RESUMO
A genomic clone encoding the Paracoccidioides brasiliensis orotidine monophosphate decarboxylase gene (PbrURA3) was isolated by screening a subgenomic plasmid DNA library of this fungus, using a PCR amplification product of the gene as a probe. Sequence analysis revealed that the gene contains an open reading frame of 855 bp with a single intron (162 bp), and encodes a putative 285 amino acids polypeptide of estimated molecular weight 31.1 kDa and isoelectric point 6.5. The deduced amino acid sequence predicted a 73.4% identity with orotidine monophosphate decarboxylase of Aspergillus nidulans. Functionality of the gene was demonstrated by transformation into a Saccharomyces cerevisiae ura3 null mutant.
Assuntos
Orotidina-5'-Fosfato Descarboxilase/genética , Paracoccidioides/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Biblioteca Gênica , Teste de Complementação Genética , Ponto Isoelétrico , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Paracoccidioides/genética , RNA Fúngico/química , RNA Fúngico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Transformação GenéticaRESUMO
Paracoccidioides brasiliensis genome has been reported as having a size of about 30 Mb. By digestion of genomic DNA from strain IVICPb 73 (ATCC 32071), we have constructed a DNA library with an insert size average of 8 kb in Escherichia coli XL1 Blue. We have fully sequenced 7 clones comprising 51,022 bp which represent 20 putative protein-coding sequences (seven of them, partial) and one tRNA. The 20 coding sequences cover 46% of the total 51,022 bp with introns present in 10 out of the 20 sequences. Database similarity analysis reveals the presence of genes conserved in other fungal species and higher organisms, including humans.