Non-satellite repetitive human DNA families. Sequence properties and evidence for occurrence in chimpanzee DNA.

Repetitive human DNA, fractionated on CsCl gradients following hydroxyapatite isolation, contains two complex DNA fractions, the 1.703 and 1.714 DNA families (Marx, K.A., Allen, J.R. and Hearst, J.E. (1976) Biochim. Biophys. Acta 425, 129-147). Biphasic Topt profiles, obtained in DNA excess hybridizations with cRNA tracers from each DNA family, have been shown to be the likely result of a fast kinetic component hybridizing at higher temperatures (67 degrees C peak) and this fast plus a slow kinetic component both hybridizing at lower temperatures (47 degrees C peak). Equilibrium CsCl gradient DNA-cRNA hybrid distributions support previous interpretations of the sequence composition of both DNA families. That is, the fast component is a relatively undiverged repetitive sequence of recent origin, while the slow component is a highly diverged, less thermally stabile, old primate sequence. This interpretation is further strengthened by cRNA tracer hybridization experiments in chimpanzee DNA excess where the fast component appears to be absent and the slow component present.

Ion competition and micrococcal nuclease digestion studies of spermidine-condensed calf thymus DNA. Evidence for torus organization by circumferential DNA wrapping.

Spermidine-condensed calf thymus DNA structures have been studied by ion competition using a sedimentation assay and by micrococcal nuclease digestion. Competitor ions Mg2+, Ca2+ and putrescine2+ show specific ion effects; but all three appear to affect the DNA condensation-decondensation equilibrium caused by spermidine3+ in a qualitatively similar manner, suggesting the spermidine3+-DNA interaction is largely electrostatic. Our data show a hysteresis in condensation and decondensation transition directions. We interpret this in terms of a kinetic block in the condensation direction with decondensation representing the equilibrium state of the system. These results agree with results obtained from related systems using different measurement techniques. Micrococcal nuclease digestion of spermidine-condensed calf thymus DNA produces broad but discrete bands in gel electrophoresis experiments. At least two bands determined to be 760 +/- 87 bp and 1355 +/- 135 bp, possess the size ratio 1:1.8 +/- 0.4 consistent with their forming the monomer and dimer fragments of an arithmetic band series. We rationalize this result in terms of a localized micrococcal nuclease cleavage model of circumferentially-wrapped DNA toruses proposed previously by Marx, K.A. and Reynolds, T.C. (Proc. Natl. Acad. Sci. (1982) 79, 6484-6488). The arithmetic series monomer band (760 +/- 87 bp), corresponding to wrapping B DNA once circumferentially about the torus, is in agreement with the electron microscopic measurements of hydrated calf thymus DNA torus circumferences presented by Marx, K.A. and Ruben, G.C. (Nucleic Acids Res. (1983) 11, 1839-1853).

High-affinity microtubule protein-higher organism DNA complexes. Many-fold enrichment in repetitive mouse DNA sequences comprised of satellite DNAs.

