1 School of Biological Sciences, University of Manchester, Manchester M13 9PT; and 2 Institute of Genetics, Queen's Medical Centre, Nottingham University, Nottingham NG7 2UH, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The movement of urea across
plasma membranes is modulated by facilitated urea transporter proteins.
These proteins are the products of two closely related genes, termed
UT-A (Slc14a2) and UT-B (Slc14a1). By genomic
library screening and P1 artificial chromosome "shotgun"
sequencing, we have determined the structure of the mouse UT-A gene.
The gene is >300 kb in length, contains 24 exons, and has 2 distinct
promoters. Flanking the 5'-region of the gene is the UT-A promoter
that regulates transcription of UT-A1 and UT-A3. The second promoter,
termed UT-A
, is present in intron 13 and regulates transcription of
UT-A2. cAMP agonists (100 µM dibutryl cAMP, 25 µM forskolin, 0.5 mM
IBMX) increased the activity of a 2.2-kb UT-A
promoter construct
6.2-fold [from 0.026 ± 0.003 to 0.160 ± 0.004, relative
light units (RLU)/µg protein] and a 2.4-kb UT-A
promoter
construct 9.5-fold (from 0.020 ± 0.002 to 0.190 ± 0.043 RLU/µg protein) above that in untreated controls. Interestingly, only
the UT-A
promoter contained consensus sequences for CREs and
deletion of these elements abolished cAMP sensitivity. Increasing the
tonicity of culture medium from 300 to 600 mosmol/kgH2O with NaCl caused a significant
increase (from 0.060 ± 0.004 to 0.095 ± 0.010 RLU/µg
protein) in UT-A
promoter activity but had no effect on the UT-A
promoter. A tonicity-responsive enhancer was identified in UT-A
and
is suggested to be responsible for mediating this effect. Levels of
UT-A2 and UT-A3 mRNA were increased in thirsted mice compared with
control animals, indicating that the activities of both promoters are
likely to be elevated during prolonged antidiuresis.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FACILITATIVE UREA TRANSPORTERS belonging to the UT-A family of proteins regulate the movement of urea across biological membranes (24, 28). In rats and humans, these proteins are the product of a single gene (Slc14a2) (1, 18). Seven cDNAs encoding four urea transporters, UT-A1, UT-A2, UT-A3, and UT-A4, have been characterized in rat kidney (2, 28), and a cDNA encoding a novel isoform, UT-A5, has been isolated from mouse testes (8).
UT-A proteins expressed in the kidney play a critical role in generating the hypertonic medulla, which is an integral component of the urinary concentrating mechanism. In rat, thirsting increases levels of UT-A2 and UT-A3 mRNA (2, 27), and parallel changes have been observed in UT-A2 protein (32). The increase in UT-A2 mRNA is mediated in part by arginine vasopressin (AVP), because Brattleboro rats, which lack endogenous AVP, do not show this response. Only when AVP or 1-desamino-8-D-arginine vasopressin is administered is the increase in UT-A2 mRNA then observed (21, 26). These changes are possibly mediated via vasopressin V2 receptors coupled to cAMP (21). A similar mechanism may also be responsible for the increase in UT-A3 mRNA, although this remains to be determined.
We have previously mapped the mouse UT-A gene to chromosome 18 and shown that it is arranged in tandem with the UT-B gene (Slc14a1) (7). To understand how UT-A mRNAs are differentially regulated, in particular during water restriction, and as a prerequisite for groups wishing to perform mouse knockout studies, we determined the structure of the mouse UT-A gene and have conducted studies to characterize its promoters.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of the mouse UT-A gene.
cDNAs encoding UT-A1 (GenBank accession no. AF366052) and UT-A2
(GenBank accession no. AF367359) were isolated from a mouse kidney
inner medulla (IM) cDNA library and sequenced as previously described
(Fenton RA, Stewart GS, Carpenter B, Howorth A, Cooper GJ, and Smith
CP, unpublished observations). A FIXII/129sv mouse genomic library
(Stratagene) and a P1 artificial chromosome (PAC) genomic library,
obtained from the Human Genome Mapping Project (HGMP Resource Centre),
were screened using the following 32P-labeled mouse UT-A
cDNA probes: probe 1, nucleotides 1-222 of mouse UT-A3
(GenBank accession no. AF258602); probe 2, nucleotides 1-176 of mouse UT-A5 (GenBank accession no. AF258601); probe 3, nucleotides 1,745-1,950 of mouse UT-A3; probe
4, nucleotides 1-799 of mouse UT-A2; and probe 5,
nucleotides 3,378-3,956 of mouse UT-A1. Filters were incubated
overnight at 48°C in hybridization solution containing 7% SDS, 0.5 M
NaHPO4 (pH 7.2), 1 mM EDTA, and 50 mM BSA, supplemented
with denatured salmon sperm DNA (10 µg/ml hybridization solution).
