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Abstract
The snail, Biomphalaria glabrata is a major intermediate host for the parasitic trematode Schistosoma mansoni the causative agent of human schistosomiasis. Efforts are underway to decipher the molecular basis of the snail - host schistosome interaction, and the
Bge embryonic cell line derived from this snail provides a unique biology for both organisms. In line with these interests, this snail has been chosen as a molluscan representative to have its genome sequenced. in vitro model cell system to assess whether interactions between the snail host and parasite affects the cell and genome
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By using the method whereby labelled nucleotides are incorporated into replicating
DNA and cells are then allowed to progress through several cell divisions in the absence of labelled nucleotides we were able to visualise, for the first time, whole individual chromosomes within interphase nuclei of the Bge cells, study their morphology and where they are located within interphase nuclei. By using bioimaging and image analysis we revealed that these chromosome territories are similar in morphology to those found in more complex organisms and are radially positioned within nuclei, non-randomly. Furthermore, specific gene loci, present as 2 copies on homologous Bge chromosomes, delineated using
BAC probes and fluorescence in situ hybridisation (FISH), were also non-randomly positioned with Bge cell nuclei.
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In order to determine whether there is a genomic response to parasitic exposure, the interphase spatial positioning of the gene loci was assessed after co-culturing live or irradiated attenuated schistosoma miracidia with the Bge cells for 30 minutes to 24 hours.
The genes investigated, actin and ferritin , are genes known to be up-regulated in the snail host when subjected to parasite infection. Most interestingly, with alteration in the expression of these genes, as determined by quantitative-PCR, gene repositioning in interphase nuclei was correlated temporally with up-regulation of gene expression and then with downregulation. Co-culture with irradiated attenuated parasite did not elicit interphase gene repositioning correlated with changes in gene expression. Thus, the parasite is able to elicit

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51 influence directly over the host genome via secreted molecules through the hosts’ own nuclear architecture.

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Introduction
Introductory paragraph about the importance of understanding host:parasite interactions and Schisto.
The host:parasite interaction between Biomphalaria glabrata and S. mansoni is initiated when a miracidium enters the snail via the surface epithelium and consequently develops into a primary sporocyst (Miller et al., 2001). Naturally, the field of research into this relationship has focused on the genetic factors and potential markers that lead to parasite elimination in some snails and not in others (Raghavan and Knight 2006). S. mansoni’s specificity for Biomphalaria species; points towards a genomic communication between these two organisms which has been developed by co-evolution (Miller et al., 2001,
Raghavan et al., 2003). An elucidation of the genetic factors which influence resistance and susceptibility in the snail host has come from research investigating the transcriptional modulation of genes in the snail upon infection (Hertel et al., 2005, Miller et al., 2001).
Investigations into B. glabrata’s relationship with S. mansoni have been aided by the development of an of S. mansoni in vitro tissue culture model to support the intramolluscan sporocyst stage
(Yoshino and Laursen 1995; Castillo and Yoshino 2002) and
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in vitro development of cercaria (Basch and DiConza 1977). Such a model system exists in the Bge cell line established in the 1970s from macerated embryonic tissue that spontaneously immortalized (Hansen, 1976). The Bge cell line is able to maintain primary sporocysts and produce secondary sporocysts via co-culturing (Coustau et al., 1997; Laursen and Yoshino
1999; Castillo et al 2007) and supports the continuous of S. mansoni in vitro propagation and differentiation
(Ivanchenko et al., 1999, Coustau and Yoshino 2000; Kapp et al., 2003).
Interestingly, bringing the cells together with parasite or parasite products gives rise to alterations in gene expression in the Bge cells. Indeed, Humphries and Yoshino (2006) utilised the excretory and secretory (ES) products from S. mansoni signalling pathway in the Bge cells (Humphries and Yoshino, 2006)
to stimulate the p38
These cell cultures are also amenable to small interference knockdown of gene expression (Jiang et al., 2006). In terms of gene expression, it is not just the host-parasite relationship which has been

