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1.
Neuroscience ; 169(2): 781-6, 2010 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-20493928

RESUMO

Estrogen (17beta-estradiol) plays key regulatory roles in a variety of physiological and biological processes. Several lines of evidence also support its role as a protective factor in Alzheimer's disease; however, the basis of this effect is unclear. Here we show that an early-onset Alzheimer's disease transgenic mouse model expressing the double-mutant form of human amyloid precursor protein (APP); Swedish (K670N/M671L) and Indiana (V717F) undergoing treatment with 17beta-estradiol show significantly lower levels of APP processing through beta-secretase and enhanced alpha-secretase processing resulting in marked reductions of APP-CTFbeta, Abeta42 and plaque burden, along with increased levels of the non-amyloidogenic sAPPalpha. Moreover, 17beta-estradiol resulted in elevated brain levels of transthyretin, which inhibits aggregation of Abeta into plaques; though the insulin-degrading enzyme, which breaks down Abeta, was significantly reduced. These results illustrate a multifaceted effect of 17beta-estradiol on the biochemical basis of Alzheimer's disease, through effects on APP processing, Abeta levels and factors that affect its clearance and aggregation. Overall, these results support the need for further long-term longitudinal studies to elucidate consequences of menopause as well as hormone therapy on Alzheimer's disease, and explore its potential as a therapeutic avenue for the disease.


Assuntos
Doença de Alzheimer/prevenção & controle , Estradiol/farmacologia , Estrogênios/farmacologia , Fármacos Neuroprotetores/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Estradiol/sangue , Estradiol/uso terapêutico , Estrogênios/sangue , Estrogênios/uso terapêutico , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Fármacos Neuroprotetores/uso terapêutico , Placa Amiloide/efeitos dos fármacos , Placa Amiloide/patologia
2.
Drug Metab Dispos ; 35(7): 1064-70, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17403913

RESUMO

Arylamine N-acetyltransferases (NAT) catalyze the biotransformation of many important arylamine drugs and procarcinogens. NAT can either detoxify or activate procarcinogens, complicating the manner in which these enzymes may participate in enhancing or preventing toxic responses to particular agents. Mice possess three NAT isoenzymes: Nat1, Nat2, and Nat3. Whereas Nat1 and Nat2 can efficiently acetylate many arylamines, few substrates appear to be appreciably metabolized by Nat3. We generated a Nat3 knockout mouse strain and used it along with our double Nat1/2(-/-) knockout strain to further investigate the functional role of Nat3. Nat3(-/-) mice showed normal viability and reproductive capacity. Nat3 expression was very low in wild-type animals and completely undetectable in Nat3(-/-) mice. In contrast, greatly elevated expression of Nat3 transcript was observed in Nat1/2(-/-) mice. We used a transcribed marker polymorphism approach to establish that the increased expression of Nat3 in Nat1/2(-/-) mice is a positional artifact of insertion of the phosphoglycerate kinase-neomycin resistance cassette in place of the Nat1/Nat2 gene region and upstream of the intact Nat3 gene, rather than a biological compensatory mechanism. Despite the increase in Nat3 transcript, the N-acetylation of p-aminosalicylate, sulfamethazine, 2-aminofluorene, and 4-aminobiphenyl was undetectable either in vivo or in vitro in Nat1/2(-/-) animals. In parallel, no difference was observed in the in vivo clearance or in vitro metabolism of any of these substrates between wild-type and Nat3(-/-) mice. Thus, Nat3 is unlikely to play a significant role in the N-acetylation of arylamines either in wild-type mice or in mice lacking Nat1 and Nat2 activities.


Assuntos
Artefatos , Arilamina N-Acetiltransferase/metabolismo , Regulação Enzimológica da Expressão Gênica , Isoenzimas/metabolismo , Acetilação , Compostos de Aminobifenil/metabolismo , Ácido Aminossalicílico/metabolismo , Animais , Arilamina N-Acetiltransferase/deficiência , Arilamina N-Acetiltransferase/genética , Feminino , Fluorenos/metabolismo , Isoenzimas/deficiência , Isoenzimas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Fatores Sexuais , Especificidade por Substrato , Sulfametazina/metabolismo
3.
Neurobiol Aging ; 23(2): 187-94, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-11804702

