RESUMO
To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Genes Reporter/genética , Vetores Genéticos/genética , Óperon Lac/genética , Plasmídeos/genética , beta-Galactosidase/genéticaRESUMO
Objective To analyze the determinants of emergency contraception non-use among women in unplanned and ambivalent pregnancies. Method Cross-sectional study with a probabilistic sample of 366 pregnant women from 12 primary health care units in the city of São Paulo, Brazil. A multinomial logistic regression was performed, comparing three groups: women who used emergency contraception to prevent ongoing pregnancies (reference); women who made no use of emergency contraception, but used other contraceptive methods; and women who made no use of any contraceptive methods at all. Results Cohabitation with a partner was the common determinant of emergency contraception non-use. No pregnancy risk awareness, ambivalent pregnancies and no previous use of emergency contraception also contributed to emergency contraception non-use. Conclusion Apart from what is pointed out in the literature, knowledge of emergency contraception and the fertile period were not associated to its use. .
Objetivo Analizar los determinantes del no uso de la anticoncepción de emergencia entre las mujeres con embarazo no planeado o ambivalente. Método Estudio transversal en una muestra probabilística de 366 mujeres embarazadas de 12 Unidades Básicas de Salud de São Paulo. Mediante regresión logística multinomial, se comparó tres grupos de mujeres: aquellas que usaron la anticoncepción de emergencia para prevenir el embarazo en curso (referencia), aquellas que usaron algún método anticonceptivo, pero no la anticoncepción de emergência; y aquellas que no usaron ningún método. Resultados Los hallazgos mostraron que vivir com la pareja fue el determinante común del no uso de la anticoncepción de emergencia. No tener conciencia del riesgo de embarazo, estar en un embarazo ambivalente y nunca tener utilizado la anticoncepción de emergencia también fueron associados con su no uso para prevenir el embarazo en curso. Conclusión Contrariamente a lo que reporta la literatura, el conocimiento de la anticoncepción de emergencia y el período fértil no mostró asociación con el no uso. .
Objetivo Analisar os determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente. Método Estudo transversal com amostra probabilística de 366 gestantes de 12 Unidades Básicas de Saúde da cidade de São Paulo. Por meio de regressão logística multinomial, compararam-se três grupos de mulheres: as que usaram anticoncepção de emergência para prevenir a gravidez em curso (referência); as que usaram algum método contraceptivo, mas não anticoncepção de emergência; e as que não usaram nenhum método. Resultados Os achados mostraram que morar com o parceiro foi o determinante comum do não uso da anticoncepção de emergência. Não ter consciência do risco de engravidar, estar em uma gravidez ambivalente e nunca ter usado anticoncepção de emergência também foram associados ao seu não uso para prevenir a gravidez em curso. Conclusão Diferentemente do que relata a literatura, o conhecimento sobre anticoncepção de emergência e sobre o período fértil não mostrou qualquer associação ao não uso. .
Assuntos
Proteínas de Ligação a DNA , Escherichia coli/genética , Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-Híbrido , Bacteriófago lambda/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/biossíntese , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/fisiologia , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Genes Reporter/genética , Fosforilação , Plasmídeos/biossíntese , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Transcrição Gênica/genética , Transcrição Gênica/fisiologia , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/fisiologia , Proteínas Virais Reguladoras e Acessórias , beta-Galactosidase/biossíntese , beta-Lactamases/biossínteseRESUMO
We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.
Assuntos
Animais , Humanos , Expressão Gênica/fisiologia , Genes fos/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Ativação Transcricional , Proteínas Elk-1 do Domínio ets/metabolismo , Western Blotting , Chlorocebus aethiops , Células COS , Indução Enzimática , Expressão Gênica/genética , Genes Reporter/genética , Genes Reporter/fisiologia , Genes fos/genética , Células HeLa , Células Jurkat , Transdução de Sinais , Ativação Transcricional , Transfecção , Proteínas Elk-1 do Domínio ets/genéticaRESUMO
Prostate cancer is a disease involving complicated multiple-gene alterations. Both NKX3.1 and p53 are related to prostate cancer and play crucial roles in prostate cancer progression. However, little is known about the relationships and interactions between p53 and NKX3.1 in prostate cancer. We found that NKX3.1 expression is down-regulated by over-expression of wild type (wt) p53 in prostate cancer LNCaP cells. NKX3.1 is down-regulated at both the mRNA and protein levels by p53 over- expression due to either transient transfection of exogenous p53 or induction of endogenous p53. p53 over-expression represses androgen-induced transactivation of NKX3.1 by inhibiting the promoter of the androgen acceptor (AR) gene and by blocking AR-DNA binding activity. In addition, transfection with the p21 expression vector (pPSA-p21) showed that p21 does not reduce NKX3.1 expression, indicating that NKX3.1 expression is not the result of nonspecific effects of cell growth arrest. Our results provide biochemical and cellular biologic evidence that NKX3.1 is down-regulated by p53 over-expression in prostate cancer cells.
