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The sequence organization of the DNA of the mollusc Aplysia californica has been examined by a combination of techniques. Close-spaced interspersion of repetitive and single copy sequences occurs throughout the majority of the genome. Detailed examination of the DNA of this protostome reveals great similarities to the pattern observed in the two deuterostome organisms previously examined in detail in this laboratory, Xenopus laevis and Strongylocentrotus purpuratus. Labeled and unlabeled Aplysia DNA were prepared from developing embryos and sheared to a fragment length of 400 nucleotides. The kinetics of reassociation were studied by means of hydroxyapatite chromatography, single-strand-specific S1 nuclease, and optical methods of assay. Aplysia DNA of this fragment length contains at least five resolvable kinetic fractions. One classification of these fractions, listed with their reassociation rate constants (l M-1 sec-1) is: single copy (0.00057), slow (0.047), fast (2.58), very fast (4000), and foldback (greater than 10(5)). Sequence arrangement was deduced from: the kinetics of reassociation of DNA fragments of length 400 or 2000 nucleotides; the hyperchromicity of reassociated fragments containing duplex regions; the size of duplex regions resistant to S1 nuclease; and the reassociation of labeled fragments of various lengths with short driver fragments. More than 80% of the single copy DNA sequences are interspersed with repetitive sequences. The maximum spacing of the repeats is about 2000 nucleotides, and the average less than 1000. The very fast fraction does not show interspersion with single copy sequences or with other kinetic fractions. The foldback fraction sequences are fairly widely interspersed. The slow fraction sequences are interspersed with the fast fraction, and possibly also with the single copy DNA. The fast fraction is the dominant interspersed repetitive fraction. Its sequences are adjacent to the great majority of the single copy sequences and have an average length of about 300 nucleotides.

The separation and purification of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis is described. Physical studies were used to compare this histone H1 molecule with H1 histones from other species. C.d. and 270 MHz n.m.r. spectroscopy indicate that, despite significant compositional differences from other sea-urchin sperm H1 histones, their secondary and tertiary structures are very similar. A large difference in helicity was, however, found between S. granularis histone H1 and calf thymus histone H1, and their n.m.r. and fluorescence spectra also differ considerably. It is concluded that secondary structure and tertiary structure have not been conserved in the evolution of the H1 histone family.

We have examined the poly(A)- and poly(A)+ polyribosomal (Ps) RNAs of developing sea urchin embryos to see whether they contain the capped structures previously observed in the poly(A)+ cytoplasmic RNA (mRNA) of a variety of eucaryotic organisms. We have found that both classes of Ps RNA contain 7-methylguanosine linked to the RNA by a 5'-5' triphosphate bridge. Capped structures derived from either class of Ps RNA appear to be of the type m7G5'ppp5'NmpNp, with only a single sugar-methylated nucleoside per cap, and we have estimated that both classes of Ps RNA are capped to the same extent. The 5' terminal 2'-0-methylated nucleosides have been identified and quantitated. The relative amounts are Am = 78%, Gm = 14%, Cm = 5%, Um = 3% for poly(A)- Ps RNA, and Am = 78%, Gm = 15%, Cm = 5%, Um = 2% for poly (A)+ Ps RNA. In addition, both classes of Ps RNA contain 2-4 internal residues of N6-methyladenylic acid per molecule.

Structural changes of histones H2B in aqueous media were studied under conditions of varying pH and ionic strength. The techniques involved intrinsic fluorescence of tyrosine residues, extrinsic fluorescence using the hydrophobic probe 8-anilinonaphthalene sulfonate and fluorescence polarization. Secondary structure predictive methods were also used to study potential effects of primary mutations. In spite of many primary structure changes, the predicted effect on a globular structure of H2B is minimal. The results indicate the formation of a major hydrophobic region, the formation of helices and the burial of tyrosine residue 83 (calf), the structural changes of which appear to be identical in all H2B's as ionic strength is increased. The effect of primary structure changes on protein-protein interaction where H2B's are concerned is likely to be negligible, whereas the major effect is likely to be the protein-DNA interaction where H2B could be at least partially responsible for differentiation.

