Identification of a transcriptionally active pseudogene in the chorion locus of the silkmoth Bombyx mori. Regional sequence conservation and biological function.

We have determined the primary structure of a 3500 base-pair part of the silkmoth chorion locus mapping in a region containing genes of late developmental specificity. This part of the locus was found to harbour a pseudogene related to one of the families of chorion genes encoding high cysteine proteins, HcB. The pseudogene exhibits an overall sequence identity of 84% to the consensus coding region of HcB chorion genes. A 95% identity was observed over a length of 190 base-pairs of its immediate 5' upstream sequences and the corresponding part of the consensus 5'-intergenic sequences of Hc gene pairs, normally encompassing 270 base-pairs. Thus, the pseudogene has retained part of the promoter region that includes sequence elements whose presence is thought to be necessary for transcriptional competence of HcB genes. The pseudogene is also characterized by the elimination of part of its first exon containing most of the 5' untranslated region, the ATG translation initiation codon and part of the signal peptide sequences. Its intron is longer than that of other HcB genes due to the insertion of a copy of a repetitive element that appears to be transcribed by RNA polymerase III. A previously characterized chorion cDNA clone, m2282, representing a rare mRNA sequence of late developmental specificity, was found to be identical to the pseudogene over its entirety spanning 65% of the pseudogene's second exon. Hybridizations of clones spanning a 260,000 base-pair domain of the chorion locus of Bombyx mori and of total genomic DNA to a subfragment of the cDNA clone containing relatively unique sequences, coupled to primer extension experiments, have demonstrated that m2282 mRNA originated from the pseudogene and that the pseudogene transcripts are initiated at the chorion cap site consensus sequence. We conclude that the 5'-flanking sequences retained by the pseudogene encompass elements necessary and adequate for correct transcriptional activation, but may not include those required for quantitative expression of the promoter. Possible reasons for the observed lack of random drift in the 5'-upstream sequences of the pseudogene and the maintenance of a functional promoter in a non-functional gene are discussed on the basis of the observation that elements resembling scaffold attachment sites are present in these sequences.