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R Gonzàlez-Duarte

Publications and source records attributed to R Gonzàlez-Duarte.

At least 37 records · Page 2Linked to original sources
PubMed2000

Novel mutations in the TULP1 gene causing autosomal recessive retinitis pigmentosa.

PURPOSE: To assess the contribution of TULP1 to autosomal recessive retinitis pigmentosa (arRP). METHODS: Fifteen exons of the gene were screened by single-strand conformation polymorphism analysis of 7 (of 49) arRP pedigrees showing cosegregation with TULP1 locus markers. RESULTS: In one of the seven families two allelic mutations, IVS4-2delAGA and c.937delC, were found in exons 5 and 10, respectively. CONCLUSIONS: Two novel mutations in TULP1 were found to be associated with arRP. That they both compromise the gene product supports their pathogenicity. This gene was present in no more than 2% of a panel of 49 Spanish families affected by arRP.

Amino Acid Sequence↗
PubMed1999

USP25, a novel gene encoding a deubiquitinating enzyme, is located in the gene-poor region 21q11.2.

We have identified a new gene, USP25, spanning over 150 kb at 21q11. 2, one of the lowest gene-density regions of the human genome. USP25 is made up of 25 exons and encodes a 1087-aa protein. Database comparisons reveal high homology with members of the ubiquitin protease family (UBP). Basal expression was observed in all human tissues tested, and two main transcripts were identified. The homologous murine gene has also been characterized. In situ hybridization in mouse embryonic brains showed a clear correlation of expression with proliferative neuroepithelial cells and postmitotic neurons. Moreover, high expression was observed in adult mouse testis. UBPs belong to a complex family of deubiquitinating enzymes that specifically cleave ubiquitin conjugates on a great variety of substrates. These enzymes have an essential role in protein degradation via the 26S proteasome and thus regulate many cellular pathways. An increase in USP25 gene dosage in Down syndrome patients could seriously disturb the balance between ubiquitinated and deubiquitinated substrates.

Amino Acid Sequence↗
PubMed1999

The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: observation of an enzyme-bound NAD-ketone adduct at 1.4 A resolution by X-ray crystallography.

Drosophila alcohol dehydrogenase (DADH) is an NAD+-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones. DADH is the member of the short-chain dehydrogenases/reductases family (SDR) for which the largest amount of biochemical data has been gathered during the last three decades. The crystal structures of one binary form (NAD+) and three ternary complexes with NAD+.acetone, NAD+.3-pentanone and NAD+.cyclohexanone were solved at 2.4, 2.2, 1. 4 and 1.6 A resolution, respectively. From the molecular interactions observed, the reaction mechanism could be inferred. The structure of DADH undergoes a conformational change in order to bind the coenzyme. Furthermore, upon binding of the ketone, a region that was disordered in the apo form (186-191) gets stabilized and closes the active site cavity by creating either a small helix (NAD+. acetone, NAD+.3-pentanone) or an ordered loop (NAD+.cyclohexanone). The active site pocket comprises a hydrophobic bifurcated cavity which explains why the enzyme is more efficient in oxidizing secondary aliphatic alcohols (preferably R form) than primary ones. Difference Fourier maps showed that the ketone inhibitor molecule has undergone a covalent reaction with the coenzyme in all three ternary complexes. Due to the presence of the positively charged ring of the coenzyme (NAD+) and the residue Lys155, the amino acid Tyr151 is in its deprotonated (tyrosinate) state at physiological pH. Tyr151 can subtract a proton from the enolic form of the ketone and catalyze a nucleophilic attack of the Calphaatom to the C4 position of the coenzyme creating an NAD-ketone adduct. The binding of these NAD-ketone adducts to DADH accounts for the inactivation of the enzyme. The catalytic reaction proceeds in a similar way, involving the same amino acids as in the formation of the NAD-ketone adduct. The p Kavalue of 9-9.5 obtained by kinetic measurements on apo DADH can be assigned to a protonated Tyr151 which is converted to an unprotonated tyrosinate (p Ka7.6) by the influence of the positively charged nicotinamide ring in the binary enzyme-NAD+form. pH independence during the release of NADH from the binary complex enzyme-NADH can be explained by either a lack of electrostatic interaction between the coenzyme and Tyr151 or an apparent p Kavalue for this residue higher than 10.0.

