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PURPOSE: Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is an inhibitor of T-cell activation, regulating intracellular signal transduction and thereby being implicated in the pathogenesis of autoimmune thyroid disease (AITD). The exact molecular mechanisms have not been fully elucidated. The aim of the present study was to quantitate DNA methylation within the PTPN22 gene promoter in children and adolescents with AITD and healthy controls. METHODS: 60 Patients with Hashimoto thyroiditis (HT), 25 patients with HT and type 1 diabetes (HT + T1D), 9 patients with Graves' disease (GD) and 55 healthy controls without any individual or family history of autoimmune disease were enrolled. Whole blood DNA extraction, DNA modification using sodium bisulfate and quantification of DNA methylation in the PTPN22 gene promoter, based on melting curve analysis of the selected DNA fragment using a Real-Time PCR assay, were implemented. RESULTS: DNA methylation in the PTPN22 gene promoter was found to be significantly higher in HT patients (39.9 ± 3.1%) in comparison with other study groups (20.3 ± 2.4% for HT + T1D, 32.6 ± 7.8% for GD, 27.1 ± 2.4% for controls, p < 0.001). PTPN22 gene promoter DNA methylation was also associated marginally with thyroid autoimmunity in general (p = 0.059), as well as considerably with thyroid volume (p = 0.004) and the presence of goiter (p = 0.001) but not thyroid function tests. CONCLUSIONS: This study demonstrates for the first time that a relationship between autoimmune thyroiditis and PTPN22 gene promoter DNA methylation state is present, thus proposing another possible etiological association between thyroiditis and abnormalities of PTPN22 function. Further expression studies are required to confirm these findings.
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Biomarcadores/análisis , Metilación de ADN , Predisposición Genética a la Enfermedad , Enfermedad de Hashimoto/diagnóstico , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Adolescente , Estudios de Casos y Controles , Niño , Femenino , Estudios de Seguimiento , Grecia/epidemiología , Enfermedad de Hashimoto/epidemiología , Enfermedad de Hashimoto/genética , Humanos , Masculino , PronósticoRESUMEN
BACKGROUND: In women with recurrent miscarriages, up to 50 % of those cases remain unexplained. In this study, we evaluated the impact of Cytosine/Guanine/Guanine (CGG) trinucleotide expansions of the fragile-X mental retardation 1 (FMR1) gene in women with unexplained recurrent miscarriages. METHODS: This is a prospective case-control pilot study involving 49 women with unexplained recurrent miscarriages and 49 age-matched controls with documented fertility. The case group consisted of women with a history of two or more consecutive miscarriages, in whom no known factor could be identified. The maximum age of recruitment was 40 years. We obtained blood samples that were checked, using polymerase chain reaction with electrophoresis, for the presence of expanded alleles of the FMR1 gene. We further evaluated using sequencing analysis, those women marked as positive. We set the limit at more than 40 repeats. RESULTS: The repeat sizes of CGG expansion in the FMR1 gene differ significantly in the two population groups (p =0.027). We found four women in the miscarriage group and one in the control group positive for carrying premutation alleles (Odds ratio: 4.267, confidence interval: 0.459-39.629). All the positive cases involved intermediate zone carriers. We found no association between the number of abortions each woman had, and her respective CGG repeat number (p =0.255). CONCLUSIONS: Many couples are desperately looking for the cause of their recurrent miscarriage suffering. The CGG expanded allele of the FMR1 gene is possibly to be blamed in some of these cases. More studies are needed to support the results of this prototype study. HIPPOKRATIA 2018, 22(3): 132-136.
