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Deletion Mapping of Four Loci Defined by N-Ethyl-N-Nitrosourea-Induced Postimplantation-Lethal Mutations Within the Pid-Hb

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Deletion Mapping of Four Loci Defined by N-Ethyl-N-Nitrosourea-Induced Postimplantation-Lethal Mutations Within the Pid-Hb Copyright 0 1993 by the Genetics Society of America Deletion Mapping of Four Loci Defined by N-Ethyl-N-Nitrosourea-Induced Postimplantation-Lethal Mutations Within thepid-Hbb Region of Mouse Chromosome 7 Eugene M. Rinchik, Donald A. Carpenter and Carol L. Long Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8077 Manuscript received May 12, 1993 Accepted for publication August 21, 1993 ABSTRACT As part of a long-term effort to refine the physical and functional maps of the Fes-Hbb region of mouse chromosome 7,four loci [1(7)1Rn, 1(7)2Rn,1(7)3Rn, 1(7)4Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutationswere mapped by means of trans complementation crosses to mice carrying lethal deletionsof the mouse chromosome-7 albino(c) locus. Each locuswas assigned to a defined subregionof the deletionmap at the distal endof the Fes-Hbb interval. Of particular use for this mapping were preimplantation-lethal deletions having distal breakpoints localized between pad and Omp. Hemizygosity or homozygosity for each of the ENU-induced lethalswas found to arrest development after uterine implantation; the specifictime of postimplantation death varied, and depended on both the mutation itself and on whether it was hemizygous or homozygous. Based on their map positions outsideof and distal to deletions that cause death at preimplantation stages, these ENU-induced mutations identify loci, necessary for postimplantation development, that could not have been discovered by phenotypic analyses of mice homozygous for any albino deletion. The mapping of these loci to specific genetic intervals definedby deletion breakpoints suggestsa number of positional-cloning strategies for the molecular isolation of these genes. Phenotypic and genetic analyses of these mutations should provide useful information on the functional composition of the corresponding segmentof the human genome (perhaps human1 lql3.5). HE overlapping deletion mutations that encom- Mice homozygous for the largest of the c deletions T pass the albino (c) locus within the Fes-Hbb die at preimplantation stages of development, presum- region of mouse chromosome7 are providing auseful ably due to the deficiency of a gene or genes at the series of reagents with which to develop correlated preimplantationdevelopment ($id) locus (LEWIS physical and functional maps of megabase regions of 1978; GLUECKSOHN-WAELSCH1979; RUSSELLand amammalian chromosome (RUSSELL,RUSSELL and RAYMER1979; RUSSELL,MONTGOMERY and RAYMER KELLY 1979; GLUECKSOHN-WAELSCH1979; RUSSELL, 1982; RINCHIKand RUSSELL1990; RINCHIKet al. MONTGOMERYand RAYMER1982; reviewed in RIN- 1993). The early lethal phenotype exhibited by these CHIK and RUSSELL1990). These deletions have been homozygotes makes it impossible to estimate the num- useful for both genetic and physical mapping of loci ber of transcription units that might map within these defined by DNA probes and/or by phenotypes (RIN- large deletions. It is likewise impossible to determine CHIK and RUSSELL1990). Moreover, the mutant phe- the phenotypic effects of genes mapping outside of notype exhibited by mice homozygous for a deletion the relatively shorter c deletions, but within the longer has, in several cases, provided insights into the genetic deletions, if these genes normally function at stages control of developmental processes involved in liver subsequent to this preimplantation block. and kidney function in thelate fetus and neonate To generate afine-structure functional/mutation (ERICKSON,GLUECKSOHN-WAELSCH and CORI 1968; map of the Fes-Hbb region in which individual genes GLUECKSOHN-WAELSCH1979), spermiogenesis and their corresponding effects on development can (LEWIS,TURCHIN and WOJTOWICZ1978), gastrulation be defined and characterized, we have been utilizing (LEWIS,TURCHIN and GLUECKSOHN-WAELSCH1976; the presumptive point-mutation inducer, N-ethyl-N- NISWANDERet al. 1988, 1989), and in cleavage of the nitrosourea (ENU),and a screening protocolbased on preimplantation embryo (LEWIS1978). Moreover, a deletion hemizygosity, toinduce and recover both particulardeletion homozygote (cz4c0s/c’4c0s) that is lethal and viable single-gene mutations that map deficient for fumarylacetoacetate hydrolase (Fah) has within a large c deletion that is lethal at preimplanta- been proposed as an animal model for human type-1 tion stages of development(RINCHIK, CARPENTER and tyrosinemia (KLEBIG,RUSSELL and RINCHIK1992; SELBY1990). This report describes the more refined RUPPERTet al. 1992). deletion mapping of four such mutations, which are Genetics 135 1117-1 123 (December, 1993) 1118 E. M. Rinchik, D. A. Carpenter and C. L. Long lsqm-qtrr g mlq14.31 ESP ??? gllp15.51 llq13.5 9FR60Hb IFR6OHb 2R145L 3R145L lOFRQL 146G, 39SAS 26DVT 12FR60Hb FIGURE .