Donald A. Carpenter

Effects of ENU dosage on mouse strains.

Abstract. The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F1 hybrids to ENU. The results show that most F1 hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU.

Mutations in the clathrin-assembly gene Picalm are responsible for the hematopoietic and iron metabolism abnormalities in fit1 mice

Recessive N-ethyl-N-nitrosourea (ENU)-induced mutations recovered at the fitness-1 (fit1) locus in mouse chromosome 7 cause hematopoietic abnormalities, growth retardation, and shortened life span, with varying severity of the defects in different alleles. Abnormal iron distribution and metabolism and frequent scoliosis have also been associated with an allele of intermediate severity (fit14R). We report that fit14R, as well as the most severe fit15R allele, are nonsense point mutations in the mouse ortholog of the human phosphatidylinositol-binding clathrin assembly protein (PICALM) gene, whose product is involved in clathrin-mediated endocytosis. A variety of leukemias and lymphomas have been associated with translocations that fuse human PICALM with the putative transcription factor gene AF10. The Picalmfit1–5R and Picalmfit1–4R mutations are splice-donor alterations resulting in transcripts that are less abundant than normal and missing exons 4 and 17, respectively. These exon deletions introduce premature termination codons predicted to truncate the proteins near the N and C termini, respectively. No mutations in the genes encoding Picalm, clathrin, or components of the adaptor protein complex 2 (AP2) have been previously described in which the suite of disorders present in the Picalmfit1 mutant mice is apparent. These mutants thus provide unique models for exploring how the endocytic function of mouse Picalm and the transport processes mediated by clathrin and the AP2 complex contribute to normal hematopoiesis, iron metabolism, and growth.

N-Ethyl-N-nitrosourea-induced prenatally lethal mutations define at least two complementation groups within the embryonic ectoderm development (eed) locus in mouse Chromosome 7

Two loci [l(7)5Rn and l(7)6Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the albino (c) locus in Chromosome (Chr) 7. Both loci were found to map to the subregion of the Mod-2-sh-1 interval that contains the eed (embryonic ectoderm development) locus. eed has been defined by the inability of embryos homozygous for certain c deletions to develop beyond the early stages of gastrulation. Evidence for at least two loci necessary for normal prenatal development, rather than one locus, that map within the eed interval came from the observation that two prenatally lethal mutations, 3354SB [l(7)5Rn3354SB] and 4234SB [l(7)6Rn4234SB], could complement each other in trans, but could not each be complemented individually by c deletions known to include the eed locus. A somewhat leaky allele of l(7)5Rn [l(7)5Rn1989SB] was also recovered, in which hemizygotes are often stillborn and homozygotes exhibit variable fitness and survival. The mapping of the loci defined by these mutations is likely to be useful for genetic, molecular, and phenotypic characterization of the eed region, and mutations at either locus (or both loci) may contribute to the eed phenotype.

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

Eleven independent, recessive, N-ethyl-N-nitrosourea-induced mutations that map to a ≈1- 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.

Genetic and physical mapping of the fitness 1 (fit1) locus within the Fes-Hbb region of mouse chromosome 7.

Mutations at the fit1 locus affect normal pre- and postnatal development by retarding growth and reducing viability. We report mapping of the fit1 locus, by trans-complementation crosses to mice carrying deletions of the albino (c) locus in Chromosome (Chr) 7, to a subregion of the c-deletion complex within the Mod2-sh1 interval. The fit1 locus, which is currently defined by five N-ethyl-N-nitrosourea (ENU)-induced mutations, was found to map in a subregion between the eed and exed loci. A restriction fragment containing a deletion breakpoint that genetically defines the proximal border of fit1 was cloned, providing a DNA probe (RN302) that maps proximal to fit1. Long-range mapping with this probe, and with a DNA probe that maps distal to the fit1 interval, established that the region containing at least part of the fit1 gene is 530 kb or less. Positioning of fit1 between deletion breakpoints, and the isolation and mapping of a DNA probe proximal to it, should facilitate the cloning and molecular characterization of fit1, as well as of the eed locus and the tightly linked l(7)5Rn and l(7)6Rn loci.

Reverse Genetics in the Mouse and Its Application to the Study of Deafnessa

Genetic variants of the laboratory mouse can serve as useful models for hereditary deafness syndromes in humans. Recessive mutations at the shaker-1 (sh-1) and whirler (wi) loci, in chromosomes 7 and 4, respectively, both result in circling behavior and a deafness syndrome. In sh-1 homozygotes this deafness is associated with neurophysiological abnormalities that may be accompanied by structural abnormalities of the inner ear. Radiation-induced deletion mutations are being used in a strategy of reverse genetics to identify the genes defined by these mutations. Genetic analyses have refined the position of sh-1 to a chromosomal interval between break points of deletions involving the closely linked albino (c) locus. A cDNA encoding olfactory marker protein (OMP) and the anonymous locus D7OR1 have also been mapped to this interval. These clones contribute to the physical map of the sh-1 region and could be important for accessing the sh-1 gene itself. Similarly, we have identified a radiation-induced deletion of the brown (b) locus that covers the wi locus and two that do not. Thus, the wi locus has been located within a chromosome 4 interval defined by structural rearrangements, which should likewise aid in identifying closely linked molecular clones.

