FEBS Letters 517 (2002) 180^184

FEBS 26007

Helicobacter pylori in North and South America before Columbus
Yoshio Yamaokaa; , Etsuro Oritob , Masashi Mizokamic , Oscar Gutierrezd , Naruya Saitoue ,Tadashi Kodamaf , Michael S. Osatoa , Jong G. Kimg , Francisco C. Ramirezh ,Varocha Mahachaii , David Y. GrahamaaDepartment of Medicine, Veterans A¡airs Medical Center and Baylor College of Medicine, Houston, TX 77030, USAb
Department of Internal Medicine II, Nagoya City University Medical School, Nagoya 467-8601, Japanc
Department of Laboratory Medicine, Nagoya City University Medical School, Nagoya 467-8601, Japand
Universidad Nacional de Colombia, Bogota, ColombiaeLaboratory of Evolutionary Genetics, National Institute of Genetics, Mishima 411-8540, Japanf
Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japang
Guro Hospital, Korea University College of Medicine, Seoul 152-050, South Korea
hCarl T. Hayden Veterans A¡airs Medical Center, Phoenix, AZ 85012, USAi
Gastroenterology Unit, Chulalongkorn Hospital, Bangkok 10330, Thailand
Received 12 February 2002; revised 17 March 2002; accepted 18 March 2002
First published online 28 March 2002
Edited by Takashi Gojobori

Abstract We present a molecular epidemiologic study, based on
an analysis of vacA, cagA and cag right end junction genotypes
from 1042 Helicobacter pylori isolates, suggesting that H.
pylori was present in the New World before Columbus. EightNative Colombian and Alaskan strains possessed novel vacA
and/or cagA gene structures and were more closely related toEast Asian than to non-Asian H. pylori. Some Native Alaskan
strains appear to have originated in Central Asia and to havearrived after strains found in South America suggesting that H.
pylori crossed the Bering Strait from Asia to the New World at
different times. ß 2002 Published by Elsevier Science B.V. on
behalf of the Federation of European Biochemical Societies.
Key words: cagA gene; vacA gene; Native American ;Helicobacter pylori

1. Introduction
Helicobacter pylori is a major pathogen etiologically involved in peptic ulcer disease and gastric cancer [1]. AlthoughH. pylori may have become established in the human stomach
thousands of years ago [2,3], it has remained virulent, possibly
because (1) the short life span of our ancestors may not haveprovided a survival advantage to lesser virulent H. pylori, and(2) the clinical manifestations of infection typically do notappear until adulthood.
Nearly 500 years ago, Europeans brought many deadly infectious diseases to the New World. Based on genotypic analyses of H. pylori strains isolated from di¡erent ethnic popu-*Corresponding author. Fax: (1)-713-790 1040.
E-mail addresses: yyamaoka@bcm.tmc.edu (Y. Yamaoka),orito@med.nagoya-cu.ac.jp (E. Orito), mizokami@med.nagoya-cu.
ac.jp (M. Mizokami), o_gutierrez0@yahoo.com (O. Gutierrez),
nsaitou@genes.nig.ac.jp (N. Saitou), saigon@mint.ocn.ne.jp(T. Kodama), mosato@bcm.tmc.edu (M.S. Osato),
franmi36715@aol.com (F.C. Ramirez),fmedvmc@md2.md.chula.ac.th (V. Mahachai),dgraham@bcm.tmc.edu (D.Y. Graham).

