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.
Institut für Molekularbiologie (Abteilung II), Universität Zürich, Switzerland.
M
M
Hagmann
R
R
Bruggmann
L
L
Xue
O
O
Georgiev
W
W
Schaffner
D
D
Rungger
P
P
Spaniol
T
T
Gerster
D000818:Animals; D001483:Base Sequence; D019175:DNA Methylation; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000196:embryology* / Q000235:genetics*; D005399:Fishes / Q000196:embryology* / Q000235:genetics*; D018390:Gene Targeting; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D015053:Zygote / Q000378:metabolism*
D017931:DNA Primers; D009841:Oligonucleotides