Molecular and Biochemical Parasitology, 36 (1989) 217-222 217 Elsevier

MOI.BIO 01198

Isolation of a female-specif ic , highly repeated Schistosoma mansoni D N A probc and its use in an assay of cercarial sex

P h i l i p p a W e b s t e r 1.2, T a g E . M a n s o u r ~ a n d D a v i d B i e b e r ~

I D~Tmrtment o/Pharmacology aml ~ Department ~l Biological Scieme~. Stanfi)rd Univer~it.v, Stan.lbrd. CA, I,:..~.A.

(Received 3 March 19F,9: accepted 26 April 1989)

A 476 bp fragment of female-specific Schi.stosoma mwl.soni genomic DNA, chine W l, represents a degenerative repeat present in more than 500 copies per female genome, and may be part of the constitutive hctcrochromatin of the W chromosome. The cloning method described can be used as a general approach for isolating sex-specilic, repeated I)NA fragments. Using V,,"I as a probe, wc have developed a rapid and accurate dot-blot assa.', for determining the sex of S, maJzsoni cercariae.

Key words: Schistosoma mansom: W chromosome: ('crcarial sex: Femalc-spccilic DNA probe: Rcpcatcd sequence

Introduction

In Schistosoma mansoni, a dioecious trema- tode, the female is the heterogametic sex [l]: the sex chromosomes in the female and male are des- ignated ZW and ZZ. respectively. While there is a clear dimorphism between adult males and fe- males, the sexes are visually indistinguishable in the miracidial and cercarial stages. During the transition through the snail intermediate host, each miracidium initiates a sporocyst which gen- eratcs asexually thousands of identical cercariae. A snail infected with a single miracidium can sur- vive and shed ccrcariae for three to six months, providing clonal populations as an cxperimcntal resourcc. It is useful to bc able to determine the sex of these populations rapidly and accurately, both for studying unisexual infections in mice and

('orre.~pomlence addre.s.~: "I':.lg E. Mansour. Depar tment ol Pharmacology, Stanford University School ol Medicine, Stan- ford. ( 'A 94305, t ' . S .A .

Note: Nucleotidc sequence data reported in this paper have been submit ted to the GenBank t'~ Data I?,ank with the acces- sion number .104665,

Abbreviations': SS('. s tandard saline citrate: SDS. sodium dodecvl sulfate: AI 'W. aged tap water.

for establishing genetic crosscs. ('ytological evidence shows the S. mansoni W

chromosome to bc approximately 45% hetero- chromatic [2]. Eukaryotic heterochromatin gen- erally contains repetitive DNA sequences: such sequences from other parasites have proven use- ful as sensitive diagnostic probes [3-6]. This re- port describes the isolation of a female-specific, highly repeated sequence from S. mansoni and its use as a probe in a quick, reliable assay for de- termining the scx of cercariae.

Materials and Methods

Cloning and characterization of WI. Unless oth- erwise noted, DNA manipulations were per- formed according to Maniatis et al. [7].

5 Ia-g of total female genomic S. mansoni DNA was digested to completion with EcoRI (New England Biolabs) and ligated with 1 gg of EcoRI- digested, phosphatased Blucscript SK- plasmid vector (Stratagene). Competent Escherichia coli cells (strain JM109) were transformed and se- lected on ampicillin plates. 500 white cokmies were picked onto selective plates, and in two re- plicas onto nitrocellulose filters (Schleicher and Schuell) overlaid on selective plates. Filters were processed according to the method of Thaycr [8].

(1166-6851:8,9:$03.50 © 19,R9 lZlsevier Science Publishers B.V. (Biomedical Division)

218

300 ng each of total female and total male gen- omic DNA (approximately I(Y' copies of each genome) were radioactively labeled [9] with [c~- 3-'P]dCTP (Amersham). One set of filters was probed with the labeled male DNA, and the other with the labeled female DNA plus 75 txg of un- labeled male D N A (approximately 2.5 × 10 * copies of the male genome). Hybridization was carried out in 50% formamide, 5 x Dcnhardt ' s solution, 5 × SSC. 0.1% SDS and l(10 p,g ml denatured salmon sperm DNA at 42°C for 40 h. Filters were washed in 1 x SSC and 0.1c7~ SDS at 68°C for 1 h, then in 0.2 × SSC and 0.1% SDS at 68~C for 1 h. Plasmid DNA was isolated from colonies corresponding to strong hybridization on the female screen and reduced hybridization on the male screen. Insert fragments were isolated and used to probe genomic Southern blots of male and female S. mansoni DNA.

