Fluorescent in situ hybridization (FISH) – using mouse chromosome paints, probes for the mouse major centromeric satellite DNA, and probes for genes on chromosomes (Chr) 16 and 17 – was employed to locate the breakpoint in a translocation used to produce a mouse model for Down syndrome. The Ts65Dn trisomy is derived from the reciprocal translocation T(16;17)65Dn. The Ts65Dn mouse carries a marker chromosome containing the distal segment of Chr 16, a region that shows linkage conservation with human Chr 21, and the proximal end of Chr 17. This chromosome confers trisomy for most of the genes in the Chr 16 segment and Ts65Dn mice show many of the phenotypic features characteristic of Down syndrome. We used FISH on metaphase chromosomes from translocation T65Dn/+ heterozygotes and Ts65Dn mice to show that the Chr 17 breakpoint is distal to the heterochromatin of Chr 17, that the Ts65Dn marker chromosome contains a small portion of Chr 17 euchromatin, that the Chr 16 breakpoint lies between the Ncam2 and Gabpa/App genes, and that the Ts65Dn chromosome contains >80% of the human Chr 21 homologs. The significance of this finding is discussed in terms of the utility of this mouse model.

In Xenopus somatic cells, the somatic-type 5S RNA genes replicate early in S phase, bind the transcription factor TFIIIA, and are expressed; in contrast, the late replicating oocyte-type genes do not bind TFIIIA and are transcriptionally inactive. These facts support a model in which the order of replication of the somatic-type versus the oocyte-type SS genes causes their differential expression in somatic cells due to sequestration of TFIIIA by the early-replicating somatic genes. Here we provide further evidence for the model by showing that in one Xenopus cell line in which some oocyte-type 5S genes are translocated, some oocyte-type 5S genes replicate early and are expressed.In eukaryotes, chromosomal regions are replicated at different times in S phase (1, 2). Funthermore, particular regions appear to be replicated at the same time during successive S phases (3 In Xenopus laevis there are two multigene families of 5S RNA genes (8): the oocyte-type 5S genes, which are expressed only in oocytes (9, 10), and the somatic-type 5S genes, which are expressed throughout development (10). There are 20,000 copies of the oocyte-type 5S genes and 400 copies of the somatic-type 5S genes per haploid genome (11), all of which are located at the distal ends of the long arms of most Xenopus chromosomes (12, 13). We found that, in somatic cells, the somatic-type genes replicate early in S phase, whereas the oocyte-type genes replicate late (14). These data show a correlation between early replication and expression for genes transcribed by RNA polymerase III that is consistent with the work noted above on genes transcribed by RNA polymerase II. The findings also provide support for the replication-expression model proposed by Gottesfeld and Bloomer (15) One prediction of the replication-expression model is that an alteration in the timing of replication of5S genes should be accompanied by an alteration in their expression. We exploited a Xenopus somatic cell line in which some of the 5S genes are translocated to a pericentric position to investigate the relationship among chromosomal position, time of replication, and expression. Pardue et al. (12) had previously shown by in situ hybridization that, in addition to typical telomeric clustering of 5S genes, this line possesses a chromosome with two 5S clusters, one telomeric and the other pericentromeric. However, they did not determine which type of 5S gene was translocated to the pericentromeric location. Interestingly, Ford and Mathieson (19) showed by fingerprint analysis that there is some oocyte-type 5S RNA synthesized in this cell line. The cell line, originally established by K. A. Rafferty in 1968, has not been named. Based on the work reported here, we refer to it as TrXo (Translocated Xenopus oocyte-type 5S genes). In the present study, we show that the translocated 5S RNA genes in TrXo are oocyte-type. In addition, the expression of some oocyte-type 5S genes in this cell line was confirmed by a new assay. Finally, we determined that in TrXo some oocyte-typ...

Although expressed at very low levels in islets of NOD mice, GAD65 is a candidate islet autoantigen. Two transgenic lines of NOD/Lt mice expressing high levels of human GAD65 from a rat insulin promoter were generated. Transgenes were integrated on proximal chromosome 15 of the A line and on the Y chromosome of the Y line. Transgenic A-line mice were obligate hemizygotes, since homozygous expression resulted in developmental lethality. A twofold higher level of hGAD65 transcripts in A-line islets from young donors was associated with higher GAD protein and enzyme activity levels. Y-line males developed diabetes at a similar rate and incidence as standard NOD/Lt males. In contrast, A-line mice of both sexes exhibited a markedly lowered incidence of diabetes. Insulitis, present in both transgenic lines, developed more slowly in A-line mice and correlated with a reduction in the ratio of gamma-interferon to interleukin-10 transcripts. Splenic leukocytes from young A-line donors transferred diabetes into NOD-scid recipients at a retarded rate compared with those from nontransgenic donors. Further, nontransgenic NOD T-cells transferred diabetes more slowly in NOD-scid recipients that were congenic for A-line transgenes as compared with standard NOD-scid recipients. Primed T-cell responses and spontaneous humoral reactivity to GAD65 failed to distinguish transgenic from nontransgenic mice. Quantitative differences in expression level or insertional mutagenesis are possible mechanisms of protection in the A line.

Abstract. The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater interkinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.

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