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MBB267 Genes, Genomes and Chromosomes > JG Microbial Genetics > Flashcards

JG Microbial Genetics Flashcards

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
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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
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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
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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
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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)
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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)

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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)
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8
Q

What is mutation rate?

A

The number of mutations per cell division

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9
Q

What is mutation frequency?

A

The ratio of number of mutant cells to total cells in the population

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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
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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

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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

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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
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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
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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
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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
Q

What are the different mutagens?

A
  • Physical: electromagnetic radiation, spontaneous tautomers
  • Chemical: analogues of bases, base modifying chemicals, intercalators
  • Biological: transposons
26
Q

What are the different types of mutations?

A
  • 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
Q

What is slip-strand mis-pairing?

A
  • 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
Q

How does insertion/deletion of DNA occur?

A
  • Homologous recombination
  • Illegitimate recombination
  • Site specific recombination
  • Replicative recombination/transposition
29
Q

Describe methyl mismatch repair

A
  • 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
Q

Describe nucleotide excision repair

A
  • 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
Q

Describe base excision repair

A
  • 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
Q

Describe recombinational repair

A
  • 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
Q

When does recombinational repair occur?

A

Occurs when DNA replication takes place before a UV-induced thymine dimer can be excised by nucleotide excision

34
Q

Describe error-prone (SOS) repair

A
  • 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

MBB267 Genes, Genomes and Chromosomes (4 decks)

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