No abstract

Mouse L929 cells were separated into enucleated cytoplasmic components (cytoplasts) and nucleated subcellular fractions (karyoplasts) in the presence of cytochalasin B. Karyoplasts from cells containing tritiated nuclei were fused, using inactivated Sendai virus, to cytoplasts from cells containing large (1.0-pm diameter) latex spheres in the cytoplasm. Mononucleated cells containing radioactive nuclei and large latex spheres in the cytoplasm were observed among the products of the fusion reaction. Some of these cells were in mitotic configurations. The results indicate that cells capable of undergoing mitosis can be reconstructed from the products of cellular enucleation in the presence of cytochalasin B.Cytochalasin B causes the nucleus of animal cells growing as monolayers in culture to segregate into an outpocketing of cytoplasm on the top of the cell (1). The process frequently develops to such an extent that the outpocketing remains attached to the main body of the cytoplasm only by a thin cytoplasmic stalk. Occasionally the stalk breaks, and the cell becomes separated into nucleate and enucleate portions (1). The frequency of this separation into nuclear and cytoplasmic parts can be greatly increased by pulling the nucleus away from the cell with centrifugal force while the cell is exposed to cytochalasin B (2, 3).The enucleated cells (cytoplasts) quickly recover from the distortion produced by cytochalasin and remain intact for 1-3 days (4). These cytoplasts continue to synthesize proteins for at least 12 hr (2) and can support synthesis activities of viruses (5, 6). Cytoplasts can also be trypsinized and replated on a new substrate (7).The nuclei removed from cells by the cytochalasin-centrifugation technique are surrounded by a thin shell of cytoplasm and a plasma membrane (8). These nucleated structures (karyoplasts) contain ribosomes, occasional mitochondria, and a few fragments of the endoplasmic reticulum (8), but always lack centrioles, microtubules, and a Golgi apparatus (4). Karyoplasts remain intact for a least 24 hr and continue to synthesize RNA for several hr after the enucleation procedure.Using inactivated Sendai virus as a fusing agent, it should be possible to reconstruct a whole cell by joining a cytoplast with a karyoplast. Development of such a technique would permit the construction of new cells in which the nucleus is derived from one parental cell type and the bulk of the cytoplasm is derived from another cell type. Such "hybrids" would be useful for analyzing nuclear-cytoplasmic interactions on a variety of cellular activities involved in the cell life cycle, cell aging, differentiation, and cell transformation.We have developed a procedure for the reconstruction of mouse L cells from nuclear and cytoplasmic parts (karyoplasts and cytoplasts). Such reconstructed cells are viable as judged by their ability subsequently to enter mitosis. The reconstruction method is described in this paper. MATERIALS AND METHODSMouse L929 cells were grown in Eagle's Minimal Essential Medium ...

Although DNA polymerase-a (DNA nucleotidyltransferase; deoxynucleoside triphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) probably functions in the nucleus, it is usually found predominantly in the nonnuclear fraction of disrupted cells. We have reexamined the intracellular location of this enzyme using cytochalasin-B-induced enucleation, a technique which avoids exposure of nuclei to extra-cellular conditions during cell fractionation. In conditions where viability of separated cell parts is high and recovery is quantitative, we find greater than 85% of total DNA polymerase-a (and DNA polymerase-P) activity in the nucleated cell fragments (karyoplasts), from which we conclude that the location in vivo of DNA polymerase-a is either nuclear or perinuclear. On the other hand, thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7. MATERIALS AND METHODS Mouse L929 cells were grown in Eagle's minimum essential medium with 10% fetal calf serum (Kansas City Biological) and were judged to be mycoplasma-free by the following tests: cytoplasmic thymidine incorporation (19), surface appearance in the scanning electron microscope (performed by J. Meek and M. Clark), and conversion of uridine to uracil by cell extracts (20). Enucleation was performed by centrifugation at 370 in medium containing 10 .g/ml of cytochalasin B (Aldrich Chemical), after a pre-spin to remove weakly attached cells, as described elsewhere (21). Cytoplasts or untreated whole cells were removed by trypsin treatment, collected by a brief centrifugation 5.min at 300 X g), and gently resuspended in phosphate-b ffered saline. The karyoplasts were gently resuspended in the enucleation flasks in phosphate-buffered saline. Concentrations of whole cells, karyoplasts, and cytoplasts in the suspensions were determined by drying drops of known weight on microscope slides; these preparations were subsequently fixed, Feulgen stained, methyl green counter-stained, and all particles from each drop were scored as whole cells, karyoplasts, or cytoplasts. After samples were removed from the suspensions for counting and determination of intactness (trypan blue exclusion), the whole cells and cell parts were collected by a final centrifugation. Examination of supernatants indicated complete recovery (>99%) in the three pellets.For

No abstract

No abstract