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Wild-Type Nonneuronal Cells Extend

Survival of SOD1 Mutant Motor
Neurons in ALS Mice
minh nguyen

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 Wild-Type Nonneuronal Cells Extend Survival of SOD1 Mutant Motor
Neurons in ALS Mice
A. M. Clement, et al.
Science 302, 113 (2003);
DOI: 10.1126/science.1086071

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 CORRECTED 24 OCTOBER 2003; SEE LAST PAGE
PAGE
domains, intracellular domain associations
would be required to bring these groups
together into an adhesive patch with a suf-
ficient number of intercellular bonds to
resist shear forces.
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www.sciencemag.org SCIENCE VOL 302 3 OCTOBER 2003
REPORTS
47. J. R. Kremer, D. N. Mastronarde, J. R. McIntosh, J. 1052, and EMD-1053. Supported by NIH grant R01
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48. We thank Y. Ji, M. Auer, and K. MacDonald for help
in preparing samples; F. Macaluso and J. Ault for
Supporting Online Material
assistance with high-pressure freezing; and K. Tay- www.sciencemag.org/cgi/content/full/302/5642/109/
lor, E. Branlund, N. Kisseberth, and D. Mastronarde DC1
Materials and Methods
for providing facilities and support for tomographic
data collection and image reconstruction. Coordi- SOM Text
nates for Fig. 2, G to I, have been deposited in the Figs. S1 to S5
Protein Data Bank with accession codes 1Q55, Tables S1 and S2
1Q5A, 1Q5B, and 1Q5C. Tomographic reconstruc- Movies S1 to S3
tions have been submitted to the electron micros- References
copy database of the European Bioinformatics In-
stitute with accession codes EMD-1051, EMD- 19 May 2003; accepted 26 August 2003
Wild-Type Nonneuronal Cells
Extend Survival of SOD1 Mutant
Motor Neurons in ALS Mice
Downloaded from www.sciencemag.org on February 4, 2011
A. M. Clement,1,3,4* M. D. Nguyen,5† E. A. Roberts,2,3
M. L. Garcia,1,3,4 S. Boillée,1,3,4 M. Rule,6 A. P. McMahon,6
W. Doucette,7 D. Siwek,8 R. J. Ferrante,8 R. H. Brown Jr.,7
J.-P. Julien,5‡ L. S. B. Goldstein,2,3 D. W. Cleveland1,3,4§
The most common form of amyotrophic lateral sclerosis (ALS), a neurodegen-
erative disease affecting adult motor neurons, is caused by dominant mutations
in the ubiquitously expressed Cu-Zn superoxide dismutase (SOD1). In chimeric
mice that are mixtures of normal and SOD1 mutant– expressing cells, toxicity
to motor neurons is shown to require damage from mutant SOD1 acting within
nonneuronal cells. Normal motor neurons in SOD1 mutant chimeras develop
aspects of ALS pathology. Most important, nonneuronal cells that do not
express mutant SOD1 delay degeneration and significantly extend survival of
mutant-expressing motor neurons.
Amyotrophic lateral sclerosis (ALS) is a pro- express mutant SOD1 develop a progressive mo-
gressive neurodegenerative disorder in which tor neuron disease that shares many features with
motor neurons die beginning in mid–adult life. human ALS; the complete absence of SOD1 in
About 10% of cases are dominantly inherited; mice does not cause such disease (7). Because
about 20% of these arise from mutations in the toxicity is neither accelerated nor ameliorated by
gene for Cu-Zn superoxide dismutase (SOD1) reducing wild-type SOD1 activity (8) and is
(1). Transgenic mice (2–4) and rats (5, 6) that either unaffected (8) or enhanced (9) by increas-
ing wild-type SOD1 activity, mutant SOD1 must
1
Ludwig Institute for Cancer Research, 2Howard Hughes
cause disease through acquisition of toxic prop-
Medical Institute and 3Department of Cellular and Mo- erties. These may include aberrant oxidative
lecular Medicine and 4Department of Neurosciences, chemistry catalyzed by SOD1-bound copper
University of California, 9500 Gilman Drive, La Jolla, CA
(10–14) or poisoning of a cellular process (or
92093– 0670, USA. 5Centre for Research in Neuro-
sciences, Research Institute of the McGill University
processes) by abundant SOD1 protein aggre-
gates (15–17). This triggers a cell death pathway
Health Care Centre, Montreal General Hospital, 1650
Cedar Avenue, Montreal, Quebec, Canada H3G 1A4. in motor neurons that includes activation of
6
Department of Molecular and Cellular Biology, Harvard caspase 3 (18, 19).
University, 16 Divinity Avenue, Cambridge, MA 02138, Damage to nonneuronal cells may be involved
USA. 7Day Neuromuscular Research Laboratory, Massa-
chusetts General Hospital–East, Charlestown, MA in toxicity. Before onset of disease in SOD1 mu-
02139, USA. 8Departments of Neurology, Pathology, tant mice, there is an inflammatory response, in-
and Psychiatry, Boston University School of Medicine, cluding activation of microglia (20–22) and astro-
Bedford VA Medical Center, Geriatric Research Educa- cytosis (3, 20); the anti-inflammatory compound
tion Clinical Center, Bedford, MA 01730, USA.
minocycline extends survival in mouse models of
*Present address: Institute for Physiological Chemis- ALS (23–25), although whether this reflects ac-
try and Pathobiochemistry, Johannes-Gutenberg-Uni-
versity, Duesbergweg 6, 55099 Mainz, Germany. tion on microglia, astrocytes, or more directly on
†Present address: Department of Pathology, Harvard motor neurons is not established (24). Although
Medical School, 200 Longwood Avenue, Boston, MA accumulation of mutant SOD1 damages motor
02115, USA. neurons in culture (26), SOD1 mutant expression
‡Present address: Centre de Recherche de l’Université
Laval (CHUL), Quebec, QC, Canada G1V 4G2.
only in neurons (27, 28) or glia (29) has not
§To whom correspondence should be addressed. E- provoked disease in mice. Thus, fundamental un-
mail: dcleveland@ucsd.edu answered questions are whether motor neuron
113
 REPORTS
death is caused by toxicity of mutant SOD1 acting
solely within motor neurons, whether cells
expressing mutant SOD1 damage neighboring
wild-type motor neurons, and whether wild-type
nonneuronal cells can protect motor neurons ex-
pressing ALS-causing SOD1 mutations.
To resolve these issues, we generated chimeric
animals composed of mixtures of normal cells and
cells that express a human mutant SOD1 polypep-
tide at levels sufficient to cause fatal motor neuron
disease when expressed systemically in mice.
Forty-two chimeras were produced by injection of
wild-type embryonic stem (ES) cells that consti-
tutively express yellow fluorescent protein (YFP)
(30) into SOD1G85R (line 148) (3) or SOD1G37R
(line 42) (4) mutant blastocysts (Fig. 1, A and B).
Percent chimerism (ranging from ⬃5 to ⬃90%
wild-type cells) was determined by multiple mea-
sures (table S1), including assessment of coat
color, immunoblotting of tail extracts for accumu-
lation of mutant SOD1 and YFP (Fig. 1B), and by
the proportion of cross-sectional area in spinal
cords with detectable YFP (Fig. 1, C and D).
Wild-type and SOD1 mutant–expressing cells
contributed to multiple cell types (fig. S1).
An additional 23 chimeras (Fig. 1H) were
produced by using aggregation (31) of morulae
from wild-type embryos with morulae carrying
transgenes for another mutant SOD1 (SOD1G93A)
(2) and for ubiquitously expressed ␤-galactosidase
(lacZ) (32). Comparable estimates were obtained
for the amount of mutant SOD1 by using either
coat color or immunohistochemistry of spinal
cord sections to identify cells expressing lacZ. For
example, animals determined to be ⬃50% chi-
meric by coat color had a corresponding ⬃50% of
spinal cord areas expressing lacZ, including large
spinal motor neurons (Fig. 1I). For comparison,
spinal cord sections from wild-type and germline
SOD1G93A mice are unstained or completely
stained for lacZ (Fig. 1, J and K).
The presence of wild-type cells in the
SOD1G37R/YFP and SOD1G85R/YFP chimeras
delayed disease onset with average extensions
of 1.6 months (P ⫽ 0.0033; n ⫽ 17; one tailed
Mann-Whitney test) for SOD1G85R and 1.2
months (P ⫽ 0.0007, n ⫽ 17) for SOD1G37R
chimeras (Fig. 1E). Compared with germline
SOD1 mutant mice, there was an average ex-
tension of life-span of 1.8 months (P ⫽ 0.04,
n ⫽ 13) for SOD1G85R and 1.1 months (P ⫽
0.0001, n ⫽ 17) for SOD1G37R chimeras with a
maximum delay of 7.8 and 3.3 months, respec-
tively; Fig. 1F). Both measures correlated well

