Individual regulation of the accumulation of H1 mRNA and core histone mRNAs in sea urchin embryos.

The relative cytoplasmic accumulation of the individual histone mRNAs in sea urchins was determined by gel analysis of 3H-labeled cytoplasmic RNA isolated from embryos of the early cleavage through the mesenchyme blastula stages. A number of separate determinations showed that H1 mRNA accumulates at a molar ratio of 0.5 or less compared with each of the H2 or H3 core histone mRNAs through approximately the first 12 h of embryonic development. After this time, the accumulation of H1 mRNA increases relative to the core histone mRNAs, and approximately equimolar amounts of the histone mRNAs are produced by about the 14-h stage. The equimolar synthesis of H1 mRNA appears to be transient, returning to 0.5-molar levels several hours later. The increase in H1 mRNA accumulation, relative to the core histone RNAs, is coincident with the transition from expression of the early (alpha) sea urchin histone gene set to the late histone genes. Since all five of the early histone genes occur in a 1:1 ratio within repeating units, the data suggest that the genes within a single repeat, or their immediate products, are individually regulated. Gel analysis of the proteins synthesized in vivo by embryos demonstrates that the pattern of synthesis of the histone proteins reflects the changing ratios of the histone mRNAs.

Persistent cytoplasmic location of a DNA polymerase beta in sea urchins during development.

A subcellular localization study of a low molecular weight DNA polymerase beta indicates that this enzyme, as well as a high molecular weight DNA polymerase alpha, is found in large quantities in the cytoplasm of Strongylocentrotus purpuratus eggs. The two enzyme activities are distinguished by DEAE-sievorptive chromatography and by their differential activities with activated DNA and oligo(dT)10 . poly(dA)200 primer-templates. Using an enucleation procedure, it is concluded that an extremely low proportion if any, of both polymerases is present in the egg nucleus. At blastula stage, a period of rapid cell proliferation, similar studies of DNA polymerase subcellular localization using two different methods of nuclear isolation indicate that the DNA polymerase beta remains largely cytoplasmic while the alpha enzyme is found to be predominantly nuclear. Since the results for the alpha enzyme agree with previous reports (Loeb, L.A. (1969) J. Biol. Chem. 244, 1672) and since one method of nuclear isolation, using hypotonic solutions, enables us to recover both DNA polymerase alpha and beta activities in isolated mouse L-cell nuclei, the enzyme quantitation of isolated sea urchin nuclei is considered accurate. Thus, although there is a translocation of the polymerase alpha from a cytoplasmic to nuclear site during early embryonic development, such a massive relocalization of the polymerase beta does not occur.

A potential DNA replication complex isolated from sea urchin embryos by renografin gradient centrifugation.

A complex containing deoxyribonucleic acid, protein and lipid has been isolated from the nuclei of the sea urchin Stronglocentrotus pupuratus by gentle lysis of nuclei and centrifugation of the lysate in a renografin gradient. The complex is similar in several respects to DNA-membrane complexes isolated by the sarkosyl M-band technique [1]. These include the following: 1, most of the cellular DNA is present in the complex but the bulk of the DNA may be removed by sonication or nuclease treatment; 2, nascent DNA is preferentially found in the complex; and 3, destruction of membrane/protein components removes DNA found in the complex.

Effects of training and visual distance on speechreading performance.

The purpose of this study was to investigate the effects of training on subjects' ability to speechread sentences at varying visual distances of 3-6 ft., 6-12 ft., and 12-18 ft. 15 college women who received training obtained higher sentence speechreading scores than the 15 untrained women. The emphasis placed on kinesthetic feedback may have assisted the trained subjects' performance; however, distance up to 18 ft. did not appear to affect subjects' ability to speechread sentences, regardless of training.

A low molecular weight DNA polymerase beta in the sea urchin Strongylocentrotus purpurantus. Partial purification, properties, and changes in development.