Determining the sex o,f cercariae using W1. PR-B albino Biomphalaria glabrata with a shell diame- ter of 5 to 7 mm were infected with a Puerto Ri- can strain of S. mansoni which is maintained in BALB/c mice. Each snail was incubated in 1 ml of A'I 'W (tap water aged 3 or more days at room temperature) along with one miracidium for 3(I to 60 rain. Snails were subsequently raised in batches of 24 in 6-1 tanks and checked for cercarial shed- ding at 7 weeks and 9 weeks post-infection. Cer- cariae were collected by incubating infected snails in 1.5 ml ATW under bright light for I to 2 h. Snails were marked for identification with col- ored nail polish.

Chmal populations ranging from approxi- mately 3(X/to more than 4(XX) cercariae, each shed from a single snail, were collected in 1.5-ml mi- crofuge tubes, frozen in dry ice, thawed, and pel- leted in a microfuge. Pellets were resuspended in 200 p.I of 10 mM "I'ris-HCI, pH 8, 50 mM EDTA, 0.25% SDS and incubated at 50°C for 1(I min with 200 p,g ml- ~ proteinase K. Preparations were ex- tracted with an equal volume of neutralized phenol, and ethanol precipitated in the presence of 0.3 M sodium acetate. Pellets were resus- pended in 20 ~1 of 10 mM Tris-14CI, p l t 8. 1 mM EI)TA. Initially, either the A>. of each sample was assessed or comparat ive yields were esti- mated by analyzing 5 p.l of each sample on an

agarosc gel and visualizing the DNA with ethid- ium bromide. Yields of DNA were found to vary over an approximately ten-fold range; this varia- lion was not enough to affect the results of the dot blot assay, and the step to determine the DNA yield was eventually omitted.

For the dot blot assay, 3 ~1 of 3 M NaCI, 0.8 N NaOH were added to 3 lal of each cercarial DNA sample, spotted onto Nvtran membrane (Schleicher and Schuell), and allowed to air-dry (alkaline bonding of the DNA to a nylon mem- brane eliminates the need for separate denatur- ation treatment and for fixation by baking [10]). Filters were prehybridized for 1 h or longer, hy- bridized with 32p-labeled WI[9] overnight, and washed in 0.1 × SSC, 0.1% SDS at 50°C for 3(I min or longer. With fresh probe, exposure times of under one hour were generally adequate.

BAI,B/c mice were infected with 21)11 putative male cercariac or 1200 putative female cercariac. Cercariae from individual snails were added to 6 ml of ATW in a jar 6 cm in diameter, and a mouse was placed in each jar for 30-45 rain. Mice were subsequently raised for 49 days. at which time the worms were pcrfused from the hepatic portal sinus using the method of Duvall and Dewitt [11}. Male and female worms could easily be distinguished under a dissecting microscope.

Results

Isolation and characterization c~[ pWl . In a plas- mid library of EcoRI fragments from female S. mansoni genomic DNA, the colony containing plasmid pWl showed strong hybridization to ra- dioactively labeled total female S. mansoni gen- omic DNA in the presence of a 250-fold excess of unlabeled total male S. mansoni genomic DNA. On replicate tilters probed with radioactively la- beled total male genomic I )NA. the cohmy con- taining pWl showed reduced hybridization com- pared to the surrounding colonies.

