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B André

Publications and source records attributed to B André.

At least 37 records · Page 2Linked to original sources
PubMed1999

NH4+-induced down-regulation of the Saccharomyces cerevisiae Gap1p permease involves its ubiquitination with lysine-63-linked chains.

Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces internalization of the general amino acid permease Gap1p and its subsequent degradation in the vacuole. An essential step in this down-regulation is Gap1p ubiquitination through a process requiring the Npi1p/Rsp5p ubiquitin ligase. We show in this report that NPI2, a second gene required for NH4+-induced down-regulation of Gap1p, codes for the ubiquitin hydrolase Doa4p/Ubp4p/Ssv7p and that NH4+-induced Gap1p ubiquitination is strongly reduced in npi2 cells. The npi2 mutation results in substitution of an aromatic amino acid located in a 33-residue sequence shared by some ubiquitin hydrolases of the Ubp family. In this mutant, as in doa4(delta) cells, the amount of free monomeric ubiquitin is at least four times lower than in wild-type cells. Both ubiquitination and down-regulation of the permease can be restored in npi2 cells by over-expression of ubiquitin. In proline-grown wild-type and npi2/doa4 cells overproducing ubiquitin, Gap1p appears to be mono-ubiquitinated at two lysine acceptor sites. Addition of NH4+ triggers rapid poly-ubiquitination of Gap1p, the poly-ubiquitin chains being specifically formed by linkage through the lysine 63 residue of ubiquitin. Gap1p is thus ubiquitinated differently from the proteins targeted by ubiquitination for proteolysis by the proteasome, but in the same manner as the uracil permease, also subject to ubiquitin-dependent endocytosis. When poly-ubiquitination through Lys63 is blocked, the Gap1p permease still undergoes NH4+-induced down-regulation, but to a lesser extent.

Amino Acid Sequence↗
PubMed1998

Extracting regulatory sites from the upstream region of yeast genes by computational analysis of oligonucleotide frequencies.

We present here a simple and fast method allowing the isolation of DNA binding sites for transcription factors from families of coregulated genes, with results illustrated in Saccharomyces cerevisiae. Although conceptually simple, the algorithm proved efficient for extracting, from most of the yeast regulatory families analyzed, the upstream regulatory sequences which had been previously found by experimental analysis. Furthermore, putative new regulatory sites are predicted within upstream regions of several regulons. The method is based on the detection of over-represented oligonucleotides. A specificity of this approach is to define the statistical significance of a site based on tables of oligonucleotide frequencies observed in all non-coding sequences from the yeast genome. In contrast with heuristic methods, this oligonucleotide analysis is rigorous and exhaustive. Its range of detection is however limited to relatively simple patterns: short motifs with a highly conserved core. These features seem to be shared by a good number of regulatory sites in yeast. This, and similar methods, should be increasingly required to identify unknown regulatory elements within the numerous new coregulated families resulting from measurements of gene expression levels at the genomic scale. All tools described here are available on the web at the site http://copan.cifn.unam.mx/Computational_Biology/ yeast-tools

Algorithms↗
PubMed1998

Benzene exposure in car mechanics and road tanker drivers.

The purpose of this study was to identify professional factors related to benzene exposure and to deduce suitable safety measures. Atmospheric benzene, urinary muconic acid (tt-MA) and leukocyte alkaline phosphatase activity (LAPA) were evaluated among 66 car mechanics, 34 road tanker drivers, and 28 nonexposed workers. Professional and medical questionnaires were filled in at the same time. Atmospheric benzene was significantly higher among road tanker drivers than among car mechanics. The arithmetic mean +/- SD, median, and geometric mean values were, respectively, 0.48 +/- 1.49, 0.14, and 0.06 mg/m3 among car mechanics and 1.88 +/- 4.18, 0.68, and 0.65 mg/m3 among road tanker drivers. In the latter case the increase was caused by transport of unleaded petrol and correlated with the volume of the tank. Among car mechanics, tobacco smoking, windy conditions, dismantling of petrol filters, and handling of petrol increased atmospheric benzene levels. Urinary muconic acid was increased significantly among car mechanics (148 +/- 137, 127, and 111 micrograms/g) and among road tanker drivers (309 +/- 420, 137, and 151 micrograms/g) as compared with the controls (49 +/- 46, 33, and 33 micrograms/g). Among road tanker drivers, alcohol intake and transportation of unleaded petrol increased the excretion of muconic acid, which was also directly related to the volume of the tank. Among car mechanics, professional factors (dismantling of petrol filters, handling of and washing of hands with petrol) and nonprofessional factors (tobacco smoking and damaged skin on the hands and forearms) increased muconic acid excretion. In the control group, tobacco smoking increased its excretion. LAPA was not significantly modified among exposed workers. There was a weak but significant linear correlation between LAPA and muconic acid. These results suggest that to reduce exposure to benzene in unleaded petrol, individual and collective safety measures should be imposed in both occupations.

