Species-specific dissociation into single cells of live sea urchin embryos by fab against membrane components of Paracentrotus lividus and Arbacia lixula

The species and stage specificities of membrane components active in promoting reaggregation of cells dissociated from embryos of the two Mediterranean sea urchin species Paracentrotus lividus and Arbacia lixula have been examined. Membrane proteins extracted with butanol either from purified membranes or from dissociated cells without significant reduction of viability promoted reaggregation of both the homologous and heterologous species. Extracts from plutei and blastulae were equally effective in promoting reaggregation of blastula cells. By contrast, Fab's prepared from IgG raised against these extracts or purified membranes are strictly species specific because they prevent reaggregation of cells and actively dissociate live embryos of only the homologous species. No corresponding stage specificity of the Fab was observed: Fab against extracts from blastula embryos also caused dissociation of plutei. Antigenic analysis of the extracts by the Ouchterlony test revealed the presence of components specific for each species as well as others common to both.     

THE SYNTHESIS OF 5S RNA AND ITS REGULATION DURING EARLY SEA URCHIN DEVELOPMENT

De novo synthesis of 5S RNA and of transfer RNA (tRNA) has been demonstrated previously to occur by mid-cleavage (128-cell stage) in sea urchin embryos (24). The present study focused on determining more precisely the time of onset of activity of the genes for 5S RNA and for tRNA during sea urchin embryogenesis by preloading the GTP precursor pools of unfertilized eggs. The results showed that newly-made 5S RNA and tRNA could be detected as early as the 32-cell stage. In order to determine whether newly-synthesized 5S RNA accumulates coordinately during development with newly-made 26S (34) and 18S ribosomal RNAs (rRNAs), the relative rates of accumulation of these three RNA molecules were measured and compared at each of several stages of sea urchin embryogenesis. In contrast to the coordinated accumulation of newly-synthesized 26S and 18S rRNAs, newly-made 5S RNA accumulated in excess at the mesenchyme blastula (9-fold excess), midgastrula (5-fold excess) and prism (3-fold excess) stages. The 5S RNA/26S RNA molar ratios only approached unity in advanced (48 hr) plutei. The non-coordinated accumulation of newly-made 5S RNA with that of 26S and 18S rRNAs suggests that the accumulation of these newly-synthesized RNAs is differentially regulated during early sea urchin development.     

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.     

High molecular weight RNA containing histone messenger in the sea urchin Paracentrotus lividus

Sea urchin RNA extracted from early and mesenchyme blastula embryos and oocytes and fractionated on denaturing sucrose density gradients, was hybridized with histone DNA recombinants of Psammechinus miliaris (clone λh22) and of Paracentrotus lividus (clone pPH70). Histone sequences are found in the 9 S and larger than 9 S regions of the formamide/sucrose density gradients. The melting of the RNA-DNA duplexes obtained by hybridization of polysomal and high molecular weight RNA of embryos of P. lividus at the stage of early blastula, suggests a degree of heterogeneity in the high M_r RNA. The high M_r RNA contains at least four of the five histone gene sequences covalently linked.     

Phosphatase active antigens in sea urchin eggs and embryos. II. A comparison between the activities in unfertilized eggs and plutei.

Phosphatase activities in sea urchin eggs and plutei were investigated by means of histochemical staining of immunoprecipitates. Two protein fractions were obtained by extraction in a hypotonic medium and by detergent treatment of the residual pellet. Three distinctly different phosphatase activities were discerned, nucleoside diphosphatase (EC 3.6.1.6.), acid phosphatase (EC 3.1.3.2.) and alkaline phosphatase (EC 3.1.3.1.). The nucleoside diphosphatase activity, which was confined to one antigen, was present in both water soluble and detergent extracts and at roughly the same concentration in eggs and plutei. By means of a monospecific antiserum the immunological identify of this antigen was established in all instances. The acid phosphatase activity, which was displayed by ten detergent extracted antigens in eggs, was only found in five detergent extracted antigens in plutei. This decrease in number of enzyme active antigens was also reflected by a general decrease in number of enzyme active antigens was also reflected by a general decrease in activity as assessed by quantitative determinations. Furthermore, by means of absorbed antisera it was established that two or three of the acid phosphatase active antigens were "egg specific". Another acid phosphatase active antigen, which was common to both developmental stages, was investigated by a monospecific antiserum. While this antigen was found in both soluble fractions, it was only enzymatically active when extracted with detergent. Alkaline phosphatase active antigens were only found in the detergent extract of plutei. However, immunoprecipitates with this activity appeared both with antiserum against unfertilized eggs and with antiserum against plutei. This suggests that the egg contained the antigens in an enzymatically inactive form.     

