Differential expression of three alpha-tubulin genes in Chinese hamster ovary cells

1985 ◽  
Vol 5 (1) ◽  
pp. 236-241
Author(s):  
E M Elliott ◽  
H Okayama ◽  
F Sarangi ◽  
G Henderson ◽  
V Ling
Keyword(s):  
Chinese Hamster Ovary ◽  
Sequence Data ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Cdna Libraries ◽  
Coding Region ◽  
Ovary Cells ◽  
Alpha Tubulin ◽  
Tubulin Genes

Chinese hamster ovary cells contain a complex family of ca. 16 unique alpha-tubulin sequences and a similar multiplicity of beta sequences. To examine which members of this multigene family are expressed, we constructed cDNA libraries from two Chinese hamster ovary cell lines according to the method of H. Okayama and P. Berg (Mol. Cell. Biol. 3:280-289, 1983). Each library consisted of 5.5 X 10(5) transformants and contained a high percentage of full-length tubulin clones. Three different alpha-tubulin genes were identified by sequence analysis of the 3' noncoding regions of these tubulin clones. The relative abundance of the transcripts corresponding to the three genes was estimated by gene-specific dot blotting of 96 cDNA alpha-tubulin clones and was found to be 71, 24, and 5%. There is little homology in the 3' noncoding sequences of these genes; however, a strong interspecies homology exists in this region for two of the Chinese hamster ovary genes with the two alpha-tubulin genes previously described in other systems. The third Chinese hamster ovary gene, with an expression frequency of 24%, is unique in that its 3' noncoding region is unlike that of the other mammalian alpha-tubulin genes. In addition, limited sequence data from the coding region of this gene indicates it codes for a unique alpha-tubulin protein.

scholarly journals Differential expression of three alpha-tubulin genes in Chinese hamster ovary cells.

1985 ◽  
Vol 5 (1) ◽  
pp. 236-241 ◽  
Author(s):  
E M Elliott ◽  
H Okayama ◽  
F Sarangi ◽  
G Henderson ◽  
V Ling
Keyword(s):  
Chinese Hamster Ovary ◽  
Sequence Data ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Cdna Libraries ◽  
Coding Region ◽  
Ovary Cells ◽  
Alpha Tubulin ◽  
Tubulin Genes

Chinese hamster ovary cells contain a complex family of ca. 16 unique alpha-tubulin sequences and a similar multiplicity of beta sequences. To examine which members of this multigene family are expressed, we constructed cDNA libraries from two Chinese hamster ovary cell lines according to the method of H. Okayama and P. Berg (Mol. Cell. Biol. 3:280-289, 1983). Each library consisted of 5.5 X 10(5) transformants and contained a high percentage of full-length tubulin clones. Three different alpha-tubulin genes were identified by sequence analysis of the 3' noncoding regions of these tubulin clones. The relative abundance of the transcripts corresponding to the three genes was estimated by gene-specific dot blotting of 96 cDNA alpha-tubulin clones and was found to be 71, 24, and 5%. There is little homology in the 3' noncoding sequences of these genes; however, a strong interspecies homology exists in this region for two of the Chinese hamster ovary genes with the two alpha-tubulin genes previously described in other systems. The third Chinese hamster ovary gene, with an expression frequency of 24%, is unique in that its 3' noncoding region is unlike that of the other mammalian alpha-tubulin genes. In addition, limited sequence data from the coding region of this gene indicates it codes for a unique alpha-tubulin protein.

Cloning of 11 α-tubulin gene sequences from the genome of Chinese hamster ovary cells

1985 ◽  
Vol 63 (6) ◽  
pp. 511-518 ◽  
Author(s):  
Elizabeth M. Elliott ◽  
Farida Sarangi ◽  
Graham Henderson ◽  
Victor Ling
Keyword(s):  
Chinese Hamster Ovary ◽  
Genomic Library ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Coding Region ◽  
Tubulin Gene ◽  
Ovary Cells ◽  
Tubulin Genes ◽  
Clonal Lines ◽  
Β Tubulin

We have analyzed the complex tubulin gene family in clonal lines of Chinese hamster ovary cells. There are approximately 16 α-tubulin genes and a similar multiplicity of β-tubulin genes. The α-tubulin genes are not closely linked to each other nor to the β-tubulin genes. A genomic library has been constructed in the vector λ charon 4A containing insert sizes of 13–20 kilobases. The library has been screened with both inter- and intra-species α-tubulin probes. Eleven α-tubulin clones with different restriction patterns have been isolated and characterized. At least seven of these clones contain the complete gene coding region. One clone appears to represent the transcribed α-tubulin gene II. The sequence of an intron from this gene is compared with that from an equivalent gene in the rat.

Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer

1989 ◽  
Vol 9 (4) ◽  
pp. 1754-1758
Author(s):  
T M Underhill ◽  
W F Flintoff
Keyword(s):  
Gene Transfer ◽  
Dna Sequences ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Ovary Cells ◽  
Ovary Cell Line ◽  
Human Specific

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.

