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D Gozalbo

Publications and source records attributed to D Gozalbo.

At least 37 records · Page 2Linked to original sources
PubMed1995

Preliminary characterization of the material released to the culture medium by Candida albicans yeast and mycelial cells.

Culture filtrate concentrates were obtained from Candida albicans yeast and mycelial cells grown in the presence of 14C-protein hydrolysate for radioactive labeling of cellular polypeptides. Both growth forms released to the medium minor but significant amounts of proteinaceous materials. The analysis of culture filtrate concentrates by means of SDS-polyacrylamide gel electrophoresis and fluorography revealed a similar and complex electrophoretic pattern, though some qualitative and quantitative differences between samples obtained from yeast and mycelial cells were observed. Materials released, mostly composed of mannoproteins as shown by their affinity towards concanavalin A, presented (i) cross-reactivity (by Western immunoblotting and ELISA) against polyclonal antisera to genuine cell wall components (among them the 58-kilodalton fibrinogen-binding mannoprotein) and (ii) high affinity for polystyrene-latex microbeads. Results presented suggest a possible common identity for the molecules shed to the medium and the cell wall protein and mannoprotein constituents.

Antibodies, Fungal↗
PubMed1994

Differential expression of SUC genes: a question of bases.

Non-coding nucleotide sequences located 5' upstream of the transcriptional start site play an essential role in gene expression as they contain binding sites for transcription and regulatory factors. The yeast SUC gene family is a useful model to study the influence that nucleotide exchanges within the promoter regions have on their expression, since (i) these genes, regulated by glucose repression, are differentially transcribed (invertase activity produced by distinct SUC genes may show variations of about 10-fold); and (ii) promoter sequences of SUC3, SUC4, SUC5 and SUC7 are more than 99% homologous, showing only six base exchanges among all of them. Comparison of these nucleotide exchanges with the expression of each SUC gene (located either on chromosomes or on multicopy and centromeric plasmids) points out that naturally occurring base exchanges as few as one nucleotide modification (G to A transition at position -497 relative to the translational start site, C to T transition at position -460 and insertion/deletion of a T at positions -590, -586 and -435) may have a strong effect on gene expression.

Gene Expression Regulation, Fungal↗
PubMed1994

Genes involved in the regulation of invertase production in Saccharomyces cerevisiae.

The expression of Saccharomyces cerevisiae SUC genes is exclusively regulated by catabolic repression, mediated by glucose. Genes involved in this process have been defined by means of mutants either unable to express invertase or with constitutive phenotype, although none of the genes is specific for invertase regulation. The affected genes in mutants unable to produce invertase are designated SNFX. These genes can be assorted into two groups considering either their function in regulation of gene expression or their epistatic relationships. Mutants with constitutive phenotype have been selected either by resistance to 2-deoxyglucose or by suppression of snf mutations. Among the different genes previously outlined, some of which code for transcription factors, only the MIG1 product, a "zinc finger" protein, shows a clear capacity of binding DNA in vitro. Besides the ON/OFF switch mechanism of the expression of SUC genes, some genes seem to play a role in modulating invertase expression, either hindering or stimulating transcription. A model to define the relationship between the different gene products involved in the regulation of transcription of the SUC genes is proposed.

Biological Transport↗
PubMed1994

Chitin synthetases in Candida albicans: a review on their subcellular distribution and biological function.

In the light of recent genetic advances, some results regarding chitin biosynthetic activities are reviewed in this paper. Genes coding for distinct enzymes displaying chitin synthetase activities have been characterized in Saccharomyces cerevisiae as well as in other fungal species including Candida albicans. Several activities seem to exist in the cells: (i) one zymogenic, located in cytoplasmic vesicles called chitosomes, although the presence of other types of vesicles with zymogenic activity cannot be completely discarded, and (ii) plasma membrane associated activities (the active enzyme and probably two distinct pools of zymogenic activity). Possible relationships between these activities, if any, remain to be determined. These multiplicity of enzymes is not surprising taking into account that chitin biosynthesis is required during very well defined temporal and spatial events of the cell cycle. A general repair function for one of the chitin biosynthetic activities is proposed as a possible salvage mechanism to warrant cell survival after wall damage has been caused, since chitin appears to be the most suitable polymer to carry out this function due to its particular physico-chemical properties.

