RESUMO
We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of â¼140â¯nt containing a 24â¯nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24â¯nt random sequence insert of N1 was abundant in guanine residues (ten out of 24â¯nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G16, -G18, -G20, -G22, and -G24). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G20 clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Pequeno RNA não Traduzido/genética , Salmonella typhimurium/crescimento & desenvolvimento , Sequência de Bases , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Plasmídeos/administração & dosagem , Plasmídeos/química , Plasmídeos/genética , Pequeno RNA não Traduzido/administração & dosagem , Pequeno RNA não Traduzido/química , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Transformação GenéticaRESUMO
BACKGROUND: The function of promyelocytic leukemia (PML) bodies is not well known but plays an important role in controlling cell proliferation, apoptosis and senescence. This study was undertaken to analyze the clinical significance of PML body expression in primary tumor samples from malignant fibrous histiocytoma (MFH) and liposarcoma patients. METHODS: We studied MFH and liposarcoma samples from 55 patients for PML bodies. Fluorescent immunostaining of PML bodies was performed in the paraffin-embedded tumor sections. RESULTS: PML body immunostaining was identified in 63.9% of MFH and 63.2% of liposarcoma samples. PML body expression rates of all sarcoma cells were 1.5 +/- 1.8% (range: 0-7.0) in MFH and 1.3 +/- 1.4% (0-5.2) in liposarcoma samples. PML body expression (p = 0.0053) and a high rate of PML body expression (p = 0.0012) were significantly greater prognostic risk factors for death than the other clinical factors in MFH patients. All liposarcoma patients without expression of PML were disease free at the end of the study. CONCLUSION: Our study suggests that the presence of PML bodies may indicate a poor prognosis for MFH and liposarcoma patients.