Investigating Gene Function Flashcards
What is the aim of forward genetics?
Identify mutations that produce a certain phenotype.
How does forward genetics find mutations?
Use a common mutagenetic agent eg EMS and radiation. This means the process is random. The mutant gene can be isolated and characterised.
What is reverse genetics?
Targeted approach to investigating gene function. Determines the phenotype that results from mutating a specific gene.
How does reverse genetics work?
Possible to induce targeted modification of a particular gene to generate a gene ‘knockout’ or ‘knock in’ (for dominant mutations).
Alternative methods include the random induction of DNA deletions and subsequent selection for deletions in a gene of interest, as well as the application of RNA interference.
How does reverse genetics work for determining bacterial gene function?
Use recombinant DNA technology to construct the mutated version of the target gene.
Transform into bacteria (eg e.coli).
Screen for antibiotic resistant transformants (eg AMP resistant gene added with the mutant target gene when you screen you get the transformed bacteria only).
Analyse the phenotype to see what the mutation did.
How are we assaying defective MMR in E. coli?
- Use Rifampicin (Rif) as effective against WT E. coli.
- Rif binds to and inhibits the B subunit of RNA polymerase encoded by rpoB gene.
- Involves AA in rpoB protein w/ aromatic side chaims. If altered due to mutation, protein can become insensitive to Rif. E. coli that is mutated can then grow on media w/ Rif.
- E. coli w/ defetive MMR should become highly mutable and in Luria-Delbruck fluctutaion assay they will show a high frequency for Rif resistance.
The procedure for assay of defective MMR in E. coli?
- Each of the strains are tested for MMR activity as follows:
- Each strain grown under a period of non-selective growth (LDFA).
- In WT strains mutations that are introduced during DNA replication are corrected by the functional MMR system.
- Some mutations in rpoB gene, that results from defective MMR, make the rpoB protein insensitive to Rif.
- Mutations are detected by plating onto LB+Rif after period of non-selective growth.
- Low no. of Rif-resistant colonies for WT but larger no. observed in mutants defective in MMR.
What mutant knockout did pRB318 help restore?
mutS
What did pRB318 help do to the mutS knockout E. coli mutation?
It will have a low mutation rate like WT as the mutS helps restore the MMR.
Why might conserved sequences indicate sections of critical function to a gene?
If the mutations are different then they are not tolerated so that cells can’t survive. So the mutations in these regions are pruned and cells are not able to replicate.
What are transcriptional fusions?
Cells/tissues in which the gene is transcribed are investigated using transgenics where the promoter is fused to a reporter gene eg GFP (fluorescence).
What are translation fusions?
Cells/tissues or the subcellular location where the protein is expressed/active are investigated using transgenics expressing a chimeric protein.
What is RNAi?
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What is the transgenic mouse?
Used in research to investigate gene function –> model for human genetic disease and investigating human gene function.
What is the SRY gene?
Male presenting gene. XX mouse could express SRY if mutations occur.
