JG Microbial Genetics Flashcards
What are the advantages of using microbes for genetics?
- 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
What are the advantages of using bacteria for genetics?
- 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
What is the method for forward genetics studies?
- Random genome wide mutagenesis
- Phenotypic screening for desired mutants
- Biochemical/physiological characterisation of the mutants
- Genetic analysis
- Gene isolation
- Gene sequence determination
What is the method for reverse genetics studies?
- 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
What are the uses of mutants?
- 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)
How is slip strand mis-pairing used in some pathogenic bacteria?
To switch expression of surface exposed proteins on or off for immune evasion (phase variation)
What are the different types of DNA repair?
- Methyl mis-match repair
- Repair of thymine dimers
- Base excision repair
- Recombinational repair
- Error-prone repair (SOS repair)
What is mutation rate?
The number of mutations per cell division
What is mutation frequency?
The ratio of number of mutant cells to total cells in the population
What are the key features of pKD46 (Lambda Red)?
- 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
What are transposons?
DNA sequences that can move from one genetic element to another and which contain genes additional to those required for transposition
What is the difference between non-replicative and replicative transposition?
Non-replicative transposition: transposable element jumps from one site to another
Replicative transposition: transposable element is copied; one copy remains in original site
What are the key properties of transposons?
- 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
Describe the mechanism for TMDH
- 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
Describe the mechanism for homologous recombination
- 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
Describe the mechanism for non-replicative transposition
- 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
Give examples of commonly used reporter genes and their functions
- 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
Describe transcriptional fusion
- 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
Describe translational fusion
- 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
How are single copy reporter fusions carried out?
- 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
What are the three key features of CRISPR systems?
- 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)
What is the historical method of reverse genetics?
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
What are point mutations?
- Transition: purine –> purine, pyrimidine –> pyrimidine
- Transversion: purine pyrimidine
What are the large mutations?
Insertion/deletion/inversion of a portion of chromosome
What are the different mutagens?
- Physical: electromagnetic radiation, spontaneous tautomers
- Chemical: analogues of bases, base modifying chemicals, intercalators
- Biological: transposons
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
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
How does insertion/deletion of DNA occur?
- Homologous recombination
- Illegitimate recombination
- Site specific recombination
- Replicative recombination/transposition
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
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
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
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
When does recombinational repair occur?
Occurs when DNA replication takes place before a UV-induced thymine dimer can be excised by nucleotide excision
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
