In Vivo Transcriptome of Plasmodium falciparum Reveals Overexpression of Transcripts That Encode Surface Proteins

Abstract

Infections with the human parasite Plasmodium falciparum continue to present a great challenge to global health. Fundamental questions regarding the molecular basis of virulence and immune evasion in P. falciparum have been only partially answered. Because of the parasite's intracellular location and complex life cycle, standard genetic approaches to the study of the pathogenesis of malaria have been limited. The present study presents a novel approach to the identification of the biological processes involved in host-pathogen interactions, one that is based on the analysis of in vivo P. falciparum transcripts. We demonstrate that a sufficient quantity of P. falciparum RNA transcripts can be derived from a small blood sample from infected patients for whole-genome microarray analysis. Overall, excellent correlation was observed between the transcriptomes derived from in vivo samples and in vitro samples with ring-stage P. falciparum 3D7 reference strain. However, gene families that encode surface proteins are overexpressed in vivo. Moreover, this analysis has identified a new family of hypothetical genes that may encode surface variant antigens. Comparative studies of the transcriptomes derived from in vivo samples and in vitro 3D7 samples may identify important strategies used by the pathogen for survival in the human host and highlight, for vaccine development, new candidate antigens that were not previously identified through the use of in vitro cultures.

The human parasite Plasmodium falciparum continues to pose a great challenge to the health of most of the world's population and to the economies of most of the world's countries [1, 2]. Patients infected with P. falciparum present with a range of outcomes, from asymptomatic parasitemia to severe disease and death. The host and parasite factors that mediate the severity of disease are only partially defined [3–9]. One approach to the identification of parasite virulence factors is the characterization of in vivo parasite biological processes. It has been demonstrated that malarial transcripts encoded on chromosome 2 from P. falciparum–infected patients' blood samples can be reliably assessed, despite the abundance of human RNA [10]. The present study reports important differences in the expression of genes detected in freshly obtained in vivo samples and the in vitro 3D7 transcriptome and provides a new approach to the study of the pathogenesis of malaria.

Patients, Materials, and Methods

Patients and samples

During transmission season in October 2003, patients with malaria were recruited from a clinic in a region of Senegal where P. falciparum is hypoendemic. The present study was conducted as part of an ongoing drug-resistance study that has been described elsewhere [11] and was approved by the institutional review boards at the Harvard School of Public Health and Cheikh Anta Diop University. Children and adults who presented with fever and whose blood smears were positive for P. falciparum were enrolled in the study. Blood (5–15 mL) was drawn into EDTA-coated Vacutainer (Becton Dickinson) tubes and, to collect serum and buffy-coat deplete, was centrifuged. RNA was stabilized within 10 min by use of 4.5 volumes of Tri Reagent BD (Molecular Research Center). Samples were stored at −80°C until RNA extraction was completed, as described elsewhere [10].

DNA extraction

Venous blood was applied to IsoCode paper for each sample, and DNA was later extracted in accordance with the manufacturer's instructions (Schleicher and Schuell). To determine the number of clones infecting each patient, genotyping was performed for the merozoite surface protein (MSP) 1 and MSP 2 alleles by use of polymerase chain reaction (PCR) with nested primers under standard PCR conditions [12]. Samples with late-stage parasites, as detected by microscopy, whose species could not be determined morphologically were subjected to PCR with species-specific 18S rRNA primers [13].

Oligonucleotide array analysis

To determine whether a sufficiently abundant number of mRNA transcripts could be identified in in vivo samples, the 5 samples that had the largest volume of blood and the highest parasitemias were analyzed by use of an oligonucleotide array, which contained probes that are based on the sequence of 3D7, the laboratory-adapted P. falciparum reference strain for which the complete genome is available [14]. Total RNA was extracted by use of Tri Reagent BD, in accordance with the manufacturer's instructions. Labeling and hybridization of total RNA was performed as described elsewhere [15].

The match-only integral distribution algorithm was used to assess the expression level for each transcript [16]. Background was assessed on the basis of the probe intensities of 100 negative control genes. Only those probe sets that had >10 probes per gene and that had a signal that was ≥ 1.5-fold higher than background were analyzed. In addition, a transcript was considered to be present if the expression level was >10 expression units and its probe signal distribution had log P < −.5. After background was subtracted, the average intensity of genes between the 30th and 90th percentiles were normalized to 200 expression units across all samples, to allow comparison [15].

We compared the expression level of each transcript in the in vivo samples and each life-cycle stage from the previously reported 3D7 reference strain cell cycle transcriptome by use of a Spearman's rank sum correlation coefficient [15]. Genes were considered to be overexpressed in vivo when the in vivo expression levels were ≥ 2-fold greater than those derived from in vitro 3D7 samples with parasites of all life-cycle stages. The statistical significance of differences between the number of transcripts expressed in each gene family for each in vivo sample compared with in vitro 3D7 was tested by use of Fisher's exact test (Stata; version 7.0; Stata Corporation).

In addition to testing for significant differences in mRNA levels for individual genes between the in vivo and in vitro 3D7-derived RNA, genes grouped on the basis of function were evaluated. To do this, the gene ontology (GO) annotation (http://www.geneontology.org) and malarial metabolic pathways from PlasmoDB (http://www.plasmoDB.org) were downloaded into Genespring (version 6.1; Silicon Genetics). A list that contained genes that were at least 2-fold overexpressed, compared with expression in in vitro 3D7 samples, was generated for each in vivo sample. These lists were tested for overlap with malarial metabolic pathways and GO annotations by use of a hypergeometric distribution equation (Bioscript Library; version 2.0; Silicon Genetics), with P < .05 considered to be significant.

Real-time reverse-transcriptase (RT) PCR

To confirm the overexpression of PF14_0752 in the in vivo samples, real-time PCR quantification of cDNA from in vivo and in vitro 3D7 samples was performed. cDNA was generated from the in vivo isolated RNA of samples 12 and 29 and of 2 additional samples, 35 and 43, that had been collected from children with similar symptoms of mild malaria. 3D7 RNA was obtained from sorbitol synchronized parasites in in vitro culture containing 10% serum under standard conditions and total RNA was isolated at ring stage by use of Tri Reagent BD [17]. cDNA was synthesized from all samples by use of the SuperScript First-Strand Synthesis System (Invitrogen). One aliquot of total RNA from each sample was treated without RT. cDNA from these 4 in vivo samples and from samples with 3D7 ring-stage parasites was subjected to PCR with primers for PF14_0752 (forward, 5′-GAATTTAAAATGACGGAGGATTGTT-3′; reverse, 5′-AAGATCTAGTATGTTCGGTTTCATT-3′). PFB0120w was used as a loading control for parasite RNA, since it demonstrated similar expression under in vivo and in vitro conditions (forward, 5′-CAGCCCTCTTAGCTCTCAACTTC-3′; reverse, 5′-AGCAACAGCAGAGGCTATAGAACT-3′). Standard curves generated from genomic DNA were used to quantify cDNA in each sample and are reported relative to PFB0120w. Duplicate reactions were analyzed for each sample by use of real-time PCR with 1 μL of cDNA, gene-specific primers, and the fluorescent dye SYBR Green (SYBR Green PCR Master Mix; Applied Biosystems) in a 50-μL reaction volume. For each sample, real-time PCR quantification of cDNA generated without RT was subtracted as background. The reactions were performed by use of an ABI Prism 7700 sequence detector (Applied Biosystems).

Results

To assess steady-state mRNA levels of P. falciparum in vivo, blood samples were obtained from 5 P. falciparum–infected patients with fever, symptoms compatible with mild malaria, and a parasitemia >1% who were evaluated at an outpatient clinic in Senegal, and the blood samples were analyzed by use of microarray-based hybridization (table 1). Microscopic examination demonstrated early ring-stage parasites in all blood smears. Sample 7 also contained rare schizont forms (<0.1% of parasite forms). Total RNA was isolated from the blood samples, fluorescently labeled, and hybridized to an array of high-density oligonucleotides [15].

Table 1. Patient and isolate characteristics of in vivo samples.

	Sample
Category, variable	6	7	8	12	29
Patient characteristic
Age, years	10	26	10	10	12
Sex	Male	Male	Male	Female	Male
Time symptomatic, days	3	3	3	3	4
Temperature, °C	39	39	38.2	39.6	38
Treatment history	Paracetamol	None	None	Chloroquine	Paracetamol
Parasitemia, %	1.8	7	1.4	3.4	8
Packed volume, mL	2	3	2	4	2
Isolate characteristic
No. of transcripts	1872	2988	2197	2140	2159
No. of transcripts overexpressed	59	553	120	124	28
No. of clonesa	1	2	1	2	1

^aNo. of clones was determined by merozoite surface protein (MSP) 1 and MSP 2 typing.

In vivo transcript detection and correlation to the 3D7 reference strain

Between 1872 and 2988 transcripts were detected in each of the in vivo samples (table 1). The expression level of each transcript was normalized between all samples and ordered from highest to lowest, to derive a transcriptome that reflects steady-state mRNA expression. Comparison of the rank correlation of the transcriptome in the in vivo samples (samples 6, 8, 12, and 29) with each life-cycle stage of the 3D7 strain grown in vitro demonstrated the highest similarity with early ring stages (correlation coefficient, 0.80–0.93) (figure 1). This is consistent with the observation that ring-stage parasites are predominant in peripheral blood of P. falciparum–infected patients. These 4 samples also had a high correlation with the 3D7 in vitro merozoite stage (correlation coefficient, 0.75–0.85) and the late schizont stage (correlation coefficient, 0.74–0.80). Sample 7 had a lower correlation with all life-cycle stages; its highest correlation was with the late schizont stage.

Figure 1.

Correlation coefficients comparing the transcriptomes of each in vivo sample with 3D7 stage-specific transcriptomes. ER, early ring; ES, early schizont; ET, early trophozoite; LR, late ring; LS, late schizont; LT, late trophozoite; M, merozoite.

The results suggest that the majority of parasite genes expressed in in vitro 3D7 samples are also expressed in vivo. In addition, the steady-state levels of mRNA for most genes are similar. This analysis suggests that the in vivo biological processes of field isolates are very similar to those of the 3D7 strain grown in laboratory culture.

Unique in vivo parasite gene expression

Although expression of the majority of genes was well correlated between the in vivo samples and in vitro 3D7 culture, we nevertheless were able to identify a number of parasite genes displaying a ≥ 2-fold expression level in vivo, compared with that in samples with all in vitro 3D7 life-cycle stages. The number of overexpressed genes ranged from 28 to 553 for each sample. These included genes that encode membrane proteins, proteins involved in metabolic processing, and hypothetical proteins (tables 2–7).

Table 2. Genes that were at least 2-fold overexpressed in vivo, compared with expression in in vitro 3D7 samples with parasites of all life-cycle stages.

		Expression units
		In vivo					In vitro 3D7
Gene	Description	6	7	8	12	29	M	ER	LR
PF11_0512a	RESA-2	530	156	379	443	626	0	0	0
PFC0050ca	Long-chain fatty acid ligase, putative	617	186	192	73	92	0	0	0
PFD0065wa	STEVOR, pseudogene	94	276	233	165	285	31	0	0
PF11_0006a	Hypothetical protein	173	1188	142	295	178	0	0	0
PF14_0116a	Hypothetical protein	16	19	13	12	15	0	0	0
MAL8P1.22	Dehydrodolichyl diphosphate synthetase, putative	26	130	20	0	14	0	0	0
PF14_0004	RIFIN	77	164	127	77	48	18	0	17
PFI0030c	RIFIN	124	185	156	120	53	33	16	21
PFI0485c	Conserved in Plasmodium yoelii, SET domain	55	289	98	144	0	0	0	0
PF14 0752	Conserved regions in other hypothetical proteins (figure 2)	837	0	314	569	351	44	76	23
PFA0720w	Hypothetical protein	31	365	86	116	0	0	0	0
PFL2525c	Hypothetical protein	38	182	25	88	0	0	0	0

NOTE. ER, early ring; LR, late ring; M, merozoite.

^aGenes overexpressed in all 5 in vivo samples; other genes were overexpressed in 4 of 5 samples.

Table 7. Sample 29 genes with transcript levels at least 2-fold greater than those in in vitro 3D7 samples.

