Fig. 1

Fig. 1 A zebrafish spi1 orthologue. (A) Comparison of functional domains of SPI.1 proteins from four species, showing amino acid identity scores for each of the three functional domains. In the box linear diagrams, numbers above domain boxes indicate the last residue of each functional domain, and numbers within indicate the percent identity to zebrafish spi1. (B) Phylogenetic analysis for amino acids demonstrates that D. rerio spi1 is an orthologue of previously described SPI.1 genes, and that the related gene family members SPI-B and SPI-C form separate clades. [GenBank GI (or Accession) numbers, top to bottom: 4507175, 6755473, 8745406, 2369863, 11245497, (AF321099), 8745412, 36562, 2586116, 8745407, 8745409, 11245499, 8745404, 8745414, 11245501, 6755618, 4557551]. The analysis was based over the DNA binding domain for all proteins commencing at the sequence GX(R/K)(R/G/K)(K/R)XR in all but human ELF5, for which the sequence starting “TSLQSS” was used. The dendrogram was constructed by using ClustalX and Treeview building on the analysis of Spi family DNA-binding domains given in Shintani et al. (2000) and Anderson et al. (2001) using human ELF-5 as an outgroup. Bootstrap values (n = 1000) are indicated at nodes as percents. (C) Only one spi1 gene exists in the zebrafish genome. Southern analysis of zebrafish genomic DNA digested with a various restriction enzymes (Lanes: 1, EcoRI; 2, BamHI; 3, BglII; 4, PstI; 5, XbaI; 6, SmaI) and hybridized to a radiolabeled probe corresponding to either the entire 1034 nucleotides (Probe A) or the first 367 nucleotides (Probe B) of the zebrafish spi1 cDNA clone. A single band in lane 6 (probe B) indicates that this spi1 gene fragment is represented in the zebrafish genome by one copy. (D) Survey of spi1 expression in early zebrafish development by RT-PCR. Templates in control lanes (a–d) are: a, plasmid DNA; b, genomic DNA; c, water; d, RT-PCR without reverse transcriptase. Developmental ages from which RNA were prepared are 2, 8, 16, and 24 hpf and 2, 3, 5, and 7 dpf.