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M Méchali

Publications and source records attributed to M Méchali.

At least 55 records · Page 3Linked to original sources
PubMed1990

Competition between transcription complex assembly and chromatin assembly on replicating DNA.

We have used a Xenopus egg extract to show that a competition exists between the assembly of transcription complexes and nucleosomes on replicating 5S DNA. This competition results in the establishment of a transcriptionally repressed state for 5S DNA that is dependent on core histones but not on the precise positioning of the cores. The repression is selective, since satellite I DNA is not significantly repressed under these conditions. We demonstrate that the efficiency of chromatin assembly compared with transcription complex assembly is an important variable in determining gene activity.

Animals↗
PubMed1990

Characterization and developmental expression of Xenopus proliferating cell nuclear antigen (PCNA).

The coding sequence of the proliferating cell nuclear antigen (PCNA) was characterized in the amphibian Xenopus laevis. The deduced protein sequence shares an extensive homology (89%) with the mammalian PCNA coding sequences. Xenopus PCNA is expressed beginning in early oogenesis and reaches a level of 3 X 10(7) transcripts per mature oocyte, whereas proliferative somatic cells contain 3 X 10(2) PCNA transcripts per cell. Most of the PCNA protein is expressed during late oogenesis and one single stage VI oocyte contains the amount of PCNA protein present in 4 X 10(5) somatic cells in culture. Thus most, if not all, of the PCNA required for early development is stored as a maternal gene product. Part of the mRNA stockpile is degraded during the cleavage stage and then new PCNA zygotic expression at the neurula stage maintains a constitutive value of 30 transcripts per cell until the tailbud stage. The maternal protein is maintained at a constant level during embryonic development at least until the swimming tadpole stage. The protein is localized in the nuclei at all stages of oogenesis and development that were examined.

Amino Acid Sequence↗
PubMed1990

Characterization and expression of a Xenopus ras during oogenesis and development.

We have characterized a cDNA which contains the entire coding sequence of a Xenopus laevis ras protein. The deduced amino acid sequence reveals a strong homology (92%) to human Ki-ras 2B protein. ras expression has been studied both qualitatively and quantitatively during Xenopus development. ras is expressed as a maternal mRNA in oocytes and early embryos at a level up to 1.5 x 10(7) copies per mature oocyte, corresponding to the level of ras mRNA found in 4 x 10(5) somatic growing cells. This level remains constant throughout the first rapid cleavage stages of the blastula before the midblastula transition (MBT). After this stage, the amount of ras RNA decreases gradually until the hatching tadpole stage, when a new zygotic expression is detected in the embryo. From that stage, a constitutive amount of 30-50 ras RNA transcripts per embryonic cell is registered, as observed in Xenopus proliferative somatic cells. The 23-kDa Xenopus ras protein has also been identified by both specific monoclonal antibody and in vitro transcription-translation experiments. It is expressed in oocytes before maturation, indicating that maturation is not the trigger for ras expression. The expression of Xenopus ras at a high level during oogenesis and early development suggests a major function of this gene both in meiosis and in mitosis events during embryonic development.

Amino Acid Sequence↗
PubMed1990

Enhanced c-myc gene expression during forelimb regenerative outgrowth in the young Xenopus laevis.

Analysis of the expression of the c-myc protooncogene has been carried out in the forelimb regenerate of the Xenopus laevis froglet. Northern blot hybridization analysis revealed the presence of a 2.5-kilobase c-myc transcript in the regenerate forelimb at a level at least 7-fold more than the one found in nonregenerating forelimbs or stumps of regenerating forelimbs. In situ hybridization analyses confirmed the relative abundance of c-myc RNA in the regenerate forelimb and provided evidence of spatial localization of high levels of c-myc RNA in specific cell layers. The deepest layers of the wound epithelium of epidermal origin showed a strong signal, whereas virtually no c-myc RNA was detected in the outermost layers. Labeling was also observed in mesenchymal cells of the blastema where it was relatively evenly distributed. This pattern of c-myc RNA in the regenerate might indicate that the expression of c-myc plays a role in the regulation of the continued proliferation of specific cells of the regenerate, whereas repression of this gene in the epidermis correlates with terminal differentiation of keratinocytes.

Amputation, Surgical↗
PubMed1990

Genes and mechanisms involved in early embryonic development in Xenopus laevis.