Interspersed repetitive sequences were isolated from sea urchin DNA by renaturing to low Cot followed by treatment with nuclease SI. Synthetic Eco RI sites were ligated onto the repetitive sequence elements, which were then inserted at the Eco RI site of plasmid RSF2124 and cloned. The repetitive sequences can be excised from the plasmid with Eco RI for further study.

Two different methods were used to isolate high-molecular-weight heavy satellite DNA from the spermatozoa of the sea-urchin Lytechinus variegratus. Purification of satellite rDNA (ribosomal DNA) by selective denaturation of the spermatozoal DNA followed by a separation of the native and denatured DNA in an aqueous mixture of poly(ethylene glycol)/dextran is affected by the molecular weight of the spermatozoal DNA. Enrichment of satellite rDNA by selective precipitation of main-band DNA by poly-L-lysine is cumbersome but more suitable.

Yolk platelets, a major organelle of sea urchin eggs and embryos, were isolated from Arbacia punctulata and biochemically characterized over the course of development to the pluteus stage. Fractionation by sucrose gradient centrifugation revealed yolk platelets in two major density classes. The low-density yolk platelet fraction could be obtained as a very homogeneous preparation and was highly enriched in acid phosphatase activity, while depleted of mitochondrial (cytochrome c oxidase) and plasma membrane (phosphodiesterase) marker enzymes. The chemical composition of low-density yolk platelets prepared from eggs and embryos at various stages of development remained unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and protein. However, analysis of the major yolk platelet glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a number of stage-specific changes. These glycoproteins were found to be major glycoproteins of crude embryo lysates and were predominantly of the polymannose, N-linked type. The predominance of polymannose-type glycoproteins in yolk platelets was further demonstrated by their staining with concanavalin A-colloidal gold in Lowicryl-embedded sections of embryos. These studies represent the first systematic biochemical characterization of intact yolk platelets and the changes in them during early embryonic development.

alpha-Tubulin subunits from trout (S. gairdneri) sperm tails, sea urchin (S. purpuratus) cilia, protistan alga (C. elongatum ) flagella and rose (Paul's Scarlet) cytoplasm have been characterized by limited proteolytic cleavage with the enzyme Staphylococcus aureus protease and electrophoresis of the digestion products on SDS-PAGE. The resulting patterns corresponded to either of two major types representative of animal and non-animal alpha-tubulins, respectively. A total of 28 alpha-tubulins have now been characterized by this method. They are classified in this paper according to the type of cleavage pattern generated by the enzyme S. aureus protease. The implications of these results for metazoan evolution are discussed.

Arrangement of repetitive and single copy DNA sequences in the DNA of 8 Echinodermata species (sea urchins, starfishes and sea-cucumber) has been studied. Comparison of the reassociation kinetics of short and long DNA fragments assayed by hydroxyapatite binding indicates that the pattern of DNA sequence organization of all these species is similar to the so called Xenopus pattern found in genomes of most animals and plants. Interspecies differences consist mainly in the quantities of sequences of various repetition degrees and their interspersion with each other and with single copy sequences. Measurements of the size of S1 nuclease resistant reassociated repetitive sequences show variability in the relative quantities of long and short repetitive sequences of different species. Difference in the arrangement of single copy and repetitive sequences between Echinodermata species are not related to their evolutionary proximity.

