RESUMEN
The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.
Asunto(s)
Metilación de ADN , Semen , Islas de CpG , Genética Forense , Humanos , Laboratorios , Análisis de Secuencia de ADNRESUMEN
The VISAGE (VISible Attributes through GEnomics) consortium aims to develop, optimize and validate prototype tools to broaden the use of DNA intelligence methods in forensic routine laboratories. This includes age estimation based on the quantification of DNA methylation at specific CpG sites. Here, we present the VISAGE basic prototype tool for age estimation targeting 32 CpGs from five genes ELOVL2, MIR29B2CHG (herein, MIR29B2C), FHL2, TRIM59 and KLF14. The assay interrogates these well described age markers by multiplex PCR for bisulfite converted DNA and massively parallel sequencing on a MiSeq FGx instrument. We describe protocol optimizations including tests on five bisulfite conversion kits and an evaluation of the assay's reproducibility and sensitivity with artificially methylated DNA standards. We observed robust quantification of methylation levels with a mean standard deviation of 1.4 % across ratios. Sensitivity tests showed no increase of variability down to 20â¯ng DNA input into bisulfite conversion with a median difference below 1.6 % between technical replicates.
Asunto(s)
Envejecimiento/genética , Islas de CpG , Genética Forense/métodos , Metilación de ADN , Elongasas de Ácidos Grasos/genética , Marcadores Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción de Tipo Kruppel/genética , Proteínas con Homeodominio LIM/genética , Reacción en Cadena de la Polimerasa Multiplex , Proteínas Musculares/genética , Reproducibilidad de los Resultados , Factores de Transcripción/genética , Proteínas de Motivos Tripartitos/genéticaRESUMEN
The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology.
Asunto(s)
ADN/análisis , Genética Forense , ARN/análisis , Piel/química , HumanosRESUMEN
The European Forensic Genetics Network of Excellence (EUROFORGEN-NoE) undertook a collaborative project on mRNA-based body fluid/skin typing and the interpretation of the resulting RNA and DNA data. Although both body fluids and skin are composed of a variety of cell types with different functions and gene expression profiles, we refer to the procedure as 'cell type inference'. Nine laboratories participated in the project and used a 20-marker multiplex to analyse samples that were centrally prepared and thoroughly tested prior to shipment. Specimens of increasing complexity were assessed that ranged from reference PCR products, cDNAs of indicated or unnamed cell type source(s), to challenging mock casework stains. From this specimen set, information on the overall sensitivity and specificity of the various markers was obtained. In addition, the reliability of a scoring system for inference of cell types was assessed. This scoring system builds on replicate RNA analyses and the ratio observed/possible peaks for each cell type [1]. The results of the exercise support the usefulness of this scoring system. When interpreting the data obtained from the analysis of the mock casework stains, the participating laboratories were asked to integrate the DNA and RNA results and associate donor and cell type where possible. A large variation for the integrated interpretations of the DNA and RNA data was obtained including correct interpretations. We infer that with expertise in analysing RNA profiles, clear guidelines for data interpretation and awareness regarding potential pitfalls in associating donors and cell types, mRNA-based cell type inference can be implemented for forensic casework.
Asunto(s)
Líquidos Corporales/metabolismo , Conducta Cooperativa , ADN/genética , ARN Mensajero/genética , Piel/metabolismo , Secuencia de Bases , Cartilla de ADN , Europa (Continente) , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology.
Asunto(s)
Sangre , ADN/genética , Menstruación , ARN/genética , Vagina/metabolismo , Líquidos Corporales/metabolismo , Femenino , HumanosRESUMEN
A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 µl saliva, 5-0.01 µl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 µl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies.
Asunto(s)
ADN/análisis , ARN/análisis , Saliva/química , Semen/química , ADN/genética , Electroforesis Capilar , Humanos , Reacción en Cadena de la Polimerasa , ARN/genéticaRESUMEN
A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 µl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.
Asunto(s)
ADN/sangre , ARN/sangre , Conducta Cooperativa , Humanos , Repeticiones de Microsatélite/genética , Reacción en Cadena de la PolimerasaRESUMEN
A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using different kits, chemistries and instrumentation. The results demonstrate that HBB is expressed abundantly in blood, SPTB moderately and PBGD significantly less. All but one of the 16 participating laboratories were able to successfully isolate and detect RNA from the dried bloodstains even though a majority of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative to conventional serological tests.
