Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and Westside Veterans Affairs Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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The
Na+/H+ exchanger (NHE) 2 belongs to a family of
plasma membrane transporters involved in intracellular pH and cell
volume regulation. We recently reported cloning of human
NHE2 (hNHE2) from a colonic cDNA library.
Northern blot analysis has identified NHE2 mRNA only in
small intestine, prostate, kidney, colon, and skeletal muscle. In this
study, we describe the structure and 5'-regulatory region of the
hNHE2 gene. The hNHE2 gene spans >90 kb
and is organized in 12 exons intervened by 11 introns. All introns
contain the conserved GT and AG dinucleotides at the donor and acceptor
sites, respectively. The hNHE2 gene was mapped to chromosome
2q11.2. Primer extension analysis revealed a single transcription
initiation site in human colonic adenocarcinoma cell lines. Analysis of
the DNA nucleotide sequences of a 1.4-kb fragment of the 5'-flanking
region shows no canonical TATA or CAAT boxes. However, the promoter
region contains several potential cis-regulatory elements
such as Sp1, early growth response-1, activator protein-2, MyoD, p300,
nuclear factor-B, myeloid zinc finger protein-1, caudal-related
homeobox (Cdx) gene A, and Cdx protein-2 binding sites. In
transient transfection studies, a reporter construct containing the
1.4-kb promoter region exhibited low luciferase activity levels.
However, after deletion upstream of
664, its activity increased
approximately threefold. Thus our data suggest that an inhibitory
element may exist in the NHE2 promoter 5'-upstream region.
exon-intron junction; chromosome 2q11.2; transcription factor; C2BBe1; transfection
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INTRODUCTION |
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STUDIES IN THE HUMAN ILEUM and colon have established the involvement of an electroneutral Na+/H+ exchanger (NHE) in the process of Na+ absorption. Recent studies (2, 7, 25, 37, 38, 41, 50, 53) have identified six molecular isoforms of the NHE gene family. NHE1 to NHE5 are plasma membrane proteins, whereas NHE6 is found in the mitochondrial membrane. Studies (14) of the NHE1 isoform have demonstrated its ubiquitous nature as well as its localization in the basolateral membrane of the polarized epithelial cells. The NHE4 isoform has also been implicated in the basolateral Na+/H+ exchange activity of the rat kidney cells (11). The NHE2 and NHE3 isoforms, on the other hand, are localized to the apical membrane and have been implicated in apical Na+ absorption (21, 50, 51). In contrast to the NHE1 isoform, the expression of the other family members is tissue and cell specific (2, 7, 12, 25, 37, 38, 50). All six isoforms show similar structural features, including a membrane-spanning domain known to be involved in ion exchange (17) and a cytoplasmic domain that is highly divergent among all isoforms and contains several putative regulatory consensus sequences (52).
Chromosomal mapping has shown a wide distribution of members of this gene family on the genome of different species. In humans, NHE1 (28, 32), NHE3 (6), and NHE5 (25) have been reported to map to chromosomes 1p35, 5p15.3, and 16q22.1, respectively. To date, only the genomic organization of the human NHE1 (hNHE1) (33) and NHE5 has been reported (2). Also the 5'-regulatory region of the NHE1 has been characterized to some extent (26, 33). However, no information is available on the other isoforms. To characterize the hNHE2 isoform and its contribution to apical Na+/H+ absorption in the intestine, we (29) have cloned and sequenced this isoform and shown that its gene product is capable of Na+/H+ exchange activity in a heterologous system. In the present study, we have determined the genomic organization of the hNHE2 gene, identified its exon-intron boundaries, and mapped the gene to the chromosome 2q11.2. We have also cloned a 3.3-kb fragment of the NHE2 promoter region. Sequence analysis of the promoter and the 5'-flanking region revealed the presence of numerous potential transcription factor response elements as well as intestinal-specific cis-acting regulatory elements that might be involved in tissue-specific expression and regulation of the NHE2 gene.
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MATERIALS AND METHODS |
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Materials. All chemicals were purchased from Sigma Chemical (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Restriction endonuclease and other modifying enzymes were from either New England Biolabs (Beverly, MA), GIBCO BRL (Gaithersburg, MD), or Promega (Madison, WI). The T-A cloning kit, JM109 competent cells, and luciferase assay system were from Promega.
