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.

Visando explorar a mutagênese insercional em Magnaporthe grisea, foram avaliadas a transformação dos protoplastos obtidos após adequação do protocolo e a eficiência da integração de pAN7-1 no genoma do ascomiceto na presença da enzima de restrição Hind III. Os protoplastos de M. grisea I-22 foram prontamente transformados para a resistência à higromicina. Quando o vetor linearizado com Hind III foi usado para transformar o fungo na presença de Hind III, a eficiência de transformação foi 1,1 a 8,1 vezes superior ao tratamento sem a adição da enzima. No geral, a melhor concentração de Hind III foi 5 unidades/reação de transformação. Tal concentração promoveu a produção média de 332 transformantes/µg de pAN7-1/10(7) protoplastos. A presença do gene de seleção hph no genoma de 18 indivíduos resistentes à higromicina foi confirmada por PCR.

To investigate insertional mutagenesis in Magnaporthe grisea, we tested the transformation of protoplasts produced after protocol optimization, and analyzed the integration efficiency of pAN7-1 into the M. grisea genome mediated by the restriction endonuclease Hind III. The I-22 protoplasts were readily transformed for hygromycin resistance. When pAN7-1 was linearized with Hind III and used to transform fungal protoplasts in the presence of the corresponding enzyme, the transformation efficiency was increased 1.1 to 8.1-fold. The optimal Hind III concentration for enhanced transformation corresponded to 5 unit/transformation mix. This concentration led to average frequency of 332 transformants/µg de pAN7-1/10(7) protoplasts. The presence of selection gene hph in the 18 transformant genome was confirmed by PCR.

Linhagens de Aspergillus nidulans que apresentam duplicação 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, não estava presente e foi encontrada uma nova banda. Foram feitas hibridizações 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 duplicação 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 obtenção dos marcadores genéticos. Os resultados obtidos nesse trabalho demonstram que a técnica de eletroforese em campo pulsado é uma ferramenta excelente para a localização de rearranjos cromossômicos.

Pulsed-field gel electrophoresis was used to characterize strains of Aspergillus nidulans with a chromosomal duplication Dp(I-II). Morphologically deteriorated and improved variants of these strains were also analyzed. The electrophoretic karyotype demonstrated that in two duplicated strains (A and B) the 4.2 Mb band, which corresponds to chromosome II, was absent and a new band was observed. Hybridization studies using the uapA (chromosome I) and wA (chromosome II) genes demonstrated that the new band corresponded to chromosome II plus the duplicated segment of chromosome I. The size of the chromosomal duplication was approximately 1.0 Mb. Analysis of the chromosomal bands of a morphologically improved strain showed that the duplicated segment of chromosome I was completely lost. The morphologically deteriorated variants V9 and V17 had the same karyotype as the duplicated strains. However, the deteriorated variant V5 lost part of chromosome I and had a rearrangement involving chromosome V. This rearrangement may have resulted from the mutagenic treatment used to obtain the genetic markers. Pulsed-field gel electrophoresis was found to be an excellent tool for locating chromosomal rearrangements.