From the Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom
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ABSTRACT |
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A library of Escherichia coli fnr
mutants has been screened to identify FNR (regulator of
fumarate and nitrate reduction) variants that are defective repressors, but competent activators. All
but one of seventeen variants had substitutions close to or within the
face of FNR that contains activating region 1 (AR1). Activating region
1 is known to contact the The FNR protein of Escherichia coli is a global
transcription regulator, controlling the expression of genes in
response to oxygen starvation. FNR is predicted to be structurally
related to the cAMP receptor protein and acts mainly as an activator of genes involved in anaerobic energy generation (1). Generally, FNR
activates transcription by binding to a site centered at about As well as acting as an activator of anaerobic gene expression, FNR
also acts as a repressor of some aerobic genes (1). Unlike the
FNR-activated promoters, there is no discernible pattern of FNR site
positioning among FNR-repressed promoters, although a common feature is
the presence of multiple FNR sites upstream of the transcription start
(1). The best characterized of the FNR-repressed genes is
ndh, which encodes a non-proton-translocating NADH
dehydrogenase that is the major primary dehydrogenase of the aerobic
respiratory chain (4). The ndh promoter has two FNR sites
centered at Error-prone Polymerase Chain Reaction Mutagenesis--
Random
mutations in the fnr gene carried by pGS24 (a derivative of
pBR322 containing the fnr gene in a
HindIII-BamHI fragment) were introduced using
Taq DNA polymerase and the following synthetic primers, as
described previously (2):
5'-GCTTATCATCGATAAGCTTCGTGAATATTTTGCCGG (fnr coordinates 1-23) and
5'-CGTAGAGGATCCAGGCTGTACGC (1625-1641), where the unique
HindIII and BamHI targets are underlined.
Following digestion with HindIII and BamHI, the
polymerase chain reaction products were ligated into the corresponding
sites of pBR322. Plasmids were isolated by standard methods, and
mutations in the fnr gene were defined by Applied Biosystems
cycle sequencing with the aid of two primers:
5'-AAACATATGGTCCCGGAAAAGCG (520-536) and 5'-GGAAACCTCGATGGTAGCTGAAATCCCGTTCG (866-898).
Identification of FNR Variants Defective in Repression--
The
library of mutagenized fnr genes in pBR322 was used to
transform JRG3701, a derivative of JRG1728
[ Partial Proteolysis--
The FNR protein was purified from a
glutathione S-transferase-FNR fusion protein as described
(15). Aliquots (20 µl, 3.4 mg/ml) of the isolated FNR were incubated
at 30 °C for up to 2 h in the presence of either 0.68 unit of
trypsin (Sigma), 1 unit of chymotrypsin (Sigma), or 0.1 unit of V8
protease (Sigma). The peptides generated were then fractionated by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes for N-terminal amino acid sequencing.
Identification of Repression Defective FNR Variants--
Previous
attempts to isolate repression defective FNR variants using the
FNR-repressible ndh promoter as a screen were unsuccessful, yielding only FNR proteins with reduced affinity for their DNA target
and therefore compromised in both activation and repression (16).
However, recent analysis of the regulation of yfiD
expression in E. coli (9) has provided an opportunity to
develop a better screen. The yfiD gene has an unusual
promoter architecture for an FNR-activated gene, with two FNR sites
centered at
Error-prone polymerase chain reaction mutagenesis was used to generate
a library of randomly mutated fnr genes in pBR322. Transformants of JRG3701 (
Nucleotide sequence analysis revealed that most of the FNR variants
encoded by the plasmids contained substitutions in the face of FNR that
contains AR1 (Table I, Fig.
