(Hypertension. 1999;33:290-297.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Genes Encoding Atrial and Brain Natriuretic Peptides as Candidates for Sensitivity to Brain Ischemia in Stroke-Prone Hypertensive Rats
From the Department of Medicine and Therapeutics and Wellcome Surgical Institute, University of Glasgow (Scotland) (M.J.B., J.S.C., B.J., C.D.N., S.M.A., H.C., I.M.M., A.F.D.); Molecular Medicine Group, MRC Clinical Sciences Centre, London, England (T.Y.A.); and the Department of Molecular Biology, Universite Libre de Bruxelles (Belgium) (P.V.V., C.S.).
Correspondence to Prof Anna F. Dominiczak, Department of Medicine and Therapeutics, Western Infirmary, 44 Church St, Glasgow G11 6NT. E-mail anna.dominiczak{at}clinmed.gla.ac.uk
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Abstract |
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Abstract—Previous studies suggested that atrial natriuretic peptide gene (Anp) and brain natriuretic peptide gene (Bnp) are plausible candidate genes for susceptibility to stroke and for sensitivity to brain ischemia in the stroke-prone spontaneously hypertensive rat (SHRSP). We performed structural and functional analyses of these 2 genes in SHRSP from Glasgow colonies (SHRSPGla) and Wistar-Kyoto rats from Glasgow colonies (WKYGla) and developed a radiation hybrid map of the relevant region of rat chromosome 5. Sequencing of the coding regions of the Anp and Bnp genes revealed no difference between the 2 strains. Expression studies in brain tissue showed no differences at baseline and at 24 hours after middle cerebral artery occlusion. Plasma concentrations of atrial natriuretic peptide (ANP) did not differ between the SHRSPGla and WKYGla, whereas concentrations of brain natriuretic peptide were significantly higher in the SHRSPGla as compared with the WKYGla (n=11 to 14; 163±21 pg/mL and 78±14 pg/mL; 95% confidence interval 31 to 138, P=0.003). We did not detect any attenuation of endothelium-dependent relaxations to bradykinin or ANP in middle cerebral arteries from the SHRSPGla; indeed the sensitivity to ANP was significantly increased in arteries harvested from this strain (WKYGla: n=8; pD2=7.3±0.2 and SHRSPGla: n=8; pD2=8.2±0.15; P<0.01). Moreover, radiation hybrid mapping and fluorescence in situ hybridization allowed us to map the Anf marker in the telomeric position of rat chromosome 5 in close proximity to D5Rat48, D5Rat47, D5Mgh15, and D5Mgh16. These results exclude Anp and Bnp as candidate genes for the sensitivity to brain ischemia and pave the way to further congenic and physical mapping strategies.
Key Words: rats, inbred SHR • peptides • genes • genetics • hybridization
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Introduction |
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The stroke-prone spontaneously hypertensive rat (SHRSP) is a good model for studying genetic determinants of hypertension and stroke. It is characterized by a high frequency of spontaneous strokes1 as well as an increased sensitivity to experimentally induced focal cerebral ischemia.2 3 4 We and others have studied this model extensively to establish the mode of inheritance and the quantitative trait loci (QTLs) for the susceptibility to stroke5 and for the sensitivity to cerebral ischemic insult.4 6 Rubattu et al5 performed a genome-wide screen in an F2 cross obtained by breeding SHRSP rats from Heidelberg colonies (SHRSPHdl) and spontaneously hypertensive rats from Heidelberg colonies (SHRHdl), thus eliminating blood pressure as a confounding variable. In their study, latency to stroke on a Japanese diet was used as a phenotype. The study identified 3 QTLs for susceptibility to stroke that were localized to rat chromosomes 1, 4, and 5. It is of interest that the QTLs on chromosomes 4 and 5 conferred a protective effect against stroke in the presence of 2 SHRSP alleles.5 Moreover, the QTL on chromosome 5 colocalized with the genes encoding atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP).5 Thus these 2 genes have become candidates for the susceptibility to stroke in the SHRSP.
