Molecular and Biochemical Parasitology, 43 (1990) 39-50
39
Elsevier MOLBIO 01399
Isolation and characterization of a repetitive DNA element from the genome of the human filarial parasite, Brugia malayi Sundareshwaran Natarajan, Craig Werner, Margaret C a m e r o n and Thiruchandurai V. Rajan Departments of Pathology and Microbiology, University of Connecticut Health Center, Farmington, CT, U.S.A. (Received 26 February 1990; accepted 29 May 1990)
The genome of the human filarial parasite Brugia malayi contains at least two major repetitive DNA elements. One, referred to as the HhaI family, consists of 104-105 tandemly arrayed copies per haploid genome of a monomer of 322 base pairs and does not contain a cleavage site for the restriction endonuclease MboI. We constructed a library of MboI-digested genomic B. malayi DNA in BamHI-cut M13mpl8 resulting in the exclusion of the HhaI repeat family from the library. Hybridization of this genomic library with nick-translated genomic DNA yielded several copies of a repeat family which we have named the BmMboI family. From sequence analysis of more than 50 monomers, which differ from each other in sequence and length, we have been able to divide the monomers into several regions based on the level of sequence conservation. Southern blot analyses of B. malayi genomic DNA digested with a variety of restriction endonucleases and probed with the isolated repeat demonstrate multiple bands of varying sizes except with HindlIl-cut DNA, where the repeat is found only in very high-molecular-weight DNA. Key words: Brugia malayi; Repetitive DNA; Isolation; Characterization
Introduction Human lymphatic filariasis is a major public health problem in a number of countries, with an estimated 100 million individuals afflicted with this disease [1]. Repetitive DNA elements offer novel diagnostic reagents. Attempts to derive probes from the genome of B. malayi for such studies have resulted in the repeated isolation of a repeat family that has been referred to as the HhaI family [3,4]. This tandemly arranged repetitive DNA family, comprising about 10% of the B. malayi genome with a monomer of 322 bp, contains cleavage sites for a number of commonly used restriction endonucleases, so that genomic libraries contain multiple representations of the tanCorrespondence address: T.V. Rajan, University of Connecticut Health Center, 263 Farmington Avenue, L-1006, Farmington, CT 06032, U.S.A.
Abbreviations: mf, microfilariae; kb, kilobases. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank TM data base with the accession number M34369
dem repeat, ranging from a unit to large multiples of the monomer to the size accommodated by the vector. However, the repeat does not have cleavage sites for the restriction endonuclease MboI, so that cleavage of the B. malayi DNA by MboI resuits in the segregation of the family as a fragment of DNA too large to be accommodated by most commonly used vectors. The use of this strategy has allowed us to exclude this tandem repeat family from the library and isolate a novel repetitive member from the B. malayi genome. Materials and Methods
Construction of B. malayi genomic DNA libraries. B. malayi-infected jirds (Meriones unguiculatus) were obtained from the University of Georgia through a U.S./Japan cooperative program in illariasis. Microfilariae (mf) were purified from the peritoneal cavity of these animals as described earlier [4]. Genomic DNA was isolated from mf, subjected to quantitative digestion with the restriction endonuclease MboI, and ligated to BamHIdigested phosphatased Ml3mpl8. From such a li-
0166-6851/90/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
40 TABLE I Results of screening different B. malayi genomic libraries with the BmMboI family repeat fragment Librarya
No. of plaques
Positives
Frequency
EcoRI.l HindlII.I Mbol.A MboI.B EcoRI.II HindlII.II
13000 30 000 20000 20000 40000 40 000
46 0 6 21 78 0
3.5 x 10-3 0 3 x 10-4 1 X 10 - 3 1.95 X 10 - 3 0
aI and II refer to different EcoRI and HindlII libraries; MboI.A and MboI.B refer to different size-fractionated libraries. brary, 5 x 105 recombinant clones were isolated and amplified. All other genomic DNA libraries were constructed in the bacteriophage A-derived vector Charon 27 [5] after cutting the phage with EcoRI, HindlII or BamHI. In the case of EcoRI and HindlII, genomic DNA was cut to completion with the respective enzyme and ligated into phosphatased vector. The BamHI-cut vector was used to construct two different MboI partial libraries - genomic DNA was subjected to partial cleavage with MboI and size fractionated into 4-6-kb and 6-9-kb aliquots, which were then separately cloned into the BamHI-cut phosphatased vector. Isolation of Mbol repeats. B. malayi genomic DNA was labeled by nick translation and hybridized to filters of the MboI M13 library [6]. A number of hybridizing plaques were isolated and plaque-purified. After several rounds of screening, five different clones, BmMbo3, BmMbo4, BmMbo8, B m M b o l 0 , and B m M b o l 1, which consistently hybridized to radiolabeled genomic DNA, were retained for analysis. Subsequently, repeat clones were isolated from the other B. malayi genomic libraries using one of the MboI library repeat members as probe. Southern blot and dot blot analysis. B. malayi genomic DNA was subjected to restriction en-
donuclease cleavage with a variety of enzymes under conditions recommended by the manufacturers. DNA was subjected to electrophoresis in Tris-borate-EDTA (TBE) gels (0.7% for 6-base cutters and 1.1% for 4-base cutters). Following denaturation and renaturation in situ, the DNA was transferred to nitrocellulose filter papers and hybridized to nick-translated or random-primer (Boehringer-Mannheim) extended BmMbo8 or B m M b o l l DNA [7]. To investigate the relatedness of individual worms, single adult male and female worms were recovered from the peritoneal cavities of infected jirds obtained from W. McCall. Each adult worm was processed separately in an Eppendorf centrifuge tube to obtain DNA. The DNA sample from each individual worm was subjected to restriction endonuclease cleavage separately, electrophoresed in a separate lane of a gel and analysed after transfer using radiolabeled repeat probe. Dot blots were performed by spotting serial dilutions of B. malayi genomic DNA on nitrocellulose filter paper as described by Kafatos et al. [8]. DNA sequencing and analysis. Sequences were obtained from the repeat clones by using the chain termination method [9,10]. The sequences were analysed using the University of Wisconsin Genetics Computer Group (UWGCG) Sequence Analysis Software Package.
