RESUMEN
The original article [1] contains errors in Table 1 affecting some of the presented oligonucleotide sequences and readthrough values in Table 1.
RESUMEN
In universal-code eukaryotes, a single class-1 translation termination factor eRF1 decodes all three stop codons, UAA, UAG, and UGA. In some ciliates with variant genetic codes one or two stop codons are used to encode amino acid(s) and are not recognized by eRF1. In Stylonychia, UAG and UAA codons are reassigned as glutamine codons, and in Euplotes, UGA is reassigned as cysteine codon. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of eRF1. Because variant-code ciliates most likely evolved from universal code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. To find out amino acid residues which confer UAR-only specificity to Euplotes aediculatus eRF1, eRFI chimeras were constructed by swapping eRF1 E. aediculatus N-terminal domain sequences with the matching ones from the human protein. In these chimeras the MC-domain was from human eRF1. Functional analysis of these chimeric eRFI highlighted the crucial role of the two regions (positions 38-50 and 123-145) in the N-terminal domain of E. aediculatus eRF1 that restrict E. aediculatus eRF1 specificity toward UAR codons. Possibly, restriction of eRF1 specificity to UAR codons might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UGA to sense codons.
Asunto(s)
Codón de Terminación , Euplotes/genética , Código Genético , Modelos Moleculares , Factores de Terminación de Péptidos/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Euplotes/fisiología , Humanos , Datos de Secuencia Molecular , Factores de Terminación de Péptidos/genética , Conformación Proteica , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The suppression levels induced by gentamicin on premature stop codons, caused by primary nonsense mutations found in muscular dystrophy patients, were assessed using a very sensitive dual reporter gene assay. Results show that: (i) the effect of gentamicin on readthrough is similar in cultured cells and in vivo in murine skeletal muscle; (ii) a wide variability of readthrough efficiency is obtained, depending on the mutation tested; (iii) due to the complexity of readthrough regulation, efficiency cannot be predicted by the nucleotide context of the stop codon; (iv) only a minority of premature stop codons found in patients show a significant level of readthrough, and would thus be amenable to this pharmacological treatment, given our present understanding of the problem. These results probably provide an explanation for the relative failure of clinical trials reported to date using gentamicin to treat diseases due to premature stop codons, and emphasize that preliminary assays in cell culture provide valuable information concerning the potential efficiency of pharmacological treatments.
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Antibacterianos/uso terapéutico , Codón de Terminación , Terapia Genética/métodos , Gentamicinas/uso terapéutico , Músculo Esquelético/enzimología , Distrofias Musculares/terapia , Células 3T3 , Animales , Codón sin Sentido , Terapia Combinada , Electroporación , Expresión Génica , Luciferasas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Distrofias Musculares/tratamiento farmacológico , beta-Galactosidasa/genéticaRESUMEN
The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3' termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence -CA(A/G)N(U/C/G)A-, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3' stop codon context on translation termination.
Asunto(s)
Codón de Terminación , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Alelos , Secuencias de Aminoácidos , Secuencia de Bases , Clonación Molecular , Codón , Biblioteca de Genes , Glicina/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico 18S/metabolismo , ARN de Transferencia/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
BACKGROUND: Translation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context. Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved. RESULTS: We have performed an in vivo analysis of translational readthrough in mouse cells in culture using a reporter system that allows the measurement of readthrough levels as low as 10(-4). We first quantified readthrough frequencies obtained with constructs carrying different codons (two Gln, two His and four Gly) immediately upstream of the stop codon. There was no effect of amino acid identity or codon frequency. However, an adenine in the -1 position was always associated with the highest readthrough levels while an uracil was always associated with the lowest readthrough levels. This could be due to an effect mediated either by the nucleotide itself or by the P-site tRNA. We then examined the importance of the downstream context using eight other constructs. No direct correlation between the +6 nucleotide and readthrough efficiency was observed. CONCLUSIONS: We conclude that, in mouse cells, the upstream and downstream stop codon contexts affect readthrough via different mechanisms, suggesting that complex interactions take place between the mRNA and the various components of the translation termination machinery. Comparison of our results with those previously obtained in plant cells and in yeast, strongly suggests that the mechanisms involved in stop codon recognition are conserved among eukaryotes.
