Maternal Effect for DNA Mismatch Repair in the Mouse

Corresponding author: Sean M. Baker, Morgan Hall 233, University of California, Berkeley, CA 94720., sbaker{at}nature.berkeley.edu (E-mail)

Communicating editor: N. ARNHEIM

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA mismatch repair (DMR) functions to maintain genome stability. Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences. Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of 10% was observed. To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F₁ progeny from Pms2^-/- female mice mated with wild-type males. Our analysis indicates that MSI in the female germ line was 9%. MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient. We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions. The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote. The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene.

MAINTENANCE of genome stability requires the coordinate expression of DNA replication, recombination, and repair proteins. The mechanism of DNA mismatch repair (DMR) functions to correct base-base and small insertion/deletion loops that arise during both DNA replication and genetic recombination (MODRICH and LAHUE 1996 ; BUERMEYER et al. 1999 ). Cells lacking DMR show increased frequency of mutation, a mutator phenotype, that is frequently observed as microsatellite instability (MSI) in eukaryotic cells (STRAND et al. 1993 ; MODRICH and LAHUE 1996 ). The observation of MSI in human tumors was a pivotal clue in determining the genetic basis of a hereditary form of colorectal cancer and further underscores the importance of mutation avoidance and genetic stability in preventing cancer (KINZLER and VOGELSTEIN 1996 ; MODRICH and LAHUE 1996 ; BUERMEYER et al. 1999 ).

In Escherichia coli, the MutHLS complex is required for methyl-directed postreplication mismatch repair, with each protein functioning as a homodimer (MODRICH 1991 ; MODRICH and LAHUE 1996 ). It has been proposed that MutL functions as a molecular matchmaker, having an essential role in the communication between two DNA-binding proteins (MutS and MutH; SANCAR and HEARST 1993 ). DMR is a conserved pathway, and structural and functional homologs of MutS (Msh1–6) and MutL (Mlh1–3, Pms1, and Pms2) proteins have been identified in yeast and mammals (BUERMEYER et al. 1999 ). The greater number of eukaryotic DMR proteins reflects in part the function of some homologs for processes other than mutation avoidance in the nuclear DNA. Eukaryotic DMR homologs have specialized roles in mitochrondial maintenance (Msh1) and during meiotic recombination (Msh4, Msh5; CHI and KOLODNER 1994 ; ROSS-MACDONALD and ROEDER 1994 ; HOLLINGSWORTH et al. 1995 ). Mutation avoidance in mammals requires mismatch recognition by either MutS (Msh2 and Msh6 dimer) or MutSß (Msh2 and Msh3 dimer; BUERMEYER et al. 1999 ). For the MutL homologs, heterodimeric protein complexes involving either Mlh1 and Pms2 (MutL) or Mlh1 and Pms1 (MutLß) have been identified (BUERMEYER et al. 1999 ). Although the in vivo signal for strand discrimination remains to be determined in mammals, a putative mechanism involving proliferating cell nuclear antigen (PCNA) has been suggested since interaction of PCNA with several components of the DMR initiation complex has been observed (UMAR et al. 1996 ; GU et al. 1998 ; FLORES-ROZAS et al. 2000 ). Interaction of Pms2 with PCNA further confirms a role for MutL homologs in promoting the formation of higher-order complexes to facilitate repair in mammals (UMAR et al. 1996 ; GU et al. 1998 ).

To experimentally address the consequences of DMR deficiency in mammals, targeted mutations of murine DMR gene homologs have been analyzed. Mice deficient in Mlh1, Pms1, or Pms2 genes show a difference in cancer predisposition, mutation spectrum, and fertility (BAKER et al. 1995 , BAKER et al. 1996 ; EDELMANN et al. 1996 ; PROLLA et al. 1998 ; YAO et al. 1999 ). Differences in fertility between Mlh1- and Pms2-deficient animals were also observed, suggesting distinct requirements for these proteins for germ-line functions. Mlh1 deficiency results in both male and female animals being sterile, whereas Pms2 deficiency results in male sterility only. The sterility of the Pms2-deficient male mice was associated with abnormalities in chromosome synapsis during meiosis, whereas Mlh1^-/- spermatocytes failed to establish reciprocal exchanges, resulting in a premature separation of homologous chromosomes and subsequent meiotic failure (BAKER et al. 1995 , BAKER et al. 1996 ).