RESUMO

Presenilin 1-null mice die at birth from brain and skeletal developmental deformities due to disrupted Notch signaling. Presenilin 1-null mice also have severely reduced gamma-secretase cleavage of betaAPP. The assumption has been that facilitation of Notch signaling and betaAPP processing by presenilin 1 are analogous functions. Here we describe a presenilin 1-targetted mouse model that expresses extremely low levels ( approximately 1% of normal) of mutant PS1-M146L. Homozygous mice have significantly reduced viability due to a Notch-like phenotype. The animals that survive have severe axial skeletal deformities and markedly diminished gamma-secretase activity and accumulation of betaAPP-C100, but no obvious abnormalities in brain development. These results suggest that, in mice, a marked reduction of PS1-facilitated gamma-secretase activity is not detrimental to normal brain development.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/crescimento & desenvolvimento , Endopeptidases/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Animais , Especificidade de Anticorpos , Ácido Aspártico Endopeptidases , Western Blotting , Encéfalo/anatomia & histologia , Encéfalo/patologia , Ensaio de Imunoadsorção Enzimática , Marcação de Genes , Genótipo , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Fenótipo , Presenilina-1 , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Medula Espinal/patologia
4.
J Biol Chem ; 276(46): 43446-54, 2001 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-11551913

RESUMO

Recent studies have shown independently that presenilin-1 (PS1) null mutants and familial Alzheimer's disease (FAD)-linked mutants should both down-regulate signaling of the unfolded protein response (UPR). However, it is difficult to accept that both mutants possess the same effects on the UPR. Furthermore, contrary to these observations, neither loss of PS1 and PS2 function nor expression of FAD-linked PS1 mutants were reported to have a discernable impact on the UPR. Therefore, re-examination and detailed analyses are needed to clarify the relationship between PS1 function and UPR signaling. Here, we report that PS1/PS2 null and dominant negative PS1 mutants, which are mutated at aspartate residue 257 or 385, did not affect signaling of the UPR. In contrast, FAD-linked PS1 mutants were confirmed to disturb UPR signaling by inhibiting activation of both Ire1alpha and ATF6, both of which are endoplasmic reticulum (ER) stress transducers in the UPR. Furthermore, PS1 mutants also disturbed activation of PERK (PKR-like ER kinase), which plays a crucial role in inhibiting translation during ER stress. Taken together, these observations suggested that PS1 mutations could affect signaling pathways controlled by each of the respective ER-stress transducers, possibly through a gain-of-function.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Estresse Fisiológico , Transdução Genética , Animais , Ácido Aspártico/química , Western Blotting , Regulação para Baixo , Fibroblastos/metabolismo , Genes Dominantes , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Plasmídeos/metabolismo , Presenilina-1 , Ligação Proteica , Dobramento de Proteína , Transdução de Sinais , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas
5.
Pediatr Res ; 48(6): 731-4, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11102538

RESUMO

The variability in intestinal disease severity in patients with cystic fibrosis (CF) has been associated with the expression of secondary modifier genes. The locus containing these modifier genes in CF patients is syntenic with a modifier locus previously associated with survival in CF transmembrane conductance regulator-knockout mice. These previous studies showed that the proportion of CF mice that survive weaning (mildly affected mice) versus those that succumb to obstruction of the small intestine (severely affected) is related to their genetic background and the expression of modifier genes. In the present work, we show that the basal transepithelial chloride transport measured across jejuna obtained from mice of mixed genetic backgrounds segregates into two groups, some mice having low and others having high, near normal chloride transport. Further, we report that the segregation of mice with respect to intestinal chloride transport correlates with their predicted segregation on the basis of genotype at the "modifier locus." These findings support the hypothesis that intestinal disease modification in CF mice correlates with improved chloride transport through non-CF transmembrane conductance regulator chloride channels.


Assuntos
Canais de Cloreto/metabolismo , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fibrose Cística/complicações , Obstrução Intestinal/etiologia , Transporte de Íons/genética , Jejuno/metabolismo , Mecônio , Animais , Animais Recém-Nascidos , Animais Lactentes , Cruzamentos Genéticos , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Dieta , Epitélio/metabolismo , Epitélio/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Jejuno/patologia , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Índice de Gravidade de Doença
6.
Proc Natl Acad Sci U S A ; 97(23): 12822-7, 2000 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-11070093