~a~T~AAT TC A T G CCACTTCTCTACTGATATTACA CCATTTCTCTACAGATTAAACA CCATTTCTCTGCAATCATAACA
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TTTGAT A A A T T C A T T T A C T C A T A TTGCAT TAATTC A T T A A T T C A T A TTCGAT C
Fig. 1. Complete nucleotide sequence (184 bp) of BmMbo8. The MboI recognition sequences (GATC)are underlined.The sequence is arranged to highlight the fact that the unit is composed of three internal repeats of approximately62 bases, as described in the text.
41 Plmll
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Isolation of the Mbol repeat family. When the MboI M 1 3 m p l 8 B. malayi library was probed with labeled B. malayi genomic D N A , a total of 13 plaques hybridized to the D N A and were picked for secondary lifts. Following the secondary and subsequent lifts, a total of five plaque-purified phages continued to hybridize to radioactive ge-
nomic D N A . These five recombinant phages were retained for further analyses. The sizes of the inserts in these recombinant phage particles ranged from 250 bp to 3.5 kb. Six other genomic libraries (two different EcoRI, two different HindlII and two different size-fractionated MboI libraries) were screened with one of the repeat members. We obtained a variable number of hybridizing plaques with the
42
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Fig. 3. Sequence a l i g n m e n t s o f repeat family members from different libraries displayed to highlight the internal structure of the monomers. Sequences 1-7 refer to members from the M13 MboI library, 8-15 refer to sequences from the Charon27 EcoRI library a n d 1 6 - 1 9 indicate members from the Charon27 MboI l i b r a r y . T h e consensus sequence is indicated at the top; a p e r i o d indicates conservation; a letter indicates substitution a n d a gap indicates deletion. The regions within the consensus are indicated at the bottom.
EcoRI and MboI libraries (ranging from 6-78), but we repeatedly failed to find any positive clone from the HindlII libraries with the same radiolabeled repeat probe. These results are illustrated in Table I. DNA sequence analysis. The sequence of the prototypic member BmMboI8 is shown in Fig. 1.
Visual inspection of the sequence revealed certain sequences that were repeated at regular intervals. This is demonstrated more clearly by a diagonal plot comparison. Fig. 2 shows representative output where the X axis represents the sequence of BmMbo8 and the Y axis represents either the sequence of BmMbo8 (Fig. 2A), its reverse complement (Fig. 2B), BmMbol0 (Fig. 2C) or BmMbol 1
44 TABLE II Size and structure of vector-insert junction of full-length repeat clones obtained from different B. malayi genomic libraries No.
Library
Clone
Size (bp)
Junctional sequence
I
M 13.MboI
BM3 Bm4 Bm8 Bml0 Bm I 1
400 700 182 3500 800
R R R R R
It
EcoRI
pBm.A pBM.B pBm.C pBm.D pBm.E pBm.F
III
ch27.MboI
R R R R R
RR R R
R R R R
8000
pBm.Mj3*
2100
R R U R
RR
R R indicates that both ends of the insert fragment consist of repeat sequence; R U indicates that one end of the insert fragment is repeat and the other is unique; *, due to the fact that there were internal (RI or Hill) sites in the other 5 clones, we were able to analyze only two genomic ends. Clones pBm.A, pBm.B, pBm.C, pBm.D and pBm.F range in size from 2-7 kb. (Fig. 2D). Internally repeated sequences produce satellite diagonals offset from the central diagonal by an a m o u n t proportional to the spacing o f the repeat. This is seen in the plot of B m M b o 8 versus itself, where, in addition to the central diagonal, two satellite diagonals can be discerned. It is worth noting that c o m p a r i s o n s o f B m M b o 8 versus the reverse c o m p l e m e n t fails to reveal a diagonal line, indicating that there are no inverted repeats in this sequence (Fig. 2B). Further scrutiny of these sequences revealed that each of our clones is in fact c o m p o s e d o f multiple m o n o m e r s . Each m o n o m e r can be further divided into seven regions as shown in Fig. 3 based on the level o f sequence conservation a m o n g the 50 or so m o n o m e r s that we have characterized, arbitrarily defining the CC doublet as the 5' end o f a m o n o m e r : a 5-bp ' A ' region, a 5-bp ' B ' region, a 4-bp ' C ' region, a 31-bp ' D ' region, a 4-bp ' E ' region, a 4-bp ' F ' region and a 9-bp ' G ' region. A histogram illustrating the pattern o f conservation o f the consensus nucleotides is shown in Fig. 4. Regions B, D and F are highly conserved while regions A, C, E and G are less so, with region B being the most conserved and region G the least. Regions G and A are the most variable in length due to the occurrence o f frequent deletions giving rise to variation in the length o f individual
m o n o m e r s ; for instance, region G consists o f a single nucleotide in 24.6% o f m o n o m e r s while in 49% it consists o f its full c o m p l e m e n t o f 9 nucleotides. Region D consists o f a 31-bp stretch
120
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100
80
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0
60"
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20
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40
Consensus Positions(I-62)
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IlllJF
Fig. 4. Histogram showing the degree of conservation of the consensus nucleotides among the monomers. Over 50 monomers have been sequenced and the degree of conservation at each position is shown graphically as a percentage. Contiguous nucleotides of similar conservation can be grouped into regions, as indicated by the horizontal bars above the histogram.