The previous analysis on germ-line microsatellite mutation was performed on spermatozoa from Pms2^-/- male mice (BAKER et al. 1995 ). Although functionally sterile, the Pms2-deficient mice produce spermatozoa that are reduced in number and abnormally shaped. Since a MSI of 10% was observed in the analysis of the genetic material in an individual spermatozoon from Pms2^-/- males, we were interested in the consequences to genome stability resulting from a maternal germ line deficient in DMR.

Therefore, we crossed Pms2-deficient females with wild-type male mice and analyzed the progeny for MSI. We used different genetic strains of male and female mice to unambiguously determine the parental origin of the alleles analyzed. Instability in the Pms2-deficient maternal germ line was 9%, a frequency similar to that observed previously in Pms2^-/- males. A surprising observation was mutation of the inherited paternal genome, resulting in animals that were mosaic for the alteration. To account for the formation of mutations, we propose that embryos from Pms2-deficient female mice are DMR deficient during the initial cell divisions. Therefore Pms2 acts as a maternal effect, one of a limited number identified in mammals and the first to involve a DNA repair gene.

	MATERIALS AND METHODS

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Animal husbandry:
The Pms2^-/- female mice for our experiments were generated by intercrossing Pms2^+/- mice that had first been backcrossed with C57BL/6J mice for five generations, substantially enriching for C57BL/6J alleles. To unambiguously determine the parental origin of the DNA, Pms2-deficient female mice were bred with wild-type male mice of a different strain. In initial experiments, wild-type male 129J mice were bred to generate 63 F₁ offspring. Using a cohort of different Pms2^-/- female mice, breedings were repeated with wild-type DBA/2J males to generate 85 F₁ offspring. Male mice used were purchased from a commercial vendor (The Jackson Laboratory, Bar Harbor, ME). Purchasing the male mice from a respected vendor minimized the aberrant introduction of targeted Pms2 alleles that could occur from genotyping errors and ensured that the offspring were heterozygous for the Pms2 mutation. Breeding of DBA/2J and Pms2-deficient female mice was not continuous. After the birth of the second litter, the DBA/2J males were removed and set up in breedings with Pms2^+/+ females, siblings to the Pms2^-/- mice, to generate control progeny. Similarly, following the birth of the second litter to the wild-type females, the DBA/2J males were returned to the original Pms2^-/- females for the birth of additional litters. Breeding of wild-type females produced 52 F₁ control progeny.

Rationale for choosing microsatellite markers:
The choice of microsatellite markers was dependent upon the presence of a dinucleotide (GT/CA)_n repeat 16 repeats or greater in number. As described previously (BAKER et al. 1995 ) and below, (GT/CA)_n was chosen for detection purposes. Polymorphic markers for C57BL/6 and DBA/2 were obtained at the following URLs: http://carbon.wi.mit.edu:8000/cgibin/mouse/index and http://www.informatics.jax.org/searches/polymorphism_form.shtml.

Microsatellite analysis:
Oligonucleotide primer pairs informative for 129J breedings D9Mit67, D16Mit4, D13Mit139, D17Mit123, D17Mit93, D1Mit355, D9Mit50, D4Mit155, D7Mit76, and D6Mit59 and for DBA/2J breedings D9Mit67, D6Mit59, D17Mit93, D1Mit355, D9Mit50, and D13Mit139 were obtained from Research Genetics (Huntsville, AL). PCRs were performed as previously described (BAKER et al. 1995 ), except that 100 ng of template DNA was used and annealing was at a temperature of 52° for 45 sec. Prior to further analysis, PCRs were analyzed by DNA agarose gel electrophoresis to confirm amplification and absence of product from negative controls. In our analysis, mutations were observed for all of the selected markers.

