scholarly journals RNA SYNTHESIS IN CHINESE HAMSTER CELLS

1968 ◽  
Vol 36 (3) ◽  
pp. 583-593 ◽  
Author(s):  
M. D. Enger ◽  
R. A. Tobey ◽  
A. G. Saponara
Keyword(s):  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Specific Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Sedimentation Analysis ◽  
Ovary Cells ◽  
Chinese Hamster Cells ◽  
Mechanical Selection ◽  
Selection Of

The incorporation of methionine-methyl-14C into 18S ribosomal RNA of cultured Chinese hamster ovary cells in early and late interphase has been determined by zone-sedimentation analysis of phenol-extracted RNA preparations. Synchronized cell cultures were prepared for these studies by thymidine treatment and by mechanical selection of mitotic cells. The specific activity of 18S RNA labeled in late interphase was found to be 1.1–1.2 times that of 18S RNA labeled in early interphase. Upon correction for increase in RNA mass, the rate of methylation of 18S RNA in late interphase is about 1.9 times that in early interphase.

scholarly journals Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

1981 ◽  
Vol 1 (10) ◽  
pp. 902-909 ◽  
Author(s):  
C B Hirschberg ◽  
R M Baker ◽  
M Perez ◽  
L A Spencer ◽  
D Watson
Keyword(s):  
Chinese Hamster Ovary ◽  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Wild Type ◽  
Ovary Cells ◽  
Replica Plating ◽  
Selection Of

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.

scholarly journals RNA synthesis in the ultrastructural and biochemical components of the nucleolus of Chinese hamster ovary cells.

1975 ◽  
Vol 66 (3) ◽  
pp. 577-585 ◽  
Author(s):  
A Royal ◽  
R Simard
Keyword(s):  
Chinese Hamster Ovary ◽  
Rna Synthesis ◽  
Biochemical Study ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Rna Molecules ◽  
Biochemical Components ◽  
Rapid Labeling ◽  
Fibrillar Region

A correlated autoradiographic and biochemical study of RNA synthesis in the nucleoli of chinese hamster ovary cells has been made. Quantitative analysis of the labeling indicates that the fibrillar ribonucleoprotein (RNP) component is labeled faster than 80S RNP and 45S RNA molecules, but approaches simultaneously a steady-state 3H to 14C ratio or grains/mum2 after 30 min of [3H]uridine incorporation. On the other hand, the 55S RNP, the 36S + 32S RNA, and the granular RNP components have the same kinetic of labeling with [3H]uridine. These results suggest that the fibrillar and granular RNP components of the nucleolus are the ultrastructural substratum of, respectively, the 80S RNP (45S RNA) and 55S RNP (36S + 32S RNA). The possibility that precursors to 80S RNP exist also in the fibrillar region of the nucleolus is strongly suggested by the rapid labeling of the fibrils on the autoradiographs.

scholarly journals Binding of [125I] wheat germ agglutinin to Chinese hamster ovary cells under conditions which affect the mobility of membrane components.

1978 ◽  
Vol 79 (3) ◽  
pp. 617-622 ◽  
Author(s):  
P Stanley ◽  
J P Carver
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Specific Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
High Specific Activity ◽  
Ovary Cells ◽  
Membrane Components

The binding of [125I]wheat germ agglutinin ([125I]WGA) of high specific activity to Chinese hamster ovary (CHO) cells has been examined over a millionfold range of WGA concentrations and correlated with the phenomena of agglutination and capping by WGA. Analysis of the binding data by the method of Scatchard gives a complex curve indicative of positive cooperativity amongst high-affinity binding sites. Binding assays performed under conditions which inhibit capping and/or agglutination, such as low temperature or glutaraldehyde fixation, give similarly complex binding curves. Thus, the gross mobility of WGA receptors in the membrane does not appear to be responsible for the cooperative binding of WGA to CHO cells.

Selection of mitotic Chinese hamster ovary cells from microcarriers

1980 ◽  
Vol 109 (2) ◽  
pp. 231-238 ◽  
Author(s):  
John J.Y. Ng ◽  
Charles L. Crespi ◽  
William G. Thilly
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Selection Of

scholarly journals Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis.

1980 ◽  
Vol 86 (3) ◽  
pp. 844-848 ◽  
Author(s):  
M Salditt-Georgieff ◽  
M Harpold ◽  
S Sawicki ◽  
J Nevins ◽  
J E Darnell
Keyword(s):  
Chinese Hamster Ovary ◽  
Rna Synthesis ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Rna Sequences ◽  
Ovary Cells ◽  
Different Types ◽  
Nuclear Rna ◽  
Specific Mrnas ◽  
Chain Synthesis

A kinetic analysis of the appearance of [3H]uridine label in RNA sequences that neighbor poly(A), as well as the incorporation of [3H]adenosine label into both the RNA chain and the poly(A) of poly(A)-containing molecules, shows that poly(A) is added within a minute or so after RNA chain synthesis in Chinese hamster ovary cells and HeLa cells. Previous conclusions by several groups (5-7) that poly(A) might be added as long as 20-30 min after RNA synthesis appear to be in error, and the present conclusion seems much more in line with several different types of recent studies with specific mRNAs that suggest prompt poly(A) addition (13-16).

scholarly journals Polyamine-mediated turnover of ornithine decarboxylase in Chinese-hamster ovary cells

1986 ◽  
Vol 236 (2) ◽  
pp. 351-357 ◽  
Author(s):  
J R Glass ◽  
E W Gerner
Keyword(s):  
Ornithine Decarboxylase ◽  
Chinese Hamster Ovary ◽  
Specific Activity ◽  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Defined Medium ◽  
Ovary Cells ◽  
Exogenous Substrate ◽  
Polyamine Content

