1. The document discusses gene expression methods used to study the var2 and var3 genes of Plasmodium falciparum, which cause malaria. 2. Microarray, northern blotting, and RT-qPCR methods are described for analyzing var gene expression at different parasite stages. Studies found var2 mRNA is expressed at higher levels, associated with more severe malaria. 3. Understanding var gene expression is important as it leads to production of PfEMP1, which allows the parasite to bind red blood cells and evade the immune system, causing infection. Interfering with var gene expression may help circumvent malaria's dangers.

1. 1
REVIEW ON THE USE OF GENE EXPRESSION DATA IN THE STUDY OF
HUMAN MALARIA DISEASE.
BY ONYIA, CHIADIBOBI E.
SCHOOL OF BIOMEDICAL SCIENCES, CARDIFF METROPOLITAN UNIVERSITY, UK.
MALARIA OVERVIEW
Malaria with symptoms like fever and headache is a human disease caused by Plasmodium
falciparum and transmitted by female Anopheles mosquito. The disease affects over 400
million people worldwide, mainly Africa, Asia and South America killing nearly one million
people per annum (Kritsiriwuthinan et al., 2011) with 91% of cases in Africa (WHO, 2012)
(Bachmann et al., 2011; Volz et al., 2012).
Plasmodium falciparum undergoes cyclic developmental life cycle, where the oocysts
produced by the fusion of the gametes develop into sporozoites in the mosquito mid-gut and
transmitted to the human host during the mosquito blood meal. The matured sporozoites in
the hepatocyte are released into the blood as merozoites (Boddey and Cowman, 2013) as
shown in fig.1.
Fig. 1a: Malaria the fourth leading deadly disease in Africa (WHO, 2012) as indicated by the arrow.

2. 2
Fig. 1b: The life cycle ofPlasmodiumfalciparum(McW Healthcare, 2008). Female Anopheles mosquito onfeedingwith on human host
bloodtransmits sporozoite tothe hepatocyte,which develop andare released toinvade the erythrocyte as merozoite. It is the initiates the
production of pfemp1 that mediate malaria infection.
The merozoites released from the hepatic region internalized the cell by bind to the
glycophorin-A receptor complex (see fig 2ii) on the erythrocyte membrane and remolds the
erythrocyte, by altering the erythrocytic normal gene expression (Zhang et al., 2014). The
reason why the parasite chose to use the red blood cell, might be because of the presence of
iron that help them mediate its transcription. The presence of haem-iron in the erythrocyte
helped merozoite with its machinery to switch on its gene expression, where by Plasmodium
falciparum erythrocyte membrane protein 1 (PfEMP1) and other proteins are produced as
they undergo various stages of development (namely; ring stage, trophozoite stage and
schizont stage) inside the human red blood cell, (Boddey and Cowman, 2013; Zhang et al.,
2014) as shown in fig 2i.
i ii
Fig.2. (i) The asexual intraerythrocyticdevelopmental cycle (IDC). From 0-8h (ring stage), 8-16h (early trophozoite), 16-24h (late
trophozoite stage),24-32h(earlyschizont),32-40h(late schizont stage), 40-48h (assembly andrelease of PfEMP1proteinforinfection) (ii)
merozoite internalization via band-3-glycophorinA complex (Rai et al., 2014; PNAS, 2010)
The PfEMP1 produced, binds to uninfected erythrocyte (rosetting) with its duffy binding like
(DBL) region, while cysteine-rich interdomain region (CIDR) mediates binding to inter
cellular adhesion molecule1 (ICAM-1) and cluster of differentiation 36 (CD36) receptors in
the endothelial of the human cells (Duffy et al., 2002; Pasternak and Dzikowski, 2009). The
rosetting prevents the cell from being destroyed by the splenic macrophage (Scherf et al.,
2008; Boddey and Cowman, 2013). But this antigenic PfEMP1 is encoded by variant (var)
gene.
Var gene family of about 60 genes is being grouped into three main upstream sequences
(ups), based on their transcriptional orientation, as upsA, upsB and upsC (Witmer, 2011;
Volz, 2012), whereby upstream sequence A (upsA) var genes and upsB genes are located in

