Contrasting Effects of ENU Induced Embryonic Lethal Mutations of the quaking Gene Roger D. Cox,* ,1 Alison Hugill,* Alexandra Shedlovsky,² Janice K. Noveroske,‡ Steve Best,* Monica J. Justice,‡ , § Hans Lehrach, ¶ and William F. Dove² *Wellcome Trust Centre For Human Genetics, Oxford University, Windmill Road, Headington, Oxford OX3 7BN, United Kingdom; ²McArdle Laboratory for Cancer Research, University of Wisconsin, 1400 University Avenue, Madison, Wisconsin 53706; ‡The University of Tennessee, Knoxville, Tennessee 37931; §Department of Molecular and Human Genetics, Baylor College of Medicine 410A, One Baylor Plaza, Houston, Texas 77030; and ¶ Max Planck Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin (Dahlem), Germany Received November 2, 1998; accepted February 25, 1999 Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previ- ous study identified a candidate gene, QKI, that con- tains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identify- ing an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal alleles qk l-1 , qk kt1 , qk k2 , and qk kt3 . The mutation in qk l-1 creates a splice site in the terminal exon of the QKI-6 isoform. Missense muta- tions in the KH domain and the QUA1 domains in qk k2 and qk kt3 , respectively, indicate that these do- mains are of critical functional importance. Al- though homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qk v differ. Compound hetero- zygous qk v animals carrying qk kt1 , qk k2 , and qk kt3 all exhibit a permanent quaking phenotype similar to that of qk v /qk v animals, whereas qk v /qk l-1 ani- mals exhibit only a transient quaking phenotype. The qk l-1 mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking pheno- type observed in qk v /qk l-1 mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentra- tion-dependent role in early myelination. This mu- tational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene. © 1999 Academic Press INTRODUCTION The mouse quaking (qk) gene has several recessive alleles causing either embryonic lethality at approxi- mately day 9 in utero, qk lethal-1 (qk l-1 ), qk kt1 , qk k2 , and qk kt3 (Shedlovsky et al., 1988; Justice and Bode, 1986, 1988), or dysmyelination in the central nervous system (CNS) and peripheral nervous system (PNS), resulting in a quaking phenotype accompanied by severe tonic/ clonic seizures in viable adult mice (qk v ; Sidman et al., 1964). The lethal alleles were induced by N-ethyl-N- nitrosourea (ENU) while the qk v allele arose spontane- ously. Compound heterozygotes of the viable allele with each lethal allele exhibit a dysmyelinating phe- notype similar to qk v homozygotes (Justice and Bode, 1988), with the exception of the qk l-1 allele, which ex- hibits a transient dysmyelinating phenotype (Shed- lovsky et al., 1988). Table 1 summarizes the different alleles of the quaking gene and their phenotypes. A candidate gene, QKI, encodes a KH domain-containing protein that binds RNA (probably as a dimer) and may be involved in some aspect of RNA metabolism (Eber- sole et al., 1996; Zorn and Krieg, 1997; Zorn et al., 1997; Caslini et al., 1997; Zaffran et al., 1997; see Vernet and Artzt, 1997). Transcription of the gene is complex, pro- ducing at least three isoforms. The 5-kb isoform en- codes a protein called QKI-5 that is localized in the nucleus of myelinating glia. QKI-5 is the only form seen in significant amounts in the early embryo, when homozygotes of the lethal alleles die (Ebersole et al., 1996; Justice and Bode, 1988; Cox et al., 1993, 1994). The 6- and 7-kb isoforms encode QKI-6 and QKI-7, and these are expressed together late in development and during myelination (Ebersole et al., 1996; Hardy et al., 1996). In the CNS these isoforms are located primarily in the cytoplasm of the glia (Hardy et al., 1996). Probes from this locus revealed that the molecular lesion in the qk v mutation involved a .1-Mb deletion. 1 To whom correspondence should be addressed. Telephone: 44- 1235-834393. Fax: 44-1235834776. E-mail: r.cox@har.mrc.ac.uk. Genomics 57, 333–341 (1999) Article ID geno.1999.5804, available online at http://www.idealibrary.com on 333 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.