Analyses of the
CYP11B
gene family in the guinea pig suggest
the existence of a primordial
CYP11B
gene with aldosterone synthase
activity
Hannes E. Bu¨ low
1,
* and Rita Bernhardt
2
1
Max-Delbru
¨
ck-Centrum fu
¨
r Molekulare Medizin, Berlin-Buch, Germany;
2
Universita
¨
t des Saarlandes, FR Biochemie, Saarbru
¨
cken,
Germany
In this study we describe the isolation of three genes of the
CYP11B family of the guinea pig. CYP11B1 codes for the
previously described11b-hydroxylase[Bu
¨
low, H.E., Mo
¨
bius,

ase; phylogeny.
Higher vertebrates regulate vital processes like volume/
electrolyte homeostasis and glucose/lipid metabolism by
means of steroid hormones, namely mineralocorticoids and
glucocorticoids. The biosynthesis of these steroids occurs
primarily in the adrenal cortex within morphologically and
functionally distinct zones. Accordingly, mineralocorticoids
are produced by the outer zona glomerulosa while gluco-
corticoids are formed in the two inner layers of the cortex,
the zonae fasciculata/reticularis. Originating from cholester-
ol they are synthesized by a number of consecutive
oxidations and dehydrogenations where all oxidative reac-
tions are catalysed by enzymes of the cytochrome P450
superfamily [2]. The first and rate-limiting step is the
conversion of cholesterol to pregnenolone by the mitochon-
drial cytochrome P450 side-chain cleavage enzyme (P450
scc
,
CYP11A1). Subsequently, pregnenolone is dehydroge-
nated and oxidized in position 17 and/or 21 to yield
11-deoxycortisol or 11-deoxycorticosterone, respectively.
Both compounds in turn are substrates for the cytochrome
P450 enzymes of the CYP11B subfamiliy, namely the
11b-hydroxylase (CYP11B1) and the aldosterone synthase
(CYP11B2). While CYP11B1 hydroxylates 11-deoxycorti-
sol in position 11 to give cortisol as the major glucocorti-
coid, the closely related aldosterone synthase forms
aldosterone as the major mineralocorticoid by means of
an 11b-hydroxylation and an 18-hydroxylation/oxidation of
11-deoxycorticosterone. Thus, the proteins of the CYP11B

Note: a website is available at
/>(Received 12 April 2002, revised 11 June 2002, accepted 26 June 2002)
Eur. J. Biochem. 269, 3838–3846 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03076.x
which factors convey this specificity and how these similar
but distinct systems evolved.
To investigate the zone-specific synthesis of mineralocor-
ticoids and glucocorticoids and the evolution of the
hormonal system in more detail we chose the guinea pig
as a model. The guinea pig is an interesting species because
its taxonomical position remains controversial [12,13].
These features should provide new insight into the evolution
and function of the hormonal system. We first cloned the
genes of the CYP11B family of the guinea pig. The 11b-
hydroxylase of the guinea pig showed higher substrate
specificity than the aldosterone synthase. In addition, the
aldosterone synthase exhibited unique properties in that
18-hydroxylase activity was strongly dependent on the
presence of high levels of reducing equivalents whereas basic
levels were sufficient for high 11b-hydroxylase activity of
this enzyme. This suggests a new regulatory level in
aldosterone synthesis that together with the higher substrate
specificity of the 11b-hydroxylase could be crucial for the
tissue-specific synthesis of steroid hormones. Phylogenetic
analyses indicate a gene duplication event of a bipotent
CYP11B ancestor gene before the mammalian radiation
with subsequent distinct evolution in different clades. This
indicates that a differential glucocorticoid and mineralocor-
ticoid synthesis is an exclusive property of mammals.
EXPERIMENTAL PROCEDURES
General procedures

