©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Down Regulated in Adenoma (dra) Gene Encodes an Intestine-specific Membrane Sulfate Transport Protein (*)

Debra G. Silberg (1), Wei Wang (2), Richard H. Moseley (2), Peter G. Traber (1)(§)

From the (1) Department of Medicine and Genetics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the (2) Department of Medicine and Veterans Affairs Medical Center, University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
REFERENCES

ABSTRACT

A gene has been described, Down Regulated in Adenoma (dra), which is expressed in normal colon but is absent in the majority of colon adenomas and adenocarcinomas. However, the function of this protein is unknown. Because of sequence similarity to a recently cloned membrane sulfate transporter in rat liver, the transport function of Dra was examined. We established that dra encodes for a Na-independent transporter for both sulfate and oxalate using microinjected Xenopus oocytes as an assay system. Sulfate transport was sensitive to the anion exchange inhibitor DIDS (4,4`-diisothiocyano-2,2`disulfonic acid stilbene). Using an RNase protection assay, we found that dra mRNA expression is limited to the small intestine and colon in mouse, therefore identifying Dra as an intestine-specific sulfate transporter. dra also had a unique pattern of expression during intestinal development. Northern blot analysis revealed a low level of expression in colon at birth with a marked increase in the first 2 postnatal weeks. In contrast, there was a lower, constant level of expression in small intestine in the postnatal period. Caco-2 cells, a colon carcinoma cell line that differentiates over time in culture, demonstrated a marked induction of dra mRNA as cells progressed from the preconfluent (undifferentiated) to the postconfluent (differentiated) state. These results show that Dra is an intestine-specific Na-independent sulfate transporter that has differential expression during colonic development. This functional characterization provides the foundation for investigation of the role of Dra in intestinal sulfate transport and in the malignant phenotype.


INTRODUCTION

Marked progress has been made in understanding the molecular pathogenesis of colorectal cancer by exploring hereditary and acquired genetic alterations (1) . Another approach to understanding the development of the malignant phenotype is to identify genes that are differentially expressed in normal and neoplastic tissue. In this regard, a gene was recently described that is expressed in normal colonic tissue but is significantly decreased or absent in adenomas and adenocarcinomas (2) . Because of this pattern of expression, the cDNA was named Down Regulated in Adenoma, or dra. In the course of examining the expression of dra in normal human tissues, dra mRNA was found to be limited to the colon, although small intestinal epithelial cells were not tested (2) . It is unclear whether the unique pattern of expression of dra in normal and neoplastic colonic tissue is related to the development or progression of neoplasia. Furthermore, there has been no function assigned to the Dra protein. The original analysis of the protein structure of Dra revealed three potential nuclear-targeting sites, a potential homeodomain motif, and a possible acidic transcriptional activation domain (2) . This analysis suggested that the Dra protein may be a transcription factor or a protein that interacts with transcription factors.

The aim of the present study was to determine the function of the protein encoded by the dra cDNA. The achievement of this goal was aided by the comparison of the sequence for Dra with nucleotide and protein data bases. A computer analysis of the amino acid sequences of Dra with these data bases revealed varying homology with a soybean nodulin clone (GMAK170), Neospora crassa sulfate permease II (Cys-14) (3) and sulfate anion transporter-1 (Sat-1) of rat hepatocytes (4) . These proteins represent membrane-spanning transport molecules, and comparison of the putative hydrophobic membrane-spanning domains suggested that Dra may be in a similar class of molecule. Furthermore, Cys-14 and Sat-1 are known transporters of sulfate ions (3, 4) , which narrowed the possibilities for the function of Dra. Recently, the gene responsible for diastrophic dysplasia (called DTDST) was found to have homology to these sulfate transporters as well (5) . The aligned amino acid sequences for Sat-1, Dra, and DTDST are illustrated in that publication (5) .

To examine whether dra encodes a transport protein, we utilized the Xenopus laevis oocyte expression system. These functional studies demonstrated that dra does encode for a Na-independent sulfate transporter with similarities to the Sat-1 liver transporter. In addition, this study describes the tissue distribution of dra mRNA and its unique developmental expression in the mouse, as well as the differential expression of dra in preconfluent and postconfluent Caco-2 cells. Our results suggest that Dra is a functional transport protein that is likely a characteristic of fully developed colon and that loss of expression in colonic neoplasia may represent reversion to an earlier developmental phenotype, although a role in the development of the malignant phenotype remains a possibility.


