- Email: [email protected]
Highly repeated DNA sequences as species-specific probes for Brugia
ParasitologyToday,vol.3, no. 12, I987
378 References
I 2 3
4
Perler, F.B. and Karam,‘M.( I986)Mol. Blochem Parmtoi. 2 I I 7 I-I 78 Shah, J.S., Karam, M.. Plessens. W.F. and Worth, D.F. Am.]. Trap. Med. Hyg. (In press) Harnett, W., Chambers, A. and Parkhouse, R.M.E. (1987) 1. Celi. Biochem. I IA (Suppl.), p. 158 Omar. M.S., Denke, A.M. and Raybould. J.N.
5 6 7 8
(I 979) Tropenmed. Porovtol. 30, 157-l 62 Duke, B.O.L. (I 966) Ann. Trap. Med. Porasrtoi. 60.495 Budden. F.H. i I9631 Trans. R. Sot Troo. Med. Hyg. 57, 64-7; ’ Clanchl, R. et al. (I 985)Acto Trap 42,34 l-35 I Flockhart H.A.. Clbulskls. R.E.. Karam. M. and Alblez. E.]. (I 986) Trans. R. Sot Trap
Med.Hyg.
80,285-292
9
Ertmann. K.D. et 01. (I 987) Nature 327, 415417
Thomas Unnasch is wfh the Dlvlslon of Geographic Medicine and Department ofMolecuTar Biology and Microbiology, Case Western ReserveUniversity,Cleveland,OH 44 106, USA.
Highly Repeated DNA Sequences as Speciesspecific Probes for Brugiu W. F. Piessens, L. A. McReynolds Several filartae of the genus Brugia are known or suspected agents of lymphatic filariasis. 6. molayi naturally infects humans, several species of nonhuman primates and various other vertebrates, and is a cause of zoonotic infections in many endemic areas of South-East Asia. 5. timori also infects humans but appears not to have a natural animal host, while 5. pohangi IS a major parasite of domestic and wild animals and has long been suspected to infect humans as well. These closely related worms occur in adjacent or overlapping areas and can be transmitted by the same mosquito vectors’,2. Traditional methods to distinguish these filariae are inadequate. Within one pedigreed ‘species’, the variation in most phenotypic markers commonly used for this purpose (morphometric and histochemical measurements) severely limits their ability to identify a particular worm in a given isolate. The use of functional criteria - microfilarial periodicity, ability to infect some types of mosquitoes, etc. - is equally prone to error, because they may reflect adaptive rather than intrinsic properties of the worms. Some existing methods are of limited practical value, because they require detection of differences among developmental stages that are difficult to Isolate from infected hosts (eg. adult male worms). As a result, many questions regarding the transmission, epidemlology and pathogenicity of brugian filariae cannot be answered until new, simple and reliable methods to identify these helminths become availablej. We have cloned and characterized repeated DNA sequences to generate species-specific probes. The rationale for this approach is as follows. A DNA probe designed to detect highly repeated DNA sequences should obviously be more sensitive than one that reacts with DNA present in low copy numbers. Further, most highly repeated
and S. A. Williams
sequences identified to date are noncoding and evolve more rapidly than the rest ofthe genome (Box 2). Thus, It should be possible to exploit the unique properties of highly repeated DNA to develop sensitive and specific probes to trace the of filarial relatedness evolutionary nematodes. Because these sequences are present in all developmental stages of the parasite, such species-specific probes may allow identification of a single filarial worm in either the vector or the vertebrate host. Our experience to date suggests that these assumptions are essentially correct.
Repeated DNA elements in Brugia Reassociation kinetics indicate that approximately 25% of the genome of 5. molayi cons&s of repeated DNA4. One family of repeated DNA elements (the Hha I repeat family) was first identified by McReynolds et aL5 as a band of approximately 320 base pairs (bp) in ethidium bromide-stained gels of genomic DNA of
Table I. Specificity of Probes Containing Highly Repeated DNA Sequences from Brugio moloyi Parasites tested
6. malayi B. pohangi 6. timofi
W. bancrofi W. kalimantani 0. vohlJlus A. viteae D. immitis I_. carinii D. repens B. boolid Cardioflaria spp.
Reactivity with clone:
pBma68
pBml5
Yes Noa
Yes N@
Yes No
Yes No
No No No No No No No
No No No Unknown No No
No
No
aSeetext for discussion.
