Investigating the human purine biosynthesis pathway in yeast cells

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Neta Agmon, Leslie Mitchell, Yizhi Cai and Jef Boeke

Johns Hopkins University, United States

The purine metabolism pathway is highly conserved from yeast to humans. This pathway includes both the de-novo biosynthesis of AMP and GMP as well as the salvage pathway that regenerates AMP and GMP from nucleic acid turnover. In humans there are more than 20 disorders associated with the purine pathway, and they include a variety of symptoms, including: Gout, autism, blindness, loss of immunity, kidney failure and different neurological abnormalities. Some of these diseases and their hallmarks were extensively studied, however some of the symptoms associated with other disorders are much less well understood. The high conservation of the purine biosynthesis pathway between yeast and humans allows the study of the pathway and the mutational effects to be done in yeast. Using a synthetic approach, we are swapping the entire purine biosynthesis pathway of the yeast with the cognate human genes. We are cloning each human codon optimized gene under its yeast ortholog promoter and terminator. Each gene will be examined for its ability to complement the corresponding deletion of the yeast gene. In addition to swapping each gene at a time, all 26 genes participating in this pathway will be added to yeast cells on a single plasmid. This will allow for future investigation of the entire pathway, and its associated genes, in a variety of yeast based high-throughput methods as well as screening for new drugs for the pathway associated diseases.