Allende M L, Amsterdam A, Becker T, Kawakami K, Gaiano N, Hopkins N
Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Genes Dev. 1996 Dec 15;10(24):3141-55. doi: 10.1101/gad.10.24.3141.
Recently our laboratory described an efficient method for generating retroviral provirus insertions in the zebrafish germ line, and we showed that provirus insertions induce embryonic mutations at a frequency of roughly one mutant per 70 insertions. To date we have isolated four insertional mutants and, using the proviruses as a molecular tag, have cloned the genes disrupted in three of them. The proviruses in all three mutants lie within or just 5' of the first coding exon, point in the opposite transcriptional orientation from the gene, and disrupt transcription. Here we present a molecular characterization of two genes identified by this method and describe the associated mutant phenotypes. The pescadillo (pes) gene is predicted to encode a protein of 582 amino acids with no recognizable functional motifs, which is highly conserved from yeast to humans. pes mRNA is expressed widely and dynamically during the first 3 days of embryogenesis. Prominent sites of expression are the eyes and optic tectum on day 1, the fin buds, liver primordium, and gut on day 2, and the branchial arches on day 3. Beginning at day 3 of embryogenesis, pes mutant embryos exhibit small eyes, a reduced brain and visceral skeleton, shortened fins, and a lack of expansion of the liver and gut, and then die on the sixth day of development. The dead eye (dye) gene encodes a protein of 820 amino acids that is homologous to genes of unknown function in human, mouse, and Xenopus, and that has weak homology with the yeast NIC96 (nucleoporin-interacting component) gene. dye mutants can be recognized on day 2 of embryogenesis by the presence of necrotic cells in the tectum and eyes. dye mutants die on day 5 of development. These results demonstrate the power of insertional mutagenesis in zebrafish for rapidly finding and characterizing novel genes essential for embryonic development. Using our current methodology, we estimate that our laboratory could screen approximately 25,000 insertions in 2-3 years, identifying perhaps 250-350 embryonic lethal genes. Assuming that all genes are accessible to proviral insertion, the wider application of this approach could lead to the rapid identification of the majority of genes that are required for embryonic development of this vertebrate.
最近，我们实验室描述了一种在斑马鱼生殖系中产生逆转录病毒原病毒插入的有效方法，并且我们表明原病毒插入以大约每70次插入产生一个突变体的频率诱导胚胎突变。到目前为止，我们已经分离出四个插入突变体，并以原病毒作为分子标签，克隆了其中三个突变体中被破坏的基因。所有三个突变体中的原病毒位于第一个编码外显子之内或其5'端，转录方向与基因相反，并破坏转录。在这里，我们展示了通过这种方法鉴定的两个基因的分子特征，并描述了相关的突变体表型。pescadillo（pes）基因预计编码一个582个氨基酸的蛋白质，没有可识别的功能基序，从酵母到人类高度保守。pes mRNA在胚胎发育的前3天广泛且动态地表达。突出的表达部位在第1天是眼睛和视顶盖，第2天是鳍芽、肝原基和肠道，第3天是鳃弓。从胚胎发育第3天开始，pes突变体胚胎表现出眼睛小、脑和内脏骨骼减小、鳍缩短以及肝和肠道没有扩张，然后在发育的第6天死亡。dead eye（dye）基因编码一个820个氨基酸的蛋白质，与人、小鼠和非洲爪蟾中功能未知的基因同源，并且与酵母NIC96（核孔蛋白相互作用成分）基因有弱同源性。dye突变体在胚胎发育第2天可通过视顶盖和眼睛中坏死细胞的存在而被识别。dye突变体在发育第5天死亡。这些结果证明了斑马鱼插入诱变在快速发现和表征胚胎发育所必需的新基因方面的强大作用。使用我们目前的方法，我们估计我们实验室在2 - 3年内可以筛选大约25000次插入，可能鉴定出250 - 350个胚胎致死基因。假设所有基因都可被原病毒插入，这种方法的更广泛应用可能导致快速鉴定出该脊椎动物胚胎发育所需的大多数基因。