Unknown
6. ma/ayi digested with Hind I I I , A/u I Rsa I or Hhal. A dimer ofthis repeat was cloned In a plasmid vector (clone pBma68). Approximately 30 000 copies of this family of direct repeats are present In the genome of 5. malayi - comprising about IO7 bp or 12% of the 8. malayi genome. Another highly repeated DNA sequence was cloned from Sau3A digests of 5. malayi DNA by Sim et al.6. The resulting clone, pBm I 5, appears to contain fourtandem repeats. Although the Hha I repeats in pBma68 do not contain a Sau3A site, dlrect comparison reveals 97.% homology between the parts of clone pBm I5 that have been sequenced so far and the t/ha I repeats in clone pBma68. This Indicates that these independently cloned DNA fragments are closely related and probably belong to the same family of highly repeated DNA elements In the genome of 6. malayi. Cloned repeated DNA fragments 32P-labelled clones pBma68 and pBm I 5 are extremely sensitive diagnostic probes. Both can detect 300-500 pg of isolated genomlc DNA from 6. malayi - less than the calculated DNA content of a single microfilaria. In addition, both clones can be used to detect a single infective larva or fewer than five microfilariae lysed in situ on nitrocellulose filters6,7. Some aspects of the specificity of either DNA clone (Table I) deserve further comment. It was origlnally reported that clone pBma68 crosshybridizes to 6. pahangi DNA at IO% of the intensity observed with 5. malayi DNAs, whereas pBm I5 hybridizes several hundredfold better to 5. malayi than to 5. @hang+. Subsequent direct comparison of the two clones indicates that this apparent discrepancy is due in part to differences in the stringency of the hybridization conditions used by our two groups. However, even under condrtions of high stringency, both @I987,
Elsewr
Publcattons, Cambr,dge
0,69-4758187@02
00
379
Parasitology Today, vol. 3, no. 12, I987
Ancestral Fllariae
clones hybridize to 6. timori, albeit less well than to 6. malayi. This suggests a greater degree of relatedness between the ‘human’ parasites 6. mu/ayi and 6. timori than between the former and the ‘animal’ parasite B. pahongi. Both clones also hybridize equally well to isolates of 6. malayi with different patterns of microfilarial periodicity, including one of the periodic variety. In marked contrast, neither DNA probe hybridizes with Wuchereria bancrofti or Onchocerca volv~~l~~s,nor with a variety of related parasites of animals.
Ancestral
Other Filariae
Brugia DNA repeat
Oligonucleotide
Second Generation Oligonucleotide DNA Probes Clone pBma68 derived from the genome of 6. malayi was used by McReynolds et al.5 to identify and isolate a corresponding family of repeated DNA sequences in the genome of 6. pahangi. Because the enzyme Hhol cleaves homologous repeated DNA in both worm species to the same unit size of approximately 320 bp, this was called the Hhal repeat family. However, since several enzymes (Hind I I I A/u I and RsaI ) cleavethe /-/ho1 family in 6. malayi but not in 5. pahangi DNA, the DNA repeats from these two parasite species are homologous but not identical. To analyse these differences at the nucleotide level, Williams et a/.* cloned and sequenced I 5 members ofthe Hhal family of each parasite species. The 322 bp repeats from both species are 80% AT-rich. The overall homology between repeats from 6. malayi and 6. pahangi is 89% - compared with 97% homology between repeats cloned from the same parasite species. However, differences in nucleotide sequence between the two Brugia species are not random, but clustered in a GC-rich region located some 250 bp from the Hhal site. This DNA sequence information has been used to species-specific construct putative oligonucleotide probes (Fig. I ) designed tcp maximize sequence divergence between the Hhal repeats of the two filarial species, and to minimize divergence within each species. These probes were made to be at least 20 nucleotides long to ensure sensitivity, and to have the maximal GC content to ensure stability. Two such probes were synthesized: both have greater than 95% homology to the corresponding parental repeat and only 65% homology with the Hhal repeat of the opposite parasite species. These two oligonucleotides are both sensitive and species specific. The 29nucleotide probe for 6. malayi shows a SOO-fold preference for 6. malayi DNA
J-
Fig. 1. Repeated DNA sequences and probes fir Brugia. A 322 base pair
probes
/1
Hha/ repeated DNA sequence is fbund only of?.er Brugia had diverged from other filarial species.A voriable region within this repeat (shown by diagonal lines in B. malayi or checks in B. pahangi) has evolved afier the two species diverged. The B. malayi variable region has Alul and Rsa/ sites (shown by A or R) while B. pahangi does not Specific and sensitiveolig+ nucleotide probes syn-
thesized from this region recognize the correct speciesin a DNA hybridiza-
li\
tion assay
over 6. pahangi DNA in dot blot assays (Box 3). The converse is true for the 2 I nucleotide probe for 6. pahangi. Either probe can detect about 500 pg of DNA, or approximately the same amount of DNA as the original cloned probes described above. These second generation DNA probes remain to be tested in the field, but we have no reason to believe that they will perform less well than their predecessors.
