Search

Despite the development of prophylactic anti-malarial drugs and practices to prevent infection, malaria remains a health concern. Preclinical testing of novel malaria vaccine strategies achieved through rational antigen selection and novel particle-based delivery platforms is yielding encouraging results. One such platform, self-assembling virus-like particles (VLP) is safer than attenuated live viruses, and has been approved as a vaccination tool by the FDA. We explore the use of Norovirus sub-viral particles lacking the natural shell (S) domain forming the interior shell but that retain the protruding (P) structures of the native virus as a vaccine vector. Epitope selection and their surface display has the potential to focus antigen specific immune responses to crucial epitopes. Recombinant P-particles displaying epitopes from two malaria antigens, Plasmodium falciparum (Pf) CelTOS and Plasmodium falciparum (Pf) CSP, were evaluated for immunogenicity and their ability to confer protection in a murine challenge model. Immune responses induced in mice resulted either in sterile protection (displaying PfCelTOS epitopes) or in antibodies with functional activity against sporozoites (displaying PfCSP epitopes) in an in vitro liver-stage development assay (ILSDA). These results are encouraging and support further evaluation of this platform as a vaccine delivery system.

Editing in plant mitochondria consists in C to U changes and mainly affects messenger RNAs, thus providing the correct genetic information for the biosynthesis of mitochondrial (mt) proteins. But editing can also affect some of the plant mt tRNAs encoded by the mt genome. In dicots, a C to U editing event corrects a C:A mismatch into a U:A base-pair in the acceptor stem of mt tRNAPhe (GAA). In larch mitochondria, three C to U editing events restore U:A base-pairs in the acceptor stem, D stem and anticodon stem, respectively, of mt tRNAHis (GUG). For both these mt tRNAs editing of the precursors is a prerequisite for their processing into mature tRNAs. In potato mt tRNACys (GCA), editing converts a C28:U42 mismatch in the anticodon stem into a U28:U42 non-canonical base-pair, and reverse transcriptase minisequencing has shown that the mature mt tRNACys is fully edited. In the bryophyte Marchantia polymorpha this U residue is encoded in the mt genome and evolutionary studies suggest that restoration of the U28 residue is necessary when it is not encoded in the gene. However, in vitro studies have shown that neither processing of the precursor nor aminoacylation of tRNACys requires C to U editing at this position. But sequencing of the purified mt tRNACys has shown that psi is present at position 28, indicating that C to U editing is a prerequisite for the subsequent isomerization of U into psi at position 28.

In plant mitochondria, editing of messenger RNA by C-to-U conversions is essential for correct gene expression as it usually improves the protein-sequence conservation between different species or sometimes affects the reading frames (for a review, see Maier et al+, 1996)+ Editing sites have been identified in mitochondrial (mt) RNA of all major groups of land plants, including Bryophytes, Pteridophytes, Prespermaphytes, and Spermaphytes (Hiesel et al+, 1994a,b;Malek et al+, 1996)+ Editing mainly affects messenger RNA, but editing sites have also been identified in three transfer RNAs+ In dicot mitochondria a C-to-U editing event corrects a C:A mismatch into a U:A base pair in the acceptor stem of tRNAPhe(GAA) (Marechal-Drouard et al+, 1993; Binder et al+, 1994)+ In the gymnosperm Larix leptoeuropaea, three C-to-U conversions restore a U:A base pair in the acceptor stem, D stem, and anticodon stem of tRNAHis(GUG), respectively (Marechal-Drouard et al+, 1996b)+ The third example described is the Oenothera berteriana mt tRNACys(GCA), where a C28:U42 mismatch is converted into a U28:U42 noncanonical base pair (Binder et al+, 1994)+ In the case of both tRNAPhe and tRNAHis, editing of precursors is a prerequisite for 59 and 39 processing to generate a mature tRNA (Marchfelder et al+, 1996; Marechal-Drouard et al+, 1996a, 1996b; Kunzmann et al+, 1998)+ The role of editing in the case of tRNACys has not been studied so far, although it has been shown that it occurs at the precursor level (Binder et al+, 1994)+ In this letter, we report an evolutionary and functional study of mt tRNACys(GCA) editing in plant mitochondria+ The cloverleaf structure of the mt tRNACys(GCA) deduced from the sequence of the single Solanum tuberosum mt trnC gene (EMBL Accession Number X93575) is identical to its counterpart in O. berteriana and reveals a weak anticodon stem with a U27:G43 noncanonical interaction, and a C28:U42 mismatch+ By analyzing RT-PCR amplified cDNAs of S. tuberosum mt tRNACys precursors (362 nt in length), we found that 7 out of 11 independent clones contained a T at position 28+ The ratio of edited versus nonedited mature tRNACys was determined by RT-mini-sequencing+ When total S. tuberosum mt tRNAs were used as template, only dATP was incorporated, demonstrating that the mature tRNACys is fully edited in vivo (Fig+ 1B)+ From an evolutionary point of view, the comparison of the S. tuberosum mt trnC gene with its counterpart in Marchantia polymorpha shows in particular two differences in the anticodon stem (Fig+ 1A)+ In M. polymorpha, an A residue at position 43 allows a T27:A43 base pairing, and a T residue is present at position 28+ Considering that this sequence is more closely related to the ancestral sequence, we postulated that the C-to-U editing site found in dicot mitochondria restores this ancestral sequence+ To confirm this hypothesis, we first tried to determine when, during the evolution of land plants, the mt trnC gene acquired a C at position 28 and when the C28-to-U28 editing event occurred+ To do so, the internal sequence of trnC (from position 25 to 52) was PCR-amplified, cloned, and sequenced in several species that belong to different groups of land plants+ A single difference could be observed in this region between the different plants tested: a T residue was present at position 28 of mt trnC in the Pteridophyte Pteris nephrolepis (Filicales order) and in the Reprint requests to: Laurence Marechal-Drouard, Institut de Biologie Moleculaire des Plantes, Centre National de la Recherche Scientifique, Universite Louis Pasteur, 12 rue du General Zimmer, F-67084 Strasbourg Cedex, France; e-mail: laurence+drouard@ibmpulp+u-strasbg+fr 2Present address: Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853-1801, USA 3Present address: Department of Molecular Biophysics and Biochemistry, Yale University, School of Medicine, New Haven, Connecticut 06520-8024, USA RNA (2000), 6:470–474+ Cambridge University Press+ Printed in the USA+ Copyright © 2000 RNA Society+

