RESUMO
Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mutants defective in peptidyl-tRNA hydrolase (Pth) activity. Mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome. Expression of wild-type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to Pth-defective cells. Two of these wild-type bar regions, barI+ and barII+, contain minigenes with similar AUG-AUA-stop codon sequences preceded by different Shine-Dalgarno (SD) and spacer regions. The induced expression of barI+ and barII+ regions from plasmid constructs resulted in similar patterns of protein synthesis inhibition and cell growth arrest. Therefore, these deleterious effects may stem from translation of the transcripts containing the minigene two-codon 'ORF' (open reading frame). To test for this possibility, we assayed the effect of point mutations within the barI minigene. The results showed that a base pair substitution within the SD and the two-codon 'ORF' sequences affected protein synthesis and cell growth inhibition. In addition, mRNA stability was altered in each mutant. Higher mRNA stability correlated with the more toxic minigenes. We argue that this effect may be caused by ribosome protection of the mRNA in paused complexes as a result of deficiency of specific tRNA.
Assuntos
Bacteriófago lambda/crescimento & desenvolvimento , Bacteriófago lambda/genética , Escherichia coli/crescimento & desenvolvimento , Genes Virais , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Genes Virais/genética , Genes Virais/fisiologia , Mutação , Plasmídeos , Biossíntese de ProteínasRESUMO
Expression of the bacteriophage lambda two-codon, AUG AUA, barI minigene (bar+) leads to the arrest of protein synthesis in cells defective in peptidyl-tRNA hydrolase (Pth). It has been hypothesized that translation of the bar+ transcript provokes premature release and accumulation of peptidyl-tRNA (p-tRNA). Inhibition of protein synthesis would then result from either starvation of sequestered tRNA or from toxicity of accumulated p-tRNA. To test this hypothesis and to investigate the cause of arrest, we used a coupled in vitro transcription-translation system primed with DNA containing bar+ and the beta-lactamase-encoding gene of the vector as a reporter. The results show that expression of bar+ minigene severely inhibits beta-lactamase polypeptide synthesis by Pth-defective extracts and partially inhibits synthesis by wild-type extracts. Fractions enriched for Pth, or a homogeneous preparation of Pth, prevented and reversed bar+-mediated inhibition. A mutant minigene, barA702, which changes the second codon AUA (Ile) to AAA (Lys), was also toxic for Pth-defective cells. Expression of barA702 inhibited in vitro polypeptide synthesis by Pth-defective extracts and, as with bar+, exogenous Pth prevented inhibition. Addition of pure tRNALys prevented inhibition by barA702 but not by bar+. Expression of bar+ and barA702 led to release and accumulation of p-tRNAIle and p-tRNALys respectively but bar+ also induced accumulation of p-tRNALys. Finally, bar+ stimulated association of methionine with ribosomes probably as fMet-tRNAfMet and the accumulation of methionine and isoleucine in solution as peptidyl-tRNA (p-tRNA). These results indicate that minigene-mediated inhibition of protein synthesis involves premature release of p-tRNA, misincorporation of amino acyl-tRNA, accumulation of p-tRNAs and possibly sequestration of tRNAs.
Assuntos
Bacteriófago lambda/genética , Genes Virais , Biossíntese de Proteínas , Aminoacil-RNA de Transferência/biossíntese , RNA de Transferência de Isoleucina/biossíntese , RNA de Transferência de Lisina/biossíntese , Hidrolases de Éster Carboxílico/metabolismo , Sistema Livre de Células , Regulação Viral da Expressão Gênica , RNA de Transferência/biossínteseRESUMO
A comparison was made of ultrasonographic diagnosis of fetal nuchal encirclement by the umbilical cord versus the gold standard in pregnant, women at labor. 114 pregnant women at labor were studied. On admission to the labor and delivery room, each patient underwent an abdominal ultrasonographic evaluation for the identification of nuchal encirclement by the umbilical cord and the diagnosis by ultrasound was compared versus direct visualization of the umbilical cord at the moment of delivery or cesarean section (gold standard). Of the 114 patients studied, the prevalence of nuchal cord diagnosed by ultrasound was 20.1%. The diagnostic test had a sensitivity of 80% (CI 95%: 72.66-87.34), specificity of 96% (CI 95%:92.91-99.09), and positive and negative predictive values of 87% and 94% respectively. The accuracy of the test was 92%. Analysis of the discordances by the McNemar's test was not significative between obstetric ultrasound and the gold standard (p = 0.7236). The likelihood ratios were 20 and 0.20 for a positive and a negative results respectively. The ultrasonographic study during labor for diagnosis of nuchal encirclement by the umbilical cord had a high specificity (96%), and this advantage permit it to be utilized like screening test for the identification of high risk pregnancies with nuchal cord.
Assuntos
Complicações do Trabalho de Parto/diagnóstico por imagem , Cordão Umbilical/diagnóstico por imagem , Adulto , Índice de Apgar , Cesárea , Interpretação Estatística de Dados , Parto Obstétrico , Feminino , Humanos , Masculino , Pescoço , Gravidez , Sensibilidade e Especificidade , UltrassonografiaRESUMO
Peptidyl-tRNA hydrolase (Pth), an enzyme essential for Escherichia coli viability, scavenges peptidyl-tRNA released during abortive polypeptide chain elongation. Bacterial strains of E coli partially defective in Pth activity are unable to maintain bacteriophage lambda growth. Phage mutations that overcome the bacterial defect have been located to several regions in the lambda genome named bar. Plasmid constructs expressing just the bar region are toxic and cause a general arrest of protein synthesis in Pth-defective cells. Inspection of the nucleotide sequence from two bar regions reveals the short coding sequence AUG AUA Stop, spaced by an AT-rich segment from a Shine Dalgarno-like sequence (S-D). These sequences have been named minigenes. Base changes altering the putative S-D, the two sense codons, or the stop codon have been found to reduce Bar-toxicity. Transcripts containing bar function as mRNA. Upon expression in pth mutants, wild-type (bar+) transcripts are found associated with ribosomes. In addition, bar+ RNA forms ternary complexes with the 30S ribosomal subunit and the initiator tRNA and can be released upon run-off translation in the same way as an authentic mRNA. A cell free system for protein synthesis reproduces the in vivo effects: bar+ expression inhibits protein synthesis, bar+ RNA sequences are associated with ribosomes in the inhibited extracts, addition of purified Pth restores synthesis, and excess of tRNA(Lys), specific for the last sense codon in a mutant toxic minigene, prevents protein synthesis inhibition. Also, bar expression promotes association of methionine with ribosomes possibly in a translation complex. These results are consistent with a model proposing tRNA starvation to explain the behaviour of a pth mutant, thermosensitive for protein synthesis.