(Hypertension. 1995;26:998-1002.)
© 1995 American Heart Association, Inc.
Articles |
Cloning, Characterization, and Genetic Mapping of the Rat Type 2 Angiotensin II Receptor Gene
From the Cardiovascular Research Center, Massachusetts General Hospital–East (G.K., E.S.W., D.M.B., H.J.J.), Charlestown; the Falk Cardiovascular Research Center, Stanford University School of Medicine (M.H., VJ.D.), Stanford, Calif; and the Département de Biologie Moléculaire, Université Libre de Bruxelles (C.S.), Rhode-St-Genèse, Belgium.
Correspondence to Howard J. Jacob, PhD, Cardiovascular Research Center, Mail Code 1494201, Massachusetts General Hospital–East, 149 13th St, Charlestown, MA 02129-2060. E-mail jacob@helix.mgh.harvard.edu.
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Abstract |
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 Top Abstract IntroductionMethodsResults and DiscussionReferences |
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Abstract The renin-angiotensin system plays an important role in blood pressure homeostasis, but the contribution of the type 2 angiotensin II receptor (AT2R) is still unclear. The reports that the AT2R gene has been mapped to the X chromosome in human and rat and the previous report of a gene, Bp3, on the X chromosome responsible for an increase in blood pressure have suggested that the rat AT2R gene (Agtr2) could be this gene. To elucidate whether Agtr2 is Bp3, Agtr2 was cloned. A simple sequence repeat in the 3'-flanking region of this gene was identified and used as a genetic marker to map Agtr2 to the X chromosome at 18.1 cM distal to the androgen receptor locus. This map position is outside the confidence interval reported for Bp3, demonstrating that Agtr2 cannot be Bp3. However, these data will enhance the research into the AT2R biology as well as the study of the X chromosome.
Key Words: receptor, angiotensin • rats • cloning, molecular
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Introduction |
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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Angiotensin II (Ang II), one of the major regulators for cardiovascular homeostasis, exerts a wide range of actions on the kidney, heart, blood vessel, adrenal gland, and central nervous system in physiological and pathophysiological states as well as during development. Multiple lines of evidence have suggested the existence of Ang II receptor subtypes. Based on their differential pharmacological and biochemical properties, two distinct Ang II receptor subtypes have been defined and designated as type 1 receptors (AT1Rs) and type 2 receptor (AT2R).1 2 Most of the well-characterized actions of Ang II are mediated by the AT1Rs. In contrast, much less has been known about the AT2R.
Recently, cDNAs and genes for the mouse3 4 5 and human AT2R6 7 8 9 have been cloned and characterized. These data have revealed the structure of the receptor and the gene. However, for the rat AT2R, only the cDNA has been reported.10 11 Functionally, the AT2R exhibits an antigrowth effect in the rat endothelial cell12 and vascular smooth muscle cell,13 and contributes to the pressure natriuresis in the rat.14 However, the contribution of the AT2R to blood pressure regulation is still unclear.
Genetic mapping of the cross between the stroke-prone spontaneously hypertensive rat (SHRSP) and the Wistar-Kyoto rat (WKY) has identified a gene responsible for hypertension, Bp3, on the rat X chromosome.15 Since Agtr2 was assigned (but not mapped) to the X chromosome in the rat,6 we hypothesized that Agtr2 was a candidate for Bp3.
Addressing our hypothesis in this study, we cloned Agtr2 and developed a genetic marker for Agtr2. We also characterized this gene in detail to reveal the gene organization and the 5'-flanking region for the study of the regulatory mechanism of gene expression. We found that Agtr2 cannot be Bp3 but determined that identification of the sequence of the 5'-flanking region (promoter region) will enhance the investigation into the role of this receptor.
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Methods |
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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Screening of Genomic Library
A rat genomic library (Stratagene Cloning Systems; independent plaques, 2.0x106), prepared by partial Sau3AI digests of genomic DNAs isolated from Fischer male rat liver and cloned into the BamHI site of Lambda DASH, was screened. The HindIII/Kpn I fragment of the cDNA for the rat AT2R10 was used as a probe. This probe was labeled with [
-32P]dCTP (3000 Ci/mmol,
Amersham Corp) with the Prime-a-Gene labeling system (Promega Corp)
and purified with a NICK column (Pharmacia LKB Biotechnology). The
genomic library was screened by plaque hybridization16
with the use of Colony/Plaque Screen (Du Pont–New England Nuclear).
Hybridization was carried out at 65°C in Church's hybridization
solution17 with a radiolabeled probe as described above.
Filters were washed at 60°C in 2x SSC (1x SSC is 150 mmol/L NaCl
and 15 mmol/L sodium citrate) and 0.1% sodium dodecyl sulfate.
Positive clones were isolated by repeated phage purification.
