Msx1 Is Present in Thyrotropic Cells and Binds to a Consensus Site on the Glycoprotein Hormone {alpha}-Subunit Promoter

Virginia D. Sarapura, Heidi L. Strouth, David F. Gordon, William M. Wood and E. Chester Ridgway

University of Colorado Health Sciences Center Denver, Colorado 80262


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our studies are aimed at identifying the transcription factors that activate the glycoprotein hormone {alpha}-subunit promoter. Therefore, we performed a Southwestern screening of a thyrotropic ({alpha}TSH) cDNA expression library, using the region of the promoter from -490 to -310 that contains sequences critical for expression in thyrotrope cells. A clone was isolated corresponding to part of the coding sequence of Msx1, which is a homeodomain-containing transcription factor that has been found to play an important role in the development of limb buds and craniofacial structures. Northern blot analysis, using the cloned Msx1 cDNA fragment as a probe, demonstrated that {alpha}-subunit-expressing thyrotrope cells ({alpha}TSH cells and TtT97 tumors) contained Msx1 RNA transcripts of 2.2 kb, while somatomammotrope (GH3) cells that do not produce the {alpha}-subunit had barely detectable levels. The presence of Msx1 protein was demonstrated by Western blot analysis in {alpha}TSH cells. We also demonstrated that transcripts encoding the closely related Msx2 factor were not detectable by Northern blot analysis in either thyrotrope or somatomammotrope-derived cells. Subfragments of the region from -490 to -310 of the {alpha}-subunit promoter were used in a Southwestern blot assay using bacterially produced Msx1 and demonstrated that binding was localized specifically to the region from -449 to -421. Deoxyribonuclease I protection analysis, using purified Msx1 homeodomain, demonstrated structurally induced differences in DNA digestion patterns between -436 and -413, and sequence analysis of this region revealed a direct repeat of the sequence GXAATTG, which is similar to the Msx1 consensus-binding site. When nucleotides at both sites were mutated, Msx1 binding was dramatically reduced, and the activity of an {alpha}-subunit promoter construct decreased by ~50% in transfected thyrotrope ({alpha}TSH) cells. These studies suggest that Msx1 may play a role in the expression of the {alpha}-subunit gene in thyrotrope cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of the glycoprotein hormone {alpha}-subunit (subsequently referred to as the {alpha}-subunit) gene in the pituitary gland requires regions of the promoter located upstream of -200 relative to the transcriptional start site (1, 2, 3, 4), which differs from placental expression, where sequences downstream of -200 appear to be sufficient (5, 6, 7, 8, 9, 10). Whereas the factors necessary for placental {alpha}-subunit gene expression have been studied extensively, only a few of those that determine {alpha}-subunit expression in pituitary thyrotropes or gonadotropes have been described. The first one identified was steroidogenic factor-1, which is present in gonadotrope-derived {alpha}T3 cells, as well as in adrenocortical and gonadal cells, and activates the region from -221 to -206 of the human {alpha}-subunit promoter, which corresponds to the region from -219 to -204 of the mouse promoter (11). A LIM-homeodomain factor, present in thyrotropes, gonadotropes, and somatomammotropes, was found to activate the {alpha}-subunit promoter through its interaction with the region from -337 to -330 (12, 13). Recently, Ptx1, which was described as an activator of the POMC gene promoter (14), was also found to activate the {alpha}-subunit promoter (15). Our laboratory has been interested in defining the factors necessary for expression of the {alpha}-subunit promoter particularly in thyrotrope cells.

Previous transfection studies from our laboratory have determined that the region of the {alpha}-subunit promoter from -480 to -254 contains sequences critical for expression in two types of thyrotropic cell models: the TtT97 tumor, a mouse TSH-producing T3-regulated tumor (16, 17, 18, 19), and {alpha}TSH cells (20), a cloned cell line derived from the MGH101A tumor, a mouse thyrotropic tumor that produces only the {alpha}- but not the ß-subunit of TSH and is not regulated by T3 (21). The current studies were aimed at determining what transcription factors present in thyrotrope cells are interacting with this functionally important region of the {alpha}-subunit promoter.

A cDNA expression library in {lambda}-Exlox, constructed from {alpha}TSH mRNA, was screened with the promoter region extending from -490 to -310. Among the proteins identified was Msx1, a homeobox protein that plays an important role during embryogenesis (22, 23, 24, 25). Msx1 was the first mammalian member of the Msx subfamily of homeobox genes that was described, and it was isolated from a cDNA library prepared from mouse 8.5-day embryos (22, 23). This clone hybridized at reduced stringency with a mouse Hox 1.6 cDNA and was initially named Hox 7.1. Analysis of the homeobox sequence demonstrated that this gene had significant similarity with the Drosophila Msh (muscle segment homeobox) gene and diverged from other Hox genes. In addition, its localization on mouse chromosome 5 is not linked to the other Hox gene clusters (Hox A, B, C, and D). Revision of the Hox gene nomenclature resulted in renaming this Hox 7.1 gene Msx1. Expression of Msx1 has been described during embryogenesis of limb buds, genital ridges, visceral arches, craniofacial structures, central nervous system, and the developing pituitary gland (24, 25). Msx1 expression in adult mouse uterus (26) and mammary gland (27, 28) has also been reported recently. The only target genes shown to be activated by Msx1 include Wnt-1, a developmental regulatory gene (29), and Msx1 itself (30). We report the presence of Msx1 mRNA and protein in pituitary thyrotropic cells but not in somatomammotropic cells and identify an Msx1- binding region in the {alpha}-subunit promoter. In addition, mutation of this binding region abrogated binding of Msx1 and decreased {alpha}-subunit promoter activity in thyrotropes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of Msx1 from an {alpha}TSH cDNA Expression Library
To identify which factors interact with the {alpha}-promoter regions critical for expression in thyrotropic cells, we carried out a Southwestern screening of an unamplified cDNA expression library prepared from mouse {alpha}TSH thyrotrope cell RNA, using a region of the mouse {alpha}-subunit promoter extending from -490 to -310. More than 1 million phage were screened. A 1000-bp cDNA was isolated that was almost identical to the published sequence of Msx1, extending from the homeobox into the 3'-untranslated region of the gene (23, 31) (clone A, Fig. 1Go). Sequencing of the clone and of a Msx1 cDNA provided by Dr. David Sassoon verified that, in agreement with Monaghan et al. (31), it contained the sequence GGCC, rather than GC, beginning at nucleotide 877 in the original sequence published by Hill et al. (23). This change in frame predicts a protein that is 26 amino acids shorter. We also verified the presence of an additional 12 bp corresponding to nucleotides 905–916 in the sequence published by Hill et al. (23) that was not present in the sequence of Monaghan et al. (31).