Jeffs et al6 used experimentally induced middle cerebral artery (MCA) occlusion to identify the genetic component responsible for large infarct volumes in the SHRSP from Glasgow colonies (SHRSPGla). In this study, a genome scan was performed in an F2 cross-derived from the SHRSPGla and the normotensive reference strain, Wistar-Kyoto rats from Glasgow colonies (WKYGla). A highly significant QTL on rat chromosome 5 was identified.6 This QTL was again blood pressure independent and was localized in the region of chromosome 5 close to that identified by Rubattu et al.5 However, in the study by Jeffs et al,6 homozygosity for the SHRSPGla alleles for the markers within the QTL was associated with larger infarct volumes than homozygosity for the WKYGla alleles.
The natriuretic peptide family consists of 3 peptides: ANP, BNP, and C-type natriuretic peptide.7 The genes encoding ANP and BNP colocalize in human, mouse, and rat on chromosomes 1, 4, and 5, respectively.8 These 2 peptides are extensively distributed in the cardiovascular system, and their predominant effects are to produce natriuresis and vasodilatation.7 Previous studies indicated that SHR and SHRSP had elevated plasma ANP concentrations as well as alterations in the number of ANP-binding sites in the kidney and brain.9 10 It has therefore become important to ascertain whether the genes encoding ANP and BNP are likely candidates for sensitivity to brain ischemia. To achieve this aim, we performed sequencing of the coding regions of the Anp and Bnp genes and assessed the mRNA expression of both genes before and after the permanent MCA occlusion in the SHRSPGla and WKYGla. Moreover, we measured circulating levels of both peptides in the 2 strains and performed functional analysis of the MCA relaxations to ANP with the use of pressure myography. In view of discrepancies between our genetic map and other genetic maps of rat chromosome 5,5 6 we used radiation hybrid mapping and fluorescence in situ hybridization (FISH) to obtain the physical map of the chromosomal region of interest.
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Methods |
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Experimental Animals
Rats were obtained from the Glasgow colonies of SHRSP and WKY. The full history of these colonies and the breeding paradigms have been described previously.11 12 13 At 12 weeks of age, systolic blood pressure and heart rate were measured by tail-cuff plethysmography in conscious, restrained rats as previously described.12 Mean systolic blood pressures (mm Hg) were: SHRSPGla males (n=16), 182±18; SHRSPGla females (n=9), 159±12; WKYGla males (n=8), 124±11; and WKYGla females (n=8), 126±12.
MCA Occlusion
Permanent MCA occlusion was performed as we have previously
described.6 Twenty-four hours after ischemia, the
animal was anesthetized, decapitated, and the whole brain
frozen rapidly in liquid nitrogen.
Gene Expression Studies
RNA was extracted from the whole brain with the use of RNAzol B.
Total RNA (10 µg) was electrophoresed on a 1.2% agarose gel and
transferred to a nylon membrane. Filters were sequentially probed with
Anp-, Bnp-, and Gapdh-labeled
fragments that had been generated in-house with the use of polymerase
chain reaction (PCR). Blots were exposed to film and the
autoradiograms analyzed with a densitometer
(Bio-Rad). The signals from Anp and Bnp were
expressed relative to the intensity of the Gapdh signal.
Sequencing of Coding Regions of Anp and
Bnp Genes
DNA was extracted from tail biopsies of 4 male SHRSP and WKY
rats.14 The exonic regions of Anp and
Bnp were amplified by PCR with the use of intronic specific
primers shown in Table 1
, designed from
sequences lodged in GenBank (K02062 and M60266). The 750-bp amplicon
containing exons 1+2 of Anp was obtained with primers 1A and
5A. Exon 3 was generated with 7A and 8A. The first 2 exons of
Bnp were generated with 1B and 3B; the third exon used 7B
and 8B. The amplicons were electrophoresed on either 1.5% or 2%
agarose gels, and the bands were excised from the gels and
electroeluted. DNA was quantified with the use of the DynaQuant
fluorometer (Hoefer) and used for cycle sequencing reactions. One
microliter of 32-P end-labeled primer was added to the cycle sequencing
mix consisting of 20 µL of water, 4 µL of reaction mix, 2 µL of
template, and 1 µL of DMSO. For each sequencing reaction, 2 µL of
terminator G, A, T, and C were added to separate tubes. Six microliters
of the cycle sequencing mix was added to the terminator tube. The cycle
sequencing reaction was performed on an MJ Thermal Cycler (MJ Research)
with a program of 94°C for 4 minutes followed by 30 cycles of 94°C
for 1 minute, 1 minute at the annealing temperature of the primer,
followed by 1 minute of 72°C. At the end of the reaction, stop
solution was added and samples resolved on a 6% polyacrylamide
gel. The gel was dried onto a Whatman filter paper and placed against
film for 4 to 8 hours.