DNA Sequencing
Double-stranded DNA prepared with the use of QIAspin kits
(Qiagen Inc) was sequenced with ABI 373A DNA sequencer (Applied
Biosystems) with the use of the manufacturer's Taq cycle
sequencing protocol. The genomic DNA clones were sequenced in both
directions.
Primer Extension Assay
The primer (5'-CTTAAAATGCAGGCTGAAGAAAGC- TTTCAATTCTGT-3') was
labeled with [
-32P]ATP (6000 Ci/mmol, Amersham Corp)
with the use of T4 kinase (Life Technologies) and annealed with 100
µg RNA in 40 µL hybridization buffer (80% formamide, 12 mmol/L
Tris-HCl [pH 7.0], and 0.56 mmol/L NaCl). Total RNA was extracted
from 16- to 18-day-old whole Sprague-Dawley rat fetuses
with the use of RNAzol (Tel-Test, Inc). After denaturation at 70°C
for 10 minutes, samples were incubated at 42°C for 16 hours,
precipitated with ethanol, and dissolved in 50 µL of 50 mmol/L
Tris-HCl (pH 8.1), 40 mmol/L KCl, 5 mmol/L MgCl2, 2
mmol/L dithiothreitol, and 0.2 mmol/L of dATP, dGTP, dTTP, and dCTP.
After addition of 2.5 U avian myeloblastosis virus reverse
transcriptase, samples were incubated at 42°C for 2 hours, alkaline
denatured with 50 µL of 0.4 mol/L NaOH at 42°C for 90 minutes, and
neutralized with 100 µL of 0.3 mol/L sodium acetate and 0.1 mol/L
Tris-HCl (pH 7.5). Finally, samples were precipitated with ethanol and
run on polyacrylamide (8%)/urea (7 mol/L) gels followed by
autoradiography. The sizes of extended products
were measured with the use of both a radiolabeled
HaeIII-digested 
X174 DNA and a sequence reaction
ladder.
Generation of the Genetic Marker for
Agtr2
A DNA sequence containing a simple sequence repeat was
analyzed with the computer program PRIMER (Lincoln
et al, unpublished results, 1991) to select polymerase chain reaction
(PCR) primers flanking the repeat that would generate a product of
100 to 300 bp, with predicted melting temperature in the range of
57°C to 63°C and no predicted potential for significant secondary
structure or primer-dimer formation.
We identified an ATT trinucleotide repeat in the 3'-flanking region of Agtr2 (2190 bp from the stop codon of the Agtr2 coding region) and designed PCR primers (forward primer, 5'-AACTCCAGTTCCAGGGGACT-3'; reverse primer, 5'-TGGAAGCAGAAGCCAGTTTT-3') flanking this (ATT)n repeat, with the expected amplified product size of 230 bp and the predicted melting temperature of 60°C. This marker was characterized for 12 different rat strains as previously described18 (Table 1).
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Genetic Mapping of Agtr2
To map Agtr2, the progeny of F2
intercross between the spontaneously hypertensive rat (SHR) and Brown
Norway rat (BN) was genotyped as previously
described.18 These SHR and BN were from Dr Michal Pravenec
in the Czech Republic: SHR/Cz and BN/Lx, respectively. After
genotyping, linkage analysis was performed using the
MAPMAKER computer package19 with the same
criteria as previously described.18 To confirm the
chromosomal location, the chromosomal assignment study was performed by
PCR amplification of the rat/mouse somatic cell hybrid panel as
described previously with minor modification.20 Primers of
mouse D-100 (D8 MIT16-F&R) (Research Genetics) were used for internal
control of PCR amplification.
Additional Genetic Markers on the X Chromosome
Previously, 1171 genetic markers were generated and 432 markers
mapped in the progeny of the F2 intercross between SHR/Cz
and BN/Lx; however, only 11 genetic markers were mapped to the X
chromosome.18 Here we add two more genetic markers,
DXMgh6 and DXMgh8, to the X chromosome as
described previously.18 PCR primers for these markers are
as follows: DXMgh6 forward primer,
5'-GTGAAGCCACTTAACCCCAA-3'; DXMgh6 reverse primer,
5'-AAAAGATGCCAACTGTTGCC-3'; DXMgh8 forward primer,
5'-AATCCCTGCTGAATGCAATT-3'; DXMgh8 reverse primer,
5'-ATGTGCAAACATTTGAACATCC-3'. The allele sizes for these markers
were determined for 12 different rat strains as described
previously18 (Table 1).
