In this study, the reproduction of the mitotic centers in the eggs of a sea urchin, Strongylocentrotus purpuratus and a sand dollar Dendraster excentricus has been studied by means of experimental designs that do not depend on the actual visualization of centrioles. The centers are defined in operational terms as potential poles. Blockage of mitosis by mercaptoethanol, it was found, inhibits the duplication of the centers, but does not inhibit the splitting and separation of centers that have already duplicated and thus potential poles could be realized as actual poles in multipolar divisions. At all times, the center is at least a duplex structure; that is, it contains two potential poles. The actual duplication process is the earliest event in a given mitotic cycle, taking place at very early interphase or in late telophase of the previous division. The splitting of the centers following duplication is a distinct process, dissociable from the duplication as such. Duplication and splitting normally occur at about the same time in the mitotic cycle, with a precession of the former. That is, as the two members of a pair of "old" centers split, each one gives rise to a new one, which remains associated with it until the next phase of splitting and duplication occurs. The results are consistent with what is termed a "generative" model of the self-reproduction of an intracellular body. According to this, the body does not immediately produce a full-fledged copy of itself, with simultaneous fission, but the primary duplication event involves only a part of the parent structure. This gives rise to a "germ" or "seed" which then grows to be equivalent to the parent body, and finally splits from it.

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1. The patterns of incorporation of radioactivity from d-[6-(3)H]-, d-[1-(14)C]-, d-[U-(14)C]- and d-[6-(14)C]-glucose and [U-(14)C]myoinositol into the neutral sugars and uronic acids of the polysaccharides synthesized in different regions of the root-tip of maize were determined. 2. The root-cap tissue synthesized a slime in which a polysaccharide that contained a high proportion of fucose (32%) and galactose (21%) was found. This polysaccharide is synthesized only by the root-cap cells, and very little polysaccharide containing fucose is synthesized in adjacent tissues. Part of the meristematic tissue of the root is surrounded by the cap cells. A section of the root that contains both these tissues can be analysed, and the polysaccharide synthesized by the meristematic region can be obtained since the contribution of the root-cap cells can be found by the amount of fucose formed. 3. It was shown that there is very little difference in the polysaccharide synthesis of the meristematic region from that of the cells immediately behind it. In the more mature cells, however, the amount of xylose synthesized relative to that of arabinose is increased, and the proportion of xylose and arabinose formed in the matrix polysaccharides is increased whereas that of galactose is decreased. 4. The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on polysaccharide synthesis was to bring about a decrease in the relative amount of galactose synthesized in the matrix polysaccharides of cells immediately adjacent to the meristematic region and also in the more mature tissue. The growth factor also increased the amount of xylose synthesized relative to that of arabinose in the more mature tissue. These metabolic effects were related to a very obvious change in the morphological appearance of the root-tips. 5. Radioactivity from [U-(14)C]myoinositol was incorporated mainly into xylose, arabinose and galacturonic acid rather than into the hexoses, although small amounts of these sugars were formed.

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The mitotic figures in dividing cells of sea urchin embryos, from first division to the onset of cilia formation, were studied with regard to the filament system and its relation to kinetochores, chromosomes, and poles, as well as to fixation conditions which would best preserve these structures. With regard to fixation, variations in the salt concentration and pH of the fixative indicated that an extraction effect on the chromosomes noted in earlier work was probably due to a combination of neutral pH and salt concentration equivalent to sea water. The presence of the 15 m# filaments depended on the presence of either of two stabilizing conditions: pH 6.1 or presence of the salts of sea water, presumably the divalent cations of Ca and Mg. Kinetochores and centrioles were unaffected by the fixative variations. The 15 m# filaments, reported earlier in the central spindle, are also found in great numbers in the asters of early cleavage divisions. However, with successive divisions and reduction in cell size, the aster disappears at about the 32 to 64 cell stage, and the 15 m~ filaments are entirely associated with the central spindle. This disappearance of the aster suggests that it may be, in fact, merely a specialization of large cells for cytokinesis.

Eggs of the sea urchin Strongy1ocentrotus purpuratus were examined by indirect immunofluorescence microscopy for tubulin-containing structures at intervals from fertilization through first cleavage . The staining revealed that the monaster is made up not only of the sperm aster but also of tubulin-staining fibers originating elsewhere in the egg . The monaster does not divide directly but is broken down first before the amphiaster or interphase asters begin to form . The interphase asters reach a peak of development at the streak stage and are in turn broken down before the formation of the mitotic apparatus . The breakdown of the monaster, interphase asters, as well as the asters of the mitotic apparatus proceeds from the cell center or aster centers to the periphery of the cell and is followed by growth of new asters, also proceeding outward from the aster centers . The pattern suggests a transient wavelike movement of some condition, or factor, which favors microtubule depolymerization . KEY WORDS microtubules sea urchin eggs indirect immunofluorescence " fertilization mitosisAlthough the study of fertilization and early cleavage of sea urchin eggs has a history of well over a hundred years, the application of new or improved techniques often warrants a reexamination of these much-studied events . Recent development of indirect immunofluorescence microscopy has provided a useful means of visualizing tubulin-containing structures in whole cells (for review see reference 15) . Although most of the studies on mitosis using this technique have been carried out on relatively flat tissue culture cells (1, 2, 15), antitubulin staining has recently been used successfully to study the meiotic divisions of the larger but clear mammalian egg (14) . It thus seemed worthwhile to apply the method to the rapidly dividing and highly synchronous fertilized sea urchin eggs, about which a great deal is already known concerning the cellular ultrastructure and the biochemistry of the cell cycle .The disadvantage of cell thickness and yolk content resulting in lowered resolution was more than compensated for by the visualization of tubulin distribution in the whole cell-a picture that would have been almost impossible to obtain by serial sectioning of individual cells for electron microscopy . In an initial report (8), we have de-J . CELL BIOLOGY

The fine structure of cells at different stages of the mitotic cycle was studied in the blastomeres of 6-hour-old embryos of the sea urchin Strongylocentrotus purpuratus. The material was fixed in 1 per cent osmium tetroxide in sea water, buffered with veronal-acetate to pH 7.5, embedded in Araldite, and sectioned with glass knives. The aster, as it forms around the centriole, has the appearance of the endoplasmic reticulum, with elements oriented radially from the centrosphere to the periphery of the cell. Anaphase structures described include the kinetochores, with bundles of fine filaments extending toward the centrioles, as well as continuous filaments passing between the chromosomes. Two cylindrical centrioles composed of parallel rods are present in each of the anaphase asters. At late anaphase, elements of the endoplasmic reticulum condense on the surface of the chromosomes to form a double membrane which already at this stage possesses pores or annuli. At telophase bundles of continuous filaments can be seen in the interzonal region. 3-hese filaments, as well as those associated with the chromosomes, have a diameter of approximately 15 m~t, and appear physically different from the astral structure.

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