lations, it has been suggested that H. pylori was ¢rst brought
to the New World from Europe [4]. The genes most commonly used as markers for genomic diversity among populations are cagA and vacA. cagA is part of the cag pathogenicity
island, an approximately 40 kbp region in the H. pylori genome [5,6]. Recently, Kersulyte et al. [4] classi¢ed the rightend of the cag pathogenicity island into ¢ve subtypes according to deletion, insertion and substitution motifs. Type I
strains were predominant among isolates from Spanish-speaking patients including Peruvian Indians. Type II strains werepredominant in China and Japan and type III strains weremost common in India. Theoretically, if H. pylori were
present in the stomachs of the ancestors of Native Peruvian
Indians, it should have been genotype II or III. Instead, investigators found Peruvian strains to be similar to H. pylori
typically found among inhabitants from the Iberian Peninsula.
Molecular epidemiology studies have also used the vacA
gene, which encodes a vacuolating cytotoxin to assess transmission pathways. The studies have been based on di¡erent
mosaic combinations of the signal (s) region (s1a, s1b, s1c ands2) and middle (m) region (m1 and m2) allelic types [7,8]. For
example, we found that the predominant vacA genotype
among Hispanics in Houston, TX, USA, was the s1b-m1 subtype, similar to strains from Hispanic America and the Iberian
Peninsula [9]. These data are consistent with the notion thattransmission of speci¢c genotypes remains conserved within
ethnic groups for at least several generations.
This study tests the hypothesis that H. pylori existed in the
New World before Columbus. To test the hypothesis, H. pylori isolated from residual populations of infected patients in
the New World were analyzed to determine whether they possessed the Asian-like cagA and/or vacA structures.
2. Materials and methods
2.1. H. pylori studied
A total of 1042 H. pylori isolates from di¡erent regions in the world
were studied, including 41 strains from native New World populations(Tables 1 and 2). All samples were obtained with informed consent
under protocols approved by the local human studies committees. The
Alaskan strains originated from 16 ‘Eskimo’ and four ‘Aleut’ patients.
The three strains from Native North Americans were from Navahos

0014-5793 / 02 / $22.00 ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
PII: S 0 0 1 4 - 5 7 9 3 ( 0 2 ) 0 2 6 1 7 - 0

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Y. Yamaoka et al./FEBS Letters 517 (2002) 180^184

181

living in Arizona. Eighteen strains were from Huitotos, a primitive,
isolated group living in the Amazonian jungles in Colombia.

Asian countries could be almost completely distinguished by
PCR-based cagA 5P and 3P region genotyping, which is inagreement with previous studies [10,12,16,17]. As genotypic
results from the 5P region and the 3P repeat region were almost
identical, we used the same notation for typing (which wedenoted the East Asian type as cagA 1a type and the nonAsian type as cagA 2a type) (Table 1). All cagA positive
Kazakh and Pakistani strains possessed the non-Asian typestructure.
We con¢rmed the ¢ndings of Kersulyte et al. [4] that thetype I cag right-junction motif was most common among
strains from ethnic European groups and type II was predominant in strains from East Asia (Table 1). Type II was also
found in Kazakhstan (three of four cag positive strains). Interestingly, H. pylori ATCC51407 that was isolated from arhesus monkey [18] possessed the type III cag right-junction
motif as well as vacA m1c/cagA 2a, which was typical ofstrains from India. Rhesus monkeys are widely distributed
throughout the Indian subcontinent and suggest that the
ATCC51407 strain may have been transmitted to monkeys
from humans.

2.2. PCR-based genotyping and sequencing of PCR fragments
Chromosomal DNA was isolated from con£uent plate cultures expanded from a single colony. PCR-based genotyping and sequencing
for the vacA, cagA and the cag right end motifs were performed aspreviously described, with modi¢cation [4,7,10^12]. We designed speci¢c primer sets which could distinguish di¡erent subtypes among thegenes. All sequences were reduced to common lengths consistingof nucleotides 6007^6229 (vacA s region, GenBank accession no.
AE000598), 7805^8260 (vacA m region, AE000598), 1472^1691 and
2877^3505 (cagA, AE000569), and 3987^5339 (cag right-junction motif, AE000569). Sequences were aligned by the CLUSTAL W softwareand con¢rmed by visual inspection [13]. Genetic distances were estimated by the six-parameter method and phylogenetic trees were constructed by the neighbor-joining method [14]. The proportion of thenucleotide di¡erence was used for distance calculation and 1000 bootstrap samplings were made to obtain bootstrap probabilities. These
analyses were carried out using the ODEN program of the National
Institute of Genetics (Mishima, Japan) [15].

