Localization of rat genes in the nitric oxide signaling pathway:
candidates for the pathogenesis of complex diseases
Jason S. Simon,
Howard J. Jacob,
Kenneth D. Bloch
The Cardiovascular Research Center, Massachusetts General Hospital-East, 149 13th Street, Charlestown, Massachusetts 02129-2060, USA
The Medical College of Wisconsin, Department of Physiology, 8701 Watertown Plank Road, P.O. Box 26509,
Milwaukee, Wisconsin 53226-0509, USA
De´partement de Biologie Mole´culaire, Universite´ Libre de Bruxelles, B-1640 Rhode-St-Gene`se, Belgium
Received: 4 August 1998 / Accepted: 28 August 1998
Nitric oxide (NO) has critical regulatory function in multiple cell
types. In the vasculature, NO is a vasodilator but also modulates
smooth muscle cell proliferation (Schmidt and Walter 1994) and
apoptosis (Pollman et al. 1996). In the nervous system, NO serves
as a neurotransmitter, whereas, in endocrine tissues, NO has im-
portant roles in the regulation of hormone release, such as insulin
and renin (Schmidt and Walter 1994). Alterations in NO produc-
tion and responsiveness could contribute to the pathogenesis of
several complex human diseases, including hypertension, diabetes
mellitus, and renal failure.
Binding of NO to soluble guanylate cyclase (sGC) stimulates
the production of cGMP, an intracellular second messenger, which
is thought to mediate many of the biological functions of NO. sGC
is a heterodimer composed of ␣ and ␤ subunits, and there are two
of each isoform encoded in the rat genome, ␣1, ␣2, ␤1, and ␤2
(GUCY1A1, GUCY1A2, GUCY1B1, and GUCY1B2, respec-
tively; Wong and Garbers 1992). The biological effects of cGMP
are mediated, in part, by cGMP-dependent protein kinase (PRKG).
There are two PRKG genes encoded in the mammalian genome,
one of which, type I (PRKG1), has two isoforms which are the
products of alternative splicing of the first exon (Ørstavik et al.
In the last decade, genetic mapping studies for complex traits
have identified several quantitative trait loci (QTLs) with various
rat strains as animal models for human diseases. With respect to
NO synthesis, genes for three isoforms of nitric oxide synthase
(NOS), neuronal (Nos1), inducible (Nos2), and endothelial (Nos3),
were mapped, and possibilities for candidate genes were addressed
(Deng et al. 1995; Deng and Rapp 1995, 1997; Hu¨bner et al. 1995).
In this study, since the pathway identification is a principal strat-
egy for finding genes contributing to complex human traits, ge-
netic markers for rat genes involved in NO responsiveness,
Gucy1b1 and Prkg1, were developed. Comparative mapping be-
tween rat and human was performed by radiation hybrid (RH)
mapping and fluorescence in situ hybridization (FISH) studies.
These genes mapped to regions containing QTLs linked to hyper-
tension, non-insulin-dependent diabetes mellitus, and renal failure,
suggesting that these genes are attractive candidates contributing
to the pathogenesis of these diseases (Deng and Rapp 1992; Harris
et al. 1995; Schork et al. 1995; Galli et al. 1996; Gauguier et al.
1996; Brown et al. 1996).
To generate genetic markers for Prkg1 and Gucy1b1, genomic
fragments containing a (CA/GT)n repeat within/near these genes
were identified with the two-step strategy. First, using cDNAs for
PRKG1 and GUCY1B1, rat genomic libraries (Stratagene for
Prkg1 and Clontech for Gucy1b1) were screened to isolate geno-
mic fragments containing these genes. A cDNA for PRKGI was
generated by reverse transcription followed by polymerase chain
reaction (RT-PCR) from rat brain mRNA with degenerate primers
(forward primer: 5Ј-CCGAATTCAGGAGCATGGGCACCYT-
GCG-3Ј; reverse primer: 5Ј-CCGGATCCTTTATRAGATCCT-
TGGA-3Ј) corresponding to amino acids shared by human and
bovine PRKG1s (Sandberg et al. 1989; Wolfe et al. 1989). A
cDNA for GUCY1B1 was obtained from Dr. Masaki Nakane (Na-
kane et al. 1990). Second, these genomic clones were screened
oligonucleotides to isolate genomic frag-
ments containing a (CA/GT)n repeat followed by sequencing.
Then, primers flanking a (CA/GT)n repeat within/near these genes
were designed by the computer program PRIMER (Lincoln, un-
published results) with the same criteria described previously
(Jacob et al. 1991): D1Mgh25 (Prkg1): forward primer 5Ј-CTT-
CACCACTAATAACTAACCC-3Ј, reverse primer 5Ј-AGAG-
GAGTGGAAGTTGGG-3Ј; D2Mgh17 (Gucy1b1): forward primer
5Ј-AAAGCGACAGAGGAATGTTCA-3Ј, reverse primer 5Ј-
GGAATTCAGATGGGCTCAGA-3Ј). Allele sizes for these mark-
ers were determined for 12 rat strains (Jacob et al. 1995), identi-
fying that these markers are informative between the Spontane-
ously Hypertensive rat (SHR) and the Brown Norway rat (BN, data
not shown). Then, the 46 progeny of an F
intercross between SHR
and BN were genotyped as previously described (Jacob et al.
1995). These SHR and BN strains were derived from the colonies
of Dr. Michal Pravenec in the Czech Republic: SHR/Cz and BN/
Lx, respectively. After genotyping, linkage analysis was per-
formed with the MAPMAKER computer package (Lander et al.
1987) with the same criteria as previously described (Jacob et al.
As shown in Fig. 1A, Prkg1 (D1Mgh25) mapped between
D1Mit6 and D1Mit7 on the rat Chr 1. Interestingly, it has been
demonstrated that this region contains QTLs for non-insulin-
dependent diabetes mellitus (NIDDM) (Galli et al. 1996; Gauguier
et al. 1996) and renal failure (Brown et al. 1996). Since NO is
known to modulate pancreatic insulin secretion and renal homeo-
stasis (Schmidt and Walter 1994), abnormalities in NO respon-
siveness could contribute to the pathogeneses of NIDDM and renal
failure. Therefore, Prkg1 is a shared candidate for the pathogenesis
of NIDDM and renal failure.
Previously, the cytogenetic position of the human PRKG1 was
identified (Ørstavik et al. 1992) at the human Chr 10q11.2. To
determine the precise position of PRKG1 in the human genome,
we designed PCR primers (forward primer: 5Ј-TTACTA-
TGGTACAGAAACTGGGC-3Ј, reverse primer: 5Ј-ATCATG-
AAACTTCTAGCACTGGT-3Ј) based on the published sequence
of the PRKGI cDNA (Sandberg et al. 1989), and the RH mapping
was carried out with Gene Bridge 4 panel. PRKG1 mapped to 4.4
Correspondence to: G. Koike in Milwaukee.
Mammalian Genome 10, 71–73 (1999).
© Springer-Verlag New York Inc. 1999