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Molecular and Biochemical Parasitology, 56 (1992) 189-196 189 © 1992 Elsevier Science Publishers B.V. All fights reserved. / 0166-6851/92/$05.00

MOLBIO 01835

Cloning and characterization of a Loa loa-specific repetitive DNA

T h o m a s G. E g w a n g , Pau l M. A j u h a n d J e a n - P a u l A k u e

International Center for Medical Research of Franceville ( CIRMF), Franceville, Gabon

(Received 21 April 1992; accepted 13 July 1992)

A Loa loa EcoRI genomic library in 2gtl 1 was screened with 32p-labeled L. loa DNA and 1 repetitive clone, LL20, was isolated. An 800-bp Rsa I fragment of LL20, which is L. loa specific, was subcloned into pUC19 and the recombinant plasmid was designated pRsa4. While the 3.8-kb Eco RI fragment of LL20 cross-hybridized to other filarial DNA under low stringency conditions, the 800-bp fragment of pRsa4 was L. loa specific under the same conditions. Further characterization of the insert of pRsa4 was therefore carried out. Its lower limit of detection is 800 pg of L. loa genomic DNA, it has a low copy number (50-100) and an interspersed distribution in the genome. As a probe it does not distinguish between simian and human L. loa DNA. The nucleotide sequence contains 69% A + T and 31% G + C and shows no notable internal repeats.

Key words: Loa loa; Repetitive DNA; Species-specific probe

Introduction

Loa loa is a human filarial parasite which afflicts some 13 million individuals in West and Central Africa [1]. Infection is transmitted by the intermediate an thropod tabanid vector of the genus Chrysops the distribution of which is restricted within the equatorial rain forest zone of Africa [2]. In the same endemic zone it is not unusual to find 2 strains of L. loa: a human strain which shows a diurnal periodicity and a simian strain with a nocturnal periodicity [3]. Although early observations suggested that human and simian strains are transmitted by different species of Chrysops [3], it remains unclear whether the simian L. loa reservoir constitutes a zoonotic risk to human subjects

Correspondence address: Thomas G. Egwang, CIRMF, BP 769, Franceville, Gabon.

Abbreviations: EDTA, disodium ethylenediaminetetra-acetate; SDS, sodium dodecyl sulphate; SSC, standard saline citrate.

Note." Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M91591.

[4]. The existence of biological variants of L. loa from different geographical regions giving rise to different clinical manifestations still remains a controversial issue [5,6]. Diagnostic tools which can differentiate between simian and h u m a n L. loa on the one hand and between different geographical isolates of human L. loa on the other remain to be developed. In this regard, repetitive DNA probes are clearly the best tools for taxonomic purposes since in Onchocerca volvulus, another filarial parasite, they can distinguish between strains of the same species [7]. In this paper we describe a L. loa-specific, low copy, and interspersed repetitive DNA present in both simian and human L. loa.

Materials and Methods

Parasites. L. loa adult worms were obtained by surgical removal f rom the eyes of patients who presented with an adult worm migrating beneath the conjunctiva as already described [8]. Live worms were rinsed in phosphate- buffered saline and stored in liquid nitrogen

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until used for DNA extraction. Microfilariae were purified from the peripheral blood of microfilaremic subjects by a previously described technic [9].

Isolation of DNA. DNA from L. loa adult worms was extracted using mixed alkyltrime- thylammonium bromide as previously described [10]. DNA from Chrysops was extracted according to the protocol of Jacobs-Lorena et al. [11]. DNA from L. loa microfilariae and from human lymphocytes was extracted by a standard technic employing SDS/EDTA and proteinase K digestion, followed by phenol/ chloroform extractions and ethanol precipitation [12]. DNA from all sources were then resuspended in 10 mM Tris-HC1, 1 mM EDTA, pH 8.0. Purified DNA from Litoso- moides carinii, Dipetalonema viteae, Dirofilaria immitis, Onchocerca volvulus, O. gibsoni, O. cervicalis, and Brugia malayi was kindly provided by Dr. Larry McReynolds (New England Biolabs, Beverly, MA). DNA from Caenorhabditis elegans N2 strain was provided by Dr. Thomas Barnes (MRC Molecular Biology Laboratory, Cambridge, UK).

