Cellular compartmentalization is vital for the evolution of all living organisms. Cells keep together the genes, the RNAs and proteins that they encode, and the products of their activities, thus linking genotype to phenotype. We have reproduced this linkage in the test tube by transcribing and translating single genes in the aqueous compartments of water-in-oil emulsions. These compartments, with volumes close to those of bacteria, can be recruited to select genes encoding catalysts. A protein or RNA with a desired catalytic activity converts a substrate attached to the gene that encodes it to product. In other compartments, substrates attached to genes that do not encode catalysts remain unmodified. Subsequently, genes encoding catalysts are selectively enriched by virtue of their linkage to the product. We demonstrate the linkage of genotype to phenotype in man-made compartments using a model system. A selection for target-specific DNA methylation was based on the resistance of the product (methylated DNA) to restriction digestion. Genes encoding HaeIII methyltransferase were selected from a 10 7 -fold excess of genes encoding another enzyme.
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Cech, T.R. 1986. A model for the RNA-catalyzed replication of RNA. Proc. Natl. Acad. Sci. USA 83: 4360–4363.
Joyce, G.F. 1989. RNA evolution and the origins of life. Nature 338: 217–224.
Eigen, M. 1992. Steps towards life. Oxford University Press, Oxford, UK.
Oparin, A.I. 1957. The origin of life on the earth. Oliver and Boyd, London.
Deamer, D.W. 1997. The first living systems: a bioenergetic perspective. Microbiol. Mol. Biol. Rev. 61: 239–261.
Gold, L., Polisky, B., Uhlenbeck, O., and Yarus, M. 1995. Diversity of oligonu-cleotide functions. Annu. Rev. Biochem. 64: 763–797.
Hager, A.J., Pollard, J.D., and Szostak, J.W. 1996. Ribozymes: aiming at RNA replication and protein synthesis. Chem. Biol. 3: 717–725.
Smith, G.P. 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315–1317.
Clackson, T. and Wells, J.A. 1994. In vitro selection from protein and peptide libraries. Trends Biotechnol. 12: 173–184.
Lissant, K.J. (ed.). 1984. Emulsions and emulsion technology. Marcel Dekker, New York.
Becher, P. 1957. Emulsions: theory and practice. Reinhold, New York.
Jeltsch, A., Sobotta, T., and Pingoud, A. 1996. Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalyt-ically inactive single mutants. Protein Eng. 9: 413–423.
Krieg, N.R. and Holt, J. 1984. Bergey's manual of systematic bacteriology. Williams and Wilkins, Baltimore, MD.
Lesley, S.A. 1995. Preparation and use off. coll S-30 extracts. Methods Mol. Biol. 37: 265–278.
Pohl, F.M., Thomae, R., and Karst, A. 1982. Temperature dependence of the activity of DNA-modifying enzymes: endonucleases and DNA ligase. Eur. J. Biochem. 123: 141–152.
Chen, L., MacMillan, A., and Verdine, G. 1993. Mutational separation of DNA binding from catalysis in a DNA cytosine methyltransferase. J. Am. Chem. Soc. 115: 5318–5319.
Fastrez, J. 1997. In vivo versus in vitro screening or selection for catalytic activity in enzymes and abzymes. Mol. Biotechnol. 7: 37–55.
Mattheakis, L.C., Bhatt, R.R., and Dower, W.J. 1994. An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. USA 91: 9022–9026.
Cull, M.G., Miller, J.F., and Schatz, P.J. 1992. Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac represser. Proc. Waft Acad. Sci. USA 89: 1865–1869.
Roberts, R. and Szostak, J. 1997. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. USA 94: 12297–12302.
Widersten, M. and Mannervik, B. 1995. Glutathione transferases with novel active sites isolated by phage display from a library of random mutants. J. Mol. Biol. 250: 115–122.
Soumillion, P., Jespers, L., Bouchet, M., Marchand, B.J., Winter, G., and Fastrez, J. 1994. Selection of beta-lactamase on filamentous bacteriophage by catalytic activity. J. Mol. Biol. 237: 415–422.
Benner, S.A. 1993. Catalysis: design versus selection. Science 261: 1402–1403.
Stemmer, W.P. 1994. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91: 10747–10751.
Leung, D.W., Chen, E., and Goeddel, D.V. 1989. A method for random mutagen-esis of a defined DNA segment using a modified polymerase chain reaction. Technique 1: 11–15.
Lowman, H.B., Bass, S.H., Simpson, N., and Wells, J.A. 1991. Selecting high-affinity binding proteins by monovalent phage display. Biochemistry 30: 10832–10938.
Zhao, H., Giver, L, Shao, Z., Affholter, J.A., and Arnold, F.H. 1998. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nature Biotechnology 16: 258–261.
Tawfik, D.S., Green, B.S., Chap, R., Sela, M., and Eshhar, Z. 1993. catELISA: a facile general route to catalytic antibodies. Proc. Natl. Acad. Sci. USA 90: 373–377.
Eisenthal, R. and Danson, M.J. (eds.). 1993. Enzyme assays—a practical approach. Oxford University Press, Oxford, UK.
McCafferty, J., Griffiths, A.D., Winter, G., and Chiswell, D.J. 1990. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348: 552–554.
Hoogenboom, H.R., Griffiths, A.D., Johnson, K.S., Chiswell, D.J., Hudson, R, and Winter, G. 1991. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 19: 4133–4137.
Wang, A.M., Doyle, M.V., and Mark, D.F. 1989. Quantitation of mRNA by the polymerase chain reaction. Proc. Watt. Acad. Sci. USA 86: 9717–9721.
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Tawfik, D., Griffiths, A. Man-made cell-like compartments for molecular evolution . Nat Biotechnol 16, 652–656 (1998). https://doi.org/10.1038/nbt0798-652
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