Molecular and Biochemical Parasitology, 34 (1989) 261-268 261 Elsevier
MBP 01139
Cloning of a polymorphic DNA fragment from the genome of Leishmania donovani
J o h n El l i s* , T e r e s a K n a p p a n d J u l i a n C r a m p t o n
Wolfson Molecular Genetics Unit, Liverpool School of Tropical Medicine, Liverpool, U.K.
(Received 19 September 1988; accepted 13 December 1988)
Recombinant DNA clones, containing highly repetitive DNA sequences, have been isolated from a Le&hmania donovani gen- omic DNA library prepared in the replacement vector kgt.WES.kB. Two clones, probably telomeric in location, have been char- acterised and show a restriction fragment size polymorphism. Evidence is presented which suggests that L. donovani is diploid for this cloned genomic locus.
Since the introduction of D N A fingerprinting for the determinat ion of human pedigrees [1], the concept of using hypervariable D N A sequences for studies on parasite speciation and in diagnosis has received wide attention. Repeti t ive D N A se- quences in particular have found use in the iden- tification of species and individual strains of a number of pathogenic protozoa [2-4]. In addi- tion, the advent of pulsed field gel electrophore- sis has allowed studies on the karyotypes of many of these organisms [5,6], which hitherto had re- mained undetermined. Linkage analysis has now become feasible with the success of these studies relying on the availability of cloned D N A se- quences as genetic markers. Detailed molecular analysis of cloned loci may provide the necessary
locus-specific probes [7]. In a previous communication, the cloning and
preliminary characterisation of a highly repetitive D N A sequence from the genome of Leishmania
donovani was reported [8]. This sequence was of particular interest for the following reasons. DNA sequence analysis had shown that the repeat se- quence was identical to the major telomeric re- peat of the trypanosomes. Fur thermore, exten- sive hypervariability in its genomic organisation between species and even strains allowed the D N A fingerprinting of Leishmania isolates. In this report , studies describing the cloning and char- acterisation of a large, apparently intact, hyper- variable D N A fragment from the L. donovani genome are described.
Materials and Methods
Correspondence address: J.M. Crampton, Wolfson Molecu- lar Genetics Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, U.K.
*Present address: Department of Parasitology, AFRC Insti- tute for Animal Health, Houghton Laboratory, Houghton, Huntingdon, U.K.
Parasites. Amastigotes of the L. donovani strain LV9 [9] were maintained by routine passage of infected spleen homogenates through cotton rats. The purified amastigotes used in this study were kindly donated by Dr. M.L. Chance. Promasti- gotes were cultured at 26°C in H O M E N medium [10] supplemented with fetal calf serum. All other parasite cell lines used in this study have been previously described [8].
General methods. Genomic DNA was isolated from amastigotes or promastigotes by lysis in SDS and proteinase K [8], followed by banding in ce- sium chloride/ethidium bromide gradients to re- move kDNA. Southern blotting and DNA hybri- disations were performed according to standard procedures [11]. All enzyme manipulations were done according to suppliers' instructions.
Library construction and screening. A genomic DNA library was established from LV9 amasti- gote genomic DNA in the vector ~gt.WES.kB [12]. Mixed partial digests of genomic DNA was ligated into the arms of )tgt.WES.kB prepared as previously described [13], packaged and plated for individual plaques on the Escherichia coli host LE392 [11]. The library was subsequently ampli- fied on tryptone plates, harvested and stored at -20°C.
Individual plaques from the library were screened on nitrocellulose filters using the tech- nique of Benton and Davis [14] and a nick-trans- lated DNA probe. Filters were washed in 2 x SSC 0.1% SDS at 65°C and autoradiographed at -70°C using a preflashed film and an intensifying screen. Plaques showing positive hybridisation to the probe were selected and rescreened to con- firm the probe hybridisation. Single positive plaques were then picked and amplified in 200-ml L-broth cultures on LE392 at 37°C. After lysis, phage were harvested by polyethylene glycol pre- cipitation [15] and banded over a cesium chloride step gradient [16]. Phage DNA was then purified according to standard procedures [16].
