Biosynthesis of Nucleotides and Nucleic Acids - …
For all microarray experiments, larvae were collected, snap-frozen and maintained at -80C prior to RNA extraction. Total RNA was extracted with TRIzol reagent (Invitrogen) followed by RNeasy (Qiagen) clear up. cRNA targets were generated and coupled to either Cy3 or Cy5 fluorophores according to 'FHCRC Genomics Resource DNA Array Laboratory' protocols. Expression profiles were performed using spotted microarrays constructed from release 1 (6 K, Figs , , , and ) or 1 and 2 (12 K, Fig ) of the D. melanogaster Gene Collection (GEO GPL4285 = '6 k' and GEO GPL1908 = '12 k'). Target label preparation and hybridization protocols are described elsewhere . Hybridization and scanning were performed by the Fred Hutchinson Cancer Research Center Genomics Resource DNA Array Laboratory. Microarray images were quantified using GenePix Pro software (Axon Instruments).
Protein synthesis starts with Deoxyribonucleic acid (DNA)
We found that starvation for dietary amino acids (AA's) leads to dynamic changes in transcript levels of many metabolic genes. The conserved insulin/PI3K and TOR signaling pathways mediate nutrition-dependent growth in Drosophila and other animals. We found that many AA starvation-responsive transcripts were also altered in TOR mutants. In contrast, although PI3K overexpression induced robust changes in the expression of many metabolic genes, these changes showed limited overlap with the AA starvation expression profile. We did however identify a strong overlap between genes regulated by the transcription factor, Myc, and AA starvation-responsive genes, particularly those involved in ribosome biogenesis, protein synthesis and mitochondrial function. The consensus Myc DNA binding site is enriched in promoters of these AA starvation genes, and we found that Myc overexpression could bypass dietary AA to induce expression of these genes. We also identified another sequence motif (Motif 1) enriched in the promoters of AA starvation-responsive genes. We showed that Motif 1 was both necessary and sufficient to mediate transcriptional responses to dietary AA in larvae.
The upstream regions of selected genes (cluster A - CG768, CG1135, CG6311, CG32486; cluster B - CG8470, CG6459, CG9539) were amplified by PCR from genomic DNA isolated from wildtype flies and subcloned into Stinger GFP transformation vector (obtained from Drosophila Genome Resource Center). The 5× motif1 reporter genes were constructed by cloning five tandem repeats of Motif1 into a Stinger GFP transformation vector . GFP mRNA levels were measured by quantitative real-time RT-PCR using RNA extracted from whole larvae.