3. 3
the two subtelomeric regions while upsC are centrally located within the telomeric region of
the chromosome.
Kyes et al., (2007) stated that most var gene expression occur during the ring stage while
Zhang et al., (2014) and Albrecht et al., 2011 on exploring var3 and var2 genes respectively
observed that their transcription peak fall within late trophozoite stage. Further study
conducted by Noble et al., (2013) on the antigenic switching of thirty-five different var genes
and their implication for malaria disease, concluded that most var gene are expressed at the
three stages of their intraerythrocytic developmental cycle. This provoked an initial debate on
whether var gene location contributes to its gene expression efficiency.
But since it has been established that var genes encode for the main virulence antigenic
PfEMP1, it is evident that var2 and var3 genes forms the bases of this review concerning
malaria disease, based on the fact that they possessed duffy binding like (DBL) core domain
within their PfEMP1 (see fig 11) which is the bases of malaria infection occurrence.
VAR GENES EXPRESSION METHODS FOR MALARIA DISEASE
For merozoite to cause malaria, PfEMP1 antigenic protein must be produced, but there are
various research works conducted on var2 and var3 genes expression. Their work showed
that these genes expresses either micro inhibitory RNA (miRNA) that does not produce
PfEMP1 or mRNA which produces the main antigenic variant (PfEMP1), which gave rise to
malaria disease in man (Pasternak and Dzikowski, 2009). In that essence, the severity of any
var gene expression lies on its ability to produce PfEMP1.
Therefore, studies on var2 and var3 gene expressions have been determined using various
methods like microarray method, northern blotting and quantitative reverse transcriptase
polymerase chain reaction (RT-qPCR) (QPCR) method as described below.
Microarray method protocol: Due to microarray competency in accessing multiple
genes at a time, this method was used to determine the likely gene expressions that are
responsible for malaria infection and probably its treatment following these protocols:
Infected and non-infected human volunteers and animal models were used, whereby total
RNA were extracted from infected and non-infected erythrocyte using TRIzol reagent
(Kritsiriwuthinan et al., 2011).
The RNA obtain were converted to mRNA poly (A) using oligo (dT) and then to cDNA using
reverse transcriptase, for easy labeling. Each cDNA used were labelled using different
fluorescent dye like (cyanine) Cy3, Cy5 monoreactive dye (Kritsiriwuthinan et al., 2011).
The labelled cDNA transcripts were transferred unto an affymetrix gene-chips micro-chip
slide, wherein several oligo nucleotides have been (embedded) incorporated for hybridization
to take place (Kulkarni et al., 2012). The cDNA differently labeled hybridized, with different
colour indication as shown in fig 3.

4. 4

5. 5
Fig.3 Microarraydataanalysis: Sample from human iRBC, reverse transcribed and labeled with cyanine dye. The labeled dyes then
hybridizedandvisualize The result showedthat the micro-chipthat have already been embeddedwith oligo nucleotides hybridizedwith the
appropriate complementary cDNA labelledstrandandfluorescence, wherein coloredblack showedun-infected (i.e. no expression and no
hybridization), green showed moderate expression and red showed high expression (Kulkarni et al., 2012).
Northern blotting protocol: This method was used to isolate the specific RNAs from
the genes of interest for more detailed study. Kyes et al., (2007) identified Plasmodium
falciparum var gene expression using northern blotting method; firstly RNAs were obtained
using Trizol reagent to lyse the parasitized synchronized red blood cell (pRBC) at ring,
trophozoite and schizont stages of parasite cycle.
Secondly the RNAs extracted from pRBC were hybridized with α-32P-dATP mega prime
probe and visualise using agarose gel that containing ethidium bromide (Kyes et al., 2007),
knowing that the band intensity is proportional to the amount of target mRNA present.
This method proved that different quantities of mRNAs were expressed at difference stages
of the parasite intraerythrocytic developmental cycle.
RT-qPCR (Real-time PCR) protocol: Albrecht et al., (2011); Bachmann et al.,
(2011); Noble et al., (2013) and Zhang et al., (2014) on their various var genes exploration,
used RT-qPCR method for var gene expression. RT-qPCR method measures PCR amplicon
as it occurs, so that the concentration of RNAs will be determined at its linear phase (Applied
Biosystem, 2010).
Sample collection for successful RT-qPCR protocol: Experimental sample, dNTP-mix,
forward and reverse primer, Taq polymerase, buffer, nuclease free water, reverse
transcriptase and real-time PCR which contains a fluorescent reporter molecule, TaqMan
probe or SYBR Green dye were collected(Applied Biosystem, 2010).
RNA isolation, confirmation and reverse transcription procedure: RNAs isolate were
obtained firstly by lysing the cell and by treating them with DNase1 to remove the whole
genomic DNA (gDNA) present and seryl-tRNA synthetase were used to confirm the absence
of gDNA which would have caused spurious result (false positive result) (Bachmann et al.,
2011). The purified polyadenylated mRNA present were reversed transcribed to double
stranded cDNA by adding oligo (dT) primer (see fig.4). Rename H were also added to

