Effect of Chromosome Location on Bacterial Mutation Rates

Richard Ellis Hudson, Ulfar Bergthorsson, John R. Roth and Howard Ochman

Department of Ecology and Evolutionary Biology, University of Arizona, Tucson;
Department of Biology, University of Utah, Salt Lake City


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
In previous comparisons of enterobacterial sequences, synonymous substitution rates were higher in genes closer to the replication terminus, suggesting that mutation rates increase with distance from the replication origin. In order to directly test for the effects of chromosomal location on the rates of point mutations, we assayed the reversion rates of two lacZ alleles inserted at four positions in the Salmonella enterica chromosome. Mutation rates at an intermediate locus were significantly higher than those at loci nearer to and farther from the replication origin. The higher reversion rates at this locus were neither the result of an overall increase in mutation rates produced by the insertion at this location nor a function of the mutations' immediate neighbors, but rather a regional effect. At all loci, regardless of chromosome location, T·A -> G·C transversions were more frequent than A·T -> G·C transitions during the exponential phase.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Models of molecular evolution are commonly based on the assumption that spontaneous mutations arise at random with respect to nucleotide type and position. However, evidence from experimental systems (Benzer 1961Citation ; Schaaper and Dunn 1991Citation ; Fujii et al. 1999Citation ) and from DNA sequence comparisons (Nussinov 1981Citation ; Blake, Hess, and Nicholson-Tuell 1992Citation ) show that mutation rates vary among sites. For example, A·T -> T·A transversions are 80-fold more common than G·C -> C·G transversions on an Escherichia coli plasmid (MacKay, Han, and Samson 1994Citation ), and the same trend is apparent in a mammalian in vitro system (Roberts et al. 1994Citation ; Izuta, Roberts, and Kunkel 1995Citation ).

Rates of replication errors, which underlie many spontaneous mutations, also vary among sites. Error rates can be affected by the specific bases adjacent to a base; for example, when the nucleotide adjacent to a thymidine is changed from a guanosine to an adenosine, T -> G errors decrease more than 30-fold, and T -> A errors increase more than 10-fold (Kunkel and Soni 1988Citation ). In addition, there are differences in the rates of errors originating on the leading and lagging strands. In a mismatch-repair–deficient strain of E. coli, G·C -> A·T transitions were four times more frequent, and A·T -> G·C transitions were two times less frequent, on the leading strand (Fijalkowska et al. 1998Citation ; Maliszewska-Tkaczyk et al. 2000Citation ), and errors attributable to misincorporations of dTTP were four times less frequent on the leading strand in the mammalian in vitro system (Roberts et al. 1994Citation ).

Rates of point mutations determined from sequence comparisons, as with those determined experimentally, depend on the particular type of mutation (Li, Wu, and Luo 1984Citation ), the adjacent nucleotides (Blake, Hess, and Nicholson-Tuell 1992Citation ), and the strand location (Lobry 1996Citation ; Francino and Ochman 1999Citation ). An additional factor influencing mutation rates, as revealed from comparisons of homologous genes of E. coli and S. enterica, is the distance from the replication origin: for genes of similar codon usage bias, those genes farthest from the origin have approximately twofold higher synonymous substitution rates than those nearest to it (Sharp et al. 1989Citation ). This difference may be solely because of the chromosomal position; for example, rates of postreplication recombinational repair may be higher for sequences closer to the origin because these sequences are more likely to be replicated and in higher copy numbers (Sharp et al. 1989Citation ). However, because different sets of genes are being compared, the inherent properties of the genes themselves, such as base composition and nearest-neighbor frequencies, might also contribute to variation in the mutation rates with chromosomal position.

