ABSTRACT
The human
CSB
gene, mutated in Cockayne's syndrome group B (partially defective in both
repair and transcription) was previously cloned by virtue of its ability to
correct the moderate UV sensitivity of the CHO mutant UV61. To determine
whether the defect in UV61 is the hamster equivalent of Cockayne's syndrome,
the RNA polymerase II transcription and DNA repair characteristics of a repair-proficient CHO cell line (AA8), UV61 and a
CSB
transfectant of UV61 were compared. In each cell line, formation and removal of
UV-induced cyclobutane pyrimidine dimers (CPDs) were measured in the
individual strands of the actively transcribed
DHFR
gene and in a transcriptionally inactive region downstream of
DHFR
. AA8 cells efficiently remove CPDs from the transcribed strand, but not from
either the non-transcribed strand or the inactive region. There was no detectable repair
of CPDs in any region of the genome in UV61. Transfection of the human
CSB
gene into UV61 restores the normal repair pattern (CPD removal in only the
transcribed strand), demonstrating that the DNA repair defect in UV61 is
homologous to that in Cockayne's syndrome (complementation group B) cells.
However, we observe no significant deficiency in RNA polymerase II-mediated transcription in UV61, suggesting that the CSB protein has
independent roles in DNA repair and RNA transcription pathways.
Prokaryotic and eukaryotic cells have a variety of DNA repair mechanisms to
maintain the integrity of the genetic material. Among these, nucleotide
excision repair (NER) is the least specific, having the capability to remove a
vast array of more or less bulky DNA adducts, including those adducts formed by
ultraviolet (UV) light. Extensive research (reviewed in
1
,
2
) into the pattern of NER within the genome has revealed that certain regions
are repaired better (or at least faster) than other regions. In general,
regions containing transcriptionally active genes are repaired preferentially
when compared with inactive regions of the genome. Moreover, within actively
transcribing genes, the transcribed strand is repaired more efficiently than
the non-transcribed strand (
3
,
4
). This is widely believed to occur through a direct link of the repair
machinery to the transcriptional apparatus due to the recent isolation and
characterization of a transcription-repair coupling factor (TRCF) in
Escherichia coli
(
5
). The identification of certain NER genes as subunits of the basal
transcription factor TFIIH (reviewed in
6
) also suggests intimate connections between transcription and repair. These
selective mechanisms, termed preferential and transcription-coupled repair (TCR), supposedly serve to allow early resumption of
transcription and thus increase the survival of cells containing DNA damage.
The importance of preferential repair and TCR is illustrated by the human DNA
repair-deficient genetic disease Cockayne's syndrome (CS). Patients with this
disease lack TCR; cells isolated from these patients repair the transcribed
strand of an active gene at the same rate as non-transcribed regions of the genome in normal cells (
7
,
8
). CS patients are also thought to have variable defects in RNA polymerase II
(pol II) transcription (
9
). Among the consequences of these defects are UV hypersensitivity, cachetic
dwarfism, neurological degeneration and premature aging. The manifestations of
CS are now believed to be caused by specific alterations in at least one of a
number of gene products involved in DNA repair, all of which appear to either
be a subunit of or interact with TFIIH.
The
CSB
(earlier known as
ERCC6
) gene, mutations in which have been shown in a CSB patient (
10
), was cloned by virtue of its ability to correct the UV sensitivity of UV61 (
11
), a Chinese hamster ovary (CHO) cell line from complementation group six of a
series of UV-sensitive mutants isolated by Busch and co-workers (
12
). UV61 cells are sensitive to UV light, but not as sensitive as other cell lines which have been shown to be completely defective
in NER (
13
). UV light introduces almost exclusively two types of damage to DNA,
cyclobutane pyrimindine dimers (CPDs) (65-80%) and (6-4) pyrimidine-pyrimidone photoproducts (20-35%). At UV doses of 10 J/m
2
and above, UV61 cells can remove (6-4) photoproducts from total genomic DNA as efficiently as wild-type cells, but have a complete deficiency in the removal of CPDs
from total genomic DNA (
13
). When CPD removal was examined in the actively transcribed dihydrofolate
reductase (
DHFR
) gene in UV61 cells, a very low level of repair (when compared with wild-type cells) of CPDs was observed in the transcribed strand, while no
repair was observed in the non-transcribed strand (similar to wild-type cells) (
14
). Thus, the UV sensitivity of UV61 cells can be attributed to a shortfall in
repair of CPDs in the transcribed strand of active genes.
