Immune-specific probes oligonucleotides

To determine what cells make the immune-specific RNA we inoculated mid-third instar Drosophila larvae with bacteria and six hours later dissected them into fat bodies and fat body-free carcass. Total RNA was collected, separated by electrophoresis, transfered to nitrocellulose, and probed with labelled Pool 1 oligonucleotide. The results showed (Fig. 5) that intact inoculated larvae accumulate the immune-specific transcript while intact control larvae do not. The tissue dissection experiment showed that fat body cells of inoculated larvae contain transcripts homologous to the immune-specific probe, but the carcass does not. We conclude that fat body cells in Drosophila larvae accumulate a transcript that has homology to sarcotoxin when they have been inoculated with bacteria. We are currently cloning the responsible immune gene. 

Figure 4. The oligonucleotide recognizes an immune-specific transcript. melanogaster were inoculated with bacteria or left as controls and incubated for 11 or 30 hours as indicated. RNA was collected and separated on the gel shown at the right stained with ethidium bromide and then hybridized to the oligonucleotide probe in a northern blot on the left.

Figure 5. Drosophila fat body cells accumulate immune-specific RNA. Drosophila larvae were inoculated with bacteria and six hours later RNA was extracted from either whole larvae, from fat body, or from the carcasses minus fat body. These RNAs were separated on the gel shown on the right stained with ethidium bromide, then northern blotted and probed with the oligonucleotide on the left.

Some of the ABs may have structural homology to antibacterial proteins isolated from the flesh fly (20,21) since an oligonucleotide probe that can encode a portion of the sarcotoxin protein recognizes an immune-specific RNA in Drosophila fat body cells, the cellular origin of Drosophila s ABs (unpubl.). The induction mechanism must work relatively rapidly since we found immune-specific RNA in fat body cells within 6 hours after inoculation. Because of its homology to sarcotoxin, we assume that the immune-specific transcript detected by the oligonucleotide probe encodes an antibacterial protein. 

---