6. 6
remove the RNA strand of the double stranded cDNA leaving only single stranded DNA as
the template cDNA ready for qPCR assay (Sellner an d Turbett, 1998).
Fig.4 Prerequisite for determination of mRNAlevel:The mRNAs are being reverse transcribed to cDNA that is more stable for PCR
amplification (Applied Biosystem, 2010)
Primer design for RT-qPCR method: Good gene specific primers were designed following
the standard method of obtaining 40-60% of GC content, melting temperature of 58-600C ,
base length of 18-25 and targeting of the 3’ untranslated region (Holland et al., no-date).
SYBR Green probe for RT-qPCR quantification: Albrecht et al., (2011) and Bachmann et
al., (2011) used Power SYBR Green Master mix as their fluorescent dye. As the primers
elongate the single stranded cDNA sequence, SYBR Green being a non-specific probe binds
to the double stranded cDNA to fluorescence (see fig. 7b) (Thomas, 2014). Knowing that
SYBR Green is non-specific, melting analysis was used to confirm the PCR product
specificity (Noble et al., 2013) should in-case primers hybridized.
TaqMan probe for RT-qPCR quantification: Others used TaqMan probe (hydrolysis
probe) which is sensitive, specific and are capable of quantitating the target PCR product.
TaqMan probe mechanism, shows that the experimental genes (cDNA of interest) at 950C
PCR temperature were denatured and the specific hybridize probe binds (anneals) to the
transcript using its 5’ to 3’ exonuclease (see fig. 5) (Sellner et al., 1998). The probe was
designed by placing reporter fluorescent and a quencher at very close proximity so that the
reporter will not be able to fluorescence except when disassociated from the quencher, as
shown in fig.5 and 7a.

7. 7
Figure 5: TaqMan Gene Expression Assayreaction steps (AppliedBiosystem,2012).DNA template denatures allowingprobe andprimer
to anneal.Primerbindingmediate Taqpolymerase toelongate,separatingthe reporter from quencher allowing reporter to fluorescence.
Threshold line Exponential stage Stationary phase
Fig 6. RT-qPCR product visualization through graph display (Adopted from, Applied Biosystem, 2010), were the gene expression
product is made visible when the emissioncrosses the thresholdline.The higher the start template the faster it crosses the threshold line.
This can be applicable to both TaqMan and SYBR Green probe only that TaqMan product will be more specific.

8. 8
a b
Fig. 7. (a) 5’ Nuclease (TaqMan Probe) Assay (b) SYBR Green Dye Assay
Here shows the differences betweenthe use of TaqManandSYBR Green probe. TaqManhas a specific oligo nucleotide with reporter dye
andquencher andbinds to a specific template,whereas SYBR Green probe binds to anydouble strandpresent makingit nonspecific in that
it can detect probe hybrid as count which is a false positive result (Applied Biosystem, 2010).
In probe designing it was made in such a way that the probe annealing temperature is 5-100C
greater than the primer annealing temperature (Sellner et al., 1998) and the temperature
variation encourages the probe to anneal first to the single stranded cDNA before the primer,
because primer annealing initiates extension. If primer anneal and extent without the probe,
photon will not be emitted.
Primer binding causes the Taq polymerase to commence extension of the transcript, which
knocks-out the probe thereby separating the reporter from the quencher and the reporter emit
photon that are detected by the fluorimeter (Sellner et al., 1998). As the cycle continues the
emission increases, displaying a graph chat as shown in fig 6, creating an exponential phase
that crosses the threshold line as the PCR product increases.
CRITICAL ANALYSIS ON VAR GENE EXPRESSED DATA
Considering the vast work conducted by many researchers in the area of Plasmodium
falciparum var gene expression, their works reveal the likely causes and possible approaches
to malaria disease, which is anchored on var gene expression and its protein sequestration as
shown in fig 8.

