RESUMO
Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) share high genetic and antigenic similarities, but exhibit marked differences in tissue tropism and neurovirulence. The amino-terminal region of glycoprotein C (gC), which is markedly different in each of the viruses, is involved in virus binding to cellular receptors and in interactions with the immune system. This study investigated the genetic and antigenic differences of the 5' region of the gC (5' gC) gene (amino-terminal) of South American BoHV-1 (n=19) and BoHV-5 (n=25) isolates. Sequence alignments of 374 nucleotides (104 amino acids) revealed mean similarity levels of 97.3 and 94.2% among BoHV-1 gC (gC1), respectively, 96.8 and 95.6% among BoHV-5 gC (gC5), and 62 and 53.3% between gC1 and gC5. Differences included the absence of 40 amino acid residues (27 encompassing predicted linear epitopes) scattered throughout 5' gC1 compared to 5' gC5. Virus neutralizing assays testing BoHV-1 and BoHV-5 antisera against each isolate revealed a high degree of cross-neutralization between the viruses, yet some isolates were neutralized at very low titers by heterologous sera, and a few BoHV-5 isolates reacted weakly with either sera. The virus neutralization differences observed within the same viral species, and more pronounced between BoHV-1 and BoHV-5, likely reflect sequence differences in neutralizing epitopes. These results demonstrate that the 5' gC region is well conserved within each viral species but is divergent between BoHV-1 and BoHV-5, likely contributing to their biological and antigenic differences.
Assuntos
Região 5'-Flanqueadora/genética , DNA Viral/genética , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5/genética , Análise de Sequência de DNA , Proteínas do Envelope Viral/genética , Animais , Antígenos Virais/análise , Bovinos , Epitopos/análise , Herpesvirus Bovino 1/imunologia , Herpesvirus Bovino 1/patogenicidade , Herpesvirus Bovino 5/imunologia , Herpesvirus Bovino 5/patogenicidade , Testes de Neutralização , Especificidade de Órgãos , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , América do Sul , VirulênciaRESUMO
Determining the insertion position of an exogenous gene in the target plant genome is one of the main issues in the transgenic plant field. This study introduced a simple, rapid, and accurate method to clone the flanking sequences of the transgenic bar gene as the anchoring gene in the transgenic maize genome using single-primer polymerase chain reaction (PCR). This method was based on the distribution of restriction sites in the maize genome and adopted the single-primer PCR method. Cloning the flanking sequences with the restriction site-anchored single-primer PCR simplified the experimental procedures by about 70% and reduced the experimental time by more than 80%. In conclusion, the restriction site-anchored single-primer PCR was a simple, rapid method to obtain the unknown flanking sequences in the transgenic plants.
Assuntos
Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/métodos , Transgenes/genética , Zea mays/genética , Região 5'-Flanqueadora/genética , Clonagem Molecular , Primers do DNA , Genoma de PlantaRESUMO
We analyzed the possible association between human leukocyte antigen-G (HLA-G) genetic variants, supposed to regulate HLA-G expression, and the susceptibility to develop rheumatoid arthritis (RA) as well as its clinical manifestations. The 5'upstream regulatory region (5'URR) and 3'untranslated region (3'UTR) regions of the HLA-G gene were screened in 127 RA patients and 128 controls: 10 5'URR and 3 3'UTR HLA-G polymorphisms as well as two haplotypes were associated with risk for RA development, while a polymorphism in the 5'URR showed an association with the degree of disease activity. These findings, although the number of cases analyzed is limited and the P-values are modest, indicate a possible association between HLA-G gene polymorphisms and susceptibility to develop RA disease and its severity.
Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Antígenos HLA-G/genética , Regiões 3' não Traduzidas/genética , Região 5'-Flanqueadora/genética , Idoso , Brasil , Análise Mutacional de DNA , Progressão da Doença , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , RiscoRESUMO
To get further insights on the estrogen regulation of the uteroglobin (UG) gene, the 5'-flanking region of the UG gene from the brown hare (Lepus capensis) (Lc) was cloned and compared with those from two phylogenetically related species: the rabbit (Orictolagus cuniculus) (Oc) and the volcano rabbit (Romerolagus diazi) (Rd). The Lc-UG gene is very similar to those from rabbits (94%) and volcano rabbits (95%), and shares a number of genetic elements, including an estrogen response element (ERE). The estrogen-regulated transcription of a series of progressive 5'-deletion mutants of the Lc-UG gene, identified a functional ERE in the promoter region exhibiting the same orientation and relative position than that previously described in rabbits. The Lc-ERE is identical to the Oc-ERE, but different from both the Rd-ERE and the consensus ERE (c-ERE) by one nucleotide. We also detected important species-specific differences in the estrogen-regulated transcription of the UG gene. A luciferase reporter driven by 333 base pairs (bp) of the Lc-UG promoter elicited a higher response to estradiol than its related counterparts when expressed in estrogen-sensitive MCF-7 cells. Several ERE-like motifs which failed to act as functional EREs were also identified; one of them exhibited two mismatches in its palindromic sequence, a characteristic exhibited in many other natural occurring EREs, including the Rd-ERE.
Assuntos
Estrogênios/farmacologia , Lebres/genética , Transcrição Gênica/efeitos dos fármacos , Uteroglobina/genética , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Coelhos , Elementos de Resposta/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Uteroglobina/metabolismoRESUMO
We explored a possible correlation of genetic instability and CpG methylation in the 5'-flanking region of the PAI-1 gene with clinicopathologic features of gastric cancer in Chinese patients and looked for molecular markers for diagnosing gastric tumor development. Microsatellite instability and loss of heterozygosity of the PAI-1 gene locus D7S515, D7S471 and pai-1 in 50 specimens of gastric cancer and relevant pericancerous tissues were detected by PCR-single strand conformation polymorphism (PCR-SSCP) with sliver staining. Methylation-specific PCR was used to detect CpG methylation in the 5'-flanking region of the PAI-1 gene. Microsatellite instability was significantly more common in the negative than in the positive serosa infiltration group of gastric cancer (42.86 vs 2.33%). The frequency of microsatellite instability was significantly lower in the cases with lymph node metastasis than in those without metastasis (18.18 vs 2.56%); however, it was significantly higher in the low differentiation group than that in the middle or high differentiation groups (21.05 vs 0.00%). CpG methylation in the 5'-flanking region of the PAI-1 gene did not differ significantly. Microsatellite instability and loss of heterozygosity of the PAI-1 gene apparently regulates the development of gastric cancer through different pathways. Microsatellite instability could be used as a molecular marker for the development of gastric cancer. CpG methylation in the 5'-flanking region of the PAI-1 gene appears not to be involved in the development of gastric cancer.
Assuntos
Região 5'-Flanqueadora/genética , Povo Asiático/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Instabilidade Genômica/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Neoplasias Gástricas/genética , Alelos , China , Loci Gênicos/genética , Predisposição Genética para Doença , Humanos , Perda de Heterozigosidade/genética , Instabilidade de Microssatélites , Reação em Cadeia da Polimerase , Neoplasias Gástricas/patologiaRESUMO
Myostatin is a negative regulator of the growth and development of skeletal muscle mass. In fish, myostatin is expressed in several organs in addition to skeletal muscle. To understand the mechanisms regulating myostatin gene expression in the sea perch, Lateolabrax japonicus, we examined the methylation status of the myostatin gene promoter region in several tissues (liver, eye, kidney, brain, and heart) isolated from adult specimens. The frequency of methylated cytosines was very low in all tissues, regardless of the level of myostatin expression, suggesting that DNA methylation is not involved in the tissue-specific regulation of myostatin expression. Southern blot analysis of genomic DNA obtained from micrococcal nuclease-treated nuclei showed that chromatin digestion occurs in tissues where the myostatin gene is actively transcribed and that the myostatin gene is protected from micrococcal nuclease in tissues where myostatin is not expressed. The chromatin structure in the myostatin gene region appears to regulate its expression without DNA methylation.
