Quantitative Measurement of Rates of 5S RNA and Transfer RNA Synthesis in Sea Urchin Embryos

Embryonic differentiation is believed to be due to a programmed expression of genes, which includes their time of activation, sequence of appearance, and amount transcribed into the immediate gene product, RNA. Differential synthesis of the major RNA classes, such as the ribosomal RNAs (28S, 18S, 5S) and transfer RNA (tRNA), characterizes many animal developing systems, including the sea urchin embryological system. Previous work has shown that the genes for 5S RNA and tRNA are active during early cleavage in sea urchin embryos. The present study focused on quantitatively measuring and comparing the rate of 5S RNA and tRNA synthesis in cleavage, early blastula, and early pluteus embryos of Arbacia punctulata. At each stage, embryos were labeled for 3 h with [8-³H]-guanosine. Total cellular RNA was extracted using the cold (4°C)-phenol-sodium dodecyl sulfate method and purified (LiCl-soluble) RNA preparations were fractionated by electrophoresis on 10% polyacrylamide gels. The amount of 5S RNA and tRNA synthesized at each stage was calculated from the radioactivity coincident with the 5S RNA and with the tRNA absorbance peaks (A_{260 nm}) on each gel, from the known guanosine monophosphate (GMP) compositions of sea urchin 5S RNA and tRNA and from the average specific radioactivity of the GTP precursor pool during each 3 h labeling period. The results showed that on a per embryo basis the rates of 5S RNA and tRNA synthesis increased slightly (about 1.4-fold) from cleavage through pluteus stages, while on a per cell basis the rates declined severalfold (about 3-fold) during embryogenesis. The rates of 5S RNA and tRNA synthesis determined here parallel previously-reported levels of RNA polymerase III in sea urchin embryos, suggesting that cellular levels of RNA polymerase III may exert some positive control over 5S RNA and tRNA synthesis during sea urchin embryogenesis.     

CANCELING EFFECT OF 2, 4-DINITROPHENOL ON THE VEGETALIZATION OF SEA URCHIN LARVAE INDUCED WITH CAFFEINE*

The treatment of the sea urchin morulae with both caffeine and 2, 4-dinitrophenol (DNP) for a couple of hours exerts no harmful effect on the development of sea urchin, whereas the tretment with caffeine alone yields vegetalized larvae. As long as the morulae are kept in the pressence of DNP alone, further development or the embryos is arrested, but the treated embryos develop normally after they are transferred into plain sea water. Hence, DNP is supposed to cancel vegetalizing effect of caffeine on the sea urchin morulae. When the embryos were kept in sea water containing respective radioactive precursors of macromolecules and caffeine, the radioactivity in the DNA fraction is slightly higher and those in the RNA and protein fraction are slightly lower than those of control ones (without the caffeine treatment). In the presence of DNP, the radioactivity in these macromolecules is very low in the caffeine-treated embroyos as well as in the control.     

After fertilization, sperm surface components remain as a patch in sea urchin and mouse embryos

Sea urchin and mouse sperm that are labeled on their surfaces with fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TMRTC) or 125I-diiodofluorescein isothiocyanate (125IFC) remain viable and can fertilize eggs. When sea urchin eggs were fertilized with 125IFC-labeled sperm, the radioactivity from the sperm was quantitatively transferred to the egg (at a ratio of one sperm equivalent per egg) and persisted in the embryo as it developed to the pluteus larval state (5 days at 12 degrees C). The radioactivity was acid-precipitable and was associated with the particulate fraction of embryo homogenates. In addition, FITC-labeled sea urchin sperm were used to fertilize eggs, and the labeled components were followed by fluorescence microscopy. In the embryo, labeled sperm components were present as a discrete patch that was partitioned unequally during early cleavages. In experiments using mouse sperm labeled with TMRTC, the labeled sperm components were also transferred to the embryo as a discrete patch that was again distributed unequally after cleavage. This physiological cell fusion system therefore has distinctive characteristics: there is limited lateral mobility of surface components, which have a low turnover rate unlike that see in other systems. In this paper, we discussed the possible morphogenetic role of this unusual behavior.     

