RESUMO
After incomplete spinal cord injury (SCI), patients and animals may exhibit some spontaneous functional recovery which can be partly attributed to remodeling of injured neural circuitry. This post-lesion plasticity implies spinal remodeling but increasing evidences suggest that supraspinal structures contribute also to the functional recovery. Here we tested the hypothesis that partial SCI may activate cell-signaling pathway(s) at the supraspinal level and that this molecular response may contribute to spontaneous recovery. With this aim, we used a rat model of partial cervical hemisection which injures the bulbospinal respiratory tract originating from the medulla oblongata of the brainstem but leads to a time-dependent spontaneous functional recovery of the paralyzed hemidiaphragm. We first demonstrate that after SCI the PI3K/Akt signaling pathway is activated in the medulla oblongata of the brainstem, resulting in an inactivation of its pro-apoptotic downstream target, forkhead transcription factor (FKHR/FOXO1A). Retrograde labeling of medullary premotoneurons including respiratory ones which project to phrenic motoneurons reveals an increased FKHR phosphorylation in their cell bodies together with an unchanged cell number. Medulla infusion of the PI3K inhibitor, LY294002, prevents the SCI-induced Akt and FKHR phosphorylations and activates one of its death-promoting downstream targets, Fas ligand. Quantitative EMG analyses of diaphragmatic contractility demonstrate that the inhibition of medulla PI3K/Akt signaling prevents spontaneous respiratory recovery normally observed after partial cervical SCI. Such inhibition does not however affect either baseline contractile frequency or the ventilatory reactivity under acute respiratory challenge. Together, these findings provide novel evidence of supraspinal cellular contribution to the spontaneous respiratory recovery after partial SCI.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Bulbo/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Proteína Oncogênica v-akt/metabolismo , Recuperação de Função Fisiológica/fisiologia , Respiração , Traumatismos da Medula Espinal/fisiopatologia , Animais , Vértebras Cervicais , Diafragma/fisiopatologia , Modelos Animais de Doenças , Feminino , Lateralidade Funcional , Bulbo/efeitos dos fármacos , Bulbo/patologia , Neurônios Motores/patologia , Neurônios Motores/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Proteína Oncogênica v-akt/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Nervo Frênico/patologia , Nervo Frênico/fisiopatologia , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Respiração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Traumatismos da Medula Espinal/patologia , Fatores de TempoRESUMO
Prader-Willi syndrome (PWS) is a neurogenetic disorder that results from the absence of a normal paternal contribution to the 15q11-13 region. The clinical manifestations of PWS are a transient severe hypotonia in the newborn period, with mental retardation, hypogonadism and obesity observed later in development. Five transcripts with exclusive expression from the paternal allele have been isolated, but none of these has been shown to be involved in PWS. In this study, we report the isolation and characterization of NDN, a new human imprinted gene. NDN is exclusively expressed from the paternal allele in the tissues analysed and is located in the PWS region. It encodes a putative protein homologous to the mouse brain-specific NECDIN protein, NDN; as in mouse, expression in brain is restricted to post-mitotic neurons. NDN displays several characteristics of an imprinted locus, including allelic DNA methylation and asynchronous DNA replication. A complete lack of NDN expression in PWS brain and fibroblasts indicates that the gene is expressed exclusively from the paternal allele in these tissues and suggests a possible role of this new gene in PWS.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Síndrome de Prader-Willi/genética , Síndrome de Angelman/genética , Animais , Northern Blotting , Mapeamento Cromossômico , Cromossomos Humanos Par 15 , Metilação de DNA , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Feminino , Humanos , Hibridização In Situ/métodos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Fenômenos Fisiológicos do Sistema Nervoso , Proteínas Nucleares/metabolismo , Distribuição TecidualRESUMO
Introduction The present study reports cross-cultural comparisons of body mass index (BMI) and growth in Prader-Willi syndrome, a neurodevelopmental disorder associated with obesity, growth restriction and mild learning disability. Our objectives were to: (1) compare rates of obesity in adults with Prader-Willi syndrome (PWS) in France, with data available from Belgium, the UK and the USA; (2) compare growth of French children with PWS with their counterparts in Germany and the USA; and (3) evaluate the contribution of genetic, medical and social parameters to obesity outcome in French children and adults with PWS. Method (1) Cross-sectional comparison of BMI of 40 French adults, 38 Belgian adults, 46 British adults and 292 North American adults; (2) Construction of growth curves for French children aged 2-20 years from longitudinal data for 150 individuals with PWS, and comparison with published growth curves from Germany and the USA; and (3) Longitudinal regression analysis of 141 French children and adults to determine the factors contributing to obesity outcome. Results A total of 82.5% French adults with PWS have BMI > 30 compared with 65.8% in Belgium (n.s.), 58.2% in the USA (P < 0.005), and 54.3% in the UK (P < 0.01). Higher rates of obesity in females vs. males were found in the USA sample (P < 0.001) but not in the other samples. In contrast to adults, growth curves for French children with PWS show similar rates of growth compared with children with PWS in Germany and the USA. The principal determining factors of BMI status in the French PWS population are age (P < 0.0001), cohort (born within the last 15 years vs. born over 15 years ago, P < 0.0002) and growth hormone replacement therapy (P < 0.0002). Significant subsidiary effects include domestic situation (P < 0.0001), genetic diagnosis (P < 0.0001) and age of diagnosis (P < 0.0001). Conclusions French adults with PWS have significantly higher rates of obesity than adults in the UK and the USA, but growth in French children with PWS is similar to the USA and Germany. Clinical management has a greater impact on obesity outcome in PWS than cultural factors.