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BACKGROUND: Recent studies provide evidence that peripheral blood monocytes have the ability to differentiate into mesenchymal-like cells. The ability of cultured monocytes to differentiate and produce insulin in vitro is analysed in the present study. METHODS: Peripheral blood monocytes were isolated from healthy donors and cultivated for fourteen days. Growth factors and liraglutide were used to induce pancreatic differentiation in most of the cultures. The growth factors were: monocyte colony-stimulating factor, interleukin-3, hepatocyte growth factor and epidermal growth factor. The rest of the cultures were cultivated only with nutrient medium and human serum. Insulin levels were measured by an enzyme-linked immunosorbent assay. Cellular morphology was observed using optical and electron microscopy. Cell membrane receptors were detected by flow cytometry. RESULTS: Monocytes were able to synthesize and excrete high levels of insulin after seven days in culture. A further increase in the excretion of insulin was observed after fourteen days. Cells were also able to differentiate and synthesize insulin, even if no growth factors were added to the culture medium. Some of the cultures were able to excrete insulin in a glucose-dependent manner. Differentiated monocytes were connected to neighbouring cells with axons and resembled the morphology of mesenchymal, dendritic and myeloid-progenitor cells. Cells retained their mature receptors and simultaneously developed immature receptors on their membrane. CONCLUSIONS: Monocytes can acquire morphological properties of multipotent cells when they are cultivated under specific conditions in vitro. Differentiated monocytes are able to synthesize and excrete insulin. Hippokratia 2015; 19 (4): 344-351.
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OBJECTIVES: To evaluate the role of HPV in oral carcinogenesis, we examined the prevalence of HPV in malignant, potentially malignant and normal oral epithelium and studied the relation of HPV prevalence with other factors obtained from the patient's records. MATERIALS AND METHODS: Our material consisted of 291 tissue specimens from 258 individuals. From every individual formalin fixed and paraffin embedded tissues were examined by nested Polymerase Chain Reaction (NPCR) for the detection of HPV DNA and by immunohistochemistry (IHC) for the in situ detection of HPV L1 protein. Positive PCR products were sequenced in order to type HPVs. Also 33 fresh tissues were obtained, fixed and used to detect HPV particles by transitional electron microscopy (TEM). RESULTS: HPV was detected in 32.9% of the tissue specimens by NPCR, in 4.7% by immunohistochemistry and in 28.1% by TEM. In detail, by nested PCR HPV L1 DNA was detected in 40% of normal tissues, 40% of fibromas, 35.8% of non-dysplastic leukoplakias, 31.6% of dysplastic leukoplakias and 22.2% of oral squamous cell carcinomas. The HPV viral load of 96.5% of the samples was very low (1 viral copy per 10(2)-10(4) cells). HPV16 prevails in all histological groups in 89-100%. CONCLUSION: We conclude that HPV does not seem, from the specific sample examined, to play a substantial role in oral carcinogenesis. However, it cannot be excluded that HPV could be involved in oral carcinogenesis only in cases with high viral load or at early stages of carcinogenesis possibly through the hit-and-run mechanism.
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Alphapapillomavirus/aislamiento & purificación , Mucosa Bucal/virología , Neoplasias de la Boca/virología , Reacción en Cadena de la Polimerasa/métodos , Alphapapillomavirus/clasificación , Alphapapillomavirus/genética , ADN Viral/análisis , Femenino , Grecia , Humanos , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Carga ViralRESUMEN
BACKGROUND: fl/d3 polymorphism in human GH receptor was correlated with the response to GH therapy in different groups of children with short stature. AIM: This is a 2-yr retrospective study which evaluates the influence of fl/d3 polymorphism to the 1st-and 2nd-year response to GH replacement therapy in Greek children with isolated GH deficiency (GHD). SUBJECTS AND METHODS: A total number of 195 pre-pubertal Greek children were studied (121 controls and 74 patients with GH peak <10 ng/ml). Patients with deficiency were treated with exogenous GH at a mean dose of 28.8 µg/kg.d. Multiplex PCR was used to genotype all children for fl/d3 polymorphism, followed by statistical analysis. The main parameters which were used to assess the association of genotype with the response to GH replacement were height SD score (SDS), height gain SDS, and growth velocity (GV) expressed as cm/yr and SDS. RESULTS: Our results revealed that the frequency of d3-homozygosity in the Greek population was 8.26%. No association was detected between the presence or abcense of GHD and genotype. Moreover, no connection between genotype and sex was observed. First-year height SDS, height gain SDS, and GV SDS were significantly higher in d3-carriers (p<0.05). However, this difference did not appear in the 2nd year of treatment. CONCLUSIONS: In our study, the d3-polymorphism seems to be associated with a higher efficacy to GH replacement, at least at the beginning of the treatment.
