Experimental DNA melting behavior of the three major Schistosoma species

Molecular and Biochemical Parasitology 107 (2000) 303 – 307 www.elsevier.com/locate/parasitology

Short communication

Experimental DNA melting behavior of the three major Schistosoma species Kenneth A. Marx a,*, Jeffrey W. Bizzaro a, R.D. Blake c,1, Meng Hsien Tsai a, Liang Feng Tao b,2 a

Center for Intelligent Biomaterials, Department of Chemistry, Uni6ersity of Massachusetts-Lowell, Lowell, MA 01854, USA b Center for Tropical Diseases and Biomedicine, Uni6ersity of Massachusetts-Lowell, Lowell, MA 01854, USA c Department of Biochemistry, Microbiology and Molecular Biology, Uni6ersity of Maine, Orono, ME 04469 -0131, USA Received 7 July 1999; accepted 21 December 1999

Keywords: Schistosoma DNA; Schistosoma mansoni DNA; Schistosoma japonicum DNA; Schistosoma haematobium DNA; DNA melting; Melting simulation; Repetitive sequences

Schistosomiasis is a debilitating tropical disease worldwide, caused by infection with three major species of a trematode (Schistosoma spp): Schistosoma mansoni, Schistosoma heamatobium and Schistosoma japonicum. An international effort lead by the WHO (UNDP World Bank) Schistosoma Genome Initiative has been mounted to sequence the genomes of Schistosoma species [1]. Little has heretofore been published concerning the whole genome physical properties of these parasitic worms. Based upon analytical ultracentrifugation and low resolution thermal denaturation measurements, the one reported DNA base composition was 34.2% (G +C) for the three * Corresponding author. 1 Present address: Department of Chemistry, WiIliams College, Williamstown, MA 01267, USA. 2 Present address: Millenium Pharmaceuticals, Inc., 215 First Street, Cambridge, MA 02142, USA.

Schistosoma species [2]. The DNA melting experiment commonly carried out is a low resolution technique providing limited information on the differences between DNA samples as well as details of heterogeneity within a sample. However, high resolution melting, has been shown to provide highly detailed information about the whole genome properties of a number of prokaryotes and eukaryotes [3–8]. Furthermore, the program MELTSIM, now allows comparison between experimental melting and melting behavior calculated from DNA sequence database information. In the only whole eukaryotic genome comparison performed to date, experimental Saccharomyces cere6iseae DNA melting was found to agree well with the calculated melting, using the complete 12 067 277 bp genome file in GENBANK™ as input data to MELTSIM [7]. Individual chromosomes were also melted to create denaturation maps along individual chromosomes in silico.

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In the present report, we compare the experimental high resolution melting behavior of S. mansoni, S. japonicum and S. haematobium DNAs. We observed significant differences in the overall% (G+C) base composition of the three genomes, as well as differences in the sequence melting temperature distribution. These differences include observation of small, sharp melting subtransitions in S. japonicum and S. haematobium, indicative of repetitive sequences. We demonstrate reasonably good agreement between the experimental melting of S. mansoni and the simulated melting of a subset of its genome available in GENBANK™. Schistosoma haematobium Egyptian adult worms were provided by NIH/NIAID supply contract AIc 55270. S. mansoni PR-1 and S. japonicum Chinese adult worms were obtained from the Center for Tropical Diseases and Biomedicine at the University of Massachusetts Lowell. Adult worm pairs from S. haematobium (14 – 15 weeks in Golden Syrian Hamsters), S. mansoni and S. japonicum (7 – 8 weeks in CD1 mice) were obtained by liver perfusion, washed 5-times with ice-cold phosphate buffered saline, and either stored at −70°C or used immediately. DNAs were isolated by a minor modification of a published procedure [9]. Frozen worms (liquid N2) were ground into a fine powder in a mortar and pestle. The powder was thawed in extraction buffer (50 mM Tris– HCl, pH 8.0, 50 mM EDTA, 100 mM NaCl) at 37°C. The suspension containing final concentrations, respectively, of 1% SDS and 100 ug/ml RNase A was incubated for 4 h at 37°C. Incubation with proteinase K at 400 mg/ml was continued for 4 h at 37°C, followed by overnight incubation at 4°C. The solution was extracted once with an equal volume of 10 mM Tris pH 8.0 buffered phenol, followed by three extractions with an equal volume of 10 mM Tris pH 8.0 buffered phenol:chloroform:iso-amyl alcohol in a 24:24:1 ratio, followed by a final extraction with an equal volume of chloroform. The DNA was ethanol precipitated and the pellet was washed repeatedly with cold 70% ethanol, then ether. Following air drying, the DNA was resuspended in 0.1 mM EDTA, 10 mM Tris buffer pH 8.0.