Applications in Lymphatic Filariasis The availability of apparently speciesspecific DNA probes that detect invariant, or at least relatively fixed, characteristics of the genome of closely related filarial parasites provides the tools needed to settle several unresolved issues. These probes can be used to determine whether 6. pahangi infects humans, as has been reported to occur in Indonesiag; to identify a potential animal reservoir of human infection in areas where zoonotic filariasis may occur; and to identify filarial larvae in mosquito vectors, thereby allowing realistic estimates of the intensity of transmission within a given area, and of the impact of control programmes on transmission. Efforts to produce these diagnostic tools have also provided new insights into the molecular biology of filarial Sequence and restriction worms. analysis of the Hhal repeat family suggest the existence of subfamilies of this highly repeated DNA sequence5s6f8. These may have evolved by a series of amplifications of single repeats that have accumulated point mutations as new species diverged from their ancestors.
This could explain why Hhal repeats cloned from within one parasite species are much more homologous than corresponding repeats from different species. Thus, further comparison of the degree of relatedness between different genera of filarial nematodes may improve our understanding of their evolutionary origins. Finally, it should be pointed out that not all segments of DNA present in multiple copy numbers in the genome have evolved as rapidly as the Hhal repeat family. This is true, for example, of the noncoding regions of ribosomal genes of filarial helminthslo. References
I 2 3
4
5
6 7 8
9 IO
Yen, P.K.E., Zaman, Y. and Mak, J.W. (I 982)). Helminthol. 56.69-80 Nelson, G.S. (I 979) Nature 300, I I36- I I39 WHO Expert CommIttee on Fllarws Fourth Report (I 984) Technical Report Series No. 702, WHO, Geneva Maina, C.V. et al. In Molecular Paradigms for Eradicating Helminth Parasites (Proc. UCLA Symp) (In press) McReynolds, L.A., DeSlmone, S.M. and Wllllams, S.A. (I 986) Proc. Nat/ Acad. Sci USA 83,797-80 I Sim. B.K., Plessens, W.F. and Worth, D.F. (I 986) Mol. Blochem. Parasitol 19, I 17-l 23 Stm, B.K. et al. (I 986)Am.J Trap Med. Hyg. 35. 559-564 Williams, S.A., DeSlmone, S.M.. Poole, C.B. and McReynolds, L.A. Am. /, Jrop. Med. Hyg. (In press) Palmieri, J, et al. (I 985) Trap. Geogr. Med 37, 239-243 Shah, J.S., Lamontagne, L., Unnasch, T.R, Wirth, D.F. and Piessens, W.F. (1986) Mol. Biochem. Parasftol. I9,67-75
Willy Piessens is at the
Deportment of Tropical
School ofPublic Health, Boston, MA 02 I 15, USA; Larry McReynolds is with New England Biolabs Inc., Beverly, MA 0 I9 15. USA; and Steven Williams is at the Department ofBiological Sciences, Smith College, Northampton, MA 0 1063, USA.
