Evidence for multiple sequences and factors involved in c-myc RNA stability during amphibian oogenesis.

To investigate the molecular mechanisms regulating c-myc RNA stability during late amphibian oogenesis, a heterologous system was used in which synthetic Xenopus laevis c-myc transcripts, progressively deleted from their 3' end, were injected into the cytoplasm of two different host axolotl (Ambystoma mexicanum) cells: stage VI oocytes and progesterone-matured oocytes (unfertilized eggs; UFE). This in vivo strategy allowed the behavior of the exogenous c-myc transcripts to be followed and different regions involved in the stability of each intermediate deleted molecule to be identified. Interestingly, these specific regions differ in the two cellular contexts. In oocytes, two stabilizing regions are located in the 3' untranslated region (UTR) and two in the coding sequence (exons II and III) of the RNA. In UFE, the stabilizing regions correspond to the first part of the 3' UTR and to the first part of exon II. However, in UFE, the majority of synthetic transcripts are degraded. This degradation is a consequence of nuclear factors delivered after germinal vesicle breakdown and specifically acting on targeted regions of the RNA. To test the direct implication of these nuclear factors in c-myc RNA degradation, an in vitro system was set up using axolotl germinal vesicle extracts that mimic the in vivo results and confirm the existence of specific destabilizing factors. In vitro analysis revealed that two populations of nuclear molecules are implicated: one of 4.4-5S (50-65 kDa) and the second of 5.4-6S (90-110 kDa). These degrading nuclear factors act preferentially on the coding region of the c-myc RNA and appear to be conserved between axolotl and Xenopus. Thus, this experimental approach has allowed the identification of specific stabilizing sequences in c-myc RNA and the temporal identification of the different factors (cytoplasmic and/or nuclear) involved in post-transcriptional regulation of this RNA during oogenesis.

Evidence for introduction of a variable G1 phase at the midblastula transition during early development in axolotl.

After fertilization in axolotl, the synchronous cell cleavages are triphasic (S, G2 and M phases). Midblastula transition (MBT) begins at the ninth cleavage and is the consequence of lengthening of cell cycles. By spectrofluorometry and incorporation of 3H thymidine into the nuclear DNA followed by autoradiography on individual cells, the time at which a G1 phase appears during early development was investigated. The present results show that the G1 phase was introduced for the first time at MBT and its duration was variable from one blastomere to another. This variability could account for lengthening of cell cycles and be required for zygotic transcriptions necessary for DNA replication. From this point of view, axolotl represents an interesting alternative amphibian model to identify regulators involved in the G1-S transition at MBT during early development.

Post-transcriptional control of c-myc RNA during early development analyzed in vivo with a Xenopus-axolotl heterologous system.

We have set up a heterologous in vivo system to study gene regulation at the post-transcriptional level during early development. This system uses two amphibian species, Xenopus laevis and Ambystoma mexicanum (axolotl), the development of which is three to four times slower than that of X. laevis. The stability of three different synthetic X. laevis c-myc transcripts was followed after injection into fertilized axolotl eggs. One transcript is 2.2 kilobases (kb) long (full-length). The second is 1.5-kb long with most of the 3' untranslated region (3'UTR) removed, and the third corresponds to the 3'UTR (0.7-kb). The behavior of the endogenous axolotl c-myc RNA was compared with the exogenous injected c-myc transcripts. Our results show the existence of several developmental timers controlling degradation of the c-myc molecules. The first is activated at oocyte maturation and affects both the endogenous and exogenous (2.2- and 1.5-kb) transcripts containing the coding regions. A second timer could be linked to the number of cell divisions since fertilization (6th-7th cleavages) and involves the endogenous c-myc RNAs. Another timer could involve the c-myc mRNA molecule itself, because when injected into axolotl eggs, the half-life of the 2.2-kb X. laevis transcript appears to be independent of the axolotl context. After injection into axolotl fertilized eggs, the behavior of this X. laevis full-length c-myc molecule reveals an unexpected increase in the intensity of its autoradiographic signals. This increase occurs independently of events linked to mid-blastula transition and preliminary investigations are discussed.