Micro-Raman and photoluminescence spectroscopies of horizontal Bridgman-grown AgGaSe2

AgGaSe2 crystals grown by the horizontal Bridgman technique were studied by room-temperature micro-Raman scattering and low-temperature photoluminescence (PL) spectroscopies. The most intense Raman line observed had the frequency of 177cm−1, corresponding to the Γ1(W1) mode. The measurements were performed along the direction of growth of the boule and the line frequency was found to be almost constant within the experimental accuracy. The average full width at half maximum of the Γ1(W1) mode was found to be 4.6cm−1 and its insignificant variation along the ingot suggests its structural and compositional uniformities. At low temperature (8K), the main PL peak at 1.772eV is due to donor-acceptor-pair recombination. A doublet in the excitonic peak, observed at that temperature, suggests a reduced inhomogeneous broadening and the better crystallinity of the sample, when compared with the previous literature. The dispersion of the various PL peaks along the length of the ingot was also found negligible, which...

Identification of mutations from phenotype-driven ENU mutagenesis in mouse Chromosome 7

We have used the new high-throughput mutation-scanning technique temperature-gradient capillary electrophoresis (TGCE) for the identification of point mutations induced by N-ethyl-N-nitrosourea (ENU) in the mouse genome. TGCE detects the presence of heteroduplex molecules formed between a wild-type gene segment and the corresponding homologous segment containing an induced mutation or a naturally occurring single nucleotide polymorphism (SNP). Partially denatured heteroduplex molecules are resolved from homoduplexes by virtue of their differential mobilities during capillary electrophoresis conducted in a finely controlled temperature gradient. Simultaneous heteroduplex analysis of 96 amplicons ranging from 150 to 600 bp in size is achieved in approximately 45 min without the need for predetermining the melting profile of each fragment. Initially, we exploited known mouse mutations to develop TGCE protocols for analyzing unpurified PCR samples amplified from crude tail-DNA preparations. TGCE was then applied to the rapid identification of three new ENU-induced mutations recovered from regional mutagenesis screens of a segment of mouse Chromosome 7. Enzyme assays and quantitative reverse transcription-PCR (qRT-PCR) methods validated these new mutations. Our data demonstrate that rapid mutation scanning with TGCE, followed by sequence verification only of detected positives, is an efficient approach to the identification of point mutations in the mouse genome.

X-ray-induced deletion complexes in embryonic stem cells on mouse chromosome 15.

Chromosomal deletions have long been used as genetic tools in dissecting the functions of complex genomes, and new methodologies are still being developed to achieve the maximum coverage. In the mouse, where the chromosomal deletion coverage is far less extensive than that in Drosophila, substantial coverage of the genome with deletions is strongly desirable. This article reports the generation of three deletion complexes in the distal part of mouse Chromosome (Chr) 15. Chromosomal deletions were efficiently induced by X rays in embryonic stem (ES) cells around the Otoconin 90 (Oc90), SRY-box-containing gene 10 (Sox10), and carnitine palmitoyltransferase 1b (Cpt1b) loci. Deletions encompassing the Oc90 and Sox10 loci were transmitted to the offspring of the chimeric mice that were generated from deletion-bearing ES cells. Whereas deletion complexes encompassing the Sox10 and the Cpt1b loci overlap each other, no overlap of the Oc90 complex with the Sox10 complex was found, possibly indicating the existence of a haploinsufficient gene located between Oc90 and Sox10. Deletion frequency and size induced by X rays depend on the selective locus, possibly reflecting the existence of haplolethal genes in the vicinity of these loci that yield fewer and smaller deletions. Deletions induced in ES cells by X rays vary in size and location of breakpoints, which makes them desirable for mapping and for functional genomics studies.

Molecular basis for hair loss in mice carrying a novel nonsense mutation (Hrrh-R ) in the hairless gene (Hr).

Animal models carrying mutations in the hairless (Hr) gene provide a rich resource for study of hair follicle biology. A spontaneous mouse mutant with a phenotype strikingly similar to rhino mutants of Hr arose spontaneously in the mouse facility at Oak Ridge National Laboratory. Sequence analysis of Hr in these mutants uncovered a nonsense mutation in exon 12, designated as Hrrh-R (rhino, Oak Ridge). The mutation led to significant reduction in Hr mRNA levels, predicted to be due to nonsense-mediated decay. Histological analysis indicated dilated hair follicle infundibula at 14 days of age that rapidly became filled with cornified material. Microarray analyses revealed that expression levels of many genes involved in keratinocyte differentiation, epidermal regeneration, and wound healing were significantly upregulated before morphological detection of the phenotype, suggesting their role in onset of the Hrrh-R phenotype. Identification of this new Hr allele and the underlying molecular alterations allows further understanding of the role of Hr in hair follicle biology.