Genomic variation in two gametocyte non-producing Plasmodium falciparum clonal lines.

BACKGROUND: Transmission of the malaria parasite Plasmodium falciparum from humans to the mosquito vector requires differentiation of a sub-population of asexual forms replicating within red blood cells into non-dividing male and female gametocytes. The nature of the molecular mechanism underlying this key differentiation event required for malaria transmission is not fully understood. METHODS: Whole genome sequencing was used to examine the genomic diversity of the gametocyte non-producing 3D7-derived lines F12 and A4. These lines were used in the recent detection of the PF3D7_1222600 locus (encoding PfAP2-G), which acts as a genetic master switch that triggers gametocyte development. RESULTS: The evolutionary changes from the 3D7 parental strain through its derivatives F12 (culture-passage derived cloned line) and A4 (transgenic cloned line) were identified. The genetic differences including the formation of chimeric var genes are presented. CONCLUSION: A genomics resource is provided for the further study of gametocytogenesis or other phenotypes using these parasite lines.

Secretion of protective antigens by tissue-stage nematode larvae revealed by proteomic analysis and vaccination-induced sterile immunity.

Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES) products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4) larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL) members, with acetylcholinesterases (ACEs) and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel repertoire of H. polygyrus proteins closely implicated in immune modulation and protective immunity.

Choice of bacterial growth medium alters the transcriptome and phenotype of Salmonella enterica Serovar Typhimurium.

The type of bacterial culture medium is an important consideration during design of any experimental protocol. The aim of this study was to understand the impact of medium choice on bacterial gene expression and physiology by comparing the transcriptome of Salmonella enterica SL1344 after growth in the widely used LB broth or the rationally designed MOPS minimal medium. Transcriptomics showed that after growth in MOPS minimal media, compared to LB, there was increased expression of 42 genes involved in amino acid synthesis and 23 genes coding for ABC transporters. Seven flagellar genes had decreased expression after growth in MOPS minimal medium and this correlated with a decreased motility. In both MOPS minimal medium and MEM expression of genes from SPI-2 was increased and the adhesion of S. Typhimurium to intestinal epithelial cells was higher compared to the levels after growth in LB. However, SL1344 invasion was not significantly altered by growth in either MOPs minimal media or MEM. Expression of SPI-2 was also measured using chromosomal GFP reporter fusions followed by flow cytometry which showed, for the first time, that the reduction in SPI-2 transcript after growth in different media related to a reduction in the proportion of the bacterial population expressing SPI-2. These data highlight the profound differences in the global transcriptome after in vitro growth in different media and show that choice of medium should be considered carefully during experimental design, particularly when virulence related phenotypes are being measured.

The transcriptional response of Caenorhabditis elegans to Ivermectin exposure identifies novel genes involved in the response to reduced food intake.

We have examined the transcriptional response of Caenorhabditis elegans following exposure to the anthelmintic drug ivermectin (IVM) using whole genome microarrays and real-time QPCR. Our original aim was to identify candidate molecules involved in IVM metabolism and/or excretion. For this reason the IVM tolerant strain, DA1316, was used to minimise transcriptomic changes related to the phenotype of drug exposure. However, unlike equivalent work with benzimidazole drugs, very few of the induced genes were members of xenobiotic metabolising enzyme families. Instead, the transcriptional response was dominated by genes associated with fat mobilization and fatty acid metabolism including catalase, esterase, and fatty acid CoA synthetase genes. This is consistent with the reduction in pharyngeal pumping, and consequential reduction in food intake, upon exposure of DA1316 worms to IVM. Genes with the highest fold change in response to IVM exposure, cyp-37B1, mtl-1 and scl-2, were comparably up-regulated in response to short-term food withdrawal (4 hr) independent of IVM exposure, and GFP reporter constructs confirm their expression in tissues associated with fat storage (intestine and hypodermis). These experiments have serendipitously identified novel genes involved in an early response of C. elegans to reduced food intake and may provide insight into similar processes in higher organisms.

Characterization of the xenobiotic response of Caenorhabditis elegans to the anthelmintic drug albendazole and the identification of novel drug glucoside metabolites.

