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Biomedical subjects

N R Ringertz

Publications and source records attributed to N R Ringertz.

At least 55 records · Page 3Linked to original sources
PubMed1979

Restoration of metabolic cooperation in heterokaryons between HGPRT-deficient mouse A9 fibroblasts and chick embryo erythrocytes.

Genetic determinants of metabolic cooperation were studied by fusing chick erythrocytes to HGPRT- mammalian cells. Heterokaryons were then tested for their ability to incorporate [3H]hypoxanthine and to transfer radioactive material to HGPRT- recipient cells. Chick erythrocytes (CE) have nuclei which are inactive but contain the HGPRT gene and some cytoplasmic HGPRT enzyme activity. They are unable, however, to cooperate with HGPRT- cells. Of the two mammalian cell lines used, the human GM29 line is HGPRT- and capable of functioning as a receptor cell in cooperation experiments with HGPRT+ cells. The HGPRT- mouse A9 line on the other hand is unable to cooperate. Immediately after fusion, both types of heterokaryons incorporated [3H]hypoxanthine, indicating the presence of some chick HGPRT enzyme contributed by the erythrocyte partner at the time of fusion. While the CE-GM29 heterokaryons participated in metabolic cooperation shortly after fusion, the CE-A9 heterokaryons did not. However, four days after fusion, i.e., at a time when the erythrocyte nucleus had been reactivated, the CE-A9 heterokaryons did cooperate. This suggests that in CE-A9 heterokaryons the genes required for metabolic cooperation are expressed by the previously dormant chick erythrocyte nucleus.

Animals↗
PubMed1978

Reconstitution of cells by fusion of cell fragments. I. Myogenic expression after fusion of minicells from rat myoblasts (L6) with mouse fibroblast (A9) cytoplasm.

Rat L6 myoblasts and mouse A9 fibroblasts (HGPRT-) were enucleated by centrifugation of monolayers in the presence of cytochalasin B. Intact cells were reconstituted by Sendai virus mediated fusion of nuclei (minicells) from enucleated rat myoblasts and cytoplasms from enucleated mouse fibroblasts. Colonies arising from proliferating reconstituted cells were distinguished from intact parental cell types on the basis of nuclear and cytoplasmic markers. In five replicate experiments, approx. 70% of all colonies found after fusion were derived from reconstituted cells, 30% arose from intact rat myoblasts contaminating the minicell preparations, and two colonies were identified as hybrids between the parental cell types. Clones derived from reconstituted cells formed myotubes which produced myosin and developed the cross-striated pattern typical of skeletal muscle. The myogenic program of the rat myoblast thus can persist through the enucleation and reconstitution procedures, and is not obviously altered by a period of exposure to mouse fibroblast cytoplasm.

Animals↗
PubMed1978

DNA content of microcells prepared from rat kangaroo and mouse cells.

Enucleation of animal cells in which nuclear fragmentation (micronucleation) has been induced by treatment with mitotic inhibitors results in the formation of subdiploid microcells consisting of one or several micronuclei, some cytoplasm and surrounded by a plasma membrane. Microcells were prepared from rat kangaroo cells (12 chromosomes) and a polyoma virus transformed mouse cell line (61 chromosomes) and analysed for DNA content. Microspectrophotometric DNA measurements and the appearance of micronuclei at mitosis show that small micronuclei contain genetic information equivalent to single chromosomes. A large proportion of the micronuclei and the microcells, however, contains DNA corresponding to several chromosomes. Heterogeneous mixtures of microcells can be fractionated by a unit gravity sedimentation procedure so as to isolate the small microcells. These can afterwards be fused with intact normal or mutant cells.

Animals↗
PubMed1978

Reconstruction of cells by Sendai virus-induced fusion of cell fragments.

Large numbers of animal cells can be enucleated by centrifugation in the presence of cytochalasin. The nuclei (minicells) obtained by this technique are surrounded by a thin rim of cytoplasm and a cell membrane. The cytoplasms (anucleate cells) contain cytoplasmic organelles and are capable of protein synthesis. By themselves, these cell fragments are not viable under usual culture conditions for longer than 2 days after enucleation. They can, however, be fused by UV-inactivated Sendai virus to form reconstituted cells. Nuclei from rat myoblasts have been combined with cytoplasms from mouse fibroblasts. The resulting reconstituted cells are viable and multiply rapidly in vitro. Enucleation or disruption of "micronucleated" cells, cells in which the genome has been fragmented into many small micronuclei by prolonged exposure to antimitotic agents results in microcells. These cell fragments are subdiploid and may contain as little as a single chromosome. Fusion of microcells from normal cells with intact mutant cells offers a method of transferring complementing chromosomes into genetically defective cells. Specific areas in which reconstitution techniques should prove useful include studies of gene expression, cell differentiation, and the dependence of mitochondria on nuclear genes. Microcell hybrids could be useful in chromosome mapping and gene complementation analysis.

Animals↗
PubMed1977

Cells reconstituted from cell fragments of two different species multiply and form colonies.

Nuclei (minicells) prepared by cytochalasin-induced enucleation of rat myoblasts (L6) were fused with inactivated Sendai virus to preparations of cytoplasms from mouse fibroblasts (A9). Cell colonies arising from such fusion mixtures were examined 5 days after fusion. In the presence of virus a new type of colony was obtained. The cells in these colonies were derived from reconstituted cells capable of undergoing repeated cell divisions.

Cell Nucleus↗
PubMed1975

Expression of SV40 T antigen during the cell cycle of sv40-transformed cells.

The expression of the nuclear SV40-induced T antigen was measured by microfluorimetry on individual, asynchronously growing SV40-transformed cells which had been stained with hamster T-antiserum by the indirect immunofluorescence method. The same individual cells were first measured for T antigen and then for DNA by Feulgen microspectrophotometry. A linear correlation was observed between the two parameters. T antigen expression was also measured in cell populations arrested at different phases of the cell cycle. Results of both types of experiments show that the expression of the gene (s) for T antigen in transformed cells increases during DNA replication and reaches its highest level in G2 nuclei. During mitosis T antigen is found in the cytoplasm.

Animals↗
PubMed1975

Reactivation of chick erythrocyte nuclei after fusion with enucleated cells.

Inactivated Sendai virus was used to fuse nucleated chick erythrocytes with mouse L and A9 cells which had been enucleated by centrifugation in the presence of cytochalasinB. The enucleation step removed the nuclei from more than 99% of the cells. During the fusion step, chick erythrocyte nuclei were introduced into 20% of the enucleated mouse cytoplasms. This resulted in the formation of a large number of "reconstituted cells" where practically all the cytoplasm originated from the mouse cell while the nucleus was of chick origin. The chick erythrocyte nuclei appeared to become well integrated into the mouse cytoplasms since they increased dramatically in size and dry mass, formed nucleolus-like bodies, and resumed RNA synthesis. This, however, did not prevent a gradual decrease in the rate of protein synthesis in the cytoplasm after the removal of the mouse nucleus. Protein synthesis decayed at a similar rate in both reconstituted and enucleated cells. The majority of these "cells" died within 48 h and all of them within 5 days after enucleation/fusion. By contrast, the small number of L cells which failed to become enucleated multiplied rapidly. The results obtained suggest that the reactivation of the chick erythrocyte nuclei is not fast enough to rescue the enucleated mouse cytoplasms.

Animals↗