Assuntos
Cromatina/genética , Metilação de DNA/genética , Miostatina/genética , Percas/genética , Regiões Promotoras Genéticas/genética , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Southern Blotting , Sequência Conservada/genética , Ilhas de CpG/genética , DNA/isolamento & purificação , Metilação de DNA/efeitos dos fármacos , Genoma/genética , Nuclease do Micrococo/farmacologia , Dados de Sequência Molecular , Oceanos e Mares , Análise de Sequência de DNA , SulfitosRESUMO
The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5'-flanking sequence (5'FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5'FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5'FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5'FS are used as promoter, efficient transcription only occurs with 44 bp of 5'FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5'FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.
Assuntos
Regulação da Expressão Gênica/genética , Túbulos Renais Proximais/metabolismo , Regiões Promotoras Genéticas/genética , Trocadores de Sódio-Hidrogênio/genética , Regiões Terminadoras Genéticas/genética , Transcrição Gênica/genética , Região 5'-Flanqueadora/genética , Animais , Didelphis , Mucosa Intestinal/metabolismo , Intestinos/citologia , Túbulos Renais Proximais/citologia , Mutação Puntual/genética , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
Steroidogenic acute regulatory protein-related lipid transfer domain containing 7 (StarD7) is a poorly characterized member of the steroidogenic acute regulatory protein-related lipid transfer proteins, up-regulated in JEG-3 cells, involved in intracellular transport and metabolism of lipids. Previous studies dealing with the mechanisms underlying the human StarD7 gene expression led us to define the cis-acting regulatory sequences in the StarD7 promoter using as a model JEG-3 cells. These include a functional T cell-specific transcription factor 4 (TCF4) site involved in Wnt-ß-catenin signaling. To understand these mechanisms in more depth, we examined the steroidogenic factor 1 (SF-1) contribution to StarD7 expression. Cotransfection experiments in JEG-3 cells point out that the StarD7 promoter is activated by SF-1, and this effect is increased by forskolin. EMSA using JEG-3 nuclear proteins demonstrated that SF-1 binds to the StarD7 promoter. Additionally, chromatin immunoprecipitation analysis indicated that SF-1 and ß-catenin are bound in vivo to the StarD7 promoter. Reporter gene assays in combination with mutations in the SF-1 and TCF4 binding sites revealed that the StarD7 promoter is synergistically activated by SF-1 and ß-catenin and that the TCF4 binding site (-614/-608) plays an important role in this activation. SF-1 amino acid mutations involved in the physical interaction with ß-catenin abolished this activation; thus demonstrating that the contact between the two proteins is necessary for an efficient StarD7 transcriptional induction. Finally, these data suggest that ß-catenin could function as a bridge between SF-1 and TCF4 forming a ternary complex, which would stimulate StarD7 expression. The SF-1 and ß-catenin pathway convergence on StarD7 expression may have important implications in the phospholipid uptake and transport, contributing to the normal trophoblast development.
Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica , Transdução de Sinais , Fator Esteroidogênico 1/metabolismo , Trofoblastos/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Região 5'-Flanqueadora/genética , Animais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Bovinos , Linhagem Celular , AMP Cíclico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator Esteroidogênico 1/química , Fator Esteroidogênico 1/genética , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos , Via de Sinalização WntRESUMO
OBJECTIVE: Preclinical and clinical studies have shown that serotonin levels might modulate susceptibility to seizures. Here we evaluated an association between 5HTTLPR and 5HTTVNTR allele variants in serotonin transporter gene and epileptogenesis in temporal lobe epilepsy (TLE). METHODS: A case-control candidate gene study evaluating the frequencies of 5HTTLPR biallelic and 5HTTVNTR allele variants in patients and healthy subjects. Genotypes were grouped according to transcriptional efficiency. Cases were 175 patients with TLE selected from the Epilepsy Outpatient Clinic of Hospital de Clínicas de Porto Alegre, classified according to the electroclinical classification of the ILAE and neuroimaging findings. The control group consisted of 155 healthy unrelated subjects selected from the same population. RESULTS: We observed that less efficient transcriptional genotypes for 5-HTT polymorphisms were more frequent in epileptic patients (O.R.=3.24; 95% C.I.=1.08-9.73; p=0.036). Our results suggest that less efficient transcriptional genotypes for serotonin transporter gene are associated with TLE. CONCLUSION: In this study we observed an association between the presence of 5HTTLPR and 5-HTTVNTR less transcriptional efficient combined genotypes and TLE. Our results suggest that modulation of the serotoninergic system might be implied in epileptogenesis in TLE.
