Muir-Torre-like syndrome in Fhit-deficient mice.

To investigate the role of the Fhit gene in carcinogen induction of neoplasia, we have inactivated one Fhit allele in mouse embryonic stem cells and produced (129/SvJ x C57BL/6J) F(1) mice with a Fhit allele inactivated (+/-). Fhit +/+ and +/- mice were treated intragastrically with nitrosomethylbenzylamine and observed for 10 wk posttreatment. A total of 25% of the +/+ mice developed adenoma or papilloma of the forestomach, whereas 100% of the +/- mice developed multiple tumors that were a mixture of adenomas, squamous papillomas, invasive carcinomas of the forestomach, as well as tumors of sebaceous glands. The visceral and sebaceous tumors, which lacked Fhit protein, were similar to those characteristic of Muir-Torre familial cancer syndrome.

Voltage-gated potassium channel genes are clustered in paralogous regions of the mouse genome.

Cloning of the Drosophila Shaker gene established that a neurological phenotype including locomotor dysfunction can be caused by a mutation in a voltage-gated potassium (K) channel gene. Shaker sequences have been used to isolate a large family of related K channel genes from both flies and mammals. Toward elucidating the evolutionary relationship between loci and the potential causal connection that K channels may have to mammalian genetic disorders, we report here the genetic mapping of 12-16 different murine, voltage-gated K channel genes. We find that multiple genes, in some cases from distantly related K channel subfamilies, occur in clusters in the mouse genome. These mapping results suggest that the K channel gene subfamilies arose through ancient localized gene duplication events, followed by chromosomal duplications and rearrangements as well as further gene duplication. We also note that several neurologic disorders of both mouse and human are associated with the chromosomal regions containing K channel genes.

Transgenic mouse model of X-linked cleft palate.

A transgenic mouse line, PyLMP.5, exhibited a sex-linked lethality not observed in any other lines expressing the transgene. In this unique line, the transgene integrated into the X chromosome, yielding a simple tandem duplication of the insert sequences with minimal, if any, additional rearrangement of the cellular sequences. The predominant phenotype was a cleft secondary palate and neonatal lethality in males. Survival of females was dependent on the mouse strain background. The disrupted cellular sequences have been mapped to the proximal region of the mouse X chromosome. The disrupted locus may represent the mouse counterpart to a human locus mutated in an X-linked cleft secondary palate syndrome.

A single cyclin A gene and multiple cyclin B1-related sequences are dispersed in the mouse genome.

Cyclin activation of protein serine/threonine kinases plays a pivotal role in regulating the cell cycle. Multiple cyclins that fall into at least five classes, A, B, C, D, and E, have been identified. In some organisms, more than one member of a single cyclin class has been observed. To gain insight into the function of cyclin multiplicity, we determined the number of cyclin A- and B1-related sequences present in the mouse genome, the relationship between these cyclin-related sequences and previously described mutations in the mouse, and cyclin A and B1 mRNA expression in mouse embryos. By genetic mapping using human cyclin A and B1 probes, we identified 1 cyclin A gene located on chromosome 3 and 10 cyclin B1-related sequences located on chromosomes 4, 5, 7, 8, 13, 14, 15, and 17. Cyclin B1-related sequences map in the vicinity of the metaphase-arrest mutation oligosyndactyly (Os) and embryonic lethal mutations associated with the albino (c) locus and the t-complex. In Northern analysis, two cyclin A-related transcripts of 2.1 and 3.4 kb and three cyclin B1-related transcripts of 1.7, 2.1, and 2.7 kb were detected in embryonic stem cells and postimplantation embryos from Day 9.5 to 15.5 of development. Identification of multiple cyclin B1-related sequences in the mouse genome and multiple cyclin B1 mRNAs raises the possibility that seemingly redundant cyclin B genes might have developmental- and/or cell-type-specific functions.

Requirement for mast cell growth factor for primordial germ cell survival in culture.

Mast-cell growth factor (MGF) is encoded by the murine steel (Sl) locus and is a ligand for the tyrosine kinase receptor protein encoded by the proto-oncogene c-kit at the murine dominant white spotting (W) locus. Mutations at both these loci affect mast cells, primordial germ cells (PGCs), haemopoietic stem cells and melanocytes. In many Sl and W mutants, the rapid proliferation of PGC that normally occurs between day 7 and 13.5 of embryonic development fails to occur. As c-kit is expressed in PGCs while MGF is expressed in the surrounding mesenchyme, MGF might promote the proliferation of PGCs. Here we report that MGF is essential for PGC survival in culture, but does not stimulate PGC proliferation. Moreover, whereas both the transmembrane and soluble proteolytic cleavage forms of MGF stimulate mast-cell proliferation, soluble MGF has a relatively limited ability to support survival of PGCs in culture, thus explaining the sterility in mice carrying the steel-dickie (Sld) mutation, which encodes only a soluble form of MGF, and providing a functional role for a transmembrane growth factor.