Alcohol Dehydrogenase↗
PubMed1999

GEM, a cluster of repetitive sequences in the Drosophila subobscura genome.

GEM is a new family of repetitive sequences detected in the D. subobscura genome. Two of the four described GEM elements encompass a heterogeneous central module, with no detectable ORF, flanked by two long inverted repeats. These elements are composed of a set of repetitive modules, which are inverted repeat (IR), direct repeat (DR), palindromic sequence (PS), long sequence (LS) and short sequence (SS). These five modules can be found either clustered or dispersed as single modules in the D. subobscura genome, in euchromatic and heterochromatic regions. In addition to the 3' region of Adh retrosequences, single IR and LS blocks were found associated with the promoter region of different genes, in particular, LS-like blocks have also been found associated with functional genes in D. melanogaster and D. virilis. Conversely, the DR block is highly similar to satellite DNAs from some other species of the obscura group. In addition, GEM elements share some structural features with IS elements described in different Drosophila species. It is likely that both GEM and IS sequences would be vestiges of an ancestral transposable element.

Alcohol Dehydrogenase↗
PubMed1999

In vivo copper- and cadmium-binding ability of mammalian metallothionein beta domain.

The beta domain of mouse metallothionein 1 (betaMT) was synthesized in Escherichia coli cells grown in the presence of copper or cadmium. Homogenous preparations of Cu-betaMT and Cd-betaMT were used to characterize the corresponding in vivo-conformed metal-clusters, and to compare them with the species obtained in vitro by metal replacement to a canonical Zn3-betaMT structure. The copper-containing betaMT clusters formed inside the cells were very stable. In contrast, the nascent beta peptide, although it showed cadmium binding ability, produced a highly unstable species, whose stoichiometry depended upon culture conditions. The absence of betaMT protein in E. coli protease-proficient hosts grown in cadmium-supplemented medium pointed to drastic proteolysis of a poorly folded beta peptide, somehow enhanced by the presence of cadmium. Possible functional and evolutionary implications of the bioactivity of mammalian betaMT in the presence of monovalent and divalent metal ions are discussed.

Animals↗
PubMed1999

Molecular characterisation of partial chromosome 21 aneuploidies by fluorescent PCR.

Although trisomy of chromosome 21 is the most prevalent human genetic disorder, data from partial 21 aneuploidies are very scanty. Eight different partial aneuploidies for chromosome 21 were characterised by fluorescence quantitative PCR. Allelic dosage analysis was performed for each patient using 25 CHLC STRs covering the entire q arm. The length of the corresponding trisomies and monosomies was ascertained for five partial trisomics and three partial monosomics. All trisomic patients carried unbalanced translocations involving chromosome 21, whereas one of the monosomic patients bore a ring chromosome 21 and another showed an interstitial deletion of chromosome 21. The chromosomal breakpoints of two partial trisomy patients could be clearly delimited. However, the other three trisomies involved most of the 21 q arm as three allelic doses were detected for each marker. Although these latter patients do not show all the features of Down syndrome, genotype/phenotype correlations agree with previously reported data. The chromosomal breakpoints observed in two partially monosomic patients helped further to define the region involved in different phenotypic features associated with chromosome 21 monosomy. Telomeric material loss was also detected in a patient bearing a ring 21 chromosome. The parental origin of the aneuploidy was assigned for each case, which allowed us to conclude that two of the monosomic cases originated from de novo chromosomal rearrangements. There was no correlation with parental sex in contrast to trisomic patients originating from meiotic nondisjunction.

