Rapid identification of causative species in patients with Old World leishmaniasis.

Conventional methods for the identification of species of Leishmania parasite causing infections have limitations. By using a DNA-based alternative, the present study tries to develop a new tool for this purpose. Thirty-three patients living in Marseilles (in the south of France) were suffering from visceral or cutaneous leishmaniasis. DNA of the parasite in clinical samples (bone marrow, peripheral blood, or skin) from these patients were amplified by PCR and were directly sequenced. The sequences observed were compared to these of 30 strains of the genus causing Old World leishmaniasis collected in Europe, Africa, or Asia. In the analysis of the sequences of the strains, two different sequence patterns for Leishmania infantum, one sequence for Leishmania donovani, one sequence for Leishmania major, two sequences for Leishmania tropica, and one sequence for Leishmania aethiopica were obtained. Four sequences were observed among the strains from the patients: one was similar to the sequence for the L. major strains, two were identical to the sequences for the L. infantum strains, and the last sequence was not observed within the strains but had a high degree of homology with the sequences of the L. infantum and L. donovani strains. The L. infantum strains from all immunocompetent patients had the same sequence. The L. infantum strains from immunodeficient patients suffering from visceral leishmaniasis had three different sequences. This fact might signify that some variants of L. infantum acquire pathogenicity exclusively in immunocompromised patients. To dispense with the sequencing step, a restriction assay with HaeIII was used. Some restriction patterns might support genetic exchanges in members of the genus Leishmania.

Identification of Bartonella (Rochalimaea) species among fastidious gram-negative bacteria on the basis of the partial sequence of the citrate-synthase gene.

The bacterial genus Bartonella (Rochalimaea) includes emerging human pathogens with five recognized species. These are fastidious gram-negative bacteria, exhibiting few phenotypic characteristics and whose identification relies upon serotyping, cellular fatty acid analysis, and molecular typing. Most of the isolates have been recovered from the blood of patients, and three of the four pathogenic Bartonella species are associated with infectious endocarditis. We performed PCR-restriction fragment length polymorphism (RFLP) analysis of the blood culture bottle supernatant for the routine identification of Bartonella species among fastidious gram-negative bacteria. The amplification of the citrate-synthase gene with primers previously reported (R. L. Regnery, C. L. Spruill, and B. D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991) yielded a 379-bp product from Bartonella species and a 382-bp product for Capnocytophaga ochracea but no product from any of the other 15 genotypically or phenotypically related species tested. We determined the sequences of the citrate-synthase gene-amplified products for Bartonella species and C. ochracea in order to predict the optimal restriction enzyme to be used in RFLP analysis. TaqI and AciI allowed identification of Bartonella species and C. ochracea. We propose that acridine orange and Gram staining, followed by PCR-RFLP analysis of the blood bottle supernatant, be included in the examination of blood samples from patients with suspected infectious endocarditis.

Ribosomal protein L25 from Trypanosoma brucei: phylogeny and molecular co-evolution of an rRNA-binding protein and its rRNA binding site.

The gene encoding ribosomal protein L25, a primary rRNA-binding protein, was isolated from the protozoan parasite Trypanosoma brucei. Hybridization studies indicate that multiple copies of the gene are present per T. brucei haploid genome. The C-terminal domain of L25 protein from T. brucei is strikingly similar to L23a protein from rat, L25 proteins from fungal species, and L23 proteins from eubacteria, archaebacteria, and chloroplasts. A phylogenetic analysis of L23/25 proteins and the putative binding sites on their respective LSU-rRNAs (large subunit rRNAs) provides a rare opportunity to study molecular co-evolution between an RNA molecule and the protein that binds to it.

The yemanuclein-alpha: a new Drosophila DNA binding protein specific for the oocyte nucleus.

The Drosophila yG 4.5 gene (now called yemanuclein-alpha gene), which maps at 98F, is a member of the yema gene cluster isolated in a search for differentially expressed maternal genes. The yemanuclein-alpha transcript (formerly yT 4.5) is specifically expressed in the female germ cells at early oogenic stages and displays a graded distribution along the antero-posterior axis of the oocyte. These provocative features are reminiscent of that of K10, bicoid and Bicaudal-D gene transcripts and lead us to hypothesize that the yemanuclein-alpha gene plays a key role in egg organization. We show in the present work that the yemanuclein-alpha is a nuclear protein highly specific for the oocyte nucleus. The sequence analysis of the 5696 bp EcoRI fragment containing the yemanuclein-alpha gene, and of 5 overlapping cDNAs, reveals a 3006 nucleotides long open reading frame (ORF) flanked by long untranslated 5' and 3' sequences. This ORF predicts a 109,215 kDa protein which is basic (pHi: 8.57), and serine rich (12.08%). It contains a 40 amino acid acidic domain in the first third of the protein with a potential alpha-helix organization; this domain has some similarity with the nucleolin acidic domain. Parts of the yemanuclein-alpha sequence are likely to form secondary structures known to interact with DNA. We demonstrate the DNA binding activity of the yemanuclein-alpha by affinity chromatography experiments. Our data indicate that the yemanuclein-alpha shares some of the features which are characteristic of genuine transcriptional activators.

Expression in the central nervous system of a subset of the yema maternally acting genes during Drosophila embryogenesis. Post-embryonic expression extends to imaginal discs and spermatocytes.

The yema gene region of Drosophila melanogaster is a cluster of maternally acting genes isolated in differential screens. At least ten transcripts are encoded by the yema gene region; most of them are produced by independent transcription units (eight different transcription units). Using RNA dot-blot analysis and in situ hybridization to tissue sections, we have realized a comprehensive survey of the temporal and spatial expression of the yema transcripts. All these transcripts are maternally expressed. Five of them display a strict maternal expression. They are found exclusively in the female germ line (nurse cells and oocyte). These transcripts are still present in the embryo as maternal information. However, a subset of the yema genes also shows an embryonic and a post-embryonic expression. Interestingly, this expression is essentially restricted to the central nervous system (CNS) throughout the fly development, to the larval and pupal imaginal discs and to a subset of cells in the male gonad, the spermatocytes. Strikingly, these expression sites mainly contain proliferating and/or differentiating cells.