RESUMEN
A prion is an infectious, altered form of a cellular protein which can self-propagate and affect normal phenotype. Prion conversion has been observed for mammalian and yeast proteins but molecular mechanisms that trigger this process remain unclear. Up to now, only post-translational models have been explored. In this work, we tested the hypothesis that co-translational events may be implicated in the conformation changes of the Ure2p protein of Saccharomyces cerevisiae. This protein can adopt a prion conformation leading to an [URE3] phenotype which can be easily assessed and quantified. We analyzed the effect of two antibiotics, known to affect translation, on [URE3] conversion frequency. For cells treated with G418 we observed a parallel increase of translational errors rate and frequency of [URE3] conversion. By contrast, cycloheximide which was not found to affect translational fidelity, has no influence on the induction of [URE3] phenotype. These results raise the possibility that the mechanism of prion conversion might not only involve alternative structures of strictly identical molecules but also aberrant proteins resulting from translational errors.
Asunto(s)
Priones/genética , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Animales , Antibacterianos/farmacología , Sistema de Lectura Ribosómico/efectos de los fármacos , Gentamicinas/farmacología , Glutatión Peroxidasa , Humanos , Fenotipo , Priones/química , Biosíntesis de Proteínas/efectos de los fármacos , Conformación Proteica , Pliegue de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.
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Mutación del Sistema de Lectura , Mutación , Biosíntesis de Proteínas , Secuencia de Aminoácidos , Secuencia de Bases , Codón , Escherichia coli/metabolismo , Genes Reporteros , Datos de Secuencia Molecular , Plásmidos , Saccharomyces cerevisiae/genética , Activación TranscripcionalAsunto(s)
Sistema de Lectura Ribosómico , Proteínas Fúngicas/fisiología , Código Genético , Modelos Genéticos , ARN Mensajero/genética , Proteínas de Unión al ARN , Ribosomas/fisiología , Proteínas de Saccharomyces cerevisiae , Transactivadores/fisiología , Codón/genética , Codón sin Sentido/genética , ADN Recombinante/genética , Factor 2B Eucariótico de Iniciación/fisiología , Genes/genética , Genes Reporteros , Operón Lac , Luciferasas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
A 2'-O-methylribooligonucleotide containing a G1.U.G3 triad modified by trans-diamminedichloro-platinum(II) was targeted to the RNA region responsible for the gag-pol frameshifting during translation of the HIV-1 mRNA. The binding of the platinated oligonucleotide to its target RNA induced a rearrangement of the (G1, G3)-intrastrand crosslink, leading to the formation of an intermolecular oligonucleotide-RNA G-A crosslink. This resulted in the selective arrest of translation of a luciferase gene placed downstream of the HIV-1 frameshift signal both in a cell-free extract (rabbit reticulocyte lysate) and in RNA-transfected cells. A specific inhibition of luciferase activity was still observed when the oligonucleotide-RNA complex was not pre-formed prior to either translation or transfection. Moreover, a selective inhibition was also observed when the oligonucleotide and the plasmid DNA encoding the luciferase and bearing the RNA gag- pol frameshifting signal were co-transfected in NIH 3T3 cultured cells. Therefore the intra-strand-->interstrand conversion of the platinum crosslink kinetically competes with the translation machinery and blocks the polypeptide elongation. These transplatin-modified oligonucleotides which operate within a live cell on a 'real-time' basis and do not need an external triggering signal constitute a promising new class of selective reactive probes.
