Characterization of a ribosomal DNA clone of Brugia malayi

Molecular and Biochemical Parasitology, 19 (1986) 67-75 Elsevier

67

MBP 00651

Characterization of a ribosomal D N A clone of

Brugia malayi

Jyotsna S. Shah, Louis Lamontagne, Thomas R. Unnasch, Dyann F. Wirth and Willy F. Piessens Department of Tropical Public Health, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115, U.S.A. (Received 8 July 1985; accepted 16 November 1985)

The structure of ribosomal DNA (rDNA) clone pBmr7 from microfilariae of the human parasite Brugia malayi has been examined in detail by Southern blot analysis and S-1 mapping techniques. The results demonstrate that this clone contains regions homologous to 28S, 18S and 5.8S rDNAs. A noncoding or 'spacer' region lies between the 3' end of 28S rDNA and the 5' end of 18S rDNA. An AccI-Sau3AI fragment of approximately 900 bp from this spacer region cross-hybridizesto genomic DNA fragments of different sizes from Brugia pahangi and Dirofilaria immitis. The differences observed in hybridization suggest that this rDNA fragment can be used to differentiate between various fllariid species. Key words: Brugia malayi; Filariasis; rDNA; S-1 mapping; Spacer regions; Sequence homology

Introduction Three species of lymph-dwelling filarial parasites c o m m o n l y infect humans: Brugia malayi, Brugia timori and Wuchereria bancrofti. They have a complex life cycle, which includes an obligatory maturation stage in h e m a t o p h a g o u s mosquitoes and a reproductive stage in the m a m malian host. M a n y mosquito vectors that transmit these parasites feed not only on humans, but also on a variety of domestic and wild animals which can be infected with other filarial species. In the genus Schistosoma, it is possible to differentiate species and strains by Southern blot analysis of restriction endonuclease digests of genomic parasite D N A , using cloned D N A fragments of S. mansoni ribosomal genes as probes [1]. M a r k e d differences in non-coding regions of ribosomal genes have also been found in other closely related organisms [2,3]. This suggested that it might be possible to use a D N A fragment from a spacer region of ribosomal genes as a probe to

Abbreviations: bp, base pairs; kb, kilobase pairs; NET buffer, sodium chloride, EDTA, Tris buffer; SCC, sodium chloride, sodium citrate solution; SDS, sodium dodecyl sulfate.

differentiate between various species of filarial helminths. To test this possibility, we characterized a cloned ribosomal gene from B. malayi, isolated a fragment from the spacer region of the r D N A , and examined whether this r D N A fragment could be used to differentiate B. malayi from other filariids. Materials and Methods

Parasites. Microfilariae of B. malayi were obtained from jirds injected intraperitoneally with 100-200 infective larvae 5-6 months before use. B. pahangi microfilariae were obtained from the peritoneal cavity of C 3 H / H e N nu/nu mice infected with this species (a kind gift from Dr. A. Vickery, University of South Florida, T a m p a , FL). Dirofilaria immitis microfilariae (a generous gift from Dr. A. Scott, John Hopkins University, Baltimore, M D ) , were isolated from blood of infected dogs. The microfilariae were freed from contaminated cells by density centrifugation on Ficoll 400 [4]. Purification o f D N A . D N A was p u r i f e d from microfilariae as follows: 2-5 x 106 parasites were suspended in 4.5 ml of N E T buffer (150 mM NaCI,

0166-6851/86/$03.50 (~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)

68 5 mM E D T A , 50 mM Tris-C1, p H 7.5). The parasites were sonicated on ice at 400 W with 6 pulses of 45 s to rupture the microfilarial sheath. Sarcosyl and proteinase K (Boehringer-Mannhelm Biochemicals, Indianapolis, IN) were added to a final concentration of 1% and 100 I~g m1-1, respectively, and the mixture was incubated at 37°C for 60 min. Ribonuclease A (Sigma Chemical, St. Louis, MO) was then added to a final concentration of 100 Izg ml 1 and the mixture was further incubated for 30 min at 37°C. The solution was sequentially extracted with an equal volume of phenol, a 1:1 (v/v) mixture of phenol and chloroform and an equal volume of chloroform. The supernatant fluid was dialyzed extensively at 4°C against 3 x 2 1 of 10 mM Tris-HC1, 1 mM E D T A , pH 8.0. D N A concentration was determined by absorbance at 260 nm. Approximately 1 mg D N A was obtained from 2-3 x 106 microfilariae.

