Analysis of DNA of Higher Primates Using Inter-SINE PCR

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1 ISSN , Russian Journal of Genetics, 2008, Vol. 44, No. 3, pp Pleiades Publishing, Inc., Original Russian Text N.L. Ryabinina, A.A. Bannikova, V.A. Sheremet eva, M.G. Chikobava, B.A. Lapin, D.A. Kramerov, 2008, published in Genetika, 2008, Vol. 44, No. 3, pp MOLECULAR GENETICS Analysis of DNA of Higher Primates Using Inter-SINE PCR N. L. Ryabinina a, A. A. Bannikova b, V. A. Sheremet eva b, M. G. Chikobava c, B. A. Lapin c, and D. A. Kramerov a a Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia; kramerov@eimb.ru b Moscow State University, Department of Vertebrate Zoology, Moscow, Russia c Research Institute of Medical Primatology, Russian, Sochi Adler, Russia Received April 20, 2007 Abstract Two types (MIR and Alu) of short interspersed repeated DNA sequences (SINEs) were used for analysis of genetic relationships among higher primates, and for detection of polymorphism in human genomic DNA. The DNA regions located between the neighboring copies of these SINEs were amplified in polymerase chain reaction with primers complementary to the MIR and Alu consensus sequences (inter-sine PCR). Comparison of the sets of amplified DNA fragments for different species or individuals provides evaluation of the relationships among them. Using inter-mir PCR technique, the relationships among the higher primates of the infraorder Catarrhini reported elsewhere were confirmed, pointing to the efficiency of the method for phylogenetic studies. No human DNA polymorphism was revealed with the help of inter-mir PCR. This polymorphism was detected by means of inter-alu PCR, which is probably associated with the continuing amplification of Alu elements in human genome. DOI: /S INTRODUCTION The genomes of mammals, as well as of other eukaryotes, are characterized by the presence of a high (10 5 to 10 6 ) number of copies of repeated DNA sequences, defined as short retrotransposons, or SINEs (short interspersed elements) [1]. These elements amplified during the evolution via retroposition, which includes transcription of these elements by RNA polymerase III and reverse transcription associated with the integration of synthesized DNA into new genomic region. Due to mutations accumulated during millions of years, the copies of each SINE, which collectively form the corresponding SINE family, usually differ from each other by 10 to 30% of the bases. Eukaryotic genome contains at most three to four SINE families. Furthermore, each SINE family is typical of one or several animal families (sometimes, orders). The genomes of higher primates contain only two SINE families, Alu and MIR [2]. The first family belongs to the unique type of SINEs, which together with the B1 SINE family of rodents is the descendant of 7SL RNA, the component of cytoplasmic signal recognition particles (SRP), involved into the polypeptide secretion out of the cells. Unlike B1, the Alu element, whose size is about 300 bp, consists of two structurally similar parts (monomers) displaying homology to 7SL RNA [3, 4]. MIR, similarly to most of SINEs from other known families, originate from the trna molecules, which is evidenced by the structure of nucleotide sequence, located in the 5'-terminal ( leading ) part of these short retroposones [5, 6]. One of the specific features of MIRs is their wide distribution. This SINE family is present in the genomes of all mammals, and probably, other vertebrates [7]. This suggests that MIR emerged at least 130 million years ago, which is confirmed by extremely high divergence level of the copies of SINEs of this family (the mean homology between the copies constitutes only 70%). For a yet unknown reason, the most conservative part of MIR element is its central (core) part, located between the trna-relative and tail parts [6, 8]. For this reason, most of the copies, unlike the full-length MIR with the size of about 260 bp, contain only this core sequence with the average size of 65 bp [7]. In recent years, to study phylogenetic relationships among the species, we have elaborated the method of inter-sine PCR [9]. This method was initially used for detection and demonstration of the wide spread of MIR element in mammals [7]. Polymerase reaction of this type results in amplification of the genomic DNA regions with the sizes lower than 3 kb, which are flanked by SINE copies. Amplified DNA fragments are separated by means of electrophoresis in polyacrylamide gel. After this, evaluation of the degree of relatedness among the taxa of species and subspecies levels is possible based on the similarities of the fragment sets obtained. The method of inter-mir PCR was found to rather efficient for evaluation of phylogenetic relation- 266