Asunto(s)
Cisplatino/química , Sistema de Lectura Ribosómico , Proteínas de Fusión gag-pol/genética , VIH-1/genética , Oligorribonucleótidos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Células 3T3 , Animales , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , Oligorribonucleótidos/química , ARN Viral/genética , Transcripción Genética/efectos de los fármacosRESUMEN
In many retroviruses, the expression of reverse transcriptase, protease, and integrase is dependent upon a -1 frameshift event. The frameshift signal is composed of a slippery sequence where the ribosome shifts, and a downstream stimulatory sequence. In most cases, the stimulatory sequence is a pseudoknot, but in some viruses, such as human immunodeficiency virus type 1 (HIV-1), a single stem-loop is involved. Here, we analyzed the precise role of the stem-loop thermodynamic stability. We tested the frameshifting stimulatory activity of a series of HIV-1-derived sequences showing a stepwise increment of the estimated deltaG degrees. These sequences were introduced at the junction of a lacZ-luc fusion gene cloned on a versatile expression vector, and the different constructs were tested in Saccharomyces cerevisiae and in mouse NIH3T3 cells. The results showed that the frameshifting efficiency was correlated directly to the stem stability between deltaG degrees = -2.5 kcal mol(-1) and deltaG degrees = -19.4 kcal mol(-1). This demonstrates the essential role of the stability of the stem-loop and does not support the involvement of a specific RNA-binding protein target sequence. However, increasing further the stem stability led to a diminution of frameshifting efficiency, suggesting that the stem-loop acts through a precise kinetic of pausing. Because the same pattern was observed in both yeast and mouse cells, it is likely that the stimulatory mechanism is conserved through evolution.
Asunto(s)
Sistema de Lectura Ribosómico , Regulación Viral de la Expresión Génica/genética , VIH-1/genética , ARN Mensajero/genética , ARN Viral/química , ARN Viral/genética , Células 3T3 , Animales , Composición de Base , Operón Lac/genética , Ratones , Conformación de Ácido Nucleico , ARN Mensajero/química , Saccharomyces cerevisiae , Termodinámica , Transfección , Transformación GenéticaRESUMEN
In many viruses and transposons, expression of some genes requires alternative reading of the genetic code, also called recoding. Such events depend on specific mRNA sequences and can lead to read through of an in-frame stop codon or to +1 or -1 frameshifting. Here, we addressed the issue of conservation of recoding rules between the yeast Saccharomyces cerevisiae and mammalian cells by establishing a versatile vector that can be used to study recoding in both species. We first assessed this vector by analysing the site of +1 frameshift of the Ty1 transposon. Two sequences from higher organisms were then tested in both yeast and mammalian cells: the gag-pol junction of human immunodeficiency virus type 1 (HIV-1) (a site of -1 frameshift), and the stop codon region of the replicase cistron from the tobacco mosaic virus (a site of UAG read through). We show that both sequences direct a high level of recoding in yeast. Furthermore, different mutations of the target sequences have similar effects on recoding in yeast and in mouse cells. Most notably, a strong decrease of frameshifting was observed in the absence of the HIV-1 stem-loop stimulatory signal. Taken together, these data suggest that mechanisms of some recoding events are conserved between lower and higher eukaryotes, thus allowing the use of S. cerevisiae as a model system to study recoding on target sequences from higher organisms.