Final washes were in 0.1× standard sodium citrate, 0.1% SDS at
65°C.
Phage clones were further analyzed by restriction digest
and Southern blotting. Four
genomic clones, MUT1 (16 kb), MUT2
(15.1 kb), MUT4 (4.5 kb), and MUT9 (15.2 kb), were identified on the
basis of their binding to the panel of cDNA probes. These were digested
with SalI, XbaI, and SacI,
respectively, subcloned into pBluescript SK(
), and
sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Ready
Reaction kit (PE Applied Biosystems).
Promoter-reporter plasmid construction.
To analyze promoter activity, promoter constructs were engineered into
the pGL3 Basic reporter vector (Promega). A 2,239-bp fragment of the
UT-A promoter (UT-A
-2.2) was generated from MUT9 by PCR using
Pfu polymerase (Stratagene) and the primer pair 5'-GACTCACTATAGGGCGTCG and 5'-XhoI-CAGCCTAGAAGACAAGTGG. A
2,438-bp (UT-A
-2.4) fragment of the UT-A
promoter was generated
from MUT1 by PCR using Pfu polymerase and primer pair
5'-XhoI-CTTCCCGTAGCTACTACCC and
5'-BglII-GTATATGACCTAACCGGTG. Primers
5'-XhoI-CGTGTCCTGCTTTCTAAGG, 5'-XhoI-CTGAGAGCCTAGACAGCCA, and
5'-XhoI-GTCAGAAGGAGAGTTTGTC, each in combination with
primer 5'-BglII-GTATATGACCTAACCGGTG, were used to generate,
respectively, the following truncated UT-A
promoter constructs: 857 bp (UT-A
-0.8), 533 bp (UT-A
-0.5), and 295 bp (UT-A
-0.3). All
products were directionally cloned into pGL3 Basic and sequenced.
Cell culture, transfection, and reporter gene assay.
Madin-Darby canine kidney (MDCK) cells were grown at 37°C/5%
CO2 in MEM (GIBCO BRL) supplemented with 10% fetal bovine
serum (GIBCO BRL), 0.5% penicillin/streptomycin (Sigma), 1%
L-glutamine (200 mM; GIBCO BRL), and 1% nonessential amino
acids (Sigma). Twenty-four hours before transfection, cells were seeded
in 24-well plates at a density of 8 × 104 cells/ml.
Cells (~60% confluent) were transfected using Transfast reagent
(Promega) with equimolar amounts of UT-A plasmid constructs (5 pmol)
and, as a control for transfection efficiency, 5 ng of pRLB19
(11). pRLB19 was a kind gift from Dr. Arlyn Garcia Perez, National Heart, Lung, and Blood Institute, National Institutes of
Health. Transfections were performed according to the manufacturer's recommended protocol. MEM was removed 24 h after transfection, the
cells were washed twice with prewarmed PBS, and 1 ml of fresh media was
added. At this point, in experiments employing hypertonic media, cells
were exposed to medium made hypertonic (600 mosmol/kgH2O) by the addition of NaCl. In experiments employing cAMP, cells were
subjected to media supplemented with dibutryl cAMP (100 µM), forskolin (25 µM), and IBMX (0.5 mM). After a further 24 h,
cells were washed with PBS, 100 µl of passive lysis buffer (Promega) was added to each well, and plates were shaken on an orbital shaker for
30 min at room temperature. The resulting cell lysate was centrifuged
at 10,000 g for 1 min and analyzed for total protein using a
protein assay kit with -globulin as a standard (Bio-Rad). Firefly
and Renilla luciferase activities, in relative light units (RLU), were determined using the Dual Luciferase Reporter Assay System
(Promega) and an EG&G Berthold Microlumat 96P luminometer.