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Radiation Hybrid Mapping
Rat whole genome radiation hybrids were obtained from Research
Genetics, Inc (Huntsville, Ala). To create the panel, a rat donor cell
line (Rat FR, a diploid fibroblast cell line derived from skin biopsy
of a fetal SD rat) was exposed to 3000 rad of x-rays and then fused
with nonirradiated thymidine kinase–deficient hamster recipient cells
(A23). The panel consists of 106 clones and has an average locus
retention rate of 28% to 30%. The presence or absence of each of 41
microsatellite markers was determined by PCR with rat primers purchased
from either Research Genetics or Genosys Biotechnology (Cambridge, UK).
In addition, 4 other control samples underwent PCR: FR DNA, A23 DNA,
WKY rat DNA, and a water blank. Six microsatellite markers gave
inconsistent amplification and were excluded from the data
analysis. The PCR amplification of the microsatellites was
carried out with an MJ Thermal Cycler; 25 ng of radiation hybrid DNA
was amplified in 20 µL of 1x reaction buffer (Promega) containing
25 µmol/L each dATP, dCTP, dGTP, dTTP, 0.25 µmol/L of
each primer, and 0.4 U of Taq polymerase (Promega). The PCR
program consisted of 4 minutes at 94°C and 35 cycles of 30 seconds at
94°C, 40 seconds at Tannealing (Table 3), and 10 seconds at 72°C. The PCR products were
separated by electrophoresis on a 3% agarose gel containing ethidium
bromide and visualized with a FluorS-Multimager (Biorad, UK). The PCR
screening of the radiation hybrid panel was done in duplicate and
scored by 2 independent observers.
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The radiation hybrid mapping programs of the RHMAP package, version 3.0
(http://www.sph.umich.edu/group/statgen/software) were used to
analyze the data. These programs assume that breakage is at
random along the chromosome, with constant intensity and no
interference.15 16 17 The RH2PT program was used to perform
a 2-point analysis. A set of best orders with the fewest
obligate chromosome breaks was defined with the use of a stepwise locus
ordering strategy with the RHMINBRK program. Because of to the high
number of markers, we constructed 5 linkage groups on the basis of the
2-point analysis and the RHMINBRK best order. The orientation
of these linkage groups was determined by reference to the genetic map.
The order within each linkage group was determined with the multipoint
maximum likelihood method with the RHMAXLIK program and a branch and
bound strategy.15 These orders were then checked with a
simulated annealing strategy with the use of a random initial locus
order. Map distance estimates, D, were calculated with the mapping
function, D=-ln (1-
), where is the breakage probability
estimate between 2 markers. D was expressed in centiRays (cR), where a
distance of 1 cR3000 corresponds to a 1%
probability of breakage between 2 markers after exposure to 3000 rads
of x-rays. The maximum likelihood method was used to calculate map
distances of individual markers within linkage groups, and the 2-point
analysis was used to calculate map distances between the
linkage groups.
Fluorescence In Situ Hybridization
FISH was done as described previously,18 19 with
cultured vascular smooth muscle cells harvested from the
SHRSPGla and
WKYGla.20 The chromosome images were
captured and treated with the ISIS imaging system (MetaSystems, D-68804
Althussheim, Germany). Only double spots (2 labeled sister chromatids)
formed by the probes were taken into account, and 2 methods were used
to determine the regional position of the signals. First, the
fractional length distance of the fluorescent signal to the
centromere relative to the total chromosome arm length was used to map
the genes, with banded rat chromosomes as references.21
Second, the position of the fluorescent spots was superimposed
on the image of DAPI-banded chromosomes, with the use of the ISIS
system. The Anf probe was a 5.2 kb KpnI fragment
from the rat gene (JA200).22 The Dsi1
probe (Genbank accession number L26461) was the 1.8 kb
SmaI-EcoRI fragment named
dsI-SR.23 The primer sequences of the markers
D5Mgh16, D5Mit2, D5Mit9, and D5Wox15 were obtained from
http://ratmap.gen.gu.se/and used to identify the P1 clones from the
chromosome 5 fraction of a rat genomic library (Genomic Systems, St
Louis, Mo). These 4 P1 clones were then used to localize the
chromosomal position of these markers with the use of FISH.