We have examined aspects of the interaction of cycled microtubule protein preparations with 35S-labeled mouse DNA tracer in a competition system with unlabelled competitor E. coli or mouse DNA. The nitrocellulose filter binding assay was used to measure interaction by scintillation counting. DNA molecular weight affected the levels of filter retained 35S-labelled mouse tracer DNA. Filter retention levels increased if 35S-labelled mouse DNA tracer size was increased, and the filter binding level decreased if competitor DNA size was increased. There was a sizeable, reproducible difference in the 35S-labelled mouse DNA tracer binding level of about 1% when E. coli or mouse DNA competitors were compared. Mouse DNA more effectively competed with 35S-labelled mouse DNA for microtubule protein binding than did E. coli DNA, suggesting that a small class of higher-organism DNA sequences interacts very strongly with microtubule protein. From other studies we know this to be the MAP fraction (Marx, K.A. and Denial, T. (1984) in The Molecular Basis of Cancer (Rein, R., ed.), Alan R. Liss, New York, in the press; and Villasante, E., Corces, V.G., Manso-Martinez, R. and Avila, J. (1981) Nucleic Acids Res. 9, 895-908). We find that this difference in competitor DNA strength is qualitatively similar under high-stringency conditions (0.5 M NaCl, high competitor [DNA]) we developed for examining high-affinity complexes. Under high-stringency conditions we isolated 1.2% and 0.6% of 35S-labelled mouse DNA at 4200 and 350 bp respective sizes as nitrocellulose filter bound DNA-protein complexes. At both molecular weights these high-affinity DNA sequences, isolated from the filters, were shown to be significantly enriched in repetitive DNA sequences by S1 nuclease solution reassociation kinetics. The kinetics are consistent with about a 4-fold mouse satellite DNA enrichment as well as enrichment in other repetitious DNA sequence classes. The high molecular weight filter-bound DNA samples were sedimented to equilibrium in CsCl buoyant density gradients and found to contain primarily mouse satellite DNA density sequences (1.691 g/cm3) with some minor fractions at other density positions (1.670, 1.682, 1.705, 1.740, 1.760 g/cm3) similar to those observed by our laboratory in previous investigations of micrococcal nuclease-resistant chromatin (Marx, K.A. (1977) Biochem. Biophys. Res. Commun. 78, 777-784). That the high-affinity microtubule-bound DNA was some 3-5-fold enriched in mouse satellite sequences was demonstrated by its characteristic BstNI restriction enzyme cleavage pattern.

Characterization of the repetitious human DNA families.

Human DNA isolated from HeLa cells or human placental tissue has been fractionated on hydroxyapatite at COt 1.0. The 25% of total DNA isolated at COt 1.0 is composed of 3% foldback DNA and 22% which renatures by second-order kinetics and can be resolved into five renatured DNA families banding at distinct densities in CsC1 gradients. The individual renatured DNA families were isolated and their physical properties including reassociation kinetics determined. A two-component kinetic analysis was used to resolve kinetic heterogeneity. The three lightest density DNA families possess satellite DNA-like properties. The two heaviest density DNA families were shown to contain reassociated highly repetitious DNA as well as single-stranded, middle-repetitious DNA sequences, suggesting interspersion. The middle repetitious DNA sequences are thought to be related in these two DNA families.

DNA bound to polypyrrole films: high-resolution imaging, DNA binding kinetics and internal migration.

We have studied the time-dependent uptake of 35S radiolabeled DNA with electrochemically prepared polypyrrole films. The two distinct polypyrrole film surfaces, a rough (solution polymeric growth face, R) and a smooth surface (electrode face, S) were characterized by low-resolution AFM and high-resolution transmission electron microscopy (TEM). These studies showed the presence of steep contours and defects in the form of large and small surface holes and valleys on the rough surface of polypyrrole. The void dimensions ranged from the nanoscale to micron size. By contrast, the smooth surface was flatter and largely devoid of significant structural defects and exhibited closer packing of the polypyrrole chains over large areas. Both surfaces were comprised largely of chains whose average diameters were 1.0-1.2 +/- 0.3 nm. The surface characterization studies were complemented by time-dependent DNA uptake studies which showed a t1/2-dependent total uptake of 35S DNA at higher levels on the rough surface compared to the smooth surface. This is consistent with the apparent higher effective surface area of the rough surface compared to the smooth. Using a proportional counter the time-dependent ratio (R/S) of the 35S DNA detected from the rough surface of the polypyrrole disk to that detected from the smooth surface suggested that DNA was migrating into the disk interior from its uptake surface. The rough side defect dimensions measured by TEM were more than sufficient to allow for the penetration and migration of DNA into the disk interior. Both R/S ratios were extrapolated and found to intersect at an R/S value close to 1.0, suggesting a kinetic process leading ultimately towards a nearly uniform radiolabeled DNA distribution in the disk. These kinetic results were in agreement with the surface characterization studies and suggest a model in which sizeable internal pores exist throughout the electrochemically prepared polypyrrole, that could account for the DNA migration effect. This was confirmed by TEM of the interior of a polypyrrole disk produced by Argon ion milling.

A quartz crystal microbalance cell biosensor: detection of microtubule alterations in living cells at nM nocodazole concentrations.