Construction and screening of a genornic DNA library. A genomic library of Eco RI-cut L. loa DNA fragments in the bacteriophage )tgt 11 was constructed as described [13]. The library was amplified once in Escherichia coli strain Y1088 and stored in 100 mM NaCI, 10 mM MgSO4, 50 mM Tris-HC1, pH 7.5, and 0.01% gelatin over chloroform at 4°C. The amplified library was screened with [~-32p]dCTP-labeled total L. loa DNA under high stringency hybridization and wash conditions according to Benton and Davis [14]. After autoradiography, strongly hybridizing clones were identified and subjected to three rounds of plaque purification. One recombinant phage clone, LL20, consistently gave a strong signal. The 3.8-kilobase (kb) insert of LL20 was released by Eco RI digestion, purified from a 1% agarose gel in 40 mM Tris-acetate/l mM EDTA using Geneclean (B10 101 Inc., La Jolla, CA), and ligated to Eco RI-treated and phosphatased pBR322 using T4 DNA ligase at

15°C overnight. The ligation mixture was used to transform competent E. coli HB101 cells and transformants carrying the recombinant plasmid was identified by colony hybridization with 32p-labeled L. loa DNA. This plasmid was designated pLL20. An 800-base pair (bp) RsaI fragment of pLL20 was subcloned into the SmaI site of pUC19 and transformants of E. coli DH5~ cells were identified by mini-plasmid analysis. The recombinant plasmid containing the 800-bp insert, which could be released by double digestion with KpnI and BamHI, was designated pRsa4.

Southern and dot blots. Southern and dot blots to characterize the 800-bp insert of pRsa4 and the 3.8-kb insert of pLL20 were carried out using Zetaprobe nylon membranes (Bio Rad Laboratories, Richmond, CA) according to the manufacturer's instructions. Capillary transfer of enzyme restricted genomic L. loa DNA from agarose to Zetaprobe was in 0.4 M NaOH. For dot blots, samples were boiled for 10 rain in 0.4 M NaOH/10 mM EDTA and filtered through the nylon membrane using a manifold filtration apparatus (Bio Rad La- boratories). The membranes were rinsed in 2×SSC ( l x S S C = 1 5 0 mM NaC1/15 mM sodium citrate), air dried, and baked in vacuo at 80°C for 30 min. The prehybridization and hybridization was carried out at 65°C in 1 mM EDTA, pH 8.0/500 mM NaHzPO4, pH 7.2/7% SDS. The membranes were washed at 65°C 2 × for 30 min each in 1 mM EDTA, 40 mM NaHzPO4 and 5% SDS and another 2 x for 30 min each in 1 ×SSC and 1% SDS. The membranes were then exposed for 2-48 h before autoradiography. The nylon membrane was stripped of the previous radioactive probe by boiling twice for 15 min in 0.1 × SSC/ 1% SDS and rehybridized with another probe.

Radiolabeling of DNA. DNA was radiola- 32 beled with [~- P]dCTP (3000 Ci mo1-1"

Amersham International, UK) by random priming using an oligolabeling kit from Pharmacia (Uppsala, Sweden). The internal primer C was 3' end-labeled with terminal deoxynucleotidyl transferase using a kit from

Boehringer-Mannheim according to the sup- plier's instructions. Unincorporated radioac- tivity was removed using Sephadex G 50 columns.

Copy number determination. The copy number of the cloned 800-bp genomic RsaI fragment was determined by a quantitative Southern-blot analysis as described [15,16]. Serially diluted samples of pRsa4 DNA were mixed with 1/~g of salmon sperm carrier DNA and digested with KpnI and BamHI. The amounts of pRsa4 used in this titration experiment corresponded to 12.5-800 mol of a single copy gene found in 1 #g of L. loa genomic DNA, assuming a haploid genomic size of 8 × 107 bp as reported for Brugia malayi [17]. Two dilutions of L. loa genomic DNA (1.0 and 0.25 #g) were digested to completion with RsaI. Both plasmid and genomic DNA were run on a 1.5% agarose gel in 40 mM Tris- acetate, 1 mM EDTA overnight at 30 V. After electrophoresis, the DNA was transfered to Zetaprobe membrane and hybridized with 32p_ labeled 800-bp insert of pRsa4. Following autoradiography, the bands in plasmid and genomic DNA were compared by densitometric scanning.

Nucleotide sequence analysis. The 800-bp KpnI/Bam HI insert of pRsa4 was subcloned into both pBluescript KS and SK vectors and sent to Ozyme (Paris, France) for custom sequencing. Sequencing reactions were performed using the dideoxychain termination method of Sanger et al. [18] with T7 DNA polymerase. The sequence was confirmed on both strands by one of us (P.M.A.) by making deletion clones of the insert in pUC19 and sequencing by the Sanger method using M13 forward and reverse sequencing primers and T7 DNA polymerase (Pharmacia). The following polymerase chain reaction (PCR) primers were custom synthesized by New England Biolabs: (A) 5'-GGATCCCCACCGAATA AATAAC- TGGAGTGACTTCT-3', (B) 5'-CGGTACC- CACTTACGCTATCATAAGACCCTGTT-3'. The internal probe C, 5'-CATTGTCGTCTA- TTGCGAATTGTCATCTAGCCTGGGG-3',

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was custom synthesized by Ozyme. The PCR was performed with the Gene Amp T M DNA amplification kit (Perkin Elmer Cetus, Nor- walk, CT) according to the manufacturer's conditions using a Perkin Elmer Cetus DNA Thermal Cycler.

Results

Isolation o f a repeated DNA clone. A L. loa EcoRI genomic 2gtll library was screened with [32p]-labeled L. loa DNA under sub- optimal probe concentrations. The rationale for this approach was that only clones carrying repeated sequences would be selected since they would produce stronger signals than single copy genes under these conditions. One recombinant phage consistently produced strong hybridization signals during 3 rounds of plaque purification. This contained a L. loa insert of 3.8 kb and was designated LL20. The insert of LL20 was subcloned into the EcoRI site of pBR322, yielding the recombinant plasmid pLL20.

Preliminary characterization of the 3.8-kb EcoRI insert demonstrated that it cross- hybridized with DNA from various filarial species (see below), thus prompting us to look for smaller fragments of the cloned DNA which might be more species specific. L. loa genomic DNA was cut to completion with RsaI, TaqI and HhaI and the Southern blot of this restricted DNA was probed with [32p]. labeled pLL20. Two RsaI fragments of 1100 and 800 bp showed the strongest reactivity, suggesting that L. loa repeated sequences were found in these fragments (data not shown). When pLL20 was cut to completion with RsaI, several L. loa-specific fragments were evident with the largest being 1100 and 800 bp respectively (data not shown). We therefore subcloned the 800-bp fragment in pUC19 on the premise that it may contain L. loa-specific repeat sequences. The recombinant plasmid was designated pRsa4.

Specificity and sensitivity. For assessment of the specificity of the 800-bp insert of pRsa4,

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DNA of L. loa and various filarial species as well as human and Chrysops DNA were dotted onto Zetaprobe and probed with 32p-labeled 800-bp insert. After hybridization and autoradiography, the probe was stripped off and the same membrane was rehybridized with the

0 ¢1 o4

- J e r . j

n

HLL

SLL

OV

OG

OC

DI

DV

LC

BM

HU

CH

pRsa4

Fig. 1. Specificity of pRsa4. Approximately 50 ng of human and simian L. loa DNA (HLL and SLL, respectively), 100 ng of DNA from other filarial parasites, 1 #g of human (HU) and Chrysops silacea (CH) DNA and 1 ng of pRsa4 as a positive control, were processed and filtered through a Zetaprobe membrane as described under Materials and Methods. The membrane was hybridized with the labeled purified insert of pRsa4. Exposure was for 6 h. OV, O. volvulus; OG, O. gibsoni; OC, O. cervicalis; DI, D. irnrnitis;

DV, D. viteae; LC, L. earinii; BM, B. malayi.

3.8-kb EcoRI insert of pLL20. As can be seen from Fig. 1, the inserts of both pLL20 and pRsa 4 do not hybridize with host and vector DNA. However, while the larger insert hybridized to DNA of other filarial species, notably O. volvulus and L. carinii, the 800-bp insert hybridized only to L. loa DNA from both simian and human strains. Both inserts, as expected, hybridized strongly with the positive control plasmid pRsa4. Longer ex- posures of the autoradiograph confirmed that the 800-bp insert hybridized only to L. loa DNA whereas the larger insert hybridized to DNA from all filarial species tested when the membrane was subjected to a more relaxed stringency wash (50°C at 1 × SSC) (data not shown). As a DNA probe, the lowest amount

2 0 0 ng

1 0 0

5 0

25

12.5

6.3

0 3.1

1.6

~ 0 . 8

i! 0 . 4

Fig. 2. Sensitivity of pRsa4. Doubling dilutions of L. loa genomic DNA in TE containing 200 ng #1 - ~ salmon sperm carrier DNA were filtered through a Zetaprobe membrane and hybridized with the labeled purified insert of pRsa4.

Numbers indicate the amount of DNA in ng.

of L. loa genomic DNA detected by the 800 bp insert was 800 pg after 12 h exposure (Fig. 2).

Copy number and sequence organisation. A quantitative Southern blot of L. loa genomic DNA cut with RsaI and various amounts of pRsa4 cut with BamHI and KpnI was probed with labeled purified insert (Fig. 3). The results suggested that the copy number of the 800-bp Rsa I fragment in the genome was about 50- 100. This was in agreement with the results of dot blot analysis and densitometric scanning (data not shown). Thus the 800-bp sequence represents about 0.05q).1% of the haploid

- I > Z 0 :31> ::U

0

625

400

200

100

50

25

12.5

193

genome of L. loa. The organisation of the 800-bp sequence in

the genome was investigated by cutting L. loa genomic DNA with various enzymes and then probing the Southern blots of the restricted DNA with purified labeled insert (Fig. 4). Complete digestion with RsaI produced a single 800-bp fragment as expected, whereas incomplete digestion with the same enzyme

k h

2

- - > ¢ ~ ~ n," ¢ "r-

5 . 6 m

4.4--

2.3-- 2.0--

r 1 I -

0.25

1.1-

0 . 6 -

Fig. 3. Copy number determination by quantitative Southern-blot analysis. The numbers above the standards represent molar equivalents of pRsa4 relating to the number of moles of a single copy gene found in 1 #g of L. loa DNA. The numbers above LL indicate ng of L. loa genomic DNA. The total amounts of pRsa 4 (3503 bp) loaded in each track, 0.55, 1.1, 2.2, 4.4, 8.8, 17.5 and 27.4 ng, correspond to 12.5, 25, 50, 100, 200, 400 and 625 times, respectively, the number of moles of a single copy gene found in 1/tg of genomic DNA. This calculation assumes a haploid genomic content of 8 x 107 bp as reported for B.

malayi [17].

Fig. 4 Southern-blot analysis of L. loa DNA (500 ng) digested with various restriction enzymes and probed with labeled purified pRsa4 insert. A 1.5% agarose gel of L. loa DNA digested either completely (first lane) or partially for 15 and 30 min (second and third lanes, respectively) with Rsa I (R); lanes RI, RV and H represent DNA digested

with Eco RI, Eco RV and Hpa I, respectively.

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produced the 800-bp fragment as well as a ladder of unevenly spaced higher molecular weight fragments (Fig. 4, lanes marked R). EcoRI produced, in addition to the 3.8-kb fragment corresponding to the cloned insert of pLL20, several fragments ranging from 1.6 to 6.4 kb (lane RI). EcoRV produced a doublet at 6.6 and 6.4 kb (lane RV) and HpaI produced several fragments ranging from 3 kb to 6.6 kb. The fact that these three 6-base cutters produce restriction fragments of about the same size (4.0-kb fragments for HpaI and EcoRI, and the doublet around 6.6 kb for all 3 enzymes) suggests that there is a higher order repeat unit

A 1 ~GATCCCCAC CGAATAAATA ACTGGAGTGA CTTCTTGTCT AGGAATATTT

[ . . . . . . . . . . Primer A . . . . . . . i 51 TCCAATCTAA TTTCACTTCC ACATGCTTCT TATTTACTTT ATTTGCACGC

i01 ATAATGAGTC TGTGAAGAAA ACCCTTCGCT ATCCTCATGA AAAAGCATTT

151 ACATGTAGAT CAAATTCTCG AAATTGCAAC GGAGACAACG AGTTTCACTT

201 CGATTGAAAC AAGCAGCATA TAAAAGTGTC ATTGTCGTCT ATTGCGAATT Primer C

251 GTCATCTAGC CTGGGGTTTA TTAATGGTTT GCTAAATGGT TATAAATATC

301 GTTGGATGAC TACAGCAGTG ATCAATTTGA TGACGTTGTT GTGTAAATAC

351 GTTGTCCCTC GTTATCCCTT CTGACTGTCG AACAAACATA AATATGCTAT

401 TCTGCCATTC TGATTATACC GTTAAAGGAG AAAATCAGAG AATCATTTAC

451 ATAAACCTAT TATTACTATT ATTATTAAAC CAAATATTTT GATATTCCAA

501 AAAGACTAAC AATAACGATG AAAGTTGAAT TTATAACTTC GGTATGTTAA

551 GTCAAAAGTA AACAACCTTT AGAAGCAATT TAATGTTAGA TATCGAGTTA

601 TAATTATATT GGAATAGATT GTTCAAATAT TGATAAGATC AAACAAAGAT

651 TATCACTGCA AATTAATCAA ATATACAGTT GATTTTTCAT TTTCGGAACT

701 TTTTAATGAA TAATGCCAGA AAGCAAGAGT TCACTGGAAT TGATTTCAAG

751 ATTACTCAGT TAACTTACTA ATAATCAAAT GATCTATAAT TAGAATGATG

801 AACAGGGTCT TATGATAGCG TAAGTGGGTA CCG

I . . . . . . . . Primer B . . . . . . . . . I

8

__.l ii O kb

I :

I . . . .

O8 kb

L i

100 bp

Fig. 5. (A) The nucleotide sequence of the insert of pRsa 4 shown 5' to 3'. The positions of the PCR primers A and B and the internal probe C are indicated. Vector sequences at the 5' and 3' ends are underscored; these sequences contain the BamHI and KpnI recognition sites respectively. (B) A partial restriction map of the insert. Note that in the pUC 19 vector (dashed lines), the 5' end Rsa I site has been lost.

of the 800-bp DNA sequence.

DNA sequence analysis of the pRsa 4 insert. The DNA sequence of the insert is 817 bp in length (Fig. 5). It is A + T rich (69% AT, 31% GC) and contains no open reading frame, nor significant internal repeats. The DNA sequence confirmed the presence of restriction sites for all the enzymes used in characterizing the insert. The enzyme MseI (which recognises the sequence TTAA) cuts 8 x whereas TspE I (which recognises the sequence AATT) cuts 11 times. There are no sites for HhaI, AvaI, or Cvi JI. There is no similarity to any known sequences in the GenBank Genetic Sequence Bank (Release 64, Los Alamos National Laboratory, Los Alamos, NM) nor in the EMBL Data Library (Heidelberg, Germany). After obtaining the nucleotide sequence,

