In early mouse embryos, the major inducible heat shock gene, hsp68, is spontaneously and transiently activated at the two-cell stage and becomes heat-inducible around blastocyst stage. We have probed mouse embryo's ability to activate the promoter of this gene during preimplantation development by expression analysis of DNA constructs containing a reporter lacZ gene driven by hsp68 (hsp70A1) 5'-regulatory sequences of various length: (i) a full-length promoter (construct phsplacZ); (ii) a heat shock element (HSE)-deleted promoter (p delta 1hsplacZ); and (iii) a minimal, proximal promoter (p delta 2hsplac Z). When analyzed in transfected L-cells, phsplacZ was heat-inducible, while neither p delta 1hsplacZ nor p delta 2hsplacZ was. Developmental activity of the full-length construct was first analyzed after genome integration in transgenic embryos and found to follow endogenous hsp68 expression in terms of spontaneous activation at the 2-cell stage, down-regulation at the 4-cell stage, and acquisition of heat inducibility at the 16/32-cell stage. In transient expression experiments, injected phsplacZ, p delta 1hsplacZ, and p delta 2hsplacZ were expressed at similar levels by 2-cell embryos, independently of construct topology and injection stage. At the 4-cell stage, however, phsplacZ and p delta 1hsplacZ were expressed at similar levels, while p delta 2hsplacZ was inactive. Only phsplacZ became heat-inducible in late morulas. We conclude that in early mouse embryos, developmental activity of episomic hsp68 promoter depends on proximal sequences at the 2-cell stage and on putative enhancer sequences at the 4-cell stage, while HSEs appear dispensable during early cleavage.

We have investigated the ability of growing dictyate oocytes and early preimplantation embryos of the mouse to process extrachromosomal DNA molecules with free ends by intranuclearly microinjecting DNA fragments containing a region of homology of various extent at either the 5' or 3' terminus. Homologous recombination of these fragments by single-strand annealing (SSA), but not other DNA recombination/joining mechanisms, resulted in the formation of a full-length hsp-lacZ-pA fusion gene that was transcriptionally activated by heat shock in growing oocytes and spontaneously at the early two-cell stage in the embryos, making it possible to quantitatively evaluate SSA activities of these cells by the beta-galactosidase produced. SSA activities of oocytes and embryos were similar in their general properties and in the activity levels observed with saturating amounts of DNA. However, embryo SSA was almost one order of magnitude less effective than that of oocytes. Oocyte and embryo 5' --> 3' exonuclease (a key function of the SSA pathway) and DNA nonhomologous end joining (NHEJ) activities were also investigated using an asymmetric PCR assay. Results showed that NHEJ is lacking in oocytes and is very prominent in the embryos, where it competes with SSA for the injected DNA.