Gene	Description	Expression units
		In vivo					In vitro 3D7
		6	7	8	12	29	M	ER	LR
Surface protein
PF10_0003	RIFIN	144	0	0	215	384	52	0	0
PF11_0512	RESA-2	530	156	379	443	626	0	0	0
Metabolic/cellular function
PF13_0045	40S ribosomal protein S27, putative	925	511	1133	1248	1431	0	342	483
PFC0050c	Long-chain fatty acid ligase, putative	617	186	192	73	92	0	0	0
MAL8P1.22	Dehydrodolichyl diphosphate synthetase, putative	26	130	20	0	14	0	0	0
MAL8P1.37	Lipoate-protein ligase, putative	10	0	0	11	13	0	0	0
Hypothetical protein
PF14_0753	Hypothetical protein	2110	432	660	1539	1655	664	323	62
PFI1640c	Hypothetical protein	695	167	350	380	876	0	130	96
PFB0255w	Hypothetical protein	585	0	0	484	825	0	347	0
PFA0700c	Hypothetical protein, conserved in P. falciparum	331	0	0	0	818	0	0	0
PF14_0734	Hypothetical protein	196	87	69	256	537	18	102	37
MAL7P1.133	Hypothetical protein	338	0	154	317	361	0	82	0
PF14_0752	Hypothetical protein	837	0	314	569	351	44	76	23
PFD0065w	Hypothetical protein	94	276	233	165	285	31	0	0
MAL8P1.160	Hypothetical protein	214	0	0	225	207	80	38	0
PF13_0210	Hypothetical protein	75	0	77	145	188	0	47	33
PF11_0006	Hypothetical protein	173	1188	142	295	178	0	0	0
PF11_0123	Hypothetical protein	0	106	0	72	128	0	37	0
PF11_0003	Hypothetical protein	0	764	0	232	117	0	0	0
PF10_0044	Hypothetical protein	0	152	0	0	76	0	0	21
PFC0405c	Hypothetical protein	0	0	0	0	41	0	0	0
PF10_0139	Hypothetical protein	0	242	0	0	38	0	0	12
PFL2020c	Hypothetical protein	0	0	11	0	19	0	0	0
PF14_0116	Hypothetical protein	16	19	13	12	15	0	0	0
PFE0230w	Hypothetical protein	10	0	0	12	15	0	0	0
PF14_0720	Hypothetical protein	17	100	0	0	15	0	0	0
PFL0985c	Hypothetical protein	0	24	11	0	12	0	0	0
MAL6P1.211	Hypothetical protein	0	15	0	13	11	0	0	0

NOTE. ER, early ring; LR, late ring; M, merozoite

We wanted to determine whether the genes overexpressed in vivo could be correlated to any malarial metabolic pathways or specific gene functions. Therefore, the list of overexpressed genes from each in vivo sample was tested for overlap with the malarial metabolic pathways annotated in PlasmoDB and with gene function families by use of the GO annotations. Analysis of these differentially expressed genes did not reveal statistically significant overrepresentation of genes in any malarial metabolic pathways. However, overrepresentation was observed in the GO plasma membrane category for samples 8 (18 genes; P = 9.18 × 10⁻⁷) and 12 (19 genes; P = 3.2 × 10⁻⁷). This plasma membrane cellular component category includes genes that encode surface antigens such as rifins and vars that are known to play important roles in infection and pathogenesis.

We then examined individual genes that were commonly overexpressed in all in vivo samples, compared with expression in vitro 3D7 samples. Three genes—RESA-2 (PF11_0512), a putative long-chain fatty-acid ligase (PFC0050c), and a stevor (PFD0065w)—were overexpressed in all in vivo samples (table 2). RESA-2 was not detected in any 3D7 stage or in previous in vitro studies of laboratory-adapted strains [18, 19]. However, RESA-2 transcript was detected in freshly obtained samples from infected patients in French Guiana [20]. This gene is a homologue of RESA (PFA0110w), which is a ring-stage surface antigen and a potential vaccine candidate [21]. Other genes overexpressed in 4 of the 5 samples include a putative diphosphate synthetase (MAL8P1.22) and 2 additional genes that encode rifins (PF14_0004 and PFI0030c).

Putative novel gene family

Five of the 12 genes we identified as being overexpressed in vivo were hypothetical proteins that did not have significant homology to other proteins of known function. The National Center for Biotechnology Information TBLASTN program was used to further characterize these genes. TBLASTN analysis revealed that PF14_0752 demonstrated high amino acid homology to 9 other P. falciparum hypothetical proteins (figure 2) [22]. Eight of these 10 homologous genes are predicted to encode proteins with a single transmembrane domain. Nine of the 10 genes are located in the subtelomeric regions of chromosomes. Whole-genome single nucleotide polymorphism analysis showed that 7 of the 10 genes are within the top 10% of all genes with respect to allelic variation (C. Kidgell, J. Borevitz, J. Johnson, S. Volkman, D. Plouffe, K. Le Roch, D. Wirth, Y. Zhou, and E. Winzeler, unpublished data). This is consistent with polymorphic DNA sequences found in other genes that encode surface proteins [23]. Recently described host targeting motifs that are present in proteins found at the red cell surface in infected erythrocytes are present in the majority of these sequences [24, 25]. The overexpression of PF14_0752 in vivo was verified by real-time PCR of cDNA from 4 in vivo samples (figure 3). An 8–28-fold overexpression was observed relative to that in samples with ring-stage 3D7 parasites. These results imply that this putative gene family encodes surface transmembrane proteins similar to genes in the GO function plasma membrane category.

Figure 2.

CLUSTALW alignment of PF14_0752 and genes displaying amino acid homology using default settings (E value threshold for TBLASTN, <1 × 10⁻⁷). Coding is as follows: shading denotes identity in at least 5 sequences; the black bar indicates a hydrophobic region; and the boxed sequences represent a host cell targeting signal. Seven genes are expressed in at least 1 sample.

Figure 3.

Confirmation of in vivo overexpression of cDNA for PF14_ 0752 in in vivo samples, compared with that in in vitro 3D7 samples with ring-stage parasites.

In vivo overexpression of genes that encode surface variant proteins

The only GO function that is significantly overrepresented in the ≥2 fold overexpressed in vivo samples gene lists contains genes that encode parasite proteins that are expressed at the red cell membrane. The members of these multigene families have polymorphic regions that are thought to play a role in immune evasion [26]. To examine the expression pattern of these gene families in detail, we determined the presence or absence of transcript for each rifin, var, and stevor gene family member by use of methods described above in each in vivo sample and in the 3D7 ring stage (figure 4). The number of rifin gene family members detected was significantly greater in all of the in vivo samples, compared with that in the in vitro 3D7 samples (P < .001). Six rifins (PF10_0394, PF10_0402, PF11_0011, PFB0060w, PFI1810w, and PFC1100w) were exclusively expressed in vivo. The rifins comprise the largest surface variant gene family. Unlike var genes, where a single transcript is expressed as a protein, multiple rifins appear to be translated [27]. It has been reported that rifin gene expression is more prevalent in wild-type isolates but is absent or faint in long-term cultured laboratory strains, which is consistent with the present data [27].

Figure 4.

Graphic representation of the no. of genes expressed in each gene family for each sample. n represents the no. of genes in each family. Each bar represents a gene family member. Red represents detection of transcript, and grey represents unexpressed transcripts. Asterisks denote in vivo samples that contain a significantly greater no. of gene family member transcripts, compared with the no. expressed in in vitro 3D7 (P < .05, Fisher's exact test).

Similarly, there was a trend toward a greater number of stevor genes being overexpressed in vivo, with sample 8 reaching statistical significance (P = .021). Stevor PF14_0767 was uniquely expressed in 1 in vivo sample and was not detected in in vitro 3D7 samples with parasites of any 3D7 life-cycle stage. Stevor proteins, which are localized to Maurer's cleft, are under immune pressure, and in vitro studies have shown multiple transcripts per single-cell parasite [28–30]. These results could have been caused by the presence of multiple clones in some samples; however, the 3 samples (samples 6, 8, and 29) that had monoclonal parasites also contained a proportionally greater number of distinct genes in the rifin and stevor families that were expressed. Conversely, a greater number of var gene transcripts were detected in in vitro 3D7 samples, compared with that in samples 6 (P = .022), 8 (P = .036), and 29 (P = .007).

Distinct in vivo transcriptome

Sample 7 was distinctive, with a lower correlation to in vitro 3D7 samples with parasites of all life-cycle stages as well as to the other in vivo samples (figure 1). This sample contained late-stage parasites in the peripheral blood that were confirmed to be P falciparum by detection of species-specific 18S rRNA (data not shown) [13]. This sample had the largest number of genes (553) that were at least 2-fold overexpressed, compared with in vitro 3D7 stage-specific transcriptomes. As with the other in vivo samples, there were no significant differences in any annotated malarial metabolic pathway, and the only GO function differences were also in the GO-annotated plasma membrane gene list (72 genes; P = 2.6 × 10⁻²¹). The number of rifin and stevor gene family members expressed in this sample was significantly greater than that in the in vitro 3D7 sample with a ring-stage parasite (P < .001) (figure 4).

Discussion

The present study has demonstrated that sufficient amounts of RNA can be extracted from in vivo samples to support a whole-genome microarray analysis of P. falciparum gene expression. An excellent correlation between expression levels of parasite transcripts in in vivo samples and in in vitro 3D7 samples was found. More importantly, this analysis was sensitive enough to detect specific differences in expression in vivo and in vitro. This approach has demonstrated the overexpression of genes that encode surface proteins and has identified a putative novel gene family that encodes surface proteins that may play a role in in vivo parasite biological processes.

The analysis of genes differentially expressed in vivo and in vitro has identified virulence genes in bacterial systems [31, 32]. This approach was adopted to study the pathogenesis of malaria in vivo. The total number of parasite transcripts detected in a small blood sample approaches the number of transcripts reported to be expressed in in vitro cultivated 3D7 ring-stage parasites [15]. For this first analysis of whole-genome in vivo steady-state mRNAs, the potential host effect on parasite selection and RNA expression was minimized through selection of samples from patients with similar demographics and disease presentation. A surprisingly high correlation between the transcriptomes in all the samples obtained from children and those in the in vitro 3D7 samples with ring-stage parasites was found by use of a rank-based correlation statistic. The high correlation between the in vivo samples and in vitro 3D7 samples with parasites in stages preceding the ring stage is notable. Although there are clearly defined morphologic differences between the schizont, merozoite, and ring stages, biological processes may be shared in contiguous stages. For example, developmental clustering between stages was noted for a set of 300 open-reading frames involved in host cell invasion in these stages [15, 33]. These data demonstrate the reproducibility of this method and validate the results.

The high correlation between the in vivo transcriptomes and the 3D7 ring-stage transcriptome, as well as the lack of significant difference in malarial metabolic pathways and most GO functions, is striking. This observation implies that the basic molecular processes of natural isolates and the 3D7 strain are highly conserved, consistent with the observation that the 3D7 strain can infect human volunteers and Anopheles mosquitoes in experimental settings [34].

The lack of overall diversity among the in vivo transcriptomes obtained from a homogenous cohort of patients suggests that distinct transcriptomes, when identified, could be informative. The transcriptome derived from the only adult patient had a lower correlation with the other in vivo samples and with 3D7 stage-specific transcriptomes. The presence of parasites in stages other than rings in the peripheral blood may account for some of these differences; however, the overexpressed gene list contains transcripts that are at least 2-fold more abundant than those for all 3D7 life-cycle stages, including the schizont stage. Similar to the other in vivo samples, there was no difference in genes that encode malarial metabolic pathways or GO functions, aside from the plasma membrane GO function. The presence of late forms, as detected by microscopy, has been shown to correlate with a more severe outcome, and it is intriguing that this was found in the sample with a distinct transcriptome [35, 36]. A larger study that enrolls patients with differences in age and disease severity will be necessary to interpret the significance of unique transcriptomes.

