Multiple pathways for Cre/lox-mediated recombination in plastids.

Plastid transformation technology involves the insertion by homologous recombination and subsequent amplification of plastid transgenes to approximately 10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subsequent role in the transformed line. We report here a feasibility study in the model plant tobacco, to test the heterologous Cre/lox recombination system for antibiotic marker gene removal from plastids. To study its efficiency, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molecular assays, we show that transgene excision occurs completely from all plastid genomes early in plant development. Selectable marker-free transplastomic plants are obtained in the first seed generation, indicating a potential application of the Cre/lox system in plastid transformation technology. In addition to the predicted transgene excision event, two alternative pathways of Cre-mediated recombination were also observed. In one alternative pathway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in the plastid genome. The other alternative pathway uncovered a plastid genome 'hot spot' of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, the resultant plastid mutations are silent, and their study provides the first insights into tRNA accumulation and trans-splicing events in higher plant plastids.

The cellular level of PR500, a protein complex related to the 19S regulatory particle of the proteasome, is regulated in response to stresses in plants.

In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of approximately 800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the "lid" subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.

Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco.

Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

High-yield production of a human therapeutic protein in tobacco chloroplasts.

Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.

Characterization of two subunits of Arabidopsis 19S proteasome regulatory complex and its possible interaction with the COP9 complex.

The nuclear localized, multi-subunit COP9 complex (or COP9 signalosome) is a key developmental modulator involved in repression of photomorphogenesis. In an effort to further define the molecular actions of the COP9 complex, a yeast two hybrid interactive screen was undertaken to identify proteins that could directly interact with one subunit of this complex, namely FUS6/COP11. This screen identified one specific interactive protein, AtS9, that is likely the Arabidopsis non-ATPase S9 (subunit 9) of the 19S regulatory complex from the 26S proteasome. AtS9 specifically interacts with FUS6/COP11 via the C-terminal domain of FUS6/COP11, which is distinct from the N-terminal domain necessary for FUS6/COP11 to interact with itself. As anticipated, AtS9 is not a member of the COP9 complex, but tightly associates with an ATPase subunit of the Arabidopsis 19S proteasome regulatory complex, AtS6A. Since all three proteins, FUS6/COP11, AtS9, and AtS6A, are present as complexed forms in vivo, the observed interaction implies that the COP9 complex may directly interact with the 19S regulatory complex of the 26S proteasome or other potential AtS9-containing complex. This notion is consistent with the parallel tissue-specific expression patterns and the similar, predominantly nuclear localization of both the COP9 complex and the AtS9 protein.

Antibody responses to capsular polysaccharide backbone and O-acetate side groups of Streptococcus pneumoniae type 9V in humans and rhesus macaques.

Streptococcus pneumoniae is responsible for high rates of pneumococcal bacteremia, meningitis, pneumonia, and acute otitis media worldwide. Protection from disease is conferred by antibodies specific for the polysaccharide (Ps) capsule of the bacteria. Of the four types of group 9 pneumococci, types 9N and 9V cause the most disease, and both types are included in the polyvalent pneumococcal vaccine. The type 9V capsule consists of repeating pentasaccharide units linearly arranged, with an average of 1 to 2 mol of O-acetate side chains per mol of repeat units, added in a complex pattern in which not all repeat units are alike. alpha-GlcA residues may be O-acetylated in the 2 (17%) or 3 (25%) position and beta-ManNAc residues may be O-acetylated in the 4 (6%) or 6 (55%) position. Under certain conditions, the O-acetate side chains are subject to oxidation, which results in subsequent de-O-acetylation of a significant number of the repeat units. This de-O-acetylation could adversely affect the efficacy of a vaccine containing the 9V Ps. A study was undertaken to compare the relative contributions of O-acetate and Ps backbone epitopes in the immune response to S. pneumoniae 9V type-specific Ps. In both an infant rhesus monkey model and humans, antibodies against the non-O-acetylated 9V backbone as well as against O-acetylated 9V Ps were detected. Functional (opsonophagocytic) activity was observed in antisera in which the predominant species of antibody recognized de-O-acetylated 9V Ps. We concluded that the O-acetate side groups, while recognized, are not essential to the ability of the 9V Ps to induce functional antibody responses.

Evidence for FUS6 as a component of the nuclear-localized COP9 complex in Arabidopsis.