Preparation of total RNA from B. malayi microfilariae. Gradient purified microfilariae (3-5 × 106) were suspended in 10 ml of 0.1% diethylpyrocarbonate-treated N E T buffer containing 1% sarcosyl. The suspension was sonicated with 4 pulses of 45 s at 400 W to break the microfilarial sheath. The solution was sequentially extracted once with 16 ml of a 1:1 (v/v) mixture of phenol and chloroform, once with 12 ml of 1:1 (v/v) phenol/chloroform and once with 10 ml of chloroform. The supernatant was brought to 0.5 M sodium acetate and precipitated with 2.5 volumes of ethanol. R N A was dissolved in 200-300 ~1 of 10 mM Tris, 1 mM E D T A , p H 8.0 and stored at - 7 0 ° C until used. Because these R N A preparations contain small amounts of genomic D N A , DNA-free total R N A to be used for some experiments was prepared by the guanidinium/cesium chloride method

[51.

cut out and electroeluted into dialysis bags filled with R N A running buffer (50 mM boric acid, 5 mM sodium borate and 10 mM sodium sulphate) [6].

Southern blot analysis. Nuclear D N A (2 I~g) or D N A from plasmid pBmr7 containing ribosmal genes was digested with 5-fold excess of various restriction enzymes and the resulting D N A fragments were separated by electrophoresis on agarose gels [5]. The D N A was partially depurinated by soaking the gel for 15-20 min in 0.1 M HCI, denatured and cleaved by soaking the gel for 30 min in 0.5 M N a O H , 1.5 M NaCI and finally neutralized by soaking for 45-60 min in 3.0 M Tris, pH 8.5. Denatured D N A was transferred to nitrocellulose filters (0.45 txm pore size, Schleicher and Schuell, Keene, NH) as described by Southern [7] and baked for 2 h in vacuo at 70°C. Hybridization was carried out with either nick-translated D N A probes [8] or with purified total RNA, 18S or 28S RNA, labelled at the 5' end with polynucleotide kinase [9]. After hybridization, the filters were washed 3 times in 0.1 x SSC (150 mM NaCI, 15 mM sodium citrate), 0.1% sodium dodecyl sulfate (SDS) for 30 min at 50°C, dried and exposed to Kodak XAR-5 film at - 7 0 ° C with or without intensifying screens.

Mapping ofplasmid pBmr7. Plasmid pBmr7 is a clone of pBR322 which contains a fragment of r D N A of B. malayi microfilariae inserted at the SphI site of pBR322 (manuscript in preparation). Plasmid D N A was prepared from cultures of this clone stored at -70°C. The plasmid was digested with restriction endonuclease SphI; the inserted D N A was separated from vector D N A by agarose gel electrophoresis and eluted from the agarose [6]. Restriction mapping of the inserted DNA was by single or double digestion procedures.

Purification of 28S and 18S RNA. Five i~g of total R N A from the phenol/chloroform preparation was electrophoresed on a 1% agarose gel containing 5 mM methyl mercuric hydroxide [5]. Following electrophoresis, the gel was stained for 30 min with 2 txg m1-1 ethidium bromide in 0.5 M ammonium acetate. Slices of agarose containing bands corresponding to 28S and 18S R N A were

End-filling of DNA. Plasmid D N A (20 I~g) was digested with restriction endonuclease EcoRI and end-filled at the 3' end using the Klenow fragment of polymerase I and [~x-32p]ATP [5]. S-1 mapping. S-1 mapping was performed essentially as described by Landfear and Wirth [10].

