RESUMO
We investigated the effects of different directions of insertion of matrix attachment region (MAR) sequences on transgenic expression in stably transformed Chinese hamster ovary (CHO) cells. The MAR sequences were inserted in forward or reverse directions into the expression vectors, and transfected into CHO cells. The expression of the chloramphenicol acetyltransferase (CAT) reporter gene and the relative copy numbers of the CAT gene were analyzed. The CAT gene expression levels in the vector with the MAR sequence inserted in the forward or reverse directions increased compared with expression without the MAR sequence. The relative copy numbers of the CAT gene with MAR sequenced vectors inserted in the reverse and forward directions were lower, than in the control group. The direction of insertion of MAR sequences had no significant effect on expression levels. The expression levels were not proportional to the copy numbers of the gene.
Assuntos
Cloranfenicol O-Acetiltransferase/genética , DNA Intergênico/genética , Vetores Genéticos/química , Regiões de Interação com a Matriz , Plasmídeos/química , Animais , Células CHO , Linhagem Celular Transformada , Cloranfenicol O-Acetiltransferase/metabolismo , Cricetulus , DNA Intergênico/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/metabolismo , Plasmídeos/metabolismo , TransgenesRESUMO
Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5' coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3' UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5' coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters.
Assuntos
Cloranfenicol O-Acetiltransferase/genética , Vetores Genéticos/genética , Neospora/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Coccidiose/parasitologia , Genes Reporter , Vetores Genéticos/metabolismo , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Neospora/crescimento & desenvolvimento , Neospora/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismoRESUMO
Genetic transformation is useful for basic research and applied biotechnology. However, genetic transformation of microalgae is usually quite difficult due to the technical limitations of existing methods. We cloned the promoter and terminator of the nitrate reductase gene from the microalga Phaeodactylum tricornutum and used them for optimization of a transformation system of the microalga Chlorella vulgaris. This species has been used for food production and is a promising candidate as a bioreactor for large-scale production of value-added proteins. A construct was made containing the CAT (chloramphenicol acetyltransferase) reporter gene driven by the nitrate reductase promoter. This construct was transferred into the C. vulgaris genome by electroporation. Expression of CAT in transgenic Chlorella conferred resistance to the antibiotic chloramphenicol and enabled growth in selective media. Overall efficiency for the transformation was estimated to be approximately 0.03%, which is relatively high compared with other available Chlorella transformation systems. Expression of CAT was induced in the presence of nitrate and inhibited in the presence of ammonium as a sole nitrogen source. This study presented an inducible recombinant gene expression system, also providing more gene regulation elements with potential for biotechnological applications.
Assuntos
Chlorella vulgaris/genética , Expressão Gênica , Transformação Genética , Cloranfenicol/farmacologia , Cloranfenicol O-Acetiltransferase/genética , Resistência ao Cloranfenicol/genética , Chlorella vulgaris/efeitos dos fármacos , Chlorella vulgaris/enzimologia , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Nitrato Redutase/genética , Nitratos/farmacologia , Organismos Geneticamente Modificados , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Seleção Genética , Transformação Genética/efeitos dos fármacosRESUMO
