cDNA sequence, genomic structure, and expression of the mouse
dematin gene (Epb4.9)
Anser C. Azim,
* Anthony C. Kim,
** Mohini Lutchman,
Luanne L. Peters,
Athar H. Chishti
Section of Hematology-Oncology Research, Department of Medicine, St. Elizabeth’s Medical Center, Tufts University School of Medicine,
Boston, Massachusetts 02135, USA
The Jackson Laboratory, Bar Harbor, Maine 04609, USA
Department of Anatomy and Cellular Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts 02111, USA
Received: 23 February 1999 / Accepted: 1 June 1999
Dematin (protein 4.9 region) is an actin-bundling phosphoprotein
of the erythroid membrane cytoskeleton (Rana et al. 1993; Azim et
al. 1995). Although much is known about the functions of the core
group of erythroid membrane proteins, such as spectrin, ankyrin,
band 3, and protein 4.1, relatively little is known concerning the
physiological role of dematin in vivo. In vitro, dematin’s actin-
bundling activity is abolished upon phosphorylation by the cAMP-
dependent protein kinase (Chishti et al. 1988). The significance of
dematin’s actin-bundling activity in erythrocytes is unknown. Ac-
tin bundles are not present in mature erythrocytes but do exist in
early erythroblasts where dematin’s catalytic activity may be func-
tionally significant (Koury et al. 1989).
The primary sequence of dematin consists of an amino-
terminal core domain of unknown function and a carboxy-terminal
domain homologous to the “headpiece” domain of villin, an actin-
binding protein of the brush border cytoskeleton (Rana et al. 1993;
Arpin et al. 1988). This domain has since been identified in several
other proteins including limatin (abLIM), supervillin, and advillin
(Roof et al. 1997; Pestonjamasp et al. 1997; Marks et al. 1998).
Transfection and mutagenesis studies of villin cDNAs revealed the
headpiece domain to be crucial in microvilli morphogenesis (Frie-
derich et al. 1992). While the normal development of microvilli in
villin knockout mice seems to contradict the importance of villin,
this observation suggests that dematin and/or other headpiece-
containing proteins may compensate for villin function in the ab-
sorptive epithelia (Pinson et al. 1998). Here we report the cDNA
sequence, genomic organization, and the mRNA expression of
mouse dematin in fetal and adult tissues. The primary character-
ization of mouse erythroid dematin is the critical first step in
generating knockout mice to study dematin function.
We used RT-PCR and RACE analysis on cDNA reverse tran-
scribed from mouse spleen mRNA to amplify overlapping frag-
ments of the complete mouse dematin cDNA. The full-length
cDNA sequence is ∼2.3 kb in length, encoding an open reading
frame of 383 amino acids (Fig. 1). This murine polypeptide cor-
responds to the 48-kDa isoform of human dematin. We also iso-
lated transcripts encoding the 52-kDa polypeptide of dematin in
which there is a 22-amino acid insertion (I-52) within the head-
piece domain (Fig. 1B; Azim et al. 1995). We have previously
demonstrated that this insertion, encoded by exon 13, binds ATP in
vitro (Azim et al. 1996) and is homologous to a segment of protein
4.2 (Azim et al. 1995). The 5Ј noncoding sequence is 149 bp in
length, while the 3Ј noncoding sequence is approximately 1.0 kb in
length. A comparison of the 3Ј noncoding nucleotide sequence of
mouse and human dematin cDNA indicates ∼60% sequence iden-
tity, suggesting a conservation of function across the two species.
The coding sequence of mouse dematin displays 95% identity with
the human sequence (Fig. 1C). Such striking similarity supports
the notion that dematin knockout mice are likely to shed light on
the physiological function of the human dematin gene.
To elucidate the mouse dematin gene organization, we used a
primer pair derived from the 3Ј noncoding region to screen a
mouse P1 genomic library (Genome Systems Inc., St. Louis, MO).
P1 clones #7227 and #7228 were determined to contain the entire
dematin gene by Southern blot analysis (data not shown). Intron-
exon boundaries were determined by amplifying genomic frag-
ments from the P1 clones with primers derived from the mouse
dematin cDNA. PCR products were subcloned into pCR2.1 (In-
vitrogen Inc., Carlsbad, CA) for sequence analysis. The mouse
dematin gene, in conservation with the human gene, is composed
of 15 exons interrupted by 14 introns (Fig. 2A; Kim et al. 1998).
All but one of the intron-exon boundaries, that between intron 7
and exon 8, conform to the eukaryotic 5Ј donor and 3Ј acceptor
consensus splice junction sequence GT-AG (data reviewed but not
shown). The positions of the intron-exon boundaries reveal the
mouse exon sizes to be identical to that of human dematin (Kim et
al. 1998). Exons 4 and 5 encode a PEST motif, while exon 8
contains a polyacidic motif. The headpiece domain of dematin is
encoded by exons 10–15. Although the precise function of the
PEST motif in dematin is not known, the proline-rich PEST sig-
nature sequence usually marks signaling proteins for regulated
proteolysis in vivo (Rana et al. 1993). Similarly, the polyacidic
motif in dematin may function in the subcellular targeting of the
protein and in the formation of dematin-mediated multiprotein
complexes (Rana et al. 1993). The approximate locations of the
proline-rich motif (PEST), polyacidic motif, and the protein 4.2
homology motif in dematin are shown in Figure 2B. In addition,
the carboxyl terminus of the headpiece domain of dematin contains
a signature actin-binding motif that is found in a number of actin-
binding proteins (Fig. 2B).
Although originally isolated as a major component of the red
cell cytoskeleton, dematin transcripts have been detected in a wide
variety of tissues (Rana et al. 1993; Kim et al. 1998). To examine
the expression pattern of mouse dematin, we performed Northern
blot analysis of mRNA from fetal brain, spleen, liver, and reticu-
locytes. Fetal spleen and reticulocytes were obtained from phen-
ylhydrazine-induced anemic mice. Interestingly, dematin expres-
sion in the fetal brain appears to be relatively low, contrary to the
predominant message seen in adult brain (Fig. 3; Kim et al. 1998).
Dematin is abundantly expressed in the fetal spleen and liver, the
primary sites of erythropoiesis during embryogenesis, but it is
Correspondence: A. Chishti at Biomedical Research ACH-404, St. Eliza-
beth’s Medical Center, 736 Cambridge Street, Boston, MA 02135, USA.
* Present address: Division of Hematology, Brigham and Women’s Hos-
pital, Harvard Medical School, Boston, MA 02115, USA.
** Present address: Department of Pharmacology, NYU Medical Center,
New York, NY 10016.
A.C. Azim and A.C. Kim contributed equally to this work.
Mammalian Genome 10, 1026–1029 (1999).
© Springer-Verlag New York Inc. 1999