Target selection for Danio rerio functional genomics.

Secreted proteins are of interest as targets for morpholino technology since they frequently mediate cell-cell interactions. Most are identified through the presence of an N-terminal signal sequence. Proteins from the zebrafish Danio rerio containing such signal sequences will be easy to detect with the publication of the complete genome, expected by the end of 2003 (Sanger Centre, 2000). Until then, most zebrafish proteins are only available as translated Expressed Sequence Tags (ESTs) that often lack N-terminal residues. Interim innovative techniques for the identification of EST-derived proteins predicted to contain signal sequences are needed. Here we describe the use of genetic homology among eukaryotes as a basis for selecting a subset of zebrafish sequences with high probability of encoding secreted proteins. We estimate there are 1,000–2,000 gene families encoding these proteins in the zebrafish genome. Zebrafish DNA sequences most based on ESTs, and proteins from the fruitfly Drosophila melanogaster genome containing N-terminal signal sequences, were obtained and compared as described in Materials and Methods. Comparing zebrafish sequences to the signal protein database, 560 zebrafish sequences (5.3%) are similar to one or more type I signal protein sequences. As shown below, if similar proteins from paralog clusters are counted as one, there are 487 unique hits. Inverting the method to compare the signal protein sequences to the zebrafish database, 659 signal protein sequences are similar to one or more zebrafish sequences. If similar proteins from paralog clusters are counted as one, there are 415 unique hits. The zebrafish and signal protein datasets were compared to themselves to quantify paralog content. The zebrafish dataset has 2,333 sequences that are similar to at least one non-identical sequence. There are 8,208 singletons and 958 paralog clusters, for a total of 9,166 clusters from 10,541 sequences. That is, if only one representative were counted from each paralog cluster, 13% of the sequences could be eliminated. The signal protein dataset has 1,357 sequences that are similar to at least one nonidentical sequence. There are 1,080 singletons and 456 paralog clusters, for a total of 1,536 clusters from 2,437 sequences. That is, if only one representative was counted from each cluster, 37% of the sequences could be eliminated. The 24 zebrafish protein sequences in the nonsecreted dataset were compared with the 2,437 sequences in the signal protein database. Five nonsecreted sequences are similar to sequences in the signal protein dataset; they and their best match are shown in Table 1. Of the five nonsecreted zebrafish proteins that match sequences in the signal protein dataset (Table 1), one, heat-shock protein 70, has strong similarity to its fruitfly homolog along the entire length of the shared sequence. In zebrafish and many other eukaryotes, this protein is not known to be secreted. The fruitfly protein may not be secreted or may be the first reported secreted Hsp-70 homolog. The remaining matches are weaker and the paired sequences differ markedly in size. The similar regions are limited to single domains in multidomain proteins: Zinc finger cerebellum 2 protein and the ci gene product share a zinc finger domain; retinoic acid receptor rxralpha and the CG16801 gene product share an rxr-alpha receptor domain; homeobox protein lim-3 and the CG5708 gene product share a lim domain; and glycogen synthase kinase and the Alk gene product share a eukaryotic protein kinase domain. Members of the ci, rxr-alpha, and homeobox lim protein families are not known to be secreted; the fruitfly members of these families may not be secreted. In contrast, the alk gene product is secreted; the eukaryotic protein kinase domain it contains is found in both secreted and nonsecreted proteins. SignalP (Nielsen et al., 1997) predicts type I signal sequences; however, proteins may also be secreted through non-type I mechanisms. Examples include the hedgehog (Lee et al., 1992) and fibroblast growth factor (FGF) (Mignatti and Rifkin, 1991) proteins; known fruitfly members of these protein families are not found in the signal sequence dataset. Subcellular localization of proteins in the zebrafish dataset is being predicted using other techniques (Emanuelsson et al., 2000). The most serious limitations of our study are the possible presence of nonsecreted proteins in the signal sequence dataset, inability to detect non-type I-secreted proteins, and inability to detect type I-secreted zebrafish proteins not