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Mutagenesis Advance Access originally published online on March 8, 2005
Mutagenesis 2005 20(2):105-110; doi:10.1093/mutage/gei014
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© The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oupjournals.org

Mutagenesis studies of the major benzo[a]pyrene N2-dG adduct in a 5'-TG versus a 5'-UG sequence: removal of the methyl group causes a modest decrease in the [G->T/G->A] mutational ratio

Arumugam Nagalingam, Kwang-Young Seo and Edward L. Loechler*

Biology Department, Boston University, Boston, MA 02215, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations primarily at the G:C base pairs (e.g. GC->TA, GC->AT, etc.). Each of these mutations can be induced by its major adduct [+ta]-B[a]P-N2-dG, where DNA sequence context appears to influence both the quantitative and qualitative pattern of mutagenesis. We noted previously that 5'-TG sequences tend to have a higher fraction of G->T mutations for both [+ta]-B[a]P-N2-dG and (+)-anti-B[a]PDE in comparison with 5'-CG, 5'-GG or 5'-AG sequences. To investigate a possible structural element for this trend, the role (if any) of the methyl group on the 5'-T is considered. Using adduct site-specific means, the [G->T/G->A] mutational ratio for [+ta]-B[a]P-N2-dG is determined to be ~1.08 in a 5'-TGT sequence, and ~0.60 in a 5'-UGT sequence. (G->C mutations are minor.) Although this modest ~1.8-fold decrease in [G->T/G->A] ratio is statistically significant (P = 0.03), it suggests that the methyl group on the 5'-T is not the main reason why a 5'-T tends to enhance G->T mutations. This study was prompted by an adduct conformational hypothesis, which predicted that the removal of the methyl group in a 5'-TG sequence would lower the fraction of G->T mutations; however, the ~1.8-fold decrease is too small to do additional experiments to assess whether this conformational hypothesis, or other hypotheses, are the true cause of the decrease, which is discussed in this paper.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Conclusions
 References
 
Benzo[a]pyrene (B[a]P) is a potent mutagen/carcinogen, and an example of a polycyclic aromatic hydrocarbon (PAH), a class of substances produced by incomplete combustion, which are found ubiquitously in the environment (1GoGoGoGoGoGo–7Go). B[a]P is metabolized in the cells to the ultimate mutagen/carcinogen (+)-anti-B[a]PDE, which gives one predominant DNA adduct [+ta]-B[a]P-N2-dG (Figure 1) (8Go,9Go). Most carcinogens are active by causing mutations, and it is generally believed that mutations induced by PAHs in general, and B[a]P in particular, could cause mutations relevant to cancer causation (10GoGoGoGo–14Go), and are important in human cancer (15Go, and references therein).



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  		Fig. 1.. Structures of (+)-anti-B[a]PDE and its major adduct [+ta]-B[a]P-N2-dG.

 
B[a]P mutagenesis has been extensively studied (16GoGoGoGo–20Go), and mutational spectra have been generated for pure (+)-anti-B[a]PDE in Escherichia coli (21Go,22Go) and in mammalian (Chinese hamster ovary, CHO) cells (23Go,24Go). In E.coli, the most prevalent mutations were at the G:C base pairs, where G:C->T:A (57%), G:C->A:T (23%) and G:C->C:G (20%) mutations were each significant, and the patterns were influenced by the DNA sequence context (21Go,22Go). Adduct site-specific mutational studies with [+ta]-B[a]P-N2-dG have reinforced this conclusion, where dramatic shifts in mutagenic pattern could occur, such as >95% G->T mutations in a 5'-TGC-3' sequence (25Go), ~95% G->A mutations in a 5'-AGA-3' sequence (26Go), and ~70% –1 frameshift mutations in a 5'-GGGA sequence (27Go).

One of these patterns is considered herein. Based on the (+)-anti-B[a]PDE mutational spectra, the most striking influence of sequence context was the fraction of G->T mutations in all 5'-TG sequences being ~10-fold higher than on average for the 5'-AG, 5'-CG and 5'-GG sequences (22Go). Studies with [+ta]-B[a]P-N2-dG reinforced this observation: G->T mutations were more dominant in a 5'-TGC-3' sequence (>95%, 25Go) than in a 5'-CGG (~60%, 28Go), 5'-CGT (~20%, 29Go), or 5'-AGA (~5%, 26Go) sequence. Evidence suggests that a 5'-T may have a similar effect in eukaryotic cells (30Go,31Go).