Assuntos
Região 5'-Flanqueadora/genética , Epilepsia do Lobo Temporal/genética , Repetições Minissatélites/genética , Polimorfismo de Nucleotídeo Único , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Adulto , Alelos , Brasil/epidemiologia , Estudos de Casos e Controles , Epilepsia do Lobo Temporal/epidemiologia , Feminino , Frequência do Gene , Estudos de Associação Genética , Genótipo , Humanos , Masculino , Mutagênese Insercional , Deleção de Sequência , Proteínas da Membrana Plasmática de Transporte de Serotonina/fisiologia , Transcrição Gênica , Adulto JovemRESUMO
Phloem-transported signals play an important role in regulating plant development and in orchestrating responses to environmental stimuli. Among such signals, phloem-mobile RNAs have been shown to play an important role as long-distance signaling agents. At maturity, angiosperm sieve elements are enucleate, and thus transcripts in the phloem translocation stream probably originate from the nucleate companion cells. In the present study, a pumpkin (Cucurbita maxima) phloem transcriptome was used to test for the presence of common motifs within the promoters of this unique set of genes, which may function to coordinate expression in cells of the vascular system. A bioinformatics analysis of the upstream sequences from 150 Arabidopsis genes homologous to members of the pumpkin phloem transcriptome identified degenerate sequences containing CT/GA- and GT/CA-rich motifs that were common to many of these promoters. Parallel studies performed on genes shown previously to be expressed in phloem tissues identified similar motifs. An expanded analysis, based on homologs of the pumpkin phloem transcriptome from cucumber (Cucumis sativus), identified similar sets of common motifs within the promoters of these genes. Promoter analysis offered support for the hypothesis that these motifs regulate expression within the vascular system. Our findings are discussed in terms of a role for these motifs in coordinating gene expression within the companion cell/sieve element system. These motifs could provide a useful bioinformatics tool for genome-wide screens on plants for which phloem tissues cannot readily be obtained.
Assuntos
Arabidopsis/genética , Cucurbita/genética , Floema/genética , Regiões Promotoras Genéticas/genética , Região 5'-Flanqueadora/genética , Biologia Computacional , Cucumis sativus/genética , DNA de Plantas/genética , Bases de Dados de Ácidos Nucleicos , Genes de Plantas/genética , Oryza/genética , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Populus/genética , RNA Mensageiro/genética , RNA de Plantas/genética , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , TranscriptomaRESUMO
PAX9 gene is a member of the family homeobox of transcription factors and performs important function in development and organogenesis. Mutations in PAX9 coding sequences have been implicated in autosomal dominant oligodontia affecting predominantly permanent molars and second premolars. Previous studies have shown that PAX9 is required for secondary palate development and teratogens have been identified as inducers of a tooth and craniofacial malformations. This work focused on the analysis on the 5'-flanking region of the PAX9 gene studying the influence of retinoic acid, dexamethasone, and vitamin D on the expression of PAX9 by expression constructs that carry the reporter gene luciferase. As results, retinoic acid and dexamethasone showed progressive decrease of PAX9 expression. PAX9-pGL3B1 and PAX9-pGL3B2 promoter was inhibited under the treatment of dexamethasone and ergocalciferol. Retinoic acid and dexamethasone did not alter PAX9-pGL3B3 behavior indicating that sequences present between -1106 and +92 were important for the transcriptional activity of PAX9 promoter. In this study, we characterized the transcriptional activity of specific regions of the PAX9 promoter gene and we demonstrated that retinoic acid and ergocalciferol can modulate the transcriptional activity of PAX9 gene.
