Molecular characterization of radiation- and chemically induced mutations associated with neuromuscular tremors, runting, juvenile lethality, and sperm defects in jdf2 mice Mitchell Walkowicz, 1, * Yonggang Ji, 2 Xiaojia Ren, 1, ** Bernhard Horsthemke, 3 Liane B. Russell, 1 Dabney Johnson, 1 Eugene M. Rinchik, 1 Robert D. Nicholls, 2 Lisa Stubbs 1, ** 1 Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8077, USA 2 Department of Genetics and Center for Human Genetics, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland OH 44106, USA 3 Institute for Human Genetics, University Clinic Essen, D-4300 Essen 1, Germany Received: 11 March 1999 / Accepted: 30 April 1999 Abstract. The juvenile development and fertility-2 (jdf2) locus, also called runty-jerky-sterile (rjs), was originally identified through complementation studies of radiation-induced p-locus mu- tations. Studies with a series of ethylnitrosourea (ENU)-induced jdf2 alleles later indicated that the pleiotropic effects of these mu- tations were probably caused by disruption of a single gene. Re- cent work has demonstrated that the jdf2 phenotype is associated with deletions and point mutations in Herc2, a gene encoding an exceptionally large guanine nucleotide exchange factor protein thought to play a role in vesicular trafficking. Here we describe the molecular characterization of a collection of radiation- and chemi- cally induced jdf2/Herc2 alleles. Ten of the 13 radiation-induced jdf2 alleles we studied are deletions that remove specific portions of the Herc2 coding sequence; DNA rearrangements were also detected in two additional mutations. Our studies also revealed that Herc2 transcripts are rearranged, not expressed, or are present in significantly altered quantities in animals carrying most of the jdf2 mutations we analyzed, including six independent ENU-induced alleles. These data provide new molecular clues regarding the wide range of jdf2 and p phenotypes that are expressed by this collection of recently generated and classical p-region mutations. Introduction The location of a gene(s) required for growth and survival, fertil- ity, and neurological function in proximal mouse Chromosome 7 (Mmu7) was originally deduced through analysis of p 6H , a radia- tion-induced recessive allele at the pink-eyed dilution (p) locus (Hunt and Johnson 1971). Although mutations that disrupt the p gene alone exert noticeable effects only on the coat and eye color of the animal, a number of radiation-induced p mutations produce a complex suite of phenotypes similar to that observed in p 6H mice (Lyon et al. 1992; Russell et al. 1995). These mutations have been termed runty-jerky-sterile (rjs) alleles by Lyon and colleagues (1992) and p-juvenile lethal (p jl ) alleles by Russell and coworkers (1995). The sterility of males homozygous for these alleles (here- after referred to as p jl alleles) is associated with reduced germ cell numbers and with the production of sperm with multiple, abnor- mally shaped acrosomes. Binucleate spermatids and “giant” sperm with deformed heads and multiple tails have also been observed (Hunt and Johnson 1971). Although p 6H /p 6H females are fertile, reproductive features associated with immaturity, such as the pres- ence of polyovular follicles and absence of corpora lutea, have been documented in ovaries of adult p 6H mutant mice (Melvold 1974). Pituitary defects observed in association with certain p- linked juvenile-lethal alleles have been suggested as contributing factors in reproductive and growth defects observed in these ani- mals (Melvold 1975; Johnson and Hunt 1975). Studies with the ENU-induced p-region mutations have since provided compelling evidence that the neurological abnormalities, genital hypoplasia, sperm head defects, and reduced fitness of p 6H and p jl -mutations are due to disruption of a single genetic locus, called juvenile development and fertility 2 (jdf2; Rinchik et al. 1995). Recently, one of the radiation-induced rjs alleles, p blind- sterile (p bs ), was identified as an interstitial deletion in a gene located just proximal of p and encoding an exceptionally large, novel protein (Lehman et al. 1998). Single-base splice junction mutations in this same large gene were identified in animals car- rying three independent ENU-induced jdf2 alleles, providing con- clusive evidence of the link to the phenotype expressed by jdf2 mice (Ji et al. 1999). The Herc2 gene, so-named because it encodes a protein with HE CT1 and RC C1 motifs similar to those contained in the guanine nucleotide exchange factor (GEF) protein Herc1, encodes a protein of 4836 amino acids and is thought to play a role in vesicular trafficking (Ji et al. 1999). Herc2 is expressed in most mouse tissues with especially high levels of expression in testis and brain (Lehman et al. 1998; Ji et al. 1999), consistent with the pleiotropic effects that jdf2 and rjs mutations produce in mutant mice. Most of the radiation-induced, prenatally or neonatally lethal p-locus mutations are now known to be deletions (Johnson et al. 1995; Lyon et al. 1992), but molecular defects have not been documented for the majority of p jl alleles. Here we report the analysis of structure and Herc2 gene expression associated with 13 radiation-induced and 6 ENU-induced jdf2 alleles. Portions of the Herc2 transcription unit are deleted or otherwise rearranged in 12 of these radiation-induced mutations, including five of six p jl al- leles and several p-region mutations that fail to complement p jl alleles but that are associated with homozygous neonatal or pre- natal lethality. One radiation-induced mutation, p 12DTR , is an in- terstitial deletion that removes 5015 bp of the 15,247 bp Herc2 transcription unit; this mutation is also associated with reduced pigmentation, suggesting that this interstitial deletion affects se- quences required for the proper expression of p. Herc2 is tran- scribed at anomalously low levels in animals carrying five differ- ent ENU-induced jdf2 mutations, including two mutations for * Present address: Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, MD 20892, USA ** Present address: Human Genome Center, Lawrence Livermore Na- tional Laboratory, 7000 East Avenue, L-452, Livermore, CA 94550, USA. Correspondence to: L. Stubbs at Livermore Mammalian Genome 10, 870–878 (1999). © Springer-Verlag New York Inc. 1999 Incorporating Mouse Genome