Knowledge of how anthelmintics are metabolized and excreted in nematodes is an integral part of understanding the factors that determine their potency, spectrum of activity and for investigating mechanisms of resistance. Although there is remarkably little information on these processes in nematodes, it is often suggested that they are of minimal importance for the major anthelmintic drugs. Consequently, we have investigated how the model nematode Caenorhabditis elegans responds to and metabolizes albendazole, one of the most important anthelmintic drugs for human and animal use. Using a mutant strain lacking the ß-tubulin drug target to minimize generalized stress responses, we show that the transcriptional response is dominated by genes encoding XMEs (xenobiotic-metabolizing enzymes), particularly cytochrome P450s and UGTs (UDP-glucuronosyl transferases). The most highly induced genes are predominantly expressed in the worm intestine, supporting their role in drug metabolism. HPLC-MS/MS revealed the production of two novel glucoside metabolites in C. elegans identifying a major difference in the biotransformation of this drug between nematodes and mammals. This is the first demonstration of metabolism of a therapeutic anthelmintic in C. elegans and provides a framework for its use to functionally investigate nematode anthelmintic metabolism.

Microarray analyses of mouse responses to infection by Neospora caninum identifies disease associated cellular pathways in the host response.

Neospora caninum is a coccidian cyst-forming parasite found in a wide range of host species such as mice, dogs and cattle. The development of methods such as vaccines to prevent abortion and fetal loss due to neosporosis would be greatly assisted by further knowledge on immunity and host responses to infection. In this study we used microarray technology to investigate the protective host responses occurring at 6h post infection in the spleen of mice infected with a prototype live N. caninum vaccine. Naive non-pregnant mice were infected with the NC-Nowra isolate as such infections are known to induce protective host responses that will prevent transplacental transmission of a challenge given using pregnancy. The expression data was analysed by SAM (significance of microarrays), ANOVA and clustering methods. Gene lists were investigated for enrichment of gene ontology terms by functional annotation using hypergeometric tests. The results show that Qs and BALB/c mice infected with NC-Nowra differ in their transcriptional responses to infection and these affect a wide range of biological and molecular processes. Transcriptional changes in the Jak-STAT signaling pathway (as well as Irf and other IFN-γ regulated molecules such as GTPases) confirmed the influence of IFN-γ in the mouse response to N. caninum. Gene ontology analyses also assigned some of the molecules involved to well known disease pathways associated with cancer, Parkinson's and Alzheimer's diseases, which were linked to the cell cycle, mitochondrial electron transport chain and coupled proton transport pathways amongst others. Although infection of mice with NC-Nowra causes little or no signs of clinical disease, the molecular functions, processes and pathways identified through these studies clearly warrant further investigation for their role in the development of protective immunity as well as pathogenesis. These studies therefore provide new, exciting leads by which to study neosporosis.

RamA, a member of the AraC/XylS family, influences both virulence and efflux in Salmonella enterica serovar Typhimurium.

The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 lacking a functional ramA or ramR or with plasmid-mediated high-level overexpression of ramA were compared to those of the wild-type parental strain. Inactivation of ramA led to increased expression of 14 SPI-1 genes and decreased expression of three SPI-2 genes, and it altered expression of ribosomal biosynthetic genes and several amino acid biosynthetic pathways. Furthermore, disruption of ramA led to decreased survival within RAW 264.7 mouse macrophages and attenuation within the BALB/c ByJ mouse model. Highly overexpressed ramA led to increased expression of genes encoding multidrug resistance (MDR) efflux pumps, including acrAB, acrEF, and tolC. Decreased expression of 34 Salmonella pathogenicity island (SPI) 1 and 2 genes, decreased SipC production, decreased adhesion to and survival within macrophages, and decreased colonization of Caenorhabditis elegans were also seen. Disruption of ramR led to the increased expression of ramA, acrAB, and tolC, but not to the same level as when ramA was overexpressed on a plasmid. Inactivation of ramR had a more limited effect on pathogenicity gene expression. In silico analysis of a suggested RamA-binding consensus sequence identified target genes, including ramR, acrA, tolC, sipABC, and ssrA. This study demonstrates that the regulation of a mechanism of MDR and expression of virulence genes show considerable overlap, and we postulate that such a mechanism is dependent on transcriptional activator concentration and promoter sensitivity. However, we have no evidence to support the hypothesis that increased MDR via RamA regulation of AcrAB-TolC gives rise to a hypervirulent strain.

Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei.

BACKGROUND: Trypanosomes undergo extensive developmental changes during their complex life cycle. Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. These developmental events are highly regulated, temporally reproducible and accompanied by expression changes mediated almost exclusively at the post-transcriptional level. RESULTS: In this study we have examined, by whole-genome microarray analysis, the mRNA abundance of genes in slender and stumpy forms of T.brucei AnTat1.1 cells, and also during their synchronous differentiation to procyclic forms. In total, five biological replicates representing the differentiation of matched parasite populations derived from five individual mouse infections were assayed, with RNAs being derived at key biological time points during the time course of their synchronous differentiation to procyclic forms. Importantly, the biological context of these mRNA profiles was established by assaying the coincident cellular events in each population (surface antigen exchange, morphological restructuring, cell cycle re-entry), thereby linking the observed gene expression changes to the well-established framework of trypanosome differentiation. CONCLUSION: Using stringent statistical analysis and validation of the derived profiles against experimentally-predicted gene expression and phenotypic changes, we have established the profile of regulated gene expression during these important life-cycle transitions. The highly synchronous nature of differentiation between stumpy and procyclic forms also means that these studies of mRNA profiles are directly relevant to the changes in mRNA abundance within individual cells during this well-characterised developmental transition.

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a species-specific response, including drug detoxification.

OBJECTIVES: The use of triclosan within various environments has been linked to the development of multiple drug resistance (MDR) through the increased expression of efflux pumps such as AcrAB-TolC. In this work, we investigate the effect of triclosan exposure in order to ascertain the response of two species to the presence of this widely used biocide. METHODS: The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L) were determined in microarray experiments. Phenotypic validation of the transcriptomic data included RT-PCR, ability to form a biofilm and motility assays. RESULTS: Despite important differences in the triclosan-dependent transcriptomes of the two species, increased expression of efflux pump component genes was seen in both. Increased expression of soxS was observed in Salmonella Typhimurium, however, within E. coli, decreased expression was seen. Expression of fabBAGI in Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH was increased. Increased expression of ompR and genes within this regulon (e.g. ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An unexpected response of E. coli was the differential expression of genes within operons involved in iron homeostasis; these included fhu, fep and ent. CONCLUSIONS: These data indicate that whilst a core response to triclosan exposure exists, the differential transcriptome of each species was different. This suggests that E. coli K-12 should not be considered the paradigm for the Enterobacteriaceae when exploring the effects of antimicrobial agents.

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport.

In this study, differences at the genetic level of 37 Salmonella Enteritidis strains from five phage types (PTs) were compared using comparative genomic hybridization (CGH) to assess differences between PTs. There were approximately 400 genes that differentiated prevalent (4, 6, 8 and 13a) and sporadic (11) PTs, of which 35 were unique to prevalent PTs, including six plasmid-borne genes, pefA, B, C, D, srgC and rck, and four chromosomal genes encoding putative amino acid transporters. Phenotype array studies also demonstrated that strains from prevalent PTs were less susceptible to urea stress and utilized l-histidine, l-glutamine, l-proline, l-aspartic acid, gly-asn and gly-gln more efficiently than PT11 strains. Complementation of a PT11 strain with the transporter genes from PT4 resulted in a significant increase in utilization of the amino acids and reduced susceptibility to urea stress. In epithelial cell association assays, PT11 strains were less invasive than other prevalent PTs. Most strains from prevalent PTs were better biofilm formers at 37 degrees C than at 28 degrees C, whilst the converse was true for PT11 strains. Collectively, the results indicate that genetic and corresponding phenotypic differences exist between strains of the prevalent PTs 4, 6, 8 and 13a and non-prevalent PT11 strains that are likely to provide a selective advantage for strains from the former PTs and could help them to enter the food chain and cause salmonellosis.