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Figure 1. Schematic Representation of the Msx1 and Msx2 cDNAs

The upper lines represent the DNA sequences of the Msx1 (A) and Msx2 (B) transcripts, with several restriction endonuclease sites indicated. The positions of the homeobox (HB), the single intron (triangle) and translational start ({Rightarrow}) and termination (<-) sites are indicated. A, Msx1 clones obtained by Southwestern (clone A) and cDNA hybridization (clone B) screening of an {alpha}TSH library are indicated, with arrows denoting the extent and direction of the DNA fragments sequenced. Msx1 probes A and C, used in Northern blot analysis, and Msx1 probe B, used in cDNA screening, are indicated. B, Msx2 probes D, E, and F used in Northern blot analysis are indicated.

 
Subsequent rescreening of the same library using a DNA fragment corresponding to the most upstream 100 bp of the Msx1 clone (probe B, Fig. 1Go) yielded a 1600-bp clone that contained the complete coding region of Msx1 (clone B, Fig. 1Go) and was also found to be identical to the published sequences (23, 31) except for the discrepancies noted above. The sequence we obtained predicts a protein containing 300 amino acids with a molecular mass of 32 kDa.

Detection of Msx1 Transcripts in Thyrotropic Cells
To demonstrate that Msx1 transcripts were present in thyrotropic cells, poly A (+) RNA from {alpha}TSH cells and TtT97 thyrotropic tumors were examined by Northern blot analysis, using the 1007-bp isolated clone as a probe. A transcript of 2.2 kb, which is the same as that described for whole mouse embryonic RNA, was detected in both thyrotrope cells. A greatly reduced signal was detected in GH3 somatotrope cells (Fig. 2Go). Messenger RNA loading and integrity were verified by the detection of approximately equal amounts of actin transcripts in GH3 cells when compared with {alpha}TSH RNA (Fig. 2Go, lower panel). Since the homeobox RNA sequence is 82% homologous to a related gene, Msx2 (32, 33), which has been found to express 2.2-kb and 1.4-kb transcripts in mouse embryonic and newborn tissue (33), we proceeded to analyze whether the transcript detected might correspond to Msx2. These results are shown in Fig. 3Go. Northern blot analysis was performed with an 800-bp fragment containing the complete coding sequence for Msx2, and a single band was detected, after prolonged exposure (17 days), in the same location as that detected using the Msx1 probe, with the same washing conditions. This band was detected in {alpha}TSH cells and very faintly in TtT97 tumors, but not in GH3 cells. When we used a 250-bp SauI fragment corresponding to the Msx2 homeobox, which has 82% homology with Msx1, the signal was readily detectable after 6 days of exposure. We then used a 150-bp SauI to HindIII fragment corresponding to the 3'-end of the coding region of Msx2, which has less homology with Msx1, and this probe did not detect a signal in either cell type after 13 days. No transcripts of 1.4 kb were detected with any of the Msx2 probes. To further demonstrate that this signal corresponded to Msx1 transcripts, we hybridized the filter with a 730-bp PstI to HindIII fragment corresponding to 600 bp of the 3'-untranslated region and 130 bp of the adjacent exon 2 of Msx1 that has no homology with Msx2. A strong signal was detected within 2 days, in the same location as described above. Therefore, we verified that the signal detected corresponded to Msx1 and not Msx2.



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Figure 2. Northern Blot Analysis of Msx1 Transcripts in Thyrotrope Cells

Left panel, Five micrograms of polyA(+) RNA from {alpha}TSH thyrotrope cells, two different TtT97 thyrotropic tumors, and GH3 somatotrope cells were separated by electrophoresis through a 0.8% agarose 6% formaldehyde gel, transferred to Nytran filters, and hybridized to a radiolabeled mouse Msx1 cDNA probe extending from the homeobox to the 3'-untranslated region (probe A, Fig. 1Go). DNA size standards are shown on the left. Right panel, Five micrograms of polyA(+) RNA from {alpha}TSH and GH3 cells were separated and probed as for the left panel, after which the filter was washed and hybridized to a mouse actin cDNA probe (lower panel). The autoradiographs were exposed for 36 h (Msx1) and 4 h (actin) at -70 C with intensifying screens.

 


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Figure 3. Northern Blot Analysis of Msx Isoform Transcripts in Thyrotrope Cells

Poly A(+) RNA from {alpha}TSH cells, TtT97 tumors, and GH3 cells was analyzed as described in Fig. 2Go, using the radiolabeled Msx1 probe C and Msx2 probes D, E, and F (Fig. 1Go). After each hybridization, the filter was washed and exposed for at least 14 days to demonstrate that the signal was eliminated. The autoradiographs were exposed for 17 days (probe D), 6 days (probe E), 13 days (probe F), 2 days (probe C), and 4 h (actin) at -70 C with intensifying screens.

 
Detection of Msx1 Protein in {alpha}TSH Cells
To demonstrate whether Msx1 protein was detectable in thyrotropic cells, total cell lysates from {alpha}TSH cells were examined by Western blot analysis using an antibody to the homeodomain of Msx1 (34). This antibody is able to cross-react with Msx2 (34). As a positive control, we used 60 ng purified Msx1-homeodomain protein (34), and we detected a single band of ~14 kDa, appropriate for the size of the homeodomain fragment. In {alpha}TSH cells, we detected a doublet at ~30 kDa (Fig. 4Go), consistent with the size of the full-length Msx1 protein. The doublet may result from the presence of a truncated form of the Msx1 protein, possibly during processing of the cell sample, from posttranslational modifications, such as phosphorylation, from a different Msx protein containing a homologous homeodomain, or from the presence of two translational start sites in the Msx1 transcript. We also cannot exclude that a homologous transcript of the same size may be giving rise to a homologous protein that differs in size.



