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Biomedical subjects

M Mechali

Publications and source records attributed to M Mechali.

17 recordsLinked to original sources
PubMed2001

A novel cell-free system reveals a mechanism of circular DNA formation from tandem repeats.

One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). High levels of eccDNA are associated with genomic instability, exposure to carcinogens and aging. We have recently reported developmentally regulated formation of eccDNA that occurs preferentially in pre-blastula Xenopus laevis embryos. Multimers of tandemly repeated sequences were over-represented in the circle population while dispersed sequences were not detected, indicating that circles were not formed at random from any chromosomal sequence. Here we present detailed mechanistic studies of eccDNA formation in a cell-free system derived from Xenopus egg extracts. We show that naked chromosomal DNA from sperm or somatic tissues serves as a substrate for direct tandem repeat circle formation. Moreover, a recombinant bacterial tandem repeat can generate eccDNA in the extract through a de novo mechanism which is independent of DNA replication. These data suggest that the presence of a high level of any direct tandem repeat can confer on DNA the ability to be converted into circular multimers in the early embryo irrespective of its sequence and that homologous recombination is involved in this process.

Animals↗
PubMed1999

Characterization of Xenopus RalB and its involvement in F-actin control during early development.

We describe the characterization and a functional analysis in Xenopus development of RalB, a small G protein. RalB RNA and protein are detectable during oogenesis and early development, but the gene is expressed only weakly in adult tissues. The RalB transcripts are processed by poly(A) extension during oocyte maturation and up to the gastrulation stage. Microinjection of wild-type or mutant RalB RNAs was performed in fertilized eggs in order to gain insight into the function of RalB during development. We show that during cleavage stages the activated GTP form of RalB specifically induces a cortical reaction that affects the localization of pigment granules. The use of different drugs suggests that this reaction is dependent on the outer cortical actin array. The relation between F-actin and RalB was shown by confocal analysis. Injection of mRNAs encoding the mutated activated form of RalB leads, at dependent doses, to a blocking of gastrulation or defects in closing of neural folding structures. In contrast, the inactivated form blocks only the closing of neural tube. Altogether, these observations suggest that RalB is part of a regulatory pathway that may affect the blastomere cytoskeleton and take part in early development.

Actins↗
PubMed1997

Conservation of structural domains and biochemical activities of the MDM2 protein from Xenopus laevis.

The Mdm2 gene is the best known cellular regulator of p53 tumor suppressor activity. We report here the cloning and characterization of Xdm2, its homolog in Xenopus laevis. Human, mouse and Xenopus MDM2 proteins are more than 65% identical in several regions which are likely to be important for the biological activities of MDM2. Region I is sufficient for binding p53 and inhibiting its G1 arrest and apoptosis functions. Region II contains most of a central acidic region required for interaction with the L5 ribosomal protein and a putative C4 zinc finger. Region III is nearly identical from Xenopus to human and comprises the RING finger domain. We show that this structural conservation is associated with the conservation of three biochemical activities of MDM2; binding to the p53 and L5 proteins and specifically to RNA. Lastly, Xdm2 expression during early development is mainly restricted from the oocyte stage I/II to the blastula stage and is possibly independent of transcriptional activation by p53. These data as well as the utilization of Xenopus laevis to investigate the roles of MDM2 and p53 during early embryogenesis are discussed.

Amino Acid Sequence↗
PubMed1995

Isolation of cDNAs from maternal mRNAs specifically present during early development.

In many animals the regulation of early embryonic development is under the control of mRNAs of maternal origin. We describe here the construction and characterization of a subtractive cDNA library to isolate genes whose expression is restricted to early development. Such genes might be potentially involved in developmental diseases, if accidentally expressed during adult life. Seven yet unknown genes preferentially expressed in the oocyte and present during early development were isolated from this library by differential screening. Their transcription patterns provide evidence for a selective expression during oogenesis and the existence of a gene family specifically implicated in functions linked to early embryogenesis.

Animals↗
PubMed1995

Selective and rapid nuclear translocation of a c-Myc-containing complex after fertilization of Xenopus laevis eggs.