Air Pollutants, Occupational↗
PubMed1998

Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae.

Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces rapid inactivation and degradation of the general amino acid permease Gap1 through a process requiring the Npi1/Rsp5 ubiquitin (Ub) ligase. In this study, we show that NH4+ induces endocytosis of Gap1, which is then delivered into the vacuole where it is degraded. This down-regulation is accompanied by increased conversion of Gap1 to ubiquitinated forms. Ubiquitination and subsequent degradation of Gap1 are impaired in the npi1 strain. In this mutant, the amount of Npi1/Rsp5 Ub ligase is reduced >10-fold compared with wild-type cells. The C-terminal tail of Gap1 contains sequences, including a di-leucine motif, which are required for NH4+-induced internalization and degradation of the permease. We show here that mutant Gap1 permeases affected in these sequences still bind Ub. Furthermore, we provide evidence that only a small fraction of Gap1 is modified by Ub after addition of NH4+ to mutants defective in endocytosis.

Amino Acid Sequence↗
PubMed1997

The nucleotide sequence of Saccharomyces cerevisiae chromosome XII.

The yeast Saccharomyces cerevisiae is the pre-eminent organism for the study of basic functions of eukaryotic cells. All of the genes of this simple eukaryotic cell have recently been revealed by an international collaborative effort to determine the complete DNA sequence of its nuclear genome. Here we describe some of the features of chromosome XII.

Base Sequence↗
PubMed1997

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.

Base Sequence↗
PubMed1997

Gzf3p, a fourth GATA factor involved in nitrogen-regulated transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, two positive transcription factors of the GATA family, Gln3p and Nil1p/Gat1p, upregulate the expression of multiple nitrogen pathway genes via upstream 5'-GATA-3' sequences. Another GATA factor, Uga43p/Dal80p, downregulates to varying degrees the expression of some nitrogen-regulated genes. Here, we report the functional analysis of a fourth GATA factor, Gzf3p/Nil2p, whose gene was discovered by systematic sequencing of chromosome X. The Gzf3 protein most closely resembles Uga43p. Similar to Uga43p, Gzf3p has the properties of a negative GATA factor. While Uga43p is active specifically under nitrogen-depression conditions, Gzf3p exerts its negative regulatory function specifically on preferred nitrogen sources: It is involved in nitrogen repression of Nil1p-dependent transcription. At least one positive GATA factor is required for the UGA43 and GZF3 genes to be expressed. The Uga43p factor negatively regulates GZF3 expression and vice versa. In addition, both Uga43p and Gzf3p moderately regulate expression of their own genes. These two proteins seem to be parts of a complex network of GATA factors which probably play a determining role in nitrogen-regulated transcription.

Amino Acid Sequence↗
PubMed1997

A C-terminal di-leucine motif and nearby sequences are required for NH4(+)-induced inactivation and degradation of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae.

The general amino acid permease, Gap1, of Saccharomyces cerevisiae is very active in cells grown on proline as the sole nitrogen source. Adding NH4+ to the medium triggers inactivation and degradation of the permease via a regulatory process involving Npi1p/Rsp5p, a ubiquitin-protein ligase. In this study, we describe several mutations affecting the C-terminal region of Gap1p that render the permease resistant to NH4(+)-induced inactivation. An in vivo isolated mutation (gap1pgr) causes a single Glu-->Lys substitution in an amino acid context similar to the DXKSS sequence involved in ubiquitination and endocytosis of the yeast alpha-factor receptor, Ste2p. Another replacement, substitution of two alanines for a di-leucine motif, likewise protects the Gap1 permease against NH4(+)-induced inactivation. In mammalian cells, such a motif is involved in the internalization of several cell-surface proteins. These data provide the first indication that a di-leucine motif influences the function of a plasma membrane protein in yeast. Mutagenesis of a putative phosphorylation site upstream from the di-leucine motif altered neither the activity nor the regulation of the permease. In contrast, deletion of the last eleven amino acids of Gap1p, a region conserved in other amino acid permeases, conferred resistance to NH4+ inactivation. Although the C-terminal region of Gap1p plays an important role in nitrogen control of activity, it was not sufficient to confer this regulation to two NH4(+)-insensitive permeases, namely the arginine (Can1p) and uracil (Fur4p) permeases.

