Plant-made vaccines: biotechnology and immunology in animal health.

The use of plants as production systems for vaccine antigens has been actively investigated over the last 15 years. The original research focused on the value of this expression system for oral delivery based on the hypothesis that plant-expressed antigens would be more stable within the digestive tract and would allow for the use of the oral route of administration to stimulate a mucosal immune response. However, while first conceived for utility via the oral route, plant-made antigens have also been studied as classical immunogens delivered via a needle to model animal systems. Antigens have been expressed in a number of whole plant and cell culture systems. Several alternative expression platforms have been developed to increase expression of antigens or to elicit preferred immunological responses. The biotechnological advances in plant expression and the immunological testing of these antigens will be reviewed in this paper focusing primarily on diseases of livestock and companion animals.

Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants.

Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes beta-galactosidase, and a polyadenylation 3'-untranslated nopaline synthase sequence from Agrobacterium. These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses. A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5' promoter tested, a 3.0 kb fragment. Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin. LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments. Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament. The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature. Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypocotyls. The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation. Staining was evident in cell types that develop lignified cell walls, e.g. trichomes, anther endothecial cells, and especially developing xylem. The chimeric gene was not expressed in primary meristems. While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged. Transgenic plants with a 600 bp promoter construct (-0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct. Transgenic plants with a -500 bp promoter had levels of expression similar to the -3.0 kb construct. The -0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 x 10(-6) to 5 x 10(-5) M 2,4-D and was responsive to as little as 5 x 10(-8) M. The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.

Regulatable endogenous production of cytokinins up to 'toxic' levels in transgenic plants and plant tissues.

The effects of expressing a chimeric gene consisting of a soybean heat shock gene promoter and a sequence that encodes an enzyme catalyzing the synthesis of a potent phytohormone, the cytokinin iPMP, have been analyzed in transgenic tobacco plants. The production of cytokinin endogenously produced several effects previously undocumented. The differentiation of shoots independent of exogenous cytokinin from heat-treated transgenic plant leaf explants demonstrates that long-term heat treatments do not interfere with complex developmental processes. This extends the potential usefulness of heat shock gene promoters to conditionally express genes during windows of development that span several weeks.

Transformation of maize (Zea mays L.) protoplasts and regeneration of haploid transgenic plants.

Transgenic haploid maize (Zea mays L.) plants were obtained from protoplasts isolated from microspore-derived cell suspension cultures. Protoplasts were electroporated in the presence of plasmid DNA containing the gus A and npt II genes encoding ß-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II), respectively. Transformed calli were selected and continuously maintained on kanamycin containing medium. Stable transformation was confirmed by enzyme assays and DNA. analysis. Stably transformed tissue was transferred to regeneration medium and several plants were obtained. Most plants showed NPT II activity, and some also showed GUS activity. Chromosome examinations performed on representative plants showed that they were haploid. As expected, these plants were infertile.

Development of a heat shock inducible expression cassette for plants: characterization of parameters for its use in transient expression assays.

A heat-inducible expression cassette has been constructed to study the conditional expression of sense or antisense orientations of any sequence of interest in transgenic plants or plant tissues. The construct includes the promoter and all but 5 bases of the mRNA leader from the soybean Gmhsp17.5-E gene, the polylinker from pUC18 (modified to remove the ATG), and a fragment that contains the polyadenylation signal and site from the nopaline synthase gene. Analysis of transient expression of a construct containing the beta-glucuronidase (GUS) coding sequence cloned in the cassette and introduced into Nicotiana plumbaginifolia protoplasts by electroporation shows that the promoter has high expression at heat shock temperatures. This construct is expressed at a roughly 80-fold higher level per unit time than a cauliflower mosaic virus 35S gene promoter-GUS construction. The heat shock promoter is regulated positively by supercoiling in this transient assay system. The level of expression of HS-GUS constructions with the polyadenylation sites from either the nopaline synthase gene or the Gmhsp17.5-E gene was similar. Constructs with a perfect fusion at the 5' end had higher levels of expression than those with the corresponding nonperfect transcriptional fusion.

Sequence and characterization of two auxin-regulated genes from soybean.

The auxin-regulated expression of two poly(A)+ mRNAs in soybean hypocotyl was demonstrated by cloning of the cDNAs and Northern blot hybridization analyses (Walker, J.C., and Key, J.L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7185-7189). The corresponding genes, designated Aux28 and Aux22, have been isolated, and the cDNAs and genes have been sequenced. The Aux28 and Aux22 genes are present at one to two copies per haploid genome, contain four and two introns, and encode hydrophilic proteins of 26.8 and 21.5 kDa, respectively. Although the cDNAs were isolated independently and do not cross-hybridize under stringent hybridization conditions, the protein coding sequences of the two cDNAs have several colinear regions of high homology at the nucleic acid (77-80%) and the amino acid (80-100%) levels; together these regions constitute approximately a third of the protein coding sequences of the cDNAs. These data, together with genomic Southern blot hybridization analysis and hybrid-select translations of mRNAs homologous to the cDNAs, show that these genes belong to two related multigene families. We have identified two sequences, TGATAAAAG and GGCAGCATGCA, that occur at similar distances upstream of the transcription start site in each gene, and the spacing between these two elements is essentially identical in the two genes. The possible significance of these sequences is under evaluation.

Auxin-regulated gene expression.

