Jacques Lefresne

Enzymes involved in DNA replication in the axolotl. II. Control of DNA ligase activity during very early development.

Abstract The light form of DNA ligase (6 S) present in the unfertilized egg of the axolotl undergoes a rapid decay as the egg enters cleavage. At the same time a heavy form of ligase (8.2 S) appears and becomes prominent. This change occurs progressively between 3 and 9 hr of development, and its control has been studied here by experimental analysis. The modification can be brought about by artificial activation of the eggs as well as by normal fertilization. The phenomenon is sensitive to cycloheximide, actinomycin D, α-amanitin, and injury of the female pronucleus by uv irradiation. These results lead to the conclusion that the shift in ligase form involves de novo protein synthesis and transcription of an intact maternal genome. The paternal genome is unable to govern a similar change in haploid androgenetic embryos. The control of ligase replacement appears, therefore, to be the consequence of a direct gene expression revealed for the first time in an egg before cleavage. This expression is differential for paternal and maternal genomes in the same cytoplasm.

Enzymes involved in DNA replication in the axolotl: I. Analysis of the forms and activities of DNA polymerase and DNA ligase during development☆

Abstract DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.

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 change in molecular form 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.

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.

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 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.

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.

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

SummaryDuring 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.

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.