AP4A-hydrolysing activity in sea urchin embryos

The enzyme activity hydrolysing diadenosine 5,5'-P1, P4-tetraphosphate (AP4A) was demonstrated in the embryonic extract of sea urchin. The enzyme activity was preferentially inhibited by ZnCl2 and by high concentrations of isobutylmethylxanthine, indicating that two types of the enzyme, (AP4A) hydrolase and non-specific phosphodiesterase, are related to the degradation of (AP4A) in sea urchin embryos. The (AP4A)-hydrolysing activity was not detectable in the unfertilized eggs because of the presence of a high-molecular weight (HMW) and thermolabile inhibitory factor. Though the enzymes were activated immediately after fertilization, no cell cycle-dependent fluctuations in their activities were observed.     

Nuclear AP4A-binding activity of sea urchin embryos changes in relation to the initiation of S phase

The AP4A-binding activity of sea urchin embryos was studied using radioactively labelled diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). Among various subcellular components that can bind [3H]AP4A, nuclei alone showed the highly specific Ap4A-binding activity which was not influenced by the presence of AP4A, AP5A and GP4G. The addition of an excess amount of ATP only slightly reduced the binding of [3H]AP4A to the nuclei. It was found that AP4A binds to the residual proteinaceous structure of nuclei which was resistant to the extraction with 2 M NaCl. The nuclear AP4A-binding activity fluctuated cyclically during each cell cycle, with a transient increase at the beginning of S phase followed by an abrupt decrease within 10 min. When the initiation of S phase was blocked, the increase in the AP4A-binding activity was also prevented. It seems that the binding of AP4A to the nuclear structural protein is involved in the initiation of S phase.     

STUDIES ON THE POLYSACCHARIDE-SULFATING SYSTEM IN SEA URCHIN EMBRYOS

The sulfating system in sea urchin embryos was examined, using the labeled precursor inorganic [³⁵S]sulfate in vivo and [³⁵S]3'-phosphoadenosine 5'-phosphosulfate ([³⁵S]PAPS) in a cell-free system. In vivo incorporation of [³⁵pS]sulfate into the trichloroacetic acid (TCA)-insolubte fraction increased gradually during sea urchin development, whereas radioactivity of [³⁵S]sulfate contained in the TCA-soluble fraction showed a conspicuous peak at the late gastrula stage.
In a cell-free system, the particulate fraction showed marked incorporation of [³⁵pS]JPAPS. This sulfating activity was highest at pH 6.4 to 7.2 and at 27°C, and it was strongly inhibited by Hg ²⁺and p-chloromercuribenzoic acid.
The sulfating activity was quite low in fertilized eggs, but then increased rapidly up to the swimming blastula stage. The activity in the particulate fraction precipitated at 10,000 xg increased gradually and that in the particulate fraction precipitated at 100,000 xg was almost constant from the swimming blastula stage to the pluteus stage.     

The Effects of Chemical Animalizing and Vegetalizing Agents and of Cell Dissociation on the Synthesis of 5S RNA and Transfer RNA in Cleaving Sea Urchin Embryos

The effect of altering normal cell associations and interactions on the synthesis of 5S RNA and transfer RNA (tRNA) was studied in cleaving embryos of the sea urchin, Arbacia punctulata. Cell interactions were altered: (1) by culturing cleaving embryos in the animalizing agent, Evans Blue, and in the vegetalizing agent, Li⁺ as LiCl and (2) by culturing dissociated cells. Control and experimental embryos each were labeled from 3 h to 6 h post fertilization with [8-³H]-guanosine. Sixteen-cell embryos, whose GTP precursor pools had been preloaded, were dissociated, labeled and cultured under conditions which prevent reaggregation. Quantitative measurements of rates of accumulation of newly synthesized 5S RNA and tRNA showed that these rates are similar in cleaving sea urchin embryos and in corresponding embryos cultured in the presence of Evans Blue and of Li⁺. In addition, cells dissociated from cleavage embryos and maintained under conditions which prevent reaggregation retained the ability to synthesize 5S RNA and tRNA. These results suggest that normal cell associations and interactions are not necessary for the synthesis of 5S RNA and tRNA to occur in cleaving sea urchin embryos.     

