Nucleic Acids Research, 1985, Vol. 13, No. 8 2943-2958
© 1985
Articles |
DNA synthesis in yeast cell-free extracts dependent on recombinant DNA plasmids purified from Escherichia coli
Department of Microbiology 131 Burrill Hall, University of illinois at Urbana-Champaign, Urbana, IL 61801, USA
+To whom correspondence should be addressed
Received February 5, 1985. Accepted March 25, 1985.
In our attempts to establish a cell-free DNA replication system for the yeast Saccharomyces cerevisiae, we have observed that recombinant DNA plas-mids purified from Escherichia coli by a common procedure (lysozyme-detergene lysis and equilibrium banding in cesium chloride ethidium bromide gradients) often serve as templates for DNA synthesis by elongation enzymes. The templates could be elongated equally well by enzymes present in the yeast cell-free extracts, by the large proteolytic fragment of E. coli DNA polymerase I or by T4 DNA polymerase. The template activity of the purified plasmids was dependent on the presence of heterologous DNA segments in the bacterial vectors. The template activity could be diminished by treatment with alkali. We propose that the ability of recombinant plasmids isolated from bacterial hosts to serve as elongation templates may lead to erroneous conclusions when these plasmids are used as templates for in vitro replication or transcription reactions.
*Present address: Department of Chemistry, California Institute of Technology, Pasadena, CA 91125, USA