45
tO
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60 •
Conservation
• Substitution I~1 Deletion. • Insertion 40
20
A
B
C
D
E
F
G
Regions of the consensus Fig. 5. Histogram showing the differential patterns of conservation, substitution, deletion and insertion among the regions of the consensus. Each region differs significantly in conservation from its neighbours. Regions A and D show a high percentage of deletions while region C shows a high frequency of substitutions.
of highly conserved nucleotides including an A in position 25 which is completely conserved among all monomers sequenced. These data are graphically indicated in Fig. 5. To determine whether the MboI repeat family had the potential to encode a protein, the translation of the repeats in all three reading frames and in both orientations were obtained using the MAP program of UWGCG; these analyses (not shown) revealed that there is no significant open reading frame in any of the six reading frames.
Southern blot analysis. To visualize the organization of the repeat family in the genome, Southern blot analyses were performed using a variety
of enzymes and radiolabeled BmMbo8 DNA (Fig. 6). A large number of bands, in excess of a number that could be readily counted, were visualized with most enzymes. Notable was the absence of the ladder like array that is seen when Southern blots are performed with a typical tandem repeat. To investigate whether there were individual differences amongst individual B. malayi worms, Southern blot analyses were performed on DNA from individual adult (male and female) B. malayi cut with PvulI, since this enzyme produced the most distinctive restriction pattern. When such Southern blots were subjected to hybridization with radiolabeled BmMbol 1 DNA, the data revealed that the worms were essentially indistin-
46
7
8
9
10
11
!i!i I
I 2 3 4 5 6 7 8 9
101112
B
Fig. 6. Southern blot analyses of B. malavi DNA (lanes 1-5) cut with a variety of restriction endonucIeases and other filarial DNAs cut with Pvull and hybridized to radiolabeled repeat probe (A) or to a cloned lYagmentof a collagen gene from B. malayi (B). The enzymes used in lanes I-5 (all B. malavi genomic DNA) are: lane 1, EcoRl; lane 2, Hindlli; lane 3, Pvull; lane 4, SspI', lane 5, Xmnl. The worms used in lanes 7-10 are (all cut with Pvull): lane 7, Acanthocheilonema vitiae; lane 8. Brugia pahangi; lane 9, Litmosoides carinii; lane 10, Setaria sp. Lane 11 is jird DNA(control) and lane 5 is the A Hindlll marker. guishable from each other (Fig. 7). Estimation o f copy number. To d e t e r m i n e the n u m b e r of copies of the repeat family in the g e n o m e , B. malayi D N A was spotted onto nitrocellulose paper in d o u b l i n g dilutions in duplicate,
starting at 600 rig/spot. O n e of the two duplicate sets was h y b r i d i z e d to a single copy g e n o m i c D N A ( a m i n o acids 1 4 9 5 - 1 5 6 0 o f the filarial body wall m y o s i n h e a v y chain) [ 12] and the other set to B m M b o 8 . The results of the dot blot are s h o w n in Fig. 8. U n d e r similar c o n d i t i o n s of exposure,
47
Fig. 8. Dot blots of B. malayi DNA hybridized either to BmMbo8 repeat or a single copy probe derived from the B. malayi myosin gene. Note that hybridization can be visualized using the repeat up to the lowest amount of DNA spotted onto the filter; in case of the single copy probe, hybridization is not visible beyond the fourth highest dilution.
the conditions of hybridization it recognizes a single copy sequence in the genome under the conditions of the hybridization (Werner et al., manuscript in preparation), and since the specific activity of the two probes was comparable, in excess of 109 cpm (/zg probe) -~, we estimate that the repeat is represented at least 100-200 times per haploid genome equivalent. Fig. 7. Southern blot analyses of individual B. malayi females, obtained from W. McCall, subjected to DNA extraction, restriction endonuclease cleavage with PvulI, electrophoresis and blotting on separate lanes. The molecular weight markers are indicated along the left margin and are HindllI fragments derived from )~ DNA. Note the identity of the bands noted in the individual worms.