Downloaded from www.sciencemag.org on February 4, 2011

Fig. 1. Wild-type cells increase survival of mice expressing ALS-linked SOD1
mutations. (A) Chimeras generated by injection of E-YFP– expressing ES cells
into blastocysts of SOD1G85R or SOD1G37R transgenic mice. (B) Degree of
chimerism determined by immunoblotting of tail extracts from mice in (A)
with antibodies having equal affinity for human and mouse SOD1 (3, 4) or to
GFP. Spinal cord sections from mice with (C) low and (D) high contribution
of wild-type cells identified with a GFP antibody (green). DH, Dorsal horn;
VH, ventral horn. (E and F) Delayed onset of disease and extended survival of
SOD1 mutant chimeras. (E) Disease onset and (F) survival times for
SOD1G37R/YFP chimeras. (G) All wild-type cells or all neurons, respectively,
identified in lumbar spinal cord sections of SOD1G37R/YFP chimeras identi-
fied with GFP (green) or neurofilament (red) antibodies. Ages shown are at
end-stage disease. (H to K) Chimeras generated by aggregation of morulas
from normal mice and mice heterozygous for SOD1G93A and lacZ transgenes.
(H to J) Spinal cords from a (I) chimera, ( J) wild-type mouse, and (K)
germline SOD1G93A, lacZ mouse after hematoxylin-and-eosin staining and
assay for lacZ (brown). Black and white arrows point to mutant and
wild-type neurons, respectively. (L) Survival versus age for SOD1G93A/lacZ
chimeras and germline SOD1G93A animals. (M) Survival of SOD1G93A/lacZ
chimeras versus percent wild-type cells. (N and O) SOD1G93A mutant motor
neurons identified in ventral horns of a SOD1G93A/lacZ chimera (N) versus
a germline SOD1G93A mouse (O). Mutant cells were identified by antibodies
specific for human SOD1 (green) (3, 4); neurons were identified with a
neurofilament antibody (SMI32) (red). Wild-type motor neurons (red ar-
rows); SOD1G93A mutant– expressing motor neurons (white arrows). A non-
neuronal, SOD1G93A-expressing cell is marked by an asterisk.

114 3 OCTOBER 2003 VOL 302 SCIENCE www.sciencemag.org


with the proportion of wild-type (YFP-express-
ing) cells within each spinal cord (Fig. 1G).
A robust extension in life-span was also seen
in the SOD1G93A/lacZ chimeras. Eleven of the 23,
including 5 with ⬍30% contribution from wild-
type cells, survived disease-free until they were
killed at ⬎10 months of age (Fig. 1, L and M), an
age at least twice that of the longest lived germline
SOD1G93A littermates (Fig. 1L). Examination of
spinal cord and motor roots of two of these re-
vealed that 67% (chimera 45; Fig. 1N) and 77%
(chimera 67) of motor neurons contained mutant
SOD1, but there was no degeneration or axonal
loss in thoracic roots of either chimera and only
the earliest signs of degeneration in some lumbar
roots of chimera 67. This contrasts with germline
SOD1G93A animals in which 100% of the motor
neurons express mutant SOD1 (Fig. 1O), and half
of these are lost by 5 months (2).
REPORTS
G37R
SOD1 mutant and hNF-L in spinal cord and C, arrows) and motor roots (Fig. 3E)
extracts revealed ⬃30 and ⬃90% mutant cells in accumulated mutant SOD1. Both of these
two SOD1G37R/hNFL chimeras (Fig. 2B). Chi- also displayed a striking left-right asymmetry
mera 7, with the higher wild-type content, did not in the proportion of wild-type (YFP-positive)
develop disease even 5 months beyond the age of
the longest-lived germline SOD1G37R mice. As
seen with antibodies specific for human SOD1
(fig. S2), hNF-L, or all neurofilaments (to iden-
tify mutant and wild-type axons), 30% of ventral
root axons (Fig. 2, C to E) were mutant. Howev-
er, there was no sign of axonal degeneration or
loss in the L5 ventral root (Fig. 2F), in which 978
axons remained, a number consistent with the
927 ⫾ 99 (n ⫽ 26) seen in age-matched wild-
type mice. Furthermore, in contrast to parental
SOD1G37R mice (Fig. 2, H, K, and N), even the
earliest pathologic signs of disease, including
astrocytosis and microgliosis, were absent in this
chimera (Fig. 2, I, L, and O), just as they were in