69

Approximately 200 ng of labelled or unlabelled plasmid DNA digested with appropriate restriction endonucleases was dissolved in 30 ixl of 80% formamide, 0.4 mM NaC1, 40 mM Pipes (pH 6.4), 1 mM EDTA. For each digestion, three reactions were set up: one containing DNA only, one containing DNA and S-1 nuclease, and one containing DNA, 2 ixg of total RNA and S-1 nuclease. DNA-RNA hybrids were formed by incubating at 55°C for 3~ h. 300 Ixl of an ice-cold solution of 30 mM sodium acetate (pH 4.6), 250 mM NaC1, 1 mM ZnSO4, 5% glycerol and 20 p.g ml-1 denatured Escherichia coli DNA was then added and the mixture was cooled on ice for 5 min. Hybrids were incubated with or without 10 units of S-1 nuclease (Bethesda Research Laboratories, Bethesda, MD) at 37°C. After 45 min at 37°C, the reaction mixture was precipitated with ethanol. For reactions using unlabelled DNA, the pellet was dissolved in 20 Ixl; of 50 mM NaOH, 1 mM EDTA, 0.025% Ficoll, 0.025% bromocresol green, and electrophoresed on a 1% alkaline agarose gel in 30 mM NaOH, 1 mM EDTA. The gel was neutralized in 1 M Tris (pH 7.6), 1.5 mM NaC1, and the DNA was blotted onto nitrocellulose. Hybridizations were performed for Southern blots. For reactions using end-labelled DNA, the pellet was dissolved in 3 Ixl H20 and 6 p.l of deionized formamide containing 0.3% xylene cyanol FF, 0.3% bromophenol blue and 0.37% EDTA, heated to 95°C for 3 min, and loaded onto an 8 M urea - 5% acrylamide gel [11]. After electrophoresis at 35 mA, the gel was dried and exposed to Kodak XAR-5 film.

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Results

Restriction mapping of cloned ribosomal gene of B. malayi. As an initial step in the study of rDNA of B. malayi, a restriction map of gel-purified rDNA fragment from plasmid pBmr7 was constructed (Fig. 1). The cloned rDNA fragment was digested with 20 restriction endonucleases in single or double digests. Restriction endonucleases BglI, HindlII, NdeI, SmaI, and XbaI had only one site, AccI, EcoRI, HinclI and PvuI had two sites, and AluI, HpalI, HaelII, Sau3AI and RsaI had many sites in the rDNA fragment (not shown in the map). Avail, BamHI, PstI, and PvulI recognized no sites in the fragment. Comparison of restriction sites in B. malayi genomic rDNA and cloned rDNA fragment. Restric-

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Fig. 1. Restriction map of cloned rDNA fragment of B. rnalayi. Enzyme sites: A (AccI), B (BglI), Bs (BstEII), E (EcoRI), H (HindlII), Hc (HinclI), N (NdeI), P (PvuI), S (Sau3AI), Sm (SmaI), Sp (SphI). The hatched bars under the restriction map indicate regions that are homologous to mature rRNA. Each bar is also labelled with the type of rRNA with which it is homologous. The direction of transcription is indicated by the arrow.

Fig. 2. Comparison of genomic rDNA and cloned rDNA. Parallel restriction digests of genomic DNA (G) and plasmid pBmr7 DNA (P) were run on a 1% agarose gel. The DNA was transferred to nitrocellulose and probed with radiolabelled purified pBmr7 insert DNA. The insert DNA was prepared by digestion of pBmr7 with SphI. Restriction enzymes used: 1, SphI; 2, SphI and EcoRI; 3, SphI and HinclI; and 4, SphI and Accl. M = molecular weight markers from HindlII digest of k and HaelII digest of ¢X174.