myosin hybridizes to the spot corresponding to 75 ng of B. malayi as intensely as the repeat does to the 1.2 ng spot. Since the length of the myosin fragment is the same as that of the repeat probe (approximately 200 bp), since we know that under
Discussion One repetitive DNA family in B. malavi, referred to as the HhaI family constitutes about 8-10% of the genome of B. malayi, is represented very highly in genomic libraries and is repeatedly isolated by the techniques commonly used to isolate repetitive DNA elements [2,3,4,11 ]. Since the HhaI monomer does not contain an MboI cleavage site, the use of this enzyme for genomic cloning has apparently allowed us to excise the tandem family as a piece too large to be accommodated
48 in M 1 3 m p l 8 and thus clone a novel repeat family from the B. malayi genome. Sequence analysis of this novel repeat has revealed an intricate arrangement of seven smaller regions within the larger unit, identified on the basis of their degree of sequence and length polymorphisms. While regions B, C, D, and F are relatively constant in length, regions A and G are very polymorphic due to the occurrence of frequent deletions, giving rise to considerable variation in the length of the monomers. The incidence of substitutions, deletions and insertions in the different regions is shown in Fig. 5. Most of our Southern blots suggest that the B m M b o I family is an interspersed repeat. However, we have conducted Southern blot analyses using HindlIl on numerous occasions, and have repeatedly failed to observe any fragment containing the repeat that is smaller than the exclusion limit of the agarose gel. An example is Fig. 6A, lane 2. Possible explanations for this observation are (a) the lane with the HindlII-digested D N A did not contain adequate amounts of DNA, or (b) the enzyme failed to cleave the DNA. To address these two possibilities we reprobed the blot shown in Fig. 6A with a fragment of a collagen gene derived from the B. malayi genome. It will be noted that this reprobing (Fig. 6B) highlights a fragment in lane 2 (B. malayi genomic D N A digested with HindlII) whose size is consistent with that expected from known sequence of this gene (manuscript submitted), and that the intensity of this fragment is comparable to that in other lanes. We are unable to come up with an explanation for an interspersed repeat that fails to generate any small fragments containing the repetitive D N A element. These results with Southern blots conducted with HindlII are consistent with the results obtained on screening the HindlII library with the repeat probe (Table I) and the structure of vector-insert junctions of several clones from different libraries (Table II). Repeated screens of the HindlII libraries (Fig. 1) have failed to isolate any m e m b e r of this family; finally, though we have sequenced 12 fulllength insert containing genomic clones (or a total of 24 genomic ends), we have obtained only one phage-insert junction that is not a repeat (Table II). Finally, we sought to determine whether we
could discern any individual-to-individual differences in a fairly well defined stock of B. malayi that has been carried in North America. This stock appears to have been brought to North America by Mak in a single infected cat about 40 years ago, and has been since maintained entirely in the laboratory by passage between mosquitoes and infected jirds [14]. We argued that such a stock should be genetically homogeneous and be ideal to insure that the level of individual-toindividual variation is low. The data from such analysis clearly show that there is no individualto-individual variation in the pattern of bands in the North American stock using the B m M b o I repeat.
Acknowledgements We wish to thank Ms. Ruth Conrod for assistance with typing this manuscript. This work was made possible by a grant from the USPHS (R22-27773) awarded to TVR.
References I Mak, J.W. (1986) Epidemiology of lymphatic filariasis. In: Symposium on Filariasis (Evered, D. and Clark, S., eds.), Ciba Foundation Symposium 127, pp. 5-14. 2 Wyman, A. and White, R. (1980) A highly polymorphic locus in human DNA. Proc. Natl. Acad. Sci. USA 77, 6754-6758. 3 McReynolds, L.A., DeSimeone, S.M. and Williams, S.A. (1986) Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugiamalayi and the animal parasite Brugiapahangi. Proc. Natl. Acad. Sci. USA 83, 797-801. 4 Sire, B.K.L., Mak, J.W., Cheong, W.H., Sutanto, I., Kurniawan, L., Marwoty, H.A., Franke, E., Campbell, J.R., Wirth, D.F. and Piessens, W.F. (1986) Identification of Brugiamalayi in vectors with a species specific probe. Am. J. Trop. Med. Hyg. 35, 559-564. 5 Cameron, M.L., Levy, P., Nutman, T., Vanamala, C.R., Narayanan, P.R. and Rajan, T.V. (1988) Use of restriction fragment length polymorphisms (RFLPs) to distinguish between nematodes of pathogenic significance. Parasitology 96, 381-390. 6 Rimm, D.L., Horness, D., Kucera, J. and Blattner, F.R. (1980) Construction of coliphage lambda charon vectors with BamHl cloning sites. Gene 12, 301-309. 7 Wahl, G.M. and Berger, S.L. (1987) Screening colonies or plaques with radioactive nucleic acid probes. Methods Enzymol. 152, 415-423. 8 Rajan, T.V., Halay, E.D., Potter, T.A., Evans, G.A., Seidman, J.G. and Margulies, D.H. (1983) H-2 hemizygous mutants from a heterozygous cell line: role of mitotic recombination. EMBO J. 2, 1537-1542. 9 Kafatos, F.C., Jones, C.W. and Efstratiadis, A. (1979) De-
49 termination of nucleic acid homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 7, 1541-1552. 10 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463. 11 Chen, E.Y. and Seeburg, P.H. (1985) Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4, 165-170. 12 Werner, C.W., Higashi. G.I., Yates, J.A. and Rajan, T.V.
(1989) Differential recognition of two cloned Brugia malayi antigens by antibody class. Mol. Biochem. Parasitol. 35, 209-218. 13 Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985) Hypervariable 'minisatellite' regions in human DNA. Nature 314, 67-74. 14 Ash, L.R. and Riley, J.M. (1970) Development of subperiodic Brugia malayi in the Jird, Merione unguiculatus, with notes on infections in other rodents, J. Parasitol. 56, 969-973.