The quartz crystal microbalance (QCM) was used to create a piezoelectric biosensor utilizing living endothelial cells (ECs) as the biological signal transduction element. ECs adhere to the hydrophilically treated gold QCM surface under growth media containing serum. At 24 h following cell addition, calibration curves were constructed relating the steady state Deltaf and DeltaR shift values observed to the numbers of electronically counted cells requiring trypsinization to be removed from the surface. We then utilized this EC QCM biosensor for the detection of the effect of [nocodazole] on the steady state Deltaf and DeltaR shift values. Nocodazole, a known microtubule binding drug, alters the cytoskeletal properties of living cells. At the doses used in these studies (0.11-15 microM), nocodazole, in a dose dependent fashion, causes the depolymerization of microtubules in living cells. This leads a monolayer of well spread ECs to gradually occupy a smaller area, lose cell to cell contact, exhibit actin stress fibers at the cell periphery and acquire a rounded cell shape. We observed the negative Deltaf shift values and the positive DeltaR shift values to increase significantly in magnitude over a 4-h incubation period following nocodazole addition, in a dose dependent fashion, with a transition midpoint of 900 nM. Fluorescence microscopy of the ECs, fixed on the gold QCM surface and stained for actin, demonstrated that the shape and cytoskeleton of ECs were affected by as little as 330 nM nocodazole. These results indicate that the EC QCM biosensor can be used for the study of EC attachment and to detect EC cytoskeletal alterations. We suggest the potential of this cellular biosensor for the real time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment, regardless of their molecular mechanism of action.

Involvement of the Na,K-ATPase in the induction of ion channels by palytoxin.

The effects of ouabain, ATP, and vanadate on palytoxin induction of ion channels were examined with the aim of elucidating the role of Na,K-ATPase in palytoxin action. Palytoxin-induced membrane depolarization of crayfish giant axons and single channel currents of frog erythrocytes and mouse neuroblastoma N1E-115 cells were examined using the intracellular microelectrode and patch-clamp techniques. External application of palytoxin in nanomolar concentrations induced depolarization in the crayfish giant axons, and the depolarization was inhibited by pretreatment of the axon with ouabain (10 microM). Internally perfused axons were less sensitive to palytoxin unless ATP (6 mM) was added internally. In patch-clamp experiments, picomolar palytoxin in the patch electrode induced single channels in both cell-attached and inside-out patches of erythrocytes and neuroblastoma cells. The induced channels had a conductance of about 10 pS, reversed near 0 mV in physiological saline solution, and was permeable to Na+, K+, Cs+, and NH4+, but not to choline. Single channel activities induced by palytoxin were inhibited by ouabain (10 microM) and vanadate (1 mM), but promoted by ATP (1 mM). The modulating effects of ouabain, vanadate, and ATP on palytoxin action suggest that the Na,K-ATPase is involved in the induction of single channels by palytoxin. Palytoxin-induced and ouabain-inhibitable single channels were observed in planar lipid bilayer incorporated with purified Na,K-ATPase. The results indicate that an interaction between palytoxin and Na,K-ATPase leads to opening of a 10-pS ion channel. They further raise the possibility that a channel structure may exist in the sodium pump which is uncovered by the action of palytoxin.

A study of phi X-174 DNA torus and lambda DNA torus tertiary structure and the implications for DNA self-assembly.

Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular phi X-174 DNA and lambda DNA preparations. Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width. Measurements of torus inner and outer circumference as well as ring thickness were performed. Observed differences in the torus dimension distributions from circular phi X-174 DNA and linear phi X-174 DNA may be related to the different topological constraints on DNA folding in these two samples (1). On the basis of annulus thickness measurements phi X-174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation. All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses. End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica. These direct measurements confirmed quaternary structure interpretations made from simple strand packing models. We compared the measured torus properties in this linear DNA size series (5386-48000 bp). With increasing DNA length the pattern of DNA strand self-assembly was found to be more varied producing lambda DNA toruses of varying shape. The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed.

The affinity of DNA-microtubule protein complexes and their disruption by tubulin binding drugs.