2 ANALYSIS OF DNA OF HIGHER PRIMATES USING INTER-SINE PCR 267 Geography and the number of samples used in the analysis Geographic attribution of the samples No. of samples Source Homo sapiens Russians, European part of Russia 13 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences Afghans, Afghanistan 1 Moscow State University, Department of Anthropology Tuvinians, Tuva, Todja raion, the settlement 1 Moscow State University, Department of Anthropology of Toora-Khem Evenks, Yakutia, the settlement of Zolotinka 1 Moscow State University, Department of Anthropology Koryaks, Koryak National District, Kamchatka 1 Moscow State University, Department of Anthropology Peninsula Indians, North America 4 Moscow State University, Department of Anthropology Vietnamese, Vietnam 1 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences Malagasy (Antandroy), the Island of Madagascar 1 Moscow State University, Department of Anthropology Nyamwezi, Tanzania 1 Moscow State University, Department of Anthropology Bambra, Mali 1 Moscow State University, Department of Anthropology Toubou, Chad 1 Moscow State University, Department of Anthropology Nigerian populations, Nigeria 1 Moscow State University, Department of Anthropology Ethnic affiliation is unknown, Russia Engelhardt Institute of Molecular Biology, Russian Academy of Sciences Apes from infraorder Catarrhini Pan troglodytes, common chimpanzee 1 Moscow Zoo Gorilla gorilla, gorilla 1 German Primate Center Pongo pygmaeus, Bornean orangutan 1 Moscow Zoo Hylobates lar, white-handed gibbon 1 German Primate Center Macaca silenus, lion-tailed macaque 1 Moscow Zoo Macaca arctoides, stump-tailed macaque 1 Research Institute of Medical Primatology, Russian Macaca mulatta, rhesus monkey 1 Moscow Zoo Papio anubis, olive baboon 1 Research Institute of Medical Primatology, Russian Papio hamadryas, hamadryas baboon 1 Research Institute of Medical Primatology, Russian Chlorocebus aethiops, green monkey 1 Research Institute of Medical Primatology, Russian ships among the species and genera of Chiroptera [10], Insectivora [9, 11, 12], and some rodents [13]. Phylogeny of primates is most thoroughly studied using various molecular techniques based on the analysis of mitochondrial and nuclear genes [14 17]. In the present study, inter-mir PCR was used for the analysis of phylogenetic relationships among higher primates (Catarrhini) in order to test whether the results of this simple and inexpensive method coincide with the data obtained using other techniques. In addition, in this study a new approach to primer design, providing analysis of total genomic DNA of primates by means of inter-alu PCR, was suggested. This PCR technique enabled detection of human DNA polymorphism. MATERIALS AND METHODS DNA isolation and inter-sine PCR. Genomic DNA was extracted from blood cells [18], or from the ethanolfixed tissues (muscles and placenta) through the lysis of tissue homogenates with proteinase K with subsequent deproteinization with phenol chloroform mixture (1 : 1). A list of DNA samples used is presented in the table. Inter-MIR PCR was carried out using two primers complementary to the MIR sequence [7]: 5'-AGT- GACTTGCTCAAGGT-3' (MIR17) and 5'-GCCT- CAGTTTCCTCATC-3' (MIL17). Both primers were labeled with (γ 32 ê)-atp (1 MBq/100 pmol oligonucleotide) with the help of polynucleotide kinase. The reaction mixture (20 µl) contained the buffer (70 mm Tris

3 268 RYABININA et al bp Fig. 1. Inter-MIR PCR of human and ape DNA samples. Homo sapiens: (1 to 24); Apes: common chimpanzee, Pan troglodytes, (25); gorilla, Gorilla gorilla, (26); Bornean orangutan, Pongo pygmaeus, (27); white-handed gibbon, Hylobates lar, (28); lion-tailed macaque, Macaca silenus, (29); stump-tailed macaque, Macaca arctoides, (30); rhesus monkey, Macaca mulatta, (31); olive baboon, Papio anubis, (32); hamadryas baboon, Papio hamadryas, (33); and green monkey, Chlorocebus aethiops, (34). Denatured DNA fragments were separated by means of electrophoresis in polyacrylamide gel with urea. The sizes of molecular size markers (DNA of FKh174, digested with HinfI) are shown to the right of electrophoregram. 150 HCl, ph 8.6; 16.6 mm (NH 4 ) 2 SO 4 ; 2.5 mm MgCl 2 ); 0.2 mm dntp; 4 pmol of each labeled primer; 1 unit of Taq polymerase (Sileks, Russia); and 25 ng genomic DNA. The conditions of inter-mir PCR were as described [7]: denaturing at 94 C for 30 s; annealing at 56 C for 45 s; and extension at 72 C for 2 min. The number of cycles was 27. The initial denaturation was performed at 94 C for 3 min, and final extension at 72 C for 5 min. Inter-Alu PCR was performed with a single primer, 5'-CCACCGCGCCCGGCCAG-3' (the underlined nucleotides are complementary to the 5' end of the Alu