Asunto(s)
Manchas de Sangre , Dermatoglifia del ADN/métodos , ARN Mensajero/sangre , Población Blanca/genética , Biomarcadores/sangre , Conducta Cooperativa , Dermatoglifia del ADN/instrumentación , Electroforesis Capilar , Humanos , Hidroximetilbilano Sintasa/análisis , Límite de Detección , Técnicas de Amplificación de Ácido Nucleico , ARN/sangre , ARN/aislamiento & purificación , ARN Mensajero/química , Juego de Reactivos para Diagnóstico/estadística & datos numéricos , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad , Espectrina/análisis , Globinas beta/análisisRESUMEN
The codling moth (Cydia pomonella L., Tortricidae, Lepidoptera) is an important pest of pome fruit with global distribution. It has adapted successfully to different habitats by forming various ecotypes and populations, often termed strains, which differ among each other in several morphological, developmental, and physiological features. Many strains of Cydia pomonella have developed resistance against a broad range of chemically different pesticides. Obviously, pesticide-resistant strains must have a genetic basis inherent to the gene pool of codling moth populations, and this deserves our particular attention. The primary intention of the present study was to contribute novel information regarding the evolutionary phylogeny and phylogeography of codling moth populations in Central Europe. In addition, we aimed at testing the hypothesis that differential biological traits and response patterns towards pesticides in codling moth populations may be reflected at a mitochondrial DNA level. In particular, we wanted to test if pesticide resistance in codling moths is associated repeatedly and independently with more than one mitochondrial haplotype. To this end, we analyzed mitochondrial DNA and constructed phylogenetic trees based on three mitochondrial genes: cytochrome oxidase I (COI), the A+T-rich region of the control region (CR), and the nicotinamide adenine dinucleotide dehydrogenase subunit 5 (ND5). The results indicate that Central European populations of Cydia pomonella are clearly divided in two ancient clades. As shown by means of a molecular clock approach, the splitting of the two clades can be dated to a time period between the lower and middle Pleistocene, about 1.29-0.20 million years ago. It is assumed that the cyclic changes of warm and cold periods during Pleistocene may have lead to the geographic separation of codling moth populations due to glaciation, giving rise to the formation of the two separate refugial clades, as already shown for many other European animal species. Due to their inclination towards developing novel detoxification gene variants, codling moth individuals from both clades independently and multifariously may have developed pesticide resistance, and this process may be ongoing. During their more recent evolutionary history, natural events such as the gradual disappearance of climate-specific geographic barriers, as well as human-aided dispersal in recent historic times, may have allowed codling moth haplotypes from the original clades to interbreed and completely merge again, creating a globally successful insect species with a gene pool capable of responding to novel selective challenges by rapid adaptation.
Asunto(s)
Haplotipos , Mitocondrias/genética , Mariposas Nocturnas/genética , Animales , Evolución Biológica , ADN Mitocondrial/genética , Diflubenzurón/farmacología , Europa (Continente) , Evolución Molecular , Marcadores Genéticos , Genotipo , Modelos Genéticos , Filogenia , Análisis de Secuencia de ADNRESUMEN
We investigated 15 polymorphic short tandem repeat (STR) loci (D1S1656, D7S1517, D8S306, D8S639, D9S304, D10S2325, D11S488, D12S391, D14S608, D16S3253, D17S976, D18S1270, D19S253, D20S161, and D21S1437) which are not included in the standard sets of forensic loci. The markers were selected according to the complexity of the polymorphic region: Of the 15 investigated loci, 7 loci showed a simple repeat structure (D9S304, D10S2325, D14S608, D16S3253, D18S1270, D19S253, and D21S1437), 3 loci (D7S1517, D12S391, and D20S161) consisted of compound repeat units, and 5 loci (D1S1656, D8S306, D8S639, D11S488, and D17S976) showed a more complex polymorphic region partly including different repeat blocks and incomplete repeat units, which resulted in a relatively high proportion of intermediate alleles. A population study on a sample of 270 unrelated persons from Austria was carried out. We did not observe significant deviations from Hardy-Weinberg expectations. The combined probability of exclusion for the 15 loci was 0.99999998. In combination with the conventional set of STR markers included in commercially available kits (no linkage was observed between these 15 loci and the Powerplex 16 System loci), these markers are approved as highly discriminating forensic tools, also suitable for the analysis of difficult paternity and kinship constellations.
