The control of gene expression by regulated nuclear transport.

Many proteins show distinct nuclear- and cytoplasmic-localization patterns. For proteins above the diffusion limit of the NPC, this localization is governed by the activity of NLSs and/or NESs contained in the protein. Structural modification of proteins can affect NLS and NES activities. Ligand binding, phosphorylation and proteolysis are each capable of modifying the nucleocytoplasmic distribution of proteins. In the case of transcription factors, control of these structural modifications affects access of the transcription factor to the chromatin. This management of cellular distribution, in turn, regulates gene expression.

Immunization of monkeys with recombinant complimentary deoxyribonucleic acid expressed zona pellucida proteins.

OBJECTIVE: To evaluate the effects of immunization with zona pellucida (ZP) proteins produced by recombinant complementary DNA (cDNA) technology for the elicitation and antibodies that inhibit sperm binding without altering ovarian function in the nonhuman primate. DESIGN: Controlled nonhuman primate study. SETTING: Controlled environment with individual housing of monkeys in facility approved by National Institutes of Health (NIH) guidelines. PARTICIPANTS: Monkeys housed and treated according to NIH regulations. INTERVENTIONS: Monkeys immunized and boosted at regular intervals with ZP proteins produced using recombinant cDNA techniques. MAIN OUTCOME MEASURE: Urinary estrogen, P, serum antibody levels, sperm-ZP binding, and ovarian morphology. RESULTS: Monkeys immunized with a recombinant rabbit 75-kd ZP protein expressed from a partial cDNA in the pEX bacteria expression system produce antibodies that interfere with ovarian follicular development and ovarian cyclicity. On the contrary, monkeys immunized with a recombinant rabbit 55-kd ZP protein develop antibodies that inhibit homologous sperm binding but do not affect ovarian follicular development or subsequent ovarian hormonal cyclicity. CONCLUSION: Monkey antibodies to the rabbit 75-kd ZP recombinant protein can be generated that inhibit ovarian cyclicity as desired for animal sterilization vaccines. Antibodies to the 55-kd ZP recombinant protein inhibit homologous monkey sperm binding to the ZP without altering ovarian endocrine function or morphology as is desired for human immunocontraception.

Nuclear import in digitonin-permeabilized cells.

Import of proteins into the nucleus of a cell is a complex process that can be reconstituted in vitro. Digitonin-permeabilized cells are washed free of cytosolic factors to provide competent nuclei. Cytosolic factors for import are provided by an extract of Xenopus ovarian cells. Fluorochrome-conjugated probes, cloned proteins fused to green fluorescent protein, or antibodies to the protein of interest are used to visualize nuclear import. The system can also be used to identify nuclear localization sequences (NLS) of imported proteins.

Immunogenicity of zona pellucida vaccines.

Development of zona pellucida (ZP) based contraceptive vaccines raises a number of complications that challenge current immunological capabilities. Our research examines two aspects of these immunological problems. First, recent studies demonstrate that one bacterially expressed rabbit ZP recombinant vaccine (rec55) induces autoantibodies in primates that prevent sperm-ZP binding and induction of the acrosome reaction in the homologous ZP in vitro. Immunization with rec55 does not induce ovarian pathology, and the duration of antibody titres indicates that this vaccine would have reversible effects on fertility. However, the immunogenicity of the rec55 protein produced in the pEX bacterial expression vector is low. We have therefore expressed the cDNA encoding rec55 using the pGEX vector for the following reasons: (i) the pGEX expressed recombinant proteins are soluble in aqueous solution; (ii) affinity purification of recombinant proteins on glutathione Sepharose columns is more effective for obtaining larger quantities of purified protein; and (iii) the availability of protease cleavage sites between the ZP and glutathione S transferase fusion proteins should eliminate the possibility of carrier-mediated suppression of immune responses. These improvements in protein production and purification yield immunogen which is more malleable to immunological studies. Second, while it is clear that ZP recombinant vaccines can eliminate the problem of ovarian pathology, it is important to understand how such pathology results from immunization with native ZP proteins and certain recombinant ZP proteins. To this end, we have initiated immunization studies in baboons comparing rabbit ZP and pig ZP immunogens in Titremax adjuvant. Unilateral ovariectomies at different time points after immunization (1.5, 2, 4 and 6 months) will allow studies on the time course of pathology within the ovary. Comparison of these results with ongoing rabbit ZP immunizations will help elucidate the differences in immunogenicity of ZP proteins isolated from the two species.

Ran-dependent signal-mediated nuclear import does not require GTP hydrolysis by Ran.

Nuclear import of classical nuclear localization sequence-containing proteins involves the assembly of an import complex at the cytoplasmic face of the nuclear pore complex (NPC) followed by movement of this complex through the NPC and release of the import substrate into the nuclear interior. This process has historically been thought to require nucleotide hydrolysis as a source of energy. We found, using hydrolysis-resistant GTP analogs and a mutant Ran unable to hydrolyze GTP, that transport of classical nuclear localization sequence containing substrate through the NPC and release of the substrate into the nucleus did not require hydrolysis of GTP by Ran. In fact, for movement of this type of import substrate into the nuclear interior we did not observe a requirement for hydrolysis of any nucleotide triphosphate. We did, however, find that a pool of free GTP (or its structural equivalent) must be added, probably because the GDP Ran that is added must be converted to GTP Ran during the import process. We found that a requirement for GTP hydrolysis can be restored to an import mixture consisting of recombinant import factors by the addition of RCC1, the Ran guanine nucleotide exchange factor.

Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model.

The studies reported here are the first to demonstrate that recombinant zona pellucida (ZP) proteins will elicit a humoral immune response that recognizes native ZP proteins. Three cDNAs encoding rabbit ZP protein antigens expressed in bacteria were used to immunize cynomolgus monkeys. Four groups of six monkeys each were immunized with bacterially expressed cro-beta-galactosidase recombinant proteins encoded by a full-length cDNA (rc55) encoding the 55-kDa rabbit ZP recombinant protein (rec55), two partial cDNAs (rc75a and rc75b) encoding two recombinant peptides (rec75a and rec75b) of the 75-kDa rabbit ZP protein, and the plasmid-encoded cro-beta-galactosidase control protein. Initial immunizations with these fusion proteins using the muramyl dipeptide adjuvant did not elicit significant levels of antibodies to native or recombinant ZP proteins. Further immunizations were therefore carried out using recombinant ZP proteins conjugated to either protein A or keyhole limpet hemocyanin. Antibodies were detected in the groups immunized with the rec55 and rec75a; however, no antibodies were generated against the rec75b protein. These antibodies have been characterized by two-dimensional PAGE immunoblotting and shown to recognize antigenic domains associated with two of the native rabbit ZP proteins. Reprobes of these immunoblots with sheep anti-total native rabbit ZP proteins, affinity-purified on pig ZP, further demonstrate that a fourth distinct rabbit ZP antigen may be present. The characterization of species-conserved antigenic domains of mammalian ZP proteins is important for studies of the functional regions of ZP proteins and is critical for the design of safe and effective contraceptive vaccines.