Our laboratory is studying genes involved in the regulation of the balance between cell growth and differentiation during embryonic development in Xenopus. We have analyzed the developmental expression of the proto-oncogenes c-myc, and KiRas 2B, the proliferating cell nuclear antigen (PCNA), and the tumor suppressor gene p53. These genes, usually expressed during cell proliferation, are expressed in the oocyte in large quantities, but the majority of their maternal RNAs are degraded by the gastrula stage. The expression of c-myc and the localization of the protein indicate that c-myc has the characteristics expected for a gene involved in the regulation of the mid-blastula transition, when zygotic expression is turned on in the embryo. Its expression during late development or during regeneration indicates that it enables the cells to remain competent for cycling during organogenesis. In vitro systems that reproduce the principal cellular functions during early development are used as model systems to understand the mechanisms involved in early embryogenesis.

Animals↗
PubMed1989

Differential expression of two Xenopus c-myc proto-oncogenes during development.

Two distinct Xenopus c-myc cDNA clones have been characterized from an oocyte cDNA library. This allowed a comparison of the c-myc protein sequence across the vertebrate phylum to be made and prominent conservations to be identified. The majority of the sequence differences between the two Xenopus c-myc cDNAs are in the 5' and 3' untranslated regions. Sequence-specific oligonucleotide probes from the 5' untranslated region were used to demonstrate the differential expression of the two c-myc mRNAs during development. One of the mRNAs corresponds to the Xenopus c-myc gene previously reported expressed as a stable maternal mRNA uncoupled from cell division during oogenesis (c-myc I). It is the major mRNA species expressed during oogenesis and is expressed again from the zygotic genome in post-gastrula embryos. In contrast, the second c-myc mRNA (c-myc II) is expressed only from the maternal genome during oogenesis. Primer extension experiments show that in the oocyte the transcriptional initiation sites for c-myc I and c-myc II are at different distances from the translational start site. The 'oocyte-specific' and 'somatic-type' developmental regulation of c-myc is reminiscent of polymerase III 5S RNA gene expression in Xenopus, and may provide new insights into the developmental regulation of genes transcribed by RNA polymerase II.

Amino Acid Sequence↗
PubMed1989

Analysis of 3'-untranslated regions of seven c-myc genes reveals conserved elements prevalent in post-transcriptionally regulated genes.

We have characterized the complete sequence of two c-myc cDNAs from the amphibian Xenopus laevis, and could thus compare the 3'-non-coding sequences of 7 myc cDNAs from 6 species spread over 350 million years of evolution. Although the size of these sequences is heterogeneous, we identified three completely conserved sequences of 10, 11 and 12 contiguous nucleotides. We observed that two of these elements may be contained in conserved stem-loop structures previously implicated in mRNA turnover. The length of these motifs, their existence in conserved predicted structures, and their presence in regulated eukaryote mRNA with a frequency greater than predicted by chance, suggest that they are functionally important.

Animals↗
PubMed1989

Mismatch repair involving localized DNA synthesis in extracts of Xenopus eggs.

Repair of heteroduplex DNA containing G.T or A.C mismatches or containing two tandem unpaired bases occurred in vitro with Xenopus egg extracts as detected by a physical assay. The repair was accompanied by a mismatch-stimulated and mismatch-localized DNA synthesis. Repaired molecules, separated from unrepaired molecules, showed a 20- to 100-fold increase in DNA synthesis in the region of the mismatch compared to regions distant from the mismatch. The remaining unrepaired heteroduplex DNA included molecules that also displayed mismatch-stimulated DNA synthesis in the mismatch-proximal regions. These may represent intermediates in the repair process. The patterns of DNA synthesis suggest that repair begins at some distance from the mismatch and that as much as 1 kilobase or more can be involved in the mismatch-stimulated synthesis.

Animals↗
PubMed1989

Translocation of a store of maternal cytoplasmic c-myc protein into nuclei during early development.

The c-myc proto-oncogene is expressed as a maternal protein during oogenesis in Xenopus laevis, namely, in nondividing cells. A delayed translation of c-myc mRNA accumulated in early oocytes results in the accumulation of the protein during late oogenesis. The oocyte c-myc protein is unusually stable and is located in the cytoplasm, contrasting with its features in somatic cells. A mature oocyte contains a maternal c-myc protein stockpile of 4 x 10(5) to 6 x 10(5) times the level in a somatic growing cell. This level of c-myc protein is preserved only during the cleavage stage of the embryo. Fertilization triggers its rapid migration into the nuclei of the cleaving embryo and a change in the phosphorylation state of the protein. The c-myc protein content per nucleus decreases exponentially during the cleavage stage until a stoichiometric titration by the embryonic nuclei is reached during a 0.5-h period at the midblastula stage. Most of the maternal c-myc store is degraded by the gastrula stage. These observations implicate the participation of c-myc in the events linked to early embryonic development and the midblastula transition.