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Trastornos del Crecimiento/tratamiento farmacológico , Trastornos del Crecimiento/genética , Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/uso terapéutico , Polimorfismo Genético , Receptores de Somatotropina/genética , Proteínas Recombinantes/uso terapéutico , Alelos , Estatura/genética , Niño , Femenino , Genotipo , Grecia , Humanos , Masculino , Receptores de Somatotropina/metabolismoRESUMEN
BACKGROUND: HPV16 E6/E7 oncoproteins are critical for cervical carcinogenesis. The corresponding oncogenes are also detected in head and neck cancer, but in lung cancer their presence is strongly debated. PCR-based detection protocols amplify different target sequences. OBJECTIVES: To examine the frequency of different length HPV16 E7 segments in lung carcinomas. STUDY DESIGN: We designed four different amplification schemes for the detection of overlapping segments of the HPV16 E7 ORF, all suitable for specific HPV detection in cervical carcinoma. In two schemes, the entire E7 ORF was targeted while in the remaining schemes internal, smaller sequences were targeted. In total, 76 specimens were used; 29 lung carcinoma specimens, 16 non-cancerous lung tissue specimens from the same patients and 31 bronchial washings from different lung cancer patients. RESULTS: Amplification of the entire HPV16 E7 ORF, using two protocols, demonstrated the absence of the specific HPV16 E7 sequences (74 samples either tested negative by the first PCR protocol or false positive by the second, based on sequencing or AvaII or PvuII digestion). However, both schemes targeting smaller E7 segments revealed the frequent presence of HPV16 E7 sequences in lung carcinoma specimens (14/23 positive by either scheme). CONCLUSIONS: HPV16 E7 sequences are frequently observed in lung carcinomas. Decreasing the size of PCR-target sequences increases the detection frequency, possibly indicating the presence of incomplete viral ORFs. Restriction endonuclease analysis is critical for verifying the reliability of the detection of these sequences.
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Carcinoma/virología , Papillomavirus Humano 16/genética , Neoplasias Pulmonares/virología , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/virología , Carcinoma/patología , Humanos , Neoplasias Pulmonares/patología , Patología Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , PrevalenciaRESUMEN
The aim of this prospective study was to analyze the expression of messenger RNA of genes, such as MDR1, MRP1, BCRP, and LRP, implicated in the mechanism of multidrug resistance (MDR) in relation to the response to induction chemotherapy and relapse and these genes' correlation with each other and with pretreatment laboratory and clinical characteristics. We prospectively studied 49 children (26 boys and 23 girls) with acute lymphoblastic leukemia (ALL) (median age, 5.5 years; range, 15 months to 12.5 years) who were treated with the BFM95 chemotherapy protocol. We used bone marrow mononuclear cells from 7 healthy children as controls. The expression of MDR genes and the beta-actin housekeeping gene was detected by the reverse transcription-polymerase chain reaction with the appropriate primers. The mean expression of each MDR gene was significantly higher in the patients than in the control group (P < .01). We found statistically significant correlations between MRP1 and LRP expression and between MRP1 or LRP expression and MDR1 expression (P < .05). High expression for the MDR1 gene was found in 18 patients (36.7%), and their prognoses were significantly worse than those with low expression (event-free survival, 55.56% versus 86.67%; P = .03, log-rank test). Expression of each of the MDR genes was independent of the initial white blood cell count, immunophenotype, National Cancer Institute risk classification, and prednisone response. Interestingly, MDR1 expression was significantly higher at relapse than at diagnosis for 4 sample pairs. Evaluation of MDR1 expression at diagnosis of childhood ALL may contribute to the early identification of patients at risk of treatment failure.
