MutantsSimon 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.

Missense mutation

Nonsense mutation

Neutral mutation

Silent mutation

Frameshift mutation

Fig 19.3 from Essential iGenetics, Russell

Point Mutations

Mutants can be studied by selecting for known phenotype or genotype.

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

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

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.

2. Knockout organisms ii

Exon NeomycinR

Intron

Knockout chromosome

(vector DNA)

Chromosome of ES cell

Plate on medium with

Neomycin

wt

2. Knockout Organisms iii

Diagram from Introduction to Genetic Analysis, Gelbart, W

et al

Parents F1 F2

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.

• Mendelian ratios of F2 mice were as expected so Mlh3 not required for development.

• F2 generation sterile.

Fig

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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.

• Mlh3 shown to be essential for reproduction and meiosis.

Fig

ure

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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)