Leishmania major chromosome 3 contains two long convergent polycistronic gene clusters separated by a tRNA gene

Leishmania parasites (order Kinetoplastida, family Trypanosomatidae) cause a spectrum of human diseases ranging from asymptomatic to lethal. The ~33.6 Mb genome is distributed among 36 chromosome pairs that range in size from ~0.3 to 2.8 Mb. The complete nucleotide sequence of Leishmania major Friedlin chromosome 1 revealed 79 protein-coding genes organized into two divergent polycistronic gene clusters with the mRNAs transcribed towards the telomeres. We report here the complete nucleotide sequence of chromosome 3 (384 518 bp) and an analysis revealing 95 putative protein-coding ORFs. The ORFs are primarily organized into two large convergent polycistronic gene clusters (i.e. transcribed from the telomeres). In addition, a single gene at the left end is transcribed divergently towards the telomere, and a tRNA gene separates the two convergent gene clusters. Numerous genes have been identified, including those for metabolic enzymes, kinases, transporters, ribosomal proteins, spliceosome components, helicases, an RNA-binding protein and a DNA primase subunit.     

Genetic variation in RPOIILS gene encoding RNA polymerase II largest subunit from Leishmania major

Leishmaniasis is a geographically widespread severe disease which includes visceral leishmaniasis, cutaneous leishmaniasis (CL). There are 350 million people at risk in over 80 countries. In the Old World, CL is usually caused by Leishmania major, Leishmania tropica, and Leishmania aethiopica complex which 90 % of cases occurring in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil, and Peru. Recently, some reports showed that some strains of L. major have internal transcribed space (ITS-1) with differential size exhibiting homology with the related gene in a divergent genus of kinetoplastida, the Crithidia. This prompted us to analyze the mentioned gene in 100 isolates obtained from patients with suspected CL. After obtaining samples from 100 patients, DNA extraction was performed and ITS-1 was analyzed using PCR–RFLP. These samples were sequenced for verifying their homology. Then, RPOIILS gene was analyzed in the samples that their ITS-1 gene exhibiting homology with the related gene in Crithidia. Results showed that 10 % of the isolates have ITS-1 exhibiting different size with the routine ones. Sequencing of them showed their similarity to the one from Crithidia fasciculata. RPOIILS gene encoding RNA polymerase II largest subunit analysis showed genetic diversity. This study might also help in solving the problems concerning Leishmaniasis outbreak currently facing in Iran and some other endemic regions of the world.     

Base compositional bias in trans-spliced sequences of unknown function in Leishmania major

Iribar, M. P., and Cruz, A. K. 2001. Base compositional bias in trans-spliced sequences of unknown function in Leishmania major. Experimental Parasitology100, 1-5     

Multilocus microsatellite typing shows three different genetic clusters of Leishmania major in Iran

Ten polymorphic microsatellite markers were used to analyse 25 strains of Leishmania major collected from cutaneous leishmaniasis cases in different endemic areas in Iran. Nine of the markers were polymorphic, revealing 21 different genotypes. The data displayed significant microsatellite polymorphism with rare allelic heterozygosity. Bayesian statistic and distance based analyses identified three genetic clusters among the 25 strains analysed. Cluster I represented mainly strains isolated in the west and south-west of Iran, with the exception of four strains originating from central Iran. Cluster II comprised strains from the central part of Iran, and cluster III included only strains from north Iran. The geographical distribution of L. major in Iran was supported by comparing the microsatellite profiles of the 25 Iranian strains to those of 105 strains collected in 19 Asian and African countries. The Iranian clusters I and II were separated from three previously described populations comprising strains from Africa, the Middle East and Central Asia whereas cluster III grouped together with the Central Asian population. The considerable genetic variability of L. major might be related to the existence of different populations of Phlebotomus papatasi and/or to differences in reservoir host abundance in different parts of Iran.     

Production of polyhydroxybutyrate by polycistronic expression of bacterial genes in tobacco plastid

Transgenic techniques are used to enhance and improve crop production, and their application to the production of chemical resources in plants has been under investigation. To achieve this latter goal, multiple-gene transformation is required to improve or change plant metabolic pathways; when accomplished by plant nuclear transformation, however, this procedure is costly and time consuming. We succeeded in the metabolic engineering of the tobacco plant by introducing multiple genes within a bacteria-like operon into a plastid genome. A tobacco plastid was transformed with a polycistron consisting of the spectinomycin resistance gene and three bacterial genes for the biosynthesis of the biodegradable polyester, poly[(R)-3-hydroxybutyrate] (PHB), after modification of their ribosome binding sites. DNA and RNA analysis confirmed the insertion of the introduced genes into the plastid genome and their polycistronic expression. As the result, the transplastomic tobacco accumulated PHB in its leaves. The introduced genes and the PHB productivity were maternally inherited, avoiding genetic spread by pollen diffusion, and were maintained stably in the seed progeny. Despite the low PHB productivity, this report demonstrates the feasibility of transplastomic technology for metabolic engineering. This "phyto-fermentation" system can be applied to plant production of various chemical commodities and pharmaceuticals.     

Patched represses the Hedgehog signalling pathway by promoting modification of the Smoothened protein

Hedgehog (Hh) signalling plays a central role in many developmental processes in both vertebrates and invertebrates [1]. The multipass membrane-spanning proteins Patched (Ptc) [2-4] and Smoothened (Smo) [5-7] have been proposed to act as subunits of a putative Hh receptor complex. According to this view, Smo functions as the transducing subunit, the activity of which is blocked by a direct interaction with the ligand-binding subunit, Ptc [8]. Activation of the intracellular signalling pathway occurs when Hh binds to Ptc [8-11], an event assumed to release Smo from Ptc-mediated inhibition. Evidence for a physical interaction between Smo and Ptc is so far limited to studies of the vertebrate versions of these proteins when overexpressed in tissue culture systems [8,12]. To test this model, we have overexpressed the Drosophila Smo protein in vivo and found that increasing the levels of Smo protein per se was not sufficient for activation of the pathway. Immunohistochemical staining of wild-type and transgenic embryos revealed distinct patterns of Smo distribution, depending on which region of the protein was detected by the antibody. Our findings suggest that Smo is modified to yield a non-functional form and this modification is promoted by Ptc in a non-stoichiometric manner.     

IL-9 is a susceptibility factor in Leishmania major infection by promoting detrimental Th2/type 2 responses

IL-9 is a cytokine produced by Th2 cells, induced during Leishmania major infection. Because the role of IL-9 in leishmaniasis is currently unknown, IL-9-deficient mice were generated by immunization with mouse IL-9 coupled to OVA. This produced strong and long-lasting neutralizing anti-IL-9 Abs in vivo. Anti-IL-9 vaccination showed protective effects, because it enabled L. major-infected nonhealer BALB/c mice to better resist to leishmaniasis with doubling the time span until pathological disease progression occurred. Increased resistance was also demonstrated by moderate footpad swelling and histopathology due to reduced parasite burden compared with sham-immunized BALB/c mice. Mechanistically, IL-9 neutralization in BALB/c mice resulted in a reduction of detrimental Th2/type 2 responses with an observed shift toward protective Th1 immune responses. This led to an alteration from alternative to classical macrophage activation with subsequent enhanced killing effector functions, as demonstrated by increased NO production but reduced arginase 1-mediated macrophage responses. Conclusively, the data show that IL-9 is a susceptible factor in leishmaniasis. They further suggest that IL-9 is able to influence Th dichotomy in leishmaniasis by promoting detrimental Th2/type 2 responses in BALB/c mice. The results extend efforts made to generate autoantibodies capable of regulating biological processes, with IL-9 a potential drug target against leishmaniasis.     

Class II major histocompatibility complex genes of the sheep

The class II genes of the sheep major histocompatibility complex (MHC) have been cloned from two unrelated heterozygous sheep into cosmid vectors. By restriction mapping and hybridization with a number of class II probes of human and mouse origin, the cloned genetic material has been assigned to seven distinct alpha genes, 10 distinct beta genes and 14 beta-related sequences. It was difficult to identify homologues of specific HLA class II genes because of a tendency for the ovine genes to cross-hybridize between HLA probes representing different loci. Such cross-hybridization was especially marked among the beta genes. While DQ and DR homologues have been tentatively identified by several criteria, no genes corresponding to DP have been identified. Cosmids containing class II alpha and beta genes have been transfected into mouse LTK- cells, and surface expression of a sheep class II molecule has been obtained.     

Comparison of the A2 gene locus in Leishmania donovani and Leishmania major and its control over cutaneous infection

In Old World Leishmania infections, Leishmania donovani is responsible for fatal visceral leishmaniasis, and L. major is responsible for non-fatal cutaneous leishmaniasis in humans. The genetic differences between these species which govern the pathology or site of infection are not known. We have therefore carried out detailed analysis of the A2 loci in L. major and L. donovani because A2 is expressed in L. donovani but not L. major, and A2 is required for survival in visceral organs by L. donovani. We demonstrate that although L. major contains A2 gene regulatory sequences, the multiple repeats that exist in L. donovani A2 protein coding regions are absent in L. major, and the remaining corresponding A2 sequences appear to represent non-expressed pseudogenes. It was possible to restore amastigote-specific A2 expression to L. major, confirming that A2 regulatory sequences remain functional in L. major. Although L. major is a cutaneous parasite in rodents and humans, restoring A2 expression to L. major inhibited its ability to establish a cutaneous infection in susceptible BALB/c or resistant C57BL6 mice, a phenotype typical of L. donovani. There was no detectable cellular immune response against L. major after cutaneous infection with A2-expressing L. major, suggesting that the lack of growth was not attributable to acquired host resistance but to an A2-mediated suppression of parasite survival in skin macrophages. These observations argue that the lack of A2 expression in L. major contributed to its divergence from L. donovani with respect to the pathology of infection.