Herein, we investigate the role that the methyl group on the 5'-T might play on the high fraction of G->T mutations in 5'-TG sequences by comparing the results for [+ta]-B[a]P-N2-dG in both a 5'-TG and a 5'-UG sequence context in E.coli. We were motivated to do this study because of a correlation between our mutagenesis and conformational modeling work, from which we proposed that a base displaced conformation, which we call Gma5, might be responsible for G->T mutations (32GoGo–34Go). Gma5 has the pyrene moiety of [+ta]-B[a]P-N2-dG stacked with the surrounding base pairs, while the dG moiety of the adduct is displaced into the major groove (Figure 2). Gma5 was computed to be relatively low in energy in the 5'-TG sequences (32Go,34Go), and in our models, the methyl group (gray in Figure 2) on the 5'-T interacts with the dG (yellow) and B[a]P (purple) moieties, which protrude into the major groove. If this interaction provided a favorable stacking energy, then removing this methyl group might destabilize Gma5 and lower the fraction of G->T mutations, if indeed Gma5 were a G->T conformation. We estimated this stabilization to be as high as ~10-fold (see Results and Discussion).



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  		Fig. 2.. Methyl group (gray) on the 5'-T (green) interacting with the B[a]P moiety (yellow) and dG moiety (purple) of [+ta]-B[a]P-N2-dG in the Gma5 conformation in a 5'-TGT sequence. Both images are shown looking into the major groove with the view differing via a rotation of ~90° around the helix axis. Only the central 5 bp are shown (5'-CTGTC/5'-GACAG). The structure was derived from a molecular dynamics simulation at 300 K with solvent and counter ions (Chandani and Loechler, unpublished).

 
We report an ~1.8-fold decrease in the [G->T/G->A] ratio for [+ta]-B[a]P-N2-dG when the methyl group is removed from a 5'-T in a 5'-TG sequence. This modest decrease suggests that the methyl group is not the main reason why 5'-TG sequences tend to promote G->T mutations. Assuming the decrease to be real, three mechanisms that explain this decrease are discussed, including the one noted in the previous paragraph, as well as those involving events during the lesion bypass by a DNA polymerase or during DNA repair.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Conclusions
 References
 
The study of [+ta]-B[a]P-N2-dG in 5'-TGT and 5'-UGT sequences is identical to our study in the 5'-TGT sequence context alone (35Go). Oligonucleotides with [+ta]-B[a]P-N2-dG in a 5'-TGT or 5'-UGT sequence context ([+ta]-B[a]P-TGT and [+ta]-B[a]P-UGT, respectively) were synthesized by reacting (+)-anti-B[a]PDE with appropriate oligonucleotides (C-TGT: 5'-GACGCTGTCATCC-3' and C-UGT: 5'-GACGCUGTCATCC-3'; Midland Certified Reagent Co., Midland, TX), using methods identical to those described previously (26Go,28Go,29Go,35Go,36Go). As determined by mass spectrometry, the (observed/expected) molecular weights were consistent with the starting oligonucleotides C-TGT [3916.1/3910.6] and C-UGT [3897.8/3896.6] having the correct base compositions, and the difference in molecular weights was consistent with the former having an extra methyl group (within experimental error). Adducted oligonucleotides were purified sequentially by reverse phase HPLC, native PAGE and denaturing PAGE, as performed previously (26Go,28Go,29Go,35Go,36Go). The presence and positioning of [+ta]-B[a]P-N2-dG in [+ta]-B[a]P-TGT and [+ta]-B[a]P-UGT was determined as described previously, and each oligonucleotide was shown to be >99.5% pure (the limit of detection) by methods described previously (26Go,28Go,29Go,35Go,36Go).