39
Elsevier MOLBIO 01399
Isolation and characterization of a repetitive DNA element from the genome of the human filarial parasite, Brugia malayi Sundareshwaran Natarajan, Craig Werner, Margaret C a m e r o n and Thiruchandurai V. Rajan Departments of Pathology and Microbiology, University of Connecticut Health Center, Farmington, CT, U.S.A. (Received 26 February 1990; accepted 29 May 1990)
The genome of the human filarial parasite Brugia malayi contains at least two major repetitive DNA elements. One, referred to as the HhaI family, consists of 104-105 tandemly arrayed copies per haploid genome of a monomer of 322 base pairs and does not contain a cleavage site for the restriction endonuclease MboI. We constructed a library of MboI-digested genomic B. malayi DNA in BamHI-cut M13mpl8 resulting in the exclusion of the HhaI repeat family from the library. Hybridization of this genomic library with nick-translated genomic DNA yielded several copies of a repeat family which we have named the BmMboI family. From sequence analysis of more than 50 monomers, which differ from each other in sequence and length, we have been able to divide the monomers into several regions based on the level of sequence conservation. Southern blot analyses of B. malayi genomic DNA digested with a variety of restriction endonucleases and probed with the isolated repeat demonstrate multiple bands of varying sizes except with HindlIl-cut DNA, where the repeat is found only in very high-molecular-weight DNA. Key words: Brugia malayi; Repetitive DNA; Isolation; Characterization
Introduction Human lymphatic filariasis is a major public health problem in a number of countries, with an estimated 100 million individuals afflicted with this disease [1]. Repetitive DNA elements offer novel diagnostic reagents. Attempts to derive probes from the genome of B. malayi for such studies have resulted in the repeated isolation of a repeat family that has been referred to as the HhaI family [3,4]. This tandemly arranged repetitive DNA family, comprising about 10% of the B. malayi genome with a monomer of 322 bp, contains cleavage sites for a number of commonly used restriction endonucleases, so that genomic libraries contain multiple representations of the tanCorrespondence address: T.V. Rajan, University of Connecticut Health Center, 263 Farmington Avenue, L-1006, Farmington, CT 06032, U.S.A.
Abbreviations: mf, microfilariae; kb, kilobases. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank TM data base with the accession number M34369
dem repeat, ranging from a unit to large multiples of the monomer to the size accommodated by the vector. However, the repeat does not have cleavage sites for the restriction endonuclease MboI, so that cleavage of the B. malayi DNA by MboI resuits in the segregation of the family as a fragment of DNA too large to be accommodated by most commonly used vectors. The use of this strategy has allowed us to exclude this tandem repeat family from the library and isolate a novel repetitive member from the B. malayi genome. Materials and Methods
Construction of B. malayi genomic DNA libraries. B. malayi-infected jirds (Meriones unguiculatus) were obtained from the University of Georgia through a U.S./Japan cooperative program in illariasis. Microfilariae (mf) were purified from the peritoneal cavity of these animals as described earlier [4]. Genomic DNA was isolated from mf, subjected to quantitative digestion with the restriction endonuclease MboI, and ligated to BamHIdigested phosphatased Ml3mpl8. From such a li-
0166-6851/90/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
40 TABLE I Results of screening different B. malayi genomic libraries with the BmMboI family repeat fragment Librarya
No. of plaques
Positives
Frequency
EcoRI.l HindlII.I Mbol.A MboI.B EcoRI.II HindlII.II
13000 30 000 20000 20000 40000 40 000
46 0 6 21 78 0
3.5 x 10-3 0 3 x 10-4 1 X 10 - 3 1.95 X 10 - 3 0
aI and II refer to different EcoRI and HindlII libraries; MboI.A and MboI.B refer to different size-fractionated libraries. brary, 5 x 105 recombinant clones were isolated and amplified. All other genomic DNA libraries were constructed in the bacteriophage A-derived vector Charon 27 [5] after cutting the phage with EcoRI, HindlII or BamHI. In the case of EcoRI and HindlII, genomic DNA was cut to completion with the respective enzyme and ligated into phosphatased vector. The BamHI-cut vector was used to construct two different MboI partial libraries - genomic DNA was subjected to partial cleavage with MboI and size fractionated into 4-6-kb and 6-9-kb aliquots, which were then separately cloned into the BamHI-cut phosphatased vector. Isolation of Mbol repeats. B. malayi genomic DNA was labeled by nick translation and hybridized to filters of the MboI M13 library [6]. A number of hybridizing plaques were isolated and plaque-purified. After several rounds of screening, five different clones, BmMbo3, BmMbo4, BmMbo8, B m M b o l 0 , and B m M b o l 1, which consistently hybridized to radiolabeled genomic DNA, were retained for analysis. Subsequently, repeat clones were isolated from the other B. malayi genomic libraries using one of the MboI library repeat members as probe. Southern blot and dot blot analysis. B. malayi genomic DNA was subjected to restriction en-
donuclease cleavage with a variety of enzymes under conditions recommended by the manufacturers. DNA was subjected to electrophoresis in Tris-borate-EDTA (TBE) gels (0.7% for 6-base cutters and 1.1% for 4-base cutters). Following denaturation and renaturation in situ, the DNA was transferred to nitrocellulose filter papers and hybridized to nick-translated or random-primer (Boehringer-Mannheim) extended BmMbo8 or B m M b o l l DNA [7]. To investigate the relatedness of individual worms, single adult male and female worms were recovered from the peritoneal cavities of infected jirds obtained from W. McCall. Each adult worm was processed separately in an Eppendorf centrifuge tube to obtain DNA. The DNA sample from each individual worm was subjected to restriction endonuclease cleavage separately, electrophoresed in a separate lane of a gel and analysed after transfer using radiolabeled repeat probe. Dot blots were performed by spotting serial dilutions of B. malayi genomic DNA on nitrocellulose filter paper as described by Kafatos et al. [8]. DNA sequencing and analysis. Sequences were obtained from the repeat clones by using the chain termination method [9,10]. The sequences were analysed using the University of Wisconsin Genetics Computer Group (UWGCG) Sequence Analysis Software Package.