Evidence is described which suggests that a large fraction of sea urchin embryo polyribosomal RNA is not polyadenylated, and that the proportion of poly(A)-containing RNA increases during embryological development. Cleavage, early blastula, and mesenchyme blastula stage embryos were treated with cordycepin, and the effects of the drug on polyribosome appearance were determined. Polyribosomes in cleaving embryos and early blastulae were unaffected by the drug. However, there was a significant reduction in the amount of polyribosomes present and in the amount of newly synthesized RNA entering polyribosomes in mesenchyme blastulae after cordycepin treatment. Polyribosomal RNA was isolated from cleavage, early blastula, and mesenchyme blastula stage embryos, and the RNA was characterized by sucrose gradient centrifugation. Cleaving embryo polyribosomal RNA has a prominent 9 S peak and a smaller 20 S peak, and the larger RNA is heterodisperse. There is no poly(A) associated with the 9 S and 20 S material, but there is poly(A) associated with the RNA sedimenting faster than 20 S. Early blastula and mesenchyme blastula polyribosomal RNA have 22 S and 26 S peaks, respectively. This later stage RNA is quite heterodisperse and poly(A) is associated with RNA in all size classes larger than 9 S. Finally, the proportion of polyadenylated light and heavy polyribosome RNA was determined for cleavage, early blastula, and mesenchyme blastula stage embryos. Approximately 20-25% of newly synthesized light polyribosome RNA is polyadenylated and this amount is relatively constant during early development. However, the proportion of newly synthesized RNA in heavy polyribosomes that is polyadenylated increases during development: 25% at cleavage, 33% at early blastula, and 48% at mesenchyme blastula stages.

When DNA isolated from freshly collected sperm of sea urchin (Lytechinus variegatus) is centrifuged to equilibrium in CsCl, 2 heavy satellite bands appear beside the main band DNA. Satellite DNA (II) appears in between the main band DNA (rho = 1.695 g/cm3) and the rDNA satellite (rho = 1.722 g/cm3). Satellite DNA (II) has a buoyant density 1.710 g/cm3, corresponding to 50% GC content. It is speculated that the satellite DNA (II), which appears to be of high mol. wt, might contain the sequences complementary to histone mRNA.

A close relationship between histone synthesis and DNA replication has been suggested for many biological systems. The presence of histonic proteins has been demonstrated in practically all kinds of eukaryotic cells. During the clevage period of sea urchins it has been postulated that histones are not present in nuclei, and that only at blastula stage they do associate with DNA. Our results suggest that the failure to isolate histonic proteins from nuclei derived from cleavage cells might be caused by the presence in those cells of proteases activated by NaHSO3. This compound has being widely used to inhibit proteolytic action in other biological systems. By changing the method for chromatin isolation, we have been able to isolate basic proteins from nuclei of gametes, zygotes and 2-4 blastomeres. These proteins behave like calf thymus histones in urea-acetic acid polyacrylamide gels, and they do not show great differences from proteins isolated from nuclei of blastula, gastrula, prism, and pluteus. The electrophoretic patterns of basic proteins obtained from eggs and zygotes are practically identical, except for one protein moving like lysine-rich calf thymus histones. This protein appears in zygote nuclei at the beginning of the first replication wave.

The tridentate pedicellaria of Salmacis bicolor consists of calcareous head and stalk, connected by a muscular neck regions. Head, neck and stalk are covered by a thin cuticle, which is non-mineralized and soft. Both the cuticle and the organic fraction of the calcareous region are resistant to acids and alkalis. Histochemical studies reveal that both the cuticle and the organic matrix of the calcified head and stalk contain protein, polysaccharide and lipids. The lipid content of the organic fraction of the mineralized matrix recalls the spicules of Pluteus larva reported in an earlier study. The protein component of the cuticle and that of the organic matrix slightly differ in that the former is rich in aromatic amino acids, namely tyrosine and tryptophane. Both cuticle protein and the protein of the organic matrix lack sulphur containing aminoacids. Another feature of similarity between the cuticle and the organic matrix is that both contain acid mucopolysaccharide and neutral mucopolysaccharide components. The role of acid mucopolysaccharide in calcification is discussed. The neutral mucopolysaccharide has been suggested to be similar to chitin, and its significance is discussed. Unlike the skeletal elements of other invertebrates, the cuticle and the organic matrix of pedicellariae are devoid of any phenolic compounds.

