Cloning and expression of cDNA encoding mouse tyrosinase

Abstract

We have isolated a pigment cell-specific cDNA clone from a B16 mouse melanoma cDNA library by differential hybridization. The mRNA of isolated cDNA is highly expressed in B16 melanoma cells and in black mouse (C57BL/6) skin, but is not detectable in mouse neuroblastoma cells nor in K1735 mouse amelanotic melanoma cells. The protein sequence deduced from the nucleotide sequence of the cloned cDNA shows significant similarity to the entire region of Neurospora tyrosinase. To know the identity of cDNA, we transfected K1735 amelanotic melanoma and COS-7 cells with the cDNA carried in a simian virus 40 vector (pKCRH2). We confirmed that the isolated cDNA encodes mouse tyrosinase by immunofluorescence staining of transfected cells using two different anti-T4-tyrosinase monoclonal antibodies. Tyrosinase is composed of 513 amino acids with a molecular weight of 57,872 excluding a hydrophobic signal peptide of 24 amino acids.

The melanoma antigen gp75 is the human homologue of the mouse b (brown) locus gene product.

“…2. There was 90% identity with amino acid sequences deduced from the mouse cDNA clone, pMT4, isolated by Shibahara et al (at amino acid positions 247-260, 333-349, and 428-441 ofpMT4) (15). Oligonucleotide probes were derived from peptide sequences of p75 and used to screen a cDNA library constructed from the human melanoma cell line SK-MEL-19.…”

“…The pMT4 clone was isolated from mouse melanocytic cells (15). Originally pMT4 cDNA was thought to code for mouse tyrosinase, which maps to the mouse c (albino) locus.…”

Molecular basis for the heterogeneity of human tyrosinase.

“…Furthermore, it was shown that the mouse tyrosinase cDNA, pmcTyrl , encodes a protein possessing tyrosine hydroxylase activity, namely functional tyrosinase. The amino acid sequence deduced from the nucleotide sequence of pmcTyrl shows about 40% homology with the entire region of mouse tyrosinase deduced from pMT4 (Shibahara et al 1986). It is therefore conceivable that there may be at least two tyrosinase isozymes encoded by two distinct genes, and that the enzyme protein encoded by pMT4 may possess dopy oxidase activity instead of tyrosine hydroxylase activity.…”

“…We have isolated a pigment cell-specific mouse cDNA, pMT4, and provided evidence that it encodes tyrosinase (Shibahara et al 1986). Recently, other groups have isolated and sequenced another cDNAs encoding mouse tyrosinase (Yamamoto et al 1987; Kwon et al 1988;Muller et al 1988) and a cDNA for human tyrosinase, Pmel 34 (Kwon et al 1987).…”

Cell-free tumor antigen peptide-based cancer vaccines

“…Two were deduced from tyrosinase (17) and tyrosinase-related protein 1 (TRP-1; ref. 18). The sequences of the tyrosinase peptides were YYVSRDTLL (amino acids 128-136) and PYLEQASRI (amino acids 468-474).…”