to another and which contain genes additional to those required for transposition
12
Q
What is the difference between non-replicative and replicative transposition?
A
Non-replicative transposition: transposable element jumps from one site to another
Replicative transposition: transposable element is copied; one copy remains in original site
13
Q
What are the key properties of transposons?
A
- They must integrate into a target site and become part of the target replicon
- Move between or within DNA molecules at low frequency
- Do not require homology between DNA sequences
- Key enzyme is transposase; carried on the mobile element
- Ubiquitous
- Transposon mutagenesis only works with bacteria that are genetically “tractable” for Tn delivery
14
Q
Describe the mechanism for TMDH
A
- TMDH uses a modified transposon with outward facing T7 and SP6 promoters
- A mutant library is created
- Genomic DNA is prepared and digested by a restriction endonuclease
- In vitro transcription generates labelled RNA run-offs from T7 and SP6 promoters
- Run-offs are hybridised to a whole genome tilling array
- Comparison of signals between RE sites allows the location of the transposon to be determined
15
Q
Describe the mechanism for homologous recombination
A
- RecBCD enters end of DNA fragment and unwinds it, when it reaches a Chi site (8 bp sequence) it nicks the DNA and continues to unwind the DNA
- A RecA filament assembles on the ssDNA, scans the dsDNA for homology and catalyses strand invasion and D-loop formation (single-strand crossover)
- RuvAB assemble at the crossover point and pull the donor and recipient strands in opposite directions (branch migration)
- Endonuclease cleaves one end of the D-loop
- Displaced ends are ligated to opposite strands
- Holliday structure is resolved by RuvC cleaving the DNA across the junction
- Ligation of broken ends completes a single crossover
16
Q
Describe the mechanism for non-replicative transposition
A
- Transposase aligns inverted repeats and flanking DNA
- One phosphodiester bond is cleaved on each strand at opposite ends of the IS element
- 3’-OH groups attack intact ends to produce a hairpin structure and host carrier DNA is ejected and repaired
- Hairpins are re-nicked and 3’-OH groups attack recipient DNA
- The IS element has moved from one DNA site to another
17
Q
Give examples of commonly used reporter genes and their functions
A
- lacZ: encodes β-galactosidase, stable therefore measures cumulative promoter activity, simple colourimetric assay
- cat: encodes chloramphenicol acetyltransferase, easily assayed and selectable; if expressed bacteria are resistant to chloramphenicol (easy screening)
- lux: encodes luciferase, allows real-time measurement, needs oxygen to function
- gfp: encodes green fluorescent protein, cell imaging for protein expression and localization, needs oxygen to mature
18
Q
Describe transcriptional fusion
A
- The 5’ promoter region of your favourite gene (yfg) is fused upstream of a promoterless lacZ gene, which retains the lacZ ribosome binding site (rbs)
- Produces wild-type β-galactosidase and a N-terminal fragment of Yfg
19
Q
Describe translational fusion
A
- The 5’ promoter region of yfg is fused in frame to the lacZ coding region
- Transcription (promoter) and translation (rbs) elements are from yfg
- A single hybrid protein is produced
20
Q
How are single copy reporter fusions carried out?
A
- Ligate your promoter of interest (POI) into a vector that contains a promoterless lacZYA operon (with translation initiation sequences) and a selectable marker (bla for ampicillin resistance)
- Transform an E. coli lacZ mutant strain and select for ampicillin resistance in the presence of X-gal to detect β-galactosidase synthesis
- Infect transformants with bacteriophage RS45, which contains the lacZ cistron deleted for the promoter-proximal two-thirds (lacZSC), wild-type versions of the lacY and lacZ and a truncated bla gene (bla’)
- The lac-bla sequence of RS45 is homologous to sequences of your pRS415 derivative
- This permits recombination (X) to generate a phage lysate containing bacteriophage genomes carrying the gene fusion
- The bacterial strain to be investigated is infected with the lysate carrying the gene fusion and grown on plates containing X-gal; blue plaques are restreaked
- Insertion of the prophage at the att is confirmed by PCR
21
Q
What are the three key features of CRISPR systems?
A
- Adaptation: insertion of new spacers into the CRISPR locus
- Expression: transcription of the CRISPR locus and processing of RNA
- Interference: detection and degradation of mobile genetic elements by CRISPR RNA and Cas protein(s)
22
Q
What is the historical method of reverse genetics?
A
Starting from the protein product then finding the responsible gene in a library by either:
- Using N-terminal sequence as a probe to detect colonies whose DNA hybridises to the degenerate oligonucleotide protein
- Using antibodies raised against purified protein which detects the colonies expressing the desired protein
23
Q
What are point mutations?
A
- Transition: purine –> purine, pyrimidine –> pyrimidine
- Transversion: purine pyrimidine
24
Q
What are the large mutations?
A
Insertion/deletion/inversion of a portion of chromosome
25
What are the different mutagens?
- Physical: electromagnetic radiation, spontaneous tautomers
- Chemical: analogues of bases, base modifying chemicals, intercalators
- Biological: transposons
26
What are the different types of mutations?
- Silent
- Missense: one codon changes to another
- Nonsense: a codon is changed to a stop
- Frameshift: in/del of a single base, altering all codons downstream
27
What is slip-strand mis-pairing?
- Common in tandem triplet repeats
- Small single stranded loop of DNA forms during replication
- Mispairing of codons
- Synthesis continues
- Longer strand of DNA in mutant than WT; leads to translational frameshift
28
How does insertion/deletion of DNA occur?
- Homologous recombination
- Illegitimate recombination
- Site specific recombination
- Replicative recombination/transposition
29
Describe methyl mismatch repair
- Incorrect base inserted which must be removed
- MutS binds to mismatch and recruits MutL and MutH
- MutL recognises the parent (methylated) strand and loops the DNA
- MutH cleaves the daughter (unmethylated) strand containing the mutation
- UvrD unwinds the cleaved strand, exonucleases remove it, and DNA pol synthesises a new strand
30
Describe nucleotide excision repair
- Thymine dimers are induced by UV damage
- UvrA and UvrB form a complex which binds to the thymine dimer
- UvrA bends the DNA and is then ejected
- UvrC is recruited to the site by UvrB, and cleaves the DNA backbone in two places (either side of damage)
- UvrD removes the ss fragment containing the damage
- DNA pol fills the gap
31
Describe base excision repair
- DNA glycosylase binds to and excises the damaged base
- An AP endonuclease cleaves the DNA backbone
- DNA pol synthesises a replacement strand
- DNA ligase seals the nicked strand
32
Describe recombinational repair
- Replication fork approaches thymine dimer
- DNA pol skips damaged region, forming a gap in the strand
- RecA binds to the sister double helices at the ss segment
- RecA-dependent recombination replaces the damaged-strand gap with a section of homologous undamaged strand
- Gap is repaired by DNA polymerase
- Thymine dimer can now be repaired by nucleotide excision
33
When does recombinational repair occur?
Occurs when DNA replication takes place before a UV-induced thymine dimer can be excised by nucleotide excision
34
Describe error-prone (SOS) repair
- Extensive damage inactivates LexA repressor protein
- Activation of many repair genes occurs
- Rapid polymerisation of DNA
- Error prone but better than no repair
- Promotes mutations, some of which could be advantageous to survival
