RESUMEN
In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.
Asunto(s)
Coprinus/enzimología , Coprinus/genética , Endonucleasas de ADN Solapado/genética , Regulación Fúngica de la Expresión Génica , Meiosis/fisiología , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Complementario/química , ADN Complementario/aislamiento & purificación , ADN de Hongos/química , ADN de Hongos/aislamiento & purificación , Endonucleasas de ADN Solapado/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Filogenia , Antígeno Nuclear de Célula en Proliferación/genética , Profase/genética , Profase/fisiología , ARN de Hongos/análisis , ARN Mensajero/análisis , Fase S/genética , Fase S/fisiología , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Coprinus congregatus synthesized and secreted high amounts of laccase under acidic culture condition (pH 4.1) transiently. We have cloned a genomic DNA fragment between the copper binding regions I and II of a laccase by PCR from C. congregatus. This fragment was used as a probe to clone a laccase cDNA from the acidic culture. The cDNA (clac2) consisted of 1817 bp which contains 1086 bp encoding 362 amino acids. The N-terminal sequence of the purified CLAC2 revealed that the first 16 amino acids were removed by the modification. The purified protein had two copper binding regions, although other fungal laccases had four. According to a Northern blotting analysis the clac2 was expressed not in neutral culture but in acidic culture only. The transcription of the clac2 as well as the laccase activity reached a maximum after 24 h upon transferring to an acidic culture, and then decreased very rapidly.
Asunto(s)
Coprinus/genética , Proteínas Fúngicas , Genes Fúngicos , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , Coprinus/enzimología , ADN Complementario , Regulación Fúngica de la Expresión Génica , Concentración de Iones de Hidrógeno , Lacasa , Datos de Secuencia Molecular , Oxidorreductasas/química , Reacción en Cadena de la PolimerasaRESUMEN
The nucleotide sequences of genes for the homolog in Coprinus cinereus of the eukaryotic ribosomal protein L41 and for tRNAThr(AGU) are reported. The gene for tRNAThr(AGU) was located upstream of the gene for the L41 ribosomal protein, and these genes were adjacent to each other but in opposite orientations. The deduced amino acid sequence of ribosomal protein L41 exhibited strong homology to those of L41 proteins of several yeasts. The 56th amino acid of the deduced protein was proline, as it is in the L41 protein of a cycloheximide-sensitive strain of yeast. The putative secondary structure of the tRNA gene resembled the characteristic cloverleaf structure of tRNAs. Elements resembling an A-box and a B-box were found in the gene for tRNAThr(AGU). These boxes are known as internal promoter elements in genes for eukaryotic tRNAs.