RESUMO
In this study, two transformation vectors (pMG101 and pMG103) for Phanerochaete chrysosporium were constructed, based on the ble phleomycin-resistance-encoding gene and a homologous histone H4 promoter. Transformation frequencies were 6-10 per micrograms of DNA. Transformed vector DNA could either exist as an unstable replicating plasmid or could be stably integrated. Integrated vector DNA from pMG101, which also contains a histone-encoding H3 gene in the promoter fragment, becomes methylated, resulting in inactivation of ble-dependent resistance. Plasmid pMG103, which lacks the H3, does not show methylation.
Assuntos
Basidiomycota/genética , Vetores Genéticos , Histonas/genética , Fleomicinas/farmacologia , Regiões Promotoras Genéticas/genética , Proteínas de Bactérias/genética , Basidiomycota/efeitos dos fármacos , Basidiomycota/crescimento & desenvolvimento , DNA Fúngico/metabolismo , Resistência Microbiana a Medicamentos/genética , Marcadores Genéticos/genética , Metilação , Transformação GenéticaRESUMO
We have investigated four distinct sequences in genomic DNA of Phanerochaete chrysosporium that show strong homology to a lignin peroxidase cDNA sequence [Tien and Tu: Nature 236 (1987) 520-523, and 328 (1987) 742]. The complete nucleotide sequence of one of these genes is presented here. Comparison of the sequenced gene, LIG1, with the published cDNA sequence reveals the presence of eight short intervening sequences. The open reading frame encodes a polypeptide of 372 amino acids (aa), which includes a 28-aa residue hydrophobic leader sequence. The predicted Mr of the mature protein product is 37,000. Partial sequencing of the other three genes reveals substantial nucleotide and amino acid homology.
Assuntos
Agaricales/genética , Genes Fúngicos , Genes , Família Multigênica , Oxigenases/genética , Agaricales/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Genes Homeobox , Dados de Sequência MolecularAssuntos
Abelhas/genética , Cloretos , DNA/isolamento & purificação , Animais , Composição de Bases , Bisbenzimidazol , Centrifugação com Gradiente de Concentração , Césio , DNA/genética , DNA Mitocondrial/genética , DNA Mitocondrial/isolamento & purificação , Eletroforese em Gel de Ágar , LiofilizaçãoRESUMO
The white rot fungus Phanerochaete chrysosporium is an efficient degrader of lignin. Development of classical genetics in this organism would require several years of work, so an alternative strategy was devised. As in the mapping of human genetic diseases, the approach depends on the presence of interallelic restriction site polymorphism, but differs in following their meiotic segregation into the haploid state. Using a set of 20 cloned sequences as probes we were able to demonstrate meiotic segregation of 14 restriction site polymorphisms from fruiting of a single dikaryotic culture and obtained evidence of genetic linkage within two groups of three and two of two. The method could be used to obtain a genetic map of this fungus and should be applicable to genetic analysis of any other organism for which haploid material is available.
Assuntos
Mapeamento Cromossômico/métodos , Ligação Genética/genética , Meiose/genética , Phanerochaete/genética , Polimorfismo de Fragmento de Restrição , AlelosRESUMO
DNA-hybridisation studies showed a close relationship between Phanerochaete chrysosporium ME446, most used in lignin degradation studies, and Sporotrichum pulverulentum Novobranova, the other standard lignin degrading strain. Two other strains of P. chrysosporium were both less related. We show that P. chrysosporium ME446 and S. pulverulentum Novobranova both have a GC-content of 59% for chromosomal DNA with the rRNA genes present as an AT-rich satellite; the mitochondrial DNA has a GC-content of 33%. The genome size estimated for P. chrysosporium ME446 is about 4-5 × 10(7) bp.
RESUMO
We describe the construction of an RFLP-based genetic map of the white rot fungus Phanerochaete chrysosporium ME446. The map is deduced from the allele distributions of 38 RFLP markers in a test set of 53 meiotically derived haploid recombinants of this strain. The map includes a cellulase gene, a matingtype locus and a family of lignin peroxidase (and related) genes that are arranged in two unlinked clusters.
Assuntos
Basidiomycota/genética , Genes Fúngicos , Família Multigênica , Peroxidases/genética , Polimorfismo de Fragmento de Restrição , Alelos , Sequência de Bases , Basidiomycota/enzimologia , Southern Blotting , Troca Genética , Ligação Genética , Marcadores Genéticos , Haploidia , Meiose , Mapeamento de Nucleotídeos , Fenótipo , Homologia de Sequência do Ácido NucleicoRESUMO
Haploid recombinant progeny of Phanerochaete chrysosporium ME446, genome compositions of which had been defined by RFLP-mapping, vary in their idiophasic behaviour. This allowed us to formulate a model of the sequence of idiophasic activities. One component of this variation, the amount of lignin peroxidase activity, is independent of the allele distributions of the lignin peroxidase gene clusters, but correlates with the allele distribution of another locus. This locus appears to control the spread of the lignin peroxidase-active state within the idiophasic mycelial mat and may be the mating-type locus. The successful determination of linkage relied on analysis of chromosome intervals rather than linkage to single markers; this approach should be generally useful for analysing quantitative characters by RFLP mapping.