复旦大学：《人类进化 Human Evolution》参考文献_分子人类学_Neandertal DNA Sequences and the Origin of Modern Humans

Cell, Vol. 90, 19-30, July 11, 1997, Copyright @1997 by Cell Press Neandertal DNA Sequences and the origin of Modern Humans Matthias Krings, * Anne Stone, t Ralf W. Schmitz, t these analyses rely on assumptions, such as the ab Heike Krainitzki, 5 Mark Stoneking, t and Svante Paabo* sence of selection and a clock-like rate of mole volution in the DNA sequences under study, whose University of Munich alidity has been questioned (Wolpoff, 1989: Templeton Box202136 992). An additional and more direct way to address the D-80021 Munich question of the relationship between modern humans nd Neandertals would be to analyze DNA sequences t Department of Anthropology from the remains of neandertals nsylvania State University The reproducible retrieval of ancient DNA sequences State College, Pennsylvania 16802 became possible with the invention of the polymerase t Rheinisches Amt fur Bodendenkmalpflege hain reaction (Mullis and Faloona, 1987: Paabo et al Endenicher Strasse 133 D-53115 Bonn ilson, 1991; Lindahl 1993a) as well as empirical studies Germany (Paabo, 1989: Hoss et al. 1996a), show that dNa in 5Hohere berufsfachschule fur fossil remains is highly affected by hydrolytic as well praparationstechnische Assistenten as oxidative damage. Therefore, the retrieval of DNA Markstrasse 185 sequences older than about 100,000 years is expected 44799 Bochum to be difficult, if not impossible, to achieve(Paabo and Germany Wilson, 1991). Fortunately, Neandertal remains fall most fail to yield ampl的hym within the age range that in principle allow uences to survive. It is noteworth at even Summary mong remains that are yo DNA was extracted from the Neandertal-type speci- 996b) In addition, contamination of ancient specimens men found in 1856 in western Germany By sequencing and extracts with modern DNA poses a serious probler clones from short overlapping PCR products, a hith (Handt et al. 1994a)thatrequires numerous precautions erto unknown mitochondrial (mt) DNA sequence was ind controls. This is particularly the case when human determined. Multiple controls indicate that this se- remains are studied, since human dna is the most com quence is endogenous to the fossil. Sequence com- mon source of contamination. Therefore, a number of arisons with human mtDNA sequences, as well as tic analyses, show that the Neandertal se- mined from extracts of an ancient specimen can be quence falls outside the variation of modern humans. taken to be genuine (Paabo et al. 1989 Lindahl, 1993b Furthermore, the age of the common ancestor of the Handt et al. 1994a: Handt et al. 1996 Neandertal and modern human mtDNAs is estimated Since 1991, the Neandertal-type specimen, found in to be four times greater than that of the common an- 1856 near Dusseldorf, Germany, has been the subject cestor of human mt DNAS. This suggests that Neander- of an interdisciplinary project of the Rheinisches tals went extinct without contributing mtDNA to mod Landesmuseum Bonn, initiated and led by R. W. s enn numan (Schmitz et al. 1995: Schmitz, 1996). As a part of thi project, a sample was removed from the Neandert specimen for DNA analysis. Here, we present the se- quence of a hypervariable part of the mtDNA control region derived from this sample. We describe the evi Neandertals are a group of extinct hominids that inhab nce in support of its authenticity and analyze the rela ited Europe and western Asia from about 300,000 to ionship of this sequence to the contemporary human 30,000 years ago. During part of this time they coexisted mtDNa gene pool with modern humans. Based on morphological compari sons, it has been variously claimed that Neandertals: (1)were the direct ancestors of modern Europeans; (2) Results contributed some genes to modern humans; or(3)were completely replaced by modern humans without con- Amino Acid racemization tributing any genes (reviewed in Stringer and Gamble, 3.5 g section of the right humerus was removed from 1993: Trinkaus and Shipman 1993: Brauer and Stringer, the Neandertal fossil(Figure 1). It has previously been 1997). Analyses of molecular genetic variation in the shown that ancient specimens exhibiting high levels of mitochondrial and nuclear genomes of contemporary amino acid racemization do not contain sufficient DNA human populations have generally supported the third or analysis(Poinar et al., 1996). To investigate wheti view, i.e., that Neandertals were a separate species that the state of preservation of the fossil is compatible with went extinct without contributing genes to modern hu- DNA retrieval, we therefore analyzed the extent of amino mans(Cann et al. 1987; Vigilant et al., 1991; Hammer, acid racemization. Samples of 10 mg were removed 1995; Armour et al., 1996 Tishkoff et al., 1996). However, from the periostal surface of the bone from the compact