The specific overexpression of genes that encode surface proteins, hypothesized to be involved in immune evasion, is consistent with the biological processes of the in vivo environment, which is rich in immune cells and factors, compared with the in vitro environment. Previous work has demonstrated changes in expression of surface-expressed proteins in P. falciparum when the parasite is in prolonged culture or under biologic or immune selection [37–39]. Gene family members that are uniquely expressed in vivo may simply represent diversity of gene expression or be required for in vivo survival. Var genes were not found to be overexpressed in vivo. Because more-recent data examining geographically distinct strains (C. Kidgell, J. Borevitz, J. Johnson, S. Volkman, D. Plouffe, K. Le Roch, D. Wirth, Y. Zhou, and E. Winzeler, unpublished data) has found marked sequence polymorphism in the region from which the var probes used in our microarray analysis were derived, our detection methods may have resulted in an underestimation of the number of var transcripts present. Because parasite-encoded surface proteins interact with the host immune system, comprehensive analysis of genes in this functional class isolated from patients who demonstrate immunity, compared with those isolated from patients who do not demonstrate immunity, may provide insight into this critical aspect of the host-pathogen interaction.

A major goal of this work was to identify, for further analysis, genes that encode hypothetical proteins that may play a role in in vivo biological processes. One gene that encodes a hypothetical protein (PF14_0752) was identified as being overexpressed in 4 of the 5 in vivo samples. Further analysis revealed additional P. falciparum homologues that encode hypothetical proteins; these homologues were predominantly located at the telomeres, with the majority containing a predicted transmembrane domain, a vacuolar export/host targeting signal, and sequence polymorphism. Taken together, these characteristics suggest a new family of surface proteins, and analysis of these genes is under way to test this hypothesis.

Parasites residing in vivo are challenged with unique features that are not present under in vitro conditions. The in vivo environment contains immune factors, endothelial ligands, and variation in microenvironments secondary to sequestration in different organs. The molecular analysis of in vivo biological processes by means of this new approach will identify genes that are important for survival of the parasite in the human host and provide additional candidates for vaccine development, to lessen disease severity and provide immunity.

Table 3. Sample 6 genes with transcript levels at least 2-fold greater than those in in vitro 3D7 samples.

Gene	Description	Expression units
		In vivo					In vitro 3D7
		6	7	8	12	29	M	ER	LR
Surface protein
PFL2585c	RIFIN	1606	245	0	2301	0	0	0	0
PFI1810w	RIFIN	177	0	0	183	0	0	0	0
PFI0030c	RIFIN	124	185	156	120	53	33	16	21
PF14_0004	RIFIN	77	164	127	77	48	18	0	17
PF11_0512	RESA-2	530	156	379	443	626	0	0	0
Cell function/metabolism
PF11_0250	High mobility group–like protein NHP2	2285	402	1081	2205	1351	317	545	563
PFC0050c	Long-chain fatty acid ligase	617	186	192	73	92	0	0	0
PF14_0183	RNA helicase	480	131	413	1373	227	92	154	128
PFB0805c	Clathrin coat assembly protein	32	0	0	0	0	0	0	0
MAL8P1.22	Dehydrodolichyl diphosphate synthetase	26	130	20	0	14	0	0	0
MAL8P1.37	Lipoate-protein ligase	10	0	0	11	13	0	0	0
Hypothetical protein
PF13_0194	Hypothetical protein	2303	820	615	2394	1690	519	822	215
PF14_0753	Hypothetical protein	2110	432	660	1539	1655	664	323	62
PFL2565w	Hypothetical protein	2093	130	845	3139	1078	340	770	207
PFB0280w	Hypothetical protein	1475	112	55	1723	166	32	259	188
chr5.rRNA-1-18s-A	Hypothetical protein	1331	719	550	1575	887	0	0	0
PFA0635c	Hypothetical protein	1170	159	317	1017	944	105	421	137
PFD0320c	Hypothetical protein	1051	78	691	1671	346	43	438	104
PF14_0752	Hypothetical protein	837	0	314	569	351	44	76	23
PFI1640c	Hypothetical protein	695	167	350	380	876	0	130	96
MAL13P1.122	Hypothetical protein	683	275	287	693	582	99	174	85
PFA0135w	Hypothetical protein	469	313	577	883	307	229	0	37
PF11_0035	Hypothetical protein	437	0	0	994	0	0	0	0
PFD1180w	Hypothetical protein	364	85	133	149	201	0	0	0
MAL7P1.133	Hypothetical protein	338	0	154	317	361	0	82	0
PFA0700c	Hypothetical protein	331	0	0	0	818	0	0	0
PF11_0037	Hypothetical protein	326	135	0	614	164	0	0	0
MAL8P1.160	Hypothetical protein	214	0	0	225	207	80	38	0
PFD1185w	Hypothetical protein	213	0	26	0	0	0	36	0
PF11_0006	Hypothetical protein	173	1188	142	295	178	0	0	0
PFI1800w	Hypothetical protein	150	95	0	119	40	0	0	0
PFI1775w	Hypothetical protein	132	185	0	67	0	0	0	0
PFA0655w	Hypothetical protein	98	0	0	102	0	0	0	0
PFD0065w	Hypothetical protein	94	276	233	165	285	31	0	0
PFI0485c	Hypothetical protein	55	289	98	144	0	0	0	0
PFA0685c	Hypothetical protein	52	85	34	0	0	0	0	0
PFL0945w	Hypothetical protein	50	127	0	47	0	0	0	12
MAL13P1.11	Hypothetical protein	46	0	0	0	0	0	0	0
MAL13P1.251	Hypothetical protein	44	0	0	11	0	0	0	0
PFL2525c	Hypothetical protein	38	182	25	88	0	0	0	0
PFA0720w	Hypothetical protein	31	365	86	116	0	0	0	0
PF08_0016	Hypothetical protein	31	39	19	0	14	11	0	0
MAL13P1.325	Hypothetical protein	23	0	0	0	0	0	0	0
PFB0610c	Hypothetical protein	22	0	18	0	0	0	0	0
PF14_0558	Hypothetical protein	19	0	27	0	0	0	0	0
PFC0075c	Hypothetical protein	18	0	16	32	0	0	0	0
PFB0470w	Hypothetical protein	18	0	10	0	0	0	0	0
PF14_0422	Hypothetical protein	18	0	0	0	0	0	0	0
PF14_0720	Hypothetical protein	17	100	0	0	15	0	0	0
PF14_0116	Hypothetical protein	16	19	13	12	15	0	0	0
MAL8P1.41	Hypothetical protein	15	0	11	0	0	0	0	0
PF08_0013	Hypothetical protein	14	0	22	0	0	0	0	0
PFC0180c	Hypothetical protein	14	0	0	0	0	0	0	0
PF11_0233	Hypothetical protein	12	0	0	0	0	0	0	0
PF14_0521	Hypothetical protein	11	0	18	0	0	0	0	0
PFL1190c	Hypothetical protein	11	40	0	0	0	0	0	0
PF11_0411	Hypothetical protein	11	163	45	0	0	0	0	0
PFD0395c	Hypothetical protein	11	0	0	0	0	0	0	0
PFE0230w	Hypothetical protein	10	0	0	12	15	0	0	0

NOTE. ER, early ring; LR, late ring; M, merozoite.

Table 4. Sample 7 genes with transcript levels at least 2-fold greater than those in in vitro 3D7 samples.

