The Cloning and Characterization of Chick Tyrosinase from a Novel Embryonic cDNA Library

Very little is known about the genes involved in the regulation of avian skin and feather pigmentation. In mammals, two gene families have been identified as being important for the regulation of melanin biosynthesis. To isolate the avian equivalents of these families, we have generated an embryonic...

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Bibliographic Details
Published in:Experimental cell research 1996-05, Vol.224 (2), p.372-378
Main Authors: April, C.S., Franz, T., Kidson, S.H.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Base Sequence
Blotting, Northern
Cells, Cultured - enzymology
Cells, Cultured - physiology
Chick Embryo
Cloning, Molecular
DNA Probes - genetics
DNA, Complementary - genetics
Epidermal Growth Factor - chemistry
Epidermal Growth Factor - genetics
Feathers - cytology
Feathers - physiology
Gene Expression Regulation, Developmental - physiology
Gene Library
Melanocytes - enzymology
Melanocytes - physiology
Mice
Molecular Sequence Data
Monophenol Monooxygenase - genetics
Monophenol Monooxygenase - metabolism
Pigment Epithelium of Eye - enzymology
Pigment Epithelium of Eye - physiology
Protein Structure, Tertiary
Restriction Mapping
RNA, Messenger - analysis
Skin - cytology
Skin Physiological Phenomena
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Summary:Very little is known about the genes involved in the regulation of avian skin and feather pigmentation. In mammals, two gene families have been identified as being important for the regulation of melanin biosynthesis. To isolate the avian equivalents of these families, we have generated an embryonic chick melanocyte cDNA library. Neural crest cells from 500 black chick embryos were cultured under conditions supportive of melanocyte differentiation and proliferation. A cDNA library was constructed and screened with a mouse tyrosinase cDNA probe. Nineteen clones were obtained, seven of which cross-hybridized to a mouse tyrosinase cDNA on Southern blots. The longest of these clones, B8.3 (1.9 kb), was sequenced and found to share 99.7% nucleotide and 99.8% amino acid sequence homology to a reported chick tyrosinase cDNA. Both Northern blot analysis andin situhybridization demonstrated that clone B8.3 was expressed in the retinal pigment epithelium of chick embryos. Our results suggest therefore that the cDNA library described here may allow the cloning of novel melanogenic genes.
ISSN:0014-4827
1090-2422
DOI:10.1006/excr.1996.0147