Biomedical subjects

E Martegani

Publications and source records attributed to E Martegani.

At least 55 records · Page 3Linked to original sources

PubMed1991

Efficient production of recombinant DNA proteins in Saccharomyces cerevisiae by controlled high-cell-density fermentation.

High levels of expression of heterologous proteins (from 5 to 15% of total cell proteins) in the budding yeast Saccharomyces cerevisiae have been obtained previously by the use of the inducible strong hybrid promoter UASGAL/CYC1, in batch as well in continuous cultures. However, in order to maximize the yield of heterologous proteins, a computer controlled fed-batch fermentation is essential. For this reason we have developed a fed-batch system based on a semiconductor gas detector that measures ethanol in the outflow gases. The optimal conditions are described for very high production (up to 1550 mg/liter), with both high productivity (up to 100-120 mg/liter/h) and high yield (up to 15 mg of protein/g of dry biomass), of heterologous protein driven by the UASGAL/CYC1 promoter in a completely computer controlled fed-batch fermentation of budding yeast. However, high production was dependent upon the addition of a large amount of galactose. The process was improved by developing a new, more easily inducible, vector system obtained by subcloning the GAL4 gene.

Biotechnology↗

PubMed1990

The glucose-induced polyphosphoinositides turnover in Saccharomyces cerevisiae is not dependent on the CDC25-RAS mediated signal transduction pathway.

Recently the polyphosphoinositides (PI) turnover has been related to the control of growth and cell cycle also in Saccharomyces cerevisiae, and the RAS2 and RAS1 gene products have been shown to be involved in the stimulation of PI turnover in G0/G1 arrested yeast cells. Here we show that addition of glucose to previously glucose-starved cells, stimulates, the PI turnover with fast kinetics also in yeast cells that were not arrested in the G0/G1 phase of the cell cycle. In addition PI turnover is equally stimulated in temperature sensitive cdc25-1 and cdc25-5 strains at restrictive temperature, as well as in ras1, ras2-ts strain, suggesting that PI turnover stimulation is not dependent on the CDC25-RAS mediated signal transduction pathway.

Cell Cycle Proteins↗

PubMed1990

Overexpression of the CDC25 gene, an upstream element of the RAS/adenylyl cyclase pathway in Saccharomyces cerevisiae, allows immunological identification and characterization of its gene product.

The product of the START gene CDC25, an upstream element of the RAS/adenylyl cyclase pathway in Saccharomyces cerevisiae, was identified using specific antibodies raised against a chimeric beta-galactosidase/CDC25 protein. The CDC25 protein is poorly expressed and can be detected only when the CDC25 gene is overexpressed under the control of the galactose-inducible GAL1-10 strong promoter elements. It has a molecular weight of 180,000, is not glycosylated and is strongly associated with the particulate fraction. After deletion of residues 1255-1550 the protein is found in the soluble fraction.

Adenylyl Cyclases↗

PubMed1989

Secretion of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae using the signal sequence from the glucoamylase-encoding STA2 gene.

The budding yeast Saccharomyces cerevisiae is a safe and widely used host for the production of recombinant DNA-derived proteins. We have used the signal sequence from the S. diastaticus STA2 gene, encoding glucoamylase II, to secrete Escherichia coli beta-galactosidase, encoded by the lacZ gene. In frame STA2/lacZ gene fusions have been constructed and expressed in S. cerevisiae under the control of either the STA2 or the galactose inducible GAL1-10 upstream promoters. Fairly high amounts of the enzyme (up to 76% of total activity, depending on the growth conditions) are secreted in the periplasmic space. Adding yeast extract and peptone to the growth medium results in a dramatic increase in both synthesis and secretion of beta-galactosidase.

Amino Acid Sequence↗

PubMed1989

Kinetics and regulation of tyrosine phosphorylation of the platelet derived growth factor receptor.

Platelet derived growth factor (PDGF) interaction with the cells induces rapid tyrosine phosphorylation of the PDGF receptor in a dose dependent manner. At 37 degrees C phosphorylation of the receptor is followed by its dephosphorylation and internalization. It is observed that the higher the ligand concentration, the more transient is the response, and the observed kinetics are explained by a simple kinetic model. At 4 degrees C the phosphorylated form of the receptor is more stable; however, if PDGF is dissociated from the cell surface-associated ligand-receptor complexes, the receptors are rapidly dephosphorylated, indicating that phosphatases specific for phosphotyrosine groups are very active within the cells. In fact, addition of orthovanadate stabilizes the phosphorylated form of the receptor and helps in recognizing possible physiological substrates of the PDGF receptor kinase. The expression of PDGF receptors on the cell surface has been investigated under different growth conditions: a positive correlation exists between the amount of PDGF receptors and the duplication times of exponentially growing cultures. Moreover, during exponential growth the PDGF receptors are scarcely expressed, and their number increases reaching a maximal value when the population enters the stationary phase.