Fluid-restriction protocol. Adult male MF1 mice were used in this study and had unrestricted access to standard 18% protein mouse chow (Special Diet Services). Control mice received water ad libitum, and thirsted mice had no access to water for 20 h. Mice were killed by cervical dislocation, and blood and urine were immediately collected, respectively, by cardiac puncture or by puncturing the urinary bladder. Kidney IMs were removed and snap-frozen in liquid N2. The osmolality of serum and urine samples was determined using a Roebling microosmometer. Serum and urine urea nitrogen concentrations were determined using the Sigma Diagnostics BUN (end point) reagent kit according to the manufacturer's protocol.
Northern blot analysis. Total RNA was isolated from individual kidney IMs by the guanidine isothyocyanate method followed by ultracentrifugation as previously described (27). Total RNA (8 µg/lane) was separated in a 1% agarose gel in the presence of 2.2 M formaldehyde and transferred to Hybond-N filters (Amersham Pharmacia Biotech). Filters were probed, using a 32P-labeled full-length mouse UT-A1 cDNA. Hybridization was for 16 h at 42°C (50% formamide) and washing at 65°C in 0.1× standard sodium citrate, 0.1% SDS. To control for equal loading of RNA, membranes were subsequently probed with a 32P-labeled mouse glyceraldehyde-3-phosphate dehydrogenase cDNA probe. Autoradiographs were analyzed by densitometry (LAS-100 camera using AIDA software, FujiFilm) to quantify the relative abundance of different mRNA transcripts under different hydration states.
Statistical analysis. For experiments involving two groups, the unpaired Student's t-test was used. For experiments involving three or more groups, one-way ANOVA combined with the Bonferroni post hoc test was used. Groups were deemed statistically significant if P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Structure of the UT-A gene.
The UT-A gene consists of 24 exons, ranging in size from 50 (exons 12 and 23) to 965 bp (exon 24),
distributed over ~300 kb of DNA (Fig.
1B). All exon/intron
boundaries contained a 5'-donor gt and a 3'-acceptor
ag (Table 1). The first five
exons of the UT-A gene are present in the transcripts UT-A1 and
UT-A31; the first three form
the 5'-untranslated region (UTR) of these transcripts. The putative
translational start site for these transcripts is located in exon
4 (sequence caaATG). Exon 6 forms the unique 5'-UTR of
the UT-A5 transcript. This is one of three unique exons not present in
the largest transcript, UT-A1. Exon 7 contains the translation start
site of UT-A5 (sequence gtgATG). Exons 8-12 are common
to UT-A1, UT-A3, and UT-A5.
|
|
UT-A promoters.
Multiple transcriptional start sites for the UT-A1 and UT-A3
transcripts were identified (Fenton RA, Stewart GS, Carpenter B,
Howorth A, Cooper GJ, and Smith CP, unpublished observations). Sequence
analysis of the 5' sequence flanking exon 1, termed UT-A, (GenBank accession no. AF367361) revealed that it resembled a TATA-less
promoter, in that it lacked a TATA-box consensus sequence. However, it
did contain three CCAAT boxes, three Sp1 transcription factor binding sites, and a consensus site for a transcriptional initiator protein [Inr; (C/T)(C/T)A(T/A)(C/T)(C/T)] (Fig.
2A). Also present were
consensus sequences for other well-characterized transcription factors
(see Fig. 2A), including two glucocorticoid response
elements (GRE) and, interestingly, a tonicity-responsive enhancer/osmotic response element (TonE/ORE) (10, 23, 29). Of note is the absence of any consensus sequences for cAMP responsive elements (CRE) or CRE binding proteins (3).
|
Functional analysis of the UT-A promoters.
The activities of the UT-A and UT-A
promoters were measured using
MDCK cells transiently transfected with different promoter constructs.
Constructs were tested for both basal and stimulated luciferase
activity. The 2.2-kb UT-A
construct (UT-A
-2.2) induced basal
promoter activity [0.026 ± 0.003 (SD) RLU/µg protein,
n = 3] 5.2-fold greater than that with pGL3 Basic
without an insert (Fig. 3B).