We have used Chinese-hamster ovary (CHO) cells maintained in a chemically defined medium to study the regulation of ornithine decarboxylase (ODC) by polyamines. Cells maintained in the defined medium had no detectable putrescine, and approx. 1-3 units of ODC activity/10(6) cells, where 1 unit corresponds to 1 nmol of substrate decarboxylated in 30 min. The defined medium is ornithine-deficient, thus limiting the exogenous substrate for ODC, and subsequently decreasing intracellular polyamine accumulation. Restoration of intracellular putrescine and increased formation of spermidine by addition of exogenous ornithine or putrescine led to a marked decrease in ODC activity, which was paralleled by a decrease in a alpha-DL-difluoromethyl[3,4-3H]ornithine (DFMO)-binding protein of Mr approx. 53,000, which is precipitable with anti-ODC antibody. Calculation of DFMO binding per unit of activity showed no change in the specific activity of the enzyme. We identified [35S]methionine-labelled peptides corresponding to ODC by immunoprecipitation of radiolabeled whole cell proteins. Only one protein was precipitated, of Mr approx. 53 000, which co-migrated with the DFMO-binding protein. Immunoprecipitation of radiolabelled proteins from cells incubated in the presence of exogenous ornithine indicated that the observed activity decrease was not due to an inhibition of ODC protein synthesis. Analysis of immunoprecipitable ODC protein from cells that had been pulse-labelled with [35S]methionine, and then treated for 5 h with 100 microM-ornithine, -putrescine or -spermidine, revealed a distinct disappearance of labelled ODC protein after restoration of intracellular polyamine pools. No detectable turnover of ODC was observed in the absence of exogenous polyamine treatment. These data support the hypothesis that ODC protein, and subsequent activity, is regulated by intracellular polyamine content through mechanisms that influence turnover of the enzyme.

scholarly journals Intracellular transport of human lysosomal α-mannosidase and α-mannosidosis-related mutants

2004 ◽  
Vol 381 (2) ◽  
pp. 537-546 ◽  
Author(s):  
Gaute HANSEN ◽  
Thomas BERG ◽  
Hilde M. F. RIISE STENSLAND ◽  
Pirkko HEIKINHEIMO ◽  
Helle KLENOW ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Active Site ◽  
Specific Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
In Vitro Mutagenesis ◽  
Single Chain ◽  
African Green Monkey Kidney ◽  
Ovary Cells ◽  
Cos Cells

Human LAMAN (lysosomal α-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a single-chain form and partly sorted to the lysosomes being subsequently cleaved into three peptides of 70, 40 and 15 kDa respectively. Both the secreted and the lysosomal forms contained endo H (endoglucosidase H)-resistant glycans, suggesting a common pathway through the trans-Golgi network. A fraction of LAMAN was retained intracellularly as a single-chain endo H-sensitive form, probably in the ER (endoplasmic reticulum). The inherited lack of LAMAN causes the autosomal recessive storage disease α-mannosidosis. To understand the biochemical consequences of the disease-causing mutations, 11 missense mutations and two in-frame deletions were introduced into human LAMAN cDNA by in vitro mutagenesis and the resulting proteins were expressed in COS cells. Some selected mutants were also expressed in Chinese-hamster ovary cells. T355P (Thr355→Pro), P356R, W714R, R750W and L809P LAMANs as well as both deletion mutants were misfolded and arrested in the ER as inactive single-chain forms. Six of the mutants were transported to the lysosomes, either with less than 5% of normal specific activity (H72L, D196E/N and R220H LAMANs) or with more than 30% of normal specific activity (E402K LAMAN). F320L LAMAN resulted in much lower activity in Chinese-hamster ovary cells when compared with COS cells. Modelling into the three-dimensional structure revealed that the mutants with highly reduced specific activities contained substitutions of amino acids involved in the catalysis, either co-ordinating Zn2+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320).

scholarly journals THE ACTION OF α-AMANITIN ON RNA SYNTHESIS IN CHINESE HAMSTER OVARY CELLS

1974 ◽  
Vol 63 (3) ◽  
pp. 831-842 ◽  
Author(s):  
Claude Kedinger ◽  
Rene Simard
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mouse Liver ◽  
Rna Synthesis ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Drug Leads ◽  
Nucleolar Rna

α-Amanitin acts in vitro as a selective inhibitor of the nucleoplasmic form B RNA polymerases. Treatment of Chinese hamster ovary (CHO) cells with this drug leads principally to a severe fragmentation of the nucleoli. While the ultrastructural lesions induced by α-amanitin in CHO cells and in rat or mouse liver are quite similar, the results diverge concerning the effect on RNA synthesis. It has been shown that in rat or mouse liver α-amanitin blocks both extranucleolar and nucleolar RNA synthesis. Our autoradiographic and biochemical evidence indicates that in CHO cells high molecular weight extranucleolar RNA synthesis (HnRNA) is blocked by the α-amanitin treatment, whereas nucleolar RNA (preribosomal RNA) synthesis remains unaffected even several hours after the inhibition of extranucleolar RNA synthesis. Furthermore, the processing of this RNA as well as its transport to the cytoplasm seem only slightly affected by the treatment. Finally, under these conditions, the synthesis of the low molecular RNA species (4–5S) still occurs, though less actively. The results are interpreted as evidence for a selective impairment of HnRNA synthesis by α-amanitin in CHO cells.

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