Perfusion Myography
MCAs were mounted on a pressure-perfusion myograph (Living
Systems) as previously described.24 The artery was imaged
with a video camera, and the internal diameter was measured with a
Video Dimension Analyzer (Living Systems), which was linked to
a chart recorder (Linseis L6512B, Belmont Instruments).
Each segment was pressurized at half the systolic blood pressure of each rat. After a 60-minute equilibration period at 37°C in oxygenated physiological salt solution, the tone was raised with 5-hydroxytryptamine (5-HT; 3x10-7 mol/L), and once a stable contraction was reached, a cumulative concentration response curve to ANP (10-11 to 3x10-7 mol/L) was performed. After a washout period, the same protocol was used for bradykinin (BK; 10-10 to 3x10-6 mol/L) and sodium nitroprusside (SNP; 10-9 to 3x10-5 mol/L). Contraction to 5H-T was calculated as a percentage decrease of the internal diameter (D) and relaxations to ANP, BK, and SNP as a percentage of previous contraction to 5-HT.
Measurements of Plasma Levels of ANP and BNP
Rat ANP was measured in plasma by radioimmunoassay, after prior
extraction on C18 Sep-Pak columns, with a modification of the method of
Richards et al.25 The antibody cross-reacted 100% with
rat ANP. Rat BNP was measured with a radioimmunoassay kit (RIK 9103,
Peninsula Labs) again after prior extraction.
Statistical Analysis
Results are expressed as mean±SEM, and n denotes the number of
animals used in each experiment. Unpaired Student's t test
was used for comparisons between 2 groups, and P<0.05 was
considered significant. The statistical analysis of the
radiation hybrid mapping is described above.
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Results |
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Sequencing Analysis of Coding Region of Anp and Bnp Genes
Sequencing the coding region of the Anp and Bnp genes revealed no difference between the SHRSPGla and the WKYGla strains. Table 2
shows 4 substitutions (2
silent) identified within the coding region of the Anp gene
of the SHRSPGla as compared with the SD sequence
but no differences between SHRSPGla and
WKYGla. The comparison between the Rattus
norvegicus sequence of the Bnp gene (GenBank accession
number M60266) and those sequences in the 2 Glasgow strains revealed no
differences.
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Expression Studies
Representative Northern blots from
SHRSPGla and WKYGla brain
tissue and the quantitative data are shown in Figure 1. We found no detectable Anp
mRNA expression and low levels of the Bnp mRNA expression at
baseline. At 24 hours after the MCA occlusion, mRNA expression was
upregulated for Anp and Bnp, but there were no
significant differences between the 2 strains.
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Perfusion Myography
Contraction to 5-HT 3x10-7 mol/L was
similar in MCA from WKYGla and
SHRSPGla rats (WKY=42.2±4.9% decrease and
SHRSP=40.0±6.2% decrease of the internal diameter).
Concentration-response curves to ANP, BK, and SNP are shown in Figure 2. Relaxation to ANP was similar in
WKYGla and SHRSPGla MCA,
with a maximum relaxation of almost 100%. However,
pD2 values (negative logarithm of the
concentration required to produce half-maximal response) were
significantly different between the 2 strains
(WKYGla pD2=7.3±0.2 and
SHRSPGla pD2=8.2±0.15;
P<0.01). Relaxations to BK and SNP were similar in
WKYGla and SHRSPGla
arteries, and there was no difference between strains in the
pD2 values for BK and SNP.
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Plasma Levels of ANP and BNP
There was no significant difference in plasma ANP concentrations
between SHRSPGla (n=14; 630±45 pg/mL) and
WKYGla (n=11; 539±50 pg/mL) (95% confidence
interval -50 to 231, P=0.19). Plasma concentration of BNP
was significantly greater in SHRSPGla (n=14;
163±21 pg/mL) compared with WKYGla (n=11; 78±14
pg/mL) (95% confidence interval 31 to 138, P=0.003).
Analysis of Radiation Hybrid Panel
The presence or absence of each of the 35 rat microsatellite
markers in 106 radiation hybrid clones was determined by PCR screening
with primers shown in Table 3
. The mean
retention frequency was 28% (range 16% to 35%); (Table 3).
