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Question 1

Why is studying MO genetics important for geneticists/biochemists?

Answer 1

• useful model systems for underlying fundamental processes common to all forms of life
• helped us to understand eg. genetic code, DNA rep, mutations

Question 2

Why is studying MO genetics important for microbiologists?

Answer 2

• ecology –> ubiquitous, occupy extreme niches, essential for nitrogen fixation, global geochem cycles
• cell biology –> complex and dynamic cell structure (can study smaller organisms)
• pathogenicity –> treatment and control of disease (must understand how MO lives)
• biotech –> antibiotics, new chemicals and enzymes

Question 3

What are the advantages of using MOs for genetics?

Answer 3

• reproduce rapidly
• simple to maintain and cultivate
• large no. prod in short time
• pops large enough to contain spontaneous mutants (freq increases w/ mutagenic agents)
• selection techniques can allow detection of 1 mutant w/in v large cell pop
• haploid so mutant phenotype seen immediately
• relatively small genome
• genetic manipulation straightforward (eg, gene KOs)
• make strains carrying desired combinations of mutations w/ relative ease

Question 4

What are the classical (forward) and reverse genetics approaches?

Answer 4

• classical is from biological function (phenotype) to gene

- reverse is from gene to biological function

Question 5

What happens during the classical (forward) genetic approach?

Answer 5

• random genome wide mutagenesis
• phenotypic screening for desired mutants
• biochem/physiological characterisation of mutants
• genetic analysis (genetic mapping/complementation test)
• gene isolation (easier w/ model organism)
• gene seq determination (study gene product)

Question 6

What are the advantages and disadvantages of the classical (forward) genetic approach?

Answer 6

Advantages:
- emphasis on desired phenotype and can find mutants w/ defects in essential genes

Disadvantages:

• slow
• may be impossible to find all genes in a species for given phenotype

Question 7

How was the reverse genetic approach done historically?

Answer 7

• started from protein product
• find gene in gene library
• EITHER via protein N-ter seq (detect colonies whose DNA hybridises to degenerate oligonucleotide probe)
• OR via antibodies raised against purified protein (detect colonies expressing proteins)

Question 8

How is the reverse genetic approach done, now that entire MO genome seqs are readily available?

Answer 8

• focus on 1 GOI
• mutate gene in vitro
• sub mutated allele for WT allele in genome
• determine phenotype of resulting mutant strain

Question 9

What are the uses of MO mutants?

Answer 9

• define genes involved in particular function –> look at how many diff genes represented in mutant library, all are except essential ones
• mutant phenotypes can be informative –> eg. blocks in pathway allow accum of intermediates, regulatory mutants allow identification of reg proteins and their sites of action on DNA
• permit matching protein to its biological function
• conditional lethal mutations –> eg. temp sensitivity, WT at permissive temps and mutant phenotype at restrictive temps, often missense mutations that destabilise protein structure/function only at higher temp
• having mutant can help clone genes –> eg. if WT is selectable phenotype, can transform mutant cells w/ gene library, mutant and WT on plasmid, complementation, then WT phenotype restored

Question 10

What are the types of mutation?

Answer 10

• point
  
  • -> transition = purine to purine or pyrimidine to pyrimidine
  • -> transversion = purine to pyrimidine or pyrimidine to purine
• insertion
• deletion
• inversion

Question 11

What are the different types of mutagens?

Answer 11

• EM radiation (1 of most common)
• spontaneous tautomers
• chemical
  
  • -> analogs of bases
  • -> base mod chemicals, eg. nitrous acid
  • -> intercalators insert between bases
• biological –> transposons

Question 12

What effects can mutations have?

Answer 12

• silent
• missense (change codon)
• nonsense (stop codon)
• frame shift

Question 13

What is slip strand mis-pairing?

Answer 13

• repetitive seq can cause slippage, leading to ss looping out of some codons
• pol extends loop, resulting in longer DNA (insertion)

Question 14

Why is slip strand mis-pairing used in some pathogenic bacteria?

Answer 14

• phase variation

- involves switching expression of surface exposed proteins on/off for immune inversion

Question 15

What are the different types of recombination?

Answer 15

• homologous (identical seq)
• nonhomologous (diff seqs, consequences depend on nature of fusion)
• site specific (carried out by integrase, eg. in phages)
• replicative recombination transposition (by transposase)

Question 16

What are the 3 types of DNA repair?

Answer 16

• methyl mismatch repair
• repair of thymine dimers
• repair of damaged bases
• recombinational repair
• error prone repair

Question 17

How is methyl mismatch repair carried out? (DNA repair)

Answer 17

• mispaired base cut out of strand (by a complex bringing mismatch and methyl group together)
• strand w/o methyl groups newer so assumed to be in error
• DNA pol resynthesises newer strand from old strand

Question 18

How are thymine dimers repaired?

Answer 18

• induced by UV
• A and B mark error, 2 nicks flank dimer and cut out by UvrAB complex
• gaps refilled by DNA pol

Question 19

How are damaged DNA bases repaired?

Answer 19

• excised by specific enzymes, eg. DNA glycosylase removes damaged base and AP endonuclease recognises gap and chops backbone
• replaced by DNA pol I and DNA ligase seals nick

Question 20

How is recombinational repair carried out and when does it occur? (DNA repair)

Answer 20

• occurs just after DNA strand rep
• rep stops and skips over damaged error
• gap recognised by RecA protein, scans homologous sister DNA for approp seq, allows formation of triple helix (D loop)
• cat by RecA recombinase
• then repaired by UvrC system

Question 21

How is error prone repair (SOS response) carried out, and in what organism is it most common?

Answer 21

• extensive DNA damage inactivates LexA (protein that binds DNA)
• activation of many repair genes (eg. 1 is rapid pol of DNA)
• activation of many repair genes
• rapid polymerisation of DNA –> error prone but better than no repair, promotes mutations, some could be beneficial
• most common in bacteria

Question 22

How is the SOS response regulated?

Answer 22

• LexA binds promoters and switches genes off
• ssDNA recognised by RecA
• RecA acts as protease, breaks down LexA so cant repress genes and their products are made

Question 23

What is the mutation rate?

Answer 23

• no. mutations per cell division

Question 24

How does the no. bacteria relate to the no. divisions?

Answer 24

• approx equal

- ie. 8 bacteria req 7 cell divisions

Question 25

What is the mutation frequency?

Answer 25

• ratio of no. of mutant cells to total cells in pop

Question 26

What are the 3 methods of phenotypically selecting bacterial mutants?

Answer 26

• direct selection
  
  • -> use media that only allows growth of mutant colonies
  • -> select for resistance to agent (eg. antibiotic)
  • -> WT killed and mutants in transport or metabolism form colonies on plates containing the analogue (eg. ONPG, a lactose analogue, toxic of transported/metabolised by cell)
• test every bacterium
  
  • -> eg. on indicator media (pH changes/chromogenic substrates)
  • -> replica plating used to detect mutants conditional on temp or medium (eg. nutritional auxotrophs)
• enrichment techniques
  
  • -> eg. penicillin enrichment
  • -> try to kill WT and allow mutants to survive to increase their no.s
  • -> incubate cells in conditions in which only WT would grow, inc penicillin which kills growing cells
  • -> wash and transfer surviving cells to medium allowing growth of mutants
  • -> mutants enriched 100 fold, can cycle again
  • -> plate out and screen for mutants (eg. replica plating)