Plasmid construction in the 5'-TGT and 5'-UGT sequences was identical to previous work in the identical 5'-TGT sequence (Figure 3, 35Go). In parallel, plasmids [+ta]-B[a]P-TGT-pRT2, [+ta]-B[a]P-UGT-pRT2, C-TGT-pRT2 and C-UGT-pRT2 were each transformed into BW310 cells, which had been SOS-induced by a procedure [UV irradiation at 254 nm; total dose: 12.6 J/m2, giving a cell survival of ~50%] that we have used consistently (26Go,28Go,29Go,35GoGo–37Go). BW310 cells were obtained from the CGSC E.coli Genetic Stock Center, and they are ung–1, thi–1, spoT1 and relA1 (38Go). Importantly, BW310 cells are ung– and, therefore, uracil DNA glycosylase deficient. Cells with the ung–1 allele are estimated to retain no more than 1/4000 of the activity of corresponding ung+ cells (39Go). BW310 was confirmed to be ung– by a method adapted from Kunkel (40Go), which began with the isolation of pRT2 from BW313 cells, which are dut–/ung– and, therefore, give plasmid DNA (‘pRT2/BW313’) with a high fraction of uracils. Subsequent transformation of ‘pRT2/BW313’ into BW310 cells (ung–) gave a higher pRT2 plasmid output (~8-fold) than when transformed into the isogenic ung+ strain KL16. Plasmid output is lower in KL16 cells, because uracil DNA glycosylase converts uracil residues to AP-sites that decrease plasmid replication efficiency, and this does not occur in BW310 cells, because they are deficient in uracil DNA glycosylase, which is what we sought to establish.



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  		Fig. 3.. The strategy to place the [+ta]-B[a]P-N2-dG in a 5'-TGT (or 5'-UGT) sequence context, which is identical to that used in Yin et al. (35Go). Steps a and b: [+ta]-B[a]P-TGT (or [+ta]-B[a]P-UGT) was formed by the reaction of C-TGT (or C-UGT) with (+)-anti-B[a]PDE, and purified by HPLC, as well as native and denaturing gel electrophoresis. Steps 1 and 2: Construction of pRT2 by the introduction of a duplex oligonucleotide (5'-GACGCTGTCATCC-3'/5'-GGATGACAGCGTC-3') into the unique Hind II site in pRE0. Steps 3 and 4: ss-pRT2 was isolated and UV irradiated to eliminate progeny from this strand (Materials and methods). Steps 5–8: ds-pRE0 was digested with Hinc II and mixed with UV irradiated ss-pRT2, and denatured/renatured to give gapped heteroduplex DNA (GHD), which was isolated and purified. Step 9: [+ta]-B[a]P-TGT (or [+ta]-B[a]P-UGT) was covalently incorporated into the GHD via ligation to give [+ta]-B[a]P-TGT-pRT2 (or [+ta]-B[a]P-UGT-pRT2).

 
Mutations at the original genome location of the [+ta]-B[a]P-N2-dG adduct eliminate the unique PflF I site in pRT2, which was used to enrich for cells containing progeny plasmids with mutations in the PflF I site exactly as described previously (35Go). After mutant enrichment, plasmid DNA was isolated from individual colonies and sequenced.

