ABSTRACT
By sequencing downstream of the cloned nitrate reductase gene (NIA1) in the phytopathogenic fungus Stagonospora (Septoria) nodorum, a second open reading frame was found. Further analysis revealed this to be the nitrite reductase gene (NII1). Both genes are transcribed in the same direction, and are separated by an intergenic region of 829-bp. The coding sequence of NII1 is interrupted by three small introns and corresponds to a predicted protein of 1141 amino acids in length. Consensus binding sites for regulatory proteins are present in the promoter region of NII1. There is no indication, however, from hybridisation or sequence analysis that the nitrate transporter gene is closely associated with the NIA1-NII1 cluster, as has been found for a number of fungi.
Subject(s)
Ascomycota/enzymology , Ascomycota/genetics , Genes, Fungal , Multigene Family , Nitrate Reductases/genetics , Amino Acid Sequence , Ascomycota/metabolism , Base Sequence , Carrier Proteins/genetics , DNA, Fungal/genetics , Fungal Proteins/genetics , Fungi/enzymology , Fungi/genetics , Genetic Linkage , Introns , Molecular Sequence Data , Nitrate Reductase , Nitrates/metabolism , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
The nitrate reductase gene (NIA1) of the phytopathogenic fungus Stagonospora (Septoria) nodorum has been cloned from a cosmid library by homologous hybridisation with a PCR-generated probe. A 6. 7-kb fragment carrying the NIA1 gene was subcloned and partially characterised by restriction mapping. Sequencing of the gene indicated a high degree of homology, both at the nucleotide and amino-acid levels, with nitrate reductase genes of other filamentous fungi. Furthermore, consensus regulatory signals thought to be involved in the control of nitrogen metabolism are present in the 5' flanking region. The cloned NIA1 gene has been used to develop a gene-transfer system based on nitrate assimilation. Stable nia1 mutants of S. nodorum defective in nitrate reductase were isolated by virtue of their resistance to chlorate. These were transformed back to nitrate utilisation with the wild-type S. nodorum NIA1 gene. Southern analyses revealed that transformation occurred as a result of the integration of transforming DNA into the fungal genome; in all cases examined, integration was targeted to the homologous sequence.
Subject(s)
Ascomycota/enzymology , Ascomycota/genetics , Genes, Fungal , Mitosporic Fungi/enzymology , Mitosporic Fungi/genetics , Nitrate Reductases/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cosmids , DNA Primers/genetics , DNA, Fungal/genetics , Genetic Markers , Molecular Sequence Data , Nitrate Reductase , Phylogeny , Polymerase Chain Reaction , Transformation, GeneticABSTRACT
Phlyctenules and catarrhal infiltrates of the human cornea have been described in association with staphylococcal blepharitis. Rabbits immunized and boosted with phenol-inactivated Staphylococcus aureus had a fourfold or greater increase in antibody titer to S aureus and delayed hypersensitivity to S aureus. After topical challenge with viable S aureus, the rabbits in this model had vascularized, elevated, nodular infiltrates of the cornea resembling phylctenules in humans and peripheral corneal infiltrates running parallel to the limbus and separated from it by a lucid interval resembling catarrhal infiltrates in humans. The nodular corneal infiltrates were found in a subepithelial location and were composed of vessels, polymorphonuclear leukocytes and mononuclear cells, including lymphocytes, plasma cells, and macrophages. The peripheral corneal infiltrates separated from the limbus by a lucid interval were found in the anterior stroma beneath the corneal epithelium and were composed of polymorphonuclear leukocytes and mononuclear cells.
