Albumin mRNA was purified from rat liver by indirect immunoprecipitation of albumin‐synthesizing polysomes. Monospecific antibodies against albumin, raised in rabbits, were added to rat liver polysomes and the antibody‐polysome complex formed was adsorbed to goat antibodies against rabbit immunoglobulins, coupled to p‐aminobenzyl‐cellulose according to Schütz et al. [Nucleic Acid Res. 4, 71–84 (1979)]. Approximately 8–10% of the polysomes were bound to the matrix. They were then eluted from the column, RNA was isolated and mRNA recovered by chromatography on oligo(dT)‐cellulose. By translation of the mRNA in vitro in a wheat germ system, it was estimated that approximately 90% of the albumin‐synthesizing mRNA was precipitated and that the albumin mRNA fraction obtained was enriched 8–10‐fold for albumin‐coding sequences. Complementary DNA was transcribed from this mRNA. Albumin‐specific cDNA was purified by hybridization of the cDNA to polysomal rat liver mRNA up to a limited rot value of 2 × 10−2 mol · 1−1· s, isolation of the double strands by hydroxyapatite chromatography and recovery of the hybridized cDNA after alkali digestion of RNA. Kinetic data obtained by hybridization of this cDNA against total cytoplasmic and purified mRNA demonstrate that more than 90% of this cDNA comprises only one sequence component. An r0t1/2 value of 2.2 × 10−3 mol · l−1· s was estimated for the immunoprecipitated mRNA fraction. This indicates already a high degree of purity of the mRNA component of this fraction, as this RNA still contains approximately 50% ribosomal RNA. By electrophoresis of glyoxal‐denatured RNA in agarose gels, a molecular weight of 780000 was found for albumin mRNA, a value which corresponds well with a sequence complexity of 880000, estimated from the kinetics of the mRNA‐cDNA hybridization. Titrations of albumin cDNA with total polysomal poly(A)‐containing RNA indicated a content of approximately 10% for albumin mRNA in this fraction. Total nuclear RNA was shown to contain approximately 0.1% albumin‐specific sequences, whereas poly(A)‐containing nuclear RNA was about 4‐fold enriched for albumin sequences.

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 lo6, 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 cot values, indicating that Calliphora mRNA is transcribed from the unique portion of the fly genome.During development of Diptera larvae, a group of closely related proteins are synthesized in the fat body [l-71 and accumulate, making up at a certain stage more than half of the proteins soluble in dilute salt solutions [I, 3,5,7]. In the blowfly Calliphora vicina, a single protein has been identified, composed of six or seven identical subunits with a molecular weight of approximately 80000 each, known as calliphorin [I, 21 ; it is immunochemically related to drosophilin and luciphilin, the proteins of Drosophila [l, 5,6] It has been demonstrated that the biosynthesis of calliphorin is restricted to 3 -5-days-old larvae [7,8] ; furthermore, translatable calliphorin mRNA appears in 3-days-old larvae, reaches maximal concentrations in 4 -5-days-old animals and then decreases, being absent in pupae or imagos [7]. It is evident that the determination of the mRNA content by means of translational activity encompasses only the functionally active mRNA. We have, therefore, in order to account for all of the RNAs containing the sequences for calliphorin, proceeded to purify the calliphorin mRNA and with the help of reverse transcriptase synthesize its cDNA. The results of these experiments will be presented in this paper. MATERIALS AND METHODS Reagen AnimalsCalliphora vicina R.D. larvae were reared on beef meat at 25 "C and 65 % relative humidity. They pupate under these conditions within 7-8 days after egg deposition. Maximal synchronization was ensured by allowing deposition of eggs only for 60 min.

No abstract