Candida albicans↗
PubMed1993

Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells of Candida albicans.

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) from C. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen, in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processes in vivo and in vitro or, alternatively, that both enzyme activities (active in vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml-1 of digitonin and 64 micrograms ml-1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 micrograms ml-1 of protease, whereas activity strongly decreases in the presence of 64 micrograms ml-1 of trypsin. Digitonin does not produce zymogen activation per se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.

Candida albicans↗
PubMed1993

Critical steps in fungal cell wall synthesis: strategies for their inhibition.

Development of new effective antifungal drugs is limited by the absence of specific target sites in the fungal cells. Knowledge of the fungal cell wall structure and biosynthesis is of interest in searching for a potential target site for new chemotherapeutic agents. Our group has demonstrated that the fungal cell wall is a metabolically active structure where interaction between distinct components occurs to give rise to the mature cell wall structure. Mannoproteins play an essential role in the cell wall organization, and there is evidence for the formation of covalent bonds between these molecules and the structural polymers (glucans and chitin) outside the plasma membrane. Such interactions, which specifically occur at the fungal cell wall, are of great interest in defining target sites for potential new chemotherapeutic agents, which may inhibit the interactions and, thus, lead to a defective cell wall formation and cell death.

Antifungal Agents↗
PubMed1992

Chitin synthetase activity is bound to the plasma membrane and to a cytoplasmic particulate fraction in Candida albicans germ tube cells.

Subcellular distribution of chitin synthetase has been studied in germ tubes of Candida albicans. Two fractions with synthetase activity were separated from cell homogenates: (i) a mixed membrane fraction where the enzyme, partly in an active form, is associated with the plasma membrane (isopycnic centrifugation of mixed membrane fraction on linear sucrose gradients resolved a unique peak of activity matching with [3H]ConA-labelled membranes at a buoyant density of 1.195 g/ml); and (ii) a cytoplasmic fraction containing fully zymogenic enzyme associated with particles whose buoyant density (determined by isopycnic centrifugation on linear sucrose gradients) depended on the cell breakage conditions. The actual cytoplasmic fraction-enzyme may correspond to particles with buoyant density 1.135 g/ml (chitosomes), whereas the enzyme particles with other densities (1.085 and 1.165 g/ml) probably originated during cell disruption, as has been reported previously to occur during the preparation of yeast cell homogenates.

Candida albicans↗
PubMed1992

Multiple copies of SUC4 regulatory regions may cause partial de-repression of invertase synthesis in Saccharomyces cerevisiae.

Transformation to generate multiple copies of regulatory DNA sequences has been used to study the interactions between regulatory proteins and their target sequences, since a high copy number of these sequences may titrate trans-acting regulatory proteins. We have analyzed the synthesis of invertase in yeast strains carrying different SUC genes transformed with the multiple-copy plasmid pSH143, a derivative of pJDB207 containing the promoter and upstream regulatory sequences of SUC4. The results obtained seem to be strain dependent. Under repressing conditions a high copy number of SUC4 promoter regions may cause increased expression of the invertase genes resulting in the synthesis of external glycosylated protein. A similar result was obtained under de-repressing conditions since transformants from some strains showed higher levels of activity. These results suggest that transcriptional regulatory (negative) factors may become limiting when the copy number of their target DNA sequences is increased. This effect may depend on the amount of active repressor molecules as well as on their affinity for SUC4 upstream sequences. This is discussed on the basis of the nucleotide sequences of SUC promoters.

Base Sequence↗
PubMed1992

Are there salvage routes within the general secretory pathway in yeast?