The specific RNA-binding activity of cytoplasmic extracts of a number of eukaryotic cells and tissues was determined by sorption of radioactive RNA on nitrocellulose filters. This activity varies within a wide range: from 0,5 up to 80 microgram of 23S-RNA per mg of extract protein. The percentage of RNA-binding proteins as measured by their adsorption on RNA-Sepharose is 0,3--60% of total protein of cytoplasmic extracts and is well-correlated with their RNA-binding activity. The variability of the RNA-binding activity of the isolated preparations of RNA-binding proteins (from 100 to 300 microgram of 23S-RNA per mg of protein) is indicative of their qualitative differences. The analysis of the polypeptide composition of RNA-binding proteins revealed that the set of the major polypeptide chains is rather simple (with few exceptions) and includes both universal components with mol. weights of about 36,000, 49,000 and 95,000 characteristic of a number of cells and tissues and tissue- and species-specific polypeptides. Upon differentiation of rabbit reticulocytes into erythrocytes the percentage of RNA-binding proteins is decreased 4-fold and one of the three main polypeptides with the mol. weight of about 95,000 disappears. After fecundation of sea urchin ovicelles the polypeptide with the mol. weight of 37,000 disappears from the preparations of RNA-binding proteins and at the morula stage two new components with the mol. weights of about 35,000 and 26,000 appear instead of it. The germination of wheat embryos results in a two-fold increase of the percentage of RNA-binding proteins without any essential changes of the set of main polypeptide chains.

The complete amino acid sequence (125 residues) of sea urchin histone H2A has been established by structural studies of peptides derived from tryptic and chymotryptic cleavage of the maleylated protein and from thermolysin cleavage of the intact protein. By comparison with calf homologous histone, the basic amino-terminal and carboxy-terminal parts of the protein show 11 substitutions and 4 deletions. The remainder of the sequence, mostly hydrophobic, is almost completely unchanged.

Tryptic digestion of histone H1 from the sperm of the sea urchin Sphaerechinus granularis leaves a limiting peptide of approx. 80 residues that is of similar size to the limit peptide from calf thymus H1 or chicken erythrocyte H5. The S. granularis limit peptide folds to form tertiary structure similar to that of the intact parent histone H1 (shown by n.m.r. spectra), but the helical content is decreased by the digestion from 64 residues to 28. In contrast, intact calf thymus H1 and chicken erythrocyte H5 histones have only about 28 helical residues, which are preserved in their limit peptides. The extra helix in S. granularis is shown to be rapidly digested away by trypsin, and its location in histone H1 is discussed. A possible relationship of this structural feature to the length of linker DNA is proposed.

Changes in the content and composition of lipids in the gonads of the Strongylocentrotus nudus females during the annual cycle of their development were studied. The qualitative composition of lipids was shown not to change. There were found cerebrosides, sulfatides, free fatty acids, diphosphatidyl glycerine, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl inositol, phosphatidyl serine, lysophosphatidyl choline, lysophosphytidyl ethanolamine, phosphatide acid, sphingomyelin and non-identified phospholipids X1 and X2 in the gonads. The relative content of individual classes of phospholipids suffers marked changes. The similarity in the content of total lipids and their individual classes at the 2nd and 5th stages confirms the existence of two generations of oocytes (Gnezdilova, 1971). The relationship between the lipid composition and the morphological state of the gonads with respect to the stage of development is discussed.

In this report the coisolation of two proteins from sea urchin smooth muscle of apparent molecular weights (Mr) 54 and 56 kD respectively, as determined on SDS-PAGE, is described. Like the intermediate filament proteins, these two proteins are insoluble in high ionic strength buffer solution. On two-dimensional gel electrophoresis and by immunological methods it is shown that these proteins are not related (by these criteria) to rat smooth muscle desmin (54 kD) or vimentin (56 kD). Furthermore, in conditions where both desmin and vimentin assemble in vitro into 10 nm filaments, the sea urchin smooth muscle proteins do not assemble into filaments. Ultrastructural studies on the sea urchin smooth muscle cell show that the thin and thick filaments organization resembles that described in the vertebrate smooth muscle. However, instead of 10 nm filaments, a network of filaments, 2-4 nm in diameter, is revealed, upon removal of the thin and thick filaments by 0.6 M KCl treatment. By indirect immunofluorescence microscopy, and in particular by immunocytochemical electron microscopy studies on the sea urchin smooth muscle cell, it is shown that the antibodies raised against both 54 and 56 kD proteins appear to specifically label these 2-4 nm filaments. These findings indicate that both the 54 and 56 kD proteins might be constituents of this category of filaments. The possible significance of this new cytoskeletal element, that we have named echinonematin filaments, is discussed.