9. 9
Fig. 8: Schematic flow chart of PfEMP1 key role in malaria pathogenicity
Parasite infectionon theerythrocyte expresses var genes which are translatedintoPfEMP1transportedby the MC tothe infected red blood
cell (iRBC) membrane. PfEMP1bindingto endothelial cells of the host, causes sequestration and cell rosetting which leads to malaria.
[Intracellular adhesion molecule 1 (ICAM-1), Chondroitin sulphate A (CSA), Parasitophorous vacuole (PV), Maurer’s cleft (MC)]
(Pasternak and Dzikowski, 2009)
Knowing that var gene encodes PfEMP1 that causes sequestration (binding) in an endothelial
part of any part of the body which determines the severity of malaria. Kraemer and Smith,
(2006) showed that the binding of PfEMP1 to ICAM-1 or chondroitin sulphate A (CSA)
using DBL domain cause the most severe malaria infection. It is evident that ICAM-1 causes
erythrocyte sequestration within the brain region which leads to cell auto-agglutination,
apoptosis and probably death of the host (Siau et al., 2007). If the sequestration was unto
CSA within the intervillous spaces of the placenta during pregnancy, it can also cause a
severe pregnancy associated malaria (PAM) that might lead to premature delivery or severe
anemia or even death of the mother and the fetus (Pasternak and Dzikowski, 2009). These
dangers can be circumvented by interfering the var gene expression. Var2 and var3 gene are
the gene that expresses the mRNA that encodes PfEMP1 within the most appropriate domain
as shown in figs. 9a and 9b were found to up regulate its gene at DBLα1.7 which is the
domain cassette 13 as shown in fig.10 . Goel et al., (2014) in their study on the most
expressing var gene, observed that var2 expressed higher quantity of mRNA (see fig. 9a)
which adherence to endothelial cell surface molecules, using CD36 and ICAM-1.

10. 10
Fig. 9a: Var gene expression.Var2 gene obtained from Plasmodium falciparum strain B, showing high gene expression product and
invariably causes more severe malaria infection (Goel et al., 2014).
Further study on var gene expression analysis by Albrecht et al., (2011) observed that out of
sixteen var gene family analyzed using RT-qPCR method only var2 gene were genuinely
expressed as was shown in fig.9. This gives an impression that all var genes are not always
on for expression based on its position on the telomere, but are switched on or off by a certain
switching agent (ligand). Knowing that var2 gene is sub telomeric located, Albrecht et al.,
(2011) disproved previous claim made by Frank et al., (2007) and Peters et al., (2007)
suggested that high gene expression product exhibited by some var genes was as a result of
their location in the telomere, stressing that centrally located genes expresses’ more than the
sub-telomeric gene as was claimed by some authors. This might be because some
subtelomeric deletion of ~100 kb in chromosome 2 which resulted in significant reduction in
the cytoadherence, as this region contains the knob-associated histidine rich protein
(KAHRP) gene, which is crucial for the assembly of a rigid knob and a clustering of PfEMP1
in the knob (Goel et al., 2014).

11. 11
Fig. 9b. Relative var transcriptlevels ofsixteen var genes usingQPCR method with var2gene as the gene expressed (Albrecht et al.,
2011). This figure showed var2 gene as the only gene that expressed its gene in this cascade of genes.
Volz et al., (2012), on exploring cascade of var gene observed that var2 gene expression was
great, but further discovered that a histone-3-lysine-4- methyltransferase (H3L4M) were
required to maintain var gene in the active state during expression. This shows that inhibition
H3L4M inhibits var2 gene expression (see fig. 13a).
Smith et al., 2001 showed that var gene expression of coding transcript region depends only
on the ability of each gene sequence to properly express the core sequence domain (see
fig.10a and 10b) that cut-across the intraerythrocytic developmental cycle (IDC) pathway.
The core regions in the var genes if properly expressed makes antigenic protein, but if not
expressed, brings miRNA transcript that cannot form proteins and can disintegrate so easily
(Claessens et al., (2012). This might be the same reason why some detected transcript
disintegrates with time, because of their inability to express the appropriate domain sequence.
It is clear from this evidence that DBL exon expression produces antigenic protein and this
also agreed with Scherf et al., (2008) which proved that in most var genes coding exons, the
first exon encode for the PfEMP1 extracellular potion for binding while the second exon
encode for the transmembrane and intracellular region of PfEMP1.
Fig.10a. PfEMP1 architecture of variants up-regulation:Eachvar gene has difference sequence of these core regions that must be up
regulatedfor mRNA transcript tobe made. [ATS=acidic terminal segment; CIDR= cysteine-rich interdomainregion; DBL= Duffy binding-
like domain; DC13=domaincassette 13; DC8= domain cassette8; NTS= N-terminal segment] Var 2 and var3 are among the genes that
transcribe the core domainfor theproductionof virulent PfEMP1. (Smithet al., 2001). ExpressionofDBL1αandDBL2βmediate for more
severe malaria infection.
Fig.10b: Plasmodiumfalciparum erythrocyte membrane protein 1(PfEMP1) conservation and polymorphism. (a) Duffy-binding-like
domain (DBL) sequence conservationis significantlyconcentratedin ten semi conservedhomologyblocks (colouredpinkandshown as the