Nomenclature
Rat strains, genes, and genetic markers were named
in accordance with the rat nomenclature committee
recommendations.18 21
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Results and Discussion |
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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Cloning and Characterization of Agtr2
We screened a genomic library of the Fischer male rat liver (1.8x106 plaques) with the HindIII/Kpn I fragment of the cDNA for the rat AT2R10 as a probe, and three independent positive clones (
J1-3) were obtained. Restriction enzyme mapping
revealed that these three clones overlapped each other (data not
shown). The region shown in Fig 1 was subcloned into the
pBluescript II SK(+) vector (Stratagene Cloning Systems) and further
analyzed. We sequenced part of this region in both directions
(Fig 2). As shown in Figs 1 and 2,
sequence comparison between genomic DNA and cDNA reveals that
Agtr2 contains three exons. The coding region in the third
exon is not interrupted by any intron, as is often found in genes for
seven-transmembrane receptors. The structure of Agtr2 is
identical to that of the mouse AT2R gene4 5
and the human AT2R gene.6 7 9 All
exon-intron junction sequences follow the GT/AG
rule.22
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Characterization of 5'-Flanking Region of
Agtr2
To further characterize the 5'-flanking region of
Agtr2, we carried out primer extension analysis
using rat fetus (18-day-old) RNA because AT2R is highly
expressed in the rat fetus.23 A primer was designed based
on the 3'-end sequence of exon 1. As shown in Fig 3,
the primer extension product was observed at a unique site (Fig 3; position +1). At 28 bp upstream of this
transcriptional initiation site, a TATA-box motif (TATAA) is
observed.22 This transcriptional initiation site is
consistent with general consensus of the eukaryotic
transcriptional initiation site. In contrast with a single initiation
site in rat, multiple transcriptional initiation sites of the mouse
AT2R gene were observed,5 24 suggesting that
transcriptional regulation of the AT2R gene might be
different between rat and mouse.
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Mapping of Agtr2
Previously, we assigned Agtr2 to the rat X chromosome
using a rat/mouse somatic hybrid panel,6 but the precise
location of this gene had not been demonstrated. Since Hilbert et
al15 reported that a gene on the X chromosome,
Bp3, was responsible for the majority of variance in blood
pressure in female progeny of an F2 intercross between
SHRSP and WKY, we set out to determine whether Agtr2 could
be a candidate for Bp3. Agtr2 would be a
candidate if it mapped in the same interval as Bp3. We
genotyped 39 female progeny from an SHR/CzxBN/Lx
F2 intercross. As shown in Fig 4, the marker
for Agtr2, named DXMgh7, was located 18.1 cM
distal to the androgen receptor locus on the X chromosome. We also
added two new anonymous genetic markers, DXMgh6 and
DXMgh8, to the rat X chromosome (Fig 4).
Previously, Bp3 was mapped between DXMgh5
(Pfkfb1) and DXMit4 (
15 cM from
DXMgh5).15 Therefore, our linkage map (Fig 4) shows that Agtr2 cannot be
Bp3.
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By use of fluorescence in situ hybridization (FISH), the regional localization of Agtr2 has recently been determined at Xq34,25 and the rat 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase gene encoding the liver and muscle isozyme (Pfkfb1) was previously assigned to Xq22-q31.26 By integration of these FISH data and our linkage map data, the orientation for the rat X chromosome linkage map is as follows: centromere-DXMit1, DXMgh3-DXMgh5(Pfkfb1, q22-q31)-DXMgh7 (Agtr2, q34)-telomere.
Characterization of DXMgh7
Although Agtr2 is not Bp3 in SHRSP,
Agtr2 could play a role in blood pressure regulation in
another hypertensive model. Therefore, we characterized several
hypertensive and normotensive inbred rats. As summarized in Table 2, DXMgh7, the genetic marker for
Agtr2, is informative in several existing rat genetic
crosses. In a cross derived from SHR/Nih (SHR from the National
Institutes of Health) and BN/Jk (BN from Dr José E. Krieger in
Brazil), blood pressure did not cosegregate with DXMgh7
(data not shown). However, this marker will be useful for other
investigators studying the genetic basis of blood pressure in other
crosses.
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Here we report the cloning, characterization, and genetic mapping of Agtr2. An (ATT)n repeat was identified in the 3'-flanking region of the Agtr2. Using PCR primers flanking this repeat as a genetic marker for the Agtr2, we mapped the Agtr2 to the X chromosome. Genetic mapping data for Agtr2 revealed that Agtr2 is not Bp3.
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Acknowledgments |
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This work was supported in part by grants from the National Institutes of Health (NIH), National Institute of Diabetes and Digestive and Kidney Diseases, and Bristol-Myers Squibb (Dr Jacob); NIH grants HL-46631, HL-35252, HL-35610, HL-48638, and HL-07708; a grant from the American Heart Association Bugher Foundation Center for Molecular Biology in the Cardiovascular System; a grant from Ciba-Geigy, Basel, Switzerland (Dr Dzau); and grants from the Fund for Scientific Medical Research (Belgium) and the Belgian program on interuniversity attraction poles initiated by the Belgian State Prime Minister's Office (Dr Szpirer). Dr Szpirer is a Research Director of the National Fund for Scientific Research, Belgium. Nucleotide sequence data from this article has been deposited in the GenBank database under accession number U22663.
Received July 6, 1995; first decision July 24, 1995; accepted August 16, 1995.
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References |
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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