Gene	Description	Expression units
		In vivo					In vitro 3D7
		6	7	8	12	29	M	ER	LR
Surface proteins
PF07_0051	PfEMP1	0	555	0	51	0	0	35	0
PF11_0007	PfEMP1	168	478	269	250	162	49	131	72
PF11_0521	PfEMP1	107	444	144	258	133	53	118	123
PFB0020c	PfEMP1, truncated	0	397	0	0	0	0	0	0
PFD0020c	PfEMP1	101	363	192	188	74	48	69	62
PFL0935c	PfEMP1	81	309	128	94	82	53	31	0
MAL6P1.4	PfEMP1	0	275	183	157	0	47	121	81
PF13_0003	PfEMP1	85	233	178	134	98	18	65	27
PFB1055c	PfEMP1	77	206	132	121	49	0	66	0
PFD0615c	PfEMP1	54	189	0	0	0	38	85	34
PFA0765c	PfEMP1	0	122	68	45	36	29	25	0
MAL6P1.316	PfEMP1	0	106	141	0	0	0	32	21
PF08_0141	PfEMP1	0	90	180	0	0	0	32	0
PFB0010w	PfEMP1	0	73	0	0	0	0	0	0
MAL6P1.313	RIFIN	0	729	0	0	0	0	0	18
PFL2605w	RIFIN	135	561	126	185	75	33	0	0
PFL2625w	RIFIN	185	486	360	328	141	52	30	49
PFL2660w	RIFIN	0	451	177	0	120	40	42	0
PF10_0404	RIFIN	100	445	170	149	82	24	25	41
PFL0015c	RIFIN	104	443	196	162	71	41	0	28
PF14_0003	RIFIN	105	440	128	227	56	0	20	0
PFD1020c	RIFIN	71	428	91	112	60	22	0	0
PF11_0515	RIFIN	0	396	174	116	0	0	0	28
PFC1100w	RIFIN (3D7-rifT3-6)	0	386	0	66	0	0	0	0
PF10_0399	RIFIN	197	356	303	185	58	52	0	32
PF08_0104	RIFIN	47	356	174	58	50	32	30	48
PFB0060w	RIFIN	0	346	0	128	0	0	0	0
PF07_0135	RIFIN	126	333	191	161	114	35	18	0
PF08_0139	RIFIN	122	318	153	121	97	57	50	32
PF14_0768	RIFIN	162	290	295	182	144	36	39	28
PF11_0010	RIFIN	0	259	93	0	0	0	0	0
PF11_0517	RIFIN	73	259	125	90	73	19	0	0
PFB1040w	RIFIN	138	251	175	116	0	50	49	0
PFB0015c	RIFIN	32	248	80	33	30	0	24	0
PFL2585c	RIFIN	1606	245	0	2301	0	0	0	0
PF14_0005	RIFIN	61	241	148	113	50	47	31	33
MAL6P1.315	RIFIN	67	237	147	99	71	25	0	0
PFI1805w	RIFIN	55	236	82	77	47	25	0	0
PF10_0400	RIFIN	43	235	0	0	0	0	0	0
PF11_0011	RIFIN	0	232	0	0	0	0	0	0
PFC1095w	RIFIN (3D7-rifT3-5)	0	227	0	0	0	0	0	0
PFC0030c	RIFIN	48	221	59	61	0	22	0	20
PFL2640c	RIFIN	0	205	82	0	0	15	0	0
MAL6P1.310	RIFIN-related protein	0	205	0	0	0	0	0	0
PF07_0134	RIFIN	0	205	0	0	22	0	0	0
PF10_0394	RIFIN	0	201	45	0	0	0	0	0
MAL6P1.9	RIFIN	34	195	62	125	55	0	14	0
PFI0030c	RIFIN	124	185	156	120	53	33	16	21
PF14_0769	RIFIN	0	182	0	164	0	0	0	0
PFD0130c	RIF pseudogene	0	176	77	76	0	30	0	0
PFD1230c	RIFIN	0	173	0	0	0	0	0	0
PFE1630w	RIFIN	41	173	90	63	27	0	0	0
PFA0740w	RIFIN	0	173	110	118	50	0	0	0
PFA0020w	RIFIN	0	173	99	0	0	22	20	29
PF14_0004	RIFIN	77	164	127	77	48	18	0	17
PFD0640c	RIFIN	0	163	0	0	0	20	25	0
PFD0645w	RIFIN	60	156	60	71	37	0	0	0
MAL7P1.57	RIFIN	0	149	70	64	0	0	0	0
PF11_0519	RIFIN	0	148	0	0	0	0	0	0
PFI0070w	RIFIN	59	144	123	108	40	38	0	0
PFD0120w	RIF pseudogene	0	143	0	0	0	0	0	0
PFB0035c	RIFIN	0	140	0	72	0	0	36	15
PFD0055w	RIFIN	46	138	137	108	38	17	0	13
PFB0040c	RIFIN	39	134	0	86	35	0	0	24
PF08_0138	RIFIN	50	129	0	83	0	25	0	0
PF13_0007	RIFIN	58	123	106	64	43	0	14	21
PFB1010w	RIFIN	45	121	115	65	0	19	0	0
PFD0050w	RIFIN	0	119	0	0	0	0	0	0
PFC0035w	RIFIN	67	114	62	50	30	23	0	0
PF08_0105	RIFIN	68	113	95	55	23	21	0	0
PF10_0004	RIFIN	40	106	78	51	58	0	20	0
MAL6P1.250	RIFIN	52	82	0	115	44	0	0	0
MAL6P1.6	RIFIN	0	44	0	0	0	0	0	10
PFB1020w	STEVOR, putative	0	980	240	294	176	0	17	33
PF07_0130	STEVOR	52	697	0	0	0	0	0	0
PFC1105w	STEVOR (3D7-stevorT3-2)	81	683	122	115	78	0	0	27
PF11_0516	STEVOR, putative	102	626	154	132	68	29	0	65
PFL2610w	STEVOR	0	595	0	234	90	0	0	0
PFB0065w	STEVOR, putative	94	548	150	139	79	82	30	100
PFD0035c	STEVOR	57	502	197	189	0	50	0	0
PFB0955w	STEVOR, degenerate, putative	0	450	174	217	0	25	19	0
PFB0025c	STEVOR, putative	0	276	147	0	0	64	0	56
PFB0050c	STEVOR isoform	0	275	80	0	37	0	0	0
MAL6P1.10	STEVOR	65	266	193	136	75	71	0	92
PFI0045c	STEVOR	46	243	0	0	0	0	0	18
PF13_0009	STEVOR	0	221	94	0	0	0	0	0
PFC0025c	STEVOR, putative	0	209	0	0	0	0	0	0
PF14_0007	STEVOR, putative	0	200	216	108	81	42	0	30
PF14_0767	STEVOR, putative	0	72	0	0	0	0	0	0
PF11_0512	RESA-2	530	156	379	443	626	0	0	0
Metabolic/cellular function
MAL13P1.82	Phosphatidylinositol synthase, putative	0	1761	0	0	0	0	0	0
PF11_0280	Small nuclear ribonucleoprotein F, putative	890	1572	1200	1408	662	85	278	229
PFC0855w	Ubiquitin-conjugating enzyme, putative	277	709	396	473	194	0	121	153
PF14_0697	Dihydroorotase, putative	81	698	160	191	97	43	87	73
MAL13P1.213	Transcription activator, putative	0	691	0	0	0	0	0	319
PF11_0311	N-acetyl-glucosamine phosphate mutase, putative	90	570	0	133	123	33	37	66
PFB0130w	Polyprenyl synthetase, putative	0	481	0	0	0	0	0	49
PF14_0331	Cytochrome c oxidase assembly protein, putative	0	457	107	147	48	58	19	40
PFL0695c	Geranylgeranyl transferase type 2 βsubunit, putative	86	457	104	130	99	0	81	76
PFE0375w	Cell differentiation protein rcd1, putative	122	450	0	137	135	93	69	173
MAL13P1.304	Malarial antigen	89	435	0	182	112	28	122	81
PFE0805w	Cation-transporting ATPase 1	0	430	0	0	0	0	0	23
PFL1150c	Ribosomal protein L24, putative	0	413	80	81	0	0	0	0
MAL13P1.322	Splicing factor, putative	135	408	64	211	140	60	82	50
PFE1265w	G-protein coupled receptor, putative	40	405	135	91	0	0	0	0
PF14_0545	Thioredoxin	0	395	120	144	66	0	47	51
PFE0665c	GTP-binding protein, putative	0	391	96	60	0	18	29	58
PF13_0324	Vesicle transport protein, putative	0	375	0	72	68	35	18	27
PFE1185w	Transporter, putative	0	373	0	0	0	0	28	0
MAL13P1.279	Cell division control protein 2 homologue	65	360	83	0	66	0	0	0
PF07_0068	Cysteine desulfurase, putative	0	354	70	67	0	0	42	55
PF14_0314	Chromatin assembly factor 1 p55 subunit, putative	0	349	98	103	34	0	27	43
PF13_0128	β-hydroxyacyl-acp dehydratase precursor	0	328	0	97	73	36	21	26
PFB0505c	β-ketoacyl-acyl carrier protein synthase III precursor, putative	0	300	93	74	29	41	0	0
PFI0425w	Transporter, putative	0	295	62	72	0	0	59	68
PFC0831w	Triosephophate isomerase, putative	0	293	0	0	0	0	0	0
PFI1110w	Glutamate-ammonia ligase (glutamine synthetase), putative	79	289	126	109	66	0	52	37
MAL6P1.58	Cyclin dependent kinase binding protein, putative	83	271	108	0	0	0	0	0
MAL13P1.324	Aldo-keto reductase, putative	0	268	0	0	0	0	0	0
PF10_0334	Flavoprotein subunit of succinate dehydrogenase	0	264	0	37	21	0	0	0
PFC0420w	Calcium-dependent protein kinase, putative	0	263	127	101	71	70	22	0
PF14_0146	Ribonucleoprotein, putative	78	260	0	119	70	72	64	93
MAL8P1.48	Small nuclear ribonucleoprotein polypeptide g, 65009–64161, putative	0	244	0	0	0	0	0	0
PF07_0115	Integral membrane protein, putative	0	236	0	0	0	0	0	0
PF10_0137	GDP-fucose synthase, putative	0	234	0	0	18	0	0	0
PFL1920c	Hydroxyethylthiazole kinase, putative	0	227	0	0	0	0	0	44
PFI0300w	Developmental protein, putative	0	225	0	0	0	0	0	0
PFI0240c	E1-E2_ATPase/hydrolase, putative	0	223	0	79	32	0	0	0
MAL6P1.68	Polypyrimidine tract binding protein, putative	149	222	169	202	121	70	94	65
MAL8P1.57	C-13 antigen	0	217	98	155	61	59	56	87
MAL6P1.194	Fe-superoxide dismutase	73	214	87	116	52	0	0	0
PF13_0100	Glycerol-3-phosphate acyltransferase, putative	0	211	0	55	0	0	0	0
PFE0045c	Kinase, putative	0	208	87	0	0	31	0	0
PF11_0483	Farnesyltransferase βsubunit, putative	0	203	0	74	0	0	28	37
PFL1940w	3-hydroxyisobutyryl-coenzyme A hydrolase, putative	76	203	0	102	62	0	28	0
PF14_0513	RNA binding protein, putative	64	203	80	80	65	34	20	0
PFL1525c	Pre-mRNA splicing factor RNA helicase, putative	0	197	0	48	40	0	45	37
PF14_0415	Dephospho-CoA kinase, putative	0	191	0	0	0	39	0	29
PF10_0379	Phospholipase, putative	0	189	0	0	0	0	0	0
PF13_0149	Chromatin assembly factor 1 subunit, putative	0	187	0	113	0	21	17	0
PF14_0224	PP1-like protein serine/threonine phosphatase	0	187	0	0	0	0	0	0
PFC0050c	Long-chain fatty acid ligase, putative	617	186	192	73	92	0	0	0
MAL13P1.326	Ferrochelatase, putative	0	183	0	0	0	11	0	0
MAL13P1.22	DNA ligase 1	0	182	102	81	33	0	0	0
PF14_0528	Hemolysin, putative	0	179	0	37	0	36	0	0
PF14_0346	cGMP-dependent protein kinase 1, βisozyme, putative	70	177	83	82	37	0	0	0
PFE1125w	50S ribosomal subunit protein L17, putative	0	176	0	0	20	0	0	0
PFD0830w	Bifunctional dihydrofolate reductase-thymidylate synthase	0	176	0	0	26	11	0	0
PF11_0240	Dynein heavy chain, putative	15	175	0	0	0	0	0	0
PFD0260c	Sequestrin	0	172	0	0	0	22	0	0
PFC0945w	Protein kinase, putative	0	170	86	98	39	0	13	0
PFA0130c	Serine/threonine protein kinase, putative	0	170	0	115	42	0	0	0
PF13_0061	ATP synthase γchain, mitochondrial precursor, putative	0	169	58	52	0	18	20	25
PF10_0009	Pseudogene, STEVOR, putative	0	169	0	68	0	0	0	18
PF11_0510	Serine/threonine protein kinase, putative	0	163	0	0	0	0	0	0
PFI0255c	Mitochondrial carrier protein, putative	0	161	62	112	0	0	0	0
PF10_0051	ADP/ATP carrier protein, putative	0	159	0	50	27	11	0	31
MAL7P1.118	PelOta protein homologue, putative	0	155	0	0	0	0	37	0
PFA0660w	Protein with DNAJ domain, dnj1/sis1 family	0	153	75	0	0	65	60	0
MAL7P1.37	sin3-associated polypeptide p18–like protein, putative	0	152	17	0	0	0	0	0
PFB0360c	Cysteine protease, putative	11	146	0	0	0	0	0	0
MAL8P1.35	Exonuclease, putative	0	146	0	64	0	0	0	0
PFE0305w	Transcription initiation factor TFiid, TATA-binding protein	0	145	0	0	0	0	0	0
PF14_0751	Fatty acyl coenzyme A synthetase-1, putative	0	143	80	0	0	0	0	0
MAL6P1.24	ATP dependent RNA helicase, putative	0	140	28	49	43	0	66	28
MAL7P1.20	Peptide chain release factor	0	140	0	0	0	0	0	0
PF13_0084	Ubiquitin-like protein, putative	0	138	0	0	0	0	0	0
PFI0860c	ATP-dependant RNA helicase, putative	0	136	0	0	0	0	38	35
PF14_0530	Ferlin, putative	0	133	0	0	0	0	0	0
PFL2165w	Kinesin-like protein, putative	11	130	0	0	0	0	0	0
PF14_0381	δ-aminolevulinic acid dehydratase	0	130	0	0	28	20	0	0
MAL8P1.22	Dehydrodolichyl diphosphate synthetase, putative	26	130	20	0	14	0	0	0
PFI1240c	Prolyl-t-RNA synthase, putative	0	128	21	0	0	0	0	0
PF14_0676	20S proteasome β4 subunit, putative	0	128	41	23	31	0	0	44
PF11_0145	Glyoxalase I, putative	0	125	17	0	0	0	0	0
PFI1100w	Para-aminobenzoic acid synthetase	12	125	0	0	0	0	0	0
PFL0560c	Minichromosome maintenance protein, putative	0	124	0	0	0	0	0	0
PF14_0358	41-2 protein antigen precursor	0	121	0	0	33	0	43	0
PFC0800w	Band 7–related protein	0	121	0	0	0	16	0	0