The pleiotropic CONSTITUTIVE PHOTOMORPHOGENIC (COP), DEETIOLATED (DET), and FUSCA (FUS) loci are essential regulatory genes involved in the light control of seedling developmental patterns in Arabidopsis. Although COP1, DET1, COP9, and FUS6 (also called COP11) have been cloned, their biochemical activities and interactions remain elusive. We have recently suggested that multiple pleiotropic COP, DET, and FUS genes may encode subunits of a large regulatory complex. In this study, we generated specific antibodies against Arabidopsis FUS6 and show that accumulation of both COP9 and FUS6 is coordinated in the pleiotropic cop, det, and fus mutant backgrounds and in wild-type plants throughout development. Both COP9 and FUS6 cofractionated into identical high molecular mass fractions in an analytical gel filtration assay, and neither was found in its monomeric form. Moreover, antibodies raised against either COP9 or FUS6 selectively coimmunoprecipitated both proteins. We have also developed an Arabidopsis protoplast immunolocalization assay and demonstrated that the COP9 complex is localized in the nucleus and that its nuclear localization is not affected by light conditions or tissue types. The integrated genetic and biochemical results strongly support the conclusion that both COP9 and FUS6 are components of the nuclear-localized COP9 complex. Therefore, we have provided the strongest evidence for the conclusion that at least some of the pleiotropic COP, DET, and FUS loci act in the same signaling pathway.

The COP9 complex, a novel multisubunit nuclear regulator involved in light control of a plant developmental switch.

Arabidopsis COP9 is a component of a large protein complex that is essential for the light control of a developmental switch and whose conformation or size is modulated by light. The complex is acidic, binds heparin, and is localized within the nucleus. Biochemical purification of the complex to near homogeneity revealed that it contains 12 distinct subunits. One of the other subunits is COP11, mutations in which result in a phenotype identical to cop9 mutants. The COP9 complex may act to regulate the nuclear abundance of COP1, an established repressor of photomorphogenic development. During the biogenesis of the COP9 complex, a certain degree of prior subunit association is a prerequisite for proper nuclear translocation. Since both COP9 and COP11 have closely related human counterparts, the COP9 complex probably represents a conserved developmental regulator in higher eukaryotes.

Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector.

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICE1 sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the ColE1 ori for maintenance in E. coli and a spectinomycin resistance gene (aadA) for selection in both systems. In addition, pNICER1 carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICER1 was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.

Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids.

Polycistronic mRNAs are the predominant form of plastid primary transcripts. To determine if there is internal initiation of translation of promoter-distal open reading frames, a promoterless uidA reporter gene was integrated into the tobacco plastid genome downstream of the rbcL gene. Monocistronic uidA mRNA does not accumulate from the promoterless uidA construct. However, due to inefficient rbcL transcription termination, a polycistronic transcription unit is created that contains the uidA gene as the second cistron. Numerous stop codons in all three reading frames between the rbcL and uidA coding regions ensure that translation of uidA initiates only from the correct start codon. The encoded reporter gene product, beta-glucuronidase (GUS) accumulates to high levels in the transplastomic plants indicating that promoter-distal cistrons can be efficiently translated in plastids.

Translation of psbA mRNA is regulated by light via the 5'-untranslated region in tobacco plastids.

The plastid psbA gene encodes the 32 kDa D1 polypeptide of photosystem II. It has previously been shown that the initiation of psbA mRNA translation in tobacco is regulated by sequences outside of the coding region. To identify the cis-acting regulatory sequences involved in the translational control, a series of chimeric uidA genes, encoding the beta-glucuronidase (GUS) reporter enzyme, were introduced into the plastid genome. GUS accumulation in response to the light (135- to 200-fold), and the arrest of uidA mRNA translation in light-grown seedlings following transfer for 2 h to the dark, was observed only if the transgenes contained the psbA 5'-untranslated leader region (UTR). Changes in GUS accumulation were accompanied by little or no changes in the uidA mRNA levels. The data indicate that the initiation of D1 translation in tobacco plastids is controlled via the psbA 5'-UTR.

Extrachromosomal elements in tobacco plastids.

The plastid genome of higher plants is a circular double-stranded DNA molecule which is present in multiple identical copies. We report here an 868-bp plastid DNA minicircle, NICE1, that formed in tobacco (Nicotiana tabacum) plastids during transformation, as an unexpected product of homologous recombination. Such extrachromosomal elements are normally absent in plastids of higher plants. We have constructed shuttle plasmids containing NICE1 sequences which are maintained extrachromosomally when reintroduced into plastids by particle bombardment. Furthermore, recombination between homologous sequences in the shuttle plasmids and the main plastid genome occurs. Recombination products were characterized after recovery of the shuttle plasmids in Escherichia coli and of recombinant plastid genomes in the progeny of transformed plants. Our findings indicate that shuttle plasmids can be used to engineer plastid genes without concomitant integration of foreign DNA and to recover plastid mutations in E. coli.

Safety and immunogenicity of Haemophilus influenzae type B-Neisseria meningitidis group B outer membrane protein complex conjugate vaccine mixed in the syringe with diphtheria-tetanus-pertussis vaccine in young Gambian infants.