The 2.4-kb UT-A
(UT-A
-2.4) construct induced basal promoter
activity (0.020 ± 0.002 RLU/µg protein) 4.0-fold greater than
that with pGL3 Basic without an insert. Interestingly, the smaller
UT-A
constructs induced higher basal promoter activity than did the
largest construct (Fig. 3B), suggesting possible negative
regulation by suppresser or silencer elements (15).
|
|
Effect of fluid restriction on UT-A mRNA expression.
To determine whether changes in UT-A promoter activity conferred
changes to UT-A mRNA levels in vivo, we compared UT-A mRNA levels in
kidney IMs from control mice and mice deprived of water for 20 h.
Urine osmolalities were significantly higher for fluid-restricted animals (3,480 ± 355 mosmol/kgH2O) compared with
control mice (1,973 ± 560 mosmol/kgH2O). Northern
blot analysis (Fig. 5) of mouse kidney IM
showed that the levels of the UT-A2 (3.1-kb) and UT-A3 (2.1-kb)
transcripts increased, compared with non-fluid-restricted controls
(P < 0.05) in response to fluid deprivation
(summarized in Fig. 6). In contrast,
neither UT-A1 mRNA (4.1-kb) nor glyceraldehyde-3-phosphate dehydrogenase mRNA levels were different between experimental groups.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By screening a FIXII/129sv genomic library, shotgun sequencing
of a 180-kb PAC clone, and primer walking of PAC clone DNA, we have
determined the structure of the mouse UT-A gene. The gene spans ~300
kb of chromosome 18 (7) and includes 24 exons.
Transcription is driven by two promoters, one proximal to exon
1 (termed UT-A
), driving transcription of UT-A1 and UT-A3, and
an internal promoter (termed UT-A
), flanked by exons 14 and 15, driving transcription of UT-A2.
UT-A1, the largest transcript derived from the UT-A gene, comprises
exons 1-5, 7-12, part of exon 13, and
exons 15-24. Exon 13 is intriguing because it is a
split exon. The first 175 bp are present in the mature UT-A1 transcript
as a coding sequence, whereas the complete exon is present in UT-A3 and
UT-A5 and codes for the terminal amino acids and the 3'-UTR containing
the polyadenylation consensus. Given its dual role, this exon is likely
to influence the transcription of UT-A1 because the efficiency of an
RNA splicing event is determined by a number of elements often present
within an exon or the flanking intron (16). These include
purine-rich splicing enhancers, which are usually located within exons,
or pyrimidine-rich enhancers that reside within introns
(16). In one class of purine-rich splicing enhancers, the
nucleotide sequence of the 5'-splice site [AGGT(A/G)AGT]
influences its splicing efficiency (4, 5). After bp 175 of
exon 13, there is a consensus GT that acts as an
intronic donor site for splicing of UT-A1. The flanking sequence of
this site (TGGTGACT) does not exactly match the consensus.
Base substitutions in flanking nucleotides, such as those apparent in
exon 13, are known to weaken the influence this consensus
motif has on splicing frequency. Thus, given the potential weakness of
this site, we suggest that UT-A3 may be the default transcript
resulting from activation of the UT-A promoter. Clearly, this
remains speculative but provides an intriguing avenue for future research.
Overall comparison of the structure of the mouse UT-A gene with that of the rat (18) revealed that the structures of the two genes are very similar; the rat UT-A gene extends for greater than 300 kb and contains 24 exons. However, the transcript encoding UT-A5 has not been identified in the rat, although a candidate 1.7-kb transcript has been reported in rat testes (14). This suggests that at least one further exon remains to be identified in the rat gene. Similarly, the transcripts UT-A1b, UT-A2b, and UT-A3b have not been identified in the mouse, although the information to test for their existence is now available in the form of genomic sequence data we have generated.
Analysis of the two UT-A gene promoters revealed that UT-A is a
TATA-less promoter and drives the transcription of UT-A1, UT-A3, and,
possibly, UT-A4 from several start sites (Fenton RA, Stewart GS,
Carpenter B, Howorth A, Cooper GJ, and Smith CP, unpublished observations). In contrast, the UT-A
promoter conforms to a classic TATA-box-containing promoter (13), and transcription
of UT-A2 is initiated from a single locus (Fenton RA, Stewart GS,
Carpenter B, Howorth A, Cooper GJ, and Smith CP, unpublished
observations). The known mouse UT-A transcripts are therefore formed as
a result of alternative pre-mRNA splicing, utilization of a split exon (exon 13) in combination with the use of an alternate polyadenylation signal, and differential promoter activity.