12. 12
same size for simplicity)flankedby tenhypervariable blocks (blue). DBL domains display type-specific differences in lengthandmolecular
mass. Size variation is illustratedas the average length difference between DBLα and δ types. Distributed unequally among ho mology
blocks are ten conservedcysteine residues (C1–C10) present both in PfEMP1 and erythrocyte-binding antigen (EBA) DBL domains.
Additional type-specificcysteine residues are also present12. Crucial EBA DBL binding residues map between C4 and C7 (Ref. 53). (b)
Cysteine-richintracellular domainregion(CIDR) domains can be dividedinto three areas (M1, M2 and M3) based on the Malayan Camp
CIDR1, in which the minimal CD36 binding region (M2) has been defined. M1 and M2 areas with greater sequence conservation are
indicatedwith boldblack lines. The M3area is extremelydegenerate (indicated with green background) and frequently contains runs of
charged residues. The general position of 13 conserved cysteine residues (C1–C13) are shown for CIDRα and β types. Additional
typespecific cysteines are colouredorange (CIDRα) orgreen (CIDRβ). (c) Comparisons of CIDRα andβ consensus motifs, with invariant
cysteine (Smith et al., 2001).
Fig. 11 Var gene transcripts from patients with cerebral malaria and uncerebral malaria in Benin
[White-bar =˂ 2%, light-gray-bar = ˂ 5% andmid-grey-bar=˂ 20%] the higherthe % gene expressedthe more severe the malaria and the
more the core geneexpressed. Since more mid-grey-bars were formedat the cerebral malaria samples section,this provedthat expression of
the core gene sequence tookplace(Bertinet al.,(2013) for cerebral malariatooccur.The arrows spottedthe core DBL expression which is
the main reason for severe malaria.

13. 13
Fig12: Var gene expression quantification against apoptosis in malariadisease. Characteristics of the three nonapoptogenic (NA1–
NA3) andthe fiveapoptogenic (A1–A5)isolates were detailedandcomparedwith strain 3D7.This shows that somevar transcript might not
be apoptogenic and cannot cause severe malaria disease. The genes with red and asterisk are genes t hat express most severe malaria
infection and the arrow indicate var2 gene with high transcript in A1-A5 strains (Siau et al., 2007)
Zhang et al., (2014) on exploring the var gene sequence of Plasmodium falciparum, observed
that var3 genes are found to conserved and encode only four domains which were identified
to be N-terminal segment (NTS), Duffy binding-like (DBL), Transmembrane region (TM)
and Acidic terminal segment (ATS). The inability to transcribe the right domain might be the
reason why some var gene expression disintegrates. These claims were further clarified when
Bertin et al., (2013) compared the cerebral malaria with the non-malaria person, which
showed that core gene region when (expressed) transcribe produces more stable transcript as
shown in fig.11, than when not properly expressed.

