Homologous recombination and DNA-end joining reactions in zygotes and early 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 recombination (HR). We have studied the relative contributions of these processes in zygotes or early embryos of species that serve as model organisms for developmental genetics. Thus, we have microinjected a linearized plasmid substrate into zygotes of zebrafish (Danio rerio) or into the posterior end of Drosophila melanogaster early embryos before pole cell formation. Similar to the situation observed previously in Xenopus zygotes/early embryos, we detected a large preponderance of DNA-end joining over homologous recombination. A comparison of end-joined junctions revealed that from the three species tested, zebrafish introduced the least number of sequence distortions upon DNA-end joining, while Drosophila produced the largest deletions (average 14 bp) with occasional nucleotide patch insertions, reminiscent of the N nucleotides at V(D)J junctions in mammalian immune receptor genes. Double-strand gap repair by homologous sequences ('homologous recombination') involving a bimolecular reaction was readily detectable in both zebrafish and Drosophila. This involved specifically designed recombination substrates consisting of a mutagenized linear plasmid and DNA fragments carrying the wild-type sequence. Our results show that the basic machinery for homologous recombination is present at early developmental stages of these two genetic model organisms. However, it seems that for any experimental exploitation, such as targeted gene disruption, one would have to inhibit or bypass the overwhelming DNA-end joining activity.

Class III POU genes of zebrafish are predominantly expressed in the central nervous system.

POU genes encode a family of transcription factors involved in a wide variety of cell fate decisions and in the regulation of differentiation pathways. We have searched for POU genes in the zebrafish, a popular model organism for the study of early development of vertebrates. Besides five putative pseudogenes we have identified five POU genes that are expressed during embryogenesis. Probes obtained by PCR were used to isolate full-length cDNAs. Four of the isolated genes encode proteins with class III POU domains. Analysis of genomic clones suggests that the fish genes in general do not contain introns, similar to class III genes of mammals. However, the C-termini of two of the encoded proteins vary due to facultative splicing of a short intervening sequence. These two genes show very strong similarities in their sequence. They have probably arisen by gene duplication, possibly as part of a larger scale duplication of part of the zebrafish genome. Analysis of the expression of the class III genes shows that they are predominantly expressed in the central nervous system and that they may play important roles in patterning the embryonic brain.