Cantharidin and calyculin A, natural toxins that are inhibitors of protein phosphatases 1 and 2A (PPI and PP2A, respectively). inhibit Neurospora crassa hyphal growth. When N. crassa was grown in the presence of either drug, abnormalities were observed at hyphal tips. In addition, both drugs induced an increase in hyphal branching. Cantharidin inhibited N. crassa hyphal growth in a temperature-dependent manner, as the effect of the drug was more pronounced at 34 degrees C than at 25 degrees C. In addition to the drug-mediated inhibition of phosphatase activity, a genetic approach was used to determine the phenotypic consequences of reduced PP2A activity. Two strains with subnormal PP2A activity were constructed. The first, in which the original pph-1 gene (encoding the PP2A catalytic subunit) was replaced with an ectopically integrated copy of pph-1, exhibited lower levels of pph-1 transcript, lower PP2A activity and increased sensitivity to cantharidin. Similarly, in a second strain, in which the pph-1 gene was cloned in an antisense orientation downstream of the inducible isocitrate lyase promoter, lower levels of pph-1 transcript, as well as of PP2A activity, and a reduction in hyphal growth were observed. The results of this study indicate that PP2A, and probably other Ser/Thr phosphatases, are involved in the regulation of hyphal growth in N. crassa.

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Methylation of specific cytosines in the DNA is generally believed to play some role in the regulation of gene expression in eukaryotes. However, some eukaryotes, such as Drosophila and yeast (S. Hattman, personal communication) seem not to contain 5-methylcytosine in their DNA. It would be interesting to test, how gene expression in such organisms would respond to the methylation of specific cytosines in the genome. As a first step towards this goal, we have introduced the gene encoding the Bacillus sphaericus R modification methylase, which methylates the internal cytosine within the recognition sequence 5'-GGCC, into yeast cells. Southern-type hybridization to DNAs isolated from the transformed yeast clones revealed that the yeast plasmid carrying the prokaryotic methylase gene, as well as the two chromosomal genes tested (his3 and leu2) were methylated, whereas the bulk of the yeast DNA remained largely unmethylated. This indicates that the Bacillus sphaericus modification methylase was expressed in yeast but it modified only certain parts of the yeast DNA.