Cell, Vol. 90, 19–30, July 11, 1997, Copyright 1997 by Cell Press Neandertal DNA Sequences and the Origin of Modern Humans Matthias Krings,* Anne Stone,† Ralf W. Schmitz,‡ these analyses rely on assumptions, such as the ab￾Heike Krainitzki, sence of selection and a clock-like rate of molecular § Mark Stoneking,† and Svante Pa¨ a¨ bo* *Zoological Institute evolution in the DNA sequences under study, whose University of Munich validity has been questioned (Wolpoff, 1989; Templeton, PO Box 202136 1992). An additional and more direct way to address the D-80021 Munich question of the relationship between modern humans Germany and Neandertals would be to analyze DNA sequences from the remains of Neandertals. †Department of Anthropology Pennsylvania State University The reproducible retrieval of ancient DNA sequences State College, Pennsylvania 16802 became possible with the invention of the polymerase ‡Rheinisches Amt fu¨r Bodendenkmalpflege chain reaction (Mullis and Faloona, 1987; Pa¨ a¨bo et al., Endenicher Strasse 133 1989). However, theoretical considerations, (Pa¨ a¨bo and D-53115 Bonn Wilson, 1991; Lindahl 1993a) as well as empirical studies Germany (Pa¨ a¨bo, 1989; Ho¨ ss et al., 1996a), show that DNA in §Ho¨ here Berufsfachschule fu¨r fossil remains is highly affected by hydrolytic as well pra¨parationstechnische Assistenten as oxidative damage. Therefore, the retrieval of DNA Markstrasse 185 sequences older than about 100,000 years is expected D-44799 Bochum to be difficult, if not impossible, to achieve (Pa¨ a¨bo and Germany Wilson, 1991). Fortunately, Neandertal remains fall within the age range that in principle allows DNA se￾quences to survive. It is noteworthy, though, that even Summary among remains that are younger than 100,000 years most failto yield amplifiable DNA sequences(Ho¨ ss et al., DNA was extracted from the Neandertal-type speci- 1996b). In addition, contamination of ancient specimens men found in 1856 in western Germany. By sequencing and extracts with modern DNA poses a serious problem clones from short overlapping PCR products, a hith- (Handt et al., 1994a) that requires numerous precautions and controls. This is particularly the case when human erto unknown mitochondrial (mt) DNA sequence was determined. Multiple controls indicate that this se- remains are studied, since human DNA is the most com￾quence is endogenous to the fossil. Sequence com- mon source of contamination. Therefore, a number of parisons with human mtDNA sequences, as well as criteria need to befulfilled before a DNA sequence deter￾phylogenetic analyses, show that the Neandertal se- mined from extracts of an ancient specimen can be quence falls outside the variation of modern humans. taken to be genuine (Pa¨a¨bo et al., 1989; Lindahl, 1993b; Furthermore, the age of the common ancestor of the Handt et al., 1994a; Handt et al., 1996). Neandertal and modern human mtDNAs is estimated Since 1991, the Neandertal-type specimen, found in 1856 near Du¨ sseldorf, Germany, has been the subject to be four times greater than that of the common an￾cestor of human mtDNAs. This suggests that Neander- of an interdisciplinary project of the Rheinisches Landesmuseum Bonn, initiated and led by R. W. S. tals went extinct without contributing mtDNA to mod￾ern humans. (Schmitz et al., 1995; Schmitz, 1996). As a part of this project, a sample was removed from the Neandertal specimen for DNA analysis. Here, we present the se￾quence of a hypervariable part of the mtDNA control Introduction region derived from this sample. We describe the evi￾dence in support of its authenticity and analyze the rela- Neandertals are a group of extinct hominids that inhab- tionship of this sequence to the contemporary human ited Europe and western Asia from about 300,000 to mtDNA gene pool. 30,000 years ago. During part of this time they coexisted with modern humans. Based onmorphological compari￾sons, it has been variously claimed that Neandertals: (1) were the direct ancestors of modern Europeans; (2) Results contributed some genes to modern humans; or (3) were completely replaced by modern humans without con- Amino Acid Racemization tributing any genes (reviewed in Stringer and Gamble, A 3.5 g section of the right humerus was removed from 1993; Trinkaus and Shipman 1993; Bra¨ uer and Stringer, the Neandertal fossil (Figure 1). It has previously been 1997). Analyses of molecular genetic variation in the shown that ancient specimens exhibiting high levels of mitochondrial and nuclear genomes of contemporary amino acid racemization do not contain sufficient DNA human populations have generally supported the third for analysis (Poinar et al., 1996). To investigate whether view, i.e., that Neandertals were a separate species that the state of preservation of the fossil is compatible with went extinct without contributing genes to modern hu- DNA retrieval, we therefore analyzed the extent of amino mans (Cann et al., 1987; Vigilant et al., 1991; Hammer, acid racemization. Samples of 10 mg were removed 1995; Armour et al., 1996; Tishkoff et al., 1996). However, from the periostal surface of the bone, from the compact