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Figure 4. Western Blot Analysis of {alpha}TSH Cells Using an Antibody to the Msx1 Homeodomain Peptide

Total cell lysates from {alpha}TSH cells were separated on a 10% polyacrylamide-0.1% SDS gel and electrotransferred to a nylon membrane, incubated with a mouse monoclonal antibody to the Msx1 homeodomain at a dilution of 1:2000, and then with horseradish peroxidase-conjugated goat-anti-mouse IgG diluted 1:5000. An ECL kit was used to detect proteins binding to the Msx1 antibody. As a control, 60 ng purified homeodomain peptide were used. Molecular weight standards are shown on the left.

 
Binding of the Full-Length Msx1 Protein to the {alpha}-Promoter
Msx1 was expressed as a GST fusion and used to localize the binding region on the {alpha}-subunit promoter. The full-length Msx1 was introduced into a pGEX construct, with a hemagglutinin (HA) epitope appended to the amino terminus of Msx1 to allow detection with an anti-HA antibody. Protein extracts from bacteria transformed with this construct, as well as bacteria transformed with the pGEX construct lacking HA-Msx1, were size-separated on a 10% acrylamide-SDS gel. Using an anti-HA antibody, we detected a signal of the appropriate size (58 kDa) for the GST-HA-Msx1 fusion protein (Fig. 5AGo). Aliquots from the same bacterial extracts were again size-separated on a 10% acrylamide-SDS gel and examined by Southwestern analysis, using a radiolabeled fragment corresponding to the {alpha}-subunit promoter region from -490 to -310, identical to that used in the {alpha}TSH cDNA expression library screening. No binding was detected in extracts expressing GST alone. A strong signal was detected, of the same size as the signal detected with the anti-HA antibody (Fig. 5BGo), indicating a strong interaction between the full-length Msx1 protein and the {alpha}-promoter region from -490 to -310. Aliquots from the same bacterial extracts were subsequently examined, again by Southwestern blot analysis, using fragments within the 180 bp {alpha}-promoter region from -490 to -310. This analysis revealed that the region from -453 to -396, and within this, the region from -449 to -421 were able to bind to Msx1, but that the regions from -484 to -446 and from -417 to -373 showed no binding (Fig. 5Go, C-F). These results strongly suggest that the region from -449 to -421 contains the binding region for Msx1.



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Figure 5. Southwestern Blot Analysis of GST-HA-Msx1 Binding to {alpha}-Promoter Fragments

A, One hundred micrograms of bacterial protein extracts containing GST alone (-) or GST-HA-Msx1 (+) were separated on a 10% polyacrylamide 0.1% SDS gel and electrotransferred to a Nytran filter. Binding with an anti-HA polyclonal antibody was visualized using a horseradish-peroxidase goat-anti-rabbit antibody, and detected a prominent signal of the appropriate size for GST-HA-Msx1 (arrow). Molecular weight standards are shown on the left. B–F, A Southwestern blot analysis was performed with proteins separated and transferred to Nytran filters as described above. Filters were subjected to denaturation by incubation in a 6 M guanidinium hydrochloride solution followed by decreasing concentrations down to 187.5 mM guanidinium hydrochloride, then incubated with radiolabeled regions of the {alpha}-promoter fragments as shown.

 
Deoxyribonuclease I (DNase I) Protection Analysis of Msx1 Binding to the {alpha}-Promoter
DNase 1 footprinting analysis was performed using 0.7 µM of a purified Msx1-homeodomain peptide (34) to saturate the binding sites and increase the ability to detect footprinted regions. This analysis revealed that Msx1 induced differences in DNA digestion, displaying enhancement of residues at positions -436, -429, and -413, with protected bands between these sites, the strongest protection corresponding to position -426 (Fig. 6Go). Since DNase I does not generate a band at each residue in the control lane, several blank regions are present that make it difficult to define the precise boundaries of the protected region in the presence of Msx1. Similar findings have been reported in studies of the origin of replication of the plasmid pSC101, where blank regions are observed in the control lane, and in the presence of increasing amounts of a partially purified 37.5 kDa protein there is increased enhancement of certain residues (35). The region of Msx1 binding is located within the -447 to -400 region footprinted by thyrotropic cell nuclear extracts (both {alpha}TSH and TtT97) as previously reported (36). Examination of this sequence revealed a direct repeat of the nucleotides GXAATTG, extending from -436 to -421, with two nucleotides (GA) separating the repeated sequences (Fig. 6Go). This motif agrees with the consensus Msx1 binding motif [C/G]TAATTG (37) and is likely to be the target site for Msx1 binding to the {alpha}-subunit promoter.



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Figure 6. DNase I Protection Analysis of {alpha}-Promoter Binding to Msx1

An {alpha}-promoter fragment extending from -490 to -310 radiolabeled at the upstream end was incubated in the presence or absence of 300 ng purified Msx1 homeodomain peptide as described in Materials and Methods. Positions of hypersensitive sites and protected bands are indicated by arrows. The sequence corresponding to the -436 to -413 fragment is indicated on the right, with the boxed nucleotides corresponding to the putative Msx1-binding sites.

 
Effect of Mutating the Direct Repeat on Msx1 Binding
To determine whether Msx1 binding was dependent on the GXAATTG sequences, we mutated the bases from -434 to -430 and from -424 to -421, disrupting both of the direct repeats, within a fragment extending from -453 to -396 (Fig. 7AGo), and performed Southwestern blot analysis of bacterially expressed Msx1. The wild type and mutated probes were radiolabeled to the same specific activity. The wild type probe demonstrated strong binding (Fig. 7BGo, left panel) similar to that previously shown (Fig. 5DGo), while equal counts of the mutated fragment showed dramatically reduced binding to Msx1 (Fig. 7BGo, right panel). The filter was then again bound to the wild type probe, showing no change in the intensity of binding compared with the first assay (not shown). These results indicated that the mutated nucleotides were essential for Msx1 binding.



