Molecular cloning and characterization of a gene expressed in mouse
developing tongue, mDscr5 gene, a homolog of human DSCR5 (Down
syndrome Critical Region gene 5)
* Yutaka Suzuki,
Todd D. Taylor,
Human Genome Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Genomic Sciences Center, Institute of Physical and Chemical Research (RIKEN-GSC): 2-1, Hirosawa, Wako-shi, Saitama 351-0106, Japan
Department of Virology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan
Received: 17 July 2000 / Accepted: 15 December 2000
Abstract. For understanding the pathogenesis of Down syndrome
(DS), it is important to identify and characterize the genes on
Chromosome (Chr) 21, especially those in the Down syndrome
critical region (DSCR) on Chr 21q22.2. Recently we have deter-
mined 33.5 Mb (more than 99%) of DNA sequence of Chr 21 and,
from these sequence data, we identified a novel gene, DSCR5
(transcript ס 0.8 kb), from DSCR by combination of computa-
tional gene prediction and cDNA screening. For functional analy-
sis of DSCR5, we identified a mouse homolog of the DSCR5
cDNA, and termed it mDscr5 (transcript length ס 0.8 kb). The
gene was mapped to mouse Chr 16 C3-C4, the syntenic region of
human Chr 21, and encodes an amino acid of 132 residues with
90% identity to DSCR5. In situ hybridization showed that mDscr5
is predominantly expressed in the developing tongue. To our best
knowledge, no other gene in DSCR is reported to be expressed in
tongue, so that DSCR5 may be the first candidate to elucidate the
pathophysiology of tongue malformation observed in DS.
Down syndrome (DS) is the most common birth defect (1 in 600–
1000 newborns) and is caused by trisomy of Chr 21 (Epstein
1995). DS manifests complex phenotypes, including flat nasal
bridge, a thick protruding tongue, high arched or narrow palate,
short and broad hands with curved fifth finger, wide cleft between
the first and second toes, joint hyperlaxity, floppy muscles, short
stature, and mental retardation (Rahmani et al. 1989; Delabar et al.
1993; Korenberg et al. 1994). These various symptoms of the
disease imply the involvement of multiple genes in the pathogen-
esis of DS. Cytogenetic and clinical studies on partial trisomy 21
DS patients suggested that a region of 21q22.2 extending from
DNA marker D21S55 to the ERG gene is critical in the pathogen-
esis of DS, and it was designated the Down syndrome critical
region (DSCR) (Rahmani et al. 1989; Korenberg et al. 1990; Dela-
bar et al. 1993; Ohira et al. 1997), although we do not exclude the
possibility that genes outside this region may contribute to the DS
phenotype (Korenberg et al. 1994). Thus, cloning and functional
characterization of genes in DSCR is an essential step in under-
standing the pathogenesis of the various phenotypic anomalies in
For these several years, we have systematically analyzed the
DSCR and identified genes that should be responsible for some of
Down syndrome’s complex features (Hattori et al. 1998; Tsuka-
hara et al. 1996; Nakamura et al. 1997; Tsukahara et al. 1998).
Recently we have determined 33.5 Mb (more than 99%) of DNA
sequence of Chr 21, which includes the 1.53 Mb of DSCR (Hattori
et al. 2000). Combining computational gene prediction and cDNA
screening, we and others identified a novel gene, DSCR5, from
21q22.1-q22.2, that is highly expressed in human heart, liver, and
skeletal muscle (Togashi et al. 2000; Shibuya et al. 2000). To
explore the pathophysiological significance of this gene in DS, it
is important to clarify its expression pattern during the embryonic
period. Therefore, in the present study, we cloned the mouse ho-
molog of DSCR5, termed mDscr5, and examined its expression
profile during murine embryogenesis. We report here that mDscr5
is predominantly expressed in tongue during development.
Materials and methods
Isolation of mDscr5 cDNA.
The human DSCR5 sequence (Accession
No. AB035742) was used to search for homologous sequences in the
mouse EST database by using the BLASTN program. Oligodeoxynucleo-
tides based upon the mouse EST sequence were used for RT-PCR to
amplify the corresponding cDNA fragment from mouse heart poly(A)+
RNA (Clontech, Polo Alto, Calif.) The cDNA (0.5 ng) was amplified by
PCR in a reaction mixture (25 l) containing 0.5 U of Ex taq,1×PCR
buffer, 0.25 m
dNTP, and 0.2
of 5Ј-primer (5Ј-CCTCAGCTCG-
GTGGGGTGGG-3Ј) and 3Ј-primer (5Ј-ATAGTATAATGTGGTATTT-
TAT-3Ј). The thermal cycling conditions of the PCR were 25 cycles of 20s
at 94°C, 1 min at 55°C, and 1 min at 72°C. For the confirmation of cDNA
sequence, direct cycle sequencing of RT-PCR products was performed to
eliminate any effect of misincorporation during PCR. Sequence data were
edited and assembled by using Sequencer 3.0 (Gene Codes, Ann Arbor,
Chromosomal localization of mDscr5 gene.
Fluorescence in situ hy-
bridization (FISH) was performed as described previously (Hirai et al.
1996) by using a 0.5-kb fragment containing nt 198–698 of the mDscr5
cDNA. The mDscr5 cDNA was biotinylated by nick-translation and hy-
bridized to G-banded chromosomes from cultured splenocytes of male
mice (BALB/c). The location of FISH signal was identified according to
the G-banding pattern.
Cell culture and transfection.
The full-length ORF cDNAs of mDscr5
were ligated into the BglII-HindIII sites of pEGFP-C1, a C-terminal GFP-
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession numbers: AF216306,
AF216307, AF216308, AF216309.
*Present address: Department of Neurology, Columbia University, New
York, NY 10032, USA.
Correspondence to: Y. Sakaki; E-mail: firstname.lastname@example.org
Mammalian Genome 12, 347–351 (2001).
© Springer-Verlag New York Inc. 2001