Using the gel shift assay system, we have measured the apparent affinity constant for the interaction of two different DNAs with MAP proteins found in both total calf brain microtubules and heat stable brain preparations. Both DNAs studied contained centromere/kinetochore sequences- one was enriched in the calf satellite DNA; the other was a large restriction fragment containing the yeast CEN11 DNA sequence. Complexes formed using both DNAs had similar Kapp values in the range of 2.1 x 10(7) M-1 to 2.0 x 10(8) M-1. CEN11 DNA-MTP complexes had by far the highest Kapp value of 2.0 x 10(8) M-1. The CEN11 DNA sequence is where the yeast kinetochore of chromosome 11 is formed and where the single yeast microtubule is bound in vivo. The CEN11 conserved region II known binding sites-(dA/dT)n runs- for mammalian MAP2 protein, are in good agreement with this higher Kapp value. The effects of the classical tubulin binding drugs colchicine, podophyllotoxin and vinblastine on the DNA-MAP protein complex stability were investigated by determining the drug concentrations where the complexes were destabilized. Only the complexes formed from total microtubule protein (tubulin containing) were destabilized over a wide drug concentration range. Heat stable brain protein complexes (no tubulin) were largely unaffected. Furthermore, it took 10-100 fold higher drug concentrations to disrupt the CEN11 DNA complexes compared to the calf thymus satellite DNA enriched complexes. These data support our previous results suggesting that there is a DNA sequence dependent interaction with MAP proteins that appears to be conserved in evolution (Marx et. al., Biochim. Biophys. Acta. 783, 383-392, 1984; Marx and Denial, Molecular Basis of Cancer 172B, 65-75 1985). In addition, these results imply that the classical tubulin binding drugs may exert their biological effects in cells at least in part by disrupting DNA-Protein complexes of the type we have studied here.

Studies of DNA organization in hydrated spermidine-condensed DNA toruses and spermidine-DNA fibres.

We have investigated in vitro spermidine-condensed DNA preparations by both biochemical and freeze-etch electron microscopic approaches. These studies lead us to the conclusion that the reversibly condensed preparations, qualitatively described by Manning's counterion condensation theory, contain disk-like torus structures largely comprised of unidirectional, circumferentially wrapped DNA. Stereoscopic measurements on stereomicrographs of DNA torus and fibre objects have demonstrated the feasibility of directly measuring DNA writhe or, for that matter, mapping any secondary, tertiary or quaternary structure features of a hydrated macromolecular array in which the features can be differentially highlighted by low replica metal shadow levels.

Solution structure of bacteriophage T4D and icosahedral capsid geometry visualized in freeze-fractured, deep-etched replicas.

The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles. Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80 degrees angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm. From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q number of 21. This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2. Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging. Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted). The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with 2% glutaraldehyde. The FDET protocols appear to reveal important structural features not seen in negative stained preparations. These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection.

Alignment of (dA).(dT) homopolymer tracts in gene flanking sequences suggests nucleosomal periodicity in D. discoideum DNA.

It has been shown that the frequency versus size distribution of A and T overlapping and non-overlapping homopolymer tracts of N > 5 in D. discoideum gene flanking and intron regions are significantly greater than in coding regions(1). In the present report, we demonstrate, that a spatial periodicity exists in long A and T tracts (N > 10) in long flanking sequences by scored alignments of those tracts (N > 10) with the nucleosomal repeat. A tract spacing was found at 185-190 bp that corresponds to a maximum alignment score. This is exactly the average spacing of D. discoideum nucleosomes determined experimentally. A majority of A and T tracts in flanking sequences are often spaced by short DNA stretches and the total length of adjacent A and T tracts plus the interrupting short DNA stretch corresponds closely to the average experimentally measured nucleosomal linker DNA size in D. discoideum-42 bp. These data suggest a model which has A and T runs of N > 10 bp in flanking DNA of D. discoideum organized in a regular phase with nonhomopolymer sequences along the DNA. This model has functional implications for A and T tracts, suggesting that they are found in nucleosomal linker DNA regions of chromatin during some necessary portion(s) of the life of the cell.

