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J C Hubert

Publications and source records attributed to J C Hubert.

At least 19 recordsLinked to original sources
PubMed1991

Complexes of mycobactin from Mycobacterium smegmatis with scandium, yttrium and lanthanum.

The interaction of cations of group IIIb elements (Sc, Y, La) with mycobactin S in ethanol leads to the formation of 1:1 complexes which closely resemble the known aluminium compound with respect to ultraviolet absorption and fluorescence emission spectra. Determination of molar stoichiometry by spectrophotometry shows that this method can be conveniently applied to the estimation of purity in mycobactin samples. Hydrolytic dissociation measurements based on aqueous extraction of the labelled complexes in heterogeneous phase indicate a pronounced gradation in cation-binding stability, which increases from La (rapid and complete dissociation) to sc (approximately 24% dissociation under similar conditions). The observed properties of the complexes are rationalized by semi-empirical model calculations, which suggest that ionic radius effects resulting from interaction of the IIIb cations with mycobactin S would not favour octahedral coordination of these elements as in the stable Fe(III) complex.

Kinetics↗
PubMed1990

Cloning and structure of the pyrE gene of Lactobacillus plantarum CCM 1904.

The pyrE gene of Lactobacillus plantarum CCM 1904, coding for the orotate phosphoribosyl transferase involved in the pyrimidine biosynthetic pathway, was cloned in Escherichia coli and sequenced. The predicted polypeptide sequence extending over 212 amino acids (MW 22,690) was compared to those of E. coli and to those of lower eukaryotes (Saccharomyces cerevisiae, Podospora anserina, Sordaria macrospora, Dictyostelium discoideum). Important conserved stretches were revealed, implying that these proteins are closely related.

Amino Acid Sequence↗
PubMed1990

Cloning and sequencing of URA10, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae.

Orotate phosphoribosyl transferase (OP-RTase) catalyses the transformation of orotate to OMP in the pyrimidine pathway. In the yeast Saccharomyces cerevisiae, the URA5 gene is known to encode this enzyme activity. In this paper we present the cloning and sequencing of a yeast gene, named URA10, encoding a second OPRTase enzyme. Comparison of the predicted amino acid sequences between URA5 and URA10 genes shows more than 75% similarity. These sequences have also been compared to those of Escherichia coli, Podospora anserina, Sordaria macrospora and Dictyostelium discoideum. Remarkable similarities in the primary structure of these proteins have been found. Gene disruption experiments revealed that URA10 gene expression is responsible for the leaky phenotype of a ura5 mutant. Assays of OPRTase activity in extracts from ura5 and ura10 mutants indicate that the URA10 product contributes only 20% of the total activity found in wild type cells.

Amino Acid Sequence↗
PubMed1990

Studies on transcription of the yeast URA2 gene.

The multifunctional protein carbamoylphosphate synthetase (CPSase)-aspartate transcarbamylase (ATCase) encoded by the URA2 gene catalyses the first two steps of the yeast pyrimidine pathway. An excess of the final product, the intracellular UTP (uridine triphosphate), inhibits both the transcription of the URA2 gene and the enzymatic activities. Results presented in this paper suggest that transcription of URA2 is negatively regulated (repression-derepression) and establish that this regulation is less efficient in the flow of the pyrimidine pathway than feedback inhibition.

Aspartate Carbamoyltransferase↗
PubMed1989

Structure and expression of the URA5 gene of Saccharomyces cerevisiae.

The URA5 gene of Saccharomyces cerevisiae encodes orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase) which catalyses the transformation of orotate to OMP in the pyrimidine pathway. We present in this paper the cloning and the sequencing of this gene, the last in the yeast pyrimidine pathway to be cloned. We have deduced the protein sequence of the OPRTase of S. cerevisiae from the DNA sequence and compared it to that of Escherichia coli, Podospora anserina and Dictyostelium discoideum. Some important similarities in the structure of these four proteins have been found. Finally, we have quantified the transcription of the URA5 gene in different physiological conditions and confirmed that it was not under the control of UTP or any intermediary product of the pathway.

Amino Acid Sequence↗
PubMed1989

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904.

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.