Amino Acid Sequence↗
PubMed1996

Dictyostelium discoideum contains a family of calmodulin-related EF-hand proteins that are developmentally regulated.

A full-length genomic DNA fragment that codes for a novel EF-hand protein Dictyostelium discoideum was cloned and sequenced. The protein is composed of 168 amino acids and contains four consensus sequences that are typical for (Ca2+)-binding EF-hand domains. The protein sequence exhibits only minor similarities to other calmodulin-type proteins from Dictyostelium. The genomic DNA harbors two short introns; their positions suggest that the gene is unrelated to the EF-hand proteins from the calmodulin group. Northern blot analysis showed that the mRNA level was significantly increased during development. Polyclonal antibodies raised against the recombinant protein recognized in Western blots a protein of about 20 kDa. Like the mRNA, also the protein was more abundant in developing cells. Overlay experiments with 45Ca2+ indicated that the EF-hands in fact have (Ca2+)-binding activity. The recent description of CBP1, another calmodulin-type Dictyostelium protein that is upregulated during development [Coukell et al. (1995) FEBS Lett. 362, 342-346], suggests that D. discoideum contains a family of EF-hand proteins that have specific functions during distinct steps of development. We therefore designate the protein described in this report as CBP2.

Amino Acid Sequence↗
PubMed1996

Approach for the smoothing of three-dimensional reconstructions of the human spine using dual Kriging interpolation.

In numerous situations, 3-D reconstructions of the spine are represented as curves in space, with the vertebral centroids as control points. Interpolation functions such as splines, polynomials or Fourier series have been used to minimise measurement errors and to perform specific calculations. A more general approach, dual Kriging, is presented which incorporates in a single formulation several methods, such as piece-wise linear interpolation, splines and least square functions as a limit case. To minimise user interaction and to control the different Kriging parameters, a computer program is developed allowing efficient use of these interpolation techniques in a clinical environment. Given different drift and covariance functions, the program determines the most suitable Kriging model for specific spine geometries and controls the amount of smoothing performed on raw data. Validation of the technique is with analytical 3-D curves, where random noise is added to represent reconstruction errors. A maximum absolute mean difference of 1.85 +/- 0.50 mm is found between the analytical and noisy curves smoothed with the Kriging technique for 200 points. Results obtained on actual 3-D reconstructions of scoliotic patients are very promising.

Algorithms↗
PubMed1995

The yeast YBR235w gene encodes a homolog of the mammalian electroneutral Na(+)-(K+)-C1- cotransporter family.

The YBR235w gene of the yeast Saccharomyces cerevisiae was found during sequencing of chromosome II. Here, we show that the 1120 aa protein (Ybr235p) encoded by this gene shares strong sequence similarity with the highly related electroneutral Na(+)-C1- and Na(+)-K(+)-C1- cotransporters of animal cells. We hypothesize that this yeast protein also mediates active uptake of C1- into the cell.

Amino Acid Sequence↗
PubMed1995

Structure/function studies on cytoskeletal proteins in Dictyostelium amoebae as a paradigm.

The actin cytoskeleton in motile non-muscle cells is being regulated by a large number of actin-binding proteins. A deeper insight into the complex nature of the dynamic rearrangements of the microfilament system during cell movement requires an experimental system that allows the combined application of biochemical, biophysical, cell biological and molecular methods. Dictyostelium amoebae are well suited especially for a genetic approach because they are amenable to gene disruption, antisense and gene tagging techniques. The actin-binding proteins profilin, hisactophilin and protovillin are being described in this context as typical examples that either bind to G-actin, or anchor the actin cytoskeleton to the plasma membrane, or are structurally similar to vertebrate proteins but distinct in their functions.

Animals↗
PubMed1995

Cis- and trans-acting elements determining induction of the genes of the gamma-aminobutyrate (GABA) utilization pathway in Saccharomyces cerevisiae.

In S. cerevisiae, gamma-aminobutyrate (GABA) induces transcription of the UGA genes required for its utilization as a nitrogen source. Analysis of the 5' region of the UGA1 and UGA4 genes led to the identification of a conserved GC-rich sequence (UASGABA) essential to induction by gamma-aminobutyrate. Alone, this UASGABA element also supported some levels of reporter gene transcription in the presence of gamma-aminobutyrate. To be effective, UASGABA requires two positive-acting proteins that both contain a Cys6-Zn2 type zinc-finger motif, namely pathway-specific Uga3p and pleiotropic Uga35p(Dal81p/DurLp). Further analysis of the UGA4 gene revealed that Gln3p, a global nitrogen regulatory protein containing a GATA zinc-finger domain, is required in order to reach high levels of gamma-aminobutyrate-induced transcription. The Gln3p factor exerts its function mainly through a cluster of 5'-GAT(A/T)A-3'(UASGATA) situated just upstream from UASGABA. The role of Gln3p is less predominant in UGA1 than in UGA4 gene expression. We propose that tight coupling between the UASGABA and UASGATA elements enables the cell to integrate, according to its nitrogen status, the induced expression levels of UGA4.

