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.     

Stability of alpha-fetoprotein messenger RNA in mouse yolk sac

Changes in the activity of DNA polymerase-α and in subcellular distribution were studied during gastrulation of the sea urchin, Hemicentrotus pulcherrimus. Although the activity of DNA polymerase-α for each embryo was constant up to the blastula stage as reported previously, the enzyme activity increased during gastrulation by about twofold prior to an increase in its DNA content. Thereafter the enzyme activity remained constant at a high level until the early pluteus stage. During gastrulation, an increase in the fraction of DNA polymerase-α was associated with the rough endoplasmic reticulum. During the period between the gastrula and pluteus stages, the cytoplasmic DNA polymerase-α activity decreased gradually with a concomitant increase of activity in the nucleus fraction. The timing of this increase in the nucleus coincided with the increase of DNA content per embryo. These results suggest that DNA polymerase-α accumulates on the rough endoplasmic reticulum during gastrulation and then translocates to the nucleus for DNA synthesis as seen before the blastula stage. DNA polymerase-α obtained from gastrula nuclei did not associate with the endoplasmic reticulum from gastrulae. DNA polymerase-α obtained from the gastrula endoplasmic reticulum membranes became bound to the salt-washed membranes from gastrulae but not to those from unfertilized eggs. Likewise, DNA polymerase-α from the rough endoplasmic reticulum of unfertilized eggs became attached to salt-washed membranes from unfertilized eggs, but not to those from gastrulae. This suggests that DNA polymerase-α is synthesized anew, and a transition of both DNA polymerase-α and endoplasmic reticulum occurs at the gastrula stage.     

Nuclear pore flow rate of ribosomal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis

The number of ribosomal RNA molecules which are transferred through an average nuclear pore complex per minute into the cytoplasm (nuclear pore flow rate, NPFR) during oocyte growth of Xenopus laevis is estimated. The NPFR calculations are based on determinations of the increase of cytoplasmic rRNA content during defined time intervals and of the total number of pore complexes in the respective oogenesis stages. In the mid-lampbrush stage (500–700 μm oocyte diameter) the NPFR is maximal with 2.62 rRNA molecules/pore/minute. Then it decreases to zero at the end of oogenesis. The nucleocytoplasmic RNA flow rates determined are compared with corresponding values of other cell types. The molecular weight of the rRNA precursor transcribed in the extrachromosomal nucleoli of Xenopus lampbrush stage oocytes is determined by acrylamide gel electrophoresis to be 2.5 × 10⁶ daltons. From the temporal increase of cytoplasmic rRNA (3.8 μg per oocyte in 38 days) and the known number of simultaneously growing precursor molecules in the nucleus the chain growth rate of the 40 S precursor RNA is estimated to be 34 nucleotides per second.     

Rates of synthesis of major classes of RNA in Drosophila embryos.

We have been successful in labeling to high specific activity (3 × 10⁵ dpm/μg) the RNA synthesized by large numbers of Drosophila embryos. Embryos of various developmental stages were rendered permeable with octane and labeled with [³H]uridine for 1 hr. At each stage the total dpm incorporated into RNA and the specific activity of the UTP pool were measured and used to calculate the absolute rate of RNA synthesis per embryo. This rate increases during embryonic development, from 1 pmole UTP/hr at 2 hr after oviposition to 6 pmoles UTP/hr at 15 hr. The rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNAs were determined by analyzing the fractionated RNAs from each stage by sucrose gradient sedimentation. There is a significant activation of nuclear RNA synthesis at the blastoderm stage (approximately 2 hr after oviposition). After blastoderm, the rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNA per embryo increase continuously; the rate of synthesis of each of these classes per nucleus, however, remains fairly constant. After making corrections for turnover during the labeling period, we find that the rates of synthesis of the major classes of RNA per nucleus at the gastrula stage are: cytoplasmic poly(A)⁺ RNA, 0.06 fg/nucleus-min; hnRNA, 0.86 fg/nucleus-min; and ribosomal RNA, 0.46 fg/nucleus-min. These rates are compared to rates of RNA synthesis in sea urchin embryos.     

