DNA replication in cell-free extracts from Drosophila melanogaster.
We have developed an efficient in vitro replication system from 0-2 h Drosophila melanogaster embryos. Demembranated Xenopus sperm DNA when incubated in such an extract first becomes enclosed in a nucleus-like structure with a nuclear envelope and a karyoskeleton. It then undergoes one round of semiconservative replication--this replication appears completely dependent on nuclear formation. Up to 30% of input DNA is nucleated in one reaction. Efficient nuclear formation and replication are dependent on a cold treatment step, prior to disruption of the embryos. They also depend on the age of the embryos used. Extracts from older embryos (0-5 h) are capable of nuclear formation, although at a much reduced efficiency, and repair synthesis, but seem to have lost the ability to initiate DNA replication. In addition to replicating sperm DNA this system appears capable of carrying out semi-conservative replication on some plasmids. However, it cannot use these to trigger nuclear formation; replication is only seen if the plasmids are coincubated with sperm DNA. The in vitro formed nuclei have not been observed to trigger nuclear envelope breakdown and entry into mitosis. However, they can re-replicate the DNA if the nuclei are permeabilized. This system should be a useful complement to the previously isolated Xenopus in vitro replication system. In addition the amenability of Drosophila to genetic study should open up new approaches not previously possible with Xenopus.
Crevel
G
G
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
Cotterill
S
S
D000818:Animals; D001345:Autoradiography; D002467:Cell Nucleus; D002474:Cell-Free System; D003080:Cold Temperature; D004261:DNA Replication; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope; D010957:Plasmids; D014981:Xenopus