PFB0220w	UbiE-like methlytransferase, putative	0	116	0	0	0	0	0	0
PF14_0297	Ecto-nucleoside triphosphate diphosphohydrolase 1, putative	0	115	26	45	0	0	0	20
PFE1430c	Cyclophilin, putative	0	115	0	0	43	0	0	0
MAL13P1.169	Syntaxin 5, putative	12	114	21	0	0	0	0	0
PF13_0229	IRP-like protein	0	108	0	0	0	0	0	0
PF14_0349	Histidine triad protein, putative	0	105	63	61	0	19	40	0
MAL6P1.165	3-oxoacyl-(acyl-carrier-protein) synthase i/ii	0	103	0	0	0	0	0	0
PF13_0052	Ribosomal RNA methyltransferase, putative	0	102	0	0	30	38	36	45
PFE1470w	Cell cycle regulator protein, putative	0	95	0	0	0	0	0	0
PFI0925w	γ-glutamylcysteine synthetase	0	92	0	0	0	0	0	20
PF14_0147	ATP-dependent protease, putative	0	89	0	0	0	0	18	0
PFE1340w	Transmembrane protein, putative	0	82	0	0	0	0	37	0
MAL7P1.151	Modification methylase–like protein	0	82	0	0	0	0	0	0
MAL7P1.159	Antioxidant protein, putative	0	81	0	0	0	0	0	0
PFE0455w	Phosphatase 1 regulatory subunit, putative	0	77	0	0	0	0	0	0
PF10_0147	FAD synthetase, putative	0	74	0	0	0	0	0	0
PFE0255w	Actin-related protein, putative	0	70	0	0	0	0	0	0
PF13_0096	Ubiquitin carboxyl-terminal hydrolase-like zinc finger protein	0	58	0	29	22	0	0	0
Hypothetical protein
PF13_0160	Hypothetical protein	1952	5135	3763	2153	1650	0	0	0
PF11_0023	Hypothetical protein	1952	5135	3763	0	0	2362	0	1333
PF10_0382	Hypothetical protein	0	5135	3763	0	0	0	0	0
PF11_0523	Hypothetical protein	0	5135	3764	2154	0	0	1679	0
mitopl_rpl14	Hypothetical protein	1952	5135	3763	2153	1650	0	1679	1333
PF13_0195	Hypothetical protein	1952	5135	0	2153	1650	0	0	1333
PF11_0032	Hypothetical protein	1646	3627	3499	2583	1071	774	393	697
MAL7P1.39	Hypothetical protein	114	2246	604	504	226	175	105	94
PFE0850w	Hypothetical protein, conserved	571	1931	892	727	619	467	591	378
PF11_0033	Hypothetical protein	284	1926	550	544	241	212	49	68
PF11_0006	Hypothetical protein	173	1188	142	295	178	0	0	0
PF10_0383	Hypothetical protein, conserved	111	1063	229	345	120	105	17	33
chr1.rRNA-1-ITS2	Hypothetical protein	147	1021	395	333	96	49	0	0
PF11_0394	Hypothetical protein	139	1006	252	286	129	64	0	0
PFI1705w	Hypothetical protein	232	988	352	514	229	101	197	150
MAL6P1.3	Hypothetical protein	0	948	0	0	0	0	0	0
PFI1395w	Hypothetical protein	164	818	226	239	111	42	77	58
PF13_0029	Hypothetical protein	97	814	158	182	143	71	0	76
PFL2410w	Hypothetical protein	105	784	133	200	67	36	30	29
PF11_0003	Hypothetical protein	0	764	0	232	117	0	0	0
PFA0185w	Hypothetical protein	171	748	288	243	245	46	209	247
PF14_0743	Hypothetical protein	119	730	224	174	84	96	0	78
PFC0970w	Hypothetical protein	481	711	259	522	351	69	300	169
PF14_0728	Hypothetical protein	0	682	0	0	0	0	0	0
PFI1135c	Hypothetical protein	0	674	0	0	0	0	0	0
PFL0720w	Hypothetical protein	76	672	201	175	86	69	0	38
PFL1455w	Hypothetical protein	0	670	92	99	34	0	0	91
PFA0075w	Hypothetical protein	0	664	259	195	0	67	0	0
PF14_0299	Hypothetical protein	112	657	372	408	138	87	45	0
MAL8P1.30	Hypothetical protein	118	639	227	206	109	52	28	42
PFA0245w	Hypothetical protein	52	626	170	197	68	39	0	0
PF14_0064	Hypothetical protein	0	615	0	121	0	0	0	0
PF11_0059	Hypothetical protein	0	612	322	216	141	42	62	75
MAL13P1.276	Hypothetical protein	0	600	0	0	0	0	0	0
MAL8P1.161	Hypothetical protein	0	597	0	0	40	0	53	149
PFC0390w	Hypothetical protein, conserved	130	573	147	247	126	0	101	57
PF11_0176	Hypothetical protein	0	570	109	104	52	77	0	55
MAL7P1.123	Hypothetical protein	83	541	161	156	74	40	34	46
PFC0505c	Hypothetical protein	76	536	130	169	74	43	30	48
PFB0965c	Hypothetical protein	0	535	0	0	0	0	0	0
PFL1210w	Hypothetical protein	59	527	182	203	61	62	45	133
PFL1965w	Hypothetical protein	116	510	180	289	105	0	0	0
PF14_0562	Hypothetical protein	0	509	135	104	0	0	0	24
MAL13P1.177	Hypothetical protein	0	506	56	69	30	42	0	0
PF10_0287	Hypothetical protein	79	493	172	143	84	0	56	42
PF11_0459	Hypothetical protein	30	475	0	57	0	0	0	0
chr8.rRNA-2-28s-pseudo	Hypothetical protein	55	474	147	137	48	62	18	52
PF14_0387	Hypothetical protein	0	455	0	0	0	0	14	39
PF14_0662	Hypothetical protein	0	450	0	81	45	0	38	59
PFD0325w	Hypothetical protein	0	449	0	0	0	0	0	0
MAL8P1.130	Hypothetical protein	0	445	137	0	53	76	110	139
PFB0725c	Hypothetical protein	0	443	0	98	0	0	0	0
PFI0390c	Hypothetical protein	0	443	0	0	0	0	0	0
MAL7P1.33	Hypothetical protein	0	440	0	0	0	0	0	0
MAL7P1.97	Hypothetical protein	0	434	0	89	0	0	0	0
PFL2315c	Hypothetical protein	0	429	0	0	54	0	0	0
PF11_0047	Hypothetical protein	93	405	133	107	87	54	53	82
PF10_0339	Hypothetical protein	0	399	0	0	0	0	0	0
PFD1075w	Hypothetical protein	0	395	0	112	67	0	0	0
PF10_0377	Hypothetical protein	0	391	0	0	68	0	41	0
PF13_0274	Hypothetical protein, conserved	0	390	0	0	0	0	38	81
PF13_0139	Hypothetical protein	0	386	48	55	0	22	0	21
PF14_0539	Hypothetical protein	43	385	133	129	49	37	26	0
PF08_0073	Hypothetical protein	90	380	179	127	0	109	21	28
PF11_0467	Hypothetical protein	0	378	82	65	27	0	0	0
PF14_0161	Hypothetical protein, conserved	0	374	0	67	0	36	0	20
PFA0720w	Hypothetical protein	31	365	86	116	0	0	0	0
PF10_0089	Hypothetical protein	226	361	149	326	208	40	131	104
MAL13P1.299	Hypothetical protein, conserved	87	361	93	130	120	36	89	92
PF11_0271	Hypothetical protein, conserved	0	360	146	139	58	0	0	71
PF10_0283	Hypothetical protein	0	358	0	0	104	0	0	0
PF14_0756	Hypothetical protein	0	357	0	0	0	0	0	0
PFA0715c	Hypothetical protein	0	353	0	0	0	0	0	0
PF14_0726	Hypothetical protein	0	349	97	199	0	23	0	0
PFA0705c	Hypothetical protein	0	349	0	57	0	0	0	0
PF13_0317	Hypothetical protein	0	346	0	0	39	0	27	0
PFL0745c	Hypothetical protein	48	346	77	98	0	23	0	0
PFI0105c	Hypothetical protein	67	346	129	118	49	0	31	37
PF14_0572	Hypothetical protein	0	344	95	111	33	0	0	0
PF08_0083	Hypothetical protein	78	343	117	120	0	134	49	0
MAL13P1.246	Hypothetical protein	0	342	0	0	0	0	0	0
PF14_0755	Hypothetical protein	0	342	0	0	0	0	0	0
PF11_0236	Hypothetical protein	58	338	130	126	57	37	25	0
PF11_0054	Hypothetical protein	0	338	0	0	0	0	0	0
MAL13P1.353	Hypothetical protein	0	335	0	0	0	0	47	0
PFE0650c	Hypothetical protein	0	333	51	78	21	0	0	0
PF14_0087	Hypothetical protein	62	332	72	147	146	0	55	34
PF14_0160	Hypothetical protein	0	331	0	0	0	0	0	0
PF10_0338	Hypothetical protein	0	327	0	0	0	0	0	0
PF11_0363	Hypothetical protein	0	312	64	57	41	22	44	27
PFL1460c	Hypothetical protein	0	311	109	88	46	0	0	0
PFI0100c	Hypothetical protein	0	311	0	55	0	0	0	0
PFC0680w	Hypothetical protein	0	308	95	75	51	0	0	19
MAL13P1.210	Hypothetical protein	0	307	0	40	0	45	0	53
PFD0370w	Hypothetical protein	82	306	0	77	52	0	63	85
PF14_0596	Hypothetical protein	0	305	0	61	0	41	0	0
PF14_0355	Hypothetical protein	0	302	0	0	0	0	0	0
MAL7P1.124	Hypothetical protein	77	298	91	120	0	48	0	0
PFL1905w	Hypothetical protein	0	296	0	0	0	62	58	0
PF14_0121	Hypothetical protein	12	295	0	0	0	0	0	0
PF14_0739	Hypothetical protein	67	295	83	86	62	0	19	28
PF10_0167	Hypothetical protein	0	294	0	60	50	0	0	0
MAL8P1.157	Hypothetical protein	19	293	42	0	0	0	0	53
PFB0950w	Hypothetical protein	0	289	100	99	0	0	0	0
PFI0485c	Hypothetical protein	55	289	98	144	0	0	0	0
PF14_0312	Hypothetical protein	0	283	0	79	0	0	54	56
PF14_0409	Hypothetical protein	0	283	0	0	0	0	0	0
PFL0200c	Hypothetical protein	0	277	70	0	0	0	0	0
PFC0450w	Hypothetical protein	0	277	0	0	0	0	0	0
PFD0065w	Hypothetical protein	94	276	233	165	285	31	0	0
MAL13P1.24	Hypothetical protein	0	275	0	0	0	0	0	41
PF08_0136	Hypothetical protein	0	275	0	0	0	0	0	0
PF14_0262	Hypothetical protein	0	274	59	64	0	21	0	0
PFI0790w	Hypothetical protein	0	273	0	0	0	0	0	0
PFE0260w	Hypothetical protein	11	272	38	0	0	31	0	0
PF14_0638	Hypothetical protein	0	271	0	0	0	0	11	0
MAL13P1.112	Hypothetical protein	0	271	95	0	0	22	0	0
PFL1130c	Hypothetical protein	0	271	69	81	70	0	58	52
PF10_0108	Hypothetical protein	19	270	0	0	0	0	26	59
PFA0355w	Hypothetical protein	0	269	0	0	0	0	0	0
MAL13P1.259	Hypothetical protein	0	267	0	0	0	0	0	0
MAL8P1.14	Hypothetical protein	0	266	0	0	0	0	0	38
PFI0710c	Hypothetical protein	0	264	0	0	0	11	0	0
PFL1300c	Hypothetical protein	20	259	0	46	0	0	0	58
PFE0510c	Hypothetical protein	0	258	45	47	0	0	0	0
PF13_0357	Hypothetical protein	0	258	0	0	0	0	0	0
MAL7P1.52	Hypothetical protein	0	258	63	92	0	0	0	0
PF11_0335	Hypothetical protein	0	254	0	0	0	36	0	0
PFL1660c	Hypothetical protein	0	253	90	48	0	23	0	20
PF11_0004	Hypothetical protein	49	253	97	152	67	35	16	0
PFL0940c	Hypothetical protein	78	247	120	88	58	22	34	20
PF07_0075	Hypothetical protein	0	244	0	0	0	0	0	0
PF08_0072	Hypothetical protein	0	244	0	0	0	76	33	0
PF14_0023	Hypothetical protein, conserved	39	244	73	68	34	0	34	46
PF10_0139	Hypothetical protein	0	242	0	0	38	0	0	12
MAL6P1.241	Hypothetical protein, conserved	0	242	0	0	0	0	0	0
PF10_0182	Hypothetical protein	0	240	0	0	0	44	0	12
PFL0375w	Hypothetical protein	0	240	0	0	48	0	0	0
MAL8P1.98	Hypothetical protein	0	239	0	50	30	0	0	0
PF14_0410	Hypothetical protein	14	238	23	0	0	0	0	42
PFI1160w	Hypothetical protein	0	238	0	0	0	0	0	0
PFE0030c	Hypothetical protein	0	238	114	100	0	18	0	0
MAL8P1.10	Hypothetical protein, conserved	0	237	0	0	0	0	0	0
PFL1575w	Hypothetical protein	0	237	0	0	0	0	0	0
MAL13P1.197	Hypothetical protein	0	236	0	0	0	0	0	0
PF14_0066	Hypothetical protein, conserved	0	235	0	46	20	0	0	20
PF13_0337	Hypothetical protein	0	234	0	0	0	0	0	0
PF07_0028	Hypothetical protein	0	228	0	57	36	40	14	27
PF11_0387	Hypothetical protein	24	227	42	0	0	12	0	13
PFB0700c	Hypothetical protein	42	225	80	94	43	29	27	28
PF14_0029	Hypothetical protein	0	223	0	0	0	0	0	0
MAL6P1.109	Hypothetical protein	0	221	0	0	0	0	0	0
PFL2270w	Hypothetical protein	67	218	0	0	67	42	37	30
PFA0240w	Hypothetical protein	0	216	0	0	50	0	0	0
PF14_0298	Hypothetical protein	96	215	163	122	0	0	22	0
PFI1165c	Hypothetical protein	0	213	0	0	0	0	0	0
PF10_0197	Hypothetical protein	0	213	0	112	79	18	50	47
PF14_0757	Hypothetical protein	0	207	0	0	0	0	0	0
PF14_0158	Hypothetical protein	0	205	44	0	0	0	0	0
PF10_0240	Hypothetical protein	0	204	57	0	0	49	0	33
