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. 2015 Nov;201(3):1263-74.
doi: 10.1534/genetics.115.181099. Epub 2015 Sep 9.

Complementation of Yeast Genes With Human Genes as an Experimental Platform for Functional Testing of Human Genetic Variants

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Free PMC article

Complementation of Yeast Genes With Human Genes as an Experimental Platform for Functional Testing of Human Genetic Variants

Akil Hamza et al. Genetics. .
Free PMC article

Abstract

While the pace of discovery of human genetic variants in tumors, patients, and diverse populations has rapidly accelerated, deciphering their functional consequence has become rate-limiting. Using cross-species complementation, model organisms like the budding yeast, Saccharomyces cerevisiae, can be utilized to fill this gap and serve as a platform for testing human genetic variants. To this end, we performed two parallel screens, a one-to-one complementation screen for essential yeast genes implicated in chromosome instability and a pool-to-pool screen that queried all possible essential yeast genes for rescue of lethality by all possible human homologs. Our work identified 65 human cDNAs that can replace the null allele of essential yeast genes, including the nonorthologous pair yRFT1/hSEC61A1. We chose four human cDNAs (hLIG1, hSSRP1, hPPP1CA, and hPPP1CC) for which their yeast gene counterparts function in chromosome stability and assayed in yeast 35 tumor-specific missense mutations for growth defects and sensitivity to DNA-damaging agents. This resulted in a set of human-yeast gene complementation pairs that allow human genetic variants to be readily characterized in yeast, and a prioritized list of somatic mutations that could contribute to chromosome instability in human tumors. These data establish the utility of this cross-species experimental approach.

Keywords: chromosome instability; human genetic variants; human–yeast cross-species complementation; model organisms and human disease; tumor-specific missense mutations.

Figures

Figure 1
Figure 1
Overview of the experimental design for the complementation screens. (A) Pipeline outlining which human–yeast complementation pairs were included in both screens. (B) Flowchart for the complementation screens. Human cDNAs were shuttled from entry clones to indicated yeast destination vectors to generate yeast expression vectors. Single or pooled expression vectors were then transformed to matched or pooled haploid convertible heterozygous diploids and maintained on −Ura media. Following sporulation, heterozygous diploids were plated on haploid selection media (MM −Ura). “Rescued” haploids were tested for plasmid dependency by replica plating on MM +5-FOA. For the pooled screen, 5-FOA-sensitive colonies were isolated for sequencing of yeast barcode and expression vectors.
Figure 2
Figure 2
Essential yeast genes that are replaceable by human genes. A total of 58 yeast genes are represented by nodes and grouped according to cellular processes (Yeastmine). Yeast CIN genes are represented by black nodes.
Figure 3
Figure 3
Analyzing features of essential yeast genes that predict replaceability including (A) localization patterns, (B) molecular function, (C) no. of genetic interactions, (D) no. of physical interactions, (E) part of macromolecular complexes, (F) yeast gene size, and (G) human–yeast sequence identity. Localization data, Gene Ontology (GO) terms, no. of genetic/physical interactions, and gene size for each yeast gene were obtained from Yeastmine and each feature is represented as a proportion of the total number of genes input for each set (n = 621 for all essential genes included in both screens and n = 58 for the complementation genes). Overall, the complementation set was enriched for yeast proteins that localize to the cytoplasm (P = 8.18E-03), have catalytic activity (P = 2.28E-02), less physical interactions (P = 5.77E-03), are less likely to be part of macromolecular complexes (P = 3.36E-07), and have smaller gene size (P = 9.16E-03). For sequence identity, “essential gene pairs” refers to the 1076 human–yeast pairs included in this study corresponding to 621 yeast genes and “complementation gene pairs” refers to the 65 complementation pairs corresponding to 58 yeast genes. The box plot highlights the median and range of sequence identity for each set of gene pairs.
Figure 4
Figure 4
hSEC61A1 complements yRFT1. Expression vectors (hRFT1 and hSEC61A1 under the control of the GPD constitutive promoter and ySEC61 under the control of the GAL-inducible promoter) were transformed into the RFT1/rft1∆ heterozygous diploid yeast strain and plated on haploid selection media (MM −Ura) following sporulation. Complementation was scored by higher than background growth on MM −Ura as shown and confirmed by testing for plasmid dependency on MM +5-FOA and tetrad dissection.
Figure 5
Figure 5
Plasmid shuffle used to generate strains expressing human cDNAs with missense mutations. Yeast haploid knockout strains covered by wild-type human cDNAs on URA3-marked vectors were transformed with the following LEU2-marked vectors (empty, wild-type human cDNA, and human cDNA with missense mutation) and maintained on −Ura −Leu media. Strains were plated on −Leu +5-FOA media to generate haploid yeast knockouts covered by LEU2-marked vectors. Strains were confirmed to have lost the URA3-marked plasmid by streaking on −Ura media to observe no growth. In the presented example, hSsrp1-K33E is able to complement pob3∆, but S481P results in a nonfunctional hSsrp1 protein.

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