4 ANALYSIS OF DNA OF HIGHER PRIMATES USING INTER-SINE PCR 269 element consensus sequence; in this case PCR occurs only with the involvement of those Alu copies, the flanking sequences of which start from dinucleotide 5'-CT-3'). Oligonucleotide was labeled as described. PCR was performed as in the case of inter-mir PCR, except that primer annealing and extension were conducted at the same temperature (72 C), and the reactions time was 3 min. Labeled PCR products after denaturing were fractionated by electrophoresis in 6% polyacrylamide gel, containing the Tris borate buffer and 8 M urea (the gel length, 50 cm; width, 28 cm; thickness, 0.4 mm). The gel was run for 7 h at 75 W. The gel was incubated in a solution of 5% acetic acid and 10% ethanol for 30 min, dried, and exposed to X-ray film for 18 to 24 h. The protocol for inter-alu PCR with unlabeled primer was analogous to that described above, except for the amount of oligonucleotide in the reaction mixture, which constituted 7.5 pmol. Amplification products were separated overnight by means of electrophoresis in 2% agarose gel (the gel length 20 cm), containing Tris borate buffer. DNA bands were stained with ethidium bromide and photographed in UV light on transilluminator. Phylogenetic analysis. Fingerprints obtained in inter-mir and inter-alu PCR were transformed into a binary matrix (1, band presence; 0, band absence), which was analyzed using maximum parsimony (MP) and neighbor-joining (NJ) procedures implemented in the PAUP*version 4.0b10 software package [19]. Reliability of the dendrogram grouping patterns was assessed by means of bootstrap analysis with 1000 reiterations. Genetic distances (D NL ) were computed according to Nei and Li [20]. NJ MP Hylobates lar Gorilla gorilla Pongo pygmaeus Homo sapiens Pan troglodytes Chlorocebus aethiops Macaca arctoides Macaca silenus Papio anubis Macaca mulatta Papio hamadryas RESULTS AND DISCUSSION PCR reaction performed simultaneously with two primers (MIR17 and MIL17) specific to MIR genetic element [7] results in amplification of the DNA regions located between the neighboring MIR copies independently from their coorientation. Figure 1 presents the results of inter-mir PCR of genomic DNA samples of 24 humans belonging to Eurasian, Asian American, and Equatorial races, and 10 apes from the families Cercopithecidae, Hylobatidae, and Hominidae. The banding patterns observed were practically identical for all human samples (lanes 1 to 24), indicating that no DNA polymorphism was detected using this method. The banding pattern revealed in anthropoid apes (Homonidae) was rather similar to that observed in humans, however, the differences increased in the series from chimpanzee to gorilla, orangutan, and gibbon. As expected, banding pattern differences in other apes (Cercopithecidae) were even higher. Using the parsimonious (MP) and distance (NJ) analyses of inter-mir PCR data, dendrograms were generated (Fig. 2). Topologies of MP and NJ dendrograms were found to be almost identical. The families Cercopithecidae and Hominidae formed two independent branches. The genera Chlorocebus, Macaca, and Papio, belonging to Cercopithecidae, were well-differentiated. The position of the green monkey (Chlorocebus aethiops) on the dendrogram was basal relative to other members of Cercopithecidae examined; Macaca mulatta was closer to M. silenus, than to M. arctoides. In the family Hominidae, gibbon (Hylobates lar) represents a basal branch. Then follows orangutan (Pongo pygmaeus), gorilla (Gorilla gorilla), chimpanzee (Pan troglodytes), and human (Homo sapiens). Although the relative position of P. pygmaeus and G. gorilla is unstable, which is expressed in low bootstrap values at the corresponding nodes, in all other respects the scheme was consistent with the phylogenetic trees of Hominidae, inferred from structural analyses of mitochondrial and nuclear genes [15 17]. Thus, inter-mir PCR Fig. 2. Unrooted dendroghram of phylogenetic relationships of the primates analyzed generated based on inter-mir PCR data and using neighbor-joining method (NJ) based on Nei and Li genetic distances. For clusters, the probability of which is higher 50%, bootstrap indices are shown (in % from 1000 reiterations) above the branches (for neighbor-joining (NJ) analysis), and below the branches (for parsimonious analysis (MP)).

