Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'.
Whole genome sequencing of several microbes has revealed thousands of genes of unknown function. A large proportion of these genes seem to confer subtle quantitative phenotypes or phenotypes that do not have a plate screen. We report a novel method to monitor such phenotypes, where the fitness of mutants is assessed in mixed cultures under competitive growth conditions, and the abundance of any individual mutant in the pool is followed by means of its unique feature, namely the mutation itself. A mixed population of yeast mutants, obtained through transposon mutagenesis, was subjected to selection. The DNA regions (targets) flanking the transposon, until nearby restriction sites, are then quantitatively amplified by means of a ligation-mediated PCR method, using transposon-specific and adapter-specific primers. The amplified PCR products correspond to mutated regions of the genome and serve as 'mutant DNA fingerprints' that can be displayed on a sequencing gel. The relative intensity of the amplified DNA fragments before and after selection match with the relative abundance of corresponding mutants, thereby revealing the fate of the mutants during selection. Using this method we demonstrate that UBI4, YDJ1 and HSP26 are essential for stress tolerance of yeast during ethanol production. We anticipate that this method will be useful for functional analysis of genes of any microbe amenable to insertional mutagenesis.
Sharma
V M
VM
Institute of Microbial Technology, Sector 39A, Chandigarh-160036, India.
Chopra
R
R
Ghosh
I
I
Ganesan
K
K
D016172:DNA Fingerprinting / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D016254:Mutagenesis, Insertional; D009154:Mutation; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development
D004251:DNA Transposable Elements; D004271:DNA, Fungal