Downloaded from www.sciencemag.org on February 4, 2011

Extended survival of mutant-expressing mo-
tor neurons was also seen in chimeras generated
by aggregation of morulae from a SOD1G37R
transgenic line (line 29, which has a later disease
onset) (4) with morulae whose wild-type neurons
were marked by expression of very low levels of
the smallest human neurofilament subunit
(hNF-L) (33) (Fig. 2A). Immunoblots for the
normal mice (Fig. 2, G, J, and M).
Extended survivals of SOD1 mutant–
expressing motor neurons in the chimeras could
arise, at least in part, from a protective effect of
wild-type motor neurons. To test this, two
SOD1G37R/YFP chimeras were identified that
developed without wild-type motor neurons; all
motor neurons of multiple lumbar levels (Fig. 3, B

Fig. 2. Absence of motor

neuron pathology or de-
generation despite 30%
SOD1G37R mutant–
expressing motor neu-
rons. (A) Chimeras gen-
erated by aggregation of
morulas derived from
hNF-L and SOD1G37R
(line 29) mice. (B) Chi-
merism was determined
by immmunoblotting
spinal cord extracts with
antibodies to hNF-L, ac-
tin, and human/mouse
SOD1. The amount of
hNF-L is a measure for
the contribution of Fig. 3. Wild-type cells that are not motor neurons
wild-type neurons. (C extend survival of SOD1 mutant motor neurons. (A)
to F) Sections of an Number of ventral horn motor neurons on both
L5 ventral root of sides of the lumbar spinal cord was determined for
SOD1G37R/hNF-L chi- germline SOD1G37R mice, age-matched wild-type
mera 7 simultaneously mice, and two chimeras with asymmetric distribu-
labeled for (C) neuro- tion of wild-type cells (n ⱖ 4 sections per animal).
filaments (antibody (***P ⱕ 0.001 with Student’s t test for paired
SMI-32) and (D) human samples; error bars are SEM). (B) Lumbar spinal
SOD1 (3, 4). (E) The ge- section demonstrating an asymmetric distribution
notype of all axons in of wild-type (green, GFP-immunopositive) cells. (C)
the same root were Higher power view of (B). Motor neurons are mu-
identified as in (C and tant (blue, stained with a human-specific SOD1
D) with antibodies spe- antibody), not wild type (GFP-specific antibody,
cific for human SOD1 green). (D) Asymmetric loss of large-caliber axons
(green), hNF-L (red) (to seen in toluidine blue–stained semithin sections of
identify wild-type ax- left and right L5 ventral roots of chimera 646. (E)
ons), and myelin basic Triple-immunofluorescence staining for (green)
protein (blue). (F) No wild-type YFP-expressing cells, (red) mutant human
sign of neurodegenera- SOD1, and (blue) myelin in an L5 ventral root of
tion was visible in a chimera 646. Green staining of a wild-type Schwann
toulidine blue–stained, cell body (left) serves as a positive control for de-
semithin section of the same root. (G to O) Spinal cord sections of (G, J, and M) wild-type, (H, K, and tection of YFP-containing wild-type cells. Examina-
N) SOD1G37R (line 29), and (I, L, and O) chimera 7 immunostained with antibodies against MAC-2 (G tion of the entire root revealed that all axons were
to I), glial fibrillary acidic protein (GFAP) ( J to L), and cyclin-dependant kinases (Cdk1, 2, 3) (M to O), mutant bearing (red) (fig. S2 for roots from other
which are markers of activated microglia, astrocytes, and proliferating cells, respectively. chimeras stained contemporaneously).

www.sciencemag.org SCIENCE VOL 302 3 OCTOBER 2003 115

 REPORTS
nonneuronal cells in the two halves of their
spinal cords. Germline SOD1G37R mice at end-
stage disease uniformly exhibit symmetric loss
of two-thirds of their large motor neurons in
both halves of the lumbar spinal cord (Fig. 3A).
However, although all motor neurons were mu-
tant in these two SOD1G37R/YFP chimeras,
there was an asymmetric loss of motor neurons
(Fig. 3A; P ⬍ 0.001; paired Student’s t test with
n ⬎ 4 sections per animal) and axons, with
more than twice as many large-caliber (⬎3.5
␮m diameter) surviving axons (Fig. 3D; 187 on
the left versus 89 on the right) in the less-
affected side. In both chimeras, the side with
higher neuronal survival had a higher propor-
tion (25 versus 2% in chimera 646; 30 versus
10% in chimera 213) of wild-type (YFP-
expressing) nonneuronal cells throughout the
lumbar cords. Thus, even when all motor neu-
signs of neurodegeneration. A hallmark for dam-
age to neurons in human patients is the appear-
ance of ubiquitin-positive protein aggregates (34,
35). These are also seen as an early sign for
damaged neurons in SOD1G85R (3, 8) and
SOD1G37R (4) mice, but do not appear in motor
neurons of wild-type mice (Fig. 4, A to C). Ubiq-
uitin aggregates appear in neuronal processes and,
less prominently, in cell bodies (Fig. 4B; fig. S3).
Similar ubiquitinated epitopes were never seen in
age-matched wild-type littermates (Fig. 4A; fig.
S3). In contrast, in both SOD1G37R and
SOD1G85R chimeras (n ⫽ 4), some wild-type
neurons (YFP-containing; arrows in Fig. 4, D and
E, and G and H) in end-stage chimeras accumu-
lated ubiquitinated epitopes in neuronal processes
(Fig. 4F, arrows) and cell bodies (Fig. 4I, arrow),
which indicates that a deficit in ubiquitin-depen-
dent protein degradation is acquired by these wild-
parental mice is not sufficient to trigger their
degeneration or the development of pathologic
abnormalities. Rather, wild-type nonneuronal
cells, in some cases representing a small minor-
ity of total cells, can ameliorate degeneration
and death of SOD1 mutant–expressing motor
neurons compared with those in parental SOD1
mutant mice. That SOD1 mutant neurons sur-
vive longer when surrounded by a wild-type
environment supports the view that damage to
adjacent nonneuronal cells by mutant SOD1 is a
major contributor to disease caused by SOD1
mutations. Damaged glial cells and neurons,
therefore, could act in concert to provoke dis-
ease, consistent with failure of mutant expres-
sion in single cell types to induce motor neuron
degeneration (27–29). It is also consistent with
the failure of increased levels of mutant SOD1
within neurons to accelerate disease caused by