The global consequence of disruption of the AcrAB-TolC efflux pump in Salmonella enterica includes reduced expression of SPI-1 and other attributes required to infect the host.

The mechanisms by which RND pumps contribute to pathogenicity are currently not understood. Using the AcrAB-TolC system as a paradigm multidrug-resistant efflux pump and Salmonella enterica serovar Typhimurium as a model pathogen, we have demonstrated that AcrA, AcrB, and TolC are each required for efficient adhesion to and invasion of epithelial cells and macrophages by Salmonella in vitro. In addition, AcrB and TolC are necessary for Salmonella to colonize poultry. Mutants lacking acrA, acrB, or tolC showed differential expression of major operons and proteins involved in pathogenesis. These included chemotaxis and motility genes, including cheWY and flgLMK and 14 Salmonella pathogenicity island (SPI)-1-encoded type III secretion system genes, including sopE, and associated effector proteins. Reverse transcription-PCR confirmed these data for identical mutants in two other S. Typhimurium backgrounds. Western blotting showed reduced production of SipA, SipB, and SipC. The absence of AcrB or TolC also caused widespread repression of chemotaxis and motility genes in these mutants, and for acrB::aph, this was associated with decreased motility. For mutants lacking a functional acrA or acrB gene, the nap and nir operons were repressed, and both mutants grew poorly in anaerobic conditions. All phenotypes were restored to that of the wild type by trans-complementation with the wild-type allele of the respective inactivated gene. These data explain how mutants lacking a component of AcrAB-TolC are attenuated and that this phenotype is a result of decreased expression of numerous genes encoding proteins involved in pathogenicity. The link between antibiotic resistance and pathogenicity establishes the AcrAB-TolC system as fundamental to the biology of Salmonella.

Detection of genome-wide polymorphisms in the AT-rich Plasmodium falciparum genome using a high-density microarray.

BACKGROUND: Genetic mapping is a powerful method to identify mutations that cause drug resistance and other phenotypic changes in the human malaria parasite Plasmodium falciparum. For efficient mapping of a target gene, it is often necessary to genotype a large number of polymorphic markers. Currently, a community effort is underway to collect single nucleotide polymorphisms (SNP) from the parasite genome. Here we evaluate polymorphism detection accuracy of a high-density 'tiling' microarray with 2.56 million probes by comparing single feature polymorphisms (SFP) calls from the microarray with known SNP among parasite isolates. RESULTS: We found that probe GC content, SNP position in a probe, probe coverage, and signal ratio cutoff values were important factors for accurate detection of SFP in the parasite genome. We established a set of SFP calling parameters that could predict mSFP (SFP called by multiple overlapping probes) with high accuracy (> or = 94%) and identified 121,087 mSFP genome-wide from five parasite isolates including 40,354 unique mSFP (excluding those from multi-gene families) and approximately 18,000 new mSFP, producing a genetic map with an average of one unique mSFP per 570 bp. Genomic copy number variation (CNV) among the parasites was also cataloged and compared. CONCLUSION: A large number of mSFP were discovered from the P. falciparum genome using a high-density microarray, most of which were in clusters of highly polymorphic genes at chromosome ends. Our method for accurate mSFP detection and the mSFP identified will greatly facilitate large-scale studies of genome variation in the P. falciparum parasite and provide useful resources for mapping important parasite traits.

Fasciola hepatica expresses multiple alpha- and beta-tubulin isotypes.

We have identified five alpha-tubulin and six beta-tubulin isotypes that are expressed in adult Fasciola hepatica. Amino acid sequence identities ranged between 72 and 95% for fluke alpha-tubulin and between 65 and 97% for beta-tubulin isotypes. Nucleotide sequence identity ranged between 68-77% and 62-80%, respectively, for their coding sequences. Phylogenetic analysis indicated that two of the alpha-tubulins and two of the beta-tubulins were distinctly divergent from the other trematode and nematode tubulin sequences described in this study, whereas the other isotypes segregated within the trematode clades. With regard to the proposed benzimidazole binding site on beta-tubulin, three of the fluke isotypes had tyrosine at position 200 of beta-tubulin, two had phenylalanine and one had leucine. All had phenylalanine at position 167 and glutamic acid at position 198. When isotype RT-PCR fragment sequences were compared between six individual flukes from the susceptible Cullompton isolate and from seven individual flukes from the two resistant isolates, Sligo and Oberon, these residues were conserved.

