Expression of human and suppression of mouse nucleolus organizer activity in mouse-human somatic cell hybrids.

Abstract

28S ribosomal RNA (rRNA) (2-4), suggesting that the absence of the human rRNA may be related to the loss of human chromosomes. It is not due to the absence of the human acrocentric chromosomes, which carry the structural genes for rRNA (5, 6), because Marshall et al. (4) found no human 28S rRNA in a large series of mousehuman hybrids that contained 2 to 11 human acrocentrics.The chromosome regions that carry the rRNA genes have been identified as the nucleolus organizer regions (NORs) (7), and these regions can be stained preferentially by the Ag-AS silver staining method (8). In human diploid cell cultures the Ag-AS method stains the short arm regions of most of the acrocentric chromosomes (9, 10). The NORs of the same human acrocentric chromosomes are not stained in a mouse-human hybrid that has lost some human chromosomes (11). There is no evidence to suggest that rRNA genes are deleted from the human acrocentrics in hybrid cells. Therefore the absence.of Ag-AS stain suggests that this method detects only chromosome regions that functioned as nucleolus organizers in the preceding interphase, and, by implication, produced rRNA.Somatic cell hybrids between either mouse peritoneal macrophages or mouse cells obtained from a teratocarcinoma and HT-1080 human fibrosarcoma cells retain human chromosomes and lose mouse chromosomes (12). If preferential chromosome elimination is closely correlated with preferential suppression of nucleolus organizer activity, these hybrids should express only human nucleolus organizer activity. This appears to be the case. METHODS BALB/c mouse peritoneal macrophages were obtained according to a modification of the procedure described by Cohn and Benson (13) and were fused with HT-1080-6TG human fibrosarcoma cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) (12) in the presence of f3-propiolactone-inactivated Sendai virus at pH 8.0 (14). The fused cultures were maintained in hypoxanthine-aminopterin-thymidine (HAT) selective medium (15). Large colonies of hybrid cells were visible 3-4 weeks after fusion. The colonies were picked and, subsequently, grown. OTT6050 mouse teratocarcinoma cells were obtained from a solid teratocarcinoma of a strain 129 mouse (16,17) by cutting in small fragments in trypsin/EDTA, resuspending in Eagle's minimal essential medium (MEM), and filtering through sterile gauze. The teratocarcinoma cells were fused with HT-1080-6TG cells in the presence of f.-propiolactone-inactivated Sendai virus. Hybrid colonies were selected in hypoxanthine-aminopterin-thymidine medium.Hybrid cells were maintained in Eagle's medium supplemented with 10% fetal calf serum. Mitotic cells were shaken from the culture flasks and transferred to a centrifuge tube containing 0.1 ml of colcemid (10 Ag/ml) for every 10 ml of medium and the tubes were centrifuged immediately at 800 rpm in an IEC clinical centrifuge for 7 min. The cell pellet was resuspended in 75 mM KCI. After 10 min the suspension was centrifuged and the cells were fixed for an hour in freshly prepa...

The origin of the rRNA precursor fromXenopus borealis, analysedin vivoandin vitro

“…The species specificity that is observed in mouse and human cell extracts is paralleled in mouse/human cell hybrids. Cell lines that are segregating human chromosomes synthesize only mouse rRNA, even when human rDNA is present (28,29), while, hybrids that are segregating mouse chromosomes synthesize only human rRNA (30,31). Nucleolar dominance is also observed in Xenopus species hybrids since only laevis rRNA is synthesised in Xenopus laevis/Xenopus borealis hybrid embryos (32).…”

Transcription of mouse rDNA and associated formation of the nucleolus organizer region after gene transfer and amplification in Chinese hamster cells.

“…The number of rRNA genes and the size of the silver-stained NORs (AgNORs) generally parallel the level of nucleolus organizer activity and, presumably, the rRNA transcriptional activity (24,40). Silver staining is restricted to potentially active NORs; it is absent from the nonexpressed rDNA clusters on chromosomes of one species in-rodent-human hybrid cells (7,27,32) and is also absent from homogeneously staining chromosomal regions containing greatly amplified and hypermethylated rDNA (28,37).Transcription of rDNA requires both RNA polymerase I and several protein cofactors; the cofactors from rodent cells do not support in vitro transcription from human rDNA and vice versa (13, 20, 23, 31 predominates (8,26,41,42), and it is unclear whether mouse and hamster rDNAs can use the same transcription cofactors.In this paper, we show that mouse rDNA can be transcribed by using only Chinese hamster components. We describe the transfer of a cloned mouse rRNA gene fragment into dihydrofolate reductase-deficient (dhfr-) hamster cells by DNA-mediated cotransformation with a constructed dhfr gene (9), coamplification of the two genes by stepwise selection of methotrexate-resistant (MTXr) sublines, faithful transcription of some of the rDNA fragments, and silver staining of a fraction of this rDNA chromatin.…”

“…The number of rRNA genes and the size of the silver-stained NORs (AgNORs) generally parallel the level of nucleolus organizer activity and, presumably, the rRNA transcriptional activity (24,40). Silver staining is restricted to potentially active NORs; it is absent from the nonexpressed rDNA clusters on chromosomes of one species in-rodent-human hybrid cells (7,27,32) and is also absent from homogeneously staining chromosomal regions containing greatly amplified and hypermethylated rDNA (28,37).…”

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“…On the other hand, NOR number polymorphism that observed in S. lendlii has been reported in many fish species. The reason for this situation could based on the differentiation of cistron numbers and transcriptional activity (Miller et al, 1976;Warburton and Henderson, 1979;Gaffaroğlu, 2003).…”