MATERIALS AND METHODS

RNA Isolation

Total RNA was extracted from mouse tissues using a modification of the technique of Chirgwin et al. (6). Small amounts of tissue were homogenized, in a solution containing 4.2 M guanidinium thiocyanate, 25 mM sodium citrate, 17 mM sodium lauryl sarcosine, and 0.7% 2-mercaptoethanol, using a Brinkmann homogenizer. The homogenate was layered on top of 4 ml of a 5.7 M cesium chloride cushion buffered with 25 mM sodium acetate, and RNA was separated by ultracentrifugation (28,000 rpm, 20 °C, 18 h). The RNA pellet was desolved in 0.3 M sodium acetate (pH 5) and ethanol-precipitated twice, then resuspended in water treated with diethyl pyrocarbonate (DEPC).() In some experiments RNA, from tissues and cell lines, was extracted as described by Chomczynski and Sacchi (7) and as described previously (8). RNA from CaCo-2 cells was extracted at days 4 and 18 after plating for preconfluent and postconfluent cells, respectively.

Cloning of the Human dra cDNA and Portion of the Mouse cDNA

Total RNA isolated from human ileum was reverse transcribed into complimentary DNA (cDNA) using oligo(dT) (0.5 µg) and MMLV reverse transcriptase, as described previously (9) with minor modifications. The MMLV reverse transcriptase and RNA were incubated at 37 °C for 1 h. The cDNA obtained was amplified by PCR using Taq polymerase with specific primers to the 5` and 3` ends of the total human cDNA (2) containing a 5` eukaryotic start and 3` stop translation site based on those described by Cavener and Ray (10) : primer 1, 5`-CGCGTTAAGCGGCCGCACCATGATTGAACCCTTTGGGAATCA-GTAT-3`; primer 2, 5`-CGCGTTAAGCGGCCGCGATTAGAATTTTGTTTCAACTGGCACC-3`. NotI restriction enzyme sites were incorporated into the primers and the PCR products were cloned into Bluescript KS (Stratagene) after digestion of the vector and PCR products with NotI. The resulting 2300-bp cDNA was sequenced as described by the manufacturer (Sequenase 2.0, U. S. Biochemical Corp.), and this was compared to the published sequence (2) . Total RNA isolated from adult mouse colon was reverse transcribed into complimentary DNA (cDNA) using random hexamer primers (125 pmol) and MMLV reverse transcriptase. The cDNA was amplified by PCR with specific primers to dra from the human sequence +1427 to +1867 from the start of translation (2) , using Taq polymerase (primer 1: 5`-TCATCTTCACCATTGTCCTGGG-3`; primer 2, 5`-TGGTATTGATTGGCTGGTCCAG-3`). The 441-bp PCR product was cloned into Bluescript KS (Strategene) by TA cloning (the vector was prepared according to the protocol of Marchuk et al.(11) ). The cDNA clone was sequenced utilizing the T3 and T7 primers of Bluescript (Sequenase).

In Vitro Transcription of dra cDNA

dra plasmid (5 mg) was linearized with XbaI at the 3` end. After phenol/chloroform extraction and ethanol precipitation, capped mRNA was synthesized using T7 RNA polymerase in the presence of the capping analog mG(5`)ppp(5`)G using a Promega Ribomax large scale RNA system T7. Unincorporated nucleotides were removed with Chromaspin-30 columns (Clontech), and synthesized mRNA was recovered by ethanol precipitation, followed by resuspension in DEPC/water for oocyte injection at a concentration of 10 ng/ml.

RNase Protection Assay

The RNase protection assay was performed essentially as described previously (8) . Single-stranded P-labeled probe for mouse dra was synthesized from pKS-dra linearized with XbaI, using T3 RNA polymerase (Promega) and [P]CTP (3000 Ci/mol). This yielded a probe of 441 bp in length. After synthesis, the P-labeled RNA probe was purified by digestion with RNase-free DNase I, extracted with phenol and chloroform, and precipitated with ethanol. Solution hybridization was performed for 16 h at 45 °C in a solution containing 10 µg of total RNA, 1 10 cpm of probe, 80% formamide, 40 mM PIPES (pH 6.4), 0.4 M NaCl, and 1 mM EDTA. Samples were then digested with RNase A and T1 for 60 min at 15 °C, extracted with phenol:chloroform (1:1), and precipitated with ethanol. The pellets were resuspended in loading buffer (80% formamide, 0.1% bromphenol blue, 0.1% xylene cyanol, and 1 mM EDTA, pH 8.0) and separated in a 6% denaturing polyacrylamide gel.

