POLYAMINES, DNA METHYLATION, AND FUNGAL DIFFERENTIATION

Mucorales constitute a group of fungi that, because of their growth characteristics, have been used extensively in the study of cell differentiation, cell morphogenesis, and stimuli perception. We have studied the role of polyamine metabolism in the development of different Mucorales, with emphasis on Mucor and Phycomyces species. It has been observed that previous to each differentiative step, the cellular levels of the most regulated enzyme of the pathway, ornithine decarboxylase (ODC), and polyamines suffer a noticeable increase. Addition of diaminobutanone (DAB), a competitive inhibitor of ODC, blocks all the corresponding differentiative phenomena. In its presence, germinating spores fail to produce germ tubes and keep growing isodiametrically; mycelia do not sporulate but continue their vegetative growth, and yeast cells are unable to engage in a dimorphic transition without alterations in their growth rate. This differential effect of the ODC inhibitor in growth and development is apparently due to the location of ODC in at least two different cell compartments, one of which is impermeable to the drug. Inhibition of development is counteracted by putrescine and more noticeably by 5-azacytidine (5AC), a strong inhibitor of DNA methylation. Methylation levels of DNA are high in spores, and they become reduced after germination. Demethylation is inhibited by hydroxyurea, which blocks DNA replication, and by DAB. The effect of the latter is reversed by 5AC. These results suggest a relationship between polyamines and DNA methylation. Analysis of metallothioneine gene (CUP) behavior and expression during spore germination has confirmed this hypothesis. The gene is methylated and not transcribed in the spore, and becomes demethylated and transcriptionally active after germination. Both processes, demethylation and transcription, are inhibited by DAB. The hypothesis has been forwarded that in Mucorales (and perhaps in other fungi), polyamine levels regulate gene expression during development, affecting DNA methylation by an as yet unknown mechanism.