Assuntos
Dexametasona/farmacologia , Ergocalciferóis/farmacologia , Regulação da Expressão Gênica , Fator de Transcrição PAX9/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Tretinoína/farmacologia , Região 5'-Flanqueadora/genética , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Camundongos , Concentração Osmolar , Fator de Transcrição PAX9/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: The AMACR gene is located in the schizophrenia susceptibility locus on chromosome 5p13, previously identified in a large Puerto Rican pedigree of Spanish origin. The AMACR-encoded protein is an enzyme involved in the metabolism of branched-chain fatty and bile acids. The enzyme deficiency causes structural and functional brain changes, and disturbances in fatty acid and oxidative phosphorylation pathways observed in individuals with schizophrenia. Therefore, AMACR is both a positional and functional candidate gene for susceptibility to schizophrenia. METHODS: The study had a two-step design: we performed mutation analysis of the coding and flanking regions of AMACR in affected members of the pedigree, and tested the detected sequence variants for association with schizophrenia in a Puerto Rican case-control sample (n=383) of Spanish descent. RESULTS AND CONCLUSION: We identified three missense variants segregating with the disorder in the family, rs2278008, rs2287939 and rs10941112. Two of them, rs2278008 and rs2287939, demonstrated significant differences in genotype (P = 4 × 10-4, P = 4 × 10-4) and allele (P = 1 × 10-4, P = 9.5 × 10-5) frequencies in unrelated male patients compare to controls, with the odds ratios (OR) 2.24 (95% CI: 1.48-3.40) and 2.25 (95% CI: 1.49-3.38), respectively. The G-C-G haplotype of rs2278008-rs2287939-rs10941112 revealed the most significant association with schizophrenia (P = 4.25 × 10-6, OR = 2.96; 95% CI: 1.85-4.76) in male subjects. There were no statistically significant differences in genotype, allele, and haplotype frequencies between female schizophrenia subjects and controls. Our results suggest that AMACR may play a significant role in susceptibility to schizophrenia in male patients.
Assuntos
Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Racemases e Epimerases/genética , Esquizofrenia/genética , Região 3'-Flanqueadora/genética , Região 5'-Flanqueadora/genética , Adulto , Alelos , Estudos de Casos e Controles , Análise Mutacional de DNA , Família/psicologia , Feminino , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Fases de Leitura Aberta/genética , Linhagem , Reação em Cadeia da Polimerase , Porto Rico , Fatores Sexuais , Irmãos/psicologiaRESUMO
To gain further insights on the genetic divergence and the species-specific characteristics of the follicle-stimulating hormone receptor (FSHR), we cloned 946 bp of the 5'-flanking region of the FSHR gene from the volcano mouse (Neotomodon alstoni alstoni), and compared its features with those from other mammalian species. The sequence of neotomodon FSHR (nFSHR) gene from the translation initiation site to -946 is 74, 71, 64, and 59% homologous to rat, mouse (129/J), human, and sheep, respectively. The nFSHR 5'-flanking region exhibits new interesting putative cis-regulatory elements including those for the SRY transcription factor, which had not been previously related to the FSHR gene. The transcriptional regulation properties of nFSHR gene were studied in mouse Sertoli (MSC-1) and non-Sertoli (H441) cell lines, and compared with those obtained with similar 129/J constructs. All constructs tested were more active in H441 than in MSC-1 cells. The low transcription levels detected in MSC-1 cells probably reflect the recruitment of Sertoli cells-specific nuclear factors that repress transcription of the FSHR gene. In H441 cells, 129/J constructs were more active than their neotomodon counterparts, indicating important species-specific differences in their transcription pattern. Functional analysis of a series of progressive 5'-deletion mutants identified regions involved in positive and negative transcriptional regulation as well as the strongest minimal promoter spanning 260 bp upstream the translation initiation site. The identification of inhibitory nuclear transcription factors, which are apparently expressed in MSC-1 cells, may contribute to a better understanding of the transcriptional regulation of the FSHR gene.