MAL6P1.205	Hypothetical protein	0	203	0	0	0	36	0	35
PFC0240c	Hypothetical protein	0	203	0	106	47	31	42	24
PF11_0276	Hypothetical protein	80	201	0	126	0	21	17	0
PFC0550w	Hypothetical protein	0	200	0	43	0	0	13	28
PFL0955c	Hypothetical protein	0	200	128	102	0	0	0	0
PFD1025w	Hypothetical protein	0	199	0	0	0	0	0	0
PFC0610c	Hypothetical protein	0	199	84	0	0	0	0	0
PF08_0022	Hypothetical protein	0	198	0	42	0	0	0	0
PFL0765w	Hypothetical protein	0	198	0	24	0	0	0	0
PFB0910w	Hypothetical protein	84	198	0	0	85	88	79	0
MAL6P1.312	Hypothetical protein	126	196	0	229	72	0	0	31
PFD0655w	Hypothetical protein	0	195	0	120	0	0	0	0
PFL1730c	Hypothetical protein	0	195	0	0	0	0	0	0
PF11_0484	Hypothetical protein	0	193	0	0	0	0	0	0
PF11_0345	Hypothetical protein	0	192	0	0	0	0	0	0
PFA0450c	Hypothetical protein	0	192	0	0	0	0	0	0
PF14_0266	Hypothetical protein	0	187	0	0	24	19	45	64
PFC0765c	Hypothetical protein	0	186	0	0	50	30	0	0
PFI1775w	Hypothetical protein	132	185	0	67	0	0	0	0
PF14_0575	Hypothetical protein	0	184	0	0	0	0	0	0
PFL1115w	Hypothetical protein	0	183	0	46	0	0	13	0
PFL2525c	Hypothetical protein	38	182	25	88	0	0	0	0
PF14_0136	Hypothetical protein, conserved	0	182	0	0	0	0	24	0
PFE1635w	Hypothetical protein	0	178	0	52	0	0	0	24
MAL8P1.97	Hypothetical protein	0	178	0	45	0	36	0	0
PF11_0287	Hypothetical protein	0	177	0	0	0	0	0	0
PF10_0032	Hypothetical protein	21	176	0	54	0	0	0	44
PF13_0225	Hypothetical protein	0	175	0	0	0	0	0	0
MAL6P1.259	Hypothetical protein	0	173	0	0	0	0	0	28
PFD0715c	Hypothetical protein, conserved	0	172	55	0	0	21	0	0
PF10_0216	Hypothetical protein	0	171	0	54	11	0	0	0
PF11_0274	Hypothetical protein	0	171	0	64	0	0	34	0
PFD0490c	Hypothetical protein	0	171	0	46	0	0	0	0
PFA0015c	Hypothetical protein	0	171	0	88	0	0	0	0
PFL1930w	Hypothetical protein	11	171	29	0	0	0	0	0
MAL13P1.340	Hypothetical protein	16	170	0	0	0	0	0	0
PFC0530w	Hypothetical protein	0	168	0	61	66	18	42	26
MAL8P1.162	Hypothetical protein	0	168	0	0	0	0	0	0
PF11_0334	Hypothetical protein	0	166	35	0	0	21	0	0
MAL13P1.194	Hypothetical protein	0	166	25	0	0	0	0	0
MAL8P1.80	Hypothetical protein	0	166	0	0	70	0	39	0
PFL1690w	Hypothetical protein	0	164	0	29	0	0	0	0
PF14_0269	Hypothetical protein, conserved	0	163	0	0	0	23	0	0
PF11_0411	Hypothetical protein	11	163	45	0	0	0	0	0
PF14_0130	Hypothetical protein	0	162	0	0	0	0	0	0
PF08_0050	Hypothetical protein	0	162	0	0	0	0	0	0
PF07_0076	Hypothetical protein	0	160	0	32	63	0	0	0
PF14_0759	Hypothetical protein	0	158	0	0	0	0	0	0
PF10_0113	Hypothetical protein	12	158	34	0	0	0	0	0
PF13_0260	Hypothetical protein, conserved	0	158	0	60	0	0	0	0
PFB0070w	Hypothetical protein	0	157	0	0	0	0	0	0
PF10_0161	Hypothetical protein	0	156	0	58	0	0	28	0
PFB0580w	Hypothetical protein	0	156	0	0	0	0	0	0
PF10_0290	Hypothetical protein	0	154	22	0	0	12	0	0
PFI0125c	Hypothetical protein	0	154	50	38	26	0	29	0
PFI0840w	Hypothetical protein	0	153	0	0	0	0	0	0
PF10_0044	Hypothetical protein	0	152	0	0	76	0	0	21
PFI0115c	Hypothetical protein	0	151	0	0	0	20	0	0
PF14_0170	Hypothetical protein	0	150	68	0	54	0	39	45
PF14_0284	Hypothetical protein	0	149	0	0	0	0	0	0
PF14_0663	Hypothetical protein	0	149	26	0	0	0	23	0
PF11_0221	Hypothetical protein	0	148	0	0	0	0	0	0
PF08_0012	Hypothetical protein	48	148	0	88	22	0	0	0
PFA0690w	Hypothetical protein	0	145	0	0	29	0	0	0
PF13_0078	Hypothetical protein	0	144	0	0	0	0	0	11
PFE1570c	Hypothetical protein	68	144	0	0	0	0	0	0
PFI0095c	Hypothetical protein	0	144	0	164	35	0	38	49
PF14_0574	Hypothetical protein	0	144	0	0	0	62	34	0
PF14_0626	Hypothetical protein	0	143	0	0	0	0	0	0
PF10_0230	Hypothetical protein, conserved	0	143	0	0	0	0	0	0
PFI1080w	Hypothetical protein	0	141	23	0	0	0	0	0
PF13_0230	Hypothetical protein	61	141	100	0	0	0	0	0
PF13_0172	Hypothetical protein	0	140	0	0	0	0	12	0
PFL1805c	Hypothetical protein	0	140	0	0	0	0	0	0
PFE1315w	Hypothetical protein	0	140	0	0	0	0	0	0
PF08_0030	Hypothetical protein	0	140	0	0	0	0	0	0
PF11_0518	Hypothetical protein	0	139	0	0	0	0	0	0
PFA0235w	Hypothetical protein	0	137	16	0	0	0	0	0
PF08_0117	Hypothetical protein	0	137	0	0	0	0	0	31
MAL6P1.43	Hypothetical protein	0	137	0	0	0	0	0	0
PF11_0178	Hypothetical protein	0	136	0	0	0	0	0	0
MAL6P1.31	Hypothetical protein, conserved	0	136	43	88	0	31	28	0
PF14_0330	Hypothetical protein	0	136	0	0	0	0	0	0
PF10_0142	Hypothetical protein	0	136	0	0	0	0	29	0
MAL6P1.20	Hypothetical protein, conserved	0	136	0	0	0	0	0	0
PF10_0042	Hypothetical protein	0	134	0	0	0	0	0	0
PFI0900w	Hypothetical protein	0	134	0	0	0	17	0	0
PF14_0287	Hypothetical protein	0	132	0	0	0	0	0	0
PFB0415c	Hypothetical protein	0	132	0	0	0	19	24	20
PF10_0284	Hypothetical protein	0	131	0	0	0	0	0	0
PFE1040c	Hypothetical protein	0	128	0	0	0	0	27	27
PFL0850w	Hypothetical protein	0	128	0	0	0	0	0	0
PFD0135w	Hypothetical protein	38	127	0	0	0	0	0	0
PFL0945w	Hypothetical protein	50	127	0	47	0	0	0	12
PFC0580c	Hypothetical protein	0	126	0	0	0	0	0	0
PFE1060c	Hypothetical protein	0	125	0	63	0	0	0	0
PFA0725w	Hypothetical protein	0	125	0	0	0	0	0	0
PFI1000w	Hypothetical protein	0	125	0	0	0	0	0	0
PF14_0379	Hypothetical protein	0	124	0	0	0	0	0	0
PF14_0169	Hypothetical protein	0	124	19	0	0	0	0	0
PF10_0027	Hypothetical protein	0	124	0	0	0	0	0	0
MAL7P1.23	Hypothetical protein	23	123	0	0	0	0	0	0
PFA0385w	Hypothetical protein	0	121	12	0	0	0	0	0
PF11_0349	Hypothetical protein	0	121	0	0	0	0	28	0
PFE0910w	Hypothetical protein	0	118	0	0	0	0	0	0
MAL13P1.348	Hypothetical protein	0	118	0	0	0	0	0	0
PFC0790w	Hypothetical protein	0	117	0	0	0	0	0	24
PF11_0089	Hypothetical protein	0	116	95	0	46	12	0	0
PFL1810w	Hypothetical protein	0	116	0	0	0	0	0	0
PFI1410c	Hypothetical protein	0	115	14	0	0	0	0	0
PF14_0264	Hypothetical protein	0	113	0	0	0	38	21	0
PF07_0127	Hypothetical protein	0	113	0	29	0	0	0	0
PFL1935c	Hypothetical protein	0	110	0	0	23	0	22	22
PF13_0355	Hypothetical protein	0	109	0	0	0	0	0	0
PF11_0366	Hypothetical protein	0	108	0	0	0	0	0	0
PF11_0527	Hypothetical protein	17	108	35	0	0	0	0	0
PFD1065c	Hypothetical protein	0	108	0	0	0	0	0	0
PFC0630w	Hypothetical protein	0	108	0	0	0	0	0	0
PFL0805w	Hypothetical protein	0	107	0	0	0	0	0	0
PFD0435c	Hypothetical protein	0	105	0	13	0	0	0	0
PFL2125c	Hypothetical protein	0	105	0	35	16	0	42	0
PF11_0063	Hypothetical protein	0	103	0	0	0	0	0	0
PFA0325w	Hypothetical protein	0	103	0	0	15	0	0	0
PFA0640c	Hypothetical protein	0	102	0	0	15	0	0	0
PF11_0360	Hypothetical protein	0	101	0	0	0	0	0	0
PF14_0720	Hypothetical protein	17	100	0	0	15	0	0	0
PF11_0468	Hypothetical protein	15	99	32	33	13	0	0	0
PFL0520c	Hypothetical protein	77	98	40	0	42	0	27	0
PF10_0131	Hypothetical protein	0	97	0	0	0	0	0	0
PF10_0059	Hypothetical protein	0	97	0	0	0	0	0	0
PFI1800w	Hypothetical protein	150	95	0	119	40	0	0	0
MAL8P1.68	Hypothetical protein	0	95	0	0	0	0	0	0
PF13_0038	Hypothetical protein	0	95	18	0	0	0	0	0
PFE1175w	Hypothetical protein	0	93	0	0	0	0	0	0
PF13_0292	Hypothetical protein	0	88	0	0	0	0	17	0
PFA0685c	Hypothetical protein, conserved in P. falciparum	52	85	34	0	0	0	0	0
PF11_0368	Hypothetical protein	0	84	0	0	0	0	0	0
PF10_0237	Hypothetical protein	17	84	25	11	0	0	0	0
PF11_0404	Hypothetical protein	0	84	13	0	0	0	16	0
PFE1525w	Hypothetical protein	18	84	0	0	0	0	0	0
PFC0460w	Hypothetical protein	0	83	0	0	0	0	0	0
PF11_0101	Hypothetical protein	11	83	0	0	0	0	0	0
PF11_0330	Hypothetical protein	0	82	0	0	0	0	0	0
PFC0905c	Hypothetical protein	0	81	0	0	0	0	0	0
PF11_0441	Hypothetical protein	0	80	0	0	0	0	0	0
PF11_0262	Hypothetical protein	0	80	0	0	0	0	0	0
PFL0760w	Hypothetical protein	0	79	0	0	0	0	0	0
PF07_0125	Hypothetical protein, conserved	0	76	0	0	0	0	0	0
PF11_0084	Hypothetical protein	0	76	0	0	0	0	0	0
PF14_0305	Hypothetical protein	0	74	0	0	0	0	0	0
PF14_0271	Hypothetical protein	0	73	0	0	0	10	0	0
PF14_0044	Hypothetical protein	0	71	0	52	0	0	0	0
MAL7P1.59	Hypothetical protein	12	71	0	14	0	0	0	0
PFI0260c	Hypothetical protein	0	70	0	0	0	0	0	0
PFA0665w	Hypothetical protein	0	68	0	29	0	0	0	0
PF14_0458	Hypothetical protein	12	67	16	0	0	0	0	0
PF14_0573	Hypothetical protein	0	67	0	0	0	0	0	0
PF14_0199	Hypothetical protein	0	67	0	0	0	0	0	0
PF08_0090	Hypothetical protein	0	66	16	0	0	0	0	0
PFI0285w	Hypothetical protein	15	65	17	0	0	0	17	0
PFD0445c	Hypothetical protein	0	65	0	0	0	0	0	0
PFD0330w	Hypothetical protein	0	64	0	0	0	0	0	0
PFI1045w	Hypothetical protein	0	59	0	0	0	0	0	0
PF11_0288	Hypothetical protein	0	57	0	0	0	0	0	0
MAL13P1.179	Hypothetical protein	0	53	28	0	0	0	0	0
PFL1515c	Hypothetical protein	0	52	0	15	0	0	0	0
PFI1620c	Hypothetical protein	0	51	0	0	0	0	0	11
MAL13P1.188	Hypothetical protein	0	49	0	0	0	0	0	0
PF07_0014	Hypothetical protein	0	47	0	0	0	0	0	0
PF14_0706	Hypothetical protein	0	43	0	0	0	0	0	0
PF14_0742	Hypothetical protein	0	42	0	0	0	0	0	0
PFL1190c	Hypothetical protein	11	40	0	0	0	0	0	0
PFD1085w	Hypothetical protein	0	40	0	0	0	0	0	0
PFE1095w	Hypothetical protein	0	39	0	16	0	0	0	0
PF08_0016	Hypothetical protein	31	39	19	0	14	11	0	0
PF13_0293	Hypothetical protein	0	37	0	0	0	0	0	0
PFC0990c	Hypothetical protein	0	34	0	0	0	0	0	0
PFD1160w	Hypothetical protein	0	31	0	0	0	0	0	0
PFL1985c	Hypothetical protein	0	26	0	0	0	0	0	0
PFI0090c	Hypothetical protein	12	25	0	0	0	0	0	0
PF14_0535	Hypothetical protein	0	24	0	0	0	0	0	0
PFL0985c	Hypothetical protein	0	24	11	0	12	0	0	0
PFI0245c	Hypothetical protein	0	22	0	0	0	0	0	0
PF14_0395	Hypothetical protein, conserved	0	20	0	0	0	0	0	0
PF14_0116	Hypothetical protein	16	19	13	12	15	0	0	0
PF11_0514	Hypothetical protein	0	18	0	0	0	0	0	0
PFL0115w	Hypothetical protein	0	18	0	0	0	0	0	0
MAL8P1.47	Hypothetical protein	0	17	0	15	0	0	0	0
PF13_0075	Hypothetical protein	0	17	11	0	0	0	0	0
MAL13P1.232	Hypothetical protein	0	16	0	0	0	0	0	0
MAL6P1.211	Hypothetical protein	0	15	0	13	11	0	0	0
PFB0190c	Hypothetical protein	0	15	0	0	0	0	0	0
MAL8P1.7	Hypothetical protein	0	14	0	0	0	0	0	0
PF14_0162	Hypothetical protein	0	11	0	0	0	0	0	0
PF14_0445	Hypothetical protein	0	11	0	0	0	0	0	0
MAL6P1.166	Hypothetical protein	0	11	11	0	0	0	0	0

