Nucleotide sequence of an unusual regionally expressed silkmoth chorion RNA : Predicted primary and secondary structures of an architectural protein ( chorion morphogenesis / chorion gene evolution / consensus sequence ) JEROME

We have sequenced DNA clones corresponding to the entire coding and 5' untranslated regions and almost all of the 3' untranslated region of a silkmoth chorion RNA which is expressed largely in a subpopulation of follicular epithelial cells (aeropyle crown region). This RNA encodes the El protein, one of two components of the prominent "ifiler" that helps mold the shape ofaeropyle crowns. The conceptually translated El sequence reveals an alternation in hydrophobic and hydrophilic stretches of amino acids that correlates with certain predictions about its secondary structure. El is unusual in revealing no sequence homology with other known chorion sequences and in having an unusually long 3' untranslated region. Sequence analysis of the 5' end of the El gene has identified an intron near the end of the signal peptide-encoding region, a feature shared with other chorion genes. Choriogenesis in silkmoths entails the sequential synthesis of more than 100 distinct proteins during its 2-day duration (1). During the first nine-tenths of choriogenesis, all cells in the chorion-producing follicular epithelium produce the same proteins at the same times and at the same rates (2). These proteins are secreted and assemble extracellularly to form a largely lamellar structure (3). Then, during the "very late" period, two major subpopulations of follicle cells become distinguishable on the basis of distinct patterns of chorion protein and RNA synthesis (ref. 2 and unpublished observations). Cells in the aeropyle crown region begin synthesizing a distinct subset of chorion proteins that assemble into prominent crown-shaped surface structures underlying each three-cell junction. At the ultrastructural level, aeropyle crowns consist of several lamellae similar to, but thinner than, those of the underlying lamellar chorion (4). Within each aeropyle crown is a ball of spongy material called "filler," which appears to have an architectural role and extends into the underlying narrow aeropyle channel (2). Filler consists of only two distinct proteins, called El and E2, whereas lamellae, including those ofthe aeropyle crowns, are biochemically much more complex and include most members of the A-, B-, and C-size classes of chorion proteins (2). The other major population of follicle cells is found in the so-called flat region. Very-late-period-specific proteins appear to be synthesized in this region as well, although their absolute rates are reduced by a factor of 10 or more relative to cells in the aeropyle crown region. In the flat region, no aeropyle crowns ever form and filler is confined to the aeropyle channels. Regier et al. (5) have described the molecular cloning of very-late-period-specific nucleic acid sequences that encode the filler proteins, El and E2. Our interest in further characterization of E sequences is threefold: (i) to search for features shared betweeen very late period E sequences and previously characterized chorion sequences that might indicate evolutionary relatedness, (ii) to begin to understand how El and E2 sequences assemble to form filler, and (iil) to describe the molecular basis of regionalization, including cell-specific expression at the RNA level and organization of regionally expressed genes. Concerning the first goal, it should be noted that the extensive chorion sequence data base (more than 20 partial or complete sequences) includes lamellar components only (6-8). Within this group, striking homologies have been found for each of the five chorion multigene families characterized. Homologies are apparent even between different gene families, so that all lamellar chorion sequences to date may be placed into a chorion "superfamily" (6, 9). In this report we present the complete El amino acid sequence as deduced from its encoding RNA, and we compare its sequence with those of lamellar chorion proteins. MATERIALS AND METHODS Isolation and Sequencing of Clones. The construction of an Antheraea polyphemus silkmoth chorion cDNA library in the plasmid pBR322 and the isolation and characterization of El-encoding cDNA clones have been described (5). To facilitate sequencing by the chemical degradation method (10), we isolated intact plasmid inserts from two of these clones and reinserted them into the plasmid pUC8 (11). Isolation of the El gene-containing chromosomal clone AP8 will be described in a future publication.