Screening of a Xgtll human melanocyte cDNA library with antibodies against hamster tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) resulted in the isolation of 16 clones. The cDNA inserts from 13 of the 16 clones cross-hybridized with each other, indicating that they were from related mRNA species. One of the cDNA clones, Pmel34, detected one mRNA species with an approximate length of 2.4 kilobases that was expressed preferentially in normal and malignant melanocytes but not in other cell types. The amino acid sequence deduced from the nucleotide sequence showed that the putative human tyrosinase is composed of 548 amino acids with a molecular weight of 62,610. The deduced protein contains glycosylation sites and histidinerich sites that could be used for copper binding. Southern blot analysis of DNA derived from newborn mice carrying lethal albino deletion mutations revealed that Pmel34 maps near or at the c-albino locus, the position of the structural gene for tyrosinase.Tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) is a copper-based oxidoreductase that catalyzes the oxidation of tyrosine to dopa and the oxidation of dopa to dopaquinone (1). It is a key enzyme in melanin biosynthesis. Oculocutaneous albinism, a group of autosomal-recessive diseases in humans (2) and animals, is characterized by reduced or absent melanin in skin, hair, and eyes. Tyrosinase-negative albino melanocytes have no tyrosinase activity in vitro.Genetic control of pigmentation has been extensively studied in mice. There is evidence that the c-albino locus at chromosome 7 codes for the structural gene for tyrosinase (3,4). Mutations at this locus affect both tyrosinase activity and coat color (5, 6).A nucleic acid probe for tyrosinase would be an invaluable tool for studies of the regulation of tyrosinase, of the molecular basis of human albinism, and of various mouse mutations affecting coat and eye color. We report here the isolation and sequence of a cDNA clone for human tyrosinase that maps at or near the mouse c-locus. ¶ MATERIALS AND METHODS Cell Culture. Normal human melanocytes, melanotic melanoma cells (LG), and neuroblastoma cells (SK-N-SH) were cultured as described (7-9). The murine neuroblastoma cell line NIE115 was obtained from X. 0. Breakefield (E. K. Shriver Center, Waltham, MA). Proteins of normal melanocytes were radiolabeled with [355]methionine (Amersham) (100 ,XCi/ml, 1390 Ci/mmol; 1 Ci = 37 GBq) as described (10).cDNA Libraries and Screening. RNA from normal human melanocytes was prepared, and poly(A)+ RNA was purified on an oligo[d(T)I-cellulose column (11,12). A cDNA li was prepared employing a Xgtll cloning vector (13-15) Xgtll library contained 1.7 x 106 independent phages immunobiological screening and analysis of the fusion teins produced by Xgtll cDNA clones were carried c described by Young and Davis (15). The rabbit anti-hal tyrosinase antibodies and their application in the stun tyrosinases have been described in detail (10, 16).RNA Blot Hybridization. ...

No abstract

No abstract

DNA samples from 100 patients with cleft lip with or without cleft palate (CL/P) were compared with those of 98 unaffected control individuals with respect to transforming growth factor alpha (TGFA) genotypes. Among the Caucasians in this population (83 CL/P, 84 controls), there was a significant difference in the restriction fragment length polymorphisms (RFLPs) observed after digestion with TaqI (chi 2 = 4.68, P = 0.03). The frequency of the C2 allele in the Caucasian CL/P population was 0.169, whereas that in the control group was 0.089. When the data for Caucasians, African-Americans, and Asians were examined jointly, the chi 2 value for the pooled sample was 5.08 (P = 0.02). This confirms the hypothesis of Ardinger et al. [1989, Am J Hum Genet, 45:348-353] that TFGA itself or a closely linked gene contributes to the development of CL/P in humans.

No abstract