Aneuploidy↗
PubMed1998

The refined crystal structure of Drosophila lebanonensis alcohol dehydrogenase at 1.9 A resolution.

Drosophila alcohol dehydrogenase (DADH; EC 1.1.1.1) is a NAD(H)-dependent oxidoreductase belonging to the short-chain dehydrogenases/reductases (SDR) family. This homodimeric enzyme catalyzes the dehydrogenation of alcohols to their respective ketones or aldehydes in the fruit-fly Drosophila, both for metabolic assimilation and detoxification purposes. The crystal structure of the apo form of DADH, one of the first biochemically characterized member of the SDR family, was solved at 1.9 A resolution by Patterson methods. The initial model was improved by crystallographic refinement accompanied by electron density averaging, R-factor=20.5%, R-free=23.8%.DADH subunits show an alpha/beta single domain structure with a characteristic NAD(H) binding motif (Rossmann fold). The peptide chain of a subunit is folded into a central eight-stranded beta-sheet flanked on each side by three alpha-helices. The dimers have local 2-fold symmetry. Dimer association is dominated by a four-helix bundle motif as well as two C-terminal loops from each subunit, which represent a unique structural feature in SDR enzymes with known structure. Three structural features are characteristic for the active site architecture. (1) A deep cavity which is covered by a flexible loop (33 residues) and the C-terminal tail (11 residues) from the neighboring subunit. The hydrophobic surface of the cavity is likely to increase the specificity of this enzyme towards secondary aliphatic alcohols. (2) The residues of the catalytic triad (Ser138, Tyr151, Lys155) are known to be involved in enzymatic catalysis in the first line. The Tyr151 OH group is involved in an ionic bond with the Lys155 side-chain. Preliminary electrostatic calculations have provided evidence that the active form of Tyr151 is a tyrosinate ion at physiological pH. (3) Three well-ordered water molecules in hydrogen bond distance to side-chains of the catalytic triad may be significant for the proton release steps in DADH catalysis.A ternary structure-based sequence alignment with ten members of the SDR family with known three-dimensional structure has suggested to define a model consisting of four groups of residues, which relates the observed low degree of sequence identity to quite similar folding patterns and nearly identical distributions of residues involved in catalysis.

Alcohol Dehydrogenase↗
PubMed1998

Shaping of Drosophila alcohol dehydrogenase through evolution: relationship with enzyme functionality.

Drosophilidae is a large, widely distributed family of Diptera including 61 genera, of which Drosophila is the most representative. Drosophila feeding is part of the saprophytic trophic chain, because of its dependence upon decomposing organic matter. Many species have adapted to fermenting fruit feeding or to artificial (man-made) fermentation habitats, such as cellars and breweries. Actually, the efficient exploitation of niches with alcohols is considered one of the reasons for the worldwide success of this genus. Drosophila alcohol dehydrogenase (ADH), a member of the short-chain dehydrogenase/reductase family (SDR), is responsible for the oxidation of alcohols, but its direct involvement in fitness, including alcohol tolerance and utilization, gives rise to much controversy. Thus, it remains unclear whether ADH differentiation through evolution is somehow associated with natural adaptation to new feeding niches, and thus maybe to Drosophila speciation, or if it is a simple reflection of neutral divergence correlated with time separation between species. To build a hypothesis which could shed light on this dilemma, we analyzed the amino acid variability found in the 57 protein ADH sequences reported up to now, identified the taxon-specific residues, and localized them in a three-dimensional ADH model. Our results define three regions whose shaping has been crucial for ADH differentiation and would be compatible with a contribution of ADH to Drosophila speciation.

Adaptation, Biological↗
PubMed1998

A new autosomal recessive retinitis pigmentosa locus maps on chromosome 2q31-q33.

Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous disease. To date, mutations in four members of the phototransduction cascade have been implicated in ARRP. Additionally, linkage of the disease to three loci on 1p, 1q, and 6p has been described. However, the majority of cases are still uncharacterised. We have performed linkage analysis in a large nuclear ARRP family with five affected sibs. After exclusion of several regions of the genome known to contain loci for retinal dystrophies, a genomic search for linkage to ARRP was undertaken. Positive lod scores were obtained with markers on 2q31-q33 (Zmax at theta = 0.00 of 4.03, 4.12, and 4.12 at D2S364, D2S118, and D2S389, respectively) defining an interval of about 7 cM for this new ARRP locus, between D2S148 and D2S161. Forty-four out of 47 additional ARRP families, tested with markers on 2q32, failed to show linkage, providing evidence of further genetic heterogeneity.

Adult↗
PubMed1998

Identification of a Drosophila presenilin homologue: evidence of alternatively spliced forms.

Some cases of Alzheimer's disease are inherited as a dominant trait in humans. To date, mutations in three genes account for some of them: the amyloid precursor protein (APP) and presenilins 1 and 2 (PS-1 and PS-2, respectively). The function of the presenilins is still unclear, although they belong to a transmembrane protein-gene family, probably involved in some signaling pathway. We report here the isolation of the Drosophila presenilin homologue using the human PS-1 and PS-2 cDNAs as probes. Only one single gene has been detected in the Drosophila genome and evidence for alternatively spliced forms is presented and compared to the isoforms reported in humans. Temporal and spatial expression has been assessed by Northern blot and in situ hybridization on embryos of different developmental stages.

Alternative Splicing↗
PubMed1997

Binding of excess cadmium(II) to Cd7-metallothionein from recombinant mouse Zn7-metallothionein 1. UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis.

A mouse metallotbionein (MT) 1 expression system has been constructed that renders recombinant MT as a high purity Zn-coordinated protein. Spectral changes in absorption and circular dichroism following the addition of up to 7 mol equivalents of Cd2+ to recombinant Zn7-MT showed that it behaves like the native protein. Exposure of Cd7-MT to Cd2+ resulted in further binding of these ions to the protein, although saturation was not achieved on the addition of up to 22 mol equivalents of Cd2+ to Zn7-MT. Spectral data are compatible with a model in which the first four additional Cd2+ ions are bound to Cd7-MT via sulfur atoms, and indicate that no further thiol groups are involved in the binding of the excess Cd(II) over 11. Cd2+ ions bound in excess to Cd7-MT appear to have lower binding constants as exposure of Cdn-MT (n > 7) species to Cbelex-100 retrieved Cd7-MT. Based on the X-ray data, the accessible surface areas of sulfur atoms in Cd5,Zn2-MT 2 were calculated. This led us to propose that the coordination of the first three additional Cd(II) ions to Cd7-MT proceeds by means of S-Met1-O-Met1, S-Cys7-S-Cys13 and S-Cys5-S-Cys26 pairs. Finally, comparison of the behavior of the entire MT with that of the recombinant alpha MT and beta MT subunits indicates that mutual influences may not be negligible.

Animals↗
PubMed1997

Drosophila alcohol dehydrogenase: evaluation of Ser139 site-directed mutants.

Drosophila alcohol dehydrogenase (DADH) belongs to the large and highly heterogeneous (15-30% residue identity) short-chain dehydrogenase/reductase family (SDR). It is the only reported member that oxidizes mainly ethanol and 2-propanol among other alcohols. To confirm the role of Ser139 we constructed two site-directed mutants, Ser139Ala and Ser139Cys, which show no enzymatic activity. Molecular replacement and data from crystallographically refined 3D structures confirm the position of Ser139, whose hydroxyl group faces the cleft of the presumed catalytic pocket, very close to Tyr152 and Lys156. Thus, consistent with the constitution of the catalytic triad of other SDR, our results suggest that Ser139 of DADH is directly involved in the catalytic reaction.

Alcohol Dehydrogenase↗
PubMed1997

A new locus for autosomal recessive retinitis pigmentosa (RP19) maps to 1p13-1p21.

