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.     

Carbonic anhydrase activity in developing sea urchin embryos

A 13-fold increase in carbonic anhydrase specific activity was found during the first 24 h in developing embryos of the sea urchin, Strongylocentrotus purpuratus. Carbonic anhydrase activity was sensitive to inhibition by 10⁻⁴ M acetazolamide. Roles for carbonic anhydrase activity in intracellular pH regulation and spicule formation are discussed.     

Subcellular localization of DNA polymerase gamma and changes in its activity in sea urchin embryos

1. Subcellular localization and changes in the activity of DNA polymerase gamma were examined in sea urchin eggs and embryos. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopycnic centrifugation. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. The gamma-activity per mitochondrial DNA remained constant during embryogenesis. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase gamma) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.     

Developmental changes of chromatin nonhistone proteins of sea urchin embryos

Summary The developmental changes in the pattern of chromatin nonhistone proteins have been investigated. The main feature of the observed changes was not the introduction of new or the disappearence of earlier existing species, but the stage-specific alterations in the rate of biosynthesis of the nonhistone protein constituents.     

Immunofluorescence studies of developmental changes in sea urchin eggs and embryos

By means of indirect immunofluorescence techniques, distinct sea urchin antigens were localized in eggs and embryos (Paracentrotus lividus). The specificity of the method was ascertained from controls in which the specific rabbit anti-sea-urchin sera were substituted by rabbit antiserum to an unrelated antigen (human serum albumin), by normal rabbit serum or by phosphate-buffered saline. The specificity of staining was also evaluated by comparing the different staining patterns obtained either with antisera to whole homogenates of eggs and embryos or with antisera to distinct antigens.     

Commitment to vegetalized development in sea urchin embryos

Summary Strongylocentrotus purpuratus embryos were reared in 0.025 M LiCl, which causes commitment to vegetalized development within 5 h after treatment begun at fertilization. Treated and control embryos were labelled with³⁵S-methionine for 3 h intervals from 2–14 h, solubilized, and subjected to 2-dimensional polyacrylamide gel electrophoresis. Comparison of autoradiographs of the gels, in which over 400 proteins can be detected, indicate that while LiCl treatment causes a short delay in the initiation or cessation of synthesis of a few proteins, no qualitative or major quantitative differences can be detected between control and treated embryos. Normal gastrulae and vegetalized exogastrulae labelled 38 h after fertilization have several differences in patterns of protein synthesis. We conclude that the early determinative events involved in vegetalization are not reflected in detectable differences in the pattern of protein synthesis.     

Replication timing: histone genes replicate during early S phase in cleavage-stage embryos of sea urchin.

Newly synthesized DNA was separated from the bulk of the DNA by pulse-labeling with BUdR and centrifugation in an alkaline CsCl buoyant density gradient. The content of histone gene in the newly synthesized DNA was determined by DNA dot hybridization. The gene contents in DNA replicated during the early half of S phase and during the whole S phase were compared. Results showed that histone genes were replicated during the first half of the S phase in embryos in the early cleavage stage.     

Exposure to ultraviolet radiation causes proteomic changes in embryos of the purple sea urchin, Strongylocentrotus purpuratus

We performed experiments to determine how environmentally relevant ultraviolet radiation (UVR) affects protein expression during early development in the sea urchin, Strongylocentrotus purpuratus. To model the protein-mediated cell cycle response to UV-irradiation, six batches of embryos were exposed to UVR, monitored for both delays in the first mitotic division and changes in the proteome at two specific developmental time points. Embryos were exposed to or protected from artificial UVR (11.5 W/m²) for 25 or 60 min. These levels of UVR are within the range we have measured in coastal waters between 0.5 and 2 m. Embryos treated with UVR for 60 min cleaved an average of 23.2 min (± 1.92 s.e.m.) after UV-protected embryos. Differential protein spot migration between UV-protected and UV-treated embryos was examined at 30 and 90 min post-fertilization using two-dimensional SDS-PAGE (2D GE). A total of 1306 protein spots were detected in all gels, including differences in 171 protein spots (13% of the detected proteome) in UV-treated embryos at 30 min post-fertilization and 187 spots (14%) at 90 min post-fertilization (2-way ANOVA, P = 0.03, n = 6). The majority of the proteins affected by UVR were subsequently identified using matrix assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS). Our results indicate UVR affects proteins from multiple cellular pathways and indicate that the mechanisms involved in UV-stress and UV-induced developmental delay in sea urchin embryos are integrated among multiple pathways for cellular stress, protein turnover and translation, signal transduction, cytoskeletal dynamics, and general metabolism.     

Ultrastructure of collagen in sea urchin embryos

Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.     

Nuclear RNA in sea urchin embryos : I. Some characteristics of ribonucleoprotein complexes

Using cesium chloride gradient analysis, we have examined the kinetics of labeling and some physical properties of nuclear ribonucleoprotein complexes in sea urchins. Our results strongly indicate that these complexes are not artifacts of procedure but are definite members of chromatin that are readily distinguishable from cytoplasmic RNP complexes. The nuclear complexes can be separated into two major classes: (1) A class in which the protein to RNA ratio is at least 4:1; (2) a second class in which the protein to RNA ratio is much less. Using [³H]uridine, long-term labeling and short pulses of [³²P], we have studied the labeling kinetics of RNA in the two classes. The nuclear RNA which is associated to a high degree with protein turns over very rapidly; over 70% of the nuclear RNA labeled in a short pulse appears in this fraction, whereas the nuclear RNA synthesized in long-term pulses is present to a greater extent in complexes containing a much smaller proportion of protein. The difference in the rate of turnover of RNA in these two classes may be significant in the regulation of RNA processing in the nucleus.     

Nuclear RNA in sea urchin embryos. II. Absence of "cRNA".

Using published procedures for the isolation of “chromosomal RNA”, a small RNA with many of the reported characteristics of cRNA can be isolated from the chromatin of sea urchin embryos. This RNA is not a degradation product of tRNA as claimed by some workers nor is it likely to be derived from ribosomal RNA (rRNA). However, we provide evidence that this RNA is in fact a degradation product of larger nuclear RNA.     

Carbonic anhydrase activity in developing sea urchin embryos with special reference to calcification of spicules

Eggs and embryos of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus did not exhibit significant changes in carbonic anhydrase activity during early development. Acetazolamide inhibited enzyme activity in homogenates of embryos and inhibited the formation of calcified spicules in a culture of micromeres at concentrations between 40 and 100 μM. Acetazolamide allowed intact embryos to develop to quasi-normal plutei but inhibited calcium deposition in the spicules. It is suggested that carbonic anhydrase contributes to CaCO₃ deposition in the spicule.     

Biomineralization of the spicules of sea urchin embryos

The formation of calcareous skeletal elements by various echinoderms, especially sea urchins, offers a splendid opportunity to learn more about some processes involved in the formation of biominerals. The spicules of larvae of euechinoids have been the focus of considerable work, including their developmental origins. The spicules are composed of a single optical crystal of high magnesium calcite and variable amounts of amorphous calcium carbonate. Occluded within the spicule is a proteinaceous matrix, most of which is soluble; this matrix constitutes about 0.1% of the mass. The spicules are also enclosed by an extracellular matrix and are almost completely surrounded by cytoplasmic cords. The spicules are deposited by primary mesenchyme cells (PMCs), which accumulate calcium and secrete calcium carbonate. A number of proteins specific, or highly enriched, in PMCs, have been cloned and studied. Recent work supports the hypothesis that proteins found in the extracellular matrix of the spicule are important for biomineralization.