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M Méchali

Publications and source records attributed to M Méchali.

At least 37 records · Page 2Linked to original sources
PubMed1995

Transition in specification of embryonic metazoan DNA replication origins.

In early Xenopus embryos, in which ribosomal RNA genes (rDNA) are not transcribed, rDNA replication initiates and terminates at 9- to 12-kilobase pair intervals, with no detectable dependence on specific DNA sequences. Resumption of ribosomal RNA (rRNA) synthesis at late blastula and early gastrula is accompanied by a specific repression of replication initiation within transcription units; the frequency of initiation within intergenic spacers remains as high as in early blastula. These results demonstrate that for rRNA genes, circumscribed zones of replication initiation emerge in intergenic DNA during the time in metazoan development when the chromatin is remodeled to allow gene transcription.

Animals↗
PubMed1995

Programming of a repressed but committed chromatin structure during early development.

The determination of chromatin for transcription during early development as well as the requirement for trans-acting factors during this period has been analysed in Xenopus. Basal transcription is repressed both during oogenesis and after the mid-blastula transition (MBT), and transactivators are required to relieve this repression. In contrast, transactivators cannot overcome the generalized transcriptional repression which occurs in embryos before MBT. However, they do bind to promoters leading to a repressed but preset chromatin structure. Experiments involving the pre-binding of TATA binding protein (TBP) or of the strong transactivator GAL4-VP16 further show that there is no limiting factor before the MBT, and that it is the recruitment and stabilization of the basal transcription machinery and not of transactivators which is repressed during early development. This multi-step process in gene activation, with activation of promoters temporally uncoupled from their commitment, may be of importance in the regulation of early embryonic events by providing molecular signposts for future determinations.

Animals↗
PubMed1995

Analysis of c-Myc and Max binding to the c-myc promoter: evidence that autosuppression occurs via an indirect mechanism.

c-myc negatively autoregulates its expression at the level of transcriptional initiation, although the precise mechanism remains debated. While conclusive evidence for c-Myc binding in its own promoter has not been found, it has been proposed that c-Myc binds to a site upstream of the human c-myc gene which may also be a component of a replication origin (Ariga et al., 1989). In an attempt to clarify this issue, sequences flanking the c-myc gene were screened for c-Myc or Max binding sites using a novel procedure to facilitate the detection of DNA binding dependent upon long distance interactions or DNA secondary structure. Since the sequence specificity of DNA binding proteins may also be mediated by interaction with other proteins or by protein modification, this affinity capture assay was used in conjunction with nuclear extracts, potentially allowing the selection of novel in vivo DNA binding specificities. Using conditions that gave strong binding to an internal control sequence, c-Myc or Max binding elements were not detected in genomic sequences extending 5.4 kb upstream of the Xenopus c-myc gene. Identical results were obtained using both purified proteins and a variety of nuclear extracts, suggesting c-myc autosuppression most likely involves an indirect pathway.

Animals↗
PubMed1994

Production of a functional full-length Xenopus laevis c-Myc protein in insect cells.

C-Myc is a nuclear phosphoprotein whose normal cellular function has not yet been clearly defined. Studies with this protein have always been constrained by the difficulty of obtaining full-length c-Myc in an active form, whatever the expression system used. We report here experimental conditions optimized to increase the solubility and the purification of c-Myc in a baculovirus expression system. Such conditions allow the production of both soluble and active full-length c-Myc. Interestingly, soluble c-Myc is found associated with a 500-kDa high-molecular-mass complex comparable to that found in human and Xenopus laevis embryos, and which may be required for its function in vivo.

Animals↗
PubMed1994

Competition between chromatin and transcription complex assembly regulates gene expression during early development.

Xenopus early development is characterized by a generalized absence of transcription, which resumes at the midblastula transition (MBT). We analyzed this regulation using a plasmid containing the c-myc promoter that is under the same developmental control when injected into fertilized eggs. We find that the repression of transcription can be relieved simply by preincubating the reporter plasmid with TATA binding protein (TBP). However, the repression of gene activity normally occurring before the MBT soon becomes dominant over this activation independent of cell cycle phases. This inactivation correlates with chromatin assembly, and titration of chromatin components not only relieves repression of TBP-dependent transcription but also permits the establishment of stable transcription during early development. Our data suggest that the large excess of histones represses gene activity during early development through a dynamic competition between chromatin assembly and transcription complex assembly.