Base substitution mutation frequency (MF) at the original genome location occupied by [+ta]-B[a]P-N2-dG in the 5'-TGT sequence of pRT2 was determined exactly as described previously (35Go). The PflF I site in pRT2 is in the lacZ' gene, which permits the {alpha}-complementation to give ß-galactosidase activity in appropriate cells (e.g. DH5{alpha}). ß-Galactosidase activity can be monitored with X-gal, which gives colonies a blue color when cleaved. Blue colonies have plasmids with lacZ' in-frame, which includes wild-type or base substitution mutations at the PflF I site, as well as in-frame deletion/insertion mutants. Approximately 10% of the colonies were white, and correspond in sequence to pRE0, which is a starting plasmid (Figure 3), is a 13 bp deletion of pRT2 and is also a PflF I resistant mutant. [Progeny pRE0 are genetic engineering-derived, side products of plasmid construction, as discussed previously (25Go,28Go).] We define: B/WR0 = [(WTb + Mb)/(Mw)]. B/WR0 is the ratio of (blue colonies/white colonies) derived from the original transformation with (e.g.) [+ta]-B[a]P-TGT-pTR2, where ‘R0’ means ‘round zero’ (i.e. before mutant enrichment). WTb represents the number of blue wild-type colonies, and Mb and Mw represent the number of blue and white mutant colonies, respectively. After three rounds (R3) of mutant enrichment, wild-type PflF I sensitive plasmids (i.e. WTb) were eliminated and the ratio [blue colonies/white colonies] corresponds to: B/WR3 = [(Mb)/(Mw)]. The apparent mutation frequency is calculated according to: MFapp = [(B/WR3)/(B/WR0)], because [(B/WR3)/(B/WR0)] = [(Mb)/(Mw)]/[(WTb + Mb)/(Mw)] = [(Mb)/(WTb + Mb)], the latter term being MF by definition. Plasmids from individual blue colonies were isolated after R3 and sequenced. In the case of the control, C-TGT-pTR2, the majority (i.e. 44/45) of the blue mutants after R3 had no 5'-GACGCTGTCATCC-3 insert, being pRE0 with an additional deletion mutation that restored the reading frame, where such mutants are derived from genetic engineering side-reactions, as noted in the past (25Go,28Go). Thus, for C-TGT-pTR2, the ratio [true mutants/total mutants] = [1/45], and the true mutation frequency is calculated from three ratios: MFtrue = [true mutants/total mutants] [(B/WR3)/(B/WR0)]. By these methods, MFtrue was determined for C=TGT-pTR2, C-UGT-pTR2, [+ta]-B[a]P-TGT-pTR2 and [+ta]-B[a]P-UGT-pTR2, where the ratio of true base substitution mutations to genetic engineering derived mutations after R3 was 1/44, and 0/107, 141/244 and 152/119, respectively. The SD of MFtrue was determined using standard statistical methods (41Go) as follows. The relevant ratios ([B/WR3], [B/WR0] and [true mutants/total mutants]) were log averaged, and the SD was taken as the square root of the variance, which was determined as the sum of the squares of the log standard deviations for each of the individual ratios (41Go). To conform to standard practice, anti-log values are reported in Table I. It is noted that MFtrue is derived from three ratios (i.e. [B/WR3], [B/WR0] and [true mutants/total mutants]), and this is the source of uncertainty in MFtrue as discussed in Results and Discussion. In contrast, the [G->T/G->A] mutational ratio is independent of all of these ratios, and simply depends on the number of G->T versus G->A mutations sequenced. The Monte Carlo method of Adams and Skopek (42Go) was used to perform the statistical comparison of the [G->T/G->A] mutational ratios for 5'-TGT versus 5'-UGT.


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  		Table I.. Base substitution mutations in the PflF I site in progeny plasmids derived from [+ta]-B[a]P-N2-dG, when adjacent either to a 5'-T or to a 5'-U, which is studied in BW310 E.coli, which are uracil DNA glycosylase deficient

 

   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Conclusions
 References
 
Before undertaking this study, we made a simplistic estimate of the interaction energy that might be lost upon the removal of the methyl group of the 5'-T in a 5'-TG sequence, and this estimate influenced our experimental strategy. The binding energy of a substrate methyl group in a protein pocket has been estimated experimentally to be 1.8–3.5 kcal/mol (43GoGoGo–46Go). Interactions with only a single side of a methyl group, however, should give a smaller effect, which we estimate to be anywhere up to ~1.3 kcal/mol, based on relative solvent accessible surfaces for Gma5 (Figure 2) in comparison to the values mentioned above for an entire protein pocket. A 1.3 kcal/mol effect might explain the ~10-fold greater tendency of the 5'-TG sequences to give G->T mutations (Introduction). If (e.g.) G->T mutations changed significantly (i.e. based on an energy difference of >~0.6 kcal/mol or ~4-fold), then a statistically significant statement about MF could probably be made (based on typical SDs in our previous experiments). However, a more modest change might not permit a statistically significant statement, because our values for MF are determined from the product of three ratios (Materials and methods). Taking note of this and wanting to assess an effect even if it were in the 2-to 4-fold range, we designed our study so that we could also evaluate a change in the [G->T/G->A] mutational ratio, which could be determined more reliably because a single ratio is being compared for a 5'-TG versus a 5'-UG sequence (Materials and methods). In this respect, a suitable DNA sequence context had to be chosen. The sequence 5'-TGC seemed inappropriate, because the fraction of G->A mutations was very low (25Go). Thus, we were faced with the question: how could we increase the fraction of G->A mutations in a 5'-TG sequence context? Our work in other sequence contexts suggested that an A:T base pair on the 3'-side of [+ta]-B[a]P-N2-dG tends to increase the fraction of G->A mutations (‘Hypothesis 3’;33). Recently, we showed that [+ta]-B[a]P-N2-dG in a 5'-TGT sequence gave a higher fraction of G->A mutations ([G->T/G->A] ~1.1; 35Go), which is consistent with this hypothesis for 5'-TG sequences. Thus, the 5'-TGT sequence seemed a reasonable choice, both because approximately equal numbers of G->T and G->A mutations could be collected, and because the methyl group on the 5'-T in the 5'-TGT sequence showed the favorable interaction in the Gma5 conformation (Figure 2), which prompted this study in the first place.

