Mitotic stability of transforming DNA is determined by its chromosomal configuration in the fungus Cochliobolus heterostrophus

Summary Cochliobolus heterostrophus was transformed with a plasmid (pH1S) containing a bacterial gene (hygB), which confers resistance to the antibiotic hygromycin B when under control of an 838-bp fragment of promoter 1 from C. heterostrophus. The plasmid integrated at either homologous (52% single copy, 33% tandemly repeated copies) or ectopic (4% single copy, 11% tandemly repeated copies) sites on different chromosomes, resulting in four distinct configurations of integrated DNA. All four configurations were highly stable during mitotic growth; virtually no loss of integrated DNA was detected after five subcultures on nonselective medium or after seven cycles of pathogenesis on maize, the normal host of this fungus. However, deletion of integrated DNA was detected after eight or more disease cycles. The frequency of deletion depended on the configuration of the recombinant chromosome. A single copy of pH1S integrated at an homologous site was flanked by direct repeats of the target sequence and was least stable; up to 50% of the population lacked integrated DNA after 12 disease cycles. A single copy integrated at an ectopic site had no repeated DNA directly associated with it and was the most stable; no deletions were detected after 12 disease cycles. Tandemly repeated copies of pH1S integrated at either homologous or ectopic sites appeared to have intermediate stability; 2–18% of each population lost at least one copy after 12 disease cycles, although in no case were all copies deleted. Cytosine residues of integrated DNA were methylated during mitotic growth, but this had no apparent effect on the expression of hygB.