Differential stability of Xenopus c-myc RNA during oogenesis in axolotl Involvement of the 3' untranslated region in vivo.

We have used the axolotl oocyte (Ambystoma mexicanum Shaw) to study the stability of exogenously injected Xenopus RNAs. Three different cellular developmental stages have been analysed: (1) the growing oocyte (stage III-IV of vitellogenesis), (2) the full-grown oocyte at the end of vitellogenesis (stage VI) and (3) the progesterone-matured stage VI oocyte. Three exogenous RNAs have been synthesized in vitro from a c-myc Xenopus cDNA clone. One transcript is 2.3 kb long (full length), the second is 1.5 kb long, with most of the 3' untranslated region (3'UTR) removed, and the third corresponds to the 3'UTR (0.8 kb). After injection or coinjection of these exogenous Xenopus RNAs into axolotl oocytes, the stability of the molecules was studied after 5 min, 6 h and 21 h by extraction of total RNA and Northern blot analysis.Results show a difference in Xenopus RNA stability during axolotl oogenesis. In growing oocytes, the three synthetic transcripts are gradually degraded. The absence of the 3'UTR is not therefore sufficient to stabilize the transcript during early oogenesis. No degradation is observed in full-grown oocytes, suggesting the existence of stabilizing factors at the end of oogenesis. When stage VI oocytes are induced to mature by progesterone, only the 2.3 and 1.5 kb Xenopus RNAs disappear. This suggests a role for germinal vesicle breakdown in this degradation process as well as the existence of a factor present in the nucleus and involved in the specific destabilization of these RNAs after oocyte maturation. This degradation might implicate several destabilizing sequences localized in the coding or in the 3'UTR of the c-myc gene. In contrast, the 0.8 kb transcript (3'UTR) is not degraded during this period and remains very stable. Therefore, degradation appears distinct from one transcript to another and from one region to another within the same molecule. During maturation, the behaviour of the 2.3 and 1.5 kb transcripts is different when coinjected with the 3'UTR, suggesting a role in trans of this untranslated molecule in c-myc stability. Our approach allows us to analyse the role of the coding and 3'UTR regions of the c-myc RNA in the control of mRNA degradation in vivo.

Evidence for a nuclear factor involved in c-myc RNA degradation during axolotl oocyte maturation.

We have previously described an in vivo heterologous system which has allowed us to study the stability of different Xenopus c-myc RNA constructs injected into axolotl oocytes. In full-grown oocytes, degradation of c-myc RNA does not occur. In mature oocytes treated with progesterone, transcripts containing the coding sequence of the gene are degraded, whereas those corresponding to the 3'UTR (untranslated region) alone are stable. In order to study the role of nuclear or cytoplasmic components in this process, degradation of injected c-myc transcripts was analysed (i) after inhibition of germinal vesicle breakdown (GVBD) in progesterone treated oocytes (ii) after induced maturation of enucleated oocytes, (iii) injection of nuclear contents into immature oocytes and (iv) after direct injection into the germinal vesicle of full-grown oocytes. Our results demonstrate the role of a nuclear factor stockpiled in the germinal vesicle of full-grown oocytes and specifically involved in the degradation of c-myc transcripts containing the coding region. Further biochemical characterization of this new nuclear component should lead to a better understanding of the post-transcriptional control of c-myc expression during oogenesis and early development.

Evidence for a change in expression of DNA ligase genes in the Pleurodeles waltlii germ line during gonadogenesis.

The expression of DNA ligase genes was studied using the nuclear transplantation approach in the germ line of Pleurodeles waltlii (P. waltlii) just before and during gonadogenesis. Germ cell (GC) nuclei were isolated from larvae of P. waltlii and transplanted into unfertilized Ambystoma mexicanum eggs. DNA ligase activity in these eggs was then analyzed after sucrose gradient fractionation. The activity of DNA ligase I (heavy form, 7.5 S) of P. waltlii was present when the transplanted GC nuclei were isolated before the first histological appearance of gonadogenesis. At the beginning of genital ridge formation and thereafter, DNA ligase I activity was replaced by that of DNA ligase II (light form, 7 S). Expression of form I was found to be sensitive to inhibitors of translation and transcription, while that of form II was not. Therefore, the change in DNA ligase activity of the transferred nuclei of P. waltlii germ cells was assumed to be the consequence of a change in gene activity, namely, the repression of the gene encoding DNA ligase I. This change in the gene-regulated state could be linked to protein modifications of the chromatin. These results indicate that, at the beginning of gonadogenesis, germ cells receive information leading to a new state of differentiation.

