Opa proteins of Neisseria meningitidis exhibit translational phase variation via addition or deletion of repetitive coding repeat units within the DNA encoding the protein leader sequence. In contrast, Opc phase variation is the result of transcriptional regulation. Transcription starts 13 nucleotides after the -10 region of an unusual promoter sequence containing a variable number of contiguous cytidine residues and lacking a -35 region. Efficient expression of Opc occurred in strains with 12 to 13 cytidine residues, intermediate expression in strains with 11 or 14 residues and no expression with < or = 10 or > or = 15 residues. This unusual regulation may have evolved because the Opc protein enables meningococcal invasion and is immunogenic.

A plasmid construct carrying the hygromycin phosphotransferase (hph) gene fused to the expression elements of the trpC gene of Aspergillus nidulans was used to obtain hygromycin B (Hyg)-resistant transformants of Neurospora crassa. The plasmid does not have any homology with the N. crassa genome. Here we demonstrate that most of the transformants arise from integration of the transforming DNA into only one of the nuclei present in the protoplasts. Furthermore, in most of the transformants the integrated transforming DNA is physically stable after growth of the transformants for about 25 nuclear divisions without Hyg selection, in spite of being present in multiple copies. In transformants carrying only a single insertion, phenotypic expression of the hph gene remains unaltered in conidial isolates obtained without Hyg selection. On the other hand, about 40% of transformants harbouring plasmid DNA integrated at more than one location yield conidial isolates showing reversible inactivation of the hph genes. Interestingly, the presence of methylated cytosine residues in the integrated DNA is strongly correlated with the number of plasmid copies. The hph genes are heavily methylated in transformants harbouring multiple copies but not in those harbouring only one copy of the plasmid. Phenotypic expression of the inactive hph genes can be restored by growing the transformants either under Hyg selection pressure or in the presence of 5-azacytidine. In the first case the hph genes are again inactivated when Hyg selection pressure is removed, while the activation of the hph gene by 5-azacytidine gives stable Hygr strains.

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Simple methods for crossing and genetic analysis of Neurospora crassa strains Simple methods for crossing and genetic analysis of Neurospora crassa strains