Gene regulation in trypanosomatids occurs mainly by post-transcriptional mechanisms modulating mRNA stability and translation. We have investigated heat shock protein (HSP) 70 gene regulation in Trypanosoma cruzi, the causal agent of Chagas' disease. The HSP70 mRNA's half-life increases after heat shock, and the stabilization is dependent on protein synthesis. In a cell-free RNA decay assay, a U-rich region in the 3' untranslated region (UTR) is a target for degradation, which is reduced when in the presence of protein extracts from heat shocked cells. In a transfected reporter gene assay, both the 5'- and 3'-UTRs confer temperature-dependent regulation. Both UTRs must be present to increase mRNA stability at 37 degrees C, indicating that the 5'- and 3'-UTRs act cooperatively to stabilize HSP70 mRNA during heat shock. We conclude that HSP70 5'- and 3'-UTRs regulate mRNA stability during heat shock in T. cruzi.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Regiões 5' não Traduzidas/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP70/genética , RNA Mensageiro/metabolismo , Trypanosoma cruzi/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Bases , Northern Blotting , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Meia-Vida , Temperatura Alta , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Trypanosoma cruzi/metabolismoRESUMO
The multidrug resistance EhPgp1 gene is constitutively expressed in drug resistant trophozoites from Entamoeba histolytica. It has been demonstrated that two CCAAT/enhancer binding sites located in the EhPgp1 gene promoter control its transcriptional activation. However, functional assays of the 5' end of its promoter showed that region from -234 to -196 bp (38 bp) is also important for the EhPgp1 gene transcription. Here, we demonstrated that in the 38 bp region putative cis-activator sequences are located. In silico analysis showed the presence of GATA1, Gal4, Nit-2, and C/EBP consensus sequences. Additionally, we identified three specific DNA-protein complexes, which were competed by a C/EBP, GATA1, and HOX oligonucleotides. Finally, we partially purified three proteins of 64.4, 56.7, and 27.4 kDa. Further investigations are currently in progress to determine the identity of these nuclear factors and how they are interacting with the EhPgp1 gene promoter.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Entamoeba histolytica/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas de Protozoários/genética , Animais , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cromatografia de Afinidade , DNA de Protozoário/metabolismo , Resistência a Múltiplos Medicamentos/genética , Entamoeba histolytica/química , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Transfecção , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
The Entamoeba histolytica Ehcp112 and Ehadh112 genes that encode the EhCPADH complex are separated by a non-coding 188pb region. Their proximity suggests a coordinated expression regulation for both genes. Here, we studied the structure and function of 996 bp (p996CAT) upstream the ATG start codon of the Ehadh112 gene. The p996CAT plasmid drove CAT transcription with a 78% of the activity showed by actin promoter. Deletion of 330 bp at the 5' end of p966CAT to produce the p776CAT plasmid, decreased activity to 40% in relation to actin promoter and to 50% of p996CAT, suggesting the presence of a silencer in this region. Interestingly, deletion of other 297 bp to the p776CAT to generate the p469CAT plasmid, augmented activity in 2.5-fold compared with p776CAT construction, showing the presence of a proximal enhancer promoter. Transcription initiation sites (-69 and -150 bp), TATA like box, GAAC, and Inr elements, as well as putative DNA binding motifs, were mapped in the -1 to -469 bp core promoter region.
Assuntos
Entamoeba histolytica/genética , Lectinas/genética , Glicoproteínas de Membrana/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas de Protozoários/genética , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , DNA de Protozoário/química , Lectinas/fisiologia , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/fisiologia , TransfecçãoRESUMO
Ehcp112 encodes the Entamoeba histolytica EhCP112 cysteine protease that is part of the EhCPADH complex. By in silico analysis we identified putative transcription factor-binding sites along 837 bp upstream the Ehcp112 gene ATG codon. A TATA-like motif (TATATAAA) was located at -36 to -29 bp, a GAAC box (GAACC) was found at -10 to -14 bp and an Inr sequence (TTCAAC) at -8 to -2 bp. These tripartite promoter elements are in non-canonical positions, downstream the transcription initiation site (-280 bp). We cloned four Ehcp112 promoter fragments in pBSCAT-ACT plasmid to obtain pI (355 bp), pII (681 bp), pIII (833 bp), and pIV (554 bp) constructs. In transfected trophozoites, only pIII drove CAT activity with 44% efficiency in relation to actin promoter activity. Our results showed the presence of a distal and weak promoter in the Ehcp112 gene. The active DNA region is inside the open reading frame of the Ehrab B gene, suggesting that expression of both genes could be coordinately regulated.