Changes in the catalytic properties of DNA ligases during early sea urchin development.

Two distinct DNA ligases are expressed during early sea urchin embryogenesis. A light form (50 kDa) is found in unfertilized eggs (oocyte form) and a heavier enzyme (110 kDa) is observed at the two-cell stage (embryonic form). The chronology of the change reveals that the embryonic form is detected 90 min after fertilization. After the two proteins were purified, their catalytic properties were studied using different substrates. The oocyte ligase acts only on deoxypolymers while the embryonic form also ligates heteropolymers. The two enzymes were found to undergo both nick and cohesive-end ligation. With different kinds of restriction sites it was observed that the embryonic enzyme could also ligate blunt-ended DNA. These catalytic properties account for sealing of exogenous DNA and concatenation following DNA injection into eggs. The role of the oocyte form of the enzyme is unclear; one speculation is a role in repair of DNA breaks which might accumulate during long-term sperm and oocyte storage in the gonad.

Evidence for a change in molecular for of DNA ligase in early development of the sea urchin Psammechinus miliaris.

A change in the molecular form of DNA ligase appears when the sea urchin egg enters cleavage. Sucrose gradient analysis and DNA cellulose chromatography show that a slower migrating form (7 S) of enzyme exists in unfertilized eggs and in sperm. A faster migrating form of DNA ligase (7.8 S) is present in developing embryos as well as in artificially activated eggs. The timing of this early biochemical event has been determined, following fertilization or activation. The change in molecular form of DNA ligase has been shown to be sensitive to drugs inhibiting protein synthesis, gene transcription, or DNA replication. Consequently the appearance of the faster migrating form of enzyme is assumed to result from expression of the corresponding gene, transcription, and translation. RNA extracted from testes and from cleaving stages, assayed in vitro and in vivo, have been shown to carry the information for, respectively, 7 S and 7.8 S DNA ligase according to the origin of the RNA.

Expression of DNA ligase genes by ram spermatid nuclei and RNA in amphibian eggs.

During animal development and gametogenesis two DNA ligases are found and successively expressed. In this study the two DNA ligases present in the axolotl egg and the two ligases present during ram sperm cell maturation were distinguished by biochemical and immunological methods. The expression of the genes for the heavy and light ram DNA ligases has been studied using transplantation of spermatid and sperm nuclei in axolotl eggs. We found that ram DNA ligases were expressed in axolotl egg cytoplasm. The exclusion phenomenon between the heavy and light form of DNA ligase is species-specific and involves a cytoplasmic mediator. In the transplanted ram germ cell nuclei the heavy ram DNA ligase expression was found to be sensitive to inhibitors of transcription while the light one was not. When mRNA was used, no exclusion process was observed and both the heavy and light enzyme expression were sensitive to cycloheximide and not to aamanitin. These results are discussed in terms of the possible stability of the gene-regulated state following nuclear transfer.

Molecular duality of DNA ligase in axolotl corresponds to distinctive transcriptional information.

Based upon the use of specific antibodies and sucrose gradient sedimentation analysis, the present work describes the use of the post-transcriptional equipment of the urodele egg to compare the information contained in two RNA samples extracted from respectively liver and activated axolotl eggs. It is shown that besides the normal DNA ligase activity present in the host Pleurodeles eggs, RNA can translate for the specific carried information revealing a difference between the two samples. Moreover, unlike in nuclear transplantation, the homologous DNA ligases are not mutually exclusive. These observations give a new convincing support of the genetic basis of the molecular duality of DNA ligases.

Control of DNA ligase molecular forms in nucleocytoplasmic combinations of axolotl and Pleurodeles.

