Three new polymorphic equine microsatellites: HLM2, HLM3, HLM5.
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Animals↗
Biomedical subjects
G Dorado
Publications and source records attributed to G Dorado.
T-vector cloning and high performance PCR with SuperTth from Thermus thermophilus.
The SuperTth DNA polymerase from Thermus thermophilus exhibits template-independent terminal transferase (extendase) activity. This enzyme is proposed as a cheap alternative for both high performance PCR as well as quick T-vector cloning of amplicons, including reverse transcription and cDNA cloning.
Base Sequence↗
Promutagen activation by fish liver as a biomarker of littoral pollution.
Metabolic activation of known promutagens by liver S9 fractions of Mugil sp. (grey mullet) from two zones of the South Atlantic Spanish littoral was determined and related to their pollution levels. Sediments from the putative contaminated area contained high concentrations of PAHs, PCBs and pesticides, and animals from the polluted site exhibited higher concentrations of metals than those from the reference area. Hepatic S9 fractions of mullets from the polluted site showed 5.1-, 18.6- and 42.8-fold higher capability to activate benzo(a)pyrene, 2-acetyl-aminofluorene and 2-aminoanthracene, respectively, than those from reference animals. Cadmium, a highly toxic metal, was one of the pollutants detected in the contaminated area. Gilthead seabream (Sparus aurata) were exposed under controlled conditions to different Cd concentrations in order to investigate the effects of Cd on fish promutagen activation capability. A clear dose-response relationship was observed between Cd concentration, EROD activity and metabolic activation of 2-aminoanthracene and benzo(a)pyrene. Our data indicate that the enhanced promutagen activation by fish S9 fractions accompanying induction of EROD activity is a sensitive and reliable index of pollution in aquatic environments.
Animals↗
Biochemical and genetic indices of marine pollution in Spanish littoral.
Increased activities of several detoxifying and antioxidant enzymes were detected in mollusc and fish from Spanish littoral areas with high metal contents. Ethanolic extracts from molluscs contained direct-acting and polar genotoxins of oxidative type, which were detected by strain TA102 of S. typhimurium and catalase-deficient strains of E. coli. Animals from contaminated sites contained less genotoxins than those from control areas. Polluted fishes displayed highly induced cytochrome P-450 activity and increased promutagen activation capabilities. In addition, specific forms of glutathione transferase and superoxide dismutase were induced, particularly highly acidic forms.
Animals↗
Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene.
A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA.
Base Sequence↗
Metal, mutagenicity, and biochemical studies on bivalve molluscs from Spanish coasts.
Three species of marine bivalve molluscs (Chamelea gallina, Ruditapes decussatus, and Crassostrea gigas) have been studied in order to evaluate the levels of pollution on the South Atlantic Spanish littoral. Several transition metals (Cu, As, Cd, Sn, Hg, Pb) were determined as a general index of total contamination. Animals from putative contaminated areas exhibited higher metal contents than those from cleaner waters. C. gigas showed 5-20-fold higher total metal content than the other two species. The mutagenicity of ethanolic extracts was assayed by using both the His reversion and the Ara forward mutation tests. Mollusc tissues from the three species did not contain genotoxins active on TA98 (frameshift mutations) or TA100 (mainly G:C base-pair substitutions), but did contain direct-acting genotoxins of a polar nature and oxidative type. This was based on the following observations: 1) mammalian metabolic activation was not required for mutagenicity, 2) mutagens were eluted with the polar fraction from XAD-2 columns, and 3) mutagenic responses were observed with Salmonella typhimurium TA102 (A:T base-pair substitutions; sensitive to oxidative damages) and Escherichia coli catalase-deficient (AraR forward mutations) strains. No relevant differences were found in the mutagenicity of mollusc extracts from areas with different pollution levels. Otherwise, our data suggest that, in general, animals living in contaminated environments had fewer genotoxins of oxidative type than those from less polluted areas. Such a result might be explained by the observation of increased levels of a number of detoxifying and antioxidant enzymes, such as glutathione-S-transferase, glutathione-peroxidase, catalase, and superoxide dismutase. Thus, contaminated animals seem to be better protected against the oxidative damages induced by metals, in agreement with their lower malondialdehyde levels. To what extent the responsible mutagenic compounds are of endogenous origins, or "Nature's pesticides" (the major toxic chemicals ingested by phytoplankton filter-feeders), and/or the result of human activities remains to be determined.