RNAse protection analyses were carried out using a
HybSpeed
TM
RPA Kit (Ambion) according to the
manufacturer’s recommendations. Briefly, specific
32
P
labelled RNA antisense transcripts (corresponding to
nucleotides 1491–1700 in the CYP11B1 cDNA [1] and
nucleotides 1511–1750 in the CYP11B2 cDNA; Fig. 2) were
hybridized with total RNA from different tissues. After
digestion of the reaction mixture with RNAse A/H
protected fragments were separated by PAGE and visual-
ized by autoradiography.
RACE
The cDNA for CYP11B2 of the guinea pig was amplified
and cloned using a MarathonÒ cDNA Amplification Kit
(Clontech) following the supplier’s recommendations. In
brief, after reverse transcription of 1 lg of polyA
+
RNA
and second-strand synthesis an adapter comprising the T7
promoter sequence combined with a NotIandaSmaIsite
was ligated to both ends of the cDNA pool. Using a
combination of a primer complementary to the adapter
(adapter primer: 5¢-CCATCCTAATACGACTCACTA
TAGGGC-3¢) and a gene-specific sense primer (5¢-GCCG
CTCGAGTTTGAGTTAGCCAGAAACTCC-3¢, XhoI
site underlined) or antisense primer (5¢-ATAC
GGGCCC

the DNA was redissolved in Tris/EDTA, pH 8.0 and sep-
arated on a 1 · Tris/borate/EDTA, 0.9% agarose gel. After
capillary transfer to Hybond
TM
nylon membranes (Amer-
sham) nucleic acids were UV cross-linked (0.24 JÆcm
)2
).
Prehybridization was performed in 5 · NaCl/Cit, 5 · Den-
hardt’s, 0.5% SDS and 50 lgÆmL
)1
sonicated salmon sperm
DNA for 2 h at 65 °C. [a-
32
P]dCTP labelled DNA probes
( 1 · 10
6
c.p.m.ÆmL
)1
) were hybridized in the same solu-
tion for 16 h. For low stringency hybridization the blot was
washed twice at room temperature in 2 · NaCl/Cit, 0.1%
SDS for 10 min followed by two 30 min washes at 50 °Cin
1 · NaCl/Cit, 0.1% SDS. Autoradiography was carried out
with Hyperfilm
TM
MP (Amersham).
Ó FEBS 2002 Guinea pig CYP11B genes (Eur. J. Biochem. 269) 3839
Construction of expression plasmids
For the construction of pCMV/11B2, pRc/CMV was

Phylogenetic analyses
Phylogenetic analyses were conducted using the
PHYLIP
package (Version 3.5c, 1993) [17].
The sequences have been submitted to GenBank under
the accession numbers AF191278, AF191279 (for
CYP11B1), AF191281, AF191280 (for CYP11B2), and
AF191282 (for CYP11B3).
RESULTS
In a previous study we isolated an 11b-hydroxylase of the
guinea pig [1] by screening an adrenal cDNA library with a
PCR amplified orthologous probe. Upon expression, the
isolated cDNA turned out to be a pure 11b-hydroxylase
with no detectable 18-hydroxylation activity suggesting the
existence of additional isoenzymes of the CYP11B subfam-
ily in the guinea pig. To investigate this notion, a Southern
blot was performed utilizing an exon-1-specific probe of
CYP11B1 under low stringency conditions and digesting the
genomic DNA with various restriction endonucleases that
did not cut within exon 1. The result (Fig. 1) strongly
suggested the existence of at least three different genes as
judged from the appearance of three bands if the DNA was,
e.g. digested with EcoRI/EcoRV, XbaI, or XbaI/HindIII.
Although guinea pigs had been sodium depleted to
stimulate the expression of a putative aldosterone synthase
as much as possible [18], repeated screening of the cDNA
library did not result in the identification of any cDNA
other than CYP11B1 (data not shown). Thus, we devised
another strategy for the identification of additional genes of
the CYP11B subfamily in the guinea pig. To this end, a

