A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells

Embryos from a female of Xenopus laevis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastrula stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest.     

Non-Coordinated Synthesis of RNA's in Pre-Gastrular Embryos of Xenopus Laevis

The rates of syntheses of 18S and 28S rRNA, 5S RNA, capped mRNA and 4S RNA were determined in isolated cells from pre- and post-gastrular embryos of Xenopus laevis. The rate of rRNA synthesis per nucleolated cell Mas about 0.2 pg/hr, or about 5.5 × 10⁴ molecules/hr at the blastula stage, and this value remained constant in later stages. At the blastula stage, about 30 molecules of 5s RNA, 10 molecules of capped mRNA and 900 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA. These values were all greatly reduced during the gastrula stage, and at the neurula stage, one molecule each of 5S RNA and capped mRNA and 10 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA.     

A cell-free assay system for the analysis of changes in RNA synthesis during the development of Xenopus laevis.

Conditions were established for the maximal synthesis of RNA by Xenopus cultured cell nuclei. These differed from those for mammalian nuclei in having a lower K⁺ optimum. The Xenopus nuclei showed all three RNA polymerase activities and processed rRNA to 28 S and 18 S species. Extracts of full-grown oocytes stimulated the rate of RNA synthesis 2.5-fold and caused it to continue linearly for at least 6 hr. This full effect could be produced by preincubation of the nuclei with oocyte extract, followed by their reisolation and assay under standard conditions, provided that the four ribonucleotide triphosphates were present during the preincubation. The stimulatory factor(s) were mainly present in the cytoplasm of the oocyte. They produced quantitatively identical stimulations of RNA synthesis in hamster nuclei. The overall stimulatory effect of cell extracts disappears in the egg, remains absent through cleavage, but reappears at the late blastula stage. This corresponds to the changes in RNA synthesis believed to occur in early development. The extracts affect polymerases I and III, but not II to a significant extent. They also stimulate the incorporation of [γ-³²P]ATP and GTP into RNA, though to a lesser extent than the incorporation of [³H]UTP. The egg extract inhibits γ-³²P incorporation. There therefore seems to be some effect on the initiation of new chain synthesis, but its magnitude is uncertain, and the effect could be indirect.     

Synthesis and transport of various RNA species in developing embryos of Xenopus laevis.

Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs.     

Enzymes of DNA biosynthesis in developing sea urchins. Changes in ribonucleotide reductase, thymidine, and thymidylate kinase activities.

The pattern of ribonucleotide reductase, thymidine kinase, and thymidylate kinase activities during development of Paracentrotus lividus eggs and the effect of actinomycin on these enzymatic activities have been studied. Ribonucleotide reductase activity is detectable, though at a low level, in the unfertilized egg; the activity increases sharply soon after fertilization and reaches a peak at the morula stage. Thereafter it decreases and remains at a lower level than that of the unfertilized egg. Actinomycin, at a concentration sufficient to inhibit messenger RNA (mRNA) synthesis does not affect the level of enzymatic activity, indicating that preexisting maternal mRNA is used for the synthesis of this enzyme. Thymidine kinase is present at a low level in the egg; it increases sharply after the hatching blastula until the pluteus stage. Actinomycin does not affect the enzyme activity from fertilization until blastula but prevents the increase in enzyme activity that is observed between blastula and pluteus. Thymidylate kinase activity shows an increase after fertilization, followed by fluctuations throughout development with a considerable decrease at the blastula stage and at the end of gastrulation. Actinomycin has no effect on the activity of thymidylate kinase regardless of when the drug is added to the embryo suspension. Possible regulatory mechanisms of DNA synthesis in sea urchin embryos are discussed: The presence in the unfertilized egg of the most important enzymes controlling the cellular flow of DNA precursors and the availability of dTTP suggest that the block in DNA synthesis observed in the unfertilized egg is due to some particular mechanism that is switched on at fertilization.     

Differential changes in nucleolar size and ribosomal RNA synthesis during diapause break by prolonged chilling in Bombyx eggs

The activity of ribosomal RNA (rRNA) genes as judged by nucleolar size and rRNA synthesis has been shown to depend upon the phase of diapause in the eggs of Bombyx mori. In the present study, we found that nucleolar size in diapausing eggs was enlarged at a very early stage during cold treatment, a procedure necessary for the termination of diapause. In contrast, the intrinsic capacity of ribosomal RNA synthesis in the chilled eggs, as examined at 25°C by radioactive precursor incorporation into rRNA, increased much later, in parallel with the break of diapause. The early phase of cold treatment is the period when the eggs undergo some important changes (the so-called diapause development), preparing for diapause termination. Thus we infer that the above mentioned increase in nucleolar size may be one of the features of diapause development.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.     

