The mouse Clc1 /myotonia gene: ETn insertion, a variable AATC repeat, and PCR diagnosis of alleles

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 provide animal models for human myotonias. It was shown that the allele adr (Clc1adr) is due to an insertion of an ETn type transposon that is transcribed and leads to multiple splicing events; the allele mto (Clc1adr-mto) 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 the Clc1adr 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 homologous 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 5' and 3' 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 3' end of intron 12, a tetranucleotide repeat (AATC)n was found that is polymorphic between mouse species Mus musculus, M. molossinus, M. castaneus, and M. spretus, and can thus be used for chromosomal mapping studies.

Characteristics of Cl− uptake in rat alveolar type I cells

“…Thus, the findings of CLC5 and CLC2 broaden the possible role for TI cells in Cl Ϫ transport. By RT-PCR, we could not detect the CLC1 isoform, which is found in skeletal muscle (56). We could, however, detect CLC6 in both TI and TII cells.…”

Chloride conductance in mouse muscle is subject to post‐transcriptional compensation of the functional Cl− channel 1 gene dosage

“…/+ and Clc 1 adr‐mto ? /+ individuals were determined by polymerase chain reaction (PCR) diagnosis (Schnülle et al 1997). The detection of the Clc 1 adr allele was done by amplification of genomic DNA with a mouse intron 12‐specific and an Etn transposon‐specific primer.…”

“…The Clc 1 adr‐mto allele differs from the wild‐type allele by two base substitutions (Gronemeier et al 1994). A PCR product of genomic DNA covering these mutations can be distinguished by single strand conformational polymorphism (Schnülle et al 1997). For physiological and biochemical analyses, mice were anaesthetized and killed by gradually increasing CO 2 concentration in accordance with German law for the protection of animals.…”

“…Hence, all myotonias in the mouse are chloride channel diseases of the recessive Becker type. We have molecularly analysed alleles Clc 1 adr (retroposon insertion; Steinmeyer et al 1991 a ; Schnülle, Antropova, Wedemeyer, Jockusch & Bartsch, 1997), Clc 1 adr‐mto (stop codon; Gronemeier, Condie, Prosser, Steinmeyer, Jentsch & Jockusch, 1994), and Clc 1 adr‐K (missense mutation; Gronemeier et al 1994). Using an antibody against a ClC‐1 peptide that reacts with the ClC‐1 protein in frozen sections, Gurnett, Kahl, Anderson & Campbell (1995) showed the ClC‐1 channel to be present on the sarcolemma and demonstrated its absence from myotonic muscle of the homozygous Clc 1 adr‐mto mutant, in which the ClC‐1 polypeptide is truncated close to its N‐terminus (Gronemeier et al 1994).…”