Dolores Gutiérrez, Amalia Vázquez, Miguel Sánchez, Victor J. Cid, César Nombela y Alberto M. Álvarez*.

Dpto. de Microbiología II, Fac. Farmacia, *Centro de Citometría de Flujo, U.C.M.

Flow Cytometry was used to characterise phenotipically cell cycle mutants from parental strain D1 (A/, ura3/ura3, leu2/leu2, trpl, his1, his3). We selected two mutants denominated 918 and 327. We knew that these mutants are bigger than D1 and witj abnormal budding formation process. Several fluorochromes were used in order to determine total proteins, total lipids, chitin, cellular activation, viability, peroxidase activity and anion superoxide.

          FITC    Rhl23   PRIMULI   RED    DCFH    Drh 12     PI      HE
                             N      NILE             3
TARGET   protein mitochond chitin lipids   pero     H2O2    death     O2
                 potential                  act             cells
EXCIT.     488    488       488     488     488      488     488      488
(nrn)
EMIS.      530    530       530     530-    530      530     630      630
(nm)                                600

These mutants have the feature of being thermosensible, so when they grow at 37º they express the mutant phenotype and die. Some of them (Lyt1) only show phenotype differences when they are at 37º, meanwhile others 918 and 327, show differences even at permissive temperature. Lyt1 has no differences compared with parental strain at permissive temperature. When grown at 37º its peroxidases increase their activity and after 2 hours the number of cells PI+ is about 40º. After 4 hours between 60-80% of cells are PI+ and PI- cells have a higher peroxidase activity than parental strain.

918 and 327 have more protein and chitin than parental cells at permissive temperature, though the striking differences are found in peroxidase activity. Their size is higher than parental strain and the shape is totally different. When they are incubated at 37º, the malformations are more evident and in the 918 mutant the peroxidase activity is up to 10 times increasing compared to parental strain. 327 mutant has lower increase. PI+ cells appeared as soon as 30' of incubation at 37º. After 4 hours the PI+ cells are 90% aprox.