A Southern blot of EcoRI-digcsted male and female S. mansoni gcnomic DNA probed with the insert isolated from pWl gave multiple bands of hybridization in the female lane but no detecta- ble hybridization in the male lane (Fig. la) in a l-h exposure. A further three-day exposure failed to show any signal in the male lane. To demon-

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cl b Fig. 1. (a) Southcrn blot of D N A isolated from (A) adult male or (B) adult female S. mansoni worms, digested with EcoRI and hybridized to radioactively labclcd WI 19] (1 h exposure). (b) The same blot. strippcd and rc-probcd with a low-copy- number sequence present in both males and females (24 h ex- posure). Specific hybridization is indicated by the arrow on the

right.

219

strate that the restriction digests were complete and that approximately equal amounts of D N A were present in each lane, the blot was stripped and re-probed with SGI, the cDNA clone of a GTP-binding protein which is present in equal cop} number in the genome of both sexes (Iltzsch, Zurita, Mansour and Bicber, manuscript in prep- aration). A single band appeared with equal in- tensity in both the male and the female lanes after 24 h (Fig. lb).

Both strands of the pWl insert were se- quenced. W1 is a 476-bp fragment bounded by an EcoRI restriction site at either end (Fig. 2). The sequence is slightly A+T-rich (59%). No signifi- cant homology was detected to nucleic acid se- quences in the EMBL or GenBank T M databases. A slot blot comparing the hybridization of radio- actively labeled WI to known amounts of female S. mansoni genomic D N A and known numbers of copies of pW1 (Fig. 3) shows approximately 500 copies of Wl-homologous sequence per female genome at high stringency and 1000 copies at lower stringency.

No detectable message was seen on a Northern blot of poly(A)" RNA isolated from adult male or female S. mansoni and probed with WI (data not shown). On genomic Southern blots of D N A from male and female S. mansoni, Schistosoma mattheei, Schistosoma haematobium, Schisto- soma japonicum, Schistosomatium douthitti, Fas- ciola hepatica (a hermaphroditic trematode), and male and female BALB/c mice, WI hybridized onh' to the female S. mansoni DNA, even at low stringency (data not shown).

W1 as a probe for determining the sex of cercar- iae. Approximately 800 snails were subjected to

1 GRRTTCGTTC 51 RTGTCTTCGC

1~1 CRTGRTGRCT 151 CRRTGRGRRT 201 TCCTTGTGRC 251 GRTGGTGTTC 3g l RTTRGGGTGT 351 TGTGCRCRTG 491 TTRRCCRTGC 451 GRRCRTTGRG

RRCRCROTOR RRTTCTTCCT RRTRTTTTOG RGTGRRRTTT GRTGTGACRG ORRTGRGGRT TGTGRRTCGG RTGTGCAGRT RCRRRGGRGT GGTGRTGCCR RCACGTGGRT TGRRTRRGCG GTGGTTGTGC TGGRCCRRTG GRCCRCCRCR RRTRRCRCRC BTTOCTTTCT CRTCRRCRCC TTGRATGTCG RGTGGTGRRT

TCRCRCRTRT CTRCCRTCCR GCTTTTCTCR TTRTRTTGTG TRTGTTGRTR TCGTSTGRGT GRGRGGTTGT GCRTPCTT3T GTTCGAGTGT TTGTGGRTGC ATGRRCRRRT GCGATGRTGC TGCRTRRTGG RRTCGTTGCT TCRRTTCRTR CTCCGTCCA- RCAGTTTGSA TTATCRTTTC TC

Fig. 2. DNA sequence of W1. The EcoRl sites delining the repeat unit arc underlined. Wl '.'.'as scqucnccd by the d~deoxynu- cleotide chain termination method 112] using Sequenase (t)nitctl States Biochemical) [13].

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g e n o m e e q u i v a l e n t s

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42 ° 60 °

1

2

3

4

5

6

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9.