1A). The only exception was
the variant M147T (which was isolated twice) representing a replacement
buried in the dimer interface (helix
The pBR322 derivatives encoding FNR variants defective in
yfiD down-regulation were transferred into JRG3917
( Substitutions Effecting FNR-mediated Repression Are Located in
Surface-exposed Regions--
There is no structure available for FNR,
but there is evidence to indicate that the predicted similarity with
the cAMP receptor protein is well founded (1). An essential feature of
any regions of FNR involved in contacting other components of the
transcription machinery is that they must be solvent exposed. Partial
proteolysis of isolated FNR with trypsin (28 possible cleavage sites,
406 possible peptides) or V8 protease (28 possible cleavage sites, 435 possible peptides) followed by N-terminal amino acid sequencing of the
resulting peptides revealed that the substitutions affecting repression
were within regions of FNR accessible to the proteases (Table
II, Fig. 1B). Following
digestion with trypsin, separation of peptides by SDS-polyacrylamide
gel electrophoresis, and blotting, 10 Coomassie Blue-stained bands were
detected. The N-terminal amino acid sequences of the peptides present
in each band were determined (Table IIa). Many of the bands contained
the same N-terminal sequences, indicating preferential cleavage at a
limited number of positions with subsequent C-terminal processing. The
preferred tryptic/chymotryptic cleavage points were: Arg6,
Lys77, Tyr79, Phe92, and
Arg184 (Fig. 1B). Treatment with V8 protease
produced four bands with the N-terminal sequences in Table IIb. These
data imply cleavage at positions Glu4, Glu38,
Glu117, and Glu123. Therefore, the region
encompassing the series of loops in the AR1 side of the Transcription can be repressed either passively by promoter
occlusion, i.e. when a regulator blocks access of RNA
polymerase to the promoter, or actively, in which the regulator makes
direct contact with RNA polymerase to inhibit transcription initiation (17). The observation that FNR and RNA polymerase can simultaneously interact with the FNR-repressible ndh promoter suggested
that, in this case, repression is unlikely to be mediated simply by promoter occlusion (6, 7). The ndh promoter has two FNR sites centered at Using yfiD-lacZ as an initial screen 17 FNR variants
defective in repressing both yfiD and ndh
promoters were identified. All but one of the variants (M147T) had
substitutions near or within the face of FNR that contains AR1 (Fig. 1,
2), a region of the protein known to contact the subunit of RNA polymerase to facilitate
transcription activation. It is now evident that this face also has a
role in FNR-mediated repression. Single amino acid substitutions at
Lys54, Gly74, Ala95,
Met147, Leu193, Arg197, or
Leu239, and double substitutions at Ser13 and
Ser145, Cys16 and Ile45,
Tyr69 and Ser133, or Lys164 and
Phe191, impaired FNR-mediated repression of ndh
without greatly affecting activation from model Class I (FNR site at
71.5) and Class II (FNR site at
41.5) FNR-activated promoters.
Although repression was impaired in a second group of FNR variants with
substitutions at Leu34, Arg72 and
Leu193, Phe92, or Ser178,
transcription activation from the simple FNR-dependent
promoters was severely reduced. However, expression from
pyfiD (FNR sites at
40.5 and
93.5) and a derivative
lacking the site at
93.5, pyfiD
/+, remained relatively
high indicating that this second group have a
context-dependent activation defect as well as a repression
defect. The prediction that the substitutions affecting repression were
likely to be in solvent exposed regions of FNR was supported by
analysis of peptides produced by partial proteolysis of FNR. Thus,
FNR-mediated repression at promoters with multiple FNR sites requires
regions of FNR that are different from, but overlap, AR1.
INTRODUCTION
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41 in
target (Class II) promoters where it makes multiple direct activating
contacts with RNA polymerase involving two discrete activating regions
of FNR (Fig. 1A, 2 and 3). Activating
region 1 (AR1) appears to contact the C-terminal domain of the RNA
polymerase
subunit, thereby preventing inhibition of transcription
activation caused by an untethered
subunit. Activating region 3 (AR3) contacts the
70 subunit of RNA polymerase to
activate transcription (2). At promoters in which the FNR site is
centered at
61 or beyond (Class I) activation depends on a different
AR-
subunit contact (3).
50.5 and
94.5, and both contribute to FNR-mediated
repression (5-7). The mechanism of FNR-mediated repression appears not
to be due to simple promoter occlusion, but rather displacement of the
RNA polymerase
subunit leads to inhibition of transcription (7).