Animals↗
PubMed1993

Stathmin gene family: phylogenetic conservation and developmental regulation in Xenopus.

The ubiquitous cytoplasmic phosphoprotein stathmin was proposed to play a general role as an intracellular relay integrating diverse signals regulating the proliferation, differentiation, and functions of cells (Sobel, A. (1991) Trends Biol. Sci. 16, 301-305). It was originally identified in mammalian cells and tissues, but antibodies directed against the mammalian protein also recognized a stathmin-like 19-kDa protein in all vertebrate classes. The immunoreactive protein in Xenopus laevis displayed, like mammalian stathmin, several nonphosphorylated and phosphorylated heat-soluble forms with distinct migration on two-dimensional polyacrylamide gel electrophoresis. Screening of Xenopus oocyte and brain cDNA libraries with a rat stathmin cDNA probe allowed us to isolate several stathmin-related cDNA clones, among which clone XO35 encodes the Xenopus homologue of stathmin whose deduced amino acid sequence is 79% identical to and displays most of the characteristic structural features of the mammalian protein. In particular, one of the cAMP-dependent protein kinase and the two "proline-directed" kinase-specific sites known to be phosphorylated in rat stathmin are also present in the Xenopus protein. Furthermore, two other sets of clones coding for related proteins belonging to the stathmin gene family were also isolated; clone SC15 encodes the Xenopus homologue of SCG10, a rat protein specifically related to neuronal differentiation; clone XB3 encodes a protein which, as SCG10 or SC15, possesses a stathmin-like domain and an additional N-terminal extension but is more distant from SCG10 than SC15. Interestingly, the mRNA transcripts of Xenopus stathmin (XO35) appear ubiquitous, like stathmin in mammals, whereas the SC15 and XB3 mRNAs appeared as markers of the nervous tissue in Xenopus. During Xenopus oogenesis, stathmin accumulates and remains stable as a maternal product throughout early development. Concurrently, its phosphorylation is regulated from essentially unphosphorylated forms to highly phosphorylated ones in the mature egg, which are then progressively dephosphorylated again from the midblastula to the tailbud stage. Altogether, our results demonstrate the high evolutionary conservation of stathmin together with the members of its related gene family, not only at the level of their molecular structures, but also of their biochemical and biological regulation. These observations are thus further in favor of a very general and likely essential role of stathmin in the normal control of cells throughout development and in the adult.

Amino Acid Sequence↗
PubMed1993

TFIIIC relieves repression of U6 snRNA transcription by chromatin.

The U6 small nuclear (sn)RNA gene (SNR6) from the yeast Saccharomyces cerevisiae is transcribed by RNA polymerase III in vivo. This gene is unusual in having a TATA box at position -30, and an essential B-block element located downstream of the T-rich termination signal. The B block is one of the two intragenic promoter elements of transfer RNA genes that are recognized by transcription factor (TF)IIIC (ref. 4). But accurate in vitro transcription of yeast U6 snRNA gene by PolIII in a purified system requires only TFIIIB components, including the TATA-box binding protein TBP. Here we report that, after nucleosome reconstitution or chromatin assembly, U6 snRNA synthesis becomes dependent on TFIIIC and on the integrity of the B-block element. This observation resolves an apparent paradox between in vitro and in vivo results concerning the necessity of the downstream B-block element and sheds light on a new role of TFIIIC in gene activation.

Animals↗
PubMed1993

Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos.

We have analysed the replication of the chromosomal ribosomal DNA (rDNA) cluster in Xenopus embryos before the midblastula transition. Two-dimensional gel analysis showed that replication forks are associated with the nuclear matrix, as in differentiated cells, and gave no evidence for single-stranded replication intermediates (RIs). Bubbles, simple forks and double Ys were found in each restriction fragment analysed, showing that replication initiates and terminates without detectable sequence specificity. Quantification of the results and mathematical analysis showed that the average rDNA replicon replicates in 7.5 min and is 9-12 kbp in length. This time is close to the total S phase duration, and this replicon size is close to the maximum length of DNA which can be replicated from a single origin within this short S phase. We therefore infer that (i) most rDNA origins must be synchronously activated soon in S phase and (ii) origins must be evenly spaced, in order that no stretch of chromosomal DNA is left unreplicated at the end of S phase. Since origins are not specific sequences, it is suggested that this spatially and temporally concerted pattern of initiation matches some periodic chromatin folding, which itself need not rely on DNA sequence.