Chinese hamster ovary cell mutants defective in the internalization of ricin

1982 ◽  
Vol 2 (5) ◽  
pp. 535-544
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Electron Microscopic ◽  
Ovary Cells ◽  
Internalization Process ◽  
In The Wild ◽  
Mutant Cells

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.

scholarly journals Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line.

1981 ◽  
Vol 1 (12) ◽  
pp. 1069-1076 ◽  
Author(s):  
R J Kaufman ◽  
R T Schimke
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Ovary Cells ◽  
Methotrexate Resistance ◽  
Gene Copies ◽  
Methotrexate Concentration ◽  
Ovary Cell Line

During stepwise increases in the methotrexate concentration in culture medium, we selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). We studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

scholarly journals Selection of specific wheat germ agglutinin-resistant (WgaR) phenotypes from Chinese hamster ovary cell populations containing numerous lecR genotypes.

1981 ◽  
Vol 1 (8) ◽  
pp. 687-696 ◽  
Author(s):  
P Stanley
Keyword(s):  
Chinese Hamster Ovary ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Ovary Cell ◽  
Mutant Type ◽  
Similar Frequency ◽  
Ovary Cells

Three distinct Chinese hamster ovary mutants selected for resistance to wheat germ agglutinin were previously described by this laboratory. In this paper, evidence is provided that each phenotype occurs at a similar frequency in an unmutagenized population of Chinese hamster ovary cells. Two novel wheat germ agglutinin resistance phenotypes (WgaR), which also appear to occur at similar frequencies were uncovered in the course of these studies. One mutant type belongs to a new, recessive complementation group (VIII), and the second belongs to a previously defined complementation group (VI). Mutants from each of the four WgaR complementation groups (I, II, III, and VIII) exhibited characteristic and unique patterns of resistance to the toxicity of a variety of plant lectins. These properties were used in developing independent selection protocols which were highly specific for the isolation of each of the mutant types.

Melanogenesis in murine B16 cells exposed to Aeromonas hydrophila cytotoxic enterotoxin

1986 ◽  
Vol 32 (10) ◽  
pp. 814-819 ◽  
Author(s):  
V. K. Bunning ◽  
R. G. Crawford ◽  
G. N. Stelma Jr. ◽  
L. O. Kaylor ◽  
C. H. Johnson
Keyword(s):  
Cholera Toxin ◽  
Chinese Hamster Ovary ◽  
Aeromonas Hydrophila ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Transferase Activity ◽  
End Points ◽  
Ovary Cells ◽  
Culture Filtrates

Specific markers (growth, melanogenesis) of B16 murine melanoma cells in culture were used as indicators of toxin production by Aeromonas hydrophila. Cytotonic enterotoxinlike activity (inhibited growth, raised tyrosinase activity, and melanin accumulation) occurred at cytotoxic end points of purified β-hemolysin and several culture filtrates. Antihemolysin rabbit serum inhibited this activity. A hemolysin-neutralized culture filtrate concentrate (10×) failed to elevate tyrosinase relative to untreated and cholera toxin treated controls. Similar dilution profiles using Chinese hamster ovary cells showed limited cell extension only at cytotoxic end points with antihemolysin inhibiting this activity. Cytotoxicity of Chinese hamster ovary cells and B16 cells was proportional to hemolytic activity, with B16 cells showing about 100-fold greater sensitivity on a per cell basis. Cell culture cytotoxicity attributed to β-hemolysin correlated with reactivity in rabbit ileal loop assays. The ADP-ribosyl transferase activity of concentrated (10×) A. hydrophila culture filtrates and fractions thereof was negative. Apparently sublethal doses of A. hydrophila β-hemolysin can nonspecifically stimulate cyclic adenosine monophosphate mediated events in melanoma and Chinese hamster ovary cell assays, producing lower activities than cholera toxin with shorter lag times.

scholarly journals Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

1984 ◽  
Vol 4 (2) ◽  
pp. 296-301 ◽  
Author(s):  
B Storrie ◽  
M Sachdeva ◽  
V S Viers
Keyword(s):  
Horseradish Peroxidase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Ovary Cell ◽  
Cell Fractionation ◽  
Marker Enzyme ◽  
Ovary Cells

We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

scholarly journals Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

1982 ◽  
Vol 2 (3) ◽  
pp. 250-257 ◽  
Author(s):  
J A Tischfield ◽  
J J Trill ◽  
Y I Lee ◽  
K Coy ◽  
M W Taylor
Keyword(s):  
Chinese Hamster Ovary ◽  
Hot Spot ◽  
Chinese Hamster Ovary Cells ◽  
Human Fibroblasts ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Adenine Phosphoribosyltransferase ◽  
Ovary Cells ◽  
L Cells ◽  
A Site

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.

scholarly journals Expression and amplification of engineered mouse dihydrofolate reductase minigenes.

1983 ◽  
Vol 3 (2) ◽  
pp. 257-266 ◽  
Author(s):  
G F Crouse ◽  
R N McEwan ◽  
M L Pearson
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Cdna Clones ◽  
Coding Region ◽  
Chromosomal Dna ◽  
Lambda Phage ◽  
Ovary Cells

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.

Sign in / Sign up

Export Citation Format

Share Document