Downloaded from www.sciencemag.org on February 4, 2011

rons are mutant, an environment having a high-
er proportion of wild-type, nonneuronal cells
reduces motor neuron mortality.
To assess whether SOD1 mutant nonneuronal
cells can influence neighboring wild-type neu-
rons, spinal cord sections of chimeric animals
were analyzed at end-stage disease for pathologic
type neurons. The intensity of such ubiquitin
staining in wild-type axons (Fig. 4F, arrows) and
motor neuron cell bodies (Fig. 4I, arrow) frequent-
ly exceeded that of neurons expressing mutant
SOD1 (Fig. 4F, boxed areas; Fig. 4I).
We found that expression of mutant SOD1
in motor neurons at levels that cause disease in
ubiquitous expression of SOD1G93A (28). In-
deed, we know of no compelling in vivo evi-
dence that the genotype of the motor neurons
themselves has any bearing on the probability of
their death in ALS; motor neuron death could in
principle be provoked solely by damage to multi-
ple types of adjacent cells such as interneurons,
astrocytes, and microglia. Further work is critical
to evaluate this possibility.

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cells. Confocal micrographs of spinal cord cross sections from the lumbar region of (A) normal (C57/B6), (B) 268 (2002).
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SOD1G85R and SOD1G37R germline animals, as well as in the cell bodies (block arrow). (D to I) Triple labeling G. A. Rouleau, J. Neurosci. 21, 3369 (2001).
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of spinal cord sections from (D to F) SOD1G37R and (G to I) SOD1G85R chimeras stained with (D and G) SMI32 4825 (2002).
to identify neurons (red), (E and H) GFP to detect wild-type cells (green), and (F and I) ubiquitin (blue). Arrows 29. Y. H. Gong, A. S. Parsadanian, A. Andreeva, W. D.
in (D to F) point to a wild-type axon with elevated levels of ubiquitin compared with a mutant axon Snider, J. L. Elliott, J. Neurosci. 20, 660 (2000).
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accumulation compared with an adjacent SOD1G85R motor neuron. Additional examples are in fig. S3. 115, 49 (2001).

116 3 OCTOBER 2003 VOL 302 SCIENCE www.sciencemag.org


31. A. Nagy, J. Rossant, in Gene Targeting: A Practical
Approach, A. L. Joyner, Ed. (Oxford Univ. Press, New
York, 1999), pp. 177–206.
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J. Physiol. 84, 50 (1990).
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36. We gratefully acknowledge A.-K. Hadjantonakis (Co-
lumbia University, New York, NY ) and A. Nagy (Mount
Sinai Hospital, Toronto) for providing the E-YFP-ES cells;
P. Hince and D. Houle for their technical help; and J.
Folmer ( Johns Hopkins, Baltimore, MD). This work was
REPORTS
supported by the grants from the NIH (NS 27036 to CIHR Senior Investigator Award. D.W.C. receives salary
D.W.C, HD 30249 to A.P.M., AG 13846 to R.J.F., AG support from the Ludwig Institute for Cancer Research.
12992 to R.H.B. and R.J.F, and NS 31248 and NS 37912 L.S.B.G. is an Investigator of the Howard Hughes Med-
to R.H.B.); the Center for ALS Research at Johns Hopkins ical Institute.
(to D.W.C and L.S.B.G.); the ALS Association (to A.P.M.
Supporting Online Material
and R.H.B.); the Canadian Institutes of Health Research
www.sciencemag.org/cgi/content/full/302/5642/113/
(CIHR) (to J.-P.J.); the Angel Fund for ALS Research; and
DC1
Project ALS (to R.H.B.); and the Veterans Administration
Materials and Methods
(R.J.F.). A.M.C. was supported in part by a fellowship
from the German Research Council (DFG, Cl-175). Figs. S1 to S4
M.D.N. is a recipient of a K. M. Hunter/CIHR Scholarship. Table S1
M.L.G. is the recipient of a postdoctoral fellowship from References and Notes
the NIH. S.B. is a recipient of a Fondation pour la
Recherche Medicale fellowship. J.-P.J. is a recipient of a 24 April 2003; accepted 13 August 2003

Thalamic Control of Visceral Mice homozygous for a null mutation of the

␣1G (CaV3.1) gene showed a functional deletion
of T-type currents and lacked low threshold burst
Nociception Mediated by T-Type firing in the thalamocortical relay neurons (13).
We measured the sensitivity of the ␣1G-deficient
Ca2ⴙ Channels mice (␣1G–/–) by delivering thermal or mechani-
cal stimuli delivered either on the palm or tail