Hexokinase activity from eggs of the sea urchin Arbacia punctulata

1. The hexokinase activity of homogenates of eggs and embryos of the sea urchin Arbacia punctulata has been measured. Expressed as micrograms glucose consumed at 20°C., per hour per milligram of protein the following values were obtained: unfertilized eggs, 67; fertilized eggs, 72; 24 hour plutei, 94; 48 hour plutei, 226. The concentration of the enzyme in the eggs is small and may be calculated to be about 0.001 per cent of the dry weight of unfertilized eggs. 2. The hexokinase activity of the egg homogenate was virtually all recovered in the supernatant fraction when the homogenate was centrifuged at 20,000 x g for 30 minutes and was found to have the following properties: The concentrations for half maximal hexokinase activity with various substrates were, approximately: Glucose, 0,00003 M; fructose, 0.00075; mannose, 0.00007; 2-desoxyglucose, 0.00025. The relative rates of phosphorylation of various sugars by the supernate fraction when saturated with substrate were, approximately: Glucose, 1.0; mannose, 1.2; fructose, 1.8; 2-desoxyglucose, 2.0; glucosamine, 0.6. Adenosinediphosphate and glucose-6-phosphate inhibited the enzyme. No evidence for more than one hexokinase in the Arbacia extracts was found.     

RNA--protein interactions in the ribosome. IV. Structure and properties of binding sites for proteins S4, S16/S17 and S20 in the 16S RNA

Proteins S4, S16/S17 and S20 of the 30 S ribosomal subunit of Escherichia coli+ associate with specific binding sites in the 16 S ribosomal RNA. A systematic investigation of the co-operative interactions that occur when two or more of these proteins simultaneously attach to the 16 S RNA indicate that their binding sites lie near to one another. The binding site for S4 has previously been located within a 550-nucleotide RNA fragment of approximately 9 S that arises from the 5′-terminal portion of the 16 S RNA upon limited hydrolysis with pancreatic ribonuclease. The 9 S RNA was unable to associate with S20 and S16/S17, however, either alone or in combination. A fragment of similar size and nucleotide sequence, termed the 9 S¹ RNA, has been isolated following ribonuclease digestion of the complex of 16 S RNA with S20 and S16/S17. The 9 S¹ RNA bound not only S4, but S20 and S16/S17 as well, although the fragment complex was stable only when both of the latter protein fractions were present together. Nonetheless, measurements of binding stoichiometry demonstrated the interactions to be specific under these conditions. A comparison of the 9 S and 9 S¹ RNAs by electrophoresis in polyacrylamide gels containing urea revealed that the two fragments differ substantially in the number and distribution of hidden breaks. Contrary to expectation, the RNA in the ribonucleoprotein complex appeared to be more accessible to ribonuclease than the free 16 S RNA as judged by the smaller average length of the sub-fragments recovered from the 9 S¹ RNA. These results suggest that the binding of S4, S16/S17 and S20 brings about a conformational alteration within the 5′ third of the 16 S RNA.To delineate further the portions of the RNA chain that interact with S4, S16/S17 and S20, specific fragments encompassing subsequences from the 5′ third of the 16 S RNA were sought. Two such fragments, designated 12 S-I and 12 S-II, were purified by polyacrylamide gel electrophoresis from partial T₁ ribonuclease digests of the 16 S RNA. The two RNAs, which contain 290 and 210 nucleotides, respectively, are contiguous and together span the entire 5′-terminal 500 residues of the 16 S RNA molecule. When tested individually, neither 12 S-I nor 12 S-II bound S4, S16/S17 or S20. If heated together at 40 °C in the presence of Mg²⁺ ions, however, the two fragments together formed an 8 S complex which associated with S4 alone, with S16/S17 + S20 in combination, and with S4 + S16/S17 + S20 when incubated with an un fractionated mixture of 30 S subunit proteins. These results imply that each fragment contains part of the corresponding binding sites.     

Cellular distribution of RNA populations in 16-cell stage embryos of the sand dollar, Dendraster excentricus

RNA was extracted from pure preparations of micromeres and meso-plus macromeres isolated from 16-cell stage embryos of Dendraster excentricus. Molecular hybridization-competition experiments disclosed that the binding of 16-cell stage labeled RNA to denatured sperm DNA was competed equally well by micromere RNA, meso-plus macromere RNA, total 16-cell RNA and unfertilized egg RNA, indicating the egg-type populations were distributed almost equally in the different blastomeres. In contrast, experiments with ³H-RNA extracted from micromeres obtained from pulse-labeled 16-cell stage embryos showed qualitative differences when unfertilized egg RNA and total 16-cell stage RNA were used as competitors. Such differences in RNA populations could not be detected in ³H-RNA isolated from the meso-plus macromere fraction.     

