Pier Paolo Puglisi

FOG1 and FOG2 genes, required for the transcriptional activation of glucose-repressible genes of Kluyveromyces lactis, are homologous to GAL83 and SNF1 of Saccharomyces cerevisiae

Abstract The fog1 and fog2 mutants of the yeast Kluyveromyces lactis were identified by inability to grow on a number of both fermentable and non-fermentable carbon sources. Genetic and physiological evidences suggest a role for FOG1 and FOG2 in the regulation of glucose-repressible gene expression in response to a glucose limitation. The regulatory effect appears to be at the transcriptional level, at least for β-galactosidase. Both genes have been cloned by complementation and sequenced. FOG1 is a unique gene homologous to GAL83, SIP1 and SIP2, a family of regulatory genes affecting glucose repression of the GAL system in Saccharomyces cerevisiae. However, major differences exist between fog1 and gal83 mutants. FOG2 is structurally and functionally homologous to SNF1 of S. cerevisiae and shares with SNF1 a role also in sporulation.

Induction of respiratory-deficient mutants in a “petite negative” yeast species Kluyveromyces lactis with N-methyl-N′-nitro-N-nitrosoguanidine

Summary Thirty-nine nitrosoguanidine-induced respiratory-deficient mutants of Kluyveromyces lactis strain CBS 2359 were isolated. These mutants do not grow on glycerol and have a lower respiration rate associated with a reduced capacity to react with tetrazolium salts. Mitochondria isolated from these mutants are altered in the oxidation of several substrates of the respiratory chain. From the spectroscopic analysis of the cytochromes it is possible to distinguish four groups of mutants in which: first group) all cytochromes are missing; second group) only cytochrome a is missing; third group) cytochromes a , b , c 1 are missing; fourth group) no cytochrome is absent. Tetrade analyses show that the respiratory-deficient mutants are of chromosomal type. The existence of these mutants demonstrates that the failure in obtaining cytoplasmic respiratory-deficient mutants in Kluyveromyces lactis does not depend on the lethality of the alteration of the respiratory chain.

Mutagenic and antimutagenic effects of acridinium salts in yeast.

Abstract Acridines and their methyl derivatives at the ring nitrogen are mutagenic during meiosis, and antimutagenic during mitosis in the yeast Saccharomyces cerevisiae . The biological activity is strongly dependent upon the physio-chemical conditions of the experiments.

Role of the mitochondrial protein synthesis in the catabolite repression of the petite-negative yeast K. lactis

Abstract The effect of glucose in two different strains of the petite-negative yeast K. lactis is studied. The results obtained show that one strain ( K. lactis CBS 2359 ) is glucose repressible for Glutamate Dehydrogenase and β-Galactosidase, whereas the other one (CBS 2360) is almost completely insensitive. The effect of Erythromycin on expression of catabolite repression in CBS 2359 is also analyzed. The results show that the dependence of catabolite repression on mitochondrial protein synthesis reflect the degree of interaction between the nuclear and mitochondrial compartments.

Identification of chloroplast associated heat-shock proteins in Nicotiana plumbaginifolia protoplasts

SummaryNicotiana plumbaginifolia protoplasts grown at 25 °C and transferred at 40 °C synthesized new temperature-dependent polypeptides called heat-shock proteins (hsp) distributed in a wide range of molecular weight. The cellular localization of hsp showed a concentration of the low molecular weight hsp in a fraction enriched in membranes and chloroplasts. Purified chloroplasts obtained by a Percoll cushion and by a discontinuous sucrose density gradient centrifugation, were therefore analyzed for hsp distribution. Several hsp were found to be associated with this subcellular compartment: in particular three hsp were associated with the soluble fraction of the chloroplasts, whereas six hsp were associated with organelle membranes. The involvment of chloroplast information in the synthesis of some of these organelle associated hsp and therefore in the response to heat-shock is discussed.

Antimutagenic activity of actinomycin D and basic fuchsine in Saccharomyces cerevisiae

SummaryThe antimutagenic activity, i.e. the capacity to decrease the spontaneous mutation rate, of actinomycin and basic fuchsine was investigated. It was found that the minimal requirement for the antimutagenic activity of compounds forming complexes with DNA is their binding to the polynucleotides rather than their stabilizing them against thermal denaturation. It is proposed that these antimutagens act through an inhibition of the exonucleolytic removal of bases from DNA during transcription.