To test how chromosomal location affects point mutation rates, we assayed mutation rates of lacZ alleles inserted at several positions on the chromosome. By assaying mutations that reside at identical positions within identical genes, but at different genomic locations, we were able to remove any confounding effects that neighboring bases or local base composition may have on mutation rates. We found that mutation rates do vary with chromosomal position, but do not increase with distance from the origin of replication, indicating that other factors underlie the observed trend in synonymous substitution frequencies.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Strain Construction and Verification
Strains used for mutation rate assays contain one of two mutant lacZ alleles—one reverting by a transition and the other by a transversion—inserted into one of four locations of the S. enterica sv. Typhimurium LT2 chromosome (table 1 ). These strains were derived from those listed in table 2 as follows: F' episomes containing the mutant lacZ alleles were moved into Typhimurium strain TT18300 by conjugation (Zinder 1960Citation ) to produce strains TT22260 and TT22265. To add a selectable marker, the lacA genes of these strains were replaced with lacA::Tn10d(Cm) from Typhimurium strain TT10604 by phage P22 (HT105/1 int-201)–mediated transduction (Schmieger 1972Citation ), producing strains TT22269 and TT22274. The chromosomal integration sites for these lacZ constructs were supplied by the MudP elements of Typhimurium strains TT17165, TT15269, TT15270, TT10604. These elements were converted into MudF elements, which contain the complete lac operon and a kanamycin (kan) resistance cassette (Sonti, Keating, and Roth 1993Citation ), by transduction from Typhimurium TT12116, yielding strains BGA through BGD. Next, the lacZlacA::Tn10d(Cm) constructs of strains TT22269 and TT22274 were transduced into the MudF elements of strains BGA through BGD, producing MudF[lacZ-lacA::Tn10d(Cm)] constructs. Finally, these constructs were transduced into the wild-type background supplied by Typhimurium TR10000.


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Table 1 Strains of S. enterica sv. Typhimurium Used in Reversion Assays

 

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Table 2 Strains Used in Constructions

 
To confirm that the constructs possessed the appropriate mutations, a 613-bp portion of each mutant lacZ allele was amplified by the polymerase chain reaction and sequenced, using primers that span the active site of this gene: 5' AAA ATC ACC GCC GTA AGC CGA CCA 3', and 5' GCT GTT CGC ATT ATC CGA ACC ATC C 3' (positions 72322 through 72345, and 72935 through 72911, respectively, GenBank sequence U73857). To determine whether the lac operons of these strains were inducible, revertants were isolated, grown to stationary phase, and then plated with and without isopropyl-ß-d-thiogalactoside (IPTG) on minimal glycerol media supplemented with 5-bromo-4-chloro-3-indolyl-ß-d-galactoside (X-gal). All strains produced blue colonies on plates with IPTG, and white or pale blue colonies on plates lacking IPTG.

Mutation Assays
For each strain, four estimates of lacZ reversion rates were derived from batches of 15–70 cultures. To make estimates statistically independent, each batch was derived from a different colony, but all cultures within a batch were from the same colony. Cultures were grown in 20 ml of M9 minimal glycerol media (Miller 1992Citation ) supplemented with 300 µM amino acids when appropriate. Each culture was inoculated with 2.0 µl of an overnight culture grown in Luria-Bertani (LB) medium. (Inocula of this size rarely contain Lac revertants and allow cultures to reach the stationary phase before sampling.) Cultures were incubated at 37°C for 24 h under moderate shaking.

Serial dilutions of one to three cultures per batch were plated on minimal glycerol (0.2%) plates to obtain total cell counts. To enumerate revertants, cultures were centrifuged for 20 min at 3000 g, then cells were resuspended in 0.4 ml of M9 salts and plated onto minimal lactose plates (0.2%) supplemented with 100 µM IPTG, 20 µg/ml X-gal, and 0.3 mM amino acids. Blue colonies visible after 3-day incubation at 37°C were scored as revertants.

To reduce the survival of nonrevertants, minimal lactose plates were pretreated overnight with ~1010 scavengers (S. enterica serovar Typhimurium strain 14028s), i.e., Lac- cells added to consume residual nutrients. Scavenger cells from an overnight culture were resuspended in 1/10 volume M9 salt solution, and 100 µl of the suspension was spread onto each plate.