3. Results and discussion
3.1. Genotypes from non-Native American strains
Strains from East Asian and non-Asian countries werereadily separated by di¡erences in the vacA s and m regions(Table 1). The vacA s1c genotype was predominant in strainsfrom East Asia (94.7%) whereas the vacA s2 genotype was notfound in strains from East Asia. Strains from East Asia werepredominantly of the m1b genotype (96.8% of m1 strains)
whereas strains from non-Asian countries were predominantlyof the m1a genotype (99% of m1 strains). The vacA m1c genotype was predominant in strains from Calcutta, India [16],and was also found in strains from ethnic Kazakhs and from
Pakistanis.
As with the vacA gene, strains from East Asian and non-3.2. Genotypes from Native American strains
Sequence analysis of the native populations in the New
World showed that 29 of the 41 strains (71%) (100% for
Arizona, 78% for Colombia and 60% for Alaska) had genotypes characteristic of non-Asian strains (combinations of
vacA (s1a, s1b or s2 and m1a or m2), cagA (2a or negative)
and cag right junction (type I or negative)).
We found that four (22%) Native Colombian strains possessed novel structures in vacA m and cagA and four (20%)
Native Alaskan strains also possessed novel vacA m structures(Table 2). Phylogenetic analysis of the vacA s region indicatedthat the nucleotide di¡erences and the bootstrap values wereTable 1
Distribution of the vacA, cagA and cag right-junction genotypes of H. pylori strains from non-Native American populations
Sources ofisolates

Number Number with indicated genotype
analyzed
vacA sEast Asia
Japan
Korea
TaiwanHong Kong
VietnamSoutheast Asia
ThailandSouth Asia
Pakistan
Central Asia
Kazakhstan
Europe
Italy
FranceAfricaSouth Africa
North America
CanadaUSA
South America
Colombia
BrazilavacA mcagAcag right junction

s1a

s1b

s1c

s2

m1a

m1b

m1c m2

1aIIIothersnegative

210
178
34
27
25

9
13
0
0
1

1
1
0
0
0

200
164
34
27
24

0
0
0
0
0

6
6
0
0
1

197
149
14
18
13

0
0
0
0
0

7a
23
20
9
11

206
174
34
27
24

0
0
0
0
0

0
0
0
0
1

192
158
32
25
21

12
14
2
2
3

2
2
0
0
0

4
4
0
0
1

8

3

0

5

0

3

3

0

2

4

4

0

4

4

0

0

26

22

4

0

0

0

0

9

17

0

26

2

0

24

0

0

5

4

0

0

1

0

0

3

2

0

4

1

3

0

0

1

34
73

13
29

11
33

0
0

10
11

13
32

0
0

0
0

21
41

0
0

24
63

24
61

0
0

0
0

0
2

10
10

13

0

11

0

2

11

0

0

2

0

12

12

0

0

0

1

20
183

16
55

3
102

0
0

1
26

12
17

0
2

0
0

8
64

0
0

20
163

20
157

0
0

0
1

0
5

0
20

149
16

60
0

62
8

0
0

27
8

104
6

0
0

0
0

45
10

0
0

133
10

130
10

0
0

2
0

1
0

16
6

FEBS 26007 12-4-02

I

cag islandII

One Japanese strain has the vacA gene with recombination of m1b and m2 genotypes.

2a

182

Y. Yamaoka et al./FEBS Letters 517 (2002) 180^184

Table 2The vacA, cagA and cag right-junction genotypes of H. pylori strains from Native American populations that were di¡erent from the non-Asian
type
StrainvacA sOriginal Native American type
Alaska-2
s1
Alaska-10, -11
s1
Colombia-NA1692
s1
Colombia-NA1764, -NA1766, -NA1768
s1
Recombination of the original Native American and Central Asian type (?)
Alaska-7
s1
Central Asian type
Alaska-6*, -13*, -19
s1a
Recombination of East Asian and Central Asian type (?)
Alaska-18
s1a

vacA mcagAcag right junction

m1d
m1e
m2c
m1d

negative
negative1c
1b

negative
negative
III
III

m1d

2b

II

m1c

2a

II

m1b

2a

II

*: Aleut. s1: Classi¢ed as s1 genotype, but not identical to s1a, s1b or s1c genotypes.