Assuntos
Cisteína Endopeptidases/genética , Entamoeba histolytica/genética , Regiões Promotoras Genéticas/genética , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , DNA de Protozoário/química , Entamoeba histolytica/enzimologia , Regulação da Expressão Gênica , Dados de Sequência Molecular , Plasmídeos/química , Regiões Promotoras Genéticas/fisiologia , Ativação Transcricional/fisiologia , TransfecçãoRESUMO
Estrogen receptor (ER)-negative breast carcinomas are often difficult to treat with antiestrogens. This work was performed to determine if the re-expression of the human ER alpha could restore the hormone response of these cells. We have transfected the human wild-type ER alpha to an ER-negative breast cancer cell line (MDA-MB-231) using a tetracycline-regulated gene expression system. We obtained a new cell line, MDA-A4-5/2. Cell count and flow cytometry "S" phase cell fraction showed that 17-beta-estradiol induced an inhibition on the proliferation of these cells; on the contrary, the antiestrogens ICI 182 780, and tamoxifen blocked this effect. Finally, we demonstrated an induction of the endogenous progesterone receptor gene when ER alpha was present. These results suggest that the re-expression of ER alpha in ER-negative breast cancer cells recreate, at least partially, a hormone-responsive phenotype and may be useful as a therapeutic approach to control this pathology.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/análogos & derivados , Receptor alfa de Estrogênio/fisiologia , Estrogênios/fisiologia , Tamoxifeno/análogos & derivados , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Cloranfenicol O-Acetiltransferase/genética , Células Clonais , Doxiciclina/farmacologia , Estradiol/metabolismo , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/genética , Citometria de Fluxo , Fulvestranto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Luciferases , Receptores de Progesterona/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fase S/efeitos dos fármacos , Tamoxifeno/farmacologia , TransfecçãoRESUMO
The poly(A)-binding protein (PABP) is a highly conserved eukaryotic protein whose synthesis is regulated at the post-transcriptional level. The binding of PABP to the poly(A)-rich element found in the 5'-untranslated region (5'UTR) of PABP mRNA specifically inhibits its own translation. In this report, we show that similar adenosine-rich elements in the 5'UTR of the chloramphenicol acetyl-transferase (CAT) gene can significantly reduce the reporter mRNA abundance and translation in human 293 cells. The reduction in mRNA level, but not CAT expression, is dependent on the size of the 5'UTR poly(A) element. Furthermore, one 5'UTR-tethered PABP molecule is enough to inhibit CAT expression without affecting its mRNA level. We propose that the control of PABP synthesis may involve mRNA decay and the repression of translation.
Assuntos
Regiões 5' não Traduzidas , Adenosina/metabolismo , Cloranfenicol O-Acetiltransferase/genética , Proteínas de Ligação a Poli(A)/genética , RNA Mensageiro/genética , Sequência de Bases , Linhagem Celular , Primers do DNA , Genes Reporter , Humanos , Dados de Sequência Molecular , RNA Mensageiro/químicaRESUMO
BACKGROUND: Chloramphenicol is one of the therapeutic options for shigellosis, but resistance to this antimicrobial is increasing. AIM: To characterize molecular mechanisms conferring resistance to chloramphenicol (Cm) in Shigella flexneri strains isolated from Chilean children with acute diarrhea. MATERIAL AND METHODS: Thirty one Shigella filexneri strains, including 22 with the Cm phenotype were analyzed. Strains were tested for antimicrobial susceptibility by plate dilution and for the presence of an internal fragment of the cat gene encoding for chloramphenicol o-acetyl-transferase, by polymerase chain reaction and Southern blot analysis. RESULTS: All Cm strains had a minimal inhibitory concentration over 64 micrograms/ml and amplified the internal fragment of the cat gene. Southern blot analyses indicated that this gene was located in the bacterial chromosome. CONCLUSIONS: Resistance to chloramphenicol in this group of Shigella flexneri strains was mediated by a chromosomally located cat gene.