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Vectores Genéticos , VIH-1/genética , ARN Mensajero/genética , Virus del Mosaico del Tabaco/genética , Animales , Secuencia de Bases , Secuencia Conservada , Mutación del Sistema de Lectura , Humanos , Mamíferos , Datos de Secuencia Molecular , Saccharomyces cerevisiae , Alineación de SecuenciaRESUMEN
A frameshift event is necessary for expression of the products of the pol gene in a number of retroviruses, including human immunodeficiency virus type 1 (HIV-1). The basic signals necessary for frameshifting consist of a shifty sequence in which the ribosome slips and a downstream stimulatory structure which can be either a stem-loop or a pseudoknot. In HIV-1, much attention has been paid to the frameshift site itself, and only recently has the role of the downstream structure been examined. Here we used a luciferase-based experimental system to analyze in vivo the cis and trans factors potentially involved in controlling frameshifting efficiency at the gag-pol junction of HIV-1. We demonstrated that high-level frameshifting is dependent on the presence of a palindromic region located downstream of the site where the frameshift event takes place. Frameshifting efficiencies were found to be identical in mouse fibroblasts and the natural host cells of the virus, i.e., CD4+ human lymphoid cells. Furthermore, no increase in frameshifting was observed upon virus infection. Previous observations have shown that viral infection leads to specific alteration of tRNAs involved in translation of shifty sites (D. Hatfield, Y.-X. Feng, B.J. Lee, A. Rein, J.G. Levin, and S. Oroszlan, Virology 173:736-742, 1989). The results presented here strongly suggest that these modifications do not affect frameshifting efficiency.
Asunto(s)
Genes gag/genética , Genes pol/genética , VIH-1/genética , Biosíntesis de Proteínas , Sistemas de Lectura/genética , Animales , Secuencia de Bases , Linfocitos T CD4-Positivos/microbiología , Línea Celular , Niño , ADN Recombinante , Regulación Viral de la Expresión Génica , Humanos , Insectos/enzimología , Insectos/genética , Luciferasas/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , TransfecciónRESUMEN
To evaluate the importance of the transcription factors known to bind to the albumin promoter as well as the parameters involved in their activity, we have used cotransfections with an albumin promoter-cat plasmid combined with expression vectors driving the expression of cDNAs coding for liver-enriched factors known to interact with this promoter. We describe the characteristics of a set of clones of hepatic origin: well differentiated, partial variants or pleiotropic dedifferentiated variants. These lines have been characterized for the accumulation of RNAs corresponding to each of the albumin promoter-binding factors. Only HNF1, and to a lesser extent C/EBP, show differences depending upon the differentiation state of the cells. Overexpression of exogenous HNF1 in these cells reveals that this factor is able to transactivate the albumin promoter only in variant cells where the endogenous protein is limiting. By contrast, if the HNF1-binding site is of weak affinity, overexpression of exogenous HNF1 stimulates the albumin promoter even in the HNF1-rich differentiated cells. Overexpression of exogenous LAP strongly transactivates an artificial promoter containing one LAP-binding site, but surprisingly in all the cell lines, it has little effect upon the albumin promoter. These results demonstrate that the transactivation potential of a given transcription factor depends on the degree of differentiation of the recipient cells, on the promoter structure, and on the affinity of the binding site for this factor.
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Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Activación Transcripcional , Albúminas/genética , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Línea Celular , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Hígado/citología , Fenotipo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Expression of the pol gene of the murine leukemia viruses is subject to translational control at the UAG termination codon of the upstream gene gag. Previous experiments have suggested that: i) Moloney murine leukemia virus infection induces a tRNA(Gln)iii) in an in vitro system using the tobacco mosaic virus as template, this tRNA is able to increase readthrough at the UAG codon [1]. Here we demonstrate that, in vivo, Moloney murine leukemia virus infection does not increase translational readthrough at either the tobacco mosaic virus or the Moloney murine leukemia virus UAG stop codons.
