Visual Pigment Gene Structure and the Severity of Color Vision Defects

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The Expand Long Template PCR System (Boehringer Mannheim) was used exactly as recommended by the manufacturer to do long PCR. The first gene in the array was amplified in long PCR with primers 5′ -GAGGCGAGGCTACGGAGT and 5′-ACGGTATTTTGAGTGGATCTGCT, which correspond to sequences 862 base pairs (bp) upstream of the first exon of the first gene (23) and to the 3′ end of intron 5 (8), respectively. The PCR product served as a template to amplify exons 2, 3, 4, and 5 as described previously (2, 8, 24)

Restriction analysis was done on exons 3, 4, and 5 from the first gene and on exons 3 and 4 from downstream L genes. The presence of an Rsa I site in exon 5 from the first gene from each male identified it as a L pigment gene (8). Whether exon 3 contains a Bso FI restriction site or not indicates whether codon 180 specifies alanine or serine, respectively (24). A Dde I site is found in exon 4 only when serine is specified by codon 233, and this was used to deduce whether the first gene or the downstream L genes were M4L5 hybrids. The results of the restriction analyses were confirmed by direct sequence analysis (24), which was also used to determine whether the sequence of exon 2 differed among the L pigment genes in individual men

For men whose L pigment genes did not differ in exon 4 (Fig. 1C), all L genes except the first gene in the array were amplified by long-PCR (17) with primers 5′ -TTAGTCAGGCTGGTCGGGAACT and 5′ -CATGATGATAGCGAGTGGGATG, which correspond to sequences 465 bp upstream of the first exon of all genes in the array except the first one (5), and to L gene-specific exon 4 sequences (24), respectively. For men whose L genes differed in exon 4 (Fig. 1C), no PCR product was obtained with this primer pair, presumably because their downstream L genes were hybrid genes with an M gene exon 4 sequence. For these men, downstream L pigment genes were selectively amplified by long PCR (17) with the use of an exon 2 primer (5′ -CCTTCGAAGGCCCGAATTA) paired with a L gene-specific exon 5 primer (24). The PCR product was used to amplify only the downstream L pigment genes with the use of the same exon 2 primer and an M gene-specific exon 4 primer (24)

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We thank P. Summerfelt for help in preparing the manuscript and G. H. Jacobs for his many helpful suggestions and comments. Supported by NIH grants EY09303, EY09620, and EY01931, and by Research to Prevent Blindness James. S. Adams Scholar Award to M.N