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Transportadoras de Casetes de Unión a ATP/genética , Resistencia a Múltiples Medicamentos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Partículas Ribonucleoproteicas en Bóveda/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Pronóstico , Estudios Prospectivos , ARN Mensajero/análisis , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Gene targeting is a powerful method for introducing mutations into the genome of embryonic stem cells. The most widely used approach is the positive-negative selection method in which a gene encoding a negative selection marker is cloned into the replacement vector to obtain an enrichment of properly targeted clones. Here, we present an alternative means to introduce any given negative selection marker at the ends of a replacement vector using a single ligation step, thereby avoiding laborious cloning procedures. Our results demonstrate that this fast and simple method consistently provides a high level of enrichment of appropriately targeted clones.
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ADN Ligasas/metabolismo , Marcación de Gen/métodos , Marcadores Genéticos , Vectores Genéticos , Selección Genética , Animales , Clonación Molecular/métodos , Factor 5 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células MadreRESUMEN
In contrast to sympathetic and sensory neurons in the peripheral nervous system, the neurotrophic requirements for neurons in the central nervous system (CNS) have not been clearly identified. The inactivation of specific neurotrophic factors and their receptors by gene targeting has shown that there are no major changes in neuron numbers in the CNS. This suggests an overlap between the action of different neurotrophic factors in the brain during development. Here we have studied the survival of hippocampal neurons prepared from embryonic rats using different culture conditions. Whereas the hippocampal neurons survive well in culture when plated at high density, they die at lower cell densities in the absence of appropriate neurotrophic factors. Under the latter conditions, both insulin-like growth factor-1 (IGF-1) and neurotrophins - brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) - rescued a large proportion of cultured neurons. In addition, hippocampal neurons from BDNF knockout mice exhibited enhanced cell death compared with cells from wild-type animals. BDNF and IGF-1 both increased the survival of the hippocampal neurons lacking BDNF, showing complementary action for these factors in supporting survival. Blocking antibodies against NT-3 and IGF-1 decreased hippocampal neuron survival at low cell densities, showing autocrine or paracrine action of the factors. At higher cell densities, however, the antibodies had no effect, demonstrating that there is a sufficient amount of endogenous factors in supporting survival. Blocking antibodies against NT-3 and IGF-1 decreased hippocampal neurons depend for survival on local neurotrophic factors such as IGF-1, BDNF and NT-3, which act in an autocrine/paracrine manner. The multifactorial support of hippocampal neurons ensures a maximal degree of neuron survival even in the absence of an individual factor
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Hipocampo/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Factores de Crecimiento Nervioso/fisiología , Neuronas/citología , Animales , Factor Neurotrófico Derivado del Encéfalo/fisiología , Recuento de Células , Supervivencia Celular/fisiología , Células Cultivadas , Sinergismo Farmacológico , Hipocampo/citología , Ratones , Ratones Noqueados , Neurotrofina 3 , RatasRESUMEN
Overexpression of the proto-oncogene bcl-2 blocks programmed cell death in sympathetic and sensory neurons that normally die after the withdrawal of neurotrophic factors. The role of endogenous bcl-2 in the development and function of the peripheral and central nervous system is, however, not known. We have found that low levels of bcl-2 messenger RNA are widely distributed in the adult and developing rat brain. In situ hybridization localized bcl-2 messenger RNA in mitral cells of the olfactory bulb, granule and pyramidal neurons of hippocampus, pontine nuclei, cerebellar granule neurons, and in ependymal cells in adult rat brain. bcl-2 messenger RNA levels were higher in late prenatal development than in postnatal and adult brain. High levels of bcl-2 messenger RNA were expressed in the neuroepithelium and in the cortical plate in prenatal cortex. During postnatal development the distribution of the message resembled that found in adult brain. We have also tested the hypothesis that induction of bcl-2 messenger RNA expression might be part of the survival-promoting action of neurotrophic factors. Brain-derived neurotrophic factor, which supports survival of cultured cerebellar granule neurons, failed to influence the levels of bcl-2 messenger RNA in these cultures.