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Figure 7. Southwestern Blot Analysis of GST-HA-Msx1 Binding to the {alpha}-Promoter Mutant Fragment

A, Sequence of the wild type and mutated {alpha}-subunit promoter fragments from -453 to -396 that were used as probes in Southwestern blot analysis. The mutated nucleotides are boxed, and the nucleotides that match the Msx1 consensus binding site are underlined. B, Southwestern blot analysis was performed as described in Fig. 3Go, using the wild type and mutated probes as shown.

 
Effect of Mutating the Direct Repeats on {alpha}-Promoter Activity
To determine the effect of disrupting the direct repeat sequences on the function of the {alpha}-subunit promoter, we examined the effect of mutating the same nucleotides (from -434 to -430 and from -424 to -421) that abrogated binding to Msx1, on the activity of the {alpha}-promoter fragment from -480 to +43 fused to a luciferase reporter, in transiently transfected {alpha}TSH cells that express Msx1 and GH3 somatomammotrope cells that do not. The results are shown in Fig. 8Go. When thyrotropic {alpha}TSH cells were transfected with the mutated promoter construct, the expression decreased by ~50% compared with the wild type promoter. In contrast, this same mutation did not affect the activity of the promoter in somatomammotropic GH3 cells. This correlated with the lack of effect of a 5'-deletion of the {alpha}-subunit promoter from -480 to -254 in somatomammotrope cells, where a lower but readily measurable level of activity was detected (36). These results suggest that the direct GXAATTG repeat is important for Msx1 binding and for {alpha}-promoter activity in thyrotropic cells.



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Figure 8. Transient Transfection Analysis of the Mutant {alpha}-Promoter Activity in Thyrotrope and Somatomammotrope Cells

Three million {alpha}TSH (thyrotropic) or GH3 (somatomammotropic) cells were transfected with a luciferase expression vector containing either the wild type or the mutated {alpha}-promoter -480 to +43 fragment, as described in Materials and Methods. Results are the mean ± SEM of n quadruplicate assays and are expressed as a percent of the activity of the wild type promoter. *, P < 0.001 vs. wild type.

 
Effect of Msx1 Cotransfection on {alpha}-Promoter Activity
Cotransfection of an Msx1 expression construct under the direction of the cytomegalovirus (CMV) promoter was performed in {alpha}TSH cells, as well as in GH3 cells that do not contain Msx1, with doses ranging from 10 ng to 20 µg. In {alpha}TSH cells, no change in the activity of the {alpha}-subunit promoter was noted with doses up to 1 µg; thereafter, a dose-dependent suppression was observed, with 5 µg, 10 µg, and 20 µg resulting in a 70%, 77%, and 90% inhibition, respectively. A similar effect was seen in GH3 cells, where the same doses resulted in a 60%, 72%, and 79% inhibition. The {alpha}-subunit promoter mutated at the Msx1 binding site was also inhibited to a similar degree. A construct containing a 5'-deletion of the {alpha}-subunit promoter to -120, which lacked any identifiable Msx1 binding sites, was inhibited by 78%, and the Rous sarcoma virus long terminal repeat (RSV) promoter was inhibited by 60% when cotransfected with 10 µg Msx1 expression construct. These results are in agreement with other studies that have demonstrated that Msx1 can interact with components of the core transcriptional complex and behave as a transcriptional repressor independent of specific DNA binding (38, 39).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We report the cloning of the homeobox protein Msx1 from a mouse {alpha}TSH cell cDNA expression library, using as a probe the region of the {alpha}-subunit promoter that is important for its activity in thyrotropic cells. The Msx1 cDNAs isolated from this library matched the previously published sequence of Msx1 (22, 23). We detected Msx1 transcripts not only in thyrotrope-derived {alpha}TSH cells from which the cDNA expression library was prepared, but also in the TtT97 thyrotropic tumor, which is a well differentiated hyperplastic tissue that, in contrast to {alpha}TSH cells, produces both TSHß- and {alpha}-subunits and is responsive to thyroid hormone. In contrast, we detected a very low signal for Msx1 transcripts in rat GH3 somatomammotropic cells. Although the possibility exists that the mouse Msx1 probes used were unable to hybridize to rat Msx1, this is unlikely due to the high degree of homology between the Msx1 genes that have been cloned. For example, the homeoboxes of mouse and human Msx1 share 94% identity (40).

Most significantly, our studies demonstrate that Msx1 is expressed in highly differentiated pituitary cells. Until recently, Msx1 was thought to be expressed exclusively during embryogenesis. Studies from the laboratory of Paul Sharpe (24, 25) have established the temporal and spatial expression patterns of Msx1. Using in situ hybridization, gene expression was detected from embryonic day 8 to 16 in brain, spinal cord, visceral arches (including heart), nasal/maxillary/mandibular processes (including teeth), eye, ear, tail, and genital ridges (including Mullerian ducts), and limb buds. Expression of Msx1 was also detected in Rathke’s pouch, the developing pituitary gland, with an intense signal by in situ hybridization at embryonic day 10 in the mouse (25), closely preceding expression of the glycoprotein hormone {alpha}-subunit gene, which is the first hormone gene to be detected during pituitary development and was reported to be detected by in situ hybridization at embryonic day 11 in the rat (41). By mouse embryonic day 12.5, Msx1 expression was localized to the pars distalis, the region that will develop into the anterior pituitary gland (24). However, Msx1 expression subsequently decreased and was found to be very low or absent throughout the mouse embryo by day 18 (42). Nevertheless, sites of postnatal Msx1 expression have been described in the mouse, including uterine epithelium, where expression decreases during pregnancy (26), in nail bed and hair follicle, where its presence may correlate with the regenerative ability in these tissues (43), and in mammary gland, where expression decreases during late pregnancy and lactation (27, 28).