14. 14
Fig 13a: Prerequisites of nucleosomeformationandcontexts of histonechaperone activity.Histone chaperones may facilitate nucleosome
formationby beinginvolvedin some or all ofthe processes I-VI indicated. Histones maybe transferredbetweenchaperones to complete all
these processes in a regulated manner (Elsasser and Arcy, 2013).
The issue of proper gene switching is attributed to histone dimerization which mediate proper
incorporation. The histone serves as chaperone that help mediate proper switching as was
explained in figs. 13a and 13b.
Fig.13b: A proposed model for the regulation of var geneexpression in theexpression site
Cytoplasm(Cy), nucleus (N), histone 3 (H3), (H2A)Histone 3 epigeneticmodifications involvedin var gene regulationwere proposed. Var
gene transcription is characterizedby H3K4trimethylation, H3K9 acetylation, andH2AZ for histonevariant.Loss of histonevariant H2AZ
from the active var promoter causes silencing of var genes. After replication, canonical histones such as histone H2A and H3 are
incorporatedtothe var promoter providinga windowof opportunity for switching and silencing. Histone H2.AZ is deposited at the var
promoterduringringstage andvargene silencinginvolves movement of thevar genelocus out ofthe expressionsite. Deacetylation is likely
mediated by SIR2 homologs and H3K9 trimethylation by a yet unknown HKMT (Volz et al., 2012).
PfEMP1 encoded by var genes, constitute the major parasite virulence factors that are made
of a long coding exon (NTS, DBL, CIDR and TM) and a short ATS exon separated by an
intron which is the domain architecture for malaria disease initiation (Zhang et al., 2014).
There are two promoters within each gene sequence, one at the upstream open reading frame
for mRNA expression and the other within the intron which produces a sterile transcript that
regulates the expression (Pasternak and Dzikowski, 2009). The histone serves as a chaperone
that initiates transcription.
There is likely speculation that if the ATS segment of the gene is blocked, the mRNA
expression product will stop, and if the mRNAs are not expressed, PfEMP1 will not be
formed thereby preventing malaria disease. It has also been established that different DBL
and CIDR binding repeats functions differently, Kraemer and Smith, (2006) established that
PfEMP1 binding on CD36 mediated by CIDR-1α causes mild malaria while PfEMP1 binding
on ICAM-1 or CSA mediated by DBL-2β and DBL-ɛ respectively causes severe malaria.
Almelli et al., (2014) knocked-out CIDR-1α gene segment prevented bind to CD36,
proposing that this knock-out procedure can be of help in preventing malaria disease. The
understanding of var gene expression has revealed some likely pathways or sequence that

15. 15
might be the target site for drugs and also epi-drug pathway that will help inhibit malaria
menace if properly implemented.
VAR GENE EXPRESSION APPROACHTO MALARIA PREVENTION
AND TREATMENT
The malaria disease approach through gene expression, has unfolded the genetic pathway to
this disease. This explained how the genes encode for merozoite surface proteins (msp)
within the hepatocyte egress (Boddey and Cowman, 2013; Gupta et al., 2013). Knowing that
epigenetic regulations mechanisms are the hallmark of malaria infection on the platform of
IDC transcriptional activity (Gupta et al., 2013).
Gupta et al., (2013) explored that most anti malaria drugs through epigeneticity, remodels
the chromatin structure and prevent transcriptional activity. Using a mice model, Darkin-
Rattray et al., (1996) discovered that apicidin (anti malaria) when administered orally inhibits
histone deacetylase (HDAC) thereby distorting the normal remodeling initiation that mediate
transcription, which invariably deregulate the IDC transcriptional cascade. This evidence on
gene expression, paved way for the invention of apicidin that inhibits transcriptional
initiation.
Usually anti-malaria drugs like chloroquine, mefloquine and sulfodoxine-pyrimethamine
usually binds to haem-iron which down regulate gene expression by inhibiting the alpha-
haematin formation, knowing that haem reduction down regulate gene expression
(Kritsiriwuthinan et al., 2011; Mok et al., 2011). Down regulation of gene, makes the
erythrocyte not to be sequestered and can be killed by macrophage. But some strains devised
other pathway where by miRNA produced up regulates gene expression thereby rendering
those drugs impotent. Based on this researcher ventured on the use of combine therapy which
might be of help to the menace.
Artemisinin combination therapy (ACT) was produced with much prolonged parasite
clearance-mean time (PCT) of 84 hours, and was recommended by the World Health
Organization as anti-malaria drug of choice (Mok et al., 2011). Recently ACT resistant strain
has occurred. The newly discovered ACT resistant parasites has a conserved exon that are
expressed at schizont stage for which histone 4 are normally use in the progressive assembly.
Therefore, more new antimalaria (epi-drugs) that may inhibit both HDAC and histone 4 and
other chromatin remodeling complexes (Prado and Aguilera, 2005; Mok et al., 2011) will be
of help in treating malaria.
Now, the knowledge of gene expression has enlightened us on the possible route the parasite
follows in the course of its normal life cycle and the stages that interference can occur so as
to inhibit malaria disease. It is now clear that var2, var3, when full expressed, give rise to
PfEMP1 which serves as both variant antigen, parasite virulence factor (Pasternak and
Dzikowski, 2009; Noble et al., 2013).

