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R Gonzàlez-Duarte

Publications and source records attributed to R Gonzàlez-Duarte.

70 records · Page 4Linked to original sources
PubMed1993

Frequency of the arylsulphatase A pseudodeficiency allele in the Spanish population.

Pseudodeficiency in arylsulphatase A (ASA) is a relatively frequent condition in healthy individuals. It produces a reduction in enzyme activity similar to that found in metachromatic leukodystrophy (MLD). Unambiguous discrimination between the two conditions cannot be achieved through conventional enzyme activity assays. A PCR method has been developed which detects the pseudodeficiency (pd) allele using a single pair of primers encompassing the mutation site and hair follicles as the source of DNA. The frequency of this allele in the Spanish population has been evaluated and correlations between different genotypes and ASA activity levels have been established. Ten out of 55 individuals were heterozygous for the ASA pd allele, while two were homozygous. The allele frequency was thus 12.7%.

Alleles↗
PubMed1992

Short-chain dehydrogenases. Proteolysis and chemical modification of prokaryotic 3 alpha/20 beta-hydroxysteroid, insect alcohol and human 15-hydroxyprostaglandin dehydrogenases.

Prokaryotic 3 alpha/20 beta-hydroxysteroid dehydrogenase exhibits one segment sensitive to proteolysis with Glu-C protease and trypsin (cleaving after Glu192 and Arg196, respectively). Cleavage is associated with dehydrogenase inactivation; the presence of NADH offers almost complete protection and substrate (cortisone) gives some protection. Distantly related insect alcohol dehydrogenase is more resistant to proteolysis, but cleavage in a corresponding segment is detectable with Asp-N protease (cleaving before Asp198), while a second site (at Glu243) is sensitive to cleavage with both Glu-C and Asp-N proteases. Combined, the results suggest the presence of limited regions especially sensitive to proteolysis and the possibility of some association between the enzyme active site and the sensitive site(s). Modification of the hydroxysteroid dehydrogenase with tetranitromethane is paralleled by enzyme inactivation. With a 10-fold excess of reagent, labeling corresponds to 1.2 nmol Tyr/nmol protein chain and is recovered largely in Tyr152, with lesser amounts in Tyr251. Tetranitromethane also rapidly inhibits the other two dehydrogenases, but they contain Cys residues, preventing direct correlation with Tyr modification. Together, the proteolysis and chemical modifications highlight three segments of short-chain dehydrogenase subunits, one mid-chain, containing Tyr152 of the steroid dehydrogenase (similar numbers in the other enzymes), strictly conserved and apparently close to the enzyme active site, the other around position 195, sensitive to proteolysis and affected by coenzyme binding, while the third is close to the C-terminus.

Alcohol Dehydrogenase↗
PubMed1992

Identification of reactive tyrosine residues in cysteine-reactive dehydrogenases. Differences between liver sorbitol, liver alcohol and Drosophila alcohol dehydrogenases.

Modification of tyrosine residues with tetranitromethane and reversible sulphite protection of cysteine residues were tested on three dehydrogenases of two families. In liver alcohol dehydrogenase no Tyr residue is appreciably labelled, while in the homologous sorbitol dehydrogenase Tyr-109 is specifically labelled; the difference corresponds to a segment correlating with subunit interactions and the different quaternary structures of the proteins. In Drosophila alcohol dehydrogenase, Tyr modification is multiple, and the results show the presence of two different states of Cys residues, reactive in the presence and absence of cupric ions, respectively. Super-activation with cyanide was also noticed after S-sulphocysteine protection. The results demonstrate the possibility of identification of specific Tyr residues in proteins with reversibly protected Cys residues.

Alcohol Dehydrogenase↗
PubMed1992

Evidence for retrotranscription of protein-coding genes in the Drosophila subobscura genome.