To test for differences in overall mutation rates among the strains, we assayed for rpsL and rpoB mutations that confer resistance to 500 µg/ml streptomycin sulfate and 100 µg/ml rifampicin, respectively. Estimates of the rates of the streptomycin-resistant mutations were derived from two or more batches of 17–70 cultures per strain grown and plated as described. Estimates of the rates of mutations to rifampicin resistance for each strain were derived from four batches of 18 cultures grown and plated as follows: tubes containing 0.3 ml LB media were inoculated with approximately 106 cells and incubated at 37°C for 6.5 h with moderate shaking. Fifteen cultures per batch were plated directly onto LB-rifampicin plates, and the remaining three were plated to enumerate the numbers of total cells.

Calculation of Mutation Rates
When calculating mutation rates, it is necessary to distinguish between the frequencies of mutants and those of mutations because each early exponential-phase mutation gives rise to several mutants. Furthermore, determination of mutation rates from information about the frequencies of mutants requires mathematical models of all the relevant mutational processes. Standard models for mutations occurring in the exponential phase predict that mutants follow a Luria-Delbrück (LD) distribution (Luria and Delbrück 1943Citation ), whereas models for stationary-phase and postplating mutations predict a Poisson distribution. As our procedures yielded cultures that were predominantly, but not exclusively, in the exponential phase, we expected that the mutants followed some particular combined Poisson-LD distribution. The distribution parameters, m, the overall mutation rate and {theta}, the proportion of the mutations that occurred in the stationary phase and after plating, were estimated for the given batches from the data (to estimate the latter with sufficient accuracy, batches of 45–70 cultures were required).

Data were analyzed using a program made available by P. Gerrish (ftp-t10.lanl.gov). This program calculates the likelihood of distributions based on different values of {theta}. Because few post–exponential-phase mutations were expected, we assumed that mutations followed a pure LD distribution ({theta} = 0), unless the best-fitting combined Poisson-LD distribution described the observed distribution much better, as indicated by a likelihood 10-fold greater than that of the pure LD distribution. When the pure LD distribution was rejected, we employed the value of {theta} that yielded the maximum likelihood. Because the estimates of {theta} for strains containing the same lacZ mutation were similar, we grouped the strains by mutation type and produced one estimate for each group by averaging those derived from two different strains.

Estimates of overall (LD + Poisson) mutations per culture for each batch were generated by a maximum-likelihood method. Batch mutation rates were used in the following formula to estimate the mutation rate of each strain:


where µstrain is the strain overall mutation rate (i.e., overall mutations per cell), mi and ni are the estimates for overall mutations and for total cells per culture of batch i, respectively, and N is the total number of batches. Confidence intervals were calculated assuming that the log-transformed data followed a normal distribution, as suggested by experiments (data not shown) and simulation studies (Stewart 1994Citation ).

Estimates of exponential-phase (LD) mutation rates and their confidence intervals were derived using the same procedures. Estimates of LD mutations per culture for each batch were generated by determining the value of this parameter that maximized likelihood, using the value of {theta} chosen for that strain.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Mutation Rates at Different Locations
To test if chromosomal location affects point mutation rates, rates of reversions were calculated for two lacZ alleles inserted at four chromosomal loci. Figure 1 shows reversion rates plotted as a function of mutation type and chromosomal location. Chromosomal location has a statistically significant effect on both exponential-phase (LD) and overall (LD + Poisson) mutation rates (table 3 ). However, this effect is the result not of an increase in mutation rates with distance from the replication origin, but rather of elevated mutation rates for alleles inserted at an intermediate locus, envZ. Rates of T·A -> G·C mutations are significantly higher in the envZ insert than in those at any other locus tested, for both overall and exponential-phase mutations (P < 0.01, Tukey-Kramer test for a posteriori comparisons of means). Although the treA locus is over 30 min farther from the replication origin than envZ, the mutation rate for the envZ-strain is 230% higher. Rates of A·T -> G·C mutations are also significantly higher for inserts at the envZ locus than for those at ilvA and metE. The average rate of this mutation at envZ is 62% higher than at treA, though this difference is not statistically significant (P = 0.30).



