We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts . Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence . For comparison, lamins and histones were localized using human autoimmune antibodies . At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components . Antibody Pl stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus . Antibody 11 detected an antigen distributed as fine granules throughout the nuclear interior . Monoclonals PI1 and P12 stained both the nuclear periphery and interior, with some characteristic differences . During mitosis, P1 and 11 were chromosome-associated, whereas P11 and P12 dispersed in the cytoplasm . Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order . With antibody 11, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to 11 may be involved in chromatin/ chromosome higher-order organization throughout the cell cycle . Antibodies P11 and P12 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase . Antibody P12 also detected antigen associated with the spindle poles.The nuclear matrix is a complex biochemical fraction consisting of nonhistone nuclear proteins and small quantities of DNA and RNA . It is obtained by sequential extraction of isolated interphase nuclei with low-and high-salt buffers, detergents, and DNAse and RNAse . Structurally, the nuclear matrix comprises the peripheral nuclear pore complex-lamina, an internal fibrogranular network and residual nucleoli . Part of the matrix has been envisaged as an interphase "nuclear skeleton" on which nuclear functions such as DNA replication, RNA transcription and processing, virus replication, and hormone response can be ordered (see references 1-3 for reviews).Several studies have suggested that the nuclear matrix is involved in mitosis (4-7). Recent work (8, 9) has also indicated a potential role for the nuclear matrix in the organization of mitotic chromosomes per se. To examine the distribution of individual nuclear matrix polypeptides throughout the cell cycle, we used monoclonal antibodies against isolated THE JOURNAL OF CELL BIOLOGY " VOLUME 99 AUGUST 1984 661-671 © The Rockefeller University Press -0021-9525/84/08/0661/11 $1 .00 nuclear matrices in an immunofluorescence study of mouse 3T3 fibroblasts . MATERIALS AND METHODSCell Culture: Mouse 3T3 fibroblasts were cultured at 37°C with 5% C02 in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 100 U/ml penicillin, 100 Ag/ml streptomycin, and 0.25 lg/ml fungizone . Mouse myel...

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High frequencies of somatic embryogenesis evaluated on the basis of average embryo yield per explant were observed on leaf explants of Solanum melongena (L.) (eggplant) after 21 days of culture on a modified Murashige and Skoog medium. Callus proliferation and embryogenesis occurred in the presence of 1-naphthaleneacetic acid (NAA), whereas only callus induction occurred in the presence of the other auxins. Maximal embryo yields were obtained at 10 mg/L NAA. Although cytokinins inhibited the NAA-induced embryogenic response, they acted synergistically to promote callus growth. The frequency of embryogenesis on leaf explants was shown to be under the control of the nitrogen content of the medium. Both NH4+ and NO3− were essential for embryogenesis and an optimal ratio of 2:1 (NO3−:NH4+) was established. The optimal sucrose concentration of the medium was 0.06 M and both elevated and reduced sucrose levels inhibited embryogenesis. Seven cultivars showed significant quantitative differences in their capacities to form embryos, although all were embryogenic. Cell suspension cultures were also embryogenic, when grown under conditions which stimulated embryogenesis in leaf explants. Somatic embryos taken from leaf callus or cell suspensions and cultured on hormone-free medium gave rise to plants which set seed when transferred to the greenhouse. Shoot organogenesis was observed on leaf explants which were cultured in the presence of either of four cytokinins. Shoots were rooted on hormone-free medium and mature plants were subsequently obtained.

In studies of the structure and development of primary walls of plant cells, little detailed attention has been given to the non-cellulosic components of the wall. Indeed, as Bonner (6, p. drous calcium chloride for at least 18 hours. Hydrolyses were carried out by heating samples of 10 to 20 mg of the various fractions with 1 ml N hydrochloric acid in a sealed tube at 1000 C for 8 hours. Chromatograms were run by the descending method (17) using one of the following solvent systems: (A) pyridine ethyl acetate : water-i : 2 : 2 (11); (B) ethyl acetate acetic acid: water-3 1: 3 (11); (C) n-butanol : pyridine: water-6 4: 3 (10). Sugars were detected on the chromatograms by the p-anisidine hydrochloride spray reagent (10) and were chromatographically identified by running samples of known sugars on the same paper sheet. At least two solvent systems were used to establish this identification. Nitrogen was determined on 15 to 25 mg samples by the micro-Kjeldahl method and protein was taken as 6 times the micro-Kjeldahl value.The supernatant and washings from the ground coleoptiles were dialyzed in Visking cellophane tubing against fresh distilled water for three 24-hour periods. A precipitate which formed inside the dialysis tube was centrifuged, washed once with water and dried to give Fraction 1 (0.5323 g; N, 9.85 % = 59 % protein). After hydrolysis, chromatography (solvents A and C) revealed the presence of arabinose, xylose, glucose and galactose in the approximate ratios shown in table I.The supernatant liquor from Fraction 1 was concentrated to 1/20 its volume and ethanol was added until precipitation was complete. The precipitate was centrifuged and dried to give Fraction 2 (0.3166 g; N, 5.26 % = 32 % protein). Hydrolysis and chromatography (solvents A and C) showed the presence of arabinose, xylose, glucose and galactose (table I).The ethanolic mother liquors from Fraction 2 were evaporated to dryness and the residue was redissolved in ethanol. Addition of ether caused precipitation of Fraction 3 which was washed once with ether and dried (0.2360 g; N, 3.6 % = 22 % protein). Arabinose, xylose and glucose were detected chromatographically (solvents A and C) after hydrolysis. Evaporation of the supernatant liquor from Fraction 3 left no residue indicating that Fractions 1, 2 and 3 together represented all of the non-dialyzable material washed from the coleoptiles by water.The washed sediment from the original ground coleoptiles was dried in air, ground through a Wiley mill (20-mesh screen), and dried to constant weight at a pressure of 0.03 mm Hg over phosphorus pentoxide. For convenience, this dried product (Fraction 4; 4.8286 g after removal of 0.0255 g for N determination; N, 1.58 % = 9.5 % protein) was regarded as the total primary cell wall material to which Fractions 5 to 9 are referred in terms of percent by dry weight (see discussion and table I).Fraction 4 was extracted in a Soxhlet apparatus 283 www.plantphysiol.org on May 10, 2018 -Published by Downloaded from

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