NOTE. ER, early ring; LR, late ring; M, merozoite; PfEMP1, erythrocyte membrane protein 1.

Table 5. Sample 8 genes with transcript levels at least 2-fold greater than those in in vitro 3D7 samples.

Gene	Description	Expression units
		In vivo					In vitro 3D7
		6	7	8	12	29	M	ER	LR
Surface proteins
PFE0005w	PfEMP1	114	209	234	184	90	34	95	61
PF08_0141	PfEMP1	0	90	180	0	0	0	32	0
MAL6P1.316	PfEMP1	0	106	141	0	0	0	32	21
PF10_0399	RIFIN	197	356	303	185	58	52	0	32
PF14_0768	RIFIN	162	290	295	182	144	36	39	28
PFL0015c	RIFIN	104	443	196	162	71	41	0	28
PFL2660w	RIFIN	0	451	177	0	120	40	42	0
PF11_0515	RIFIN	0	396	174	116	0	0	0	28
PFI0030c	RIFIN	124	185	156	120	53	33	16	21
PFD0055w	RIFIN	46	138	137	108	38	17	0	13
PF14_0004	RIFIN	77	164	127	77	48	18	0	17
PFB1010w	RIFIN	45	121	115	65	0	19	0	0
PF13_0007	RIFIN	58	123	106	64	43	0	14	21
PFA0050c	RIFIN	0	0	86	96	0	0	0	0
PFE0025c	RIFIN	0	0	83	73	0	0	0	0
PFD0130c	RIF pseudogene	0	176	77	76	0	30	0	0
PF10_0394	RIFIN	0	201	45	0	0	0	0	0
PFA0090c	STEVOR	197	263	364	245	0	52	36	0
PF14_0007	STEVOR, putative	0	200	216	108	81	42	0	30
PFD0035c	STEVOR	57	502	197	189	0	50	0	0
PFB0955w	STEVOR, degenerate, putative	0	450	174	217	0	25	19	0
PFB0025c	STEVOR, putative	0	276	147	0	0	64	0	56
PF11_0512	RESA-2	530	156	379	443	626	0	0	0
Metabolic/cellular function
PF13_0045	40S ribosomal protein S27, putative	925	511	1133	1248	1431	0	342	483
PF14_0183	RNA helicase, putative	480	131	413	1373	227	92	154	128
PFC0855w	Ubiquitin-conjugating enzyme, putative	277	709	396	473	194	0	121	153
PFC0050c	Long-chain fatty acid ligase, putative	617	186	192	73	92	0	0	0
PFE1265w	G-protein coupled receptor, putative	40	405	135	91	0	0	0	0
PFI1110w	Glutamate-ammonia ligase (glutamine synthetase)	79	289	126	109	66	0	52	37
PF14_0751	Fatty acyl coenzyme A synthetase-1, putative	0	143	80	0	0	0	0	0
PFI0255c	Mitochondrial carrier protein, putative	0	161	62	112	0	0	0	0
PFI0415c	Ribosomal RNA methyltransferase, putative	23	0	42	22	0	0	0	0
MAL8P1.22	Dehydrodolichyl diphosphate synthetase, putative	26	130	20	0	14	0	0	0
MAL7P1.92	Cysteine repeat modular protein 2 homologue	0	0	13	0	0	0	0	0
MAL7P1.164	Adaptor-related protein, putative	0	0	10	18	0	0	0	0
Hypothetical protein
PF11_0032	Hypothetical protein	1646	3627	3499	2583	1071	774	393	697
PFA0620c	Hypothetical protein	569	211	2950	122	1383	0	913	444
MAL7P1.39	Hypothetical protein	114	2246	604	504	226	175	105	94
PFA0135w	Hypothetical protein	469	313	577	883	307	229	0	37
PF11_0033	Hypothetical protein	284	1926	550	544	241	212	49	68
chr1.rRNA-1-ITS2	Hypothetical protein	147	1021	395	333	96	49	0	0
PF14_0752	Hypothetical protein	837	0	314	569	351	44	76	23
PFA0075w	Hypothetical protein	0	664	259	195	0	67	0	0
PFD0065w	Hypothetical protein	94	276	233	165	285	31	0	0
PF10_0383	Hypothetical protein, conserved	111	1063	229	345	120	105	17	33
MAL8P1.30	Hypothetical protein	118	639	227	206	109	52	28	42
PF14_0743	Hypothetical protein	119	730	224	174	84	96	0	78
PFL0720w	Hypothetical protein	76	672	201	175	86	69	0	38
PFA0245w	Hypothetical protein	52	626	170	197	68	39	0	0
PF14_0298	Hypothetical protein	96	215	163	122	0	0	22	0
PFL1225c	Hypothetical protein	0	0	158	0	0	61	0	58
PF11_0271	Hypothetical protein, conserved	0	360	146	139	58	0	0	71
PF11_0006	Hypothetical protein	173	1188	142	295	178	0	0	0
PF14_0562	Hypothetical protein	0	509	135	104	0	0	0	24
PF14_0539	Hypothetical protein	43	385	133	129	49	37	26	0
PFC0505c	Hypothetical protein	76	536	130	169	74	43	30	48
PFL0955c	Hypothetical protein	0	200	128	102	0	0	0	0
PFE0030c	Hypothetical protein	0	238	114	100	0	18	0	0
PF13_0230	Hypothetical protein	61	141	100	0	0	0	0	0
PFB0950w	Hypothetical protein	0	289	100	99	0	0	0	0
PFI0485c	Hypothetical protein	55	289	98	144	0	0	0	0
PF11_0004	Hypothetical protein	49	253	97	152	67	35	16	0
PF11_0089	Hypothetical protein	0	116	95	0	46	12	0	0
PFL1660c	Hypothetical protein	0	253	90	48	0	23	0	20
PFA0720w	Hypothetical protein	31	365	86	116	0	0	0	0
PFE0095c	Hypothetical protein	0	0	70	0	0	0	0	0
MAL7P1.52	Hypothetical protein	0	258	63	92	0	0	0	0
MAL6P1.233	Hypothetical protein	0	0	54	0	0	0	0	0
PF11_0411	Hypothetical protein	11	163	45	0	0	0	0	0
PF14_0158	Hypothetical protein	0	205	44	0	0	0	0	0
PF13_0151	Hypothetical protein	0	0	43	0	0	0	0	0
mitopl_rpoC	Hypothetical protein	0	0	38	0	0	0	0	0
PFB0395w	Hypothetical protein	0	0	37	0	0	0	0	0
PF07_0109	Hypothetical protein	0	0	34	0	0	0	0	0
PFB0690w	Hypothetical protein	0	0	34	0	0	10	0	0
MAL7P1.89	Hypothetical protein	0	0	30	22	0	0	0	0
MAL13P1.179	Hypothetical protein	0	53	28	0	0	0	0	0
PF14_0558	Hypothetical protein	19	0	27	0	0	0	0	0
PFL2525c	Hypothetical protein	38	182	25	88	0	0	0	0
PF08_0013	Hypothetical protein	14	0	22	0	0	0	0	0
PFB0110w	Hypothetical protein	0	0	20	0	0	0	0	0
PF11_0421	Hypothetical protein	0	0	19	15	0	0	0	0
PFI1785w	Hypothetical protein	0	0	18	0	0	0	0	0
PF13_0012	Hypothetical protein	0	0	18	0	0	0	0	0
PF14_0521	Hypothetical protein	11	0	18	0	0	0	0	0
PF13_0038	Hypothetical protein	0	95	18	0	0	0	0	0
PF14_0202	Hypothetical protein	0	0	17	0	0	0	0	0
PFE0190c	Hypothetical protein	0	0	16	0	0	0	0	0
PF10_0206	Hypothetical protein	0	0	16	0	0	0	0	0
PFC0075c	Hypothetical protein	18	0	16	32	0	0	0	0
MAL13P1.32	Hypothetical protein	0	0	16	0	0	0	0	0
PFL2385c	Hypothetical protein	0	0	15	20	0	0	0	0
PF08_0070	Hypothetical protein	0	0	15	14	0	0	0	0
PFB0960c	Hypothetical protein	0	0	15	0	0	0	0	0
PF11_0410	Hypothetical protein	0	0	13	11	0	0	0	0
PF14_0049	Hypothetical protein	0	0	13	0	0	0	0	0
PF11_0064	Hypothetical protein	0	0	13	0	0	0	0	0
PF14_0116	Hypothetical protein	16	19	13	12	15	0	0	0
PFI0815c	Hypothetical protein, conserved	0	0	13	0	0	0	0	0
PF10_0055	Hypothetical protein	0	0	12	0	0	0	0	0
PFC0215c	Hypothetical protein	0	0	12	0	0	0	0	0
PFA0385w	Hypothetical protein	0	121	12	0	0	0	0	0
PFB0946c	Hypothetical protein	0	0	12	0	0	0	0	0
MAL6P1.187	Hypothetical protein	0	0	12	0	0	0	0	0
PFE0610c	Hypothetical protein	0	0	12	13	0	0	0	0
PF10_0158	Hypothetical protein	0	0	12	0	0	0	0	0
PFE0085c	Hypothetical protein	0	0	11	0	0	0	0	0
MAL8P1.41	Hypothetical protein	15	0	11	0	0	0	0	0
MAL6P1.166	Hypothetical protein	0	11	11	0	0	0	0	0
PF13_0075	Hypothetical protein	0	17	11	0	0	0	0	0
PFB0510w	Hypothetical protein	0	0	11	23	0	0	0	0
PF14_0534	Hypothetical protein	0	0	11	0	0	0	0	0
PFE1180c	Hypothetical protein	0	0	11	10	0	0	0	0
PFL0985c	Hypothetical protein	0	24	11	0	12	0	0	0
PFL2020c	Hypothetical protein	0	0	11	0	19	0	0	0
PFI1305w	Hypothetical protein	0	0	11	0	0	0	0	0
PF11_0462	Hypothetical protein	0	0	11	0	0	0	0	0
PFB0470w	Hypothetical protein	18	0	10	0	0	0	0	0
PFD0870w	Hypothetical protein	0	0	10	0	0	0	0	0
PFC0130c	Hypothetical protein	0	0	10	0	0	0	0	0

NOTE. ER, early ring; LR, late ring; M, merozoite; PfEMP1, erythrocyte membrane protein 1

Table 6. Sample 12 genes with transcript levels at least 2-fold greater than those in in vitro 3D7 samples.

