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1.
Neuropediatrics ; 43(4): 194-200, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22911481

RESUMO

Inflammation and genetics may play a role in the pathogenesis of febrile seizures. The aim of this study was to investigate the spontaneous and lipopolysaccharide (LPS)-induced production of IL-1ß and IL-10, and the association between IL-1ß (-511) and IL-10 (-1082) single nucleotide polymorphisms with LPS-induced cytokine production. The study included 92 febrile seizure patients and 132 healthy controls. First, we isolated genomic DNA and by using PCR-RFLP we genotyped the individuals for the cytokines gene polymorphism. Second, peripheral mononuclear cells of the individuals were isolated and stimulated with LPS to measure secretion capacity of IL-1ß and IL-10 using specific ELISA kits. We found that both the IL-1ß and IL-10 production was increased in febrile seizures. The rapid increase of IL-1ß production in the supernatants of the LPS-induced cells was significantly higher at the fourth and the twenty-fourth hours in febrile and complex febrile seizures, respectively. The distribution of IL-10 (-1082) G allele differs significantly between cases and controls. The IL-1ß (-511) G/A and the IL-10 (-1082) G/A genotype combination was found to be higher in patients with febrile seizure. Our results showed that IL-1ß and IL-10 production was not influenced by the single nucleotide polymorphisms in the pathogenesis of febrile seizures.


Assuntos
Interleucina-10/genética , Interleucina-1beta/genética , Polimorfismo de Nucleotídeo Único/genética , Convulsões Febris/genética , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Criança , Pré-Escolar , Citocinas/metabolismo , Análise Mutacional de DNA , Feminino , Frequência do Gene , Genótipo , Humanos , Lactente , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Polissacarídeos/farmacologia , Convulsões Febris/patologia , Fatores de Tempo
2.
J Pediatr Endocrinol Metab ; 23(12): 1289-97, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21714463

RESUMO

BACKGROUND: Vitamin D receptor (VDR) gene is regarded as one of the candidate genes for type 1 diabetes mellitus (T1D) susceptibility and of some genetic factors involved in the development of osteoporosis in this group. STUDY DESIGN: We characterized the VDR gene polymorphism (BsmI, ApaI, TaqI, FokI and Cdx-2 binding site) in a group of Turkish patients with T1D (n=90) and correlated respective VDR genotypes with the bone mass and some parameters of bone turnover. RESULTS: There were no differences in the genotype frequencies of the BsmI, ApaI, TaqI and Cdx-2 polymorphisms in patients and control subjects. We found a significantly higher prevalence of the F allele/the FF genotype in the patients compared to controls (p=0,0031, odds 1.96 (1,27-3,01)). We observed no difference in markers of bone turnover (Serum levels of osteocalcin, PINP and alkaline phosphatase, urinary levels of calcium/ creatinine and N-telopeptid) among different VDR genotypes. No correlation was found between VDR polymorphisms and DEXA measurements of these patients. CONCLUSIONS: Although the FF genotype was found to be a risk factor in a Turkish population, elucidation of this result is necessary in other larger study groups drawn from the same ethnic population.


Assuntos
Densidade Óssea , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Polimorfismo Genético , Receptores de Calcitriol/genética , Fator de Transcrição CDX2 , Criança , Pré-Escolar , Feminino , Genótipo , Proteínas de Homeodomínio/fisiologia , Humanos , Masculino , Transativadores/fisiologia , Turquia
3.
J Eur Acad Dermatol Venereol ; 22(8): 950-7, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18355201

RESUMO

BACKGROUND: Genetic factors that predispose individuals to Behçet's disease (BD) and periodontal disease. Tumour necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of both BD and periodontal disease. The relationship with periodontitis and the pathogenesis of BD has not yet been determined. OBJECTIVE: The aim of the present study was to investigate the possible relation of the periodontal scores and single nucleotide polymorphism of TNF-alpha-1031T/C with BD compared with healthy controls (HC) and recurrent aphtous stomatitis (RAS). We also sought to determine the effects of periodontal condition and TNF-alpha-1031T/C polymorphism on clinical severity of BD. METHODS: Eighty-two unrelated patients with BD, 42 RAS patients and 77 HC were enrolled in the study. Periodontal status of all subjects was evaluated according to the World Health Organization community periodontal index of treatment needs (CPITN). For genotyping, polymerase chain reaction-restriction fragment length polymorphism was employed. RESULTS: The mean CPITN was observed to be higher in patients with BD compared with HC and RAS (P < 0.001). TNF-alpha-1031C allele was significantly higher in patients with BD (P = 0.023) and RAS (P = 0.007) compared with HC. Mean CPITN was higher in CC genotype compared with other genotypes (P = 0.004). Moreover, CPITN and CC genotype was found to be correlated with the severity of the disease. CONCLUSIONS: Our data indicate that the TNF-alpha-1031T/C gene polymorphism (CC genotype) is a risk factor for periodontitis, RAS and BD patients and also suggests that long-term periodontal follow-up and education of oral hygiene in patients with BD may help to prevent the development and/or progression of the disease.