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos: VI. Occurrence in mature oocytes and unfertilized eggs

Occurrence of a factor(s) which can selectively inhibit ribosomal RNA synthesis in isolated neurula cells of Xenopus laevis was examined in oocytes, unfertilized eggs, and embryos of Xenopus laevis. It was found that acid-soluble materials from full-sized oocytes, white-banded mature oocytes, unfertilized eggs, and pregastrular embryos were all active in significantly reducing the relative ratio of the [³H]uridine incorporation into 18S and 28S ribosomal RNA to that into 4S RNA from the control value. These results suggest that the inhibitor appears in the terminal step of oogenesis and, hence, may be assumed as a maternal regulator.     

A method of preparing Escherichia coli 16 S RNA possessing previously unobserved 30 S ribosomal protein binding sites

A method of preparing 16 S RNA has been developed which yields RNA capable of binding specifically at least 12, and possibly 13, 30 S ribosomal proteins. This RNA, prepared by precipitation from 30 S subunits using a mixture of acetic acid and urea, is able to form stable complexes with proteins S3, S5, S9, S12, S13, S18 and possibly S11. In addition, this RNA has not been impaired in its capacity to interact with proteins S4, S7, S8, S15, S17 and S20, which are proteins that most other workers have shown to bind RNA prepared by the traditional phenol extraction procedure (Held et al., 1974; Garrett et al., 1971; Schaup et al., 1970,1971).We have applied several criteria of specificity to the binding of proteins to 16 S RNA prepared by the acetic acid-urea method. First, the new set of proteins interacts only with acetic acid-urea 16 S RNA and not with 16 S RNA prepared by the phenol method or with 23 S RNA prepared by the acetic acid-urea procedure. Second, 50 S ribosomal proteins do not interact with acetic acidurea 16 S RNA but do bind to 23 S RNA. Third, in the case of protein S9, we have shown that the bound protein co-sediments with acetic acid-urea 16 S RNA in a sucrose gradient. Additionally, a saturation binding experiment showed that approximately one mole of protein S9 binds acetic acid-urea 16 S RNA at saturation. Thus, we conclude that the method employed for the preparation of 16 S RNA greatly influences the ability of the RNA to form specific protein complexes. The significance of these results is discussed with regard to the in vitro assembly sequence.     

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.     

Occurrence of heat-dissociable ribosomal RNA in insects: the presence of three polynucleotide chains in 26 S RNA from cultured Aedes aegypti cells

When RNA extracted from a mixture of cultured mosquito (Aedes aegypti) and hamster (BHK) cells is heated at 60 °C for five minutes the 26 S mosquito RNA but not the 28 S BHK RNA is converted to 18 S products. These products are not separable from each other or from pre-existent 18 S RNA on 2.4% acrylamide gels and have molecular weights near 0.7 × 10⁶. The large ribosomal RNA from insects belonging to ten different orders shows a similar conversion, although this property is absent in two species of aphid.A. aegypti 26 S RNA dissociates over a narrow temperature range. The reaction equilibrium favours dissociation and is dependent on ionic strength, showing a 6 deg. C change in T_m′ (the temperature of 50% dissociation) with tenfold change in salt concentration. Although the T_m of 26 S RNA from Drosophila melanogaster and A. aegypti is markedly different, reflecting the difference in base composition, the T_m′ of the two RNA species was virtually the same.High molecular weight ribosomal RNA from Escherichia coli, BHK cells and A. aegypti cells was terminally labelled with [³H]isonicotinic acid hydrazide. The specific activities of the large RNA species show the presence of one, two and three polynucleotide chains in 23 S, 28 S and 26 S RNA, respectively. A. aegypti 26 S RNA contains a small, heat-dissociable “IRNA” similar in relative amount and mobility to that found in BHK cells.     

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.     

Conserved pattern of embryonic actin gene expression in several sea urchins and a sand dollar

An examination of the size and relative abundance of actin-coding RNA in embryos of four sea urchins (Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, Arbacia punctulata, Lytechinus variegatus) and one sand dollar (Echinarachnius parma) reveals a generally conserved program of expression. In each species the relative abundance of these sequences is low in early embryos and begins to rise during late cleavage or blastula stages. In the four sea urchins, actin-coding RNAs increase between approximately 9- and 35-fold by pluteus or an earlier stage, and in the sand dollar about 5.5-fold by blastula. A major actin-coding RNA class of 2.0-2.2 kilobases (kb) is found in each species. A smaller actin-coding RNA class, which accumulates during embryogenesis, is also present in S. purpuratus (1.8 kb), S. droebachiensis (1.9 kb), and A. punctulata (1.6 kb), but apparently absent in L. variegatus and E. parma. In S. droebachiensis, actin-coding RNA is relatively abundant in unfertilized eggs and drops sharply by the 16-cell stage. This is in contrast to the other sea urchins where the actin message content is relatively low in eggs and does not change substantially in the embryos throughout early cleavage. The observations in this study suggest that the pattern of embryonic expression of at least some members of this gene family is ancient and conserved.