Genomic Southern blot analysis of pooled amphioxus DNA suggests that AmphiRAR, AmphiRXR and AmphiTR2/4 are single genes. Aliquots (10 mg) of genomic DNA were each digested with a different restriction enzyme, subjected to electrophoresis and transferred to Hybond N⁺ (Amersham Life Sciences, Cleveland, OH) according to L. Z. Holland et al. (1995; see text). This blot was sequentially hybridized with probes for (A) AmphiRAR, (B) AmphiTR2/4 and (C) AmphiRXR under moderately low stringency (55°C). Between each hybridization, the blot was stripped with boiling 0.5% SDS. Probes were a 401 bp PstI, BstxI fragment of the amphioxus RAR cDNA, a 481 bp SfiI, HindII fragment of the amphioxus TR2/4 cDNA and a 435 bp SphI, BamHI fragment of the amphioxus RXR cDNA. All probes corresponded to the coding region. After each hybridization, the blot was stripped with boiling 0.5% SDS. Numbers at top indicate digestion in: 1, XhoI; 2, XbaI; 3, StuI; 4, SalI; 5, PvuI; 6, NcoI; 7, NotI; 8, PstI; 9, KpnI; 10, HindIII; 11, BstXI; 12, EcoRI; 13, EcoO109I; 14, BstEII; 15, BglI; 16, BamHI. Molecular size standards are given in kilobases on the left. For AmphiRAR (A), a single band ranging from 2 to 10 kb was revealed with eight out of 16 enzymes. Multiple bands with three enzymes might result from polymorphism or cutting within an intron. Amphioxus genes are highly polymorphic; on the average, there is one base difference in every 100 bp within coding sequences and one base difference in every 50 bp in non-coding sequences (H. E., N. D. H., H. G., V. L., L. Z. H., unpublished ). Similarly, for AmphiTR2/4, a single major hybridizing band was seen with four enzymes, while a single major band with additional fainter bands, probably resulting from

Phylogenetic tree showing the placement of AmphiRAR (A), AmphiRXR (B) and AmphiTR2/4 (C). The RAR tree was rooted with the thyroid hormone receptors. The other two trees were rooted with insect homologs. Amino acid sequences were aligned using the SEAVIEW package, which allows manual alignment (Galtier et al., 1996). GenBank Accession Numbers of the sequences used are available upon request from hector.escriva.garcia@ens-lyon.fr. Alignments included only the relatively conserved domains: the C domain (the DNA-binding domain), the D domain (a hinge that typically contains nuclear localization signals) and the E domain (the ligand-binding domain) (reviewed by Laudet, 1997); subsequent analysis was based on only the C and E domains. The data set was analyzed using the neighbor-joining method (NJ option of PHYLO_WIN) with a global gap removal option and 1000 bootstrap replicates. The RAR tree was rooted with the thyroid hormone receptors, the most closely related sequences in the nuclear receptor superfamily (Laudet, 1997). The RXR tree was rooted with the cnidarian Tripedalia RXR and the TR2/4 tree with Drosophila DHR78 and Tenebrio THR6, two insect homologs of TR2 and TR4 orphan receptors. In all three trees, the amphioxus sequence is located before the split, giving rise to the three (RAR and RXR) or two (TR2/4) paralogs. The placements of AmphiRAR and AmphiRXR is supported by high bootstrap values (the number above each branch is the bootstrap value). For the TR2/4 proteins, AmphiTR2/4 and the sea urchin (Strongylocentrotus) homolog group together at the base of the vertebrate homologs. This grouping may reflect a close relationship between the two invertebrate deuterostome homologs, neither of which has undergone duplication like its vertebrate counterparts. However, the grouping could be due to long-branch attraction, although the high bootstrap values suggest this is unlikely. Taken together, the phylogenetic analyses are consistent with the results from cloning and Southern blot analyses that AmphiRAR, AmphiRXR and AmphiTR2/4 represent unique amphioxus homologs of the respective vertebrate RARs, RXRs and TR2/4 genes