Re-examination of histone changes during development of newt embryos

Embryos of Triturus pyrrhogaster (BOIE) were labeled with Na₂¹⁴CO₃ and the incorporation of radioactivity into histone fractions was determined by the electrophoresis of the acid-soluble protein from isolated nuclei on a polyacrylamide gel with or without Triton X-100. The results supported the previous observation that the content of H1 histone might be low in blastulas and increased during development but they did not confirm the displacement of blastula H1 by other H1 molecular species in later embryos. The rate of H2b or H2a histone synthesis did not change much during development which contrasted sharply with the case of histone synthesis in sea urchin embryos. By changing the label duration or by culturing various durations after the label it was suggested that the histone fractions were synthesized or degraded as a set and any particular fraction that had a markedly long or short life could not be detected. The results were discussed in relation to the possible functions of H1 histone and to the histone synthesis in sea urchin embryos.     

Sea urchin embryos do not synthesize diadenosinetetraphosphate

Sea urchin embryos were labelled with [³H]adenine at two different developmental stages (cleavage and prism). Analysis by DEAE-Sephadex 7 M urea and DEAE cellulose columns of the acidsoluble nucleotide pool revealed no radioactivity under the region of the Ap₄A used as internal marker. We conclude that there is no appreciable de novo synthesis of Ap₄A during these two developmental stages.     

Formation, size, and solubility in chloroform/methanol of products of protein synthesis in isolated mitochondria of rat liver and Zajdela hepatoma.

The number, size, solubility in chloroform/methanol and some aspects of the formation of the components labeled by radioactive amino acids in isolated mitochondria of rat liver and Zajdela hepatoma were studied. Isolated mitochondria were labeled with radioactive amino acids under various conditions, and the distribution of radioactivity in sodium dodecylsulfate-polyacrylamide gels after electrophoresis of mitochondrial membrane fraction was analysed. 1. Isolated mitochondria of rat liver and Zajdela hepatoma incroporated radioactive amino acids almost exclusively into the membrane fraction. Electrophoretic analysis of this fraction revealed the presence of 15 distinct peaks of radioactivity with corresponding apparent molecular weights of 10 000 to 58 000. The electrophoretic mobility of the labeled components was identical and the general pattern of the radioactivity distribution in the gel for the rat liver and the tumour mitochondria was very similar. 2. Components of the membrane fraction of rat liver mitochondria labeled in vitro displayed an unequal solubility in acidic (2 mM HC1) chloroform/methanol (2/1) mixture; as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis a single labeled component with apparent molecular weight of 10 000 was soluble in neutral chloroform/methanol. 3. Inverse relation was observed between amino acid incorporation activity of isolated mitochondria and the portion of the label incorporated into the component with apparent molecular weight 10 000. The identity of this component with that soluble in neutral chloroform/methanol mixture has been indicated. 4. The rate of incorporation of [3H]leucine by isolated Zajdela hepatoma mitochondria into the components with lower (10 000-25 000) apparent molecular weights decreased with time, whereas that into components with higher (above 25 000) apparent molecular weight remained approximately constant within the time interval tested (30 min). 5. From the total radioactivity incorporated into the membrane fraction during 5-min pulse labeling of isolated Zajdela hepatoma mitochondria by [3H]leucine up to 25% was recovered in the region of the gel corresponding to a component with apparent molecular weight 10 000. After 25 min chase the radioactivity in this region decreased about 3.5 times while the specific radioactivity of the total membrane fraction did not change significantly. The pattern of radioactivity distribution observed after the pulse was preserved by chloramphenicol. 6. Unlabeled sonicated mitochondria or postribosomal supernatant from rat liver regenerating in the presence of chloramphenicol were incubated with neutral chloroform/methanol extract of in vitro with [14C]leucine labeled rat liver mitochondria. After this incubation several labeled components with apparent molecular weights above 10 000 were recovered in the electrophoreograms of the originally unlabeled fractions.     

