The cloning and characterisation of the chicken tyrosinase-related protein gene family

Doctoral Thesis


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University of Cape Town

Very little is known about the molecular and genetic mechanisms controlling pigmentation within the bird kingdom. The aim therefore, of this study was to contribute towards the understanding of the genetic regulation of avian pigmentation by the cloning and characterisation of the chicken Tyrosinase-related protein (TRP) gene family. To accomplish this goal, neural crest cells from 500 black chick embryos were cultured under conditions supportive of melanocyte differentiation and proliferation. Using RNA extracted from these pigmented melanocyte cultures, a novel embryonic chick cDNA library was constructed. Screening of this library for chicken equivalents of the mammalian TRP gene family yielded more than 200 cDNA clones. After sequencing, three of these clones, 88.3, pcTRP- 1.6 and pcTRP -2. 10, were found to encode chicken Tyrosinase (Tyr), Tyrosinase-related protein-1 (Tyrp1) and Tyrosinase-related protein-2 (Tyrp2), respectively. In addition, a chicken Microphthalmia (Mi) cDNA clone (M156) was isolated using a mouse Mi cDNA probe. Comparative analyses revealed that chicken Tyr, Tyrp1 and Tyrp2 share approximately 68%, 72% and 70% amino acid sequence identity with their vertebrate orthologues. Northern blot hybridisation analysis demonstrated that the chicken TRPs are expressed in RNA from cultured retinal pigment epithelial (RPE) cells as well as in whole eye RNA. The major transcript sizes for the chicken Tyr, Tyrp1 and Tyrp2 genes are 2.5 kb, 2.3 kb and 3.5 kb, respectively. In situ hybridisation studies confirmed that both chicken Tyr and Tyrp2 genes are expressed in a pigment cell-specific fashion with signals detected in both the skin and RPE of chick embryos. Genomic Southern blot hybridisation analyses strongly suggested that all three chicken TRP genes contain several introns that are likely to be conserved within the vertebrate TRP gene family. Furthermore, the chick Tyr, Tyrp1 and Tyrp2 genes were found to span approximately 5-19 kb, 5-11 kb and 15-30 kb, respectively of the chicken genome. Comparisons between a black and white chick breed at the Tyr and Tyrp1 loci revealed no gross rearrangements at either of these loci. However, 1-2 kb alterations were observed between the same breeds at the Tyrp2 locus. The nature and significance of this alteration is not known. The cloning of the chicken Tyr, Tyrp1 and Tyrp2 cDNAs constitutes the first molecular cloning and characterisation of any avian TRP gene family. Taken together therefore, this study contributes towards the further understanding of the molecular mechanisms regulating pigmentation as well as the evolution of gene families.