Oligonucleotide synthesis. The oligonucleotide 5 '-TTTTTCTCTCCACT(T/G)AT(T/C)CTT-3' was synthesised using the [3-cyanoethylamidate chemistry adapted for use with controlled-pore glass supports [17]. Deprotection and cleavage of the oligonucleotide from the glass support was performed with concentrated ammonia solution. The oligonucleotide was end-labelled using 3,-ATP (Amersham, 5000 Ci, mmol 1) and T4 polynu- cleotide kinase, desalted on Sephadex G-100 (Pharmacia) to remove unincorporated nucleo- tides and low molecular weight failure products from the organic synthesis. Hybridisation of the oligo-probe was performed at 37°C for 2 h in
6xSSC, 10xDenhardt's, 10 ixg/ml -~ yeast tRNA, 100 ixg/ml -~ denatured, sonicated herring sperm DNA. The blot was then briefly washed at room temperature in 2xSSC, 0.1% SDS and autora- diographed at -70°C with a preflashed film and an intensifying screen.
Results
Construction and screening of a genomic DNA li- brary. Amastigote genomic DNA from the L. donovani strain LV9 was digested with the re- striction enzyme EcoRI to either 10, 50 or 90% of a complete digestion and the products pooled and size fractionated on a 10-40% sucrose gra- dient [13]. Partial EcoRI fragments of size 7-14 kb were selected by monitoring the gradient frac- tions by agarose gel electrophoresis. These DNA fragments were ligated into the arms of the vec- tor )tgt.WES.kB [12], packaged and plated for plaques on the E. coli host LE392. The library was amplified on tryptone plates and stored in phage buffer at 4°C. Greater than 90% of the clones isolated from this clone bank were judged to con- tain a Leishmania insert. Furthermore, screening of several thousand clones from this library with a total kinetoplast DNA probe derived from the same strain failed to reveal any clones containing kinetoplast sequences.
In an earlier communication [8] the isolation and preliminary characterisation of a highly re- petitive sequence from the L. donovani genome was described. This sequence was of particular interest since because of its extreme hypervaria- bility in its genome organisation it allowed the DNA fingerprinting of Leishmania species. The recombinant plasmid pRs2A contains a string of this repeating sequence (5'CCCTAA-3') cloned into the plasmid vector pAT153 [8]. In order to further study the organisation of these sequences in the Leishmania genome, genomic DNA clones were sought in the amastigote genomic DNA li- brary.
40000 clones of the amplified library were screened by the procedure of Benton and Davis using a nick-translated pRs2A DNA probe. Ap- proximately 26 clones were identified by low tem- perature autoradiography as successfully hybri- dising to this probe at high stringency (0.1xSSC,
0 . 1 % x S D S , 65°C) in this plaque screen. Details corresponding to two recombinant clones (kRs2A and kRs3A) are presented here. These two clones showed the most intense hybridisation to the probe in the library screen.
Characterisation of genomic clones ARs2A and ARs3A. Phage DNA from recombinants hRs2A and hRs3A was purified from a 200-ml lysed cul- ture (details in Materials and Methods). These 2 clones were subsequently mapped according to their restriction sites for the enzymes XhoI, SalI and BamHI, chosen because of the low fre- quency of the recognition sequence for these en- zymes within the genome. Phage DNA from the host )tgt.WES-)tB was run as a control in sepa- rate digestions to identify h-specific fragments. The partial restriction maps determined for these 2 clones are presented in Fig. 1. The restriction sites for the above mentioned enzymes were con- firmed by gel purification of the insert DNAs from the 2 clones followed by restriction mapping of the fragments using single and double enzyme diges- tions.
Comparisons of the restriction maps of the two recombinants revealed that they were similar
with respect to the position of restriction sites across their entire sequence. However, these 2 clones differed in the size of a SalI-SalI restric- tion fragment adjacent to the EcoRI terminus (labelled Fragment A, Fig. 1). The similarity in their restriction maps implied that these 2 cloned DNA sequences may be homologous variants of a single cloned chromosomal locus. To confirm this assumption, hRs2A was nick-translated and used as a probe onto a blot containing digests of both clones. Hybridisation of this probe occurred to all fragments of both of these clones at high stringency (0 .1xSSC 65°C) confirming that the insert sequences were indeed homologous in these 2 clones. The realisation that hRs2A and hRs3A may be overlapping fragments produced by the preparative partial EcoRI digestions used during cloning, was dismissed due to the absence of any other sites for this enzyme within hRs3A. It was concluded that the clones hRs2A and hRs3A were indeed probably variants of the same cloned lo- cus.
Since the two clones under consideration had
Kb
Rs2A
RsaA
R S S B X S t T • • ~ t
263
SR Y!