Animals↗
PubMed1993

Stabilization and expression of high levels of p53 during early development in Xenopus laevis.

We previously isolated a p53 cDNA from a Xenopus oocyte library. To determine if p53 has a function in the developmental period, we have studied its expression at the RNA and protein levels during the early development of Xenopus laevis. Two p53 transcripts (3 and 2.2 kb) are expressed from the beginning of Xenopus oogenesis, and the major one (2.2 kb) reaches a level of 7 x 10(5) to 7 x 10(6) transcripts per mature oocyte. After fertilization only the 2.2-kb RNA is detected, but its level decreases and at the neurula stage p53 RNA becomes undetectable. The p53 protein is highly expressed during Xenopus development, in contrast to an undetectable level in Xenopus cells in culture. Most of the p53 protein is synthesized during late oogenesis and a stage VI oocyte contains 7 x 10(11) molecules of p53 protein. This maternal p53 store is maintained at a constant level during Xenopus development, at least until the tadpole stage. This high level of expression is mainly due to stabilization of the p53 protein. Unusually for p53, the protein is strictly located in the cytoplasm of oocytes and this localization might indicate that it is stored in an inactive form at this stage. These data are discussed relative to previous observations made in transgenic mice.

Animals↗
PubMed1992

Plasmid replication in Xenopus eggs and egg extracts: a 2D gel electrophoretic analysis.

We have examined the replication patterns of ribosomal DNA plasmids in vivo and in vitro using Xenopus eggs. Plasmids carrying different parts of the Xenopus ribosomal DNA sequence were allowed to replicate either in vitro in an egg extract or in vivo after microinjection into unfertilized eggs. The replication intermediates were analyzed by the 2D gel electrophoretic technique of Brewer and Fangman (1), using original or modified electrophoresis conditions. With standard electrophoresis conditions, the patterns obtained for restriction fragments larger than 5 kb were unreliable because of artefactually distorted Y arcs and unrecognizable bubble arcs. Interpretable patterns could nevertheless be obtained using suitably modified electrophoresis parameters. Under these conditions, replication was found to initiate and terminate at multiple, random locations on each plasmid both in vivo and in vitro. However, only one or very few of these potential initiation sites are used during the replication of an individual plasmid molecule. We discuss the possible artefacts and misinterpretations that can result when the 2D electrophoresis parameters are not adapted to the size of the fragment examined. We also discuss the relevance of the random replication mode to the mechanisms and the control of DNA replication in eukaryotes.

Animals↗
PubMed1992

Localization of ras proto-oncogene expression during development in Xenopus laevis.

The expression of the ras protooncogene was investigated in Xenopus laevis, throughout development, by in situ hybridization using a 35S-labelled antisense RNA probe. During oogenesis, the ras RNA was strongly expressed in the cytoplasm of previtellogenic oocytes and further diluted between yolk platelets; no specific localization of transcripts was observed. The signal density was particularly weak over embryo sections until the tailbud stage. On the other hand, a high level of ras RNA expression was detected on sections through the young tadpoles. An intense labelling was observed in several areas, including the branchial apparatus, gut, somites, nervous system, and lens. It is noteworthy that the heterogeneity of labelling increases as tadpoles grow older. Together, these results are discussed in relation to cellular events appearing throughout the early development.

Animals↗
PubMed1992

Comparative analysis of the intracellular localization of c-Myc, c-Fos, and replicative proteins during cell cycle progression.

In eukaryotic cells, nucleus-cytoplasm exchanges play an important role in genomic regulation. We have analyzed the localization of four nuclear antigens in different growth conditions: two replicative proteins, DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), and two oncogenic regulatory proteins, c-Myc and c-Fos. A kinetic study of subcellular localization of these proteins has been done. In cultures in which cells were sparse, these proteins were detected in the nucleus. When proliferation was stopped by the high density of culture cells or by serum starvation, these proteins left the nucleus for the cytoplasm with different kinetics. DNA polymerase alpha is the first protein to leave the nucleus, with the PCNA protein, c-Fos, and c-Myc leaving the nucleus later. In contrast, during serum stimulation c-Fos and c-Myc relocalize into the nucleus before the replicative proteins. We also noticed that in sparse cell cultures, 10% of the cells exhibit a perinuclear staining for the DNA polymerase alpha, PCNA, and c-Myc proteins but not for c-Fos. This peculiar staining was also observed as an initial step to nuclear localization after serum stimulation and in vivo in Xenopus embryos when the G1 phase is reintroduced in the embryonic cell cycle at the mid-blastula stage. We suggest that such staining could reflect specific structures involved in the initiation of the S phase.