~a~T~AAT TC A T G CCACTTCTCTACTGATATTACA CCATTTCTCTACAGATTAAACA CCATTTCTCTGCAATCATAACA
ATATCACT~T ATATCACT~TT ATATCACTAGAAGACAT
TTTGAT A A A T T C A T T T A C T C A T A TTGCAT TAATTC A T T A A T T C A T A TTCGAT C
Fig. 1. Complete nucleotide sequence (184 bp) of BmMbo8. The MboI recognition sequences (GATC)are underlined.The sequence is arranged to highlight the fact that the unit is composed of three internal repeats of approximately62 bases, as described in the text.
41 Plmll
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Isolation of the Mbol repeat family. When the MboI M 1 3 m p l 8 B. malayi library was probed with labeled B. malayi genomic D N A , a total of 13 plaques hybridized to the D N A and were picked for secondary lifts. Following the secondary and subsequent lifts, a total of five plaque-purified phages continued to hybridize to radioactive ge-
nomic D N A . These five recombinant phages were retained for further analyses. The sizes of the inserts in these recombinant phage particles ranged from 250 bp to 3.5 kb. Six other genomic libraries (two different EcoRI, two different HindlII and two different size-fractionated MboI libraries) were screened with one of the repeat members. We obtained a variable number of hybridizing plaques with the
42
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EcoRI and MboI libraries (ranging from 6-78), but we repeatedly failed to find any positive clone from the HindlII libraries with the same radiolabeled repeat probe. These results are illustrated in Table I. DNA sequence analysis. The sequence of the prototypic member BmMboI8 is shown in Fig. 1.
Visual inspection of the sequence revealed certain sequences that were repeated at regular intervals. This is demonstrated more clearly by a diagonal plot comparison. Fig. 2 shows representative output where the X axis represents the sequence of BmMbo8 and the Y axis represents either the sequence of BmMbo8 (Fig. 2A), its reverse complement (Fig. 2B), BmMbol0 (Fig. 2C) or BmMbol 1
44 TABLE II Size and structure of vector-insert junction of full-length repeat clones obtained from different B. malayi genomic libraries No.
Library
Clone
Size (bp)
Junctional sequence
I
M 13.MboI
BM3 Bm4 Bm8 Bml0 Bm I 1
400 700 182 3500 800
R R R R R
It
EcoRI
pBm.A pBM.B pBm.C pBm.D pBm.E pBm.F
III
ch27.MboI
R R R R R
RR R R
R R R R
8000
pBm.Mj3*
2100
R R U R
RR
R R indicates that both ends of the insert fragment consist of repeat sequence; R U indicates that one end of the insert fragment is repeat and the other is unique; *, due to the fact that there were internal (RI or Hill) sites in the other 5 clones, we were able to analyze only two genomic ends. Clones pBm.A, pBm.B, pBm.C, pBm.D and pBm.F range in size from 2-7 kb. (Fig. 2D). Internally repeated sequences produce satellite diagonals offset from the central diagonal by an a m o u n t proportional to the spacing o f the repeat. This is seen in the plot of B m M b o 8 versus itself, where, in addition to the central diagonal, two satellite diagonals can be discerned. It is worth noting that c o m p a r i s o n s o f B m M b o 8 versus the reverse c o m p l e m e n t fails to reveal a diagonal line, indicating that there are no inverted repeats in this sequence (Fig. 2B). Further scrutiny of these sequences revealed that each of our clones is in fact c o m p o s e d o f multiple m o n o m e r s . Each m o n o m e r can be further divided into seven regions as shown in Fig. 3 based on the level o f sequence conservation a m o n g the 50 or so m o n o m e r s that we have characterized, arbitrarily defining the CC doublet as the 5' end o f a m o n o m e r : a 5-bp ' A ' region, a 5-bp ' B ' region, a 4-bp ' C ' region, a 31-bp ' D ' region, a 4-bp ' E ' region, a 4-bp ' F ' region and a 9-bp ' G ' region. A histogram illustrating the pattern o f conservation o f the consensus nucleotides is shown in Fig. 4. Regions B, D and F are highly conserved while regions A, C, E and G are less so, with region B being the most conserved and region G the least. Regions G and A are the most variable in length due to the occurrence o f frequent deletions giving rise to variation in the length o f individual
m o n o m e r s ; for instance, region G consists o f a single nucleotide in 24.6% o f m o n o m e r s while in 49% it consists o f its full c o m p l e m e n t o f 9 nucleotides. Region D consists o f a 31-bp stretch
120
A_.~_-L_G._
..K_~-E--
100
80
i e-
0
60"
0
40"
20
i lO
20
3O
40
Consensus Positions(I-62)
50
IlllJF
Fig. 4. Histogram showing the degree of conservation of the consensus nucleotides among the monomers. Over 50 monomers have been sequenced and the degree of conservation at each position is shown graphically as a percentage. Contiguous nucleotides of similar conservation can be grouped into regions, as indicated by the horizontal bars above the histogram.