RESUMO

A direct pathophysiological role of Familial Alzheimer's Disease (FAD)-associated Presenilin 1 (PS1) mutations in neuronal vulnerability remains a controversial matter. We evaluated the relationship between PS1 and excitotoxicity in four different experimental models of neurotoxicity by using primary neurons from (i) transgenic (tg) mice overexpressing a human FAD-linked PS1 variant (L286V mutation), (ii) tg mice overexpressing human wild-type (wt) PS1, (iii) PS1 knockout mice, and (iv) wt mice in which PS1 gene expression was knocked down by antisense treatment. We found that primary neurons overexpressing mutated PS1 showed an increased vulnerability to both excitotoxic and hypoxic-hypoglycemic damage when compared with neurons obtained from either mice overexpressing human wt PS1 or in wt mice. In addition, reduced excitotoxic damage was obtained in neurons in which PS1 expression was absent or diminished. Data obtained in in vivo experimental models of excitotoxicity partially supported the in vitro observations. Accelerated neuronal death was demonstrated in the hippocampus of mice overexpressing mutated PS1 after peripheral administration of kainic acid in comparison with wt animals. However, measurement of the infarct volume after middle cerebral artery occlusion did not show significant difference between the two animal groups. The results altogether suggest that expression of FAD-linked PS1 variants increases the vulnerability of neurons to a specific type of damage in which excitotoxicity plays a relevant role. In addition, they support the view that reduction of endogenous PS1 expression results in neuroprotection.


Assuntos
Doença de Alzheimer/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Proteínas de Membrana/metabolismo , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Humanos , Infarto da Artéria Cerebral Média/fisiopatologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Degeneração Neural , Neurônios/citologia , Neurônios/patologia , Neurônios/fisiologia , Presenilina-1
7.
J Biol Chem ; 275(47): 36794-802, 2000 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-10962005

RESUMO

Absence of functional presenilin 1 (PS1) protein leads to loss of gamma-secretase cleavage of the amyloid precursor protein (betaAPP), resulting in a dramatic reduction in amyloid beta peptide (Abeta) production and accumulation of alpha- or beta-secretase-cleaved COOH-terminal fragments of betaAPP (alpha- or beta-CTFs). The major COOH-terminal fragment (CTF) in brain was identified as betaAPP-CTF-(11-98), which is consistent with the observation that cultured neurons generate primarily Abeta-(11-40). In PS1(-/-) murine neurons and fibroblasts expressing the loss-of-function PS1(D385A) mutant, CTFs accumulated in the endoplasmic reticulum, Golgi, and lysosomes, but not late endosomes. There were some subtle differences in the subcellular distribution of CTFs in PS1(-/-) neurons as compared with PS1(D385A) mutant fibroblasts. However, there was no obvious redistribution of full-length betaAPP or of markers of other organelles in either mutant. Blockade of endoplasmic reticulum-to-Golgi trafficking indicated that in PS1(-/-) neurons (as in normal cells) trafficking of betaAPP to the Golgi compartment is necessary before alpha- and beta-secretase cleavages occur. Thus, although we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role in general protein trafficking. These results are more compatible with a role for PS1 either as the actual gamma-secretase catalytic activity or in other functions indirectly related to gamma-secretase catalysis (e.g. an activator of gamma-secretase, a substrate adaptor for gamma-secretase, or delivery of gamma-secretase to betaAPP-containing compartments).


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/fisiologia , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Biomarcadores , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Compartimento Celular , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Presenilina-1
9.
Nat Med ; 5(2): 164-9, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9930863

RESUMO

The presenilin proteins are components of high-molecular-weight protein complexes in the endoplasmic reticulum and Golgi apparatus that also contain beta-catenin. We report here that presenilin mutations associated with familial Alzheimer disease (but not the non-pathogenic Glu318Gly polymorphism) alter the intracellular trafficking of beta-catenin after activation of the Wnt/beta-catenin signal transduction pathway. As with their effect on betaAPP processing, the effect of PS1 mutations on trafficking of beta-catenin arises from a dominant 'gain of aberrant function' activity. These results indicate that mistrafficking of selected presenilin ligands is a candidate mechanism for the genesis of Alzheimer disease associated with presenilin mutations, and that dysfunction in the presenilin-beta-catenin protein complexes is central to this process.


