Identification of genes up- and down-regulated in the nuc-2A mutant strain of Neurospora crassa.

GRAS DE; SILVEIRA HC; MARTINEZ-ROSSI NM; ROSSI A

Asilomar

Conferencia; 24th Fungal Genetics Conference at Asilomar; 2007

Genetics Society of America

Microorganisms have evolved complex signal transduction networks that enable them to make optimal use of the nutrient sources available. Inorganic phosphate (Pi), an essential nutrient for all organisms, is required for the biosynthesis of nucleic acids, phospholipids and cellular metabolites, as well as for the energy transduction and metabolic signaling responses. The phosphorus acquisition system in Neurospora crassa includes four regulatory genes: nuc- 2+, preg+, pgov+, and nuc-1+. Under limiting Pi, NUC-2 inhibits the functioning of the complex PREG-PGOV through the interaction of ankyrin-like repeats, allowing the activation of the wide domain transcription regulator NUC-1. In an attempt to identify genes involved in metabolic responses to exogenous Pi sensing, we employed suppression subtractive hybridizations (SSH) between RNA isolated from the wild type (St.L.74A) and nuc-2A (FGSC#1996) grown under Pi starvation, pH 5.4. Following SSH, expression of clones was examined using dot-blot macro-arrays. Of the 900 clones arrayed from the SSH cDNA library, approximately 21% were differentially regulated. A total of 66 differentially up-regulated and 124 down- regulated clones were identified and sequenced. Genes encoding proteins involved in signal transduction and regulation were identified among the up-regulated sequences, including translation initiation factor eIF3, ubiquitin and kinases. Among the genes found to be down regulated in nuc-2A, six were involved in gluconeogenesis and oxidative phosphorylation. Virtual and Northern blot analyses of randomly selected genes confirmed the differential gene expression result. Thus, the identification of differentially-regulated genes in nuc-2A is key to the understanding of molecular events involved in phosphorus sensing.