Physical mapping and promoter structure of the murine cAMP-specific
phosphodiesterase pde4a gene
Aileen E. Olsen, Graeme B. Bolger
Medical Service, Department of Veterans Affairs Medical Center, and Department of Medicine (Division of Oncology) and Department of Oncologic
Sciences, University of Utah Health Sciences Center, 500 Foothill Blvd, Salt Lake City, Utah 84148, USA
Received: 10 June 1999 / Accepted: 16 September 1999
Abstract. The Pde4a gene is a mammalian homolog of the dunce
learning and memory gene of Drosophila melanogaster and en-
codes cAMP-specific phosphodiesterases, targets for drugs with
antidepressant and anti-inflammatory actions in humans. We have
analyzed the intron/exon and promoter structure of the murine
Pde4a gene. Pde4a encodes at least two different transcripts, each
generated by alternative mRNA splicing and the use of alternative
promoters. The majority of Pde4a exons are tightly clustered at the
3Ј end of the gene. The 5Ј region of the gene contains at least one
widely separated exon, which encodes the 5Ј end of a distinct
mRNA transcript and contains a separate promoter and transcrip-
tional start site. Analysis of YAC clones determined that the Pde4a
gene maps to the 4-cM region of Chromosome (Chr) 9, close to
Ldlr and Epor, in a region syntenic to human PDE4A.
Cyclic adenosine monophosphate (cAMP) is an important intra-
cellular signaling molecule that plays a role in many physiologic
processes, including those in the immune/inflammatory system,
airway smooth muscle, and the brain. cAMP is generated by ad-
enylyl cyclases, which in turn are regulated, at least in part, by
seven transmembrane receptors acting through G-proteins. cAMP
in turn exerts its biological effects by interacting directly with
several downstream effectors, including the cAMP-dependent pro-
tein kinase (PKA), several ion channels, and the recently discov-
ered GTP exchange factors for the rap1 signaling protein (Ka-
wasaki et al. 1998; de Rooij et al. 1998). cAMP and the related
second messenger cGMP are degraded by cyclic nucleotide phos-
phodiesterases (PDEs), which comprise a large and diverse evo-
lutionarily related family of enzymes (Beavo 1995). There are at
least 30 different mammalian PDEs, which have been subdivided
into ten families (PDE1 through PDE10), based on their substrate
preferences (cAMP vs cGMP), the ability to be inhibited by vari-
ous classes of drugs, and the amino acid sequence of the catalytic
regions of the enzymes (Beavo 1995; Soderling et al. 1998a,
1998b). The cAMP-specific PDEs, or PDE4 enzymes, can be dis-
tinguished from other PDEs by sequence homology in the catalytic
region of the enzymes (Houslay et al. 1998) and by their ability to
be specifically inhibited by the drug rolipram. PDE4 inhibitors in
turn have antidepressant, anti-inflammatory and pro-apoptotic ac-
tivity in humans (Houslay et al. 1998; Jiang et al. 1996; Barnette
et al. 1998). The mammalian PDE4 genes are also the closest
mammalian relatives of the dunce gene of Drosophila melanogas-
ter, which was first isolated as a learning and memory mutant in
that organism (Qiu et al. 1991; Davis 1996).
There are at least 15 different PDE4 isoforms, which are en-
coded by four genes in mammals (PDE4A, PDE4B, PDE4C, and
PDE4D), with additional diversity being generated by alternative
mRNA splicing [see (Houslay et al., 1998) for a detailed review].
The chromosomal locations of the four human genes have been
mapped previously to four widely separate locations on three dif-
ferent chromosomes [PDE4A at 19p13.2; PDE4B at 1p31, PDE4C
on 19, and PDE4D at 5q12; (Milatovich et al. 1994; Szpirer et al.
1995; Sullivan et al. 1998)]. The four murine genes are located at
correspondingly conserved locations in the mouse genome [Pde4a
in the 5-cM interval near the centromere of Chr 9, Pde4b on Chr
4 (extremely close to db), Pde4c on 8B3-C (near Lpl), and Pde4d
on Chr 13, respectively (Milatovich et al. 1994; Lee et al. 1996)].
To date, no diseases/mutant phenotypes have been mapped to these
loci, but the strong evolutionary conservation of these genes and
the widespread distribution of PDE4 isoforms in tissues argues
strongly for their having important functional roles.
As a first step in determining how the various PDE4A tran-
scripts are generated, we wished to determine the intron/exon
boundaries and overall structure of the murine Pde4a gene. Our
data demonstrate that each of the PDE4A isoforms is likely to be
generated off a separate promoter. The use of separate promoters
allows independent transcriptional regulation of each isoform,
which in turn explains the marked differences that we have ob-
served in the tissue expression of the various isoforms. We also
provide additional physical mapping of the murine Pde4a gene.
Materials and methods
A murine embryonic stem cell genomic DNA library,
cloned into the XhoI site of the lambda FIX II cloning vector (Stratagene,
La Jolla, CA; Sambrook et al. 1989), was generously provided by Kirk R.
Thomas (Howard Hughes Medical Institute and Department of Medicine,
University of Utah). The cloning procedure used to generate the library
removed the XhoI sites, but left intact the SalI, SacI, and NotI sites on each
side of the insert.
Screening, subcloning and sequencing of Pde4a genomic clones.
Initially, the lambda library was screened with pooled cDNA probes
derived from all PDE4 genes. Specifically, the full inserts of the rat clones
PDE4A5 (pRPDE6; Bolger et al. 1994), PDE4B1 (pDPD; Colicelli et al.
1989), PDE4C1 (pRPDE36; Bolger et al. 1994), and PDE4D3 (pRPDE3;
Bolger et al. 1994) were used. Radioactive probes were generated from
these clones, and the library was screened with standard techniques (Sam-
brook et al. 1989). Final washes during the hybridization were performed
in 0.3 × SSC (Sambrook et al. 1989), 0.1% SDS at 60 °C. As each of the
four rat probes cross-hybridized to all four murine Pde4 genes under these
Correspondence to: G.B. Bolger
The nucleotide sequence data reported in this paper have been submitted to
GenBank under the accession numbers AF142643, AF142644, AF142645,
Mammalian Genome 11, 41–45 (2000).
© Springer-Verlag New York Inc. 2000