-Deletion 1 mapping of four ENU-induced lethal mutations within the pid-Hbb region of mouse chromosome 7. The map of the Fes-Hbb region of chromosome 7 presented in RINCHIKet al. (1993) (accompanying report) has been modified by incorporating the complementation data from Table 1. The loci l(7)lRn through 1(7)4Rnare defined by ENU-induced prenatally lethal mutations described in the text and areshown in larger type along with D7Rn6 and Omp (RINCHIKet al. 1993). Below the map are shown the extents of a subset of c-locus mutations that are most relevant for the mapping these four loci. (Table 1 lists additional c deletions that are not pictured here.) The thicker lines represent deletions that genetically bracket pid-Hbb-region loci defined by the ENU-induced mutations. The bracket above 1(7)2Rnand 1(7)4Rn indicates that these two loci cannot yet be ordered with respect to each other; likewise, the relative order of the l(7)IRn and D7Orl loci with respect to Omp and sh-I is not known. Estimates of the genetic distance (in cM) between c (Tyr) and Hbb ranges from 3.46 k 0.64 to 6.19 k 0.65 (DAVISSON,RODERICK and DOOLITTLE1989), and the c~~~~~ deletion has been estimated to be 6-1 1 cM in length (RUSSELL,MONTGOMERY and RAYMER 1982;RINCHIK, CARPENTER and SELBY 1990).The boxes above the map denote known human homology regions; human 1 1 q homologies are based on data of EVANSet al. (1993). lethal shortly after implantation, to a subregion of the nonalbino progeny were assumed to carry an ENU-induced Fes-Hbb interval distal to the pid locus. Thus, these viable mutation closely linked to the c locus. mutations define loci within the c-deletion complex The mutant c 1 chromosomes, recovered from cCh+/c 1 (light-chinchilla) Gn siblings, were propagated by crossing cCh that could never have been identified by analysis of +/c 1 mice to mice of the inbred strain 47BS-cch/cch, which deletion homozygotes. Moreover, the deletion map- exhibit the full (darker) chinchilla color. The c mutation ping reported here,in conjunction with the molecular serves as a marker for all l's, and because it can potentially analysis of a number of preimplantation-lethal c dele- be separated from 1 by crossing over in cCh+/c 1 carriers, cch +/c 1 males in each generation were first crossed to cCh+/ tions (RINCHIK et al. 1993), suggests strategies for c26DVT identifying these loci on the physical map of the females to progeny-test for the presence of 1. Males pid- that produced zero or one albinos in at least 30 progeny of Hbb region. this testcross were considered to be proved cCh+/c 1 carriers (RINCHIK, CARPENTERand SELBY 1990). Lethal albino deletions [Dxc)] were maintained by cross- MATERIALS ANDMETHODS ing light-chinchilla heterozygotes [c"'/DfTC)] to mice of the non-inbred, chinchilla stock 2A-cCh/cch.New ENU-induced The origin of ENU-induced c-region mutations has been described in detail elsewhere (RINCHIK,CARPENTER and lethal mutations were mapped with respect to breakpoints SELBY1990). Briefly, each mutation was induced by treat- of lethal albino deletions by crossing proved carrier males (cCh+/c ment of BALB/cRl(c/c) Generation-0 (Go)males with a total I) with females heterozygous for lethal albino dele- (but fractionated) dose of 400 mg/kg of ENU to spermato- tions [cCh/Dj'c)].Specific ENU-induced lethal mutations used gonial stem cells. Newly induced mutations that are closely for these mapping analyses include: 1(7)IRn'8'SB;1(7)2Rn'75SB; linked to c were identified by crossin GI females (+/c) with 1(7)3Rn677Sn;and 1(7)4Rn"08Sn(RINCHIK, CARPENTER and males carrying the Dfl;Mod-2 sh-IpDVTdeletion (abbrevi- SELBY1990). ated c26DVT;see Figure 1) opposite the cch (chinchilla) marker. To determine the approximate time of death caused by GI females that yielded no GPalbino progeny were hemizygosity for each of the four new ENU-induced lethal considered to carry an ENU-induced lethal mutation, de- mutations, proved carrier males (cch +/c 1) were crossed to noted here as "1," mapping within the limits of the c~~~~~ cCh+/c~~~~ females. At 14.5 days post coitum (E14.5) (where deletion. Likewise, GI females that gave rise to GP albino the morning of finding a vaginal plug was considered to be progeny manifesting abnormal phenotypes (e.g., differential E0.5), the females were sacrificed, and the ovaries and size, abnormal balance, etc.) that were not observed in uterus were removed. The number of corpora lutea within Deletion Mapping in Mapping Deletion the Mouse 1119 the ovary was determined, and the contents of the uterus stages) caused by hemizygosity for each of the four were examined as described RUSSELL in and RAYMER(1 979). mutationsmapped in Figure1 was determined by Some of the experiments designed to estimate the time of death of embryos/fetuses homozygous for a particular 1 crossing cch +/c~~~~females to proved cch +/c 1 males. involved crossing proved carrier males (cch+/c I) to proved As a control, cch +/c~~~~females were crossed to cch carrier females; these proved females were obtained by a +/c ~h-1’~’~males, which carry an ENU-induced mu- pro en testcross of presumed cCh +/c 1 females to cCh +/ tation at theshaker-1 locus (RINCHIK,CARPENTER and c,’ c,’ :ales.
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