Assuntos
Síndrome de Behçet/genética , Doenças Periodontais/genética , Polimorfismo de Nucleotídeo Único , Fator de Necrose Tumoral alfa/genética , Adulto , Alelos , Análise de Variância , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Progressão da Doença , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Modelos Logísticos , Masculino , Polimorfismo de Fragmento de Restrição , Estudos Prospectivos , Fatores de Risco , Turquia
4.
Br J Dermatol ; 155(2): 350-6, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16882174

RESUMO

BACKGROUND: Genetic factors that predispose individuals to Behçet's disease (BD) are considered to play an important role in development of the disease. The tumour necrosis factor (TNF)-alpha gene, which is closely linked to the HLA-B51 gene, is involved in susceptibility for BD. Recently, a polymorphism at position -1031 within the TNF-alpha promoter region was demonstrated to be responsible for susceptibility to BD in a British population. However, the functional effects of this polymorphism have not yet been determined. OBJECTIVES: To investigate the possible relation of the TNF-alpha-1031 T/C polymorphism with susceptibility to BD in a Turkish population and to determine the functional importance of this polymorphism. METHODS: Ninety-nine unrelated patients (47 women, 52 men; mean +/- SD age, 34.10 +/- 10.53 years) with BD and 103 ethnically matched healthy controls (52 males, 51 females; mean +/- SD age, 40.25 +/- 14.15) were enrolled in the study. For genotyping, polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis was employed. The functional importance of TNF-alpha-1031 T/C polymorphism was determined with an enzyme-linked immunospot (ELISPOT) assay. For this purpose, mononuclear cells obtained from BD patients and controls were analysed for TNF-alpha and interferon (IFN)-gamma production. RESULTS: A significant difference was observed between BD patients and controls with respect to the allele frequency of TNF-alpha-1031C [P = 0.018, OR = 1.83, 95% confidence interval (CI) = 1.07-3.13]. When the allele frequencies were analysed according to the clinical features, the T allele in patients with positive skin pathergy test (SPT) was significantly increased when compared with those of patients without these findings (P = 0.004, OR = 2.75, 95% CI = 1.3-5.86). To demonstrate the frequency of TNF-alpha and IFN-gamma producing cells, mononuclear cells from four representative individuals of each genotype were used and the spontaneous and stimulated TNF-alpha and IFN-gamma values (spot numbers) were analysed. Compared with the control groups, a significant increase was observed in the number of cells producing TNF-alpha obtained from BD patients (P < 0.001). Moreover, the stimulation index for TNF-alpha [bacterial lipopolysaccharide (LPS) stimulated/unstimulated] was higher for the CC genotype (9 +/- 9.5) with respect to the other genotypes (TT; 1.3 +/- 0.3 and TC; 1.2 +/- 0.2). While the difference in the spontaneous IFN-gamma values between groups were not statistically significant, the stimulated IFN-gamma values were found to be significantly increased in the BD group when compared with the healthy control group (P = 0.004). CONCLUSIONS: Our results showed that, in the Turkish population the TNF-alpha-1031C allele is associated with susceptibility to BD. On the other hand, carrying the T allele may render patients more prone to developing a positive skin pathergy test. In addition, ELISPOT assays revealed that BD patients exhibited a significantly higher number of mononuclear cells producing TNF-alpha, and cells obtained from patients with a CC genotype had a stronger response to LPS stimulation. The strong IFN-gamma response upon LPS stimulation in BD patients supports the previous findings that BD is a Th1 driven disease. These findings suggest that the TNF-alpha-1031 polymorphism may have a functional effect and could explain the reason for high levels of TNF-alpha production observed in BD patients.