Amino Acid Sequence↗
PubMed1989

Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction.

A small 2.1-kb plasmid called pLP1 was extracted from Lactobacillus plantarum CCM 1904 (ATCC 8014) and cloned into the Escherichia coli pUC19 plasmid. As determined by DNA-DNA Southern hybridization with a pLP1-radioactively labeled probe, other lactic acid bacteria such as L. curvatus, L. sake, Carnobacterium, and Leuconostoc mesenteroides harbor pLP1-related plasmids. Shuttle vectors based on the pLP1 replicon were constructed by inserting the erythromycin-resistance gene from pVA891 into the various pUC19-pLP1 constructions. pLP1-based shuttle vector transformation efficiencies (TE) by electroporation were compared to TE of a broad-host-range plasmid pGK12 in different lactobacilli strains. Expression of the pUC19-pLP1 plasmids in Escherichia coli maxicells showed that pLP1 encodes for a 37,000 MW protein which can act in trans allowing the replication of plasmids in which this protein is truncated. The pLP1-based shuttle vectors producing this protein replicate in lactobacilli and also in Bacillus subtilis. A pLP1-free strain was obtained by incompatibility with a pLP1-based shuttle vector introduced in L. plantarum CCM 1904 by electroporation. The absence of pLP1 has no incidence on the strain phenotype suggesting that pLP1 is not essential for the strain in our laboratory conditions.

Cloning, Molecular↗
PubMed1989

Cloning and analysis of the gene for the major outer membrane lipoprotein from Pseudomonas aeruginosa.

The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage lambda EMBL3 vector and subsequently subcloned in the low copy-number, wide host-range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular-weight protein (8 kD) that was found to be associated with the outer membrane. Sequence analysis showed an open reading frame of 83 amino acids with a putative N-terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY-CYS-SER-SER (residues 19-22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram-negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.

Amino Acid Sequence↗
PubMed1988

Structure of the Saccharomyces cerevisiae URA4 gene encoding dihydroorotase.

The URA4 gene of Saccharomyces cerevisiae, coding for the third enzyme of the pyrimidine pathway, has been cloned through phenotypic complementation of a ura4 mutant of S. cerevisiae. Subcloning of an original 9 kb DNA fragment, carrying the yeast URA4 gene, allowed us to localize the gene on a 2 kb ClaI--BamHI fragment. The sequence of the URA4 structural gene and surrounding DNA was determined by the dideoxynucleotide chain termination method. The URA4 gene encodes a dihydroorotase subunit of calculated molecular weight 40,600. S1 nuclease mapping indicated that transcription of URA4 is initiated at four major start sites located at positions -41, -30, -22 and -18. A set of potentially significant sequences was identified in the 5' OH non-coding region of the gene. The deduced amino acid sequence of dihydroorotase was examined and compared with homologous amino acid sequences of Salmonella typhimurium, Escherichia coli and Drosophila melanogaster. S. cerevisiae dihydroorotase shows 40% homology with the S. typhimurium and E. coli enzymes and 23% homology with the D. melanogaster enzyme. A potential active site has been predicted for dihydroorotase from these comparisons.

Amidohydrolases↗
PubMed1987

Nucleotide sequence of the pyrimidine specific carbamoyl phosphate synthetase, a part of the yeast multifunctional protein encoded by the URA2 gene.

Yeast URA2 encodes a multifunctional carbamoyl phosphate synthetase-aspartate transcarbamylase of 220,000 molecular weight. We determined the nucleotide sequence of the 5' proximal part of the gene which is responsible for the glutamine amide transfer function of the carbamoyl phosphate synthetase activity. Alignment of the enzyme sequence derived from URA2 with sequences from Escherichia coli carA carB and yeast arginine-specific CP A1 CP A2 indicates that monofunctional and bifunctional carbamoyl phosphate synthetases are probably homologous. The URA2-derived enzyme organization is NH2-carbamoyl phosphate synthetase-aspartate transcarbamylase-CO2H.

Amino Acid Sequence↗
PubMed1984

Yeast regulatory gene PPR1. I. Nucleotide sequence, restriction map and codon usage.