Hemi-nested PCR:
Amplification of D9Mit67 from approximately one genome equivalent template DNA was performed as described previously (BAKER et al. 1995 ) with the following modifications. The second round of PCR was performed using 1 µl reaction mixture from the first round. Conditions for both rounds of PCR were 1 cycle of 94° for 3 min; 30 cycles of 94° for 45 sec, 60° for 45 sec, and 72° for 45 sec; and 1 cycle of 72° for 3 min.

Nonisotopic detection of microsatellite alleles:
Conditions for microsatellite detection were described previously (LEEFLANG et al. 1994 ; BAKER et al. 1995 ) except a nonisotopic procedure for detection was developed. Following PCR, 2 µl of product was analyzed on a 7% acrylamide sequencing gel under denaturing conditions. The DNA was transferred overnight to Genescreen Plus membrane treated with 0.4 M NaOH. The blot was hybridized for 1 hr at 50° in hybridization solution (7% SDS, 0.25 M NaCl, 0.13 M NaPO₄, pH 7.4) containing a 5' biotinylated (CA)₁₀ oligo probe (Operon, Alameda, CA) at a final concentration of 1 pmol/ml. The CA probe in hybridization solution was stable when stored at 4° and was used repeatedly for a period of up to 4 weeks. The blot was washed twice with 6x SSC, 0.1% SDS for 10 min at 50°. Next the blot was hybridized for 15 min at 25° with Streptavidin-horseradish peroxidase conjugate (Strep-HRP; Pierce, Rockford, IL), prepared at a final concentration of 0.5 µg/ml in 5% SDS, 125 mM NaCl, 25 mM NaPO₄, pH 7.2. Following binding of Strep-HRP conjugate, the blot was washed twice with 0.5% SDS, 12.5 mM NaCl, 2.5 mM NaPO₄, pH 7.2 for 10 min at room temperature. The diluted Strep-HRP solution was stable when stored at 4° and was used repeatedly for 1 week before a significant loss in activity occurred. Detection was performed with Renaissance Western Blot Chemiluminescence Reagent Plus (New England Nuclear, Boston) according to the manufacturer's instructions.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Experimental design and mutation analysis:
To determine the effect of DMR deficiency on genetic stability in the maternal germ line, we mated Pms2^-/- female mice (C57BL/6J) with either 129J or DBA/2J wild-type males to generate 63 and 79 F₁ offspring, respectively. Breeding the Pms2-deficient female mice (substantially enriched for C57BL6/J alleles) with wild-type male mice from different strains introduced genetic variation to clearly show the parental origin of the allele being analyzed. DNA isolated from the tail of each offspring was analyzed with a panel of microsatellite markers known to be polymorphic between the maternal and paternal strains. As anticipated, we observed repeat length alterations in maternal alleles and, unexpectedly, alterations were also observed in paternal alleles. We tabulated the results depending upon the parental origin of the allele involved and the type of mutation (see Fig 1). MSIs in maternal alleles, class II (Fig 1, Fig 2A, lane 4; 2b, lanes 1, 8, and 18; and 2c, lane 1), are mutations most likely to have occurred during mitotic cell divisions preceding oocyte formation. Class II products were observed in 74/816 alleles for an average frequency of 9.1% (Fig 1B). The observed MSI frequency for the female germ line is similar to that reported previously in the Pms2^-/- male germ line (BAKER et al. 1995 ). As a control, breedings between DBA/2J males and Pms2^+/+ females (siblings to the Pms2-deficient mice) were performed. In the analysis of 260 alleles from 52 F₁ control offspring born to wild-type Pms2 females, no mutations were observed (data not shown). The lack of mutations in microsatellite markers from the control animals is consistent with previous data estimating the MSI frequency in wild-type mice to be 1/22,000 per locus per generation (DIETRICH et al. 1992 ).