Downloaded from www.sciencemag.org on February 4, 2011

Daesoo Kim, Donghyun Park, Soonwook Choi, Sukchan Lee, (supporting online material). No significant differ-
Minjeong Sun, Chanki Kim, Hee-Sup Shin* ence was observed between the mutants and their
wild-type littermates in these assays (Fig. 1, A to
Sensations from viscera, like fullness, easily become painful if the stimulus persists. Mice C). Hyperalgesia to cutaneous pain, as measured
lacking ␣1G T-type Ca2⫹ channels show hyperalgesia to visceral pain. Thalamic infusion by the relative enhancement of the pain response
of a T-type blocker induced similar hyperalgesia in wild-type mice. In response to by a subcutaneous injection of complete Freund’s
visceral pain, the ventroposterolateral thalamic neurons evoked a surge of single spikes, adjuvant (CFA) before pain tests (14), also did not
which then slowly decayed as T type–dependent burst spikes gradually increased. In significantly differ between the wild type and the
␣1G-deficient neurons, the single-spike response persisted without burst spikes. These mutant (Fig. 1D). Next, we examined the sensi-
results indicate that T-type Ca2⫹ channels underlie an antinociceptive mechanism tivity of the mice to visceral pain induced by
operating in the thalamus and support the idea that burst firing plays a critical role in intraperitoneal administration of either acetic acid
sensory gating in the thalamus. (Fig. 1E) or MgSO4 solution (Fig. 1F) as previ-
ously described (15). The wild-type mice showed
Low voltage–activated (LVA) T-type Ca2⫹ chan- sensory inputs, they respond in dual firing typical pain behaviors characterized by writhing,
nels play crucial roles in the control of cellular modes: either in singular action potentials or in such as abdominal stretching and constriction in
excitability under diverse physiological and a burst of action potentials clustered together as response to these two chemicals, with MgSO4-
pathological processes (1, 2). Recently, studies a high-frequency discharge (8–10). T-type induced pain responses terminated earlier than
revealed a novel role of T-type Ca2⫹ channel in Ca2⫹ channels are known to excite hyperpolar- those by acetic acids (15). However, compared
the pain sensory pathway by showing that this ized thalamic neurons to generate bursts of
channel facilitates pain signals in peripheral noci- action potentials. There has been much debate
ceptors (3, 4) and in the spinal cord (5). T-type about the role of the thalamic burst firing in the National Creative Research Initiative Center for Cal-
channels are also highly expressed in the thalamus sensory processing (11, 12). Therefore, whether cium and Learning, Korea Institutes of Science and
(6), through which noxious signals from the spi- thalamic T-type channels would contribute to Technology, Seoul 136-791, Korea.
nal cords must pass before reaching the cortex (7). the nociceptive signal processing as a signal *To whom correspondence should be addressed. E-
When the thalamocortical relay neurons receive enhancer or a suppressor is an open question. mail: shin@kist.re.kr

Fig. 1. Pain responses

of ␣1G⫺/⫺ mice to
noxious stimuli. (A)
Responses to mechan-
ical stimuli with von
Frey filaments. (B) Tail
flick responses to ther-
mal stimuli. (C) Paw
withdrawal responses
to infrared thermal
stimuli at two differ-
ent intensities. (D) One day after injection of CFA (1⫻) in the left paw, infrared
thermal stimuli were delivered either to the injected paw (ipsilateral) or the opposite
uninjected paw (contralateral). Visceral pain induced by intraperitoneal injection of
either acetic acid (E) or MgSO4 solution (F). Writhing responses were examined for 20
min after acetic acid injection or for 10 min after MgSO4 injection. Error bars indicate
SEM. Two-tailed t test, *P ⬍ 0.01; **P ⬎ 0.05.

www.sciencemag.org SCIENCE VOL 302 3 OCTOBER 2003 117

C O R R E C T I O N S A N D C L A R I F I C AT I O N S

ERRATUM post date 24 October 2003

REPORTS: “Wild-type nonneuronal cells extend survival of SOD1 mu-

tant motor neurons in ALS mice” by A. M. Clement et al. (3 Oct. 2003,
p. 113). The word “inherited” was deleted from the first sentence of
the abstract. It should read as follows: “The most common inherited
form of amyotrophic lateral sclerosis (ALS), a neurodegenerative dis-
ease affecting adult motor neurons, is caused by dominant mutations
in the ubiquitously expressed Cu-Zn superoxide dismutase (SOD1).”

Downloaded from www.sciencemag.org on February 4, 2011

www.sciencemag.org SCIENCE Erratum post date 24 OCTOBER 2003 1

 Letters to the Editor
Letters (~300 words) discuss material published
in Science in the previous 6 months or issues
of general interest. They can be submitted by
e-mail (science_letters@aaas.org), the Web
(www.letter2science.org), or regular mail
(1200 New York Ave., NW, Washington, DC
20005, USA). Letters are not acknowledged
upon receipt, nor are authors generally
consulted before publication. Whether
published in full or in part, letters are subject
to editing for clarity and space.
A Serendipitous
Purchase
I RECENTLY ORDERED A BOOK, BASIC COLOR
Terms: Their Universality and Evolution
(1), through an online book dealer. The
compelling evidence for the control of
phytoplankton productivity by dissolved
iron (K. O. Buesseler, P. W. Boyd, “Will
ocean fertilization work?”, Perspectives, 4
Apr., p. 67) (1).
However, there is an intriguing conun-
drum when interpreting the annual develop-
ment of primary production in this region. In
autumn, about 14 million km2 of the
Southern Ocean freezes over, providing a
seasonally variable habitat for marine organ-
isms (2, 3). If iron is limiting to Southern
Ocean phytoplankton growth, and sea ice is
formed from the same iron-deficient waters,
it seems reasonable to conclude that the ice-
based primary production should also be iron
LETTERS
School of Ocean Sciences, University of Wales,
Bangor, Menai Bridge, Anglesey LL59 5AB, UK. E-
mail: d.thomas@bangor.ac.uk
References
1. S. W. Chisholm, P. G. Falkowski, J. J. Cullen, Science
294, 309 (2001).
2. D. N.Thomas, G. S. Dieckmann, Science 295, 641 (2002).
3. A. S. Brierley, D. N.Thomas, Adv.Mar.Biol. 43, 171 (2002).
4. H. Eicken, Polar Biol. 12, 3 (1992).
5. S. F. Ackley, C.W. Sullivan, Deep-Sea Res. 41, 1583 (1994).
6. K. R. Arrigo, D. L. Worthen, M. P. Lizotte, P. Dixon, G. S.
Dieckmann, Science 276, 394 (1997).
European Union R&D
Spending
LIKE MANY WHO SHARE PIERRE PAPON’S

