Karen Artzt

A major testicular cell protein specified by a mouse T/t complex gene

Abstract The technique of two-dimensional gel electrophoresis was used to identify a major testicular cell protein, p63/6.9, which is specified by a gene (p63) within the mouse T/t complex on chromosome 17. A wild-type gene causes the expression of one form of this protein, p63/6.9b. All lethal and semilethal t haplotypes derived from wild mice cause the expression of an apparently identical alternate allelic form of the p63/6.9 protein. This protein, p63/6.9a, represents the first t haplotype-specific molecule to be biochemically identified. A dominant haplotype ( T Hp ) acts as a null allele of the p63 gene; this unique behavior provides additional evidence for the interpretation of T Hp as a deletion within the T/t region of chromosome 17. Limited proteolysis of viable testicular cells causes selective cleavage of the p63/6.9 proteins, relative to other detergent-soluble testicular cell proteins known to be internal. This result strongly suggests that p63/6.9 proteins are located on the cell surface. Qualitative and quantitative estimates indicate that p63/6.9 is one of the most prominent proteins on the testicular cell surface. p63/6.9 is expressed in all other mouse cell types analyzed but at greatly reduced levels. Partial t haplotypes obtained from infrequent recombination events were used to map the p63 gene close to the dominant mutation T and separate from the lethal factors of t haplotypes. A 100% correlation was observed between the expression of p63/6.9a and the genetic presence of the tail interaction factor of t haplotypes. The significance of this correlation in terms of the evolution of t haplotypes among wild mice is discussed.

Gene mapping within the T/t complex of the mouse. I. t-Lethal genes are nonallelic.

The t haplotypes of mouse chromosome 17 are natural polymorphisms in wild populations that contain mutations that affect or control such diverse functions as tail length, embryonic lethality and maturation and function of male germ cells. The major impediment to dissecting the genetics of this complex region has been its unusual property of recombination suppression in heterozygotes with wild-type chromosomes. Recently it was shown that recombination suppression does not occur in heterozygotes containing two different t haplotypes, which suggested that t chromosomes may be mismatched with respect to wild-type but share sequences that permit crossing-over between them. Thus for the first time questions of allelism and map positions of the t-lethal mutations can be addressed. We report here the results of three experiments that analyzed the tw12 haplotype trans to either tw5, tw32 or tw18. In all cases these lethal mutations were nonallelic to tw12. These results, together with evidence for functional relatedness, suggest the t-lethals may be a gene family spread out over more than 15 centiMorgans of chromosome 17.

Genetic structure and origin of t haplotypes of mice, analyzed with H-2 cDNA probes

We investigated the genetic organization and evolutionary origin of t chromosomes of mice by examining the restriction fragment patterns of DNA from t haplotypes and normal chromosomes with cDNA probes to H-2 class I genes. On genomic DNA blots, the restriction fragments containing H-2-related sequences were highly variable among different inbred strains of mice, whereas they were very similar among different t haplotypes even when the t haplotypes carried serologically different H-2 haplotypes. These observations suggest that all t haplotypes have a common origin and are not products of independent mutational events. We also mapped the position of several restriction fragments characteristic of t DNA by using a battery of recombinant t haplotypes, defined with respect to their t-lethal factors and H-2 haplotypes. We thus show that restriction fragments containing H-2-related sequences map to the left of the H-2 class I genes in t chromosomes, a region in which the tw32 b-lethal factor also maps. The cloning of these fragments can be expected to provide an entry for the structural analysis of t DNA.

Carbohydrate changes in pre- and peri-implantation mouse embryos as detected by a monoclonal antibody

We have examined the tissue and embryonic distribution of an antigen on a large polysaccharide that is recognized by a monoclonal antibody, IIC3, prepared against F9 teratocarcinoma cells. By immunofluorescence the antigen is first detected on compacted morulae and early blastocysts. It is strongly expressed on the primary endoderm and trophoblast of expanded blastocysts, but then disappears from the trophoblast of attached blastocysts in vitro. The binding of the antibody is completely inhibited by D-galactose and N-acetylgalactosamine. Fluoresceinated lectins were used to study further the changes in cell surface carbohydrates on trophoblast during implantation. Ricinus I, specific for terminal galactose, binds to preimplantation stages but does not bind to the trophoblast of the attached blastocyst. On the other hand, wheat germ agglutinin, specific for N-acetylglucosamine and sialic acid, binds to all preimplantation embryos and also to attached blastocysts (embryo proper and trophoblast). Neuraminidase treatment of blastocyst outgrowths enhances binding of both IIC3 and Ricinus I to the trophoblast; conversely, the binding of wheat germ agglutinin is decreased by this treatment. The results obtained in this study show changes of cell surface carbohydrates during early mouse development and suggest that sialic acid may be masking molecules on the surface of the trophoblast at the time of implantation.

Relationship of F9 antigen and genes of theT/t complex.

AbstractSerological studies relating F9 antigen of embryonal carcinoma cells to

Cis-trans test shows a functional relationship between non-allelic lethal mutations in the T/t-complex

+ ^{t^{12} }

Characterization of the F9 antigen(s) isolated from teratocarcinoma cell culture medium

at the murineT/t complex have been extended and confirm that only the lethal haplotype t12- and none of the other five lethal haplotypes-affects the quantitative expression of F9 antigen on sperm. Cytotoxicity tests on preimplantation embryos show that t12 homozygotes are less susceptible to antiF9 serum than tw5 homozygotes, and that using specific antimutant haplotype antisera prepared against sperm, t12 antigen is detectable on morulae, whereas tw5 antigen is not.

Rescue of embryonic cells homozygous for a lethal haplotype of the T/t complex: tw12.

As a first step in the study of the possible relationship between the T/t and H-2 complexes, the H-2 antigenic composition of the strains carrying factors t12, tw32, tw2, tw3, t1, t0, t6, tw1 tw71 tw73 tw12, tw5, tw75, and t38 was studied by using a battery of antisera containing antibodies against inbred-derived H-2 antigens. In addition, five t strains (t12, t6 tw5, tw1 and tw2) were selected for the production of antisera against the H-2 complexes carried by t chromosomes. Spleen, lymph node, and thymus cells from H-2b/t heterozygotes and tw2/tw2 homozygotes were injected into appropriate F1 hybrids between two inbred strains that carried the inbred-derived H-2 antigens of the donor. Four new H-2 antigens and one Ia antigen were uncovered and were assigned the symbols H-2.106 through H-2.109, and Ia.101, respectively. Three new H-2 haplotypes were also described, based upon the H-2 antigenic pattern of three t factors, t12, tw1, and tw5. These new haplotypes were given the symbols H-2t12, H-2tw1 and H-2tw5. When the t factors were grouped according to their H-2 haplotypes, their distribution, with certain exceptions, corresponded to the complementation groups. Thus, t chromosomes in the same complementation group carried similar, if not identical, H-2 haplotypes, despite the fact that these chromosomes were derived from widely separated geographic areas. Such an association between the t and H-2 complexes is most unusual in light of what is known of the polymorphism of H-2 haplotypes in wild mice populations. It suggests more than a casual relationship, at least at the population level, between the t and H-2 loci.