Vitellogenesis in the insect Dacus oleae. Isolation and characterization of yolk protein mRNA

Abstract

The two major yolk polypeptides (YP1 and YP2) of Dacus oleae have been isolated from egg extracts. Their molecular weights (as determined by SDS-PAGE) are 47,000 and 49,000, respectively, and they are synthesized in vivo by ovaries and fat bodies of female flies; none of the two polypeptides were detectable in male haemolymph. The mRNAs for the YP1 and YP2 polypeptides were isolated from female flies. Purification was achieved, using three successive cycles of sucrose gradient centrifugation under denaturing conditions and hybridization to yolk protein clones immobilized on DBM paper. The purity of the yolk protein mRNA was judged by translation in a cell-free protein-synthesizing system derived from rabbit reticulocytes. Purified yolk protein mRNA migrated, following denaturing agarose gel electrophoresis, as a single band, slightly faster than the 16S rRNA. Yolk protein complementary DNA (cDNA) was transcribed from purified yolk protein mRNA. The resultant cDNA was an almost full length copy as defined by denaturing gel electrophoresis following hybridization-arrested translation. Hybridization analysis of the yolk protein gene transcripts using the synthesized yolk protein cDNA as a probe, showed that these are present only in the ovaries and fat bodies of female flies but not in the fat bodies of male flies.