Figure 4.
atm-1 and
atl-1 are not required for RNR expression. (A) The relative transcriptional levels of
rnr-1 and
rnr-2 were normalized using
eft-2 expression. Total RNAs were isolated from L4 and 3-day-old hermaphrodites of wild-type,
atm-1, and
atl-1 homozygotes. Real-time PCR experiments were performed in triplicate for each sample. The following primer sets were used to amplify
rnr-1: (forward) 5'-GCG AGT CGA GAA GGA TCA AG and (reverse) 5'-GGC TTC GTA TTT GGC GTA AA,
rnr-2: (forward) 5'-ATC AGT GCC GAA ACA CTC ATC and (reverse) 5'-CAG CTC ATT CAC CTC CGA CT, and
eft-2 as described in the legend of Figure 1. Vertical bars indicate standard error. (B) Western blot analysis of RNR-1 and RNR-2 protein levels. Wild-type,
atm-1 and
atl-1 homozygous mutant hermaphrodites (3- day-old) and
rnr-1 and
rnr-2 RNAi hermaphrodites were boiled for 5 min in 23 SDS-loading buffer (63 mm Tris-HCl [pH 6.8], 4% SDS, 5% b-mercaptoethanol, 20% glycerol) and then homogenized with a sonicator for 30 sec. The RNAi samples were obtained from the young adults (3 days from L1) that had been fed from the L1 larval stage on E. coli HT115 (DE3) expressing
rnr-1 dsRNA (541- 1559) or
rnr-2 dsRNA (254-1397) (Kamath et al. 2001). Total protein from 25 adult hermaphrodites was separated by discontinuous 8 or 12% SDS-PAGE and then electrotransferred to Hybond ECL nitrocellulose membranes (GE Healthcare). Immunochemical hybridization was performed using anti-RNR-1 (Santa Cruz Biotechnology) and anti-RNR-2 (Calbiochem) polyclonal antibodies, with horseradish peroxidase-coupled secondary antibodies of donkey anti-goat IgG (AP-180P, Chemicon) and goat anti-rabbit IgG (GE Healthcare), respectively. Signals were visualized using the ECL Plus Western blotting detection system (GE Healthcare) and quantified with a Fuji LAS 1000 digital image analyzer (Fuji film). Coomassie Brilliant Blue (CBB) staining of major proteins was used as an internal standard. (C) The ATP/dATP ratios of wild-type hermaphrodites and
atl-1(
tm853) homozygous mutants were measured using LC-MS/MS. Approximately 1000 wild-type hermaphrodites and
atl-1(
tm853) homozygous mutants (3 days old) were suspended in PBS containing 2% SDS and boiled for 5 min, followed by homogenization with a sonicator for 30 sec. The extract was deproteinized with TCA (final concentration of 5%), vortexed, placed in an ice bath to incubate for 10 min, and then revortexed. The supernatant was collected, neutralized with potassium carbonate, and filtered through a 0.45 mm membrane filter. Finally, the extract was purified by centrifugal ultrafiltration using a 30,000 MW cutoff membrane (Amicon YM-3, Millipore).