FNR sites located beyond the region of DNA occupied by RNA polymerase
have also been shown to be necessary for efficient repression of the
fnr and narX promoters (8). Furthermore, FNR
occupation of the far upstream FNR site of the yfiD promoter
(FNR sites at
40.5 and
93.5) down-regulates yfiD
expression (9). These observations indicated that FNR-mediated repression of these promoters requires multiple FNR binding sites and
thus repression may arise a consequence of interactions between two or
more FNR dimers, or between FNR dimers (or tetramers) and RNA
polymerase. Therefore a library of fnr mutants was screened for FNR variants defective in repression. Two types of variant were
identified: type A was a poor repressor but good activator; type B was
a poor repressor but failed to activate transcription from simple
FNR-activated promoters. However, the type B variants did activate
transcription from the complex yfiD promoter. All the
variants isolated, except one, contained amino acid substitutions overlapping the face of FNR that contains AR1, indicating that this
surface may have a role in repression as well as in activation and
anti-inhibition.
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(lacIPOZYA)X74 galU galK rpsL
(ara-leu)
(tyrR-fnr-rac-trg)17
zdd-230::Tn9] containing a compatible
yfiD-lac reporter plasmid, pGS1000 (10). Transformants were
tested for enhanced yfiD-lacZ expression on
L-agar containing: 5-bromo-4-chloro-3-indoyl
-galactoside (40 µg/ml), ampicillin (200 µg/ml), and
tetracycline (35 µg/ml). The plates were incubated under anaerobic
conditions at 37 °C for 16 h, after which they were exposed to
air. The colonies were monitored and those that became blue first were
noted and recovered. The levels of yfiD-lacZ expression were
determined by measuring
-galactosidase activity (11) with cultures
that had been grown anaerobically for 16 h at 37 °C in sealed
bottles containing L broth supplemented with glucose (0.4% w/v),
ampicillin (200 µg/ml), and tetracycline (35 µg/ml). The pBR322
derivatives containing the mutant fnr genes were isolated
and were then used to transform JRG3917, a JRG1728 derivative
containing a pRW2-based ndh-lacZ reporter, pndh
(2), and the degree of repression conferred by the FNR variants was estimated by measuring
-galactosidase activity as above. As
anaerobic ndh-lacZ expression of fnr strains is
enhanced during growth on rich medium (6), the effect of the FNR
variants on ndh-lacZ expression was also determined
following growth on a glucose minimal medium supplemented with leucine
(12) and appropriate antibiotics. Similarly, two simple FNR-activated
promoters, the Class I FF-71.5pmelR (FNR site at
71.5) and
Class II FFpmelR (FNR site at
41.5), as well as
pyfiD promoter mutants with impaired FNR sites at either
93.5 (pyfiD
/+; pGS1062) or
40.5 (pyfiD+/
;
pGS1063) fused to lac in pRW50 were used to determine the
effects of the selected amino acid substitutions on FNR-mediated
activation (9, 13). Western blotting of the soluble fraction of sonic
cell-free extracts with polyclonal anti-FNR serum has been described
previously (14). The relative amount of each FNR variant was estimated
by quantitative densitometry using a Vilber-Lourmat imaging system.
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40.5 and
93.5 relative to the transcript start.
Multiple FNR sites are usually found in FNR-repressed promoters; for
example, the ndh gene has FNR sites centered at
50.5 and
94.5 (1). Expression of yfiD is dependent upon an
activating FNR dimer centered at
40.5, but occupation of the upstream
FNR site (
93.5) down-regulates expression (9). Therefore, screening a
library of randomly mutagenized fnr genes for those that
allow increased yfiD-lacZ expression (i.e. FNR
variants that still activate from position
40.5, but fail to act as
repressors at
93.5) should ensure that the FNR proteins selected are
not compromised in DNA binding activity.