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83 investigated in Bge cells but also stress responses such as heat-shock (Laursen et al., 1997;
Yoshino 1998) and chemokinetic/tactic response to molecules such as cytokines (Steelman and Connors 2009). Thus, even though the Bge cells now have some issues with aneuploidy
(Odoemelam et al., 2009), they provide a manageable and responsive
Here we utilise the Bge cell in vitro in vitro in which to study molluscan host:parasite interactions, stress responses and chemotaxis.
co-culture system to determine spatio-temporal affects on specific Bge genes in the nuclei of cells that have been co-cultured with miracidia.
model system
S. mansoni
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The cell nucleus, of many organisms, is a highly organised structure, with complex and dynamic architecture that controls the behaviour and function of the genome through regulating gene expression. Interphase chromosomes are not found in an unravelled state but as individual entities known as chromosome territories (Cremer et al., 2007; Meaburn and Misteli 2008). The highly compartmentalized structure of the eukaryotic cell nucleus and the dynamic organisation of chromosome territories, and the gene loci within them, is believed to play an integral role in controlling gene expression (Kumaran et al., 2008). In a change in status to a cell that requires or induces altered gene expression, chromosome territories and/or individual gene loci within nuclei can be functionally spatially repositioned i.e. during differentiation (Skalnikova et al., 2000; Kosak et al., 2002; Kuroda et al., 2004;
Chambeyron and Bickmore 2004; Ragoczy et al., 2006; Foster et al., 2005; Szczerbal et al.,
2009; Solovei et al., 2009), in disease (Cremer et al., 2003; Zink et al., 2004; Meaburn et al.,
2007; Meaburn et al., 2009; Li et al., 2009;), and in cellular proliferation (Bridger et al., 2000;
Branco et al., 2008; Mehta et al., 2010). This can either be whole chromosome territories being repositioned or the activated gene loci looping away from the main body of the chromosome territory. A number of studies using mammalian models have correlated regional positions of gene loci within the nucleus with levels of gene expression (Volpi et al.,
2000, Mahy et al., 2002, Williams et al., 2006, Finlan et al., 2008, Meaburn and Misteli,
2008, Ballabio et al., 2009, Szczerbal et al., 2009; Takizawa et al., 2008a), with activation of

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121 a gene being correlated with the movement of a gene towards the nuclear interior (for review see Takizawa et al., 2008b; Denauid and Bickmore 2009; Elcock and Bridger 2010). During embryogenesis, a vital transcription factor Mash (AsII), is required for the production of neuronal precursor cells (Williams et al., 2006). In embryonic stem cells, the Mash 1 gene is transcriptional repressed, however during neuronal differentiation it is preferentially repositioned from the nuclear periphery to the nuclear interior and is transcriptional upregulated by more than 100 fold (Williams et al., 2006). Indeed, the impact of cellular differentiation upon specific gene loci repositioning and transcriptional activation has also been reported in porcine mesenchymal stem cells (Szczerbal et al., 2009). Seven genes involved in adipogenesis were found to adopt a more internal position upon the induction of adipogenesis and transcriptional up-regulation. In support of gene activation being connected to a movement of gene loci to the nuclear interior one study by Takizawa et al reveals that copies of monoallelically expressed genes localise in different nuclear compartments to each other, with the active allele more centrally located in nuclei (Takizawa et al., 2008a).
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Historically, the nuclear periphery has been associated with down-regulation of gene activity and gene silencing since there are many data that support repositioning of genes to the nuclear periphery with gene inactivation and transcriptional repression (for review see
Shaklai et al., 2007). When genome-wide screens of chromatin attached at the nuclear periphery were performed the resultant fractions were generally gene-poor regions of the genome and heterochromatic in both Drosophila and human cells (Pickersgill et al., 2006;
Guelen et al., 2008). However, as a caveat towards the dogma of the nuclear periphery being an exclusive region of gene suppression, some genes relocated to the nuclear periphery are still actively transcribed (Denaiud and Bickmore 2009), the INF

gene in mouse is located at the nuclear periphery whether it switched on or off (Hewitt et al., 2004) and in yeast and Drosophila , transcriptionally active genes have been found located around