~_ FRAGMENT B FRAGMENT A >
0 1 2 3 4 5 6 7 8 9 10 11 12
Fig. 1. Restriction maps of ~.Rs2A and XRs3A. The ~Rs2A (top) and )tRs3A (bottom) recombinant clones were mapped for the following restriction enzyme sites according to the procedures described in Materials and Methods: R, EcoRI; S, SalI: B, BamHI; X, Xhol. The restriction fragments, Frag-
ments A and B, are as described in the text.
been identified by virtue of their hybridisation to the recombinant plasmid pRs2A, it seemed per- tinent to ask to which restriction fragment(s) did the selective hybridisation occur? Southern hy- bridisation of radiolabelled pRs2A to digests of )tRs2A and ~Rs3A revealed that hybridisation occurred only to fragment A of both clones un- der either low or high stringency (2xSSC or 0.1 ×SSC at 65°C, respectively).
In an earlier communication [8] it was reported that a 21-bp repeat sequence was commonly found associated 5' to the repeating unit (CCCTAA)n. It was suggested that this association may be a common feature of the Leishmania genome. One would predict that if this was the case, then Frag- ment A should be expected to contain this se- quence. The presence or absence of the 21-mer was addressed as follows. A 21-mer oligonucleo- tide sequence was synthesised on a Coder 2 au- tomatic DNA synthesiser (Dupont) incorporating the mixed bases as indicated in Materials and Methods. Following purification, the oligonu- cleotide was end-labelled with 32p and further purified as described in Methods. Southern hy- bridisation of this radiolabelled probe in 1 M NaC1 to X clone digestions revealed specific hybridisa- tion to Fragment A only of both clones, confirm- ing the prediction that this sequence would be present (Fig. 2).
264
oo ILl
• 9"5 Kb
• 3"5
: ~::ii i/~ii!il ~̧ ili::~:(~)'!ii
Fig. 2. Oligonucleotide hybridisation. A 32p-labelled 21-mer oligonucleotide conforming to the conserved sequence de- scribed in the text, was hybridised for two hours to restriction digests of hRs2A as described in Materials and Methods. Au- toradiography was overnight at -70°C with a preflashed film
and an intensifying screen.
Investigation of flanking sequences. To examine the structure of sequences flanking FragmentA, Fragment B (Fig. 1) was gel-purified and used as
A B C D E F G H
• 2 3 K b
.~4.0
a probe onto a Southern blot containing HindIII digestions of promastigote genomic DNA iso- lated from 4 different Leishmania species. Hy- bridisation occurred under high stringency to a large number of restriction fragments, the hybri- disation being species specific in its pattern (Fig. 3). Subsequently, this blot was cleared of probe with dilute alkali and then reprobed with radio- labelled pRs2A to identify restriction fragments associated with the CCCTAA repeat. Compari- son of the 2 autoradiographs obtained revealed that fragment B hybridised to the same set of re- striction fragments detected by the pRs2A probe. Furthermore, Fragment B hybridised specifically to a HindIII fragment of 4 kb, to which no hy- bridisation of the pRs2A probe could be de- tected, even at low stringency. It therefore ap- pears that in these Leishmania strains studied, Fragment B sequences are frequently found closely linked to the putative telomeric repeated sequence. There is however, at least one locus which is either more distant from the abundant
A B C
• 2 1 ' 5 K b
• 9"6
Fig. 3. Southern hybridisation using Leishmania genomic DNAs. HindlII-restricted genomic DNAs from L. donovani chagasi strain M4192 (A,E); L. donovani strain DD8 (B,F); L. donovani infantum strain LEM235 (C,G); and Leishmania adleri strain LV36 (D,H) were probed with either a Fragment B probe (A-D) or nick-translated pRs2A DNA (E-H). The filters were washed in 0.1xSSC 0.1% SDS at 65°C and au-
toradiographed overnight.
Fig. 4. Southern blot analysis of L. donovani chagasi DNA. EcoRI-restricted DNA was hybridised to a radiolabelled Fragment B probe or nick-translated pRs2A under low strin- gency. Hybridisation was visualised following these washing conditions: (A) pRs2A, 0.1xSSC, 0.1% SDS, 65°C; (B) Fragment B, 0.1xSSC, 0.1% SDS, 65°C; (C) Fragment B,
2xSSC, 0.1% SDS, 65°C.
