The transcriptome of Toxoplasma gondii
© Radke et al; licensee BioMed Central Ltd. 2005
Received: 06 October 2005
Accepted: 02 December 2005
Published: 02 December 2005
Toxoplasma gondii gives rise to toxoplasmosis, among the most prevalent parasitic diseases of animals and man. Transformation of the tachzyoite stage into the latent bradyzoite-cyst form underlies chronic disease and leads to a lifetime risk of recrudescence in individuals whose immune system becomes compromised. Given the importance of tissue cyst formation, there has been intensive focus on the development of methods to study bradyzoite differentiation, although the molecular basis for the developmental switch is still largely unknown.
We have used serial analysis of gene expression (SAGE) to define the Toxoplasma gondii transcriptome of the intermediate-host life cycle that leads to the formation of the bradyzoite/tissue cyst. A broad view of gene expression is provided by >4-fold coverage from nine distinct libraries (~300,000 SAGE tags) representing key developmental transitions in primary parasite populations and in laboratory strains representing the three canonical genotypes. SAGE tags, and their corresponding mRNAs, were analyzed with respect to abundance, uniqueness, and antisense/sense polarity and chromosome distribution and developmental specificity.
This study demonstrates that phenotypic transitions during parasite development were marked by unique stage-specific mRNAs that accounted for 18% of the total SAGE tags and varied from 1–5% of the tags in each developmental stage. We have also found that Toxoplasma mRNA pools have a unique parasite-specific composition with 1 in 5 transcripts encoding Apicomplexa-specific genes functioning in parasite invasion and transmission. Developmentally co-regulated genes were dispersed across all Toxoplasma chromosomes, as were tags representing each abundance class, and a variety of biochemical pathways indicating that trans-acting mechanisms likely control gene expression in this parasite. We observed distinct similarities in the specificity and expression levels of mRNAs in primary populations (Day-6 post-sporozoite infection) that occur prior to the onset of bradyzoite development that were uniquely shared with the virulent Type I-RH laboratory strain suggesting that development of RH may be arrested. By contrast, strains from Type II-Me49B7 and Type III-VEGmsj contain SAGE tags corresponding to bradyzoite genes, which suggests that priming of developmental expression likely plays a role in the greater capacity of these strains to complete bradyzoite development.
Toxoplasma gondii belongs to the phylum Apicomplexa, which comprises a diverse group of protozoa, considered to share much of the biology underlying obligate occupation of a host cell and responsible for disease in a range of host species. Toxoplasma is distinct from most members of the large coccidian family contained in this phylum owing to the exceptional number of animals that are able to serve as host including virtually all warm-blooded animals. While T. gondii completes the definitive life cycle in a single animal host (feline), the capacity of oocysts (shed from the feline host) as well as tissue cysts to infect multiple hosts has enabled T. gondii to increase the host range for the intermediate life cycle. This rare modification to the heteroxenous (two host) life cycle is thought to have occurred relatively recently and may be responsible for the expansion of this parasite to nearly every continent . Parasite transmission via the oocyst stage has resulted in epidemics of human toxoplasmosis [2–6] and widespread infections of livestock that can also lead to human infections through the consumption of tissue cyst-contaminated food [7, 8]. Together, oocyst and tissue cyst sources contribute to rates of human exposure such that the risk of infection in the U.S. is one in three by age 50 (25% for >20 yrs of age ; and nearly 100% by the end of childhood in other parts of the world ).
Given the importance of Toxoplasma infections to human populations, understanding developmental mechanisms leading to tissue cyst formation is critical for ultimately controlling transmission and chronic disease. Based on cat bioassays, tissue cysts are first detected in mouse tissues approximately one week from the time of oral inoculation of oocyst (containing sporozoites) or tissue cyst material (containing bradyzoites) [11, 12]. The invariant course of T. gondii primary infections in animals suggests that developmental mechanisms initiated by either the sporozoite or bradyzoite stage are similar and are likely the consequence of an unfolding parasite genetic program. Studies of sporozoite- and bradyzoite-initiated development in vitro [13, 14] support this view, as parasites emerging from infections of human foreskin fibroblasts (HFF) follow a defined course of development evident by nearly synchronous changes in growth and stage-specific gene expression that result in the emergence of bradyzoites 7–10 days later [13, 14]. The key to this developmental pathway in T. gondii may lie in a shift to slower growth that occurs following a limited number of divisions in sporozoite-infected cultures, and is well documented in all studies of bradyzoite differentiation [13–17]. The link between cell cycle mechanisms and bradyzoite development is unknown, but is characterized by a transient slowing of S phase that leads to mature bradyzoites, which possess a uniform genome content consistent with cell cycle arrest in G1/G0 (1N DNA content) [13, 14]. These studies suggest that a developmental timer system in primary T. gondii infections (oocyst or tissue cyst) may regulate tissue cyst development in the intermediate host.