Transfection of the human
CSB
gene into either UV61 or CSB cells both complements their UV sensitivity and
restores nearly normal recovery of RNA synthesis following UV irradiation (
10
), suggesting that UV61 cells might have a genetically homologous defect to that
of CSB cells. However, whether transfection of
CSB
actually restores the specific TCR defect in CSB or UV61 cells has not been
demonstrated. To clarify the role of the
CSB
gene in repair processes, we have measured the induction and removal of CPDs in
both strands of actively transcribed genes and in an inactive region from AA8
(wild-type repair), UV61 and UV61 cells transfected with the
CSB
gene. We have also measured
in vitro
pol II transcription by extracts and partially purified fractions of these
three cell lines. Our results indicate that UV61 completely lacks TCR and that
transfection of the
CSB
gene restores the wild-type pattern of repair. However, in contrast to CSB cells (Dianov
et al
., unpublished observations), transcription is not defective in UV61 (when
compared with AA8 or the
CSB
transfectant of UV61), indicating that any role of the CSB protein in pol II
transcription can be uncoupled from its role in TCR.
UV61 cells were obtained from D.Busch and L.Thompson. PT5, a
CSB
transformant of UV61 (
11
), was a kind gift from C.Troelstra and J.Hoeijmakers. AA8 cells, the wild-type parental strain of UV61, were obtained from the ATCC. All cells were
grown in monolayer in DMEM/Ham's F-10 (1:1) supplemented with fetal bovine serum (10%), penicillin (100 U/ml)
and streptomycin (100 [mu]g/ml).
For the clonogenic survival assay (
15
), AA8, UV61 and PT5 cells were seeded in complete medium at densities ranging
from 10
2
to 10
6
cells/dish. After attachment of cells, the dishes were washed once with
phosphate-buffered saline (PBS). After PBS was removed, the cells were irradiated
with UV light (254 nm) at doses of from 0 to 14 J/m
2
, then incubated in complete medium until the appearance of colonies (6-7 days). The colonies were then rinsed with PBS, fixed with methanol and
stained with methylene blue (0.02%). Colonies containing greater than ~50 cells were counted. After correction for plating efficiency, survival
was determined by comparing the number of colonies in irradiated dishes to the
number of colonies in unirradiated controls.
Techniques for treatment of cells in culture with DNA damaging agents and for
subsequent isolation of genomic DNA were essentially as described (
16
). Briefly, cells were pre-incubated with [
3
H]thymidine (50-90 Ci/mmol) to label DNA. During exponential growth, cells were either
untreated or irradiated with UV light (20 J/m
2
at 254 nm), then harvested immediately (0 h) or given fresh medium containing
bromodeoxyuridine and fluorodeoxyuridine and harvested after 8 or 24 h. Lysis
of cells for isolation of genomic DNA was achieved using a proteinase K, SDS,
Tris, pH 8.0, solution.
Total genomic DNA was isolated by a NaCl extraction procedure (
17
). Treatment of DNA with RNase (100 [mu]g/ml) and restriction enzyme (
Kpn
I) was as described (
16
).
Kpn
I digestion (5 U/[mu]g DNA) yields a unique 14 kilobase (kb) fragment containing the 5'-end of the
DHFR
gene. Unreplicated DNA was isolated from replicated DNA (containing BrdU) after
fractionation of CsCl density gradients, dialyzed against TE buffer (10 mM
Tris, pH 8.0, 1 mM EDTA), ethanol precipitated and finally resuspended in TE.
All DNA concentrations were determined by measuring UV absorbance at 260 nm
using a Pharmacia/LKB Ultrospec III spectrophotometer.
The location of CPDs present in DNA samples isolated from UV-irradiated cells was detected by treatment of the DNA with a CPD-specific enzyme, T4 endonuclease V. Briefly, unreplicated DNA
samples (5 [mu]g) from various time points were incubated with T4 endonuclease V in buffer
containing 10 mM Tris, pH 8.0, 100 mM NaCl and 10 mM EDTA for 15 min at 37oC and the reactions stopped by the addition of formamide loading dye.
Parallel reactions in the same buffer minus T4 endonuclease V were performed to
determine the original distribution of restriction fragments.