Solid CsCl, along with 0.2 mg added ethidium bromide, was added to the DNA dissolved in buffer to a final concentration of 1.10 g/ml. DNA was spun at 45 000 rpm in a 70 Ti rotor in a Beckman L8-55 Ultracentrifuge for 30 h. Then the DNA band was slowly removed through a 16 G needle, the ethidium bromide was extracted and CsCl was removed from the DNA by extensive dialysis into 0.0716 M NaCl, 0.0038 M cacodylic acid, 0.00015 M EDTA, pH 6.7. Immediately prior to melting, high molecular weight poly [d(A).d(T)] marker was added and the DNA was filtered through a 0.45 mm filter. High resolution melting curves were obtained as previously described [3,4,6,8]. The program MELTSIM was used to calculate the melting curves of GENBANK™ sequences. This is a 32-bit Microsoft Windows program that calculates derivative melting curves with a one-dimensional Ising lattice algorithm, parameterized with experimentally derived nearest neighbor base pair enthalpies and entropies. MELTSIM has been described in detail elsewhere [8]. Multiple DNA isolations were performed using adult worms of: S. mansoni, PR-1 strain (3); S. japonicum, Chinese strain (1); S. haematobium, Egyptian strain (2). The samples were then subjected to multiple high resolution meltings: S. mansoni (5); S. japonicum (3); S. haematobium (2). All of the independent melting curves for each species were averaged and the results are presented in Fig. 1. The melting curves are shown as du/ dT representations versus T. This is the first derivative of a traditional u (fraction single stranded) versus T sigmoid shaped plot of DNA melting. The du/ dT representation more clearly defines the melting behavior of any given sample. For example, the Tm is the point of maximum slope in a u versus T plot, and zero slope in a du/ dT plot. Therefore, Tm values correspond to the zero slope, peak maxima in the du/ dT plot. This allows much clearer visualization of individual components in complex melting samples, where multiple additive sigmoid transitions would not be discernible. Fig. 1 displays the averaged du/ dT curves for the S. mansoni, S. japonicum and S. haematobium genomes, offset from the du/ dT zero point for

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ease of comparison. The absolute magnitude of the du/ dT values for each curve are not important in our discussion. However, the T or thermal stability positions and relative intensities for any individual thermal transition are important. They indicate the sequence %(G+ C) composition and its percentage of the genome. It is clear from Fig. 1 that the melting curves of S. mansoni and S. haematobium are centered at similar T values, while that of S. japonicum is centered at a lower T. In Table 1, we present the Tm values, calculated for each species, along with the corresponding calculated %(G+C) values for the DNAs. S japonicum possesses a %(G+C) value of 26.1%, significantly different from the 28.5 and 29.4% of S. haematobium and S. mansoni, respectively. All three melts possess a similar skewed melting distribution. While the low T region of the curve is relatively sharp, there is a significant melting

Fig. 1. Comparison of the averaged first derivative experimental melts of S. mansoni (5 melts), S. japonicum (3 melts) and S. haematobium (2 melts) DNAs presented offset from each other. Lowest panel compares the MELTSIM calculated first derivative melt of S. mansoni total sequences contained in GENBANK™. The left hand scale in this figure corresponds to that for the S. haematobium melt. Thick vertical lines indicate the calculated Tm values of the entire melt.