Assuntos
Arvicolinae/genética , Arvicolinae/metabolismo , Regiões Promotoras Genéticas , Receptores do FSH/química , Receptores do FSH/genética , Região 5'-Flanqueadora/genética , Sequência Rica em At , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Genes Reporter , Genes sry , Masculino , Camundongos , Camundongos da Linhagem 129 , Dados de Sequência Molecular , Receptores do FSH/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Células de Sertoli/metabolismoRESUMO
The Pitx3 gene is a homeobox transcription factor. This gene is expressed only in midbrain dopaminergic-neurons in the central nervous system, where its expression is important for development and survival of mesencephalic-dopaminergic neurons. The promoter region of the Pitx3 gene is not yet completely delimited. We used the Cre-loxP system to demonstrate the efficiency and specificity of a 4.2-kbp sequence in the 5'-flanking region of the Pitx3-gene promoter inserted in the 5'-terminus of the Cre-recombinase gene. A Cre-recombinase-reporter assay indicated that this 5'-flanking region possesses promoter activity. The cell-specific gene regulation of the Pitx3 promoter in neurons was demonstrated by a reverse-transcription polymerase chain reaction (RT-PCR) and Western blot detection of Cre-recombinase in SH-SY5Y cells but not in MCF7 cells. Functionality of the Cre-recombinase was confirmed in vitro. The Pitx3-promoter-Cre cassette here described can be used to develop transgenic mice with the specific expression of Cre-recombinase in midbrain-dopaminergic neurons to elucidate the gene function in which the conventional knockout leads to an early lethal phenotype. Such specific expression of the Pitx3 promoter may be used for gene therapy studies of Parkinson's disease.
Assuntos
Proteínas de Homeodomínio/genética , Integrases/genética , Integrases/metabolismo , Neuroblastoma/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Região 5'-Flanqueadora/genética , Linhagem Celular Tumoral , Clonagem Molecular , Regulação Neoplásica da Expressão Gênica , Humanos , Recombinação Genética/genética , Transcrição GênicaRESUMO
In this study we investigated a possible role for the single nucleotide polymorphism C1858T of the PTPN22 (protein tyrosine phosphatase nonreceptor 22) gene in determining the susceptibility to Trypanosoma cruzi infection, as well as in development of chagasic heart disease. This study included 316 patients with Chagas' disease and 520 healthy individuals from Colombia and Peru. Genotyping of PTPN22 was performed by the real-time polymerase chain reaction technology, using the TaqMan 5' allelic discrimination assay. No statistically significant differences in the frequency of PTPN22 C1858T gene polymorphism between chagasic patients and controls or between asymptomatic and cardiomyopathic individuals were observed. Our findings suggest that the PTPN22 polymorphism analyzed does not play a major role in the development of Chagas' disease in the Colombian and Peruvian populations.
