Interphase cytosol extracts prepared from Xenopus laevis eggs are active in RNA polymerase III (Pol III) transcription. Addition of recombinant B1 cyclin to these extracts activates mitotic protein kinases that repress transcription. Affinity-purified p34cdc2-cyclin B kinase (mitosis-promoting factor) is sufficient to effect this repression in a simplified Pol III transcription system. This mitotic repression involves the direct phosphorylation of a component of the Pol III transcription initiation factor TFIIIB, which consists of the TATA box-binding protein (TBP) and associated Pol III-specific factors. The transcriptional activity of the TFIIIB-TBP fraction can be modulated in vitro by phosphorylation with mitotic kinases and by dephosphorylation with immobilized alkaline phosphatase.

Abstract. A normal consequence of mitosis in eukaryotes is the repression of transcription. UsingXenopus egg extracts shifted to a mitotic state by the addition of purified cyclin, we have for the first time been able to reproduce a mitotic repression of transcription in vitro. Active RNA polymerase l/I transcription is observed in interphase extracts, but strongly repressed in extracts converted to mitosis. With the topoisomerase II inhibitor VM-26, we demonstrate that this mitotic repression of RNA polymerase III transcription does not require normal chromatin condensation. Similarly, in vitro mitotic repression of transcription does not require the presence of nucleosome structure or involve a general repressive chromatin-binding protein, as i~bition of chromatin formation with saturating amounts of non-specific DNA has no effect on repression. Instead, the mitotic repression of transcription appears to be due to phosphorylation of a component of the transcription machinery by a mitotic protein kinase, either cdc2 kinase and/or a kinase activated by it. Mitotic repression of RNA polymerase Ill transcription is observed both in complete mitotic cytosol and when a kinase-enriched mitotic fraction is added to a highly simplified 5S RNA transcription reaction. We present evidence that, upon depletion of cdc2 kinase, a secondary protein kinase activity remains and can mediate this in vitro mitotic repression of transcription.

Immune surveillance depends on lymphocyte access to tissue. Lymphocytes emigrate from blood when adhesion receptors such as L-selectin and the ␣ 4 ␤ 7 integrin on these cells bind to ligands expressed on venular endothelium. Among transgenic mouse lines expressing an oncoprotein (Tag) in islet ␤ cells, some recognize Tag as nonself. In these mice, Tag expression elicits both ␤ cell hyperplasia with subsequent progression to tumors and lymphocytic infiltration. Endothelial ligands for L-selectin and ␣ 4 ␤ 7 were upregulated in infiltrated islets in these transgenic mice. These ligands were not expressed in tumors, which were devoid of lymphocytic infiltration. In contrast, the adhesion molecules PECAM-1, ICAM-1, and VCAM-1 were expressed on endothelium in both noninfiltrated tumors and infiltrated islets.

Abstract. Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of ~, DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the ~ DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with or-amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205-217). In contrast, chromatin formed from phage h DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chrornatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed. WITH the advent of cell-free extracts capable of forming nuclei from chromatin or naked DNA templates, it is now possible to study discrete steps in nuclear assembly. Such extracts permit the analysis of the intermediate chromatin structures involved, as well as the components required for the assembly of functional nuclei in vitro. Several nuclear reconstitution extracts are available, among them one prepared from cultured mammalian ceils (Burke and Gerace, 1986) and one from the eggs of Xenopus laevis. The latter is especially amenable to analysis since the Xenopus egg contains large pools of the components required for nuclear assembly stored in a soluble form for later embryonic development (for rev...

The cotyledons of the soybean (Glycine max [L.] Merrill cv Amsoy 71) were examined for proteolytic activities capable of degrading soybean seed proteins. Three distinct activities were identified that attack the native Kunitz soybean trypsin inhibitor of Amsoy 71, Ti'. Protease Kl cleaves Ti5 to Ti',, the inhibitor form lacking the five carboxyl-terminal amino acid residues relative to Ti'. Protease Kl is a cysteine protease that peaks in activity on day 4 after the beginning of imbibition, with maximal activity toward Ti5 at pH 4. The characteristics of protease Kl are consistent with the involvement of this protease in the initial proteolysis of the Kunitz inhibitor during germination. Protease K2 also degrades Ti5 at pH 4 but produces no electrophoretically recognizable products. It peaks later in seedling growth, at day 8. Protease K3 degrades Ti' to products other than Ti5.. However, it is active at pH 8. Two proteolytic activities were identified that attack the major storage protein, glycinin. Protease Gl (which appears by 4 days after imbibition) specifically cleaves the acidic polypeptides of glycinin at pH 4, yielding a product approximately 1.5 kilodaltons smaller. Protease Gl is inhibited by metal chelators as well as by reagents reactive toward thiols. Protease G2 also degrades the glycinin acidic chains at pH 4, but without the appearance of electrophoretically recognizable products. Protease G2, while present at low levels in the dry seed, is found primarily in the cotyledons after 8 days of growth.Dicot, and particularly legume, seeds contain reserve proteins that are degraded during the germination and the subsequent early growth of the seedling. The proteolysis of these storage molecules supplies amino acids and their derivatives for the biosynthetic and energy needs of the developing plant (1). The soybean, Glycine max (L.) Merrill contains two major reserve globulins, glycinin and , 3-conglycinin (12, 17). In addition, the soybean typically contains large amounts of protein proteinase inhibitors, in particular KSTI.2We have recently examined the early stages of degradation of these proteins in the Amsoy 71 cultivar of soybean. KSTI is initially cleaved at the carboxyl-terminus, removing five amino acid residues from the native inhibitor, KSTI-Tia, to cinin exhibit similar, initially limited, proteolysis, with one or more specific intermediates accumulating, at least transiently, during the degradation of these proteins (24).We have now identified at least three proteolytic activities that are capable of degrading KSTI in vitro. One enzyme converts KSTI-Tia to KSTI-Tiam, suggesting that this protease is responsible for this reaction in vivo. Another protease has been identified that catalyzes a limited specific proteolysis of the acidic chains of glycinin. MATERIALS AND METHODS Plant Materials and Extracts. Soybeans, Glycine max (L.)Merrill cv Amsoy 71, were purchased from May Seed and Nursery Co., Shenandoah, IA. Germination and growth were carried out as previously described (24) ...