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throughout a higher T range by sequences of relatively high %(G+ C) composition. Similar behavior has been observed for the melting of a number of eukaryotic DNAs, including: human, ox, S. cere6iseae and D. discoideum [4–8]. This is in contrast to the melting of bacterial DNAs, which possess narrow and nearly symmetric shaped melting curves [4]. Asymmetric melting curves in eukaryotes are due to: the gross organization of higher vertebrate DNAs into ‘Bernardi isochores’ of different average %(G+ C) base composition; the presence of repetitive DNA interspersed with single copy sequences; and tandemly repetitious satellite DNA sequences. DNA sequences can melt in highly cooperative segments of as little as a few hundred base pairs while the flanking DNA sequences remain intact. Therefore, the temperature distribution of sequence melting accurately reflects the thermodynamic stability of individual melting regions. This behavior stands in contrast to CsCl buoyant density gradient centrifugation, where the equilibrium CsCl density position of an individual DNA molecule reflects the averaged behavior of its individual sequence regions. In S. japonicum and S. haematobium we observed a few sharp melting subtransitions. S. mansoni only possesses broad subtransitions in its averaged melting curve, although in some individual melting curves a few small subtransitions were observed. We summarize the observed repetitive DNA melting features for all three species in Table 1. These subtransitions correspond to specific repetitive DNA sequence families whose monomer sequence elements melt cooperatively and whose high copy number result in the well defined peaks observed. Using the program MELTSIM, it should soon be possible to relate repetitive sequences in GENBANK™ to the specific sharp subtransition melting features identified in Table 1. Currently, the GENBANK™ database (retrieved 6/11/98) only has a sizeable number of sequence documents for the S. mansoni species(4777 documents; 1 594 696 bp). Notwithstanding the overrepresentation of coding sequences in the database, we demonstrate that the statistical mechanical program MELTSIM, de-

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Table 1 Experimental melting properties of three Schistosoma species’ genomic DNAs Species

Tm (°C)a

% (G+C)b

D%(G+C)c

S. mansoni total S. japonicum total S. haematobium total

76.4 75.0 76.0

29.4 26.1 28.5

0 3.3 0.9

78 (broad) 79 (broad) 70 (shoulder) 78.4 (sharp) 84 (broad) 72 (shoulder) 76.4 (sharp) 77.1 (sharp) 77.4 (sharp) 79.1 82 86 (broad)

34 36 14 34.3 48 19 29.6 31.3 32.0 36.0 44 55

– – – – – – – – – – – –

Indi6idual subtransitions S. mansoni S. japonicum

S. haematobium

a

Tm values are determined by the area method and represent the weighted average midpoint for the entire averaged (all melts) genomic melting curve under the 0.075 M Na+ condition. b Calculated from the Tm using the Marmur–Schildkraut–Doty equation: Tm(°C)= 193.67−(3.09−FGC)(34.64−2.83 ln [Na+]). c D%(G+C) represents the difference in %(G+C) base composition between two species’ genomes where S. mansoni is the constant basis for comparison.

scribed in the Methods section, with the available GENBANK™ files as input data, can be used to calculate the melting of S. mansoni DNA. In Fig. 1(lowest panel) we present the MELTSIM calculated melting curve of all the S. mansoni sequence files compared to the S. mansoni experimental melting curve above it. The total calculated sequence population melts at a higher T than does the experimental genome melt since the GENBANK™ database contains only 0.59% of the estimated S. mansoni genomic sequences, with an overrepresentation in higher melting coding sequences (unpublished results) compared to the actual genome composition. The lower melting flanking DNA sequence population begins melting at low T values, around 71°C, similar to the T values observed for the initiation of the experimental melting curve(unpublished results). These facts suggest that MELTSIM is accurately simulating the melting behavior of the available S. mansoni DNA sequences.