70

tion digest patterns of genomic rDNA and cloned rDNA of B. malayi were compared by Southern blot analysis using labelled pBmr7 plasmid as a probe (Fig. 2). Genomic rDNA and the plasmid pBmr7 were digested with SphI alone or with Sphl plus another restriction endonuclease, so that a comparison of restriction sites could be made, since clone pBmr7 was made from a SphI digest of genomic DNA. Restriction endonucleases EcoRI and AccI cut the cloned rDNA twice, resulting in fragments of 2.1 kilobase pairs (kb), 2.4 kb, 3.3 kb and 1.7 kb, 2.45 kb and 3.7 kb, respectively. Extra fragments of, respectively, 3.3 and 3.8 kb are present in EcoRI-SphI and AccISphl digests of genomic DNA. These results suggest that probably more than one class of ribosomal genes exists in the genome of the B. malayi. Restriction endonucleases EcoRI and AccI alone cut the genomic rDNA twice, resulting in fragments of 3.3 kb, 4.5 kb and 2.45 kb, 5.4 kb, respectively (results not shown). This suggests that the ribosomal genes are tandemly repeated.

Separation of coding regions into 18S and 28S domains. The approximate sizes of the ribosomal RNA (rRNA) of B. malayi microfilariae were de-

Fig. 3. The denaturing gel pattern of total microfilarial Total R N A (Rt) was isolated and electrophoresed scribed in Materials and Methods. Shown is a picture ethidium bromide stained gel. M = molecular weight ers as in Fig. 2.

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termined by running total RNA on 1% agarose gels under denaturating conditions. Two bands of 4.3 and 1.9 kb, representing 28S and 18S rRNA were seen (Fig. 3). These two bands were gel purified by electro-elution, labelled with [-¢3ZP]ATP and polynucleotide kinase and used separately to probe two identical Southern blots. Results of the Southern blot probed with 28S rRNA are shown in Fig. 4. SphI-AccI digestion fragments of 3.8 and 1.6 kb (lane 2) hybridize to 28S rRNA. The 3.8 kb fragment hybridizes very strongly, suggesting that most of the 28S rRNA coding region spans from the Sph! to the AccI site at the 5' end and that a smaller coding region is present at the at 3' end of the SphI-Acc| region of the clone (lane 3). The SphI-EcoRI fragment of 2.4 kb also hybridizes very strongly, which confirms these results. An AccI-AccI fragment of 2.45 kb does not hybridize to 28S rRNA, but hybridizes very strongly to total RNA (Fig. 7) and to 18S rRNA (not shown). Since the size of the 18S RNA is 1.9 kb, the 18S rRNA coding region

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Fig. 4. Identification of the coding regions homologous to purified 28S rRNA. (A) Ethidium bromide staining pattern of restriction digests of plasmid pBmr7 separated on 1% agarose gel; (B) the same gel transferred onto nitrocellulose and probed with radioactive 28S rRNA. Restriction enzymes used: 1, SphI; 2, SphI and Accl; 3, Sphl and EcoRI; 4, SphI and HinclI. M = molecular weight markers as in Fig. 2.

71

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SphI fragments (Fig. 4) whereas 18S rRNA hybridizes only to the 2.45 kb AccI fragment (results not shown). Thus, it can be concluded that the 28S rRNA coding regions span up to 2.9 and 1.4 kb, respectively, from the SphI sites (Fig. 1). The exact position of the 18S coding region and the direction of transcription was investigated by S-1 mapping of 3' end labelled fragment. Plasmid pBmr7 was digested with restriction endonuclease EcoRI. end-filled with [cx-32p]ATP and Klenow fragment of DNA polymerase I. digested with restriction endonuclease AccI and the AccIEcoRI fragments were gel purified. AccI-EcoRI fragments were hybridized to total RNA and digested with S-1 nuclease. The protected frag-

M Fig. 5. Location of 28S and 18S rDNA as determined by S-1 mapping. Plasmid pBmr7 AccI 2.45 kb fragment (lane 1) or pBmr7 digested with SphI (lane 2) was denatured, hybridized with total RNA (2 txg), and incubated with S-1 nuclease. The digests were then separated on a 1% alkaline agarose gel, blotted onto nitrocellulose and hybridized with nick-translated pBmr7 as described in Materials and Methods. The molecular weight markers, indicated in kilobase pairs are as in Fig. 2.

