The purification and properties of a lipid-containing bacteriophage, 06, are described. The phage contains a lipid envelope which is probably essential for infection. Infectivity of 06 was lost in the presence of organic solvents, sodium deoxycholate, and phospholipase A. The fatty acid composition of the phage lipid was similar to that of the Pseudomonas phaseolicola host cells. The phage was composed of about 25% lipid, 13% RNA, and 62% protein. The buoyant density of 06 was 1.27 g/ml in cesium chloride. The morphology of 06 was unusual; it had a polyhedral head of about 60 nm surrounded by a membranous, compressible envelope which appeared to assume an elongated configuration upon attachment to pili. The adsorption rate constant was 3.3 x 10-1o ml/min in a semi-synthetic medium and 3.8 x 10-10 ml/min in a nutrient broth-yeast extract medium. The latent period was shorter in the former medium (80-115 min compared with 120-160 min), and the average burst size was larger (250-400 compared with 125-150). The eclipse period coincided with the latent period. Bacteriophage 46 was isolated during an investigation of bacteriophages of phytopathogenic pseudomonads. It was extremely sensitive to organic solvents and a detergent, which suggested that it might contain lipid. Phage PM2 (4) is the only lipid-containing bacteriphage previously described. The present paper describes the isolation and purification of phage 06. In addition, electron microscopy of the phage and some of its biological and biochemical properties are presented.

Several receptor tyrosine kinases generate soluble ligand binding domains either by differential splicing resulting in a truncated RNA transcript, or by proteolytic cleavage. Although the exact role in vivo of these soluble extracellular domains is unclear, proteolysis may function to down-regulate the receptor, and soluble extracellular domains (ECD) may compete with the intact receptor binding to ligand. Axl is a member of a new class of receptor tyrosine kinases characterized by an ECD resembling cell adhesion molecules and unique sequences in the kinase domain. In addition, Axl is transforming in both fibroblast and hematopoietic cells, and appears to be involved in mesenchymal development. We now find that Axl is post-translationally processed by cleavage in a 14 amino acid region immediately NH2-terminal to the transmembrane domain resulting in a soluble ECD and a membrane bound kinase domain. The sequence of this putative cleavage site shares no homology with recognition sites of known proteases. Characterization of this proteolytic processing shows that it does not require protein synthesis or transport but is augmented by phorbol ester treatment. Since the cleavage of Axl enhances turnover of the kinase on the cell surface, we suggest that proteolytic processing down-regulates Axl kinase activity.

Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted from Saccharomyces cerevisiae using a yeast alpha-factor leader to direct secretion. However, approximately 10-20% of the IGF-I was in a monomeric form, the remaining materials being disulfide-linked aggregates. When the purified material was subjected to reverse-phase high-performance liquid chromatography (rp-HPLC), it gave two doublet peaks, I and II. Upon reduction, doublet peaks I and II converged to one doublet peak. This suggests that peaks I and II result from different disulfide structures, and the doublet feature of each peak results from other causes. Different disulfide structures between peaks I and II were also suggested from the near UV circular dichroism of these proteins. Only the peak II was biologically active, indicating that peak II has the correct disulfide structure. Concanavalin A affinity chromatography of the purified peak II doublet showed binding of the subpeak with an earlier rp-HPLC retention time, indicating that it was glycosylated. Sequence analysis of tryptic peptides suggested that Thr29 was the site of glycosylation. Site-directed mutagenesis was used to convert Thr29 to Asn29. This substitution reduced, but did not eliminate IGF-I glycosylation, suggesting additional glycosylation sites. The site of carbohydrate addition was consistent with the model that O-glycosylations occur on hydroxyl amino acids near proline residues in beta-turns.

The three double-stranded ribonucleic acid (dsRNA) segments of the bacteriophage 46 were isolated and shown to have similar melting temperatures and base compositions. RNA: RNA hybridization experiments with the isolated segments eliminate the possibility that the two smaller dsRNA segments arise from a cleavage of the large dsRNA segment. The two smaller RNA segments reanneal rapidly even at low temperatures; in contrast, the large dsRNA reannealed only at higher temperatures. Evidence is also presented which suggests that the dsRNAs may contain a short single-stranded RNA tail. Several plant and animal viruses contain a double-stranded ribonucleic acid (dsRNA) genome. In most cases, these dsRNAs have a guanosine plus cytosine (G + C) content of 38 to 44%, and each virion contains several species of dsRNA with molecular weights ranging from 0.2 x 106 to 2.9 x 106 (11). Previously, we reported the isolation and general characteristics of a morphologically unique lipid-containing bacteriophage, 46, of Pseudomonas phaseolicola (10). The phage genome was composed of at least three dsRNA segments with estimated molecular weights of 2.2 x 106, 2.8 x 106, and 4.5 x 106 (8). The G + C content of the total dsRNA was 56 to 58%, which is higher than that reported for other dsRNA viruses except for the mycovirus from Penicillium chrysogenum (7). The present report describes additional characteristics of the 06 dsRNA segments. MATERIALS AND METHODS Virus culture, purification, and nucleic acid extraction. The host, strain HB1OY of P. phaseolicola, was grown in a semisynthetic medium (SSM) as previously reported (10). The phage was isolated from 7-or 25-liter lysates and was purified by CsCl equilibrium sedimentation (10). The dsRNA was isolated from the purified phage by the single-phase phenol procedure of Diener and Schneider (4) as described previously (8). Phage 06 32P-dsRNA was prepared by adding 50 mCi of H332PO4 at the start of a 7-liter fermentation in SSM modified by a 10-fold reduction in the phosphate concentration. Centrifugation procedures. Sedimentation coefficients were estimated in linear-log sucrose density gradients (2) equilibrated with 0.3 M NaCl and 0.03 'Journal paper no. 3729. Nebraska Agricultural Experiment Station.