Animals↗
PubMed1989

[Role of c-myc protein in the early embryonic development of Xenopus].

Microinjections of antibodies directed against the protein encoded by the c-myc protooncogene strongly inhibit or arrest the early cell cleavage stage of Xenopus laevis embryos. Injections in one blastomere of a two cell stage embryo inhibit the segmentation of this blastomere. The cleavage of the uninjected blastomere behaves normally. Injections of control rabbit immunoglobulins do not alter the embryonic development.

Animals↗
PubMed1988

Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity.

Undiluted extracts from eggs or oocytes of Xenopus laevis support the assembly of chromatin with physiologically spaced nucleosomes. Micrococcal nuclease and DNase I digestion experiments show that nucleosome formation as well as supercoiling of circular DNA concomitant to assembly do not require ATP or Mg2+. However these factors are essential for the stability and the physiological spacing of the assembled chromatin. gamma-S-ATP can substitute for ATP in this process. With topoisomers of defined linking number topological interconversions proceed by steps of unity, both in vitro as well as in vivo, indicating that topoisomerase I is dominantly acting in this process. Novobiocin sensitivity occurred only with diluted extracts and was unrelated to an inhibition of topoisomerase II. Finally, nucleosome assembly occurs efficiently on linear DNA although the assembled DNA is less stable than with circular DNA. From these results we propose that mature chromatin is formed in a two-step reaction. In the first step, nucleosome deposition occurs independently of ATP and Mg2+. Thus, nucleosome formation can be uncoupled from their spacing. In this step, topoisomerase activity is involved in the relaxation of the topological constraints generated by chromatin assembly rather than in the process of assembly itself. The second step, requiring ATP and Mg2+, generates properly spaced chromatin.

Adenosine Triphosphate↗
PubMed1988

Xenopus egg extracts: a model system for chromatin replication.

A cell-free system derived from Xenopus eggs enables in vitro reproduction of the steps occurring during eukaryotic DNA replication. With a circular single-stranded DNA template, extracts obtained from high-speed centrifugation perform complementary DNA strand synthesis coupled to chromatin assembly. Nucleosomes are formed on the newly replicated DNA and the overall reaction mimics the events occurring during chromosomal replication on the lagging strand at the replication fork. ATP is necessary at all steps examined individually, including RNA priming, elongation of DNA strands and chromatin assembly. Although not required for nucleosome formation, ATP is involved in the correct spacing of nucleosomes and the stability of the assembled chromatin. Replication of double-stranded DNA was observed only with extracts obtained from low-speed centrifugation using demembraned sperm nuclei as substrate. Nuclei are reconstituted around the DNA and then undergo a series of events characteristic of a cell cycle. In contrast, neither DNA elongation or chromatin assembly require formation of the nucleus, and both are independent of the cell cycle.

Adenosine Triphosphate↗
PubMed1988

Oligonucleotide site-directed mutagenesis in Xenopus egg extracts.

Addition of M13mp18 single-stranded DNA annealed with an oligonucleotide to a Xenopus egg extract results in a rapid and efficient incorporation of the oligonucleotide in a complete double-stranded supercoiled molecule. Both the efficiency of DNA synthesis and the recovery of complete double-stranded molecules are increased relative to the reaction carried out by the classical technique using the E. coli Klenow DNA polymerase, DNA ligase, dNTPs, ATP and ions. Site specific mutagenesis was assayed by reverting a point mutation in the lacz region of M13mp18. The color assay described by Messing and sequencing of the DNA extracted from isolated plaques was used to check for the reversion. A 2 hr incubation of the heteroduplex carrying the mutagenic oligonucleotide in the Klenow-ligase-dNTP mixture allows a recovery of 6% mutant phage after transformation of competent cells with the reaction products. Using the Xenopus egg extract, 83% mutant phage were recovered after the same incubation time, in reactions entirely performed in parallel. The Xenopus extract is stable and contains all components required for the assay, including all ionic and protein factors; thus the only addition is the annealed DNA. Such an eukaryotic system is therefore an attractive alternative to the reconstituted prokaryotic DNA polymerase-DNA ligase system for site specific mutagenesis.

