Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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ABSTRACT |
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Intestinal and renal absorption of inorganic phosphate (Pi) is critical for phosphate homeostasis in mammals. We have isolated a cDNA that encodes a type III Na-dependent phosphate cotransporter from mouse small intestine (mPit-2). The nucleotide sequence of mPit-2 predicts a protein of 653 amino acids with at least 10 putative transmembrane domains. Kinetic studies, carried out in Xenopus oocytes, showed that mPit-2 cRNA induces significant Na-dependent Pi uptake with an apparent Michaelis constant (Km) for phosphate of 38 µM. The transport of phosphate by mPit-2 is inhibited at high pH. Northern blot analysis demonstrated the presence of mPit-2 mRNA in various tissues, including intestine, kidney, heart, liver, brain, testis, and skin. The highest expression of mPit-2 in the intestine was found in the jejunum. In situ hybridization revealed that mPit-2 mRNA is expressed throughout the vertical crypt-villus axis of the intestinal epithelium. The presence of mPit-2 in the mouse intestine and its unique transport characteristics suggest that multiple Na-dependent cotransporters may contribute to phosphate absorption in the mammalian small intestine.
Na-Pi-III; amphotropic murine retrovirus receptor; Ram-1; sodium-dependent phosphate cotransporter; sodium-phosphate transporter; Pit-2
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INTRODUCTION |
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PLASMA AND INTRACELLULAR PHOSPHATE (Pi) play a fundamental role in many metabolic processes and are necessary for normal cellular and organ functions. Pi homeostasis is achieved through endocrine regulation that affects the physiological activity of bone, kidney, and small intestine. The Na-dependent phosphate (Na-Pi) transport mechanism has been shown to play a major role in phosphate absorption in the intestine (2, 24, 26). The Na-Pi cotransporter has been extensively characterized by transport experiments using brush-border membrane vesicles (BBMV) isolated from the small intestine of a variety of species. These results indicated that the intestinal brush-border Na-Pi cotransporter exhibits an apparent affinity [Michaelis constant (Km)] for phosphate of 0.1-0.2 mM and a coupling stoichiometry of 2 Na ions to 1 phosphate molecule (4, 11, 28, 30). The Na-Pi transport mechanism was also described in the basolateral membrane of the intestinal epithelium using basolateral membrane vesicles (BLMV). Studies in several species, including mouse, rat, and human, suggested a higher-affinity Na-Pi cotransporter, with a Km of 14-93 µM, in the basolateral membrane (13, 17, 26).
In addition, pH was shown to have different effects on the expression of Na-Pi transport in the intestine. Studies using rat duodenal and jejunal BBMV suggested that Pi transport activity was higher at pH 6.0 than at pH 7.4 (2, 18). In contrast, Shirazi-Beechey et al. (30) found that intestinal Pi transport activity increased with increasing pH Na-Pi. Moreover, BBMV uptake studies in rabbit and chick small intestine indicated a pH-independent Pi transport mechanism (11). It was also demonstrated that both electroneutral (2, 4, 28) and electrogenic (11, 30) Na-Pi cotransporters were present in the small intestinal epithelium. Therefore, the different functional properties of intestinal Na-Pi transport systems suggest that multiple Na-Pi cotransporter isoforms may exist in the mammalian intestine. To date however, the number and individual functional characteristics of intestinal Na-Pi cotransporters are not known.
Over the past few years, three types of Na-Pi cotransporters have been identified (types I, II, and III). Type I Na-Pi cotransporters (Na-Pi-I) were originally cloned from rabbit kidney and are expressed in kidney, liver, and brain. The precise physiological role of Na-Pi-I cotransporters is not clear (34). Type II Na-Pi cotransporters (Na-Pi-II) are highly characterized, renal specific proteins that are involved in proximal tubular Pi reabsorption (7, 25). Type III Na-Pi cotransporters (Na-Pi-III) were originally identified as retroviral receptors for gibbon ape leukemia virus (called Pit-1) and for rat amphotropic virus (called Pit-2), and only later were functionally characterized as Na-Pi cotransporters (16, 23, 27). Na-Pi-III cotransporters were suggested to be expressed in many tissues (32). However, these cotransporters have been cloned only from nonintestinal tissues to date, and their function has not been completely characterized.
