Molecular analysis of the cDNAs encoded by the p un and p J alleles of the pink-eyed dilution locus

Abstract

The mouse pink-eyed dilution locus (p), one of the oldest described mouse mutations, is located on Chromosome (Chr) 7. Recessive mutations at this locus are characterized by hypopigmentation ranging from slight pigment diminution of the coat with dark eyes to a near albino coat with pink eyes. The gene responsible for this pigmentation phenotype in mice has been identified and cloned as has its human homolog, associated with oculocutaneous albinism type II (OCA2) (Gardner et al. 1992; Rinchick et al. 1993). Both the mouse and human genes encode a protein with 12 membrane-spanning domains (Gardner et al. 1992; Rinchick et al. 1993) localized to the melanosome membrane (Rosemblat et al. 1994). In this report, we characterize the transcripts encoded by two spontaneously arising mutant (null) alleles at this locus: pink-eyed unstable, pun (Wolfe 1963; Melvold 1971; Gondo et al. 1993; Brilliant and Gondo 1992) and pink-eyed dilution-Jackson, pJ (Lyon et al. 1992). Both exhibit a light coat and pink eyes, like the original p allele (Silvers 1979; Green 1989; Lyon et al. 1992). The p,,Z allele is a 70-kb, head-to-tail tandem duplication of a region of transcribed sequence within the p gene (Brilliant et al. 1991; Gondo et al. 1993) and (unlike other mutant p alleles) is associated with a high reversion frequency. Up to 3.5% of the offspring of homozygous p~n mice exhibit patches of wild-type coat color (Melvold 1971) resulting from somatic reversion of the mutant genotype and concomitant loss of duplicated sequences (Brilliant et al. 1991; Gondo et al. 1993). Gardner et al. (1992) have shown that the p,n allele expresses a p transcript that is 1.3-kb larger than the wild-type 3.3-kb mRNA and that the pJ allele expresses a p transcript that is 0.3 kb smaller than the wild-type allele, suggesting the duplication and deletion of coding sequences in the transcript in these mutations respectively. In previous studies, we identified and determined the sequence of the p gene (Gardner et al. 1992). The approximate position of the pun duplication relative to the exons of the gene has been previously described (Gondo et al. 1993; Gardner et al. 1992). Primers were synthesized (Fig. 1) and used in RT-PCR analyses to locate the regions within p,n cDNA that were variant in size compared with wild type as a result of the mutation (Fig. 1). The pJ allele was suspected to have a deletion around exon 20 of the p transcript, because in Southern hybridizations a probe specific for p exon 19 (Dra400; Gardner et al. 1992) hybridized to pJ DNA, but a probe further 3' did not. Therefore, primers flanking the region around exon 20 were selected to define and clone the boundary of the pJ deletion (Fig. 1). As expected, primers 99 and 100 generated a 560-bp product in both wild-type and p"" cDNA (Fig. 2); 92 and 99 produced no products in either cDNA (Fig. 2), as these primers are positioned such that a PCR product cannot be made in the wild-type p cDNA (Fig. 1). Primers 94 and 99 are also positioned such that no product can be made in wild-type cDNA. However, in the pU,~ cDNA, the duplication has rearranged the sequence such that primers 94 and

DOI: 10.1007/s003359900089

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@article{Oakey1996MolecularAO, title={Molecular analysis of the cDNAs encoded by the p un and p J alleles of the pink-eyed dilution locus}, author={Rebecca J. Oakey and N. M. Keiper and Ada S. Ching and Murray H. Brilliant}, journal={Mammalian Genome}, year={1996}, volume={7}, pages={315-316} }