Animals↗
Molecular analysis of ultraviolet-induced mutations in a xeroderma pigmentosum cell line.
We have isolated and characterized 47 ultraviolet light-induced hprt mutants from a simian virus 40-transformed excision-repair-deficient xeroderma pigmentosum cell line (complementation group A). Twenty-one independent mutations were found, of which the majority were point mutations. Eleven of these were identified as base changes, nine of which could be attributed to ultraviolet damage on the transcribed DNA strand. Both transitions and transversions were found among the single base changes. A large proportion of the mutations (13/21) resulted in aberrant splicing of the hprt gene, suggesting that the target size for mutations resulting in aberrant splicing must be quite large. A small number of spontaneous mutations were identified, most of which were large deletions. Our data provide a spectrum for the intrinsic mutations resulting from ultraviolet damage in human cells in the absence of repair.
Base Sequence↗
Biochemical effects of environmental pollution in fishes from the Spanish South-Atlantic littoral.
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Animals↗
L-arabinose resistance test with Salmonella typhimurium as a primary tool for carcinogen screening.
The L-arabinose resistance test with Salmonella typhimurium (Ara test) is a forward mutation assay which selects a single phenotypic change (from L-arabinose sensitivity to L-arabinose resistance) in a unique tester strain (an araD mutant). The present study examined the ability of the Ara test to identify as mutagens different classes of chemical carcinogens, including six with detection problems in most bacterial assays. A noncarcinogen related in chemical structure to the selected carcinogen was included whenever possible. A total of 25 organic compounds was assayed by means of a standard mutagenesis procedure: the plate-incorporation test in the presence (if required) of 10% S9 from rat liver induced with Aroclor 1254. Chemicals giving negative or inconclusive results were then retested using other common in vitro mutagenesis conditions. The Ara test detected as mutagens all but one (Urethane) of the 15 established carcinogens and six out of the nine chemicals with questionable or nonassayed carcinogenicity. The compound mutagenic at the lowest dose was ethionine (0.57 nmol), one of the carcinogens undetected by the popular histidine reverse mutation assay (Ames test) or by the SOS chromotest. Actually, only benzo(a)pyrene was found mutagenic at a dose (0.77 nmol) close to that of ethionine. The data reported in this paper suggest that the Ara forward mutation test is less prone than the His reverse mutation test or the SOS chromotest to misclassify as negative carcinogenic compounds. Consequently, it should be considered as an alternative to other bacterial assays in general, massive, and primary screening for genotoxic carcinogens.
Arabinose↗
Study of the causes of direct-acting mutagenicity in coffee and tea using the Ara test in Salmonella typhimurium.
The mutagenic activities of 6 of the chemicals identified in coffee solutions were assayed with the Salmonella Ara test, under experimental conditions optimized for coffee mutagenicity. Caffeine was the only non-mutagenic compound. Among the other 5 chemicals, hydrogen peroxide was the strongest mutagen and chlorogenic acid the weakest; methylglyoxal, glyoxal and caffeic acid exhibited intermediate mutagenicities. The minimal mutagenic doses of these components correlated negatively with their relative concentrations in coffee. It was concluded that chlorogenic acid, caffeic acid, glyoxal and methylglyoxal cannot contribute alone to the mutagenicity of coffee in the Ara test, since their minimal mutagenic concentrations were much higher than their respective levels in the coffee samples assayed. By contrast, 40-60% of the mutagenic activity in coffee and also in tea could be attributed to their H2O2 contents. Catalase abolished more than 95% of the mutagenic activity of coffee, as detected by the Ara test. A similar sensitivity to catalase has been reported by other authors in relation to the coffee mutagenicity identified by the Salmonella His test. Nevertheless, the results presented in this paper suggest that the Ara forward and the His reverse mutation tests are sensitive to the mutagenicity of different constituents in coffee solutions. We propose that the His test, sensitive at high coffee doses, mainly recognizes the mutagenicity of methylglyoxal, whilst the Ara test, sensitive at low coffee doses, mainly detects the mutagenic activity of hydrogen peroxide. The data reported also suggest that the direct-acting mutagenicity(ies) detected by the Ara test in tea solutions is (are) based on similar, if not identical, mechanisms.
