Xl-fli, the Xenopus homologue of the fli-1 gene, is expressed during embryogenesis in a restricted pattern evocative of neural crest cell distribution.

The Xenopus laevis fli cDNA, belonging to the ets family of transcription factors, was isolated from a library prepared from unfertilized eggs. It encodes a polypeptide with extensive homology to murine and human Fli proteins. The long 3'-untranslated region contains five nuclear polyadenylation signals and three cytoplasmic polyadenylation elements, as well as many A/T rich elements. Two polyadenylated transcripts appear at the early neurula and accumulate up to the tadpole stage. In situ hybridization reveals an expression in territories invaded by neural crest cells. In the head region, fli is expressed in the peri-ocular zone, in the branchial buds and at the level of the brain floor. In the trunk, a metamerized expression is detected in the dorsum. At a lower level, the tailbud and the peri-cardiac region also appear positive.

The c-ets-1 proto-oncogenes in Xenopus laevis: expression during oogenesis and embryogenesis.

We previously reported the cloning and sequencing of two cDNAs derived from the Xenopus laevis ets-1 gene (Stiegler et al., 1990). The Xl-ets-1a cDNA encodes a polypeptide highly homologous to known ets-1 proteins. The 3'-UTR contains two AATAAA polyadenylation signals together with three copies of the TTTTTAT sequence thought to confer a maturation-specific polyadenylation and implicated in the deadenylation of dormant mRNAs. Several transcripts with maternal characteristics were detected in oogenesis and early embryogenesis. A marked augmentation of the major transcript in the poly(A)+ fraction was detected at fertilization. Ets-1 transcripts were observed at constant levels during the cleavage stages but decreased abruptly at gastrulation, to reappear from neurulation to late embryogenesis. The possible contribution of 3'-UTR sequence elements to this behavior is discussed.

Cloning, sequencing, and expression of two Xenopus laevis c-ets-2 protooncogenes.

In a general approach to identify genes important in the control of genetic expression during development of Xenopus laevis, two complementary DNAs corresponding to two different c-ets-2 genes were cloned and sequenced. One of these complementary DNAs appears to be almost full length. The two variant genes differ in their overlapping sequences by 87 nucleotide substitutions, leading to 17 amino acid modifications in the proteins, 8 of them being conservative. All but one of these changes map outside of the 142 COOH-terminal residues, a region critical for nuclear localization and DNA binding in the ets proteins. Features potentially important for the biological activity of the gene products are conserved. Two transcripts (3.2 and 1.7 kilobases) with maternal characteristics are detected at a constant level from stages II/III of oogenesis to stage 10 of embryogenesis. They later decline to hardly detectable levels at stages 30-40. Variable amounts of the same transcripts are observed in many adult tissues. All of these characteristics support the idea that the ets-2 gene products play an important role during embryogenesis, as well as in adult life. Indeed, they act as ubiquitous transcriptional activators, as recently demonstrated by several investigators (C. V. Gunther, J. A. Nye, R. S. Bryner, and B. J. Graves, Genes & Dev., 4: 667-679, 1990; R. Bosselut, J.F. Duvall, A. Gegonne, M. Bailly, A. Hemar, J. Brady, and J. Ghysdael, EMBO J., 9: 3137-3144, 1990; R. Wasylyk, C. Wasylyk, P. Florès, A. Bègue, D. Leprince, and D. Stéhelin, Nature (Lond.), 346: 191-193, 1990).

Repair of acetyl-aminofluorene modified pBR322 DNA in Xenopus laevis oocytes and eggs; effect of diadenosine tetraphosphate.

Using Xenopus laevis oocytes and unfertilized eggs, we have developed a system which allows the study of DNA repair upon microinjection of pBR 322 DNA which has been previously modified in vitro by N-acetyl-aminofluorene, under controlled conditions. In unfertilized eggs, an efficient repair of pBR-18AAF DNA takes place, leading to a restoration of the transforming activity of the plasmid DNA towards Escherichia coli. The repaired DNA is even efficiently replicated, the egg being "activated" by the microinjection. In the oocyte, a partial repair is observed as shown by the incorporation of labelled dCTP in the modified plasmid DNA, even in the presence of aphidicolin, an inhibitor of DNA polymerase alpha. However, the repair appears to be very limited, since it does not restore the transforming activity of the modified plasmid DNA. This inefficient repair in the oocyte may be due to the rapid packaging of foreign DNA into a minichromosome and/or to a very low level of DNA polymerase beta. This system was used to study the effect of diadenosine tetraphosphate (Ap4A) on DNA repair. Ap4A seems not to interfere with repair processes in the oocyte, but significantly inhibits the replication following the repair of AAF-modified plasmid DNA in unfertilized eggs. These results suggest that Ap4A could be involved in switching off the replication machinery when DNA is badly damaged, thus helping to avoid the perpetuation of DNA modifications in the daughter cells. This hypothesis is consistent with many previous reports on the accumulation of dinucleoside polyphosphates under stress conditions, which are known to result in modification of DNA.