Assuntos
Antibacterianos/farmacologia , Resistência ao Cloranfenicol/genética , Cloranfenicol/farmacologia , Shigella flexneri/efeitos dos fármacos , Doença Aguda , Southern Blotting , Criança , Cloranfenicol O-Acetiltransferase/genética , Cromossomos Bacterianos/genética , Diarreia/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Shigella flexneri/genéticaRESUMO
BACKGROUND: Recently, we synthesized a nonviral gene vector capable of transfecting cell lines taking advantage of neurotensin (NT) internalization. The vector is NT cross-linked with poly-L-lysine, to which a plasmid DNA was bound to form a complex (NT-polyplex). Nigral dopamine neurons are able to internalize NT, thus representing a target for gene transfer via NT-polyplex. This hypothesis was tested here using reporter genes encoding green fluorescent protein or chloramphenicol acetyl transferase. MATERIALS AND METHODS: NT-polyplex was injected into the substantia nigra. Double immunofluorescence labeling was used to reveal the cell type involved in the propidium iodide-labeled polyplex internalization and reporter gene expression. RESULTS: Polyplex internalization was observed within dopamine neurons but not within glial cells, and was prevented by both hypertonic sucrose solution and SR-48692, a selective nonpeptide antagonist of NT receptors. Reporter gene expression was observed in dopamine neurons from 48 hr up to 15 days after NT-polyplex injection, and was prevented by SR-48692. However, no expression was seen when the NT-polyplex was injected into the ansiform lobule of the cerebellum, which contains low- but not high-affinity NT receptors. Neither internalization nor expression was observed in cultured glial cells, despite the NT-polyplex binding to those cells that was prevented by levocabastine, a low-affinity NT receptor antagonist. CONCLUSIONS: These results suggest that high-affinity NT receptors mediate the uptake of NT-polyplex with the subsequent reporter gene expression in vivo. NT polyfection may be used to transfer genes of physiologic interest to nigrostriatal dopamine neurons, and to produce transgenic animal models of dopamine-related diseases.
Assuntos
Dopamina/metabolismo , Técnicas de Transferência de Genes , Neurônios/metabolismo , Receptores de Neurotensina/metabolismo , Substância Negra/metabolismo , Animais , Cloranfenicol O-Acetiltransferase/genética , DNA/administração & dosagem , Imunofluorescência , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Masculino , Neuroglia/metabolismo , Pirazóis/administração & dosagem , Quinolinas/administração & dosagem , Ratos , Ratos Wistar , Substância Negra/citologiaRESUMO
Post-transcriptional regulatory mechanisms have been suggested to be the main point of control of gene expression in kinetoplastid parasites. We have previously shown that Trypanosoma cruzi SMUG mucin mRNA steady-state level is developmentally regulated by post-transcriptional mechanisms, being stable in the epimastigote insect vector stage, but unstable in the trypomastigote infective stage of the parasite. Its turnover is controlled by an AU-rich element (ARE) localized in the 3'-untranslated region, since a reporter gene lacking this sequence was stable in the trypomastigote stage (Di Noia, J. M., D'Orso, I., Sanchez, D. O., and Frasch, A. C. (2000) J. Biol. Chem. 275, 10218-10227). Here, we show by gel mobility shift assay that the 44-nt ARE sequence interacts with a set of stage-specific AU-rich element RNA-binding proteins (ARE-BPs). The epimastigote stage AU-rich element RNA-binding protein, named E-ARE-BP, and the trypomastigote stage ARE-BPs, named T-ARE-BPs, are efficiently competed by poly(U). UV cross-linking analysis showed that E-ARE-BP has an apparent molecular mass of 100 kDa and is different from the 45-50-kDa ARE-BPs present in other stages of the parasite. Transfection experiments allowed the identification of a novel cis-element that might be responsible for a positive effect on mRNA stability. It is a G-rich element, named GRE, composed by two contiguous CGGGG pentamers. The factors that recognize GRE were different from the ones that bind to ARE, in both molecular masses and subcellular localization. Thus, ARE and GRE are functionally different cis-elements, which might regulate mucin expression throughout the parasite life cycle.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/genética , RNA de Protozoário/genética , Proteínas de Ligação a RNA/metabolismo , Trypanosoma cruzi/genética , Adenina , Animais , Composição de Bases , Sequência de Bases , Sítios de Ligação , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Guanina , Dados de Sequência Molecular , Transfecção , Trypanosoma cruzi/crescimento & desenvolvimento , UracilaRESUMO
Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.
