Isolation and characterization of the tyrosinase gene from Neurospora crassa.
A precursor form of Neurospora crassa tyrosinase has been identified by Western transfer from crude protein extracts and by immunoprecipitation of in vitro translated tyrosinase mRNA. The molecular weight of protyrosinase (75,000) exceeds that of mature tyrosinase (46,000) by about 50%. In order to deduce the primary structure and the nature of the extension, the tyrosinase gene was cloned. Poly(A) RNA isolated from tyrosinase-induced cultures of N. crassa was used as a template for cDNA synthesis, primed by a tyrosinase-specific, 32-fold degenerate heptadecanucleotide. Based on this sequence, a unique 21-mer was synthesized and used to screen a cDNA library constructed from tyrosinase-enriched mRNA. A partial genomic DNA library from wild-type strain TS and a genomic library from strain OR were screened using a 400-base pair nick-translated SalI fragment from a tyrosinase-positive cDNA clone as hybridization probe. The DNA sequences obtained revealed the presence of two allelic forms of this enzyme. The coding regions are interrupted by two short introns, of 52 and 99 base pairs. The encoded proteins differ in 3 out of 621 amino acid residues. A comparison of the deduced amino acid sequence with the known primary structure of mature tyrosinase alleles (Rüegg, C., Ammer, D., and Lerch, K. (1982) J. Biol. Chem. 257, 6420-6426) showed that the enzyme is synthesized as a precursor. Protyrosinase exceeds the mature protein by 213 amino acids at its carboxyl terminus. The possible involvement of carboxyl-terminal processing in enzyme activation is discussed.
Kupper
U
U
Biochemisches Institut der Universität Zürich, Switzerland.
Niedermann
D M
DM
Travaglini
G
G
Lerch
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D004156:Catechol Oxidase / Q000235:genetics*; D003001:Cloning, Molecular; D015342:DNA Probes; D004271:DNA, Fungal / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000235:genetics*; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D015183:Restriction Mapping
D015342:DNA Probes; D004271:DNA, Fungal; D011498:Protein Precursors; D012333:RNA, Messenger; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase