Importing statistical measures into Artemis enhances gene identification in the Leishmania genome project

BACKGROUND: Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces. RESULTS: Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODONUSAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence. CONCLUSION: An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation

Expression of biopterin transporter (BT1) protein in Leishmania

The present work focuses on the growth phase regulated expression of biopterin transporter gene (BT1) from the LD1 locus on chromosome 35 of Leishmania donovani. Antiserum against recombinant BT1 detected a polypeptide of 45 kDa of equal intensity at lag, log and stationary phases of promastigote growth, both in L. donovani strain LSB-7.1 (MHOM/BL/67/ITMAP263), and strain LSB-146.1 (HOM/IR/95/X81), a natural isolate from Isfehan, Iran that caused cutaneous leishmaniasis. However, in both these strains an additional polypeptide of higher molecular mass (50 kDa) was also observed during lag phase only. In addition, polypeptides of 40, 20, 18 and 16 kDa were seen only during the lag and log phases of both strains. Analysis of L. donovani single, double and triple (null) BT1 knockout mutants confirmed that the 45-kDa polypeptide was the BT1 gene product, as it was absent in the null mutant. These results indicate that 45-kDa BT1 protein in Leishmania is consistently and constitutively expressed in all the growth stages of the parasite

Histone acetylations mark origins of polycistronic transcription in Leishmania major

<p>Abstract</p> <p>Background</p> <p>Many components of the RNA polymerase II transcription machinery have been identified in kinetoplastid protozoa, but they diverge substantially from other eukaryotes. Furthermore, protein-coding genes in these organisms lack individual transcriptional regulation, since they are transcribed as long polycistronic units. The transcription initiation sites are assumed to lie within the 'divergent strand-switch' regions at the junction between opposing polycistronic gene clusters. However, the mechanism by which Kinetoplastidae initiate transcription is unclear, and promoter sequences are undefined.</p> <p>Results</p> <p>The chromosomal location of TATA-binding protein (TBP or TRF4), Small Nuclear Activating Protein complex (SNAP₅₀), and H3 histones were assessed in <it>Leishmania major </it>using microarrays hybridized with DNA obtained through chromatin immunoprecipitation (ChIP-chip). The TBP and SNAP₅₀binding patterns were almost identical and high intensity peaks were associated with tRNAs and snRNAs. Only 184 peaks of acetylated H3 histone were found in the entire genome, with substantially higher intensity in rapidly-dividing cells than stationary-phase. The majority of the acetylated H3 peaks were found at divergent strand-switch regions, but some occurred at chromosome ends and within polycistronic gene clusters. Almost all these peaks were associated with lower intensity peaks of TBP/SNAP₅₀binding a few kilobases upstream, evidence that they represent transcription initiation sites.</p> <p>Conclusion</p> <p>The first genome-wide maps of DNA-binding protein occupancy in a kinetoplastid organism suggest that H3 histones at the origins of polycistronic transcription of protein-coding genes are acetylated. Global regulation of transcription initiation may be achieved by modifying the acetylation state of these origins.</p

Role of biopterin transporter (BT1) gene on growth and infectivity of Leishmania

Leishmania are known to be auxotrophic for pteridines that are known to play a critical role in the parasites survival. In the present work the role of biopterin transporter in the growth of the parasite and infectivity in to macrophages has been worked out. The role of biopterin transporter in the susceptibility of Leishmania to antimonial compounds has also been demonstrated. This role has been verified by using attenuated strains of Leishmania with single, double, and triple (null) biopterin (BT1) mutants made by targeted gene replacement with specific antibiotic markers. Growth analysis of these mutants revealed that wild type, single and double knock out cell lines maintained high growth rates in the medium supplemented with biopterin and folate, whereas the triple knock out or null BT1 mutants were unable to grow in the absence of supplemental biopterin. Using wild type and null BT1 mutants, we examined the role of BT1 gene in infectivity and parasite survival. The cell lines with amplified BT1 gene showed increased infectivity and survival in the macrophages where as the cell lines with disrupted BT1 gene showed reduced infectivity and survival in the macrophages. We also examined the interaction between pteridine and antimonial compounds using recombinant Leishmania strains with reduced or absent biopterin transporter gene (BT1) alleles. No difference in susceptibility to Pentostam or Glucantime was observed in both wild type and BT1-knock out strains. However, pterin or folate supplementation resulted in reversal of Glucantime but not Pentostam susceptibility in both wild type and BT1-knock out strains. The reversal of Glucantime susceptibility by pterins in BT1-knock out strains suggests that the effect may be exerted independently of biopterin transporter, possibly by blocking Glucantime uptake

Widespread variation in transcript abundance within and across developmental stages of Trypanosoma brucei

<p>Abstract</p> <p>Background</p> <p><it>Trypanosoma brucei</it>, the causative agent of African sleeping sickness, undergoes a complex developmental cycle that takes place in mammalian and insect hosts and is accompanied by changes in metabolism and cellular morphology. While differences in mRNA expression have been described for many genes, genome-wide expression analyses have been largely lacking. Trypanosomatids represent a unique case in eukaryotes in that they transcribe protein-coding genes as large polycistronic units, and rarely regulate gene expression at the level of transcription initiation.</p> <p>Results</p> <p>Here we present a comprehensive analysis of mRNA expression in several stages of parasite development. Utilizing microarrays that have multiple copies of multiple probes for each gene, we were able to demonstrate with a high degree of statistical confidence that approximately one-fourth of genes show differences in mRNA expression levels in the stages examined. These include complex patterns of gene expression within gene families, including the large family of variant surface glycoproteins (VSGs) and their relatives, where we have identified a number of constitutively expressed family members. Furthermore, we were able to assess the relative abundance of all transcripts in each stage, identifying the genes that are either weakly or highly expressed. Very few genes show no evidence of expression.</p> <p>Conclusion</p> <p>Despite the lack of gene regulation at the level of transcription initiation, our results reveal extensive regulation of mRNA abundance associated with different life cycle and growth stages. In addition, analysis of variant surface glycoprotein gene expression reveals a more complex picture than previously thought. These data provide a valuable resource to the community of researchers studying this lethal agent.</p

Functional genomics in sand fly-derived Leishmania promastigotes

BACKGROUND: Leishmania development in the sand fly gut leads to highly infective forms called metacyclic promastigotes. This process can be routinely mimicked in culture. Gene expression-profiling studies by transcriptome analysis have been performed with the aim of studying promastigote forms in the sand fly gut, as well as differences between sand fly-and culture-derived promastigotes. FINDINGS: Transcriptome analysis has revealed the crucial role of the microenvironment in parasite development within the sand fly gut because substantial differences and moderate correlation between the transcriptomes of cultured and sand fly-derived promastigotes have been found. Sand fly-derived metacyclics are more infective than metacyclics in culture. Therefore, some caution should be exercised when using cultured promastigotes, depending on the experimental design. The most remarkable examples are the hydrophilic acidic surface protein/small endoplasmic reticulum protein (HASP/SHERP) cluster, the glycoprotein 63 (gp63), and autophagy genes, which are up-regulated in sand fly-derived promastigotes compared with cultured promastigotes. Because HASP/SHERP genes are up-regulated in nectomonad and metacyclic promastigotes in the sand fly, the encoded proteins are not metacyclic specific. Metacyclic promastigotes are distinguished by morphology and high infectivity. Isolating them from the sand fly gut is not exempt from technical difficulty, because other promastigote forms remain in the gut even 15 days after infection. Leishmania major procyclic promastigotes within the sand fly gut up-regulate genes involved in cell cycle regulation and glucose catabolism, whereas metacyclics increase transcript levels of fatty acid biosynthesis and ATP-coupled proton transport genes. Most parasite's signal transduction pathways remain uncharacterized. Future elucidation may improve understanding of parasite development, particularly signaling molecule-encoding genes in sand fly versus culture and between promastigote forms in the sand fly gut. CONCLUSIONS: Transcriptome analysis has been demonstrated to be technically efficacious to study differential gene expression in sand fly gut promastigote forms. Transcript and protein levels are not well correlated in these organisms (approximately 25% quantitative coincidences), especially under stress situations and at differentiation processes. However, transcript and protein levels behave similarly in approximately 60% of cases from a qualitative point of view (increase, decrease, or no variation). Changes in translational efficiency observed in other trypanosomatids strongly suggest that the differences are due to translational regulation and regulation of the steady-state protein levels. The lack of low-input sample strategies does not allow translatome and proteome analysis of sand fly-derived promastigotes so far.The authors thank the Ramón Areces Foundation for a contract. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Strength and deformation characteristics of carbon fibre reinforced composite wrapped aluminium foam beams

Sandwich structures fabricated from an aluminium skinned foam enclosed within a carbon fibre reinforced composite structure have the potential application for high-performance on- and off-road automotive vehicles. The deformations and failure of these types of structures are presented, and results indicate that the application of aluminium face sheets with aluminium foam (AF) aids to prevent the delamination of the outer layers of carbon fibre reinforced polymers (CFRP). The load carrying capacity has been increased by utilising a manufacturing method to maintain the adhesion between the core and the skins until the failure stage is reached. The core shear and de-bonded issue associated with this type of sandwich structure can be addressed by this manufacture method. The peak average flexure load capacity of an aluminium foam sandwich structure (AFSS) with a completely wrapped around CFRP skin was 2800 N with a mass of 191 g. This compares favourably with previously used AFSS without the skins, which had a peak average load of 600 N and a mass of 125 g. An initial finite element model for comparison purposes has been developed to represent the structure’s behaviour and predict the associated failure loads. It is proposed that CFRP wrapped around AFSS enhances the structural performance without significant weight gain

Strength and deformation characteristics of carbon fibre reinforced composite wrapped aluminium foam beams

Sandwich structures fabricated from an aluminium skinned foam enclosed within a carbon fibre reinforced composite structure have the potential application for high-performance on- and off-road automotive vehicles. The deformations and failure of these types of structures are presented, and results indicate that the application of aluminium face sheets with aluminium foam (AF) aids to prevent the delamination of the outer layers of carbon fibre reinforced polymers (CFRP). The load carrying capacity has been increased by utilising a manufacturing method to maintain the adhesion between the core and the skins until the failure stage is reached. The core shear and de-bonded issue associated with this type of sandwich structure can be addressed by this manufacture method. The peak average flexure load capacity of an aluminium foam sandwich structure (AFSS) with a completely wrapped around CFRP skin was 2800 N with a mass of 191 g. This compares favourably with previously used AFSS without the skins, which had a peak average load of 600 N and a mass of 125 g. An initial finite element model for comparison purposes has been developed to represent the structure’s behaviour and predict the associated failure loads. It is proposed that CFRP wrapped around AFSS enhances the structural performance without significant weight gain

Gene expression in Leishmania is regulated predominantly by gene dosage

ABSTRACT Leishmania tropica, a unicellular eukaryotic parasite present in North and East Africa, the Middle East, and the Indian subcontinent, has been linked to large outbreaks of cutaneous leishmaniasis in displaced populations in Iraq, Jordan, and Syria. Here, we report the genome sequence of this pathogen and 7,863 identified protein-coding genes, and we show that the majority of clinical isolates possess high levels of allelic diversity, genetic admixture, heterozygosity, and extensive aneuploidy. By utilizing paired genome-wide high-throughput DNA sequencing (DNA-seq) with RNA-seq, we found that gene dosage, at the level of individual genes or chromosomal “somy” (a general term covering disomy, trisomy, tetrasomy, etc.), accounted for greater than 85% of total gene expression variation in genes with a 2-fold or greater change in expression. High gene copy number variation (CNV) among membrane-bound transporters, a class of proteins previously implicated in drug resistance, was found for the most highly differentially expressed genes. Our results suggest that gene dosage is an adaptive trait that confers phenotypic plasticity among natural Leishmania populations by rapid down- or upregulation of transporter proteins to limit the effects of environmental stresses, such as drug selection. IMPORTANCE Leishmania is a genus of unicellular eukaryotic parasites that is responsible for a spectrum of human diseases that range from cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) to life-threatening visceral leishmaniasis (VL). Developmental and strain-specific gene expression is largely thought to be due to mRNA message stability or posttranscriptional regulatory networks for this species, whose genome is organized into polycistronic gene clusters in the absence of promoter-mediated regulation of transcription initiation of nuclear genes. Genetic hybridization has been demonstrated to yield dramatic structural genomic variation, but whether such changes in gene dosage impact gene expression has not been formally investigated. Here we show that the predominant mechanism determining transcript abundance differences (>85%) in Leishmania tropica is that of gene dosage at the level of individual genes or chromosomal somy