The molecular identity of mammalian intestinal Na-Pi cotransporters was unknown until the Na-Pi-IIb cotransporter, an isoform of the renal Na-Pi-II cotransporter, was recently identified in the mouse and human intestine (14, 35). This transporter was shown to be an electrogenic Na-Pi cotransporter with low pH dependency, which showed slightly higher activity at acidic pH. Expression of Na-Pi-IIb mRNA was detected in various tissues, and immunohistochemical studies suggested that this transporter was expressed at the apical membrane of the small intestinal epithelium (14). The cloning and functional characterization of the Na-Pi-IIb cotransporter is an initial step toward understanding the molecular mechanisms of intestinal Pi absorption. However, since several intestinal Na-Pi cotransporter isoforms likely exist, identification of new isoforms from the mammalian intestine will help us to better understand the Pi transport phenomenon.
In the current communication, we report the isolation and functional characterization of a cDNA encoding a mouse intestinal Na-Pi-III cotransporter, which we have designated mPit-2 since it has high homology with rat Pit-2 (rPit-2). Kinetic studies suggested that mPit-2 is a high-affinity Na-Pi cotransporter with a Km of 38 µM for phosphate. Transport activity of mPit-2 is significantly inhibited by alkaline pH, which is opposite to that of the mouse intestinal Na-Pi-IIb transporter. mPit-2 mRNA is expressed in several tissues including the intestine, where levels are highest in the jejunum. Molecular identification and functional characterization of mPit-2 suggest that multiple Na-dependent cotransporters are involved in controlling Pi absorption in the mammalian intestine.
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METHODS |
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RNA purification and cDNA-library construction and screening. Total RNA was prepared from male C57/BL mouse intestine by the method of Chomczynski and Sacchi (5), and poly(A)+ RNA was purified with the FastTrack 2.0 kit (Invitrogen, San Diego, CA) (9). A small intestinal cDNA library was constructed in the vector pSPORT1 (Superscript Plasmid System; Life Technologies) as described previously (1). The cDNA library contains 5 × 107 independent clones.
First-strand cDNA was subjected to PCR amplification, with degenerate oligonucleotide primers corresponding to a highly conserved region in the Na-Pi-III cotransporters: sense 5'-ga(c/t)cc(t/g)gttcc(a/t/c)aa(c/t)gg(c/t)(c/t)t-3' (amino acid position 193-199 of mouse Pit-1); antisense 5'-ttgta(g/c)a(a/g)(c/g/t)cc(a/c)ga(g/a)tc(t/c)t-3' (amino acid position 386-392 of mouse Pit-1). PCR products of the expected size (~550 bp) were gel purified and subcloned into the pGEM-T vector (Invitrogen). One of five positive clones, with homology to mouse Pit-1 (47%) and rat Pit-2 (94%), was sequenced. Subsequently, two screening primers specific for mouse intestinal Pit-2 were designed in this region for PCR-based screening of the cDNA library to isolate a full-length clone (sense 5'-agatagcaggcagcttagaga-3', bp 981-1001 of mPit-2 cDNA sequence; antisense 5'-acgtgaccgtcattcctcatg-3', bp 1268-1288; Fig. 1). Approximately 10,000 recombinants were screened by sib selection (21). Bacteria were grown on solid media at all times during library screening. Replicas of each master plate were made onto nitrocellulose membranes (S&S BA85), and the colonies were allowed to grow overnight. The nitrocellulose facilitates recovery of colonies when purifying plasmid DNA. The colonies from each plate were scraped from the nitrocellulose, and the plasmid DNA was purified using Qiaprep spin columns (Qiagen) according to the manufacturer's directions. PCR was performed with mPit-2 specific primers, starting with 20 pools of ~500 clones each. Once a positive pool was identified, the clones of the respective master plate were subdivided into 20 pools of 25 colonies each until a single, positive cDNA clone was isolated.
