Purification and reverse transcription of the messenger RNA coding for the insect protein, calliphorin, isolated from larvae of the blowfly, Calliphora vicina R.-D

Abstract

Starting from poly(A)-containing RNA prepared from the fat body of larvae of the blowfly Calliphora vicina, we have purified an mRNA coding for the protein calliphorin, which is a major blowfly protein accounting for approximately 9% of the total poly(A)-containing mRNA activity in the fat body of 5-days-old larvae, as demonstrated by translation in vitro. The mRNA for this protein was purified by sucrose gradient centrifugation under denaturing conditions and identified by cell-free translation. The peak fraction of the gradient shows three bands of about 20 S after formamide/acrylamide gel electrophoresis. Using reverse transcriptase and Calliphora mRNA isolated directly from the acrylamide gel electrophoresis or from sucrose gradients, we synthesized a cDNA probe. This cDNA hybridizes with the template, showing a major kinetic component with a molecular weight of 2.8 x 10(6), fitting well with the three bands observed in the electrophoresis. Hybridization of the cDNA with total sonicated Calliphora DNA shows annealing only at very high c0t values, indicating that Calliphora mRNA is transcribed from the unique portion of the fly genome.