tends to be degraded and damaged to an extent that makes amplification of segments of mtDNA longer than 100-200 bp difficult(Paabo, 1989). Therefore, two prim ers(L16, 209, H16, 271) that amplify a 105-bp-segment of the human mtDNA control region(including primers were used to perform amplifications from the bone ex- tract as well as from an extraction control. a tion product was obtained in the bone extract but not in the control(data not shown). In a subsequent experi- ment, this was repeated and the same results were ob Sequence Variation of the Amplification Product igure 1. Sample Removed from the Right Humerus of the Neander- The two amplification products were cloned in a plasmid tal-Type Specimen vector and 18 and 12 clones, respectively were se- uenced(Figure 2, extract A). Twenty-two of the 30 cortical bone and from the endostal surface of the mar clones contained seven nucleotide substitutions and row cavity. Samples were also removed from remnants one insertion of an adenine residue, when compared to of a varnish, with which the specimen has been treated the standard human reference sequence(Anderson et at least twice. The samples were hydrolyzed under acid al., 1981). Three of these eight differences to the refer conditions, and the released amino acids were analy. ence sequence were individually lacking in a total of five using high performance liquid chromatography and fluo- of the clones. In addition, among the 27 clones were rescent detection(Poinar et al., 1996). Table 1 shows nine differences that each occurred in one clone, three that the total amounts of the amino acids detected in differences that occurred in two clones and one that the neandertal bone are 20%-73% of those in modern occurred in three clones, respectively. Such changes bone and more than two orders of magnitude higher that are present in only a few clones are likely to be than in the varnish, indicating that the results do not due to misincorporations by the DNA polymerase during more, the absolute and relative amounts of the amino DNA. In addition some of these could be due to mito acids analyzed (e.g, the ratio of glycine to aspartic acid chondrial heteroplasmy, which may be more common are similar in the three Neandertal samples and compa in humans than often assumed(Comas et al. 1995 rable to those of a contemporary bone. Most impor Ivanovet al 1996)and is abundant in some mammalian tantly, the ratio of the D to the L enantiomers of aspartic species(Petriet al 1996) Of theremaining three clones, acid in the three Neandertal samples varies between two were identical to the reference sequence, and the 0 11 and 0. 12, which is in the range compatible with third clone differed from the reference sequence at one DNA survival( Poinar et al. 1996). Thus, the extent of position amino acid racemization in the Neandertal fossil sug Thus, the amplification product was composed of two gests that it may contain amplifiable DNA three clones that is similar to the human reference se- DNA Extraction and Amplification quence, and another class represented by 27 clones DNA was extracted from 0. 4 g of the cortical compact hat exhibits substantial differences from it. the former bone. Previous experience shows that ancient dna class of molecules probably reflects contamination of Table 1. Racemization Results for Three Neandertal Bone Samples, Varnish from the Neandertal Fossil, and Modern Bone Periostal Surface Compact Bone Endostal surface Modern bone Valine(%) 23 0.11 0.114 0.110 D/L alanine 0.006 0.007 0.004 008 D/L leucine amino acid analysis of the Neandertal bone, va emoved from the bone surface, and a two-year-old bone sample no acid compositions in percentages of the eight yzed, and the D/L-ratios for three amino acids. NI electable d form

Cell 20 tends to be degraded and damaged to an extent that makes amplification of segments of mtDNA longer than 100–200 bp difficult (Pa¨a¨ bo, 1989). Therefore, two prim￾ers (L16,209, H16,271) that amplify a 105-bp-segment of the human mtDNA control region (including primers) were used to perform amplifications from the bone ex￾tract as well as from an extraction control. An amplifica￾tion product was obtained in the bone extract but not in the control (data not shown). In a subsequent experi￾ment, this was repeated and the same results were ob￾tained. Sequence Variation of the Amplification Product Figure 1. Sample Removed from the Right Humerus of the Neander- The two amplification products were cloned in a plasmid tal-Type Specimen vector and 18 and 12 clones, respectively, were se￾quenced (Figure 2, extract A). Twenty-two of the 30 cortical bone, and from the endostal surface of the mar- clones contained seven nucleotide substitutions and row cavity. Samples were also removed from remnants one insertion of an adenine residue, when compared to of a varnish, with which the specimen has been treated the standard human reference sequence (Anderson et al., 1981). Three of these eight differences to the refer- at least twice. The samples were hydrolyzed under acid ence sequence were