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cDNA sequencing and analysis. Both strands of the cDNA were sequenced by the Biotechnology Resource Facility at the University of Arizona, utilizing sequence-specific oligonucleotide primers. A cycle-sequencing protocol using Taq FS DNA polymerase and fluorescent, dideoxy chain termination were followed. The resulting DNA fragments were electrophoresed and analyzed using an automated Applied Biosystems 373A Stretch DNA Sequencer. The sequence was assembled and analyzed, and sequence comparisons were run using Omiga software (version 1.1.3) and GenBank Blast searches.
Transport experiments. Initially, Xenopus laevis frogs (NASCO) were anesthetized in ice water with benzocaine for 15 min. Then stage V and VI oocytes were dissected from the frogs and defolliculated, as described previously (1). The oocytes were injected with 50 ng of mPit-2 cRNA 1 day following isolation. Additionally in some studies, oocytes were injected with Na-Pi-IIa cRNA (the renal type II isoform). The oocytes were maintained at 18°C in Barth's solution containing 50 mg/ml gentamicin sulfate, 2.5 mM sodium pyruvate, and 5% heat-inactivated horse serum. Transport of KH232PO4 (DuPont-NEN) was measured 3 days after injection as described (1, 6). Oocyte uptake buffers consisted of: 100 mM NaCl (Na buffer) or 100 mM choline chloride (choline buffer), with 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES-Tris, pH 7.5. Inhibitors were prepared as concentrated stock solutions and then diluted to their final concentration in Na buffer. For the transport assays, groups of five oocytes were washed briefly in choline buffer to remove serum and then incubated in 0.4 or 0.7 ml (kinetic experiments) of the appropriate transport buffer as described in the figure legends. After the indicated time period, uptake was stopped with four 4-ml washes of ice-cold choline buffer. For inhibition studies, 5-min uptake of 100 µM phosphate was measured in the presence or absence of 1 mM experimental inhibitor. Individual oocytes were transferred to scintillation vials and dissolved in 0.5 ml 10% SDS, and the radioactivity was measured by scintillation counting. Counts in control, uninjected oocytes were subtracted from counts in cRNA-injected oocytes. Calculations of kinetic constants were done by nonlinear regression to the Michaelis-Menten or Hill equations using the SigmaPlot program (Jandel Scientific). All uptake experiments were repeated three times, and statistical analysis (t-test or analysis of variance) was also done using the SigmaPlot program.
Northern blot analysis.
A mouse, multiple-tissue Northern blot was purchased from Origene
(Rockville, MA). Each lane was equally loaded with 2 µg mRNA
from different tissues. The Northern blot of different segments of
intestine was prepared using 10 µg of mouse duodenal, jejunal, ileal,
and colonic mRNA (8). Equal loading and transfer
of mRNA was confirmed by detection of 18S rRNA on the blot after transfer utilizing an ultraviolet gel documentation system. The blots
were hybridized with [32P]dCTP-labeled mPit-2
cDNA-specific probes generated by PCR (nucleotides 981-1288, which
represent a low-homology, central hydrophilic domain; Fig.
2). The filter was hybridized overnight
at 42°C in 50% formamide and washed under high stringency conditions
(0.1× sodium chloride-sodium citrate, 0.1% SDS at 65°C) for
1 h (10).
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In situ hybridization of murine small intestine. mPit-2 PCR products (corresponding to nucleotides 981-1522 of the mPit-2 cDNA sequence) were subcloned in both orientations into the pCR II plasmid vector using the TA cloning kit (Invitrogen). cRNA probes were transcribed from these templates with T7 RNA polymerase using the MAXIscript in vitro transcription kit (Ambion, Austin, TX). Depending on the orientation of the insert, either sense (used as a negative control) or antisense (used as experimentals) probes were generated. Integrity of cRNA transcripts was confirmed by denaturing agarose gel electrophoresis. The cRNA transcripts were then labeled with biotin using the BrightStar Psoralen-Biotin labeling kit (Ambion). The biotinylated probes were hybridized to intestinal sections following the mRNAlocator-hyb kit protocol (Ambion). Briefly, mouse jejunum was fixed in 10% neutral buffered Formalin at 4°C for 6 h and dehydrated with alcohol and xylene, and then embedded in paraffin. Five-micron sections were predigested with 40 µg/ml of proteinase K for 30 min and then hybridized with 5 µg/ml of either antisense or sense cRNA probes at 55°C for 4 h. Posthybridization washes were carried out at 55°C three times for 4 min. The probes were detected using Ambion's mRNAlocator-biotin kit, and slides were photographed by standard light microscopy.
