The mouse Clc1/myotonia gene: ETn insertion, a variable AATC
repeat, and PCR diagnosis of alleles
Jo¨rg W. Bartsch
Developmental Biology Unit, W7, University of Bielefeld, D-33501 Bielefeld, Germany
Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia
Received: 13 October 1996 / Accepted: 10 May 1997
Abstract. Myotonias are muscle diseases in which the function of
the muscular chloride channel ClC-1 is impaired. Null alleles of
the corresponding Clc1 gene on mouse chromosome (Chr) 6 pro-
vide animal models for human myotonias. It was shown that the
allele adr (Clc1
) is due to an insertion of an ETn type transpo-
son that is transcribed and leads to multiple splicing events; the
allele mto (Clc1
) involves a stop codon near the N-terminus.
We have determined the genomic organization of the mouse Clc1
gene and the sequence requirements for the transposon insertion in
allele. The mouse Clc1 gene is composed of 23 exons,
ranging from 39 to 372 bp, and spans approximately 23 kb of
genomic DNA. The exon/intron organization is highly homolo-
gous to that of the human CLCN1 gene; the homology of the
coding sequence is 97% to rat and 89% to human. In the adr allele
the ETn transposon is inserted into intron 12, the largest intron.
Whereas the 5Ј and 3Ј LTR sequences of the ETn transposon are
homologous to those reported for other insertional mutations of the
mouse, no consensus motif for an insertion target site could be
defined. On the basis of flanking sequences, we provide duplex
PCR diagnoses for the adr, adr-mto, and wild-type alleles of Clc1.
Close to the 3Ј end of intron 12, a tetranucleotide repeat (AATC)
was found that is polymorphic between mouse species Mus mus-
culus, M. molossinus, M. castaneus, and M. spretus, and can thus
be used for chromosomal mapping studies.
Myotonic mouse mutants (Heller et al. 1982; Watkins and Watts,
1984; Mehrke et al. 1988) have served a pioneer role (Jockusch
1990a) in the localization (Abdalla et al. 1992; Koch et al. 1992)
and molecular characterization (Steinmeyer et al. 1994) of the
human Thomsen/Becker myotonia gene. In mouse and human, the
affected locus is the structural gene for the muscular chloride
channel ClC-1 (Steinmeyer et al. 1991a; Koch et al. 1992; George
et al. 1993), which codes for a 110-kDa transmembrane protein
with 12 putative membrane-spanning domains (Steinmeyer et al.
1991b). The high resting Cl
of mature skeletal
muscle is predominantly mediated by ClC-1. A drastically lowered
causes electrical instability that results in a myotonic pheno-
type (Ru¨del and Lehmann-Horn 1985; Mehrke et al. 1988).
Spontaneous and 1-ethylnitrosourea (ENU)-induced mutations
at the chloride channel 1/myotonia locus have recurred several
times in the mouse (Jockusch and Bertram 1986; Jockusch 1990b),
and three of these have been defined on the cDNA level: the
standard allele Clc1
(formerly called adr, ‘‘arrested develop-
ment of righting response’’) is due to the insertion of a retroposon
of the ETn type (Steinmeyer et al. 1991a), the Clc1
(formerly mto; Heller et al. 1982) is due to a stop codon that
truncates the ClC-1 polypeptide to a 46-amino acid, N-terminal
fragment (Gronemeier et al. 1994). The ENU-induced allele
(Neumann and Weber 1989) is a missense mutation,
changing Ile to Thre at position 553 in exon 13 (Gronemeier et al.
1994). Mouse models for myotonia are of great value for the study
of developmental and physiological aspects of myotonia (Fu¨cht-
bauer et al. 1988; Mehrke et al. 1988; Wischmeyer et al. 1993;
Gurnett et al. 1995; Bardouille et al. 1996) and the role of excit-
ability for gene expression in muscle (Klocke et al. 1994).
Early transposons (ETn) are a family of murine retrotranspo-
son-like elements, of which approximately 1000 copies are present
in the mouse genome (Bruˆlet et al. 1983). These elements are
transcribed in early mouse embryogenesis, but they contain no
significant open reading frames for proteins involved in their trans-
position (Sonigo et al. 1987). ETn insertions by which gene func-
tion is impaired were found in only a few mouse chromosomal loci
such as the immunoglobulin heavy-chain switch region in the mu-
rine plasmocytoma cell line P3.26Bu4 (Shell et al. 1987, 1990) and
the lymphoproliferation mutant (lpr) mouse (Adachi et al. 1993).
This mutation causes an autoimmune phenotype and is due to an
ETn insertion in the second intron of the antiapoptotic fas gene
leading to aberrant transcripts. In both examples, sequences of the
ETn insertion site and the flanking long terminal repeats (LTR)
have been obtained and compared with the ETn sequence (Sonigo
et al. 1987).
In view of the few well-characterized loss-of-function muta-
tions caused by ETn insertions, a study on additional mutations
should provide clues on structural requirements favoring retro-
transposition in the mouse genome. Therefore, we have sequenced
the respective genomic regions of the mouse wild-type (Clc1
the myotonia (Clc1
) alleles, containing the ETn 5Ј and 3Ј direct
and inverted repeats, together with LTRs and the genomic flanking
sequences. These data were compared with the ETn insertional
mutants mentioned above. To facilitate the identification of adr/
adr cells, embryos, neonatal mice and +/adr carriers, we have used
the genomic sequences to develop a rapid PCR diagnosis for adr
and wild-type alleles of Clc1, whereas cDNA information on exon
1 was sufficient to develop a diagnosis for the mto allele. Further-
more, detailed information about the exon—intron organization of
the mouse Clc1 gene has been obtained that allows for conclusions
on the evolutionary conservation of the chloride channel 1 gene.
Materials and methods
The origin of the A2G-adr mouse stock is described in Gronemeier
and colleagues (1994). The Mus spretus SEG/1 stock was obtained in 1987
from O. v. Deimling, (Freiburg, Germany). SWR/J mice carrying the mto
allele (now Clc1
), Mus molossinus, and Mus castaneus are from The
Correspondence to: J.W. Bartsch
The nucleotide sequences in this publication were submitted to the EMBL
database and have been assigned the accession numbers Z95127–Z95149.
Mammalian Genome 8, 718–725 (1997).
© Springer-Verlag New York Inc. 1997