Assuntos
Masculino , Humanos , Proteína Supressora de Tumor p53/genética , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacos , Elementos de Resposta , RNA Mensageiro/genética , Neoplasias da Próstata/genética , Regiões Promotoras Genéticas/genética , Plasmídeos/genética , Proteínas de Homeodomínio/genética , Genes Reporter/genética , Regulação para Baixo , Linhagem Celular Tumoral , Androgênios/farmacologiaRESUMO
A shuttle vector for Escherichia coli and Giardia lamblia was modified to produce a reporter plasmid, which monitors the expression of prescribed gene in G. lamblia by measuring its luciferase activity. Promoter regions of the gap2 gene, one of the genes induced during encystation, were cloned into this plasmid, and the resultant constructs were then transfected into trophozoites of G. lamblia. Transgenic trophozoites containing one of the 3 gap2-luc reporters were induced to encystation, and characterized with respect to gap2 gene expression by measuring their luciferase activities. Giardia containing a gap2-luc fusion of 112-bp upstream region showed full induction of luciferase activity during encystation.
Assuntos
Animais , Transfecção/métodos , Fatores de Tempo , Proteínas Recombinantes de Fusão/análise , Regiões Promotoras Genéticas/fisiologia , Plasmídeos , Luciferases/genética , Estágios do Ciclo de Vida/fisiologia , Giardia lamblia/genética , Engenharia Genética/métodos , Genes Reporter/genética , Genes de Protozoários/genética , Ordem dos Genes , Expressão Gênica/genética , Proteínas Ativadoras de GTPase/genética , Southern Blotting/métodosRESUMO
With the availability of complete genome sequences of several organisms, the focus has shifted from structural genomics to functional genomics, specifically in plants where the complete genomic sequences are becoming available i.e., Arabidopsis and rice. Agrobacterium mediated transformation which is exploited for transgenic technology is also being used as an effective mutagen and as a tool for functional genomics in higher plants. Besides the fact that the insertion of T-DNA element into a gene can lead to loss or gain of function, ingenious use of a variety of vectors have led to the identification of genes and regulatory elements in Arabidopsis. In this review, we highlight the progress made in the field of functional genomics of Arabidopsis using T-DNA tagging. Since this strategy has been very successfully employed in Arabidopsis and is now being extended to other plant species, we discuss the various vectors and experimental approaches employed to tag, identify and clone genes and promoter elements in Arabidopsis using T-DNA as a tool.
Assuntos
DNA Bacteriano/genética , Arabidopsis/genética , Mutagênese Insercional , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Reporter/genética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Vetores Genéticos/genéticaRESUMO
We investigated expression profiles and biological effects of the naked DNA vectors in the heart. To this end, naked DNA vector was injected into the apex of the beating rat heart after thorocotomy. When the expression of LacZ reporter was examined by reverse transcription-PCR and histochemical staining for b-galactosidase, LacZ expression was detected only in the heart, suggesting limited dissemination of the injected vector in vivo. Even within the heart, LacZ expression was limited to the injection area (apex). Similar observations were made with other transgenes such as VEGF and basic fibroblast growth factor (bFGF), where 77% and 69% of the total transgene exprssion were detected in the heart segments containing the apex. Although VEGF and bFGF expressions were detected until 2 weeks after DNA injection, the highest levels of VEGF and bFGF were observed on day 5 and day 1, respectively. The optimal doses of the vectors were 10 mg and 25 mg for the VEGF and bFGF vectors, respectively. Interestingly, injection of bFGF vector led to 50% increase in the level of endogenous murine VEGF expression. Consistent with this finding, the number of vessels that stained positive for alpha-smooth muscle actin was increased in the bFGF vector-injected heart. These results suggest that simple injection of naked DNA vector may be sufficient to induce significant angiogenesis in the myocardium and that naked DNA gene therapy may be a feasible approach for the treatment of ischemic heart disease.
Assuntos
Animais , Masculino , Ratos , Vasos Coronários/metabolismo , DNA/genética , Fator 2 de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Genes Reporter/genética , Vetores Genéticos/genética , Óperon Lac/genética , Miocárdio/metabolismo , Ratos Sprague-Dawley , Fatores de Tempo , Transgenes/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Thrombospondin-1 (TSP-1) level is tightly regulated at the transcriptional level. To determine the detailed molecular mechanisms of TSP-1 expression, nine serial 5'-deletion constructs of the human genomic tsp-1 promoter (nucleotides -2,220 to +756) were prepared, inserted into luciferase reporter plasmids, and transiently transfected into the Hep3B human hepatocarcinoma cell. Among the nine 5'-deletion constructs, pTSP-Luc-4 (-767~+756) had consistently decreased luciferase activity with or without PMA stimulation, whereas a further truncated construct [pTSP-Luc-4' (-407~+756)] had increased levels of expression. By searching the nucleotides from -767 to -407, a consensus binding sequence (5'-CCATTTT-3') for the repressor Yin Yang-1 (YY-1) at nucleotide -440 was identified. The suppression induced by this site was weakened in the presence of the region upstream of nucleotide -767 (pTSP-Luc-1 and -2). Nuclear protein directly bound to an oligonucleotide containing the repressive YY-1 sequence but the binding capacity of the sequence was decreased by the increased c-Jun levels. Moreover, proteins immunoprecipitated with anti-YY-1 revealed an interaction between c-Jun and YY-1 factor. These data suggest that the repressive YY-1 site of the tsp-1 promoter could not be functional via activating positive cis-elements on the upstream from this site and weakened via c-Jun/YY-1 interactions.