As previously described (
16
), T4 endonuclease V-treated (or mock-treated) DNA samples were electrophoresed under alkaline conditions
and the DNA was quantitatively transferred by posiblot (Stratagene) to nylon
membranes (Oncor). Membranes were then conditioned with 2* SSPE (0.36 M NaCl, 20 mM NaH
2
PO
4
, 2.2 mM EDTA, buffered to pH 7.4 with NaOH), vacuum dried and treated with
Hybrisol I (Oncor) at least 4 h prior to hybridization.
Double-stranded DNA and single-stranded RNA probes for hybridization were synthesized using a
random primed DNA labeling kit (Boehringer Mannheim) with radiolabeled [[alpha]-
32
P]dCTP (3000 Ci/mmol) and the T7/SP6 RNA Polymerase Transcription kit
(Boehringer Mannheim) with [[alpha]-
32
P]CTP (3000 Ci/mmol) respectively. A subcloned fragment was used to make the
probe for the non-transcribed region 3' of the
DHFR
gene (cs14DO), as previously described (
18
). The fragment used to synthesize the double-stranded DNA probe for exons 2 and 3 of the mouse c-
myc
gene was purchased from Lofstrand Labs. The transcribed and non-transcribed strand probes of the
DHFR
gene were synthesized as described (
4
). Membranes were hybridized with probe, washed to remove non-hybridized probe and then subjected to autoradiography or radioactive
detection using a phosphorimager (Molecular Dynamics). By comparison of
untreated and T4 endonuclease V-treated samples, the average number of CPDs per fragment was determined
from the zero class of the Poisson distribution. Percent repair was calculated
by dividing the amount of CPDs removed by a given time point by the initial CPD
frequency.
In vitro
transcription-competent whole cell extracts were prepared as described (
19
) from AA8, UV61 and PT5 cells. To enhance pol II transcription from cellular
components (
20
), the extracts were also fractionated by phosphocellulose column
chromatography, then specific fractions [PC-FI (flow-though at 0.1 M KCl) and PC-FII (1.0 M KCl eluate)] were recombined. Whole cell extracts
(50 [mu]g protein) or recombined fractions in various ratios (totaling 40 [mu]g protein) were incubated for 1 h at 30oC with 2 [mu]g supercoiled plasmid pML(C
2
AT) containing the adenovirus major late promoter (
21
), which directs pol II-dependent transcription of a G-less cassette (
22
). The reactions (50 [mu]l) were carried out in transcription buffer which contained ATP (500 [mu]M), CTP (500 [mu]M), UTP (5 [mu]M), [[alpha]-
32
P]UTP (20 [mu]Ci), RNase inhibitor (20 U), phosphocreatine (8 mM) and creatine
phosphokinase (2.5 [mu]g) in HEPES-KOH buffer, pH 7.9, containing dithiotheitol (1.5 mM), EDTA (0.5 mM),
glycerol (8.5%), MgCl
2
(8.5 mM) and KCl (50 mM). The samples were treated with RNase T
1
(10-20 U) for 10 min at room temperature to digest any transcripts not
specified by the adenovirus promoter/G-less cassette construct, then with SDS (0.5%) and proteinase K (50 [mu]g) for 20 min at 30oC. After the addition of carrier tRNA (20 [mu]g), RNA samples were ethanol precipitated, resuspended and
heated (95oC for 2 min) in formamide dye (10 [mu]l) and electrophoresed on a 5% polyacrylamide-7 M urea gel. Gels were washed (in H
2
O for 20 min) and vacuum dried prior to autoradiography or phosphorimaging
analysis.
Measurement of the survival of cells following DNA damaging treatments roughly
reflects their capacity to repair the inflicted damage. Cells from CHO
complementation group six (which includes UV61) have intermediate levels of UV
resistance when compared with NER-proficient and completely NER-deficient cell lines. Using clonogenic survival assays, the effect
of UV irradiation on the survival of the AA8, UV61 and PT5 cell lines was
measured (Fig.
1
). As expected, AA8 has a range of UV sensitivity typical of most normal cells,
while UV61 is significantly more sensitive. PT5 cells appear only slightly more
UV sensitive than AA8, indicating that transfection of DNA containing the
CSB
gene restores a near wild-type level of UV survival to UV61.