Northern Analysis

Samples of total RNA were size-separated by electrophoresis in 2.2 M formaldehyde, 1% agarose gels. The RNA was transferred to Hybond-N (Amersham) nylon membranes, UV cross-linked, and hybridized. The cloned mouse dra fragment was labeled with Klenow enzyme (Boehringer Mannheim) using [P]dGTP and hybridized following the protocol of Virca et al.(12) .

Oocyte Isolation and Injection

Mature X. laevis females were purchased from Xenopus I, Inc. (Ann Arbor, MI). Frogs were anesthetized by immersion for 15 min in ice-cold water containing 0.3% 3-aminobenzoic acid ethyl ester. Oocytes were removed and incubated at room temperature for 3 h in a solution containing, in mM, 98 NaCl, 2 KCl, 1 MgCl, and 5 HEPES/NaOH (pH 7.5) supplemented with 2 mg/ml collagenase (Life Technologies, Inc.). Oocytes were then washed with frog Ringer's (in mM, 98 NaCl, 2 KCl, 1.8 CaCl, 1 MgCl, and 5 HEPES/NaOH (pH 7.5)), and stage V and VI oocytes were selected. After overnight incubation at 18 °C in frog Ringer's supplemented with penicillin (500 units/ml) and streptomycin (100 µg/ml), healthy oocytes were injected with 500 pg of mRNA derived from in vitro transcription of dra or DEPC-treated water (total volume: 50 nl). Subsequently, oocytes were cultured for 4 days at 18 °C with a daily change of antibiotic-supplemented frog Ringer's.

Transport Assay

[S]Sulfate (carrier-free) and [C] oxalate were obtained from DuPont NEN. For sulfate uptake studies, 12-25 oocytes were washed once in a sodium-free and sulfate-free uptake solution (in mM, 100 choline chloride, 2 KCl, 1 CaCl, 1 MgCl, 10 HEPES/Tris, pH 7.5). The oocytes were then incubated in 500 µl of uptake solution containing 1 mM [S] sulfate (20 µCi/ml) for the designated time interval. Uptake was stopped by washing the oocytes three times with ice-cold uptake solution containing 5 mM KSO. Individual oocytes were then transferred to scintillation vials, dissolved in 0.5 ml of 10% sodium dodecyl sulfate, and after addition of scintillation fluid, oocyte-associated radioactivity determined in a Beckman LS 1801 liquid scintillation counter. For oxalate uptake studies, uptake solution contained, in mM, 100 choline chloride, 2 KCl, and 10 HEPES/Tris, pH 7.5; oocytes were incubated in 500 µl of uptake solution containing 1 mM [C] oxalate (2 µCi/ml); and uptake was stopped by washing the oocytes three times with ice-cold uptake solution containing 5 mM oxalate.

RESULTS

Cloning of the Human cDNA and a Portion of the Mouse cDNA of dra

Initially, we cloned the human dra cDNA from the known sequence and a portion of the mouse cDNA for use in expression studies in mice. First, RNA isolated from normal human ileum was reverse transcribed and the complete translated region of the cDNA was cloned by PCR using amplification primers based on the published sequence (2) . To obtain a clone with an efficient eukaryotic start and stop translation site, we modified the primers according to those described by Cavener and Ray (10) . After sequencing, the cDNA had the identical sequence to the previously reported clone (2) . A 441-bp fragment of the mouse cDNA of dra was cloned from normal mouse colonic RNA using the reverse transcription-polymerase chain reaction. The mouse dra cDNA corresponded to bases +1427 to +1867 of the human cDNA (numbered from the translational start site) and contained the 12th and last putative membrane-spanning domain (3) . The murine and human cDNAs, in this region, had 88% identity in the amino acid sequence.