very low even among the established genotypes (e.g. bootstrap
value of 44% between s1c and s2) (data not shown). Theseeight novel Native American strains clustered di¡erently fromthe established vacA s genotypes (e.g. Colombia-NA1692,
Alaska-2 and -7 had a cluster that was closely related tos1c: bootstrap value of 71%), however we did not classify
this cluster as a new genotype because of the low bootstrap
value. This might result from the short sequence sizes and by
the fact that vacA exhibited free recombination whereas recombinants involving the 5P and 3P region of cagA were therarest of any bacterial species [17].
Phylogenetic analysis of the vacA m region showed that ¢ve
Native American strains (Colombia-NA1764, -NA1766,-NA1768, Alaska-2 and -7) formed one cluster with a bootstrap value of 98% that was closely related to the East Asiantype m1b (which we denoted as m1d) (Fig. 1A). It has beensuggested that the vacA m2 genotype can be divided into m2a(both East Asia and non-Asia) and m2b (East Asia) [7], however Colombia-NA1692 was completely di¡erent from m2a
and m2b with a bootstrap value of 100% (we denoted asm2c). Phylogenetic analysis of the cagA gene also showedthat four Native Colombian strains (Colombia-NA1692,-NA1764, -NA1766 and -NA1768) formed clusters that weresimilar but not identical to the East Asian cluster (which wedenoted as cagA 1b for Colombia-NA1764, -NA1766 and-NA1768 and cagA 1c for Colombia-NA1692) (Fig. 1B).
A Native Alaskan strain (Alaska-7) formed a separate cluster and a search of GenBank revealed that two Dutch strains
and one U.S. strain (J166) had similar sequences to Alaska-7in the cagA 5P region. Achtman et al. reported that the cagA
5P region of strain J166 formed a cluster that di¡ered fromboth the East Asian and non-Asian clusters which theytermed ‘clone 2’ [17]. However, our analyses indicated thatthese strains could be classi¢ed as a subgroup of the nonAsian cluster (which we denoted as cagA 2b).
Overall, four Native Colombian strains (ColombiaNA1764, -NA1766, -NA1768 and -NA1692) had similar (i.e.
related) but not identical structures of vacA m and cagA ofisolates from East Asia. A cagA negative Native Alaskan
strain (Alaska-2) also possessed speci¢c vacA m structuresthat were closer to structures from East Asia than to thosefrom non-Asian countries. These ¢ndings suggest that these¢ve strains did not originate from modern East Asian people
and suggest that they are more ancient or they were not recently transmitted from modern East Asian people. This isalso consistent with the hypothesis that H. pylori accompanied

humans when they crossed the Bering Strait from Asia to the
New World.
There was lack of uniformity among the genotypes of the
Native Alaskan strains and this may have bearing on thecontroversy concerning the timing, place(s) of origin, andnumber of ‘waves’ of migration into the New World. A recent
model for peopling of the New World is based on the ‘threewave’ hypothesis, which states that there were three separate
migrations of people into the New World from Asia, corresponding to the three di¡erent language groups [19]. Other
studies suggest that only a single major migration occurred[20] or that as many as four major migration waves occurred[21]. Comparison of the vacA genotypes of three Native Colombian strains and two Native Alaskan strains showed them
to be very similar with respect to the vacA m region structure(m1d) suggesting a relationship between these two populations. These strains were similar, but not identical, to thestrains from East Asia suggesting that they separated fromthe original Asian strain long ago. In contrast, three Native
Alaskan strains (Alaska-6, -13 and -19) and the Kazakh (Central Asian) strains fell within the same cluster in the vacA,
cagA and cag right-junction motifs (vacA s1a-m1c, cagA 2a,
cag right-junction type II). Although we found the vacA m1c
type strains in individuals from Pakistan, all Pakistani vacA
m1c strains had type III cag right-junction motifs. Recent
reports also showed that most strains in Calcutta, India,
had the vacA m1c genotype, however the type II cag rightjunction motifs were very rare (1%) [4,16]. These ¢ndings
suggest that these Native Alaskan strains may have originatedin Central Asia. Some Native Alaskan strains and Central
Asian strains fell within the same cluster suggesting that theCentral Asian type Native Alaskan strains crossed throughBeringia after they had evolved into the present types (i.e.
more recently than the original Native Colombian strains).
Testing this hypothesis will require evaluation of additionalstrains from Central Asia. Two Native Alaskan strains (Alaska-10 and -11) had unique vacA genotypes that were considerably di¡erent from others and these genotypes were notfound in strains from any other region we studied. The originof this genotype may be di¡erent from the other two types
and may support the multi-wave models for peopling of the
New World.
It remains unclear why some Native Alaskan and Kazakh
strains possessed the non-Asian type cagA structure althoughthese strains have Asian or Asian-like vacA genotypes. There
are recent data [22] showing evidence of genetic exchange

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Y. Yamaoka et al./FEBS Letters 517 (2002) 180^184

183

Fig. 1. Phylogenetic tree analysis of the vacA m region (A) and cagA 3P repeat region (B) nucleotide sequences of H. pylori. Genetic distanceswere estimated by the six-parameter method and phylogenetic trees were constructed by the neighbor-joining method. The strain name with
GenBank accession numbers show the reference strains. Bootstrap values are shown along each main branch. The lengths of the horizontal
bars indicate the number of nucleotide substitutions per site.

