Fig. 2. Cloning and mapping of zebrafish nls/raldh2. (A) Comparison of the predicted RALDH2 amino acid sequence of human (Accession Number, 094788), mouse (Q62148), chick (093344) and zebrafish proteins. Zebrafish RALDH2 protein has 79% amino acid identity and 91% similarity to the human protein. Sequence conservation within the first 20 amino acids suggests that RALDH2 proteins may be translated from the first methionine, rather than methionine 20; the 19 N-terminal amino acids for the mouse and chick proteins were derived from their cDNA sequences. Underlined sequences correspond to primers used for RT-PCR cloning. Alignment was performed using PILEUP. (B) Schematic of part of linkage group 7 (LG7), showing the nls/raldh2 map position in relation to SSLP markers. Data were combined with those from the meiotic and LN54 radiation hybrid panels to determine the position of the raldh2 EST and nls. (C) Linkage analysis with SSLP primers on genomic DNA from nls, outcrossed to the WIK strain. In parental strains, primer pair z11119 amplified a single band of 205 base pairs (bp) in the heterozygous nls carriers and two bands of 195 bp and 130 bp, in WIK. In F₂ progeny, these primers amplified the 205 bp fragment in only homozygous nls/nls embryos, demonstrating linkage to this marker. Likewise, SSLP marker z4999 amplifies a fragment of 210 bp in heterozygous nls/+ parents, and a fragment of 200 bp in WIK (a fragment of 180 bp is amplified in both strains). These primers amplified only the 210 bp fragment in pools of homozygous F₂ nls embryos, whereas sibling embryos contained both fragments. Thus, nls is closely linked to SSLP markers z11119 and z4999, and to z11894 and z8693 (not shown, see Materials and Methods). (D) A single point mutation in all of six independently subcloned cDNAs within the nls open reading frame, generates a missense mutation of Gly²⁰⁴ to Arg²⁰⁴ (asterisk), located in a loop structure. (E) PstI restriction of PCR-fragments, amplified using raldh2-specific primers, of cDNA pools of 40 London wild-type (+/+) and nls/nls embryos, respectively, generates RFLPs of 0.6 and 0.77 kb in nls/nls, and of 1.46 kb in wild type. M, molecular weight marker.