CHO cells, in general, show a low level of repair of CPDs when repair is
measured over the entire genome. However, within actively transcribing strands
of certain genes, the level of repair of CPDs 24 h after UV irradiation has
been shown to be as high as 89% for CHO cells with normal NER capabilities (
3
). UV61 cells have been shown to repair (6-4) photoproducts efficiently, but have little or no repair of CPDs when
measured at the level of the overall genome (
13
). We sought to determine whether UV61 had detectable repair of CPDs at the
level of the gene and whether transfection of the
CSB
gene into UV61 changed the repair capability of the cells. Several previous
studies have measured repair in the house-keeping
DHFR
gene in other wild-type and NER-deficient cells, so we felt that
DHFR
would be the best model gene to study. Moreover, the preparation of both
transcribed and non-transcribed strand-specific probes allows the measurement of repair in each individual
strand of the
DHFR
gene. Therefore, we measured the induction and removal of CPDs in the
individual strands of the
DHFR
gene and in a non-transcribed region (cs14DO) downstream (3') of
DHFR
in the AA8 (parental wild-type), UV61 and PT5 (
CSB
transfectant of UV61) cell lines. Quantitative Southern blots measuring the
amount of CPDs in the individual strands of the
DHFR
gene over time after irradiation are shown in Figure
2
. For each cell line, the number of CPDs in both strands and in the cs14DO
region over time after irradiation is presented in Table
1
and the level of repair of CPDs in the transcribed strand of the
DHFR
gene is depicted in Figure
3
A. As in other CHO repair-proficient cell lines, the repair of CPDs in AA8 appears limited to only
the transcribed strand of
DHFR
(Table
1
). Specifically, the repair of CPDs in the transcribed strand of the
DHFR
gene is ~70% complete by 24 h (Fig.
3
A); there is no detectable repair in the non-transcribed strand after 24 h. In contrast, there is no measurable repair
of either the transcribed or non-transcribed strand in the UV-sensitive UV61 cell line (Table
1
and Fig.
3
A). When repair was measured in PT5, the
CSB
transfectant of UV61, again no repair of the non-transcribed strand of
DHFR
was observed. However, the transfectant had regained the ability to repair the
transcribed strand of the
DHFR
gene. Within experimental error, both the rate and extent of repair of the
transcribed strand in PT5 are similar to that of AA8 (Fig.
3
A and Table
1
). No repair of CPDs in the inactive region downstream of the
DHFR
gene was detected in any of the three cell lines (Table
1
). Thus, transfection of the human repair gene (
CSB
) into a repair-deficient CHO cell restores the wild-type pattern of repair of CPDs.
Our results showing essentially no repair of the transcribed strand of the
DHFR
gene contradict earlier findings by Lommel and Hanawalt (
14
), who reported a low level of repair (33% after 24 h) for the same DNA
sequence. Since NER of CPDs in CHO cells appears to be intimately linked to
transcription, the study of repair in a more highly transcribed gene might
clarify the extent of the CPD repair defect in UV61. As our candidate gene we
chose c-
myc
, which, in CHO cells, is transcribed at 5-10 times the level of the
DHFR
gene (
23
). The identical Southern blots used above were reprobed with a double-stranded mouse c-
myc
probe that hybridizes to a unique fragment of ~9 kb in
Kpn
I-restricted CHO genomic DNA. Results from these experiments (Table
1
and Fig.
3
B) show reasonably efficient levels of CPD repair in the c-
myc
gene in the AA8 (wild-type) and PT5 (
CSB
transfectant of UV61) cell lines. The average level of damage present in both
strands is detected by this probe, so the lower level of repair in AA8 and PT5
(when compared with the
DHFR
results) is a reflection of strong repair of the transcribed strand averaged
with zero repair of the non-transcribed strand. However, no detectable repair in the c-
myc
gene was observed in UV61, which indicates that there was no substantial repair
even in the transcribed strand of the c-
myc
gene. Thus, our data demonstrate that there is little or no repair of CPDs in
UV61 cells.
