Functional annotation of mammalian genomic DNA sequence by chemical mutagenesis: A fine-structure genetic mutation map of a 1- to 2-cM segment of mouse chromosome 7 corresponding to human chromosome 11p14-p15

Abstract

Eleven independent, recessive, N-ethyl-N-nitrosourea-induced mutations that map to a Ϸ1-to 2-cM region of mouse chromosome (Chr) 7 homologous to human Chr 11p14-p15 were recovered from a screen of 1,218 gametes. These mutations were initially identified in a hemizygous state opposite a large p-locus deletion and subsequently were mapped to finer genomic intervals by crosses to a panel of smaller p deletions. The 11 mutations also were classified into seven complementation groups by pairwise crosses. Four complementation groups were defined by seven prenatally lethal mutations, including a group (l7R3) comprised of two alleles of obvious differing severity. Two allelic mutations (at the psrt locus) result in a severe seizure and runting syndrome, but one mutation (at the fit2 locus) results in a more benign runting phenotype. This experiment has added seven loci, defined by phenotypes of presumed point mutations, to the genetic map of a small (1-2 cM) region of mouse Chr 7 and will facilitate the task of functional annotation of DNA sequence and transcription maps both in the mouse and the corresponding human 11p14-p15 homology region.N-ethyl-N-nitrosourea ͉ regional mutagenesis ͉ allelic series ͉ seizures and runting ͉ prenatal and juvenile abnormalities T he supermutagenicity of N-ethyl-N-nitrosourea (ENU) (1) has made it possible to recover heritable mutations at high frequency from mouse spermatogonial stem cells. Consequently, it is possible to use phenotype-driven strategies to recover new ENU-induced mutations in specific, preselected loci for the generation of new alleles (1-4), in whole-genome screens for genes comprising components of complex pathways or phenotypes (5-10), or, with the use of chromosomal rearrangements, in a wide variety of genes within specific chromosomal regions (11-18). The phenotyping protocols used in such strategies can range from simple to complex and can be narrowly or broadly focused, with the output of any experiment dependent largely on the discriminating power of the phenotyping used. The extensive homologies between the genomes of mouse and human make it feasible to use the mouse for discovery of new mutations and phenotypes that aid in the functional annotation of the corresponding human DNA sequence and transcription maps.The region of mouse chromosome (Chr) 7 surrounding the pink-eyed dilution (p) locus that shares homology with human genomic regions 15q11-q13 and 11p14-p15 is being characterized by both genetic and molecular approaches (15,(19)(20)(21)(22)(23)(24)(25)(26). We previously reported initial results of a hemizygosity-screen strategy for recovering ENU-induced recessive mutations within the 4-to 5-cM Del(ru2 p) 46DFiOD deletion (15); this strategy is similar to a mutation-recovery experiment carried out for a 6-to 11-cM deletion at the more distally mapping Chr 7 tyrosinase [Tyr; previously, albino (c)] locus (14,16). The initial phase of this ''p-region screen'' (like the entire Tyr-region screen) used simple phenotyping criteria such as recognizing lethal...

An essential gene mutagenesis screen across the highly conserved piebald deletion region of mouse chromosome 14

“…We isolated seven recessive lethal mutations yielding one mutant mouse line for every 136 genomes screened. Our results are comparable with the SLT deletion-based ENU mutagenesis screens covering the albino and pink-eyed dilution regions of chromosome 7 that realized mutation recovery rates of 1/147 and 1/111, respectively (Rinchik and Carpenter, 1999;Rinchik et al, 2002).…”

“…Recently, regional screens using balancer chromosomes have cataloged hundreds of essential loci on chromosomes 4, 5, and 11 (Hentges et al, 2007;Kile et al, 2003;Nishijima et al, 2003;Wilson et al, 2005). A number of regional screens have also been performed using the deletion complex resources generated by the specific locus test (SLT) (Rinchik and Carpenter, 1999;Rinchik et al, 2002). The SLT has a rich genetic legacy, including establishing ENU as the most potent point mutagen in the mouse and building the nested panels of deletions centered on the seven SLT loci (Davis and Justice, 1998;Hitotsumachi et al, 1985;Russell, 1951;Russell et al, 1979).…”

N -Ethyl- N -Nitrosourea Mutagenesis: Boarding the Mouse Mutant Express

“…2 (41,45,47). In this scheme, coat color alleles of various severity for albino (c) or pink-eyed dilute (p) were used to distinguish between chromosomes contributed by different parents and, thus, to allow identification of progeny carrying new mutations in the interval of interest (43,46). In this manner, embryonic lethal, developmental mutations, such as eed and fit-1, were first recovered (39,44).…”

The mouse genome

“…Some of the chemicals and most of the radiations that were used in these experiments produced deletions of various sizes. These deletions, provided they are viable in the heterozygous state, are very precious tools for a detailed analysis of some specific parts of the genome (Rinchik et al 2002).…”