RESUMO
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.
Assuntos
Catecol Oxidase/genética , Monofenol Mono-Oxigenase/genética , Neurospora crassa/enzimologia , Neurospora/enzimologia , Precursores de Proteínas/genética , RNA Mensageiro/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sondas de DNA , DNA Fúngico/genética , Dados de Sequência Molecular , Peso Molecular , Neurospora crassa/genética , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/genética , Mapeamento por RestriçãoRESUMO
Uncoated recA-DNA complexes were imaged with the scanning tunneling microscope (STM). The images, which reveal the right-handed helical structure of the complexes with subunits clearly resolved, are comparable in quality to STM images of metal-coated specimens. Possible conduction mechanisms that allow STM imaging of biological macromolecules are discussed.
Assuntos
DNA/metabolismo , Microscopia Eletrônica , Recombinases Rec A/metabolismo , Acetatos , Ácido Acético , Adsorção , Silicatos de Alumínio , Eletroquímica , Substâncias Macromoleculares , Magnésio , Cloreto de Magnésio , Estrutura Molecular , Conformação ProteicaRESUMO
A link between scanning tunneling microscopy (STM) and conventional transmission electron microscopy has been established for biological material by applying STM on freeze-dried recA-DNA complexes coated with a conducting film. The topography of the complexes observed by means of STM revealed a right-handed single helix composed of about six recA monomers per helical turn.
