Improved statistical analysis of budding yeast TAG microarrays revealed by defined spike-in pools.

Saccharomyces cerevisiae knockout collection TAG microarrays are an emergent platform for rapid, genome-wide functional characterization of yeast genes. TAG arrays report abundance of unique oligonucleotide 'TAG' sequences incorporated into each deletion mutation of the yeast knockout collection, allowing measurement of relative strain representation across experimental conditions for all knockout mutants simultaneously. One application of TAG arrays is to perform genome-wide synthetic lethality screens, known as synthetic lethality analyzed by microarray (SLAM). We designed a fully defined spike-in pool to resemble typical SLAM experiments and performed TAG microarray hybridizations. We describe a method for analyzing two-color array data to efficiently measure the differential knockout strain representation across two experimental conditions, and use the spike-in pool to show that the sensitivity and specificity of this method exceed typical current approaches.

Measurement of glutamate carboxypeptidase II (NAALADase) enzyme activity by the hydrolysis of [³H]-N-acetylaspartylglutamate (NAAG).

The peptide N-Acetylaspartylglutamate (NAAG) is hydrolyzed by N-Acetylated-alpha-linked-acidic dipeptidase (NAALADase, glutamate carboxypeptidase II) into N-Acetylated aspartate (NAA) and glutamate. Hydrolysis can be measured as described in this unit by employing radiolabeled NAAG (NAA-[(3)H]glu) as the substrate. The occurrence of NAALADase activity in a wide range of tissues has implications for a variety of physiological purposes. The assay described here is useful for the analysis of NAALADase activity and its inhibition in brain synaptosomal preparations, tissue homogenates and tissue culture cell pellets.