View larger version (16K): In this window In a new window Download PPT slide   Figure 1. The types and frequencies of classes observed in the analysis of progeny from Pms2-deficient female mice. (a) Diagram demonstrating differences among the five types of classes. Class I lacks genetic instability, while class II is unstable microsatellites in genetic material transmitted through the maternal germ line. Class III represents instabilities in the paternal genetic material with the appearance of a novel allele. Class IV is events in which the novel allele occurs in sequences of maternal origin. Mice in class V show an altered paternal allele in the absence of the expected paternal allele. All repeat length alterations are represented by thick lines and can occur as either an increase or decrease in size. (b) A table showing the frequency of classes observed from crossing Pms2-deficient females with 129J and DBA/2J wild-type male mice. The number of progeny generated, number of alleles analyzed, and the number of products are shown for each class. The overall frequency for each class is included and was calculated by dividing the number of events for each class by the total number of alleles analyzed.

View larger version (53K): In this window In a new window Download PPT slide   Figure 2. Examples of genetic instabilities observed in the offspring analyzed from breeding 129J and DBA/2J males with Pms2 (C57BL/6J)-deficient female mice. (a) Examples from breeding the 129J male mice. Lane 2 shows a class III event (I25 in Table 1), while lane 4 represents a class II allele. A class V event is shown in lane 7 (I27 in Table 1). Lanes 1–4 are alleles at the D9Mit67 locus, while lanes 5–8 are alleles at the D16Mit4 locus. (b) Examples from breeding the DBA/2J male mice. Lanes 1, 8, and 18 show maternal germ-line instability representing class II events. Class III events are shown in lanes 13 and 14 (Di22 and Di23, respectively; Table 1), while class V is represented in lane 5 (Di14, Table 1). The D6Mit59 locus was used for analysis. (c) Lane 9 shows an example of a class IV event (Di63 in Table 1) at the D1Mit355 locus.

Mosaic alleles occur in the offspring of Pms2-deficient female mice:
Class III (Fig 2A, lane 2, and 2b, lanes 13 and 14) and class IV products (Fig 2C, lane 9) indicate the presence of a novel allele in addition to the expected maternal- and paternal-sized alleles. Class V products (Fig 2A, lane 7, and 2b, lane 5) are indicative of an altered paternal allele in the absence of a wild-type allele.

The intensity of the new parental alleles in classes III and IV suggested that the event responsible for the new allele occurred during postzygotic development and that the animals were mosaic for the mutation. To determine the number of alleles present, DNA from one animal with a class II mutation (I25, Fig 2A, lane 2; Table 1) was diluted to approximately one genome equivalent and subjected to PCR (LEEFLANG et al. 1994 ; BAKER et al. 1995 ). Our analysis revealed three alleles: one normal maternal-sized allele, one normal paternal-sized allele, and a novel presumptive paternal allele (Fig 3). The presence of three independent alleles indicates mosaicism for repeat length alterations in the somatic tissue from this animal.

View larger version (19K): In this window In a new window Download PPT slide   Figure 3. Confirmation of mosaicism in somatic tissue from a class III animal. Each lane represents a PCR using a single genome equivalent of tail DNA from a class III animal (I25, Fig 2A, lane 2; Table 1). The altered paternal-sized alleles are observed in lanes 2, 4, 6, 9, 13, and 15. However, lanes 4 and 9 are reactions where only the novel 129J allele was amplified, indicating that the repeat length alteration is independent of the other parental alleles. Amplification was specific for the D9Mit67 locus.

Mosaic alleles exist in the germ line of F₁ progeny of Pms2-deficient females:
Mosaicism in the germ line of class III and IV animals was determined by subsequent breeding of the animals with wild-type mice. For all class III and IV mice a mosaic germ line was confirmed by the transmission of the altered allele in addition to the expected parental alleles (Fig 4 and Table 1). Breeding of animals with class V-type mutations was performed and for one animal (I27, Fig 2A, lane 7; Table 1) only the altered 129J alleles were transmitted. In contrast, when breeding the other class V animal (Di14, Fig 2B, lane 5; Table 1), both altered and normal-sized DBA/2J alleles were transmitted to the F₂ offspring, indicating that the animal was a germ-line mosaic.