Assuntos
Síndrome de Behçet/genética , Polimorfismo de Nucleotídeo Único , Fator de Necrose Tumoral alfa/genética , Adulto , Síndrome de Behçet/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Interferon gama/biossíntese , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/biossíntese , Turquia
5.
Clin Exp Dermatol ; 31(5): 699-701, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16901312

RESUMO

Having considered the impact of the function of TLR2 in the recognition of several microorganisms that are thought to have an association with Behçet's disease (BD), we aimed to determine a possible association between the TLR2 Arg753Gln polymorphism and susceptibility to BD. We genotyped 83 patients with BD, 95 ethnically matched healthy controls, 12 patients with recurrent aphthous stomatitis (RAS) and 21 patients with rheumatoid arthritis (RA) by restriction fragment length polymorphism after PCR amplification of the genomic region encompassing the polymorphic site. Comparison of the TLR2 Arg753Gln A allele and A/G genotype frequencies did not show a significant difference between patients with BD and healthy controls (1.2% vs. 0.6%, and 2.1% vs. 1.1%, respectively). None of the patients from the RAS and RA groups had the A allele or A/G genotype. Our results indicate that the TLR2 Arg753Gln polymorphism does not play a role in the aetiopathogenesis of BD.


Assuntos
Arginina/genética , Síndrome de Behçet/genética , Glutamina/genética , Polimorfismo de Nucleotídeo Único/genética , Receptor 2 Toll-Like/genética , Adulto , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Turquia
6.
Clin Exp Dermatol ; 30(5): 546-50, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16045690

RESUMO

Genetic factors predisposing individuals to Behçet's disease (BD) are considered to play important roles in the development of the disease. Patients with BD exhibit elevated levels of pro-inflammatory cytokines, and affected organs show a significant neutrophil and lymphocyte infiltration. Current evidence suggests that the activated lymphocytes contribute to neutrophil and endothelial cell activation in these patients. The cytotoxic T lymphocyte-associated antigen (CTLA)-4 molecule plays an important role in immune regulation by downregulating T-cell activation, and the CTLA-4 49A/G polymorphism in the exon 1 has been shown to be associated with a number of autoimmune diseases. In an attempt to demonstrate whether there is an association of the CTLA-4 49A/G polymorphism with BD in the Turkish population, we genotyped 59 Turkish patients and 99 healthy individuals for single-nucleotide polymorphisms. For this purpose, genomic DNA was obtained from the peripheral blood of individuals and the region of interest was amplified using PCR. Genotyping was performed using the BbvI restriction endonuclease. It was shown that the distribution of the CTLA-4 exon 1 49A/G allele and genotype frequencies did not differ between patients with BD and healthy controls. However, allele and genotype frequencies of CTLA-4 49 A and A/A were significantly higher in patients with ocular involvement compared with patients without these symptoms (90.6% vs. 65.1%, odds ratio (OR) = 9.67, P = 0.011; and 81.25% vs. 39.5%, OR = 9.56, P = 0.015, respectively). A statistically significant difference in the A allele frequency was observed in patients with erythema nodosum-like lesions (86.1% vs. 65.8%, OR = 6.24, P = 0.04). There was also an increase in A/A genotype frequency, but the difference was not statistically significant (72.2% vs. 41.5%, OR = 6.5, P = 0.068). Our data suggest that BD patients with ocular involvement and erythema nodosum-like lesions have a higher frequency of both the A allele and the A/A genotype at position 49 of exon 1 of CTLA-4. These results may also indicate that CTLA-4 is a disease-modifying rather than a susceptibility gene for BD.


Assuntos
Antígenos de Diferenciação/genética , Síndrome de Behçet/genética , Polimorfismo de Nucleotídeo Único , Adulto , Antígenos CD , Síndrome de Behçet/etnologia , Síndrome de Behçet/imunologia , Antígeno CTLA-4 , Eritema Nodoso/genética , Eritema Nodoso/imunologia , Oftalmopatias/genética , Oftalmopatias/imunologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Turquia
7.
J Biol Chem ; 274(38): 26894-900, 1999 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-10480898