A low molecular weight DNA polymerase which sediments at 3.3 S on sucrose gradients has been purified from total cell homogenates of rapidly dividing embryos of the sea urchin Strongylocentrotus purpuratus. In the presence of 2 mM N-ethylmaleimide, it is the major polymerase activity in whole cell homogenates when assayed with an oligo(dT)10.poly(dA)200 template; a template which it uses about 200 times more efficiently than activated DNA. The requirement for N-ethylmaleimide exists only in crude cell fractions where it acts to inhibit a template digesting nuclease activity. The polymerase is highly stable if maintained in the presence of 20% glycerol, is completely dependent on added template, and shows no end addition activity. The physical and enzymatic properties of this enzyme clearly distinguish it from the DNA polymerase previously described by Loeb (Loeb, L. A. (1969) J. Biol. Chem. 244, 1672-1681) which sediments as a high moeluclar weight (5.6 to 6.6 S) enzyme and prefers the activated DNA template. In addition, these two DNA polymerase enzymes show distinctive chromatographic properties using DEAE-cellulose and phosphocellulose columns as well as their sensitivity to N-ethylmaleimide. The properties of the low molecular weight polymerase indicate close similarity to the beta-polymerase isolated from mammalian cells. These low molecular weight enzymes are both sensitive to phosphate salt and able to utilize the artificial ribohomopolymer template oligo(dT)10.poly(rA)200. A quantitative analysis of the low molecular weight DNA polymerase during early embryonic development indicates that the activity of this enzyme increases at least 2-fold immediately following fertilization and again during early blastula stage (hatching). Such quantitative changes in a beta enzyme activity are in contrast to findings with the alpha-polymerase which remains constant during early development.

Distribution of messenger ribonucleic acid in polysomes and nonpolysomal particles of sea urchin embryos: translational control of actin synthesis.

We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.

Utilization of maternal and embryonic histone RNA in early sea urchin development.

Histone RNA in early sea urchin embryos is derived from maternal stores and from new transcription. We show that the sedimentation of maternal free RNPs, containing histone RNA, is somewhat more rapid than the sedimentation of the newly made histone RNPs. Yet, prior to the 2- to 4-cell stage, both the maternally derived and the newly synthesized histone RNA are localized to the same extent in the non-polysomal-free RNPs, and the timing of their recruitment into embryonic polysomes appears to be the same. The levels of hybridization of histone probe to RNAs in cleaving embryos increases severalfold in intensity, and the increase occurs primarily in the polysomes. These data suggest that new transcription may provide an important contribution to the total histone RNA mass by as early as the 32- to 64-cell stage of development.

Pressure-induced dissociation of sedimenting ribosomes: effect on sedimentation patterns.

Anomalous sedimentation patterns arise when free ribosomes from sea urchin eggs are centrifuged at high speeds. Pressure-induced dissociation of the ribosomes during sedimentation can explain the peculiar behavior; the assumption of such dissociation also yields estimates of the equilibrium constant (as a function of KCl concentration) and the change in molecular volume (500 +/- 100 ml/mol) in the reaction: subunits right arrow over left arrow ribosome. Such dissociation during centrifugation may explain many experiments in which apparent reduced sedimentation coefficients for ribosomes, and increased coefficients for the subunits, have been ascribed to conformational changes.

Nonrandom distribution of histone mRNAs into polysomes and nonpolysomal ribonucleoprotein particles in sea urchin embryos.

In the early sea urchin embryo, newly synthesized cytoplasmic histone mRNA is found both on polysomes and free of ribosomes as nonpolysomal messenger ribonucleoprotein particles (free RNPs). The distribution of newly synthesized histone mRNAs between translating and nontranslating compartments is nonrandom and dependent on the developmental stage. Gel electrophoresis and autoradiography of polysomal and free RNP RNA from embryos at various stages show that (i) the fraction of total newly synthesized histone mRNA that is in polysomes is greater than the fraction of total newly synthesized poly(A)+mRNA that is in polysomes, at all stages examined, and (ii) among the five histone mRNAs. H1 mRNA and H4 mRNA are relatively more enriched in the free RNPs than are the mRNAs for H2A, H2B, and H3. These data suggest that histone mRNA, as a class, is more efficiently utilized as a template than the average mRNA and, of the cytoplasmic histone mRNAs, the mRNAs for histones H2A, H2B, and possibly H3 are selected more frequently for translation than those for H1 and H4. Cell-free translations of polysomal and free RNP RNAs yield different ratios of in vitro histone products, consistent with the RNA distribution data. To test the possibility that the in vivo distribution of the histone mRNAs is the consequence of different intrinsic initiation capabilities of the individual mRNAs, ribosome-binding assays were carried out and unequal binding abilities of the histone mRNAs in the reticulocyte lysate were shown. A translational level component in the regulation of histone synthesis in the sea urchin embryo is indicated.

