We report for the first time in chickens, as well as in vertebrates, a mutation of the tyrosinase gene that involves intron 4 and does not affect the coding sequence of any of the 5 exons. Indeed, the albino alleles described at the TYR
locus are usually due to a single nucleotide substitution or a single nucleotide insertion/deletion in the coding sequence [11
], but not in the intronic sequence. As an example, the dark-eyed albino mutation(c44H
) in the mouse [13
] presents a high phenotypic similarity with the recessive white chicken because it exhibits a white coat color, with pigmented eyes. This mutation was found to be due to a single base point mutation in exon 1 of the mouse tyrosinase gene, but no information was provided regarding the intron sequences. Another case of a white-coat animal with pigmented eyes was described in rabbits [14
], and could not be associated to any mutation in the coding sequence. Furthermore, a screening of 120 cases of human oculocutaneous albinism (OCA) revealed the absence of any mutation in the coding sequence of the tyrosinase gene for 35 cases [15
], 24 of these 35 were found in probands with the OCA1B condition, where minimal-to-moderate amounts of cutaneous and ocular pigment could be found. Our results show that it may not be possible to elucidate the mechanism of a tyrosinase mutation in some phenotypes showing white hairs or white coat and eye pigmentation, unless intron structure, and, if possible, intron sequence, are studied.
The retroviral insertion found in intron 4 of the tyrosinase gene may modify the gene's transcription pattern. Indeed, the 3'UTR results revealed that aberrant transcripts were generated in the recessive white chicken by alternative splicing and using three alternate polyadenylation sites that are present in the normal intron 4 sequence (Figure ). It is most likely that the retroviral insertion plays a very important role in the splicing procedure and causes aberrant mRNA.
Tyrosinase exon 5 is the carboxyl terminal membrane spanning domain [16
] which has an important role for the proper positioning of the enzyme in the melanosome [19
]. A defect in the trafficking of the tyrosinase protein may affect the biogenesis of melanosomes [20
] but melanosomal transfer to the keratinocytes is not well characterised. Disruption of this process would be expected to have severe consequences for pigmentation [21
]. Misrouting of tyrosinase was observed in the platinum allele at the mouse albino locus, which is characterized by the occurrence of an abnormal stop codon in position 489 within exon 5 of the TYR
]. This mutation is responsible for an extremely diluted coat color and pink eyes. In a human case, a single base insertion located in the transmembrane domain of the tyrosinase gene eliminated a portion of the transmembrane region and the carboxy terminus, and resulted in an inactive enzyme causing tyrosinase-negative oculocutaneous albinism [23
]. In the case of the chicken recessive white mutation, the lack of exon 5 in the transcript could affect the translation of the membrane spanning domain although the conserved copper-binding regions are retained; this transcript may therefore encode a cytoplasmic, rather than a membrane-bound enzyme, and this would disturb melanogenesis. Although the truncated transcripts do not have a stop codon before the polyadenylation, they are still the major transcripts in the recessive white chicken. Previous studies of tyrosinase in the mouse suggested that shorter cDNA resulting from alternative splicing may have non functional tyrosinase activity [19
]. Translation of the recessive white chicken alternative transcripts would be expected to result in a truncated protein. A previous study of the tyrosinase enzyme suggested that both C*C
(recessive white) and C*A
(albino) alleles could produce tyrosinase-like molecules that are inactive due to a change that is electrophoretically and antigenically "silent" [24
The difference observed between the eye and the feather pigmentation will need further consideration. The retinal pigment epithelium (RPE) derives from the optic cup [25
] and arises from different precursor cells than the skin melanocytes. Furthermore, the eye pigment cells do not transfer their pigment [26
]. Thus, the consequences of a defect in pigment synthesis may be expected to differ in the eye and in the feathers. For instance, the dominant white mutation of the chicken has been found to be a defect of the PMEL17
] which codes for a membrane protein of the melanosome, this mutation suppresses black pigment from the feathers but does not affect eye pigmentation. The fact that some pigment appears in the eyes of recessive white chickens indicates some tyrosinase activity. In the case of the mouse dark-eyed albino mutation (c44H
), the TYR
gene expression was not affected by the point mutation, but the phenotypic consequences of this mutation were less severe in the eyes than in the coat. If a partially truncated tyrosinase protein is produced in the recessive white chicken, this may cause disorganization of pigment into melanosomes and may lead to an improper transfer to the feather keratinocyte, but it might not affect skin pigmentation, and could explain why the White Silky exhibits black skin with white plumage in the presence of a mutated tyrosinase gene. An alternative interpretation of the mechanism of the recessive white mutation is that the aberrant transcript does not give any functional protein at all, but that pigmentation is maintained in some tissues since a small proportion of the normal transcript is still produced in the mutant (see Figure ). Maybe this is a sufficient amount in some tissues (especially in eyes) but not in the growing feather. The splicing may also be different according to tissues.
Retroviral insertions are known to be responsible for insertional mutagenesis in mice, as illustrated by the 'dilute' coat color mutation [28
] and the hairless mutation [29
]. In chickens, the henny-feathering mutation (Hf) results from an abnormal expression pattern of the aromatase gene, under the control of a retroviral long terminal repeat [30
]. The sex-linked late-feathering mutation, K, is associated with the insertion of a full-size retroviral sequence, named ALVE
21, together with the duplication of the insertion site [31
]. Both Hf and K are dominant mutations. The K mutation exhibits some peculiar features due to its association with ALVE
21: (i) revertants have been observed which carry a solitary long terminal repeat (LTR) in place of the full retroviral element [32
], (ii) carriers of the K mutation exhibited a higher susceptibility to exogenous leucosis infection in some chicken strains such as White Leghorns, this phenomenon could be partly explained by the production of a full viral particle due to the expression of the ALVE
21 genome. No such observation has been made for the recessive white mutation, which could suggest that the insertion found within intron 4 is quite stable and that the retroviral genome is not expressed or not sufficient to induce an interaction with exogenous retroviruses. Since the recessive white mutation has never been described as unstable, it may be questioned whether the insertion in inverted orientation may limit the occurrence of reversion events. Revertants could have been detected in our diagnostic test if only a solitary LTR was remaining at the site of insertion, then a larger band would have been amplified between the Diag05-nor-up and Diagnostic05-dw primers in some colored chickens, but this was not observed.