Assuntos
Comparação Transcultural , Deficiências do Desenvolvimento/epidemiologia , Obesidade/epidemiologia , Síndrome de Prader-Willi/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Bélgica/epidemiologia , Índice de Massa Corporal , Criança , Pré-Escolar , Estudos de Coortes , Comorbidade , Estudos Transversais , Feminino , França , Predisposição Genética para Doença , Alemanha/epidemiologia , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Distribuição por Sexo , Fatores Socioeconômicos , Reino Unido/epidemiologia , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Imprinted genes are considered to play an important role in growth and early development but much of the research is based on animal studies. AIM: This study reports clinical data from a French population concerning prenatal, perinatal and postnatal complications in Prader-Willi syndrome (PWS), a genetically imprinted neurodevelopmental disorder associated with growth retardation, intellectual impairment and obesity. STUDY DESIGN: Data from family health records concerning prenatal, perinatal and postnatal complications were collected from 52 French people with the deletion form (DEL), and 34 French people with the maternal disomy form of PWS (UPD) and compared against national norms and between groups. RESULTS: Significant findings include: a history of miscarriage, high rate of polyhydramnios (12/34 UPD, 11/52 DEL), a high rate of induced labour, a high rate of Caesarian section (20/34 UPD, 26/52 DEL), small gestational age (10/34 UPD, 22/52 DEL), hypotonia (34/34 UPD, 49/52 DEL), and suckling deficit (25/34 UPD, 46/52 DEL). Significant differences between genetic subtypes include a higher rate of induced labour in UPD (27/34 UPD, 25/52 DEL), an increased risk of premature term in UPD (9/34 UPD vs. 4/52 DEL), raised maternal age in UPD (36.4 years vs. 29.3 years), low birth weight for newborns with a deletion form of PWS (girls 2.8 kg, boys 2.7 kg), a positive correlation between parental weight and offspring birth weight only for patients with UPD (UPD maternal: r=0.62, paternal: r=0.51). CONCLUSION: The results indicate significant intrauterine disturbance in PWS, particularly in PWS due to UPD, but a more significant weight disturbance for PWS due to deletion.
Assuntos
Retardo do Crescimento Fetal/etiologia , Impressão Genômica , Síndrome de Prader-Willi/complicações , Adolescente , Adulto , Criança , Pré-Escolar , Aberrações Cromossômicas , DNA/análise , Feminino , Retardo do Crescimento Fetal/diagnóstico , Humanos , Lactente , Recém-Nascido , Doenças do Recém-Nascido/etiologia , Masculino , Pessoa de Meia-Idade , Síndrome de Prader-Willi/genética , Gravidez , Complicações na GravidezRESUMO
By successive screenings of cDNA libraries prepared from human tumours and from human foreskin keratinocytes, we have isolated overlapping cDNAs coding for a novel protein which we call Ron, with sequence characteristics of a receptor protein tyrosine kinase. Ron is a 1400 amino acid protein structurally similar to the 1408 amino acid product of the C-MET proto-oncogene, the receptor for hepatocyte growth factor and scatter factor. The two proteins have 63% overall sequence identity in their intracellular regions. We have localised the RON gene to human chromosome region 3p21, a region frequently deleted in small cell carcinoma of the lung and in renal cell carcinoma, and which is believed to harbour unidentified tumour suppressor genes. Interestingly, normal lung tissue contains transcripts of the RON gene.
