Kinetoplast minicircle DNA from Leishmania (Viannia) lainsoni.

To investigate the phenomenon that PCR of Leishmania (V.) lainsoni minicircles using primers B1 and B2 gives anomalous small-sized products, unlike all other members of the Leishmania Viannia subgenus, cloned kDNA minicircles from L. (Viannia) lainsoni were sequenced using fluorescent dye terminator reactions. The sequence of L. (V.) lainsoni where the primer B2 would be expected to bind, was different from the other members of the L. Viannia subgenus, matching in only 7 out of 19 bases with the sequence of L. (V.) braziliensis at this position. The sequence obtained from the cloned minicircles enabled the design of a new primer which, when combined with B1, allowed the amplification of full sized minicircles in L. (V.) lainsoni, but not other members of the L. Viannia subgenus. Comparison of the sequence obtained for Leishmania (V.) lainsoni with other Leishmania minicircle DNA confirms that Leishmania (V.) lainsoni is more similar to members of the L. Viannia subgenus than to other Leishmania, but is distinctly different.

Transcriptional repression due to high levels of Wingless signalling.

Extracellular signals can act at different threshold levels to elicit distinct transcriptional and cellular responses. Here, we examine the transcriptional regulation of the Wingless target gene Ultrabithorax (Ubx) in the embryonic midgut of Drosophila. Our previous work showed that Ubx transcription is stimulated in this tissue by Dpp and by low levels of Wingless signalling. We now find that high levels of Wingless signalling can repress Ubx transcription. The response sequence within the Ubx midgut enhancer required for this repression coincides with a motif required for transcriptional stimulation of Dpp, namely a tandem of binding sites for the Dpp-transducing protein, Mad. Indeed, Wingless-mediated repression depends on low levels of Dpp, although apparently not on Mad itself. In contrast, high levels of Dpp signalling antagonize Wingless-mediated repression. This suggests that transcriptional activation of Ubx is subject to competition between Dpp-activated Mad and another Smad whose function as a transcriptional repressor depends on high Wg signalling. Finally, we show that Wingless can repress its own expression via an autorepressive feedback loop that results in a change of the Wingless signalling profile during development.

Functional intertwining of Dpp and EGFR signaling during Drosophila endoderm induction.

Endoderm induction in Drosophila is mediated by the extracellular signals Decapentaplegic (Dpp) and Wingless (Wg). We discovered a secondary signal with a permissive role in this process, namely Vein, a neuregulin-like ligand that stimulates the epidermal growth factor receptor (EGFR) and Ras signaling. Dpp and Wg up-regulate vein expression in the midgut mesoderm in two regions overlapping the Dpp sources. Experiments based on lack of function and ectopic stimulation of Dpp and EGFR signaling show that these two pathways are functionally interdependent and that they synergize with each other, revealing functional intertwining. The transcriptional response elements for the Dpp signal in midgut enhancers from homeotic target genes are bipartite, comprising CRE sites as well as binding sites for the Dpp signal-transducing protein Mad. Of these sites, the CRE seems to function primarily in the response to Ras, the secondary signal of Dpp. We discuss the potential significance of why an inductive process might use a secondary signal whose function is intertwined with that of the primary signal.

The genomic fingerprinting of the coding region of the beta-tubulin gene in Leishmania identification.

We have demonstrated the polymorphism of the beta-tubulin gene region in Leishmania and its value in the identification of the parasite. In this work we have shown that the coding region of the gene has sufficient variation to accurately discriminate these parasites at the subgenus level. Nevertheless, intrasubgenus diversity, for particular restriction enzymes, was found in New World Leishmania belonging to the Leishmania subgenus. For instance, differences were found between mexicana and amazonensis strains. A unique pattern at the species level was found in particular species of both subgenera, e.g. L. (L.) major strain P and L. (L.) tropica belonging to the Leishmania subgenus, and L. (V.) panamensis strain LS94 from the Viannia subgenus. Particular endonucleases are diagnostic in Leishmania species discrimination as in the case of PvuII for the mexicana and amazonensis. This variation evidenced in the beta-tubulin gene region of Leishmania also occurred in other Kinetoplastida e.g. Trypanosoma cruzi, Leptomonas spp. and Crithidia spp. Moreover, these organisms showed a different genomic fingerprinting for the beta-tubulin gene among them and also Leishmania. Thus, the polymorphism of the coding region of the beta-tubulin gene can be used as a molecular marker for the identification of Leishmania.

A CREB-binding site as a target for decapentaplegic signalling during Drosophila endoderm induction.

Decapentaplegic (Dpp) is an extracellular signal of the transforming growth factor-beta family with multiple functions during Drosophila development. For example, it plays a key role in the embryo during endoderm induction. During this process, Dpp stimulates transcription of the homeotic genes Ultrabithorax in the visceral mesoderm and labial in the subjacent endoderm. Here, we show that a cAMP response element (CRE) from an Ultrabithorax enhancer mediates Dpp-responsive transcription in the embryonic midgut, and that endoderm expression from a labial enhancer depends on multiple CREs. Furthermore, the Drosophila CRE-binding protein dCREB-B binds to the Ultrabithorax CRE, and ubiquitous expression of a dominant-negative form of dCREB-B suppresses CRE-mediated reporter gene expression and reduces labial expression in the endoderm. Therefore, a CREB protein may act as a nuclear target, or as a partner of a nuclear target, for Dpp signalling in the embryonic midgut.

LEF-1, a nuclear factor coordinating signaling inputs from wingless and decapentaplegic.

wingless and decapentaplegic signal during endoderm induction in Drosophila to regulate expression of the homeotic gene Ultrabithorax. Here, we define a minimal wingless response sequence in the midgut enhancer of Ultrabithorax. We show that this sequence is recognized by the murine transcription factor LEF-1 (lymphocyte enhancer binding factor 1) in a ternary complex with armadillo protein, the cytoplasmic target of the wingless signaling pathway. In stable transformants, transcriptional stimulation of the Ultrabithorax enhancer by LEF-1 depends on armadillo. Furthermore, overexpression of LEF-1 bypasses the need for wingless signaling and causes phenotypes in the midgut, notum, and wing that mimic wingless hyperstimulation. Finally, efficient transcriptional stimulation by LEF-1 in the midgut depends also on the decapentaplegic response sequence and is limited spatially by decapentaplegic signaling. Thus, LEF-1 coordinates inputs from multiple positional signals, consistent with its architectural role in regulating the assembly of multiprotein enhancer complexes.

decapentaplegic, a target gene of the wingless signalling pathway in the Drosophila midgut.

dishevelled, shaggy/zeste-white 3 and armadillo are required for transmission of the wingless signal in the Drosophila epidermis. We show that these genes act in the same epistatic order in the embryonic midgut to transmit the wingless signal. In addition to mediating transcriptional stimulation of the homeotic genes Ultrabithorax and labial, they are also required for transcriptional repression of labial by high wingless levels. Efficient labial expression thus only occurs within a window of intermediate wingless pathway activity. Finally, the shaggy/zeste-white 3 mutants revealed that wingless signalling can stimulate decapentaplegic transcription in the absence of Ultrabithorax, identifying decapentaplegic as a target gene of wingless. As decapentaplegic itself is required for wingless expression in the midgut, this represents a positive feed-back loop between two cell groups signalling to each other to stimulate each other's signal production.

Leishmania (Viannia) lainsoni occupies a unique niche within the subgenus Viannia.

Leishmania lainsoni has recently been recognized as a new peripylarian species belonging to the subgenus Viannia and the L. braziliensis complex. It has been isolated from its sandfly vector, reservoir host and cutaneous lesions of human patients. Microscopical examination has shown characteristics which are different from those of other members of the L. braziliensis complex. Nuclear deoxyribonucleic acid (DNA) hybridization patterns with a beta tubulin probe and kinetoplast DNA buoyant density measurements show close similarities with other species of the L. braziliensis complex. However, kinetoplast DNA restriction enzyme fragment patterns of L. (V.) lainsoni isolates show similarities to L. mexicana complex species as well as weak cross hybridization. L. (V.) lainsoni is also amplified with L. braziliensis complex specific polymerase chain reaction (PCR) primers but it requires a lower annealing temperature and gives a 300 base pair PCR product. A possible model for the binding of PCR primers to the L. (V.) lainsoni kinetoplast DNA minicircle is proposed.

Identification and diagnosis of Leishmania mexicana complex isolates by polymerase chain reaction.

Following cloning of Leishmania (L.) amazonensis kinetoplast DNA two recombinant clones were identified: one specific for L. (L.) amazonensis and the other specific for L. (L.) amazonensis and closely related isolates. DNA sequences from these clones were compared with those of other kinetoplastids and oligonucleotide primers were designed to be used in the polymerase chain reaction. A pair of these primers has been shown not only to be highly specific for L. mexicana complex isolates but can also be used to distinguish between L. (L.) mexicana and L. (L.) amazonensis isolates. These primers have been tested with water-lysed cultures, crude DNA extracts from human patients, potential host reservoirs, sandfly vectors and with cell pellets after isoenzyme characterization. The results of these tests indicate that the primers can be used specifically in the presence of excess host DNA originating from the majority of South American countries.

A small chromosome of Leishmania (Viannia) braziliensis contains multicopy sequences which are complex specific.

Orthogonal Field Alternating Gel Electrophoresis (OFAGE) has been used to show a band of approximately 260 kb which is stained intensely with ethidium bromide in Leishmania (V.) braziliensis stock M2903. This small chromosome (sc-2903), as well as a 50 kb and a 200 kb chromosome seen in L. (L.) mexicana and L. (L.) amazonensis, respectively, are stably maintained and linear. When used as a hybridisation probe, sc-2903 showed homology to large chromosomal DNA bands and to a multiplicity of genomic fragments in all braziliensis stocks tested, indicating either different sequences, different copy numbers or both but no hybridisation to mexicana stocks. It is possible that these sequences are present in all members of the braziliensis complex and are not related to LD1 or any other previously published small chromosome sequences. However, at least one clone isolated from a sc-2903 library recognised genomic DNA of stocks belonging to the braziliensis, mexicana and donovani complexes. Our results suggest that the clone carries sequence(s) that are repeated and shared between stocks of different complexes but with a variable genomic distribution.