Assuntos
Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Genoma Bacteriano , Proteínas Virais , Acetiltransferases/genética , Bactérias/efeitos dos fármacos , Cloranfenicol/farmacologia , Cloranfenicol O-Acetiltransferase/genética , DNA Recombinante , Resistência a Múltiplos Medicamentos/genética , Escherichia coli/efeitos dos fármacos , Deleção de Genes , Marcadores Genéticos , Vetores Genéticos , Gentamicinas/farmacologia , Integrases/genética , Canamicina/farmacologia , Canamicina Quinase/genética , Óperon Lac/genética , Mutagênese Insercional , Plasmídeos/genética , UDPglucose 4-Epimerase/genéticaRESUMO
The promoter of the human fragile mental retardation gene (FMR1) was functionally analyzed in order to identify elements responsible for its regulation. Plasmids carrying the wild type or different deleted-promoter sequences driving the chloramphenicol acetyl transferase gene (CAT) were transiently transfected into the SK-N-SH cells and the CAT activity was assessed. Deletion studies suggested that major regulatory elements are present in a DNA region between positions -123 and -51. Gel mobility shift and footprinting assays using a DNA fragment encompassing that promoter region showed that SP1 and AP2 transcription factors could be involved in the functioning of the FMR1 promoter.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Regiões Promotoras Genéticas/fisiologia , Proteínas de Ligação a RNA , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/fisiologia , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , DNA/genética , Pegada de DNA , Proteína do X Frágil da Deficiência Intelectual , Deleção de Genes , Humanos , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição AP-2 , TransfecçãoRESUMO
TcP2beta ribosomal protein genes in Trypanosoma cruzi are encoded by four different loci, H6.4, H1.8, H1.5 and H1.3. All loci have a similar organization, except for H1.8 that harbors two TcP2beta genes arranged in tandem and separated by a short repetitive sequence, named SIRE (short interspersed repetitive element), which is also found upstream of the first gene of the tandem and downstream of the second. In this locus the trans-splicing signal of TcP2beta is located within the SIRE element, while in the other loci it is positioned within the first 50bases upstream of the AUG with an AG acceptor site at position -12 respective to the initiation codon. Transient transfection experiments were used to evaluate the efficiency of these two different trans-splicing regions to drive CAT activity. The region named HX1 located upstream the TcP2beta H1. 8 gene was clearly more efficient than the SIRE sequence contained in the region named HX2. Therefore, we decided to use the HX1 region to ameliorate the performance of the cryptic trans-splicing signal present in the T. cruzi expression vector pRIBOTEX (Martinez-Calvillo, S., López, I., Hernandez, H., 1997. pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene 199, 71-76). By insertion of the region HX1 downstream of the ribosomal promoter of pRIBOTEX, we constructed pRHX1CAT40 that, in stable transfected cells, was able to drive CAT activity 2760 times more efficiently than the control plasmids. Based on this, a novel plasmid vector was conceived, named pTREX-n, which retains the neo gene of pRIBOTEX as a positive selectable marker and replaces the CAT-SV40 cassette in pRHX1CAT40 by a multiple cloning site.
Assuntos
DNA de Protozoário/genética , Vetores Genéticos/genética , Proteínas Ribossômicas/genética , Trypanosoma cruzi/genética , Animais , Sequência de Bases , Southern Blotting , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , DNA de Protozoário/química , Regulação da Expressão Gênica , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
Glucocorticoid gene regulation can be carried out through direct binding of glucocorticoid receptor to glucocorticoid responsive elements (GRE), regulating directly gene transcription and modulating some signaling pathways. The human immunodeficiency virus type 1 (HIV-1) expression can be activated by different immunomodulators through binding of particular nuclear factors to its long terminal repeat (LTR). In order to investigate the effect of glucocorticoids in pathways that activate HIV-1 expression, we transfected promonocyte (U937) and T lymphocyte (CEM-T4) cell lineages with a plasmid containing the chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 LTR. In U937 cells, dexamethasone (DEX) downregulates CAT expression induced by either phorbol myristate acetate (PMA), tumor necrosis factor alpha (TNFalpha) or granulocyte/macrophage-colony stimulating factor (GM-CSF). In CEM-T4 cells the CAT activity was slightly upregulated by DEX following the induction by either PMA or TNFalpha. Interestingly, in both cell lines transactivation of this reporter gene by transactivator protein (TAT) was downregulated by DEX. When the CAT gene was under control of HIV-1 enhancer isolated from its LTR background, the CAT activity induced by PMA was not affected by the presence of glucocorticoids. In all experiments, comparable data were obtained when DEX was replaced by hydrocortisone (HC). Our results show that, depending on the cell line, glucocorticoids can differently affect HIV-1 expression, probably by interfering in cellular pathways involved in virus expression. Moreover, the target of this regulation in LTR is probably not the enhancer region itself.