The observation that repetitive and single copy sequences are interspersed in animal DNAs has suggested that repetitive sequences are adjacent to single copy structural gene sequences. To test this concept, single copy DNA sequences contiguous to interspersed repetitive sequences were prepared from sea urchin DNA by hydroxyapatite fractionation (repeat-contiguous DNA fraction). These single copy sequences included about one third of the total nonrepetitive sequence in the genome as determined by the amounts recovered during the hydroxyapatite fractionation and by reassociation kinetics. 3H-labeled mRNA from sea urchin gastrula was prepared by puromycin release from polysomes and used in DNA-driven hybridization reactions. The kinetics of mRNA hybridization reactions with excess whole DNA were carefully measured, and the rate of hybridization was found to be 3-5 times slower than the corresponding single copy DNA driver reassociation rate. The mRNA hybridized with excess repeat-contiguous DNA with similar kinetics relative to the driver DNA. At completion 80 percent of that mRNA hybridizable with whole DNA (approximately 65 percent) had reacted with the repeat-contiguous DNA fraction (50 percent). This result shows that 80-100 percent of the mRNA molecules present in sea urchin embryos are transcribed from single copy DNA sequences adjacent to interspersed repetitive sequences in the genome.

A heat labile toxic protein of 25,000 molecular weight was isolated (LD50 0.85 mg/kg. i.p. injection into mice) from the pedicellariae of the sea urchin Tripneustes gratilla. Pretreatment of the mice with antihistaminics did not prevent the toxic effects.

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.

The 125 residues of the histone H2A from the sperm cells of the sea urchin Parechinus angulosus have been positioned. The N terminus is blocked by an acetyl group. Compared to the bovine histone, the sea urchin protein differs in 14 positions.

Calmodulin was purified to apparent homogeneity from sea urchin spermatozoa by heat-treatment at 85 degrees C, ammonium sulphate precipitation at pH 4.2, DEAE-Sephacel chromatography and gel filtration on Sephadex G-100. Approximately 8.3 micrograms calmodulin were recovered per 10(10) sperm cells. The sperm calmodulin had an apparent molecular weight of 17 800. The purified calmodulin activated calmodulin-deficient phosphodiesterase from pig coronary arteries, with half-maximal activation occurring at approximately 40 ng calmodulin/ml. Trifluoperazine also inhibited the sperm calmodulin activity. These results demonstrate that calmodulin is present in high amounts in sea urchin spermatozoa, and that it is essentially the same as the calmodulin isolated from various other tissues.

Total histones from the sperms and embryos of the sea urchins Paracentrotus lividus, Arbacia lixula, Psammechinus microtuberculatus and Sphaerechinus granularis hae been fractionated into the component molecules by electrophoretic analyses in SDS, in urea-acetic acid and in Triton-urea-acetic acid. Sperm H2B histones are in all cases different from those of the corresponding embryonic chromatins. Each sea urchin species has distinctive variants of the sperm H2B histones that are fractionated by electrophoresis in SDS acrylamide gel into two to four components forming a new class of lower mobility. This analytical method shows that individuals of the same species have different assortments of the H2B components. Electrophoretic analyses in Triton-urea also show multiple components for H2B but the patterns are similar in the different individuals.

1. An analysis was made of lipids extracted from the spermatozoa of the sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina. 2. Nearly all the lipids from both species consisted of phospholipids (about 80%) and cholesterol (about 14%). Triglyceride and cholesterol ester were present in trace amounts. 3. The fatty acid composition of each phospholipid was determined by gas-liquid chromatography. In both species, the fatty acid consisting of phosphatidylethanolamine was of the unsaturated type for the most part, while cardiolipin was comprised to a considerable degree of saturated fatty acids. In phosphatidylcholine and phosphatidylserine from H. pulcherrimus sperm, unsaturated fatty acid content was somewhat higher than that in phospholipids from A. crassispina sperm.