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S-1 mapping. The boundaries of the coding regions of rDNA of B. malayi microfilariae were determined more accurately by S-1 mapping. Initial mapping was performed with plasmid pBmr7 digested with restriction endonuclease SphI and with the gel-purified AccI digestion fragment of 2.45 kb. The SphI fragment (representing the whole rDNA clone) and the AccI fragment were hybridized to total R N A and treated with S-1 nuclease. Fragments that were protected from digestion with S-1 nuclease were separated on alkaline agarose gels. The protected DNA fragments were blotted onto nitrocellulose and the blots were hybridized with nick-translated plasmid pBmr7. The results are shown in Fig. 5. Three fragments, 2.9, 1.9 and 1.4 kb in length, were protected from the 7.8 kb SphI fragment (lane 2). A fragment of 1.9 kb is also protected from S-1 digestion from the AccI fragment of 2.45 kb (lane 1). 28S rRNA hybridizes to both the AccI and

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Fig. 6. Location of boundaries of 18S and 28S rDNA by S-1 mapping with labelled DNA fragments. S-1 mapping was performed with 3' end-labelled probes Accl-EcoRI (1.4 kb fragment) (lanes 1-3) or Accl-EcoRI (550 bp fragment) (lanes 4-6). Lanes 1 and 4, DNA only; lanes 2 and 5, DNA and S1 nuclease; lanes 3 and 6, DNA, RNA and S-1 nuclease. After digestion with S-1 nuclease, hybrids were resolved on a 4% acrylamide-8 M urea gel. Molecular weight markers (M) indicated in bases are from a 5' end-labelled HaelII digest of ~bX174.

72 ments were sized on urea-acrylamide gels. As shown in Fig. 6, a fragment of 525 base pairs (bp) is protected from the approximately 1400 bp long AccI-EcoRI fragment and 200 bp is protected from the 550 bp AccI-EcoRI fragment. These results suggest that the 3' end of the 18S and 28S r D N A are 525 and 200 bp from the EcoRI sites, respectively (see Fig. 1).

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5.8S RNA coding region. Results obtained by others indicate that 5.8S R N A sequences are highly conserved between different organisms [12]. This property was used to locate 5.8S R N A of filariae, Total R N A was separated on 8% acrylamide urea gel, electroblotted onto Zeta-Probe membrane (0.45 txm pore size, BioRad, Richmond, CA) according to the manufacturer's specifcations and probed with a nick-translated EcoRI-XbaI fragment from a Drosophila r D N A clone pDml03-A1 (a gift from Dr. D. Peattie, Stanford University, CA) that contains 5.8S RNA sequences [13]. The same blot was washed and

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Fig. 8. Comparison of restriction maps of B. malayi, B. pahangi and D. immitis genomic rDNA. Enzyme sites are labelled as follows:A (AccI), E (EcoRI), S (Sau3AI), Sp (SphI).

reprobed with the SphI-EcoRI fragment from the 3' end of pBmr7. As is shown in Fig. 7, a band of about 250 bp hybridizes strongly to both the EcoRI-Xbal fragment of clone pDml03-A1 and the SphI-EcoRI fragment of pBmr7. These results suggest that the 5.8S R N A sequences are present between the 3' end of 18S and 5' end of 28S rDNA. It is of interest that the size of 5.8S R N A from B. malayi microfilariae is about 250 bp, instead of 150-160 bp found in most organisms. When total R N A prepared by the phenol/chloroform method was run on acrylamide gel and probed as above, an extra band of 150 bp was seen (results not shown). Thus, it is possible that this R N A fragment of 250 bp is a precursor of 5.8S RNA.

Comparison of restriction endonuclease patterns of B. malayi, B. pahangi and D. immitis rDNA. Restriction maps of genomic r D N A of B. malayi, B. pahangi, and D. immitis, were constructed by Southern blot analysis of restriction endonuclease patterns of the genomic DNAs with pBmr7 as a probe. Genomic DNAs were digested with SphI alone or with SphI plus another restriction endonuclease, blotted onto nitrocellulose and probed with nick-translated pBmr7. The size of the SphI fragment of r D N A is slightly different in the three species of filarial worms (Fig. 8). B. malayi and B. pahangi r D N A have the same restriction endonuclease patterns, whereas D. immitis r D N A has different AccI and Sau3AI sites. The AccI-Sau3AI fragment that spans the spacer region is about 100 bp smaller in B. pahangi than in B. malayi (Fig. 8).

73

Is the spacer region of ribosomal genes of B. malayi different from that of other filarial species? Spacer region sequences differ widely in size and base composition, whereas coding sequences of r D N A are highly conserved between different species and organisms [2,3]. The studies reported so far indicated that a spacer region was present in clone p B m r 7 between the 5' end of 28S and 3' end of 18S. This was confirmed by digesting plasmid p B m r 7 with different restriction endonucleases, blotting the fragments onto nitrocellulose paper and probing the blots with kinased total R N A . Restriction endonuclease digestion patterns of plasmid pBmr7 are shown in Fig. 9A. The X b a I - P v u I fragment of 1.7 kb (lane 3) which contains some sequences of 18S and 28S r D N A hybridizes much less strongly than other fragments to total R N A . An A c c I - S a u 3 A I fragment of approximately 900 bp between the 3' end of 28S and 5' end of 18S (Fig. 1) does not hybridize to total R N A (lane 2, Fig. 9B). This fragment was gel

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Fig. 9. Determination of the spacer region of rDNA. (A) Ethidium bromide staining of restriction digests of plasmid pBmr7 resolved on 1.35% agarose gel; (B) hybridization pattern of the Southern blot prepared from the same gel probed with radioactive total rRNA. Restriction enzymes used: 1, AccI; 2, AccI-Sau3AI; and 3, XbaI-PvuI. M--molecular markers as in Fig. 2.

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Fig. 10. Homology between spacer regions from different filarial species. (A) Hybridization pattern of the Southern blots of AccI-Sau3AI digests of B. rnalayi, B. pahangi, D. immitis genomic DNA probed with AccI-Sau3AI fragment. (B) Hybridization of the same Southern blot with clone pBmr7. Genomic DNA used: m, B. rnalayi; p, B. pahangi; d, D. immitis. M = molecular weight markers as in Fig. 2. purified and used to probe genomic D N A from microfilariae of B. malayi, B. pahangi and D. immitis and genomic D N A from mosquitoes (Aedes aegyptii). Filarial D N A , digested with restriction endonuclease AccI and Sau3AI, was blotted onto nitrocellulose and probed with nick-translated AccI-Sau3A1 fragment. Genomic D N A of B. malayi, B. pahangi and D. immitis all hybridized to the AccI-Sau3AI fragment (Fig. 10A). An identical hybridization pattern was observed even under the most stringent washing conditions (3 times, 30 min washes at 65°C with 0.1 x SSC and 0.5% SDS). When the same blot was washed and hybridized to plasmid pBmrT, the restriction enzyme patterns for B. malayi and B. pahangi r D N A were similar. However, D. immitis r D N A has some different AccI and Sau3AI sites (Fig. 10B). Mosquito D N A and human D N A do hybridize very slightly to total plasmid pBmr7, and not at all to the AccI-Sau3AI fragment (results not shown).

