We are investigating the molecular mechanisms controlling initiation of DNA replication in eggs of the frog Xenopus laevis. Our approach makes use of new techniques and findings established in this laboratory, these include: 1) Development of a technique which permits injection of materials into eggs without activating them; 2) Discovery that injected Type 1 Bovine Papilloma Virus (BPV1) DNA is only replicated after eggs are activated, but replication is made more efficient by injection prior to activation; 3) Observation that vectors which lack the BPV1 origin of replication do not display the same improvement in replication efficiency. These results provide the first clear indication that Xenopus eggs initiate DNA replication via use of a known eukaryotic origin. Experiments aimed at further characterizing the egg-plasmid interactions are proposed: A) Determination of the optimal amount of DNA/egg which yields synchronous initiation of 1st round replication of the maximum number of molecules: B) Confirmation that double stranded DNA is replicated in activated eggs via a semiconservative mechanism; C) Determination, of the role of RNA transcription in the initiation of DNA replication. D) Determination of whether plasmids which lack origins of replication inhibit the efficient replication of those which contain functional origins. Experiments along these lines will help to test our working model which postulates that the protein factors in the cytoplasm of the unactivated egg interact with origin containing DNA sequences. The successful completion of these experiments will allow greater insight into the mechanisms and cellular factors necessary for the faithful initiation and replication of eukaryotic DNA in in vivo conditions.