between two di¡erent H. pylori strains during a natural mixed
infection. When recombination of the cag pathogenicity island
occurred, the cag pathogenicity island including the cag left
and right junctions was thought to transfer together as agroup [23]. If these Native Alaskan and Kazakh strains originated from post-Colombian recombination events between
Asian and non-Asian type strains, the strains would be expected to have the type I cag right-junction motif typical of

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Y. Yamaoka et al./FEBS Letters 517 (2002) 180^184

strains from non-Asian countries. These strains had a type II
cag right-junction motif suggesting that these strains originally possessed the non-Asian type cagA structure. Two Native strains (Alaska-7 and -18) also possessed the type II cag
right-junction motif with the non-Asian-like structure ofcagA. These two strains had an East Asian-like or East Asianstructure of vacA suggesting that recombination of the original Native American type or East Asian type and Central
Asian type strains might have occurred.
Finally, it remains unclear why most H. pylori from Native
Americans have genotypes typical of strains from non-Asian
countries even in sites where Western in£uence appears tohave been minimal. While further study will be necessary tode¢ne the mechanism(s) responsible for the in vivo loss of the
Native American strains when in competition with Old World
strains, preliminary data suggest that they may di¡er withrespect to colonization. For example, in a preliminary study,
we found that both Native Colombian and Western strains
colonized mice, but only Western strains could be recovered
following inoculation of both strains into mouse stomachs(Yamaoka et al., unpublished data). These ¢ndings are consistent with the notion that strains from the Old World may
be hardier or more e⁄cient colonizers than the original Native
American strains.
3.3. Data deposition
The sequences have been deposited in GenBank with accession numbers: AB057106^AB057226 (vacA s region), AB057227^AB057341 (vacA m region), AB056898^AB057002(cagA 5P region), AB057003^AB057105 (cagA 3P region) andAB057432^AB057349 (cag right-junction motif).
Acknowledgements: We thank J. Parkinson (Alaska), C.L. Perng (Taiwan), J.Y. Sung (Hong Kong), R.N. Agnes Walsh (Thailand), Z.
Nurgalieva (Kazakhstan), G. Realdi (Italy), A. Simjee (South Africa),
J.J. Pedrazzoli (Brazil), and S. Mendonca (Brazil) for H. pylori
strains. We thank M. Gomez (Colombia) for assistance in obtaining
samples from Native Colombians. This material is based upon work
supported in part by the O⁄ce of Research and Development Medical
Research Service Department of Veterans A¡airs and by Public
Health Service Grant DK-53659. We also acknowledge the generous
support of Hilda Schwartz.

References[1] Graham, D.Y. (1997) Gastroenterology 113, 1983^1991.
[2] Covacci, A., Telford, J.L., Del Giudice, G., Parsonnet, J. and
Rappuoli, R. (1999) Science 284, 1328^1333.
[3] Blaser, M.J. (1997) J. Physiol. Pharmacol. 48, 307^314.
[4] Kersulyte, D., Mukhopadhyay, A.K., Velapatino, B., Su, W.,
Pan, Z., Garcia, C., Hernandez, V., Valdez, Y., Mistry, R.S.,
Gilman, R.H., Yuan, Y., Gao, H., Alarcon, T., Lopez-Brea,
M., Balakrish Nair, G., Chowdhury, A., Datta, S., Shirai, M.,

