Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells

The spatial distribution of replication sites was studied by a sensitive method in cells cultured in vitro. Exponentially growing Chinese hamster ovary cells were permeabilized and pulse labeled in the presence of deoxyribonucleoside triphosphates, dTTP being replaced by biotin-11-dUTP as a substrate for DNA replication. The distribution of replication sites was visualized in isolated nuclei by fluorescent microscopy of samples taken periodically after short-term (2 min) in vitro labeling and pulse-chase experiments. Propidium iodide and 4,6-diamino-2-phenylindole served as fluorescent probes for total cellular DNA. Avidin-fluorescein isothiocyanate and biotinylated goat antiavidin antibody were used in an amplification procedure to fluorescently label the incorporated biotin-11-dUTP. Similar experiments using synchronized cells showed the distribution of replicons at different stages of S phase.     

Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells

Summary The spatial distribution of replication sites was studied by a sensitive method in cells cultured in vitro. Exponentially growing Chinese hamster ovary cells were permeabilized and pulse labeled in the presence of deoxyribonucleoside triphosphates, dTTP being replaced by biotin-11-dUTP as a substrate for DNA replication. The distribution of replication sites was visualized in isolated nuclei by fluorescent microscopy of samples taken periodically after short-term (2 min) in vitro labeling and pulse-chase experiments. Propidium iodide and 4,6-diamino-2-pheny-lindole served as fluorescent probes for total cellular DNA. Avidin-fluorescein isothiocyanate and biotinylated goat antiavidin antibody were used in an amplification procedure to fluorescently label the incorporated biotin-11-dUTP. Similar experiments using synchronized cells showed the distribution of replicons at different stages of S phase.     

Initiation of DNA replication in chromosomes of Chinese hamster ovary cells

The initiation of DNA replication and the subsequent chain elongation were studied using Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by a combination of mitotic selection and treatment with 5-fluorodeoxyuridine (FdU). The use of this drug at a concentration of 10^–5 M was found to effectively prevent the leakage of cells into S phase. Reversal of the FdU block by supplying thymidine resulted in the synchronous onset of initiation at multiple sites in each cell. The length of the nascent chains, as determined by autoradiography and velocity sedimentation in alkaline gradients, increased linearly with time during the first twenty minutes of S phase after release. — We applied these procedures to study the effects of the length of an FdU block on the number of functional origins per cell, the rate of chain growth, and the rate of DNA synthesis per cell following reversal of the block. Although no change was noted in the rate of DNA synthesis in cells held at the beginning of S phase from 10.5 to 24 h after division, the rate of chain growth decreased from 0.94 to 0.28 microns per min. This decrease indicated that the number of functional origins increased markedly with length of FdU block. The calculated number of utilized origins per cell increased from 1,900 to 5,700. We also presented arguments that 1,900 origins per cell represents the approximate number of origins utilized by any cell held at the beginning of S phase for less than 10.5 h after division.     

Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells.

Eukaryotic expression vectors have been used successfully in viral LT-expressing cell lines (ie. COS) to clone cDNAs encoding proteins that can be detected through their bio-activity or reactivity with specific antibodies. Since Chinese hamster ovary cells (CHO) have been used extensively for the isolation and characterization of somatic cell mutants, we felt it would be an advantage to develop an expression cloning system in CHO cells. We have modified the eukaryotic expression vector CDM8 by replacing the polyoma and SV40 origins of replication with the 427bp non-coding region of the Syrian hamster papovavirus. Wild-type CHO cells and the CHO glycosylation-mutant Lec4A were transfected with plasmids bearing the early genes of either polyoma virus or hamster papovavirus in order to establish stable, LT antigen-expressing cell lines designated CHOP or CHOH, respectively. CHOP cell lines expressing polyoma LT antigen supported efficient replication of CDM8, but replicated pMH poorly. Conversely, CHOH cells expressing the hamster papovavirus LT antigen supported replication of pMH, and at a lower efficiency, CDM8. Replication of CDM8 and pMH vectors were equally efficient in selected CHOP and CHOH cell lines, respectively and comparable to that of CDM8 replication in COS-1 cells. A bacterial beta-galactosidase fusion gene inserted into the multiple cloning site of a CDM8 derivative was efficiently expressed when transiently transfected into CHOP and CHOH cells but not CHO cells since only the former supports autonomous plasmid replication. These results show that expression-cloning in CHO cells expressing either polyoma virus or hamster papovavirus LT antigens is possible using either the CDM8 or the pMH vectors, respectively.     

Pesticide clastogenicity in Chinese hamster ovary cells

Paraquat, alachlor, butachlor, phorate and monocrotophos, several of the most extensively used pesticides in Taiwan, were investigated for their clastogenicity using chromosome aberration (CAb) induction in Chinese hamster ovary (CHO) cells. Significance levels of the binomial trend analysis and binomial mutagenicity data test were two criteria for the summary judgement of the pesticide clastogenicity. Except for phorate, all pesticides tested were clastogenic to CHO cells in the absence of in vitro metabolic activation by S9. 5 microliters/ml rat-liver extract, S9, were used as the source of in vitro metabolic activation. 3 different outcomes were found after the addition of S9. Paraquat: significant decrease in induced CAbs. Monocrotophos: concomitant occurrence of decreased cytotoxicity and increased clastogenicity. Alachlor, butachlor and phorate: increased cytotoxicities with no sign of enhancement in clastogenicity.     