The histone 2A proteins of the sea urchin Strongylocentrotus purpuratus are compared with those of the mouse. While the major H2As in these two organisms do not comigrate on two-dimensional gels, the sea urchin contains a protein that comigrates with the minor histone 2A variant H2A.Z from mammals. H2A.Z is of particular interest because its sequence homology with other H2As is quite low, and it is not phosphorylated as are other H2As. A comparison of the tryptic peptide patterns of several H2As from sea urchin blastulae and mouse L1210 cells show that, while the patterns of the H2A.Zs differ greatly from the patterns of the other H2As, the patterns of the mouse and sea urchin H2A.Zs are very similar. Since the H2A.Zs have only one or two peptides in common with the other H2As, the conservation of their sequence indicates that H2A.Zs have evolved under somewhat different selective pressures from other H2As. Unlike all the other sea urchin H2As whose syntheses either turn on or off during early development, H2A.Z seems to be synthesized continuously throughout this period.U

We present an improved procedure for detecting poly A tracts in situ by hybridization of 3H poly U. Glutaraldehyde fixation achieves significantly higher retention of RNA and better morphologic preservation than does Carnoy's. A dramatic increase in signal to noise is obtained by prehybridization treatment of glutaraldehyde-fixed sections with proteinase K and acetic anhydride. Measurement of the increase in poly A concentration after fertilization by solution titration and by in situ hybridization are in excellent agreement indicating that in situ measurements yield accurate relative estimates of local RNA concentrations in sections. Examination of the grain density distribution in section of sea urchin eggs and cleaving embryos reveals no major cytoplasmic localization of poly A+ RNA, although nuclei show much less labelling and micromeres of 16-cell embryos have a small, but significant, reduction in poly A concentration.

The most abundant glycoprotein in the coelomic fluid of sea urchin Anthocidaris crassispina was purified and its subunit structure, molecular form in the native state, amino acid composition, and electron micrographic image were studied. The results showed that the protein in its native state was basically a tetramer with a total molecular weight of about 700,000, which was in equilibrium with a high molecular weight form corresponding to an octamer. The electron micrograph of the tetramer showed two ellipsoidal units aligned in parallel with a wide gap in between. The subunits all had the same molecular weight of 180,000 +/- 10,000 and were disulfide bonded in pairs. The carbohydrate content was about 16% with mannose and fucose as the two most abundant sugars. Although this protein accounted for 70% of the total protein in the coelomic fluid, it did not take part in the known activities of the fluid, namely hemagglutination and coagulation. Despite its structural similarity to the mammalian alpha-2-macroglobulin or reptilian and avian ovomacroglobulins it did not interact with bovine trypsin or chymotrypsin. This protein showed immunological cross reactivity with 23S glycoprotein purified from sea urchin eggs which, we believe, corresponds to the previously described 22-27S protein particles in eggs.

Two chromatin fractions were isolated from rat liver nuclei and from sperm of the sea urchin, Strongylocentrotus intermedius, by chromatin treatment with DNAase II with subsequent fractionation of the resultant fragments via their precipitation with MgCl2 solution. The analysis of distribution between these fractions of rapidly in vivo labeling RNA associated with chromatin and the in vitro investigation of template activity of isolated chromatin fractions allowed to conclude that one of the fractions is transcriptionally active and the other is inactive. The active chromatin fraction, similarly to the inactive one, contains all five histones. Unlike the latter, however, the former fraction is enriched in non-histone proteins and impoverished in histone H1. It is shown that the active chromation fraction of rat liver is composed of small fragments heterogenous in size. Rapidly synthesizing RNA in this fraction is mainly associated with mononucleosomal particles. Data on electrophoretic mobility of DNA fragments isolated from the active chromatin fraction from rat liver show that transcriptionally active sites of chromatin retain the nucleosomal structure.