Assuntos
Cardiomiopatia Chagásica/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Tirosina Fosfatases/genética , Região 5'-Flanqueadora/genética , Adulto , Idoso , Alelos , Animais , Colômbia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peru , Proteína Tirosina Fosfatase não Receptora Tipo 22 , Trypanosoma cruziRESUMO
Smac/DIABLO is a mitochondrial protein that participates in apoptotic cell death by means of sequestering several members of the inhibitor of apoptosis protein family. This action allows caspase activation, cleavage of key cellular substrates and death. Release from mitochondria is considered the main regulatory step of Smac/DIABLO activity. Nevertheless, the fact that at least one isoform, Smac-beta, does not reside in this organelle implies that transcriptional regulation could also be important. cAMP is a well known second messenger with important apoptotic effects. To analyze if cAMP could be involved in Smac/DIABLO gene regulation, we analyzed 2903 base pairs upstream of the coding sequence and characterized the minimal promoter, which contains a consensus CRE site. We found that cAMP/PKA/CREB pathway is indeed an important regulator of Smac/DIABLO transcription, since exposure to the cAMP analog 8-CPT-cAMP, the adenylyl cyclase activator forskolin, the inhibitor of phosphodiesterase isobutylmethylxanthine or by hindering PKA activation with H89, regulated the promoter activity, as shown by gene reporter and RT-PCR assays. Additionally, the results of site-directed mutagenesis revealed that the consensus CRE site was biologically functional and required for cAMP-induced promoter activity in human HeLa cells. Supporting these results, a negative dominant version of the protein kinase A responsive factor, KCREB, reduced basal Smac/DIABLO expression and rendered the promoter unresponsive to cAMP. Reducing Smac expression using an antisense approach blocked the apoptosis effects of cAMP in cervical cancer cells. These results show that cAMP is an important modulator of the apoptotic threshold in cancer cell by means of regulating Smac/DIABLO expression.
Assuntos
Apoptose/efeitos dos fármacos , AMP Cíclico/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Mitocondriais/genética , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Região 5'-Flanqueadora/genética , Proteínas Reguladoras de Apoptose , Sequência de Bases , Clonagem Molecular , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Deleção de SequênciaRESUMO
Using the information about the sequence from a differentially expressed clone (designated as HbSSH10) encodes a protein specifying a cysteine-rich sequence containing a putative "RING finger" or "C3HC4" consensus motif that was cloned recently by the subtractive hybridization between latex and leaves from rubber tree (Hevea brasiliensis). A full-length cDNA encoding C3HC4 type zinc-finger protein was isolated and characterized from rubber tree. Sequence analysis revealed that the ORFs of HbRZF encode 156 amino acid residues with a total predicted molecular mass of 17.2 kD, HbRZF protein having a putative "RING finger" segment (amino acid residues 100-144). The deduced amino acid sequences of HbRZF showed high identities of 48%, 52% and 50% to those of the ring zinc protein from Poncirus trifoliata, Arabidopsis thaliana, Thellungiella halophila. The result of Northern blot analysis indicated that the transcripts of the HbRZF were expressed more in the latex than in the leaves, whereas little expression was detected in roots and flowers. The transcription of HbRZF was induced by jasmonic acid, whereas ethylene had little effect.
Assuntos
Região 5'-Flanqueadora/genética , Hevea/genética , Proteínas de Plantas/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA de Plantas/química , DNA de Plantas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hevea/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The aim of the present study was to identify and characterize polymorphisms within the 5' flanking region, first exon and part of first intron of the bovine growth hormone gene among different beef cattle breeds: Nelore (n = 25), Simmental (n = 39), Simbrasil (n = 24), Simmental x Nelore (n = 30), Canchim x Nelore (n = 30) and Angus x Nelore (n = 30). Two DNA fragments (GH1, 464 bp and GH2, 453 bp) were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. Within the GH1 fragment, five polymorphisms were identified, corresponding to three different alleles: GH1.1, GH1.2 and GH1.3 (GenBank: AY662648, AY662649 and AY662650, respectively). These allele sequences were aligned and compared with bovine GH gene nucleotide sequence (GenBank: M57764 and AF118837), resulting in the identification of five insertion/deletions (INDELs) and five single nucleotide polymorphisms (SNPs). In the GH2 fragment two alleles were identified, GH2.1 and GH2.2 (GenBank: AY662651 and AY662652, respectively). The allele sequences were compared with GenBank sequences (M57764, AF007750 and AH009106) and three INDELs and four SNPs were identified. In conclusion, we were able to identify six new polymorphisms of the bovine GH gene (one INDEL and five SNPs), which can be used as molecular markers in genetic studies.
