SUPREME-Project: Sequence of tyrosinase related protein-1 (TRP-1) in alpaca

摘要

Molecular bases of pigmentation in mammals have been deeply studied in the last years and a number of results have been obtained in relation to gene structure and expression in melanogenesis. Many loci are involved in the determination of colour pattern and several mutants have been characterised over the years. The tyrosinase gene family plays a key role in biosynthetic pathway of melanin. Three proteins are included: tyrosinase, encoded by albino locus, which is the rate-limiting enzyme, tyrosinaserelated protein-1 (TRP-1), encoded by brown locus and tyrosinase related protein-2 (TRP-2), encoded by slaty locus. Their genes have been isolated and sequenced in humans and in mice as well as in other vertebrates showing a similarity of about 40% between each other with various homologous regions. Moreover phylogenetic analysis showed a high degree of conservation among species. According to these results it was possible to design specific degenerated primers for PCR amplification able to work with nucleic acids obtained from different higher vertebrates (Jackson et al., 1994). Utilising these primers we were able to obtain a DNA fragment from Alpaca. Sequence analysis was performed and a comparison with known TRPs showed a high similarity of the fragment with mouse and human TRP-1. On the base of the obtained sequence new primers were designed and rapid amplification of cDNA end (RACE) was performed permitting the isolation and sequencing of the 3' end of the gene. The next step will be to obtainthe 5' part and to clone the whole TRP-1 in order to have enough purified quantity of the gene to be used as a probe in Southern analysis protocols.