Isolation of hNHE2 genomic clones.
Two recombinant phage clones containing hNHE2 genomic DNA
sequences were obtained by screening a placental genomic library (Stratagene, La Jolla, CA) with a 32P-labeled
NHE2 cDNA fragment. These clones were characterized by
restriction enzyme analysis and Southern blot hybridization (data not
shown) using standard methods (1). The clones, NHE2A and
NHE2B, contained 13.7- and 19-kb genomic DNA inserts,
respectively, and overlapped by 4 kb. Together, they spanned ~28 kb
of genomic DNA harboring the 3' end of the hNHE2 cDNA. To
reveal the gene structure, we subcloned the genomic DNA insert of the
phage clones into the cloning vector pGEM-13 using Not I
restriction enzyme sites that flanked the 5' and 3' ends of the
inserts. The new constructs containing 13.7- (pRD137) and 19.0-kb
(pRD190) genomic DNA fragments were used for partial sequence analysis
of the genomic DNA, using primers designed based on the
hNHE2 cDNA (29). Comparison of the nucleotide
sequences of the genomic clones and the cDNA revealed that the 13.7-kb
genomic insert contained sequences corresponding to exons 8-12 and
that the 19-kb DNA fragment contained sequences from exons 6 to 9. Our
attempts to isolate the remaining genomic DNA region corresponding to
the 5' end of the gene by using the same DNA library and screening
method were unsuccessful. Therefore we used a long-distance extension
PCR method to clone the remaining introns.
Identification of introns by PCR.
The introns were identified and cloned by PCR amplification using the
Expand long template PCR kit (Boehringer) in 50 mM Tris · HCl,
pH 9.2, 16 mM (NH4)2SO4, 1.75 mM
MgCl2, 0.35 mM of each dNTPs, 10 µM of each primer, 2.5 U
rTth polymerase, and 200 µg human genomic DNA (Clontech).
PCR amplification conditions were as follows: one cycle of
preamplification denaturation at 95°C for 2 min followed by 35 cycles
of two-step cycling at 94°C for 30 s and 5 min of annealing and
extension at 68°C followed by a final extension of 10 min at 68°C.
If needed, the annealing temperature and/or extension time was changed
based on primer specification and size of the amplification products,
where a three-step PCR cycling procedure was employed. The PCR primers (Table 1) were chosen from the vicinity
of the NHE2 cDNA sequences that resembled the known splice
junction sequences at the exon level (35).
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Chromosomal localization. Fluorescence in situ hybridization (FISH) was performed by Genome Systems (St. Louis, MO) using a hNHE2 genomic DNA clone (pRD190) as the hybridization probe. The labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes from peripheral blood lymphocytes. The initial experiment resulted in specific labeling of the long arm of a chromosome believed to be chromosome 2 on the basis of size, morphology, and banding pattern. A second experiment was conducted in which a genomic probe from the N-myc locus, which has previously (43) been mapped to 2p23, was cohybridized with the NHE2 probe. This experiment resulted in the specific labeling of the short and long arms of chromosome 2. Eighty metaphase cells were analyzed, with sixty-six exhibiting specific labeling.
To confirm the chromosomal localization, genomic DNA from a rodent-human somatic cell hybrid cell line containing only the human chromosome 2, NA11712 (Coriel Cell Repository) was subjected to PCR amplification using hNHE2-specific oligonucleotides. The primers used were sense primer from exon 2, 5'-CCATCTGTATCACAAGTTCG-3', and antisense primer from intron 2, 5'-GTCATGCAGTGTGAATGTG-3'. The thermocycling parameters were an initial denaturation for 2 min at 95°C, followed by 35 cycles at 94°C (30 s) and 54°C (30 s) and 2 min at 68°C. As control genomic DNA from parental lines, a normal human genomic DNA (NAIMR91) and mouse A9 DNA (NA00346B) also were subjected to the PCR amplifications using the same primers and PCR conditions.RNA isolation. Total RNA was isolated from the postconfluent T84 or C2BBe1 cells using RNA STAT-60 (Tel-Test, Friendswood, TX) according to the manufacturer's suggested protocol.