16. 16
Having known all these facts about malaria and its var gene expression, these evidences
suggest that we can ameliorate the infection by either preventing human contact with female
anopheles mosquitoes where the first phase of the parasites develops, or mimic the
merozoites binding at glycophorin-A receptor to block the main parasitic merozoite from
internalizing the cell or by developing more new combine therapy that can interfere and
remodel various chromatin complexes at all IDC stages thereby incapacitating all
transcriptional activities. Knowing that PfEMP1 is the main virulent protein, production of
anti-malaria drugs that will serve as chaperone to misfold this protein at the post translational
stage thereby producing pseudo-PfEMP1.
References
Applied Biosystem (2010). Available at: www.appliedbiosystem.com. Accessed date 28-10-
2014.
Applied Biosystem (2012). Available at: www.appliedbiosystem.com. Accessed date 28-10-
2014.
Albrecht, L., Moll, K., Blomqvist, K., Normark, J., Chen, Q., et al., (2011). Var gene
transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium
falciparum clone FCR3S1.2. Malaria Journal, 10:17-25.
Almelli, T., Ndam, N.T., Ezimegnon, S., Alao, M.J., Ahouansou, C., et al., (2014).
Cytoadherence phenotype of Plasmodium falciparum infected erythrocytes is associated with
specific pfemp-1 expression in parasites from children with cerebral malaria. Malaria Journal,
13(1):333-342.
Bachmann, A., Predehl, S., May, J., Harder, S., Burchard, G.D., et al., (2011). Highly co-
ordinated var gene expression and switching in clinical Plasmodium falciparum isolates from
non-immune malaria patients. Cellular Microbiology, 13(9):1397-1409.
Bertin, G.I., Lavstsen, T., Guillonneau, F., Doritchamou, J., Wang, C.W., et al., (2013).
Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1
is associated with cerebral malaria in Benin. PLOS ONE 8 (7): e68368.
Boddey, J.A., and Cowman, A.F., (2013). Plasmodium nesting: remarking the erythrocyte
from the inside out. Annual Review of Microbiology, 67:243-269.
Bustin, S.A., (2009). The MIQE guidelines: Minimum information for publication of
quantitative real-time PCR experiments. Clinical Chemistry, 55(4):611-622.
Claessens, A., Adams, Y., Ghumra, A., Lindergard, G., Buchan, C.C., et al., (2012). A subset
of group A-like var genes encodes the malaria parasite ligands for binding a human brain
endothelial cell. Proceedings of the National Academy of Science of USA, 109(26):1772-
1781.

17. 17
Darkin-Rattray, S.J., Gurnett, A.M., Myers, R.W., Dulski, P.M., Crumley, T.M., et al.,
(1996). Apicidin: a novel antiprotozoal agent that inhibits parasite histone deacetylase.
Proceeding of the National Academy of Science, USA, 93:13143-13147.
Duffy, M.F., Brown, G.V., Basuki, W., Krejany, E.O., Noviyanti, R., et al., (2002).
Transcription of multiple var genes by individual, trophozoite-stage Plasmodium falciparum
cells expressing a chondroitin sulphateA binding phenotype. Molecular Microbiology,
43:1285-1293.
Elsasser, S.J., and Arcy, S.D., (2013). Towards a mechanism for histone chaperones.
Biochimica et Biophyscica Acta (BBA), 18(9):211-221.
Frank, M., Dzikowski, R., Amulic, B., and Deitsch, K., (2007). Variable switching rates of
malaria virulence genes are associated with chromosomal position. Molecular Microbiology,
64: 1486-1498.
Goel, S., Muthusamy, A., Miao, J., Cui, L., Winzeler, E.A., et al., (2014). Targeted disruption
of a ring infected erythrocyte surface antigen (RESA)-like export protein gene in Plasmodium
falciparum confers stable chrondroitin 4-sulfate cytoadherence capacity. The Journal of
Biology Chemistry, M114.615393.
Gupta, A. P., Chin, W.H., Zhu, L., Mok, S., Luah, Y., et al., (2013). Dynamic epigenetic
regulation of gene expression during the life cycle of malaria parasite Plasmodium
falciparum. PLOS Pathogens, 9(2): e1003170.
Holland, P.M., Abramson, R.D., Watson, R., and Gelfand, D.H., (?). Detection of specific
polymerase chain reaction product by utilizing the 5’ to 3’ exonuclease activity of Thermus
aquaticus DNA polymerase. Proceeding of the National Academy of Science, USA, 88:7226-
7280.
Kraemer, S.M., and Smith, J.D., (2006). The family affair: var gene, PfEMP1 binding and
malaria disease. Current Opinion in Microbiology, 9:374-380.
Kritsiriwuthinan, K., Chaotheing, S., Shaw, P.J., Wongsombat, C., Chavalitshewinkoon-
Petmitr, P., et al., (2011). Global gene expression profiling of Plasmodium falciparum in
response to the anti-malarial drug pyronaridine. Malaria Journal, 10:242-252.
Kulkarni, P., Shiraishi, T., Rajagopalan, K., Kim, R., Mooney, S.M., et al., (2012). Schematic
diagram of an immunotherapeutic approach to treating urological malignancies that utilizes
immunogenic peptides corresponding to CTAs. Nature Reviews Urology, 9:386-396.
Kyes, S., Christodoulou, Z., Pinches, R., Kriek, N., Horrocks, P., et al., (2007). Plasmodium
falciparum var gene expression is developmentally controlled at the level of RNA
polymerase II-mediated transcription initiation. Molecular Microbiology, 63(4):1237-1247.
McW Healthcare, (2008). Available at www.mcwhealthcare.com. Accessed date, 29-10-14.