Evidence is provided for the presence of retrosequences (also named retroposons) arising from Adh in the Drosophila subobscura genome. Restriction analysis and primary structure of two different retrosequence-containing clones, S812 and S135, are reported. The fact that these retrosequences lack introns and a recognizable promoter strongly supports their retrotranscriptional origin. Adjacent to the two retrosequences analyzed, a middle repetitive DNA element has been found which bears no clear similarity to any sequence reported to date in the GenBank/EMBL Data Library. A comparative analysis of these retrosequences with the functional Adh gene of D. subobscura is presented. In addition, a model concerning the origin, functionality, and propagation of these genome elements is discussed.

Alcohol Dehydrogenase↗
PubMed1992

Linkage disequilibrium detected between myotonic dystrophy and the anonymous marker D19S63 in the Spanish population.

We used the following polymorphic markers: APOC2 (BanI, BglI, TaqI), CKMM (NcoI, TaqI), and D19S63 (PstI) to haplotype 33 Spanish myotonic dystrophy (DM) families. We analysed the allele and haplotype frequencies of our sample, and the possible association of alleles or haplotypes with the disease. We found a slight linkage disequilibrium between APOC2 (BanI) and DM, but no disequilibrium when using all other APOC2 and CKMM RFLPs; this agrees with data previously reported. In addition, we found a very strong linkage disequilibrium when using D19S63 (PstI), the + allele being associated with the DM locus. This disequilibrium in the Spanish population indicates that D19S63 is very close to the DM locus.

Alleles↗
PubMed1992

Progressive redistribution of alcohol dehydrogenase during vitellogenesis in Drosophila melanogaster: characterization of ADH-positive bodies in mature oocytes.

The use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH) provides a powerful tool in the analysis of the tissue and temporal patterns of Adh gene expression. Immunocytochemical techniques at the light- and electron-microscopic levels have been used to determine the distribution of ADH in the ovarian follicles of D. melanogaster during oogenesis. In the early stages of oogenesis, small amounts of ADH are detectable in the cystocytes. At the beginning of vitellogenesis (S7), ADH appears to be located mainly in the nurse cells. From stage S9 onwards, the ADH protein is evenly distributed in the ooplasm until the later stages of oogenesis (S13-14), when multiple ADH-positive bodies of varying size appear in the ooplasm. This change in distribution is a result of the compartmentalization of the ADH protein within the glycogen yolk or beta-spheres. Yolk becomes enclosed within the lumen of the primitive gut during embryonic development, and thus our results suggest a mechanism for the transfer of maternally-inherited enzymes to the gut lumen via yolk spheres.

Alcohol Dehydrogenase↗
PubMed1992

The Drosophila subobscura Adh genomic region contains valuable evolutionary markers.

We have sequenced 4 kb of the genomic region comprising the Adh (Alcohol dehydrogenase) gene of Drosophila subobscura. In agreement with other species which belong to the same subgenus, two structural genes, Adh and Adh-dup, are contained in this region. The main features of these two genes of D. subobscura have been inferred from the sequence data and compared with the homologous region of D. ambigua and D. pseudoobscura. Drosophila subobscura Adh and Adh-dup differ from those of D. ambigua at a corrected estimation of 10.1% and 12.5%, respectively, while from those of D. pseudoobscura they differ by 9.5% and 8.1%, respectively. Our data suggest that Adh and Adh-dup are evolving independently, showing a species-specific pattern. Moreover, particular features of some regions of these genes make them valuable evolutionary hallmarks. For instance, replacement substitutions in the third exon of Adh may indicate the branching of the melanogaster-obscura groups, whereas replacement substitutions in the third exon of the Adh-dup could be used to assess speciation within the obscura group.

Alcohol Dehydrogenase↗
PubMed1992

Developmental profile and tissue distribution of Drosophila alcohol dehydrogenase: an immunochemical analysis with monoclonal antibodies.