Characteristics of the large (dA).(dT) homopolymer tracts in D. discoideum gene flanking and intron sequences.

D. discoideum, the slime mold, is one of the most AT rich eukaryotic genomes known. In this paper we examine this organism's database for overlapping N-tuples of high frequency and find A and T tracts possess among the highest frequencies in flanking sequences but not in coding sequences. We examined both overlapping and non-overlapping frequencies of the A, T, G and C homopolymer tracts of 2 < N < 6. Overlapping (dG).(dC) and (dA).(dT) tracts occur at greater frequencies than expected, based on random occurrence. Long (dA).(dT) tracts of N > 10 occur at well above expected frequencies in flanking and intron regions, while (dG).(dC) tracts above N = 5 are rarely found. Some of the implications of these findings for tract origins in slip-strand replication and for chromatin structure are discussed.

Comparison of experimental to MELTSIM calculated DNA melting of the (A+T) rich Dictyostelium discoideum genome: denaturation maps distinguish exons from introns.

The slime mold, Dictyostelium discoideum, possesses an (A+T) rich eukaryotic genome that is being sequenced in the Human Genome Project. High resolution melting curves of isolated total and fractionated nuclear D. discoideum DNA(AX3 strain) were determined experimentally and are compared to melting curves calculated from GENBANK sequences (1.59% of genome) by the statistical thermodynamics program MELTSIM (1), parameterized for long DNA sequences (2,3). The lower and upper temperature limits of calculated melting agree well with the observed melting of total DNA. The experimental curve is unusual in that it contains a number of sharp peaks. MELTSIM allowed us to calculate positional denaturation maps of D. discoideum GENBANK sequence documents containing the 26S, 5.8S and 17S rDNA gene sequences, a major satellite DNA and repetitive sequence family present in 100-200 copies/nucleus. These denaturation maps contain subtransitions that correspond with a number of the experimentally observed peaks, some of which we show to correspond with rDNA gene enriched CsCl gradient fractions of D. discoideum DNA. MELTSIM calculated curves of coding, intron and flanking sequences indicate that both intron and flanking sequences are extremely (A+T) rich and account for most of the low temperature melting. There is no temperature overlap between thermal stabilities of these sequence domains and those of coding DNA. The latter must satisfy triplet codon constraints of higher (G+C) content. These large stability property differences enable a denaturation mapping feature of MELTSIM to clearly distinguish exon positions from those of introns and flanking DNA in long D. discoideum gene containing sequences.

Quartz crystal microbalance measurement of self-assembled micellar tubules of the amphiphilic decyl ester of D-tyrosine and their enzymatic polymerization.

Amphiphilic decyl derivatives of D-tyrosine self-assemble into long rodlike or tubular aggregate structures in aqueous buffered solution. In this report we demonstrate the novel use of the quartz crystal microbalance (QCM) to measure the presence in solution, and subequent enzymatic polymerization, of long rodlike monomer aggregates of the decyl ester of D-tyrosine (DEDT) as a function of their formation and increasing surface binding level as pH values increase from 3 to 7. From these data, using the Sauerbray equation to calculate the effective elastic mass surface binding of deprotonated DEDT aggregates, a pKapp of 8.3 is obtained for the DEDT alpha-NH2 group protonation-deprotonation and subsequent aggregation equilibrium. Furthermore, once aggregates are bound to the QCM surface, we initiate and subsequently monitor enzymatic polymerization of the DEDT monomers by horseradish peroxidase through the measurement of significant changes in the quartz crystal frequency and motional resistance. Following the onset of polymerization, the viscoelastic properties of the bound monomer aggregates change. A final polymerized state is achieved in which the altered physical properties of the polymerized rodlike aggregates make the solution immediately above the QCM surface-solution interface behave as a Newtonian fluid, producing a nearly pure viscosity-density energy dissipative effect on the measured crystal frequency and motional resistance values.

The quartz crystal microbalance as a continuous monitoring tool for the study of endothelial cell surface attachment and growth.