Primer extension analysis.
The transcription initiation site of hNHE2 was determined by
primer extension using SuperScriptII RT (GIBCO BRL). An antisense oligonucleotide complementary to nucleotides 172-195 upstream from
the translation initiation codon was synthesized and end labeled with
[-32P]ATP and T4 polynucleotide kinase. Free
[
-32P]ATP was removed by using mini Quick Spin oligo
columns (Boehringer Mannheim). For primer extension reaction,
105 counts/min of the end-labeled oligonucleotides and 15 µg of total RNA from T84 or C2BBe1 cells were coprecipitated and
dissolved in diethyl pyrocarbonate-treated water and heated at 75°C
for 5 min and then brought to 42°C. The reaction was complemented with 200 µM dNTPs and reaction buffer to a final concentration of 100 mM Tris · HCl (pH 8.3), 150 mM KCl, 6 mM MgCl2, 10 mM dithiothreitol, and 200 U of SuperScript II in a reaction volume of
20 µl. The reaction was carried out for 50 min at 42°C. The
extension products were phenol-chloroform extracted and ethanol
precipitated, and pelleted nucleic acids were dissolved in stop
solution (US Biochemical). The samples were heated at 90°C for 3 min
and analyzed on a 6% polyacrylamide-7 M urea denaturing gel. The gel
was dried and exposed to X-Omat AR film. A sequencing ladder was used
as a size marker to determine the size of the extended primer.
Cloning of 5'-flanking region. The 5'-regulatory region of the NHE2 gene was cloned using the Genome Walker kit (Clontech). PCR amplifications were performed with genomic DNA fragment pools as template, an anchor primer that hybridizes to the 5' end of the genomic fragments, and a gene-specific primer from hNHE2 cDNA. This resulted in amplification of a 1.4-kb DNA fragment. After gel purification, the 1.4-kb DNA fragment was cloned in pGEM-T cloning vector (Promega) and designated pJM1.4N2. Subsequent to similar nested PCR amplifications, a larger fragment (3.3 kb) of the 5'-flanking region of the NHE2 gene was cloned in pGEM-T and named pJM3.3 N2. DNA nucleotide sequences at the 3' end of the 3.3-kb fragment were determined and compared with the 5'-untranslated region (UTR) of the hNHE2 cDNA. This confirmed that the 3.3-kb fragment was the 5'-flanking region of the NHE2 gene.
Reporter plasmid construction.
Plasmids used for transfection were generated using pGL2-Basic
(Promega), which contains a promoterless luciferase reporter gene. To
clone the 1.4-kb DNA fragment in pGL2-Basic, pJM1.4N2 was digested with
Nco I restriction enzyme, which cuts the NHE2 sequence at the translation initiation site, and the ends were filled
in with Klenow (1) and digested again with Sac
I to release the 1.4-kb DNA fragment. After gel purification, this DNA fragment was cloned upstream from the luciferase structural gene in
pGL2-Basic that was cut at the Sac I site and blunt ended at
the Hind III site. This clone was named pJM1.4-N2P.
pJM664-N2P was constructed by digesting the 1.4-kb fragment of the
5'-flanking region with Sma I, which generates a 664-bp
fragment (bases 415 to +249), and subcloning into pGL2-Basic.
Cell culture and transfections.
C2BBe1 cells, a subclone of the Caco-2 cell line, were obtained from
Dr. M. Rao of the Department of Physiology, University of Illinois at
Chicago. The C2BBe1 cells were maintained in collagen-coated culture
dishes in DMEM supplemented with 10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, 10 µg/ml transferrin, and 2 mM glutamine. Transfection experiments were performed in six-well plates on preconfluent cells using Lipofectamine Plus reagent (GIBCO BRL) according to the manufacturer's instructions. The cells were
cotransfected with one of the NHE2 constructs and
pRSV-gal. The latter plasmid served as an internal control for
transfection efficiency. After 48 h, cells were lysed using a kit
from Promega and total cell extracts were harvested. Luciferase
activity was assayed using a Packard liquid scintillation analyzer
(1900 TR) and normalized to
-galactosidase activity.
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RESULTS |
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Genomic organization of hNHE2 gene.
