This study examined the effects of spontaneous mutations on fitness in the yeast Saccharomyces cerevisiae by analyzing 32 mutation-accumulation lines of three different cell types: haploid a, haploid alpha and diploid. The lines were generated and their mating types identified. A micro-colony growth assay was used to measure the growth rates of each strain to determine the effects of mutations on fitness. Some strains showed no halo in mating type tests but were genetically haploid, demonstrating false positives. The results will provide insights into how spontaneous mutations impact yeast fitness.

1. Fitness study on mutation-accumulation lines of
Saccharomyces cerevisiae
Xiangyu Peng and Mark L. Siegal
New York University, Center for Genomics and Systems Biology, Department of Biology
INTRODUCTION
It was generally believed that the major effects of spontaneous mutation areIt was generally believed that the major effects of spontaneous mutation are
deleterious. Dr. Fisher R. A. proposed this theory in 1930, introducing a geometricdeleterious. Dr. Fisher R. A. proposed this theory in 1930, introducing a geometric
model that implied the deleterious effects of most mutations. Moreover, somemodel that implied the deleterious effects of most mutations. Moreover, some
molecular data on the ratio of nonsynonymous to synonymous substitution ratesmolecular data on the ratio of nonsynonymous to synonymous substitution rates
pointed out that most nonsynonymous mutations have deleterious effects on fitnesspointed out that most nonsynonymous mutations have deleterious effects on fitness
(Eyre-Walker(Eyre-Walker et al.et al., 2002). However, some recent studies suggest that spontaneous, 2002). However, some recent studies suggest that spontaneous
mutations might have beneficial effects more commonly than we thought beforemutations might have beneficial effects more commonly than we thought before
(Sarah B. Joseph and David W. Hall, 2004). From analysis of a 1012-generation(Sarah B. Joseph and David W. Hall, 2004). From analysis of a 1012-generation
mutation-accumulation (MA) lines of yeast, D. W. Hallmutation-accumulation (MA) lines of yeast, D. W. Hall et al.et al. found that 5.75% of thefound that 5.75% of the
fitness-alerting mutations were beneficial.fitness-alerting mutations were beneficial.
To further understand the affects of spontaneous mutations on yeast and quantifyTo further understand the affects of spontaneous mutations on yeast and quantify
the beneficial rates, I performed fitness assays for 32 strains of MATα, MATa andthe beneficial rates, I performed fitness assays for 32 strains of MATα, MATa and
MATα/MATa, respectively. The MATα, MATa and MATα/MATa refer to sexual cellMATα/MATa, respectively. The MATα, MATa and MATα/MATa refer to sexual cell
types of yeast — haploid α, haploid a and diploid — which are determined by thetypes of yeast — haploid α, haploid a and diploid — which are determined by the
MATα and MATa alleles at the mating type locus (MAT)MATα and MATa alleles at the mating type locus (MAT) （（ Amar J. S. Klar, 2010Amar J. S. Klar, 2010 ））
METHODS
1. Generating 32 MATα and MATα/MATa strains from 32 MATa MA strains
There are three major steps: transformation, switching and halo test.
Haploid MATa strains were transformed with the pGal-HO plasmid to allow
switching to the MATa and MATα/MATa types. Then switching was induced by
using galactose medium, which may produce colonies of all three types. After
switching, I performed halo tests to determine the mating types of the colonies,
and select one representative haploid α and one representative diploid for each
original MA strain.
2. Measurement of fitness
After the preparation of the MA lines, a 96-well master plate containing 32 strains
of mating type a in the first four columns, mating type α in the middle four columns
and diploid in the last four columns. Then the growth-rate distributions of each of
the 96 strains will be determined by the microscope-based micro-colony growth
assay developed in the lab (Levy et al., 2012).
RESULTS
CONCLUSIONS
The 32 strains of three different cell types, haploid a, haploid alpha and diploid, are valid to study the effects of spontaneous mutations on fitness in budding yeast,
Saccharomyces cerevisiae. There were some false positive strains, which showed no halo in halo test, but genetically they are haploids.
RC634a (mating type a)
Halo Test
TX1-20A (mating type alpha)
Micro-colony Growth Assay
Yeast FACS Analysis with Sytox Green
Haploid (control) Diploid (control)
MAD.0 MAD.11