Joseph R. Kates

Periodic increases in enzyme activity in synchronized cultures of Chlamydomonas reinhardtii.

Abstract During synchronized growth of Chlamydomonas reinhardtii on a 12-h light-12-h dark cycle, net protein synthesis occurs approximately linearly in the light period but no appreciable increase is observed during the dark period of growth. Increases in activity of alanine dehydrogenase ( l -alanine:NAD oxidoreductase, EC 1.4.1.1), glutamate dehydrogenase ( l -glutamate:NAD(P) oxidoreductase, EC 1.4.1.3), phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylyase (phosphorylating), EC 4.1.1.31), aspartate carbamoyltransferase (carbamoylphosphate: l -aspartate carbamoyltransferase, EC 2.1.3.2) and ornithine carbamoyltransferase (carbamoylphosphate: l -ornithine carbamoyl transferase, EC 2.1.3.3) occur within characteristic periods of time during the life cycle of the cell. The activity of alanine dehydrogenase and aspartate carbamoyltransferase increases primarily in the light period, whereas the activity of the other three enzymes increases during the dark period of growth.

The construction of viable nuclear-cytoplasmic hybrid cells by nuclear transplantation

Using the mouse L-cell line as a model system, a generalized approach is presented for nuclear transplantation in cultured cells resulting in the construction of cytoplasmic-nuclear hybrid cells. Techniques were developed for the preparation of cytoplast and karyoplasts having minimum contamination by parent whole cells. Sendai virusmediated fusion was performed in a manner which maximized the formation of the desired fusion products-cells having one cell equivalent of cytoplasm from one parent and a nucleus from a second parent. The viability of the fusion products was established by examination of photographic records of the developing cultures. Using these techniques, we found that nuclei could be introduced routinely into 10-30% of a cytoplast culture. From determinations of the increase in cell number with time, it was estimated that at least 30% of the reconstructed cells were capable of division. The approach was next applied to the formation of hybrid cells from L-cell cytoplasts and A9 cell karyoplasts. The A9 cell line is an azaguanine-resistant derivative of L cells. Thus any whole cells remaining in the culture of fused cells were readily eliminated by treatment with the purine analogue. The culture of remaining cytoplasmic-nuclear hybrid cells grew to confluence in the presence of azaguanine. The applicability of the approach to the construction of hybrid cells using parent lines from different organisms is briefly discussed.

Purification and characterization of regenerating mouse L929 karyoplasts

Within 72-96 hr after preparation, about 10% of the karyoplasts made from mouse L929 cells regenerated to reform whole viable cells. As soon as 30 hr after preparation, however, nearly all of the remaining 90% of karyoplasts were dead. By separating living and dead karyoplasts at 30 hr, therefore, that fraction destined to complete regeneration was effectively purified. Complete separation was accomplished by sedimentation through Ficoll-paque (Pharmacia), a patented preparation originally developed for the separation of monocytes from whole blood. With the addition of this technique to the previously reported purification scheme for karyoplasts, various biochemical and morphological studies were attempted. Of particular importance are results indicating that karyoplasts that regenerate do not initially contain any more cytoplasm than the average karyoplasts in a preparation--that is, about 10% of the cytoplasm within a whole cell. Electron microscopy of karyoplasts immediately after preparation indicated an unequal partitioning of cytoplasmic organelles at the time of enucleation. For example, karyoplasts initially contain about 11.4% of the mitochondrial volume of whole cells, but only 2.9% of the Golgi apparatus. The size of the karyoplasts and the volume occupied by a variety of organelles was followed throughout the process of regeneration. Although there was an approximately linear increase in the diameter of regenerating karyoplasts, there appeared not to be a simple concordant increase in the volume occupied by all cellular organelles. An extensive investigation was performed to determine whether or not karyoplasts contained centrioles. Immediately after enucleation, 15,000 random thin sections through karyoplasts, which represented about 100 complete bodies, were examined for the presence or absence of centrioles. No centrioles were observed. Examination of the cytoplasts revealed that they contained a sufficient number of centrioles to account for all of the centrioles that were present in the whole cells before enucleation. Centrioles were first detected in karyoplasts in 24 hr after preparation, about the same time that karyoplasts regained the ability to adhere to the surface of tissue culture dishes. At this time, however, the average karyoplast had less than one centriole. By 72 hr, the regenerated karyoplasts had approximately the same number of centrioles as whole cells.

Protein turnover and macromolecular synthesis during growth and gametic differentiation in Chlamydomonas reinhardtii

Abstract Initiation of DNA replication in Chlamydomonas reinhardtii requires the synthesis of a protein and is inhibited by both actinomycin D and chloramphenicol when added a short time before the normal initiation of DNA. Protein turnover occurs in synchronized cultures of this alga during the dark period and during gametic differentiation. Protein turnover occurs in the arginine-less mutants of C. reinhardtii in the light only if NH + 4 is absent from the culture medium. C. reinhardtii possesses a relaxed control of RNA synthesis during amino acid starvation. Under these conditions both tRNA and rRNA are synthesized. After prolonged periods of starvation, tRNA may be synthesized preferentially over rRNA. However, when the alga grows in a non-synchronous culture there is a tight control of RNA synthesis during an energy “step-down”.

Chapter 20 Nuclear Transplantation with Mammalian Cells

Publisher Summary This chapter describes the techniques required to construct large, viable, homogeneous populations of true cytoplasmic-nuclear hybrids. These techniques are being applied to the construction of novel systems for investigating gene expression and its control in mammalian cells. Thus, the cytoplasts and karyoplasts prepared from various differentiated and transformed cell lines that exhibit distinct morphological and/or biochemical traits are fused to form true cytoplasmic-nuclear hybrid cells. The hybrids and their progeny are analyzed for the maintenance, loss, or acquisition of the parental cell traits. The chapter outlines the methods for the Preparation and Characterization of Karyoplasts and of Cytoplasts. The procedure for nuclear transplantation involves the preparation of Sendai virus, virus-mediated fusion, characterization of hybrids, and genetic selection techniques. The selection procedure should be applicable for the selection of cytoplasmic-nuclear hybrids. Thus, before fusing cytoplasts and karyoplasts derived from cell lines of interest, the mutants resistant to appropriate drugs, and toxins can first be selected.

NUCLEAR TRANSPLANTATION WITH MAMMALIAN CELLS

Using the technique of cytochalasin-induced enucleation in a centrifugal field, cultures of cytoplasts and karyoplasts were prepared from many mammalian cell lines, including mouse L929, A9 and neuroblastoma cells and Chinese hamster ovary cells. Detailed characterization of karyoplasts from the L929 and A9 lines indicated that they contained no more than 10-20% of the cytoplasm found within whole cells. After separation of karyoplasts from contaminating cytoplasmic fragments and whole cells, they were fused to cytoplasts by Sendai virus-mediated fusion. Up to 30% of an L-cell cytoplast culture could be re-nucleated using karyoplasts from either L929 or A9 cells. The reconstituted and hybrid cells were viable, that is, capable of division. Consideration of these results and of observations made using other cell lines demonstrated that the techniques should be of general applicability in the investigation of cytoplasmic-nuclear interactions.