Caffeic Acids↗
Participation of Drosophila melanogaster alcohol dehydrogenase (ADH) in the detoxification of 1-pentene-3-ol and 1-pentene-3-one.
The participation of the ADH enzymes in the detoxification by D. melanogaster of 1-pentene-3-ol (also called pentenol) and its oxidized product, 1-pentene-3-one (usually known as ethyl-vinyl-ketone or pentenone) have been studied using the LR lines. For this purpose flies of AdhS AdhS (SS) and AdhF AdhF (FF) genotypes were independently pretreated with a 2 per cent isopropanol (2-propanol) solution and the survivors exposed to water, to a 0.0075 per cent pentenol solution or to a 0.00375 per cent pentenone solution. After one day in these solutions, the ability to tolerate both compounds was checked and the ADH activity of the surviving flies was measured and compared with those of control flies not pretreated with isopropanol. Additionally, the effects of pentenone on ADH enzymes have been studied by comparing them with those of acetone. Our results show that, in contrast to acetone, pentenone neither reduced significantly the ADH activity in vivo nor altered the normal proportion of ADH isozymes of either SS or FF flies. Our findings also demonstrate that the isopropanol pretreatment implied a considerable decrease in sensitivity not only to pentenol (60 and 91 per cent for SS and FF flies, respectively) but also to pentenone (72 and 80 per cent for SS and FF flies, respectively). After isopropanol pretreatment, FF flies continued exhibiting higher ADH activities than SS ones. However, FF pretreated flies displayed higher tolerance to pentenol and a similar tolerance to pentenone than SS animals. Our results suggest that pentenol (unsaturated secondary alcohol) and isopropanol (saturated secondary alcohol) may be detoxified by slightly different processes (both ADH-activity-dependent), and that pentenone could not be accumulated in the fly but transformed into another compound(s) by means of some ADH-independent mechanism(s).
1-Propanol↗
Mutagenicity of red wine in the L-arabinose resistance test with Salmonella typhimurium.
The forward mutation assay to L-arabinose resistance (Ara test) in Salmonella typhimurium detected a Spanish red table wine (Rioja) as a direct-acting mutagen. The best mutagenic response was obtained by preincubating strain BA13 with the wine sample in the presence of sodium phosphate buffer and in the absence of any external metabolic activation. In fact, the S9 mixture abolished most of the mutagenic activity of red wine in the Ara test. Such an inactivating capacity seems to be independent of microsomal enzymes and mediated through some kind of heat-stable component(s) in the S9 fraction. Both regular wine (directly from the bottle) and lyophilized wine were strong mutagens in the Ara test, inducing 4914 and 2739 AraR mutants/ml. Both pKM101 and delta uvrB were critical factors in the detection of the mutagenicity of wine, exhibiting a synergic effect in strain BA13. The mutagenicity of red wine was somehow pH-dependent, increasing with the pH value of the preincubation mixture. In comparison with the Ara test, the His reverse mutation assay (Ames test) was much less sensitive to the mutagenicity of lyophilized red wine, TA102 being the most (448 His+/ml) and TA98 the least (38 His+/ml) sensitive strain. TA100, TA104 and TA97 manifested intermediate mutagenicities to red wine. Previous reports have identified strain TA98 as the His strain most sensitive to the apolar fraction (e.g. XAD-2-bound) of red wine. Based on these results we propose that TA98 mainly detects glycosides of mutagenic flavonols present in red wine (quercetin, rutin, etc.), which do not constitute the major direct-acting mutagens detected with the Ara test.(ABSTRACT TRUNCATED AT 250 WORDS)
Arabinose↗
Coffee is highly mutagenic in the L-arabinose resistance test in Salmonella typhimurium.