The frequency of bradyzoite switching (tachyzoite to bradyzoite, bradyzoite to tachyzoite) varies among Toxoplasma isolates and may influence the level of parasite expansion in animals. As such, defining the changes in gene expression that accompany this development pathway is important for understanding the molecular events that contribute to chronic as well as acute disease. EST projects have been undertaken in Toxoplasma [18, 19] for the purpose of gene discovery, but have also confirmed earlier studies that first demonstrated that novel gene expression is associated with the major intermediate life cycle stages [20, 21]. EST sequencing has led to the development of a limited Toxoplasma cDNA array [22–24] that focused on tachyzoite-bradyzoite transitions in cell culture models of bradyzoite differentiation  and explored gene expression in mutants that are unable to differentiate [23, 24]. It is not possible given the small size of these arrays (~600 genes) to draw global themes about Toxoplasma gene expression; however, these studies are important in that they confirm that changes in mRNA levels correlate with the expression of known bradyzoite protein antigens (supporting transcriptional mechanisms) and provide some evidence that a hierarchal progression of gene expression may govern development in this parasite . At present, the Toxoplasma genome has been sequenced to 10X-coverage of the Me49B7 Type-II strain  with plans to extend coverage to Type I and III strains. Even in the absence of whole genome sequence for all three lineages, a complete map of the parasite transcriptome will allow us to begin analysis of development and inter-strain variation.
In this paper, we report a comprehensive investigation into the whole cell changes in the levels of mRNAs occurring during progression of parasite populations through the T. gondii intermediate life cycle. Additionally, we have examined laboratory strains representing the three major genotypes and demonstrate that specific patterns of gene expression are uniquely shared between laboratory strains and the primary parasite stages characteristic of specific transitions in the T. gondii intermediate life cycle.
Results and discussion
Whole cell analysis of gene expression in the protozoan, Toxoplasma gondii
SAGE Tag Datasets
10 × Gen. Match
No Gen. Match
SAGE tag counts: ALL
Unique SAGE tag counts: ALL
AVG FREQ SAGE tag counts: ALL
SAGE tag counts: 2 × 2
Unique SAGE tag counts: 2 × 2
AVG FREQ SAGE tag counts: 2 × 2
ToxoplasmamRNA pools have a distinctive composition with respect to complexity, stage-specificity and anti-sense transcripts
To estimate gene number represented by the SAGE project, we assembled genome sequence flanking SAGE tags from the 2 × 2 set (8,188 unique tags) using 2,000 bp 5'-upstream of each tag (cap3 assembly). This bracket was conservative with respect to gene overlap given the >4,000 bp average gene size. Combined, contigs and singletons for this assembly comprise 6,372 sequences, which is proportional in size to the total set of 7,793 genes predicted for the whole genome (Mackey and Roos, unpublished results) given that parasite stages from the feline host were not included in the SAGE project. Analysis of the 2 × 2 dataset demonstrated that ~25% of the unique tags annotated with sequence from the NCBI-nr database (representing 50% of the 2 × 2 tag counts) with an average frequency of 50 tags per annotated sequence (Table 1). When comparing this set to the unassembled ESTs (125,769 ESTs) , 44% of the unique SAGE tag sequences matched an EST (80.6% of total tag counts). Roughly 50% of unique SAGE tag sequences (20% by frequency) do not match any EST, likely reflecting the 5' versus 3' preference of these methods as well as the relative greater depth of sequencing in the SAGE project.
