Expression of Neurospora crassa laccase under the control of the copper-inducible metallothionein-promoter.

Laccase from the ascomycete Neurospora crassa is an inducible secretory enzyme. In vegetatively growing cultures its biosynthesis is repressed but can be induced by different protein synthesis inhibitors. Transformation of the N. crassa wild-type strain Singapore with a fusion gene consisting of the N. crassa copper-metallothionein promoter and the laccase gene are described in this report. Correct integration of the 3.6 kilobase (kb) promoter-fragment fused with the laccase gene containing a 5' consensus region leads to copper-dependent expression of the enzyme during the vegetative growth phase. The enzyme is glycosylated and secreted, and high amounts of extracellular activity can be detected. The regulation of laccase biosynthesis of one examined transformant, followed at both the transcriptional and the translational level, indicates co-induction of both copper-metallothionein and laccase. The data presented show that expression of the recombinant laccase gene is exclusively regulated by the transformed N. crassa metallothionein-promoter.

Expression of tyrosinase in vegetative cultures of Neurospora crassa transformed with a metallothionein promoter/protyrosinase fusion gene.

Wild-type Neurospora crassa, strain Singapore, was transformed with a N. crassa metallothionein promoter/protyrosinase fusion gene. Transformants produced tyrosinase during vegetative growth, as determined by Western analyses and activity assays. This is in sharp contrast to wild-type strains, where this enzyme is only expressed in situations of starvation or sexual differentiation. Complete integration of a 400 bp metallothionein promoter-fragment leads to constitutive expression of protyrosinase, whereas a 3.6 kb promoter-fragment conferred copper inducibility on the reporter gene in four transformants. A transformant with high constitutive tyrosinase levels was able to produce melanin on complete medium agar plates supplemented with 1 mg/ml L-tyrosine.

ATP-induced protyrosinase synthesis and carboxyl-terminal processing in Neurospora crassa.

The effects of 3'-5' cyclic AMP and ATP upon tyrosinase induction in Neurospora crassa were examined. Northern analysis of total cellular RNA revealed rapid de novo synthesis of protyrosinase after addition of these substances to stationary-phase mycelia. The maturation of protyrosinase in crude extracts of mycelia was followed by Western analysis. Polyclonal rabbit antiserum directed against the denatured carboxyl-terminal extension of protyrosinase does recognize the proform and several intermediate forms of different molecular weight but not mature tyrosinase. Disruption of ATP-induced mycelia in sodium phosphate buffer (pH 6.0) demonstrate processing at the carboxyl-terminal end of protyrosinase. The activity assays revealed that protyrosinase is an inactive precursor and that at least two active forms of slightly different molecular weight are present in crude extracts. Maturation of protyrosinase thus involves specific and sequential proteolytic cleavage at the carboxyl-terminus. These results suggest the presence of a tyrosinase activator in Neurospora crassa mycelia, which is kept apart from protyrosinase in the intact mycelium.

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.

[Unusual CT skull findings after preventive CNS treatment of acute leukemia in children]. / Auffällige CT-Befunde des Schädels nach präventiver ZNS-Behandlung akuter Leukämien bei Kindern.

CT examinations of the cranium were carried out among 15 children who had previously received cranial radiation and i.th. methotrexate during leukemia management. Surprisingly, 10 of 15 patients had abnormal CT findings. Only 2 of the 10 "CT-positive" children were clinically-neurologically conspicuous. The visible CT alterations of brain structure seemed especially marked and frequent the more time had elapsed since CNS-treatment. The most striking CT pathologica were found among children up to 5 years of age, who were also conspicuous through convulsions and delayed development.