Complexity and diversity of polyadenylated mRNA in the silk gland of Bomyx mori: Changes related to fibroin production

Abstract

Poly(A) containing RNA was isolated from the posterior silk gland of Bombyx mori at the end of the fourth molt when the gland does not produce fibroin (stage V 0) and at the middle of the fifth larval instar when fibroin synthesis is massive (stage V 6). The kinetics of hybridization of these mRNAs with their complementary DNA (cDNA) were markedly different. The mRNA from V 0 stage consists of two abundance classes comprising, respectively, 38 and 2915 different sequences. V 6 mRNA-cDNA hybridization could not be analyzed properly by standard calculations since we found that the messenger RNA coding for fibroin (FmRNA) was about eight times less transcribed than the other mRNAs. This low yield of reverse transcription was assumed to be due to the exceptional length of FmRNA (16,000 nucleotides) as compared to the average length of the other silk gland mRNAs (1400 nucleotides). Therefore we separated the FmRNA from the rest of polyadenylated RNA (F ⊝mRNA) by sucrose gradients and analyzed the two preparations separately. The kinetic complexity of FmRNA was very low for its size and can be accounted for by a 66-nucleotide-long driving sequence. This result agrees with the existence of a short repetitive sequence known to represent 60% of the molecule. The V 6 F ⊝mRNA preparation was resolved into four classes containing, respectively, 1, 20, 319, and approximately 2600 sequences. Cross hybridizations have been performed with each of the cDNA abundance classes from V 0 stage and V 6 F ⊝mRNA. They demonstrated that almost all the sequences present at the V 0 stage are also detected at V 6 stage, but their distribution among the abundance classes was modified. The relative concentration of 26 species of the abundant class and 291 species of the rare class of the V 0 stage was increased at V 6 and therefore appears to be implicated in the building of the fibroin synthesizing machinery, whereas 12 mRNA species from the V 0 abundant class decreased in relative concentration and are probably subject to another mode of control. Finally a large number of mRNA species remained in a stable low concentration at both stages and may be attributed to housekeeping functions. Our data show that the spectacular silk gland cell adaptation to fibroin production is characterized by massive accumulation of FmRNA and of an unknown mRNA species, and that stage variations in mRNA populations are related to relative quantitative variations rather than qualitative differences.