Downloaded from www.sciencemag.org on February 4, 2011

book was simply advertised as a “used
book, first edition, dust jacket, good condi-
tion.” When it arrived, I opened the front
cover and found a bookplate from The
Rockefeller University and a tag with the
words “Laboratory book—Dr. Hartline.”
It has been 20 years since Haldane Keffer
Hartline passed away. Hartline shared the
Nobel Prize in Physiology or Medicine in
1967 with Ragnar Granit and George Wald.
He was a mentor to many neuroscientists at
the University of Pennsylvania, Johns
Hopkins University, and Rockefeller
University. I am a direct “academic descen-
dant” of his. My graduate advisor lineage can
be traced back through Maureen Powers,
Stephen Easter Jr., and Edward
MacNichol Jr. to “Keff ” Hartline.
Words cannot express my feelings
about owning a book that was owned by
my graduate adviser’s adviser’s adviser’s
adviser. We in science need to give
thanks to our mentors and those who
have blazed the trails before us in our
respective fields; we also need to
continue to be cognizant of our students
and how we may influence them directly or
indirectly. In the words of Hartline from his
banquet speech at the Nobel ceremony, “Tack
sa mycket.” Thank you very much.
CARL J. BASSI
College of Optometry, University of Missouri–St.
Louis, St. Louis, MO 63121, USA. E-mail:
bassi@umsl.edu
Reference
1. B. Berlin, P. Kay, Basic Color Terms: Their Universality and
Evolution (Univ. of California Press, Berkeley, CA, 1969).
Iron Limitation in the
CREDITS: D. N. THOMAS
limited. Some phytoplankton species become vision of a more interlinked future for
caught up in the ice. Maximum growth of science among countries in the European
these “ice algae” is often concentrated on the Union (EU)—a “European Research
bottom few centimeters of ice floes (2, 4), Area”—I greatly appreciated his recent
where replenishment of inorganic nutrients Editorial, “A challenge for the EU” (1
from the underlying water sustains high Aug., p. 565). The following points may
standing crops, several orders of magnitude add some relevant texture.
greater than those measured in the water Papon begins by emphasizing the dispari-
column (3–5). If this replenishment is with ties in spending on R&D between the United
States, Japan, and the EU: 2.8%,
3.0%, and 1.9% of GDP, respec-
tively. Against this background,
Papon outlines the European
Commissioners’ plan to increase
EU R&D spending to 3.0% of
GDP and then devotes much of
the rest of his Editorial to EU
proposals about spending on
basic research in general and for
a European Research Council
(ERC) in particular. To the
contrary of the impression that
Papon’s Editorial may uninten-
tionally give, science base
spending (1) is slightly higher in
Sea ice in the Southern Ocean.
the EU than in the United States,
iron-deficient water, the growth of these although lower than in Japan: 0.65%, 0.63%,
algae presumably would be iron limited. In and 0.85% of GDP, respectively. The differ-
fact, sea ice algal growth is rapid within new ences in the R&D figures arise almost
sea ice (2–6), and in spring and summer, entirely from differences in spending by the
iced-based primary production remains high private sector (business and industry),
(4, 5). This suggests that the ice-based mainly on Development rather than
primary production is not iron limited. Research. And within the EU countries,
Blooms of sea ice algae occur throughout the there is considerable variation, with several
pack and are not restricted to ice overlying spending significantly more on their science
iron-rich coastal waters. base than the United States: Germany,
Although logistically difficult, iron 0.73%; France, 0.80%; Netherlands, 0.88%;
fertilization experiments extended to and Sweden, 0.95%, compared with, for
measure the fate of a “fertilized” patch example, UK, 0.59%, and Spain, 0.42%.
when frozen into sea ice, or the “fertiliza- Studies of research output, whether meas-

Southern Ocean
LARGE-SCALE “IRON FERTILIZATION” EXPERI-
ments in the Southern Ocean provide
tion” of water just prior to freezing, would ured by numbers of papers, citations, or major
help a more complete interpretation of iron international prizes, in relation to science base
limitation of phytoplankton growth in spending 3 to 5 years earlier, reveal very big
seasonally ice-covered waters. differences—up to a factor of five—among
DAVID N. THOMAS Organisation for Economic Co-operation and

www.sciencemag.org SCIENCE VOL 302 24 OCTOBER 2003 565

 LETTERS
ADVERTISER DIRECTORY
Development (OECD) countries, with the top
performers being Switzerland, Sweden, and
Israel (2, 3). It is not simply how much you
spend, but how you spend it.
The existing European Science Found-
ation (ESF) has modest funding, but I think it
uses it wonderfully well for its designated
task of creating collegial networks in
response to theme proposals. Along with
Framework VI’s Marie Curie, Human
Resources and Mobility, and other postdoc-
toral programs that enable the best young
researchers in the EU to move freely among
the best laboratories, this is a powerful force
for breaking down hierarchical organizations
and, indeed, creating a European research
area. But any ERC, roughly aimed as a pan-
EU analog of the U.S. National Science
Foundation (NSF), should, in my view, meet
several stringent criteria: It must fulfil clearly
identified scientific needs that are not
currently being met, be based on clear prin-
The following organizations ciples of scientific excellence, have
have placed ads in the minimum bureaucracy, complement existing
Special Advertising Section organizations, and not be at the expense of
national funding. Given the huge variety of
Drug Discovery and scientific cultures currently within the EU
Biotechnology Trends
countries, fulfilment of these criteria cannot
DNA and
be lightly assumed. I look forward to seeing
how these issues are dealt with by the rele-
vant EU Panel, chaired by Federico Mayor,
Biochips 3: former Director General of UNESCO.
These observations are offered in a
constructive spirit and with real enthusiasm
Smaller, Faster, Better for the ideal of “one Europe” in science.
The EU postdoctoral mobility schemes,
ADVERTISER Page despite the considerable bureaucracy too
Affymetrix, Inc. ....................... 669
often associated with them, are truly
building the scientific Europe of tomorrow.
American Type
Culture Collection At this stage, however, I would put more
(ATCC) ...................................... 673 emphasis on unleashing this creativity of
Ciphergen the young in Europe, especially in countries
Biosystems, Inc. .................... 671 where current hierarchical structures are
Fluidigm Corporation .......... 675 correlated with relatively poor average
Roche return on research spending, and less on
Applied Sciences .................. 666 creating large EU-level research councils.
SANYO Sales & Marketing ROBERT M. MAY
Corporation / SANYO Department of Zoology, University of Oxford,
Electric Biomedical Co., Ltd. .. 676
South Parks Road, Oxford OX1 3PS, UK.
Takara Bio, Inc. ...................... 668
References and Notes
1. The term “science base” describes all research and
postgraduate training undertaken in universities,
government-funded laboratories, and private
nonprofit organizations (charities or foundations)
funded both from public and nonpublic sources. For
more information on the science base and difficulties
Turn to page 667 in its estimation [it is not a conventional OECD
statistic, and the usual R&D spending by institutions
of Higher Education (HERD) is not always a good
measure of it], see reference (9) in (4).
2. R. M. May, Science 275, 793 (1997).
3. See reference (1) in (4).
4. R. M. May, Science 281, 48 (1998).
Response
I THANK MAY FOR HIS COMMENTS ON MY
Editorial. I wanted, indeed, to emphasize the
growing gap in R&D funding between the
24 OCTOBER 2003 VOL 302
EU and the United States. Indicators reveal
that Europe is investing globally less in
research than the United States, and the
recent trend is not favorable to Europe: U.S.
industry has continuously increased its
R&D funding (but not in basic science),
while the NIH, for example, has doubled its
budget during the last 5 years. As far as the
figures regarding spending for basic science
that May cites, I think they must be consid-
ered carefully. For example, May points out
that France’s science base funding corre-
sponds to 0.80% of the GDP, with lower
figures for Germany and the UK and higher
figures for Sweden and the Netherlands.
Actually, those figures for public expenses
correspond to different types of spending in
different countries: For France, the figure
includes budgets for academic science
(CNRS, universities, etc.) and also for
Downloaded from www.sciencemag.org on February 4, 2011
space, nuclear energy, and defense activities
(roughly half of the 0.80%) that are not, for
the most part, science-related. I suspect that
the situation is the same for Germany, which
has an important space budget, and probably
for the UK, which has kept an important mili-
tary R&D effort, although the military/space
spending is probably a lower proportion of the
total than in France. The United States, which
has by far the biggest military R&D in the
world, spends a rather fair proportion of this
effort on basic science; this is not the case in a
country such as France. Furthermore, funds
provided by foundations to academic science
are certainly higher in the United States than
in Europe (the UK being a positive exception
in Europe).
I agree that research output varies
considerably within Europe and that the
way money is spent is also important. The
performance of Swedish and Swiss science
is certainly very good, but those countries
have focused their activities on a limited
number of areas (biomedicine, for
example) in which they can manage to have
a better concentration of material and
human means.
I fully agree also with May’s remarks
about the ERC, and the criteria that he
proposes are those that were highlighted in the
ESF high-level expert group’s report to which
I refer in my Editorial. An ERC should react
more quickly to the science evolution than the
present R&D Framework Programme, which
has been able to launch positive initiatives
as the Marie Curie fellowships. Lastly, I
agree with May that the absolute priority
for Europe and the European Research
Area is to support the young generation of
scientists. It should be also the task of an
ERC.
PIERRE PAPON
Ecole Supérieure de Physique et de Chimie
Industrielles, CNRS, 10 Rue Vauquelin, Paris 75005,
France.
SCIENCE www.sciencemag.org