We report here unusual features of c-Myc specific to early embryonic development in Xenopus laevis, a period characterized by generalized transcriptional quiescence and rapid biphasic cell cycles. Two c-Myc protein forms, p61 and p64, are present in large amounts in the oocyte as well as during early development. In contrast, only p64 c-Myc is present in Xenopus somatic cells. p61 c-Myc is the direct translation product from both endogenous c-myc mRNAs and c-myc recombinant DNA. It is converted to the p64 c-Myc form after introduction into an egg extract, in the presence of phosphatase inhibitors. p61 and p64 belong to two distinct complexes localized in the cytoplasm of the oocyte. A 15S complex contains p64 c-Myc, and a 17.4S complex contains p61 c-Myc. Fertilization triggers the selective and total entry of only p64 c-Myc into the nucleus. This translocation occurs in a nonprogressive manner and is completed during the first cell cycles. This phenomenon results in an exceptionally high level of c-Myc in the nucleus, which returns to a somatic cell-like level only at the end of the blastulation period. During early development, when the entire embryonic genome is transcriptionally inactive, c-Myc does not exhibit a DNA binding activity with Max. Moreover, embryonic nuclei not only prevent the formation of c-Myc/Max complexes but also dissociate such preformed complexes. These peculiar aspects of c-Myc behavior suggest a function that could be linked to the rapid DNA replication cycles occurring during the early cell cycles rather than a function involving transcriptional activity.

Animals↗
PubMed1989

Detection of proto-oncogenes in the genome of the amphibian Xenopus laevis.

The Xenopus laevis genome was probed by Southern Blot analysis for the presence of sequences homologous to mammalian or avian proto-oncogenes. Hybridization conditions were strictly defined with a known proto-oncogene to detect a positive signal with DNA sequences having at least 60 to 64% homology. In such conditions thirteen genes representing different oncogene families exhibited positive hybridizations with specific DNA restriction fragments. Members of the protein kinase oncogene family were detected including abl, erbB, fes, fms, ros, raf and mos. Ets, rel, and the steroid hormone related receptor erbA also gave positive signals with specific Xenopus DNA fragments. Proto-oncogenes raf and the ras family, N-ras, H-ras and c-ral, gave the strongest hybridizations and the signals remained positive in high stringency wash conditions. This study confirms the relative conservation of these genes during evolution and opens the possibility of studying their role in one of the best characterized systems of embryonic development.

Animals↗
PubMed1988

Localization of c-myc expression during oogenesis and embryonic development in Xenopus laevis.

The expression of the proto-oncogene c-myc during oogenesis and embryonic development was followed by in situ hybridization using a cytological protocol adapted to amphibian embryos. The c-myc RNA was highly expressed in the cytoplasm of young oocytes and was further diluted during oocyte growth without specific localization. From the neurula stage on, new myc transcripts were detected and the whole embryo appeared positive with antisense myc RNA probes relative to control sense RNA probes. In addition, a spatial localization of high levels of the transcript was also observed in specific areas of the developing embryo, including the epidermis, gill buds, optic vesicles and lens placodes. These observations might indicate a specific role of the c-myc gene during the differentiation of these tissues. Alternatively, this high level of myc expression might prevent such tissues from entering into terminal differentiation during the growth of the embryo.

Animals↗
PubMed1986

Xenopus myc proto-oncogene during development: expression as a stable maternal mRNA uncoupled from cell division.

A Xenopus cDNA clone highly homologous to the proto-oncogene c-myc has been isolated and used to derive a homologous probe to study myc expression during embryonic development. Myc RNA is identified as a member of the class of maternal mRNAs expressed before fertilisation. It is highly accumulated from early oogenesis and an unfertilised egg contains 8 pg, about 10(5)-fold the myc content of proliferative somatic cells. After fertilisation a post-transcriptional regulation of the gene is induced and the accumulated myc RNA is degraded (t1/2 = 4 h 20 min) to reach a level at gastrula of 10 transcripts per cell; a value maintained during subsequent embryonic development. The Xenopus myc protein has also been identified by both myc-specific antibodies and hybrid selection experiments. Translation in vitro of Xenopus myc RNA shows that it encodes a 62-kd protein which is also recognised by myc antibodies in oocyte extracts. This protein is accumulated in late oogenesis. The results indicate an unusual uncoupling of myc expression and cell proliferation linked to a stabilisation of the RNA product.

