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DNA pools (plate pools, then row and column pools) from an arrayed zebrafish PAC library (19) were screened with redundant primers (Hox-F: 5′-CGNAAGAARMGNGTNCCNTAYAC-3‴; Hox-R: 5‴-CATNCKNCKRTTYTGRAACCANAT-3‴; Hox-Fi: 5‴-CTNGARYTNGARAARGARTTYCA), where N is A, C, T, or G, R is A or G, M is A or C, Y is C or T, and K is T or G. Ends of positive PACs were sequenced and specific primers used to find overlapping clones. Positive PACs were amplified with redundant primers; products were cloned and sequenced, and gene specific primers were used to obtain sequence directly from PAC DNA.
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Unambiguously alignable sequences were obtained using CLUSTAL X (http://www-igbmc.u-strasbg.fr/ BioInfo/ClustalX/Top.html) and trees were generated by the neighbor-joining method [N. Saitou and M. Nei, Mol. Biol. Evol. 4, 406 (1987)]. A lamprey (Petromyzon marinus) cDNA library screened with redundant hox gene primers provided an outgroup. For accession numbers, see (11).
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See Science Online (www.sciencemag.org) for a catalog of zebrafish hox genes, alignments for the phylogenetic trees in Fig. 2, accession numbers of loci used to construct the phylogenetic trees, mapping primers for zebrafish genes, and mapping data for newly mapped loci.
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Supported by NIH grant R01RR10715 (J.H.P.), NIH grant PHS P01HD22486 (J.H.P. and M.W.), a Medical Research Council of Canada grant (M.E.), and NSF grant IBN-9614940 (C.A.). We thank J. Miles for technical assistance.
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