The arrangement of the mouse UT-A and UT-A
promoters is
comparable to the two equivalent promoter regions identified in the rat
(18, 19). As we have shown in the mouse, the rat UT-A
promoter is also a TATA-less promoter and contains three CCAAT boxes,
two GREs, and a single TonE/ORE (19). Furthermore, the rat
UT-A
promoter, like its mouse counterpart, is a typical
TATA-containing promoter and contains four CREs, compared with five
found in mouse, and a single GRE (18).
Functional analysis of the two murine promoters using luciferase
reporter constructs and reporter gene assays showed that both promoters
initiate gene transcription and have a low basal level of promoter
activity when transiently transfected into MDCK cells. We also found
that the activity of both promoters was significantly enhanced by cAMP.
Truncations of the mouse UT-A promoter indicated that activation by
cAMP was mediated by CREs. The CRE at
2,207 (see Fig. 3) was found to
have the greatest affect on promoter activity. In the rat, four CRE
consensus sequences have been identified in the UT-A
promoter, and
mutagenesis studies have confirmed that these CREs are responsible for
mediating the response of the rat UT-A
promoter to cAMP
(18). In agreement with these studies, our results
strongly suggest that CRE consensus sequences in the UT-A
promoter
are responsible for regulating the activity of UT-A2 expression in
response to cAMP.
Intriguingly, we observed that the activity of the UT-A promoter,
containing no CREs, was increased in response to cAMP. In addition to
acting directly through CREs, cAMP can also increase transcription by
interacting with other regulatory sequences such as activator protein
(AP)-1 (6), AP-2 (20), Sp1
(31), and inverted CCAAT motifs (22). The
UT-A
promoter contains three AP-1 motifs, three Sp1
motifs, and four inverted CCAAT motifs; therefore, it is possible that
UT-A
promoter activity increases in response to cAMP because of an
interaction between cAMP and one or more of these consensus motifs. In
the rat, a 1.3-kb UT-A
promoter construct showed no increase in
transcriptional activity after stimulation by cAMP (18).
This indicates that the mouse and rat UT-A
promoters have different
responses to cAMP, or, alternatively, that the element responsible for
cAMP stimulation in the rat UT-A
promoter is upstream of the
sequence contained in the reporter construct analyzed in studies by
Nakayama et al. (18).
The activity of the UT-A promoter, containing a consensus TonE/ORE
regulatory sequence (12, 23), was significantly increased in response to hypertonicity, whereas increased tonicity had no effect
on the activity of the UT-A
promoter that lacks a TonE/ORE motif.
These findings agree with those reported by Nakayama and colleagues
(19) for the rat UT-A promoters. Increasing tonicity, by
the addition of NaCl, caused an increase in UT-A
, but not UT-A
,
promoter activity. As we discovered in the mouse, the rat UT-A
promoter has a TonE/ORE motif, whereas the UT-A
promoter does not.
Mutation of the rat TonE consensus abolished the response to
hypertonicity, and gel-shift assays revealed that the TonE binding
protein (17) binds to the rat UT-A
promoter
(19). In light of these findings, we conclude that the
mouse UT-A
promoter is responsive to changes in tonicity and suggest
that this effect is mediated by a TonE consensus sequence.
Our analysis in the mouse and that of others in the rat (18,
19) suggested that both UT-A promoters are capable of responding to stimuli associated with antidiuresis, i.e., increased intracellular cAMP, mediated by vasopressin binding to the V2 vasopressin
receptor, and elevated tonicity in the kidney medullary interstitium,
caused by the action of the countercurrent multiplier. Therefore, we determined whether predicted increased activity of the UT-A promoters induced by fluid restriction led to commensurate changes in UT-A mRNA
levels in vivo. Our analysis showed that mouse UT-A2 and UT-A3 mRNA
levels increased after 20 h of fluid restriction. From this, we
concluded that stimulation of the UT-A promoters by cAMP and of the
UT-A promoter by increased medullary tonicity leads to an increase
in UT-A2 and UT-A3 mRNA.
In summary, we have determined the genomic structure of the mouse
UT-A gene and shown that activity of both promoters is increased by
cAMP, whereas only UT-A promoter activity is increased by hypertonicity. We suggest that the increased levels of UT-A2 and UT-A3 mRNA, caused by fluid restriction, are a direct result of increases in UT-A
and UT-A
promoter activity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Ged Brady and Joan Ferraris for helpful comments.