It is generally accepted that both extracellular protein secretion and plasma membrane expansion in yeast occur basically as in higher eukaryotic cells. In addition to the constitutive (default) secretory pathway, some specialized mammalian cells possess a regulated route which at present has not been detected in yeast. However, there is a body of experimental results suggesting that under certain circumstances export of integral plasma membrane and exocellular proteins may take place through alternative (salvage) pathways. The existence of these latter routes would enable the yeast cell to adapt more efficiently to distinct or adverse conditions requiring the secretion of discrete amounts of specific sets of proteins.

Carrier Proteins↗
PubMed1991

Stability of chitin synthetase in cell-free preparations of a wild-type strain and a 'slime' variant of Neurospora crassa.

Chitin synthetase activity in cell-free preparations from a wild-type strain and a 'slime' variant of Neurospora crassa was monitored over many days in samples stored at 0 degrees C. Total activity in whole-cell-free extracts and low-speed supernatants from both organisms was very unstable, losing more than 90% of the initial activity on storage at 0 degrees C for 96 h. Chitin synthetase detection was not masked by chitinase activity present in the preparations. Gel-filtration chromatography of these preparations increased the stability of the activity from the 'slime' variant, whereas removal of particulate structures by high-speed centrifugation stabilized the chitin synthetase activity in the supernatant, particularly in the wild type. These results suggest that factor(s) involved in the regulation of chitin synthetase may be differentially located or altered in 'slime' cells.

Cell-Free System↗
PubMed1991

Evidence for the involvement of acylglycerides on chitin synthetase activity in Candida albicans.

The effect of a lipase activity (EC 3.1.1.3) on the chitin synthetase from Candida albicans has been studied, both on the active and the trypsin activated enzyme. Removal of fatty acids from acylglycerides by lipase has an inhibitory effect on the activity as well as on the 'in vitro' activation process by trypsin in the membrane-bound enzyme and in the chitosomes. This would indicate that an adequate lipid environment is required for both the activation process and proper function of the synthetase activity.

Acylation↗
PubMed1989

The naturally occurring silent invertase structural gene suc2 zero contains an amber stop codon that is occasionally read through.

The yeast invertase structural gene SUC2 has two naturally occurring alleles, the active one and a silent allele called suc2 zero. Strains carrying suc2 zero are unable to ferment sucrose and do not show detectable invertase activity. We have isolated suc2 zero and found an amber codon at position 232 of 532 amino acids. However, transformants carrying suc2 zero on a multicopy plasmid were able to ferment sucrose and showed detectable invertase activity. Full-length invertase was found in gels stained for active invertase and in immunoblots. Therefore we concluded that the amber codon is occasionally read as an amino acid. The calculated frequency of read-through is about 4% of all translation events.

Alleles↗
PubMed1989

A deletion of the PDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations.

We deleted most of the pyruvate decarboxylase structural gene PDC1 from the genome of Saccharomyces cerevisiae. Surprisingly, mutants carrying this deletion allele showed a completely different phenotype than previously described point mutations. They were able to ferment glucose and their specific pyruvate decarboxylase activity was only reduced to 45% of the wild type level. Northern blot analysis revealed that a sequence in the yeast genome homologous to PDC1 and formerly designated as a possible pseudogene is expressed and may code for a different but closely related pyruvate decarboxylase. The products of the two PDC genes seem to form hybrid oligomers, however both homooligomers have enzyme activity. Thus, the product of the PDC1 gene is not absolutely necessary for glucose fermentation in yeast.

Blotting, Southern↗
PubMed1989

Comparison of the nucleotide sequences of a yeast gene family. I. Distribution and spectrum of spontaneous base substitutions.