fnr
lac,
containing a compatible yfiD-lacZ reporter plasmid,
pGS1000) were screened for elevated yfiD-lacZ expression on
5-bromo-4-chloro-3-indoyl
-galactoside plates. Approximately 4000 colonies were screened, and 19 were found to contain plasmids that
enhanced yfiD expression (Table
I). Estimation of the
-galactosidase
activities of anaerobic cultures indicated that all of the FNR variants
encoded by the plasmids were defective in the down-regulation of
yfiD expression. Most of the FNR variants were produced in
normal amounts, as judged by Western blotting (Table I). Expression of
fnr is autoregulated, partially by binding at a site
overlapping the transcript start such that FNR acts as a molecular
brick. Hence, the amount of each FNR variant may reflect their relative
affinities for this site as well as any effects that the particular
substitutions may have on protein stability. Even those produced at
lower levels were expressed well enough for the screening protocol,
because, although chromosomal expression yielded least FNR (<5% of
that obtained with pGS24), it was still sufficient to allow regulation
of yfiD expression comparable with that observed with
multicopy fnr (Table I). This reflects the relative
abundance of the reporter plasmid (2-5 copies per cell) compared with
the fnr-encoding plasmid (15-20 copies per cell).
Therefore, the failure to down-regulate yfiD is probably not
due to a lack of FNR in the cell.
Transcription regulation of FNR-repressed and FNR-activated promoters
by 17 FNR variants
-galactosidase driven by the indicated FNR variants
was measured in JRG1728 (
lac
fnr)
transformed with pyfiD, pndh, FFpmelR,
or FF-71.5pmelR fused to lac. Values (which
varied by no more than 10%) are the mean of duplicate assays of at
least two independent anaerobic cultures of each strain, grown on rich
medium in sealed bottles at 37 °C for 16 h. Expression of
ndh-lacZ was also determined in minimal medium to eliminate
the enhancement of ndh expression associated with anaerobic
growth on rich medium in the absence of FNR. Aerobic expression from
the test promoters for all the variants was similar to that observed
for FNR: 100 Miller units for pyfiD; 2100 for
pndh; 110 for pyfiD
/+; 150 for
FFpmelR; and 150 for FF-71.5pmelR. Expression of
each variant was assessed by Western blotting using anti-FNR serum and
is given as a percentage (±20%) of that observed with cultures
expressing fnr from an equivalent plasmid (pGS24, row
labeled FNR). The figures in parentheses indicate the number of
independent isolates.
C). There is no
obvious reason why such a substitution should result in the properties
observed; however, it should be noted that the M147T variant still
retained significant repressing ability (Table I). Many of the
substitutions (K54E, Y69C, R72L, G74C, F92S, A95P) were clustered in a
series of loops that form the AR1 side of the FNR
-roll. A second
cluster was evident in the region encompassing
D to
E (S178F, F191L, L193P, R197H). Four variants (G74C,
F92S, A95P, and L193P) had been identified in a previous screen, and it
was suggested that the defect in yfiD down-regulation was
due to an altered AR1-containing surface in these variants (9). Five of
the 17 variants identified contained two substitutions, but in each
case at least one of the replacements was of a residue predicted to be
close to or part of the AR1-containing surface.
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Fig. 1.
Positions of amino acid replacements that
compromise FNR-mediated repression and locations of solvent exposed
regions of FNR. A, predicted structure of an FNR
monomer based on that of the cAMP receptor protein showing the
positions of amino acid replacements that impair FNR-mediated
repression of ndh-lacZ and yfiD-lacZ expression.
The helix-turn-helix motif ( D-
F) in the
DNA-binding domain, the essential cysteine residues that act as ligands
for the [4Fe,4S] cluster (ringed), and the previously identified
activating regions AR1 and AR3 (or 85-loop) are also indicated. B,
surface-exposed regions of FNR. Predicted structure of an FNR monomer
showing the sites of cleavage by trypsin and V8 protease.