individually lacking in a total of five conditions, and the released amino acids were analyzed using high performance liquid chromatography and fluo- of the clones. In addition, among the 27 clones were rescent detection (Poinar et al., 1996). Table 1 shows nine differences that each occurred in one clone, three differences that occurred in two clones and one that that the total amounts of the amino acids detected in the Neandertal bone are 20%–73% of those in modern occurred in three clones, respectively. Such changes bone and more than two orders of magnitude higher that are present in only a few clones are likely to be than in the varnish, indicating that the results do not due to misincorporations by the DNA polymerase during reflect the amino acid content of the varnish. Further- PCR, possibly compounded by damage in the template more, the absolute and relative amounts of the amino DNA. In addition, some of these could be due to mito￾acids analyzed (e.g., the ratio of glycine to aspartic acid) chondrial heteroplasmy, which may be more common are similar in the three Neandertal samples and compa- in humans than often assumed (Comas et al., 1995; rable to those of a contemporary bone. Most impor- Ivanov et al., 1996) and is abundant in some mammalian tantly, the ratio of the D to the L enantiomers of aspartic species (Petri et al., 1996). Of theremaining three clones, acid in the three Neandertal samples varies between two were identical to the reference sequence, and the 0.11 and 0.12, which is in the range compatible with third clone differed from the reference sequence at one DNA survival (Poinar et al., 1996). Thus, the extent of position. amino acid racemization in the Neandertal fossil sug- Thus, the amplification product was composed of two gests that it may contain amplifiable DNA. classes of sequences, a minor class represented by three clones that is similar to the human reference se￾DNA Extraction and Amplification quence, and another class represented by 27 clones DNA was extracted from 0.4 g of the cortical compact that exhibits substantial differences from it. The former bone. Previous experience shows that ancient DNA class of molecules probably reflects contamination of Table 1. Racemization Results for Three Neandertal Bone Samples, Varnish from the Neandertal Fossil, and Modern Bone Periostal Surface Compact Bone Endostal Surface Varnish Modern Bone Total (ppm) 23,167 83,135 53,888 145 113,931 Aspartic acid (%) 7.8 8.3 7.4 10 8.3 Serine (%) 0.7 0.7 0.7 2 0.6 Glutamic acid (%) 20.2 20.1 20.2 22 19.9 Glycine (%) 49.5 49.0 50.2 22 51.8 Alanine (%) 14.4 14.0 14.0 11 11.1 Valine (%) 3.5 3.9 3.9 23 3.9 Isoleucine (%) 0.5 0.5 0.6 1 0.7 Leucine (%) 3.4 3.3 3.2 9 3.6 Glycine/aspartic acid 6.3 5.9 6.8 2.1 6.2 D/L aspartic acid 0.117 0.114 0.110 ND 0.05 D/L alanine 0.006 0.007 0.004 0.08 0.01 D/L leucine 0.005 ND ND ND ND Comparison of the amino acid analysis of the Neandertal bone, varnish removed from the bone surface, and a two-year-old bone sample. Given are the total amounts of the amino acids analyzed (ppm, parts per million), the amino acid compositions in percentages of the eight amino acids analyzed, and the D/L-ratios for three amino acids. ND, no detectable D form

CAGCAATCRACCCTCAACTATCACACATCAACTOCAACTCcRAAGCCACCCCT-CRCCC c5001002040 Figure 3. Quantitation of the Putative Neandertal mtDNA with a 12 bp of cd LWcompetitor molecules added are indicated. The control R17: amplification (C)contained neither competitor nor Neandertal ex- 1996). Therefore, the number of template molecules rep resenting the putative Neandertal sequence in the ex tract was determined by quantitative PCR. To this end a molecule representing the putative Neandertal se- quence but carrying a 12 bp deletion was constructed To each step in a dilution series of this construct, a constant amount of extract was added and amplifica tions were performed using primers that are specific for a 104 bp product of the putative Neandertal sequenc Figure 2. The DNA Sequ of Clones Derived from Four Amplifi and that do not amplify contemporary human se- cations of the Mitochondrial Control Region from the Neandertal uences. The results(Figure 3)show that on the order of 10 putative Neandertal molecules exist per microliter man reference sequence(Anderson e of extract and thus that amplifications starting from 5 al, 1981)given above. The clone designations consist of a letter(A, I of extract are initiated from approximately 50 template B, C)indicating the DNA extract followed by a number indicating ion, as well as a number after the period molecules. however due to variation in the efficien dentifying the particular clone. Extracts A and B were performed at the University of Munich: extract C, at Penn State University number of template molecules added to an individual Clones derived from different amplifications are separated by a amplification, some amplifications may start from fewer (or even single)molecules. This makes nucleotide misin- one clone differs from the majority of sequence corporations