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Very little is as yet known about how molluscs organise their genome in interphase nuclei but since organisms such as Hydra (Alexandrova et al., 2003) and Caenorhabiditis elegans (pers comm. Dr Kentaro Nabeshima, University of Michigan) contain individual chromosome territories then we predicted that molluscs will too since both the former organisms have evolved along the same evolutionary line. Indeed, the Red Abalone mollusc displays telomeric repeats throughout interphase nuclei which strongly suggests a non-Rabl distribution of chromosomes (Gallardo-Escarate et al., 2005), which is corroborated by studies in the King Scallop where repetitive DNA was found localised in discrete regions also throughout interphase nuclei (Biscotti et al., 2007). However we cannot postulate on whether these territories might be organised in a non-random or random fashion and if it is nonrandom whether it is a radial organisation as in more complex organisms and correlated with gene density or chromosome size as in mouse and human nuclei (Boyle et al., 2001; Foster and Bridger 2005; Mayer et al., 2005; Bolzer et al., 2005; Meaburn et al., 2008; Mehta et al.,
2007; Mehta et al., 2010).
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We have determined for in vitro
B. glabrata , using the Bge cells, that their interphase chromosomes are organised as individual territories which are non-randomly organised in a radial distribution, as were specific gene loci Actin , Ferritin , Piwi and determined that there is specific temporal repositioning of gene loci within interphase nuclei
schistosome exposure. We found dramatic gene loci re-positioning with nuclei tightly correlated with gene expression, with one gene moving to the nuclear interior and one gene moving to the nuclear periphery with up-regulated gene expression. These gene loci dynamics were not elicited when attenuated parasite were used.
BgPrx . We further

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Methods and Materials
Culturing of Bge cells
Bge cells used in this study were derived from Han sen’s original Bge cell line (Hansen,
1976), and were grown in the absence of carbon dioxide, at 26 o C in sterile medium that comprised of 22% Schneider’s Drosophila medium (Invitrogen), 0.13% galactose
(Invitrogen), 0.45% lactalbumin hydrolysate (Invitrogen), 14.11M phenol red and 20µg/ml gentamicin (Invitrogen). The Bge medium was made complete by adding 10% heat inactivated FBS (v/v, Hyclone).
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For the parasite exposure experiments; the Bge cells in T75 flasks were exposed to five S. mansoni miracidia for 0, 0.5, 2, 5, 24 hr. The cells were then centrifuged at 400g at 15 o C, and the pellet resuspended in hypotonic potassium chloride solution (0.05 M) with subsequent fixation with methanol and acetic acid (3:1 v/v). The cells were then dropped onto damp glass microscope slides.
170 Irradiation of miracidia. (20krad) was performed as described by Ittiprasert et al, (2009)
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172 Incorporation and visualisation of Bromodeoxyuridine
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The Bge cells were grown on sterile 13 mm diameter circular glass coverslips and seeded at a density of 2.5 x 10 5
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The cells were grown in complete Bge medium overnight before the addition of 0.1% 5-bromo-2-deoxyuridine (BrdU) and 5-fluoro2’-deoxyuridine (FUrd) (Sigma-
Aldrich) for 48 hours after which the medium containing the BrdU and FUrd was removed and replaced with complete Bge medium. The Bge cells were subsequently allowed to grow for 10 days in complete Bge medium (devoid of BrdU and FUrd) before fixation.
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The Bge medium with the thymidine analogs was removed from the dish and the cells washed thrice with 1 x phosphate buffered saline. The cells were then fixed with 10 ml of ice cold 1:1 methanol:acetone (v/v) for 4 min. After which, the fixative was removed and the