4-Aminobutyrate Transaminase↗
PubMed1995

Two mutually exclusive regulatory systems inhibit UASGATA, a cluster of 5'-GAT(A/T)A-3' upstream from the UGA4 gene of Saccharomyces cerevisiae.

The S. cerevisiae Uga43(Dal80) protein down-regulates the expression of multiple nitrogen pathway genes. It contains a zinc-finger motif similar to the DNA-binding domain of the vertebrate GATA family of transcription factors; this domain is known to direct binding to 5'-GATA-3' core sequences. The inducible UGA4 gene, which encodes the specific gamma-aminobutyrate permease, undergoes strong repression by Uga43p. This study shows that the 5' region of UGA4 contains a UAS element made of four directly repeated 5'-CGAT(A/T) AG-3' sequences. This element, called UASGATA, can potentially confer to the UGA4 gene high-level expression in the absence of inducer, but this potential activity is inhibited by two distinct repression systems. One system is Uga43p-dependent; it operates in cells grown on a poor nitrogen source. The other is the nitrogen repression system, which relies on Ure2p and glutamine and operates when a good nitrogen source is present. Nitrogen repression also blocks the synthesis of Uga43p, making the two repression systems mutually exclusive. Previous studies have shown that expression supported by 5'-GATA-3'-containing UAS elements requires Gln3p, another global nitrogen regulatory factor containing a GATA zinc-finger domain. Although Gln3p contributes to UASGATA activity, evidence suggests that a second factor can potentially direct expression through UASGATA. Expression conferred by this putative factor is subject to both Uga43p- and Ure2p-mediated repression. The role of UASGATA in the expression of the UGA4 gene is discussed in relation to its sensitivity to the two distinct repression systems.

Amino Acid Sequence↗
PubMed1995

Radioimmunoassay for plasma histamine: a study of false positive and false negative values.

In order to achieve a correct diagnosis of drug anaphylaxis using a radioimmunoassay devoid of interferences, we have studied factors leading to false positive or false negative values of plasma histamine. Different steps in sample collection were studied systematically in 30 normal volunteers. False positive values were found in haemolysed samples, with histamine concentrations being correlated with haemoglobin concentrations, and where plasma was aspirated from the white-cell layer. There was no significant increase when a tourniquet or vacuum tubes were used, or when blood tubes were left at 4 degrees C overnight. In 12 patients who experienced an anaphylactic reaction, histamine disappeared from blood 10 times more slowly than expected. False negative values were found in two pregnant women and one heparinized patient. Histamine was remarkably stable in vitro in blood or plasma samples, whereas it disappeared rapidly when plasma from a pregnant woman or a heparinized patient was added to the sample. We conclude that false positive and false negative values are rare when using this radioimmunoassay.

Adolescent↗
PubMed1995

NPl1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase.

When yeast cells growing on a poor nitrogen source are supplied with NH4+ ions, several nitrogen permeases including the general amino acid permease (Gap1p) are rapidly and completely inactivated. This report shows that inactivation by NH4+ of the Gap1 permease is accompanied by its degradation. A functional NPl1 gene product is required for both inactivation and degradation of Gap1p. Molecular analysis of the NPl1 gene showed that it is identical to RSP5. The RSP5 product is a ubiquitin-protein ligase (E3 enzyme) whose physiological function was, however, unknown. Its C-terminal region is very similar to that of other members of the E6-AP-like family of ubiquitin-protein ligases. Its N-terminal region contains a single C2 domain that may be a Ca(2+)-dependent phospholipid interaction motif, followed by several copies of a recently identified domain called WW(P). The Npi1/Rsp5 protein has a homologue both in humans and in mice, the latter being involved in brain development. Stress-induced degradation of the uracil permease (Fur4p), a process in which ubiquitin is probably involved, was also found to require a functional NPl1/RSP5 product. Chromosomal deletion of NPl1/RSP5 showed that this gene is essential for cell viability. In the viable npi1/rsp5 strain, expression of NPl1/RSP5 is reduced as a result of insertion of a Ty1 element in its 5' region. Our results show that the Npi1/Rsp5 ubiquitin-protein ligase participates in induced degradation of at least two permeases, Gap1p and Fur4p, and probably also other proteins.

Amino Acid Sequence↗