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Fig. 1.—Mutation rates at different locations of the Salmonella chromosome. Distance from the origin of replication is given as the log of the number of minutes. Overall mutation rates (A) and exponential-phase mutation rates (B) at each position, calculated for the T·A -> G·C transversion ({blacktriangleup}) and the A·T -> G·C transition ({blacksquare}), are averages of four replicates shown with 95% confidence intervals

 

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Table 3 Analysis of Variance (ANOVA) of Reversion Rates as a Function of Location and Mutation Type

 
Despite differences in mutation rates at certain chromosomal locations, there is no systematic increase in mutation rates with distance from the replication origin. Mutation rates for the treA inserts were not significantly different from those of the ilvA inserts, or from those of the metE inserts, although the distance hypothesis predicts that the mutation rate at treA should be about twice that at ilvA and should exceed that at metE by about 75%. Such differences, given the number of revertants recovered in these experiments, should be readily evident: calculations of power indicate that the differences among mutation rates of 65% or more should achieve significance more than 95% of the time.

Effects of Growth Phase and Mutation Type
The majority of A·T -> G·C mutations, but few if any of T·A -> G·C mutations, occur after the exponential phase: {theta}, the frequency of mutations arising postexponentially, is 0.52 for A·T -> G·C mutations and 0.00 for T·A -> G·C mutations. For strains reverting by A·T -> G·C, the likelihood is maximized at {theta} = 0.51 for a batch of 70 cultures of strain TT22912 and at {theta} = 0.53 for a batch of 69 cultures of strain TT22911 (fig. 2 ); {theta} was 0 for strains reverting by all other mutations tested. The combined Poisson-LD distributions based on these {theta} values fit the data much better than the pure LD distributions, with likelihood ratios exceeding 22. Conversely, the pure LD distributions fit the data well for strains reverting by T·A -> G·C, yielding an estimate for {theta} of 0 for both a batch of 70 cultures of strain TT22907 and a batch of 48 cultures of strain TT22910. In the former case, the pure LD distribution had the highest likelihood, but in the latter, {theta} was set to 0 because the likelihood of the optimum combined distribution exceeded that of the pure distribution by only a very small amount. To investigate the reliability of our procedures for estimating {theta}, we analyzed the distribution of revertant colonies arising on the fourth through the eighth day of plate incubation, which most likely reflect postplating mutations; as expected, the pure Poisson distribution ({theta} = 1.00) best fit these data.



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Fig. 2.—Actual versus theoretical distributions of revertants per culture for strain TT22911. Cumulative number of cultures based on experimental data ({diamondsuit}), theoretical LD distribution ({triangleup}), and combined LD-Poisson distribution ({square}) are plotted. The LD distribution accounts only for mutations occurring in the exponential phase; the combined distribution accounts for those arising in the exponential phase, in the stationary phase, and after plating. The combined distribution fits the data better: it has a likelihood 32 times greater than that of the pure LD distribution, and yields a higher value for goodness of fit (G test; P = 0.75 for combined LD-Poisson, 0.18 for pure LD; data are pooled such that the expected number of each category >3)

 
As the overall rates of the two mutation types are similar, but the proportion of T·A -> G·C mutations occurring in the exponential phase is higher, the exponential-phase mutation rates are significantly higher for the T·A -> G·C mutation (fig. 1 and table 3 ). The higher postexponential rates of the transition can be attributed to a higher stationary-phase mutation rate or a greater production of postplating (adaptive) revertants. To distinguish between these alternatives, we compared the mutation rate of TT22914 cultures grown for 17 h (i.e., not reaching stationary phase) with that of TT22914 cultures grown for 24 h. The Poisson mutation rate of the 17-h cultures was virtually identical to that of the 24-h cultures (data not shown), indicating that post–exponential-phase mutations are predominantly caused by postplating mutations.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
To test for the effects of chromosomal location and mutation type on the mutation rate, we assayed the reversion rates of two lacZ alleles inserted at four positions in the S. enterica chromosome. Reversion rates for inserts at a locus 8.6' from the replication origin, envZ, were significantly higher than those at loci nearer to and farther from it. Although it was originally observed that the evolutionary rates of synonymous substitutions increased with distance from the replication origin (Sharp et al. 1989Citation ), T·A -> G·C mutation rates for the insert at treA, which is very close to the replication terminus, are significantly lower than those for inserts at envZ and similar to those at two loci close to the origin. Moreover, the same pattern was observed for the rates of A·T -> G·C mutations at different locations.

