A cDNA clone from an abundant nonribosomal 35S RNA in salivary glands of Chironomus tentans

Salivary glands in Chironomus tentans larvae provide a model system whereby tissue-specific gene expression can be studied. While studying the transport of RNA from the nucleus to the cytoplasm, others detected a radiolabeled 35S RNA. The authors became interested in 35S RNA because its relative abundance in salivary glands suggests that it may be a tissue-specific mRNA. When extracts of total salivary gland RNA are fractionated on 0.75% agarose gels in the presence of methyl mercury and stained with ethidium bromide, several discrete bands can be seen which migrate slower than mouse 28S rRNA. Since these bands were approximately the size of nuclear precursors to C. tentans rRNA, they wanted to determine if they had any sequence homology to cloned rDNA. Thus, individual stained bands were excised from one gel rerun in parallel lanes on a second gel, blotted onto nitrocellulose and hybridized with various genomic rDNA clones labeled by nick-translation. Autoradiograms demonstrated that 38S and 30S RNA bands were the predicted (5) rRNA precursors and that 35S RNA was a nonribosomal RNA. 35S RNA was subsequently purified from agarose gels and used as a template for randomly primed synthesis of cDNA. The cDNA was copied into double-stranded DNA and inserted into the Pst I site of pBR322 via homopolymeric tailing. Recombinant plasmids were detected by antibiotic resistance and screened by colony hybridization with radiolabeled 35S RNA