Assuntos
Doença de Alzheimer/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/genética , Mutação , Transativadores , Doença de Alzheimer/metabolismo , Transporte Biológico/genética , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Presenilina-1 , Presenilina-2 , Ligação Proteica , Transdução de Sinais/genética , beta Catenina
10.
Hum Mol Genet ; 6(7): 1153-62, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9215687

RESUMO

We have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient of the endogenous protein. In this model, expression of the endogenous Cftr gene was disrupted and replaced with a human CFTR cDNA by a gene targeted 'knock-in' event. Animals homozygous for the gene replacement failed to show neither improved intestinal pathology nor survival when compared to mice completely lacking CFTR. RNA analyses showed that the human CFTR sequence was transcribed from the targeted allele in the respiratory and intestinal epithelial cells. Furthermore, in vivo potential difference measurements showed that basal CFTR chloride channel activity was present in the apical membranes of both nasal and rectal epithelial cells in all homozygous knock-in animals examined. Ussing chamber studies showed, however, that the cAMP-mediated chloride channel function was impaired in the intestinal tract among the majority of homozygous knock-in animals. Hence, failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice. These results emphasize the need to understand the tissue-specific expression and regulation of CFTR function when animal models are used in gene therapy studies.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Camundongos Transgênicos/genética , Alelos , Animais , Colforsina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Eletrofisiologia , Homozigoto , Humanos , Intestinos/efeitos dos fármacos , Intestinos/fisiologia , Camundongos , Fenótipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Transgenes
11.
Genomics ; 45(3): 554-61, 1997 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-9367680

RESUMO

Cloning and characterization of the cystic fibrosis transmembrane conductance regulator (CFTR) gene led to the identification and isolation of cDNA and genomic sequences that cross-hybridized to the first nucleotide binding fold of CFTR. DNA sequence analysis of these clones showed that the cross-hybridizing sequences corresponded to CFTR exon 9 and its flanking introns, juxtapositioned with two segments of LINE1 sequences. The CFTR sequence appeared to have been transcribed from the opposite direction of the gene, reversely transcribed, and co-integrated with the L1 sequences into a chromosome location distinct from that of the CFTR locus. Based on hybridization intensity and complexity of the restriction fragments, it was estimated that there were at least 10 copies of the "amplified" CFTR exon 9 sequences in the human genome. Furthermore, when DNA segments adjacent to the insertion site were used in genomic DNA blot hybridization analysis, multiple copies were also detected. The overall similarity between these CFTR exon 9-related sequences suggested that they were derived from a single retrotransposition event and subsequent sequence amplification. The amplification unit appeared to be greater than 30 kb. Physical mapping studies including in situ hybridization to human metaphase chromosomes showed that multiple copies of these amplified sequences (with and without the CFTR exon 9 insertion) were dispersed throughout the genome. These findings provide insight into the structure and evolution of the human genome.


Assuntos
Mapeamento Cromossômico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Éxons , Amplificação de Genes , Humanos , Células Híbridas , Hibridização In Situ , Dados de Sequência Molecular , Análise de Sequência de DNA
12.
Nat Genet ; 12(3): 280-7, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8589719

RESUMO

Mice that have been made deficient for the cystic fibrosis transmembrane conductance regulator (Cftr) usually die of intestinal obstruction. We have created Cftr-deficient mice and demonstrate prolonged survival among backcross and intercross progeny with different inbred strains, suggesting that modulation of disease severity is genetically determined. A genome scan showed that the major modifier locus maps near the centromere of mouse chromosome 7. Electrophysiological studies on mice with prolonged survival show that the partial rectification of Cl- and Na+ ion transport abnormalities can be explained in part by up-regulation of a calcium-activated Cl- conductance. Identification of modifier genes in our Cftr(m1HSC)/Cftr(m1HSC) mice should provide important insight into the heterogeneous disease presentation observed among CF patients.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Fibrose Cística/genética , Animais , Sequência de Bases , Linhagem Celular , Cloretos/metabolismo , Mapeamento Cromossômico , Colo/patologia , Fibrose Cística/patologia , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Primers do DNA , Modelos Animais de Doenças , Feminino , Íleo/patologia , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Dados de Sequência Molecular , Mutagênese , Técnicas de Patch-Clamp , Sobreviventes , Aumento de Peso
13.
Biochem Biophys Res Commun ; 219(3): 753-9, 1996 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-8645253

