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

Abstract

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. A defined mutant human glucose-6-phosphate-dehydrogenase library was constructed which contains all possible single nucleotide missense mutations in the eight-residue glucose-6-phosphate binding peptide of the enzyme. Mutant human enzymes were expressed in a zwf1 (gene encoding yeast homologue) deletion strain of Saccharomyces cerevisiae. Growth rates of the 54 mutant strains arising from this library were measured in parallel in conditions selective for active hG6PD. Several residues were identified which tolerated no mutations (Asp200, His201 and Lys205) and two (Ile199 and Leu203) tolerated several substitutions. Arg198, Tyr202, and Gly204 tolerated only 1-2 specific substitutions. Generalizing from the positions of tolerated and non-tolerated amino acid substitutions, hypotheses were generated about the functional role of specific residues, which could, potentially, be tested using higher resolution/lower throughput methods.

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

“…Each of the nine mutant p53 strains tested expressed the p53 monomer at a similar level although their activities spanned the entire range. mRNA transcription rates from the p415CYC1 expression vector have been previously demonstrated to vary little from clone to clone (Merritt et al 2003;Merritt et al 2005). Therefore, similar p53 monomer concentrations among the mutants tested indicate similar rates of protein translation and degradation.…”

“…Initially, plasmid DNA was isolated from culture samples using the Yeast RPM Plasmid Kit (Qbiogene). This plasmid DNA was then used as the template in the polony reactions described below as described in the literature (Merritt et al 2003;Merritt et al 2005). Single Base Extension (SBE) Sequencing of the sequence tags (STs) ( Fig.…”

“…Single Base Extension (SBE) Sequencing of the sequence tags (STs) ( Fig. 2A) was used to quantify polonies arising from each mutant strain in the mixed culture as described in the literature (Merritt et al 2003;Merritt et al 2005). …”

“…Gene expression analysis of individual strains was conducted essentially to verify the expression level of the p53 gene as described (Merritt et al 2003;Merritt et al 2005;Mikkilineni et al 2004). The ratio of ADE2:p53 was calculated and the % WT activity for each mutant tested was calculated using a calibration curve (R 2 = 0.93) prepared from the ADE2/p53 ratios of mutants with previously reported activity (Leu330Ala, Leu330His, Gln331Ala and Ile332Ala).…”

“…(Jia et al 1997)) and, in fact, utilized a reporter strain developed for this application (Tomso et al 2005). Unlike these assays, which utilize separate colony growth on solid agar to identify and isolate functional and non-functional p53 expressing strains and standard DNA sequencing to identify a specific inactivating mutation, we utilized mixed mutant growth competitions, polymerase colony (polony) (Mitra and Church 1999) and primer extension sequencing technology ) similar to methods we have reported previously (Merritt et al 2003;Merritt et al (2005). The primary advantage of our methodology is that mutant enrichment (via mixed strain growth competition) and identification of the associated mutation(s) (polony based) are highly parallel.…”