The one previous measurement of the base compositions of S. mansoni, S. haematobium and S. japonicum reported a 34.2 %(G + C) composition for all. This very different value from ours was determined from DNA melting and equilibrium buoyant density gradient centrifugation in CsCl [2]. We feel that our measurements more accurately represent the true base compositions of these three genomes. The earlier report does not utilize high resolution melting. And the CsCl gradient centrifugation method, using a density marker DNA, 0.050 g/cm3 away from the unknown in the CsCl gradient, cannot yield accurate base compositions. Being whole genome dependent fit parameters, our Tm values suggest that S. mansoni and S. haematobium are more closely related to each other than each is to S. japonicum. Comparing different genomic %(G+ C) values as D%(G+ C) values in Table 1, we observe the low value of 0.9 D%(G+ C) for the S. mansoni S. haematobium genomes comparison as opposed to the

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higher 3.3 D%(G+C) value for the S. mansoni S. japonicum genomes comparison. The greatery genomic differences in base composition of the latter species pair suggest that they diverged from a common ancestor much earlier than the S. mansoni S. haematobium pair divergence. This interpretation of our results is in agreement with classical morphological species differences in egg structure as well as more recent qualitative molecular evidence for species divergence based upon ribosomal RNA sequence differences [10]. Biased nucleotide DNA replication and repair represents one possible mechanism that can drive non-coding sequences to higher or lower %(G+ C) base composition. Therefore, biased DNA repair provides a rationale to explain the overall %(G + C) base differences observed between the three Schistosoma species’ genomes. According to this idea, since their divergence from a common ancestor, the S. japonicum genome has undergone a higher rate of biased repair than have the other two species, resulting in its lower genomic Tm value compared to the other two species’ DNA. Evidence for biased DNA repair operating in S. mansoni has been presented in a study of the heterogeneity of codon usage, by an examination of the third codon position base distribution in a group of 88 S. mansoni genes [11].

Acknowledgements The authors acknowledge support from the Center for Intelligent Biomaterials and the Center for Tropical Diseases and Biomedicine at the University of Massachusetts.

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References [1] http: / / www.nhm.ac.uk / hosted – sites/schisto/informatics/ Schisto DB – info.html [2] Hillyer GV. Buoyant density and thermal denaturation profiles of schistosome. DNA J Parasitology 1974;60:725 – 7. [3] Delcourt SG, Blake RD. Stacking energies in DNA. J Biol Chem 1991;266:15160– 9. [4] Blake RD. Denaturation of DNA. In: Myers RA, editor. Encyclopedia of Molecular Biology and Molecular Medicine, vol. 2. Basel: VCH Press, 1996:1 – 19. [5] Marx, K.A., Bizzaro, J.W., Assil, I., Blake, R.D., 1997. Comparison of experimental and theoretical melting behavior of DNA, In: Statistical Mechanics in Physics and Biology, Proceedings, Materials Research Society, Pittsburgh, PA., 463, pp. 147-152. [6] Marx KA, Assil I, Bizzaro J, Blake RD. Comparison of experimental to MELTSIM calculated DNA melting of the (A +T) rich Dictyostelium discoideum genome: denaturation maps distinguish exons from introns. J Biomol Struct Dyn 1998;16:329 – 39. [7] Bizzaro, J.W., Marx, K.A., Blake, R.D., 1998 Comparison of experimental with theoretical melting of the yeast genome and individual yeast chromosome denaturation mapping using the program MELTSIM. In: Materials Science of the Cell, Proceedings, Materials Research Society, Inc., Pittsburgh, PA., 489, pp. 73 – 78. [8] Blake RD, Bizzaro JW, Blake JD, Day GR, Delcourt SG, Knowles J, Marx KA, SantaLucia J. Statistical mechanical simulation of polymeric DNA melting with MELTSIM. Bioinformatics 1999;15:370 – 5. [9] Jeffs AS, Simpson AJ. Extraction and purification of Nucleic Acids from schistosomes. In: Hyde JE, editor. Methods in Molecular Biology. In: Protocols in Molecular Parasitology, vol. 21. Totowa, NJ: Humana Press, 1993:145 – 54. [10] Blair SC, Barker D. Molecular phylogeny of Schistosoma species supports traditional groupings within the genus. J Parasitol 1996;82:292 – 8. [11] Musto H, Romero H, Rodriguez-Mased H. Heterogeneity in codon usage in the flatworm Schistosoma mansoni. J Mol Evol 1998;46:159 – 67.