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Summary Northern analysis of RNAs, extracted from normal sugarbeet callus cultures (grown with growth regulators) and from leaves derived from the mother strain, when hybridized with a Xenopus c-Ki- ras probe, reveal the presence of two transcripts in the plant material. The level of RNA detected is slightly higher (approximately 2 fold) in callus culture, when compared with leaves from the mother strain. Involvement of cellular oncogenes in the control of plant cells cultured in vitro is suggested.

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.

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.

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.

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.

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.

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.

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.

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.

There are currently available in the axolotl several genes that affect development at times ranging from cleavage to larval stages. These genes would be extremely useful as markers in nuclear transplantation studies of nuclear differentiation. It is also possible that by the use of such genes the analysis of nuclear differentiation might eventually be refined to detect changes in the genes themselves, if they occur. In the past the exploitation of the axolotl material for work of this type has not been feasible because axolotl eggs, like those of other urodeles, have generally given very poor results in nuclear transplantation experiments. The present paper presents an analysis of the factors which determine whether or not nuclear transfers succeed in this species. This analysis shows that the conditions required for the transfers to be successful are: (1) activation of the egg by heat shock (35° C for 5 minutes)7; (2) enucleation by ultraviolet irradiation; and (3) nuclear transfer at 2 to 4 hours following the application of the heat shock. A method based on this analysis is described in detail on pages 151–154. Tests of the method were made by transplanting blastula nuclei into activated and enucleated eggs. Ninety-six per cent of the eggs cleaved and 77% developed into complete blastulae of normal appearance. The majority of these blastulae (66%) developed to postneurula stages. The validity of the method was tested by transplanting nuclei carrying the dominant gene D (for black pigmentation pattern) into enucleated eggs from a recessive white (dd) female. A total of 37 larvae were obtained. All showed the black phenotype, proving that the nuclear transfers were successful.

Une technique est decrite permettant d'obtenir des images chromosomiques particulierement favorables fournies par des blastulas d'Axolotl. A partir de ce materiel le caryotype de cette espece est analyse.

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.

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.

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.

International audience; 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.

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.

Publisher Summary This chapter discusses the control of the expression of genes for DNA ligase in eukaryotes. The definite molecular form of an enzyme represents the terminal product of a specific gene activity. The presence and the amount of active substance offer a model for the regulation of gene expression. The situation encountered with DNA ligase appeared to be especially favorable for an experimental approach of this matter, and the results accumulated over the past 5 years constitute the basis of the chapter. The chapter discusses tests for the different levels of gene expression. The best-suited method for determining the pattern of DNA ligase activity is fractionation following centrifugation on sucrose gradient. At least three categories of mechanisms may be involved in any change of enzymatic pattern. First, an epigenetic modification of the elaborated product might activate a preexisting inactive form already present in the cytoplasm. Second, de novo synthesis of a new enzyme might occur, directed by masked messengers of maternal origin accumulated during oogenesis. Third, complete gene expression involving transcription and translation might account for the production of a new molecular form.

Nuclei from various regions of axolotl neurulae were transplanted into eggs which had previously been electrically activated and irradiated with UV to eliminate the egg chromosomes. The majority of the nuclei, regardless of their region of origin, elicited genuine cleavage but usually failed to promote development beyond blastula or early gastrula stages. A cytological study of the recipient embryos, fixed at various times following nuclear transfer, has revealed pronounced mitotic abnormalities in approximately 90% of the cases examined. Modifications of the transplantation medium, calculated to reduce the risk of technical damage to the nuclei, do not improve their capacity to promote the development of the recipient eggs. Triploid neurula nuclei do not differ from diploid nuclei in ability to promote the development of test eggs. Nuclei from the most recently differentiated (caudal) end of the notochord and endoderm, compared with nuclei from the more cephalic regions of the same structures, display a greater capacity to promote postgastrula development of the recipient eggs. The Discussion is focused mainly on the mitotic abnormalities exhibited by the majority of the transplanted somatic nuclei. The evidence at hand suggests that these abnormalities are expressions of a differentiated state of the nuclei, affecting their capacity to proceed normally through mitosis following transfer into egg cytoplasm. However, their exact mode of origin, and the problems they pose in the interpretation of nuclear transplantation experiments, remain to be worked out.