Assuntos
Dexametasona/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Repetição Terminal Longa de HIV/efeitos dos fármacos , HIV-1/genética , Hidrocortisona/farmacologia , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Genes Reporter , Humanos , Monócitos , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes/metabolismo , Linfócitos T , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Células U937RESUMO
The natural steroid 11beta-hydroxyprogesterone is not only a modulator of 11beta-hydroxy-steroid dehydrogenase activity, but also an efficient inducer of tyrosine aminotransferase activity in hepatocytes. In contrast with the low affinity for the mineralocorticoid receptor. 11beta-hydroxyprogesterone binds well to both the glucocorticoid receptor and the carrier protein transcortin. It is accepted that the introduction of a 1:ene double bond into 3-keto 4:ene steroids increases the glucocorticoid potency, so that 3-keto-1,4:diene steroids show improved chemical stability and are more potent glucocorticoids than their respective 4:ene analogs. The steroid pregna-1,4-diene-11beta-ol-3,20-dione (deltaHOP) had previously been described as an anti-inflamatory compound and an inhibitor of macromolecular biosynthesis in thymocytes and lymphocytes. In such studies, deltaHOP also exhibited some particular glucocorticoid properties which made it attractive as a tool for the study of the mechanism of action of glucocorticoids. In the present paper we show that deltaHOP possesses some classical biological actions of glucocorticoids such as deposition of glycogen in rat liver, induction of TAT activity in hepatocytes, and inhibition of the uptake of leucine and thymidine by thymocytes. It also exhibits minimal sodium-retaining properties. Consistent with these biological effects, deltaHOP shows a 70 times lower relative binding affinity for the mineralocortioid receptor than aldosterone, but a reasonable affinity for the glucocorticoid receptor, and is as efficient as dexamethasone in dissociating the 90 kDa heat shock protein from the glucocorticoid receptor heterocomplex. However, the inhibition of the uptake of amino acids and nucleotides observed in the presence of deltaHOP is not efficiently blocked when thymocytes are coincubated in the presence of steroids with known antiglucocorticoid activity. deltaHOP is similarly inefficient in inducing chloramphenicol-acetyl transferase activity in cells transfected with a plasmid that possesses two canonical glucocorticoid-responsive elements. Unlike most glucocorticoids, deltaHOP does not induce the fragmentation of DNA in a regular pattern characteristic of apoptosis and it does not reduce thymus weight. This unusual dissociation of glucocorticoid parameters makes deltaHOP a useful tool to discriminate between mechanisms of action by which steroids can exert their biological effects.
Assuntos
Glucocorticoides/metabolismo , Hidroxiprogesteronas/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/genética , Genes Reporter , Glucocorticoides/química , Glucocorticoides/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Hidroxiprogesteronas/química , Hidroxiprogesteronas/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Cinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Modelos Moleculares , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Timo/efeitos dos fármacos , Timo/metabolismo , TransfecçãoRESUMO
We report herein the synthesis of a novel DNA delivery system and in vitro evidence of its ability to transfect cell lines by binding to the high-affinity neurotensin receptor and subsequent internalization of ligand-receptor complexes. The targeting vehicle consisted of neurotensin crosslinked with poly-L-lysine via N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). The SPDP-derivatives with either neurotensin or poly-L-lysine were purified by gel filtration. The conjugate resulting of the reaction of neurotensin-SPDP with HS-SPDP-poly-L-lysine was purified through Biogel A 1.5. The neurotensin-SPDP-poly-L-lysine conjugate was able to bind plasmidic DNAs (pSV2cat and pGreen Lantern-1) at optimal molar ratios of 1:5 and 1:6 (DNA: conjugate), respectively. The conjugate internalized those plasmids in the cell lines (N1E-115 and HT-29) bearing the high-affinity neurotensin receptor. Expression of the plasmid products, chloramphenicol acetyltransferase and green fluorescent protein, was observed in such cell lines. Both internalization and expression of the plasmids transferred by the neurotensin-SPDP-poly-L-lysine conjugate were prevented by neurotensin (1 microM) and SR-48692 (100 nM), a specific antagonist of the high-affinity neurotensin receptor. The neurotensin-SPDP-poly-L-lysine conjugate was unable to transfect cell lines lacking the neurotensin receptor (COS-7 and L-929). In rat brain, the high-affinity neurotensin receptor is expressed by specific neurons such as those of the nigrostriatal and mesolimbic dopaminergic systems. Therefore, the neurotensin-SPDP-poly-L-lysine conjugate could be a useful tool for gene delivery to those neuronal systems.
Assuntos
Marcação de Genes , Vetores Genéticos , Neurônios/química , Neurotensina/química , Polilisina/química , Succinimidas/química , Animais , Cloranfenicol O-Acetiltransferase/genética , Reagentes de Ligações Cruzadas , Proteínas de Ligação a DNA/química , Endocitose , Código Genético , Neuroblastoma/química , Ratos , Células Tumorais CultivadasRESUMO
Glucocorticoids inhibit insulin expression in cultured pancreatic islet cells. In this study, we provide evidence that transcriptional downregulation of insulin gene expression by glucocorticoids is the result of synergistic interaction between various elements of the insulin promoter. Similar synergistic effects on insulin gene transcription were previously reported for other key insulin regulators, cyclic adenosine monophosphate (cAMP) and glucose. Transfection of CAT constructs containing different segments of the insulin promoter into the pancreatic cell line, HIT T-15 2.2.2, demonstrated that dexamethasone decreased CAT activity in all constructs tested. However, differences were found in the relative sensitivities of the various constructs. Glucocorticoid inhibition of expression from plasmids containing A elements may result from decreased expression of the pancreatic homeodomain protein STF-1. However, a different mechanism must be invoked for insulin promoter constructs lacking A sites, which nevertheless still demonstrated negative regulation. Glucocorticoid-induced inhibition of one of these regions (-882 to -342) was seen to require pancreas-specific factors, because inhibition was observed in HIT T-15 2.2.2 cells but not in the nonpancreatic COS-1 cells. We conclude, therefore, that the human insulin gene contains multiple transcriptional elements that respond to glucocorticoids, some of which require beta cell-specific factors.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Insulina/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Células COS/metabolismo , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , AMP Cíclico/farmacologia , Dexametasona/farmacologia , Sinergismo Farmacológico , Glucose/farmacologia , Humanos , Ilhotas Pancreáticas/metabolismo , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/genética , TransfecçãoRESUMO
Silencing of fibronectin (FN) expression seems to be one of the key mechanisms underlying metastatic behaviour. An inverse correlation exists between FN expression levels and the metastatic potential of two related murine mammary adenocarcinomas, M3 and MM3. Primary cultures of M3 tumour, which is moderately metastatic to lung (40% incidence), show a conspicuous FN extracellular matrix (ECM) and high levels of FN mRNA, while primary cultures of the highly metastatic MM3 tumour (95% lung incidence) are negative for FN in immunofluorescence and show at least 40-fold lower levels of FN mRNA, only detectable by RT-PCR, with a different pattern of alternatively spliced EDI isoforms compared to M3 cells. We show that the FN promoter sequence is not altered in MM3 cells. Transfection experiments with CAT constructs indicate that silencing occurs at the transcriptional level, involving the 220-bp proximal promoter region.