calculated molecular weight of 57.7 kDa (Fig. 2). After
Leu24 a cleavage site for the matrix-associated protease was
predicted resulting in a mature mitochondrial protein of
55 kDa. The deduced amino acid sequence showed 81%
similarity to the guinea pig CYP11B1 and 80% similarity to
the human CYP11B2, respectively (see below). The 3¢-UTR
comprised 1079 bp with a canonical polyadenylation site
16 bp upstream of the polyA tail with no indications for the
existence of alternative poly adenylation sites (Fig. 2).
We next investigated the expression of the CYP11B
genes. A Northern blot probed with a CYP11B2-specific
probe showed a single band of 2.9 kb (data not shown)
which is consistent with the length of the isolated cDNA for
CYP11B2 assuming a polyA tail of  200–300 adenine
residues. To see where the CYP11B genes were expressed
Fig. 1. Southern blot analyses with a CYP11B1 exon 1-specific probe.
Fifteen micrograms of guinea pig genomic DNA was digested with
the indicated endonucleases. After transfer, membranes were probed
under low stringency conditions with an exon 1-specific probe of
CYP11B1 (nucleotides 1–141; see Experimental procedures for
details). Sizes of fragments are indicated on the right.
3840 H. E. Bu
¨
low and R. Bernhardt (Eur. J. Biochem. 269) Ó FEBS 2002
and whether they played a role during postnatal develop-
ment we used a highly sensitive RNAse protection assay
with RNAs from different tissues and developmental stages.
As shown in Fig. 3, expression of both the 11b-hydroxylase
and the aldosterone synthase was exclusively in the adrenal
gland. Moreover, there was no difference in expression

Fig. 2. Sequence of the CYP11B2 cDNA of the guinea pig. The nucleotide sequence and the deduced amino acid sequence are both shown. The ORF
(putative start and stop codon underlined) encodes a mitochondrial preprotein with a calculated molecular mass of 57.7 kDa. An arrowhead
indicates the presumptive cleavage site for the mitochondrial matrix associated protease. Numbers on the left denote amino acids, those on the right
indicate nucleotides. A canonical polyadylation site is shown boldface.
Ó FEBS 2002 Guinea pig CYP11B genes (Eur. J. Biochem. 269) 3841
for androstenedione (fourfold) and lowest for 11-deoxy-
cortisol (Fig. 4). This shows a higher substrate specifity of
CYP11B1 which could be due to differences in the active
centre and/or the entry channel. Moreover, it could be
important for tissue-specific synthesis of glucocorticoids
given the differences in expression levels of the two
enzymes.
We next asked whether other accessory proteins might
contribute to the zone-specific synthesis of steroid hor-
mones. A good candidate is adrenodoxin, an iron sulfur
containing electron donor protein that is required for the
function of mitochondrial cytochrome P450 proteins [19]
and has been shown to interact directly with the cyto-
chromes. To test its significance we carried out an experi-
ment where adrenodoxin was either cotransfected or
omitted. After transfection, cells were incubated with
11-deoxycorticosterone as a substrate. As shown in Fig. 5,
the omission of Adx leads to a sharp decrease in the activity
for the 11b-hydroxylase, CYP11B1. Intriguingly, however,
the 11b-hydroxylase activity of the aldosterone synthase
CYP11B2 was basically unaffected whereas the 18-hydroxy-
lation and oxidation potential were abrogated almost
completely. These results indicate clear structural differences
on the surface of these proteins involved either in glucocor-
ticoid or in mineralocorticoid biosynthesis despite a high

)1
[
3
H]androstendione (B), or 2.5 l
M
11-deoxycortisol
including 0.5 lCiÆmL [
3
H]11-deoxycorticosterone (C). Subsequently,
steroids were extracted and separated by TLC [16]. In culture medium
incubated substrates served as an additional control (substrate).
Positions of cold standards are denoted on the left. On the right
percentage of total radioactivity or relative activity is given ± SD; data
are from at least two different experiments performed in triplicate.
Fig. 3. Tissue and age-specific RNAse protection assays. Different
amountsoftotalRNAfromdifferenttissuesandstagesasindicated
were hybridized in solution with CYP11B1 and CYP11B2-specific
probes. Both probes were chosen from the 3¢ untranslated regions of
the genes where sequence divergence was maximal between the two
isoenzymes. Following RNAse digestion the probes protected a 210
nucleotide fragment of CYP11B1 (corresponding to nucleotides 1491–
1700 [1]) or a 240 nucleotide fragment for CYP11B2 (corresponding to
nucleotide 1511–1750; Fig. 2), respectively. A control lane without
RNAse (–RNAse) shows the corresponding undigested riboprobes of
242 nucleotides (for CYP11B1) and 272 nucleotides (for CYP11B2). A
molecular size marker is given on the left. Different developmental
stages are denoted on the right: P1, postnatal day 1; adult.
3842 H. E. Bu
¨
low and R. Bernhardt (Eur. J. Biochem. 269) Ó FEBS 2002