Rates of synthesis and turnover of 5′ cap structures of hnRNA and mRNA and their changes during sea urchin development

The relationship between heterogeneous nuclear RNA (hnRNA) and messenger RNA (mRNA) synthesis has been studied as a function of the development of the sea urchin embryo through the use of methyl incorporation. Several parameters in the metabolism of capped hnRNA and mRNA of early blastula and late gastrula stages have been investigated by measuring the kinetics of transfer of methyl groups from S-adenosylmethionine to the 5′ cap structures in nuclear and cytoplasmic RNA:

• 1 The rate constants for the decay of hnRNA caps and the synthesis of mRNA caps are equal to within experimental error. This equality indicates a flux of precursor hnRNA caps to mRNA caps with a very high degree of conservation of the hnRNA caps. This conservation holds for each embryonic stage.
• 2 From literature data on the labeling kinetics of GTP and mRNA, we have calculated the decay constant of a putative mRNA precursor component of hnRNA. The value of this constant is very close to that for the decay constant of hnRNA caps. Hence, all hnRNA caps and some portion of their associated hnRNA sequences behave kinetically as the pre-mRNA fraction. This kinetically ascribed pre-mRNA comprises approximately 30% of the hnRNA mass.
• 3 The part of the hnRNA which does not serve as precursor to mRNA turns over at least twice as rapidly as the pre-mRNA fraction.
• 4 During development from early blastula to late gastrula, the rate of hnRNA cap synthesis drops from 2 × 10³ molecules/min/cell to half of this value. This decline is parallel to the decline in total hnRNA synthesis and thereby confirms the constant degree of capping of hnRNA, as previously reported. We infer that the pre-mRNA fraction of hnRNA remains nearly constant during this developmental period.

     

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 radioautographic study of protein synthesis in loach blastoderm]

The dynamics of protein synthesis in the loach embryos has been studied by means of autoradiography at the stages of cleavage, blastula and gastrula. During the synchronous cleavage divisions, nuclear proteins are mainly synthesized. From the early blastula stage until the early gastrula stage, the intensity of nuclear protein synthesis increases 2.5 times whereas the intensity of cytoplasmic and total protein synthesis is low and relatively constant. After the onset of gastrulation the intensity of nuclear and cytoplasmic protein synthesis increases 3-4 times and at the late gastrula stage it decreases twice as compared with that at the midgastrula stage. During blastulation, no regional differences in the intensity of nuclear and cytoplasmic protein synthesis were found. With the onset of gastrulation, a vegeto-animal gradient of labeled aminoacid incorporation into nuclear and cytoplasmic proteins appears. During gastrulation, reliable differences were found between the intensity of labeled aminoacid incorporation into proteins of the cells of intact and dissociated blastoderms. During this period, the intensity of protein synthesis in embryonic shield is higher than that in the extraembryonic part of blastoderm.     

The Kinetics of the Synthesis of Ribosomal RNA in E. coli

The kinetics of the synthesis of ribosomal RNA in E. coli has been studied using C¹⁴-uracil as tracer. Two fractions of RNA having sedimentation constants between 4 and 8S have kinetic behavior consistent with roles of precursors. The first consists of a very small proportion of the RNA found in the 100,000 g supernatant after ribosomes have been removed. It has been separated from the soluble RNA present in much larger quantities by chromatography on DEAE-cellulose columns. The size and magnitude of flow through this fraction are consistent with it being precursor to a large part of the ribosomal RNA.

A fraction of ribosomal RNA of similar size is also found in the ribosomes. This fraction is 5 to 10 per cent of the total ribosomal RNA and a much higher proportion of the RNA of the 20S and 30S ribosomes present in the cell extract. The rate of incorporation of label into this fraction and into the main fractions of ribosomal RNA of 18S and 28S suggests that the small molecules are the precursors of the large molecules. Measurements of the rate of labeling of the 20, 30, and 50S ribosomes made at corresponding times indicate that ribosome synthesis occurs by concurrent conversion of small to large molecules of RNA and small to large ribosomes.

     

Specific changes in the biosynthesis and acetylation of nucleosomal histones in the early stages of embryogenesis of sea urchin.

Histones of the sea urchin Strongylocentrotus droebachiensis were labelled in vivo with [¹⁴C]protein hydrolysate and [¹⁴C]sodium acetate at the developmental stages of blastula, gastrula and pluteus. They were further resolved by electrophoresis in 15% polyacrylamide gels in the presence of 0.9 M acetic acid, 8 M urea as well as in 20% polyacrylamide gels containing the same concentrations of acetic acid and urea, but in addition, 4.3 mM Triton X-100. On comparison of electrophoretic patterns with autoradiograms it was shown that during the early stages of differentiation the synthesis of several new subfractions of some of the histones takes place, namely of two of H1, of two of H2b and of nine of H2a. At the same time the electrophoretic pattern of the arginine-rich histones remained constant, H3 consisting of three and H4 of four subfractions. The basic reason for this heterogeneity seems to be the specific post-synthetic acetylation of the histones. At the blastula stage all the newly synthesized H4 molecules are equally acetylated, while the histone H3 molecules are acetylated to varying extents. After gastrulation both H3 and H4 are also subject to a different level of acetylation.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

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.     

