Metabolomics, genetics and biochemistry were combined to obtain the first complete map of the nucleotide degradation and ribose salvage pathway in yeast. This pathway promotes yeast survival in starvation and oxidative stress.

Summary Allostery and covalent modification are major means of fast-acting metabolic regulation. Their relative roles in responding to environmental changes remain, however, unclear. Here we examine this issue, using as a case study the rapid decrease in pyruvate kinase flux in yeast upon glucose removal. The main pyruvate kinase isozyme (Cdc19) is phosphorylated in response to environmental cues. It also exhibits positively-cooperative (ultrasensitive) allosteric activation by fructose-1,6-bisphosphate (FBP). Glucose removal causes accumulation of Cdc19’s substrate, phosphoenolpyruvate. This response is retained in strains with altered protein-kinase-A or AMP-activated-protein-kinase activity or with CDC19 carrying mutated phosphorylation sites. In contrast, yeast engineered with a CDC19 point mutation that ablates FBP-based regulation fail to accumulate phosphoenolpyruvate. They also fail to grow on ethanol and slowly resume growth upon glucose upshift. Thus, while yeast pyruvate kinase is covalently modified in response to glucose availability, its activity is controlled almost exclusively by ultrasensitive allostery.

It is quickly becoming apparent that situating human variation in a pathway context is crucial to understanding its phenotypic significance. Toward this end, we have developed a general method for finding pathways associated with traits that control for pathway size. We have applied this method to a new whole genome survey of coding SNP variation in 187 patients afflicted with Parkinson disease (PD) and 187 controls. We show that our dataset provides an independent replication of the axon guidance association recently reported by Lesnick et al. [PLoS Genet 2007;3:e98], and also indicates that variation in the ubiquitin-mediated proteolysis and T-cell receptor signaling pathways may predict PD susceptibility. Given this result, it is reasonable to hypothesize that pathway associations are more replicable than individual SNP associations in whole genome association studies. However, this hypothesis is complicated by a detailed comparison of our dataset to the second recent PD association study by Fung et al. [Lancet Neurol 2006;5:911–916]. Surprisingly, we find that the axon guidance pathway does not rank at the very top of the Fung dataset after controlling for pathway size. More generally, in comparing the studies, we find that SNP frequencies replicate well despite technologically different assays, but that both SNP and pathway associations are globally uncorrelated across studies. We thus have a situation in which an association between axon guidance pathway variation and PD has been found in 2 out of 3 studies. We conclude by relating this seeming inconsistency to the molecular heterogeneity of PD, and suggest future analyses that may resolve such discrepancies.

We have described molecular inversion probe technologies for large-scale genetic analyses. This technique provides a comprehensive and powerful tool for the analysis of genetic variation and enables affordable, large-scale studies that will help uncover the genetic basis of complex disease and explain the individual variation in response to therapeutics. Major applications of the molecular inversion probes (MIP) technologies include targeted genotyping from focused regions to whole-genome studies, and allele quantification of genomic rearrangements. The MIP technology (used in the HapMap project) provides an efficient, scalable, and affordable way to score polymorphisms in case/control populations for genetic studies. The MIP technology provides the highest commercially available multiplexing levels and assay conversion rates for targeted genotyping. This enables more informative, genome-wide studies with either the functional (direct detection) approach or the indirect detection approach.

Pyrosequencing is a DNA sequencing method based on the principle of sequencing-by-synthesis and pyrophosphate detection through a series of enzymatic reactions. This bioluminometric, real-time DNA sequencing technique offers unique applications that are cost-effective and user-friendly. In this study, we have combined a number of methods to develop an accurate, robust and cost efficient method to determine allele frequencies in large populations for association studies. The assay offers the advantage of minimal systemic sampling errors, uses a general biotin amplification approach, and replaces dTTP for dATP-apha-thio to avoid non-uniform higher peaks in order to increase accuracy. We demonstrate that this newly developed assay is a robust, cost-effective, accurate and reproducible approach for large-scale genotyping of DNA pools. We also discuss potential improvements of the software for more accurate allele frequency analysis.