differential regulatory circuits.
We next asked when the aforementioned gene duplication
event might have occurred. To this end we conducted
phylogenetic analyses with all known sequences of the
CYP11B family of proteins. Including the sequences of the
guinea pig with its highly controversial taxonomical posi-
tion into this highly homologous family of proteins could
possibly give new insights into both its taxonomical
classification and the evolutionary relationships within this
protein family. Also, these analyses might indicate when the
gene duplication event occurred that subsequently led to
isoenzymes harbouring different enzymatic activities like,
for example, those in humans or to an exclusively differen-
tial regulation like seen in cattle. Amino acid sequences of 16
CYP11B proteins were subjected to phylogenetic analyses
using two fundamentally different methods. The use of
various methods should provide an estimate of methodical
errors. On the one hand, two distance matrix methods, the
UPGMA (unweighted pairgroup method using arithmetic
mean) and the neighbor joining method (reviewed in [21]),
were used. The distance matrices for the calculation of
phylogenetic trees were produced with three different
algorithms for amino acid exchanges, namely the Dayhoff
model [22], Kimura’s model [23] and the categories model
developed by Felsenstein [24]. On the other hand, the
maximum parsimony method as a single character state
algorithm was used to evaluate the phylogenetic relation-
ships between these proteins. This approach assumes the
most probable phylogeny to be the one that requires the
fewest nucleotide exchanges [17]. The frog was used as an

Shown is the complete genomic structure of CYP11B1 and CYP11B2.
Exon and intron boundaries are indicated. Scale bar represents 1000
nucleotides.
Table 1. Pair-wise sequence similarities of CYP11B proteins. Similarities for the CYP11B proteins were determined pair-wise using the
PALIGN
program (PcGENE, Intelligenetics) for the proteins from human (HS), mouse (MM), rat (RN) hamster (MA), guinea pig (CP), sheep (OA), cow
(BT), pig (SS) and frog (RC).
HSB1HSB2MMB1MMB2RNB1RNB2MAB1MAB2CPB1CPB2OAB0BTB0SSB0 RCB0
CP B1 74% 75% 72% 76% 73% 76% 72% 74% – 81% 73% 73% 72% 58% A
CP B2 80% 80% 75% 80% 77% 79% 76% 79% 81% – 77% 77% 77% 58% B
Ó FEBS 2002 Guinea pig CYP11B genes (Eur. J. Biochem. 269) 3843
with artiodactyls and primates, i.e. supported the paraphyly
of the order rodentia. The bootstrapping probabilities were
however, comparatively low (Fig. 7D) using the neighbor
joining approach with 49, 52 or 68% for the categories
model, the Dayhoff model or Kimura’s model, respectively.
Using UPGMA the probabilities were slightly higher;
between 70 and 82%. Moreover, the hamster proteins were
now assigned to their rat and mouse paralogues. Interest-
ingly, Kimura’s model consistently produced the highest
bootstrapping probabilities for a given topology.
DISCUSSION
In this paper, we describe the isolation and characterization
of the CYP11B genes of the guinea pig. In an earlier study
[1] we isolated a single cDNA using an orthologous probe
that had been obtained using degenerated primers to screen
a guinea pig adrenal cDNA library. This cDNA proved to
code for the abundantly expressed 11b-hydroxylase,
CYP11B1, of the guinea pig which exhibited exclusive
11b-hydroxylase activity [1]. Although cloning strategies