During the 1960s a wide range of studies provided an information base that led to the suggestion that auxin-regulated cell processes--especially cell elongation--may be mediated by auxin-regulated gene expression. Indirect evidence from our work, based on the influence of inhibitors of RNA synthesis (e.g. actinomycin D) and of protein synthesis (e.g. cycloheximide) on auxin-induced cell elongation, coupled with correlations of the influence of auxin on RNA synthesis and cell elongation, provided the basis for this suggestion. With the availability of techniques for DNA-DNA and DNA-RNA hybridization, mRNA isolation-translation, in vitro 2D gel analysis of the translation products, and ultimately the cloning by recombinant DNA technologies of genomic DNA and copy DNAs (cDNAs) made to poly(A)+ mRNAs, we and others have provided direct evidence for the influence of auxin on the expression of a few genes (i.e. poly(A)+ RNA levels). Our laboratory has provided evidence for auxin's both down-regulating and up-regulating the level of a few poly(A)+ mRNAs out of a population of about 4 X 10(4) sequences that are not significantly affected by auxin. In our studies on auxin-regulated cell elongation, two cDNA clones (pJCW1 and pJCW2) were isolated which corresponded to poly(A)+ mRNAs that responded during growth transitions in a way consistent with a potential role of their protein products in cell elongation. These mRNAs are most abundant in the elongating zone of the soybean hypocotyl. Upon excision and incubation in the absence of auxin, these mRNAs deplete in concert with a decreasing rate of cell elongation. Addition of auxin to the medium results in both increased levels of these mRNAs and enhanced rates of cell elongation. These mRNAs do not deplete if auxin is added to the medium at the onset of excised incubation, and cell elongation rates remain high. We have isolated and sequenced genomic clones that are homologous to these cDNAs. Of the two genes sequenced, both genes are members of small multigene families. There are regions of high amino acid homology even though the nucleotide sequences are sufficiently different in these regions for cross-hybridization of the clones not to be observed. More recently others, especially Guilfoyle's laboratory, have shown that auxin selectively and rapidly influences the level of certain mRNAs and proteins. We have worked on other gene systems such as ribosomal proteins and possible cell wall proteins that are responsive to auxin; again the nature of regulation of expression of these genes is not known.(ABSTRACT TRUNCATED AT 400 WORDS)

var1 Gene on the mitochondrial genome of Torulopsis glabrata.

We have cloned and sequenced a region of the Torulopsis glabrata mitochondrial genome homologous to the Saccharomyces cerevisiae var1 gene (var1Sc). An open reading frame that could encode a protein of 339 amino acids was found with 72.7% amino acid and 85.3% nucleotide sequence homology to the S. cerevisiae var1 gene. The T. glabrata gene (var1Tg) is transcribed yielding two stable RNAs, a more abundant 13.5 S RNA and a less abundant 18 S species. We have also identified a candidate for a T. glabrata var1 protein among mitochondrial translation products labeled in isolated mitochondria. The var1Tg gene is even more A + T-rich (93%) than var1Sc (89.6%) and has conserved the strong codon bias of var1Sc. Major differences between the two sequences were found. Significant among these are that no GC clusters are found in var1Tg and the sequences surrounding each of the sites where known polymorphisms exist in var1Sc have deletions at the corresponding sites in var1Tg. These data are discussed with respect to possible origins of these var1 genes and translocation of GC clusters in S. cerevisiae mitochondrial DNA.

Expression of GC clusters in the yeast mitochondrial var 1 gene. Translation and secondary structure implications.

Alleles of the yeast mitochondrial var 1 gene, which encode a protein (var 1) associated with the small mitochondrial ribosomal subunit, contain one or two identical GC clusters within the coding region that are transcribed and retained in the putative var 1 mRNA (Zassenhaus, H.P., and Butow, R. A. (1984) J. Biol. Chem. 259, 8417-8421). By comparing peptide fragments generated by defined chemical and enzymatic cleavages of the products of these alleles, we show that these GC clusters encode amino acids in the var 1 protein. First, there is a strict correlation between the presence of an optional GC cluster in the var 1 gene and a corresponding increase in size of the peptide that would contain the "extra" amino acids encoded by that GC cluster. Second, we find proline residues in specific peptides of var 1 that, from DNA sequence, would only be present if the GC clusters were translated. Thus, although the yeast mitochondrial genome contains 70-100 GC clusters similar to those in var 1, the var 1 protein is the only mitochondrial translation product now known to contain amino acids encoded by these elements. We have also examined predictions of var 1 secondary structure and find little resemblance to the secondary structures predicted for most other ribosomal proteins. Finally, our analysis suggests a significant conformational difference between the var 1 protein containing amino acids encoded by the optional GC cluster and the form of the protein lacking those amino acids.

Location and structure of the var1 gene on yeast mitochondrial DNA: nucleotide sequence of the 40.0 allele.

Alleles of the var1 locus on yeast mitochondrial DNA specify the size of var1 ribosomal protein. We report the nucleotide sequence of a var1 allele that determines the smallest var1 protein. It contains an open reading frame of 396 codons, which we identify as the structural gene for var1 protein. The var1 protein specified by this allele has an amino acid composition in close agreement with that predicted by the DNA sequence. The var1 coding region is highly unusual: it is 89.6% AT and contains a 46 bp GC-rich palindromic cluster that accounts for 38% of the total GC residues. Our results strongly suggest that like mammalian mitochondria but unlike those from Neurospora, yeast mitochondria use AUA as a methionine codon. Comparison with the sequence of a var1 allele specifying a larger protein suggests that some size polymorphism of var1 protein results from in-frame insertions of a variable number of AAT (Asn) codons.