Function of dra in Xenopus Oocytes

On the basis of significant homology between the Dra sequence and the recently cloned rat canalicular sulfate transporter Sat-1 (4) , RNA derived from in vitro transcription of dra was injected into a Xenopus oocyte expression system and sulfate transport activity assayed. Na-independent [S] sulfate uptake into RNA-injected oocytes was significantly greater than uptake in water-injected oocytes (Fig. 1). Moreover, the expressed Na-independent [S] sulfate uptake was sensitive to the anion exchange inhibitor, 4,4`-diisothiocyano-2,2`-disulfonic acid stilbene (DIDS) and oxalate (Fig. 2), exhibiting a cis-inhibition pattern similar to that for Sat-1 (4) . Since oxalate appears to be a substrate for hepatic (13) , intestinal (14, 15) , and renal sulfate transporters (16), and showed cis-inhibition of sulfate transport (Fig. 2), oxalate transport was also directly examined in RNA-injected oocytes. Na-independent [C]oxalate uptake into RNA-injected oocytes was also significantly greater than uptake in water-injected oocytes (Fig. 3).


Figure 1: Time course of sulfate uptake in Xenopus oocytes. Oocytes were injected with either 500 pg cRNA () or DEPC-treated water () (total volume: 50 nl). Four days after injection, uptake of 1 mM [S] sulfate was determined at 25 °C over 2 h. Uptake values represent the means ± S.E. of four separate oocyte preparations using 8-12 oocytes for each determination. **, p < 0.005.




Figure 2: cis-Inhibition of sulfate uptake in Xenopus oocytes. Oocytes were injected with either 500 pg cRNA () or DEPC-treated water () (total volume: 50 nl). Four days after injection, 2-h uptake of 1 mM [S] sulfate was determined at 25 °C in the presence or absence of 1 mM DIDS or 5 mM oxalate. Uptake values represent the means ± S.E. of 13-28 determinations from two separate oocyte preparations. *, p < 0.05.




Figure 3: Time course of oxalate uptake in Xenopus oocytes. Oocytes were injected with either 500 pg cRNA () or DEPC-treated water () (total volume: 50 nl). Four days after injection, uptake of 1 mM [C] oxalate was determined at 25 °C over 3 h. Uptake values represent the means ± S.E. of three separate oocyte preparations using 8-12 oocytes for each determination. **, p < 0.005.



Tissue-specific Expression of dra in the Mouse

In the initial report of the cloning of dra, expressed mRNA was found only in the colon when examined in several human tissues (2) . To examine the distribution of expression more completely and to directly compare the small intestine to the colon, an RNase protection assay of RNA isolated from different mouse tissues was performed using the 441-bp mouse cDNA as a probe. The expression of dra was limited to the small intestine and colon (Fig. 4). In the small intestine, there was low expression of dra along the entire length of the intestine. The most intense expression was found in the cecum with a proximal to distal gradient in the colon. Importantly, there was no expression in multiple other mouse tissues.


Figure 4: Tissue-specific expression of dra in the mouse. RNA extracted from tissues of adult mice were analyzed by RNase protection assay. The arrow reflects the probe binding to dra. Only intestinal tissues express DRA with the highest level of expression seen in the cecum.



Expression of dra in Developing Normal Mouse Tissue

Because of its differential expression in normal and neoplastic colonic tissue, we examined dra expression during intestinal development in the mouse. Since many important developmental events in the mouse small intestine and colon occur after birth, RNA was isolated from the small intestine and colon of normal mice at postnatal day 0, day 9, and adult. A Northern blot was prepared from the RNA and probed with the 441-bp cDNA fragment of dra. In the colon, a low level of dra mRNA was present at birth, with a marked increase observed during postnatal development (Fig. 5). In contrast, dra mRNA remained at a low constant level throughout postnatal development in the small intestine. Quantification of the increase in colonic expression revealed that there was a 134-fold increase in expression in the adult colon as compared to the newborn mouse.


Figure 5: Expression of DRA in developing normal mouse tissue. RNA extracted from postnatal day 0, day 9, and day 40 colon and small intestine were characterized by Northern blot analysis. The membrane was hybridized with a 441-bp mouse cDNA probe. The probe identified an mRNA, at the expected size for dra, of 3.2 kilobases. dra expression is minimal at birth in the colon and increases with development. Small intestine expression of dra is present at birth and continues to adulthood.



Differential Expression of dra in Preconfluent and Postconfluent Caco2 Cells and Other Tumor Cell Lines

Postconfluent Caco-2 cells are a model for differentiated enterocytes (17) and as such are a potential system to evaluate differentiation in tissue culture. Northern analysis of Caco-2 cell RNA revealed a band of 2.8 kilobases, the predicted size of dra mRNA, in the normal human ileum control and only in the postconfluent cells. There was also a smaller unidentified band in the postconfluent lane (Fig. 6). In addition, the Colo DM cells, another human colon cancer cell line, do not express dra. Hep G2 cells, derived from a hepatoma, do not express dra at the expected size, but a smaller transcript is identified.