Animals↗
PubMed1988

Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs.

A cell-free system from Xenopus eggs mimics the reaction occurring at the eukaryotic replicative fork in vivo when chromatin assembly is coupled to complementary strand synthesis of DNA. DNA synthesis on single-stranded circular DNA promotes supercoiling and the replicated molecule sediments as a discrete nucleoprotein complex. Micrococcal nuclease digestion reveals a characteristic pattern of multiples of 200 bp of DNA. The kinetics of chromatin assembly and DNA synthesis are coincident and both processes occur with a rate comparable with chromosomal replication in vivo in early embryos. The DNA synthesis reaction can be uncoupled from the assembly reaction. Thus, titration of chromatin proteins by preincubation of the extract with double-stranded DNA prevents the supercoiling of replicated DNA without affecting the rate of synthesis. In contrast, chromatin assembly performed on unreplicated double-stranded DNA is a slower process as compared with the assembly coupled to DNA synthesis. Supercoiled molecules are detected after 30 min replication whereas at least 2 h are required to observe the first form I DNA with unreplicated double-stranded DNA. Such a system where chromatin assembly is promoted by DNA synthesis should be valuable for studying the interaction of specific factors with DNA during chromatin assembly at the replicative fork.

Animals↗
PubMed1988

Proto-oncogenes and embryonic development.

The role of proto-oncogenes in embryonic development was investigated using one of the most characterized vertebrates, the amphibian Xenopus laevis. Genes which belong to the major proto-oncogene families have been detected in Xenopus genome. The developmental control of the myc gene was assayed using a characterized Xenopus myc probe and specific antibodies. The myc gene is highly expressed as a stable maternal mRNA in oocyte, and an unfertilized egg contains 5 X 10(5)-fold the myc RNA content of a proliferative somatic cell. The myc RNA store is evenly distributed in the oocyte and the egg. Fertilization triggers a post-transcriptional control of the gene and the RNA store is progressively degraded to a constitutive value of 10 to 30 myc RNA copies registered per gastrula embryonic cell. The 62K myc protein is accumulated late in oogenesis. This uncoupling of myc expression and cell proliferation appears as a specific developmental regulation of the myc gene, adapted to the series of rapid cell cleavages occurring after fertilization.

Animals↗
PubMed1987

Cloning and characterization of a cDNA from Xenopus laevis coding for a protein homologous to human and murine p53.

A Xenopus laevis oocyte cDNA library was screened with a murine p53 cDNA probe for the presence of p53-related clones. Several such clones were isolated and analysed. The nucleotide sequence of the largest cDNA clone (2.2 kb) showed a high degree of homology with the human (68%) and murine (70%) p53 coding sequences. This clone contains a single large open-reading frame, coding for a protein of 363 amino acids, which is 51% homologous to human p53 and 57% homologous to murine p53. Furthermore, five highly conserved internal regions were found in all three proteins. The three proteins have a highly similar amino acid composition (including, notably, the presence of a high proportion of proline residues), and they display a comparable distribution of charged amino acids and hydropathic index profile. The in vitro transcription-translation products of the X. laevis clone were successfully immunoprecipitated by human anti-p53 sera, demonstrating that there is at least one epitope in common between the X. laevis protein and human, and possibly murine, p53.

Amino Acid Sequence↗
PubMed1984

Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast.

We examined the controversial question concerning DNA sequences required for replication in Xenopus eggs. First we used yeast to isolate ARS elements from the Xenopus genome. They show a striking sequence homology with the yeast ARS consensus sequence. The cloning vector and the ARS-containing plasmids replicate equally after injection into Xenopus eggs. Second, we compared a wide range of DNA templates from procaryotes and eucaryotes. All DNA molecules tested replicate as monomeric molecules, and the efficiency is proportional to their size for templates between 4 and 12 kb. Third, we re-examined two reports of replication origins from the Xenopus genome. In both cases, the vector and the recombinant molecules replicate equally under all conditions tested. The apparent lack of sequence specificity for replication in Xenopus eggs does not prevent the injected molecule from being under cellular temporal control of replication. These results are compared with those from yeast.

Animals↗