RESUMO
The CRISPR-Cas system is involved in bacterial immunity, virulence, gene regulation, biofilm formation and sporulation. In Salmonella enterica serovar Typhi, this system consists of five transcriptional units including antisense RNAs. It was determined that these genetic elements are expressed in minimal medium and are up-regulated by pH. In addition, a transcriptional characterization of cas3 and ascse2-1 is included herein.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA Helicases/genética , Regulação Bacteriana da Expressão Gênica/genética , RNA Antissenso/genética , Salmonella typhi/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Transcrição Gênica/genética , Ativação Transcricional/genética , Regulação para Cima/genéticaRESUMO
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
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
DNA replication mechanisms are poorly understood in most of trypanosomatids, in particular the replication of the peculiar mitochondrial DNA, the kinetoplast DNA (kDNA). To contribute to the knowledge on the mechanism of kDNA replication in Trypanosoma cruzi, we have previously characterized the Universal Minicircle Sequence Binding Protein of this parasite (TcUMSBP), which was first called PDZ5 [E.R. Coelho, T.P. Urmenyi, J. Franco da Silveira, E. Rondinelli, R. Silva, Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi, Int. J. Parasitol. 33 (2003) 853-858]. In this work, we describe two highly polymorphic alleles of the TcUMSBP locus in the T. cruzi reference clone CL Brener and the differential expression pattern of these alleles. A 62 bp sequence in the TcUMSBP upstream intergenic region in one of its alleles affects the efficiency of polycistronic RNA processing and the polyadenylation sites, and therefore regulates the differential expression of TcUMSBP alleles of this locus.
Assuntos
Alelos , DNA de Cinetoplasto , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Polimorfismo Genético , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Southern Blotting , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , Replicação do DNA , DNA de Cinetoplasto/genética , Eletroporação , Regulação da Expressão Gênica , Genoma , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , TransfecçãoRESUMO
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
Many types of evidence support a role of the sympathetic nervous system in the regulation of thyroid function, although there is no general consensus on the type of influence that catecholamines exert. Depending on the experimental approach, epinephrine and norepinephrine (NE) can stimulate, inhibit, or fail to act on thyroid function. The aim of this study was to determine the effect of NE on thyroglobulin (Tg) synthesis and gene expression in FRTL-5 cells. Tg content, measured by immunoprecipitation with a specific antibody, showed that NE caused a 45% inhibition of thyrotropin (TSH) effect. The content of Tg mRNA was analyzed by Northern blot, the relative inhibition in total Tg mRNA levels from NE-treated cells, compared to TSH alone, ran parallel with inhibition in Tg content, while total RNA did not change after incubation with NE. There was no alteration in Tg mRNA stability by NE. When plasmids harboring different sequences of Tg promoter fused to the CAT reporter gene were transfected into FRTL-5 cells, TSH treatment stimulated promoter activity while NE diminished this effect by 43%-55%. Northern blots were performed to analyze mRNA for thyroid transcription factors (TTF1, TTF2, Pax8), and no significant changes were observed with the different treatments. In conclusion these results suggest that NE inhibits Tg synthesis at the transcriptional level.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Norepinefrina/farmacologia , Tireoglobulina/biossíntese , Tireoglobulina/genética , Glândula Tireoide/metabolismo , Animais , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Processamento de Imagem Assistida por Computador , Metionina/metabolismo , Regiões Promotoras Genéticas/genética , RNA/biossíntese , RNA Mensageiro/biossíntese , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Tireotropina/biossíntese , TransfecçãoRESUMO
BACKGROUND: Estrogens exert profound effects on target tissues. These effects are mediated by two estrogen receptors (ER(alpha) and ER(beta)) that bind to specific DNA sequences in estrogen-dependent genes. Other molecules such as growth factors, transcription factors and some oncoproteins might interact with the estrogen receptors and thus regulate the transcription of these genes. Currently there is no adequate cellular model to study these interactions. METHODS: We transfected the human wild-type ER(alpha) to an ER-negative rat epithelial endometrial cell line (Rentr01) using a tetracycline-regulated gene expression system. The exogenous receptor was correctly translated, had an appropriate hormone-binding affinity, and bound well to estrogen response elements containing DNA. RESULTS: We obtained a new stable cell line that is ER(beta) negative but ER(alpha) positive (R1-49E1). The expression of receptor alpha can be regulated in a dose-response manner by addition of tetracycline in the culture medium. Estradiol treatment of ER(alpha)-containing cells apparently diminished cellular proliferation, and the exogenous receptor can induce the transcription of the endogenous progesterone receptor isoform B (PgR-B) gene. CONCLUSIONS: This epithelial cellular model may be useful to study the interaction between estrogens and other cell signaling pathways in epithelial endometrial cell physiology.