10

Fig. 3. Slots (1-10) Serial dilutions of S. manson i fcnmlc gcn- omic I)NA and pW1 hybridized to radioactivcly labeled WI [9]. Each slot also contains 1 gg salmon sperm DNA. Slot (a) l0 ~ gcnomc equivalents of male S. manson i DNA. Slot (h) 1 Ixg salmon sperm I)NA. Slot (c) I(I ~' copies of plasmid vector. In the gcnomic lanes, 42"C reprcscnts the low stringcncy wash. 6{F(" the high stringency wash (both in ILl x SSC and (|.1'; SI)S for 90 rain). Hybridization in the plasrnid lane was iden- tical under both wash conditions. Nvtran liltcrs (Schlcichcr and

Schucll) ,,,,'ere exposed for 15 h at room temperaturc.

monomiracidial infection. Of these, 370 (45%) were shedding cercariae by 9 weeks; the rest were presumed to be uninfected. The sex of cercariae generated by the monomiracidial infections was determined by dot-blotting total DNA isolated from each clonal population of cercariae and probing with radioactively labeled WI (Fig. 4). Under stringent washing conditions, the I )NA from male cercariae gave no signal, while the DNA from female cereariae hybridized strongly to W I, producing a signal in less than 1 h.

Predictions of sex were verified by infecting mice and checking the sex of the adult worms seven weeks later. Of the 370 infected snails ana- lyzed, all predictions were accurate. 181 had fe- male infections, and 189 had male infections. It is worth noting that our results concerning the sex ratio of the miracidial infections of the snails con- flict with those of l,iberatos [14], who reported a significant male bias in the ratio of snail infec- tions done under similar conditions using similar strains.

The entire procedure, from shedding the in- fected snails to developing the exposed X-ray fihn, can be done in 9-10 h by reducing the hybridi- zation time to 2 h. However, we prefer to do the hybridization overnight and sort the snails the following day. The markings used tO identify in- dividual snails last for approximately a week.

Discussion

W1 is a 476-bp EcoRl restriction fragment of S. mansoni genomie DNA which, at the level of

1 2 3 4 5

6 7 8 9 10

Fig. 4. Dot blot of DNA isolated from tile cercariae shed by snails infected with a single S. manson i miracidium and hy- bridized to radioactively labeled WI (1 h exposure). Snails 1. 2, 5, 8. 9 and 10 have female infections; 3, 4. 6 and 7 have

male infections.

Southern blot analysis, can only be detected in females. The hybridization pattern in Fig. la is suggestive of tandemly repeated arrays of se- quence homologous to Wl . There is also evi- dence of Wl-homologous sequence present on EcoRl restriction fragments of greater than 20 kb.

The S. mansoni genome is estimated to be 2.7 × 10 s bp [15]; at 1000 copies per genome. WI- homologous I )NA accounts for approximately 0.2% of the female genome. The repeat unit is probably not transcribed; Northern analysis shows no transcription in the adult stage of the life cycle.

Most eukaryotic genomes contain some tan- demly organized, highly repeated sequences. The sex chromosomes of many species arc enriched with repetitive sequences: the Y or the W chro- mosome in the hctcrogamctic sex is often pre- dominantly hetcrochromatic [16]. The high copy number and female-specificity indicate that WI is likely to be part of the constitutive heterochro- matin of the W chromosome of S. mansoni.

Tone ct al. [17] suggest that there are at least two types of repetitive sequences associated with W and Y chromosomes: (1) scqucnccs which arc relatively enriched in the sex chromosome and widely distributed arnong many species of ani- mals: and (2) sequences which arc highly local- ized to the sex chromosome and found within a limited taxonomic group. The first class is exem- plified by a satellite fraction of DNA from Bun- garus fasciams (snakc family Colubridac) con- raining sequences which hybridize along the length of the snake W chromosome [18]. This satellite fraction also hybridizes to Drosophila, bird, mouse and human DNA. Epplen et al. [19] have described the isolation and cloning of a satellite sequence, pe t s5 , from another Colubrid snake species, Elaphe radiata, which shows homology with chicken, mouse, and human DNA. I)ata from in situ hybridization experiments using mouse metaphase chromosome spreads suggests that the pErs5-homologous sequence is enriched 13-fold in the mousc Y chromosome compared to the mouse autosomes.