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Immunological detection of BrdU incorporation requires the pre-treatment of the Bge cells with acid. The coverslips were washed with 10 ml of 2N HCl for 30 min. After which the acid was removed and slides washed 10 times in 1 X PBS so as to eliminate any latent acid.
Mouse anti-BrdU antibody (Beckton and Dickenson) was diluted 1:100 in 1% Newborn calf serum (NCS) in 1 % PBS (v/v). The coverslips were incubated with the anti-BrdU for 1 hour at room temperature. The cells were then washed thrice in 1 X PBS before the addition of the secondary antibody, donkey anti-mouse fluorescein isothiocyanate (FITC, Jackson laboratories) 1:80 dilution 1% NCS/1 X PBS for 1 hour at room temperature. The cells were mounted in Vectorshield anti-fade mountant containing 4’ 6’-diamidino-2-phenylindole
(Vectorlabs).
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Quantitative PCR
The Real time PCR was performed using Applied Biosystems 7300 Real Time PCR System
(Applied Biosystem, Foster City, CA). Reactions were performed in a one step format with total Bge cell RNA (80 ng). Synthesis of first strand cDNA and amplification by PCR were performed sequentially in a single tune using Full velocity SYBR Green QRT-PCR Master mix accord ing to the manufacturers’ instructions (Stratagene). Reactions (25µl final volume) contained the following; 200nM of specific B. glabrata primers for ferritin (F: 5'-
CTCTCCCACACTGTACCTATC-3'; R: 5'-CGGTCTGCATCTCGTTTTC-3'), actin (F: 5'-
GGAGGAGAGAGAACATGC-3'; R: 5'-CACCAATCTGCTTGATGGAC-3'). A parallel reaction was performed with the stably expressed myoglobin gene (50 nM of B. glabrata specific myoglobin primers F: 5’- GATGTTCGCCAATGTTCCC-3’; R:
5’AGCGATCAAGTTTCCCCAG-3’) was used was used to assess the comparability of samples and confirm that template cDNA was used in equivalent amounts for each amplification reaction. All reactions contained 300 nM of reference dye, 1X of Full Velocity

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SYBR Green QRT-PCR master mix containing RT-PCR buffer, SYBR green I dye, MgCl and nucleotides. The amplification protocol included an initial incubation at 48 for cDNA synthesis and a 95
58 o o o amplification period. All amplifications were run in triplicate and the fluorescence threshold value (Ct) was determined using the 7300 System v1.3.1 SDS software (Applied
,
C for 45 min
C initial denaturation for 10 sec, and annealing/ amplification at
C for 1 min. Detection of the fluorescent product was carried out at the end of the
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Biosystems). Comparison of the expression of the ferritin and actin genes between pre and post exposure Bge cells was determined using deltadelta (ΔΔ) Ct. Results were transformed into ‘fold increase’ according to the following formula:
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− ΔΔ Ct
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−
[ (Ct Prx, exposed
−Ct myoglobin, exposed
)−(Ct
Prx, unexposed
−Ct myoglobin, unexposed ) ]
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In order to determine the significance of differences ( P < 0.01) and ( P < 0.05 ) in gene expression for the different time points the Pvalue was calculated by comparing delta Ct values using the Student’s ttest between the exposed and unexposed Bge cells.
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Fluorescence in situ Hybridisation (FISH).
DNA was isolated from BAC clones the B. glabrata (BS90) BAC DNA library as previously described (Raghavan et al., 2007). The DNA was extracted using a Qiagen midi kit (Qiagen).
The BAC DNA was labelled with Biotin-14 – dATP via Nick translation using the BioNickTM kit (Invitrogen). 500 ng of the labelled gene probe and 3 µg of herring sperm DNA as carrier was combined with 40 µg of sonicated Bge genomic DNA. These constituents were ethanol precipitated together at -80 o C for 30 min. After washing in 70% ethanol twice, the DNA was subsequently dissolved in
12 µl of hybridisation buffer at room temperature for 24 hr. The probes were denatured at 75 o C for 5 min and then incubated at 37 o min prior to their use in the FISH.
C between 30 and 120