Nakazawa, T., Ally, R., Segal, I., Wong, B.C., Lam, S.K., Olfat,
F.O., Bore¤n, T., Engstrand, L., Torres, O., Schneider, R., Thomas, J.E., Czinn, S. and Berg, D.E. (2000) J. Bacteriol. 182,3210^3218.
[5] Alm, R.A., Ling, L.S., Moir, D.T., King, B.L., Brown, E.D.,
Doig, P.C., Smith, D.R., Noonan, B., Guild, B.C., deJonge,
B.L., Carmel, G., Tummino, P.J., Caruso, A., Uria-Nickelsen,M., Mills, D.M., Ives, C., Gibson, R., Merberg, D., Mills,
S.D., Jiang, Q., Taylor, D.E., Vovis, G.F. and Trust, T.J.
(1999) Nature 397, 176^180.
[6] Tomb, J.F., White, O., Kerlavage, A.R., Clayton, R.A., Sutton,
G.G., Fleischmann, R.D., Ketchum, K.A., Klenk, H.P., Gill, S.,
Dougherty, B.A., Nelson, K., Quackenbush, J., Zhou, L., Kirkness, E.F., Peterson, S., Loftus, B., Richardson, D., Dodson, R.,
Khalak, H.G., Glodek, A., McKenney, K., Fitzegerald, L.M.,
Lee, N., Adams, M.D., Hickey, E.K., Berg, D.E., Gocayne,
J.D., Utterback, T.R., Peterson, J.D., Kelley, J.M., Cotton,
M.D., Weidman, J.M., Fujii, C., Bowman, C., Watthey, L.,
Wallin, E., Hayes, W.S., Borodovsky, M., Karp, P.D., Smith,
H.O., Fraser, C.M. and Venter, J.C. (1997) Nature 388, 539^
549.
[7] Atherton, J.C., Cao, P., Peek, R.M., Tummuru, M.K.R., Blaser,
M.J. and Cover, T.L. (1995) J. Biol. Chem. 270, 17771^17777.
[8] van Doorn, L.J., Figueiredo, C., Me¤graud, F., Pena, S., Midolo,P., Maria de Magalhaes Queiroz, D., Carneiro, F., Vanderborght, B., Pegado, M.D., Sanna, R., De Boer, W., Schneeberger, P.M., Correa, P., Ng, E.K., Atherton, J.C., Blaser, M.J. andQuint, W.G. (1999) Gastroenterology 116, 823^830.
[9] Yamaoka, Y., Malaty, H.M., Osato, M.S. and Graham, D.Y.
(2000) J. Infect. Dis. 181, 2083^2086.
[10] Yamaoka, Y., Kodama, T., Kashima, K., Graham, D.Y. andSepulveda, A.R. (1998) J. Clin. Microbiol. 36, 2258^2263.
[11] Yamaoka, Y., Kodama, T., Kita, M., Imanishi, J., Kashima, K.
and Graham, D.Y. (1998) Helicobacter 4, 241^253.
[12] Yamaoka, Y., El-Zimaity, H.M.T., Gutierrez, O., Figura, N.,
Kim, J.G., Kodama, T., Kashima, K. and Graham, D.Y.
(1999) Gastroenterology 117, 342^349.
[13] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) Nucleic
Acids Res. 22, 4673^4680.
[14] Saitou, N. and Nei, M. (1987) Mol. Biol. Evol. 4, 406^425.
[15] Ina, Y. (1994) Comput. Appl. Biosci. 10, 11^12.
[16] Mukhopadhyay, A.K., Kersulyte, D., Jeong, J.Y., Datta, S., Ito,
Y., Chowdhury, A., Chowdhury, S., Santra, A., Bhattacharya,
S.K., Azuma, T., Nair, G.B. and Berg, D.E. (2000) J. Bacteriol.
182, 3219^3227.
[17] Achtman, M., Azuma, T., Berg, D.E., Ito, Y., Morelli, G., Pan,
Z.J., Suerbaum, S., Thompson, S.A., van der Ende, A. and van
Doorn, L.J. (1999) Mol. Microbiol. 32, 459^470.
[18] Drazek, E.S., Dubois, A. and Holmes, R.K. (1994) J. Clin. Microbiol. 32, 1799^1804.
[19] Greenberg, J.H., Turner, C.G. and Zegura, S.L. (1986) Curr.
Anthropol. 27, 477^497.
[20] Underhill, P.A., Jin, L., Zemans, R., Oefner, P.J. and CavalliSforza, L.L. (1996) Proc. Natl. Acad. Sci. USA 93, 196^200.
[21] Torroni, A., Neel, J.V., Barrantes, R., Schurr, T.G. and Wallace,
D.C. (1994) Proc. Natl. Acad. Sci. USA 91, 1158^1162.
[22] Kersulyte, D., Chalkauskas, H. and Berg, D.E. (1999) Mol. Microbiol. 31, 31^43.
[23] Akopyants, N.S., Clifton, S.W., Kersulyte, D., Crabtree, J.E.,
Youree, B.E., Reece, C.A., Bukanov, N.O., Drazek, E.S., Roe,
B.A. and Berg, D.E. (1998) Mol. Microbiol. 28, 37^53.

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