RESUMO
Detoxification of the pea phytoalexin pisatin via demethylation, mediated by a cytochrome P-450 monooxygenase, is thought to be important for pathogenicity of the fungus Nectria haematococca on pea. To isolate a fungal gene encoding pisatin demethylating activity (pda), we transformed Aspergillus nidulans with a genomic library of N. haematococca DNA constructed in a cosmid which carried the A. nidulans trpC gene. Transformants were selected for Trp+ and then screened for pda. One transformant among 1250 tested was Pda+ and was less sensitive to pisatin in culture than Pda- A. nidulans. The cosmid containing the gene (PDA) conferring this activity was recovered by phage lambda packaging of transformant genomic DNA. When A. nidulans was transformed with the cloned cosmid, 98% of the Trp+ transformants were Pda+. RNA blots probed with a 3.35 kb subclone carrying PDA indicated that the gene is expressed constitutively in A. nidulans but is inducible by pisatin in N. haematococca.
Assuntos
Ascomicetos/genética , Aspergillus nidulans/genética , Benzopiranos/metabolismo , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Genes Fúngicos , Genes , Oxirredutases O-Desmetilantes/genética , Oxirredutases/genética , Pterocarpanos , Transcrição Gênica , Ascomicetos/efeitos dos fármacos , Ascomicetos/enzimologia , Benzopiranos/farmacologia , Cosmídeos , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Inativação Metabólica , Oxirredutases O-Desmetilantes/metabolismo , Mapeamento por RestriçãoRESUMO
Two genomic fragments capable of driving the expression of the hygromycin B resistance gene (hph) were isolated from the phytopathogenic ascomycete Gibberella pulicaris (anamorph Fusarium sambucinum) using a "promoter-probe library" strategy. Two libraries consisting of random, 0.5-2.0-kb fragments of genomic DNA inserted 5' of a promoterless hph gene were constructed and used for transformation of G. pulicaris. Both libraries transformed G. pulicaris at a low frequency. Transformants tolerated up to 800 micrograms/ml of hygromycin B, while untransformed colonies were inhibited completely by 50 micrograms/ml of the antibiotic. Plasmids were re-isolated from transformants by simply digesting, the genomic DNA with KpnI, which cuts once in the polylinker 5' to the insert, and transforming E. coli with the re-ligated DNA. The recovered plasmids transformed G. pulicaris with a frequency of up to 4.4 transformants/micrograms of DNA. Both promoter fragments were sequenced and found to contain TATA and CAAT boxes as well as CT-rich sequences. This method makes it possible to easily isolate many fragments with promoter activity from filamentous fungi, and should facilitate the investigation of the promoter structures necessary for the expression of fungal genes.
Assuntos
DNA Fúngico/isolamento & purificação , Fusarium/genética , Regiões Promotoras Genéticas , Sequência de Bases , Sondas de DNA , DNA Fúngico/genética , Escherichia coli/genética , Dados de Sequência Molecular , Plasmídeos , Transformação GenéticaRESUMO
ATP citrate lyase (ACL) catalyzes the formation of cytosolic acetyl-CoA, which is mainly used for the biosynthesis of fatty acids and sterols. In this paper, we show for the first time that in filamentous fungi two different subunits of ACL are encoded by two separate genes. This is in contrast to animals where ACL is encoded by a single gene. Data are presented on acl genes from the filamentous fungi Sordaria macrospora and Gibberella pulicaris. In S. macrospora, both genes, acl1 and acl2, are clustered within a region of 10 kb and are divergently transcribed.