No abstract

We have isolated and partially characterized subprotoplasts containing nuclei, i.e. miniprotoplasts, and enucleated subprotoplasts, i.e. cytoplasts, from freshly isolated protoplasts of cultured cells from Hyoscyamus muticus, Nicotiana tabacum and especially Zea mays. Protoplasts were fragmentated by centrifugation through discontinuous iso‐osmotic density gradients containing colloidal silaca gel (Percoll), calcium chloride and mannitol. Using this method metabolically active miniprotoplasts and highly purified cytoplast fractions with less than 4% contamination with nucleated protoplasts were obtained. The cytoplasts prepared by our method are suitable for use in fusion experiments aimed at transferring nuclear and cytoplasmic genetic information separately.

The development of thiolase in various rat tissues from 7 days before birth to adulthood was studied. Enzyme activities in brain, liver, and kidney during the perinatal period reflect the nutritional environment, whereas those in the adult tissues do not.

Continuous cultures, established 10 days after pollination from endosperms of inbred A636 Zea mays (L.) were extracted 21 months later with aqueous ethanol. The solubilized proteins were analyzed by polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Two protein bands co-migrated with zein, the major storage protein of maize. Immunoblotting of the gel followed by incubation of the immobilized proteins with anti-zein IgG provided evidence that the polypeptides were in fact zein. Electron microscopic studies showed that the cultures contained cells with protein bodies as found in developing endosperms. The protein bodies could be isolated from the cultures and were shown to contain zein. We conclude that the long term cultures described here synthesize zein and deposit it in the form of protein bodies of the type found in developing endosperms. Thus, certain endosperm characteristics and the production of tissue-specific proteins are retained in prolonged culture.Tissue cultures which express tissue-specific proteins are highly desirable for studying the regulatory mechanisms leading to the differential expression of these proteins. In higher plants such culture systems have, to our knowledge, not been described, mainly because few plant proteins have been studied for their tissue specificity. Among the better known ones are storage proteins of seeds accumulating in developing endosperms of cereal grains (14) or in cotyledons of legumes (17). Attempts to establish long term cultures of cereal endosperms have been described (9). The first report of a successful, continuous culture of maize endosperm appeared in 1949 (16). Several more reports on endosperm cultures followed some ofwhich have been shown to produce anthocyanin (7, 25) and starch (2). Production of storage proteins in such cultures has not been investigated. Zein, which constitutes more than 50% of the seed storage proteins in maize, is synthesized on membrane-bound polysomes and deposited in the form ofprotein bodies (1, 1 1). Immunohistochemical studies have shown that zein is specifically localized in the endosperm (5). Because of its tissue specifity and the rapidly expanding knowledge of its biochemistry and molecular biology (8,13,14,20) Initiation and Maintenance of Endosperm Cultures. After carefully removing all husks and silks, ears were surface-sterilized in 0.01% mercuric chloride for 20 min and rinsed three times with sterile distilled H20. The tops of the 10-d-old kernels were cut off with a scalpel, and the endosperms were scooped out with forceps carefully avoiding the embryo. Isolated endosperms were immediately placed on 6-cm-diameter plastic Petri dishes containing 10 ml agar medium and the dishes were sealed with Parafilm. Strauss medium (26) modified according to Shannon and Batey (23) to contain 2 g/l L-asparagine and 2 g/l yeast extracts (Merck, Munich, F.R.G.) was used. All the cultures were grown in the dark at 25°C and subcultured every 3 to 4 weeks.Electron Microscopy. Two mm3 sections of each culture were pr...

Mature endosperms from opaque-2 mutant seeds with various genetic backgrounds (B 37, W 64A, R 802A and Oh 43) contained twice as much globulin than the corresponding normals, reduced amounts of zein and an increased amount of albumin. The latter is caused by a diminished disappearance of albumins in mutant endosperms during the final phases of development as compared to normal endosperms. Albumins from the opaque-2 endosperms appeared on gel electrophoresis as a similar but heterogenous polypeptide population comparable to that of normal endosperms except that in all opaque-2 forms a 70-kDa polypeptide was increased. Similarly, one to three specific globulin polypeptides (47 kDa, 52 kDa and 58 kDa) were, depending on the genotype, increased in the mutant lines. The accumulation of albumins, globulins and zeins, studied in developing W 64.4 opaque-2 mutant and corresponding normal maize kernels from 10 days after pollination until maturity, demonstrated two-phasic accumulation patterns for all proteins in the mutant, the first phase extending to about 30 days post pollination and the second one from there on until maturity. The transition time-point from first to second phase was characterized in mutant endosperm by a sudden reduction in accumulating albumins (also seen in normal endosperm), by an enhanced accumulation of globulins and the cessation of further accumulation of zein. Preferential accumulation of certain globulins has been found in the mutant during the second phase of globulin formation. The increased accumulation of globulins in the opaque-2 mutant endosperm is considered a response to the arrest in zein synthesis.Since the discovery of the high lysine mutant in Zea mays (L.) opaque-2 [l] and the finding that the main storage protein, zein, was quantitatively [2] and qualitatively [3 -51 reduced, most investigations have focused on elucidating the characteristics of this protein in terms of physical properties [4,6,7], site and mode of deposition in the endosperm in vivo and in vitro [5,8,9] and most recently on the genes of zein themselves [lo-121. Other storage proteins, such as albumins, globulins or glutelins have not received nearly as much attention as zeins. Few and mostly earlier reports, however, dealt with these non-zein proteins. Glutelins, next to zein the major group of endosperm proteins, are said to form a cytoplasmic matrix in the cells [13]; they require strong alkali and detergents for solubilization and this complicates physicochemical studies [14]. Albumins and globulins, on the other hand, are soluble in salt solutions and therefore easier to study. Hence, proteins of salt-solution extracts of corn grains were shown to be heterogenous as to charge in electrophoretic studies by Foster et al. [IS] and Mertz and coworkers [16] and they could be resolved into a large number of constituents by starch gel electrophoresis [17]. An actual separation of albumins from globulins was done the first time by Paulis and Wall [IS] who analyzed them subsequently on starch gel electrophores...