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RESULTS |
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Molecular properties of mPit-2. A 546-bp DNA fragment, generated by PCR amplification using degenerate oligonucleotide primers, was subcloned into pGEM-T vector and sequenced. Two specific primers were designed in this fragment region for PCR-based screening (see METHODS). A single cDNA clone of 2,251 bp, which we called mPit-2, was isolated after a total of three rounds of screening. The nucleotide and predicted amino acid sequence of mPit-2 is shown in Fig. 1. mPit-2 cDNA contains an open reading frame of 1,959 nucleotides, and it encodes a protein of 653 amino acids with a predicted mass of 72 kDa. Hydropathy analysis (29) suggests the presence of at least 10 membrane-spanning domains, with a large hydrophilic region near the center of the molecule (Fig. 1). The predicted topological profile differs from that of type I and type II Na-Pi cotransporters, which are predicted to contain between six and eight transmembrane domains (20, 34). This central hydrophilic domain contains several consensus phosphorylation sites for protein kinase C and casein kinase II (Fig. 1). A potential N-glycosylation site was also found in the amino terminus. A Na-binding domain (Gly-X-X-X-X-Leu-X-X-X-Gly-Arg), which is present in many Na-dependent cotransporters (3), was also identified in mPit-2 at amino acid residues 544-554.
The amino acid sequence of mPit-2 has no homology with Na-Pi-I and Na-Pi-II cotransporters but has high sequence homology with other Na-Pi-III cotransporters, including rat Pit-2 (95%) and human Pit-2 (91%), and has moderate sequence homology with mouse Pit-1 (58%). Alignment of the sequences of these four proteins is shown in Fig. 2. One potential N-glycosylation site in the amino terminus and two consensus phosphorylation sites in the central hydrophilic domain are conserved in all four proteins, which indicates that these sites may play important roles in the regulation of type III Na-Pi cotransporters (Fig. 1).Functional characterization of mPit-2.
Transport characteristics of mPit-2 were determined in oocytes
expressing mPit-2 cRNA. We used noninjected oocytes as controls for
uptake, because our preliminary data showed that phosphate uptake from
noninjected oocytes is not different from that of water-injected
oocytes (data not shown). Expression of Na-dependent phosphate uptake
was linearly related to the concentration of mPiT-2 cRNA injected up to
50 ng, after which there was maximal expression of phosphate transport
(results not shown). Phosphate transport was detectable in oocytes 1 day after injection of mPiT-2 cRNA, and the highest expression was seen
at day 3 (data not shown). Radiotracer uptake at pH 7.5 showed that mPit-2 cRNA induced a 74-fold stimulation of phosphate
transport (154 ± 11 pmol · oocyte1 · h
1),
compared with noninjected control oocytes (2.0 ± 1.2 pmol · oocyte
1 · h
1; Fig.
3). Substitution of Na with choline in
the uptake buffer abolished transport of phosphate. Kinetic experiments
indicated that mPit-2-mediated transport is dose dependent and
saturable (Fig. 4). In one experiment
depicted in Fig. 4, the apparent affinity constant
(Km) for phosphate was 44 µM, and the maximum
velocity (Vmax) was 1,305 pmol · oocyte
1 · h
1. In
three separate batches of oocytes, the Km for
phosphate was 38 ± 12 µM and the Vmax
was 1,219 ± 109 pmol · oocyte
1 · h
1. The
transport kinetics were similar to those of mouse intestinal epithelium, where the Km for Pi was
between 20 and 200 µM (26).
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Tissue distribution of mPit-2 mRNA.