First, a 2-point analysis was used with the program RH2PT, with
a lod score threshold of 8, followed by a stepwise locus ordering
strategy with the RHMINBRK program. These analyses have allowed
us to divide the 35 microsatellite markers into 5 linkage groups, with
the orientation of these groups determined from the most comprehensive
rat genetic map currently available (Reference 26 and
Figure 3). The order within each linkage
group was then determined with a branch and bound
strategy15 within the multipoint maximum likelihood method
(RHMAXLIK program). The best order together with map distances
calculated as described in "Methods" are shown in Table 4. A comparison with an integrated
genetic map of the same region of rat chromosome 5 is shown in Figure 3. The 5 linkage groups covered a total distance of 1304
cR3000, corresponding to a genetic distance of 78
centimorgans (cM). The maximum likelihood analysis allows for
the determination of the likelihood ratios, which are defined as the
ratios of the overall maximum likelihood of the comprehensive map to
the maximum likelihood of a given local order (Table 4).
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Fluorescence In Situ Hybridization
The 4 microsatellite markers D5Mit9, D5Mit2, D5Wox15, and D5Mgh16,
as well as the Anp gene and the Dsi1 locus, were
unambiguously localized by FISH with either
SHRSPGla or WKYGla cells
(identical results were obtained with the 2 cell types). D5 Mit9 maps
at 5q24, D5 Mit2 maps somewhat more distally, namely in the 5q24-q31
interval, D5Wox15, and Dsi1 colocalize at 5q36.2, whereas
D5Mgh16 and Anf colocalize at the end of the chromosome, at
5q36.3 (Figure 4). These localizations
thus establish several anchor points between the cytogenic map and the
genetic linkage map and confirm the telomeric localization of the
Anp gene as demonstrated by the radiation hybrid
mapping.
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Discussion |
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We analyzed Anp and Bnp as possible candidate genes for the increased sensitivity to a focal brain ischemic insult in the SHRSPGla. This analysis has been performed at several levels, starting from sequencing of the coding regions of both genes, through the assessment of the mRNA expression of Anp and Bnp, before and after the MCA occlusion, to measurements of the circulating concentrations of both peptides and the functional responses of the MCAs to ANP ex vivo.
Sequencing of the coding regions of both genes revealed no difference
between SHRSPGla and the
WKYGla strains. Furthermore, we compared these
sequences with the rat sequences entered into the Genbank. There were
no sequence differences for the Bnp gene but there were 4
substitutions (2 silent) within the coding region of the Anp
gene. One of the substitutions is located within the exon 2 of the gene
and has been previously reported to be present in the
SHRSPHdl by Rubattu et al27 in
an abstract. This G
A substitution in position 1125 results in serine
instead of glycine. Rubattu et al27 suggested that this
substitution together with differential mRNA expression of
Anp between SHRHdl and
SHRSPHdl were consistent with a putative
role for Anp in the pathogenesis of cerebrovascular disease
in the SHRSP. This is not confirmed by the current study in which
coding sequences of both Anp and Bnp genes were
identical and the brain mRNA expression did not differ between the
SHRSPGla and the WKYGla.