To analyze Drosophila alcohol dehydrogenase gene (Adh) expression and tissue distribution at various developmental stages, we devised several immunochemical techniques making use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH), which had been obtained previously. We here report their application to analyze the expression of Adh in a wild-type strain of D. melanogaster. s-ELISA tests were performed to evaluate fluctuations in ADH content and specific activity during development in individual organs as well as in whole individuals. In all cases, ADH specific activity appeared to be quite constant, which implies that variations in enzyme activity reflect differences in protein content. Immunoblottings of crude homogenates revealed immunoreactive low relative molecular mass peptides in addition to the 27 KD monomeric band, showing a conserved banding pattern in different organs and developmental stages. Immunohistochemical assays on whole organs were used to analyze the general pattern of ADH distribution. Immunoperoxidase staining of cryosections proved to be of crucial relevance, as it yielded full details of the tissue localization of ADH within the ADH-positive organs. We have shown not only that ADH displays a specific distribution in some organs but also that the enzyme is restricted to certain cell types.

Alcohol Dehydrogenase↗
PubMed1991

The Adh in Drosophila: chromosomal location and restriction analysis in species with different phylogenetic relationships.

Restriction analysis of the genomic region containing the Adh gene and in situ hybridization assays were performed in six Drosophila species belonging to three different subgenera: D. ambigua, D. subobscura, D. madeirensis and D. guanche (sg. Sophophora); D. immigrans (sg. Drosophila); and D. lebanonensis (sg. Pholadoris). In agreement with previous observations, comparison of restriction maps of the Adh region shows that D. subobscura and D. madeirensis are very closely related. Partial homology is also observed with the rest of the obscura group species. Nevertheless, no resemblance at the restriction map level is detected when more distantly related species are compared. In D. ambigua, D. immigrans and D. lebanonensis in situ hybridization assays reveal a single chromosomal location for Adh, which in D. lebanonensis appears to be sex linked. In contrast, in D. subobscura, D. madeirensis and D. guanche multiple sites of hybridization with homologous and heterologous probes are observed. For example, in D. subobscura and D. madeirensis the functional Adh gene is located on the U chromosome and additional homologous retrosequences are found on the E chromosome.

Alcohol Dehydrogenase↗
PubMed1991

The Adh genomic region of Drosophila ambigua: evolutionary trends in different species.

The study of individual genes is essential to a comprehensive understanding of genome evolution. The wealth of information on alcohol dehydrogenase (Adh) in Drosophila makes this gene particularly suitable for such analysis. We have characterized more than 4 kb of the genomic Adh region in Drosophila ambigua and compared this region to Drosophila mauritiana and Drosophila pseudoobscura. The presence of two genes, Adh and 3'ORF (open reading frame), has been confirmed and some of their essential features have been inferred from primary structural analysis. Inter- and intraspecific comparisons have led us to support that both genes may have diverged from an ancient precursor. They appear to be evolving independently, and show a species-specific pattern. The Adh in the obscura group species lacks amino acids three and four when compared to the species of the melanogaster group and has accumulated most of its amino acid replacements in the third exon. Neither characteristic is observed when any other group species are compared, which suggests that these may be particular features of the evolution of the obscura group. The 3'ORF is highly conserved among the three species analyzed, although variability in the length of the third exon and the nucleotide substitution rate, which is much higher than in Adh, are worth noting. According to our data, both mutation/fixation rates and the distribution of mutations vary over time, which makes it difficult to predict the evolutionary dynamics of specific genome regions.

Alcohol Dehydrogenase↗
PubMed1990

Synthesis of Drosophila melanogaster alcohol dehydrogenase in yeast.

Expression systems for the heterologous expression of Drosophila melanogaster alcohol dehydrogenase (ADH) in Saccharomyces cerevisiae have been designed, analyzed and compared. Four different yeast/Escherichia coli shuttle vectors were constructed and used to transform four different yeast strains. Expression was detectable in ADH- yeast strains, from either a constitutive promoter, yeast ADH1 promoter (ADCp), or a regulated promoter, yeast GALp. The highest amount of D. melanogaster ADH was obtained from a multicopy plasmid with the D. melanogaster Adh gene expressed constitutively under the control of yeast ADCp promoter. The D. melanogaster enzyme was produced in cell extracts, as assessed by Coomassie blue staining and Western blotting after polyacrylamide-gel electrophoresis and it was fully active and able to complement the yeast ADH deficiency. Results show that D. melanogaster ADH subunits synthesized in yeast are able to assemble into functional dimeric forms. The synthesized D. melanogaster ADH represents up to 3.5% of the total extracted yeast protein.