fnr
lac, containing a compatible
FNR-repressed ndh-lacZ reporter, pndh; Ref. 2). All the variants failed to repress ndh expression normally
indicating that the yfiD and ndh promoters may
share a common mechanism of FNR-mediated repression (Table I). As
anaerobic expression of ndh is known to respond to nutrient
quality in fnr strains (6), ndh-lacZ expression
was also determined for cultures grown in a defined minimal medium
(Table I). The data obtained confirmed the FNR repression defects
observed in rich medium. Tests with equivalent strains carrying the
FNR-activated Class II FFpmelR-lacZ reporter plasmid, which
should be substantially unaffected by substitutions in AR1, indicated
that all but four of the seventeen variants were capable of activating
transcription. It was expected that all the variants would be competent
activators of Class II promoters, because the basis of the original
screen depended on FNR activation from a site at
40.5 in the
yfiD promoter. Therefore, the response of the simple Class
II promoter FFpmelR defines two types of FNR variant: Type
A, which fails to repress but activates normally, and Type B, which has
both repression and activation defects. This assignment was confirmed
using a pyfiD
/+ reporter (pGS1062) with an impaired FNR
site at
93.5 (Table I). The Type B variants exhibited much reduced
activity at this promoter compared with the Type A proteins. Studies
with the Class I promoter FF-71.5pmelR, which requires an
AR1-
contact for activation, indicated that the Type A substitutions
could be neutral for (R197H), or improve (K54E, A95P) or impair (I45T,
Y69C, S133P, G74C, M147T, L193P, L239P, L239E), the AR1 contact.
However, any improvement in the AR1 contact was insufficient to allow
activation from a yfiD reporter with an impaired FNR site at
40.5, pyfiD
/+ (not shown). Furthermore, the variants
G74C and L193P, which activated the Class II promoter well, but were
poor at Class I, indicated that the anti-inhibition contact made
between the RNA polymerase
subunit and the AR1 face of FNR
(required at Class II promoters) is different to the activating AR1
subunit contact made at Class I promoters.
-roll was
particularly susceptible to proteolytic cleavage. These results
indicate that the regions identified as important for FNR-mediated
repression are solvent exposed and also provide further evidence to
support the cAMP receptor protein-based predicted FNR structure,
because the equivalent positions are known to be surface exposed in the
cAMP receptor protein.
Partial proteolysis of FNR
DISCUSSION
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50.5 and
94.5, and it has been proposed that FNR
occupation of these sites prevents RNA polymerase
subunit from
interacting with DNA (7). Such a repression mechanism may require
direct protein contacts between the two FNR dimers and/or FNR and RNA
polymerase. Previous attempts to identify FNR variants compromised for
repression of ndh-lacZ were unsuccessful, because the
transformants recovered contained plasmids encoding FNR proteins with
defects in DNA binding (16). However the yfiD-lacZ reporter
provided an opportunity to screen out FNR variants defective in
DNA-binding, because this promoter, although down-regulated by FNR
occupation of a site centered at
93.5, requires an activating FNR
dimer (at
40.5) for expression.
subunit
of RNA polymerase and thereby facilitating transcription activation
(3). Two types of contact can be made depending on the architecture of the activated promoter. At Class I promoters (FNR site at or beyond
61) AR1 of the downstream FNR monomer makes an activating contact with the
subunit, whereas at Class II promoters (FNR site at or
about
41) AR1 makes an anti-inhibition contact (3). It is now
apparent that the same face of FNR can be involved in repressing transcription at promoters that contain multiple FNR sites and that the
regions involved are solvent exposed. This is supported by the
observations that (i) the positions of two substitutions (Arg72 and Phe191, indicated in
yellow on Fig. 2) are common
to FNR variants with altered AR1 or repression properties; (ii) all the
repression defective variants (with the exception of the M147T) contain
a substitution in the AR1 side of the FNR monomer that forms two clusters overlapping the previously defined positions of AR1 (Fig. 2);
(iii) most of the repression defective variants display altered activation from a model Class I (AR1-dependent) promoter
(Table I); and (iv) that several amino acids close to those substituted in repression defective variants (Leu34, Gly74,
Phe92, Ser178) are sufficiently exposed to
allow proteolytic attack (Glu38, Lys77,
Phe92, and Arg184). Thus, the simplest
explanation for the repression defective phenotype displayed by the FNR
variants is that they possess an altered surface that is different from
but overlaps AR1 which has a role in repression of promoters with
multiple FNR sites.
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Fig. 2.
