RESUMEN
This study aims to elucidate the mechanism of sexual development of basidiomycetous mushrooms from mating to fruit body formation. Sequencing analysis showed the TRP1 gene of basidiomycete Schizophyllum commune encoded an enzyme with three catalytic regions of GAT (glutamine amidotransferase), IGPS (indole-3-glycerol phosphate synthase), and PRAI (5-phosphoribosyl anthranilate isomerase); among these three regions, the trp1 mutant (Trp(-)) had a missense mutation (LâF) of a 338th amino acid residue of the TRP1 protein within the IGPS region. To investigate the function of IGPS region related to sexual development, dikaryons with high, usual, and no expression of the IGPS region of TRP1 gene were made. The dikaryotic mycelia with high expression of the IGPS formed mature fruit bodies earlier than those with usual and no expression of the IGPS. These results showed that the IGPS region in TRP1 gene promoted sexual development of S. commune.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Schizophyllum/crecimiento & desarrollo , Schizophyllum/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Clonación Molecular , Proteínas Fúngicas/química , Ácidos Indolacéticos/farmacología , Indoles/farmacología , Mutación , Estructura Secundaria de Proteína , Schizophyllum/efectos de los fármacos , Schizophyllum/metabolismo , Alineación de Secuencia , Transcripción Genética , Triptófano/farmacologíaRESUMEN
The caffeine biosynthetic pathway is composed of three methylation steps, and N-methyltransferase catalyzing each step has high substrate specificity. Since the amino acid sequences among coffee 7-methylxanthosine synthase (CmXRS1), theobromine synthase, and caffeine synthase are highly homologous to each other, these substrate specificities seem to be determined in a very restricted region. The analysis of site-directed mutants for CmXRS1 that naturally acts at the initial step, i.e., 7-N methylation of xanthosine, revealed that the activity of 3-N methylation needs a histidine residue at corresponding position 161 in the CmXRS1 sequence. We succeeded in producing the mutant enzyme which can catalyze the first and second methylation steps in caffeine biosynthesis.
Asunto(s)
Cafeína/biosíntesis , Metiltransferasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clarkia/enzimología , Clarkia/genética , Café/genética , Café/metabolismo , Cartilla de ADN , Metilación , Metiltransferasas/genética , Datos de Secuencia Molecular , Mutagénesis , Plásmidos , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A nuclease was purified from the fruit body of Tricholoma matsutake. The molecular mass was 38 kDa. The optimum pH of the nuclease was about 8.0. Its activity was inhibited by GTP. The nuclease cleaved RNA and heat-denatured DNA endonucleolytically to produce 5'-mononucleotide, and showed 3'-nucleotidase activity. The amino acid sequence up to seven amino acids from the N-terminus was NH(2)-APPPSSN.
Asunto(s)
Esterasas/aislamiento & purificación , Esterasas/metabolismo , Tricholoma/enzimología , ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , ARN/metabolismo , TemperaturaRESUMEN
The ind1 and cfn1 mutations of Schizophyllum commune express resistance to high concentrations of indole and caffeine respectively, and also affect sexual development. To clarify molecular events caused by the mutations, it was investigated how cAMP levels in S. commune strains respond to externally supplied indole and caffeine. Both compounds increased the cAMP levels in wild-type strains under several culture conditions. During sexual development of the ind1 mutant, the cAMP level in an early stage (hyphal aggregation) was highly increased by addition of indole, and the phenomenon disappeared in a later stage (fruit body formation). For the cfn1 mutants, the incremental increase in cAMP levels by addition of caffeine was smaller than that of wild-type strains.
