Disruption, replacement, and cosuppression of nitrate assimilation genes in Stagonospora nodorum.

We used Stagonospora (Septoria) nodorum to explore gene disruption as a general method of fungicide target validation. Nitrate reductase was chosen as a model target because the gene (NIA1) has been cloned from S. nodorum and disruptants should have a readily detectable phenotype (chlorate resistant and nitrate nonutilizing). We have succeeded in disrupting the NIA1 gene by both integration of an unselected vector during cotransformation and one-step gene replacement. Around 2% of transformants from the cotransformation approach became nitrate nonutilizing and Southern analysis confirmed disruption of the resident NIA1 gene. Half of the transformants with the gene replacement vector showed the nitrate nonutilizing phenotype expected from disruption. However, Southern analyses of 14 of these transformants showed that only 6 contained the expected NIA1 gene replacement. Of the remaining transformants, 6 had integrated multiple copies of the vector elsewhere in their genome and still had a functional nitrate reductase gene. Their inability to utilize nitrate was due to a lack of nitrite reductase activity. How this phenotype arose is not clear, but it might involve cosuppression of the nitrite reductase gene as the vector carried 1. 1 kb of the coding region and the complete 5' region of this gene which is adjacent to NIA1. Mutants of both types retained full pathogenicity in detached leaf assays, thereby invalidating both nitrate and nitrite reductase as fungicide targets.

Cloning of the nitrate reductase gene of Stagonospora (Septoria) nodorum and its use as a selectable marker for targeted transformation.

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.

Molecular analysis of the Septoria nodorum beta-tubulin gene and characterization of a benomyl-resistance mutation.

The complete nucleotide sequence of a benomyl-resistant allele of the Septoria nodorum beta-tubulin gene (tubAR) has been determined including 745 and 1024 nucleotides 5' and 3' to the tubAR coding region, respectively. tubAR encodes a 447 amino acid polypeptide which shows a high degree of homology with other fungal beta-tubulins. The gene contains three introns at codons 4, 12 and 53, uses 48 of the possible 61 sense codons and has a GC content of 59.1% in its coding region. S1 nuclease mapping has identified two transcriptional start sites 80 bp and 83 bp upstream of the translation start, and a transcriptional termination site 192 bp downstream of the stop codon. The two transcriptional start sites lie just 8 bp and 5 bp downstream of a CT motif consisting of 18 pyrimidine nucleotides interrupted by a single adenine. The wild-type allele tubA+ has been cloned using the polymerase chain reaction and the mutation producing the benomyl-resistant phenotype of tubAR mapped to a C to T transition at the first position of codon 6, resulting in a histidine to tyrosine amino acid substitution.

Variation in electrophoretic karyotype between strains of Septoria nodorum.

The electrophoretic karyotype of 11 strains of the phytopathogenic fungus Septoria nodorum has been established by pulsed field gel electrophoresis with the CHEF DRII system. Each strain had a similar overall karyotype with 14-19 chromosomes being resolved in the size interval between approximately 0.5 and 3.5 megabase pairs (Mb). However, there were clear differences in karyotype both between and within groups of strains adapted to wheat or to barley. Considerable karyotype variation was apparent even among 6 wheat-adapted strains isolated from the same population. Only 2 strains possessed identical karyotypes; these were isolated from the same leaf and were heterokaryon-compatible and are probably independent isolates of the same clone.

Effect of therapeutic radiation on peripheral blood lymphocytes in patients with carcinoma of the breast.

Effects of radiation therapy for carcinoma of the breast on responsiveness to mitogens of blood lymphocytes of patients at different clinical stages were analysed. Patients at different clinical stages had comparable numbers and responses of lymphocytes. During therapy the mean lymphocyte numbers decreased to 31 per cent of the pre-treatment value and mean responses to mitogens in whole-blood cultures decreased to 15 to 39 per cent of pre-treatment values. Similar decreases occurred whether or not mastectomy was performed.