We showed that Msx1, but not Msx2, is expressed in thyrotropic cells. Msx2, initially designated Hox 8.1 (31, 33), was found to be expressed during development in a pattern overlapping that of Msx1 (31, 44, 45), although Msx2 has not been described in the developing pituitary gland. Msx2 expression has been detected in adult human osteoblastic cells where up-regulation by 1,25-(OH)2D3 was demonstrated (46) and in rat osteoblastic cell lines (47), where it appears to play a role in osteocalcin gene expression (46, 47), as well as in adult mammary gland stroma, where estrogen up-regulated its expression (27, 28). Southern analysis of mouse genomic DNA had suggested three related Msx genes and perhaps others (23). A third member of the Msx gene family, Msx3, was amplified from mouse DNA by PCR (32). Recently, Msx3 was cloned by RACE (rapid amplification of cDNA ends)-PCR from embryonic mouse RNA and was found to be expressed exclusively in the developing dorsal neural tube, between embryonic days 8 and 12.5 (48). Whether or not Msx3 is present in pituitary cells was not examined in the current studies. The homeodomains share >95% amino acid homology among the three Msx types, suggesting a basis for functional redundancy (49), although the distinct patterns of expression favor distinct roles for each Msx subtype. Although the nature of these roles still remains to be elucidated, several studies have suggested that Msx1 promotes cell growth and maintains a de-differentiated state (50, 51, 52, 53, 54), while Msx2 represses proliferation and promotes cell death (52).

In contrast to the extensive number of studies that have examined the distribution of Msx1 and Msx2 transcripts, little if any has been reported regarding the distribution of the corresponding proteins. We report the presence of Msx protein in {alpha}TSH cells, although we did not prove that this protein was Msx1 because the antibody is known to cross-react with Msx2. However, in the absence of detectable Msx2 transcripts, it is unlikely that the protein we detected was Msx2. In one study that used an Msx2-specific antibody, Msx2 transcript and protein expression were found to correlate precisely (45). Therefore it is more likely that the protein we detected was Msx1, although the possibility that Msx3 (which is 98% homologous in the homeodomain region) (32), or another as yet undescribed Msx isoform, is present in thyrotropic cells has not been excluded.

The homeodomain structure of Msx1 contains a helix-turn-helix motif, suggesting specific DNA-binding activity that mediates control of specific gene expression, and the binding site preference is determined by the N-terminal arm and helix III of the homeodomain (55). However, there are few identified target genes for Msx1 interaction. The only previous report of a naturally ocurring Msx1-binding motif with demonstrated binding activity for Msx1 is that present on the promoter for the developmental protein Wnt-1 (29). This promoter contained two core consensus Msx1-binding motifs in close proximity (Fig. 9Go) (29). Putative Msx1-binding sequences have also been recognized on the Msx1 promoter itself (30) and on the myoD enhancer (56, 57). In addition, the rat osteocalcin gene promoter contains a motif that appears to be a target for Msx2 (34, 47), and the promoter for another bone-specific gene, the collagen type I (COL1A1) gene, contains the sequence TAATTA, that is similar to the consensus Msx1-binding motif and is able to bind to Msx2 (58). In the current studies, we demonstrated high-affinity binding of Msx1 protein to the {alpha}-promoter region from -449 to -421 by Southwestern blot analysis, and DNase I protection assays demonstrated protein-induced changes in the pattern of DNA digestion in the presence of Msx1 within the region from -436 to -413. Sequence analysis revealed that this region contains a direct repeat of the sequence GXAATTG, which is homologous to the consensus binding motif for Msx1, [C/G]TAATTG, defined by random oligonucleotide selection (37), and is also similar to the binding site found on the Wnt-1 promoter (Fig. 9Go). A comparison between the mouse {alpha}-subunit promoter and the corresponding regions of the rhesus (59) and the pig (60) {alpha}-subunit promoters reveals that there is partial conservation of this element, and potential Msx1-binding elements are identified at positions -430 (CTAAATAG) and -453 (CTAATAG), respectively. Another potential Msx1 binding site is also identified in the rhesus {alpha}-subunit promoter, at position -494 (AATTG), in a region that is not conserved in the mouse or the pig {alpha}-subunit promoters. Analysis of the binding and functional characteristics of these sites would determine whether Msx1 may also be important for {alpha}-subunit promoter activity in other species.



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Figure 9. Comparison of the Sequences of the Msx1-Binding Sites on the {alpha}-Subunit and the Wnt-1 Promoters

The sequences shown are those corresponding to the binding sites identified on the promoters indicated on the left. Vertical lines indicate conserved nucleotides. Boxes indicate the nucleotides that match the core consensus Msx1-binding motif (37).

 
There is even less information regarding the function of Msx1 on its target genes. Other studies have shown that human chromosome 4 (containing the MSX1 locus) repressed MyoD expression in 10T1/2 cell hybrids, a function that is consistent with the role of Msx1 in inhibition of myogenesis (54) and that this repression was relieved by antisense Msx1 (56). In the case of Msx2, the activity of the osteocalcin promoter was decreased by mutations within the Msx2-binding motif (34), and mutation of the collagen type 1 (COL1A1) gene motif capable of binding Msx2 reduced bone-specific expression (58). We demonstrated that mutation of the Msx1-binding sites decreased {alpha}-subunit promoter activity in thyrotropic {alpha}TSH cells by ~50% when compared with the wild type promoter. In contrast, we showed no effect of the same mutation in GH3 cells that lack Msx1. This correlated with the lack of effect in GH3 cells of deleting the region of the promoter from -480 to -254, thus eliminating the Msx1-binding sites, while a similar deletion resulted in a 20-fold decrease in activity in {alpha}TSH cells (36). These results predicted that Msx1 would stimulate the activity of the {alpha}-subunit promoter in thyrotropes. However, we have observed that when cotransfecting an Msx1 expression construct in Msx1-containing {alpha}TSH cells and in Msx1-lacking GH3 cells, there was an inhibitory effect on {alpha}-subunit promoter activity. In addition, the {alpha}-subunit promoter mutated at the Msx1 binding region, or truncated so that it lacked that region, was inhibited by cotransfected Msx1 to a similar extent. A general inhibitory effect of Msx1 was also seen when other promoters were tested in cotransfection experiments. The phenomenon of promoter repression by overexpression of cotransfected Msx isoforms has been reported by others. For example, Msx2 cotransfection suppressed osteocalcin promoter activity in one type of osteoblastic cell line, although this effect was not seen in another type of osteoblastic cell line (47). In addition, when Msx2 was stably overexpressed in osteosarcoma cells, it decreased COL1A1 promoter activity (58), and divergent gene expression of Msx2 and COL1A1 was also observed, suggesting that endogenous Msx2 was also inhibitory to COL1A1 gene expression (58). These findings may be explained by recent reports that demonstrate that both Msx1 and Msx2 can behave as transcriptional repressors, independent of specific DNA binding and apparently mediated by their ability to interact directly with components of the core transcriptional complex (38, 39). These observations may indicate that the net functional activity of Msx1 on a gene promoter may depend on the balance between a DNA-independent repressive effect and a stimulatory effect that relies on specific DNA binding. It is possible that interactions with other nuclear proteins, which may be in place with the endogenous Msx1 and the endogenous {alpha}-subunit promoter, are not correctly aligned with exogenous cotransfected Msx1 and {alpha}-subunit promoter constructs, thus modifying the balance that results in activation of target genes.