The unique composition of Toxoplasma mRNA pools that are populated with apicomplexa-specific genes may be mirrored by the parasite transcriptional machinery, and while the full repertoire of general and specific transcription factors has not been determined for Toxoplasma, it has been noted that sequence divergence of transcriptional activator proteins is strongly correlated to evolutionary distance . This view is supported by the very recent discovery that general transcription factors in Plasmodium are highly divergent when compared to their counterparts in the crown group of eukaryotes [45, 46].
Pairwise comparisons of normalized tag frequencies for all nine SAGE libraries.
The unique tag sequence and polarity with respect to mRNA sequence allows for the determination of global sense and antisense transcripts by placing tags within the orientation of predicted open-reading-frames within the genome. Similar analyses using SAGE tags derived from P. falciparum mRNAs identified antisense transcripts (12%) that were inversely related to the nearest sense transcription . Here, antisense transcription has been independently confirmed , although the function of these transcripts has yet to be established. In order to determine whether antisense transcription was conserved in Toxoplasma, we performed a global analysis of tag frequency (using tags that matched once in the genome) and orientation in comparison to four different predicted gene sets obtained from annotation of the Toxoplasma genome . The range of antisense transcription obtained with each gene set was similar, with an average of 21.5% of the total SAGE tags observed to encode a predicted opposite strand transcript. As was found in P. falciparum , there was a strong inverse relationship between the frequency of antisense transcripts and the level of sense transcription detected within the predicted gene sequence, and as such, the higher a sense transcript was expressed, the lower we observed the level of potential antisense transcription (Fig. 2D). Thus, this unusual feature of transcription in P. falciparum extends to a distantly related member of the Apicomplexa phylum and opens the door to studying this important biological mechanism, taking advantage of the Toxoplasma experimental model.
SAGE tags contain single nucleotide polymorphisms (SNPs) and reflect differential polyA+-choices
The extent of differential splicing or alternative termination of mRNAs in Toxoplasma is largely unknown. Alternate splicing of myosin  and hypoxanthine-xanthine-guanine-phosphoribosyltransferase  transcripts are examples where protein isoforms arise as the result of these mechanisms. The structural analysis of dihydrofolate reductase-thymidylate synthase (DHFR-TS) mRNA expression represents the only case where sites of polyA+-addition have been mapped . The SAGE datasets have the potential to reveal significant information about alternate transcripts provided Nla-III sites (the anchor site for all SAGE tags in these libraries) are available to discriminate mRNA species. To explore the question of PolyA+-choice, we evaluated the clustering of SAGE tags using a 500 bp sequence bracket to gather genome proximal tags around each unique tag. Nearly two thirds of SAGE tags (61%) had no second tag within the 1 kbp sequence window while 20.6% had a second tag and 18.3% had ≥ 2 tags. The largest cluster comprised 10 tags. Interestingly, the tag frequency distributions of specific clusters sometimes varied significantly indicating that the underlying mRNA transcripts might be stage or strain specific. To test whether SAGE tag clusters might reflect differential PolyA+-site addition, we compared the specificity and relative ratio of cDNA fragments generated from 3'-RACE reactions to the nearest corresponding SAGE tag frequency of dense granule protein, GRA7. GRA7 shows a distinct pattern of tags that differentiates the three canonical Toxoplasma strains (Fig. 3C). RACE products generated from these RNA sources match reasonably well the relative SAGE tag patterns with Type I mRNA, yielding two major 3'-RACE products, and Type II RNA and Type III RNA, producing multiple or single RACE products respectively. Sequence analysis of the GRA7 3'-RACE cDNA fragments from all three strains confirmed that they were derived from GRA7 mRNA and correspond to authentic poly-adenylation sites in the GRA7 3'-UTR. Thus, the multiple SAGE tags for GRA7 are not the result of partial digests in SAGE library construction, but reflect true sites of polyA+-addition that are differentially regulated.