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The slides of Bge metaphase spreads were aged for 2 days at room temperature and then dehydrated through a series of ethanol solutions of 70%, 90% and 100% (5 min each). The cells were denatured in a solution of 70% formamide/2 X SSC at 70 o before an additional 90% and 100% ethanol cycle. The slides were allowed to dry on a hot block (37 o C) before the addition of the denatured probe.
C for 1.5 min.
Immediately after denaturation, the slides were immersed in ice cold 70% ethanol for 5 min
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Eight microliters of the biotin-labelled probe was placed onto the slide, covered with a 24 x
40 mm coverslip and sealed with rubber cement. The denatured slides and probes were hybridised overnight (12 –16 hr) in a humidified chamber at 37oC. Following hybridisation, the rubber cement and coverslips were removed and the slide was washed three times for 5 min in a neutral buffered solution of 50% formamide and 2 X SSC at 45oC. A second wash followed. The slides were transferred to Coplin jars containing pre-warmed 0.1 X SSC at
60oC, which were transferred to a 45oC water bath. The slides were washed three times for
5 min. Subsequently, the slides were placed in a solution of 4 X SSC at room temperature for 10 min after which 100 µl of blocking solution was added to each slide (4% Bovine serum albumin, BSA, in 4 X SSC). A 22 x 50 mm coverslip was placed on the slide and these were left for 10 min at room temperature. The coverslips were then removed and
100 µl of streptavidin conjugated to cyanine 3 in 1% BSA/4 X SSC (1:200 dilution) was added to each slide and a coverslip applied. The slides were incubated at 37oC for 30 min in the dark. After this incubation, the slides were washed three times for 5 min in 4 X SSC with 0.1% Tween
20 (v/v) at 42oC in the dark. A brief rinse in deionised distilled water was followed by the addition of the counterstain. The slides were counterstained with DAPI in Vectorshield antifade mountant (Vectorlabs).
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Image analysis
Digital images were captured using an epifluorescence microscope and X100 oil immersion objective (Zeiss, Axioplan 2). The images were captured using a charged coupled device

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(CCD) camera (RS Photometrics Sensys camera model KAF1401E G2) and the program
Smart capture 3.00 (Digital Scientific). 50-60 images of BrdU labelled chromosome territories and the hybridized genes was analysed by partitioning DAPI image of the nuclei into five concentric shells of equal area from the nuclear periphery to the nuclear centre with background removal by subtracting the mean pixel intensity of the nuclei. The data were normalised by division of the chromosome/gene signal intensity with the DAPI intensity measurement for each of the five shells.
B. glabrata genes in interphase nuclei of the Bge cell line were analysed using an erosion analysis script developed by Dr Paul Perry in IPLab software (Croft et al.,
1999) and was kind gift from Prof. Wendy Bickmore (MRC HGU, Edinburgh). The interphase positioning of both the BrdU labelled chromosome territories and the mapped B. glabarata
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276 Results
277 Presence of non-random radially-positioned chromosome territories in Bge cells
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To date there have been no studies on how the genome is organised in interphase nuclei of molluscan organisms, whether individual chromosome territories are present and how do specific gene loci behave. Presently there are no B. glabrata , snail or molluscan chromosome painting probes that could be employed to elucidate chromosome positioning in the Bge cells by straight forward fluorescence in situ hybridisation (FISH) or ZOO-FISH.
However, there is another method that can be used to analyse individual chromosomes and that involves the incorporation of exogenous labelled nucleotides into cells (Visser et al.,
1998; Zink et al., 1999). During the first cell cycle after addition of the nucleotides to the cell culture medium, one strand of the entire genome becomes peppered with incorporated nucleotides. Due to random chromosomal segregation into daughter cells, after a number of cell divisions there are only a few whole chromosomes that can be identified as containing exogenous nucleotides. This is the method we adopted to analyse interphase chromosomes in the Bge interphase nuclei. By using bromo-deoxy uridine (BrdU) we were able to use indirect immunofluorescence to delineate the labelled chromosomes (Figure 1A). The interphase chromosomes within the Bge nuclei were indeed found in chromosome territories of a similar morphology and structure to those of mammalian cells. Such that the chromatin strands of the chromosome territories are located within the same nuclear space as has been determined by the globule fractal model (Lieberman-Aiden et al., 2009) in chromosome territories (Cremer et al., 2007) and not located throughout the nucleoplasm. The B. glabrata chromosome territories contain invaginations at their edges which allow access to the core regions of the territories. Indeed from the representative image used in Figure 1A the two large chromosome territories, which will most likely not be homologues, are located with a portion adjacent to the nuclear periphery (large arrow) and abut a chromatin-poor region of the nucleus, most probably the nucleolus (small arrow). This implies that the chromosomes have interactions with the nuclear envelope and nucleolus as they do in other species.

