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1Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain
2Centro de Citometría de Flujo y Microscopía Confocal, Universidad Complutense, 28040-Madrid, Spain
3Department of Biochemistry, Comenius University, Faculty of Natural Sciences, Mlynská dolina CH-1, 842 15 Bratislava, Slovakia
ABSTRACT
Summary: The non-sporulating diploid strain V327 of Saccharomyces cerevisiae was previously isolated in a search for thermosensitive autolytic mutants. This strain is very efficient at releasing intracellular proteins into the medium when incubated at high temperatures. The expression of this lytic phenotype depends on a morphogenetic defect, consisting of the appearance of elongated chains of cells. Transmission electron microscopy revealed a mislocalization of septa at semi-permissive temperatures and a total lack of septation together with abnormal cell wall architecture at a non-permissive temperature. The septin-encoding CDC10 gene was cloned by complementation of the pleiotropic phenotype of the V327 mutant. Rescue and sequencing of CDC10 alleles from V327 revealed a point mutation that created a single amino acid change in a region which is well conserved among septins. This new allele was named cdc10-11. The construction of a cdc10-11 haploid strain by substituting the CDC10 gene with the rescued allele permitted further genetic analyses of the mutation and allowed the construction of new homozygous cdc10-11 diploid strains that showed a reduced ability to sporulate. Fusing both the wild-type and the cdc10-11 alleles to green fluorescent protein (GFP) demonstrated that the mutation does not affect the localization of this septin to the bud neck at the standard growth temperature of 24 °C, although the morphogenetic phenotype at 37 °C parallels the disappearance of Cdc10-GFP at the ring encircling the septum area.
Author for correspondence: Miguel Sanchez. Tel: +34 1 3941746. Fax: +34 1 3941745. e-mail: misanper@eucmax.sim.ucm.es
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