Parasites emerging from the sporozoite-infected cell retain significantsporozoite gene expression
Bradyzoite development begins with the post-growth shift at Day-7 following sporozoite inoculation and is accompanied by progressive changes in gene expression
We have previously reported that a slowing of the VEG strain tachyzoite replication rate roughly 20 division cycles after sporozoite-infection of HFF cells precedes the immediate onset of bradyzoite development . We predicted that mRNA pools at Day-7 in the developmental series would reflect the transition of these parasites between rapidly-growing parasites at Day-6 and the full emergence of bradyzoites by Day-15, and in the pH-shifted populations. For this reason, Day-7 post-sporozoite populations were critical to our analyses of the developmental transcriptome because they represent the earliest observation of the growth-shifted population that ultimately commits the parasite to bradyzoite differentiation. In characterizing the differences between Day-7 and Day-6 mRNA pools, it was evident that nearly 1,181 SAGE tags in the Day-7 population were altered from the Day-6 population. Approximately 750 and 420 of all Day-7-tags were up-regulated or down-regulated (± 2.5-fold), respectively, when compared to the frequency of SAGE tags in the Day-6 population. From the up-regulated tags in the Day-7 library, 490 tags were completely absent from the Day-6 library, and conversely, 251 tags that were down-regulated in the Day-7 population were also absent in Day-6. Figure 4C displays all SAGE tags that were observed to be Day-7-specific in comparison to the other five developmental libraries without regard to the three laboratory strains.
Congruent with the observed induction of the slow growth that leads to initiation of the bradyzoite development pathway, levels of some mRNAs encoding proteins necessary for nuclear replication and cell division were reduced in the Day-7 population. For example, the mRNA encoding PCNA1 (down 3-fold)  and the mRNAs for DHFR-TS and the adenosine transporter – each providing dTTP and dATP, ultimately to be used in DNA synthesis  – were down-regulated in the Day-7 population. Consistent with the reduced replication rate at Day-7, both Rab5 and Rab11, which were up-regulated in the Day-6 population, were down-regulated 4- and 3-fold, respectively, suggesting a reduced need for endocytosis and cytokinesis at this stage of development [57, 58]. We also observed reduced levels of mRNAs encoding many of the energy-related proteins up-regulated in the rapidly growing Day-6 population. For example, we observed a distinct reduction in the level of mRNAs encoding fructose-1,6-bisphoshatase , GAPDH and 3-phosphoglycerate kinase. We also observe the down-regulation of mRNAs encoding several molecules in mitochondrial electron transport including cytochrome b (down 8-fold) and cytochrome c oxidase (down 6-fold).
The presence together of tachyzoite and some bradyzoite specific markers in the Day-7 population is likely reflective of their position between two distinctly different stages of development, the rapidly replicating tachyzoite and the slowly growing or growth arrested bradyzoite. However, there is little expression of the most well studied bradyzoite genes (Day-7 vs. pH-shift libraries r = 0.0888, Table 2) and this is consistent with earlier measurements of BAG1 protein in these populations (2% BAG1;). Marker co-expression extends to the tachyzoite-specific surface antigen SAG1 (up 20-fold) [60, 61] and the mRNA encoding MIC10 (up 8-fold), while the increased level of the mRNA encoding the ROP4 protein (up 4-fold) is consistent with reports of higher levels of this transcript in the bradyzoite stage . The appearance of the mRNA encoding hsp90 in the Day-7 population, which has previously been demonstrated to be up-regulated in the slow or non-replicating bradyzoite form , suggests that this mRNA may be an early bradyzoite marker. It is intriguing that drugs that target hsp90 prevent bradyzoite differentiation in laboratory strains , indicating an important role for this factor in initiating bradyzoite development.
Based on SAGE data, parasites from Day-15 post-sporozoite infections were a mixture of tachyzoite and bradyzoite forms with reduced growth rates  that express additional bradyzoite markers. Thus, for the first time in this population, we observed SAGE tags corresponding to bradyzoite markers SAG4.2  and ENO1 , and although tachyzoite genes such as SAG1 and SAG2  remain present, they show a decreased expression of these mRNAs compared to Day-7 parasites, indicating that the switch to the bradyzoite stage is continuing to progress. Figure 4D shows bradyzoite-specific genes that are shared between Day-15 and pH-shifted populations. The pH-shifted parasite populations represent the best-defined bradyzoite phenotype we can achieve in tissue culture host cells. These mRNA pools were representative of a definitive phenotypic change in the growth and development of VEG strain parasites along the life-cycle continuum in the intermediate host . Similar to the significant changes observed in the Day-7 to Day-15 transition (15%), we observed that 1,441 of the SAGE tags analyzed were altered by more than 2.5-fold from the Day-15 population to the mature bradyzoite induced by alkaline stress (Fig. 4D). In this comparison, 695 tags analyzed were up-regulated in the pH-shifted library, while 746 were down-regulated. As expected, genes associated with cellular growth continue to be down-regulated in the pH-shifted library, consistent with the arrest of growth in these populations and the G1/G0 state of parasites from in vivo cysts [14, 26]. We observed a dramatic disappearance of the mRNA encoding the inner membrane complex protein IMC-1 [63–65], which, notably, was also absent from the growth arrested sporozoite stage. Levels of mRNAs encoding other microtubule and/or cytoskeletal proteins including β tubulin [66, 67] and various myosin precursor(s) were also down from the levels observed in the Day-7 and Day-15 parasite populations. Genes encoding DHFR-TS and PCNA1 and 2, which are up-regulated in the Day-4 and -6 populations, were also expressed at lower levels in the pH-shifted population as they were in the Day-7 population.
