Maria Helena Juliani

Developmental changes in translatable RNA species and protein synthesis during sporulation in the aquatic fungus Blastocladiella emersonii

Protein synthesis during sporulation in Blastocladiella emersonii is developmentally regulated as revealed using [35S]methionine pulse labeling and two-dimensional gel electrophoresis. A large increase in the synthesis of several proteins is associated with particular stages. A large number of basic proteins are synthesized exclusively during late sporulation. Changes in translatable mRNA species were also detected by two-dimensional gel electrophoresis of the polypeptides produced in a cell-free rabbit reticulocyte lysate primed with RNA prepared at different stages of sporulation. The synthesis of several proteins during sporulation seems to be transcriptionally controlled. Most of the sporulation-specific messages are not present in the mature zoospores.

Increased phosphorylation of a ribosomal protein during aggregation of the slime mold Dictyostelium discoideum

A single ribosomal protein (M r 32 000) becomes phosphorylated during differentiation of Dictyostelium discoideum amoebae. This protein is tentatively identified as the 40 S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two‐dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in growing cells. Upon starvation, S6 is progressively phosphorylated, the degree of phosphorylation being maximal during the aggregation phase of the developmental cycle.

Induction of ϱ− mutants in Saccharomyces cerevisiae by guanidine hydrochloride: I. Genetic analysis

Guanidine hydrochloride (GuHCl) induced in Saccharomyces cerevisiae cytoplasmic petite mutants (rho-) of the suppressive type. However, it was unable to induce the neutral type, even after prolonged incubation or increased drug concentration. No correlation was found between the degree of suppressiveness and the time of incubation of yeast cells with guanidine hydrochloride. The suppressiveness of rho- induced was not altered by further treatment with GuHCl, whereas it was reduced upon treatment with ethidium bromide (EtBr). Some mitochondrial genetic information was lacking in the rho- mutants induced by GuHCl, as demonstrated by the loss of the gene for erythromycin resistance and by reduced buoyant density of mitochondrial DNA of some rho-. There was no correlation between the degree of suppressiveness of the rho- induced by GuHCl and the buoyant density of the mutant mitochondrial DNA.

Non-chromosomal respiratory deficient mutants induced by guanidine hydrochloride in Saccharomycescerevisiae

Abstract Non-chromosomal petites can be produced in Saccharomyces cerevisiae by treatment with guanidine hydrochloride, a protein denaturing agent. Its efficiency in inducing petite mutants is comparable to the action of ethidium bromide. The high frequency of petite mutants observed is due to an induction effect rather than to a selection of preexisting mutants. Induction of petites by guanidine hydrochloride occurs even in non growing conditions, indicating that even parental cells are transformed in petites. Transformation depends upon the physiological properties of the cells, since repressed cells, cultivated in the presence of glucose, are more easily transformed than cells cultivated in ethanol.

Changes in cyclic AMP binding and protein kinase activities during growth and differentiation of Blastocladiella emersonii

Multiple protein kinases in the water mould Blastocladiella emersonii are described. A cyclic AMP-independent protein kinase which prefentially phosphorylates casein remains unchanged during vegetative growth of the cells and in the two phases of differentiation: germination and sporulation. In contrast, cyclic AMP-dependent protein kinase activity and cyclic AMP binding components are induced during the sporulation.

Regulation of tubulin and actin synthesis and accumulation during Blastocladiella emersonii development

Actin and alpha and beta-tubulin have been identified in Blastocladiella emersonii by two-dimensional gel electrophoresis and Western blotting. The kinetics of synthesis of these proteins were compared by pulse-labeling experiments with [35S]methionine and with the accumulation of their corresponding mRNAs, translated in a cell-free system. Large increases occur in the rates of actin and alpha- and beta-tubulin biosynthesis during sporulation and there is an accumulation of the corresponding mRNAs. In parallel to the increased synthesis, these cytoskeletal proteins accumulate during the late stage of sporulation.

Induction of ϱ− mutants in Saccharomyces cerevisiae by guanidine hydrochloride II. Conditions that prevent ϱ− induction

Abstract The induction of ϱ − “petite” mutants by guanidine hydrochloride (GuHCl) is inhibited in several conditions. Anaerobiosis inhibited the induction either with or without cell multiplication. Both nalidixic acid (NA) and cycloheximide (CH) inhibited the induction of mutants. On the other hand, chloramphenicol (CAP) produced a dual effect: at low concentration it stimulated, at high concentration it inhibited, the induction. The effect of these different inhibitors on the transformation of ϱ + mother cells into ϱ − by GuHCl is discussed.

Induction of cytoplasmic petite in yeast by guanidine hydrochloride: Combined treatment with other inducing agents

We have studied the induction of rho- mutants by guanidine hydrochloride (GuHCl) in combination with other known inducers: ethidium bromide (EB), berenil and ultraviolet light. Competition was observed when cells were simultaneously treated with optimal concentrations of EB and GuHCl; on the other hand, treatment of cells with EB in the presence of non-inducing concentrations of GuHCl resulted in the stimulation of rho- induction of EB. Furthermore, using a strain which upon treatment with high EB concentrations shows recovery of respiratory competence, the presence of GuHCl did not interfere either with the early phase of induction or with the recovery phase, but it did interfere in a competitive fashion with the final irreversible phase of EB induction. In the case of berenil, a synergistic effect was seen when cells were pretreated with GuHCl. A synergistic induction was also observed when cells were submitted to UV prior to GuHCl treatment. These results suggest that GuHCl, EB and berenil act via some common step in their rho- induction pathways. Moreover, GuHCl may somehow be decreasing the efficiency of dark repair of ultraviolet lesions on mitochondrial DNA.

Effect of heat shock on S6 phosphorylation during the development of Blastocladiella emersonii

SummaryChanges in phosphorylation of ribosomal protein S6 during heat shock, induction of thermotolerance and recovery from heat shock at different stages of Blastocladiella emersonii development were investigated. Independently of the initial state of S6 phosphorylation (maximal or intermediate), a rapid and complete dephosphorylation of S6 is induced by heat shock and S6 remains unphosphorylated during the acquired thermotolerance. During recovery from heat shock rephosphorylation of S6 occurs always to the levels characteristic of that particular stage, coincidently with the turn off of heat shock protein synthesis.