Figure 3 shows the strategy to study mutagenesis by [+ta]-B[a]P-N2-dG in 5'-TGT and 5'-UGT sequences, which is identical to our previous work in the identical 5'-TGT sequence (35Go). Double-stranded plasmids with [+ta]-B[a]P-N2-dG in 5'-TGT and 5'-UGT sequences ([+ta]-B[a]P-TGT-pTR2 and [+ta]-B[a]P-UGT-pTR2) were constructed (Materials and methods), along with unadducted controls (C-TGT-pTR2 and C-UGT-pTR2). A double-stranded plasmid was used, because this study needed to be comparable to our past studies on [+ta]-B[a]P-N2-dG and (+)-anti-B[a]PDE, which were done in ds-plasmids (Introduction).

[+ta]-B[a]P-TGT-pTR2, [+ta]-B[a]P-UGT-pTR2, C-TGT-pTR2 and C-UGT-pTR2 were each transformed in parallel into SOS-induced BW310 cells, which are uracil DNA glycosylase deficient (Materials and methods) and were used to curtail potential uracil-related base excision repair. Mutant plasmids were isolated and sequenced (Table I). MF was determined at the original genome location of the adduct (Materials and methods), and it is lower for the control plasmids (C-TGT-pTR2 and C-UGT-pTR2) than for the adduct-containing plasmids (Table I). MF for [+ta]-B[a]P-N2-dG in the 5'-TGT sequence is slightly higher than for the 5'-UGT sequence, but the values have overlapping SDs (Table I), implying that no statistically significant statement can be made about MF changes. Approximately equal numbers of G->T and G->A mutations were obtained in BW310 cells for [+ta]-B[a]P-N2-dG in the 5'-TGT sequence (Table I), as we previously observed in KH2 cells (35), although MF in the latter was ~2-fold lower (not statistically significant). The [G->T/G->A] ratio was ~1.08 for [+ta]-B[a]P-N2-dG in the 5'-TGT sequence and ~0.60 for [+ta]-B[a]P-N2-dG in the 5'-UGT sequence (Table I). This ~1.8-fold decrease in [G->T/G->A] is statistically significant (P = 0.03; Materials and methods).

The observed ~1.8-fold decrease in [G->T/G->A] ratio suggests that the methyl group is at most a small contributor to the reason why 5'-TG sequences tend to enhance the fraction of G->T mutations. Currently, we can offer no alternative hypothesis, although B[a]P-N2-dG adducts show significantly greater flexibility when flanked by A:T base pairs, and in a 5'-TGT sequence several conformers appeared to be in slow equilibrium on the NMR time-scale (47Go), where these observations may be relevant.

The magnitude of the decrease in the [G->T/G->A] mutational ratio is unfortunate, since it is too large to be considered to be of no effect, but too small to investigate its underlying mechanistic basis. However, if one assumes that the effect is real (it is statistically significant), then there are several mechanisms (discussed next) by which the presence versus the absence of this methyl group might influence the mutagenic process, involving lesion bypass DNA polymerases, DNA repair or adduct conformational equilibria.

