Slide 1: Mutants
Simon Bishop 613545
An organism or cell with a mutation. A mutation is a DNA base pair change or chromosome change. Mutants defective in a biological process can be used to increase our understanding of that process.
Slide 2: Point Mutations
Missense mutation Nonsense mutation Neutral mutation Silent mutation Frameshift mutation
Fig 19.3 from Essential iGenetics, Russell
Mutants can be studied by selecting for known phenotype or genotype.
Slide 3: Model organisms
• • • • • • • •
MICE Share homology with Humans Easier to study as fewer ethical regulations and shorter life cycle BUT hard to study development YEAST Single celled eukaryotes Fewer control mechanisms? Share cell division mechanism
Slide 4: 1. The Cell Cycle Engine
• Temperature-sensitive mutants of Schizosaccharomyces pombe selected which initiate M-phase at a reduced size to wild type • Led to studies of wee and cdc phenotypes • Led to discovery of mechanism for M & S phase regulation. • Has practical uses in study of Cancer. • Paul Nurse is former Chief Executive of Cancer Research UK.
Figure 2 from Nature vol 256 (1975) p549
Slide 5: 2. Knockout organisms i
• Gene Replacement: mutant allele replaces wild type, avoiding the so-called position effect • Gene Knockout: specific transgene replaces homologous gene in genome, replacing or removing gene function.
Slide 6: 2. Knockout organisms ii
Intron
Plate on medium with Neomycin NeomycinR Chromosome of ES cell
Exon
Knockout chromosome wt (vector DNA)
Slide 7: 2. Knockout Organisms iii
Parents
F1
F2 Diagram from Introduction to Genetic Analysis, Gelbart, W et al
Slide 8: 2. Knockout Organisms iv
• Mlh3 belongs to a family of proteins known to have roles in meiosis and DNA mismatch repair, but the function of Mlh3 itself was unknown.
Figure 1a from Nature Genetics vol 31 p385
• Mendelian ratios of F2 mice were as expected so Mlh3 not required for development. • F2 generation sterile.
Slide 9: 2. Knockout Organisms v • Male testes were smaller with no viable spermatocytes • Female ovaries unaltered but no viable oocytes • Mlh3 shown to be required to hold chromosomes together at chiasmata during meiosis.
Figure 4g,h from Nature Genetics vol 31 p385
• Mlh3 shown to be essential for reproduction and meiosis.
Slide 10: Summary • Two methods of mutant study: • Selection by phenotype (wee1 mutants)
• Nurse, P. Nature vol 256 (1975)
• Selection by genotype (Mlh3), using knockout methods
• Lipkin, S et al. Nature Genetics vol 31 (2002)