SrfB, a member of the Serum Response Factor family of transcription factors, regulates starvation response and early development in Dictyostelium.

The Serum Response Factor (SRF) is an important regulator of cell proliferation and differentiation. Dictyostelium discoideum srfB gene codes for an SRF homologue and is expressed in vegetative cells and during development under the control of three alternative promoters, which show different cell-type specific patterns of expression. The two more proximal promoters directed gene transcription in prestalk AB, stalk and lower-cup cells. The generation of a strain where the srfB gene has been interrupted (srfB(-)) has shown that this gene is required for regulation of actin-cytoskeleton-related functions, such as cytokinesis and macropinocytosis. The mutant failed to develop well in suspension, but could be rescued by cAMP pulsing, suggesting a defect in cAMP signaling. srfB(-) cells showed impaired chemotaxis to cAMP and defective lateral pseudopodium inhibition. Nevertheless, srfB(-) cells aggregated on agar plates and nitrocellulose filters 2 h earlier than wild type cells, and completed development, showing an increased tendency to form slug structures. Analysis of wild type and srfB(-) strains detected significant differences in the regulation of gene expression upon starvation. Genes coding for lysosomal and ribosomal proteins, developmentally-regulated genes, and some genes coding for proteins involved in cytoskeleton regulation were deregulated during the first stages of development.

Altered phenotype and gene transcription in endothelial cells, induced by Plasmodium falciparum-infected red blood cells: pathogenic or protective?

Severe malaria is associated with sequestration of Plasmodium falciparum-infected red blood cells (PRBC) in the microvasculature and elevation of intercellular adhesion molecule-1 (ICAM-1) and TNF. In vitro co-culture of human umbilical vein endothelial cells (HUVEC), with either PRBC or uninfected RBC, required the presence of low level TNF (5pg/ml) for significant up-regulation of ICAM-1, which may contribute to increased cytoadhesion in vivo. These effects were independent of P. falciparum erythrocyte membrane protein-1 (PfEMP-1)-mediated adhesion but critically dependent on cell-cell contact. Further changes included increases in IL8 release and soluble TNF receptor shedding. Microarray analysis of HUVEC transcriptome following co-culture, using a human Affymetrix microarray chip, showed significant differential regulation of genes which defined gene ontologies such as cell communication, cell adhesion, signal transduction and immune response. Our data demonstrate that endothelial cells have the ability to mobilise immune and pro-adhesive responses when exposed to both PRBC and TNF. In addition, there is also a previously un-described positive regulation by RBC and TNF and a concurrent negative regulation of a range of genes involved in inflammation and cell-death, by PRBC and TNF. We propose that the balance between positive and negative regulation demonstrated in our study will determine endothelial pathology during a malaria infection.

An H-NS-like stealth protein aids horizontal DNA transmission in bacteria.

The Sfh protein is encoded by self-transmissible plasmids involved in human typhoid and is closely related to the global regulator H-NS. We have found that Sfh provides a stealth function that allows the plasmids to be transmitted to new bacterial hosts with minimal effects on their fitness. Introducing the plasmid without the sfh gene imposes a mild H-NS(-) phenotype and a severe loss of fitness due to titration of the cellular pool of H-NS by the A+T-rich plasmid. This stealth strategy seems to be used widely to aid horizontal DNA transmission and has important implications for bacterial evolution.

A new environmentally resistant cell type from Dictyostelium.