18. 18
Mok, S., Imwong, M., Mackinnon, M.J., Sin, J., Ramadoss, R., et al., (2011). Artemisinin
resistance in Plasmodium falciparum is associated with an altered temporal pattern of
transcription. BioMed Central Genomics, 12:391-405.
Noble, R., Christodoulou, Z., Kyes, S., Pinches, R., Newbold, C.I., et al., (2013). The
antigenic switching network of Plasmodium falciparum and its implications for the immune-
epidemiology of malaria. E Life Science, 2: 1-19.
Pasternak, N.D., and Dzikowski, R., (2009). PfEMP1: An antigen that plays a key role in the
pathogenicity and immune evasion of the malaria parasite Plasmodium falciparum. The
International Journal of Biochemistry and cell Biology, 41:1463-1466.
Peters, J.M., Fowler, E.V., Krause, D.R., Cheng, Q., and Gatton, M.L., (2007). Differential
changes in Plasmodium falciparum var transcription during adaptation to culture. Journal of
Infectious Disease, 195:748-755.
Prado, F., and Aguilera, A., (2005). Partial depletion of histone H4 increases homologous
recombination-mediated genetic instability. Molecular and Cellular Biology, 25:1526-1536.
Rai, R., Zhu, L., Chen, H., Gupta, A.P., Sze, S.K., et al., (2014). Genome – wide analysis in
Plasmodium falciparum reveals early and late phases of RNA polymerase II occupancy
during the infectious cycle. Biomed Central Genomics, 15:959-978.
Scherf, A., Riviere, L., and Lopez-Rubio, J.J., (2008). Snapshot: var gene expression in the
malaria parasite. Cell, 134:190.
Sellner, L.N., and Turbett, G.R., (1998). Comparism of three RT-PCR methods.
Biotechnologies, 25(2):230-234.
Smith, J.D., Gamain, B., Baruch, D.I., and Kyes, S., (2001). Decoding the language of var
gene and Plasmodium falciparum sequestration. Trends in Parasitology, 17(11):538-545.
Steiner, L.A., Maksimova, Y., Schulz, V., Wong, C., Raha, D., et al., (2009). Chromatin
architecture and transcription factor binding regulate expression of erythrocyte membrane
protein genes. Molecular and Cellular Biology, 29(20):5399-5412.
Thomas, A., (2014). Gene expression analysis. On blackboard. Accesses date 22-10-14.
Volz, J.C., Bartfai, R., Petter, M., Langer, C., Josling, G.A., et al., (2012). PfSET10, a
Plasmodium falciparum methyltransferase, maintains the active var gene in a poised state
during parasite division. Cell Host and Microbe, 11:7-18.
World Health Organization (WHO) (2014). Vox Media, Inc. accessed date 20-11-14.
Zhang, Y., Jiang, N., Chang, Z., Wang, H., Lu, H., et al., (2014). The var3 genes of
Plasmodium falciparum 3D7 strain are differentially expressed in infected erythrocytes.
Parasite Journal, 21:19-25.