The unique list of up-regulated genes in the Day-15/pH-shifted libraries (graphed in Fig. 4D) included most of the well studied markers of bradyzoite differentiation. The Toxoplasma mRNA encoding lactate dehydrogenase 2 (bradyzoite-specific) , was present (while lactate dehydrogenase 1 was reduced 8-fold), as was mRNAs encoding Hsp30/BAG1 (7-fold up), p18 (SAG4.2) bradyzoite surface antigen, and Toxoplasma enolase 1, with all of these mRNAs substantially higher in the pH-shifted population when compared to Day-15. NTPase 1 and 3 mRNAs were down-regulated while a novel mRNA encoding a bradyzoite-specific NTPase (brady-NTPase) was observed to be dramatically up-regulated. The gene encoding Brady-NTPase (chr. X, TGG_994683) contains a single exon of 645 amino acids (~70 kDa) and is related (41% identity) to other well studied NTPases [69, 70]. SAGE tag frequencies for Brady-NTPase indicates that it is an abundant mRNA in the pH-shift library, and we have confirmed mRNA expression by RT-PCR and the presence of bradyzoite-specific cis-elements in the 5'-intergenic region flanking this novel NTPase (Radke and White, unpublished). The NTP3 promoter contains tachyzoite-specific cis-elements , and thus Brady-NTPase may represent a bradyzoite-specific isoform of this enzyme.
Laboratory adapted parasite strains possess gene expression that is characteristic of specific points in the parasite developmental pathway
Comparisons of SAGE datasets from the Type I, II and III laboratory strains with the VEG sporozoite-developmental series described above demonstrate correlations that may be associated with the capacity of each strain to differentiate. As expected, few of the genes specifically regulated in sporozoite/Day-4 populations were shared with the three tissue culture-adapted strains (Fig 4A), especially Type I and II strains. By contrast, a significant number of genes up-regulated in the Day-7 post-sporozoite populations were also found to be expressed at higher levels in all three lab strains (Fig 4B). Comparative similarity in mRNA pools was observed to rapidly diverge when the laboratory strains were compared to populations prior to and following the initiation of bradyzoite differentiation (Day-15 and pH-shifted populations). There is a striking correlation in the specificity and expression levels of a set of up-regulated SAGE tags (120 tags, Fig. 4B) specific for the Day-6 post-sporozoite populations that are also expressed in the RH laboratory strain (0.785 correlation), but largely not observed in the other developmental populations or the other laboratory strains. This unique relationship in gene expression may reflect a shared biology; Day-6-VEG populations, like RH parasites, lack any evidence of sporozoite or bradyzoite mRNA expression and grow with a similarly fast doubling time [13, 26]. In contrast, SAGE libraries constructed from Type II-Me49B7 and Type III-VEGmsj parasites do not have elevated Day-6 SAGE tags (0.343 and 0.049 correlations, respectively, Table 2), but unlike the RH/Day-6 datasets, SAGE tags corresponding to bradyzoite genes are found in these libraries (e.g. basal levels of SAG4.2, Brady-NTPase and ENO1 are detected; see Fig. 4D). Baseline expression of bradyzoite genes might be the result of a minor population that had differentiated, although we have not observed this population in Type III-VEGmsj using known bradyzoite markers (not shown). Interestingly, elevated basal expression of bradyzoite genes has also been recently detected in the microarray analysis of avirulent laboratory strains that are developmentally competent, but presumed to be growing as tachyzoites (Boyle and Boothroyd, unpublished). The basal expression of bradyzoite genes that we and others have observed is likely to be related to the greater capacity of VEGmsj and Me49B7 parasites and other competent strains to enter the bradyzoite developmental pathway in vitro (and in animals), and thus the mRNA patterns in laboratory-adapted strains appear to mirror characteristics from the natural development pathway with their comparative position with respect to Day-7 post-sporozoite populations. Strains of which the mRNA profile is more consistent with development patterns that are earlier