Dependence of cytoplasmic on mitochondrial protein synthesis inK. lactis CBS 2360

SummaryIn the “petite negative” yeastKluyveromyces lactis (strain CBS 2360) growth is inhibited by the same Erythromycin concentrations on fermentable and non-fermentable carbon sources. The minimal inhibitory concentration of Erythromycin does not affect respiration, but completely inhibits total aminoacid incorporation by the cells in conditions in which permeability effects can be ruled out.The effect of the antibiotic on the “in vitro” protein synthesis by cytoplasmic and mitochondrial systems shows that inhibition is effective only at the mitochondrial level.Results suggest that the inhibition of growth and of total protein synthesis is a consequence of the inhibition of mitochondrial protein synthesis.

Erythromycin and cycloheximide sensitivities of protein and RNA Synthesis in sporulating cells of Saccharomyces cerevisiae: Environmentally induced modifications controlled by chromosomal and mitochondrial genes.

SummaryThe purpose of the experiments reported below was to examine the response in sporulation medium of the three diploid cell types MATα MATα, MATα MATα (asporogenic diploids) and MATα MATα (sporogenic diploid) to erythromycin, a specific inhibitor of mitochondrial protein synthesis (MPS) in vegetative cultures, and cycloheximide, a specific inhibitor of cytosol protein synthesis (CPS) in vegetative cultures. When MATα MATα diploids are transferred to sporulation medium a significant fraction of total protein synthesis (CPS + MPS) becomes sensitive to erythromycin in contrast to the behavior of MATa MATa and MATα MATα diploids in which the resistance of CPS to erythromycin is maintained. The decompartmentalization of erythromycin sensitivity is thus cell type specific. Erythromycin stimulates total RNA synthesis of MATα MATα cells in sporulation medium but not of MATα MATα and MATα MATα cells. Cycloheximide inhibits protein synthesis and stimulates RNA synthesis in all three diploid cell types. An erythromycin resistant mutant, shown to be due to a mutation of the mitochondrial genome, exhibited only partial resistance of CPS to erythromycin in sporulation medium in the background of the MATα MATα mating type genotype. Total RNA synthesis in this mutant was not stimulated. The results reported indicate that mitochondrial functions during sporulation are not restricted to those involving respiratory metabolism.

Effects of temperature on nucleo-mitochondrial interactions in the yeast Saccharomyces cerevisiae

Abstract The temperature, as well as several antibacterial antibiotics could be used to differentiate mitochondrial protein synthesis (MPS) from the cytoplasmic (CPS) one in the yeast Saccharomyces cerevisiae . In fact MPS and CPS have respectively the optimum at 30°C and 36°C. A series of cellular processes, as the mitotic reproduction in presence of non-fermentable carbon sources, the synthesis of galactose pathway enzymes and the meiotic process have the same optimal temperature (30°C), whereas the growth of the wild type in presence of fermentable carbon sources and of a galactose repressor constitutive mutant (i-) have the optimal temperature of 36°C, in agreement with our previous hypothesis in which the expression of some sections of the nuclear genetic complement is dependent on regulatory functions controlled by MPS.

Effect of chloramphenicol, antimycin A and hydroxamate on the morphogenetic development of the dimorphic ascomycete Endomycopsis capsularis

Mitochondrial protein synthesis, primary (antimycin-sensitive) respiration and secondary (antimycin-insensitive, salicyl-hydroxamate-sensitive) respiration, have been characterized in the dimorphic yeastEndomycopsis capsularis.The inhibition by chloramphenicol (CAP) of the morphogenetic development from the yeast-like form to the mycelial structure in this yeast could represent the intervention in the morphogenetic process of mitochondrial protein synthesis, since chloramphenicol blocks in vivo and in vitro mitochondrial protein synthesis. In fact, other functions such as primary and secondary respiration, do not seem to play a role in the morphogenetic development since their inhibition by antimycin A (AA) or by salicyl-hydroxamic acid (SHAM) does not affect the process. In addition, mitochondrial protein synthesis has been shown to be uninhibited by the two respiratory inhibitors.