The higher reversion rates of alleles inserted at the envZ locus could reflect either a genome-wide increase in mutation rates caused by the inactivation of envZ or a locally elevated mutation rate at envZ. These alternatives were distinguished by determining the rates of streptomycin- and rifampicin-resistance mutations, which arise via mutations within the rpsL and the rpoB genes, respectively (Jin and Gross 1988Citation ; Lisitsyn, Monastyrskaya, and Sverdlov 1988Citation ; Timms et al. 1992Citation ; Ito et al. 1994Citation ), in each of the lacZ strains. Neither of the mutations conferring antibiotic resistance occurred at a significantly higher rate in strains harboring inserts at the envZ locus (table 4 ), although power calculations indicate differences equal to or greater than 50% and 30%, respectively, which would have been significant at the 5% level. Thus, the increase in reversion rates in these strains was specific to the inserts at envZ.


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Table 4 Control Mutation Rates as a Function of Chromosome Location

 
The elevated reversion rates of mutations of inserts situated within envZ cannot be caused by their immediate nucleotide neighbors because they are located within the same inserted construct as are the other mutations: sequences of the 224 bases downstream and the 385 bases upstream of all mutations subject to reversion assays are identical. Therefore, the elevated rate is a regional effect, caused either by sequences not immediately adjacent to the mutation site or by the relative position of this site with respect to other chromosomal locations. Moreover, in strains of E. coli harboring lac operons inserted at one of the nine chromosomal positions, there was a regional elevation in the mutagen-induced frequency of Lac- mutants at 58' and 60' (Van Brunt and Edlin 1975). This regional effect is not apparent from the sequence comparisons: the rates of divergence at synonymous sites of the envZ gene and the neighboring genes, as calculated from comparisons of Salmonella and E. coli homologs, are no higher than average (Ks = 0.85). A local elevation in mutation rates could occur if mismatch repair was less efficient in the region, which might be associated, for example, with a relative lack of Dam methylation sites.

Whereas mutation rates are influenced by the orientation of a gene (Fijalkowska et al. 1998Citation ), this does not contribute to the variation in mutation rates observed in this study. At each locus, the orientation of the inserted constructs, the disrupted gene, and the leading-strand replication all coincide. Although it is also possible that the increased mutation rates observed in the inserts at the envZ locus were caused by elevated transcription from the disrupted gene's promoter, this hypothesis is inconsistent with other data. Transcription increases the rate of this A·T -> G·C reversion over twofold more than that of the T·A -> G·C reversion (unpublished data). However, the increased rates observed for the envZ inserts follow a different pattern, with the former being augmented only about one-third as much as the latter.

Although we detected no systematic increase in mutation rates with distance from the replication origin, the frequency of synonymous substitutions among enterobacterial genes, as determined from sequence comparisons, increases with distance (Sharp et al. 1989Citation ). This disparity could be the result of the differences in mutation rates under certain conditions, such as prolonged starvation or anaerobiosis, which might be evolutionarily relevant but not tested in our experiments. Alternatively, the differences in the inherent properties of the genes included in the substitution-rate comparisons might underlie the variation in these rates. The analysis of Sharp et al. (1989)Citation controlled for the effects of codon usage bias, thereby eliminating this factor as the cause of the variation in substitution rates with chromosomal position; however, genes closer to the origin may more often possess other properties associated with more stringent selective constraints or lower mutation rates.