The nucleotide sequences of closely related members of a gene family can be used to investigate spontaneous mutations. Here we analyse the sequences of different yeast invertase genes which are more than 93% identical in the coding region and share some very similar, but not identical sequences in the noncoding flanking regions. Since all except one of the invertase genes are active, most of the base substitutions are silent. Within the coding region the base substitutions are unevenly distributed, indicating that parts of the genes were homogenized, probably via gene conversion. Transitions occurred more frequently than transversions in both, coding and noncoding regions. In the coding region pyrimidine transitions were the most abundant event due to silent changes mainly in the third codon position. In the noncoding region pyrimidine and purine transitions were found at equal frequencies. Transversions inverting base pairs (A-T and G-C) outnumber transversions changing base pairs (A-C and G-T). While the spectrum of mutations in the coding region is influenced by selective pressure to maintain the amino acid sequence, the spectrum in the noncoding region may be much less affected by selective pressure.

Base Sequence↗
PubMed1989

Comparison of the nucleotide sequences of a yeast gene family. II. Analysis of spontaneous deletions and insertions.

We compared the nucleotide sequences of 3 yeast invertase genes in regions where the homology is better than 90%. In the noncoding region 40 gaps of 1-61 bases were found. This is about half as much as the nucleotide substitutions in the same sequences. We grouped the gaps into 5 categories by their length and the characteristics of their sequences. Group I gaps are about 20 nucleotides long and are flanked by repeated sequence of 6 bases which may trigger the deletion of one of the repeats and the sequence between the repeats. Group II gaps are characterized by a small repeated sequence which is missing in one of the invertase genes. Gaps which occur in sequences exclusively made up of one of the 4 bases are summarized in group III. The 4 gaps in group IV do not show any of these sequence characteristics and they are all just one base long. A 61 nucleotide sequence found in only one of the invertase genes seems to be of complex origin. We conclude that small repeated sequences or monotonous sequences are prone to deletion or insertion mutations.

Base Sequence↗
PubMed1988

Structural analysis of the 5' regions of yeast SUC genes revealed analogous palindromes in SUC, MAL and GAL.

In the yeast Saccharomyces cerevisiae six unlinked structural genes for invertase, the SUC genes, are known. We sequenced about 800 bp of the 5' non-coding region and the first 220 bp of the coding region of the genes SUC1, SUC3, SUC4 and SUC5 and compared them with the previously sequenced genes SUC2 and SUC7 (Sarokin and Carlson 1985a). All are highly homologous within the coding region but in the non-coding region SUC1 shows some differences and SUC2 is more highly diverged. Two different kinds of TATA boxes were identified: the more strongly expressed genes SUC1, 2 and 4 have the sequence TATAAA and the more weakly expressed genes SUC3, 5 and 7 have TACAAA. Though the SUC1 sequence is in general more homologous to the other SUC genes, the region between -140 and +100 of SUC1 is nearly identical to SUC2. This could be due to a gene conversion between SUC1 and the silent suc2 degrees allele which occurs in the strains carrying SUC1. Within the upstream regions of all the SUC genes three regions with palindromic sequences analogous to stem and loop structures were identified. Comparable structures could be detected in similar positions in the upstream sequences of the divergently transcribed yeast gene pairs MAL6S-MAL6T and GAL1-GAL10. Implications for the importance of these structures in the regulation and initiation of transcription are discussed.

Amino Acid Sequence↗
PubMed1987

Contribution of polyadenylate sequences to the translational efficiency of globin messenger RNAs.

mRNAs from reticulocyte polysomes were fractionated by chromatography on poly(U)-Sepharose and thermal elution. The molar ratio of alpha- to beta-globin mRNA was found to be 2:1 and 1:1 respectively in short- and long-poly(A) size classes. Translational analyses indicated that the globin mRNAs containing long poly(A) tracts (with a mean length of about 70 nucleotides) directed protein synthesis with higher rates than did mRNA containing short poly(A) tracts (15-35 nucleotides). Experiments performed with sub-saturating mRNA concentrations showed that the digestion with RNAase H induced a decrease in the translational capacity of both globin mRNAs and an increase in the alpha- to beta-globin synthesis ratio. No correlation was observed between the size of the poly(A) tail in mRNA and the optimal K+ requirement for translation.

Base Sequence↗