Assuntos
Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 3 , DNA/química , DNA/genética , DNA/isolamento & purificação , Expressão Gênica , Humanos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/química , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-met , Receptores de Superfície Celular/químicaRESUMO
The Bmx sequence was identified and cloned during our search for novel tyrosine kinase genes expressed in human bone marrow cells. Bmx cDNA comprises a long open reading frame of 675 amino acids, containing one SH3, one SH2 and one tyrosine kinase domain, which are about 70% identical with Btk, Itk and Tec and somewhat less with Txk tyrosine kinase sequences. The amino terminal sequences of these four tyrosine kinases are about 40% identical and each contains a so-called pleckstrin homology domain. The 2.7 kb Bmx mRNA was expressed in endothelial cells and several human tissues by Northern blotting and an 80 kD Bmx polypeptide was detected in human endothelial cells. Immunoprecipitates of COS cells transfected with a Bmx expression vector and NIH3T3 cells expressing a Bmx retrovirus contained a tyrosyl phosphorylated Bmx polypeptide of similar molecular weight. The BMX gene was located in chromosomal band Xp22.2 between the DXS197 and DXS207 loci. Interestingly, chromosome X also contains the closest relative of BMX, the BTK gene, implicated in X-linked agammaglobulinemia. The BMX gene thus encodes a novel nonreceptor tyrosine kinase, which may play a role in the growth and differentiation of hematopoietic cells.
Assuntos
Proteínas Tirosina Quinases/genética , Cromossomo X , Células 3T3 , Sequência de Aminoácidos , Animais , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Humanos , Camundongos , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência de AminoácidosRESUMO
Prader-Willi syndrome (PWS) is a neurogenetic disorder resulting from the loss of paternal expression of gene(s) localized in the 15q11-q12 region. A new human gene encoding a putative protein with high homology to the mouse NECDIN protein has recently been characterized and mapped to chromosome 15q11-q12. It is expressed from the paternal allele only, suggesting its potential involvement in PWS. We now report the localization of the mouse Necdin gene in a region of conserved synteny to the human PWS region. We demonstrate the paternal specific expression of Necdin in the mouse central nervous system, and show that parental alleles display a differential methylation profile in the coding region. Finally, fluorescence in situ hybridization analysis reveals an asynchronous pattern of replication at the Necdin locus. These results clearly demonstrate imprinting of the mouse Necdin gene. Mouse models will be powerful tools in the study of human PWS phenotype and imprinting mechanisms.
Assuntos
Mapeamento Cromossômico , Regulação da Expressão Gênica no Desenvolvimento/genética , Impressão Genômica/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Síndrome de Prader-Willi/genética , Alelos , Animais , Sequência de Bases , Encéfalo , Química Encefálica , Sequência Conservada/genética , Cruzamentos Genéticos , Metilação de DNA , Replicação do DNA , Feminino , Feto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Muridae , RNA Mensageiro/análiseRESUMO
Imprinting defects in 15q11-q13 are a rare but significant cause of Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Patients with an imprinting defect have apparently normal chromosomes 15 of biparental origin, but are recognised by @parental DNA methylation at D15S63 (PW71) or SNURF-SNRPN exon 1. We have investigated the methylation status of five additional loci in 12 such patients with or without a deletion in the imprinting centre. In each patient, the imprinting defect affected all loci tested. During routine diagnostic testing we identified four patients who had a normal methylation pattern at SNURF-SNRPN exon 1, but an abnormal pattern at D15S63. In two of these patients, who were suspected of having PWS, this change was restricted to D15S63. In two patients suspected of having AS, several but not all loci were affected. Using a newly developed methylation-specific PCR test for D15S63 we found that these methylation changes are rare in patients suspected of having AS. Although we can not prove that the methylation changes in the four patients are causally related to their disease, our findings demonstrate that spatially restricted changes in methylation can occur. In some cases, these changes may reflect incomplete imprint spreading.