Changes in "template-bound" and "free" RNA polymerase activities in isolated nuclei from Xenopus laevis embryos

Nuclei have been isolated from Xenopus laevis embryos and incubated under conditions allowing RNA synthesis to proceed for more than 3 h. The RNA molecules synthesized on the endogenous template are stable, heterogeneous in size and correspond to the activities of the three RNA polymerases.In these in vitro conditions we have determined the extent of activity of the three RNA polymerases during the embryonic development from blastula to swimming tadpole. Our results on isolated nuclei are in good agreement with the changes in RNA synthesis which take place during normal embryonic development.We have measured both the “template-bound” and the “free” activities of each of the three RNA polymerases during development. Amongst the total RNA polymerase activities engaged on the template, the proportion of polymerase I increases as development proceeds: at the blastula stage, there is practically no RNA polymerase I engaged on the template, whereas in swimming tadpoles, RNA polymerase I amounts to about 90% of the RNA polymerases bound to the DNA. Conversely, RNA polymerase I represents the major part of free RNA polymerases in blastula nuclei.Autoradiography of incubated nuclei shows that, at least in swimming tadpoles nuclei, both “free” and “template-bound” RNA polymerase I are localized in the nucleoli.The evolution of “template-bound” RNA polymerase II activity during development is quite different from that of RNA polymerase I: RNA polymerase II activity represents 75% of engaged polymerase activity in blastulae and only 47% at the swimming tadpoles stage.The results suggest that part of the “free” RNA polymerase I activity might progressively become “template-bound” during embryogenesis.     

RNA transcription and translation in sea urchin oocytes and eggs

The steady-state concentrations and absolute rates of synthesis of ribosomal RNA (rRNA) molecules were measured in oocytes, eggs, embryos, and larvae of the Hawaiian sea urchin Tripneustes gratilla. The steady-state concentration per genome of the RNA precursor sequences measured by hybridization to a cloned rDNA fragment was approximately 100- to 300-fold greater in the RNA obtained from oocytes and eggs than in the RNA extracted from embryos and larvae. Since the rate of processing of the rRNA precursor at different stages is not greatly different, the rates of rRNA synthesis must be considerably greater in oocytes than in embryo cells. The absolute rate of RNA synthesis in oocytes and embryos was determined from the incorporation of [³H]guanosine into cellular GTP pools and into both precursor and mature rRNA species. The data indicate an approximately 40-fold higher rate of rRNA synthesis in oocytes than that measured in embryos or previously in larvae (J. Griffith and T. Humphreys, 1979, Biochemistry18, 2178–2185). Together these results indicate that the ribosomal genes are transcribed much more rapidly during sea urchin oogenesis than during embryogenesis or larval stages.     

Sea urchin small RNA ribonucleoprotein particles: identification, synthesis, and subcellular localization during early embryonic development.

Small RNAs in sea urchins were examined in order to characterize developmental changes in their level, subcellular localization, synthesis, and association with proteins and other RNAs. Small RNAs such as the U snRNAs, 5S and 5.8S rRNAs, and 7S RNAs were identified by their mobility on highly cross-linked acrylamide gels. In addition, 7SL and U1 RNAs were identified by northern blot hybridization to cloned human and sea urchin probes, respectively. The level, subcellular localization, and association with proteins or RNA do not change for most small RNAs from fertilization to blastula, even though this is the time when the stored maternal pool of many small RNAs is being supplemented and replaced by embryonically synthesized RNAs. New embryonic synthesis of small RNAs was first detected at the 8-12 hr blastula stage. Although the predicted subsets of the total small RNA pool can be found in the appropriate subcellular compartments, newly synthesized small RNAs have a predominantly cytoplasmic localization: All of the newly synthesized small RNAs were found to be constituents of small RNPs. The RNPs containing newly synthesized small RNAs had sedimentation rates indistinguishable from their maternal counterparts. Thus, on the basis of sedimentation rate, no gross differences could be detected between maternal and embryonic small RNP pools. These small RNPs include a cytoplasmic RNP containing newly synthesized U1 snRNA and the sea urchin signal recognition particle (SRP) containing the 7SL, RNA. We have also identified a small RNP bearing the 5S rRNA which is present in both eggs and embryos. The presence of multiple, abundant, small RNAs and RNPs that are maintained at constant levels in particular subcellular fractions throughout development suggests that small RNAs may be involved in many more cellular activities than have so far been described.     