RESUMO

The Cftr (Cystic Fibrosis Transmembrane Conductance Regulator) gene codes for an epithelial chloride (C1) channel essential for fluid secretion into the respiratory and gastrointestinal tract and from exocrine glands. Mice lacking CFTR function due to a disruption of Cftr exon 10 or exon 1 (Cftr (m1UNC/m1UNC) or Cftr(m1HSC/m1HFC) mice, respectively) generally suffer from severe gastrointestinal disease resulting in death shortly after birth or at the time of weaning. However, a subgroup of the Cftr(m1HSC/m1HSC) mice have been characterized which exhibit relatively mild intestinal pathology resulting in a noncompromised lifespan compared to the more severely affected Cftr(m1UNC/m1UNC) mice. We compared the ion transport capacity of the intestinal mucosa of the mildly and severely affected CF mice using the in vivo technique of rectal potential difference (PD) measurement and found that the net calcium-activated chloride conductance toward the lumen was much greater in the rectum of mildly affected mice than in the severely affected mice. Hence, the milder phenotype may be related to the expression of a factor which enhances the net calcium-activated chloride conductance into the lumen of the intestinal tract.


Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fibrose Cística/fisiopatologia , Amilorida/farmacologia , Animais , Cruzamentos Genéticos , Fibrose Cística/genética , Fibrose Cística/metabolismo , Epitélio/fisiologia , Epitélio/fisiopatologia , Éxons , Feminino , Homozigoto , Mucosa Intestinal/fisiologia , Mucosa Intestinal/fisiopatologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Mucosa Nasal/fisiologia , Mucosa Nasal/fisiopatologia , Reto
14.
J Biol Chem ; 267(9): 5735-8, 1992 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-1348246

RESUMO

We report the cloning of a human gene encoding the 5-hydroxytryptamine1B receptor. The receptor has the characteristics of a G-protein-linked receptor and is most homologous to the human 5-HT1D receptor. This human 5-HT receptor gene, most abundantly expressed in striatum, is localized on chromosome 6, at 6q13, and the gene encoding the 5-HT1D receptor is localized on chromosome 1. Radioligand studies indicate that the affinity of [3H]5-HT is 16 +/- 2 nM. Drug competition studies indicate that the receptor displays high affinity (i.e. less than 40 nM) for 5-HT, 5-carboxyamidotryptamine, methiothepin, and metergoline.


Assuntos
Encéfalo/metabolismo , Cromossomos Humanos Par 6 , Hipófise/metabolismo , Polimorfismo de Fragmento de Restrição , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Sequência de Aminoácidos , Animais , Autorradiografia , Sequência de Bases , Ligação Competitiva , Northern Blotting , Southern Blotting , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Biblioteca Genômica , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Especificidade de Órgãos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ratos , Receptores de Serotonina/análise , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Serotonina/metabolismo , Trítio
15.
J Biol Chem ; 266(36): 24471-6, 1991 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-1722205

RESUMO

To identify the transcription regulatory elements of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, DNA fragments located in the 5'-upstream region were fused with the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and transfected into various cell lines to test for promoter activity. The results of these studies suggested that there were at least two positive and one negative cisacting elements involved in CFTR transcription initiation. One of them was a proximal, positive element delimited by the 5' deletion constructs -226 base parts upstream of the transcription start site. This minimal promoter sequence (-226 to +98) alone seemed to be sufficient to direct cell-specific CAT expression. The sequences immediately upstream of -227, on the other hand, appeared to contain a negative regulatory element; inclusion of this sequence with the proximal element (e.g. a construct containing sequences -345 to +98) rendered the CFTR promoter inactive. This negative regulatory element could also suppress the activity of a heterologous promoter. In addition, the DNA transfection study suggested the existence of another positive regulatory element outside the CFTR promoter region examined, as the inability of this region (e.g. -658 to +98) to function in a CAT assay could be overcome by the presence of a viral enhancer element.


Assuntos
Fibrose Cística/genética , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , DNA/metabolismo , Elementos Facilitadores Genéticos , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Plasmídeos , RNA/genética , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Transcrição Gênica , Células Tumorais Cultivadas
16.
Genomics ; 10(3): 547-50, 1991 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1716243

RESUMO

We have cloned the mouse homolog of the human cystic fibrosis transmembrane conductance regulator (CFTR) using clones isolated from a mouse lung cDNA library and using amplification of cDNA to isolate specific regions. The cDNA was 6304 bp in length and encoded a polypeptide of 1476 amino acids. Comparison of the deduced amino acid sequence showed that the mouse protein has high homology to the human protein; overall identity was 78.3%. The amino acid identity was high for both transmembrane domains (first transmembrane domain, 86.7%; second transmembrane domain, 81.1%) and for both ATP-binding folds (first ATP-binding fold, 80.5%; second ATP-binding fold, 83.9%), suggesting the functional importance of these regions. On the other hand, the R domain was less well conserved (68.9% identity). All of the published missense mutation sites and the site of the common delta F508 mutation were conserved between human and mouse.


