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.2003 Aug 1;31(15):e84.
doi: 10.1093/nar/gng084.

Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies

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Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies

Joshua Merritt et al. Nucleic Acids Res..

Abstract

We describe a strategy to analyze the impact of single nucleotide mutations on protein function. Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools and polyacrylamide gel immobilized PCR. A system was constructed in which the yeast PGK1 gene was expressed from a plasmid-borne copy of the gene in a PGK1 deletion strain of Saccharomyces cerevisiae. Using this system, we demonstrated that the enrichment or depletion of PGK1 point mutants from a mixed culture was consistent with the expected results based on the isolated growth rates of the mutants. Enrichment or depletion of individual point mutants was shown to result from increases or decreases, respectively, in the specific activities of the encoded proteins. Further, we demonstrate the ability to analyze the functional effect of many individual point mutations in parallel. By functional complementation of yeast deletions with human homologs, our technique could be readily applied to the functional analysis of single nucleotide polymorphisms in human genes of medical interest.

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Figures

Figure 1
Figure 1
Saccharomyces cerevisiae growth as a function of the flux in the phosphoglyerate kinase reaction. The growth rate was calculated using a FBA model of the core metabolic pathways inS.cerevisiase as described (13). The growth rate was normalized to the optimal growth rate without flux limitations. The flux in the phosphoglycerate reaction was normalized to the optimal value.
Figure 2
Figure 2
PGK1 mRNA expression levels of several strains used in this work. Expression from plasmids utilizing the CYC1 promoter was approximately equal in the three representative strains tested. The ratio of expression from the ADH and CYC1 promoters was 2.5:1 and agreed approximately with previously reported results. AKY2 mRNA constitutively expressed from the genomic copy of the gene was used to normalize PGK1 expression.
Figure 3
Figure 3
Representative polony images. Lys131Glu mutant time series from left to right: (A) 0 h, (B) 51.75 h and (C) 97 h. Polonies were initially extended with cyanine-5-dCTP (A and C) or cyanine-5-dUTP (B). The slides were then imaged and the extended primers were removed. The procedure was then repeated with cyanine-5-dCTP used to extend (B) and cyanine-5-dUTP used to extend (A) and (C). A positive extension with cyanine-5-dUTP indicates that the wild-type nucleotide is present at position 391 of PGK1 (red), while positive extension with cyanine-5-dCTP indicates the mutant nucleotide (green) at this position.
Figure 4
Figure 4
Fraction of each mutant strain in culture as a function of time during competitive growth experiments. Fractions were measured using polony gels and single base primer extension sequencing. The fraction of the wild-type strain was determined by subtracting the sum of the four mutant strains from 100%. Measured data are presented as the average of three to six polony slides (symbols); error bars are ±1 SD in the data at a given point. Curve fits (lines) were determined using a least squares error minimization routine as described in the text. (A) Initial growth competition run in fermentor; mutations located in three distinct regions of the PGK1 gene. (B) Second growth competition run in shake flasks; all mutations lie within a single 300 bp region of the PGK1 gene.
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References

    1. Venter J.C., Adams,M.D., Myers,E.W., Li,P.W., Mural,R.J., Sutton,G.G., Smith,H.O., Yandell,M., Evans,C.A., Holt,R.A. et al. (2001) The sequence of the human genome. Science, 291, 1304–1351. - PubMed
    1. Lander E.S., Linton,L.M., Birren,B., Nusbaum,C., Zody,M.C., Baldwin,J., Devon,K., Dewar,K., Doyle,M., FitzHugh,W. et al. (2001) Initial sequencing and analysis of the human genome. Nature, 409, 860–921. - PubMed
    1. Masood E. (1999) US firm’s bid to sequence rice genome causes stir in Japan … as consortium plans free SNP map of human genome. Nature, 398, 545–546. - PubMed
    1. Steinmetz L.M., Scharfe,C., Deutschbauer,A.M., Mokranjac,D., Herman,Z.S., Jones,T., Chu,A.M., Giaever,G., Prokisch,H., Oefner,P.J. et al. (2002) Systematic screen for human disease genes in yeast. Nature Genet., 31, 400–404. - PubMed
    1. Winzeler E.A., Shoemaker,D.D., Astromoff,A., Liang,H., Anderson,K., Andre,B., Bangham,R., Benito,R., Boeke,J.D., Bussey,H. et al. (1999) Functional characterization of the S.cerevisiae genome by gene deletion and parallel analysis. Science, 285, 901–906. - PubMed

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