Development and application of highly-parallel yeast functional assays for the analysis of mutant human proteins
The ability to use an individual's genome sequence to predict and treat disease is one of the most important goals of the study of human genetics and the Human Genome Project, Sequencing the human genome was the first milestone accomplished to this end. However, a second goal, interpreting the sequence and the impact of genetic diversity in the population, is critical to understanding the basis of genetic disease and to the development of therapies. Large scale efforts are currently underway which aim to map all human Single Nucleotide Polymorphisms (SNPs). The mapping of SNPs is an important undertaking; SNPs account for a large percentage of genetic variation in humans and, therefore, may be responsible for different susceptibility to disease, potential for developing cancer, etc. among individuals. However, identifying SNPs is an intermediate goal; functional characterization of SNPs and other functionally important mutations is essential to understanding their contribution to human health. Therefore, it is critical to develop experimental tools that can be used to functionally screen the vast number of SNPs expected to be identified and to screen human genes, in general, for functionally important novel mutations.
Yeast functional assays are a promising group of technologies for the functional analysis of mutant human proteins. Most applications of yeast functional assays have been low throughput, i.e. analysis of approximately 1-10 mutant proteins. This level of throughput is not well suited to the task of characterizing the approximately 8000 possible single amino acid mutants which might arise from a single average-sized gene. In order to characterize entire genes with a reasonable number of experiments, e.g. 1-10, throughput increases of approximately 2-3 orders of magnitude would be required. With this goal in mind, several yeast functional assays were modified such that they could be used to analyze approximately 100-1000 mutants in parallel.
In initial proof-of-concept experiments, an assay was developed for the parallel analysis of the impact of single nucleotide mutations on protein function.
In a second set of experiments, the functional complementation assay was modified to increase throughput.
In a final set of experiments, modifications to the functional complementation assay used to analyze hG6PD mutants were applied to an alternative assay designed to screen the activity of transcription activator proteins. (Abstract shortened by UMI.)