in earby cycles of the amplification reaction pper amplifications (performed at the University of Munich) primers 16, 209(5'-CCC CAT GCT TAC AAG CAA GT-3)and H16, 271(5 ikely to affect a large proportion of the molecules in the ers NL16, 230(5-GCA CAG CAA TCA ACC TTC AAC TG-3) and carry miscoding base modifications(Hoss et al. 1996a) NH16, 262(5'-GTA GAT TTG TTG ATA TCC TAG TGG GTG TAA-3) To detect this type of sequence change, amplifications were used were performed such that each sequence position to be determined was covered by at least two independent PCR reactions. The products of each PCR reaction were the specimen, which is likely to have occurred during independently cloned and the sequences determined handling and treatment of the specimen during the 140 from multiple clones years since its discovery. The other class of sequences is not obviously of modern origin. Further experiments Authenticity of Sequences were therefore performed to determine if this class is The inadvertent amplification of small amounts of con- endogenous to the Neandertal fossil temporary DNA is a major source of erroneous results in the study of ancient DNA sequences(Paabo et al 1989: Lindahl, 1993b; Handt et al. 1994a. Such contami- Quantitation of putative Neandertal dna lation may result in the amplification of not only contem Amplifications that start from more than 1000 ancient porary organellar mtDNA template molecules tend to yield reproducible results of mtDNA(Collura and Stewart, 1995; van der Kuyl et while amplifications starting from fewer molecules tend al., 1995; Zischler et al., 1995). Several experiments were to yield results that vary between experiments, due to performed in order to exclude modern DNA, including a misincorporations during the early cycles of the PCR as nuclear insertion of mtDNA, as the source of the putative ell as due to sporadic contamination(Handt et al

Neandertal mtDNA Sequence 21 Figure 3. Quantitation of the Putative Neandertal mtDNA A dilution series of a competitor construct carrying the putative Neandertal sequence with a 12 bp deletion was added to 2.5 ml of extract A from the fossil. Primers used were specific for the putative Neandertal sequence. Above the lanes, the approximate numbers of cd LWcompetitor molecules added are indicated. The control amplification (C) contained neither competitor nor Neandertal ex￾tract. 1996). Therefore, the number of template molecules rep￾resenting the putative Neandertal sequence in the ex￾tract was determined by quantitative PCR. To this end, a molecule representing the putative Neandertal se￾quence but carrying a 12 bp deletion was constructed. To each step in a dilution series of this construct, a constant amount of extract was added and amplifica￾tions were performed using primers that are specific for a 104 bp product of the putative Neandertal sequence Figure 2. The DNA Sequences of Clones Derived from Four Amplifi- and that do not amplify contemporary human se￾cations of the Mitochondrial Control Region from the Neandertal quences. The results (Figure 3) show that on the order Fossil of 10 putative Neandertal molecules exist per microliter Dots indicate identity to a human reference sequence (Anderson et of extract and thus that amplifications starting from 5 al., 1981) given above. The clone designations consist of a letter (A, ml of extract are initiated from approximately 50 template B, C) indicating the DNA extract followed by a number indicating molecules. However, due to variation in the efficiency the amplification reaction, as well as a number after the period of individual primer pairs, and stochastic variation in the identifying the particular clone. Extracts A and B were performed number of template molecules added to an individual at the University of Munich; extract C, at Penn State University. Clones derived from different amplifications are separated by a amplification, some amplifications may start from fewer blank line. Asterisks identify sequence positions where more than (or even single) molecules. This makes nucleotide misin￾one clone differs from the majority of sequences. For the three corporations in early cycles of the amplification reaction upper amplifications (performed at the University of Munich) primers likely to affect a large proportion of the molecules in the L16,209 (59-CCC CAT GCT TAC AAG CAA GT-39) and H16,271 (59- final amplification product. Such misincorporations may GTG GGT AGG TTT GTT GGT ATC CTA-39) were used. For the bottom amplification (performed at Penn State University) the prim- be frequent since the template molecules are likely to ers NL16,230 (59-GCA CAG CAA TCA ACC TTC AAC TG-39) and carry miscoding base modifications (Ho¨ ss et al., 1996a). NH16,262 (59-GTA GAT TTG TTG ATA TCC TAG TGG GTG TAA-39) To detect this type of sequence change, amplifications were used. were performed such that each sequence position to be determined was covered by at least two independent PCR reactions. The products of each PCR reaction were the specimen, which is