Public Health,
Harvard
378 References
I 2 3
4
Perler, F.B. and Karam,‘M.( I986)Mol. Blochem Parmtoi. 2 I I 7 I-I 78 Shah, J.S., Karam, M.. Plessens. W.F. and Worth, D.F. Am.]. Trap. Med. Hyg. (In press) Harnett, W., Chambers, A. and Parkhouse, R.M.E. (1987) 1. Celi. Biochem. I IA (Suppl.), p. 158 Omar. M.S., Denke, A.M. and Raybould. J.N.
5 6 7 8
(I 979) Tropenmed. Porovtol. 30, 157-l 62 Duke, B.O.L. (I 966) Ann. Trap. Med. Porasrtoi. 60.495 Budden. F.H. i I9631 Trans. R. Sot Troo. Med. Hyg. 57, 64-7; ’ Clanchl, R. et al. (I 985)Acto Trap 42,34 l-35 I Flockhart H.A.. Clbulskls. R.E.. Karam. M. and Alblez. E.]. (I 986) Trans. R. Sot Trap
Med.Hyg.
80,285-292
9
Ertmann. K.D. et 01. (I 987) Nature 327, 415417
Thomas Unnasch is wfh the Dlvlslon of Geographic Medicine and Department ofMolecuTar Biology and Microbiology, Case Western ReserveUniversity,Cleveland,OH 44 106, USA.
Highly Repeated DNA Sequences as Speciesspecific Probes for Brugiu W. F. Piessens, L. A. McReynolds Several filartae of the genus Brugia are known or suspected agents of lymphatic filariasis. 6. molayi naturally infects humans, several species of nonhuman primates and various other vertebrates, and is a cause of zoonotic infections in many endemic areas of South-East Asia. 5. timori also infects humans but appears not to have a natural animal host, while 5. pohangi IS a major parasite of domestic and wild animals and has long been suspected to infect humans as well. These closely related worms occur in adjacent or overlapping areas and can be transmitted by the same mosquito vectors’,2. Traditional methods to distinguish these filariae are inadequate. Within one pedigreed ‘species’, the variation in most phenotypic markers commonly used for this purpose (morphometric and histochemical measurements) severely limits their ability to identify a particular worm in a given isolate. The use of functional criteria - microfilarial periodicity, ability to infect some types of mosquitoes, etc. - is equally prone to error, because they may reflect adaptive rather than intrinsic properties of the worms. Some existing methods are of limited practical value, because they require detection of differences among developmental stages that are difficult to Isolate from infected hosts (eg. adult male worms). As a result, many questions regarding the transmission, epidemlology and pathogenicity of brugian filariae cannot be answered until new, simple and reliable methods to identify these helminths become availablej. We have cloned and characterized repeated DNA sequences to generate species-specific probes. The rationale for this approach is as follows. A DNA probe designed to detect highly repeated DNA sequences should obviously be more sensitive than one that reacts with DNA present in low copy numbers. Further, most highly repeated
and S. A. Williams
sequences identified to date are noncoding and evolve more rapidly than the rest ofthe genome (Box 2). Thus, It should be possible to exploit the unique properties of highly repeated DNA to develop sensitive and specific probes to trace the of filarial relatedness evolutionary nematodes. Because these sequences are present in all developmental stages of the parasite, such species-specific probes may allow identification of a single filarial worm in either the vector or the vertebrate host. Our experience to date suggests that these assumptions are essentially correct.
Repeated DNA elements in Brugia Reassociation kinetics indicate that approximately 25% of the genome of 5. molayi cons&s of repeated DNA4. One family of repeated DNA elements (the Hha I repeat family) was first identified by McReynolds et aL5 as a band of approximately 320 base pairs (bp) in ethidium bromide-stained gels of genomic DNA of
Table I. Specificity of Probes Containing Highly Repeated DNA Sequences from Brugio moloyi Parasites tested
6. malayi B. pohangi 6. timofi
W. bancrofi W. kalimantani 0. vohlJlus A. viteae D. immitis I_. carinii D. repens B. boolid Cardioflaria spp.
Reactivity with clone:
pBma68
pBml5
Yes Noa
Yes N@
Yes No
Yes No
No No No No No No No
No No No Unknown No No
No
No
aSeetext for discussion.