either CYP11B1 or CYP11B2 during postnatal develop-
ment of the guinea pig using RNAse protection assays.
Instead, we saw exclusive adrenal expression of both genes.
This does, however, not rule out expression in other tissues
at lower levels. For example, expression of CYP11B genes in
the rat has been demonstrated in brain [27] and in the heart
[28] using very sensitive RT-PCR and in situ hybridization
techniques. The physiological significance of this low level
expression remains unclear.
To compare the catalytic activities of the guinea pig
CYP11B isoenzymes we cloned the cDNAs downstream of
a cytomegalovirus promoter to drive expression in COS-1
cells. This system has been proven suitable for the
characterization of enzymes of the steroidogenic pathway
[15]. These analyses demonstrated a potent 18-hydroxyla-
tion and 18-oxidation activity of CYP11B2 using various
substrates thus showing it to be the aldosterone synthase
of the guinea pig. It was interesting to note that CYP11B2
of the guinea pig had a considerably higher enzymatic
activity in terms of 11b-hydroxylated product formed
than the guinea pig CYP11B1. These results are in contrast
with findings in other species. For example, the human
CYP11B1 has a 20-fold higher activity towards 11-deoxy-
cortisol than CYP11B2 [29]. Moreover, the activity of the
guinea pig CYP11B2 compared to CYP11B1 could be
correlated to the size of the C17 substituent of the
substrate. Thus, the differences were most pronounced
with androstenedione and least with 11-deoxycortisol as a
substrate. These results might indicate steric hindrance in
the entry channel of the cytochrome P450 enzymes with

have a basic residue (arginine or lysine, respectively) at the
corresponding position. These two basic residues have been
shown in site-directed mutagenesis experiments to be of
significance to the electrostatic interaction of bovine
CYP11A1 (cytochrome P450
SCC
), a closely related mito-
chondrial protein with adrenodoxin [30]. Accordingly, the
11b-hydroxylase activity of CYP11B2 was hardly affected
by omission of cotransfected adrenodoxin. However, the
subsequent enzymatic reactions involving the 18-hydroxy-
lation and oxidation were severely impaired. This could
indicate altered binding affinities for adrenodoxin after
the 11b-hydroxylation. In this regard it is interesting to
note that in vitro experiments with purified bovine
CYP11B0 indicate a conformational change of the protein
due to rearrangement of the substrate after the first
hydroxylation step [31]. This might account for an altered
binding site for adrenodoxin or modified binding affini-
ties. Also, experiments in a reconstituted system with
bovine CYP11B0 using mutant forms of adrenodoxin that
have increased electron transfer capabilities showed a shift
in the spectrum of products formed towards compounds
modified at position 18 [32]. Interestingly, studies with the
microsomal cytochrome P450 enzyme 17a-hydroxylase/
17,20-lyase demonstrated a dependence of the more electron
consuming lyase reaction on the presence of high concen-
trations of the electron donor protein [33]. Taken together,
our results with the guinea pig suggest a new regulatory level
of aldosterone synthesis by the availability of reducing

bination between the genes for which close association has
been shown in mice [4] and humans [34]. Indeed, in humans
an unequal crossover between CYP11B genes, fusing the
CYP11B2 gene undercontrol of the CYP11B1 promoter and
vice versa, has been demonstrated to cause glucocorticoid
remediable aldosteronism, an autosomal dominant disorder
leading to severe hypertension [34], or congenital adrenal
hyperplasia [35]. In this context it is interesting to note that
the crucial determinants for regio-specificity have been
shown to reside in exon 5 [29]. Moreover, analysis of
breakpoints in patients suffering from glucocorticoid reme-
diable aldosteronism indicate that important regulatory
elements are contained within intron 2 [36]. Thus, a scenario
is conceivable where recombination between the two genes in
intron 2 and intron 5 eventually lead to both distinct regio-
specificities and/or differential regulation.
We next sought to determine when this gene duplication
event might have occurred. To this end, we conducted
phylogenetic analyses with 16 sequences of the CYP11B
family of proteins. To assess possible methodical problems
we used different algorithms, namely the maximum
parsimony and two distance matrix methods. The maxi-
mum parsimony method consistently grouped the guinea
pig with a bootstrapping probability of 98% into one clade
with rodents thus favouring monophyly of the order
rodentia. This is in contrast with the findings of Graur and
colleagues [12] who postulated paraphyly of the order. Our
results are however, in accordance with the results of
Hasegawa et al. [13] who questioned the paraphyly of the
order rodentia using the same data as Graur. In contrast,

ttner and members of the Bernhardt lab
for helpful discussions and advice. We also thank W. Oelkers and
V. Ba
¨
hr for guinea pig adrenal tissues. This work was supported by the
Deutsche Forschungsgemeinschaft Grant DFG Be 13436-1.
Ó FEBS 2002 Guinea pig CYP11B genes (Eur. J. Biochem. 269) 3845
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