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Encéfalo/metabolismo , Neuronas/metabolismo , ARN Mensajero/biosíntesis , Envejecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Muerte Celular/fisiología , Factor Neurotrófico Ciliar , Regulación de la Expresión Génica , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/fisiología , Sondas ARN , Ratas , Ratas WistarRESUMEN
Fibroblast growth factor-5 (FGF-5) is a member of the fibroblast growth factor gene family, which has a signal sequence characteristic of secretory proteins. FGF-5 mRNA has previously been shown to be present in the adult mouse brain. Here we demonstrate that recombinant FGF-5 has neurotrophic activity on cultured rat septal cholinergic and raphe serotonergic neurons. The effect of FGF-5 on serotonin uptake was stronger than that evoked with either brain-derived neurotrophic factor or neurotrophin-3. FGF-5 also increased the choline acetyltransferase activity of cultured rat septal cholinergic neurons, the effect being additive to that of nerve growth factor. In situ hybridization experiments and immunohistochemistry using a specific anti-FGF-5 antibody demonstrated that FGF-5 is expressed in rat hippocampal neurons. Like nerve growth factor mRNA, the levels of FGF-5 mRNA in the rat hippocampus increased substantially during early postnatal development. In addition, injection of the muscarinic receptor agonist pilocarpine elevated FGF-5 mRNA. The presence of the secretory FGF-5 in the rat hippocampus, a target field of septal cholinergic and raphe serotonergic neurons, suggests that FGF-5 acts as a trophic factor for these neurons also in vivo.
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Encéfalo/citología , Factores de Crecimiento de Fibroblastos/biosíntesis , Factores de Crecimiento de Fibroblastos/farmacología , Sustancias de Crecimiento/farmacología , Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Núcleos del Rafe/citología , Serotonina/metabolismo , Envejecimiento/metabolismo , Animales , Encéfalo/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colina O-Acetiltransferasa/metabolismo , Embrión de Mamíferos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor 5 de Crecimiento de Fibroblastos , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotrofina 3 , ARN Mensajero/metabolismo , Núcleos del Rafe/efectos de los fármacos , Núcleos del Rafe/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacologíaRESUMEN
Glutamate dehydrogenase (GLUD) is a key metabolic enzyme of the mitochondrion, playing an important role in mammalian neuronal transmission. GLUD deficiency has been associated with certain forms of neurodegeneration in the human cerebellum. Genomic DNA blot hybridization analysis and identification of a large number of GLUD-specific genomic clones have suggested that human GLUD is encoded by a multigene family consisting of at least six members. A functional GLUD gene, GLUD1, has been mapped to chromosome 10q22.3-23 and a full-length "processed" GLUD gene, GLUDP1, to chromosome Xq22-23. In the context of studing the structure, the role, and the chromosomal organization of the other family members, we have analysed in detail, a cosmid clone solely reactive with the 3' region of the GLUD cDNA. Structure and expression analysis of its GLUD-specific region suggests that it represents a truncated "processed" GLUD pseudogene. Fluorescence in situ hybridization using the entire cosmid as a probe, mapped this GLUD gene locus, termed GLUDP5, to chromosome 10p11.2.