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Since more complex organisms organise their genome in interphase nuclei in a radial non-random manner we wished to determine if this was conserved in the snail cells. Due to the methodology used we have no way to determine which chromosome territories are likely to correspond to which chromosome in the Bge karyotype (Odoemelam et al., 2009).
However, it is possible to select them by size and we subdivided them into two clear categories – large and small – since the medium sized chromosomes were hard to categorise since they may represent larger chromosomes that are more condensed than others and so we did not analyse this group. The cells were fixed so that when they were dropped onto microscope slides they were flattened. This allowed us to use a well-tested chromosome position script that enables the position of chromosome location in 50-100 images (Croft et al., 1999; Bridger et al., 2000; Meaburn et al., 2007; Mehta et al., 2010).
The script outlines the cell nuclei in which the DNA is stained by DAPI and uses erosion to create five shells of equal area in which the signal intensity of the chromosome fluorescence and the DAPI fluorescence is measured (Figure 1B). The chromosome signal data are normalised by division with DAPI signal measurement for each shell, for each nucleus.
When the data are plotted as histograms it became apparent that Bge chromosome territories are organised in a non-random way with large chromosomes towards an intermediate position and the small chromosomes towards the interior, implying a size correlated distribution. However, since the genome sequencing project is not yet complete we cannot attempt any chromosome position correlation with gene density, as is seen in humans (Boyle et al., 2000), amphibians and reptiles (Federico et al., 2006).
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325 Biomphalaria glabrata gene loci are positioned non-randomly in Bge cell nuclei.
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Even though chromosomal painting probes do not yet exist for B. glabrata , specific gene sequences have been cloned and placed into BACs. By using BAC probes specific for particular genes labelled by Nick translation with labelled nucleotides it is also possible to

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339 delineate specific gene loci by FISH. Using this approach we have delineated 4 genes in interphase Bge cell nuclei. They are infection, are actin and ferritin
Piwi and BgPrx (Knight et al., 2009), both of which have been mapped to two homologous chromosomes in the Bge karyotype (Odoemelam et al.,
2009). The two other genes that we studies that are upregulated after a Schistosoma
. Figure 2 displays the hybridisation by FISH of the four gene loci onto mitotic chromosomes derived from Bge cells. Although, these cells are aneuploid they contain only two signals on two homologous chromosomes for all four of these genes.
The actin gene is localised on a single pair of large acrocentric chromosomes (Figure 2A) and ferritin is localised onto a pair of large metacentric chromosomes (Figure 2B). The chromosomal mapping of Piwi and BgPrx have been shown before (Odoemelam et al.,
2009) but are displayed again (Figure 2C and D, respectively).
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All four gene loci were also revealed in interphase Bge cell nuclei, displaying two signals (Figure 3A-D) and 50+ images subjected the radial positioning script as the chromosomes were with the erosion analysis script in figure 2B. Excitingly, all the four gene loci displayed non-random radial positioning in interphase nuclei of Bge cells, with
(Figure 3E), interior and
Piwi (Figure 3G) and ferritin
BgPrx appeared slightly more random than the others but the majority of signal was found in shell 4 revealing a more interior distribution. actin
(Figure 3H) all being located towards the nuclear
being located very much at the nuclear periphery (Figure 3F). Actin
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Gene positioning is altered when Bge cells are co-cultured with Schistosoma miracidia and is correlated with gene expression.
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It is known that infection of B. glabrata with Schistosoma miracidia modulates host gene expression and certain genes are up-regulated such as actin , ferritin and hsp70
(Ittiprasert et al, 2009). We took advantage of the Bge cell:miracidia co-culture model system of infection (Ivenchenko et al, 1999; Coustau et al, 2003) to analyse how these genes