The PPR1 gene of Saccharomyces cerevisiae controls the transcription of two unlinked structural genes URA1 and URA3. The primary structure of this eukaryotic regulatory gene and its flanking regions has been established by the dideoxynucleotide chain termination method. Our data show an open reading frame of 2712 nucleotides, corresponding to 904 amino acid residues. The 3' untranslated messenger RNA region presents consensus yeast termination and polyadenylation sequences. The pattern of codon usage in the gene is clearly random. This result is discussed in relation to protein abundance and is compared with the codon usage in 20 yeast structural and regulatory genes and with that found for Escherichia coli genes.

Amino Acid Sequence↗
PubMed1983

Complete sequence of a eukaryotic regulatory gene.

Dihydroorotase, the third enzymatic activity of the pyrimidine pathway, is encoded in Saccharomyces cerevisiae by a single gene URA4, which is induced at the transcriptional level by accumulation of ureidosuccinic acid. A regulatory gene PPR2 (pyrimidine pathway regulatory 2) acting specifically on this step, has been characterized, cloned and sequenced. The main open reading frame is 384 nucleotides long and potentially codes for a basic protein, favoring a molecular mechanism involving direct binding of a regulatory protein to DNA. The short length of the PPR2 polypeptide chain and the presence of seven cysteine residues suggest that the active form of the protein is an oligomer assembled through disulphide bonds. An uninducible allele has been cloned and sequenced. The mutation corresponds to an A leads to T transversion changing a lysine triplet into an ochre codon. The uninducible phenotype of this mutant is completely suppressed by an ochre suppressor, strengthening the hypothesis that PPR2 acts on URA4 transcription through the synthesis of a regulatory protein.

Amidohydrolases↗
PubMed1982

Cloning and restriction mapping of the yeast URA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex.

Two yeast DNA pools inserted in a hybrid Escherichia coli-yeast vector pFL1 were used to transform E. coli and yeast aspartate-transcarbamylase-less strains to prototrophy. From the first pool--a BamHI yeast DNA digest--a 6.4 kb BamHI fragment was recovered that gave good complementation of the E. coli auxotrophy but poor complementation of the yeast auxotrophy. From the second pool--a partial Sau3A yeast DNA digest--five independent plasmids complementing either E. coli, yeast, or both were recovered. Each of the five plasmids possessed sequences in common with the 6.4 kb BamHI fragment. One of these plasmids, which complemented the two URA2 activities in yeast and which produced a carbamyl-phosphate synthetase, aspartate-transcarbamylase complex sensitive to UTP feedback inhibition contained the full URA2 gene. A restriction map of the URA2 gene has been constructed and seven different consecutive segments have been recloned in pBR322 to measure their hybridization with URA2 messenger RNA, allowing us to estimate the limits of the gene.

Aspartate Carbamoyltransferase↗
PubMed1976

[Regulation of aspartate-ammonia-lyase (aspartase) biosynthesis in Pseudomonas fluorescens].

Variations in aspartasic activity in various media are due to aspartate-ammonium lyase induction and to regulation of the biosynthesis of this enzyme. Evidence for neosynthesis of the enzyme is provided by labelling and separation of the protein. The inducer appears to be aspartic acid. The biosynthesis is subject to pronounced catabolic repression. The physiological function of aspartate-ammonium lyase is discussed.

Ammonia-Lyases↗
PubMed1975

[Regulation and physiological significance of aspartate-ammonium lyase (aspartase) of Pseudomonas fluorescens type R (author's transl)].

The biosynthesis of aspartate-ammonium lyase, the enzyme which is induced by aspartic acid, is specifically repressed by fumaric acid. In the presence of aspartate, the enzyme permits the deamination of this compound by the cell. Aspartic acid is converted into fumaric acid which is an intermediate in the Krebs'cycle. The reaction may be considered as an anaplerotic sequence. In the absence of aspartic acid in the culture medium, its role is anabolic; the enzyme catalyses the biosynthesis of this amino acid. Therefore it appears that the reversible reaction fumarate + NH3 = aspartate catalysed by aspartase is included in amphibolic processes.

Ammonia-Lyases↗