SummaryType I interferons (IFNs) inhibit viral replication and cell growth and enhance the immune response, and therefore have many clinical applications. IFN-α 2b ranks third in world market use for a biopharmaceutical, behind only insulin and erythropoietin. The average annual cost of IFN-α 2b for the treatment of hepatitis C infection is $26 000, and is therefore unavailable to the majority of patients in developing countries. Therefore, we expressed IFN-α 2b in tobacco chloroplasts, and transgenic lines were grown in the field after obtaining

A tandemly repeated DNA sequence organized predominantly, if not entirely, in a specific manner on the human X chromosome has been cloned in pBR322 and characterized. The sequence was detected as a 2-kilobase band in ethidium bromidestained agarose gels ofBamHI-digested total human nuclear DNA. Although in situ hybridization of the cloned sequence to human metaphase chromosomes showed a single major site of hybridization at the centromere region ofthe X chromosome and minor sites of hybridization at several autosomal centromeres, Southern blot analysis of restricted total human DNA indicated that the cloned probe is related to other repeated DNAs, particularly the human alphoid DNAs. Restriction enzyme analysis ofthe cloned fragment revealed an internal repeat structure based upon multiples of 170 base pairs, confirming this relatedness. All available data, however, suggest that the 2-kilobase spacing ofBamHI sites within the repeat may be specific to the X chromosome.The human X chromosome is a particularly interesting system for the molecular investigation of mammalian gene structure expression. A large number ofmedically significant genetic loci have been mapped to it; and one X chromosome in female eutherian mammals exhibits the phenomenon of inactivation, apparently to accomplish dosage compensation of X-linked gene functions (1). Thus, with appropriate DNA probes, this system provides the opportunity to analyze molecular properties ofthe same gene in the active as well as the inactive state in the same cell.Recent advances in DNA technology in conjunction with modem genetic methods have permitted the identification or isolation of X-chromosome specific human DNA sequences. Beauchamp et al. (2) used Southern blot analysis to identify a 2-kilobase-pair (kb) fragment generated by BamHI digestion of rodent-human somatic cell hybrid DNA present in hybrids containing the human X chromosome and which bears homology to human satellite III DNA. Recently, several single-copy sequences from mammalian X chromosomes have been cloned. These include cDNA of the human glucose-6-phosphate dehydrogenase gene (3), a partial cDNA clone of the mouse hypoxanthine phosphoribosyltransferase gene (4), an unidentified human single-copy DNA sequence (5), and two A libraries constructed from flow-sorted chromosomes enriched in human X (6, 7). To date, however, no cloned repetitive sequences that are located predominantly or entirely on the human X chromosome have been reported.We report the cloning and characterization of a 2-kb fragment of human DNA which exists in tandem arrays and is concentrated at or near the centromere region ofthe human X chromosome. The spacing of BamHI sites argues that it is homologous to the sequence noted by Beauchamp et al. (2). Restriction enzyme analysis of the DNA provides data to support the hypothesis that the organization of this sequence is predominantly, if not entirely, specific to the X chromosome. In addition, the DNA displays homology to the human EcoRI dimer and similarities to othe...

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The role of synthesis in the regulation of abscisic acid accumulation was investigated in the developing maize seed. To do this, expression and regulation of the abscisic acid biosynthetic enzyme phytoene desaturase were examined. Comparison of the gene sequence encoding phytoene desaturase and its transcript in the wild-type and viviparous-5 mutant showed that the mutant gene contains multiple insertions and deletions, resulting in the synthesis of a larger transcript. In addition, the 55-kDa phytoene desaturase protein was not detectable in the viviparous-5 mutant, indicating that this phenotype results from a mutation at the phytoene desaturase locus. Levels of phytoene desaturase transcript and protein were compared to abscisic acid levels during development to determine whether phytoene desaturase might regulate abscisic acid accumulation. In the endosperm, transcript levels were initially high and declined during late maturation and dormancy, while protein levels remained high throughout development. In the embryo, transcript levels were low and constant, while protein levels declined. Both temporal and tissue-specific expression of phytoene desaturase were unrelated to abscisic acid levels. An abscisic acid mutant (viviparous-2) deficient in phytoene desaturation was used to determine whether the wild-type protein encoded by Viviparous-2 regulates phytoene desaturase. Phytoene desaturase transcript and protein levels were compared in wild-type and viviparous-2 mutant embryos and endosperm. Normalized levels of phytoene desaturase were similar in wild-type and mutant tissues, suggesting that the wild-type Viviparous-2 protein does not regulate phytoene desaturase transcript or protein levels.

Transgenic plants have significant potential in the bioproduction of complex human therapeutic proteins due to ease of genetic manipulation, lack of potential contamination with human pathogens, conservation of eukaryotic cell machinery mediating protein modification, and low cost of biomass production. Tobacco has been used as our initial transgenic system because Agrobacterium-mediated transformation is highly efficient, prolific seed production greatly facilitates biomass scale-up, and development of new "health-positive" uses for tobacco has significant regional support. We have targeted bioproduction of complex recombinant human proteins with commercial potential as human pharmaceuticals. Human protein C (hPC), a highly processed serum protease of the coagulation/anticoagulation cascade, was produced at low levels in transgenic tobacco leaves. Analogous to its processing in mammalian systems, tobacco-synthesized hPC appears to undergo multiple proteolytic cleavages, disulfide bond formation, and N-linked glycosylation. Although tobacco-derived hPC has not yet been tested for all posttranslational modifications or for enzymatic (anticlotting) activity, these results are promising and suggest considerable conservation of protein processing machinery between plants and animals. CropTech researchers have also produced the human lysosomal enzyme glucocerebrosidase (hGC) in transgenic tobacco. This glycoprotein has significant commercial potential as replacement therapy in patients with Gaucher's disease. Regular intravenous administration of modified glucocerebrosidase, derived from human placentae or CHO cells, has proven highly effective in reducing disease manifestations in patients with Gaucher's disease. However, the enzyme is expensive (dubbed the "world's most expensive drug" by the media), making it a dramatic model for evaluating the potential of plants to provide a safe, low-cost source of bioactive human enzymes. Transgenic tobacco plants were generated that contained the human glucocerebrosidase cDNA under the control of an inducible plant promoter. hGC expression was demonstrated in plant extracts by enzyme activity assay and immunologic cross-reactivity with anti-hGC antibodies. Tobacco-synthesized hGC comigrates with human placental-derived hGC during electrophoretic separations, is glycosylated, and, most significantly, is enzymatically active. Although expression levels vary depending on transformant and induction protocol, hGC production of > 1 mg/g fresh weight of leaf tissue has been attained in crude extracts. Our studies provide strong support for the utilization of tobacco for high-level production of active hGC for purification and eventual therapeutic use at potentially much reduced costs. Furthermore, this technology should be directly adaptable to the production of a variety of other complex human proteins of biologic and pharmaceutical interest.