The Structure of
D. radiodurans
IN THEIR RECENT REPORT (“RINGLIKE STRUC-
ture of the Deinococcus radiodurans
genome: a key to radioresistance,” 10 Jan.,
p. 254), S. Levin-Zaidman et al. propose
that single genomes of D. radiodurans
assume a tightly packed toroidal morphology,
each within its own cellular compartment.
They suggest that this structure passively
protects D. radiodurans from DNA double-
strand breaks by preventing the ends of
adjacent DNA fragments from diffusing
apart during a first stage of repair. In
thinking about the organism and the model,
we believe that several additional consider-
ations should receive attention.
First, we do not consider transmission
electron microscopy images alone suffi-
cient evidence to justify categorizing the
nucleoid as toroidal. Second, the D. radio-
durans genome is divided into four circular
genetic elements that are repaired with
equal efficiency. It is difficult to envision
how all four segments of the genome could
conform to the proposed structure and
repair model. Third, it is unclear why the
authors refer to the tetracoccus as a single
cell with four compartments. Every
previous study has concluded that the tetra-
coccus represents four separate cells, each
with four or more genome equivalents of
DNA. The DNA content detected in earlier
studies argues for much more than one
genome per compartment. Fourth, the work
of Hud and Downing (1), cited by Levin-
Zaidman et al., does not address diffusion
of DNA fragments within a toroid. The
authors argue that the restricted diffusion
might allow for error-free end-joining as a
repair process. However, DNA ends
damaged by radiation have a variety of struc-
tures and may have missing nucleotides. They
are thus unlikely to be spliced together in an
error-free manner, regardless of restricted
diffusion.
We share the authors’ fascination with
this unusually adaptable bacterium. New
efforts at quantitative examination of the
morphological features of this bacterium,
complemented by work in other disci-
plines, may help to resolve these issues.
JOHN R. BATTISTA,1 MICHAEL M. COX,2 MICHAEL J.
DALY,3 ISSAY NARUMI,4 MIROSLAV RADMAN,5
SUZANNE SOMMER6
1Biological Sciences, Louisiana State University, 202
Life Sciences, Baton Rouge, LA 70803, USA. E-mail:
jbattis@lsu.edu. 2Department of Biochemistry,
University of Wisconsin–Madison, Madison, WI
53706, USA. 3Department of Pathology, Uniformed
Services University of the Health Sciences,
Bethesda, MD 20814, USA. 4Biotechnology
Laboratory, Department of Ion-beam-applied
www.sciencemag.org SCIENCE
LETTERS
Biology, Takasaki Radiation Chemistry Research
Establishment, Japan Atomic Energy Research
Institute, 1233 Watanuki, Takasaki, Gunma 370-
1292, Japan. 5Laboratoire de Génétique
Moléculaire Evolutive et Médicale, INSERM U
571–2ème étage, Faculté de Médecine Necker-
Enfants Malades Université René, Descartes-Paris
V, 75730 Paris Cedex 15, France. 6Institut de
Génétique et Microbiologie, Université Paris Sud,
Bâtiment 409, 91405 Orsay Cedex, France.
Reference
1. N. V. Hud, K. H. Downing, Proc. Natl. Acad. Sci. U.S.A.
98, 14925 (2001).
Response
THE PROPOSED CORRELATION BETWEEN
DNA repair in Deinococcus radiodurans
and the toroidal chromosome organization
exhibited by this radioresistant bacterial
strain has instigated heated debates.
Downloaded from www.sciencemag.org on February 4, 2011
In the most general terms, we do not
claim that efficient and probably unique
enzymatic pathways do not contribute to the
exceptional resistance of D. radiodurans.
Rather, we contend that error-free repair of
multiple double-stranded DNA breaks
represents an informational problem that
cannot be solved solely by such pathways.
The next issue concerns the actual pres-
ence, uniqueness, and physiological rele-
vance of the chromatin toroidal morphology
in D. radiodurans cells. This morphology
should be assessed in light of our knowl-
edge on DNA packaging modes in other
bacterial strains and the techniques used to
identify these modes. D. radiodurans cells
were prepared for transmission electron
microscopy by several fixation methods,
including chemical and ultrafast cryo tech-
niques, which are generally considered as
highly reliable (1). When these techniques
were applied on actively growing E. coli,
salmonella, B. subtilis, M. xanthus, and
other bacterial strains, they invariably indi-
cated an amorphous and irregularly
dispersed chromatin organization. The fact
that an identical array of fixation methods
regularly reveals distinct DNA toroids in D.
radiodurans attests to both the credibility
of the observation and the uniqueness of
this packaging mode. But does this organi-
zation represent indeed a decisive factor in
promoting DNA repair?
We proposed that the tight toroidal
organization prevents free DNA ends from
diffusing away from each other, based not
only on the high extent of compactness and
order that characterize in vitro DNA
toroids, but also on two additional observa-
tions. DNA repair in D. radiodurans is
markedly promoted by freezing and desic-
cation (2) that slow down molecular diffu-
sion. Repair is, in contrast, strongly attenu-
ated at high temperatures (3) that accelerate
diffusion. Moreover, conditions that induce
VOL 302 24 OCTOBER 2003
 LETTERS
Explore toroidal DNA packaging have been shown
to dramatically stimulate DNA ligation.
Greenland These findings highlight the relevance of
restricted diffusion within DNA toroids that
& Iceland! act as a “molecular cage.” Within the tightly
packed DNA toroids, water content is
Spectacular Landscapes
reduced, resulting in a decreased formation
Wildlife & History of reactive radicals, as well as in altered
September 13-26, 2004 photochemical properties of DNA (4). This,
and the close proximity of free DNA ends
within the toroids, is likely to substantially
reduce the probability of nucleotide modifi-
cations at these ends. The observation that
repair is enhanced by desiccation (2) further
substantiates this claim.
The last point concerns the morphology of
a D. radiodurans cell and its relevance to
DNA repair. In their seminal structural
studies, Murray et al. demonstrated that D.
radiodurans strains separate into what the
and toroidal chromatin packaging, implying
that these factors contribute to radioresis-
tance.
ABRAHAM MINSKY, AJAY K. SHARMA, JOSEPH
ENGLANDER
Department of Organic Chemistry, The Weizmann
Institute of Science, Rehovot 76100, Israel, and
Laboratory of Molecular Pharmacology, National
Cancer Institute/NIH, Bethesda, MD 20892, USA.
References:
1. C. Robinow, E. Kellenberger, Microbiol. Rev. 58, 211
(1994).
2. A. Venkateswaran et al., Appl. Environ. Microbiol. 66,
2620 (2000).
3. S. Kitayama, A. Matsuyama, J. Radiat. Res. 21, 257
(1980).
4. M. H. Patrick, D. M. Gray, Photochem. Photobiol. 24,
507 (1976).
5. R. G. Murray, M. Hall, B. G. Thompson, Can. J. Microbiol.
29, 1412 (1983).
6. See http://science.nasa.gov/newhome/headlines/
ast14dec99_1.htm.
7. V. Norris, M. S. Madsen, J. Mol. Biol. 253, 739 (1995).