Adenosine 5'-tetraphosphate is synthesized by the histidine alpha 142----asparagine mutant of Escherichia coli succinyl-CoA synthetase.

Recently, we described the properties of a mutant (H142N) of Escherichia coli succinyl coenzyme A (CoA) synthetase in which His-142 of the alpha-subunit was changed to Asn (Luo, G.-X., and Nishimura, J.S. (1991) J. Biol. Chem. 266, 20781-20785). The mutant enzyme was practically devoid of ability to catalyze the overall reaction but was able to catalyze half-reactions at significant rates. Thus, phosphorylation by ATP and dephosphorylation by ADP of the mutant enzyme occurred at rates that were at least 10 times greater than those with wild type enzyme, and dephosphorylation by succinate plus CoA (succinyl-CoA formation) proceeded with a Vmax of 10% that of wild type, with no change in Km for succinate and very little change in Km for CoA. In the present work, it has been shown that incubation of 32P-labeled H142N with ATP caused a rapid depletion of label from the enzyme and incorporation of radioactivity into a nucleotide species that was neither ATP nor ADP. This reaction was catalyzed at comparatively negligible rates by wild type enzyme. Analysis of the labeled product by high pressure liquid chromatography and 31P NMR revealed that it was adenosine 5'-tetraphosphate (AP4). Incubation of labeled H142N with the ATP analog beta,gamma-methylene adenosine triphosphate also gave a product that appeared to be the corresponding tetraphosphate. The reaction in which AP4 was formed was greatly stimulated by the addition of phosphoenolpyruvate plus pyruvate kinase and strongly inhibited by ADP and by CoA plus succinate. The results are consistent with binding of ATP to, and reaction with, phosphorylated succinyl-CoA synthetase to form AP4. In this reaction, it was determined that the Km for ATP and the turnover number of phosphorylated enzyme were 14.5 microM and 0.024 s-1, respectively.     

Synthesis of 2',5'-oligoadenylate by rat liver nuclear matrix protein

Nuclear matrix was prepared from unstimulated rat liver by treatment of nuclei with DNAse and 0.4 M NaCl and was further extracted with 2.0 M NaCl. Proteins were bound to poly(rI):(rC)-agarose, incubated with (alpha-32P) adenosine 5'-triphosphate and 2',5'-linked oligoadenylate was isolated from the supernatant. The substance inhibited amino acid incorporation in a reticulocyte translation system and was identified after enzymatic treatment followed by thin-layer chromatography on PEI-cellulose. The possible function of 2',5'-oligo(A) synthetase in the maturation of pre-mRNA associated with nuclear matrix is discussed.     

Exploration of long and short repetitive sequence relationships in the sea urchin genome.

Long and short repetitive sequences of sea urchin DNA were prepared by reassociation of 2000 nucleotide long fragments to Cot 4 and digestion with the single strand specific nuclease S1. The S1 resistant duplexes were separated into long repetitive and short repetitive fractions on Agarose A50. The extent of shared sequences was studied by reassociating a labeled preparation of short repetitive DNA with an excess of unlabeled long repetitive DNA. Less than 10% of the long repetitive DNA preparation was able to reassociate with the short repetitive DNA. Thus the long and short repetitive elements appear to be principally independent sequence classes in sea urchin DNA. Precisely reassociating repetitive DNA was prepared by four successive steps of reassociation and thermal chromatography on hydroxyapatite. This fraction (3% of the genome) was reassociated by itself or with a great excess of total sea urchin DNA. The thermal stability of the products was identical in both cases (Tm=81 degrees C), indicating that precisely repeated sequences do not have many imprecise copies in sea urchin DNA.     