Significance of dinucleoside tetraphosphate production by cultured tumor cells exposed to the presence of ethanol.

The use of 30 to 50% ethanol solutions to extract the nucleotides from HTC and A-459 cells results in dinucleoside tetraphosphate (Ap4X) levels 3-30-fold as high as those obtained by 5% classical trichloracetic acid extraction, while ATP levels are identical in both cases. The amplification factor varies with the percentage of ethanol and duration of contact between the cells and the extraction mixture. It remains constant for the HTC cells during cell growth, but exhibits a maximum for the A-459 cells towards the end of the exponential growth period. The incorporation of radioactivity in Ap4X when [alpha-32P]ATP is added to the extraction mixture suggests an Ap4X neosynthesis in the presence of ethanol. The results carried out in the presence of pyrophosphate, EDTA and zinc acetate strongly suggest that aminoacyl-tRNA synthetases could be responsible for the increase in Ap4A content with ethanol treatment. Nevertheless, the effect of ethanol is probably not the result of an activation of these enzymes, but rather, as already suggested by earlier results in our laboratory, the result of a fast inactivation of the degradation enzymes.

Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth.

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.

In vitro evidence that hypothyroidism modifies glucocorticoid receptors.

We have previously reported that hypothyroidism is accompanied by modifications of the cytosolic rat liver glucocorticoid receptors. In the present study we have used molybdate, which stabilizes the glucocorticoid receptor from hypothyroid rats, allowing its characterization. We confirm a decrease in the number of glucocorticoid binding sites in the liver of hypothyroid rats as compared to that in normal rats (8000 per cell versus 23,000 per cell for normal rats). At 22 degrees C and in the absence of molybdate, thermal activation shows the same kinetics in both complexes from normal and hypothyroid rats, though activated hormone-receptor complexes from hypothyroid rats are progressively degraded (t 1/2 = 230 min). At the same temperature the dissociation rate of native complexes from hypothyroid rats is slower (k-1 = 8 X 10(-3) min-1) than normal (k-1 = 16 X 10(-3) min-1). Competitive dissociation studies using [3H]dexamethasone indicate that native complexes from hypothyroid rats show altered affinities for agonist and antagonist. Hypothyroidism decreases the number of rat liver glucocorticoid receptors and alters their properties, as evidenced by their greater instability and differences in steroid binding. These effects may be due to regulating factors and/or slight, probably post-transcriptional, modifications of the binding protein.

Cell cycle variations of dinucleoside polyphosphates in synchronized cultures of mammalian cells.

Zajdela hepatoma culture cells (ZHC) and mouse embryo fibroblasts (Swiss 3T3) were synchronized in G1 or S phase by serum deprivation and aphidicolin treatment, respectively, to study the variations in adenylyl nucleotide (Ap4X) pool size during the progress of the cell cycle. Only minor variations, which never exceeded a factor of 2, were observed when the Ap4X concentrations were expressed on a cellular basis. The variations were found to be strictly parallel to the ATP variations. Upon release from an aphidicolin block, the minor variations of Ap4X followed DNA synthesis and preceded cytokinesis. When the nucleotide content was compared with the amount of proteins, the faint specific cell cycle changes were almost completely damped when the cells were synchronized by serum deprivation, but remained practically unchanged in the case of aphidicolin synchronization. These results suggest that the observed variations could reflect the accumulation of some nucleotides before cell division. It is not clear yet whether the variation in Ap4X concentration is significant by itself or is simply a phenomenon resulting from changes in the ATP pool.

Lack of correlation between extensive accumulation of bisnucleoside polyphosphates and the heat-shock response in eukaryotic cells.