The discrepancies between the current study and data presented
in the abstract by Rubattu et al27 could be best
explained by the different nature of the phenotype studied. The
major phenotype used in the current study and in our previous
genetic analyses4 6 is the volume of infarction
after permanent occlusion of the MCA, whereas Rubattu et
al5 27 used the latency to spontaneous stroke (both
ischemic and hemorrhagic) on a high salt diet. These 2
phenotypes are hardly comparable and thus the candidate genes
for each of the phenotypes might be different despite the 2
QTLs being localized on rat chromosome 5. It is also likely that there
is some genetic heterogeneity between the
SHRSPGla and the SHRSPHdl,
as documented by the results of previous studies.12 13
These uncertainties can be resolved by the construction of the
appropriate congenic lines and sublines to narrow down the chromosomal
region of interest. The alternative strategies include search for
recombinants, which may lead to the reduction of the limits of the QTL
and the homology mapping, which uses syntenic regions between rat,
mouse, and human genome as illustrated by Julier et
al.28
Moreover, we showed no differences in circulating concentrations of ANP, but there was a significant increase in BNP concentration in the SHRSPGla. Previous studies suggested altered metabolism, release, and function of the ANP in the SHR and the SHRSP.29 30 31 Concentrations of BNP have been shown previously to correlate well with the degree of left ventricular hypertrophy in human hypertension.7 It is likely that the difference in circulating levels of BNP seen in the current study is a reflection of a very significant left ventricular hypertrophy that we have previously documented in the SHRSPGla.32
Previous studies by Volpe et al33 and Russo et al34 examined vasorelaxant responses in aortas and basilar and carotid arteries of the SHRSPHdl as compared with the SHRHdl. The first study showed reduced endothelium-dependent relaxations to acetylcholine and substance P in the SHRSP, but responses to the natriuretic peptides were not tested.33 The second study examined vasorelaxations to ANP and BNP and found them to be attenuated in the SHRSPHdl as compared with the SHRHdl and the WKYHdl.34 The above experiments used mostly large vessels, and it is noteworthy that high salt, low potassium, and low protein (Japanese style) rat chow was given for at least 4 weeks before experimentation. The current study used the MCA and perfusion myography to examine this small cerebral vessel, which is central to the pathogenesis of brain ischemia in our model. We demonstrated no differences in vasorelaxation to SNP (endothelium independent) and to BK (endothelium dependent). Moreover, maximum relaxation to ANP was similar in the SHRSPGla and the WKYGla. There was, however, a small but significant difference in the sensitivity to ANP, with the arteries from the SHRSPGla displaying a greater sensitivity to the peptide. Previous extensive studies on large-vessel endothelial function in the SHRSPGla demonstrated significantly reduced nitric oxide availability at the level of vascular endothelium, and this relative nitric oxide deficiency seemed almost entirely accounted for by the excessive production of the superoxide anion by the endothelial cells.35 36 It is therefore possible that endothelial function might differ between vascular beds. However, major differences between the 2 colonies of the SHRSP might also be a factor. The last important consideration is the requirement for the Japanese style diet to obtain abnormalities in vascular relaxations. Previous studies showed that salt-induced hypertension in rats is associated with endothelial dysfunction and that this is reversed by antihypertensive treatment.37 Therefore a major contribution of the Japanese style diet to endothelial dysfunction cannot be excluded.
In view of the largely negative results of the sequencing, mRNA expression, and the functional studies, as well as the significant disagreement between the genetic map constructed in our F2 experiment6 and other genetic maps of the same region,5 26 we decided to perform physical mapping of the relevant region of rat chromosome 5. First, radiation hybrid mapping was used, and we were able to map successfully 35 microsatellite markers covering 78 cM of the chromosome. The Anf microsatellite marker was mapped between D5Rat48 and D5Rat47, which corresponds to the telomeric end of the chromosome. Furthermore, we performed FISH on cells isolated from the SHRSPGla and WKYGla and again mapped the Anf marker at 5q 36.3, which corresponds to the telomeric end of the chromosome. Therefore, 2 physical mapping methods have given identical results and are in agreement with the genetic map published by other groups.5 26 The difference between our genetic map6 and the physical mapping described in the current study remains unexplained but stresses the importance and the superior resolution of the physical mapping methods, which provide a firm base for the future positional cloning strategies.
In summary, we performed structural and functional analysis of the Anp and Bnp genes as possible candidates for sensitivity to brain ischemia in the SHRSPGla. The results largely exclude these 2 genes as putative candidates causally related to the phenotype under investigation. Moreover, we developed a detailed physical map of the relevant region of rat chromosome 5 and confirmed a telomeric position of the Anf marker.
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Acknowledgments |
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This work was supported by the British Heart Foundation (PG96175, PG97077, RG97009), The Wellcome Trust (44670/95), The Cunningham Trust and the Robertson Trust, EURHYPGEN II Concerted Action for the BIOMED 2 and BIOTECH Program of the European Community, and the Fund for Scientific Medical Research (Belgium). We thank J.J. Morton for performing ANP and BNP radioimmunoassay, A. Glazier for introduction to radiation hybrid techniques, and M. Nemer and D. Steffen for the gift of the rat Anf and Dsi1 probes for FISH.
Received September 17, 1998; first decision October 12, 1998; accepted October 23, 1998.
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