than Day-7 parasites, such as RH, may be more removed from bradyzoite differentiation, while gene expression profiles consistent with populations that have progressed beyond Day-7 (i.e. evidence of basal bradyzoite gene expression) may indicate that the parasites are primed to enter the bradyzoite developmental pathway. The level of annotation in the RH/Day-6 mRNA cluster is low (~80% are unique), providing relatively few clues to the biochemical pathways that are transiently expressed during VEG development but in RH are permanently activated. It is notable, however, that SAGE tags in this cluster correspond to ROP1 transcripts given that a minor QTL associated with RH acute virulence has been closely mapped to this locus in the T. gondii genome . In addition, DHFR-TS is also found in this pool, which may be consistent with the well known relationship between DHFR and eukaryotic replication. It is likely not a coincidence that a single QTL associated with parasites that display the characteristic rapid cell cycle rates of Type I strains has been preliminarily mapped to the DHFR-TS locus on chromosome XII (Jerome, Behnke and White, unpublished results). Thus, the 120 Day-6/RH-specific SAGE tags may represent an important mRNA group that is associated with the virulent and rapidly growing RH phenotype, and the down-regulation of these mRNAs during development adds support to the concept that developmental gene expression in T. gondii determines the observed stage-specific phenotypes that change in a predetermined hierarchical order .
Parasite culture and isolation of total RNA
RH strain tachyzoites were maintained by serial passage in human foreskin fibroblasts (HFF) and these cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL, Grand Island NY) supplemented with 1% (v/v) newborn calf serum (Hyclone Laboratories Inc., Logan UT) as previously described . To harvest total RNA for SAGE library construction, parasites were scraped from cultured cells, needle-passed and filter-purified from host cell debris using a 3 μM nucleopore membrane. Parasites were pelleted and total RNA was extracted from the pellet twice using 10 and then 5 ml of TRIzol according to the manufacturer's protocol (Gibco BRL, Rockville MD). Total RNA was also isolated from VEG strain oocysts that were obtained by sucrose flotation from cat feces as previously described [53, 83].
SAGE library construction
SAGE libraries were constructed from cDNA that was synthesized from 1 μg of total RNA using the Smart™ cDNA synthesis reagents (Clontech, Palo Alto, CA). Briefly, a biotinylated oligo-dT primer (5'-AAGCAGTGGTAACAACGCAGAGTAC(T)30VN-3' where V = A, C or G, and N = T, C, G or A) and Superscipt II reverse transcriptase (Life Technologies, Framingham MA) were used to make 1st strand cDNA. Second-strand synthesis and cDNA amplification was completed by PCR using the Advantage-2 Polymerase Mix (ClonTech, Palo Alto CA), and a switching-primer (5'-AAGCAGTGGTAACAACGCAGAGTACGCGGG-3') in combination with the original biotinylated oligo-dT. Approximately 22 amplification cycles provided 10 μg of double-stranded cDNA used to construct(s) the library of SAGE tags according to standard protocols [27, 29, 84]. To improve the cloning efficiency in the final stage of construction, we separated SphI-digested DNA by cDNA size-exclusion chromatography (manual #235612, Stratagene, La Jolla, CA) using the Sepharose CL-2B matrix (450 bp minimum cutoff, Sigma-Aldrich, St Louis, MO). We have determined that this method will cleanly separate linear cDNA fragments from circular DNA that forms during concatemerization and are not linearized in the SphI digestion (and are therefore unclonable; Radke and White, unpublished). Concatemer fragments eluted from the column were collected in twelve 100 μl fractions and the 1 to 2 kbp fragments were isolated from fractions 4, 5 and 6 by overnight precipitation in ethanol at -20°C. Purified fragments were cloned into pZero plasmid vector (Invitrogen, Carlsbad CA) that had been linearized with SphI. Following electroporation of DH10B cells (Invitrogen, Carlsbad CA), transformed colonies were plated on low LB-agarose containing zeocin (50 μg/ml) and picked for sequence analysis by standard methods.
