The size of pulse-labeled fibroin messenger RNA

A method has been developed for the isolation of fibroin gene transcripts from total RNA of the silkworm Bombyx mori. It is based on affinity chromatography using Sephadex-bound polynucleotides capable of selectively hybridizing with fibroin mRNA sequences. In vivo pulse labeling of the posterior silk gland for periods of 10-35 min produces labeled heterogeneous nuclear RNA of high molecular weight (greater 40S). Fibroin gene transcripts can be selected from the total hnRNA population by two consecutive passages through the affinity column. Analysis of the column-bound material in denaturing polyacrylamide-agarose gels reveals that the size of pulse-labeled fibroin mRNA is essentially the same (within 5%) as that of mature cytoplasmic mRNA. This holds true for pulses as short as 6 min, where even nascent mRNA can be observed. However, a small shoulder of material is present on the heavy side of the pulse-labeled mRNA, which could be indicative of an extremely short-lived precurosr species. The purified pulse-labeled mRNA (10 min incorporation) has been further analyzed by chromatography in oligo(dT)-cellulose. The data show that the mRNA is polyadenylated within a few minutes after synthesis.