Abstract, Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin Ltti did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.

Abstract. We analysed the soluble form in which the nudear pore complex protein p68 is stored in Xenopus /acpis eggs and its involvement in pore complex assembly processes, We have shown previously that p68, which is the major wheat germ agglutinin (WGA)-binding glycoprotein 01' nudear pore complexes from Xcnopus 00-cytes, is located in the pore channel and partieipates in mediated transport 01' karyophilic proteins. Using a monodonal antibody direeted against p68 (PlI) we re11l0ved this protein from XCIlOPUS egg extract by immunoadsorption, On addition 01' lambda DNA the immunodepleted extraet supported reconstitution ofnudei which were surrounded by a continuous double-membrane envdope but lacked pore complexes and were unable to import karyophilic pro teins such as nudeoplasmin or lamin L m , Essentially identical results were obtained with extract depleted 01' WGA-binding proteins. Our finding that both the anti-p68 antibody and WGA efficiently removed components from the extraet neeessary lor pore complex assembly but did not interfere with nudear membrane formation demonstrates that these processes are independent 01' each other. Analysis 01' the immunoprecipitate on silver-stained SDS-polyaerylamide gels indicated that the antibody adsorbed other proteins besides p68, notably two high molecular weight components. By sucrose gradient centrifugation and gel Illtration we showed that p68 together with associated protein(s) forms a stable, approximately globular eomplex with an M, 01' 254,000, a Stokes radius 01' 5.2 nm and a sedimentation coelTicient 01' 11.3 S. Our tinding that p68 occurs in the form 01' larger maeromolecular assemblies offers an explanation for the distinetly punctale immunofluorescence pattern observed in the eytoplasm 01' mitotic cells after staining with antibodies to p68.

Mesembryanthemum crystallinum plants were irrigated with 400 mol m"^ NaCl to induce CAM and levels of leaf starch, and activities of starch-degrading enzymes were measured. During Crassulacean acid metabolism (CAM) induction, daily starch turnover gradually became more pronounced and was three-to four-fold greater than in leaves of C3 plants after 3 weeks. Activities of a-and (3amylase, D-enzyme and starch phosphorylase all increased 10-to 20-fold within 3 weeks of the start of salt treatment. Activities of a-and f3-amylase increased more than fourfold within the first 24 h of salt treatment, which is the fastest increase in enzyme activities so far measured during the induction of CAM with salt solution in intact plants of this species. Most enzyme activities were partially chloroplastic; however, the principal starch-degrading activity was constituted by an extra-chloropiastic Pamylase. CAM starch-phosphorylase activity, which was mainly chloroplastic, exhibited a two-to three-fold diurnal change in parallel with starch content. CAM induction in M. crystallinum is clearly associated with greater starch turnover and enhanced starch-degrading enzyme activities, which as catalysts of the initial reaction to release carbon for synthesis of phosphoenolpyruvate (PEP) appear highly significant for the functioning of the CAM pathway. The diurnal rhythm of phosphorylase activity may be of particular significance.

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