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Codón/genética , Virus de la Leucemia Murina de Moloney/genética , Células 3T3 , Animales , Secuencia de Bases , Transformación Celular Viral , ADN Viral/genética , Genes gag , Genes pol , Ratones , Datos de Secuencia Molecular , Biosíntesis de Proteínas , ARN de Transferencia de Glutamina/genéticaRESUMEN
In order to evaluate the ability of an exogenous tissue-specific promoter to undergo the same dynamic changes in activity as the endogenous one, a 400-base pair fragment of the rat albumin proximal promoter, upstream of the bacterial gpt gene, has been introduced into rat hepatoma cells. Four clones containing a single integrated copy of the construct and producing substantial amounts of albumin and of xanthine phosphoribosyltransferase were isolated. These clones were subjected to two treatments known to result in silencing of the albumin gene: selection for dedifferentiated variants, and fusion with L-cell fibroblasts. In most cases, the albumin-negative progeny obtained no longer expressed the gpt gene: the exogenous promoter of 400 base pairs must contain the sequences required to respond to the mechanisms that block activity of the endogenous gene. However, exceptions were observed: the albumin-deficient variants of one clone remained xanthine phosphoribosyltransferase positive, and some of the albumin-negative hybrids from a different clone continued to produce xanthine phosphoribosyltransferase. These cases of dissociation in expression of the endogenous and the exogenous genes indicate that the site of integration of the alb-gpt construct in one clone renders the sequences insensitive to the mechanisms responsible for albumin gene silencing in dedifferentiated variants, and in the other clone to the mechanism of extinction. Consequently, the mechanisms causing gene silencing in variants and in intertypic hybrids must be different.
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Regulación de la Expresión Génica , Neoplasias Hepáticas Experimentales/genética , Regiones Promotoras Genéticas , Albúmina Sérica/genética , Aflatoxina B1 , Aflatoxinas/farmacología , Animales , Diferenciación Celular , Células Híbridas , Técnicas In Vitro , Células L , Ratones , Ratas , Transfección , Células Tumorales CultivadasRESUMEN
Translational errors are necessary so as to allow gene expression in various organisms. In retroviruses, synthesis of pol gene products necessitates either readthrough of a stop codon or frameshifting. Here we present an experimental system that permits quantification of translational errors in vivo. It consists of a family of expression vectors carrying different mutated versions of the luc gene as reporter. Mutations include both an in-frame stop codon and 1-base-pair deletions that require readthrough or frameshift, respectively, to give rise to an active product. This system is sensitive enough to detect background errors in mammalian cells. In addition, one of the vectors contains two unique cloning sites that make it possible to insert any sequence of interest. This latter vector was used to analyse the effect of a DNA fragment, proposed to be the target of high level slippage at the gag-pol junction of HIV. The effect of paromomycin and kasugamycin, two antibiotics known to influence translational ambiguity, was also tested in cultured cells. The results indicate that paromomycin diversely affects readthrough and frameshifting, while kasugamycin had no effect. This family of vectors can be used to analyse the influence of structural and external factors on translational ambiguity in both mammalian cells and bacteria.
Asunto(s)
Aminoglicósidos , Mutación del Sistema de Lectura , Proteínas de Fusión gag-pol/genética , Vectores Genéticos , VIH-1/genética , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Secuencia de Bases , Células Cultivadas , Clonación Molecular , ADN Viral/química , Escherichia coli/genética , Proteínas de Fusión gag-pol/biosíntesis , Genes Virales , VIH-1/efectos de los fármacos , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Paromomicina/farmacología , Biosíntesis de Proteínas , Reproducibilidad de los ResultadosRESUMEN
We present a strategy to elucidate the rate-limiting steps in activation of carcinogenic compounds by cytochromes P450. The principle was to select Reuber rat hepatoma cells for resistance to a procarcinogen. The hypothesis was that resistant cells should be systematically deficient in the P450 enzyme(s) involved in the activation process. Here we present an example of the use of this approach using aflatoxin B1 (AFB1), a potent hepatocarcinogen, as the selective agent. Parental cells as well as individual and pooled colonies selected for AFB1 resistance from three independent rat hepatoma lines were characterized for their content of 1) mRNA hybridizing to cDNA and/or oligonucleotide probes for cytochromes P450IIB1, P450IIB2 and albumin; and 2) aldrin epoxidase activity. Parental aflatoxin B1-sensitive cells were shown to express P450IIB1 but not P450IIB2. The majority of the aflatoxin B1-resistant clones failed to accumulate cytochrome P450IIB1 mRNA and expressed no or only very low aldrin epoxidase activity. Albumin mRNA levels remained unchanged, demonstrating that loss of expression of cytochrome P450IIB1 was not a consequence of a general dedifferentiation event. A revertant population showing restoration of both cytochrome P450IIB1 mRNA accumulation and aldrin epoxidase activity was fully sensitive to aflatoxin B1. The correlation between expression of cytochrome P450IIB1 and sensitivity to aflatoxin B1 in both parental cells and revertants strongly suggests that cytochrome P450IIB1 is a major contributor to the activation of aflatoxin B1 in rat hepatoma cells. The kind of strategy described here could be applied to other compounds that become cytotoxic for hepatoma cells following activation by cytochromes P450.