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358 behave after the addition of parasite to the tissue culture media. Since genes can move quite rapidly with the nuclear environment upon activation (Volpi et al., 2001; Szczerbal et al.,
2009) we took time points at 0, 30 minutes, 2 hours, 5 hours and 24 hours. Figure 4 displays graphically the gene positioning of position of the actin actin
gene loci move from the nuclear interior towards the nuclear periphery at
30 mins, when there is a bimodal distribution, which could imply one allele has moved or a proportion of the cells have responded (Figure 4B); and in the two hour sample where the gene loci are almost all found towards the nuclear periphery (Figure 4C). In the 5 hour sample post-co-culture, the
(Figure 4D). However at 24 hours there is further gene repositioning back to the nuclear periphery (Figure 4E). To correlate the alteration in spatial positioning with gene expression, real time quantitative- PCR using RNA isolated from cells co-cultured with miracidia for the same time points was performed (Figure 4F). There is a direct correlation with the upregulation of gene expression as shown by RT-PCR and repositioning to the nuclear periphery for the 2 and 24 hour samples. There is no correlation with up-regulation for the the 30 minute bimodal redistribution of interphase gene position; however, there was an upregulation of 3X for shown) and so the actin actin actin gene loci are repositioned back in the nuclear interior
gene expression at 15 minutes as shown by q-PCR (data not
loci may well be repositioning back to the default interior position after moving to the nuclear periphery.
at the different time points. Most excitingly, the
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Irradiated attenuated miracidia do not elicit a gene repositioning response in Bge cells.
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In order to determine if the gene repositioning is purely a reaction to the presence of miracidia rather than a response to active infection mechanism elicited by the parasite, live irradiated miracidia were added to the dishes of Bge cells. The nuclei were collected from cells that had been treated with attenuated miracidia at 0 minutes, 30 minutes, 2 hours, 5