Achromosomal Division of Early Starfish Embryos Cultured in the Presence of Actinomycin D

Embryos of the starfish Asterina pectinifera were examined for their ability to undergo the early events of embryonic development in the presence of actinomycin D, a most widely used inhibitor of RNA synthesis. Fertilized eggs continued to divide eight or nine times in the presence of 25 μg ml⁻¹actinomycin D, although delay of development was observed. Chromatin disintegrated in the blastomeres of actinomycin D-treated embryos specifically at the 32-cell stage and the nucleus was undetectable at later stages. Before the 32-cell stage, RNA synthesis was not affected by the presence of actinomycin D whereas DNA synthesis was severely inhibited. The stage when achromosomal divisions cease and embryos begin to die corresponds to the period just before onset of blastulation, suggesting that the presence of the nucleus and chromosomes is a prerequisite for blastula formation and development beyond the 512-cell stage in this species.     

Changes in Ribosome-associated Proteins during Sea Urchin Development

Ribosomes isolated from unfertilised eggs of the sea urchin, Strongylocentrotus purpuratus , have a higher protein: RNA ratio than ribosomes extracted from blastula stage ribosomes. Approximately 64 additional protein equivalents are found per ribosome. Most of the proteins are of high molecular weight and are tightly bound, being resistant to high-salt and EDTA treatment. The majority of the proteins appear to be basic in nature and remain associated with the 40S subunit on dissociation of the ribosomes. The possible physiological significance of the additional proteins is discussed in terms of the activation of protein synthesis following fertilisation. Sea urchin ribosomes, isolated from various stages of development, showed differential protein-labelling patterns. The high molecular-weight proteins had preferentially higher specific activities and one ribosomal protein was particularly highly labelled, reaching a maximum at the gastrula stage of development. The functional role of this highly labelled protein during development is discussed.     

Activation of RNA synthesis in loach embryos after injection of a nuclear extract obtained by using 0.35 M NaCl

An extract mainly containing chromatin nonhistone proteins was obtained by means of 0.35 M NaCl from nuclei isolated from loach (Misgurnus fossilis L.) embryos at the 18 hour developmental stage (late gastrula). Injection of a concentrated nuclear extract into the loach eggs was followed by intensified (1.5--2.0 fold) incorporation of radioactive precursors [3H]uridine or 14CO2 into RNA. The stage at which natural activation of the RNA synthesis occurs (6 hours, mid blastula) remains unchanged, but the rate of incorporation after the onset of synthesis activation (8 hours, late blastula) becomes greater.     

Replacement of maternal 40S rRNA precursor by newly synthesized precursor in early embryos of Xenopus laevis

The amount of intact 40S rRNA precursor was followed by Northern hybridization in the course of the early embryogenesis of wild-type Xenopus laevis and its anucleolate mutant. The total amount of 40S rRNA precursor did not alter appreciably until the midblastula stage, decreased at the late blastula stage, and then increased. In the anucleolate mutant, in which no rRNA synthesis occurs, the 40S rRNA precursor decreased at the late blastula stage and disappeared after the gastrula stage. In the nuclear fraction of the wild type, the 40S rRNA precursor was detectable after the midblastula stage. Therefore, the 40S rRNA precursor in the pre-blastula embryos is maternal and decreases at the late blastula stage. New synthesis of 40S rRNA precursor apparently occurs after the midblastula stage.     

Fate of amplified nucleoli in Xenopus laevis embryos

We have followed the fate of two components of extrachromosomal nucleoli, amplified ribosomal DNA (rDNA) and 7.5 kb precursor rRNA, during early embryogenesis of Xenopus laevis. Other workers have shown that the amount of amplified rDNA accumulated during oogenesis remains unchanged through the 16-cell stage of embryogenesis. Here we show that as embryonic cleavage continues, the amount of amplified rDNA decreases until it is no longer detectable in the early gastrula embryo. In contrast, the amount of 7.5 kb precursor rRNA in eggs, early cleavage stage embryos, or blastula stage embryos is the same as in oocyte nuclei. Since no rRNA synthesis occurs during these early stages, we conclude that the precursor rRNA sequences synthesized in the oocyte are neither processed nor degraded during early development. The amplified rDNA is not replicated in the early embryo even though the chromosomal DNA of the embryo replicates every 30 min during the first 7.5 hr of embryogenesis. When amplified rDNA is purified and then injected into cleaving embryos, however, we find that it is replicated. This finding suggests that some factor(s) prevents the endogenous amplified rDNA from responding to the cellular replication signals. We show that methylation of cytosine in the rDNA is not related to the DNA's capacity for replication in this system since amplified (unmethylated) and chromosomal (methylated) rDNA are both replicated when injected into embryos. The methylation pattern of these rDNAs appears to be maintained after replication in the embryo.