C - Chemistry – Metallurgy – 07 – H
Patent
C - Chemistry, Metallurgy
07
H
195/1.22, 195/1.
C07H 15/12 (2006.01) C12N 7/00 (2006.01) C12N 9/02 (2006.01) C12N 15/00 (2006.01) C12P 13/22 (2006.01) C12P 21/02 (2006.01) C12Q 1/26 (2006.01) C12Q 1/68 (2006.01) A61K 38/00 (2006.01)
Patent
CA 1293940
ABSTRACT This discovery resulted when a .lambda. gtll cDNA library of normal human melanocytes were screened with antibodies directed against purified hamster tyrosinase. Sixteen independent clones which gave a positive signal were isolated from 5 x 105 independent plaques. cDNA inserts of 13 clones among the 16 candidates cross-hybridized with each other, indicating that they were from related mRNA species. mRNA homologous to a representative cDNA .lambda. mel 34 was expressed specifically in melanocytes, detecting an approximately 2.4 kb mRNA species of human melanocytes. The nucleotide sequence of the three overlapping cDNA inserts spanning 1.88 kb was determined and an amino acid sequence was deduced. The human tyrosinase is composed of 548 amino acids with a molecular weight of 62,160 excluding a hydrophobic signal peptide. Mouse genomic DNA blot analysis revealed that the gene for mel 34 was deleted in albino mouse homozygous for the deletion at and around the albino locus on chromosone 7. It is concluded that A mel 34 contained cDNA encoding human tyrosinase. Moreover, the cDNA gene for human tyrosinase was recovered from the .lambda. mel 34 and can be used for many purposes including the production of pure human tyrosinase. In addition, the three clones of the 11 not represented by .lambda. mel 34 are represented by .lambda. mel 17-1. The gene for .lambda.mel 17-1 cDNA was not mapped at the albino locus, detected single hybridizing restriction fragment in human and mouse DNA, and was highly conserved from mouse to human. The abundance of .lambda. mel 17-1 cRNA paralleled the melanin content in human and mouse melanocytes. The expression of .lambda. mel 17-1 cRNA was elevated after stimulation of mouse and human melanoma cells with MSH or/and IBMX, and U-V light (such as suntan). This was also closely correlated with the elevation of melanin content. The fact that the .lambda. mel 17-1 gene is conserved evolutionarily indicates that the molecule encoded by the .lambda. mel 17-1 has biologiacally important functions. The expression of that gene is controlled by hormones (MSH) or U-V light and positively correlated with the melanin content. These data indicate that the gene is involved in melanin biosynthesis in addition to tyrosinase. Current studies suggest that .lambda. mel 17-1 gene product act on melanin biosynthesis's pathway distal to tyrosinase.
548655
Borden Ladner Gervais Llp
Donald Guthrie Foundation For Medical Research Inc.
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