Isolation and Identification of Yeasts from Natural ... - Library Science

CHAPTER 3
Mutagenesis in Yeast
John F. T. Spencer and Dorothy M. Spencer
1. Introduction
Mutations of numerous types can be induced in yeast. The basic
principle is to bring the yeast in contact with the mutagen (UV light, X-rays,
EMS, MMS, nitrous acid, nitrosoguanidine [NNG], ICR- 170, nitrogen
mustard, and so on), for long enough to bring about 50-95% killing,
after which the mutagen is removed. The yeast strain may be grown in
nutrient medium for a round of cell division to fix the mutation in the
cells (a “recovery” step), and the cells are plated out on complete medium.
Finally, the cells are replica plated to minimal medium, if auxotrophic
mutants are desired, or on various other media designed to detect muta-
tions such as resistance to a number of compounds (canavanine, cyclo-
heximide, heavy metals, and similar toxic compounds). Recessive
mutations are generally induced in haploid strains, but dominant muta-
tions, including many of the mutations to resistance, can be induced in
cells of higher ploidy. Mutations in the mitochondrial (mt) DNA form a
special class of mutations in Succharomyces cerevisiae, the classicpetite
coEonie mutation, which manifests itself as a small colony on agar, and
which lacks the ability to grow on nonfermentable substrates. The
mtDNA in this mutation is nonfunctional or absent, giving rise to the
respiratory deficiency, and the mutation occurs spontaneously, or is
induced by manganese, and by many carcinogenic drugs, such as acriflavin,
ethidium bromide, adriamycin, daunomycin, and benzidine, to name a
few. Mutations conferring resistance to such antibiotics as chloram-
From Methods m Molecular Biology, Vol 53 Yeast Protocols
Edited by: I. Evans Humana Press Inc , Totowa, NJ
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