Assuntos
Região 5'-Flanqueadora/genética , Bovinos/genética , Marcadores Genéticos/genética , Hormônio do Crescimento/genética , Polimorfismo Genético/genética , Alelos , Animais , Bovinos/classificação , Primers do DNA/química , Éxons/genética , Frequência do Gene , Ordem dos Genes , Íntrons/genética , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples , Alinhamento de Sequência/veterináriaRESUMO
The Nrf2 (nuclear factor-erythroid 2 p45-related factor 2) transcription factor regulates gene expression of the GCLC (glutamate-cysteine ligase catalytic subunit), which is a key enzyme in glutathione synthesis, and GSTs (glutathione S-transferases) via the ARE (antioxidant-response element). The Mrp2 (multidrug-resistance protein 2) pump mediates the excretion of GSH and GSSG excretion as well as endo- and xeno-biotics that are conjugated with GSH, glucuronate or sulphate. Considering that Mrp2 acts synergistically with these enzymes, we hypothesized that the regulation of Mrp2 gene expression is also dependent on Nrf2. Using BHA (butylated hydroxyanisole), which is a classical activator of the ARE-Nrf2 pathway, we observed an increase in the transcriptional activity of Mrp2, GCLC and Gsta1/Gsta2 genes in the mouse liver. A similar pattern of co-induction of Mrp2 and GCLC genes was also observed in mouse (Hepa 1-6) and human (HepG2) hepatoma cells treated with BHA, beta-NF (beta-naphthoflavone), 2,4,5-T (trichlorophenoxyacetic acid) or 2AAF (2-acetylaminofluorene), suggesting that these genes share common mechanism(s) of transcriptional activation in response to exposure to xenobiotics. To define the mechanism of Mrp2 gene induction, the 5'-flanking region of the mouse Mrp2 gene (2.0 kb) was isolated, and two ARE-like sequences were found: ARE-2 (-1391 to -1381) and ARE-1 (-95 to -85). Deletion analyses demonstrated that the proximal region (-185 to +99) contains the elements for the basal expression and xenobiotic-mediated induction of the Mrp2 gene. Gel-shift and supershift assays indicated that Nrf2-protein complexes bind ARE sequences of the Mrp2 promoter, preferentially to the ARE-1 sequence. Overexpression of Nrf2 increased ARE-1-mediated CAT (chloramphenicol acetyltransferase) gene activity, while overexpression of mutant Nrf2 protein repressed the activity. Thus Nrf2 appears to regulate Mrp2 gene expression via an ARE element located at the proximal region of its promoter in response to exposure to xenobiotics.
Assuntos
Regulação da Expressão Gênica/genética , Proteínas Mitocondriais/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Região 5'-Flanqueadora/genética , Animais , Antioxidantes/metabolismo , Sequência de Bases , Bile/efeitos dos fármacos , Bile/metabolismo , Hidroxianisol Butilado/farmacologia , Domínio Catalítico , Linhagem Celular Tumoral , Sequência Conservada , Feminino , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteína 2 Associada à Farmacorresistência Múltipla , Fator 2 Relacionado a NF-E2/genética , Ligação Proteica , Elementos de Resposta/genética , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Ativação Transcricional , beta-Naftoflavona/farmacologiaRESUMO
To better understand the phylogenetic divergence and the species-specific characteristics of the Clara cell secretory protein (CCSP), we cloned the cDNA encoding the neotomodon CCSP (nCCSP) and analyzed its tissue-specific expression. The full-length cDNA is 451bp long and predicts an amino acid sequence of 93 residues. Northern blot analysis from different neotomodon tissues demonstrated that the mRNA of CCSP appears to be solely expressed in the lung. To study the transcriptional regulation of the CCSP gene, we cloned the 5'-flanking region of the nCCSP gene and compared its features with those previously reported for the hamster gene. The neotomodon and hamster genes share 89% sequence homology in their promoter region as well as a number of conserved cis-acting elements. However, in H441 cells the expression of a reporter gene driven by the nCCSP promoter was about 4-fold greater than its hamster counterpart. Functional analysis of progressive 5'-deletion mutants identified a region involved in the higher transcriptional activity of the neotomodon promoter.