The Dominant white, Dun and Smoky color variants in chicken are associated with insertion/deletion polymorphisms in the PMEL17 gene

Abstract

Dominant white, Dun, and Smoky are alleles at the Dominant white locus, which is one of the major loci affecting plumage color in the domestic chicken. Both Dominant white and Dun inhibit the expression of black eumelanin. Smoky arose in a White Leghorn homozygous for Dominant white and partially restores pigmentation. PMEL17 encodes a melanocyte-specific protein and was identified as a positional candidate gene due to its role in the development of eumelanosomes. Linkage analysis of PMEL17 and Dominant white using a red jungle fowl/White Leghorn intercross revealed no recombination between these loci. Sequence analysis showed that the Dominant white allele was exclusively associated with a 9-bp insertion in exon 10, leading to an insertion of three amino acids in the PMEL17 transmembrane region. Similarly, a deletion of five amino acids in the transmembrane region occurs in the protein encoded by Dun. The Smoky allele shared the 9-bp insertion in exon 10 with Dominant white, as expected from its origin, but also had a deletion of 12 nucleotides in exon 6, eliminating four amino acids from the mature protein. These mutations are, together with the recessive silver mutation in the mouse, the only PMEL17 mutations with phenotypic effects that have been described so far in any species.

Figure 1.—

Birds expressing the wild-type, Dominant white, and Smoky phenotypes.

Figure 2.—

Alignment of the PMEL17 amino acid sequence associated with the wild-type (I*i) allele present in the red jungle fowl (RJF), and the Dominant white (I*I), Dun (I*D), and Smoky (I*S) alleles in chicken in comparison with human (S73003) and mouse (NM_021882) sequences including the mouse silver allele (AF119092). Sequence identities are indicated by dashes and insertion/deletion differences are indicated by dots. The signal sequence, the four copies of the 24-amino-acid repeat in chicken, the transmembrane, and the cytoplasmic region are indicated. The arrow indicates the proteolytic cleavage site that generates an aminoterminal Mα and a carboxyterminal Mβ fragment. The insertion/deletion polymorphisms associated with Dominant white, Dun, and Smoky are boxed.

Figure 3.—

Integration of PMEL17 transmembrane segments into microsomal membranes. (A) Wild-type Lep has two N-terminal TM segments (H1, H2) and a large luminal domain (P2). PMEL17-derived segments corresponding to the transmembrane region (PMEL) were inserted between residues 226 and 253 in the P2 domain. Glycosylation acceptor sites (G1, G2) were placed in positions 96–98 (Asn-Ser-Thr) and 258–260 (Asn-Ala-Thr), flanking the PMEL segment. For constructs with a PMEL segment that integrates into the membrane, only the G1 site is glycosylated (top left), whereas both the G1 and G2 sites are glycosylated for PMEL segments that do not integrate in the membrane (top right). (B) Membrane integration of PMEL17-derived transmembrane segments. Plasmids encoding the Lep-PMEL17 constructs were transcribed and translated in vitro in the absence (−RM) and presence (+RM) of dog pancreas rough microsomes. 0G, unglycosylated protein; 1G, singly glycosylated protein; 2G, doubly glycosylated protein. Molecular weight markers are shown in the first lane. WT, wild type; DW, Dominant white.

Figure 3.—

Integration of PMEL17 transmembrane segments into microsomal membranes. (A) Wild-type Lep has two N-terminal TM segments (H1, H2) and a large luminal domain (P2). PMEL17-derived segments corresponding to the transmembrane region (PMEL) were inserted between residues 226 and 253 in the P2 domain. Glycosylation acceptor sites (G1, G2) were placed in positions 96–98 (Asn-Ser-Thr) and 258–260 (Asn-Ala-Thr), flanking the PMEL segment. For constructs with a PMEL segment that integrates into the membrane, only the G1 site is glycosylated (top left), whereas both the G1 and G2 sites are glycosylated for PMEL segments that do not integrate in the membrane (top right). (B) Membrane integration of PMEL17-derived transmembrane segments. Plasmids encoding the Lep-PMEL17 constructs were transcribed and translated in vitro in the absence (−RM) and presence (+RM) of dog pancreas rough microsomes. 0G, unglycosylated protein; 1G, singly glycosylated protein; 2G, doubly glycosylated protein. Molecular weight markers are shown in the first lane. WT, wild type; DW, Dominant white.