DNA replication in Chinese hamster ovary cells made permeable to nucleotides by tween-80 treatment

We have developed a method for permeabilizing CHO cells to nucleotides under conditions which allow most cells to remain viable. Permeabilized cells can carry out ATP-dependent, semiconservative synthesis of DNA. The data are consistent with the continuation of DNA synthesis in those cells in S phase at the time of treatment, possibly limited to completion of replicon synthesis without new initiations.     

Characterization of chromosome replication during S-phase with bromodeoxyuridine labelling in Chinese hamster ovary and HeLa cells

The same C-banded human polymorphic chromosomes were observed in the light microscope (LM) and then in the scanning electron microscope (SEM) to investigate the structural changes produced by the C-banding technique. C-banded regions, which stained positively in LM, were highly condensed with tightly packed chromatin fibres, resembling non-banded chromosomes. In striking contrast, adjacent non-C-banded regions were represented by loosely arranged fibres, resembling G-banded chromosomes. The significance of these observations in relation to current theories on the effects of C-banding on chromosome structure is discussed.     

Effects of mercuric chloride on synchronized Chinese hamster ovary cells: survival and DNA replication

Chinese hamster ovary (CHO) cells in vitro were treated with HgCl2 at various stages in the cell cycle and the effects of this chemical on cell survival, DNA replication, and cell division were observed. In terms of survival the early G1 cells were the most sensitive to treatment, followed by late G1 and early S, while mid S and late S-G2 treated cells were the least sensitive. Treatment with HgCl2 also resulted in reduced rates of DNA replication and delays in cell division. The early G1 treated cells showed substantially reduced rates of DNA replication followed by 4--5 h division delay. The early S and late S-G2 treated cells had some reduction in their rates of DNA replication followed by corresponding division delay of 2.5 h in the early S treated cells and 1 h in the late S-G2 treated cells.     

Dolichol metabolism in Chinese hamster ovary cells.

The addition of oligosaccharide to asparagine residues of soluble and membrane-associated proteins in eukaryotic cells involves a polyisoprenoid lipid carrier, dolichol. In Chinese hamster ovary cells, the major isomer of this polyisoprenol has 19 isoprenyl units, the terminal one being saturated. Our laboratory has developed a procedure to analyze the levels and nature of the cell's dolichyl derivatives. Chinese hamster ovary cells contain predominately activated, anionic dolichol derivatives, such as oligosaccharyl pyrophosphoryldolichol, monoglycosylated phosphoryldolichols, and dolichyl phosphate. Our studies show that in growing cells there is continual synthesis of total dolichol. Also, preliminary data suggest there is no catabolism or secretion of this lipid. The level of dolichyl phosphate did not change significantly under a variety of conditions where the levels of enzyme activities utilizing dolichyl phosphate did change. These results suggested that these enzymes had access to the same pool of dolichyl phosphate and had similar Km values for this lipid.     

Mitotic spindle proteomics in Chinese hamster ovary cells

Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.     

Cycloheximide-resistance in Chinese hamster ovary cells and human fibroblast cells

Summary A total of 3000 men living in Yamaguchi were screened for glucose-6-phosphate dehydrogenase (G6PD) deficiency using Beutler's spot test and three types of starch gel electrophoresis. These electrophoresis used a phosphate buffer system at pH 7.0, a TRIS-EDTA-borate buffer system at pH 8.6, and a TRIS-hydrochloride buffer system at pH 8.8. Fifteen G6PD-deficient variants were found at the rate of 0.5% and classified into four groups. As new variants, G6PD Konan, Kamiube, and Kiwa were identified. These three variants had a mild to moderate G6PD deficiency and were not associated with any clinical signs. G6PD Konan had fast electrophoretic mobility as compared with normal levels, G6PD Kiwa had slightly elevated electrophoretic mobility, and G6PD Kamiube had normal electrophoretic mobility. These three variants had normal levels of Km G6P, Km NADP, and Ki NADPH, normal utilizations of both 2-deoxy-G6P and deamino-NAPD, normal heat stability, and a normal pH curve. The other variant was G6PD Ube, which we had previously found in Yamaguchi (Nakashima et al., 1977). One boy with G6PD Ube was Korean.     

Regulation of polyamine transport in Chinese hamster ovary cells

Control Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with alpha-difluoromethylornithine in CHO cells or via polyamine starvation of CHODC- cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutamine activity (-(γ-glutamyl)lysine crosslinking enzme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of the soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20–70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutaminase activity (epsilon-(gamma-glutamyl)lysine crosslinking enzyme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20--70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

The transport of L-arginine in Chinese hamster ovary cells

The transport of L-arginine has been characterized in Chinese hamster ovary cells (CHO). In the absence of Na+ the influx of the amino acid decreased. Both in the presence and in the absence of Na+ L-arginine influx was trans-stimulated and cis-inhibited by cationic amino acids. The amino acid entered CHO cells through an apparently non saturable mechanism and a single saturable agency whose Km increased in the absence of Na+. These results indicate that the agency devoted to transport cationic amino acids in CHO cells resembles system y+, the Na+-independent route that transports cationic amino acids in a number of mammalian models, although its activity is lowered by the replacement of extracellular sodium.