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Aflatoxinas/farmacología , Biotransformación , Carcinógenos , Sistema Enzimático del Citocromo P-450/metabolismo , Aflatoxina B1 , Aflatoxinas/metabolismo , Animales , Secuencia de Bases , Resistencia a Medicamentos , Neoplasias Hepáticas Experimentales , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , ARN Mensajero/biosíntesis , Ratas , Células Tumorales CultivadasRESUMEN
Gene expression in heterologous species is a powerful tool for the cloning and characterization of genes. Here, we present a family of expression shuttle vectors which work in the budding yeast, Saccharomyces cerevisiae, and also in mammalian cells. The quantity of product expressed by the gene under study can be modulated in yeast by virtue of the control over plasmid copy number and culture conditions.
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Expresión Génica , Vectores Genéticos , Plásmidos , Saccharomyces cerevisiae/genética , Animales , Secuencia de Bases , Línea Celular , Codón/genética , Técnicas Genéticas , Células L/enzimología , Ratones , Datos de Secuencia Molecular , Mapeo Restrictivo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
We previously described a family of dispersed palindromic sequences highly repeated in Escherichia coli and Salmonella typhimurium genomes. These sequences, called PU (palindromic units), are located outside structural genes. Conflicting results have been reported on the effects of different PU in transcription termination. Two PU located between co-transcribed genes in S. typhimurium were found not to cause transcription termination [25]. One PU located between convergently transcribed genes in E. coli behaved as bidirectional transcription terminators [12]. In the present paper, we show that three PU located between co-transcribed genes in E. coli are not a transcription terminator. From the literature, we define a subfamily of PU, which we called PU, located between convergently transcribed genes which we implicate in bidirectional transcription termination. This plus the analysis of another PU which terminates transcription suggest that pecularities in the sequence or in the sequence environment of PU determine their role in transcription termination.
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Escherichia coli/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
The structure and expression of the distal part of the malK-lamB operon in Escherichia coli was studied. DNA sequencing was performed as far as a HinfI restriction site located 1313 base-pairs downstream from gene lamB. The open reading frame, formerly called molA, which begins 245 base-pairs downstream from gene lamB, is longer than was initially thought, and was renamed malM. It could encode a protein of 306 amino acid residues. The complete malM open reading frame was cloned under control of the tac 12 promoter. In maxicells, the resulting plasmid permitted tac12-promoted synthesis of two polypeptides, encoded by gene malM, with apparent molecular weights of 37 X 10(3) and 34.5 X 10(3). We provide strong evidence that the 34.5 X 10(3) Mr protein is derived from the 37 X 10(3) Mr protein by processing at the amino-terminal end, and that this processed form is located in the periplasmic space. We show that the chromosomal malM gene is expressed as part of the malK-lamB operon, and that its product is periplasmic. Finally, we demonstrate with nuclease S1 mapping experiments that the mRNA terminates at a typical rho-independent terminator located about 45 base-pairs beyond the end of gene malM, which is thus the last gene of the malK-lamB operon.