Green Flashcards
Why is studying MO genetics important for geneticists/biochemists?
- useful model systems for underlying fundamental processes common to all forms of life
- helped us to understand eg. genetic code, DNA rep, mutations
Why is studying MO genetics important for microbiologists?
- 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
What are the advantages of using MOs for genetics?
- 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
What are the classical (forward) and reverse genetics approaches?
- classical is from biological function (phenotype) to gene
- reverse is from gene to biological function
What happens during the classical (forward) genetic approach?
- 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)
What are the advantages and disadvantages of the classical (forward) genetic approach?
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
How was the reverse genetic approach done historically?
- 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)
How is the reverse genetic approach done, now that entire MO genome seqs are readily available?
- focus on 1 GOI
- mutate gene in vitro
- sub mutated allele for WT allele in genome
- determine phenotype of resulting mutant strain
What are the uses of MO mutants?
- 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
What are the types of mutation?
- point
- -> transition = purine to purine or pyrimidine to pyrimidine
- -> transversion = purine to pyrimidine or pyrimidine to purine
- insertion
- deletion
- inversion
What are the different types of mutagens?
- EM radiation (1 of most common)
- spontaneous tautomers
- chemical
- -> analogs of bases
- -> base mod chemicals, eg. nitrous acid
- -> intercalators insert between bases
- biological –> transposons
What effects can mutations have?
- silent
- missense (change codon)
- nonsense (stop codon)
- frame shift
What is slip strand mis-pairing?
- repetitive seq can cause slippage, leading to ss looping out of some codons
- pol extends loop, resulting in longer DNA (insertion)
Why is slip strand mis-pairing used in some pathogenic bacteria?
- phase variation
- involves switching expression of surface exposed proteins on/off for immune inversion
What are the different types of recombination?
- 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)
What are the 3 types of DNA repair?
- methyl mismatch repair
- repair of thymine dimers
- repair of damaged bases
- recombinational repair
- error prone repair
How is methyl mismatch repair carried out? (DNA repair)
- 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
How are thymine dimers repaired?
- induced by UV
- A and B mark error, 2 nicks flank dimer and cut out by UvrAB complex
- gaps refilled by DNA pol
How are damaged DNA bases repaired?
- 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
How is recombinational repair carried out and when does it occur? (DNA repair)
- 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
How is error prone repair (SOS response) carried out, and in what organism is it most common?
- 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
How is the SOS response regulated?
- 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
What is the mutation rate?
- no. mutations per cell division
How does the no. bacteria relate to the no. divisions?
- approx equal
- ie. 8 bacteria req 7 cell divisions
What is the mutation frequency?
- ratio of no. of mutant cells to total cells in pop
What are the 3 methods of phenotypically selecting bacterial mutants?
- 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)
