A role for the miR396/GRF network in specification of organ type during flower development, as supported by ectopic expression of Populus trichocarpa miR396c in transgenic tobacco.

The MIR396 family, composed of ath-miR396a and ath-miR396b in Arabidopsis, is conserved among plant species and is known to target the Growth-Regulating Factor (GRF) gene family. ath-miR396 overexpressors or grf mutants are characterised by small and narrow leaves and show embryogenic defects such as cotyledon fusion. Heterologous expression of ath-miR396a has been reported in tobacco and resulted in reduction of the expression of three NtGRF genes. In this study, the precursor of the Populus trichocarpa ptc-miR396c, with a mature sequence identical to ath-miR396b, was expressed under control of the CaMV35S promoter in tobacco. Typical phenotypes of GRF down-regulation were observed, including cotyledon fusion and lack of shoot apical meristem (SAM). At later stage of growth, transgenic plants had delayed development and altered specification of organ type during flower development. The third and fourth whorls of floral organs were modified into stigmatoid anthers and fasciated carpels, respectively. Several NtGRF genes containing a miR396 binding site were found to be down-regulated, and the cleavage of their corresponding mRNA at the miR396 binding site was confirmed for two of them using RACE-PCR analysis. The data obtained agree with the functional conservation of the miR396 family in plants and suggest a role for the miR396/GRF network in determination of floral organ specification.

[Human resistance to African trypanosoma infections]. / Résistance à l'infection par les trypanosomes Africains chez l'homme.

African trypanosomes (prototype: Trypanosoma brucei) are protozoan flagellates that infect a wide range of different mammals. In humans, these parasites have to counteract innate immunity because human serum possesses efficient trypanolytic activity. Resistance to this activity has arisen in two T. brucei subspecies, termed T. b. rhodesiense and T. b. gambiense, allowing them to infect humans where they cause sleeping sickness in East and West Africa respectively. The study of the mechanism by which T. b. rhodesiense escapes lysis by human serum led to the identification of the trypanolytic factor, which turned out to be an ionic pore-forming apolipoprotein associated with some HDL particles.

[Trypanolytic factor of human serum]. / Le facteur trypanolytique du sérum humain.

African trypanosomes (prototype : Trypanosoma brucei) are protozoan flagellates that infect a wide range of different mammals. In humans these parasites have to counteract innate immunity because, unlike other mammals, human serum possesses efficient trypanolytic activity. Resistance to this activity has arisen in two T. brucei subspecies, termed T. b. rhodesiense and T. b. gambiense, allowing them to infect humans where they cause sleeping sickness in East and West Africa respectively. The study of the mechanism by which T. b. rhodesiense escapes lysis by human serum led to the identification of the trypanolytic factor, which turned out to be an ionic pore-forming apolipoprotein associated with some HDL particles.

The VSG expression sites of Trypanosoma brucei: multipurpose tools for the adaptation of the parasite to mammalian hosts.

The variant surface glycoprotein (VSG) genes of Trypanosoma brucei are transcribed in telomeric loci termed VSG expression sites (ESs). Despite permanent initiation of transcription in most if not all of these multiple loci, RNA elongation is abortive except in bloodstream forms where full transcription up to the VSG occurs only in a single ES at a time. The ESs active in bloodstream forms are polycistronic and contain several genes in addition to the VSG, named ES-associated genes (ESAGs). So far 12 ESAGs have been identified, some of which are present only in some ESs. Most of these genes encode surface proteins and this list includes different glycosyl phosphatidyl inositol (GPI)-anchored proteins such as the heterodimeric receptor for the host transferrin (ESAG7/6), integral membrane proteins such as the receptor-like transmembrane adenylyl cyclase (ESAG4) and a surface transporter (ESAG10). An interesting exception is ESAG8, which may encode a cell cycle regulator involved in the differentiation of long slender into short stumpy bloodstream forms. Several ESAGs belong to multigene families including pseudogenes and members transcribed out of the ESs, named genes related to ESAGs (GRESAGs). However, some ESAGs (7, 6 and 8) appear to be restricted to the ESs. Most of these genes can be deleted from the active ES without apparently affecting the phenotype of bloodstream form trypanosomes, probably either due to the expression of ESAGs from 'inactive' ESs (ESAG7/6) or due to the expression of GRESAGs (in particular, GRESAGs4 and GRESAGs1). At least three ESAGs (ESAG7, ESAG6 and SRA) share the evolutionary origin of VSGs. The presence of these latter genes in ESs may confer an increased capacity of the parasite for adaptation to various mammalian hosts, as suggested in the case of ESAG7/6 and proven for SRA, which allows T. brucei to infect humans. Similarly, the existence of a collection of slightly different ESAG4s in the multiple ESs might provide the parasite with adenylyl cyclase isoforms that may regulate growth in response to different environmental conditions. The high transcription rate and high recombination level that prevail in VSG ESs may have favored the generation and/or recruitment in these sites of genes whose hyper-evolution allows adaptation to a larger variety of hosts.

A receptor-like flagellar pocket glycoprotein specific to Trypanosoma brucei gambiense.