Asunto(s)
Dermatoglifia del ADN , Repeticiones de Microsatélite/genética , Paternidad , Adolescente , Adulto , Austria , Niño , Femenino , Frecuencia de los Genes , Humanos , Funciones de Verosimilitud , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Polimorfismo Genético/genética , Valor Predictivo de las Pruebas , Población Blanca/genéticaRESUMEN
A multiplex PCR was designed for the loci D2S1338, D16S539, D18S51, TH01 and FGA using redesigned primers in order to reduce the lengths of the amplification products compared to the designs used in commercially available multiplex PCR kits, also including amelogenin. The new PCR primers were used to amplify highly degraded DNA from casework samples, which had shown no or only poor results for these loci in previous analyses with standard primer sets. The application of the new miniSTR-multiplex resulted in an increased overall typing success rate for degraded DNA samples. In a concordance study between the conventional and the newly designed primers, no genotype differences were revealed in 124 randomly selected individuals.
Asunto(s)
Degradación Necrótica del ADN , Dermatoglifia del ADN/métodos , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetidas en Tándem , Amelogenina/genética , Cartilla de ADN , Desoxirribonucleasa I , HumanosRESUMEN
Unusually large variant alleles were observed in the short tandem repeat (STR) systems D3S1358 and D21S11, both of which are included in the international standard set of loci (ISSOL) and routinely typed in National DNA intelligence databases worldwide. The observed alleles fell within the size range of the adjacent STR marker, which could easily cause problems with respect to correct allele assignments for both loci concerned. We compared the amplification and potential interpretation with three different commercially available kits, which are frequently used in forensic work. PCR products were cloned and sequenced in order to determine the structure of these unusual allele variants and confirm their size and designation (D3S1358 allele 26, D21S11 allele 46). In the locus D21S11 we observed an as yet undescribed partial duplication of the constant region.
Asunto(s)
Alelos , Variación Genética , Secuencias Repetidas en Tándem , Dermatoglifia del ADN , Duplicación de Gen , Humanos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
The vacuolar ATPase is a multifunctional enzyme that consists of several subunits. Subunit B is part of the catalytic domain of the enzyme and is present in two isoforms in fish as well as in mammals. Possibly, these two isoforms - vatB1 (kidney isoform) and vatB2 (brain isoform) - serve different functions. A localization of the two isoforms was attempted in swimbladder gas gland cells of the European eel Anguilla anguilla by immunohistochemistry. Two antibodies were produced by immunization of rabbits with synthetic peptides. Specificity of the antibodies, on the one hand, an isoform-specific antibody for vatB1 and, on the other hand, an antibody that recognizes both isoforms (vatB1 and vatB2), was confirmed by western blot analysis using recombinant proteins produced in a bacterial expression system. The immunohistochemical localization with the antibody directed against both isoforms of the B subunit revealed a positive staining in apical membranes of swimbladder gas gland cells as well as in the basolateral membranes. Significant staining was observed in vesicles located near the apical membrane. Staining with the vatB1-specific antibody resulted in a similar picture in the apical region of the cells. In contrast to the staining with the first antibody, only a poor signal was observed in the basal region. The nature of the vesicles in the apical region of the gas gland cells was determined by using an antibody directed against surfactant protein D.
Asunto(s)
Sacos Aéreos/enzimología , Anguilla/fisiología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Sacos Aéreos/química , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/inmunología , Inmunohistoquímica , Isoenzimas/inmunología , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Sistema Respiratorio/metabolismo , ATPasas de Translocación de Protón Vacuolares/inmunologíaRESUMEN
Determination of sex using the amelogenin sex test is well established in the forensic field especially for casework and DNA databasing purposes. The sex test is part of commercially available PCR kits. Among 29,432 phenotypic male individuals stored in the Austrian National DNA database, 6 individuals were found to lack the amelogenin Y-specific PCR product which was confirmed using alternative amelogenin primers. The amplification of eight Y-chromosomal STR markers resulted in full profiles in five out of the six samples, one sample failed to amplify Y-STRs at all. The amplification of a fragment of the SRY gene gave positive results in all six samples, confirming the male phenotype of the individuals. The observed failure rate of the amelogenin sex test was 0.018% in this study.