View larger version (24K): In this window In a new window Download PPT slide   Figure 4. Germ-line transmission of mosaic alleles (locus D13Mit139). Lanes 1–6 are offspring while control DNA include 129J (lane 7) and the mosaic parent (lane 8, I30; Table 1). Altered 129J alleles have been transmitted to offspring represented in lanes 1, 3, and 5. The transmissions of the wild-type 129J alleles (lanes 2 and 4) and a C57BL/6J allele (lane 6) from the mosaic parent were also observed.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our experiments were designed to determine the effect of maternal germ-line DMR deficiency on MSI. Previous analysis in Pms2^-/- male mice indicated a MSI frequency of 10% in the germ line. Our results showed that mutation in the Pms2^-/- maternal germ line occurs at a frequency similar to that observed in spermatozoa. Our strategy of being able to distinguish parental contribution by breeding mice of distinct genetic origin allowed us to demonstrate that both parental genomes were subject to mutation in the heterozygous embryos from Pms2^-/- mothers and that the mutated alleles were predominantly mosaic.

To account for the observed paternal mutation and mosaicism, we propose the following model illustrated in Fig 5. Cells generated in the Pms2^-/- maternal germ line, including the oocytes, lack functional Pms2 protein. Fertilization by a wild-type sperm produced a one-cell Pms2^+/- zygote that is DMR deficient. If the paternal Pms2 allele remains transcriptionally silent until the two-cell stage (WANG and LATHAM 1997 ), the first round of DNA replication occurs without DMR. Preliminary RT-PCR data indicate that Pms2 transcription is first detected at the two-cell stage of development (V. E. GURTU and S. M. BAKER, unpublished results). Therefore, repeat length alterations occurring during the first and second rounds of DNA synthesis can result in mutations in one of the two daughter cells arising at each division. Our data further suggest that mismatches leading to mosaicism of parental alleles form predominantly during the first round of DNA replication. If the mismatch occurred during either the first or second cell division, then, on average, it is likely that one-quarter or one-eighth of the cells would contain the alteration by the four- and eight-cell stages of development, respectively (Fig 5). Therefore, 25 or 12.5% of the cells in the animal are expected to contain the mutation if the replication error occurred in the first or second cell divisions, respectively (Fig 5). An overall frequency of 21% for transmission of class III and IV alleles was observed, a result consistent with DNA replication errors occurring primarily in the first cell division. It should be noted, however, that these numbers are population estimates for classes III and IV based on transmission ratios and that a wide variation in transmission ratios between individuals is observed, consistent with random segregation of cells into various embryonic and extraembryonic tissues. Furthermore, mating Pms2-deficient female and wild-type male mice produces one-cell embryos that are genotypically heterozygous for Pms2, but phenotypically deficient in DMR. We propose that the early cell cleavages of the embryo are susceptible to mutation since the onset of Pms2 transcription from the paternal genome is expected to restore microsatellite mutation frequencies to normal levels. Indeed, the later in development the microsatellite mutation occurs, the less likely the mutation will be detected in analysis of normal tissue. For example, Pms2-deficient mice are expected to exhibit accumulation of microsatellite mutations due to the absence of DMR (BAKER et al. 1995 ; YAO et al. 1999 ). However the analysis of tail DNA in mismatch repair-deficient animals reveals only the expected C57BL/6J alleles (BAKER et al. 1995 ) due to the low frequency of mutation at any single microsatellite locus during a single round of replication. The majority of the maternal alleles, especially in the germ line, are normal-sized C57BL/6J alleles (Fig 1), despite the lack of DMR during the majority of the development of the animal. The altered C57BL/6J alleles present in the germ line of Pms2-deficient female mice are not reflected in analysis of the tail DNA, suggesting that microsatellite mutations arise later in development of the germ line after the early cleavage divisions. Analysis of multiple tissues in Pms2-deficient mice indicates that 10% of the microsatellite alleles contain repeat length alterations (BAKER et al. 1995 ; YAO et al. 1999 ). Additional control experiments performed from mixing parental DNA indicate that alleles present at <12% are not efficiently detected (data not shown).

