Correction to: In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study.

The original article [1] contains errors in Table 1 affecting some of the presented oligonucleotide sequences and readthrough values in Table 1.

[How translation termination factor eRF1 Euplotes does not recognise UGA stop codon].

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.

Inhibition of in vitro and ex vivo translation by a transplatin-modified oligo(2'-O-methylribonucleotide) directed against the HIV-1 gag-pol frameshift signal.

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.

Tumor x host cell hybrids in the mouse: chromosomes from the normal cell parent maintained in malignant hybrid tumors.

We investigated whether the malignancy of hybrids between normal and malignant cells could be correlated with the loss of specific genes borne by specific chromosomes from the normal parent cells. Tumors produced in mice by the inoculation of Cl.1D cells (an L cell derivative) contained tumor x host cell hybrids. Hybrid cell populations isolated from 14 tumors were injected into 123 mice, of which 108 (87%) developed tumors. Metaphases of growing hybrid cell tumors were analyzed by use of a trypsin-Giemsa banding technique. The chromosomes contributed by the host (normal) parent cell could be distinguished from Cl.1D chromosomes, since the latter exhibited morphologic differences due to rearrangements. In the 14 hybrid tumors analyzed, we found that any one of the chromosomes of the host cell might be present, which indicated that none of the chromosomes from the normal cell bore genetic information capable of suppressing the malignancy of Cl.1D cells. Absence of complimentation in the hybrids suggested that, even if the accumulation of several mutations were necessary for malignant tumor growth of Cl.D1 cells, none of these mutations is recessive.

Presence and cell growth-related variations of glycogen in human colorectal adenocarcinoma cell lines in culture.

The presence and kinetics of intracellular glycogen levels were studied, in relationship to cell growth, in asynchronous and in synchronized cultures of four human colorectal adenocarcinoma cell lines (HT-29, HRT-18, SW-480, and Caco-2). The results show that a specific pattern of glycogen accumulation occurs during the process of cell growth of the studied cell lines. The kinetics of glycogen accumulation in asynchronous cultures were similar from one cell line to another and were characterized by a low amount in the exponential phase of growth, followed by a 3- to 4-fold increase in the stationary phase. The quantities found in either phase were specific for each cell line. The maximum values found in Caco-2, HRT-18, HT-29, and SW-480 cells were, respectively, 258.5 +/- 6.9 (S.D.), 88.9 +/- 2.6, 87.5 +/- 3, and 17.5 +/- 1.8 microgram of glycogen per mg of proteins. The kinetics of glycogen accumulation during the cell cycle was also studied in synchronized cultures of HT-29 and HRT-18 cell lines. Both cell lines exhibited a common pattern of low glycogen quantities during S, G2, and M followed by an increase beginning with G1 and peaking (2.5 to 3 times the initial values) in the middle of this phase. This was followed by a symmetrical decrease in the second half of G1.

malM, a new gene of the maltose regulon in Escherichia coli K12. II. Mutations affecting the signal peptide of the MalM protein.

malM is the last gene of the malK-lamB-malM operon of Escherichia coli K12. It encodes a periplasmic protein. Mutations affecting the hydrophobic core of the N-terminal extension of the MalM protein have been isolated. They result in an increase in amount and specific activity of a MalM-LacZ hybrid protein. This result confirms that the signal peptide of the MalM protein is functional.

malM, a new gene of the maltose regulon in Escherichia coli K12. I. malM is the last gene of the malK-lamB operon and encodes a periplasmic protein.

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.

UAG readthrough in mammalian cells: effect of upstream and downstream stop codon contexts reveal different signals.

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.

Of mice and yeast: versatile vectors which permit gene expression in both budding yeast and higher eukaryotic cells.

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.

UAG readthrough is not increased in vivo by Moloney murine leukemia virus infection.

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.

Versatile vectors to study recoding: conservation of rules between yeast and mammalian cells.

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.

Translational frameshifting at the gag-pol junction of human immunodeficiency virus type 1 is not increased in infected T-lymphoid cells.

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.

Cell phenotype, binding affinity and promoter structure modulate transactivation by HNF1 and LAP.

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.

Impact of the six nucleotides downstream of the stop codon on translation termination.

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.

Hybrids between F9 nullipotent teratocarcinoma and thymus cells produce multidifferentiated tumors in mice.

Hybrids between F9 teratocarcinoma and mouse thymus cells (FT2 hybrids) were obtained. All of the seven FT2 hybrid clones produced tumors containing varying amounts of differentiated tissues in addition to embryonal carcinoma. This is in contrast to the F9 parental cells which gave tumors formed exclusively of embryonal carcinoma cells. Two clones produced either undifferentiated tumors or tumors that had only small foci of ectoderm and/or endoderm derivatives. The five other FT2 clones differentiated into derivatives of the three primitive germ layers. Among these five clones, two produced tumors with large amounts of differentiated tissues which resembled the well-differentiated teratocarcinoma tumors produced by pluripotent embryonal carcinoma cell lines. On the whole, 71% of the observed sections of the 26 tumors that were thoroughly examined at 0.5-mm intervals showed ectoderm derivatives, 40% mesoderm derivatives, and 46% endoderm derivatives. This result shows that hybridization with thymus cells allows expression of pluripotentiality that was blocked in F9 teratocarcinoma cells.

Translational errors as an early event in prion conversion.

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.

Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment.

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.

Phenotype and surface antigens of mouse teratocarcinoma x fibroblast cell hybrids.

Numerous colonies of hybrids between PCC4-aza 1 teratocarcinoma cells and fibroblasts of the heteroploid Cl.1D cell line were examined. All of the hybrids were fibroblasts showing extinction of the multiple developmental potentialities of the teratocarcinoma cell parent, irrespective of whether the teratocarcinoma parent was diploid or tetraploid. The hybrids did not show loss of any specific chromosome contributed by the PCC4-aza 1 cell parent. In contrast with the PCC4 parental cells which carry F9 antigens and do not express H-2b, the hybrids do not express F9 antigens and carry H-2 alloantigens of both parental specificities. These results suggest that in hybrids whose phenotype is that of the Cl.1D parent, a change may occur in the genetic program of the teratocarcinoma cells.

Developmental potentialities and surface antigens of mouse teratocarcinoma x lymphoid cell hybrids.

Hybrids between PCC4-aza 1 teratocarcinoma cells and either thymus cells (PcT and RPcT hybrids) or Lc lymphocytic leukemia cells (PcLc hybrids) are carcinoma cells and when injected into hosts they produce tumors which are teratocarcinomas. PcT and RPcT hybrids) or Lc lymphocytic leukemia cells (PcLc hybrids) are carcinoma cells and when injected into hosts they produce tumors which are teratocarcinomas. PcT and RPcT hybrid tumors are well differentiated and include a large variety of somatic tissues. In most PcLc tumors, neuroectoderm differentiation is predomonant. Like the PCC4 parental cells, PcT, RPcT, and PcLc hybrids carry F9 embryonic antigens and do not express perceptible amounts of H-2 alloantigens. Nonexpression of the H-2d haplotype of the thymus cell parent in PcT hybrids is not due to the loss of chromosome 17 which carried the major histocompatibility complex.

A subfamily of E. coli palindromic units implicated in transcription termination?

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.