Figure 6: Differential expression of DRA in preconfluent and postconfluent Caco-2 cells and other tumor cell lines. RNA was extracted from Caco-2 cells at day 4 (preconfluent) and day 18 (postconfluent) from plating, HepG2 cells, Colo DM cells, and human small intestine. The RNA was characterized by Northern blot analysis with a 441-bp cDNA probe from mouse dra. The postconfluent Caco-2 cells express dra with an identical size transcript (3.2 kilobases) as the human small intestine. There is also a small transcript of unknown identity, which also appears to be present in HepG2 cells. The Colo DM cells do not express dra.



DISCUSSION

We demonstrate that dra encodes for a protein that mediates Na-independent, DIDS- and oxalate-sensitive sulfate uptake and conclude that Dra is an intestine-specific sulfate (and oxalate) transporter which is expressed predominantly in the colon. Thus, the similarity in amino acid sequence shared by Dra and Sat-1 (4, 5) is also reflected in a functional similarity for transported molecules. These findings make it highly likely that the recently described gene that is responsible for diastrophic dystrophy, which also has sequence similarity to Sat-1 and Dra, is a functional sulfate transporter (5) . In fact, sulfate transport is defective in fibroblasts from patients with this inherited disease. The mRNA for the putative sulfate transporter that is mutated in diastrophic dystrophy is expressed in many cells and tissues (5) . Therefore, it appears that there will be multiple sulfate transporters, some of which are highly restricted to specific tissues and others that serve a more generalized cellular function. What is the potential role of dra in the intestine, and what is the significance of its pattern of expression in colonic neoplasia and development?

Sulfate transport has been well characterized in the rat liver. In rat liver canalicular membrane vesicles, Meier et al.(13) characterized a bicarbonate-sulfate exchanger that, given the bicarbonate gradient that exists at the canalicular lumen, would mediate the excretion of sulfate and other substrates. Substrate specificity experiments demonstrated that thiosulfate and oxalate and, to a lesser degree, succinate could substitute for sulfate, but chloride, nitrate, phosphate, acetate, lactate, glutamate, aspartate, bile acids, and reduced and oxidized glutathione were not substrates for this transporter. Since furosemide and the disulfonic derivatives SITS and DIDS also inhibited transport, sulfonated drugs as well as sulfate-conjugated metabolites might represent substrates for this transport process, although the latter were not examined. A similar transport process was described in rat and rabbit renal basolateral membrane vesicles (16, 18) . Subsequently, the rat liver canalicular sulfate carrier (sulfate anion transporter-1 or Sat-1) was functionally expressed (19) and cloned (4) using Xenopus oocytes. Northern hybridization (4) and hybrid depletion experiments using antisense oligonucleotides derived from the Sat-1 cDNA sequence (20) demonstrated that rat renal basolateral Na-independent sulfate transport activity is closely related to Sat-1. No hybridization signal was detected in duodenum, ileum, proximal colon, or distal colon (4) .

Less is known about sulfate transport in the small intestine, but both basolateral and apical transport processes have been partially characterized. Sulfate transport in rat and rabbit small intestinal basolateral membranes, which is different from that in renal basolateral membranes, has been described (21, 22) . Although this basolateral transport was DIDS- and oxalate-sensitive, chloride, and not bicarbonate, exchanges for sulfate in this transport process (21, 22) . In addition to this transporter, a sulfate:bicarbonate exchanger that also transports oxalate has been identified on the basolateral membrane of rabbit ileal villus enterocytes, but not crypt cells (23) . Apical sulfate transport (absorption) by both the kidney and the intestine was thought to be mediated by an entirely different transport process, Na:sulfate cotransport, which has also been cloned recently (20, 24) . However, demonstration that sodium-stimulated sulfate uptake in the intestine exhibited sensitivity to pH (25, 26) led to the characterization of a sulfate:OH exchanger in rabbit ileal brush-border membrane vesicles (14, 27) . Sulfate:OH exchange and Na:sulfate cotransport were shown to be separate carriers on the basis of inhibition studies (14) . The substrate specificity of intestinal brush-border sulfate:OH exchange is similar to that described for canalicular bicarbonate-sulfate exchange in that probenecid, DIDS, and oxalate cis-inhibit sulfate uptake (15); however, superimposition of a bicarbonate gradient does not further stimulate sulfate uptake over a pH gradient alone (27) .