74 Discussion

Differentiation of species and strains of the genus Schistosoma has been achieved by Southern blot analysis of genomic parasite D N A digested with different restriction endonucleases, using cloned D N A segments of S. mansoni ribosomal genes as probes [1]. With the aim of isolating a r D N A fragment that could be used to differentiate filarial species, we characterized in detail the ribosomal genes of B. malayi. When E c o R l and AccI digests of genomic B. malayi D N A were compared to restriction digests of cloned r D N A (Fig. 2), an extra restriction fragment was observed in genomic D N A digests. These results suggest that more than one class of ribosomal genes may be present in B. malayi. In another parasitic nematode, Ascaris lumbricoides, 95% of ribosomal genes also are organized in two main classes of 8.8 and 8.4 kb [141. In the present study we made use of the fact that mature R N A does not have spacer regions and, therefore, could be used to identify spacer regions in the cloned r D N A fragment. It is well established that ribosomal genes of eukaryotes are divided into three classes: 28S, 18S and 5.8S. S-1 mapping shows that the 28S r D N A of B. malayi is cut into two fragments of 2.9 and 1.4 kb by restriction endonuclease SphI, whereas the 18S rDNA gene remains intact (Fig. 4). In the genome, the ribosomal genes appear to be tandemly repeated. From detailed S-1 mapping using endfilled E c o R I fragments the exact position of the 18S and 28S coding regions were determined to within 50 bp. The coding regions for 28S R N A spans from the SphI site (5' end) to 525 bp past the EcoRI site and extends 1.4 kb from the SphI site at the 3' end. The coding region of 18S R N A spans approximately 300 bp from the AccI site to 200 bp past the EcoRI site (see Fig. 1). In most organisms, the 5.8S R N A coding fragment of 150-160 bp is present between the 3' end of 18S and the 5' end of 28S coding regions and is highly conserved [2,3]. The results we obtained by using a nick-translated EcoRI-XbaI fragment from a Drosphila r D N A clone that contains 5.8S RNA sequences and a SphI-EcoR! fragment from

the 3' end of pBmr7 to probe total R N A confirmed the presence of 5.8S R N A sequences between the 3' end of 18S and the 5' end of 28S coding regions in B. malayi. Measurement of r D N A : R N A hybrids of D. melanogaster by electron microscopy showed that the size of the 5.8S R N A gene is about 230 bp [13], which is greater than the sum of 2S and 5.8S R N A reported by Jorden et al. [15]. It is interesting to note that the size of 5.8S R N A of B. malayi is about 250 bp, rather than 150--160 bp found in most organisms. Possibly this 250 bp R N A fragment is a precursor of 5.8S RNA. There may be a 2S R N A species present in B. malayi, but this possibility was not examined in the present study. Spacer region sequences differ widely in size, base composition and secondary structure, whereas 5.8S, 18S and 28S sequences of r D N A molecules are highly conserved between different species and organisms [2,3]. However, for several closely related Drosophila species [16] and Caenorhabditis elegans [17] the spacers are as homologous as the gene regions. We found that the spacer regions of ribosomal genes from several species of filarial worms ( B. malayi, B. pahangi and D. immitis) are as homologous as the coding regions. Restriction endonuclease sites are highly conserved between B. malayi and B. pahangi rDNAs. The only difference was that the length of the AccI-Sau3AI fragment from the spacer region is slightly smaller in B. pahangi than in B. malayi. An interesting observation was that D. immitis genomic D N A hybridizes more strongly to pBmr7 than B. malayi or B. pahangi genomic D N A (results not shown). This suggests that D. immitis may have more copies of ribosomal genes than B. malayi and B. pahangi. The spacer region from B. malayi did not have sites for any of the restriction endonucleases included in this study. Since the AccI-Sau3AI fragment of 900 bp from the spacer region does not hybridize to mosquito or human genomic DNA, it may be a useful tool to determine the rate of overall infection by filarial worms. In addition, preliminary experiments with microfilariae from B. malayi, treated with SDS show that less than 5 microfilariae can be detected by clone pBmr7.

75

Acknowledgements The authors thank Dr. A. Vickery, University of South Florida, Tampa, for B. pahangi microfilariae, Dr. A. Scott, Johns Hopkins University, Baltimore for D. immitis microfilariae, Dr. B.K.L. Sim for extracting DNA from B. pahangi and D. immitis microfilariae, Dr. D. Peattie, Stanford University, CA for clone pDml03-Al. We acknowledge Drs. C. French and S. Landfear for their helpful suggestions and R. Gonski, P.

Morgan, and D. Condon for typing the manuThis was script. work supported by UNDPiWORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases, NIAID grant A120102; NIH Program Project A122794; the John D. and Catherine T. MacArthur Foundation; the Agency for International Development, DPE-5542-G-SS-4046-00 and the Rockefeller Foundation. D.F.W. is a Burroughs-Wellcome Scholar.

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