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Abstract
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. Amino acid Gln331 tolerated essentially all mutations. Importantly, highly parallel mutagenesis and cloning techniques were utilized which, in conjunction with recently reported highly parallel DNA sequencing methods, would be capable of increasing throughput an additional 2-3 orders of magnitude.
Highlights
The ability to use genotype data to understand complex phenotypes, for example the ability to predict the susceptibility to infectious disease or the likelihood of developing cancer based upon one’s genome sequence, was one of the early dreams of the Human Genome Project
The concentrations of each mutant bearing strain in culture was measured at several time points using polonies and single base extensions to identify the unique tag associated with each mutant p53 gene
All elements of l were allowed to vary in order to minimize the sum of the square of the error (R(Error)2) between the calculated and measured matrix according to the equation: X (Error
Summary
The ability to use genotype data to understand complex phenotypes, for example the ability to predict the susceptibility to infectious disease or the likelihood of developing cancer based upon one’s genome sequence, was one of the early dreams of the Human Genome Project. The publication of the human genome sequence (Lander et al 2001; Venter et al 2001) and ongoing research devoted to describing genetic variation within the human population (Masood 1999) are initial steps in this effort. One of the major steps in the Human Genome Project is to understand the physiological effect of genetic diversity in the human population. How do we identify functionally important mutations? We developed a highly parallel functional assay based upon the ability of the human p53 (p53) protein to initiate
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