Positions of amino acid substitutions in FNR
defining activating regions and repression defective variants
superimposed on the backbone of a cAMP
receptor protein monomer. The position of amino acids forming AR1
(see Refs. 3 and 18) important in transcription at Class I promoters
(Arg72, Ser73, Thr118,
Met120, Phe181, Arg184,
Phe186, Ser187, Pro188, and
Ala225; blue), AR3 (19) required for the
activation of Class II promoters (Ile81, Gly85,
Asp86, and Phe112; green); and
repression-defective variants (Ile45, Lys54,
Tyr69, Gly74, Phe92,
Ala95, Ser178, Leu193, and
Arg197; red) are indicated. The yellow
spheres indicate positions (Arg72 and
Phe191) that are common to AR1 activation and repression.
The AR3 defining residues of FNR are clustered on the left
(green); the AR1 defining amino acids (blue and
yellow) form a large surface along the opposite face of FNR
to that containing AR3. The repression-defective mutations encode
substitutions along the AR1 surface (red and
yellow residues) and a further region falling between AR3
and AR1 (red residues only) forming a sharks fin-like
pattern of substitutions. The repression defective variants with
substitutions at Ser13, Leu34, and
Leu239 lie in regions of FNR beyond that encompassed by the
cAMP receptor protein structure, Met147 in the dimer
interface, and the Ser133, Ser145, and
Lys164 (isolated as double mutations with partners in the
AR1 face) are omitted for clarity.
It has been suggested recently that transcription regulators can be viewed as catalysts (20). It is envisaged that activators lower the activation energy associated with one or more steps in the reaction pathway leading to transcription initiation, whereas repressors could increase the energy barriers to be overcome in forming an open complex, thereby inhibiting transcription initiation (20). Thus, it is possible that the specific configurations of two FNR dimers at the ndh and yfiD promoters effectively jam RNA polymerase in one of the intermediate states between the closed and open complex. Indeed, FNR-mediated inhibition of open complex formation at the ndh promoter has been observed previously (6). The data presented here suggest that this could be achieved by direct protein-protein contacts involving the face of FNR containing AR1.
The architecture of a promoter is clearly crucial in determining the
effect of a particular regulator (or combination of regulators) on
transcription (21). It is not yet established if the repression specific components of the AR1 face participate in FNR-FNR or FNR-RNA
polymerase interactions, but it is likely that the various contacts
made by the AR1 containing face are subtly different. Indeed, there is
good evidence to suggest that, for the cAMP receptor protein the
anti-inhibition and activating contacts made by AR1 are different (22).
The GalR protein is perhaps the best example of an "active"
repressor (20). Like FNR, GalR can act either as an activator (of
gal promoter 2, galP2) or as a repressor (of gal promoter 1, galP1) and a characteristic
GalR-RNA polymerase-gal ternary complex is formed at each
promoter because of putative GalR-RNA polymerase interactions.
Mutations in the region of the C-terminal domain of the subunit of
RNA polymerase thought to be involved in GalR-mediated activation also
relieve GalR-mediated repression (20). However, the activator complex
at galP2 is an open complex, whereas the repressing complex
at galP1 is a closed complex and thus the context of the
GalR-RNA polymerase contacts are different. These context effects are
proposed to be sufficient to allow a single regulatory protein to act
as a both a repressor and an activator while maintaining similar
regulator-polymerase contacts (20). Therefore, it is suggested that the
context in which FNR finds itself at the yfiD and
ndh promoters favors the formation of a ternary complex
incorporating FNR-polymerase contacts that render the complex
incompetent for transcription activation.
In conclusion, the FNR variants identified here provide the first
indication that specific regions (amino acids) of FNR that overlap AR1
may be required for transcription repression at promoters with multiple
FNR sites. Mutational analysis of the C-terminal domain of the RNA
polymerase subunit should determine whether FNR can repress
transcription via direct contact between the AR1 containing face and
RNA polymerase or whether FNR-FNR contacts are the key to FNR-mediated
repression at promoters sharing the ndh architecture.
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ACKNOWLEDGEMENTS |
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We thank J. R. Guest, S. J. W. Busby, and H. J. Wing for many helpful discussions and J. Keen for amino acid sequencing.
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Sciences Research Council of the United Kingdom and the Royal Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-114-222-4403;
Fax: 44-114-272-8697; E-mail: jeff.green{at}sheffield.ac.uk.
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