Expression of mPit-2 mRNA was analyzed by Northern blots, with a
specific probe designed from the central hydrophilic, low-homology region, under high stringency conditions. A transcript of ~3.8 kb was
observed in all analyzed tissues except spleen and thymus (Fig.
7A), indicating wide tissue
expression of this transporter. Segmental expression of mPit-2 in the
intestine was also analyzed by Northern blots (Fig. 7B).
Interestingly, two transcripts of ~3.8 kb and 2.3 kb were found in
jejunum and colon, and a single transcript of 2.3 kb was found in
duodenum and ileum. Furthermore, the overall expression of mPit-2 in
the intestine was highest in jejunum.
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In situ hybridization of murine small intestine.
In situ hybridization was used to determine the exact distribution of
mPit-2 mRNA in the intestinal epithelium. In jejunum, mPit-2 antisense
cRNA exemplified homogeneous expression in the epithelium of the
villus-crypt vertical axis (Fig. 8),
whereas sense cRNA showed no significant hybridization.
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DISCUSSION |
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Phosphate transport in the intestine occurs by mechanisms similar to those that have been extensively described in the kidney. The transport in the intestine is Na dependent with a high affinity for substrate. The Km of phosphate uptake in BBMV ranges from 0.1 to 0.2 mM (2, 30) and the Km for BLMV ranges from 0.02 to 0.09 mM (13, 17, 26). In the mammalian intestine, multiple Na-Pi cotransporter isoforms have been suggested based on functional studies in BBMV and BLMV. The recently cloned Na-Pi-IIb was the first identified intestinal Na-Pi cotransporter, with an apparent Km of 50 µM and little pH dependency (14). Na-Pi-IIb is an apical intestinal Na-Pi cotransporter and is expressed in many tissues. Conversely, the type III Na-Pi cotransporter was suggested to be a basolateral membrane transporter due to its nearly ubiquitous expression and the fact that blood-borne, murine retroviruses utilize this transporter as a receptor (33). In addition, mPit-2 has a very high affinity for phosphate, which resembles the high-affinity Na-Pi cotransporter found in the mouse jejunal BLMV (26). Thus mPit-2 may represent a basolateral isoform of intestinal Na-Pi cotransporter, although immunohistochemical studies are necessary to precisely define the location of mPit-2 in the intestine.
Although two isoforms of Pit-2 have been cloned from human and rat, little is known about their transport characteristics (16, 23). Rat Pit-2 has a high affinity for phosphate with a Km of 25 µM when expressed in oocytes and 200 µM when expressed in mammalian cells (16). Increasing pH inhibited phosphate uptake by rat Pit-2. There is no reported transport characterization of human Pit-2. In comparison, mPit-2 has a slightly lower affinity for phosphate than the rat Pit-2 when expressed in oocytes, but they are both significantly inhibited by alkaline pH. Furthermore, mPit-2 does not transport sulfate, citrate, glucose, and L-alanine. Other studies showed that arsenate and PFA, known specific inhibitors of intestinal and renal Na-Pi cotransporters, show significant inhibition on mPit-2 transport, suggesting that the function of mPit-2 resembles that of the previously described Na-Pi cotransporter in the intestine. The Hill coefficient measured in Na-activation experiments with mPit-2 is 1.3, which suggests that at least one Na ion is coupled with one substrate molecule during transport. However, to determine the exact coupling number of this Na-Pi cotransporter, one would need to measure electrical current under voltage-clamped conditions.
An important aspect of Na-Pi cotransport across the intestinal epithelium concerns the extent of its dependence on the hydrogen ion concentration. This information would reveal any preference of the transporter for monovalent or divalent phosphate ions. Early studies suggested that the divalent form was most readily transported across the renal brush-border membrane, whereas the monovalent form was preferentially utilized in the intestine (2, 24). Moreover, phosphate transport by mPit-2 was significantly inhibited by alkaline pH, suggesting that the preferred substrate is monovalent phosphate (2). Currently, this mechanism for inhibition at alkaline pH is not understood; however, it may be due to decreased concentrations of monovalent phosphate. Alternately, a separate proton-binding site may be present in the transporter, so that substrate binding may occur regardless of whether a proton is present. In this case, the translocation of the bound anion across the membrane could only occur when a proton was also bound (22).
