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Mutations (Definition/Frequency/repair system (mutations = inheritable…
Mutations
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Types of mutants
point mutations: leading to AA change or not, possible faulty protein, protein truncation
Insertion/deletion:leading to frame shifts (different codons) --> different protein, loss of function of original protein, may create new functionality
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Tracking mutations
Selection
Selecting streptomycin resistant mutants
- Str binds to 16rRNA of 30S subunit in ribosome --> prevents translation of proteins --> bacteria die
- Mutation in a ribosomal protein gene (rpsL) prevents binding to Str. (Str can't kill the mutant) while ribosomal protein is still acitve
--> mutant bacteria survive in prescence of Str, WT dies.
--> can select them by plating on Str plates.
- Str resistant strains often used for animal pathogenesis studies (marker distinct from endogenous flora, competition studies, rare mutation not occurring in animal w/o Str pressure.
Penicillin selection of His auxotrophs
- Penicillin proteins that are involved in PG synthesis (so binds to ones that are growing)
- Add penicillin and no His --> mutant cant grow so it doesnt bind to Penicillin while all the others bind to penicillin and are killed
- only the His mutants survive --> then add his medium --> spontaneous His mutant will grow well in rich media
Screening
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involves examining each colony (from WT and mutant mix) and test for different phenotype.
Example: loss of motility, sensitivity to detergent or antibiotic (can't use selection that would kill mutant, as opposed to resistance)
Colorimetric screening: Add colorimetric marker to growth medium to detect mutants (when WT grows)
- example: blue/white screening for mutations in enzyme B galactosidase that catalyzes X-Gal substrate and generates a coloured reaction produce (blue colonies = WT and white colonies = mutants)
Transformations
Definition
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One of the main natural process that bacteria uses to exchange genetic material
- transformation
- conjugation
- transduction
Leads to enhanced genetic diversity by modifying the genotype (including mutations) and acquisition of new phenotypes.
Few prokaryotes are naturally transformable, but very responsible for lateral gene transfer
Roles: nutrition, blocks for DNA repair, acquisition of advantageous traits
What's being picked up?
Pieces of DNA taken up are 8-12 Kb - not much compared to genome avg 4Mb = 10 coplete genes (assuming 1Kb per gene)
Where do the pieces of DNA come from?
- lysis of bacteria: DNA pours out
- long chromosome very fragile
DNA sequences reorganized for binding to the uptake machinery --> some specificity for which DNA comes in, varies across bacteria
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Mechanism
- Binding: Double stranded DNA binds to outer cell surface
- Movement: DNA moves across membrane and cell wall
- Degradation of one of the strands
- Translocation of remaining single strand of DNA across cytoplasmic membrane
- Stable integration by homogous recombination ito receipiet chromosome
This is for small pieces of linear DNA
mechanism different fro plasmids that they stay circular and do not integrate into chromosome
Discovery by Dr Griffith using Streptococcus pneumoniae (late 1920s)
- smooth capsules = infectious
- rough - without capsules = noninfectious
- Smooth cell capsules were heat killed
- A mixture of heat-killed smooth cells and rough cells were administered to the mice
- Results were that they were infectious!
This means that the dead smooth cells gave off a transforming principal and transformed the rough strains in a new type.
The transforming principle = DNA
Ames to test assess chemicals for mutagenicity
* used to estimate carcinogenic potential in chemical and food industries
- Test if rate of reversion of auxotrophic mutation increases upon exposure to a potential mutagen.
- use His- mutant = requires His to grow
- Plate bacteria on a plate W/O His = cannot grow
- Put potential mutagen on filter in middle
- if compound induces reverting mutations, then mutant bacteria can grow.
- count frequency of revertants around the filter disk.
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