Autosomal recessive retinitis pigmentosa (arRP) is characterized by considerable allelic and nonallelic heterogeneity. Mutations have been described in the rhodopsin gene (RHO), the genes encoding the alpha and beta subunits of rod phosphodiesterase (PDEA and PDEB), and the gene encoding the alpha subunit of the cGMP-gated channel (CNCG). In addition, linkage studies in single extended pedigrees have defined two new arRP loci, at 1q and 6p. To identify the disease gene in a Spanish consanguineous arRP family, a linkage analysis was undertaken. After testing 102 polymorphic markers, a significant positive lod score (Zmax = 3.64 at theta = 0) was obtained with marker D1S188 at 1p13-p21, the same region where the Stargardt and fundus flavimaculatus (FFM) loci were previously defined. Exhaustive ophthalmologic examination of the patients clearly distinguished the disease from the Stargardt and FFM phenotypes and revealed an atypical form of arRP with choroidal atrophy as a distinctive feature.

Aged↗
PubMed1997

Recombinant synthesis of mouse Zn3-beta and Zn4-alpha metallothionein 1 domains and characterization of their cadmium(II) binding capacity.

Genetic engineering, coupled with spectroscopic analyses, has enabled the metal binding properties of the alpha and beta subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn4-alpha MT and Zn3-beta MT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to alpha fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the native protein. Titration of Zn3-beta MT with CdCl2 results in the formation of Cd3-beta MT. The addition of excess Cd(II) leads to Cd4-beta MT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd9-beta MT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd3-beta MT species is stable in the presence of this strong metal-chelating agent.

Animals↗
PubMed1997

Putative association of a mutant ROM1 allele with retinitis pigmentosa.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous form of retinal degeneration. Several genes and loci have been shown to be involved in the disease, although each of them only accounts for a few cases. Mutations in the gene encoding ROM1, a rod-specific protein, have been putatively associated with several forms of RP. Here we describe a double-mutant allele of this gene, P60T and T108M, present in two affected sibs and also in two healthy members of a Spanish RP family. The same double-mutant allele was previously considered to be responsible for autosomal dominant RP in one family. We now report data that question the potential pathogenicity of these two ROM1 mutations.

Adolescent↗
PubMed1997

Characterization and molecular analysis of Adh retrosequences in species of the Drosophila obscura group.

Retrosequences, genes, and pseudogenes originated by retrotranscription are frequent components of vertebrate genomes, but they have only occasionally been described in invertebrates. In Drosophila, very few retrosequences have been reported, among them those of alcohol dehydrogenase (Adh) and phosphoglyceromutase (Pglym). Although 52 Adh gene sequences are available for comparison, Adh retrosequences have been described only in the sibling species D. teissieri and D. yakuba (melanogaster subgroup) and in D. subobscura (obscura subgroup). Here, we report the presence of Adh retrosequences in two closely related species of D. subobscura: D. madeirensis and D. guanche. Extensive sequence comparisons with their functional paralogs suggest separate retrotranscriptional events: one in the melanogaster subgroup in the ancestor of D. teissieri and D. yakuba, and the other in the obscura subgroup before the radiation of the lineages leading to D. subobscura, D. madeirensis, and D. guanche. In the former, the Adh retrotranscript originated a new expressed gene, named jingwei. However, in the obscura Adh retrosequences, retention of codon bias and higher Ks than Ka values, both distinctive evolutionary features supporting functionality, have to be considered together with a frameshift, premature stop codons, and other nucleotide substitutions, which, added to the lack of the original promoter elements, suggest that they are pseudogenes. At least two different Adh retrosequences have been characterized in each of the obscura species, and their phylogenetic analysis indicates that paralogs and their flanking genomic regions share a higher degree of similarity than orthologous sequences. Two alternative hypotheses could explain this current organization and structure: either a multiplication event occurred independently in each species, or gene conversion events should be invoked after a single duplication in the species ancestor. The significance of retrotranscriptional events in the evolution of invertebrate genomes is discussed.

Alcohol Dehydrogenase↗