View larger version (21K): In this window In a new window Download PPT slide   Figure 5. A model for generating mosaic alleles in progeny from a Pms2-deficient maternal germ line. In the one-cell embryo, the parental alleles can be distinguished on the basis of size differences, represented here as an 8-bp difference. Two possibilities are proposed. One mechanism involves the mismatch forming in the first mitotic division (left side of diagram). The second mechanism occurs if the mismatch arises in the second round of DNA replication and cell division (right side of diagram). For each mechanism, the presence of a mismatch is represented by one-half of the affected nucleus being black. At the next cell division the repeat length alteration is fixed, which is shown by the nucleus being entirely black. Upon analysis of DNA from the adult animal a novel-sized allele is observed by gel analysis as indicated by the arrow.

To measure the frequency of microsatellite mutation in the maternal germ line, we performed microsatellite analysis on progeny of Pms2-deficient females. However, a limitation of our analysis is that we do not sample all of the cells produced during development since the extraembryonic tissues are not analyzed. At the blastocyst stage it is only those cells present in the inner cell mass that contribute to the embryo. As microsatellite mutations are typically without functional effects, we propose that the mutations formed in the early cell division should produce cells that randomly segregate throughout the embryo, without any apparent bias. Animals with a low transmission frequency of mutated alleles, 10%, for example, are mice in which cells containing alterations make up only a small fraction of the inner cell mass of the developing embryo. To confirm that microsatellite mutation does not confer a systematic bias with respect to the embryonic/extraembryonic segregations, experiments that analyze both the embryo and extraembryonic tissues are being performed.

Mutation to the maternal genome is expected (class II) since maternal alleles were subjected to replication in mismatch repair-deficient stem cells prior to meiosis. In our present analysis we determined that a similar mutation frequency exists between paternal (class III) and maternal alleles (class IV; Fig 1). Therefore mismatches occurring in the last premeiotic S phase in the maternal genome occur infrequently. Class V products, indicative of novel paternal alleles in the absence of a normal paternal allele, are consistent with both mutation during development of the spermatogonia and postzygotic mutation. The predicted frequency is 1/22,000 per locus per generation for mutation to microsatellite markers in wild-type animals (DIETRICH et al. 1992 ). We observed two class V products in 816 alleles analyzed. While such events are rare, their occurrence is nonetheless intriguing, raising the possibility that class V microsatellite mutation may also occur in the early postzygotic divisions. We are conducting further experiments to address the formation class V products.

Maternal effects loci, in which a mutation in the mother impacts embryonic development, have been described in other organisms, for example, Drosophila melanogaster and Caenorhabditis elegans (MORISATO and ANDERSON 1995 ; BOWERMAN 1998 ). In mice the limited number of maternal effect loci is associated with defects in gene expression due to imprinting, for example, the Tme locus (TSAI and SILVER 1991 ), or uterine environment failing to support embryo development, for example, LIF deficiency (STEWART et al. 1992 ). Our observations indicate that Pms2-deficient female mice produce embryos that are subject to mutations in the postzygotic divisions of early embryonic development. Therefore the absence of Pms2 protein in early embryonic development can be considered, in a broad sense, to be a maternal effect and the first to involve a DNA repair process. Additional candidates for maternal effect could be identified in mice with targeted mutation in other genes involved in DNA metabolism. In humans, children homozygous for a deleterious mutation in another DMR gene, MLH1, manifest secondary mutations in NF1 during postzygotic development (RICCIARDONE et al. 1999 ; WANG et al. 1999 ). For example, one child exhibited an early inactivation of the NF1 locus, resulting in mosaicism to a large proportion of the body. The identification of human patients with MSI in somatic tissues (PARSONS et al. 1995 ; HACKMAN et al. 1997 ; MIYAKI et al. 1997 ) suggests that women from these families could produce a zygote that is phenotypically deficient in DMR. Therefore postzygotic mutations, similar to that observed in the offspring of Pms2-deficient female mice, could be observed in humans.

	FOOTNOTES

¹ Present address: Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, OR 97201.

	ACKNOWLEDGMENTS

We thank J. Leivers for technical assistance; A. B. Buermeyer, S. Deschênes, G. Margison, and J. Sweasy for helpful comments on the manuscript; and D. Hinkle for a healthy dose of skepticism in the initial stages of the experiments. The experiments were supported in part by a grant from the National Institutes for Health (5R01GM57525).

Manuscript received June 8, 2001; Accepted for publication October 5, 2001.

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