Animals↗

PubMed1989

Yeast 2 micron vectors replicate and undergo recombination in Torulaspora delbrueckii.

In order to develop a procedure for transformation of the industrial yeast Torulaspora delbrueckii, we have constructed a set of recombinant plasmids carrying Saccharomyces cerevisiae ARS and 2 microns origin of replication and kanamycin-G418 resistance gene of Tn903(601) as a selective marker. In this paper we show that S. cerevisiae ARS vectors can replicate autonomously and that vectors bearing the whole S. cerevisiae 2 microns sequence yield stable transformants. We also present evidence to show that 2 microns vectors undergo an FLP-mediated inter- and intramolecular recombination, which suggests that T. delbrueckii can support the amplification and partition mechanisms of these plasmids.

Blotting, Southern↗

PubMed1989

Cell size modulation by CDC25 and RAS2 genes in Saccharomyces cerevisiae.

A detailed kinetic analysis of the cell cycle of cdc25-1, RAS2Val-19, or cdc25-1/RAS2Val-19 mutants during exponential growth is presented. At the permissive temperature (24 degrees C), cdc25-1 cells show a longer G1/unbudded phase of the cell cycle and have a smaller critical cell size required for budding without changing the growth rate in comparison to an isogenic wild type. The RAS2Val-19 mutation efficiently suppresses the ts growth defect of the cdc25-1 mutant at 36 degrees C and the increase of G1 phase at 24 degrees C. Moreover, it causes a marked increase of the critical cell mass required to enter into a new cell division cycle compared with that of the wild type. Since the critical cell mass is physiologically modulated by nutritional conditions, we have also studied the behavior of these mutants in different media. The increase in cell size caused by the RAS2Val-19 mutation is evident in all tested growth conditions, while the effect of cdc25-1 is apparently more pronounced in rich culture media. CDC25 and RAS2 gene products have been showed to control cell growth by regulating the cyclic AMP metabolic pathway. Experimental evidence reported herein suggests that the modulation of the critical cell size by CDC25 and RAS2 may involve adenylate cyclase.

Adenylyl Cyclases↗

PubMed1989

Identification of proteins cross-reactive to phosphotyrosine antibodies and of a tyrosine kinase activity in boar spermatozoa.

We used two different anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins in a germ cell, the boar spermatozoon. Ejaculated spermatozoa presented three major polypeptides, of Mr 43,000, 40,000 and 36,000, respectively, that were immunorecognized on Western blots. These proteins were selectively enriched in the Triton X-100-soluble fraction and were released neither after an A23187-induced acrosome reaction nor after sperm homogenization. These findings suggest the presence of the three proteins in plasma membrane regions not involved in the acrosomal vesiculation. When epididymal boar spermatozoa were investigated, Western blot analysis of the detergent-soluble fractions from caput sperm did not reveal any detectable 43,000, 40,000 and 36,000 Mr proteins cross-reactive with phosphotyrosine antibodies, whereas the detergent-soluble fractions from cauda sperm yielded very strong immunoreactive signals. Labelling of freshly ejaculated spermatozoa with [32P]orthophosphate yielded a wide range of labelled phosphoproteins, but we failed to identify specific tyrosine phosphorylation under the experimental conditions employed. Tyrosine phosphorylation occurred when specific synthetic polymers of tyrosine, commonly used for studying tyrosine protein kinases, were assayed as substrates against both the Triton-soluble and Triton-insoluble sperm fractions. This is the first immunological and biochemical report on the presence of phosphotyrosine-containing proteins and protein kinase activities that phosphorylate tyrosine residues in a mammalian mature spermatozoon.

Amino Acid Sequence↗

PubMed1987

Translational regulation of the expression of zein cloned in yeast under an inducible GAL promoter.

Yeast cells transformed by a plasmid containing a zein sequence fused to an hybrid yeast promoter GAL1-10/CYC1 accumulate, during a batch growth in galactose minimal medium, large amounts of zein only during a growth-limited phase that precede the entering into the stationary phase. We found that zein is fairly stable in yeast cells and the increased accumulation of zein polypeptide depends mainly upon a marked increase of its rate of synthesis. The increase of the rate of heterologous protein synthesis is not dependent on variation in the plasmid copy number and it is not related to the relative level of zein mRNA, indicating the existence of a postranscriptional regulation that modulates the translatability of this messenger RNA in function of the growth conditions. A possible explanation of this modulation is discussed in terms of a codon bias effect that slow-down the translation of heterologous mRNAs during the exponential phase of growth.

