scholarly journals Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding.

1985 ◽  
Vol 101 (3) ◽  
pp. 755-765 ◽  
Author(s):  
T J Mitchison ◽  
M W Kirschner
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Lag Phase ◽  
Ovary Cells ◽  
Tubulin Binding ◽  
Mixed Polarity ◽  
Tubulin Immunofluorescence

We have isolated chromosomes from Chinese hamster ovary cells arrested in mitosis with vinblastine and examined the interactions of their kinetochores with purified tubulin in vitro. The kinetochores nucleate microtubule (MT) growth with complex kinetics. After an initial lag phase, MTs are continuously nucleated with both plus and minus ends distally localized. This mixed polarity seems inconsistent with the formation of an ordered, homopolar kinetochore fiber in vivo. As isolated from vinblastine-arrested cells, kinetochores contain no bound tubulin. The kinetochores of chromosomes isolated from colcemid-arrested cells or of chromosomes incubated with tubulin in vitro are brightly stained after anti-tubulin immunofluorescence. This bound tubulin is probably not in the form of MTs. It is localized to the corona region by immunoelectron microscopy, where it may play a role in MT nucleation in vitro.

scholarly journals The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

1977 ◽  
Vol 73 (3) ◽  
pp. 601-615 ◽  
Author(s):  
RR Gould ◽  
GG Borisy
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Carbon Coated ◽  
Pericentriolar Material ◽  
And Function

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.

Mutagenic Activity of p-Toluenesulfonhydrazide

1992 ◽  
Vol 8 (6) ◽  
pp. 369-376 ◽  
Author(s):  
David H. Blakey ◽  
Earle R. Nestmann ◽  
Janet M. Bayley ◽  
K. Laurie Maus ◽  
George R. Douglas
Keyword(s):  
Chinese Hamster Ovary ◽  
Mutagenic Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Gene Mutations ◽  
Metabolic Activation ◽  
High Volume ◽  
Ovary Cells

Toluenesulfonhydrazide (TSH) is a high volume production chemical for which there is relatively little toxicological data. In this study, the mutagenic activity of TSH was determined in the Salmonella/mammalian microsome assay and the in vitro chromosomal aberration assay using Chinese hamster ovary cells. TSH induced gene mutations both with and without metabolic activation in the Salmonella/mammalian microsome assay but that it did not induce chromosomal aberrations in Chinese hamster ovary cells. The results of this study indicate that TSH is an in vitro mutagen and should be assessed for in vivo mutagenicity.

Activity and stability of centrosomes in Chinese hamster ovary cells in nucleation of microtubules in vitro

1984 ◽  
Vol 66 (1) ◽  
pp. 277-295
Author(s):  
R. Kuriyama
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Ovary Cells ◽  
Protease Digestion ◽  
The Difference

Mitotic centrosomes were prepared from Chinese hamster ovary cells and their capacity to nucleate microtubules in vitro was demonstrated by incubation with exogenous brain microtubule protein. The number of microtubules polymerized onto centrosomes was directly counted by electron microscopy of whole-mount preparations. This simple and accurate quantitative assay has permitted characterization of the microtubule nucleating activity of centrosomes in vitro. The number of microtubules polymerized onto centrosomes varied according to the structure of the centrosome. The activity was roughly proportional to the centriole number. The number and length of microtubules nucleated by centrosomes depended both on the concentration of tubulin and the incubation time with tubulin. Under saturating conditions, an average of 200–250 microtubules were initiated by single centrosomes. Centrosomal activity is unstable (t 1/2 = 8 h) and could easily be irreversibly disrupted by a medium of high ionic strength. The activity is stabilized by the addition of glycerol. Centrosomes can be stored at −80 degrees C. The optimum pH for microtubule nucleation is 6.8. Activity is sensitive to protease digestion, but neither DNase or RNase affected the nucleating activity of centrosomes. The activity is temperature-sensitive, but addition of phenylmethylsulphonyl fluoride (PMSF) induces thermal stability. At an optimal concentration of 0.5 mg/ml, this drug increased the half-life of the activity (t 1/2 = 95 h) and made it resistant to salt extraction. Protease inhibitors other than PMSF or dansyl fluoride did not have any stabilizing effect on the activity. The difference between the centrosomal structure of polymerized microtubules in vivo and in vitro is discussed.

Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase

1983 ◽  
Vol 3 (8) ◽  
pp. 1468-1477
Author(s):  
K D Mehta ◽  
R S Gupta
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Form ◽  
Single Step ◽  
Adenosine Kinase ◽  
Cross Resistance ◽  
Ovary Cells ◽  
Cell Extracts

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.

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