Stoichiometry and stability of the adduct formed between human 4-aminobutyrate aminotransferase and 4-aminohex-5-enoate: sequence of a labelled peptide

The reaction between human 4-aminobutyrate aminotransferase and the anti-epileptic drug 4-aminohex-5-enoate, an irreversible inhibitor of the enzyme, has been studied using the radiolabelled compound. The inactivated enzyme was found to lose radiolabel over a period of a few days at 37 degrees C but even in the presence of the coenzyme, pyridoxal phosphate, no enzyme activity returned. At 4 degrees C the radiolabelled inhibitor remained stably bound. The amount of enzyme-bound 4-aminohex-5-enoate was significantly less than would be expected if one mol of inhibitor was bound per mol of active site. Reversed phase chromatography of a tryptic digest of the labelled enzyme showed that, apart from material eluting at the front of the chromatogram, all of the radioactivity was in a single fraction. This fraction contained a peptide, the sequence of which indicated that it included the lysine that binds the coenzyme and that the major release of radioactivity occurred in an Edman degradation cycle corresponding to this residue.     

Change in phosvitin kinase activity during early development of the sea urchin

Protein kinase, which phosphorylated phosvitin at the expense of ATP but did not phosphorylate casein, protamine, and histone mixture, was obtained by DEAE-cellulose column chromatography of the extract from the embryos of the sea urchin, Strongylocentrotus intermedius. This enzyme, partially purified by DEAE-cellulose column, reversibly catalyzed the reaction of phosvitin phosphorylation. This indicates that the sea urchin embryos contain phosvitin kinase. Phosvitin kinase in sea urchin embryos is somewhat different from that found in the other types of cells, which are able to phosphorylate casein as well as phosvitin. In unfertilized eggs, the activity of this enzyme was found only in the supernatant fraction obtained by centrifuging the homogenate at 10,000g for 20 min. The activity in the embryos at the swimming and the mesenchyme blastula stage was higher than in unfertilized eggs, and was localized in the sedimentable fraction obtained by centrifuging the homogenate of the embryos at 10,000g for 20 min. The highest activity of phosvitin kinase was observed in the embryos at the mesenchyme blastula stage, and the enzyme activity became quite low at the late gastrula stage. The activity and the intracellular distribution of phosvitin kinase changed during the development. The enzyme in this sedimentable fraction was not solubilized with 1% Triton X-100 but was extracted by 1 M NaCl.     

Lysine 480 is an essential residue in the putative ATP site of lamb kidney (Na,K)-ATPase. Identification of the pyridoxal 5'-diphospho-5'-adenosine and pyridoxal phosphate reactive residue

Pyridoxal 5'-diphospho-5'-adenosine (AP2PL) inhibits lamb kidney (Na,K)-ATPase and that inhibition and covalent modification is blocked by the presence of ATP. After trypsin digestion of the labeled, purified alpha subunit and subsequent peptide mapping of the fluorescently labeled peptides by means of high performance liquid chromatography, the main labeled peptide was further purified and analyzed by amino acid composition analysis and peptide sequencing. The obtained peptide had the sequence Ile470-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-Lys480-Tyr-Gln-Le u-Ser-Ile-His- Lys487. Lysine 480 is the residue modified by AP2PL in the absence, but not in the presence of ATP. The beta subunit is not differentially labeled by AP2PL in the presence or absence of ATP. Interestingly, the same results were obtained using pyridoxal phosphate as the labeling and inactivation reagent, indicating that the specificity of labeling by these reagents is not due to the presence of the adenosine moiety, but instead that the initial recognition of nucleotides by the ATP-binding site of (Na,K)-ATPase may be due to recognition of the phosphate moiety. The amino acid sequence surrounding this lysine residue labeled by both reagents is highly conserved in (Na,K)-ATPase and the related (H,K)-ATPase sequences thus far obtained, which may signify a functional importance for this region of the putative ATP-binding site in these transport proteins.     