Assuntos
Proteínas de Membrana/genética , Camundongos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Regulador de Condutância Transmembrana em Fibrose Cística , DNA/genética , Genes , Humanos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie
17.
Genomics ; 10(1): 214-28, 1991 May.
Artigo em Inglês | MEDLINE | ID: mdl-1710598

RESUMO

The gene responsible for cystic fibrosis, the most common severe autosomal recessive disorder, is located on the long arm of human chromosome 7, region q31-q32. The gene has recently been identified and shown to be approximately 250 kb in size. To understand the structure and to provide the basis for a systematic analysis of the disease-causing mutations in the gene, genomic DNA clones spanning different regions of the previously reported cDNA were isolated and used to determine the coding regions and sequences of intron/exon boundaries. A total of 22,708 bp of sequence, accounting for approximately 10% of the entire gene, was obtained. Alignment of the genomic DNA sequence with the cDNA sequence showed perfect colinearity between the two and a total of 27 exons, each flanked by consensus splice signals. A number of repetitive elements, including the Alu and Kpn families and simple repeats, such as (GT)17, (GATT)7, and (TA)14, were detected in close vicinity of some of the intron/exon boundaries. At least three of the simple repeats were found to be polymorphic in the population. Although an internal amino acid sequence homology could be detected between the two halves of the predicted polypeptide, especially in the regions of the two putative nucleotide-binding folds (NBF1 and NBF2), the lack of alignment of the nucleotide sequence as well as the different positions of the exon/intron boundaries does not seem to support the hypothesis of a recent gene duplication event. To facilitate detection of mutations by direct sequence analysis of genomic DNA, 28 sets of oligonucleotide primers were designed and tested for their ability to amplify individual exons and the immediately flanking sequences in the introns.


Assuntos
Fibrose Cística/genética , Proteínas de Membrana/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 7 , Regulador de Condutância Transmembrana em Fibrose Cística , DNA , Éxons , Variação Genética , Humanos , Íntrons , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Splicing de RNA , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Alinhamento de Sequência
19.
Nature ; 347(6288): 80-3, 1990 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-1975640

RESUMO

Receptors for dopamine have been classified into two functional types, D1 and D2. They belong to the family of receptors acting through G (or guanine nucleotide-binding) proteins. D2 receptors inhibit adenylyl cyclase, but D1 receptors stimulate adenylyl cyclase and activate cyclic AMP-dependent protein kinases. Dopamine D1 and D2 receptors are targets of drug therapy in many psychomotor disorders, including Parkinson's disease and schizophrenia, and may also have a role in drug addiction and alcoholism. D1 receptors regulate neuron growth and differentiation, influence behaviour and modify dopamine D2 receptor-mediated events. We report here the cloning of the D1 receptor gene, which resides on an intronless region on the long arm of chromosome 5, near two other members of the G-linked receptor family. The expressed protein, encoded by 446 amino acids, binds drugs with affinities identical to the native human D1 receptor. The presence of a D1 receptor gene restriction fragment length polymorphism will be helpful for future disease linkage studies.


Assuntos
Cromossomos Humanos Par 5 , Receptores Dopaminérgicos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Benzazepinas/metabolismo , Química Encefálica , Bovinos , Clonagem Molecular , Glicosilação , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fosforilação , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos , Receptores de Dopamina D1 , Mapeamento por Restrição , Distribuição Tecidual , Transfecção
20.
Science ; 245(4922): 1066-73, 1989 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-2475911

RESUMO

Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7. Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF. The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding. A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.


Assuntos
Fibrose Cística/genética , DNA/isolamento & purificação , Genes Recessivos , Genes , Proteínas de Membrana/genética , Peptídeos/genética , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Clonagem Molecular/métodos , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística , Humanos , Canais Iônicos/patologia , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
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