Asunto(s)
Proteínas del Esmalte Dental/genética , Análisis para Determinación del Sexo , Amelogenina , Austria , Bases de Datos de Ácidos Nucleicos , Medicina Legal , Humanos , Masculino , Reproducibilidad de los Resultados , Cromosoma YRESUMEN
Gas gland cells of the European eel (Anguilla anguilla) were cultured on collagen-coated coverslips, and intracellular pH was measured using the pH-sensitive fluorescent probe 2',7'-bis-(2-carboxypropyl)-5-(6)-carboxyfluorescein (BCPCF). The contributions of various proton-translocating mechanisms to homeostasis of intracellular pH (pHi) were assessed by adding specific inhibitors of the various proton-translocating mechanisms at a constant extracellular pH (pHe) of 7.4 and after artificial acidification of the cells using the ammonium pulse technique. The greatest decrease in pHi was observed after addition of 5-(N-ethyl-N-isobutyl)-amiloride (MIA), an inhibitor of Na(+)/H(+) exchange. Na(+)/H(+) exchange was active under steady-state conditions at an extracellular pH of 7.4, and activity increased after intracellular acidification. Incubation of gas gland cells with 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of anion exchange, also caused a decrease in pHi, but this decrease was not as pronounced as in the presence of MIA. Furthermore, at low pHi, the effect of DIDS was further reduced, suggesting that bicarbonate-exchanging mechanisms are involved in maintaining a steady-state pHi but that their importance is reduced at low pH. Bafilomycin A(1), a specific inhibitor of the V-ATPase, had no effect on steady-state pHi. However, recovery of intracellular pH after an artificial acid load was significantly impaired in the presence of bafilomycin. Our results suggest that Na(+)/H(+) exchange and anion exchange are important for the regulation of pHi at alkaline values of pHe. When pHi is low, a situation probably often encountered by gas gland cells during gas secretion, Na(+)/H(+) exchange continues to play an important role in acid secretion and a V-ATPase appears to contribute to proton secretion.
Asunto(s)
Sacos Aéreos/fisiología , Amilorida/análogos & derivados , Concentración de Iones de Hidrógeno , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Sacos Aéreos/citología , Amilorida/farmacología , Anguilla , Animales , Aniones , Bicarbonatos/metabolismo , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes , Homeostasis , Cinética , Cloruro de Sodio/farmacología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidoresRESUMEN
Y-chromosomal short tandem repeats (Y-STRs) are useful forensic DNA markers in investigation of sexual assault cases when a mixture of male and female DNA (e.g., in vaginal swabs) is present in a sample, especially when DNA of the male contributor is present only in very small amount compared to the DNA of the female victim. With autosomal STR analysis of male and female DNA, male DNA in mixtures can usually be detected and correctly interpreted only when it exceeds 5%. However, the amplification of some Y-STRs is known to result in polymerase chain reaction (PCR) products that are not associated with the Y-chromosome, but derive from the X-chromosome and/or autosomal regions. This can cause problems in the interpretation of results, particularly when female DNA is present in excess. Consequently, more specific and sensitive Y-STR primers and PCR conditions are needed. This paper presents two casework examples in which sensitive Y-STR multiplexes (with the addition of PCR enhancer) were successfully used in the analysis of mixtures of male and female DNA, the male component not interpretable by standard autosomal STR typing.
Asunto(s)
Dermatoglifia del ADN/métodos , Reacción en Cadena de la Polimerasa/métodos , Delitos Sexuales , Frotis Vaginal/normas , Adolescente , Secuencia de Bases , Femenino , Medicina Legal/métodos , Humanos , Labio/citología , Masculino , Datos de Secuencia Molecular , Sensibilidad y Especificidad , Acoso Sexual , Secuencias Repetidas en Tándem , Cromosoma YRESUMEN
The poly(A)(+) RNA of swimbladder gas gland cells of the European eel Anguilla anguilla was isolated and used for cDNA synthesis. Using a pair of degenerate PCR primers directed towards the evolutionary highly conserved central part of the B subunit of vacuolar type H(+)-ATPase (V-ATPase) a fragment of 388 bp was amplified. By sequencing the cloned PCR products two different amplicons with a sequence identity of about 86% were obtained. BLASTN searches revealed a high degree of similarity of both to V-ATPase B subunits of other species. The sequences were completed by performing rapid amplification of cDNA ends PCR, subsequent cloning, and sequencing of the obtained products. The expression of two different isoforms of the V-ATPase B subunit is already demonstrated for Homo sapiens and Bos taurus. This is the first report that attributes the same phenomenon to a non-mammalian species, A. anguilla. The first isoform found in eel (vatB2) shows the highest degree of amino acid sequence homology with the human brain isoform (98.2%), the second one (vatB1) with the B subunit sequence of rainbow trout (Oncorhynchus mykiss) gill and kidney (98, 6%). The alignment of the deduced amino acid sequences of vatB1 and vatB2 shows that the highest sequence variation between these two isoforms is found at the amino-terminus, where vatB1 is nine amino acids shorter than vatB2, while at the carboxy-terminus it is two amino acids longer than vatB2. This has also been reported for the human and bovine kidney isoforms when compared with the brain isoforms. Northern blot analysis using specific hybridization probes revealed the expression of two mRNA's with lengths of about 2.9 kb and 3.5 kb for vatB1 and vatB2, respectively. For mammals, it is well known that V-ATPases containing the kidney isoforms of the B subunit are responsible for the extrusion of protons across the plasma membranes of several cell types. The fact that eel vatB1 seems to share structural features with the kidney isoforms in mammals supports the hypothesis that in gas gland cells a V-ATPase contributes to the acidification of the blood in the swimbladder.