The nucleotide sequences at the exon-intron junctions and the sizes of
introns were determined as described in MATERIALS AND METHODS. On the basis of comparison of the nucleotide sequences at the exon-intron junctions to the hNHE2 cDNA sequence
(29), the genomic structure of the hNHE2 gene
was established. Figure 1 depicts the
genomic organization of the hNHE2 gene. This gene consisted
of 12 exons, which were interrupted by 11 introns. The first exon
contained the 5'-untranslated region (UTR), as well as the
transcription and translation initiation sites. Exon 12 contained the
last 122 amino acids, the stop codon, and the 3'-UTR. Exons 1-5
and a portion of exon 6 code for the NH2-terminal domain of
the polypeptide containing the transmembrane segments, and exons
6-12 encode the COOH-terminal regulatory domain (Fig.
2). The sizes of the introns were
determined by sequencing or PCR amplification of the human genomic DNA
and are presented in Table 2. All introns
were located within the coding region, and all splice junctions conform
to the GT/AG splice donor/acceptor rule (8, 44) (Table 2).
Introns 2, 5, 6, 9, and 10 are in phase 0; introns 1, 4, and 11 are in
phase 1; and introns 3, 7, and 8 are in phase 2 (Table 2). Therefore
only exons 6, 8, and 10 are symmetrical, whereas all other exons are
asymmetrical (39).
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Chromosomal localization.
The chromosomal localization of the NHE2 gene was
determined by FISH using the hNHE2 genomic DNA clone,
pRD190, as a probe. These studies resulted in the specific labeling of
a position immediately adjacent to the centromere on the long arm of
chromosome 2 (Fig. 3A).
The identity of the chromosomes exhibiting a specific signal
was confirmed by cohybridization with N-myc
(43) genomic DNA, which is a chromosome 2-specific probe
(Fig. 3B). The specifically hybridized chromosomes
demonstrated that NHE2 was located in an area that
corresponds to band 2q11.2 (Fig. 3C). To further confirm the
results of the FISH assignment, we analyzed genomic DNA from the
somatic cell hybrid cell line NA11712 by PCR using a forward primer
from exon 2 and a reverse primer from the 5' end of intron 2 (Fig.
3D, lane 1). Control genomic DNA from parental
cells, a human genomic DNA (NAIMR91; Fig. 3D, lane
3), and genomic DNA from mouse A9 (NA00346B; Fig. 3D,
lane 4) were also subjected to PCR amplification using the
same parameters. The expected PCR reaction product of 485 bp was
obtained from the hybrid cell line NA11712, which retains only human
chromosome 2, and from parental human genomic DNA, whereas no specific
amplification product was produced using mouse A9 genomic DNA as a
template. In agreement with our chromosomal mapping studies, Szpirer et
al. (48) had assigned the NHE2 gene to the
human chromosome 2, using a rat NHE2 cDNA as probe.
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Determination of transcription initiation site.
The transcription initiation site of the hNHE2 gene was
determined using total RNA from intestinal epithelial cell lines T84, Caco-2, and C2BBe1. However, before primer extension experiments, the
expression of the NHE isoforms NHE1, NHE2, and NHE3 in these cell lines
was investigated by RT-PCR as shown in Fig.
4. The results of these experiments
indicated that both NHE2 and NHE3 genes were
expressed in C2BBe1 (Fig. 4, lanes 3 and 4) and
Caco-2 cells (Fig. 4, lanes 11 and 12) but not in
the mouse fibroblast NIH/3T3 cell line (Fig. 4, lanes 7 and
8). The NHE1 isoform was expressed in all three cell lines
(Fig. 4, lanes 2, 6, and
10). The T84 cell line was shown to express the
NHE2 isoform by Northern blot analysis (data not shown).
Subsequently, primer extension analysis was performed using an
antisense oligonucleotide corresponding to cDNA sequences at
172-195 bases upstream from the translation start codon and total
RNA from the T84, Caco-2, and C2BBe1 cells. A 143-nucleotide primer
extension product was identified in all three cell lines, whereas no
signal was found in control yeast transfer RNA (Fig.