The quartz crystal microbalance (QCM) was used to monitor endothelial cell (EC) adhesion on the gold surface of an oscillating quartz crystal contained in a QCM device. A number of parameters were investigated. First, we observed differential QCM O-ring toxicities for ECs. Second, appropriate conditions for cell culture and QCM cell environment were identified that can eliminate large-scale frequency oscillations in the measurements. These artifacts are not due to added cells but originate in the time-dependent evaporation of water. Having eliminated these artifacts, we then demonstrated that the measured steady-state crystal frequency shift, Delta f, and motional resistance shift, DeltaR, were determined by the number of firmly attached ECs requiring trypsinization from the crystal surface. Last, following steady-state attachment of ECs, the EC growth stimulation by fibroblast growth factor was monitored in a continuous fashion by measuring f and R values over a 72 h. period. We observed the Delta f values to increase in a way that reflected the increase in EC number bound to the QCM surface. Following addition of ECs to the QCM, the time-dependent increase in DeltaR can be interpreted in terms of increase by the ECs of the energy dissipation properties of the solution at the solution-gold surface interface. This effect is due to their rapid surface attachment and the elaboration of their cytoskeletal properties. These results indicate that the QCM technique can be used for the study of EC attachment and growth and suggest its potential for the real time study of per unit surface area cell mass distribution dynamics and viscoelastic properties and the cells' responses to stresses or perturbations brought about using biologically active molecules.

Chemiluminescence-based inhibition kinetics of alkaline phosphatase in the development of a pesticide biosensor.

The use and application of the enzyme alkaline phosphatase in a chemiluminescence assay are discussed. The enzyme catalyzes the hydrolysis of a macrocyclic phosphate compound generating a chemiluminescence signal. On the basis of inhibition of this signal, a methodology for the detection and quantitation of organophosphorus-based pesticides has been developed. The methodology is studied with alkaline phosphatase in the bulk aqueous phase, and detection of the signal is accomplished by a simple optical setup. Parts per billion level detection of paraoxon and methyl parathion in bulk solutions is achieved. The technique is rapid and sensitive and is applicable to the detection of most organophosphorus-based pesticides. The results from kinetic studies indicate a mixed type of inhibition of the enzyme by paraoxon and methyl parathion. The detection methodology forms an integral part of a biosensor under development and is adaptable to incorporating optical fibers for remote detection of pesticides.

Micrococcal nuclease digestion study of spermidine-condensed DNA.

Spermidine-condensed lambda DNA tertiary structures have been studied by micrococcal nuclease digestion. Broad but discrete DNA bands were observed in gel electrophoresis experiments of digests at sizes of: 1003 +/- 115 bp, 1972 +/- 190 bp and 3100 +/- 350 bp. These bands comprise an arithmetic series, similar to, but larger than, arithmetic DNA band series sizes we have observed previously in calf thymus and phi x-174 DNA condensates. The 1003bp monomer lambda DNA band size corresponds to wrapping B DNA once circumferentially about the toroidal-shaped tertiary structures, the predominant condensed structures present in these preparations, and is consistent with the measured electron microscopic dimensions for hydrated lambda DNA toruses previously presented. DNA fragment length stability was determined by release from the digested condensates. Fragments of 80-85bp and sizes below are thermodynamically unstable in the lambda DNA condensates. This fragment size agrees well with a recent determination of the cooperativity size in DNA condensates.

Randomly amplified polymorphic DNA (RAPD) polymerase chain reaction assay for identification of Schistosoma mansoni strains sensitive or tolerant to anti-schistosomal drugs.

The genetic differences between Schistosoma mansoni strains from different geographic areas that were reportedly resistant or sensitive to anti-schistosomal drugs were studied with randomly amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) polymerase chain reaction (PCR) assays. Of the 20 RAPD primers we chose, 19 showed the capacity to produce a medium to high level of amplification and 6 revealed difference PCR bands between drug-resistant and drug-sensitive strains. One particular primer, 5'-CAGCGACAAG-3', showed 2 major difference bands between praziquantel (PZQ)-resistant and PZQ-sensitive strains from the endemic area of Egypt. These results demonstrate that defined sequence primers could be applied as a useful tool for differentiating drug-resistant and -sensitive schistosome parasites in the field.