Table 1
Certain types of DNA damage subject to removal by NER are repaired with the
highest priority when present in the transcribed strand of an active gene
(TCR). For instance, in human cells, UV-induced CPDs are removed much more rapidly from the transcribed strand of
active genes than from the non-transcribed strand of the same genes or from an inactive region of the
genome (
3
,
24
). In an extreme example of this type of repair bias, wild-type CHO cells have efficient repair of UV-induced CPDs in the transcribed strand of active genes but little or
no repair of these adducts in non-transcribed parts of the genome (
3
,
4
,
25
, this work). In contrast, repair of UV-induced (6-4) photoproducts is rapid over the entire genome in NER-proficient CHO cells (
13
). The UV sensitivity of CHO mutant UV61 is intermediate between wild-type CHO cells and CHO mutants completely defective in NER (
13
). Since UV61 has normal repair of (6-4) photoproducts (
13
), the partial UV sensitivity of UV61 is due to defective repair of CPDs. An
earlier study (
14
) had shown a very low level of repair in the transcribed strand of the
DHFR
gene in UV61 cells. In contrast, the studies presented here demonstrate that
UV61 cells do not detectably remove CPDs from either the transcribed strand of
the
DHFR
gene or both strands of the highly active c-
myc
gene. Our results indicate that the partial UV sensitivity of UV61 cells is due
to the complete absence of CPD repair. Thus, both (6-4) photoproducts and CPDs contribute to UV sensitivity in CHO cells and
the effect of CPDs on cell survival correlates with their persistence in the
transcribed strand of active genes.
Recently, mutations in the human
CSB
gene were documented in cells from complementation group B of CS (
10
). CS cells (complementation groups A and B) repair all DNA sequences at the
slow rate observed for inactive regions (
7
,
8
). Specifically, TCR is absent in CS cells (
26
-
28
), which may explain their moderate UV sensitivity and slow recovery of RNA
synthesis after UV irradiation (
29
). Transfection of a normal
CSB
gene into CS cells (complementation group B) complements the UV sensitivity and
improves RNA synthesis recovery (
10
), suggesting that TCR is restored. The
CSB
(originally
ERCC6
) gene was first cloned by virtue of its ability to correct the intermediate UV
sensitivity of UV61 (
11
). In this report, we have measured CPD repair in a
CSB
transfectant of UV61 and found that the wild-type repair pattern, i.e. efficient repair of CPDs only in the transcribed
strand of active genes, was restored. This is the first direct demonstration
that transfection of
CSB
corrects the specific TCR defect in either hamster or human cells and
conclusive evidence that a hamster
CSB
homolog is defective in UV61. The complete restoration of TCR by transfection
of CSB into UV61 correlates well with levels of complementation of UV
sensitivity observed previously (
10
,
11
). Surprisingly, the human CSB protein appears to be completely interchangeable
with the hamster protein with regard to its function in the TCR pathway.
Moreover, the
CSB
gene product may be the functional analog of
E.coli
TRCF (the protein product of the
mfd
gene), which has been shown to directly link NER to active transcription (
5
). Recently, the
CSB
gene product has been shown to bind to the CSA protein, which, in turn,
interacts with a subunit (p44) of TFIIH (
30
). Thus, the direct link of repair to the transcribed strand of active genes
(absent in CS cells) might occur via the binding of a CSA-CSB protein complex to TFIIH.
The potential binding of a CSA-CSB complex to TFIIH (which participates in both transcription and
repair) suggests that defects in human or hamster CSA and/or CSB homologs might
affect transcription as well as repair. Indeed, many of the clinical
manifestations of CS have been attributed to defects in transcription rather
than repair (
9
). In agreement with this hypothesis, extracts from certain CS (groups A and B)
cells display reduced levels of pol II-directed transcription (Dianov
et al
., unpublished observations). However, when we compared pol II-dependent transcription, using the same assay, in the putative
CSB
hamster mutant (UV61) with its parental cell line (AA8) and the human
CSB
transfectant of UV61 (PT5), no significant differences were found. Although the
CSB
mutation in UV61 causes a complete defect in TCR, it causes little or no change
in transcription. Thus, the established role of the
CSB
gene in TCR and any putative role in pol II-dependent transcription appear to be independent of one another, at least
in CHO cells. Intriguingly, the CSB homolog from
Saccharomyces cerevisiae
, RAD26, is necessary for TCR, but has no essential role in transcription (
31
). By analogy, the normal mammalian
CSB
gene may have no direct function in transcription, but certain (dominant
negative) mutations could cause perturbations in transcription, perhaps through
sequestration of TFIIH in the repair pathway, accounting for the clinical CS
symptoms. One prediction from this hypothesis is that many
CSB
mutations (as in UV61) cause only defects in TCR (resulting in probably a mild
UV-sensitive phenotype) and would not be detectable by screening for certain
CS symptoms. Another possibility is that the
CSB
gene product (in mammalian cells) has an auxiliary role in transcription, and
mutations in the
CSB
gene can independently affect either its repair or transcription function.