Assuntos
Síndrome de Angelman/genética , Cromossomos Humanos Par 15/genética , Metilação de DNA , Síndrome de Prader-Willi/genética , Southern Blotting , DNA/genética , DNA/metabolismo , Saúde da Família , Feminino , Impressão Genômica , Humanos , Masculino , Repetições de MicrossatélitesRESUMO
To construct a DNA probe specific for protozoa that cause visceral leishmaniasis, we cloned Pst I fragments of Leishmania infantum genomic DNA into a Bluescript II SK vector. A clone of 4.3 kb that contained a highly repetitive sequence was isolated and cut with three restriction enzymes: Hae III, Rsa I, and Sau 3A. After a new molecular cloning step, we isolated and sequenced a 140-basepair (bp) fragment. Two oligonucleotides were synthesized to be used as primers for a polymerase chain reaction. Using this probe, we detected an amount of DNA equivalent to one promastigote of L. infantum. This probe showed a high specificity; all protozoa tested that cause visceral leishmaniasis and L. major (one of the causative agents of Old World cutaneous leishmaniasis) showed a 100-bp amplified sequence, whereas other Leishmania strains showed a signal of a different size or else no signal. Moreover, no amplified sequence was obtained with other pathogenic parasites tested (Trypanosoma brucei, T. cruzi, Plasmodium falciparum, Pneumocystis carinii, and Toxoplasma gondii).
Assuntos
DNA de Protozoário/química , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Sondas de DNA , DNA de Protozoário/isolamento & purificação , Cães , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Humanos , Leishmaniose Visceral/diagnóstico , Dados de Sequência Molecular , Oligonucleotídeos/química , Mapeamento por Restrição , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
Prader-Willi syndrome (PWS), first described in 1956, is considered as a paradigm of a neurodevelopmental disorder with severe and early obesity with hyperphagia and impaired satiety. The improved knowledge in the natural history and recent data on genetics offer new perspectives for understanding the metabolic and endocrine dysfunctions and possibly for treatment. Natural history of the disease has been described due to the early diagnosis performed in the first months of life and various nutritional phases have been described. In addition, there is clear evidence that the abnormal feeding behavior is included in the behavioral problems. Brain imaging studies have shown that some brain regions may be important in PWS. The role of SNORD116 gene cluster is detailed and its links with circadian rhythm and brain and hypothalamus development. Pathophysiology of the abnormal ghrelin levels and of OT dysfunction is documented. While no effect on appetite and weight regulation has been reported with ghrelin antagonists, OT has been shown to improve some of the behavioral problems in adults. We discuss our hypothesis of an abnormal ghrelin/OT/dopamine pathway which may explain the switch of nutritional phases and behavior. These new aspects offer an opportunity for therapeutic use and possible early intervention.
Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Grelina/metabolismo , Hiperfagia/metabolismo , Síndrome de Prader-Willi/metabolismo , Comportamento Alimentar , HumanosRESUMO
NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proteínas de Neoplasias/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Animais , Fertilidade , Marcação de Genes , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/patologia , Camundongos , Camundongos Knockout , Neurônios Motores/citologia , Mutação/genética , Proteínas de Neoplasias/deficiência , Sistema Nervoso Simpático/citologia , Gânglio Trigeminal/anormalidadesRESUMO
This report investigates the case of a patient with a duplication of the long arm of the X chromosome containing the putative X inactivation center (XIC) and the recently isolated XIST (X inactive-specific transcript) gene which is thought to play a role in the X inactivation process. Based on replication studies, a two-fold higher than normal phosphoglycerate kinase activity and absence of XIST expression, we conclude that the duplicated region is not subject to X-inactivation, despite the presence of two XICs. These results are discussed with respect to different models of X inactivation.
Assuntos
Mecanismo Genético de Compensação de Dose , RNA não Traduzido , Fatores de Transcrição/genética , Cromossomo X , Anormalidades Múltiplas/genética , Sequência de Bases , Células Cultivadas , Pré-Escolar , DNA de Cadeia Simples , Feminino , Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , RNA Longo não CodificanteRESUMO
The gene for human glycerol kinase deficiency (GK) maps in Xp21.3 in a critical region of about 50-250 kb located distal to the Duchenne muscular dystrophy gene (DMD) by analysis of patient deletions and YAC contigs. We have used a genomic exon amplification strategy to isolate potential exons from two cosmids which mapped to this interval. The genomic exons were used to isolate six overlapping cDNA clones from human fetal liver which encode the X-linked glycerol kinase gene. The cDNA clones map to cosmids, YAC clones and deletions in patients which define the GK critical region and also hybridize to several autosomal fragments and one Xq fragment in genomic DNA. The GK gene is expressed most in human liver with three transcript sizes of 1.85, 2.7, and 3.7 kb. Sequence analysis of 1.5 kb of several overlapping liver cDNA clones predicted a protein with approximately 63% similarity to the E. coli and B. subtilis glycerol kinase genes. The liver cDNA clones have sequence identity with four genomic exons and the 3' untranslated region from an Xp21.3 cosmid thus indicating that this is the expressed GK gene which when deleted in patients gives rises to GK deficiency.