likely to have occurred during independently cloned and the sequences determined handling and treatment of the specimen during the 140 from multiple clones. years since its discovery. The other class of sequences is not obviously of modern origin. Further experiments Authenticity of Sequences were therefore performed to determine if this class is The inadvertent amplification of small amounts of con￾endogenous to the Neandertal fossil. temporary DNA is a major source of erroneous results in the study of ancient DNA sequences (Pa¨ a¨bo et al., 1989; Lindahl, 1993b; Handt et al.,1994a). Such contami￾Quantitation of Putative Neandertal DNA nation may result inthe amplification of not only contem￾Amplifications that start from more than 1000 ancient porary organellar mtDNA but also of nuclear insertions template molecules tend to yield reproducible results, of mtDNA (Collura and Stewart, 1995; van der Kuyl et while amplifications starting from fewer molecules tend al., 1995; Zischler et al., 1995). Several experiments were to yield results that vary between experiments, due to performed in order to exclude modern DNA, including a misincorporations during the early cycles of the PCR as nuclear insertion of mtDNA,as the source of the putative well as due to sporadic contamination (Handt et al., Neandertal sequence

Since nuclear insertions are less numerous than mito from 0. 4 g of the bone. When the primers L16, 209 and hondrial genomes in the organelles, any single insertion H16, 271 were used in an amplification from this extract sequence is expected to represent a major proportion and the product cloned( Figure 2, extract B), ten clones of an amplification product only in cases where a prime arried the eight differences from the reference se- favors the amplification of an insertion sequence over quence observed in the amplifications from the first ex the corresponding mtDNA sequence. This occurs when tract, as well as two changes affecting single clones. mismatches to the primer in the mtDNA make the prim- In addition, three sequence positions carried changes ing of an insertion more efficient than that of the organel occurring in five and four clones. These changes were lar mtDNA(Handt et al., 1996). Therefore, the preferential not observed in combination in the previous four amplifi amplification of an insertion sequence is expected to cations covering this sequence segment. Since they oc- be restricted to a particular primer. In order to elucidate curred in only one amplification product, they are proba whether the putative Neandertal sequence is seen only bly due to polymerase errors in the early cycles of the when a particular primer is used, primers were ex- PCR, possibly compounded by in vitro recombination changed such that first the 5 primer was replaced by induced by damage and degradation of template DNA a primer located outside the previous amplification prod- molecules(Paabo et al. 1990). Four clones were similar ct(L16, 122), and 13 clones of this amplification product to the human reference sequence. Thus, although the were sequenced (Figure 4, clones A7 1-13). All 13 clones amplification products clearly derive from few template showed the same eight differences from the reference molecules, the putative Neandertal sequence is present sequence that were previously observed, as well as nine in a DNA extract independently prepared from the fossil differences in the region that was not included in the To further investigate whether the results are due to earlier amplification. In addition, one difference was laboratory-specific artifacts or contamination, an addi observed in one clone, as well as length variation in tional bone sample of 0. 4 g was sent to the Anthr a homopolymer of cytosine residues, previously de- cal Genetics Laboratory at Pennsylvania State Univer- scribed to be of variable length in humans( Bendall and sity where a DNA extraction was performed. When the Sykes, 1995) primers(L16, 209 and H16, 271), which had previous The 3'-primer from the first amplification was the replaced by a primer (H16, 379)located outside the initial Neandertal sequence and contemporary human mtDNA amplification product, and 13 clones of this amplification sequences(Figure 2)were used in amplifications from product were sequenced(Figure 5, clones A121-13). this extract, 15 of the resulting clones yielded a DNA All 13 clones contained the same eight differences in sequence that was identical to the experimenter(A s ) the region overlapping the previous amplifications, as while two yielded sequences that differed by one and well as seven differences in the region not covered in two substitutions from the reference sequence, respec- the previous amplifications. In addition, two substitu- tively. However, when primers specific for the putative tions and one deletion occurred in one clone, and one Neandertal sequence(NL16, 230 and NH16, 262)were other substitution occurred in a different clone furthe used, 5 out of 5 clones yielded the putative Neandertal more, in