5). The primer extension product overlaps
with a deoxyadenosine residue 315 nucleotides upstream from the
translation initiation site. The nucleotide pair at the transcription
start site, an A residue preceded by a C, is the most common
dinucleotide pair at the eukaryotic transcription initiation sites
(10, 23). Based on these data, we have designated this
adenosine residue as +1 for the transcription initiation site of the
hNHE2 gene (Fig. 6).
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Cloning and sequence analysis of 5'-flanking region.
Using the genome walking technique, we cloned a 3.3-kb DNA
fragment upstream of the NHE2 translation initiation site. Nucleotide sequence analysis of the 3' end of this clone confirmed that the cloned
DNA fragment overlaps with the 5'-UTR of the hNHE2 cDNA reported previously (29). Subsequent to sequence
determination of 1.4 kb of the 5'-flanking region of the
hNHE2 gene, a databank homology search revealed that this
DNA sequence overlapped with nucleotide sequences of a 139-kb clone of
the human chromosome 2 deposited into GenBank (accession no. AC007239).
The nucleotide sequence of our clone was in complete agreement with
that of AC007239 except for a single discrepancy at position 689
(Fig. 6), where a G replaces a T in the chromosome 2 sequence. This G
residue was present in two independent clones that we sequenced.
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Functional promoter analysis of 5'-flanking region.
To investigate whether the putative promoter region is functional, we
cloned the 1.4-kb DNA fragment 5' of the luciferase reporter
gene in pGL2-Basic (pJM1.4-N2P). This fragment contains 1 kb of the 5'
promoter region and 315 bp of the 5'-UTR. In addition, a Sma
I fragment harboring nucleotides 415 to +249 was also subcloned into pGL2-Basic (pJM664-N2P). The resultant fusion plasmids and the
control vectors, pGL2-Basic, pGL2-Promoter, and PGL2-Control, were used
for transient transfection of the C2BBe1 cells. As shown in Fig.
8, pJM1.4-N2P containing sequences from
1050 to +318 exhibited a moderate promoter activity, which was
threefold of that obtained with the promoterless vector. However,
deletion of sequences upstream from position
415 resulted in higher
luciferase activity in C2BBe1 cells transfected with pJM664-N2P. The
luciferase activity of the NHE2 promoter construct
containing bases
415 to +249 was 13% of that found with
pGL2-Promoter vector, which uses the SV40 promoter and was
significantly lower, over 2-log range, than the pGL2-Control containing
the SV40 promoter and enhancer sequences (data not shown). The moderate
increase in expression of luciferase activity in the construct
containing the bases
415 to +249 of the NHE2 promoter may
be attributed to the loss of an inhibitory element contained within the
deleted DNA region. Several putative response elements for
transcription factors are located within this region (Fig. 6), and the
question of whether any of these regulatory factors is involved in
transcriptional repression of the NHE2 promoter will require
further study.
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DISCUSSION |
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In the present study, we established the complete structure of the hNHE2 isoform, including the transcription initiation site. Using a combination of genomic DNA library screening and PCR amplification techniques, we have determined the position and sizes of all the NHE2 introns. These studies revealed that the hNHE2 gene encompasses a region >81 kb of the genome and consists of 12 exons and 11 introns. The first exon contains the 5'-UTR, as well as the transcription and translation initiation sites. The last exon contains the last 122 amino acids of the polypeptide, the stop codon, and the 3'-UTR.
Similar to hNHE2, the NHE1 isoform is also composed of 12 exons and 11 introns (33), and exon-intron junctions are well conserved between the two genes (Fig. 2). Moreover, with respect to intron splice phasing, the two genes are totally identical (Table 2). In all higher eukaryotes, the intron splice phasing type 0, in which introns are located between two codons, is found more frequently than types 1 and 2, in which introns interrupt the reading frame (19). In both NHE2 and NHE1, by insertion of introns 1, 4, and 11, the coding triplets are interrupted between the first and second nucleotides (type 1) and between the second and third nucleotides by introns 3, 7, and 8 (type 2). On the other hand, there are no interruptions in the coding triplets by insertion of the introns 2, 5, 6, 9, and 10 (type 0). These data indicate that, despite differences in the amino acid sequences and the spatial localization of the NHE2 and NHE1 proteins, the genomic organization of their respective genes is quite similar. Unlike hNHE2 and hNHE1, the rat NHE3 and hNHE5 are organized in 17 exons and 16 introns (24) and 16 exons and 15 introns (2), respectively. These studies, along with further elucidation of the gene structure of the other NHE isoforms, may be useful for an understanding of the path of molecular evolution of the members of the NHE gene family.