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391 hours and 24 hours. The nuclei were subjected to FISH with labelled BACs containing ferritin . Figure 5 displays the data for the positioning of the ferritin gene in timed samples prepared for FISH. The ferritin gene loci are normally located at the nuclear periphery at 0 hours (Figure 4A) but with co-culture with live miracidia the interphase gene positioning is altered dramatically at 5 hours with the gene loci being located exclusively in the 3 rd and 4 th shells of the erosion analysis i.e. towards the interior of the nucleus (Figure 5D). By 24 hours the gene loci have returned to a more peripheral location. This dramatic and very specific relocation from the periphery to the interior is once again correlated with gene expression as determined by q-PCR. Indeed, a large increase in gene expression is seen at 5 hours
(Figure 5F). However, when irradiated miracidia are used in the co-culture system there is no induction of ferritin expression and subsequently no relocation of the ferritin gene loci.
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394 Discussion
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Using the we have revealed major gene repositioning in the interphase nuclei of the snail Bge cells.
Both genes, actin tightly correlated with up-regulation of gene expression as determined by quantitative RT-
PCR. However, instead of both genes moving to the nuclear interior, or, becoming more internally located, as was expected from other studies, one moved to the interior and one moved towards the nuclear edge. The peripherally located during co-culture with parasite at the time when gene expression was up-regulated.
However, the actin became more peripheral. Re-positioning towards the nuclear periphery with gene expression has not been seen in higher organisms, and may be a novel snail specific response in kin with simpler organisms. Indeed, active genes become associated with and/or are located at the nuclear periphery in yeast (Brickner and Walter 2004; Casolari et al., 2005; Taddei et al.,
2006; Brown and Silver 2007) and
Most interestingly, when causative agent in vitro
and
co-culture assay employing Bge cells and schistosome miracidia ferritin var positioning data shows that
are up-regulated in the cell assay and the repositioning is
Drosophila (
B. glabrata ferritin
genes in the apicomplexan parasite
became more interior
gene, which is internally located within interphase nuclei, upon activation
Mendjan et al., 2006; Pickersgill et al., 2006).
Plasmodium falciparum of the tropical disease, malaria, are activated they are located at the nuclear periphery (Duraisingh et al., 2005). Whereas in mammalian cells tethering of parts of the genome to the nuclear membrane or lamina either results in down-regulation of expression of genes or to no change in the level of expression (reviewed in Denaiud and Bickmore
2009; Reddy et al., 2008; Finlan et al., 2008; Kumaran and Spector 2008). Our gene periphery as regions for gene expression and would, therefore, make a good model system in providing information between yeast/ Drosophila and higher organisms to study these
, the
may use both the nuclear interior and the nuclear different nuclear compartments and the genes that use them for expression.
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420
Instead of individual genes moving towards different nuclear compartments, a global reorganisation of the whole genome upon exposure to parasite, may also explain the

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426 repositioning of genes (Strasak et al., 2009). This however is unlikely since the repositioning of the genes is non-random and even though the hsp70 gene is up-regulated the gene loci do not change their nuclear localisation at any of the time points (data not shown). Thus, we believe it is a specific response elicited for specific genes. However, it will be important to not only look at only spatial repositioning but also the activated genes ’ chromatin modifications and alterations in association with regions of specific types of chromatin.
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This study is also the first study to reveal chromosome territories in molluscs and they seem similar to territories found in higher eukaryotes with a similar morphology
(Meaburn and Misteli 2008). We also provide some evidence that they are non-randomly positioned with a bias towards size. It is really early days to make predictions about how the snail genome will be organised in interphase nuclei since the genome sequencing project needs to be much more advanced to correlate gene density with position, the community needs to have chromosome painting probes and the Bge cells have many extra small chromosomes (Odoemelam et al., 2009) which may be inactive in these cells and give a false impression of a size-correlated chromosome positioning. However, if movement of genes to the nuclear periphery and the nuclear interior is involved in gene up-regulation, a gene-density correlated positioning of chromosome territories may not make biological sense in this organism.
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This is the first example of spatial reorganisation of a genome after exposure to a parasite, although viral infection has been shown to elicit response whereby the nucleus is remodelled (Li et al., 2009). Since these organisms have co-evolved and would benefit from being able to influence each others’ genomes for their own benefit its seems possible that this spatial re-positioning is initiated by the parasite rather than being only a response by the host. Thus we should be able to identify the factors in the miracidial ES product that are responsible for this rapid communication with the snail host nucleus. By examining ES products from normal and attenuated miracidia, and also by subtractive cDNA cloning strategies, we may be in a position to isolate this parasite soluble factor.

448
449 Legends to Figures
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454 a. Fig 1 Chromosome territories Non-random , erosion analysis - b. Fig 2 – Gene mapping for ferritin and actin on mitotic chromosomes c. Fig 3 Gene positioning Non-random actin, ferritin, piwi, prx d. Fig 4 – co-culture Actin repositioning with RT-PCR e. Fig 5 – co-culture Ferritin with irradiated miracidia
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