We cloned the carB and carRA genes involved in ␤-carotene biosynthesis from overproducing and wild-type strains of Blakeslea trispora. The carB gene has a length of 1,955 bp, including two introns of 141 and 68 bp, and encodes a protein of 66.4 kDa with phytoene dehydrogenase activity. The carRA gene contains 1,894 bp, with a single intron of 70 bp, and encodes a protein of 69.6 kDa with separate domains for lycopene cyclase and phytoene synthase. The estimated transcript sizes for carB and carRA were 1.8 and 1.9 kb, respectively. CarB from the ␤-carotene-overproducing strain B. trispora F-744 had an S528R mutation and a TAG instead of a TAA stop codon. The overproducing strain also had a P143S mutation in CarRA. Both B. trispora genes could complement mutations in orthologous genes in Mucor circinelloides and could be used to construct transformed strains of M. circinelloides that produced higher levels of ␤-carotene than did the nontransformed parent. The results show that these genes are conserved across the zygomycetes and that the B. trispora carB and carRA genes are functional and potentially useable to increase carotenoid production.

We determined the nucleotide sequence of a 4599-bp DNA genomic fragment including the gamma-actin encoding gene from Blakeslea trispora, showing an open reading frame of 1561 bp interrupted by four introns with fungal consensus splice-site junctions. The untranslated regions of the actA gene contain a consensus TATA box, a CCAAT motif, a large pyrimidine stretch, and the polyadenylation sequence AATAAA. The predicted protein (375 amino acids) revealed high identity to gamma-actins from fungi (>90%), and gene phylogenies support the grouping of B. trispora actin close to those from the majority of the filamentous fungi. actA transcript (1.4 kb) level in beta-carotene producing conditions was faintly higher than carRA (1.9 kb) and slightly lower than carB (1.8 kb) beta-carotene biosynthetic genes. The use of the actA promoter (PactA) for heterologous gene expression was ascertained by the transformation of gene fusions with the bleomycin resistance gene (bleR) from Streptoalloteichus hindustanus and the geneticin resistance marker (aphI) from Tn903, into Escherichia coli and Acremonium chrysogenum.

Os dous primeiros relatos ("O peso do meu desexo" e "Ela, eu, todos os demais") e mais o último ("A ferida") foron publicados en A ferida (Xerais, 2004); o terceiro ("Reset") apareceu na revista Tempos Novos.