Similarly, specific mutations in the
XPD
gene product (a subunit of the basal transcription factor TFIIH) can affect NER
and transcription differentially (
32
-
34
).
Mammalian NER is obviously very complex, with apparent changes in mechanism and
kinetics of damage removal depending on the type of DNA adduct. Cells
completely defective in NER (CHO complementation group 2 and xeroderma
pigmentosum group A, for example) cannot repair either CPDs or (6-4) photoproducts (
35
,
36
), indicating that both major UV adducts are subject to NER. However, the
kinetics and compartmentalization of repair of these adducts in normal cells
are very different. In wild-type CHO cells, (6-4) photoproducts are repaired rapidly and thoughout the entire
genome, while CPDs are repaired more slowly and only in the transcribed strand
of active genes. The
CSB
mutation in UV61 cells eliminates the transcribed strand-specific repair of CPDs (this work), but does not affect repair of (6-4) photoproducts (
13
). This suggests that all repair of CPDs in CHO cells occurs through coupling to
transcription, explaining the lack of repair of CPDs outside the transcribed
strand of actively transcribed genes. In contrast, repair of (6-4) photoproducts can occur (via the overall genome repair pathway) without
being coupled to transcription, although a recent study (
37
) indicates that, when overall genome repair is deficient (in XPC cells), (6-4) photoproducts are repaired in a strand-specific manner. Perhaps the
CSA
and/or
CSB
gene products enhance recognition of certain bulky adducts (such as CPDs) by
linking transcription and NER processes. An analogous situation exists in
E.coli
, where the low specificity of (A)BC excinuclease for CPDs is augmented by
photolyase or TRCF (
5
,
38
). Alternatively, mutation of the
CSB
homolog in UV61 may affect damage recognition subtly but directly, influencing
repair of some adducts (such as CPDs) while not affecting repair of others [(6-4) photoproducts]. This explanation seems unlikely, however, based on the
close connections to transcription and the partial repair of CPDs in the
overall genome by human cells with a defective
CSB
gene (
7
,
26
). Also, the
CSB
gene product is not required for repair of either UV photoproducts or
cholesterol adducts by human cell extracts
in
in vitro
repair assays (
39
,
40
). Hopefully, future characterization of the purified CSB protein (and its
interactions) will clarify its roles in NER and transcription.
We thank Drs Christine Troelstra and Jan Hoeijmakers for their contribution of
the
CSB
transfectant of UV61 and their preliminary analysis of its properties. We
appreciate the interaction with the Danish Center for Molecular Gerontology. We
also thank Drs Carleen Cullinane and Collette apRhys for critical reading of
the manuscript and Jennifer Jones and Irina Dianova for technical assistance.
Cell line
DNA fragment
Time post-UV irradiation (h)
0
8
24
AA8
Transcribed
1.50 +- 0.05
a
0.84 +- 0.25
0.46 +- 0.21
Non-transcribed
1.53 +- 0.08
1.62 +- 0.18
1.58 +- 0.11
cs14DO
1.34 +- 0.09
1.38 +- 0.05
1.47 +- 0.22
c-
myc
0.66 +- 0.07
0.55 +- 0.05
0.42 +- 0.07
UV61
Transcribed
1.56 +- 0.01
1.69 +- 0.15
2.06 +- 0.46
Non-transcribed
1.67 +- 0.05
1.54 +- 0.03
2.04 +- 0.37
cs14DO
1.63 +- 0.17
1.63 +- 0.20
2.48 +- 0.01
c-
myc
0.67 +- 0.05
0.74 +- 0.11
0.91 +- 0.19
PT5
Transcribed
1.80 +- 0.16
0.79 +- 0.21
0.36 +- 0.08
Non-transcribed
1.92 +- 0.20
1.84 +- 0.19
1.82 +- 0.07
cs14DO
1.54 +- 0.07
1.40 +- 0.10
1.43 +- 0.19
c-
myc
0.71 +- 0.10
0.55 +- 0.03
0.36 +- 0.14
REFERENCES
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