Autoradiography↗

PubMed1986

Biochemical studies on proacrosin and acrosin from epididymal boar spermatozoa: in vitro translation of boar testicular proacrosin mRNA.

An inactive form of acrosin was extracted from epididymal boar spermatozoa utilizing acid pH conditions. When subjected to activation in alkaline environment, this form turns into an enzymatically active species, which exhibits close-related electrophoretic characteristics. Both the precursor and the activated species, when incubated in the presence of thermolysin, give rise to two fastly moving acrosin molecular forms. In order to establish the nature of the true acrosin zymogen, we isolated poly(A+)-RNA from boar testicles, performed its translation in vitro in the presence of [35S]-methionine and reticulocyte lysate, immunoprecipitated the translation products with anti-boar acrosin antibody, and analyzed them by SDS-polyacrylamide gel electrophoresis and autoradiography. A single translation product of molecular weight 55,000 was detected. It is concluded that the polypeptide chain of the boar zymogen is of 55,000; increases in molecular weight are due to post-translational modifications, like glycosylation.

Acrosin↗

PubMed1986

Transcription and expression of zein sequences in yeast under natural plant or yeast promoters.

Maize genomic fragments containing the regulatory and coding regions of a zein gene for a low size class 23-kd protein have been inserted in an interspecific Escherichia coli-Saccharomyces cerevisiae expression vector in different constructions. The presence of the inducible GAL1-10 upstream activation site (UAS) allows us to regulate differentially by carbon sources the transcription of the zein gene both under the plant promoter and under the yeast CYC-1 promoter. We found that the zein promoter region is properly recognized at the correct transcription start, while different termination points occur during transcription. The yeast UAS was also shown to function as a typical eukaryotic enhancer regardless of its distance or orientation with respect to the plant promoter. Yeast cells transformed by a plasmid containing a zein sequence fused to a short piece of the CYC-1 gene produced a fused polypeptide, of expected mol. wt, in variable amount from 0.2 to 5% depending on the growth phase conditions.

Journal Article↗

PubMed1986

Interaction of cAMP with the CDC25-mediated step in the cell cycle of budding yeast.

Addition of exogenous cAMP to cultures of the start mutant cdc25-1 of Saccharomyces cerevisiae shifted to restrictive temperature causes a partial reversion of the mutated phenotype, with a marked increase of the percentage of budded cells. This effect is coupled to a progression in the cell cycle, as demonstrated by DNA histograms obtained by flow cytometry. Moreover cdc25 cells have a high intracellular cAMP content also at restrictive temperature, and no change in the cAMP content was seen during a transition from restrictive to permissive temperature. These data suggest that CDC25 gene product allows cell proliferation by interacting with a cAMP-mediated mechanism.

Cell Cycle↗

PubMed1986

Cell cycle modelling.

Models able to describe the events of cellular growth and division and the dynamics of cell populations are useful for the understanding of functional control mechanisms and for the theoretical support for automated analysis of flow cytometric data and of cell volume distributions. This paper reports on models that we have developed with this aim for different kinds of cells. The models are composed by two subsystems: one describes the growth dynamics of RNA and protein, and the second accounts for DNA replication and cell division, and describe in a rather unitary frame the cell cycle of eukaryotic cells, like mammalian cells and yeast, and of prokaryotic cells. The model is also used to study the effects of various sources of variability on the statistical properties of cell populations, and we find that in microbial cells the main source of variability appears to be an inaccuracy of the molecular mechanism that monitors cell size. In normal mammalian cells another source of variability, that depends upon the interaction with growth factors which give competence, is apparent. An extended version of the model, which comprises also this additional variability, is presented and used to describe the properties of mammalian cell growth.

Amoeba↗

PubMed1986

The interaction with PDGF as a source of G1 phase heterogeneity in normal mouse fibroblasts.

In normal mouse fibroblasts exponentially growing at suboptimal serum concentration the population of cells having a presynthetic (G1) DNA content is heterogeneous, since there are present both competent and non-competent cells (Sturani et al, 1984). We show here that SV40 transformed 3T3 fibroblasts, which have lost growth control and serum requirement, lack the G1 non-competent phase. These findings are confirmed by the different kinetics of exit from the G1 compartment of transformed and normal cells. In addition a positive correlation is reported between the fraction of non-competent cells and the average abundance of the phosphorylable PDGF receptor on the cell surface of normal cells.

