Parallel analysis of tetramerization domain mutants of the human p53 protein using PCR colonies.

A highly-parallel yeast functional assay, capable of screening approximately 100-1,000 mutants in parallel and designed to screen the activity of transcription activator proteins, was utilized to functionally characterize tetramerization domain mutants of the human p53 transcription factor and tumor suppressor protein. A library containing each of the 19 possible single amino acid substitutions (57 mutants) at three positions in the tetramerization domain of the human p53 protein, was functionally screened in Saccharomyces cerevisiae. Amino acids Leu330 and Ile332, whose side chains form a portion of a hydrophobic pocket that stabilizes the active p53 tetramer, were found to tolerate most hydrophobic amino acid substitutions while hydrophilic substitutions resulted in the inactivation of the protein.

Parallel analysis of mutant human glucose 6-phosphate dehydrogenase in yeast using PCR colonies.

We demonstrate a highly parallel strategy to analyze the impact of single nucleotide mutations on protein function. Using our method, it is possible to screen a population and quickly identify a subset of functionally interesting mutants. Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools, and polymerase colonies.

Detecting changes in the relative expression of KRAS2 splice variants using polymerase colonies.

Because splice variants of a gene with multiple isoforms give rise to proteins with different functions, it seems plausible that changes in the expression levels of the splice variants could be a contributing factor to disease. In fact, recent examples in the literature clearly illustrate that altered expression levels of splice variants may play an important role in disease. Furthermore, these works demonstrate that changes in expression levels could potentially be used to (1) monitor disease progression, (2) diagnose disease, and/or (3) determine disease state.

Detection of allelic variations of human gene expression by polymerase colonies.

Background: Quantification of variations of human gene expression is complicated by the small differences between different alleles. Recent work has shown that variations do exist in the relative allelic expression levels in certain genes of heterozygous individuals. Herein, we describe the application of an immobilized polymerase chain reaction technique as an alternative approach to measure relative allelic differential expression.

Co-expression of molecular chaperones does not improve the heterologous expression of mammalian G-protein coupled receptor expression in yeast.

The limitations to high-level expression of integral membrane proteins are not well understood. The human A(2)a adenosine receptor (A(2)a) and mouse Substance P receptor (SPR) were individually expressed in S. cerevisiae to identify potential cellular bottlenecks for G-protein coupled receptors.