![]() |
FOOTNOTES |
---|
This work was supported by Biotechnology and Biological Sciences Research Council Grant 34/D10935, National Environment Research Council Grant GR3/10585, Wellcome Trust Grant 043322/Z/94, and the Royal Society (C. P. Smith).
Address for reprint requests and other correspondence: C. P. Smith, School of Biological Sciences, Univ. of Manchester, G.38, Stopford Bldg., Oxford Rd., Manchester M13 9PT, UK (E-mail: cpsmith{at}man.ac.uk).
1 Mouse UT-A4 has not been isolated. Given the similarity of the other UT-A isoforms between mouse and rat, mouse UT-A4 is presumed to consist of the same relative exons as rat UT-A4 (14).
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.
10.1152/ajprenal.00264.2001
Received 20 August 2001; accepted in final form 30 October 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bagnasco, SM,
Peng T,
Janech MG,
Karakashian A,
and
Sands JM.
Cloning and characterization of the human urea transporter UT-A1 and mapping of the human Slc14a2 gene.
Am J Physiol Renal Physiol
281:
F400-F406,
2001
2.
Bagnasco, SM,
Peng T,
Nakayama Y,
and
Sands JM.
Differential expression of individual UT-A urea transporter isoforms in rat kidney.
J Am Soc Nephrol
11:
1980-1986,
2000
3.
Benbrook, DM,
and
Jones NC.
Different binding specificities and transactivation of variant CRE's by CREB complexes.
Nucleic Acids Res
22:
1463-1469,
1994[Abstract].
4.
Dirksen, WP,
Hampson RK,
Sun Q,
and
Rottman FM.
A purine-rich exon sequence enhances alternative splicing of bovine growth hormone pre-mRNA.
J Biol Chem
269:
6431-6436,
1994
5.
Dirksen, WP,
Sun Q,
and
Rottman FM.
Multiple splicing signals control alternative intron retention of bovine growth hormone pre-mRNA.
J Biol Chem
270:
5346-5352,
1995
6.
Englaro, W,
Rezzonico R,
Durand-Clement M,
Lallemand D,
Ortonne JP,
and
Ballotti R.
Mitogen-activated protein kinase pathway and AP-1 are activated during cAMP-induced melanogenesis in B-16 melanoma cells.
J Biol Chem
270:
24315-24320,
1995
7.
Fenton, RA,
Hewitt JE,
Howorth A,
Cottingham CA,
and
Smith CP.
The murine urea transporter genes Slc14a1 and Slc14a2 occur in tandem on chromosome 18.
Cytogenet Cell Genet
87:
95-106,
1999[ISI][Medline].
8.
Fenton, RA,
Howorth A,
Cooper GJ,
Meccariello R,
Morris ID,
and
Smith CP.
Molecular characterization of a novel UT-A urea transporter isoform (UT-A5) in testis.
Am J Physiol Cell Physiol
279:
C1425-C1431,
2000
10.
Ferraris, JD,
Williams CK,
Jung KY,
Bedford JJ,
Burg MB,
and
Garcia-Perez A.
ORE, a eukaryotic minimal essential osmotic response element. The aldose reductase gene in hyperosmotic stress.
J Biol Chem
271:
18318-18321,
1996
11.
Ferraris, JD,
Williams CK,
Martin BM,
Burg MB,
and
Garcia-Perez A.
Cloning, genomic organization, and osmotic response of the aldose reductase gene.
Proc Natl Acad Sci USA
91:
10742-10746,
1994
12.
Ferraris, JD,
Williams CK,
Ohtaka A,
and
Garcia-Perez A.
Functional consensus for mammalian osmotic response elements.
Am J Physiol Cell Physiol
276:
C667-C673,
1999
13.
Gill, G.
Transcriptional initiation. Taking the initiative.
Curr Biol
4:
374-376,
1994[ISI][Medline].
14.
Karakashian, A,
Timmer RT,
Klein JD,
Gunn RB,
Sands JM,
and
Bagnasco SM.
Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4.
J Am Soc Nephrol
10:
230-237,
1999
15.
Laimins, L,
Holmgren-Konig M,
and
Khoury G.
Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus.
Proc Natl Acad Sci USA
83:
3151-3155,
1986[Abstract].
16.
Lopez, AJ.
Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation.
Annu Rev Genet
32:
279-305,
1998[ISI][Medline].
17.
Miyakawa, H,
Woo SK,
Dahl SC,
Handler JS,
and
Kwon HM.
Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity.
Proc Natl Acad Sci USA
96:
2538-2542,
1999
18.
Nakayama, Y,
Naruse M,
Karakashian A,
Peng T,
Sands JM,
and
Bagnasco SM.
Cloning of the rat Slc14a2 gene and genomic organization of the UT-A urea transporter.
Biochim Biophys Acta
1518:
19-26,
2001[ISI][Medline].
19.
Nakayama, Y,
Peng T,
Sands JM,
and
Bagnasco SM.
The TonE/TonEBP pathway mediates tonicity-responsive regulation of UT-A urea transporter expression.
J Biol Chem
275:
38275-38280,
2000
20.
Park, K,
and
Kim KH.
The site of cAMP action in the insulin induction of gene expression of acetyl-CoA carboxylase is AP-2.
J Biol Chem
268:
17811-17819,
1993
21.
Promeneur, D,
Bankir L,
Hu MC,
and
Trinh-Trang-Tan MM.
Renal tubular and vascular urea transporters: influence of antidiuretic hormone on messenger RNA expression in Brattleboro rats.
J Am Soc Nephrol
9:
1359-1366,
1998[Abstract].
22.
Rangan, VS,
Oskouian B,
and
Smith S.
Identification of an inverted CCAAT box motif in the fatty-acid synthase gene as an essential element for modification of transcriptional regulation by cAMP.
J Biol Chem
271:
2307-2312,
1996
23.
Rim, JS,
Atta MG,
Dahl SC,
Berry GT,
Handler JS,
and
Kwon HM.
Transcription of the sodium/myo-inositol cotransporter gene is regulated by multiple tonicity-responsive enhancers spread over 50 kilobase pairs in the 5'-flanking region.
J Biol Chem
273:
20615-20621,
1998
24.
Sands, JM,
Timmer RT,
and
Gunn RB.
Urea transporters in kidney and erythrocytes.
Am J Physiol Renal Physiol
273:
F321-F339,
1997
25.
Scherf, M,
Klingenhoff A,
and
Werner T.
Highly specific localization of promoter regions in large genomic sequences by PromoterInspector: a novel context analysis approach.
J Mol Biol
297:
599-606,
2000[ISI][Medline].
26.
Shayakul, C,
Smith CP,
Mackenzie HS,
Lee WS,
Brown D,
and
Hediger MA.
Long-term regulation of urea transporter expression by vasopressin in Brattleboro rats.
Am J Physiol Renal Physiol
278:
F620-F627,
2000
27.
Smith, CP,
Lee WS,
Martial S,
Knepper MA,
You G,
Sands JM,
and
Hediger MA.
Cloning and regulation of expression of the rat kidney urea transporter (rUT2).
J Clin Invest
96:
1556-1563,
1995[ISI][Medline].
28.
Smith, CP,
and
Rousselet G.
Urea transporters.
J Membr Biol
183:
1-14,
2001[ISI][Medline].
29.
Takenaka, M,
Preston AS,
Kwon HM,
and
Handler JS.
The tonicity-sensitive element that mediates increased transcription of the betaine transporter gene in response to hypertonic stress.
J Biol Chem
269:
29379-29381,
1994
30.
Tatusova, TA,
and
Madden TL.
BLAST 2 sequences, a new tool for comparing protein and nucleotide sequences.
FEMS Microbiol Lett
174:
247-250,
1999[ISI][Medline].
31.
Venepally, P,
and
Waterman MR.
Two Sp1-binding sites mediate cAMP-induced transcription of the bovine CYP11A gene through the protein kinase A signaling pathway.
J Biol Chem
270:
25402-25410,
1995
32.
Wade, JB,
Lee AJ,
Liu J,
Ecelbarger CA,
Mitchell C,
Bradford AD,
Terris J,
Kim GH,
and
Knepper MA.
UT-A2: a 55-kDa urea transporter in thin descending limb whose abundance is regulated by vasopressin.
Am J Physiol Renal Physiol
278:
F52-F62,
2000