Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.