A light form of DNA ligase (EC 6.5.1.2), the only form present in oocytes of the axolotl (Ambystoma mexicanum), has been shown to be replaced by a heavy form of the enzyme when the egg enters cleavage. This early biochemical event has been assumed to rely on direct nuclear input. Sucrose gradient analysis permits discrimination between enzymes from axolotl and the sharp-ribbed salamander (Pleurodeles waltlii) for both heavy and light enzymatic forms of DNA ligase. Genetic activity of blastula nuclei transplanted in activated cytoplasm has been tested by determination of the enzymatic forms and specific types of DNA ligases when the implanted egg enters cleavage. A blastula nucleus of Pleurodeles in axolotl cytoplasm determines a heavy ligase of the Pleurodeles type. Conversely, a haploid androgenetic nucleus of Pleurodeles in axolotl cytoplasm controls a light ligase of the Pleurodeles type. Reciprocal experiments give homologous results. To our knowledge, this is the earliest nucleus-dependent synthesis revealed in development for any system. The heavy ligase of one species may coexist with the light form of the other species but not with the light form of its own specific type. Inhibition of the production of the heavy form for one genome results in the expression of the light form. We conclude that genetic control of DNA ligase in very early development involves structural genes for heavy and light forms of enzyme, with an exclusion process operating an alternative expression of corresponding genes. This exclusion relationship between nonallelic genes is species specific.

Isolation of the messenger RNA for 8S DNA ligase in early developing axolotl egg and its cell free translation.

A new DNA ligase activity is expressed when the Axolotl eggs enter cleavage. The messenger RNA can be labelled by [3H] uridine thereby indicating its de novo synthesis. This new genetic expression is occurring just before cleavage and is the earliest found during Amphibian development. The newly synthesized [3H] mRNA can be translated in vitro in the rabbit reticulocyte lysate system. The resulting product is a 160 K protein specifically immunoprecipitated with the antiserum directed against 8S DNA ligase. This in vitro translated polypeptide exhibits 8S DNA ligase activity specific of activated or fertilized eggs but does not display 6S DNA ligase activity of non activated eggs.

DNA ligase in Axolotl egg: a model for study of gene activity control.

Replacement of the light form of DNA ligase (6 S) by the heavy form (8 S) in activated egg of Axolotl has been studied as a model for change in genetic activity exerted by the female pronucleus. Nuclear transplantation shows that a blastula nucleus is able to govern the replacement of the light ligase by the heavy one. The result is not the same if the grafted nucleus is taken from an androgenetic embryo, devoid of the heavy enzyme. Therefore the change in the properties of the female pronucleus appears stable and autoreproducible. Gamma irradiations delivered at different times after activation establish that the replacement of the ligase forms depends on an intact nucleus up to 3 hr 30 min after activation, and thereafter is achieved independently of any nuclear damage. Inhibitors of DNA replication impede the change of enzymatic form in reversible process, suggesting new chromatin synthesis as prerequisite for expression of the new genetic activity. The quantitative level of DNA ligase activity does not show any dose effect when one or many nuclei are present in the same cytoplasm. However, a change in nucleotide concentration results in a change in DNA ligase activity, indicating cytoplasmic control of enzymatic regulation.

Evidence for a DNA ligase change related to early cleavage in axolotl egg.

A definite change in the forms of DNA ligase appears when the axolotl egg enters cleavage. Sucrose gradient and phosphocellulose chromatography show that the a 6S form of DNA ligase exists before division, i.e. in unfertilised and fertilised egg, and a 8.2S form is present at the first division. N-ethylmaleimide sensitivity and heat stability are different for the two forms. The possible significance of this early change is discussed.

[Demonstration of RNA synthesis during segmentation in the Axolotl embryo]. / Mise en évidence d'une RNA synthèse en cours de segmentation chez le germe d'Axolotl

Study of the incorporation of 3H-uridine in cleaving embryo of Axolotl has shown a nuclear RNA synthesis during the period of synchronous cleavage (6th cycle) as well as after the onset of asynchronous divisions (9th or 10th cycle). In the early development of the Axolotl, the extent of the transcription phase looks to be an essential element of the quantitative control of gene activity.