Assuntos
Linhagem Celular , Endométrio/citologia , Receptor alfa de Estrogênio/biossíntese , Tetraciclina/farmacologia , Animais , Diferenciação Celular , Proliferação de Células , Cloranfenicol O-Acetiltransferase/metabolismo , Meios de Cultura/farmacologia , DNA/química , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Genes Reporter , Humanos , Imuno-Histoquímica , Camundongos , Células NIH 3T3 , Ligação Proteica , Ratos , Receptores de Progesterona/metabolismo , Elementos de Resposta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
The multidrug resistance EhPgp5 gene promoter is active in drug resistant clone C2 trophozoites and its activity increases when trophozoites are cultured in the presence of emetine, suggesting that the EhPgp5 gene shows an inducible drug dependent mechanism. We analyzed different promoter fragments to detect those regions that activate transcription in the presence of emetine. Trophozoites were transfected with p375Pgp5, p259Pgp5, p187Pgp5, and p76Pgp5 plasmids and incubated with different emetine concentrations. p375Pgp5 and p259Pgp5 plasmids were able to drive CAT expression in A and C2 trophozoites only in the presence of emetine. CAT activity was turned off in the absence of drug. Interestingly, no CAT activity was detected in the presence or in the absence of emetine with p187Pgp5 plasmid in which 59 bp were deleted at the 5' end of the EhPgp5 minimal promoter (p259Pgp5). These results suggest that the overexpression of the EhPgp5 gene is a consequence of transcriptional activation of the gene promoter by putative drug responsive elements, located within the -111 to -170 bp of the transcription initiation site.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Amebicidas/farmacologia , Emetina/farmacologia , Entamoeba histolytica/efeitos dos fármacos , Proteínas de Protozoários/genética , Ativação Transcricional/fisiologia , Animais , Cloranfenicol O-Acetiltransferase/metabolismo , Resistência a Múltiplos Medicamentos/genética , Eletroporação , Entamoeba histolytica/genética , Entamoeba histolytica/crescimento & desenvolvimento , Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , TransfecçãoRESUMO
The Human T-cell leukemia virus type I (HTLV-I) non-structural protein Tax plays a crucial role in cellular transformation. It activates the transcription factors of various cellular genes and interacts with cellular proteins. There is limited data available on the interaction between specific T-cell transcription factor GATA3 and Tax. Implications for the significance of GATA3 in T-cell development and function, T helper2 (Th2) differentiation, and a role of GATA3 during the immune response have been reported. To determine the effect of the Tax protein on GATA3 gene expression, we investigated the interaction between this protein and the GATA3 promoter and repressor regions. Results demonstrated an interaction between Tax and the GATA3 promoter via the transcription factor Sp1 and a role for Tax in the negative regulation of GATA3 expression, through its interaction with the repressor ZEB. This interaction may be involved in the pathophysiology of adult T-cell leukemia/lymphoma (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM).