Assuntos
Clonagem Molecular/métodos , Glicerol Quinase/genética , Cromossomo X , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Fúngicos , Sequência Consenso , Cosmídeos , DNA/genética , Escherichia coli/genética , Éxons , Biblioteca Gênica , Genoma Humano , Glicerol Quinase/deficiência , Humanos , Fígado , Masculino , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
A human gene with strong homology to the MAGE gene family located in Xq27-qter has been isolated by using exon-trapping of cosmids in the Xp21.3 region. We have mapped and sequenced cDNA and genomic clones corresponding to this gene, MAGE-Xp, and shown that the last exon contains the open reading frame and is present in a minimum of five copies in a 30-kb interval. MAGE-Xp is expressed only in testis and, unlike the Xq27-qter MAGE genes, it is not expressed in any of 12 different tumor tissues tested. However, the gene and predicted protein structure are conserved, suggesting a similar function. MAGE-Xp is located in the 160-kb critical interval defined for the locus involved in sex determination within Xp21 and is 50 kb distal to the DAX-1 gene, which is responsible for X-chromosome-linked adrenal hypoplasia congenita.
Assuntos
Hominidae/genética , Família Multigênica , Proteínas de Neoplasias , Proteínas/genética , Cromossomo X , Glândulas Suprarrenais/anormalidades , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Sequência Conservada , Cosmídeos , Primers do DNA , DNA Complementar , Éxons , Biblioteca Gênica , Ligação Genética , Humanos , Hipogonadismo/genética , Íntrons , Masculino , Dados de Sequência Molecular , Neoplasias/genética , Neoplasias/metabolismo , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Testículo/metabolismoRESUMO
A new human brain cDNA molecule was mapped by in situ hybridization to the 11q21-q23.1 region of the human genome, probably to the 11q22 band. An EcoRI restriction site and a (GT)n repeat element within the gene were shown to be polymorphic. Both polymorphisms were readily studied by the polymerase chain reaction. A two-allele polymorphism was described for the EcoRI restriction site, whereas four different alleles were detected for the second genetic marker. The observed heterozygosities were 37% and 42% for the former and the latter polymorphism, respectively. The combined heterozygosity index was estimated to be 0.56. These new genetic markers will be useful for linkage analysis of neurogenetic diseases that have been mapped to this chromosomal region.
Assuntos
Química Encefálica/genética , Cromossomos Humanos Par 11 , Polimorfismo Genético , Adulto , Alelos , Sequência de Bases , DNA de Cadeia Simples/química , Desoxirribonuclease EcoRI , Biblioteca Gênica , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido NucleicoRESUMO
Olfactory receptors are believed to be encoded by an extremely large subfamily of G protein-coupled receptors. A human gene (OLFR1) encoding a member of this family was cloned from a genomic library by cross-hybridization with a gene fragment obtained by the polymerase chain reaction. The nucleotide sequence of a 3.4-kb EcoRI fragment was determined, and the protein sequence was deduced from the single open reading frame. The gene was assigned by in situ hybridization of metaphase chromosomes to the 17p13-->p12 region of the human genome, in proximity to the tumor-suppressor gene encoding p53. When used as a probe on Southern blots under moderately stringent conditions, it hybridizes to at least three closely related genes. A restriction fragment length polymorphism was detected after MspI digestion. Mendelian segregation of the gene was assessed in three CEPH families, and linkage analysis confirmed the localization of the locus.