In contrast to hepatic, renal, and ileal sulfate transport, colonic transport of sulfate has not been examined in any systematic manner. Accumulation of sulfate by mouse intestine is maximal in the area adjacent to the ileocecal valve, and during development there is a continuous increase in accumulation in the terminal ileum, whereas accumulation diminishes in the remainder of the gut, including colon (28). The colon, however, has been shown to be an important site for oxalate absorption and is required for enteric hyperoxaluria to occur (29) .

Where might the Dra sulfate transporter fit in the physiology of the intestinal and colonic ion transport? From the present data, it is impossible to determine whether Dra is the apical sulfate:OH exchanger, basolateral Cl:sulfate, or basolateral sulfate:HCO exchanger. Moreover, since these transporters were characterized in the ileum, it is possible that Dra represents a transport process that has not as yet been physiologically characterized. Since all the characterized transport processes have similar kinetic features and substrate specificities, determination of the potential role of Dra will have to await development of specific antibodies and immunolocalization studies. Once the protein is localized to either the apical or basolateral membrane, the Caco-2 cell line may prove a useful model to examine its function. Furthermore, expression in eukaryotic cells will be necessary to study its transport properties.

dra was discovered because of the intriguing property of loss of expression in neoplastic colon cells, which led to the speculation that it might represent a tumor suppressor gene involved in colon carcinogenesis (2) . With the discovery that dra encodes for a sulfate transporter, it is possible that its expression represents a phenotypic characteristic of a mature colonocyte. In a neoplastic cell, expression of this gene may be lost as the cell reverts to a more immature phenotype. It is well described in colon carcinomas that genes expressed in fetal colonocytes are re-expressed in the cancer cells (30, 31, 32, 33, 34, 35) . The developmental expression of dra that we have shown in mice would be consistent with the hypothesis that a phenotype more close to a fetal colonocyte might lack dra expression. In this scenario, the dra gene would not necessarily have a functional role in the acquisition of the malignant phenotype.

It is not immediately obvious how the loss of a sulfate transporter protein would be important in the development of a malignant cells. It is interesting to note that sulfate transport activity in the HTC hepatoma cell line is also markedly reduced (36) . The identification of tumor suppressor genes that are integral to the process of colon carcinogenesis such as APC (37) , which interacts with catenins (38) , and DCC, which is a member of the cell adhesion molecule family of proteins (39) , has emphasized the potential importance of interactions of the cell with the extracellular environment. The importance of a sulfate transporter in a osteochondrodysplasia syndrome (diastrophic dysplasia) has shown that alteration of sulfate transport may change the sulfation of proteoglycans, which results in abnormal morphogenesis of joints (5) . In addition, it is known that proteoglycans in liver and kidney tumors have decreased sulfation (40, 41) . It is possible that abnormal synthesis of sulfated proteoglycans may play a role in colon cell functions as proteoglycans bind many growth factors (42) . Sulfation is also decreased in mucins associated with colorectal cancer (43-45) and in the rectum of inflammatory states such as ulcerative colitis (46) . In lung fibroblasts carrier-mediated sulfate transport at the plasma membrane is rate-limiting for macromolecular sulfation (47) . Further investigation is required to determine the function of Dra in the colon and whether loss of function of the sulfate transporter may be involved in the development of the malignant phenotype or simply a lost function that is of little consequence to the tumor cell.


FOOTNOTES

*
This research was supported by National Institutes of Health RO1 DK46704 (to P. G. T.), the Penn Training Program in Gastrointestinal Sciences (Grant DK07066 to P. G. T.), a VA Merit Review (to R. H. M.), and an American Gastroenterological Association Foundation Merck Senior Fellow Research Award (to D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprints should be addressed: 600 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-6144. Tel.: 215-898-0154; Fax: 215-573-2024; E-mail: traberp@mail.med.upenn.edu.

The abbreviations used are: DEPC, diethyl pyrocarbonate; MMLV, Moloney murine leukemia virus; PCR, polymerase chain reaction; bp, base pair(s); PIPES, 1,4-piperazinediethanesulfonic acid; DIDS, 4,4`-diisothiocyano-2,2`disulfonic acid stilbene; SITS, SITS, 4-acetamido-4`-isothiocyano-2,2`-disulfonic stilbene.


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