Trypanosoma brucei gambiense and T. b. rhodesiense are protozoan parasites causing sleeping sickness in humans due to their resistance to lysis by normal human serum (NHS). Based on the observation that the resistance gene of T. b. rhodesiense encodes a truncated form of the variant specific glycoprotein (VSG), we cloned a similar gene in T. b. gambiense using reverse transcription-linked polymerase chain reaction with VSG-specific primers. This gene, termed TgsGP for T. gambiense-specific glycoprotein, was found to be specific to T. b. gambiense. It is located close to a telomere and is transcribed by a pol II RNA polymerase, only at the bloodstream stage of the parasite development. TgsGP encodes a 47-kDa protein consisting of a N-terminal VSG domain presumably provided with a glycosylphosphatidylinositol (GPI) anchor sequence, similar to the pESAG6 subunit of the trypanosomal transferrin receptor. TgsGP is located in the flagellar pocket, and contains the linear N-linked polyacetyllactosamine characteristic of the endocytotic machinery of T. brucei. These observations strongly suggest that TgsGP is a T. b. gambiense specific receptor. Since stable expression of this protein in T. b. brucei did not confer resistance to NHS, TgsGP may either need another factor to achieve this purpose or fulfils another function linked to adaptation of the parasite to man.

Organization of telomeres during the cell and life cycles of Trypanosoma brucei.

The genome of Trypanosoma brucei contains about 120 chromosomes, which do not visibly condense during mitosis. We have analyzed the organization and segregation of these chromosomes by in situ hybridization using fluorescent telomere probes. At the onset of mitosis, telomeres migrate from their nuclear peripheral location and congregate into a central zone. This dense group of telomeres then splits into two entities that migrate to opposite nuclear poles. Segregation continues until the double-sized nucleus divides and, before cytokinesis occurs, the telomeres reorganize into the discrete foci observed at interphase. During migration, the telomeres are located at the free end of the mitotic spindle. Treatment with the microtubule polymerization inhibitor rhizoxin prevents telomere clustering and chromosomal segregation. In the insect-specific procyclic form as well as in the non-dividing bloodstream stumpy form, telomeres tend to cluster close to the nuclear periphery at interphase. In contrast, in the proliferative bloodstream slender form the telomeres preferentially locate in the central zone of the nucleus. Thus, telomeres are closer to the nuclear periphery during those life cycle stages where the telomeric expression sites for the variant surface glycoprotein are all inactive, suggesting that transcriptional inactivation of these sites is related to their subnuclear localization.

Characterization of a Trypanosoma brucei SR domain-containing protein bearing homology to cis-spliceosomal U1 70 kDa proteins.

The protozoan parasite Trypanosoma brucei relies on trans-splicing of a common spliced leader (SL) RNA to maturate mRNAs. Using the yeast two-hybrid system a protein (TSR1IP) was identified that interacts with the T. brucei serine-arginine (SR) protein termed TSR1. TSR1IP shows homology to U1 70 kDa proteins, and contains an SR rich domain as well as an acidic/arginine domain homologous to the U1 70 kDa poly(A) polymerase inhibiting domain. This protein is localized in the nucleoplasm and excluded from the nucleolus in trypanosomal bloodstream and procyclic forms. Based on structural modelling predictions and on the identification of a RNA recognition motif (RRM), it was possible to demonstrate by the yeast three-hybrid system that TSR1IP interacts with the 5' splice region of the SL RNA. All the above characteristics suggest that TSR1IP could be involved in trans-splicing.

Characterization of a SR protein from Trypanosoma brucei with homology to RNA-binding cis-splicing proteins.

The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs. A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs. The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes. Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3' splicing site (U2AF35/U2AF65). In both procyclic and bloodstream forms of T. brucei, TSR1 was found to localize in the nucleus. In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing. Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system. These and other observations suggest that TSR1 may be involved in trans-splicing in T. brucei.

Use of two-hybrid methodology for identifying proteins of interest in endocrinology.

During the last 10 years, much progress has been made in understanding signal transduction. However, the function of many newly identified proteins remains unknown. The protein/protein interactions have emerged as a major biochemical mechanism of signal transduction. They are of major interest to elucidate the role of a protein in one or another cellular process. The two-hybrid system is especially well designed for such investigation. Here we show that the contribution of this technique already is and will be essential in dissecting the molecular mechanism of transduction pathways in many cell types.

A protein linked to mitochondrion development in Trypanosoma brucei.

The use of the two-hybrid system in yeast allowed us to isolate a new mitochondrial protein of Trypanosoma brucei, termed PIE8, for putative protein interacting with ESAG8. This protein was found to localize progressively in the single mitochondrion of the parasite during the mitochondrial reactivation needed to adapt the parasite from the glycolysis-based metabolism in the mammalian host, to the cytochrome-mediated respiration in the fly vector. Once this reactivation is established, PIE8 is lost from the mitochondrion. Thus, the temporary presence of PIE8 in the mitochondrion is linked to mitochondrial reactivation.

Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity.

We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem. 227, 780-786).