5 270 RYABININA et al. Alu Alu PCR Fig. 3. Scheme of inter-alu PCR variant suggested. Shown are the two copies of Alu element (black boxes) with headto-head localization in the genome. The primer chosen initiates DNA synthesis only in the case that its 3' dinucleotide pairs to the nucleotides in the flanking region. in fact provides adequate evaluation of phylogenetic relationships. Being simple, not labor-consuming, and inexpensive technique, inter-mir PCR seems promising for the analysis of phylogenetic relationships among mammals at the levels of species, genera, and families. It is still unclear why in rather close species some of inter-mir PCR bands are different, and what these differences reflect. MIR is an ancient retrotransposon, which long ago ceased to amplify in placental mammals [5, 7, 8]. For this reason, the differences in the inter-mir PCR banding patterns between the species can not be explained in terms of the integration of new MIR copies in the genome. It seems likely that the differences in the inter-mir PCR banding patterns in different species can be explained by mutations in primercomplementary regions of MIRs, which appeared during the evolution, along with insertions/deletions in the sequences located between the MIR copies. (It should be noted in this respect that it is still unclear, which of these two factors, nucleotide substitutions, or insertions/deletions, makes higher contribution to the change of the banding pattern). As mentioned above, the banding patterns obtained in inter-mir PCR in humans and anthropoid apes are very similar. The genetic distance between humans and chimpanzee is the same as between the Old World monkeys and baboons, D NL = This is much lower than the value of genetic variation revealed using inter-mir PCR in small mammals at the generic level (D NL = ; hedgehogs and wing-handed mammals), and rather corresponds to the genetic distance between the geographically distant populations of these animals (D NL = ) [9, 10]. This finding can probably be explained by lower mutation rate in higher primates, which is caused by longer generation time in higher primates compared to small mammals [21]. Inter-Alu PCR The Alu element is much evolutionary younger than MIR. Contrary to the latter, it remains retropositionaryactive till present, although, according to some estimates, Alu activity has been nearly 100 times reduced during the last 30 million years [22]. The abilty of Alu elements to produce new copies in the human genome gives hope that it will be possible to detect human DNA Fig. 4. Inter-Alu PCR of human and ape genomic DNA samples. Denatured DNA fragments were separated by means of electrophoresis in polyacrylamide gel with urea. Homo sapiens: Indians (1 to 4); Evenk (5); Antandroy (6); Tuvinian (7); Nyamwezi (8); Bambara (9); Toubou (10); Nigerian (11); Vietnamese (12); Caucasoid (13); Negroid of the unknown national affiliation (14). Apes: Pan troglodytes (15); Gorilla gorilla (16); Pongo pygmaeus (17); Hylobates lar (18); Macaca silenus (19); M. mulatta (20); M. arctoides (21); Papio anubis (22); Chlorocebus aethiops (23). thezones of variable fragments are indicated by the arrows.