To ensure compliance and to reduce costs it is important, especially in less developed countries, that programs of child immunization should require as few clinic attendances and as few injections as possible. Therefore we have investigated whether a Haemophilus influenzae type b conjugate vaccine could be given safely and effectively with diphtheria-tetanus-pertussis vaccine (DTP). One hundred twenty-six Gambian infants were given both polyribosylribitol phosphate (PRP)-outer membrane protein complex (PedvaxHIB) and DTP on the same day at 8, 12 and 16 weeks of age; 60 were given the vaccines mixed in the syringe and 66 were given the vaccines separately. To minimize the injection volume the dose of PRP-OMPC used in both groups was 7.5 micrograms, which is half the usual dose. There were no significant differences in anti-PRP antibody titers between the groups after 1, 2 or 3 doses. The geometric mean titers of antibody for the two groups combined were 0.29 micrograms/ml 1 month after the first dose, 1.03 micrograms/ml after the second dose and 1.11 micrograms/ml after the third dose. Concentrations of antibodies to diphtheria, tetanus and pertussis 1 month after the third dose were not significantly different between the two groups. Systemic side effects were reported with equal frequency in the two groups and were similar to those reported elsewhere for DTP. Tenderness at the injection site was more common where the combined injection (0.75 ml) had been given than where DTP alone (0.5 ml) had been given. The main drawback to the use of these 2 vaccines together is the complexity of the mixing procedure used in this clinical trial.

Mammalian mitochondrial DNA sequences can function as in vivo bacterial transcription terminators.

We have used a prokaryotic terminator identification vector, pDR721, to isolate regions from rat mitochondrial DNA (mtDNA) that can act as transcription terminators in vivo. Three independent fragments having terminator capability have been mapped to three general regions of the mitochondrial genome. Two terminators, pRMT1 and pRMT3, are found within and around the D-loop and cytochrome b gene, respectively, while the third, pRMT5, is located at the 3'-end of the 16S ribosomal RNA gene. After subcloning into host cells which carried temperature sensitive mutations in the termination factor, rho protein, galactokinase assays at the permissive and non-permissive temperatures suggested that pRMT3 acted as a rho-independent termination element while the other two, pRMT1 and pRMT5, were dependent on rho protein (or a rho-like protein) for efficient transcription termination.

Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA.

The plastid psbA mRNA is present in all tissues, while the encoded 32 kDa D1 protein of photosystem II accumulates tissue-specifically and in response to light. To study the regulation of D1 accumulation, a chimeric uidA gene encoding beta-glucuronidase (GUS) under control of the psbA 5'- and 3'-regulatory regions (224 and 393 bp, respectively), was integrated into the tobacco plastid genome. A high level of GUS accumulation in leaves and the lack of GUS in roots, with uidA mRNA present in both tissues, indicated tissue-specific accumulation of the chimeric gene product. Light-regulated accumulation of GUS in seedlings was shown. (i) Light-induced accumulation (100-fold) of GUS in etiolated cotyledons was accompanied by only a modest increase in mRNA levels. (ii) Inhibition of GUS synthesis was observed in cotyledons when light-grown seedlings were transferred to the dark, with no reduction in mRNA levels. Tissue-specific and light-regulated accumulation of GUS indicates that D1 accumulation is controlled via cis-acting regulatory elements in the untranslated region of the psbA mRNA. We propose that in tobacco, control of translation initiation is the primary mechanism regulating D1 protein accumulation.

Immunogenicity of conjugate vaccines consisting of pneumococcal capsular polysaccharide types 6B, 14, 19F, and 23F and a meningococcal outer membrane protein complex.

In an effort to prepare pneumococcal (Pn) capsular polysaccharide (Ps) vaccines that would be immunogenic in infants, covalent conjugates were prepared for Pn types 6B, 14, 19F, and 23F. Each Ps type was covalently bound to an outer membrane protein complex from Neisseria meningitidis serogroup B and evaluated for immunogenicity in mice and infant monkeys. The conjugates induced specific anti-Ps antibody responses in mice and in infant rhesus and African green monkeys; a conjugate of 6B and outer membrane protein complex was immunogenic at Ps doses as low as 20 ng. Although low levels of the Pn group-common cell wall polysaccharide were present in all type-specific Ps preparations, anti-cell wall polysaccharide responses induced by covalent conjugates were < 1% of the total anti-Ps response after two doses of vaccine. In contrast, the anti-cell wall polysaccharide response of a noncovalent conjugate represented 41% of the anti-Ps response after two doses. Relative T-cell dependence, a requirement for the human target population of infants less than 18 months old, was demonstrated for all four Pn Ps conjugates in an athymic mouse model. Therefore, these Pn Ps-outer membrane protein complex conjugate vaccines are excellent candidates for evaluation in human infants.

Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation.

We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome. A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12. Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker. Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector.