Molecular and Biochemical Parasitology, 56 (1992) 189-196 189 1992 Elsevier Science Publishers B.V. All fights reserved. / 0166-6851/92/$05.00
Cloning and characterization of a Loa loa-specific repetitive DNA
Thomas G. Egwang, Paul M. A juh and Jean-Paul Akue 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
Loa loa is a human filarial parasite which afflicts some 13 million individuals in West and Central Africa . Infection is transmitted by the intermediate anthropod tabanid vector of the genus Chrysops the distribution of which is restricted within the equatorial rain forest zone of Africa . 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 . Although early observations suggested that human and simian strains are transmitted by different species of Chrysops , 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 TM data base with the accession number M91591.
. 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 human 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 . 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 from the eyes of patients who presented with an adult worm migrating beneath the conjunctiva as already described . Live worms were rinsed in phosphate- buffered saline and stored in liquid nitrogen
until used for DNA extraction. Microfilariae were purified from the peripheral blood of microfilaremic subjects by a previously descri- bed technic .
Isolation of DNA. DNA from L. loa adult worms was extracted using mixed alkyltrime- thylammonium bromide as previously descr- ibed . DNA from Chrysops was extracted according to the protocol of Jacobs-Lorena et al. . 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 precipita- tion . 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 . 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 4C. 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 . After autoradiogra- phy, 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
15C 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 2SSC ( lxSSC=150 mM NaC1/15 mM sodium citrate), air dried, and baked in vacuo at 80C for 30 min. The prehybridization and hybridization was carried out at 65C in 1 mM EDTA, pH 8.0/500 mM NaHzPO4, pH 7.2/7% SDS. The membranes were washed at 65C 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 mem- brane 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 num- ber 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 . 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 densitomet- ric 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 per- formed using the dideoxychain termination method of Sanger et al.  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',
was custom synthesized by Ozyme. The PCR was performed with the Gene Amp TM DNA amplification kit (Perkin Elmer Cetus, Nor- walk, CT) according to the manufacturer's conditions using a Perkin Elmer Cetus DNA Thermal Cycler.
Isolation of 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,
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 auto- radiography, the probe was stripped off and the same membrane was rehybridized with the
0 1 o4
- J e r . j
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 (50C at 1 SSC) (data not shown). As a DNA probe, the lowest amount
~ 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
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
- - > ~ ~ n," "r-
r 1 I -
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.
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 (fir...