The second class of repetitive sequence is ex- emplified by two W-chromosome specific repeats which have been isolated from domestic chicken [17,20]. These sequences, approximately (I.7 and 1.1 kb in length, are estimated to ¢~cur 14000 and

221

60(10 times, respectively, per female genome. Together they comprise about 46c/c of the W chromosomal DNA of Gallus gallus domesticus. The repeats are reported to cross-hybridize with DNA from several other species within the same genus, but not to any other closely related gen- era.

Within these classifications, WI falls into the second category of sex chromosome-related re- peats. It is specific to the female, and therefore likely to be localized exclusively to the W chro- mosome. It does not hybridize to DNA from mice or from five other members of the other Dige- nea, including another member of the family Schistosomatidac and three other members of the genus Schistosoma.

Several methods have bccn published previ- ously for sexing S. mansoni cercariac. Libcratos and Short described a cytological method involv- ing C-banding interphasc nuclei to detect the het- erochromatic region of the W chromosome [21]. Spotila et al. [22] have reported on a DNA probe which recognizes a restriction-fragment length polymorphism (RFLP) on a gcnomic Southern of EcoRl-restricted female and male S. mansoni DNA. Identification of sex is based on the pres- ence of an extra band of hybridization in the fe- male digest. The sequence of this probe is not homologous with that of W1.

In our hands, the dot-blot assay is more accu- rate than the cytological method described above. Dot-blotting allows the analysis of small and rel- atively impure preparations of DNA and is more rapid than using the RFLP marker , which is re- ported to take 3 to 6 days.

The approach used in cloning W1 should bc applicable for cloning repeated fragments of the W or the Y chromosomes in the hetcrogametic sex of other species. Mileham ct al. [23] recently re- ported using a similar approach to clone a male- specific porcine DNA sequence. WI itself may prove useful as a starting point for cloning other sequences on the S. mansoni W chromosome.

Addendum. After this paper was submitted, Walker et al. [24] published a description of a fe- male-specific S. mansoni D N A fragment which is approximately 4[)(I bp in length and present in 75 copies per female genome. We have been unable to verify whether this fragment is the same as WI.

222

Acknowledgements

The authors wish to thank J. Bautista, R. Fen- hetty, J. Mansour and L. Trzesniewski for expert technical assistance: Dr. P. Basch and N. Basch for providing animals infected with S. mattheei and S. douthitti; Dr. J. Donelson and Dr. G. Newport for providing S. japonicum genomic DNA: Dr. J. Bruce and the World Health Or- ganization for providing lyophilized S. haemato-

References

l Menzel. M.Y. and Short, R.B. (1960) Pachytene chro- mosomes in three species of sehistosomes: sex ,rod auto- somal bivalents in males and females. J. Hered. 51 .2-12 .

2 Short. R.B. and Grossman, A.I. (19811 Conventional giemsa and C-banded karyotypes of Schisto.soma mansotti and S. rodhaini. J. Parasitol. 67. 661-671.

3 Williams. S.A. . DeSimone, S.M. and McRcynolds, I,.A. (19881 Species-specific oligonucleotide probes for the identification of human filarial parasites. Mol. Biochem. Parasitol. 28, 163-170.

4 Kukla. B.A. , Majiwa. P .A.O. . Young. J.R. and Moloo, S . K and ole-MoiYoi. O. (19871 Use of species-specific DNA probes for the detection and identilication of trv- panosome infection in tsetse flies. Parasitology 95. 1-.16.

5 Barker Jr.. R. I t . , Suebsaeng, 1,.. Rooney. W., Alecrim. G.C. , [)ourado. ILV. and Wirth. D.F. (19861 Specific I )NA probe for the diagnosis of Plusmodium falciparum malaria. Science 231, 1434-1436.

6 Scholield, C.J. (19871 DNA probes in the field. Parasitol. Today 3. 371-383.

7 Maniatis, "I.. Fritsch, E.F. and Sambrook. J. (19821 Mo- lecular Cloning. A Laboratory Manual. Cold Spring Har- bor Laboratory, Cold Spring Harbor, NY.

8 Thayer , R.E. (19791 An improved method for detecting foreign DNA in plasmids of l-scherichia coli. Anal. Biochem. 98, 60 63.