Animals↗
PubMed1985

Chromosome replication in early Xenopus embryos.

Early embryos of X. laevis achieve exceptional rates of DNA replication and chromatin assembly. These processes have been studied by microinjecting DNA templates into eggs or by incubating DNA in egg extracts. The egg is able to regulate replication of injected DNA without requiring specialized DNA sequences. Assembly of DNA into the nucleosome subunits of chromatin involves interaction of DNA with complexes containing two classes of acidic protein, namely nucleoplasmin and N1/N2.

Animals↗
PubMed1982

Vive le replicon!

Explore the source record for details and available documents.

DNA, Bacterial↗
PubMed1980

Eukaryotic DNA polymerase alpha. Structural analysis of the enzyme from regenerating rat liver.

The DNA polymerase alpha from regenerating rat liver has been purified to near homogeneity. The most highly purified fraction gave a single stained band on native polyacrylamide gel electrophoresis, and DNA polymerase alpha activity was coincident with this band. On pore gradient gel electrophoresis, a molecular radius of 72 A was obtained for the enzyme. On denaturing sodium dodecyl sulfate-polyacrylamide gel, five polypeptides were reproducibly resolved, corresponding to molecular weights of 156,000, 64,000, 61,000, 58,000, and 54,000. The catalytic subunit correlated with the 156,000-dalton polypeptide. In the native state, the separated 54,000- to 64,000-dalton polypeptides interacted among themselves to constitute a hetero oligomer of apparent high molecular weight without DNA polymerase activity. Electron microscopy studies of the enzyme confirmed the biochemical results. The specific activity of the isolated catalytic subunit was in all cases lower than when it was associated with the 54,000- to 64,000-dalton structure.

Animals↗
PubMed1979

DNA polymerase-alpha from regenerating rat liver. Catalytic properties of the highly purified enzyme.

DNA polymerase-alpha from the cytosol of regenerating rat liver has been highly purified by a procedure which includes affinity chromatography. The purified enzyme sediments at 7.4 S in high ionic strength and at 9--10 S in low ionic strength, i.e. under in vitro polymerization conditions. This enzyme has all the properties of the other mammalian DNA polymerases-alpha: sensitivity to sulfhydryl-blocking agents, to heparin, and to the level of salt in the assay, neutral pH optimum, use of ribonucleotide-initiated DNA templates, and inability to copy the ribostrand of hybrids. After chromatography on denatured DNA-cellulose, the alpha-polymerase is completely devoid of exo- and endonuclease activities. Template competition experiments indicate that the binding of the enzyme to the template can be distinguished from the polymerization itself and that the in vitro synthesis catalyzed by this alpha-polymerase is not distributive in a classical sense. These facts are discussed.

Animals↗
PubMed1975

Co-fractionation of an endonuclease activity during the purification of DNA polymerase-alpha from regenerating rat liver. Properties and separation from DNA polymerase.

The presence of endonuclease activity associated with DNA polymerase was detected during the purification of high-molecular-weight DNA polymerase-alpha from regenerating rat liver by the use of a highly sensitive test. This endonuclease activity co-fractionated with DNA polymerase in a great variety of purification procedures involving ion-exchange chromatographies or molecular weight fractionation, but was further completely separated from DNA polymerase activity by using affinity chromatography on DNA-cellulose. The endonuclease acted on native or denatured DNA by introducing single-strand nicks in the DNA molecules; its enzymatic properties indicate that it could act in polymerisation conditions in vitro.

Animals↗
PubMedDate not supplied

Transforming potential of the v-myb oncogene from avian myeloblastosis virus: alterations in the oncogene product may reveal a new target specificity.

Transfection of brown leghorn chicken embryo fibroblasts by DNA containing v-myb sequences cloned either in a complete AMV proviral DNA or in a retroviral derived vector has led to the isolation of two kinds of transformed cells. A characterization of the proviral sequences retained and expressed in these transformed cells revealed that they contained either new or altered v-myb-related RNA species. The experiments presented in this paper also show that both types of transformants expressed truncated myb-related polypeptides, suggesting that alterations of the v-myb product may result in a new target specificity, leading to the transformation of chicken embryo fibroblasts.

Animals↗