Rate of synthesis of βL-lipovitellin in the liver of immature chicks treated with 17β estradiol

Using a combination of radioimmunoprecipitation and SDS-polyacrylamide gel electrophoresis of the immunoprecipitate we studied the rate of synthesis of the heavy chain of β-lipovitellin in the liver of immature chicks. In male and female chicks the base-line synthesis of β_L-lipovitellin¹ was about 30 molecules per minute and per cell. Four days after a single injection of 40 mg estradiol/kg, as many as 48,000 molecules of β_L-lipovitellin were synthesized per minute and per diploid liver cell. The increase in the rate of β_L-lipovitellin synthesis could be correlated with an increase in membrane bound mRNA coding for β_L-lipovitellin.     

Surface activity and locomotion of Fundulus deep cells during blastula and gastrula stages

The surface activity and locomotion of deep cells of the Fundulus blastoderm were studied in vivo with time-lapse cinemicrography. During late cleavage, the surfaces of the blastomeres begin to undulate gently. By early blastula, these undulations increase gradually in amplitude and hemispherical surface protrusions called blebs appear. These blebs form and retract rapidly, and at mid blastula some may be seen adhering to the surfaces of other cells. At the same time, they often expand into elongate lobopodia. Cell locomotion is first evident in mid blastula and continues throughout gastrulation. During locomotion, the leading edge of a deep cell behaves in various ways. When blebs and lobopodia adhere to a substratum (other deep cells, the undersurface of the enveloping layer, or the periblast) and retract, the cell may move in the direction of the shortening cell process. Alternatively, blebs and lobopodia may adhere, but not shorten. Locomotion is accomplished rather by protoplasmic flow into the protrusion. Blebs and lobopodia also may flatten and spread on the substratum as lamellipodia. Variations in the contact and locomotory behavior of deep cells and in the rate of their movement during blastula and gastrula stages are described in detail.     

CONTROL OF 5S RNA SYNTHESIS DURING EARLY DEVELOPMENT OF ANUCLEOLATE AND PARTIAL NUCLEOLATE MUTANTS OF XENOPUS LAEVIS

Ribosomes of all eukaryotes contain a single molecule of 5S, 18S, and 28S RNA. In the frog Xenopus laevis the genes which code for 18S and 28S RNA are located in the nucleolar organizer, but these genes are not linked to the 5S RNA genes. Therefore the synthesis of the three ribosomal RNAs provides a model system for studying interchromosomal aspects of gene regulation. In order to determine if the synthesis of the three ribosomal RNAs are interdependent, the relative rate of 5S RNA synthesis was measured in anucleolate mutants (o/o), which do not synthesize any 18S or 28S RNA, and in partial nucleolate mutants (p^l-1/o), which synthesize 18S and 28S RNA at 25% of the normal rate. Since the o/o and p^l-1/o mutants have a complete and partial deletion of 18S and 28S RNA genes respectively, but the normal number of 5S RNA genes, they provide a unique system in which to study the dependence of 5S RNA synthesis on the synthesis of 18S and 28S RNA. Total RNA was extracted from embryos labeled during different stages of development and analyzed by polyacrylamide gel electrophoresis. Quite unexpectedly it was found that 5S RNA synthesis in o/o and p^l-1/o mutants proceeds at the same rate as it does in normal embryos. Furthermore, 5S RNA synthesis is initiated normally at gastrulation in o/o mutants in the complete absence of 18S and 28S RNA synthesis.     

Epibolic extension of the presumptive ectodermal layer of embryos of the newt Cynops pyrrhogaster before and during gastrulation.

Epibolic extension of the presumptive ectodermal layer (PEL) was investigated in embryos of the newt Cynops pyrrhogaster before and during gastrulation. The PEL was composed of only one layer of columnar cells at all stages examined. The cells of the PEL became elongated from the blastula to the early gastrula stage. They were most elongated at the early gastrula stage and then shortened during gastrulation. Present observations suggest that changes in cell shape of the PEL play an important role in the control of the epibolic extension of the newt embryos. The morphology and movement of the isolated cells from the PEL were examined in an attempt to elucidate the role of cell movement in epibolic extension of the PEL. Blebbing and vermiform cells which showed active cell movement appeared at the early blastula stage. The blebbing cells, which formed large hyaline blebs that moved around the circumference of each cell, appeared in large numbers at the early blastula stage. The frequency of the blebbing cells decreased from the early blastula to the early gastrula stage and increased again during gastrulation. The vermiform cells, which had an elongated cell body and moved in a worm-like manner, increased in frequency from the early blastula to the early gastrula stage. The relative number of such vermiform cells was maximal at the early gastrula stage and decreased abruptly during gastrulation. These results suggest that the elongation of the cells of the PEL is controlled by the active cell movement which resembles that of a worm.