Chromosomal localization of seven members of the murine TGF-beta superfamily suggests close linkage to several morphogenetic mutant loci.

Chromosomal locations have been assigned to seven members of the TGF-beta superfamily using an interspecific mouse backcross. Probes for the Tgfb-1, -2, and -3, Bmp-2a and -3, and Vgr-1 genes recognized only single loci, whereas the Bmp-2b probe recognized two independently segregating loci (designated Bmp-2b1 and Bmp-2b2). The results show that the seven members of the TGF-beta superfamily map to eight different chromosomes, indicating that the TGF-beta family has become widely dispersed during evolution. Five of the eight loci (Tgfb-1, Bmp-2a, Bmp-2b1, Bmp-2b2, Vgr-1) mapped near mutant loci associated with connective tissue and skeletal disorders, raising the possibility that at least some of these mutations result from defects in TGF-beta-related genes.

An interspecific backcross linkage map of mouse chromosome 8.

We have established a 67-cM molecular genetic linkage map of mouse chromosome 8 by interspecific backcross analysis. Genes that were mapped in this study include Act-6, Aprt, Aprt-ps1, Emv-2, Es-N, Hp, Insr, Mt-1, Plat, Psx-8, Ucp, and Zfp-4. New regions of homology were established between mouse chromosome 8 and human chromosomes 8 and 19. A conserved linkage group was identified between mouse chromosome 8 and human chromosome 16. The map will be useful for establishing linkage of other markers to mouse chromosome 8.

Studies of the mechanism of spontaneous germline ecotropic provirus acquisition in mice.

SWR/J--RF/J hybrid mice spontaneously acquire new germline ecotropic proviruses at high frequency. We have performed ovarian transplantation and in situ hybridization studies to delineate the mechanism and developmental stage of germline provirus acquisition. In addition, we have developed a novel, efficient and simple method to introduce single copy proviruses into the mouse germline. The results reported here have direct implications for understanding how proviruses are acquired in the germline, for using murine leukemia viruses as insertional mutagens, and for using retroviral vectors to introduce foreign genes into the mouse germline.

Methylation of the Hprt gene on the inactive X occurs after chromosome inactivation.

DNA sequences have previously been identified in the first intron of the mouse Hprt gene that are methylated on the inactive but not the active X chromosome. The temporal relationship between methylation of these sequences and X-inactivation was studied in teratocarcinoma cells and postimplantation mouse embryos: the sequences are unmethylated prior to X-inactivation and do not become methylated on the inactive X in most fetal cells until several days postinactivation. Such inactive X-specific methylation occurs in a significantly smaller proportion of the cells in the extra-embryonic tissues, yolk sac mesoderm and endoderm, than in the fetus. These data suggest that the inactive X-specific methylation of sequences such as those in the first intron of the Hprt gene does not play any role in the primary events of X-inactivation, but may function as part of a secondary, tissue-specific mechanism for maintaining the inactive state.

Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.

It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt gene.

Lack of an indication of mutagenic effects of dinitrotoluenes and diaminotoluenes in mice.

The mutagenic effects of 2,4-dinitrotoluene (2,4-DNT), purified and technical grades, 3,5-DNT, 2,4-diaminotoluene (2,4-DAT), and 2,5-DAT were tested in mice using one or more of the following: the dominant lethal assay, the sperm morphology test and/or the recessive spot test. Compounds were administered by both intraperitoneal (IP) injection and gavage, and comparisons were made between controls and treated groups as well as between routes of administration. None of the five compounds tested produced a significant response in any of the systems employed. Treatment with purified 2,4-DNT and 3,5-DNT resulted in marked reductions in the percent of fertile matings. These data indicate a lack of mutagenicity of these compounds in the test systems employed here. The observed fertility effects are consistent with previously published data on DNT-induced testicular damage.

Increases in morphologically abnormal sperm in rats exposed to Co60 irradiation.

We have investigated the effects of testicular exposure to different doses of Co60 radiation on sperm morphology in F-344 rats. The results indicate that from 150 rad to 500 rad gamma irradiation causes statistically significant, dose-related increased in 1) the percent of morphologically aberrant sperm and 2) the frequency of tailless sperm. Both of these effects were detectable in sperm which were derived from treated spermatid, spermatocytes, and spermatogonial cells. These data indicate that the development of a sperm morphology assay in rats is feasible.