Assuntos
Cromossomos Humanos Par 17 , Receptores Odorantes , Células Receptoras Sensoriais/fisiologia , Olfato/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , Clonagem Molecular , Genes , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Alinhamento de SequênciaRESUMO
The selective amplification by polymerase chain reaction (PCR) of gene fragments corresponding to new G-protein-coupled receptors resulted in the cloning of 18 orphan members of this gene family. Of these, three human clones amplified from genomic DNA (HGMP03, HGMP04, and HGMP05) were shown to be structurally related. Genomic clones corresponding to HGMP03 and HGMP05 were isolated and their putative coding region sequenced. Following the characterization of two interleukin 8 (IL-8) receptors, HGMP03 appeared to encode the high-affinity IL-8 receptor, whereas the partial clone HGMP04 encodes the low-affinity IL-8 receptor. Comparison with the cDNA sequence suggests that the high-affinity receptor gene is split by an intron in the 5' untranslated region. The high-affinity receptor gene was mapped by in situ hybridization to the 2q33-q36 region of the human genome. The HGMP05 locus turned out to be a pseudogene for the low-affinity IL-8 receptor (87% identity), with multiple frameshifts and point mutations introducing stop codons. Southern blotting on genomic DNA did not allow the further detection of related loci in the human genome.
Assuntos
Cromossomos Humanos Par 2 , Pseudogenes , Receptores Imunológicos/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA/genética , Genoma Humano , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , Receptores Imunológicos/metabolismo , Receptores de Interleucina-8A , Homologia de Sequência do Ácido NucleicoRESUMO
Prader-Willi syndrome (PWS) is a complex neurogenetic disorder. The phenotype is likely to be a contiguous gene syndrome involving genes which are paternally expressed only, located in the human 15q11-q13 region. Four mouse models of PWS have been reported but these do not definitively allow the delineation of the critical region and the associated genes involved in the aetiology of PWS. Moreover, targeted mutagenesis of mouse homologues of the human candidate PWS genes does not appear to result in any of the features of PWS. Therefore, the isolation of new genes in this region remains crucial for a better understanding of the molecular basis of PWS. In this manuscript, we report the characterization of MAGEL2 and its mouse homologue Magel2. These are located in the human 15q11-q13 and mouse 7C regions, in close proximity to NDN / Ndn. By northern blot analysis we did not detect any expression of MAGEL2 / Magel2 but by RT-PCR analysis, specific expression was detected in fetal and adult brain and in placenta. Both genes are intronless with tandem direct repeat sequences contained within a CpG island in the 5'-untranscribed region. The transcripts encode putative proteins that are homologous to the MAGE proteins and NDN. Moreover, MAGEL2 / Magel2 are expressed only from the paternal allele in brain, suggesting a potential role in the aetiology of PWS and its mouse model, respectively.
Assuntos
Mapeamento Cromossômico , Síndrome de Prader-Willi/genética , Proteínas/genética , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias , Sequência de Bases , Encéfalo/metabolismo , Cromossomos Humanos Par 15 , Ilhas de CpG , Expressão Gênica , Impressão Genômica , Humanos , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Linhagem , Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Sequências de Repetição em TandemRESUMO
A viable duplication in the proximal long arm of the X chromosome in a boy with a malformative syndrome was delineated with molecular biology techniques using 14 probes from the X cen-Xq21 region. This analysis allowed us to refine the physical map of the X cen-Xq13 region.
Assuntos
Família Multigênica , Cromossomo X , Anormalidades Múltiplas/genética , Adulto , Southern Blotting , Células Cultivadas , Criança , Bandeamento Cromossômico , Feminino , Marcadores Genéticos , Humanos , Masculino , Sondas MolecularesRESUMO
We report a patient with X-linked adrenal hypoplasia congenita and hypogonadotrophic hypogonadism in whom there were no clinical or biochemical features of either glycerol kinase deficiency or Duchenne muscular dystrophy. (The adrenal hypoplasia congenita and glycerol kinase loci map in Xp21 distal to Duchenne muscular dystrophy, and proximal to DXS727). DNA isolated from our patient was analysed by PCR amplification with primers for appropriate loci in the Xp21 region. This analysis revealed the absence of DXS319, which lies near the adrenal hypoplasia congenita deletion critical region, and the presence of DXS727, which is distal to the gene. The absence of glycerol kinase deficiency biochemically and clinically was consistent with the presence of one glycerol kinase exon product from PCR primers P17/P18 which lies within the glycerol kinase gene. The hypogonadotrophic hypogonadism is universally found in X-linked adrenal hypoplasia congenita and is thought to be pituitary in origin. These findings suggest that a gene locus resulting in hypogonadotrophic hypogonadism is present in the Xp21 region and is an integral part of the adrenal hypoplasia congenita gene or in close relationship to it.