The present study shows that the L-arabinose resistance test in Salmonella typhimurium detects coffee as a strong mutagen in the absence of mammalian microsomal activation. The response of the Ara forward mutation assay was 8.5 times higher than that of TA104, which is the most sensitive to coffee of the tester strains of the Ames test. Both the mutagenesis protocol (preincubation test) and the additional genetic characteristics of the bacterial tester strain (excision repair deficiency, normal lipopolysaccharide barrier, and the presence of plasmid pKM101) were critical factors in the optimal induction by coffee of forward mutations to L-arabinose resistance. All ten samples of roasted coffee analyzed with the Ara assay were highly mutagenic: one cup of coffee (150 ml) was calculated to induce 3-4 X 10(6) AraR mutants. In contrast, coffee prepared from unroasted beans (green coffee) had no mutagenic activity. Regular- and sugar-roasted coffees showed similar mutagenicities, but the specific mutagenic activity of instant coffees (1559 AraR mutants/mg) was almost 2 times that of noninstant ones (834 AraR mutants/mg). The Ara assay allowed the direct testing of coffee, although it was demonstrated that lyophilization has no effect on the mutagenicity of this beverage. Like roasted coffee, roasted barley induced a large number of AraR mutants per mg (227), though its specific mutagenic activity was approximately 4 and 7 times lower than that of noninstant and instant coffees, respectively.
Arabinose↗
Intergenotypic effect of isopropanol ingestion in the further detoxification of ethanol and isopropanol in Drosophila melanogaster.
The effect of isopropanol ingestion on a further tolerance to ethanol and isopropanol, and its relationship with the Adh locus, have been studied using Drosophila melanogaster selected for tolerance to ethanol. For this purpose, AdhF AdhF, AdhF AdhS and AdhS AdhS flies were independently pretreated with 2 per cent isopropanol and then further exposed to solutions of 10 per cent ethanol or of 2 per cent isopropanol. Afterwards, the ability to tolerate both alcohols, and the ADH activities of the surviving flies were compared with those of flies not pretreated with isopropanol. After isopropanol ingestion, the flies of all three Adh genotypes shown much higher sensitivity to ethanol than to isopropanol although the opposite results were observed in flies not pretreated with isopropanol. Isopropanol treatment decreased the ADH activity in flies of all three genotypes within a range varying from 73 per cent (females FF) to 93 per cent (males FS), the remaining ADH activity being between 2 to 3 times higher in FF than in FS and SS flies. The reduction in ADH activity was associated with the phenomenon of ADH isozyme interconversion. After the isopropanol pretreatment, the most isopropanol tolerant flies (FF) were also the most ADH active ones. Therefore, the adaptative significance of the isozyme conversion is questioned.
1-Propanol↗
Relation between tolerance to ethanol and alcohol dehydrogenase (ADH) activity in Drosophila melanogaster: selection, genotype and sex effects.
The suggestion of Oakeshott et al. (1984) that selection at the Adh locus, as a response to ethanol, is restricted to D. melanogaster laboratory-adapted populations, is tested in this paper with the "Lagar de los Reyes" (LR) lines. For this purpose, homozygous lines for the AdhF and the AdhS alleles were maintained on food supplemented with ethanol. After the selection, the ethanol tolerance and the ADH activity of the selected flies (LRSeF and LRSeS) were determined and compared with those of the control flies (LRCF and LRCS), maintained on standard medium. Then, the effects of the selection, genotype and sex, and the relation between ethanol tolerance and ADH activity were analysed. Our results fail to show a consistent correlation between ethanol tolerance and ADH activity in the adults of LR lines. Our findings also indicate that adaptation of D. melanogaster to ethanol-containing food could be accomplished without significant changes on the ADH activity in the adults. The possibility that the adaptation of D. melanogaster to environmental ethanol could be independent of the Adh locus is discussed.
Alcohol Dehydrogenase↗