HOMOLOGOUS RECOMBINATION AND DNA-END JOINING REACTIONS IN ZYGOTES ANDEARLY EMBRYOS OF ZEBRAFISH (DANIO-RERIO) AND DROSOPHILA-MELANOGASTER

A linear DNA with partial sequence redundancy can be recircularized in cells by either nonhomologous end joining (NEJ) or by homologous reco mbination (HR), We have studied the relative contributions of these pr ocesses in zygotes or early embryos of species that serve as model org anisms for developmental genetics. Thus, we have microinjected a linea rized plasmid substrate into zygotes of zebrafish (Danio rerio) or int o the posterior end of Drosophila melanogaster early embryos before po le cell formation. Similar to the situation observed previously in Xen opus zygotes/early embryos, we detected a large preponderance of DNA-e nd joining over homologous recombination, A comparison of end-joined j unctions revealed that from the three species tested, zebrafish introd uced the least number of sequence distortions upon DNA-end joining, wh ile Drosophila produced the largest deletions (average 14 bp) with occ asional nucleotide patch insertions, reminiscent of the N nucleotides at V(D)J junctions in mammalian immune receptor genes. Double-strand g ap repair by homologous sequences ('homologous recombination') involvi ng a bimolecular reaction was readily detectable in both zebrafish and Drosophila. This involved specifically designed recombination substra tes consisting of a mutagenized linear plasmid and DNA fragments carry ing the wildtype sequence. Our results show that the basic machinery f or homologous recombination is present at early developmental stages o f these two genetic model organisms. However, it seems that for any ex perimental exploitation, such as targeted gene disruption, one would h ave to inhibit or bypass the overwhelming DNA-end joining activity.