RESUMO

Previous work has established that the melanocyte-specific tyrosinase-related protein-1 (TRP-1) promoter is regulated positively by the microphthalmia-associated transcription factor Mitf, acting through the conserved M box and negatively by the T-box factor Tbx2, which can bind two "melanocyte-specific elements" termed the MSEu and MSEi. Both the MSEu and MSEi, which share a 6-base pair GTGTGA consensus, are also recognized by a previously unidentified melanocyte-specific factor, MSF. Here we show using a combination of DNA binding assays, proteolytic clipping, and anti-Pax3 antibodies that MSF is indistinguishable from Pax3, a paired homeodomain transcription factor implicated genetically in melanocyte development and the regulation of the Mitf promoter. Consistent with Pax3 being able to bind the TRP-1 promoter, Pax3 is expressed in melanocytes and melanomas, and TRP-1 promoter activity is up-regulated by Pax3. The results identify a novel role for Pax3 in the expression of TRP-1, and the potential role of Pax3 in the melanocyte lineage is discussed.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Glicoproteínas de Membrana , Oxirredutases , Regiões Promotoras Genéticas , Proteínas/genética , Fatores de Transcrição , Animais , Sequência de Bases , Diferenciação Celular , Melanócitos/citologia , Melanócitos/metabolismo , Melanoma/metabolismo , Camundongos , Dados de Sequência Molecular , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Transfecção , Células Tumorais Cultivadas
8.
J Cell Sci ; 112 ( Pt 13): 2223-32, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10362552

RESUMO

Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.


Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Repressoras/fisiologia , Animais , Apoptose/genética , Carcinoma de Ehrlich/genética , Carcinoma de Ehrlich/patologia , Carcinoma de Ehrlich/fisiopatologia , Proteínas Correpressoras , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Expressão Gênica , Células HeLa , Humanos , Camundongos , Proteínas Nucleares , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/genética , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia
9.
Int J Cancer ; 80(4): 600-5, 1999 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-9935163

RESUMO

The retinal pigment epithelium (RPE) is essential for eye development by interacting with the overlaying neuroepithelium. Regulatory sequences of the gene encoding for tyrosinase-related protein 1 (TRP-1), linked to the lacZ reporter gene, lead to strong and specific beta-galactosidase expression in the RPE. We asked how the oncogene ret would affect this epithelial cell type during mouse development. We used the TRP-1 promoter to express ret in the developing RPE, and obtained transgenic mouse lines, which showed mild to severe microphthalmia. During development, the RPE changed to a stratified epithelium with reduced or absent pigmentation from E10.5 onward. In addition, proliferation of RPE cells and tumor formation were observed from E12.5 onward. These early events prevent closure of choroid fissure and lead to microphthalmia and secondary malformations after birth. We conclude that ret transgene expression in the RPE prevents normal differentiation of this epithelial layer and induces proliferation and tumor formation. The appearance of the microphthalmic phenotype underlines the requirement of a normally developed RPE for eye development.


Assuntos
Proteínas de Drosophila , Neoplasias Oculares/genética , Glicoproteínas de Membrana , Microftalmia/genética , Oxirredutases , Epitélio Pigmentado Ocular , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Neoplasias Oculares/embriologia , Neoplasias Oculares/patologia , Feminino , Expressão Gênica , Óperon Lac , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Epitélio Pigmentado Ocular/embriologia , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
10.
Mol Cell Biol ; 18(9): 5099-108, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9710594

RESUMO

Previous work has demonstrated that two key melanocyte-specific elements termed the MSEu and MSEi play critical roles in the expression of the melanocyte-specific tyrosinase-related protein 1 (TRP-1) promoter. Both the MSEu and MSEi, located at position -237 and at the initiator, respectively, bind a melanocyte-specific factor termed MSF but are also recognized by a previously uncharacterized repressor, since mutations affecting either of these elements result in strong up-regulation of TRP-1 promoter activity in melanoma cells. Here we demonstrate that repression mediated by the MSEu and MSEi also operates in melanocytes. We also report that both the MSEu and MSEi are recognized by the brachyury-related transcription factor Tbx2, a member of the recently described T-box family, and that Tbx2 is expressed in melanocyte and melanoblast cell lines but not in melanoblast precursor cells. Although Tbx2 and MSF each recognize the TRP-1 MSEu and MSEi motifs, it is binding by Tbx-2, not binding by MSF, that correlates with repression. Several lines of evidence tend to point to the brachyury-related transcription factor Tbx2 as being the repressor of TRP-1 expression: both the MSEu and MSEi bind Tbx2, and mutations in either element that result in derepression of the TRP-1 promoter diminish binding by Tbx2; the TRP-1 promoter, but not the tyrosinase, microphthalmia, or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) promoter, is repressed by Tbx2 in cotransfection assays; a high-affinity consensus brachyury/Tbx2-binding site is able to constitutively repress expression of the heterologous IE110 promoter; and a low-affinity brachyury/Tbx2 binding site is able to mediate Tbx2-dependent repression of the G3PDH promoter. Although we cannot rule out the presence of an additional, as yet unidentified factor playing a role in the negative regulation of TRP-1 in vivo, the evidence presented here suggests that Tbx2 most likely is the previously unidentified repressor of TRP-1 expression and as such is likely to represent the first example of transcriptional repression by a T-box family member.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fetais , Melanócitos/metabolismo , Glicoproteínas de Membrana , Oxirredutases , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Proteínas/genética , Proteínas com Domínio T , Animais , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Primers do DNA , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica , Camundongos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Reticulócitos/metabolismo , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção
11.
Genes Dev ; 12(14): 2188-99, 1998 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-9679063