Association of 7 SL RNA and an SRP-like particle with polysomes and endoplasmic reticulum in the developing sea urchin embryo.

We have identified the sea urchin cognate of the mammalian signal recognition particle (SRP). This particle contains the diagnostic 7 SL small RNA, sediments at a similar velocity to that reported for the mammalian particle, and is found associated with the ER and polysomes. We have examined its subcellular localization during embryogenesis in order to determine whether it could serve in a translational regulatory capacity for a subset of the stored maternal mRNAs. In these studies the 7 SL RNA was used as a marker for the particle, since we determined that the 7 SL RNA exists exclusively within the SRP-like particle at all developmental stages. The relative distribution of the SRP among cytoplasmic structures changes dramatically during development. This represents an actual change in subcellular localization because the 7 SL RNA level remains nearly constant per embryo until the pluteus stage, when it increases slightly. In eggs, the SRP exists almost entirely free in the cytoplasm as an 11 S particle. Very soon after fertilization and throughout development there is an increase in the association of the particle with rapidly sedimenting structures, until by the pluteus stage greater than 90% of the SRP exists in a bound state. The nature of the associations is complex, and the bound structures include, at least in part, ribosomes, polysomes, and microsomes. The SRP is associated with microsomal membranes in gastrula (36 hr) but not in blastula (12 hr) or earlier embryos. Using the criteria of sensitivity to Triton X-100, we determined that 16% of the SRP in a 10,000g cytoplasmic fraction was bound to membranes in a microsomal (endoplasmic reticulum)-containing fraction in the gastrula. In contrast, less than 1% was membrane associated in the blastula. The SRP was also found in a ribosome-polysome fraction in 12-, 36-, and 48-hr embryos, but not in eggs. Finally, a small but significant portion of the SRP was found associated with monosomes in cleavage stage embryos. The possible role the SRP could play in the elongation arrest of stored maternal messages for secreted proteins is discussed.

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.

Polyubiquitin RNA characteristics and conditional induction in sea urchin embryos.

A cDNA of the sea urchin Strongylocentrotus purpuratus was identified as encoding polyubiquitin and used to detect a single gene with transcripts containing multiple ubiquitin coding units. Polyubiquitin transcripts exist as a 3.2-kb RNA in polyribosomes and as three higher molecular weight RNAs in purified nuclei. The amount of polyubiquitin RNA is essentially constant at 10(4) -10(5) transcripts per embryo during the egg-to-blastula period and then declines during further development. Heat shock elicits a transient increase in the level of polyubiquitin RNA, while Zn(II) ions induce a sustained accumulation, that is influenced by developmental parameters: One round of Zn(II) induction elicits the accumulation of the nuclear 7.6- and 5.6-kb RNAs, as well as the 3.2-kb polysomal RNA; however, a second round of induction yields only the 5.6- and 3.2-kb RNAs, suggestive of a change in pre-mRNA size or processing. Polyubiquitin RNA is expressed equally in ectodermal and mesoendodermal tissues and is induced in both tissue fractions by treatment of pluteus larvae with Zn(II). However, in isolated and cultured tissue fractions, polyubiquitin RNA is not inducible by Zn(II), in contrast to the full inducibility of metallothionein mRNAs. Polyubiquitin RNA induction thus appears to be conditioned by the integrity of the embryo, as well as by previous exposure to inducer.

Mitotic abnormalities in sea urchin embryos exposed to Dactinomycin.

Dactinomycin at concentrations of 10, 20, 25, and 50 mug/ml causes mitotic abnormalities in sea urchin embryos. Sister chromosome separation is impaired and anaphase bridges are frequently formed. The result is an unequal distribution of the chromosome complement to daughter cells. Possible explanations are discussed.