265
repeating sequence, or simply found elsewhere in the Leishmania genome. Clearly, this can be eas- ily assessed by determining the sensitivity of this fragment in experiments using BAL 31 exonu- clease digestion of intact genomic DNA.
The nature of Fragment B sequences were studied further in L. donovani chagasi (strain M4192). Fragment B was nick-translated and used as a DNA probe onto a Southern blot of EcoRI- digested promastigote genomic DNA from this species (Fig. 4). Following a low stringency wash- ing (2 × SSC 65°C), extensive hybridisation of this probe to the genomic DNA could be detected, confirming the previously described results. Sub- sequent washing of this same filter under condi- tions of higher stringency (0.1×SSC 65°C) re- duced the overall intensity of hybridisation. However, all the bands visualised under low stringency were still detectable after this latter washing. The main feature of this result was, however, the presence of intense hybridisation to 2 dominant EcoRI restriction fragments of sizes 21.5 and 9.5 kb, the intensity of which was not reduced by an increase in washing stringency. In this strain of L. donovani chagasi, Fragment B sequences therefore appear to be organised into at least 2 major clusters within the genome. This evidence implies that the Leishmania may well be diploid for this cloned genomic locus.
Discussion
The cloning of hypervariable DNA sequences has characteristically been fraught with many problems. Simple repetitive sequences are prone to deletion and or recombination in many of the currently used E. coli cloning systems. For ex- ample, in one study on the basic and expression linked variable surface glycoprotein genes of Try- panosoma, the cloning of basic copy genes was reported. However, it was concluded that the presence of repetitive sequences in the 'uncutta- ble' regions surrounding the telomeres could be the cause of selection against telomeric clones in the cosmid banks [18]. Fragments shorter than 4 kb containing these sequences were also found to interfere with propagation in a RecA E. coli host. It is of interest to note that there are now many examples of the successful cloning of previously
unstable DNA sequences ]19]. In this study, the establishment of a genomic DNA library from amastigotes of L. donovani strain LV9 has been reported. Furthermore, the screening of several thousand clones from this library with a radiola- belled probe for a highly repetitive sequence has allowed the isolation and subsequent character- isation of cloned sequences located at a single hy- pervariable locus in the Leishmania genome.
Restriction mapping of two recombinant clones has shown that the hypervariability demonstrated by these 2 clones can be accounted for by a re- striction fragment size polymorphism. Evidence from hybridisation studies indicates that this frag- ment contains repetitive DNA sequences. Even with the uncertainty as to what is the cause of the polymorphism, the similarity of these 2 recom- binants with the schematic telomeric structure presented for Trypanosoma brucei [21] implies that these DNAs may well represent cloned te- lomeres of the Leishmania.
Telomeric restriction fragments are commonly reported to be variable in length, forming diffuse bands in agarose gels [22]. The variable numbers of repetitive sequences found at these locations is just one explanation for this phenomenon [23]. It is quite probable that the restriction fragment length polymorphisms described in this study may well result from such a difference. Indeed, pre- liminary restriction mapping by the procedure of Smith and Birnsteil (1976) [24] has revealed that changes within the terminal 2 kb adjacent to the EcoRI site appear to be responsible for the po- lymorphism.
The nature of the sequences flanking the highly repetitive sequence has been investigated by Southern blot analysis, and found to be repetitive in nature. The consistent hybridisation of a Frag- ment B probe to the same restriction fragments recognised by a probe containing the CCCTAA repeat implies that there is a strong conservation of sequence organisation at these specialised sites within the Leishmania genome. This is in keeping with the known organisation of telomeres, which generally appear to contain repeats adjacent to the terminal sequences. In some protozoa, these additional sequences are thought to represent origins of replication and/or be actively involved in chromosomal recombination [25,26].
266
Finally, the identification of variant restriction fragments from the same genomic locus implies that L. donovani strain LV9 may well be diploid for this locus. Furthermore, the observation that Fragment B sequences appear to be organised into 2 major clusters within the L. donovani chagasi genome does lend some weight to this argument. However, neither of these strains are derived from a cloned cell line. It is quite possible therefore, that these variants arise from different members of the cell population. Alternatively, it is still fea- sible that these clones arise from different chro-
mosomal sites. Further work is clearly required to elucidate the nature and extent of this chro- mosomal variation within the Leishmania gen- o m e .
Acknowledgements
We thank Dr. M.L. Chance for the supply of Leishmania parasites, Dr. H. Townson for his support of this project, and finally the Wolfson Foundation for their financial assistance.
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