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Produtos do Gene tax/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Regiões Promotoras Genéticas/genética , Transativadores/genética , Adulto , Cloranfenicol O-Acetiltransferase/metabolismo , Fator de Transcrição GATA3 , Humanos , Células Jurkat , Fator de Transcrição Sp1/fisiologia , Linfócitos T/metabolismo , Transcrição GênicaRESUMO
Secreted as well as surface exposed proteins are assumed to play major roles in bacterial virulence. In this report we describe the construction of an N-terminal protein-capturing system and its use for the isolation of Brucella abortus S2308 genes coding for putative surface exposed or secreted proteins. For this purpose, a cloning vector that generates gene fusions to a ribosome binding site and start codon deficient Chloramphenicol Acetyl Transferase (CAT) reporter gene was constructed and the resulting library introduced into B. abortus S2308 and virB mutant strains. Secreted translational fusions were identified by determining CAT activity in culture supernatants. Secretion was confirmed by Western Blot using a polyclonal anti-CAT antibody. A total of 864 clones were screened and 10 genes encoding putative secreted/surface exposed proteins were identified. Seven are Brucella proteins with an assigned function, whereas three are hypothetical proteins. The number of amino acid residues that promotes CAT secretion varies from 5 to 386 and no conserved motifs were detected. Secretion in a virB mutant background of some of the isolated fusion proteins was also determined. Interestingly, some hybrid proteins seemed to require a full VirB system for their secretion.
Assuntos
Proteínas de Bactérias/genética , Brucella abortus/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Brucella abortus/metabolismo , Brucella abortus/patogenicidade , Clonagem Molecular , Vetores Genéticos , Mutagênese Insercional , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5'-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3beta or HNF3beta plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3beta is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3beta can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3beta binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação Enzimológica da Expressão Gênica , Insulina/metabolismo , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Bases , Sítios de Ligação , Southern Blotting , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Genes Dominantes , Vetores Genéticos , Células HeLa , Fator 3-beta Nuclear de Hepatócito , Humanos , Dados de Sequência Molecular , Mutação , Fatores de Transcrição NFI , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA/química , RNA Mensageiro/metabolismo , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
ZO-2 is a membrane-associated guanylate kinase (MAGUK) protein present at the tight junction (TJ) of epithelial cells. While confluent monolayers have ZO-2 at their cellular borders, sparse cultures conspicuously show ZO-2 at the nuclei. To study the role of nuclear ZO-2, we tested by pull-down assays and gel shift analysis the interaction between ZO-2 GST fusion proteins and different transcription factors. We identified the existence of a specific interaction of ZO-2 with Fos, Jun and C/EBP (CCAAT/enhancer binding protein). To analyze if this association is present "in vivo", we performed immunoprecipitation and immunolocalization experiments, which revealed an interaction of ZO-2 with Jun, Fos and C/EBP not only at the nucleus but also at the TJ region. To test if the association of ZO-2 with AP-1 (activator protein-1) modulates gene transcription, we performed reporter gene assays employing chloramphenicol acetyltransferase (CAT) constructs with promoters under the control of AP-1 sites. We observed that the co-transfected ZO-2 down-regulates CAT expression in a dose-dependent manner. Since ZO-2 is a multidomain protein, we proceeded to determine which region of the molecule is responsible for the modulation of gene expression, and observed that both the amino and the carboxyl domains are capable of inhibiting gene transcription.
Assuntos
Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/metabolismo , Cães , Células Epiteliais/citologia , Expressão Gênica , Genes Reporter , Glutationa Transferase/metabolismo , Guanilato Quinases , Proteínas de Membrana/química , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Junções Íntimas/enzimologia , Junções Íntimas/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Proteína da Zônula de Oclusão-2RESUMO
Two clearly separated transcription start points (tsp) have been reported within the Trypanosoma cruzi rDNA (DNA encoding rRNA) gene spacer region. These sites are separated by 270 bp, a distance compatible with the occurrence of two core promoters. To characterize the individual participation of these two elements, a deletion analysis was carried out. Different versions of the promoter regions were assayed in a transient transfection analysis of epimastigotes, using the chloramphenicol acetyl transferase gene (cat) as a reporter. The data indicate that the so-called distal tsp-associated region (relative to the small subunit rRNA 5' terminus coding region) comprises most (80%) if not all of the observed activity. In addition, an associated locus specific repeated element showed a modest upregulating activity, since its presence stimulated the cat reporter gene by about 20%. The data here presented should be valuable in the design of expression vectors for T. cruzi, where the rRNA gene promoter has been an important functional element.