Kinetoplast DNA replication: mechanistic differences between Trypanosoma brucei and Crithidia fasciculata.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles. We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks. One difference is in the location of maxicircles in situ. In C. fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk. In contrast, T. brucei maxicircles generally fill the entire disk. Unlike those in C. fasciculata, T. brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts. T. brucei and C. fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network. In C. fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment. In T. brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions. Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T. brucei.

The structure of replicating kinetoplast DNA networks.

Kinetoplast DNA (kDNA), the mitochondrial DNA of Crithidia fasciculata and related trypanosomatids, is a network containing approximately 5,000 covalently closed minicircles which are topologically interlocked. kDNA synthesis involves release of covalently closed minicircles from the network, and, after replication of the free minicircles, reattachment of the nicked or gapped progeny minicircles to the network periphery. We have investigated this process by electron microscopy of networks at different stages of replication. The distribution of nicked and closed minicircles is easily detectable either by autoradiography of networks radiolabeled at endogenous nicks by nick translation or by twisting the covalently closed minicircles with intercalating dye. The location of newly synthesized minicircles within the network is determined by autoradiography of network is determined by autoradiography of networks labeled in vivo with a pulse of [3H]thymidine. These studies have clarified structural changes in the network during replication, the timing of repair of nicked minicircles after replication, and the mechanism of division of the network.

The attachment of minicircles to kinetoplast DNA networks during replication.

Kinetoplast DNA (kDNA), the trypanosomatid mitochondrial DNA, is a network containing several thousand interlocked minicircles. During kDNA synthesis, minicircles dissociate from the network, and after replication their progeny reattach to the network periphery. Using electron microscopy autoradiography, we found that newly synthesized 3H-labeled minicircles, after short labeling periods, are concentrated in two peripheral zones on opposite sides of the network. These must be minicircle attachment sites, adjacent to the two diametrically opposed complexes of replication proteins observed previously. From the pattern of radiolabeling during longer pulses, we reached the unexpected conclusion that minicircle attachment around the entire network periphery may be due to a relative movement of the kinetoplast and the two complexes. The kinetoplast probably rotates between two fixed complexes.

Characterization of kinetoplast DNA from Phytomonas serpens.

The restriction enzyme digestion of kinetoplast DNA from four Phytomonas serpens isolates shows an overall similar band pattern. One minicircle from isolate 30T was cloned and sequenced, showing low levels of homology but the same general features and organization as described for minicircles of other trypanosomatids. Extensive regions of the minicircle are composed by G and T on the H strand. These regions are very repetitive and similar to regions in a minicircle of Crithidia oncopelti and to telomeric sequences of Saccharomyces cerevisiae. Conserved Sequence Block 3, present in all trypanosomatids, is one nucleotide different from the consensus in P. serpens and provides a basis to differentiate P. serpens from other trypanosomatids. Electron microscopy of kinetoplast DNA evidenced a network with organization similar to other trypanosomatids and the measurement of minicircles confirmed the size of about 1.45 kb of the sequenced minicircle.

The absence of supercoiling in kinetoplast DNA minicircles.

Crithidia fasciculata kinetoplast DNA is a mitochondrial DNA composed of 5000 minicircles and approximately 25 maxicircles, all catenated into a giant network. By comparing the linking number of minicircles released from the network by limited sonication with that of control minicircles, we demonstrate that not only does the elaborate catenation of the network not cause supercoiling, but that there is no minicircle supercoiling at all. The absence of catenation-induced supercoiling is explained by our finding [using electron microscopy (EM) and gel electrophoresis] that network minicircles are joined by only one interlock; single interlocking can be accommodated without helix distortion. EM revealed that propidium diiodide supertwists all the network minicircles and thereby condenses the network into a much smaller size while maintaining its planarity. At high dye concentration the network is condensed to a size comparable to that found in vivo. Nevertheless, network minicircles bind less propidium than free minicircles, indicating that catenation into a network restricts the supercoiling of individual rings. These studies show that the mitochondrion of trypanosomatids may be a unique niche in nature where a covalently-closed circular DNA is not supercoiled. This absence of supercoiling may be a major factor in promoting the formation of the network.

A short DNA sequence from lambda phage inhibits protein synthesis in Escherichia coli rap.

The Escherichia coli rap mutant inhibits vegetative growth of bacteriophage lambda. Phage mutations termed bar, which overcome the rap defect, have been mapped to three genetic loci in the pL operon. Plasmids with a lambda wild-type bar DNA segment cloned downstream from an active promoter cannot be maintained in rap mutant bacteria. The viability of a rap mutant strain decreases rapidly after induction of transcription through bar regions present on plasmids. Under these (restrictive) conditions the expression of plasmid-encoded beta-lactamase and plasmid DNA replication are arrested, but plasmid RNA synthesis continues for several hours. Analysis of protein extracts from E. coli rap cells containing bar plasmids revealed that both plasmid and bacterial protein synthesis are inhibited under restrictive conditions. In addition, unlike other RNAs tested, the chemical half-life of bar RNA increases 3.5-fold relative to the half-life of bar RNA under permissive conditions. We propose that transcription through the bar region, or the accumulation of bar RNA, results in an irreversible defect in cellular mRNA translation. This defect eventually kills the rap cells, and thus prevents bar plasmid maintenance.