Based on our estimates for {theta}, T·A -> G·C transversions are more frequent than A·T -> G·C transitions during the exponential phase, but less frequent in other phases. In the exponential phase, chromosomal T·A -> G·C transversions occur approximately twofold more frequently than A·T -> G·C transitions, in agreement with the mutation rates determined using plasmids (MacKay, Han, and Samson 1994Citation ) and with the analyses of spontaneous mutational spectra (Hutchinson 1996Citation ). Because in mismatch-repair–deficient cells, T·A -> G·C transversions are generated at a lower frequency than are A·T -> G·C transitions (Fujii et al. 1999Citation ), these results suggest either that mismatch repair is more efficient at correcting the mismatches that lead to this transition or that DNA repair creates more T·A -> G·C than A·T -> G·C mutations.

Although half of the A·T -> G·C transitions detected in this study were attributed to postplating mutations, none of the T·A -> G·C transversions were. The strains reverting by A·T -> G·C transitions have higher postplating reversion rates because they are leakier, that is, they produce more wild-type lacZ proteins via mis-transcription or mis-translation (Cupples and Miller 1988Citation ; Andersson, Slechta, and Roth 1998Citation ). In contrast to these results, postplating Tcoding strand·A -> Gcoding strand·C mutations were detected using a hisG428 reversion assay; however, these arose on plates lacking scavengers after prolonged incubation (Prival and Cebula 1992Citation ).

The spontaneous mutation rates that we estimated for the lacZ reversions on the S. enterica chromosome are lower than those previously reported for the same alleles. Reversion rates on an E. coli episome are approximately 10-fold higher than those observed in this study (MacKay, Han, and Samson 1994Citation ), and the reversion rate of the A·T -> G·C transversion on the E. coli chromosome is 100-fold higher (Fijalkowska et al. 1998Citation ). The differences are consistent with data reporting higher mutation rates in nutrient-rich media (Smith 1992Citation ) and lower mutation rates in media that enable cells to skip the mutagenic steps of glycolysis (Lee and Cerami 1990Citation ). The low mutation rates that we observed are not the result of plating high numbers of Lac- cells because the average number of colonies formed on minimal lactose plates by lacZ+ revertants plated in the presence or absence of 1010 Lac- cells was similar (36.2 vs. 33.6 per plate, respectively; difference was not significant by Student's t test: t = 1.30, P = 0.23, df = 8).

The volume of cultures that we employed in these studies permitted the detection of frequencies of mutants well below the threshold of other protocols. In addition, our methods yielded more accurate mutation rates by providing the following advantages: (1) statistical power was enhanced by using a maximum-likelihood–estimation procedure (Stewart 1994Citation ); (2) the confounding effects of post–exponential-phase mutations on the estimates of exponential-phase mutation rates were removed by partitioning the mutations into LD and Poisson fractions; and (3) spurious significant differences, which can arise, for example, when different strains have different carrying capacities, were avoided by the use of multiple batches to estimate the mutation rate of each strain and by enumerating the total cells.

Applying an experimental approach, we have shown that both the chromosomal location and the type of mutation affect the rates of spontaneous point mutations. However, a general increase in mutation rate with distance from the origin of replication, as suggested by sequence divergence of homologous genes in E. coli and S. enterica, is not supported by our results. Variation in mutation rates among sites can also be influenced by other factors, such as frequency of transcription and direction of DNA replication (Beletskii and Bhagwat 1996, 1998Citation ; Francino and Ochman 1997Citation ); therefore, future work will exploit this system to establish the effects of additional factors on the rates of different point mutations.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Frank Stewart and Phil Gerrish for assistance and for providing computer programs. This research was supported by NIH grants GM 55535 (to H.O.) and GM 27068 to (J.R.R.).


    Footnotes
 
Julian Adams, Reviewing Editor

Keywords: chromosomal location molecular evolution Salmonella enterica spontaneous mutations base substitutions Back

Address for correspondence and reprints: Howard Ochman, Department of Ecology and Evolutionary Biology, 310 Biosciences West, University of Arizona, Tucson, Arizona 85721. hochman{at}email.arizona.edu . Back


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 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Accepted for publication September 7, 2001.