Workshop: Yeast Genetics

Yeast, in particular Saccharomyces cerevisiae, is used as a model to study aging, the cell cycle, apoptosis, gene expression, metabolism and many other areas. Many cause disease an so studying their virulence, metabolism and/or other properties may help us to develop therapeutics. Genetics and strain improvement is key to using yeast for biotechnology.

Yeast and fungi are extremely diverse, both genetically and phenotypically. Uses of yeast and fungi in biotechnology include fermentations, production of biopharmaceuticals, biocatalysts and recombinant proteins.

Saccharomyces cerevisiae, aka baker’s/brewer’s yeast is either diploid or haploid. Diploid cells are able to sporulate. S. cerevisiae has a doubling time of approximately 90 minutes at 30 degrees. S. cerevisiae is easily genetically manipulated for use in transformation, mutagenesis or useful auxotrophic markers.

Tetrad analysis is useful for mapping mutations and constructing strains among other uses. This is where a and alpha strains are crossed and meiosis occurs creating 4 haploid progeny which are dissected and analysed. May be ordered eg in Neurospora or unordered eg in Saccharomyces.

Parental ditype (PD) = same configuration

Nonparental ditype = completely recombinant progeny

Tetratype = one of each class

In a cross between ade- his+ x ade+his-, if 50% of offspring are ade-his+ and 50% are ade+his-, the tetrad is parental ditype.

In a cross between ade- his+ x ade+his-, if offspring are ade-his- and ade+his+ (neither of the parental genotypes), the tetrad is nonparental ditype.

In a cross between ade- his+ x ade+his-, if the offspring are one of each possible genotype, the tetrad is a tetratype.

If more tetrads are parental ditype than nonparental ditype, the observed genetic loci are linked.

If PD:NPD:T ratio is 1:1:4, independent assortment is indicated (no gene linkage).

If loci are linked to their respective centromeres, the proportion of T decreases.

If genes are linked (PD>NPD), you can estimate (for D < 35 cM) map distance (D) based on number of recombinants.

D = 100 (6NPD + T)/2 (PD + T + NPD)

Homologous recombination can be used to knock-out or knock-in genes. CRISPR-Cas technologies are used for gene editing.

Yeast two-hybrid experiments use ‘bait’ and ‘prey’ proteins fused to GAL4 domains. the interaction of the bait and prey proteins leads to transcription of the reporter gene. The reporter gene is the gene required for amino acid biosynthesis. This leads to growth. There are many variations on this theme eg one-hybrid, three-hybrid, different proteins, promoters and organisms etc. However there are many false-positives and false-negatives. The Takara system has 4 different promoters to reduce false positives.

Protein localisation is important for function. Genes are fused to gfp or another fluorescent reporter gene for use in fluorescence microscopy. Immunofluorescence can also be used. The position of the tag at the N or C terminal is important as is the choice of reporter gene. Co-localisation can be studied using different fluorescent reporters. However co-localisation doesn’t necessarily imply interaction.

Random or targeted mutagenesis can lead to the development of new isolates which demonstrate improved productivity and/or growth. By understanding yeast metabolism and biosynthetic pathways of specific products, we can design genetic engineering strategies for optimising production yields.

Host strain for artemisinin semi-synthesis = chassis for heterologous expression of Artemisia annua genes.