In vivo preparation of [32P]cAMP and thin-layer chromatography of cAMP-related compounds.

A simple and rapid method for preparing [32P]adenosine 3'5'-cyclic monophosphate (cAMP) is described. A culture of an Escherichia coli mutant which excretes cAMP about 150 times faster than does a wild-type strain was incubated overnight with [32P]orthophosphate of high specific activity (e.g., 4000 Ci/mol (1 Ci = 37 GBq). The [32P]cAMP which accumulated extracellularly was then purified to 99.9% radiochemical purity in less than 4 h by adsorption to charcoal and alumina column chromatography. A two-dimensional chromatography system using a PEI-cellulose plate is also described which should prove useful for studying cAMP metabolism with 32P- or 3H-labeled cAMP or ATP.     

Synthesis of 125I-labeled N-3-(4-hydroxyphenyl)propionyl aminoacyl transfer ribonucleic acids.

A method for the isolation and labeling to high specific radioactivity of individual isoaccepting tRNAs is described. After blocking reactive minor bases by acetylation and iodination of the crude tRNA, a single family of isoacceptors was aminoacylated. Individual isoacceptors were separated by chromatography on RPC-5 and then acylated with the 3-(4-hydroxyphenyl)propionyl ester of N-hydroxysuccinimide. The product was purified by chromatography on BD-cellulose and RPC-5. This derivatized tRNA was then iodinated with 125I- and Chloramine-T to give a product containing between 5 X 10(7) and 3 X 10(8) dpm/microgram. The suitability of such labeled tRNAs for hybridization to homologous DNA in solution and cytological preparations of chromosomes is discussed with particular reference to Drosophila melanogaster.     

Effects of 5 azacytidine on DNA methylation and early development of sea urchins and ascidia

5-azacytidine (5-azaCR), an analogue of cytidine, inhibits nuclear DNA methylation in early sea urchin embryos. This inhibition is specific and dose-dependent. Exposure of sea urchin embryos at any stage between one-cell and blastula, to micromolar quantities of 5-azaCR invariably inhibits development beyond the blastula stage. In a substantial number of embryos arrested at the blastula stage, spicule formation proceeds although other morphological differentiation is lacking. No significant effect on development is seen if sea urchin embryos are exposed to 5-azaCR at post-blastula stages. 5-azaCR also inhibits the development of a mosaic egg such as the ascidian Phallusia mammilata at the blastula stage, indicating that both regulative (sea urchin) and mosaic (ascidian) embryos respond more or less similarly to 5-azaCR treatment.     

Identification and characterization of bone morphogenetic protein 2/4 gene from the starfish Archaster typicus

A bone morphogenetic protein 2/4 (BMP2/4) gene has been cloned from the starfish, Archaster typicus, for the purpose of investigating the expression pattern of the BMP4 gene in echinoderm embryos which do not produce micromeres. The isolated gene (named AtBMP2/4) contained two exons that encoded the entire coding region. The deduced AtBMP2/4 protein sequence contained 509 amino acids. Sequence comparison showed that it shared high amino acid similarity with sea urchin BMP2/4 and Xenopus BMP2 and BMP4. Northern blot analyses indicated that AtBMP2/4 mRNA initially appears at the blastula stage and has a maximal expression level at the gastrula stage. Whole-mount in situ hybridization revealed that AtBMP2/4 mRNA is expressed in the archenteron, coelomic vesicles, and ectodermal cells of gastrula stage embryos. The observed spatial distribution pattern vastly differs from that of sea urchin SpBMP2/4, which is expressed mainly in the oral ectoderm region of the mesenchyme blastula and early gastrula embryos.