S C H HLL

SLL BH MP Ol DV LC OC 01] OV

CE

Fig. 6. Confirmation of the specificity of pRsa 4 insert using the polymerase chain reaction (PCR). (Top) An ethidium bromide-stained 1.5% agarose gel electrophoresis of the PCR products of various genomic DNA. S, ~bX174 HaeIII molecular weight markers; C, Chrysops; H, human; and MP, Mansonella perstans. The rest of the abbreviations are as explained in Fig. 1. (Bottom) An autoradiograph of a Southern transfer of the same gel onto a Zetaprobe membrane and probed with 3' end-labeled primer C. The

autoradiograph was exposed for 30 min at -70°C.

oligonucleotide primers defining the 5' and 3' ends of the insert were synthesized and the species specificity of the cloned DNA was confirmed by PCR using genomic DNA from various filarial parasites, the human and Chrysops hosts, and Caenorhabditis elegans strain N2 as templates. A specific band of the expected size was observed only in reactions containing human and simian L. loa (Fig. 6). The internal probe hybridized only to these bands.

Discussion

We have cloned and characterized a repetitive 817-bp sequence of human L. loa which appears to be specific to the human and simian strains of L. loa. This repetitive sequence has a low copy number, is interspersed within the genome, and, significantly, does not cross- hybridize with DNA from either man or Chrysops, the arthropod intermediate host. The cloned sequence hybridized with DNA from the simian strain of L. loa under very stringent conditions. This suggests that the same sequences are found in both human and simian strains. Cloning and sequencing of the simian L. loa homologue may reveal subtle strain differences which may be exploited for the development of strain-specific oligonucleotide probes. These probes would be invaluable for distinguishing between human and simian strains of L. loa microfilariae or infective larvae in the Chrysops vectors. Studies along these lines have been initiated. Recently Klion et al. described another L. loa-specific repetitive DNA of similar size [19]. However, apart from the fact that both share an interspersed distribution in the genome, the 2 repetitive DNA are quite distinct from one another with regards to copy number, sensitivity, and restriction enzyme sites.

The cloned DNA detected as little as 800 pg of L. loa DNA. This limit of detection suggests that the cloned sequence as a diagnostic probe may miss detecting a single microfilaria whose DNA content is about 700 pg [20]. The low level of sensitivity of this cloned DNA

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compared with the high sensitivity of repetitive sequences reported for L. loa and other filarial species [19,21-23] is not surprising, given its low copy number in the genome. However, the fact that the cloned DNA is L. loa-specific suggests that it could be exploited for the development of a diagnostic DNA probe. In this regard, the amplification power of the polymerase chain reaction will undoubt- edly enhance the sensitivity to the point where a single microfilaria may be detected in blood samples and a single infective larva may be detected in Chrysops vectors. Investigations along these lines are currently on going.

Repetitive DNA reported in filarial parasites currently fall into 2 major categories. In the first category are repetitive DNA organised in tandem arrays and characterized by a small unit size and high copy number. These repetitive DNA have been reported in B. malayi and B. pahangi [17], O. volvulus [21,24] and O. armillata [23]. In the second category are repetitive DNA with an interspersed distribution in the genome and characterized by a large unit size and moderate to high copy number. These repeats have been described in O. volvulus [24], IV. bancrofti [22] and L. loa [19]. The L. loa repetitive DNA reported in this paper represents yet another type of filarial interspersed repeat characterized by a large unit size and low copy number. It remains to be established whether tandem repeats such as those reported for Brugia and Onchocerca spp. exist in L. loa.

Acknowledgements

We are grateful to Dr. Margaret Pinder for her encouragement and support and for supplying Loa loa and Mansonella perstans microfilariae and Chrysops flies used for the DNA studies. The technical assistance of Ibrahim Nguiandoungou and Viviane Ngon- di-Bayinda is gratefully acknowledged. CIRMF is 70% supported by the State of Gabon and 30% by Elf Gabon.

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