Animals↗

PubMed1986

Molecular cloning and regulation of the expression of the MET2 gene of Saccharomyces cerevisiae.

The MET2 gene of Saccharomyces cerevisiae, which codes for homoserine-O-acetyltransferase, a key enzyme in methionine biosynthesis, was isolated by complementation of a met2 mutant strain of S. cerevisiae with a yeast gene bank. A 3.9-kb genomic fragment contains the entire gene, as demonstrated by genetic and molecular analysis of the integrative transformants. A polyadenylated mRNA of 1700 nt is detected by Northern blot hybridization with a MET2 probe. The level of this mRNA decreases by addition of exogenous methionine or of S-adenosylmethionine, suggesting a transcriptional regulation. The level of specific mRNA and the enzyme activity found in transformants that bear the MET2 gene on a multicopy plasmid suggest that also a post-transcriptional regulatory mechanism may be operative in budding yeast.

Acetyltransferases↗

PubMed1984

Macromolecular syntheses in the cell cycle mutant cdc25 of budding yeast.

A major control point of the cell cycle in Saccharomyces cerevisiae is a G1 event called 'start'. At start a yeast cell integrates external and internal signals and decides to progress toward mitosis or to choose alternative pathways such as sporulation, conjugation etc. cdc25 is a class II temperature-sensitive start mutant that blocks at restrictive temperature in G1 as round unbudded cells. The arrest of the cell cycle appears to be independent of the carbon and nitrogen sources, and the cell wall of cdc25-arrested cells shows changes similar to those found in cells undergoing entry in to the stationary phase. After a shift to 36 degrees C the increase in cell number of cdc25 cultures is gradually inhibited. The nuclear division cycle appears to be inhibited immediately after the shift and the percentage of budded cells decreases, while cytoplasmic growth, monitored either as increase of adsorbance at 450 nm or as protein accumulation, continues for many hours leading to a progressive increase of mean cell volume and mean protein content per cell. The stable RNA accumulation instead is immediately inhibited and this is partially due to a 50% inhibition of ribosomal RNA synthesis, while the rate of synthesis of ds-killer RNA is relatively unaffected. These data suggest that the CDC25 gene product could be a part of a mechanism that leads yeast cells to choose between the progression towards DNA replication and cell division or to enter into the stationary phase. This mechanism appears to turn off both rRNA accumulation and cell-cycle progression and to activate differentiative pathways in response to environmental restriction.

Cell Cycle↗

PubMed1984

A computer algorithm for the analysis of protein distribution in budding yeast.

Flow cytometry gives relevant data on cellular parameters such as DNA, RNA, and protein contents of individual cells and is therefore a powerful tool for analyzing microbial population dynamics. Relevant information about growth dynamics may be obtained from protein distribution. In fact, protein distribution is related to age distribution and depends on the law of growth of the population and the law of growth of the single cell. To extract the available information from protein distribution, we developed a computer algorithm starting from a model for growth of Saccharomyces cerevisiae. This algorithm quantitatively fits experimental protein distributions, allows a deconvolution of these distributions, and thus yields information about temporal parameters of the cell cycle and structure of yeast populations.

Cell Cycle↗

PubMed1984

G1 phase heterogeneity in exponentially growing Swiss 3T3 mouse fibroblasts.

The growth rate of normal cultured Swiss 3T3 fibroblasts is function of serum concentration and the fraction of G1 cells, and hence the average residence time in G1, increases with the generation time. Serum contains two sets of factors: competence factors, essentially platelet-derived growth factor (PDGF), which induces competence in quiescent fibroblasts and prevents replicating cells from entering G0, and plasma, which allows progression. The increase in the duplication time and the duration of G1 at low serum concentration could hence be due to the fact that competence factors become limiting. The fraction of non-competent cells, operationally defined as those G1 cells unable to leave G1 in the presence of plasma alone, was determined in populations exponentially growing at serum concentrations between 5 and 20%. To do so exponentially growing cultures were shifted to plasma plus colcemid: one part of the cell population progressed through the cycle and accumulated with a G2 DNA content, whereas non-competent cells remained in G1. Analysis of the DNA distributions performed 24 h after the shift showed that as serum concentration was lowered more cells were found in the non-competent state: they were less than 5% in 20% serum and almost 50% in 5% serum. The non-competent cells constitute a dynamic fraction of the population, since in the presence of serum they can leave G1 and progress in the cycle. These data indicate that one of the steps limiting exponential growth is the acquisition of competence and that this event gives rise to heterogeneity within the G1 population.

Animals↗