Alcohol Dehydrogenase↗
PubMed1989

Inter-specific analysis of Drosophila alcohol dehydrogenase by an immunoenzymatic assay using monoclonal antibodies.

Three Drosophila alcohol dehydrogenase monoclonal antibodies have been prepared and characterized. These antibodies cross-react with alcohol dehydrogenase from different species as revealed by immunoblotting assay. An enzyme-linked immunosorbent assay has been devised to quantify alcohol dehydrogenase in several species, different strains and individual larval organs. The assay detects alcohol dehydrogenase via a double-antibody sandwich assay technique giving strictly proportional values for antigen concentration and optical densities in the range of 3-30 ng of antigen per 100 microliters of sample. When alcohol dehydrogenase specific activity is compared in different larval organs a remarkable similarity is observed, whereas protein distribution varies substantially. Larval fat body and larval alimentary canal contribute 63% and 26% respectively to recovered alcohol dehydrogenase.

Alcohol Dehydrogenase↗
PubMed1988

A cytological and molecular analysis of Adh gene expression in Drosophila melanogaster polytene chromosomes.

Analysis of puffing patterns in Drosophila melanogaster salivary gland chromosomes indicates the existence of a developmentally specific puff in the 35B region. This puff seems to originate from bands 35B2 or 35B3, where Adh is located, and it is expanded in more than 60% of the nuclei examined. The presence of RNA polymerase II in this puff as well as its ability to incorporate tritiated uridine shows that it corresponds to a transcriptionally active site. RNA blotting and in situ hybridization experiments indicate that Adh is transcribed, although not very actively, in salivary glands during the third larval instar. However, this tissue does not display detectable levels of ADH activity. By contrast, we have found that in midgut polytene chromosomes the 35B region is not visibly puffed in spite of the high levels of Adh transcripts detected. These results seem to suggest that puffing at the 35B region could be mainly promoted by genes closely linked to Adh, possibly with a minor contribution of this gene.

Alcohol Dehydrogenase↗
PubMed1985

Purification and molecular characterization of alcohol dehydrogenase from Drosophila hydei: conservation in the biochemical features of the enzyme in several species of Drosophila.

Alcohol dehydrogenase (ADH) has been purified from Drosophila hydei. Biochemical investigations show that the native enzyme is a dimer consisting of two identical subunits with molecular weight 27,000. The pH optimum values of pure enzyme preparations are 7.9 and 9.4. The pI values are 8.83 and 8.41. Substrate specificities have been characterized. Km(app) values are lowest for propan-2-ol and butan-2-ol and Vmax(app) values are highest for these two substrates. The amino acid composition has been determined. Peptide mapping experiments performed after trypsin digestion of the enzyme allow the identification of 24 peptides. Peptides comprising 64% of the amino acid residues have also been purified by high-performance liquid chromatography (HPLC), and their N-terminal residues and amino acid composition determined. Results are compared with the amino acid sequence of ADH from D. melanogaster Adhs [Thatcher, D. R. (1980). Biochem. J. 187:875]. When data on the biochemical and structural characterization of ADH from D. hydei are compared with data from other species of Drosophila, clear homologies are observed.

Alcohol Oxidoreductases↗
PubMed1985

Metabolic response to ethanol and isopropanol in D. funebris and D. immigrans.

Ethanol at low concentration increases larval viability in D. funebris and D. melanogaster and decreases it in D. immigrans. Isopropanol decreases viability in all three species. Isopropanol depresses alcohol dehydrogenase activity. This is accompanied by a change in the relative proportion of electrophoretic forms of the enzyme. Acetone appears in the medium when larvae of D. funebris and D. immigrans develop in the presence of isopropanol. It can be transformed again into isopropanol on reaching a threshold value. It seems that this transformation cannot be carried out by alcohol dehydrogenase alone.

1-Propanol↗