Accentuated expression of silk fibroin genes in vivo and in vitro

Abstract

Fibroin mesenger RNA accumulates in the posterior silk gland of Bombyx mori at an almost constant rate (7 to 10 molecules/gene per min) throughout the fifth larval instar. During this period ribosomal RNA synthesis decreases from 7 to 0·2 molecules/gene per minute and synthesis of heterogeneous RNA also decreases. Thus, fibroin mRNA comprises about 1, 3 and 8% of the RNA pulse-labeled for 30 minutes in vivo on the second, fourth and seventh day, respectively. Pulselabeling experiments on the seventh day of the fifth instar indicate that a single fibroin gene is transcribed at a rate 50-fold higher than an rRNA gene. In contrast, fibroin mRNA was not detectable in RNA labeled for 30 minutes during the preceding fourth moulting stage. Posterior silk gland nuclei have an extremely ramified structure and attain about 4×105 ploidy in the late fifth instar. These nuclei have been mechanically fragmented and 90 to 95% of the cytoplasmic contaminants removed. When pulse-labeled for 30 minutes in vivo before nuclear isolation, most of the unlabeled fibroin mRNA and about half of the pulse-labeled mRNA were found in the cytoplasmic fraction. In vitro synthesis of total RNA by endogeneous RNA polymerase in the disrupted nuclei was almost linear for 30 minutes, and some reinitiation was observed. The in vitro product labeled for 30 minutes yielded a profile similar to that of RNA labeled in vivo when fractionated on a Bio-Gel A50m column. Hybridization of the in vitro RNA to B. mori DNA under conditions which allow transscripts of repetitions sequences as well as fibroin mRNA to hybridize was completely competed for by total posterior gland RNA. By sequence analysis about 2% of the in vitro transcripts labeled in 30 minutes were identified as fibroin mRNA of apparent full size length. In vitro fibroin mRNA synthesis was abolished by 0·2 μg of α-amanitin/ml suggesting that RNA polymerase II is responsible for fibroin gene transcription.