Two copies of the chloroplast-like tRNA(Asn) gene, trnN1 and trnN2, are expressed in potato mitochondria. While Northern-blot analysis revealed only mature tRNA(Asn), RT-PCR indicated that trnN1 is co-transcribed with trnY and nad2 (exons c, d and e). Using primer-extension and capping experiments, four transcription initiation sites have been mapped in the vicinity of these genes. The first site, responsible for the co-transcription of trnN1, trnY and nad2 (exons c, d and e), gives rise to a primary transcript of at least 7000 nt. A second site, 58 nt downstream from trnY, corresponds to an alternative promoter specific for nad2. In both cases, only the CRTA core motif of the consensus CRTAaGaGA of dicot mitochondrial promoters was found. Finally, two transcription initiation sites were identified 135 and 128 nt upstream of trnN2 in a region which shows no sequence homology with this consensus motif.

We have analyzed 67 sequences surrounding transcription initiation sites identified in higher plant mitochondria. The sequences were classified, independently for monocots and dicots, according to the presence of the CRTA core element found upstream of the first transcribed nucleotide and previously reported as an essential element of plant mitochondrial consensus promoters. This compilation provides new elements concerning the structure of consensus promoters and the relative importance of non-conserved promoters in plant mitochondria. It can be emphasized that promoter regions exhibit several differences between monocot and dicot mitochondria, presumably reflecting a divergent evolution: The sequences classified among consensus promoters as well as the distance between the first transcribed nucleotide and the core element are highly conserved in dicots while more plasticity is observed in monocots. It also appears that the proportion of promoters with neither the conserved promoter sequence nor any conserved motif is far greater in dicots than in monocots.

Two identical "chloroplast-like" tRNAAsn genes, trnN1 and trnN2, have been identified in the potato (Solanum tuberosum) mitochondrial genome. The flanking sequences of trnN1 are unrelated to the corresponding authentic potato chloroplast regions, whilst those of trnN2 are very similar to the chloroplast sequences. The trnN1 copy is present in the mitochondrial genome of various plants whereas the second copy, trnN2, is absent from all the other plant genomes studied so far. Interestingly, both trnN copies are expressed in potato mitochondria. Sequences flanking the chloroplast-like tRNAHis gene (trnH), present as a single copy in the potato mitochondrial DNA, are unrelated to the corresponding chloroplast sequences, whereas chloroplast-derived sequences have been maintained in the vicinity of the maize chloroplast-like mitochondrial trnH gene. However, both the potato and the maize trnH are expressed in mitochondria.

Editing in plant mitochondria consists in C to U changes and mainly affects messenger RNAs, thus providing the correct genetic information for the biosynthesis of mitochondrial (mt) proteins. But editing can also affect some of the plant mt tRNAs encoded by the mt genome. In dicots, a C to U editing event corrects a C:A mismatch into a U:A base pair in the acceptor stem of mt tRNA(Phe) (GAA). In larch mitochondria, three C to U editing events restore U:A base pairs in the acceptor stem, D stem and anticodon stem, respectively, of mt tRNA(His) (GUG). For both these mt RNA(Phe) and tRNA(His), editing of the precursors is a prerequisite for their processing into mature tRNAs. In potato mt tRNA(Cys) (GCA), editing converts a C28:U42 mismatch in the anticodon stem into a U28:U42 non-canonical base pair, and reverse transcriptase minisequencing has shown that the mature mt tRNA(Cys) is fully edited. In the bryophyte Marchantia polymorpha this U residue is encoded in the mt genome and evolutionary studies suggest that restoration of a U28 residue is necessary when it is not encoded in the gene. However, in vitro studies have shown that neither processing of the precursor, nor aminoacylation of tRNA(Cys), requires C to U editing at this position. But sequencing of the purified mt tRNA(Cys) has shown that Psi is present at position 28, indicating that C to U editing is a prerequisite for the subsequent isomerization of U into Psi at position 28.

A method is presented for the partial purification of a plant mitochondrial active chromosome (MAC). This method is based on the presence of the mitochondrial chromosome in the insoluble mitochondrial fraction which allows for its rapid purification from the bulk of detergent-solubilized proteins by ultra-centrifugation. The resuspended MAC carrying DNA and RNA-binding proteins retains DNA synthesis and transcription activities comparable to the ones found in isolated mitochondria. In comparison, tRNA-nucleotidyl terminal transferase taken as an example of RNA modifying activities remains in the soluble fraction. MAC purification is proposed as a rapid and efficient first step in the purification of DNA-binding proteins involved in DNA replication and transcription.