Assuntos
DNA Espaçador Ribossômico/genética , Genes de RNAr , Regiões Promotoras Genéticas , Transcrição Gênica , Trypanosoma cruzi/genética , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/metabolismo , DNA de Protozoário/genética , DNA Espaçador Ribossômico/química , Expressão Gênica , Genes de Protozoários , Genes Reporter , Dados de Sequência Molecular , Alinhamento de Sequência , Deleção de Sequência , TransfecçãoRESUMO
The tandemly arranged CPB genes of Leishmania mexicana are polycistronically transcribed and encode cysteine proteases that are differentially stage-specific; CPB1 and CPB2 are expressed predominantly in metacyclics, whereas CPB3-CPB18 are expressed mainly in amastigotes. The mechanisms responsible for this differential expression have been studied via gene analysis and re-integration of individual CPB genes, and variants thereof, into a CPB-deficient parasite mutant. Comparison of the nucleotide sequences of the repeat units of CPB1 and CPB2 with CPB2.8 (typical of CPB3-CPB18) revealed two major regions of divergence as follows: one of 258 base pairs (bp) corresponding to the C-terminal extension of CPB2.8; another, designated InS, of 120 bp, with insertions totaling 57 bp, localized to the intercistronic region downstream of CPB1 and CPB2. Cell lines expressing CPB2.8 or CPB2 with the 3'-untranslated region and intercistronic sequence of CPB2.8 showed up-regulation in amastigotes. Conversely, metacyclic-specific expression occurred with CPB2 or CPB2.8 with the 3'-untranslated region and intercistronic sequence of CPB2. Moreover, the InS down-regulated expression in amastigotes of a reporter gene integrated into the CPB locus. It is proposed that the InS mediates metacyclic-specific stage-regulated expression of CPB by affecting the maturation of polycistronic pre-mRNA. This is the first well defined cis-regulatory element implicated in post-transcriptional stage-specific gene expression in Leishmania.
Assuntos
Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Leishmania mexicana/enzimologia , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Cloranfenicol O-Acetiltransferase/metabolismo , Cisteína Endopeptidases/química , Regulação para Baixo , Gelatina/química , Regulação da Expressão Gênica , Genes , Genes Reporter , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Poli A/metabolismo , Estrutura Terciária de Proteína , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , TransfecçãoRESUMO
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
We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines
Assuntos
Animais , Camundongos , Células 3T3 , Actinas/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Echinococcus/genética , Regiões Promotoras Genéticas/fisiologia , Sequência de Bases , Técnicas de Cultura de Células , Clonagem Molecular , Expressão Gênica , Genes Reporter , Glicocálix , Regiões Promotoras Genéticas/genética , Transfecção/genéticaRESUMO
We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.
Assuntos
Células 3T3/enzimologia , Actinas/fisiologia , Cloranfenicol O-Acetiltransferase/metabolismo , Echinococcus/genética , Proteínas de Helminto/fisiologia , Regiões Promotoras Genéticas/fisiologia , Actinas/química , Actinas/genética , Animais , Sequência de Bases , Clonagem Molecular , Expressão Gênica , Genes Reporter , Proteínas de Helminto/química , Proteínas de Helminto/genética , Camundongos , Regiões Promotoras Genéticas/genética , Relação Estrutura-AtividadeRESUMO
The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA-protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.
Assuntos
Glicoproteínas/metabolismo , Proteínas da Gravidez/genética , Glicoproteínas beta 1 Específicas da Gravidez , Transcrição Gênica , Animais , Células COS , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Pegada de DNA , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Glicoproteínas/genética , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Família Multigênica , Placenta/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Timidina Quinase/genética , 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.