3T3 Cells↗
PubMed1992

Nuclear distribution of PCNA during embryonic development in Xenopus laevis: a reinvestigation of early cell cycles.

The immunocytological distribution of the proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication, has been examined during the early development of Xenopus laevis. The protein is uniformly detected in nuclei during early stages up to the neurula stage. PCNA is detected by its distinctive cyclical pattern during early development, remaining detectable only during the period of S phase of each cell cycle. Immunological detection of PCNA is therefore a useful and specific non-isotopic marker of S-phase cells in the embryo. PCNA associates with typical karyomeric structures, suggesting that DNA replication starts before the nuclear compartment is entirely formed. At the midblastula transition, a new pattern of PCNA staining becomes apparent. First, a new type of PCNA staining is detected at the nuclear periphery. Second, mitotic clusters with different PCNA distributions suggest that the onset of desynchronization of the cell cycle at this stage is not random.

Animals↗
PubMed1992

Nerve-dependent expression of c-myc protein during forelimb regeneration of Xenopus laevis froglets.

The consequences of denervation on the expression of c-myc protein have been analyzed on the regenerating forelimb of young froglets of Xenopus laevis. The level of c-myc expression, low in control limbs and enhanced in the regenerate, is transiently increased after a three-hour total denervation. For this protein, the level of expression is not a function of the quantity of nerve in the regenerate. Four days after denervation, c-myc signal is back to its base level observed in the regenerate. A different pattern of expression is obtained for an S phase marker (PCNA protein) taken as a control in the same experimental conditions. The data presented here show that the nervous system normally exerts a negative control on the expression of c-myc and PCNA proteins in the limb regenerate of Xenopus.

Animals↗
PubMed1991

Posttranscriptional regulation of c-myc RNA during early development of Xenopus laevis.

The remarkable stability of c-myc during oogenesis contrasts with its degradation during the early developmental period in Xenopus laevis. Three evolutionary conserved motifs found in the 3'-untranslated region of Xenopus c-myc RNAs have been analyzed for a possible role in c-myc RNA degradation. No specific degradation was observed when these sequences were cloned downstream of a reporter gene and the corresponding RNAs were injected into fertilized eggs. The relation between polyadenylation and degradation of c-myc mRNA has been examined during early development. c-myc is adenylated during early oogenesis, and a dramatic de-adenylation occurs in full grown oocytes. Consequently, the de-adenylation of c-myc mRNA that occurs in eggs might be a requirement for its degradation after fertilization, but is not sufficient to trigger its degradation.

Animals↗
PubMed1991

Transcription complex disruption caused by a transition in chromatin structure.

Chromatin structure is known to influence class III gene expression in vitro. We describe the active transcription of Xenopus class III genes following replication and assembly into chromatin by using Xenopus egg extracts. Changes in the structure of this active chromatin dependent on the presence of exogeneous Mg2+ ATP or on the addition of a mixture of histones H2A and H2B are shown to lead to the selective repression of Xenopus 5S RNA genes. Preexisting transcription complexes on 5S DNA are disrupted following the reorganization of a "disordered" histone-DNA complex into a structure consisting of physiologically spaced nucleosomes. Thus, we demonstrate that chromatin structural transitions can have dominant and specific effects on transcription.

Adenosine Triphosphate↗
PubMed1990

Chromatin assembly on replicating DNA in vitro.

Replicating single-stranded DNA is preferentially assembled into chromatin in Xenopus egg extracts relative to non-replicating double-stranded DNA. We have examined the molecular basis of this phenomenon. Single-stranded DNA itself is not a favored template for nucleosome assembly in comparison to double-stranded DNA. Complementary strand synthesis is required for the rapid assembly of nucleosomes. We present evidence that the assembly of chromatin on replicating DNA is a two step phenomenon. The first step involves the replication of DNA and the assembly of an intermediate structure, the second step involves the sequestration of histones H2A/H2B onto DNA. Histones H2A/H2B are preferentially sequestered onto replicated DNA in comparison to non-replicated DNA incubated in the extract.

Animals↗