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The messenger RNA for the lipoprotein of the E. coli outer membrane was found to code for a putative precursor, prolipoprotein, which has 20 additional amino acid residues extending from the amino terminus of the lipoprotein. Using the prolipoprotein synthesized in an E. coli cell-free system directed by purified messenger RNA for the lipoprotein, the complete amino acid sequence of the amino-terminal precursor region was determined to be as follows: Met-Lys-Ala- Thr-Lys-Leu-Val-Leu-Gly-Ala-Val-Ile-Leu-Gly-Ser-Thr- Leu-Ala-Gly-. In the present paper, we analyze the product synthesized in a cell-free system directed by the purified mRNA. We have found that the cell-free product has an extension of 20 additional amino acid residues at the amino terminus of the lipoprotein. The complete amino acid sequence of the peptide extension of the product, prolipoprotein, was determined. The unique features of this sequence have led us to propose plausible functions for this region of the prolipoprotein. MATERIALS AND METHODSCell-Free Protein Synthesis. Cell-free protein synthesis was carried out as previously described (7). The mRNA for the lipoprotein was purified by Sephadex G-200 filtrations as previously described (7), and in most cases the SF-2 fraction was used unless otherwise mentioned. The cell-free protein synthesis was carried out in a 300 ,Al reaction mixture with 30 1ACi of a radioactive amino acid (7). After 30 min incubation at 37°, the reaction mixture was put on ice and 1.5 ml of 3 mM magnesium acetate was added. The mixture was then centrifuged at 150,000 X g for 30 min. By this centrifugation about 30% of nonradioactive proteins were removed as a pellet but about 80% of cell-free products remained in the supernatant. To the supernatant was added 1.5 ml of 10% trichloroacetic acid and the mixture was incubated for 30 min in a boiling-water bath. The resultant precipitate was collected by centrifugation and washed three times with 2 ml of ether/ethanol (1:1) and two times with 2 ml of ether. The final precipitate was dried with an air jet. In some cases, the cell-free products were precipitated with antisera as described previously (7). The immunoprecipitate was suspended in 2 ml of 5% trichloroacetic acid, incubated in a boiling-water bath for 30 min, and washed as described above. Protein synthesis in toluene-treated cells was carried out as described previously (3, 10), and the prolipoprotein from the toluene-treated cells was purified by immunoprecipitation followed by sodium dodecyl sulfate/gel electrophoresis as described previously (11

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The World Health Organization's International Programme on Chemical Safety (IPCS), the Organization for Economic Cooperation and Development (OECD), and the U.S. Environmental Protection Agency have developed a collaborative partnership to foster integration of assessment approaches for human health and ecological risks. This paper presents the framework developed by that group. Integration provides coherent expressions of assessment results, incorporates the interdependence of humans and the environment, uses sentinel organisms, and improves the efficiency and quality of assessments relative to independent human health and ecological risk assessments. The paper describes how integration can occur within each component of risk assessment, and communicates the benefits of integration at each point. The goal of this effort is to promote the use of this internationally accepted guidance as a basis for harmonization of risk assessment.