9 Fcinberg. A.P. and Vogelstein. B. (19831 A technique for radiolabcling DNA restriction endonuclease fragments to high specitic activity. Anal. Bioehem. 132, ~'~13.

Ill Reed. K C . and Mann. D.A. (19851 Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13. 7207-7221.

11 Duvall, R.I1. and DeWitt , W.B. (1967~ An improved per- fusion technique for recovering adult sehistosomes from laboratory animals. Am. J. Trop. Med. t lyg. 16. 483-486.

12 Sanger. F.. Nicklen, S. and Cc, ulson. A.R. (19771 DNA sequencing with chain terminating inhibitors. Proc. Nat[. Acad. Sci. USA 74. 5463-5467.

13 Tabor, S. and Richardson. C.C. (19871 DNA sequence analysis with a modified bacteriophage T7 DNA polymcr- ase. Proe. Natl. Acad. Sci. USA 84, 4767-4771.

biurn worms; Dr. M. lltzsch for providing clone SG1; and Dr. J. Mulligan for helpful discussions. Computer resources were provided by BIO- NET TM, whose funding is provided by NIH Grant 1 U41 RR-01685. This work was supported by grants from the John D. and Catherine T. MacArthur Foundation and USPHS Grant AI 16501 to T . E . M . P . W . is a MacArthur Founda- tion Predoctoral Fellow.

14 l,iberatos, J.D. (1987) Schistosoma mansoni: male-hiascd sex ratios in snails and mice. Exp. Parasitol. 64, 165-177.

15 Simpson. A.J . ( ; . , Sher. A. and McCutchan, T.F. (19821 The genomc of Schi.stosoma mansoni: isolation of DNA. its size, bases and rcpetitive sequences. Mol. B iochem Parasitol. 6, 125-137.

16 Ohno. S. (1%71 Sex Chromosomes and Sex-l,inkcd Genes, Springcr-Vcrlag, Berlin, Heidelberg, New York.

17 Tone, M.. Sakaki, Y.. Itashiguchi, T. and Mizuno, S. (19841 Genus specificity and extensive mcthylation of the W chromosome-specific repetitive DNA sequcnccs from the domcstic ford, Gallu.~ gallus domesticus. Chromosonaa 89, 228-237.

18 Singh. 1,., Purdom. I.F. and Jones, K.W. (198(I) Con- served sex-chromosomc-associated nueleotidc sequences in eukap.otcs. Cold Spring t larbor Symp. Quant. Biol. 45. 8I}5-814.

19 Epplen, J.T., McCarrey. J .R. . Sutou, S. and Ohno. S. (1982) Base sequence of a cloned snake W-chromosome DNA fragment and idcntilication of a malc-speeific puta- tive m R N A in the mouse. F'roe. Natl. Acad. Sei. USA 79, 3798-38O2.

20 Kodama, H.. Saitoh. !1., Tone, M.. Kuhara, S.. Sakaki, Y. and Mizuno, S. (1987) Nucleotide sequences and un- usual clcetrophoretic behavior of the W chromosome-spe- cific rcpeating DNA units of the domestic fowl. Gallu.s gallus domesticus. Chromosoma 96, 18-25.

21 l,iberatos, J.D. and Short, R.B. (19831 Identification of sex of schistosome larval stages. J. Parasitol. 69, 1084-1(189.

22 Spotila, L.D., Rekosh, D.M., Boucher. J.M. and Lo Verde, P.T. (1987) A cloned DNA f, robe identilies the sex of 5chi,stosoma mansoni cercariae. Mol. Biochem. Paras- itoI. 26, 17-20.

23 Mileham, A.J. . Siggcns, K W . and Plastow, G.S. (19881 Isolation of a porcine male specific DNA sequence. Nu- cleic Acids Res. 16. 11842.

24 Walker, T K . , Rollinson. D. and Simpson, A.J .G. (19891 A I)NA probe from Schistosoma mansoni allows rapid de- termination of the sex of larval parasites. Mol. B iochem Parasitol. 33.93-100.