RESUMEN
The knowledge of translation start sites is crucial for annotation of genes in bacterial genomes. However, systematic mapping of start codons in bacterial genes has mainly relied on predictions based on protein conservation and mRNA sequence features which, although useful, are not always accurate. We recently found that the pleuromutilin antibiotic retapamulin (RET) is a specific inhibitor of translation initiation that traps ribosomes specifically at start codons, and we used it in combination with ribosome profiling to map start codons in the Escherichia coli genome. This genome-wide strategy, that was named Ribo-RET, not only verifies the position of start codons in already annotated genes but also enables identification of previously unannotated open reading frames and reveals the presence of internal start sites within genes. Here, we provide a detailed Ribo-RET protocol for E. coli. Ribo-RET can be adapted for mapping the start codons of the protein-coding sequences in a variety of bacterial species.
Asunto(s)
Codón Iniciador , Biología Computacional/métodos , Escherichia coli/genética , Ribosomas/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Codón Iniciador/efectos de los fármacos , Diterpenos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Genoma Bacteriano , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta , Biosíntesis de Proteínas/efectos de los fármacosRESUMEN
Peptide nucleic acid (PNA) is an amide based structural nucleic acid mimic with potential applications in gene therapeutic drug discovery. In the present study, we evaluated and compared the effects on gene expression, cell viability and apoptosis of two antisense PNA-d-octaarginine conjugates, targeting sequences at the AUG translation start site or the 5'-UTR of the TdT (terminal deoxynucleotidyl transferase) gene, as well as a sense oligomer corresponding to the 5'-UTR-antisense, in Molt-4 cells. The protein level of TdT was determined by flow cytometry, and qPCR was used for mRNA expression analysis. Mismatch PNAs were used as control to address the sequence/target spcifity of the biological effects. The results showed that treatment with the AUG- and to slightly lesser extent with the 5'-UTR-antisense PNAs reduced the TdT mRNA as wel as the protein level, whereas only very low effect was observed for the 5'-UTR-sense PNA. A parallel effect was observed on reduced cell survival and increased rate of apoptosis. Our findings suggest that antisense PNAs can inhibit expression of the TdT gene and induce apoptosis in Molt-4 cells.
Asunto(s)
ADN Nucleotidilexotransferasa/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Oligonucleótidos Antisentido/farmacología , Oligopéptidos/farmacología , Ácidos Nucleicos de Péptidos/farmacología , Regiones no Traducidas 5'/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Codón Iniciador/efectos de los fármacos , ADN Nucleotidilexotransferasa/genética , Ensayos de Selección de Medicamentos Antitumorales , Inducción Enzimática/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/antagonistas & inhibidores , ARN Neoplásico/biosíntesis , ARN Neoplásico/genéticaRESUMEN
OBJECTIVE: To evaluate the inhibitory effect of heparanase antisense oligodeoxynucleotide (AS-ODN) on the invasiveness of human mammary carcinoma cell line MDA435. METHODS: The AS-ODN complementary to the start codon region of heparanase mRNA and its control, scrambled nonsense oligodeoxynucleotide (NS-ODN) were designed and synthesized and phosphorothioated. The ODNs were embedded in cationic liposome Lipofectin and transfected into MDA435 cells. The total RNAs and proteins were extracted from the cells 48 hours after transfection and then semi-quantitative RT-PCR and Western blotting were performed to evaluate the heparanase gene and protein expression levels respectively. The invasiveness of transfected MDA435 cells were measured quantitatively by Matrigel invasion assays. RESULTS: The heparanase gene and protein expression and invasiveness of MDA435 cells treated with AS-ODN of different final concentrations were significantly decreased compared with that of the controls (P < 0.01). Besides, the inhibitory effects were significantly different between the cells treated with AS-ODN of different concentrations (P < 0.01). The invasiveness inhibition rates were 34.0%, 57.8% and 79.7% at the cells treated with AS-DON of the final concentrations of 0.1 micro mol/L, 0.2 micro mol/L, and 0.4 micro mol/L, respectively. CONCLUSION: Heparanase AS-DON complementary to the start codon region of heparanase mRNA has a significant inhibitory effect on the invasiveness of human mammary carcinoma cell line in a dose-dependent manner.
Asunto(s)
Neoplasias de la Mama/patología , Glucuronidasa/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Antineoplásicos/farmacología , Codón Iniciador/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glucuronidasa/biosíntesis , Humanos , Invasividad Neoplásica/prevención & control , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transfección , Células Tumorales CultivadasRESUMEN
Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with all-phosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3'-end (PPS-C(16)). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3'- and 5'-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS-C(16) ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS-C(16) ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS-C(16) ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.
Asunto(s)
Regiones no Traducidas 3'/química , Éteres de Glicerilo/química , Oligorribonucleótidos/farmacología , Tionucleótidos/síntesis química , Tionucleótidos/farmacología , Animales , Codón Iniciador/efectos de los fármacos , Codón Iniciador/metabolismo , Femenino , Genes ras/genética , Humanos , Ratones , Ratones Desnudos , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Oligorribonucleótidos/química , Oligorribonucleótidos Antisentido/metabolismo , Oligorribonucleótidos Antisentido/farmacología , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Proteína Oncogénica p21(ras)/biosíntesis , Fenotipo , ARN Mensajero/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Relación Estructura-Actividad , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
We previously suggested that the degree of polyamine stimulation of oligopeptide-binding protein (OppA) synthesis is dependent on the secondary structure and position of the Shine-Dalgarno (SD) sequence of OppA mRNA. To study the structural change of OppA mRNA induced by polyamines and polyamine stimulation of initiation complex formation, four different 130-mer OppA mRNAs containing the initiation region were synthesized in vitro. The structural change of these mRNAs induced by polyamines was examined by measuring their sensitivity to RNase T(1), specific for single-stranded RNA, and RNase V(1), which recognizes double-stranded or stacked RNA. In parallel, the effect of spermidine on mRNA-dependent fMet-tRNA binding to ribosomes was examined. Our results indicate that the secondary structure of the SD sequence and initiation codon AUG is important for the efficiency of initiation complex formation and that spermidine relaxes the structure of the SD sequence and the initiation codon AUG. The existence of a GC-rich double-stranded region close to the SD sequence is important for spermidine stimulation of fMet-tRNA binding to ribosomes. Spermidine apparently binds to this GC-rich stem and causes a structural change of the SD sequence and the initiation codon, facilitating an interaction with 30 S ribosomal subunits.