Isolation of giant silk fibroin polysomes and fibroin mRNP particles using a novel ribonuclease inhibitor, hydroxystilbamidine.

Abstract

Hydroxystilbamidine isethionate, a dye capable of binding to both DNA and RNA, has been found to be á powerful inhibitor of cellular ribonucleases . A procedure has been developed that, with the aid of this compound, permits the preparative isolation of giant silk fibroin polyribosomes from the posterior silk gland of Bombyx mori . The polyribosomes contain^-45-112 ribosomal particles, as judged by electron microscopy .Treatment of giant fibroin polyribosomes with EDTA releases a particle that sediments at 125S . This mRNP particle contains biologically active silk fibroin mRNA, as judged by cell-free translation in an mRNA-dependent reticulocyte cell-free system .The isolation and characterization of cytoplasmic messenger ribonucleoprotein particles (mRNP) containing specific mRNA's has so far been achieved in relatively few biological systems. The two best studied examples are globin mRNP and myosin mRNP (see reference 1 for review).Insects remain relatively unexploited as model systems for the study ofmRNP structure and function . A promising system for such studies is the posterior silk gland of Bombyx mori, in which silk fibroin mRNA is by far the major mRNA present in the cell.The isolation of fibroin polyribosomes has so far proven difficult because of the lability of the very long mRNA molecule (2-4). In this paper I describe a method that utilizes a novel ribonuclease inhibitor, hydroxystilbamidine isethionate, to achieve the isolation of giant silk gland polyribosomes containing undegraded fibroin mRNA. I also present a preliminary characterization of fibroin-specific mRNP particles obtained from the giant polyribosome fraction . The fibrom mRNA present in these mRNP particles is biologically active . MATERIALS AND METHODSHydroxystilbamidine isethionate was a gift from May and Baker, Ltd., Degenham, Essex, England and from Merrell-National Laboratories, Cincinnati, Ohio. The yellow powder was dissolved in small aliquots in sterile H2O (25-50 mM) and stored up to 4 mo at -20°C in the dark . Sodium heparin was obtained from Sigma Chemical Co., St . Louis, Mo.Silkwork larvae (Gunka x Hushun strain) were raised in the laboratory as described (5). Larvae on the fourth or fifth day of the fifth instar (-4.2 g body weight) were injected with 35 pl of a solution of 10 mg/ml cyclobeximide in H2O. After 5 min, the animals were immobilized in ice, and posterior silk glands were dissected and washed in ice-cold 0.15 M NaCl, 0.015 M Na citrate, 100 hg/ml cycloheximide. Washed glands from two larvae were placed in a Dounce homogenizer (Kontes Co., Vineland, N.J .) containing 4.7 ml of 40 mM triethanolamineHCl, pH 7.5, 0.15 M sucrose, 0.1 M KCI, 3 mM MgClz, 2 mM reduced glutathione, l0 ug/ml cycloheximide, 750 Wg/ml Escherichia coli tRNA, and an appropriate concentration of RNase inhibitor (sodium heparin, 1.5 mg/ml or hydroxystilbamidine isethionate, 1 .5 mM). Homogenization was accomplished by seven slow strokes of a large-clearance Dounce plunger, followed by the addition of 0.3 ml of 2.5 M KCI, 16 .7 mM M...

The Ultrastructure and Functions of the Silk Gland Cells of Bombyx mori

“…Fibroin is synthesized 60 times faster than serum albumin is synthesized by liver cells, for an equal weight of tissue (Suzuki, 1977). Lizardi (1980) isolated the giant silk fibroin polyribosomes from the posterior silk gland of B. mario The polyribosomes contained between 45 and 112 ribosomal particles. Treatment of giant fibroin poly ribosomes with EDT A released a particle that sedimented at 125 S. This mRNP particle contained biologically active silk fibroin mRNA, as judged by cell-free translation in an mRNA-dependent, reticulocyte system.…”

Electron microscopic visualization of a discrete class of giant translation units in salivary gland cells of Chironomus tentans

“…The number of ribosomes per polysome determined for globin chains (Warner et al, 1962), ovalbumin (Palacios et at., 1972) Roskam et al, 1976). Silk fibroin with 'v5000 amino acids, is formed by polysomes containing 45-112 ribosomes (Lizardi, 1980) which gives amino acids/ribosome. It is therefore possible that the spacing between ribosomes increases with polysome size.…”