In summary, we have cloned an Msx1 cDNA from a thyrotropic library and determined that Msx1 protein binds to a sequence on the {alpha}-subunit promoter from -436 to -421, which contains a direct repeat of the Msx1-binding consensus sequence GXAATTG. Mutation of nucleotides within both sites disrupted binding to Msx1 and decreased activity of the {alpha}-promoter in thyrotropic cells. We hypothesize that Msx1, or a similar protein, plays a role in the expression of the {alpha}-subunit gene in thyrotropic cells, possibly by interacting with other transcription factors present in thyrotropes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
{alpha}TSH cDNA Library Construction
A mouse {alpha}TSH cDNA expression library was constructed from twice purified polyA(+)RNA into {lambda}EXlox (61) using the method of Gubler and Hoffman (62). First-strand synthesis used 4 µg RNA, an oligo dT primer, 5-methyl-dCTP, and Moloney murine leukemia virus reverse transcriptase (GIBCO/BRL, Gaithersburg, MD). Directional EcoRI/HindIII linkers were added to blunt-ended fragments as described (63). Size-selected cDNA fragments cleaved with EcoRI and HindIII that were larger than 800 bp were ligated into {lambda}EXlox (Novagen, Madison, WI) containing EcoRI and HindIII arms, packaged into phage with Gigapack II Gold extracts (Stratagene, La Jolla, CA), amplified once, and titered using Escherichia coli ER1647 host cells. The unamplified library contained approximately 3.8 x 106 independent recombinants.

Southwestern Expression Library Screening
Plating of the library, induction of protein production, transferring of proteins to filters, protein denaturation, and binding reactions were performed as previously described (64) with the following modifications. A mouse {alpha}-subunit promoter fragment from -490 to -310 (65) was subcloned into pGEM7Zf+ and isolated by digestion with EcoRI and HindIII. Five hundred nanograms of that fragment were radiolabeled using 20 µCi each of all four {alpha}[32P]deoxynucleoside triphosphates (dNTPs) in an end-filling reaction using avian myeloblastosis virus (AMV) reverse transcriptase (Promega Biotec, Madison, WI), under conditions recommended by the supplier, to a specific activity of 109 cpm/µg. The radiolabeled probe was added to the binding reaction at a concentration of 1.5 x 106 cpm/ml. Positive phage were purified by three additional rounds of screening.

DNA Library Screening
A mouse {alpha}TSH cDNA library was plated onto 25 150-mm plates at a density of ~55,000 plaque-forming units (pfu)/plate using E. coli BL21(DE3)pLysE cells and screened for Msx1 sequences by the plaque hybridization method (66) using a 100-bp probe consisting of an EcoRI fragment at the 5'-end of the first Msx1 clone obtained (probe B, Fig. 1Go). Positive phage were purified with three additional rounds of screening.

Complementary DNA Cloning
Recombinant plasmids were produced by autoexcision of plaque-pure recombinant phage as previously described (64) and analyzed by restriction endonuclease digestion. The DNA inserts were sequenced by the chain termination method (67) using Sequenase (United States Biochemicals, Cleveland, OH) and primers corresponding to SP6 (GATTTAGGTGACACTATA) and T7gene10 (TGAGGTTGTAGAAGTTCCG) that border the multicloning region of pEXlox. DNA sequences were compared with GenBank sequences using the BLAST protocol (68).

Northern Blot Analysis
RNA isolation, purification of polyA(+)RNA, and analysis of steady state mRNA levels were performed using methods that have been previously described (69), with the following modifications. The mRNA was separated on a 0.8% agarose/6% formaldehyde denaturing gel, transferred by diffusion to nylon membranes (Nytran, 0.2-mm pore size, Schleicher & Schuell, Keene, NH) using the Turboblotter system (Schleicher & Schuell), UV cross-linked by exposure to UV light (254 nm) for a total dose of 120 mJ/cm2 (70), and hybridized using the QuickHyb hybridization solution (Stratagene) to DNA probes that were radiolabeled by nick-translation (71) using {alpha}[32P]dCTP to achieve a specific activity of ~1 x 109 cpm/µg. The probes used included: a 1007-bp EcoRI to HindIII fragment (probe A, Fig. 1Go), a 730 bp PstI to HindIII fragment corresponding to 130 bp of the 3'-portion of the coding sequence, and 600 bp of the 3'-untranslated region of Msx1 (probe C, Fig. 1Go), an 800-bp fragment corresponding to the complete coding sequence of Msx2 (probe D, Fig. 1Go), a 250-bp SauI to SauI fragment corresponding to the Msx2 homeobox (probe E, Fig. 1Go), and a 150-bp SauI to HindIII fragment corresponding to the 3'-end of the coding region of Msx2 (probe F, Fig. 1Go). The Msx2 probes were obtained as follows: the region from the translational start site to the termination codon (31) was amplified by PCR using Vent DNA polymerase (New England Biolabs, Beverly, MA) from a plasmid kindly provided by Dr. David Sassoon, using the sense oligonucleotide 5'-GCCGGATCCGCCACCATGGCTGACTACAAGGACGACGATGACAAGGCGGCTTCTCCGACTAAAGG-3' and the antisense oligonucleotide 5'-GCCGGATCCTTAGGATACATGGTAGA-3', both containing BamHI sites at their 5' ends, and subcloned into the BamHI site of pBluescriptSK(-) (Stratagene). The sense oligonucleotide contains a start site consensus sequence (72), the sequence for the Flag-1 epitope (underlined), the three nucleotides coding for the amino acid alanine, and 17 nucleotides corresponding to the Msx2 gene immediately downstream from the translational start site (31). The plasmid pBluescriptMsx2 was sequenced by the chain termination method (67) using Sequenase (United States Biochemicals) to verify all junctions. The fragments described above were isolated by restriction endonuclease digestion from this plasmid.