45
tO
t~ 03
100
CO O3 ',d"
¢; 80 ~O t.t) ¢.O O3 O,I
60 •
Conservation
• Substitution I~1 Deletion. • Insertion 40
20
A
B
C
D
E
F
G
Regions of the consensus Fig. 5. Histogram showing the differential patterns of conservation, substitution, deletion and insertion among the regions of the consensus. Each region differs significantly in conservation from its neighbours. Regions A and D show a high percentage of deletions while region C shows a high frequency of substitutions.
of highly conserved nucleotides including an A in position 25 which is completely conserved among all monomers sequenced. These data are graphically indicated in Fig. 5. To determine whether the MboI repeat family had the potential to encode a protein, the translation of the repeats in all three reading frames and in both orientations were obtained using the MAP program of UWGCG; these analyses (not shown) revealed that there is no significant open reading frame in any of the six reading frames.
Southern blot analysis. To visualize the organization of the repeat family in the genome, Southern blot analyses were performed using a variety
of enzymes and radiolabeled BmMbo8 DNA (Fig. 6). A large number of bands, in excess of a number that could be readily counted, were visualized with most enzymes. Notable was the absence of the ladder like array that is seen when Southern blots are performed with a typical tandem repeat. To investigate whether there were individual differences amongst individual B. malayi worms, Southern blot analyses were performed on DNA from individual adult (male and female) B. malayi cut with PvulI, since this enzyme produced the most distinctive restriction pattern. When such Southern blots were subjected to hybridization with radiolabeled BmMbol 1 DNA, the data revealed that the worms were essentially indistin-
46
7
8
9
10
11
!i!i I
I 2 3 4 5 6 7 8 9
101112
B
Fig. 6. Southern blot analyses of B. malavi DNA (lanes 1-5) cut with a variety of restriction endonucIeases and other filarial DNAs cut with Pvull and hybridized to radiolabeled repeat probe (A) or to a cloned lYagmentof a collagen gene from B. malayi (B). The enzymes used in lanes I-5 (all B. malavi genomic DNA) are: lane 1, EcoRl; lane 2, Hindlli; lane 3, Pvull; lane 4, SspI', lane 5, Xmnl. The worms used in lanes 7-10 are (all cut with Pvull): lane 7, Acanthocheilonema vitiae; lane 8. Brugia pahangi; lane 9, Litmosoides carinii; lane 10, Setaria sp. Lane 11 is jird DNA(control) and lane 5 is the A Hindlll marker. guishable from each other (Fig. 7). Estimation o f copy number. To d e t e r m i n e the n u m b e r of copies of the repeat family in the g e n o m e , B. malayi D N A was spotted onto nitrocellulose paper in d o u b l i n g dilutions in duplicate,
starting at 600 rig/spot. O n e of the two duplicate sets was h y b r i d i z e d to a single copy g e n o m i c D N A ( a m i n o acids 1 4 9 5 - 1 5 6 0 o f the filarial body wall m y o s i n h e a v y chain) [ 12] and the other set to B m M b o 8 . The results of the dot blot are s h o w n in Fig. 8. U n d e r similar c o n d i t i o n s of exposure,
47
Fig. 8. Dot blots of B. malayi DNA hybridized either to BmMbo8 repeat or a single copy probe derived from the B. malayi myosin gene. Note that hybridization can be visualized using the repeat up to the lowest amount of DNA spotted onto the filter; in case of the single copy probe, hybridization is not visible beyond the fourth highest dilution.
the conditions of hybridization it recognizes a single copy sequence in the genome under the conditions of the hybridization (Werner et al., manuscript in preparation), and since the specific activity of the two probes was comparable, in excess of 109 cpm (/zg probe) -~, we estimate that the repeat is represented at least 100-200 times per haploid genome equivalent. Fig. 7. Southern blot analyses of individual B. malayi females, obtained from W. McCall, subjected to DNA extraction, restriction endonuclease cleavage with PvulI, electrophoresis and blotting on separate lanes. The molecular weight markers are indicated along the left margin and are HindllI fragments derived from )~ DNA. Note the identity of the bands noted in the individual worms.