The accumulation in large amounts of bisnucleoside polyphosphates (Ap4X) after heat shock in Xenopus laevis oocytes or cultured hepatoma cells (HTC cells) is observed after exposure to temperatures of 45 degrees C or higher. The accumulation is a transient phenomenon, with the collapse in cellular ATP concentration severely affecting the rate of synthesis of Ap4X, allowing degrading activities to empty the pool of these compounds under prolonged heat shock. This accumulation of Ap4X to high levels, compared to the basic content, is only observed under conditions leading to irreversible damage, ultimately resulting in the death of the cell. It is shown that the increase in Ap4X after hyperthermia is due to the partial or almost complete inhibition of their degradation pathways, rather than to a stimulation of their rate of synthesis. Finally, the synthesis of heat-shock proteins could be observed under conditions which do not lead to important accumulation of Ap4X, therefore ruling out the possibility that these adenylylated nucleotides would behave as chemical signals ("alarmones") triggering the synthesis of heat-shock proteins. Nevertheless, on the basis of our earlier results (Guédon, G., Sovia, D., Ebel, J. P., Befort, D., and Remy, P. (1985) Embo J. 4, 3743-3749), it cannot be excluded that Ap4X might play a role in the regulation of the heat-shock response; this would, however, rely on variations in Ap4X concentrations which do not exceed a factor of 2.

Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression.

Complete complementary DNA of rat tyrosine aminotransferase messenger RNA. Deduction of the primary structure of the enzyme.

The primary structure of rat tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5), a liver-specific enzyme involved in gluconeogenesis, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 2362 nucleotides long (excluding the poly(A) tail) and codes for a polypeptide of 454 amino acids with a molecular weight of 50634. Unambiguous identification was obtained by comparison of this sequence with the amino acid sequences of several peptides obtained from the purified enzyme.

Effects of thyroidectomy on heart and liver rat tRNAs: study of chromatographic and electrophoretic behaviour.

Modifications of tRNAs in various physiological or experimental conditions are well documented. We have compared isoacceptor tRNAs extracted from target organs (heart and liver) from thyroidectomized rats to those of control animals. Nine liver aminoacyl-tRNAs and eight heart aminoacyl-tRNAs from thyroidectomized and control rats were analysed by RPC-5 chromatography. Quantitative differences were demonstrated in the relative proportions of the various liver tRNA isoacceptors for glycine, lysine, methionine, phenylalanine and serine and of the heart isoacceptor tRNAs for glycine, lysine, methionine, phenylalanine and valine. A qualitative variation was noted only for tRNATyr from the heart and liver of thyroidectomized rats. Isoacceptor tRNAs were obtained at a high resolution using a two-dimensional polyacrylamide gel electrophoresis. Isoacceptor tRNAs corresponding to 14 amino acids for the liver and 12 amino acids for the heart were identified. Although for most of the tRNAs examined the number of isoacceptors remained unchanged, the number of spots corresponding to tRNAGlu and tRNAHis from the liver and tRNAAla from the heart was different after thyroidectomy. Furthermore the change in electrophoretic behaviour of tRNATyr from the liver of thyroidectomized rats suggests a structural modification of one of the isoacceptors in relation to the change in thyroid status. Thus, thyroid hormones appear to induce some modification of the isoacceptor tRNAs in their target organs.

Impairment of enzyme induction by glucocorticoids in Zajdela hepatoma cells.

Whereas glucocorticoids induce TAT, TRP, GPT in liver and only TAT in HTC cells, no hormonal effect on the synthesis of these enzymes was found in Zajdela hepatoma cells grown in vivo as an ascitic tumor, or in vitro as layer cultures. Although these cells remain uninducible, the hormone penetrates normally, but a strong decrease of the specific binding of cytosol and nuclear proteins with the hormone was observed. The impairment at the level of the hormone receptors could account for the non-inducibility of enzyme synthesis in ZHC cells.

Inhibition of viral multiplication by homologous methylated ribonucleic acids. V. Inhibition of myxoviruses in mouse.

Mice respond to intravenous injection of homologous methylated RNA by the production of a circulating interferon-like substance. The treatment with modified RNA induces a significant protection against viral infection. This effect is optimum when the treatment occurs a few hours before viral infection. The protective effect is a peculiar property of homologous methylated RNA as heterologous RNAs do not induce any resistance to the infection with influenza virus.

Study of the role of the acceptor stem in the interactions between tRNAs and aminoacyl-tRNA synthetases.

Several studies have clearly demonstrated that the end of the acceptor stem was a very important area determining the aminoacylation properties of tRNAs. However the attempts to measure the contribution of this region to the binding of tRNAs to aminoacyl-tRNA synthetases have led to contradictory results. We report here the stepwise degradation of yeast tRNA-Phe and tRNA-Val from their 3' terminus, up to the seventh nucleotide : the affinity of each of the degraded-tRNA for their cognate aminoacyl-tRNA synthetase was compared to that of intact tRNA and it was found that these affinities are not significantly decreased when compared to those of the intact tRNAs.