RESUMO
Two cDNA sequences representing putative allelic variants of the Phanerochaete chrysosporium cbhII gene were isolated by hybridization to the Trichoderma reesei cbhII gene. Both of the equivalent genomic sequences were subsequently isolated by the inverse PCR technique. DNA sequencing showed that the cbhII open reading frame of 1,380 bp codes for a putative polypeptide of 460 amino acids which is interrupted by six introns. The domain structure found in T. reesei cbhII is conserved in the equivalent P. chrysosporium protein. The overall similarity between the two gene products is 54%, with the region of highest conservation being found in the cellulose-binding domain (65%). Unlike the cbhI gene of P. chrysosporium, cbhII does not appear to be a member of a class of closely related genes. CBHII is a new member of family B of the beta-1, 4-glucanases. Alignment of the P. chrysosporium and T. reesei CBHII protein sequences showed that all of the residues important for the formation of the extended loops of the catalytic domain and those residues that are involved in the catalytic action of the T. reesei enzyme are also present in the P. chrysosporium equivalent. The profiles of cbh gene expression in P. chrysosporium reveal that while cbhI.1 and cbhI.2 could be coregulated, cbhII can be independently controlled. The latter is so far the only cellulase gene found to be expressed when the fungus is grown on oat spelt arabinoxylan, suggesting that it may play an active role in the xylanolytic as well as the cellulolytic systems.
Assuntos
Basidiomycota/genética , Genes Fúngicos/genética , Glicosídeo Hidrolases/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Celulose 1,4-beta-Celobiosidase , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The genome of Phanerochaete chrysosporium strain ME446 contains multiple, non-allelic, cellobiohydrolase I (CBHI)-like sequences, at least two of which are expressed in a cellulose-dependent manner. Each of the expressed genes contains two identically positioned introns within its coding region. The lengths and sequences of these introns are different and one is not excised from all transcripts, raising the possibility that subtly different protein products may be expressed from a common gene. Introns are also present upstream of both genes but these differ in number and position, as well as sequence and length. Endoglucanase-like sequences could not be identified and it is suggested that variant CBHI-like proteins may provide endoglucanase activity in this fungus.
Assuntos
Basidiomycota/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Glicosídeo Hidrolases/genética , Isoenzimas/genética , Lignina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Basidiomycota/enzimologia , Celulose 1,4-beta-Celobiosidase , DNA Complementar/genética , DNA Fúngico/genética , Indução Enzimática , Proteínas Fúngicas/biossíntese , Glicosídeo Hidrolases/biossíntese , Íntrons , Isoenzimas/biossíntese , Dados de Sequência MolecularRESUMO
We seek to define more fully how Phanerochaete chrysosporium degrades its natural substrate, lignocellulose. This contribution concerns several relevant topics. Mineralisation of [14C]DHP, as a model for lignin degradation, showed that a set of genetically defined meiotically derived products of strain ME446 differed in their degradative ability and also that, under optimum conditions for mineralisation, extracellular lignin peroxidase activity was absent. Xylanolytic and xylosidase/beta(1-->3) glucanase activities are also described. The complexity of the CBHI gene family is described and differential splicing of a CBHI gene transcript is proposed. In contrast to the multiplicity of CHBI genes there is a single CBHII gene. PCR methods were developed to analyse differential gene expression on different substrates. We have also developed a transformation system involving a reporter construct for the analysis of CBHI promoter function.
Assuntos
Basidiomycota/enzimologia , Celulose/metabolismo , Lignina/metabolismo , Basidiomycota/genética , Biodegradação Ambiental , Celulose 1,4-beta-Celobiosidase , Genes Fúngicos/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA Fúngico/análise , RNA Mensageiro/análise , Transformação GenéticaRESUMO
The Phanerochaete chryososporium trpC gene has been isolated by complementation of an Escherichia coli trpC mutant. The full extent of the fungal gene, determined by sequence analysis, was found to be 2414bp. This includes a single intron of 50bp, the presence of which was confirmed by RNA-primed polymerase chain reaction analysis. This features makes the P. chrysosporium gene unique when compared to equivalent genes from other filamentous fungi. The P. chrysosporium trpC gene encodes a single protein containing three enzyme activities involved in tryptophan biosynthesis arranged in the order: NH2-GAT-IGPS-PRAI-COOH. This order is conserved in all filamentous fungi so far examined and, indeed, is the gene order within the E. coli trp operon.