This paper describes the serendipitous discovery and first characterization of a new resistant cell type from Dictyostelium, for which the name aspidocyte (from aspis: Greek for shield) is proposed. These cells are induced from amoebae by a range of toxins including heavy metals and antibiotics, and were first detected by their striking resistance to detergent lysis. Aspidocytes are separate, rounded or irregular-shaped cells, which are immotile but remain fully viable; once the toxic stress is removed, they revert to amoeboid cells within an hour. Induction takes a few hours and is completely blocked by the protein synthesis inhibitor cycloheximide. Aspidocytes lack a cell wall and their resistance to detergent lysis is active, requiring continued energy metabolism, and may be assisted by a complete cessation of endocytosis, as measured by uptake of the dye FM1-43. Microarray analysis shows that aspidocytes have a distinct pattern of gene expression, with a number of genes up-regulated that are predicted to be involved in lipid metabolism. Aspidocytes were initially detected in a hypersensitive mutant, in which the AMP deaminase gene is disrupted, suggesting that the inductive pathway involves AMP levels or metabolism. Since aspidocytes can also be induced from wild-type cells and are much more resistant than amoebae to a membrane-disrupting antibiotic, it is possible that they are an adaptation allowing Dictyostelium cells to survive a sudden onslaught of toxins in the wild.

Proteomic and microarray analyses of the Dictyostelium Zak1-GSK-3 signaling pathway reveal a role in early development.

GskA, the Dictyostelium GSK-3 orthologue, is modified and activated by the dual-specificity tyrosine kinase Zak1, and the two kinases form part of a signaling pathway that responds to extracellular cyclic AMP. We identify potential cellular effectors for the two kinases by analyzing the corresponding null mutants. There are proteins and mRNAs that are altered in abundance in only one or the other of the two mutants, indicating that each kinase has some unique functions. However, proteomic and microarray analyses identified a number of proteins and genes, respectively, that are similarly misregulated in both mutant strains. The positive correlation between the array data and the proteomic data is consistent with the Zak1-GskA signaling pathway's functioning by directly or indirectly regulating gene expression. The discoidin 1 genes are positively regulated by the pathway, while the abundance of the H5 protein is negatively regulated. Two of the targets, H5 and discoidin 1, are well-characterized markers for early development, indicating that the Zak1-GskA pathway plays a role in development earlier than previously observed.

Analysis of ESTs from Lutzomyia longipalpis sand flies and their contribution toward understanding the insect-parasite relationship.

An expressed sequence tag library has been generated from a sand fly vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed from whole adults and 16,608 clones were sequenced from both ends and assembled into 10,203 contigs and singlets. Of these 58% showed significant similarity to known genes from other organisms, <4% were identical to described sand fly genes, and 42% had no match to any database sequence. Our analyses revealed putative proteins involved in the barrier function of the gut (peritrophins, microvillar proteins, glutamine synthase), digestive physiology (secreted and membrane-anchored hydrolytic enzymes), and the immune response (gram-negative binding proteins, thioester proteins, scavenger receptors, galectins, signaling pathway factors, caspases, serpins, and peroxidases). Sequence analysis of this transcriptome dataset has provided new insights into genes that might be associated with the response of the vector to the development of Leishmania.

Developmental timing in Dictyostelium is regulated by the Set1 histone methyltransferase.

Histone-modifying enzymes have enormous potential as regulators of the large-scale changes in gene expression occurring during differentiation. It is unclear how different combinations of histone modification coordinate regimes of transcription during development. We show that different methylation states of lysine 4 of histone H3 (H3K4) mark distinct developmental phases of the simple eukaryote, Dictyostelium. We demonstrate that the enzyme responsible for all mono, di and tri-methylation of H3K4 is the Dictyostelium homolog of the Set1 histone methyltransferase. In the absence of Set1, cells display unusually rapid development, characterized by precocious aggregation of amoebae into multicellular aggregates. Early differentiation markers are abundantly expressed in growing set1 cells, indicating the differentiation program is ectopically activated during growth. This phenotype is caused specifically by the loss of Set1 catalytic activity. Set1 mutants induce premature differentiation in wild-type cells, indicating Set1 regulates production of an extra-cellular factor required for the correct perception of growth conditions. Microarray analysis of the set1 mutants reveals genomic clustering of mis-expressed genes, suggesting a requirement for Set1 in the regulation of chromatin-mediated events at gene clusters.