A series of mono- and dinucleosomal DNAs characterized by an about ten-base periodicity in the size were revealed in the micrococcal nuclease digests of Drosophila chromatin which have 180 +/- 5 base pair (bp) nucleosomal repeat. 20, 30, and 40 bp spacers were found to be predominant in chromatin by trimming DNA in dinucleosomes to the core position. Among several identified mononucleosomes (MN), MN170, MN180 and MN190 were isolated from different sources (the figures indicate the DNA length in bp). The presence of the 10, 20, and 30 bp long spacers was shown in these mononucleosomes by crosslinking experiments. The interaction of histone H3 with the spacer in the Drosophila MN180 particle was also shown by the crosslinking /5/. We conclude from these results that the 10 n bp long intercore DNA (n = 2, 3 and 4) is organized by histone H3, in particular, and together with the core DNA forms a continuous superhelix. Taken together, these data suggest that Drosophila chromatin consists of the regularly aligned and tightly packed MN180, as a repeating unit, containing 10 and 20 bp spacers at the ends of 180 bp DNA. Within the asymmetric and randomly oriented in chromatin MN180, the cores occupy two alternative positions spaced by 10 bp.

The coelomocytes of the sea urchin, Strongylocentrotus droebachiensis, may be prevented from clotting with 50 mM ethylene glycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetate, 50 mM Tris-HCl, pH 7.8 and subsequently separated into various cell types on sucrose gradients. One cell type, the petaloid coelomocyte, spontaneously undergoes a striking morphological transformation to a form exhibiting numerous, t-in cytoplasmic projections (filopodia). Moreover, the transformation is reversible. Ultrastructurally, the formation of the filopodia results from a progressive reorganization of actin-containing filaments into bundles that are radially oriented. The formation of the filament bundles is initiated at the cell's periphery and proceeds inward. Simultaneously, the cytoplasm in between the bundles is withdrawn, exposing finger-like filopodia. Ultimately, the filopodia can be extended by up to four times their original length. Biochemically, actin is the most abundant protein in while cell homogenates and is extractable in milligram quantities via acetone powders. An actomyosin complex may also be isolated from these cells and is presumed to be active in producing the various forms of motility observed.

Rat liver and sea urchin embryo nuclear matrices were found to differ in composition and in the strength of the association of their structural elements. Apart from the qualitative differences in composition, the embryonic matrices retained greater amounts of nuclear proteins and DNA, and were less susceptible to ultrasonic treatment than those of rat liver. They were essentially resistant to mild sonication, by which the rat liver matrix structure was resolved into two distinct fractions, referred to by Berezney (1980) as matricin and ribonucleoprotein (RNP). Both sub-fractions exhibited a protein kinase activity; the phosphorylating capacity of the RNP-associated protein kinases was found to be higher than that of the matricin-bound enzyme. The preferred substrate was among the secondary matrix proteins. In sea urchin embryos, sonication introduced no change in the type and lesion of the matrix proteins phosphorylated by the associated enzyme.

Accessory cells of the sea-urchin ovary were studied histologically and histochemically. Different types of globules in these cells were distinguished. Histochemical observations suggest that the accessory cells synthesize neutral mucosubstances, sulfo- and sialopolysaccharides, which might be precursors for jelly formation.

From the Triton-treated cortex fraction of sea urchin eggs, a high molecular weight actin binding protein (260K protein) was solubilized by a high salt solution and purified. A cosedimentation assay revealed that the 260K protein binds to actin filaments in a concentration-dependent manner. The low-shear viscosity of actin solutions largely increased in a concentration-dependent manner after addition of 260K protein. Electron microscopy showed that this protein induces the formation of large curled bundles of actin filaments. Different from fascin-induced actin bundles, no clear striations were observed within the actin bundles formed by the 260K protein. Antibodies against the 260K protein were raised in a rabbit and affinity purified. Immunoblotting analysis of Triton-solubilized cortex and various subcellular fractions showed that first only a single band reacted with the antibody and second that the 260K protein exclusively localized in the cortex fraction. Indirect immunofluorescence microscopy localized the protein in the cortex and the region of the cleavage furrow. After double staining, the fluorescence images for actin filaments and the 260K protein well correlate with each other.