Asunto(s)
Adenosina Trifosfatasas/genética , Anguilas/genética , Adenosina Trifosfatasas/química , Sacos Aéreos/citología , Sacos Aéreos/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/química , Anguilas/metabolismo , Isoenzimas/química , Isoenzimas/genética , Datos de Secuencia Molecular , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Vacuolas/enzimologíaRESUMEN
Species identification was carried out by nucleotide sequence analysis of the cytochrome b (cytb) gene. The aim of the study was to identify biological specimens from diverse vertebrate animals by extracting and amplifying DNA from 44 different animal species covering the 5 major vertebrate groups (i.e. mammals, birds, reptiles, amphibians and fishes). The sequences derived were used to identify the biological origin of the samples by aligning to cytb gene sequence entries in nucleotide databases using the program BLAST. All sequences were submitted to the GenBank including new species which were not observed in the databases. The applicability of this method to the forensic field is demonstrated by simulated casework conditions where different types of samples including problematic specimens such as hair, bone samples, bristles and feathers were investigated to identify the species.
Asunto(s)
Grupo Citocromo b/genética , Antropología Forense/métodos , Especificidad de la Especie , Vertebrados/genética , Animales , ADN Mitocondrial , Toma de Decisiones Asistida por Computador , Biblioteca de Genes , Humanos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Species of the family Tubificidae represent a major faunal element in benthic freshwater communities throughout the world. Some of them are considered particularly tolerant of the influence of toxicants such as cadmium. One of the most abundant species, "Tubifex tubifex," is frequently used as an indicator of environmental pollution, despite considerable taxonomic problems caused by phenotypic plasticity and genetic heterogeneity. Our study provides a phylogeny of "T. tubifex" based on a segment of the mitochondrial 16S rDNA and presents a rapid PCR-based method of genotype screening which was then applied in cadmium toxicity studies on natural populations. Phylogenetic analysis identified five major mitochondrial lineages, some of them separated by large genetic distances (up to 13%) but morphologically indistinguishable, thus highly suggestive of the existence of cryptic species. All lineages were present at different frequencies in the European river populations studied, with a tendency of the more resistant lineages to occur at higher frequencies in the more tolerant populations. In fact, lineage-specific toxicity experiments showed that individuals of different mitochondrial lineages consistently varied in cadmium resistance, suggesting that in benthic oligochaetes, evolution seems to proceed predominantly through natural selection acting on physiological, rather than morphological, characters. In consequence, toxicological studies involving "T. tubifex" as a monitoring or test organism should allow for the possibility of genetic inhomogeneity of this mudworm group by combining both toxicological and genetic methods.
Asunto(s)
Cadmio/toxicidad , ADN Mitocondrial/genética , Oligoquetos/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Oligoquetos/genética , Filogenia , Especificidad de la EspecieRESUMEN
The pH dependence of acid production and of acid release has been analyzed in cultured gas gland cells of the European eel using a cytosensor microphysiometer. Total acid release of gas gland cells showed an optimum at pH 7.4-7.6, with only a minor reduction at acidic (pH 7.0) as well as at alkaline pH (pH 8.0). The acid production was largely dependent on the availability of extracellular glucose and was almost completely abolished if glucose was replaced by succinate, alanine, or even pyruvate. Phloretin, an inhibitor of glucose uptake, significantly reduced acid release of gas gland cells with a Ki of approximately 1 x 10(-5) M, irrespective of pH. Although the glucose dependence of acid production was not modified by pH, acid release became increasingly sodium dependent with decreasing pH, but at low pH significantly higher sodium concentrations were necessary to achieve maximal rate of proton secretion. This sodium-dependent proton secretion could only in part be inhibited by application of 5-(N-methyl-N-isobutyl)-amiloride. Removal of extracellular potassium caused a slow reduction in the rate of acid secretion. A similar reduction was observed in the presence of ouabain, a specific inhibitor of Na(+)-K(+)-adenosinetriphosphatase, and both effects significantly increased with decreasing pH. The results demonstrate a significant pH dependence of the mechanisms of acid release in swim bladder gas gland cells and indicate that sodium-dependent pathways become especially important at low pH.