myosin hybridizes to the spot corresponding to 75 ng of B. malayi as intensely as the repeat does to the 1.2 ng spot. Since the length of the myosin fragment is the same as that of the repeat probe (approximately 200 bp), since we know that under
Discussion One repetitive DNA family in B. malavi, referred to as the HhaI family constitutes about 8-10% of the genome of B. malayi, is represented very highly in genomic libraries and is repeatedly isolated by the techniques commonly used to isolate repetitive DNA elements [2,3,4,11 ]. Since the HhaI monomer does not contain an MboI cleavage site, the use of this enzyme for genomic cloning has apparently allowed us to excise the tandem family as a piece too large to be accommodated
48 in M 1 3 m p l 8 and thus clone a novel repeat family from the B. malayi genome. Sequence analysis of this novel repeat has revealed an intricate arrangement of seven smaller regions within the larger unit, identified on the basis of their degree of sequence and length polymorphisms. While regions B, C, D, and F are relatively constant in length, regions A and G are very polymorphic due to the occurrence of frequent deletions, giving rise to considerable variation in the length of the monomers. The incidence of substitutions, deletions and insertions in the different regions is shown in Fig. 5. Most of our Southern blots suggest that the B m M b o I family is an interspersed repeat. However, we have conducted Southern blot analyses using HindlIl on numerous occasions, and have repeatedly failed to observe any fragment containing the repeat that is smaller than the exclusion limit of the agarose gel. An example is Fig. 6A, lane 2. Possible explanations for this observation are (a) the lane with the HindlII-digested D N A did not contain adequate amounts of DNA, or (b) the enzyme failed to cleave the DNA. To address these two possibilities we reprobed the blot shown in Fig. 6A with a fragment of a collagen gene derived from the B. malayi genome. It will be noted that this reprobing (Fig. 6B) highlights a fragment in lane 2 (B. malayi genomic D N A digested with HindlII) whose size is consistent with that expected from known sequence of this gene (manuscript submitted), and that the intensity of this fragment is comparable to that in other lanes. We are unable to come up with an explanation for an interspersed repeat that fails to generate any small fragments containing the repetitive D N A element. These results with Southern blots conducted with HindlII are consistent with the results obtained on screening the HindlII library with the repeat probe (Table I) and the structure of vector-insert junctions of several clones from different libraries (Table II). Repeated screens of the HindlII libraries (Fig. 1) have failed to isolate any m e m b e r of this family; finally, though we have sequenced 12 fulllength insert containing genomic clones (or a total of 24 genomic ends), we have obtained only one phage-insert junction that is not a repeat (Table II). Finally, we sought to determine whether we
could discern any individual-to-individual differences in a fairly well defined stock of B. malayi that has been carried in North America. This stock appears to have been brought to North America by Mak in a single infected cat about 40 years ago, and has been since maintained entirely in the laboratory by passage between mosquitoes and infected jirds [14]. We argued that such a stock should be genetically homogeneous and be ideal to insure that the level of individual-toindividual variation is low. The data from such analysis clearly show that there is no individualto-individual variation in the pattern of bands in the North American stock using the B m M b o I repeat.
Acknowledgements We wish to thank Ms. Ruth Conrod for assistance with typing this manuscript. This work was made possible by a grant from the USPHS (R22-27773) awarded to TVR.
References I Mak, J.W. (1986) Epidemiology of lymphatic filariasis. In: Symposium on Filariasis (Evered, D. and Clark, S., eds.), Ciba Foundation Symposium 127, pp. 5-14. 2 Wyman, A. and White, R. (1980) A highly polymorphic locus in human DNA. Proc. Natl. Acad. Sci. USA 77, 6754-6758. 3 McReynolds, L.A., DeSimeone, S.M. and Williams, S.A. (1986) Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugiamalayi and the animal parasite Brugiapahangi. Proc. Natl. Acad. Sci. USA 83, 797-801. 4 Sire, B.K.L., Mak, J.W., Cheong, W.H., Sutanto, I., Kurniawan, L., Marwoty, H.A., Franke, E., Campbell, J.R., Wirth, D.F. and Piessens, W.F. (1986) Identification of Brugiamalayi in vectors with a species specific probe. Am. J. Trop. Med. Hyg. 35, 559-564. 5 Cameron, M.L., Levy, P., Nutman, T., Vanamala, C.R., Narayanan, P.R. and Rajan, T.V. (1988) Use of restriction fragment length polymorphisms (RFLPs) to distinguish between nematodes of pathogenic significance. Parasitology 96, 381-390. 6 Rimm, D.L., Horness, D., Kucera, J. and Blattner, F.R. (1980) Construction of coliphage lambda charon vectors with BamHl cloning sites. Gene 12, 301-309. 7 Wahl, G.M. and Berger, S.L. (1987) Screening colonies or plaques with radioactive nucleic acid probes. Methods Enzymol. 152, 415-423. 8 Rajan, T.V., Halay, E.D., Potter, T.A., Evans, G.A., Seidman, J.G. and Margulies, D.H. (1983) H-2 hemizygous mutants from a heterozygous cell line: role of mitotic recombination. EMBO J. 2, 1537-1542. 9 Kafatos, F.C., Jones, C.W. and Efstratiadis, A. (1979) De-
49 termination of nucleic acid homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 7, 1541-1552. 10 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463. 11 Chen, E.Y. and Seeburg, P.H. (1985) Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4, 165-170. 12 Werner, C.W., Higashi. G.I., Yates, J.A. and Rajan, T.V.
(1989) Differential recognition of two cloned Brugia malayi antigens by antibody class. Mol. Biochem. Parasitol. 35, 209-218. 13 Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985) Hypervariable 'minisatellite' regions in human DNA. Nature 314, 67-74. 14 Ash, L.R. and Riley, J.M. (1970) Development of subperiodic Brugia malayi in the Jird, Merione unguiculatus, with notes on infections in other rodents, J. Parasitol. 56, 969-973.