With respect to lesion bypass during DNA replication, the methyl group is on the 5'-T, such that it would be in the single-stranded region of the template strand when a dNTP was being incorporated opposite the adduct. In the case of the lesion-bypass DNA polymerase Dpo4 (48Go), as well as T7 DNA polymerase (49Go) and rat DNA polymerase ß (50Go), the equivalent 5'-base appears to face stack with a protein residue (i.e. with L293, H607 and H34, respectively, based on coordinates in files 1JX4, 1T7P and 1BPY in the RCSB Protein Data Bank). In principle, this face stacking could be different for a 5'-T versus a 5'-U. Although it is not clear how this would translate into an effect on differential insertion opposite the lesion itself, it is not impossible, for example if different lesion-bypass DNA polymerases were involved in the G->T versus the G->A mutagenic pathway, and they each had a different affinity for 5'-T versus 5'-U. The notion that different lesion-bypass DNA polymerases might insert different bases when bypassing the same lesion is sensible, based on current thinking, including those with B[a]P-N2-dG adducts (discussed extensively in the Introduction of 35). Recently, we obtained evidence that in E.coli, DNA polymerase V is involved in the G->T pathway (but not the G->A pathway) with [+ta]-B[a]P-N2-dG in this same 5'-TGT sequence context (35Go), a finding that is consistent with in vitro studies showing that Pol V inserts dA opposite [+ta]-B[a]P-N2-dG in a 5'-CGA sequence context (51Go). In principle, experiments could be done to pursue whether the methyl group interacts with the DNA polymerase V during G->T mutagenesis.

The methyl group (or its absence) could influence the probability of DNA repair by interaction with a protein in a repair complex, for example nucleotide excision repair (NER). In particular, the adduct repair rate might be affected differently in the 5'-TG versus the 5'-UG sequence, although the mechanism is not readily apparent, since the presence of the 5'-T versus 5'-U would have to differentially affect the G->T versus G->A mutagenesis pathway. In principle, repair rate effects could be investigated using appropriate repair-deficient E.coli strains.

The presence of the methyl group on the 5'-T of [+ta]-B[a]P-N2-dG could also affect the mutagenic pattern by affecting the ratio of different DNA conformations, if these different adduct conformations caused different mutations. We have suggested that the G->T versus G->A mutagenic pathways might involve the bypass of different conformations of [+ta]-B[a]P-N2-dG (22Go,32Go,52Go). If the ratio of the G->T versus the G->A adduct conformation were affected by a 5'-T versus a 5'-U, then this could be the underlying mechanism responsible for the effect described herein. The presence versus absence of an analogous methyl group on a 5'-C in a 5'-CG sequence caused a change in the dominant conformation with B[a]P-N2-dG adducts (53Go,54Go). Regarding the conformational mutagenesis hypothesis, we proposed that the displaced conformation Gma5 (Figure 2) might be responsible for G->T mutations (Introduction; 32Go–34Go), and, if true, then removing this methyl group might decrease the Gma5 stability, and lower the fraction of G->T mutations. While this prediction is consistent with the observed decrease in the [G->T/G->A] ratio (Table I), the effect is too small to consider it in support of this (or any other) mechanism.


   Conclusions
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
References
 
A change from a 5'-TG sequence to a 5'-UG sequence did not affect MF statistically significantly, although it resulted in an ~1.8-fold decrease in the [G->T/G->A] mutagenesis ratio with [+ta]-B[a]P-N2-dG (Table I). This decrease suggests that the methyl group is not the primary reason why 5'-TG sequences tend to enhance G->T mutations. There are at least three mechanisms that might account for this decrease, based on the methyl group: (i) interacting with a relevant DNA polymerase(s) during lesion bypass, (ii) interacting with a protein during DNA repair, or (iii) influencing the ratio of adduct conformations, thereby affecting the mutational pattern (if different adduct conformations do indeed cause different mutations). The modest decrease in [G->T/G->A] ratio makes it less intriguing, less tractable, and too difficult to pursue which of these mechanistic possibilities is most likely.


   Acknowledgments
 
This work was supported by United States Public Health Services Grant R01CA50432 and R01ES03775.


   Notes
 
* To whom correspondence should be addressed. Tel: +1 617 353 9259; Fax: +1 617 353 6340; Email: loechler{at}bu.edu

All the authors contributed equally to this work.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 

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Received on December 22, 2004; revised on February 2, 2005; accepted on February 2, 2005.


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