Western Blot Analysis
This was performed as previously described (69) with the following modifications. Total cell lysates from {alpha}TSH cells were prepared by suspending 10 million cells in 100 µl PBS-0.1% Triton X-100 (PBS-0.1%T) containing 0.5 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol, and 1 mM benzamidine. The suspension was sonicated, undissolved material was sedimented by centrifugation at 2000 x g for 5 min, and the supernatant was adjusted to 10% glycerol and stored at -70 C. The supernatant (50 µg of protein) was combined with an equal volume of 2 x loading buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol), boiled for 2 min, separated on a 10% acrylamide-SDS gel, and electrotransferred for 18 h at 4 C to a nylon membrane (Nytran, 0.2-µm pore size, Schleicher & Schuell). The filter was incubated for 1 h at room temperature with a mouse monoclonal antibody to the Msx1 homeodomain (kindly supplied by Dr. Cory Abate-Shen, University of Medicine and Dentistry of New Jersey, Piscataway, NJ) (34) at a dilution of 1:3000 in 20 mM sodium phosphate, pH 7.5, 150 mM NaCl (PBS-0.1%T). The filter was washed and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat-anti-mouse IgG (GIBCO-BRL) diluted 1:6000 in PBS-0.1%T supplemented with 0.2% milk (Carnation, non-fat instant milk). After washing the filter, an enhanced chemiluminescent (ECL) kit (Amersham, Arlington Heights, IL) was used to detect proteins binding to the Msx1 antibody.

Production of Msx1 in a Bacterial Expression System
Msx1 protein was produced in a bacterial expression system as a fusion protein with the carboxyl terminus of glutathione S-transferase as previously described (69) with the following modifications. To construct the Msx1 expression vector, a fragment extending from nucleotides 127-1050 of the Msx1 gene (23) was amplified by PCR using Vent DNA polymerase (New England Biolabs) from a plasmid containing the complete coding sequence of Msx1, kindly provided by Dr. David Sassoon, using the sense oligonucleotide 5'-GCCGGATCCGCCACCATGGCTTACCCATACGATGTTCCGGATTACGCTGCGGCCCCGGCT-GCTGCTAT-3' and the antisense oligonucleotide 5'-GCCGGATCCAGGTGACTCTGGACCCACCTA-3', both containing BamHI sites at their 5'-ends, and subcloned into the BamHI site of pGEX-2T (Pharmacia, Piscataway, NJ). The sense oligonucleotide contains a start site consensus sequence (72), the sequence for a hemagglutinin (HA) epitope (underlined), the three nucleotides coding for the amino acid alanine, and 17 nucleotides corresponding to the Msx1 gene immediately downstream from the most upstream in-frame ATG, 18 nucleotides upstream from the translation start site previously indicated by Hill et al. (23), and coinciding with that reported by Monaghan et al. (31). The plasmid pGEX-HA-Msx1 was sequenced by the chain termination method (67) using Sequenase (United States Biochemicals) to verify all junctions, then transformed into E. coli DH5{alpha} cells, grown in 100 ml LB-ampicillin to an optical density of 1.1 at 600 nm, and induced for 2 h with 1 mM isopropyl-ß-D-thiogalactopyranoside (Boehringer-Mannheim, Indianapolis, IN). Cells were pelleted, resuspended in 1 ml of PBS-1%T, and sonicated on ice in 0.5-ml aliquots for 5–10 sec. The undissolved material was sedimented by centrifugation at 2000 x g for 5 min, and the supernatant was adjusted to 10% glycerol and stored at -70 C. Control bacterial supernatants were produced from E. coli DH5{alpha} transformed with pGEX-2T. Protein concentrations were determined by the method of Bradford (Bio-Rad, Richmond, CA) (73) using BSA (Boehringer Manheim, Indianapolis, IN) as a standard. A Western blot with 100 µg bacterial supernatant protein was performed as described above (see Western Blot Analysis) using a monoclonal anti-HA antibody (Babco, Richmond, CA) at a dilution of 1:10000 and horseradish peroxidase-conjugated goat-anti-mouse IgG (GIBCO-BRL) diluted 1:5000.

Southwestern Blot Analysis
Bacterial supernatants (100 µg of protein/lane) stored at -70 C were thawed and combined with an equal volume of 2 x loading buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol), boiled for 2 min, and separated on a 10% acrylamide-SDS gel and electrotransferred to a nitrocellulose filter (BA85, Schleicher & Schuell). Filters were allowed to air dry for 20 min at room temperature and then were subjected to denaturing conditions with incubation in guanidinium hydrochloride as described above (see Southwestern Expression Library Screening). Probes used for Southwestern blot analysis were generated as follows: the fragment from -490 to -310 was generated as described above (see Southwestern Expression Library Screening), and the fragments from -484 to -446, from -449 to -421, and from -417 to -373 were synthesized as single-stranded complementary sequences with the addition of half-sites for the restriction endonuclease SalI at each end, then annealed to form double-stranded oligonucleotides. The fragments from -453 to -396, wild type and mutant, were generated by PCR amplification from pSelectm{alpha} (-480/+43) and pSelectm{alpha} (-480/+43 mut) (see below, Luciferase Expression Constructs) using as primers the sense oligonucleotide 5'-GCGTCGACGATGCCTGTTAATTTAAG-3' and the antisense oligonucleotide 5'TAGTCGACTTCAACAGGAAACAG-3', which contain half-sites for the restriction endonuclease SalI (underlined), and subcloned into the SalI site of pGEM5Zf+ (Promega Biotec) for further isolation. One hundred nanograms of each mouse {alpha}-promoter fragment were radiolabeled using 20 µCi each of all four {alpha}[32P]deoxynucleoside triphosphates in a fill-in reaction using AMV reverse transcriptase under conditions recommended by the supplier, to a specific activity of 109 cpm/µg. Aliquots of all probes were separated on a nondenaturing 5% polyacrylamide gel followed by autoradiography demonstrating that they were intact. Filters were incubated for 4 h at 4 C in binding buffer (see Southwestern Expression Library Screening) with 10 µg/ml native salmon sperm DNA, 10 µg/ml boiled and denatured salmon sperm DNA, and 3 x 106 cpm/ml of the radiolabeled probe. Filters were then washed twice for 20 min in binding buffer, dried at room temperature for 20 min, and exposed to x-ray film.

