Gel mobility shift scanning of pectin-inducible promoter from Penicillium griseoroseum reveals the involvement of a CCAAT element in the expression of a polygalacturonase gene

Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.

Easy detection of green fluorescent protein multicopy transformants in Penicillium griseoroseum

Penicillium griseoroseum, a deuteromycete fungus producer of pectinolytic enzymes, was transformed with a gene encoding for green fluorescent protein (GFP). The selection of transformants was based on the homologous nitrate reductase gene (niaD). Protoplasts of a P. griseoroseum Nia mutant (PG63) were co-transformed with the plasmids pNPG1 and pAN52-1-GFP. The plasmid pNPG-1 carries the homologous niaD gene and pAN52-1-GFP carries the SGFP-TYG version of GFP. The highest transformation efficiency (102 transformants/µg of pNPG1) resulted from the utilization of equimolar amounts of transforming and co-transforming vectors. Analysis of pAN52-1-GFP insertions into the genomic DNA of the transformants revealed single and multiple copy integrations. The transformants possessing a single copy of the gfp gene showed a low level of fluorescence, whereas multicopy transformants displayed strong fluorescence under visualization with fluorescent light. The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.

Electrophoretic characterization of Aspergillus nidulans strains with chromosomal duplications

Linhagens de Aspergillus nidulans que apresentam duplicaçäo cromossômica Dp(I-II) foram caracterizadas por eletroforese em campo pulsado. Foram analisados variantes morfologicamente deteriorados e melhorados. O cariótipo eletroforético demonstrou que em ambas as linhagens duplicadas (A e B) a banda de 4,2 Mb, que corresponde ao cromossomo II, näo estava presente e foi encontrada uma nova banda. Foram feitas hibridizaçöes usando os genes uapA (cromossomo I) e wA (cromossomo II), que demonstraram que a nova banda corresponde ao cromossomo II mais o segmento duplicado do cromossomo I. O tamanho da duplicaçäo foi determinado como aproximadamente 1,0 Mb. A análise das bandas cromossômicas da linhagem morfologicamente melhorada mostrou que o segmento duplicado do cromossomo I foi completamente perdido. Os variantes morfologicamente deteriorados V9 e V17 mostraram o mesmo cariótipo eletroforético apresentado pelas linhagens duplicadas. Contudo, o variante deteriorado V5 perdeu parte do cromossomo I e apresentou um rearranjo envolvendo o cromossomo V. Esse rearranjo pode ter resultado do tratamento mutagênico usado para a obtençäo dos marcadores genéticos. Os resultados obtidos nesse trabalho demonstram que a técnica de eletroforese em campo pulsado é uma ferramenta excelente para a localizaçäo de rearranjos cromossômicos.