SAGE tag extraction and construction of SAGE datasets
Delineation of sequence, extraction of SAGE tag information, frequency analyses and tag sequence annotation were all completed using Perl 5.6.1  running in a UNIX RedHat 7.2 environment  with Perl scripts written and developed in this laboratory. Each cloned concatemer contains nucleotide sequence of repeating units of SAGE ditags, separated by a single NlaIII restriction endonuclease consensus sequence (CATG). We extracted SAGE tag sequences using CATG landmarks and the regular alternating 28 to 33 base nucleotide sequence defining each ditag. Ditag sequence was processed using software previously developed to extract individual SAGE tag information, record tag frequency and correct sequence error in the raw dataset by nearest neighbor analysis . Tag frequencies for each library were normalized by multiplying the tag count by the ratio of adjusted library size, divided by the actual size – where the adjusted size was equal to 50,000 tags.
The resulting dataset was stored and organized using MySQL  with web-based access via the Apache web server . Queries of raw SAGE tags or those corrected for sequencing error, normalized or annotated tags (as defined by BLAST score), may be performed at TgSAGEDB . Queries to TgSAGEDB datasets result in an ordered list of SAGE tags ranked by frequency and library choice with individual frequencies displayed across columns for each of the nine SAGE libraries. Dynamic links within the page connect the individual to the position of each tag within the Toxoplasma chromosome maps  or within the assembled genomic contigs and linkage to ApiDots  via tag sequence cross-connects SAGE tags to the Toxoplasma EST collection. Each tag in the results page is linked to other possible positions in the Toxoplasma genome (the initial 2 × 2 choice limits this to ≤ 2 hits), or to nearest neighbor tags (which identifies putative SNPs) that differ by a single nucleotide. Tag clusters are displayed in the Toxoplasma genomic contigs via a defined bracket set by the user (2 kbp, 5 kbp or 10 kbp on either side of the specific tag chosen). A link to the 2 kbp genomic sequence immediately adjacent to a tag and in the same strand orientation is provided along with information on tBLASTx annotations (if any). A final link takes the individual to a gene ontology site where BLAST results may be reviewed with respect to GO assignments. From the Toxoplasma GO database the individual can link back to the SAGE results via related gene products in order to assess co-regulation of specific pathways.
SAGE tags and their normalized frequencies were imported into GeneSpring 7.2 (Agilent Technologies Inc., Palo Alto, CA) used for additional analyses including the generation of standard correlations between SAGE library datasets. GeneSpring export file(s) can be downloaded from the TgSAGEDB website . Gene expression comparisons across developmental and strain libraries were performed in GeneSpring 7.2 by filtering gene lists by expression level in the 2 × 2 dataset normalized with ratio mode (signal/control). These gene lists were compared by clustering analysis using Pearson correlation as a similarity measurement. Standard k-means clustering using 100 iterations and k = 7 (k>8 resulted in progressive separation of genes into redundant clusters) generated gene lists that were highly similar to those generated by the change in fold expression (results based on fold change are shown in Fig. 4).
In order to annotate SAGE tag sequence(s), and assign gene function where available, tag sequences were compared to the 10 × Toxoplasma gondii genome obtained from the Me49B7 strain. For exact sequence matches in either strand (+ or -), the matching genomic contig number, sequence position in the contig and + or - strand orientation were recorded. Since the tag sequences have a greater bias toward the 3' end of the mRNA, we extracted 2,000 nucleotides directly 5' of each SAGE tag in the contig in order to associate a larger portion of the potential coding sequence with each tag. This dataset was blasted locally against the non-redundant database of protein sequences (nr, NCBI) using the BLASTall/BLASTx program. Each sequence was annotated using the BLAST alignment with the lowest expected value (≤ 1 × 10-6) in those alignments where the reading frame was in the positive orientation. Additionally, the ten best alignments that met these criteria were also associated with the sequence in order to provide expanded annotation information. To estimate the number of unique transcripts in the SAGE dataset (accounting for multiple polyA+-additions), we used cap3  to assemble the 2,000 nucleotide sequences 5' to each tag. Total assembled contigs and singletons (individual sequences that did assemble into any one contig) were used to predict the number of unique transcripts in the SAGE dataset.