There is no significant overall homology between mPit-2 and the Na-Pi-I- or Na-Pi-II-related proteins. The sequence and predicted secondary structure of mPit-2 are similar to those of the human and rat amphotropic virus receptor (hPit-2 and rPit-2, respectively) and the mouse gibbon ape leukemia virus receptor (mPit-1). There is ~60% sequence identity between Pit-1 and Pit-2. The greatest differences between the sequences is found in the putative intracellular loop between the sixth and seventh transmembrane domains of mPit-2, which shows only 25% amino acid identity. This may suggest different kinase regulation between Pit-1 and Pit-2, since several consensus phosphorylation sites were found in this region. Another feature shared by mPit-2 and other Na-dependent cotransporters is the presence of a proposed Na-binding domain identified by Deguchi et al. (12). The Na binding sequence G-X-X-X-X-L-X-X-X-G-R was found in the eighth transmembrane domain of mPit-2, which was suggested to be critical for ape leukemia virus infection (15, 31). It was further suggested that productive infection of type C ape leukemia virus reduced phosphate transport into the cells through Pit-1 (27). Thus the conserved Na binding sequence in this region may also be critical for transport function of these proteins.
In contrast to Na-Pi-II cotransporters that are expressed exclusively in kidney, mPit-2 is widely distributed. Using a probe derived from the low-homology central hydrophilic domains, Northern blotting detected two transcripts of ~2.3 and 3.8 kb in the intestine and a single transcript of 3.8 kb in other tissues. The 3.8-kb transcript is consistent with previous observations in rat tissues (23), whereas the 2.3-kb transcript in the intestine is consistent with the length of the cloned mPit-2 cDNA. These data suggest that the two transcripts may involve alternative polyadenylation or alternative splicing or may represent a closely related isoform of mPit-2. By comparing the relative expression of mPit-2, we found that the jejunum has the highest expression in the intestine, which is consistent with the previous observation that the jejunum is the principal site for intestinal phosphate transport (2, 24, 26). Moreover, relatively low expression of mPit-2 mRNA was also found in colon. This supports the previous conclusion that the large intestine may have the ability to absorb phosphate (19).
The exact distribution of mPit-2 in the intestinal epithelium was examined by in situ hybridization. We observed homogeneous expression of mPit-2 mRNA in the epithelial cells from within the crypts to the villi tips. This finding exemplifies expression of this transporter in differentiated and undifferentiated enterocytes. Furthermore, expression was limited to only the epithelial cell layer with no staining apparent in other cell types. This pattern of expression is in contrast to several intestinal brush-border transporters such as the Na-Pi-IIb transporter and the apical Na/H exchangers, NHE-2 and NHE-3, which are expressed only in mature enterocytes from midvillus to the villus tip (8, 10, 14). The cellular localization of mPit-2 may then strengthen previous suggestions that intestinal, type III Na-Pi transporters are located at the basolateral membrane.
In conclusion, the cDNA coding for a murine type III intestinal Na-Pi cotransporter has been cloned and functionally analyzed. mPit-2 is a high-affinity phosphate cotransporter with significant pH dependency. Northern blot analysis demonstrated wide expression of mPit-2 mRNA, with the highest expression in the intestine being in the jejunum. The distinct structure of the predicted protein and epithelial expression of mPit-2 mRNA suggest that mPit-2 is the second identified mammalian intestinal Na-Pi cotransporter.
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ACKNOWLEDGEMENTS |
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We thank Adam K. Ghishan and Kareem Shehab for help in purifying DNA.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2R01-R37DK-33209 and by the W. M. Keck Foundation.
The nucleotide sequence reported in this study has been submitted to the GenBank/European Molecular Biology Laboratories data bank with accession no. AF196476.
Address for reprint requests and other correspondence: F. K. Ghishan, Professor and Head, Dept. of Pediatrics, Director, Steele Memorial Children's Research Center, Univ. of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, Arizona 85724 (E-mail: fghishan{at}peds.arizona.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 February 2000; accepted in final form 4 May 2000.
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