DNase I Protection Analysis
A mouse {alpha}-subunit promoter fragment from -490 to -310 (65), which was subcloned into pGEM7Zf+ and isolated by digestion with EcoRI and Mlu II, was uniquely 3'end-labeled using {alpha}[32P]dATP and {alpha}[32P]dTTP in a fill-in reaction using AMV reverse transcriptase (Promega Biotec), under conditions recommended by the supplier, to a specific activity of 108 cpm/µg. Thirty thousand counts per min of this labeled fragment were incubated with 300 ng purified Msx1 homeodomain peptide (kindly supplied by Dr. Cory Abate-Shen) (34) for a final protein concentration of 0.7 µM and subjected to DNase I digestion as previously described (36, 74). DNA fragments were extracted with phenol-chlorophorm and ethanol precipitated, and separated by electrophoresis on a 7% polyacrylamide-8 M urea gel, followed by autoradiography for 72 h at -70 C using intensifying screens.

Luciferase Expression Constructs
pA3m{alpha}(-480/+43)LUC, pA3m{alpha} (-120 to +43)LUC, and pA3RSVLUC were generated as previously described (1). The mutagenesis of the {alpha}-promoter was carried out as follows. A SmaI to HindIII fragment containing the {alpha}-promoter fragment from -480 to +43 was isolated from pA3m{alpha} (-480/+43)LUC and subcloned into pSelect linearized with SmaI and HindIII, to constitute pSelectm{alpha} (-480/+43). PCR amplification was carried out with Vent DNA polymerase (New England Biolabs) using the following primers: one reaction, with the SP6 primer and the sense oligonucleotide 5'-CCTGTTAATTTAAGAGGCCTGAGCAGGCCTTTTATTTTTCTGTTTCC-3', that corresponds to the fragment from -449 to -403 of the {alpha}-promoter, where the nucleotides from -434 to -430 and from -424 to -421 have been replaced by GGCCT and GGCC (underlined), and another reaction with the T7 primer and with the antisense oligonucleotide 5'-GGAAACAGAAAAATAAAAGGCCTGCTCAGGCCTCTTAAATTAACAGG-3', complementary to the sense mutated {alpha}-promoter oligonucleotide described above. Both amplified fragments were then hybridized, and a third PCR was carried out using the T7 and SP6 primers, resulting in a mutated {alpha}-fragment extending from -480 to +43 (-480/+43 mut) flanked by the multicloning sites of pSelect. This fragment was digested with BamHI and HindIII and subcloned into pSelect linearized with BamHI and HindIII. pSelect m{alpha} (-480/+43 mut) was sequenced by the chain termination method (67) using Sequenase (United States Biochemicals) to verify the sequence of the mutated region and all junctions. The fragment SmaI to HindIII was then isolated and subcloned into pA3LUC linearized with SmaI and HindIII to constitute pA3m{alpha} (-480/+43 mut)LUC.

Transient Transfections
{alpha}TSH and GH3 cell cultures were maintained as previously described (36) and placed in medium containing 10% charcoal-stripped FCS for 48 h before transfection (36). Three million cells were mixed with DNA and transfected by electroporation, using 10 µg of a luciferase expression construct containing either the wild type or mutated {alpha}-subunit promoter, and 1 µg of a ß-galactosidase expression vector as an internal control. For cotransfection experiments, 10 ng, 100 ng, 1 µg, 5 µg, 10 µg, or 20 µg of a CMV-directed Msx1 expression vector, and a similar vector lacking the Msx1 gene in amounts required to make up for the difference in DNA added, were mixed with the cells at the time of electroporation. The Msx1 expression vector was constructed by ligating a fragment containing the complete coding sequence of Msx1 (produced as described under Production of Msx1 in a Bacterial Expression Vector), into a 3700 bp NotI to NotI fragment containing the sequence of the human cytomegalovirus immediate early promoter/enhancer obtained from pCMVß (Clontech, Palo Alto, CA), preceded by a fill-in reaction with AMV reverse transcriptase (Promega). Cells were then incubated in medium containing 10% charcoal-stripped FCS for 44 h, and cell lysates were assayed for luciferase and ß-galactosidase activities as previously described (69). Results of luciferase activity were expressed relative to ß-galactosidase activity, except in cotransfection experiments, where results were expressed relative to the volume assayed, because ß-galactosidase activity was affected by cotransfected Msx1. Transfections were performed in quadruplicate, and experiments were performed using two to four different plasmid preparations of each luciferase construct.


    ACKNOWLEDGMENTS
 
We are grateful to the University of Colorado Cancer Center Tissue Culture Core Laboratory for providing us with cell lines for our studies.


    FOOTNOTES
 
Address requests for reprints to: Virginia D. Sarapura, M.D., University of Colorado Health Sciences Center, Box B-151, 4200 East 9th Avenue, Denver, Colorado 80262.

This work was supported by NIH Grants P30-CA46934 (to the University of Colorado Cancer Center Tissue Culture Core Laboratory), DK-02169 (to V.D.S.), and CA-47411 (to E.C.R.) and by a generous gift from the Lucille P. Markey Charitable Trust.

Received for publication June 13, 1997. Revision received July 29, 1997. Accepted for publication July 31, 1997.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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