A single transcription initiation site was mapped to an adenosine
residue 315 bp upstream from the ATG translation start codon. The
5'-flanking region of the hNHE2 gene lacks a canonical TATA box (8) or CAAT sequence (13) in the expected
proximity of the transcription initiation site. However, the region
spanning from a Sma I restriction enzyme site at 415 to
ATG translation start codon at +316 is composed of 77% G + C
nucleotides. The presence of such a GC-rich sequence within the
5'-flanking region is a feature of TATA-less promoters that usually
contain Sp1 binding sites (40). In fact, two Sp1 binding
sites were identified in close proximity to the transcription start
site (Fig. 6), both of which interact with Sp1 transcription factor
family members (Malakooti and Ramaswamy, unpublished data). Thus the
NHE2 gene promoter belongs to the subclass of TATA-less RNA
polymerase II promoters.
We have also identified several other potential cis-acting regulatory elements in the promoter region. Of particular interest was the identification of the CdxA and Cdx-2 homeodomain protein binding sites. CdxA, a chicken homeobox gene, belongs to the caudal family of vertebrate homeobox genes. The expression of these genes is restricted to the endoderm-derived epithelia during embryognesis (15). Cdx-2 is expressed in the intestinal epithelial cells and has been reported (31) to be involved in differentiation of these cells. The binding site for Cdx-2 was identified in the 5'-regulatory region of several genes that are specifically expressed in enterocytes, such as sucrase isomaltase (46) and lactase-phlorizin hydrolase (49) genes. Because NHE2 mRNA is predominantly found in epithelial cells of the gastrointestinal tract, the Cdx homeodomain family members may be involved in the regulation of the NHE2 gene expression during enterocyte differentiation.
A comparion of hNHE2 and rat NHE2 promoter sequences demonstrated that several transcription factor binding sites were conserved between the two species. A knowledge of the identified conserved elements as well as of those not conserved in the hNHE2 and rat NHE2 promoters may provide the basis for detailed studies leading to the identification of the mechanisms regulating the expression of the NHE2 gene.
Transient transfection studies revealed significant activity of
the NHE2 promoter in C2BBe1 cells. The expression of the
reporter constructs containing different lengths of the NHE2
promoter fragment suggested that an inhibitory element is present
between 1050 to
415 bp from the transcription start site. The
identification of a repressor element in the upstream DNA region
parallels the finding for the rat NHE2 gene
(36), in which a similar decrease in gene expression was
noted on deletion of the upstream promoter sequences.
The mapping of NHE2 to 2q11.2 did not indicate linkage to any known diseases. A defect in NHE function has been implicated in human diseases such as essential hypertension (22) and congenital diarrhea (5). Studies by Lifton et al. (27) on families exhibiting essential hypertension have ruled out the involvement of hNHE1 in this disease. Other NHE gene products that may be involved in these disorders are NHE2 and NHE3, both of which have been suggested to play major roles in transepithelial Na+ absorption. Recently, Schultheis et al. (42) have demonstrated in NHE2 knockout mice that the NHE2 isoform may be required for the parietal cell long-term viability and not for its acid secretion. However, as yet no human disease has been mapped definitely to any of the NHE isoforms.
In summary, the genomic structure and the promoter region of the hNHE2 gene were characterized and mapped to the chromosome 2q11.2. Knowledge of the genomic organization, chromosomal loci, and the promoter region of all the NHE isoforms will facilitate the search for mutations in these genes and identification of their role in human diseases, including essential hypertension, congenital diarrhea, and other diseases of the gastrointestinal tract.
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ACKNOWLEDGEMENTS |
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We thank V. C. Memark for technical help.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33349 and by the Department of Veterans Affairs.
Address for reprint requests and other correspondence: J. Malakooti, Section of Digestive and Liver Diseases, Dept. of Medicine, Univ. of Illinois, 840 S. Wood St., Chicago, IL 60612 (E-mail: malakoot{at}uic.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 17 July 2000; accepted in final form 24 October 2000.
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