6 ANALYSIS OF DNA OF HIGHER PRIMATES USING INTER-SINE PCR 271 M Fig. 5. Inter-Alu PCR of human and ape genomic DNA samples. Denatured DNA fragments were separated by means of electrophoresis in 2% agarose gel and stained with ethidium bromide. Homo sapiens: Russians (1, 2, 12 to 16); Indians (3 to 5); Koryak (6); Evenk (7); Nyamwezi (8); Bambra (9); Toubou (10); Nigerian (11); Negroid of the unknown national affiliation (17). Apes: 18, Pan troglodytes; 19, Pongo pygmaeus; 20, Macaca silens. M, DNA fragment molecular size marker. Specific bands are indicated by the black arrows. White arrows mark the band absence detected by means of inter-alupcr in most of humans. polymorphism with the help of inter-alu PCR. Our early experiments performed with the primer complementary to the 5' end of the Alu consensus sequence produced too many bands in sequencing polyacrylamide gels, which was impossible to analyze. Actually, this finding was associated with the presence of a great number of Alu copies ( ) in the human genome, along with their low divergence, compared to MIR copies [2]. In order to reduce the number of bands produced in inter-alu PCR, the primer complementary to the 5'-terminal region of the Alu consensus sequence, but containing an additional 3'-terminal dinucleotide, was used. This dinucleotide was expected to pair with the first two nucleotides of the Alu-flanking DNA sequence (Fig. 3). In such reaction, only part of the Alu copies can serve as a template, thereby reducing the number of PCR products. Figure 4 shows the results of inter-alu PCR with the primer containing additional 3'-terminal AG dinucleotide. The 32 P-labeled PCR products were fractionated by means of electrophoresis in denaturing polyacrylamide gel. In this experiment, a total of 14 human DNA samples, representing different races, and 9 DNA samples from other higher primates were analyzed. PCR products obtained for human and ape DNA samples were remarkably different. PCR banding patterns obtained for different human DNA samples were similar. However, in contrast to inter-mir PCR, some bands present in a number of human samples, were absent from the other ones (marked in the figure). Thus, inter- Alu PCR enables identification of human DNA polymorphism. Another inter-alu PCR variant, which was less expensive and simpler, was also tested. This PCR variant was conducted using the same unlabeled primer, while double-stranded PCR products were fractionated in agarose gel. This modification enabled the analysis of longer DNA fragments (400 to 3000 bp), compared to the first PCR variant, where good band separation was limited to the fragments of about 500 bp in size. Figure 5 demonstrates the results of such analysis of the DNA samples obtained from 17 humans and three ape species. Some of the human samples demonstrated the presence of specific bands, including a 950-bp fragment detected in the Indians and the Koryak (lanes 3 to 6), somewhat longer fragment in some of the Caucasoids (12, 14, 15), as well as the 650- and 750-bp fragments detected in the Nigerian and the Evenk, respectively. There were also the bands absent in the patterns of some humans. For instance, 600-bp fragments were absent in one Indian (3), the Evenk (7), and the Negroid (17). Thus, this inter-alu PCR variant also provided identification of human DNA polymorphism. Concerning individual variable bands, it is still unclear, which changes of the DNA structure underlay this variability. Theoretically, appearance (disappearance) of certain band in the experiments on inter-alu PCR can be caused by the following reasons as follows: (1) integration of new Alu copies in the genome; (2) mutation within the Alu region complementary to the primer used; (3) large deletions or insertions within the DNA regions located between the Alu copies, served for PCR priming. Further sequencing of the variable DNA fragments is hoped to shed the light on