RESUMO

DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-strand break repair and V(D)J recombination, is comprised of a DNA-targeting component termed Ku and an approximately 465-kD catalytic subunit, DNA-PKcs. Although DNA-PK phosphorylates proteins in the presence of DSBs or other discontinuities in the DNA double helix in vitro, the possibility exists that it is also activated in other circumstances via its association with additional proteins. Here, through use of the yeast two-hybrid screen, we discover that the recently identified high affinity DNA binding protein C1D interacts with the putative leucine zipper region of DNA-PKcs. Furthermore, we show that C1D can interact with DNA-PK in mammalian cells and that C1D is a very effective DNA-PK substrate in vitro. Finally, we establish that C1D directs the activation of DNA-PK in a manner that does not require DNA termini. Therefore, these studies provide a function for C1D and suggest novel mechanisms for DNA-PK activation in vivo.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Zíper de Leucina , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular Transformada , DNA Super-Helicoidal , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares , Peptídeos/metabolismo , Fosforilação , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato
12.
Gene ; 165(1): 93-6, 1995 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-7489924

RESUMO

We describe the construction of a new vector, pWITCH, designed to facilitate the characterisation of proteins encoded by novel cDNAs isolated using either a one- or two-hybrid assay. Expression of directionally cloned cDNAs is directed in vivo in Saccharomyces cerevisiae from the inducible GAL10 promoter and in vitro from the T7 promoter, while translation of the expressed cDNAs results in proteins which are tagged in vitro with a specific epitope and in vivo with both the epitope and the VP16 transcription activation domain. The principle of using multiple promoters each able to operate under different conditions to express different combinations of protein domains without the need for subcloning should be generally applicable.


Assuntos
DNA Complementar/genética , Proteínas Fúngicas/genética , Vetores Genéticos , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular , DNA Complementar/isolamento & purificação , Epitopos/genética , Epitopos/metabolismo , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular
13.
Oncogene ; 10(1): 123-34, 1995 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-7824265

RESUMO

Little is known of the molecular mechanisms underlying the differentiation of the melanocyte from the melanoblast or the progression from the melanocyte to a malignant melanoma. Since the adenovirus E1A products have proved a useful tool for understanding control of differentiation in other systems, we explored the possibility of using E1A as a probe for factors controlling melanocyte-specific gene expression and differentiation. The results obtained show that the adenovirus E1A 13S, but not the 12S, product can transform the highly pigmented and TPA-dependent melanocyte cell line melan-a. Transformation is characterised by a morphological change, loss of TPA-dependence, the ability to grow in soft agar and strikingly, loss of pigmentation which correlates with loss of expression of the melanocyte-specific TRP-1 and tyrosinase genes. Cotransfection assays demonstrated that repression of TRP-1 by E1A correlated with E1A binding to p105Rb and p300, with the target in the TRP-1 promoter being the M-box, and 11 bp basic-Helix-loop-Helix (bHLH) factor-binding motif conserved between melanocyte-specific promoters. Consistent with the M-box acting as a target for E1a-mediated transcription repression, we also show that the basic-helix-loop-helix-leucine zipper (bHLH-LZ) protein (Mi) encoded by the microphthalmia gene (mi), which is required for pigment cell differentiation, is a positive acting transcription factor which can interact with the retinoblastoma product in vitro and activate the TRP-1 promoter. Moreover, expression of the mi gene was reduced around 50-fold in the non-pigmented E1a-transformed melan-a cells compared to the nontransformed melan-a cell line, with ectopic expression of Mi able to prevent repression of the tyrosinase and TRP-1 promoters in the presence of E1A. Mi therefore appears to play a crucial role in melanocyte-specific gene expression. The parallels between repression of myogenesis and muscle cell bHLH factors, and Mi and melanocyte differentiation are discussed.


