Temperature Sensitive Mutant Experiment

Mutant proteins are usually unstable. They are called temperature sensitive mutants because at lower temperatures they are normal (wild-type), but melt apart (denature) at higher temperatures.

Thus, it is possible to easily compare and contrast the wild-type and mutant phenotypes simply by adjusting the temperature. Important genes can be identified and understood. At a lower temperature, the mutant will be wild-type; at a higher temperature, the mutant will reveal its mutant phenotype.

The temperature-sensitive mutant experiment described below identified genes relevant to cellular division in S. cerevisiae (baker's yeast). Temperature sensitive mutants are often denoted with a ^ts superscript.

Mutagen

Add a mutagen to a liquid culture of yeast, then distribute the culture into smaller aliquots. Incubate at 23°C for 5 hours. Next, plate each aliquot onto an agar plate and incubate overnight at 23°C. This provides colonies which will later be screened for temperatures sensitivity.

Screen

Make two replica-plates of each plate prepared earlier: one will be incubated at 23°C; the other will be incubated at 36°C. Colonies which form at 23°C (the permissive temperature) but not at 36°C (the non-permissive temperature) represent temperature-sensitive mutants.

Assay

Colonies are microscopically examined to find microbes that are alive, but arrested at some point in the cell cycle. Some colonies may show no signs of division, while other colonies may be stuck midway through division.

Complement

Complementation tests determine whether different recessive mutations are in the same gene. It was accomplished by mating together temperature-sensitive haploid yeasts, then plating the diploid offspring and examining for growth at 23°C and 36°C.

Mating of Colony 1 and Colony 2	Mating of Colony 2 and Colony 3
This formed wild-type progeny that grew at both 23°C and 36°C. Being wild-type, the diploid progeny had at least one healthy allele for every gene. Thus, the mutations in Colony 1 and Colony 2 were on different genes.	This formed mutant progeny that only grew at 23°C. Being mutant, the diploid progeny had a gene with two non-functioning alleles. Thus, the mutations in Colony 2 and Colony 3 were on the same gene.

Transformation

Wild-type yeast DNA was used to form a plasmid library, which was then used to transform the temperature-sensitive cell cycle mutants isolated earlier. These transformed cells were plated and grown at 35°C -- if growth was observed, then the mutant must have been transformed with a functional version of its nonfunctional gene. The plasmids were then extracted and sequenced so that the genes involved in cell cycle mutations could be compiled.

Results

First, this experiment identifies any temperature-sensitive mutation that inhibits colony formation. However, these mutations are not just those which directly stop cell division; they could also be lethal mutations. Thus, temperature-sensitive mutants were examined microscopically to ensure that they were indeed alive but unable to divide. Some mutants were even arrested midway through division. Next, these temperature-sensitive mutants of cell division were consolidated via complementation. This allowed mutants to be organized based on whether they contain a mutation in the same gene. However, the mutated gene was still unknown. Lastly, transformation with a plasmid library allowed identification of the precise genes that had blocked the cell cycle.