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Cromosomas Humanos Par 10 , Glutamato Deshidrogenasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cósmidos , Femenino , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Masculino , Datos de Secuencia Molecular , Familia de Multigenes , Sondas de Oligonucleótidos , Placenta , Seudogenes , ARN Mensajero/análisis , Análisis de Secuencia de ADN , TransfecciónRESUMEN
Glutamate dehydrogenase is a mitochondrially located, key metabolic enzyme. In addition to its general metabolic role, GLUD is important in neurotransmission. Significant alterations in GLUD enzymatic activity have been associated with certain neurodegenerative human disorders. Although a single species of human GLUD cDNA molecule has been identified so far, both genomic DNA Southern and cytogenetic analyses have indicated the presence of a GLUD gene family. Screening of a human genomic lambda-phage library with the GLUD cDNA, led us to the isolation of several clones divided into five structurally distinct contigs. We have confirmed the presence of all GLUD-specific sequences in the human genome by detailed genomic Southern analysis. This study allowed the identification of the entire functional GLUD gene, named GLUD1. The GLUD1 gene is about 45 kb long and it is organized into 13 exons. Its nucleotide sequence, exon-intron boundaries, and transcription start sites were determined. Potential binding sites for various regulatory factors such as Sp1, AP-1, and AP-2 were recognized at the promoter region of the gene. The members of the other contigs showed an organization clearly different from GLUD1. Two distinct GLUD-specific gene loci, termed GLUDP2 and GLUDP3, possibly represent truncated pseudogenes. Their high degree of similarity to GLUD1 is limited to the region surrounding exons 2, 3, and 4. Finally, two additional GLUD-specific genomic sequences, termed GLUDP4 and GLUDP5, are structurally similar with the 3' part of the GLUD cDNA sequence. These loci probably represent truncated GLUD pseudogenes generated by retrotransposition. The data presented here suggest that all human GLUD pseudogenes have diverged recently in evolution.
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Glutamato Deshidrogenasa/genética , Familia de Multigenes , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN/genética , Exones , Genes Reguladores , Humanos , Intrones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Transcripción GenéticaRESUMEN
We isolated and analysed a full-length mouse brain glutamate dehydrogenase (GLUD) cDNA as a preliminary step to use the mouse model for the investigation of GLUD function in neurotransmission and neurodegeneration. GLUD coding sequences were found highly conserved among mouse, human and rat. Northern blots revealed two transcripts with different ratios in different mouse organs implying some mechanism of tissue-specific expression. In contrast to human, mouse GLUD gene family appears not to contain an intronless member.
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ADN/genética , Glutamato Deshidrogenasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Química Encefálica , Clonación Molecular , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , ARN/análisis , Ratas , Transcripción GenéticaRESUMEN
Glutamate dehydrogenase (GLUD) plays an important role in mammalian neuronal transmission. In human, GLUD is encoded by a small gene family. To determine whether defects in Glud genes are associated with known neurological mutations in the mouse and to contribute to the comparative mapping of homologous genes in man and mouse, the chromosomal location of genes reactive with a mouse brain GLUD cDNA were determined. Genomic Southern analysis of a well-characterized panel of Chinese hamster x mouse somatic cell hybrids identified two GLUD-reactive loci, one residing on mouse Chromosome 14 and the other on Chromosome 7. Progeny of an intersubspecies backcross were used to map one of these genes, Glud, proximal to Np-1 on Chromosome 14, but no restriction fragment polymorphisms could be identified for the second locus, Glud-2.
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Mapeo Cromosómico , Glutamato Deshidrogenasa/genética , Animales , Secuencia de Bases , Southern Blotting , Cruzamientos Genéticos , Ligamiento Genético , Glutamato Deshidrogenasa/metabolismo , Ratones , Familia de Multigenes , MutaciónRESUMEN
We have isolated a series of human liver cDNA clones encoding glutamate dehydrogenase. The cDNA-derived protein sequence specifies a single 558-amino acid long polypeptide including a cleavable signal sequence of 53 amino acids. Blotting analysis of RNA from human, monkey, and rabbit showed that glutamate dehydrogenase mRNA is present in various amounts in all tissues tested. Glutamate dehydrogenase mRNAs are of four sizes and are found in different ratios in different tissues; the predominant ones are approximately 3.5 and approximately 2.9 kilobases. Blot hybridization of human genomic DNA to nonoverlapping cDNA fragments revealed multiple bands, many of which hybridize with two or more probes in a manner inconsistent with the existence of a single GLUD gene. Moreover, two separate 36-base synthetic oligonucleotides corresponding to the coding region hybridize to multiple genomic fragments, confirming the existence of more than one GLUD-related gene in human.