a subsequent amplification from another ex- sequence(Figure 2, extract C). Thus, while this third tract where both primers (L16, 254-H16, 379)differed independent extract contains a larger amount of con- from the initial amplification, all four differences located temporary human DNA, probably stemming from labora in the segment included in the first amplifications were ory contamination, it confirms that the putative Nean observed in all 8 clones sequenced ( Figure 5, clones dertal sequence is present in the fossil specime B13.1-8). Thus, the retrieval of the putative Neandertal quence is not dependent on the primers used. Fu tive Neandertal sequence does not originate from a nu thermore, most primer combinations yield a large ex- clear mtDNA insertion and that it is endogenous to the cess of clones representing the putative Neandertal se fossil. it furthermore falls outside the variation of the quence over clones similar to contemporary human mtDNa gene pool of modern humans (see below). We tDNA therefore conclude that it is derived from the mitochon To further exclude the possibility that the sequence drial genome of the Neandertal individuaL. may represent a nuclear insertion, primers for the puta tive Neandertal sequence were constructed that do not amplify human mtDNA In control experiments where Determination of the Neandertal mtDNA Sequence various amounts of a cloned copy of the putative Nean. The entire sequence of hypervariable region I of the dertal sequence were mixed with human DNA, these mtDNA control region(positions 16,023 to 16, 400: An erson et al. 1981) was determine cloned sequence in 50 ng of total human DNA, i.e., less preservation of the DNA allowed only short fragments to than one copy per genome equivalent(data not shown). be amplified, this was achieved by several overlapping When these primers were used to amplify DNA isolated amplifications. Furthermore, since the quantitation ex- from 15 Africans, 6 Europeans, and 2 Asians, no amplifi eriments indicated that some al ations might start cation products were obtained(data not shown), indicat- from single molecules, and thus that misincorporations ing that this sequence is not present in the genome of in early cycles of the amplification might be misinter modern humans reted as sequence differences(Handt et al. 1996), all To test whether the extraction and amplification of sequence positions were determined from at least two the putative Neandertal sequence is reproducible, an independent amplifications At five sequence positions dditional independent DNA extraction was performed two amplifications yielded discordant results, i.e., all

Cell 22 Since nuclear insertions are less numerous than mito- from 0.4 g of the bone. When the primers L16,209 and chondrial genomes inthe organelles, any single insertion H16,271 were used in an amplification from this extract sequence is expected to represent a major proportion and the product cloned (Figure 2, extract B), ten clones of an amplification product only in cases where a primer carried the eight differences from the reference se￾favors the amplification of an insertion sequence over quence observed in the amplifications from the first ex￾the corresponding mtDNA sequence. This occurs when tract, as well as two changes affecting single clones. mismatches to the primer in the mtDNA make the prim- In addition, three sequence positions carried changes ing of an insertion more efficient than that of the organel- occurring in five and four clones. These changes were lar mtDNA (Handt et al., 1996). Therefore, thepreferential not observed in combination in the previous four amplifi￾amplification of an insertion sequence is expected to cations covering this sequence segment. Since they oc￾be restricted to a particular primer. In order to elucidate curred in only one amplification product, they are proba￾whether the putative Neandertal sequence is seen only bly due to polymerase errors in the early cycles of the when a particular primer is used, primers were ex- PCR, possibly compounded by in vitro recombination changed such that first the 59 primer was replaced by induced by damage and degradation of template DNA a primer located outside the previous amplification prod- molecules (Pa¨a¨bo et al., 1990). Four clones were similar uct (L16,122), and 13 clones of this amplification product to the human reference sequence. Thus, although the were sequenced (Figure 4, clones A7.1–13). All 13 clones amplification products clearly derive from few template showed the same eight differences from the reference molecules, the putative Neandertal sequence is present sequence that were previously observed, as well as nine in a DNA extract independently prepared from the fossil. differences in the region that was not included in the To further investigate whether the results are due to earlier amplification. In addition, one difference was laboratory-specific artifacts or contamination, an addi￾observed in one clone, as well as length variation in tional bone sample of 