7 272 RYABININA et al. what are the most frequent reasons for polymorphism observed. Up to now, the method of inter-alu PCR was mostly used for mapping of long cloned fragments of the human genome [23]. The variant of inter-alu PCR proposed in the present study makes it possible to use total genomic DNA as a template. This method seems to be promising for the search of human DNA polymorphism, as well as for the analysis of the relationships between the populations. In addition, the approach to inter-alu PCR primers selection used can be also applied in case of the other, relatively young SINEs with extremely high copy number in the genome. ACKNOWLEDGMENTS We are thankful to the staff of the Moscow Zoo and the German Center of Primatology for providing blood and DNA samples. This work was supported by the Russian Foundation for Basic Research (grant nos and ) and the Program Gene Pool Dynamics. REFERENCES 1. Kramerov, D.A. and Vassetzky, N.S., Short Retroposons in Eukaryotic Genomes, Int. Rev. Cytol., 2005, vol. 247, pp Lander, E.S., Linton, L.M., Birren, B., et al., Initial Sequencing and Analysis of the Human Genome, Nature, 2001, vol. 409, pp Deininger, P.L., Jolly, D.J., Rubin, C.M., et al., Base Sequence Studies of 300 Nucleotide Renatured Repeated Human DNA Clones, J. Mol. Biol., 1981, vol. 151, pp Ullu, E. and Tschudi, C., Alu Sequences Are Processed 7SL RNA Genes, Nature, 1984, vol. 312, pp Smit, A.F. and Riggs, A.D., MIRs Are Classic, trna- Derived SINEs that Amplified before the Mammalian Radiation, Nucl. Acids Res., 1995, vol. 23, pp Gilbert, N. and Labuda, D., CORE-SINEs: Eukaryotic Short Interspersed Retroposing Elements with Common Sequence Motifs, Proc. Natl. Acad. Sci. USA, 1999, vol. 96, pp Jurka, J., Zietkiewicz, E., and Labuda, D., Ubiquitous Mammalian-Wide Interspersed Repeats (MIRs) Are Molecular Fossils from the Mesozoic Era, Nucl. Acids Res., 1995, vol. 23, pp Gilbert, N. and Labuda, D., Evolutionary Inventions and Continuity of CORE-SINEs in Mammals, J. Mol. Biol., 2000, vol. 298, pp Bannikova, A.A., Matveev, V.A., and Kramerov, D.A., Using Inter-SINE-RCR to Study Mammalian Phylogeny, Russ. J. Genet., 2002, vol. 38, no. 6, pp Matveev, V.A., Systematics of the Old World Chiropterans Inferred from Dispersed DNA Repeat Analysis, Cand. Sci. (Biol.) Dissertation, Moscow: Moscow State Univ., Bannikova, A.A., Kramerov, D.A., Vasilenko, V.N., et al., DNA Polymorphism in Erinaceus Hedgehogs and Polytypicism of E. concolor (Insestivora, Erinaceidae), Zool. Zh., 2003, vol. 82, pp Bannikova, A.A., Lavrenchenko, L.A., and Kramerov, D.A., Phylogenetic Relationships between Afrotropical and Palaearctic Crocidura Species Inferred from Inter-SINE PCR, Biochem. System. Ecol., 2005, vol. 33, pp Brandler, O.V., Lyapunova, E.A., Kramerov, D.A., et al., Comparative Analysis of Application of Different Molecular Genetic Markers in the Study of the Marmot (Marmota, Sciuridae, Rodentia) Phylogeny and Taxonomy, in Teriofauna Rossii i sopredel nykh territorii (Theriofauna of Russia and Adjacent Territories), Moscow: KMK, Goodman, M., Porter, C.A., Czelusniak, J., et al., Toward a Phylogenetic Classification of Primates Based on DNA Evidence Complemented by Fossil Evidence, Mol. Phylogenet. Evol., 1998, vol. 9, pp Purvis, A., A Composite Estimate of Primate Phylogeny, Philos. Trans. R. Soc. Lond. Biol. Sci., 1995, vol. 348, pp Arnason, U., Gullberg, A., Janke, A., et al., Pattern and Timing of Evolutionary Divergences among Hominoids Based on Analyses of Complete mtdnas, J. Mol. Evol., 1996, vol. 43, pp Mathew, C.C., The Isolation of High Molecular Weight Eukaryotic DNA, in Methods in Molecular Biology, Walker, J.M., Ed., New York: Humana, 1984, pp Swofford, D.L., Phylogenetic Analysis Using Parsimony (and Other Methods), Version 4, Sunderland: Sinauer, Nei, M. and Li, W.H., Mathematical Model for Studying Genetic Variation in Terms of Restriction Endonucleases, Proc. Natl. Acad. Sci. USA, 1979, vol. 76, pp Li, W.H., Ellsworth, D.L., Krushkal, J., et al., Rates of Nucleotide Substitution in Primates and Rodents and the Generation Time Effect Hypothesis, Mol. Phylogenet. Evol., 1996, vol. 5, pp Britten, R.J., Evidence That Most Human Alu Sequences Were Inserted in a Process That Ceased about 30 Million Years Ago, Proc. Natl. Acad. Sci. USA, 1994, vol. 91, pp Kass, D.H. and Batzer, M.A., Inter-Alu Polymerase Chain Reaction: Advancements and Applications, Anal. Biochem., 1995, vol. 228, pp