Unknown
6. ma/ayi digested with Hind I I I , A/u I Rsa I or Hhal. A dimer ofthis repeat was cloned In a plasmid vector (clone pBma68). Approximately 30 000 copies of this family of direct repeats are present In the genome of 5. malayi - comprising about IO7 bp or 12% of the 8. malayi genome. Another highly repeated DNA sequence was cloned from Sau3A digests of 5. malayi DNA by Sim et al.6. The resulting clone, pBm I 5, appears to contain fourtandem repeats. Although the Hha I repeats in pBma68 do not contain a Sau3A site, dlrect comparison reveals 97.% homology between the parts of clone pBm I5 that have been sequenced so far and the t/ha I repeats in clone pBma68. This Indicates that these independently cloned DNA fragments are closely related and probably belong to the same family of highly repeated DNA elements In the genome of 6. malayi. Cloned repeated DNA fragments 32P-labelled clones pBma68 and pBm I 5 are extremely sensitive diagnostic probes. Both can detect 300-500 pg of isolated genomlc DNA from 6. malayi - less than the calculated DNA content of a single microfilaria. In addition, both clones can be used to detect a single infective larva or fewer than five microfilariae lysed in situ on nitrocellulose filters6,7. Some aspects of the specificity of either DNA clone (Table I) deserve further comment. It was origlnally reported that clone pBma68 crosshybridizes to 6. pahangi DNA at IO% of the intensity observed with 5. malayi DNAs, whereas pBm I5 hybridizes several hundredfold better to 5. malayi than to 5. @hang+. Subsequent direct comparison of the two clones indicates that this apparent discrepancy is due in part to differences in the stringency of the hybridization conditions used by our two groups. However, even under condrtions of high stringency, both @I987,
Elsewr
Publcattons, Cambr,dge
0,69-4758187@02
00
379
Parasitology Today, vol. 3, no. 12, I987
Ancestral Fllariae
clones hybridize to 6. timori, albeit less well than to 6. malayi. This suggests a greater degree of relatedness between the ‘human’ parasites 6. mu/ayi and 6. timori than between the former and the ‘animal’ parasite B. pahongi. Both clones also hybridize equally well to isolates of 6. malayi with different patterns of microfilarial periodicity, including one of the periodic variety. In marked contrast, neither DNA probe hybridizes with Wuchereria bancrofti or Onchocerca volv~~l~~s,nor with a variety of related parasites of animals.
Ancestral
Other Filariae
Brugia DNA repeat
Oligonucleotide
Second Generation Oligonucleotide DNA Probes Clone pBma68 derived from the genome of 6. malayi was used by McReynolds et al.5 to identify and isolate a corresponding family of repeated DNA sequences in the genome of 6. pahangi. Because the enzyme Hhol cleaves homologous repeated DNA in both worm species to the same unit size of approximately 320 bp, this was called the Hhal repeat family. However, since several enzymes (Hind I I I A/u I and RsaI ) cleavethe /-/ho1 family in 6. malayi but not in 5. pahangi DNA, the DNA repeats from these two parasite species are homologous but not identical. To analyse these differences at the nucleotide level, Williams et a/.* cloned and sequenced I 5 members ofthe Hhal family of each parasite species. The 322 bp repeats from both species are 80% AT-rich. The overall homology between repeats from 6. malayi and 6. pahangi is 89% - compared with 97% homology between repeats cloned from the same parasite species. However, differences in nucleotide sequence between the two Brugia species are not random, but clustered in a GC-rich region located some 250 bp from the Hhal site. This DNA sequence information has been used to species-specific construct putative oligonucleotide probes (Fig. I ) designed tcp maximize sequence divergence between the Hhal repeats of the two filarial species, and to minimize divergence within each species. These probes were made to be at least 20 nucleotides long to ensure sensitivity, and to have the maximal GC content to ensure stability. Two such probes were synthesized: both have greater than 95% homology to the corresponding parental repeat and only 65% homology with the Hhal repeat of the opposite parasite species. These two oligonucleotides are both sensitive and species specific. The 29nucleotide probe for 6. malayi shows a SOO-fold preference for 6. malayi DNA
J-
Fig. 1. Repeated DNA sequences and probes fir Brugia. A 322 base pair
probes
/1
Hha/ repeated DNA sequence is fbund only of?.er Brugia had diverged from other filarial species.A voriable region within this repeat (shown by diagonal lines in B. malayi or checks in B. pahangi) has evolved afier the two species diverged. The B. malayi variable region has Alul and Rsa/ sites (shown by A or R) while B. pahangi does not Specific and sensitiveolig+ nucleotide probes syn-
thesized from this region recognize the correct speciesin a DNA hybridiza-
li\
tion assay
over 6. pahangi DNA in dot blot assays (Box 3). The converse is true for the 2 I nucleotide probe for 6. pahangi. Either probe can detect about 500 pg of DNA, or approximately the same amount of DNA as the original cloned probes described above. These second generation DNA probes remain to be tested in the field, but we have no reason to believe that they will perform less well than their predecessors.