0.4 g was sent to the Anthropologi￾a homopolymer of cytosine residues, previously de- cal Genetics Laboratory at Pennsylvania State Univer￾scribed to be of variable length in humans (Bendall and sity where a DNA extraction was performed. When the Sykes, 1995). primers (L16,209 and H16,271), which had previously The 39-primer from the first amplification was then resulted in a product that contained both the putative replaced by a primer (H16,379) located outside the initial Neandertal sequence and contemporary human mtDNA amplification product, and 13clones of this amplification sequences (Figure 2) were used in amplifications from product were sequenced (Figure 5, clones A12.1–13). this extract, 15 of the resulting clones yielded a DNA All 13 clones contained the same eight differences in sequence that was identical to the experimenter (A. S.), the region overlapping the previous amplifications, as while two yielded sequences that differed by one and well as seven differences in the region not covered in two substitutions from the reference sequence, respec￾the previous amplifications. In addition, two substitu- tively. However, when primers specific for the putative tions and one deletion occurred in one clone, and one Neandertal sequence (NL16,230 and NH16,262) were other substitution occurred in a different clone. Further- used, 5 out of 5 clones yielded the putative Neandertal more, in a subsequent amplification from another ex- sequence (Figure 2, extract C). Thus, while this third tract where both primers (L16,254-H16,379) differed independent extract contains a larger amount of con￾from the initial amplification, all four differences located temporary human DNA, probably stemming from labora￾in the segment included in the first amplifications were tory contamination, it confirms that the putative Nean￾observed in all 8 clones sequenced (Figure 5, clones dertal sequence is present in the fossil specimen. B13.1–8). Thus, the retrieval of the putative Neandertal In summary, these experiments indicate that the puta￾sequence is not dependent on the primers used. Fur- tive Neandertal sequence does not originate from a nu￾thermore, most primer combinations yield a large ex- clear mtDNA insertion and that it is endogenous to the cess of clones representing the putative Neandertal se- fossil. It furthermore falls outside the variation of the quence over clones similar to contemporary human mtDNA gene pool of modern humans (see below). We mtDNA. therefore conclude that it is derived from the mitochon￾To further exclude the possibility that the sequence drial genome of the Neandertal individual. may represent a nuclear insertion, primers for the puta￾tive Neandertal sequence were constructed that do not amplify human mtDNA. In control experiments where Determination of the Neandertal mtDNA Sequence various amounts of a cloned copy of the putative Nean- The entire sequence of hypervariable region I of the dertal sequence were mixed with human DNA, these mtDNA control region (positions 16,023 to 16,400; An￾primers were able to detect about 20 copies of the derson et al., 1981) was determined. Since the state of cloned sequence in 50 ng of total human DNA, i.e., less preservation of the DNA allowed only short fragments to than one copy per genome equivalent (data not shown). be amplified, this was achieved by several overlapping When these primers were used to amplify DNA isolated amplifications. Furthermore, since the quantitation ex￾from 15 Africans, 6 Europeans, and 2 Asians, no amplifi- periments indicated that some amplifications might start cation products were obtained (data not shown),indicat- from single molecules, and thus that misincorporations ing that this sequence is not present in the genome of in early cycles of the amplification might be misinter￾modern humans. preted as sequence differences (Handt et al., 1996), all To test whether the extraction and amplification of sequence positions were determined from at least two the putative Neandertal sequence is reproducible, an independent amplifications. At five sequence positions, additional independent DNA extraction was performed two amplifications yielded discordant results, i.e., all

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Neandertal mtDNA Sequence 23 Figures 4. The DNA Sequences of Clones Used to Infer the Sequence of the Hypervariable Region I of the Neandertal Individual Above, the modern human reference sequence (Anderson et al., 1981) is given, below the sequence inferred for the Neandertal individual, numbered according to the reference sequence. The designations and sequences of primers used (reversed and complemented when the letter H occurs in the designations) are given for the first clone of each amplification, except for primers L16,022 (59-CTA AGA TTC TAA TTT AAA CTA TTC CTC T-39) and H16,401 (59-TGA TTT CAC GGA GGA TGG TG-39). For primers L16,209 and H16,271 and further details, see legend to Figure 2. Ambiguities in the sequencing reactions are indicated by standard abbreviations