Assuntos
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Melanócitos/metabolismo , Glicoproteínas de Membrana , Oxirredutases , Proteína do Retinoblastoma/metabolismo , Transativadores , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Primers do DNA , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Proteína p300 Associada a E1A , Melanócitos/enzimologia , Camundongos , Fator de Transcrição Associado à Microftalmia , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/genética , Proteínas Nucleares/metabolismo , Proteínas/genética , Proteína do Retinoblastoma/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
14.
Mol Cell Biol ; 14(5): 3494-503, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-8164694

RESUMO

For a gene to be transcribed in a tissue-specific fashion, expression must be achieved in the appropriate cell type and also be prevented in other tissues. As an approach to understanding the regulation of tissue-specific gene expression, we have analyzed the requirements for melanocyte-specific expression of the tyrosinase-related protein 1 (TRP-1) promoter. Positive regulation of TRP-1 expression is mediated by both an octamer-binding motif and an 11-bp element, termed the M box, which is conserved between the TRP-1 and other melanocyte-specific promoters. We show here that, consistent with its ability to activate transcription in a non-tissue-specific fashion, the M box binds the basic-helix-loop-helix factor USF in vitro. With the use of a combination of site-directed mutagenesis and chimeric promoter constructs, additional elements involved in regulating TRP-1 expression were identified. These include the TATA region, which appears to contribute to the melanocyte specificity of the TRP-1 promoter. Mutational analysis also identified two repressor elements, one at the start site, the other located at -240, which function both in melanoma and nonmelanoma cells. In addition, a melanocyte-specific factor, MSF, binds to sites which overlap both repressor elements, with substitution mutations demonstrating that binding by MSF is not required for repression. Although a functional role for MSF has not been unequivocally determined, the location of its binding sites leads us to speculate that it may act as a melanocyte-specific antirepressor during transcription of the endogenous TRP-1 gene.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Melanócitos/metabolismo , Glicoproteínas de Membrana , Oxirredutases , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/metabolismo , Proteínas de Ligação a DNA/biossíntese , Humanos , Melanoma Experimental , Camundongos , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Biossíntese de Proteínas , Proteínas/genética , Transfecção , Células Tumorais Cultivadas
15.
Mol Cell Biol ; 12(8): 3653-62, 1992 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1321344

RESUMO

Melanocytes are specialized cells residing in the hair follicles, the eye, and the basal layer of the human epidermis whose primary function is the production of the pigment melanin, giving rise to skin, hair, and eye color. Melanogenesis, a process unique to melanocytes that involves the processing of tyrosine by a number of melanocyte-specific enzymes, including tyrosinase and tyrosinase-related protein 1 (TRP-1), occurs only after differentiation from the melanocyte precursor, the melanoblast. In humans, melanogenesis is inducible by UV irradiation, with melanin being transferred from the melanocyte in the epidermis to the surrounding keratinocytes as protection from UV-induced damage. Excessive exposure to UV, however, is the primary cause of malignant melanoma, an increasingly common and highly aggressive disease. As an initial approach to understanding the regulation of melanocyte differentiation and melanocyte-specific transcription, we have isolated the gene encoding TRP-1 and examined the cis- and trans-acting factors required for cell-type-specific expression. We find that the TRP-1 promoter comprises both positive and negative regulatory elements which confer efficient expression in a TRP-1-expressing, pigmented melanoma cell line but not in NIH 3T3 or JEG3 cells and that a minimal promoter extending between -44 and +107 is sufficient for cell-type-specific expression. Assays for DNA-protein interactions coupled with extensive mutagenesis identified three factors, whose binding correlated with the function of two positive and one negative regulatory element. One of these factors, termed M-box-binding factor 1, binds to an 11-bp motif, the M box, which acts as a positive regulatory element both in TRP-1-expressing and -nonexpressing cell lines, despite being entirely conserved between the melanocyte-specific tyrosinase and TRP-1 promoters. The possible mechanisms underlying melanocyte-specific gene expression are discussed.


Assuntos
Genes Reguladores , Melanócitos/fisiologia , Glicoproteínas de Membrana , Oxirredutases , Regiões Promotoras Genéticas , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Expressão Gênica , Genes Virais , Biblioteca Genômica , Humanos , Íntrons , Melanoma Experimental/genética , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Mapeamento por Restrição , Simplexvirus/genética
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