A subtle defect underlies altered lamellar orientation in the Gr16 chorion mutant of Bombyx mori

Gr16 (chromosome 2, 6.9 cM) is a dominant, grey egg mutation of the domestic silkmoth, Bombyx mori, which produces an opaque instead of a translucent chorion. To determine the underlying cause of this defect, we examined the effects of the mutation on chorion ultrastructure, protein accumulation, and biosynthesis patterns, and on the restriction patterns of early chorion structural genes, using the presumed wild-type progenitor strain, Old European 16 (OE16), for comparison. Cross-sections of mature chorions examined by scanning electron microscopy showed that the inner lamellae were oriented perpendicular, rather than parallel, to the egg surface, with a smaller vertical zone bordered by normally oriented lamellae in the heterozygote, suggestive of codominant expression. This defect first appeared in transmission electron micrographs early in choriogenesis, probably during framework formation and/or expansion stages. Mature chorion protein patterns resolved by isoelectric focusing or two-dimensional polyacrylamide gel electrophoresis, synthetic profiles of virtually all chorion proteins following pulse-labeling with tritiated or [14C]glycine, and restriction patterns of early chorion genes examined by Southern blot hybridization were identical between mutant, wild-type, and heterozygote. These observations ruled out a major deletion of chorion structural genes or defects in the production and accumulation of normal chorion proteins as mechanisms underlying the mutation, unlike other putative Gr mutants mapping in its vicinity. The sole difference that we detected was a set of minor, early protein doublets of related size but differing isoelectric point, which showed reciprocal labeling intensities in Gr16 vs OE16 and were codominantly expressed. Preliminary pulse-chase experiments suggested that these proteins may be post-translationally modified forms which undergo differential processing in mutant and wild-type. Based on these observations, we postulate that the Gr16 mutation affects the primary structure of a minor, previously unidentified chorion or follicular cell component which is critical for determining lamellar orientation, and discuss models of how this might function.