To determine the presence and level of potential antisense transcription, SAGE tags that matched the genome once and had a sum frequency of = 2 across all libraries were matched to predicted gene annotation. Four distinct gene prediction datasets (GeneFinder, TwinScan, TigrScan and Glimmer) were available for comparison from ToxoDB [25, 92]. In these comparisons, for each predicted gene, tags matching the (+) strand were defined as 'sense' and those matching on the (-) strand 'antisense'; and frequencies were recorded separately.
SNP and polyA+ analysis
SNP analyses were completed using the EMBOSS program 'fuzznuc' [93, 94] to identify individual SAGE tag sequences with a 1 nucleotide mismatch where present in the dataset. A single base mismatch across tag sequence(s) from one or more strain types was defined as a potential SNP. To test whether these mismatched bases would accurately identify the presence of a real SNP, predicted tags where the single-base mismatch occurred in the consensus sequence for a unique restriction endonuclease were identified and flanking primers used to amplify these regions from genomic DNA by polymerase chain reaction (PCR). Two such Type II strain tag sequences were selected for this analysis: (1) the SUL1 (MnI) SAGE tag, CATGACATCGAGGA (present at nucleotide position 332,392 in genomic contig TGG_994387), and (2) the AcI I-SNP tag CATGCTCCGCTACA (nucleotide position 39,009, contig TGG_994300). These were amplified separately from Type I (RH), II (Me49B7) and III (VEG) strain genomic DNAs (MnI; F-AAGAGTGTGCTTTGGTGCCTTATTG and R-ACACTCCGCGATTGCACACAC, and AcI; F-GCCTCGCACAATCACTGGTTTGAC and R-CGGAAGAGGAAGAAGTTGCCG). Amplified products were purified by spin column according to standard protocols (Stratagene, La Jolla, CA) and digested with the appropriate restriction enzyme (AcI or MnI), and fragments were resolved by 6% polyacrylamide gel electrophoresis.
To assess differential polyA addition to mRNAs encoding GRA7, total RNA was isolated by spin column (QIAGEN, Alameda, CA) from each of the three strain types (as described above) and subjected to 3'-RACE  using a modified oligo-d(T) primer, containing attB2 recombinational cloning sequence, and a GRA7-specific primer containing the attB1 sequence  5'-GGGGACCACTTTGTACAAGAAAGCTGGGTC(T)30VN-3' and GRA7 5'-GGGGAGAAGTTTGTACAAAAAAGCAGGCTTCCGAGCAAGAGGTGCCTGAATCAGG-3'). Amplified products from each PCR reaction were resolved by 6% polyacrylamide gel electrophoresis. RACE products from three distinct cycle profiles (15, 20, 25 cycles) were compared to verify that the relative abundance of the amplified products was not influenced by cycle number (not shown). Additionally, amplified products were cloned by recombination into p221DONOR vector (Gateway™ Invitrogen Co., Carlsbad, CA) and sequence analyses used to verify the GRA7 product. To demonstrate the distance from the NlaIII restriction endonuclease consensus sequence (in the nearest upstream SAGE tag) to the polyA+-addition site(s), 3'-RACE products were compared to that predicted using the distance between this NlaIII site and the GRA7-specific primer. All predicted fragment lengths were compensated to include the 90 additional nucleotides contributed by the attB1 and attB2-oligo-d(T)30 primer sequences incorporated into the RACE fragments.
This work was supported in part by grants from USDA-CRS and the NIH to MWW, DSR and JRR including COBRE NCRR P20 RR-020185 and R21 AI53815. A special thanks to Matthew White and Maria Jerome for parasite culture and molecular biology support and to Dr. Bill Sullivan for contributions to the editing of this manuscript. Preliminary genomic and/or cDNA sequence data were accessed via http://ToxoDB.org and/or http://www.tigr.org/tdb/t_gondii/. Genomic data were provided by The Institute for Genomic Research (supported by the NIH grant #AI05093), and by the Sanger Center (Wellcome Trust). EST sequences were generated by Washington University (NIH grant #1R01AI045806-01A1). This manuscript is a contribution from the Montana State University Agricultural Experiment Station.
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