Applications in Lymphatic Filariasis The availability of apparently speciesspecific DNA probes that detect invariant, or at least relatively fixed, characteristics of the genome of closely related filarial parasites provides the tools needed to settle several unresolved issues. These probes can be used to determine whether 6. pahangi infects humans, as has been reported to occur in Indonesiag; to identify a potential animal reservoir of human infection in areas where zoonotic filariasis may occur; and to identify filarial larvae in mosquito vectors, thereby allowing realistic estimates of the intensity of transmission within a given area, and of the impact of control programmes on transmission. Efforts to produce these diagnostic tools have also provided new insights into the molecular biology of filarial Sequence and restriction worms. analysis of the Hhal repeat family suggest the existence of subfamilies of this highly repeated DNA sequence5s6f8. These may have evolved by a series of amplifications of single repeats that have accumulated point mutations as new species diverged from their ancestors.
This could explain why Hhal repeats cloned from within one parasite species are much more homologous than corresponding repeats from different species. Thus, further comparison of the degree of relatedness between different genera of filarial nematodes may improve our understanding of their evolutionary origins. Finally, it should be pointed out that not all segments of DNA present in multiple copy numbers in the genome have evolved as rapidly as the Hhal repeat family. This is true, for example, of the noncoding regions of ribosomal genes of filarial helminthslo. References
I 2 3
4
5
6 7 8
9 IO
Yen, P.K.E., Zaman, Y. and Mak, J.W. (I 982)). Helminthol. 56.69-80 Nelson, G.S. (I 979) Nature 300, I I36- I I39 WHO Expert CommIttee on Fllarws Fourth Report (I 984) Technical Report Series No. 702, WHO, Geneva Maina, C.V. et al. In Molecular Paradigms for Eradicating Helminth Parasites (Proc. UCLA Symp) (In press) McReynolds, L.A., DeSlmone, S.M. and Wllllams, S.A. (I 986) Proc. Nat/ Acad. Sci USA 83,797-80 I Sim. B.K., Plessens, W.F. and Worth, D.F. (I 986) Mol. Blochem. Parasitol 19, I 17-l 23 Stm, B.K. et al. (I 986)Am.J Trap Med. Hyg. 35. 559-564 Williams, S.A., DeSlmone, S.M.. Poole, C.B. and McReynolds, L.A. Am. /, Jrop. Med. Hyg. (In press) Palmieri, J, et al. (I 985) Trap. Geogr. Med 37, 239-243 Shah, J.S., Lamontagne, L., Unnasch, T.R, Wirth, D.F. and Piessens, W.F. (1986) Mol. Biochem. Parasftol. I9,67-75
Willy Piessens is at the
Deportment of Tropical
School ofPublic Health, Boston, MA 02 I 15, USA; Larry McReynolds is with New England Biolabs Inc., Beverly, MA 0 I9 15. USA; and Steven Williams is at the Department ofBiological Sciences, Smith College, Northampton, MA 0 1063, USA.
Public Health,
Harvard