Downloaded from www.sciencemag.org on February 4, 2011

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authors specified as individual tetrads,
whereby the compartments are not fully sepa-
rated but rather “remain in communication”
(5). We probed a very large number of cells at CORRECTIONS AND CLARIFICATIONS
different growth phases. All stationary D. Research Articles: “LMO2-associated clonal T cell
radiodurans cells were scored as tetrads, as proliferation in two patients after gene therapy for
were ~85% of exponentially growing SCID-X1” by S. Hacein-Bey-Abina et al. (17 Oct., p.
bacteria. The rest appeared as sextets or 415). The second and third authors, C.Von Kalle and
M. Schmidt, should have had asterisks after their
octets, corresponding to cells at various states
of division. The tetrad morphology of D.
names, to indicate shared first authorship with S.
Hacein-Bey-Abina. The asterisks were inadvertently
radiodurans was highlighted in a light micro- omitted because of an editorial error.
graph contributed by M. Daly, where the cells
were defined as “tetrad growth units” (6). Reports: “Wild-type nonneuronal cells extend
Our studies revealed that chromosomal survival of SOD1 mutant motor neurons in ALS
copies in a D. radiodurans cell are segregated mice” by A. M. Clement et al. (3 Oct., p. 113). The
word “inherited” was deleted from the first
in the four compartments. Because chromo- sentence of the abstract. It should read as follows:
somal segregation has been proposed to “The most common inherited form of amyotrophic
promote different extents of DNA packaging lateral sclerosis (ALS), a neurodegenerative disease
per genome (7), we claim that the tetrad affecting adult motor neurons, is caused by domi-
morphology is relevant to DNA repair, inas- nant mutations in the ubiquitously expressed Cu-
Zn superoxide dismutase (SOD1).”
much as it allows for coexistence of dispersed
and tightly packed toroidal chromosomes in a
Reports: “A dearth of dark matter in ordinary
single D. radiodurans cell. Indeed, prelimi-
nary studies conducted in our laboratory on
elliptical galaxies” by A. J. Romanowsky et al. (19
Sept., p. 1696). In the third column on page 1697,
the highly resistant strains D. radiopugnans in the 21st line, the number should be 7.1 ± 0.6,
and D. radiophilus revealed DNA segregation not 6.4 ± 0.6.
TECHNICAL COMMENT ABSTRACTS
COMMENT ON “14C Dates from Tel Rehov: Iron-Age Chronology, Pharaohs,
and Hebrew Kings”
Israel Finkelstein and Eli Piasetzky
We contest the interpretation by Bruins et al. (Reports, 11 April 2003, p. 315) of the Tel Rehov 14C data from the
points of view of method, provenance, interpretation of the calibration, and historical analysis.These data can be
interpreted as supporting the Low Chronology for Iron Age IIA strata in the Levant.
Full text at www.sciencemag.org/cgi/content/full/302/5645/568b

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RESPONSE TO COMMENT ON “14C Dates from Tel Rehov: Iron-Age Chronology,
Pharaohs, and Hebrew Kings”
Hendrik J. Bruins, Johannes van der Plicht, Amihai Mazar
The entire 10th century B.C.E. is represented in the consistent Groningen radiocarbon series of Tel Rehov: Phases
D3 and D2, and Strata VI, V, and even IV in its upper range. The results contradict Finkelstein’s Low Chronology,
but do support a Revised Traditional Chronology for the Iron Age in the Southern Levant.
Full text at www.sciencemag.org/cgi/content/full/302/5645/568c

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