Figure 3.
ORK1 is expressed in the excitable tissues of adult D. melanogaster. (a) Northern blot analysis of ORK1 mRNA. D. melanogaster mRNA (5 μg, adult, Clontech) was resolved in a formaldehyde/agarose gel, blotted to nitrocellulose, and probed with 32P-labeled 2.4-kb ORK1 XhoI fragment overnight at 65°C. The blot was exposed to x-ray film for 46 hr at −70°C with an intensifying screen. (b) In situ hybridization to polytene chromosomes identifies a band on 1–10A1-2 by the method of Todd Laverty using biotin-labeled probes produced by nick-translation and peroxidase-based visualization (Enzo Diagnostics, New York). (c) In situ hybridization in whole adult fly preparations reveals expression of ORK1 mRNA in excitable tissues in the head and thorax (Upper) and abdomen (Lower) with antisense but not control cRNA. Digoxigenin-labeled antisense RNA (to the terminal 300 nucleotides of the coding region and 400 nucleotides of the 3′ untranslated region) or a kit-supplied control RNA were hybridized to 18-μm sections of quick-frozen, OCT-embedded, D. melanogaster CS overnight at 65°C and visualized by an alkaline phosphatase-catalyzed reaction (Boehringer Mannheim).