Gene	Description	Expression units
		In vivo					In vitro 3D7
		6	7	8	12	29	M	ER	LR
Surface proteins
PFL2585c	RIFIN	1606	245	0	2301	0	0	0	0
PFA0710c	RIFIN	104	162	132	309	74	26	0	21
PF10_0003	RIFIN	144	0	0	215	384	52	0	0
PFI1810w	RIFIN	177	0	0	183	0	0	0	0
PF14_0769	RIFIN	0	182	0	164	0	0	0	0
PFL0015c	RIFIN	104	443	196	162	71	41	0	28
PFB0060w	RIFIN	0	346	0	128	0	0	0	0
MAL6P1.9	RIFIN	34	195	62	125	55	0	14	0
PFI0030c	RIFIN	124	185	156	120	53	33	16	21
PFA0740w	RIFIN	0	173	110	118	50	0	0	0
MAL6P1.250	RIFIN	52	82	0	115	44	0	0	0
PF10_0402	RIFIN	0	0	0	114	0	0	0	0
PFD0055w	RIFIN	46	138	137	108	38	17	0	13
PFA0050c	RIFIN	0	0	86	96	0	0	0	0
PF14_0004	RIFIN	77	164	127	77	48	18	0	17
PFD0130c	RIF pseudogene	0	176	77	76	0	30	0	0
PFE0025c	RIFIN	0	0	83	73	0	0	0	0
PFB1015w	RIFIN	0	0	0	73	0	0	0	0
PFD0645w	RIFIN	60	156	60	71	37	0	0	0
PFC1100w	RIFIN (3D7-rifT3-6)	0	386	0	66	0	0	0	0
PFB1020w	STEVOR, putative	0	980	240	294	176	0	17	33
PFB0955w	STEVOR, degenerate, putative	0	450	174	217	0	25	19	0
PFD0035c	STEVOR	57	502	197	189	0	50	0	0
PF14_0007	STEVOR, putative	0	200	216	108	81	42	0	30
PF11_0512	RESA-2	530	156	379	443	626	0	0	0
Metabolic/cellular function
PF11_0250	High mobility group–like protein NHP2, putative	2285	402	1081	2205	1351	317	545	563
PF14_0690	Histone deactylase, putative	1207	142	609	1929	390	80	305	138
PF11_0280	Small nuclear ribonucleoprotein F, putative	890	1572	1200	1408	662	85	278	229
PF14_0183	RNA helicase, putative	480	131	413	1373	227	92	154	128
PF13_0045	40S ribosomal protein S27, putative	925	511	1133	1248	1431	0	342	483
PFC0855w	Ubiquitin-conjugating enzyme, putative	277	709	396	473	194	0	121	153
PF14_0423	Serine/threonine protein kinase, putative	119	68	0	145	106	0	68	0
PF14_0469	Transcription factor IIIb subunit, putative	89	0	0	136	0	0	55	0
MAL6P1.194	Fe-superoxide dismutase	73	214	87	116	52	0	0	0
PF13_0149	Chromatin assembly factor 1 subunit, putative	0	187	0	113	0	21	17	0
PFI0255c	Mitochondrial carrier protein, putative	0	161	62	112	0	0	0	0
PFI1110w	Glutamate-ammonia ligase (glutamine synthetase), putative	79	289	126	109	66	0	52	37
PFL1940w	3-hydroxyisobutyryl-coenzyme A hydrolase, putative	76	203	0	102	62	0	28	0
PFC0050c	Long-chain fatty acid ligase, putative	617	186	192	73	92	0	0	0
PF10_0275	Protoporphyrinogen oxidase, putative	0	0	19	29	0	0	0	0
MAL7P1.164	Adaptor-related protein, putative	0	0	10	18	0	0	0	0
MAL8P1.37	Lipoate-protein ligase, putative	10	0	0	11	13	0	0	0
Hypothetical protein
PFL2565w	Hypothetical protein	2093	130	845	3139	1078	340	770	207
PF13_0194	Hypothetical protein	2303	820	615	2394	1690	519	822	215
PFB0280w	Hypothetical protein	1475	112	55	1723	166	32	259	188
PFD0320c	Hypothetical protein	1051	78	691	1671	346	43	438	104
chr5.rRNA-1-18s-A	Hypothetical protein	1331	719	550	1575	887	0	0	0
PF14_0753	Hypothetical protein	2110	432	660	1539	1655	664	323	62
PF11_0035	Hypothetical protein	437	0	0	994	0	0	0	0
PFB0279w	Hypothetical protein	578	0	0	968	101	31	242	93
PFA0135w	Hypothetical protein	469	313	577	883	307	229	0	37
MAL13P1.122	Hypothetical protein	683	275	287	693	582	99	174	85
PF11_0037	Hypothetical protein	326	135	0	614	164	0	0	0
PF14_0752	Hypothetical protein	837	0	314	569	351	44	76	23
PF11_0033	Hypothetical protein	284	1926	550	544	241	212	49	68
PF14_0760	Hypothetical protein	203	164	154	524	107	139	98	51
MAL7P1.39	Hypothetical protein	114	2246	604	504	226	175	105	94
PF08_0133	Hypothetical protein	142	168	0	346	0	0	0	0
PF10_0383	Hypothetical protein, conserved	111	1063	229	345	120	105	17	33
chr1.rRNA-1-ITS2	Hypothetical protein	147	1021	395	333	96	49	0	0
PF10_0089	Hypothetical protein	226	361	149	326	208	40	131	104
PF11_0006	Hypothetical protein	173	1188	142	295	178	0	0	0
PF14_0734	Hypothetical protein	196	87	69	256	537	18	102	37
PF11_0003	Hypothetical protein	0	764	0	232	117	0	0	0
MAL6P1.312	Hypothetical protein	126	196	0	229	72	0	0	31
MAL8P1.160	Hypothetical protein	214	0	0	225	207	80	38	0
MAL8P1.30	Hypothetical protein	118	639	227	206	109	52	28	42
PFA0245w	Hypothetical protein	52	626	170	197	68	39	0	0
PFC0505c	Hypothetical protein	76	536	130	169	74	43	30	48
PFD0065w	Hypothetical protein	94	276	233	165	285	31	0	0
PFI0095c	Hypothetical protein	0	144	0	164	35	0	38	49
PF11_0004	Hypothetical protein	49	253	97	152	67	35	16	0
PFI0485c	Hypothetical protein	55	289	98	144	0	0	0	0
PF14_0539	Hypothetical protein	43	385	133	129	49	37	26	0
PF11_0276	Hypothetical protein	80	201	0	126	0	21	17	0
PFD0655w	Hypothetical protein	0	195	0	120	0	0	0	0
PFI1800w	Hypothetical protein	150	95	0	119	40	0	0	0
PFA0720w	Hypothetical protein	31	365	86	116	0	0	0	0
PFC0240c	Hypothetical protein	0	203	0	106	47	31	42	24
PF14_0562	Hypothetical protein	0	509	135	104	0	0	0	24
PFL0955c	Hypothetical protein	0	200	128	102	0	0	0	0
PFA0655w	Hypothetical protein	98	0	0	102	0	0	0	0
PFE0030c	Hypothetical protein	0	238	114	100	0	18	0	0
PFB0950w	Hypothetical protein	0	289	100	99	0	0	0	0
PF10_0229	Hypothetical protein, conserved	0	0	0	99	0	0	0	0
MAL7P1.52	Hypothetical protein	0	258	63	92	0	0	0	0
MAL7P1.97	Hypothetical protein	0	434	0	89	0	0	0	0
PFL2525c	Hypothetical protein	38	182	25	88	0	0	0	0
PF08_0012	Hypothetical protein	48	148	0	88	22	0	0	0
PFI1775w	Hypothetical protein	132	185	0	67	0	0	0	0
MAL7P1.43	Hypothetical protein	0	0	0	62	0	0	0	0
PFA0705c	Hypothetical protein	0	349	0	57	0	0	0	0
PF14_0129	Hypothetical protein	0	0	13	55	0	0	0	0
PF14_0044	Hypothetical protein	0	71	0	52	0	0	0	0
PFL1660c	Hypothetical protein	0	253	90	48	0	23	0	20
PFL0945w	Hypothetical protein	50	127	0	47	0	0	0	12
PFL1115w	Hypothetical protein	0	183	0	46	0	0	13	0
PF08_0022	Hypothetical protein	0	198	0	42	0	0	0	0
PFC0075c	Hypothetical protein	18	0	16	32	0	0	0	0
PFA0665w	Hypothetical protein	0	68	0	29	0	0	0	0
PF07_0127	Hypothetical protein	0	113	0	29	0	0	0	0
PFE0210c	Hypothetical protein	0	0	0	26	0	0	0	0
PF10_0007	Hypothetical protein	0	0	0	24	0	0	0	0
PFB0925w	Hypothetical protein	0	0	15	24	0	0	0	0
PFB0510w	Hypothetical protein	0	0	11	23	0	0	0	0
PFL2385c	Hypothetical protein	0	0	15	20	0	0	0	0
PF14_0383	Hypothetical protein	0	0	0	16	0	0	0	0
MAL8P1.47	Hypothetical protein	0	17	0	15	0	0	0	0
PF11_0421	Hypothetical protein	0	0	19	15	0	0	0	0
PF08_0070	Hypothetical protein	0	0	15	14	0	0	0	0
PF10_0008	Hypothetical protein	0	0	0	13	0	0	0	0
PFE0610c	Hypothetical protein	0	0	12	13	0	0	0	0
MAL6P1.211	Hypothetical protein	0	15	0	13	11	0	0	0
PFL0905c	Hypothetical protein	0	0	0	13	0	0	0	0
PFE0230w	Hypothetical protein	10	0	0	12	15	0	0	0
PF14_0116	Hypothetical protein	16	19	13	12	15	0	0	0
PF11_0228	Hypothetical protein	0	0	0	11	0	0	0	0
PF11_0410	Hypothetical protein	0	0	13	11	0	0	0	0
PF14_0385	Hypothetical protein	0	0	0	11	0	0	0	0
MAL7P1.98	Hypothetical protein	0	0	0	11	0	0	0	0
PF11_0528	Hypothetical protein	0	0	0	11	0	0	0	0
PFE1180c	Hypothetical protein	0	0	11	10	0	0	0	0
PFL0090c	Hypothetical protein	0	0	0	10	0	0	0	0
PF14_0710	Hypothetical protein	0	0	0	10	0	0	0	0

NOTE. ER, early ring; LR, late ring; M, merozoite.