scholarly journals Transfection of a human gene that corrects the Lec1 glycosylation defect: evidence for transfer of the structural gene for N-acetylglucosaminyltransferase I.

1989 ◽  
Vol 9 (12) ◽  
pp. 5713-5717 ◽  
Author(s):  
R Kumar ◽  
P Stanley
Keyword(s):  
Structural Gene ◽  
Chinese Hamster Ovary ◽  
Human Gene ◽  
Chinese Hamster ◽  
Dna Fragments ◽  
Human Dna ◽  
Glycosylation Defect

Chinese hamster ovary (CHO) glycosylation mutants provide an approach to cloning mammalian glycosyltransferases by transfection and gene rescue. In this paper, complementation of the lec1 CHO mutation by human DNA is described. Lec1 transfectants expressed human N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and possessed common human DNA fragments. Cloning of GlcNAc-TI should therefore be possible.

Transfection of a human gene that corrects the Lec1 glycosylation defect: evidence for transfer of the structural gene for N-acetylglucosaminyltransferase I

1989 ◽  
Vol 9 (12) ◽  
pp. 5713-5717
Author(s):  
R Kumar ◽  
P Stanley
Keyword(s):  
Structural Gene ◽  
Chinese Hamster Ovary ◽  
Human Gene ◽  
Chinese Hamster ◽  
Dna Fragments ◽  
Human Dna ◽  
Glycosylation Defect

Chinese hamster ovary (CHO) glycosylation mutants provide an approach to cloning mammalian glycosyltransferases by transfection and gene rescue. In this paper, complementation of the lec1 CHO mutation by human DNA is described. Lec1 transfectants expressed human N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and possessed common human DNA fragments. Cloning of GlcNAc-TI should therefore be possible.

Isolation and characterization of an extragenic suppressor of the low-density lipoprotein receptor-deficient phenotype of a Chinese hamster ovary cell mutant

1989 ◽  
Vol 9 (11) ◽  
pp. 4799-4806
Author(s):  
P Reddy ◽  
M Krieger
Keyword(s):  
Structural Gene ◽  
Chinese Hamster Ovary ◽  
Low Density Lipoprotein ◽  
Chinese Hamster ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Ovary Cell ◽  
Low Density ◽  
Extragenic Suppressor ◽  
Extragenic Suppression

ldlC cells are low-density lipoprotein (LDL) receptor-deficient Chinese hamster ovary cell mutants which express pleiotropic defects in Golgi-associated glycosylation reactions. The dramatically reduced stability of the abnormally glycosylated LDL receptors in ldlC cells was shown to be due, in part, to rapid proteolysis and release of a large extracellular fragment of the receptor into the medium. A set of spontaneously arising LDL receptor-positive revertants of ldlC cells has been isolated. One of these, RevC-13, exhibits the glycosylation defects characteristic of the original ldlC mutant, suggesting that restoration of receptor activity was due to extragenic suppression. This suppression was due to a dramatic increase in the rate of LDL receptor synthesis rather than to an increase in the stability of the abnormally glycosylated receptors. Increased receptor synthesis was not due to receptor gene amplification. The increased LDL receptor activity was subject to normal sterol regulation. Analysis of the RevC-13 extragenic suppressor activity in a series of hybrid cells showed that RevC-13 suppression was a codominant trait that acted in cis to the LDL receptor structural gene (ldlA). Thus, the extragenic suppression in RevC-13 cells has defined a genetic element which is either part of or linked to the LDL receptor structural gene and which can control LDL receptor expression.

Moderate-level gene amplification in methotrexate-resistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene

1984 ◽  
Vol 4 (1) ◽  
pp. 69-76
Author(s):  
W F Flintoff ◽  
E Livingston ◽  
C Duff ◽  
R G Worton
Keyword(s):  
Gene Amplification ◽  
Structural Gene ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chromosome 2 ◽  
Dhfr Gene ◽  
Cell Biol ◽  
A Site

In previous studies, we have described several classes of methotrexate-resistant Chinese hamster ovary cell lines. Although the RI class is resistant because of an altered target enzyme, dihydrofolate reductase, the RIII class derived from RI cells is somewhat more resistant because of a moderate amplification of the altered dhfr structural gene (Flintoff et al., Mol. Cell. Biol. 2:275-285, 1982). In one RIII line, a translocation between the short arm (p) of chromosome 2 and the long arm (q) of chromosome 5 was observed, and the amplified RIII gene complex was mapped to the p arm of the 2p-marker chromosome derived from the translocation (Worton et al., Mol. Cell. Biol. 1:330-335, 1981). We tested the hypothesis that chromosomal translocation is a general feature of RIII cells and that such translocation involves a site at or near the dhfr structural gene. Thus, we examined four independently derived RIII-type mutants and found that each had a moderate amplification of the dhfr gene sequences, and karyotype analysis revealed that each carried a translocation involving the 2p arm at or near band 2p25. That this chromosomal rearrangement involves a site near the dhfr locus was demonstrated by mapping the altered but unamplified structural gene coding for the RI phenotype to the short arm of an unaltered chromosome 2. This suggests that a highly specific rearrangement involving an exchange at or near the site of the unamplified gene is a necessary prerequisite for the amplification process. A model for gene amplification involving chromosomal rearrangements and sister chromatid exchange is described.

Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells

1987 ◽  
Vol 7 (5) ◽  
pp. 2024-2030
Author(s):  
B Kaina ◽  
A A Van Zeeland ◽  
C Backendorf ◽  
H W Thielmann ◽  
P Van de Putte
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Human Fibroblasts ◽  
Chinese Hamster ◽  
Native Form ◽  
Ovary Cells ◽  
Human Dna ◽  
Human Genes ◽  
Partial Inactivation ◽  
High Doses

Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA.

scholarly journals Moderate-level gene amplification in methotrexate-resistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene.

1984 ◽  
Vol 4 (1) ◽  
pp. 69-76 ◽  
Author(s):  
W F Flintoff ◽  
E Livingston ◽  
C Duff ◽  
R G Worton
Keyword(s):  
Gene Amplification ◽  
Structural Gene ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chromosome 2 ◽  
Dhfr Gene ◽  
Cell Biol ◽  
A Site

In previous studies, we have described several classes of methotrexate-resistant Chinese hamster ovary cell lines. Although the RI class is resistant because of an altered target enzyme, dihydrofolate reductase, the RIII class derived from RI cells is somewhat more resistant because of a moderate amplification of the altered dhfr structural gene (Flintoff et al., Mol. Cell. Biol. 2:275-285, 1982). In one RIII line, a translocation between the short arm (p) of chromosome 2 and the long arm (q) of chromosome 5 was observed, and the amplified RIII gene complex was mapped to the p arm of the 2p-marker chromosome derived from the translocation (Worton et al., Mol. Cell. Biol. 1:330-335, 1981). We tested the hypothesis that chromosomal translocation is a general feature of RIII cells and that such translocation involves a site at or near the dhfr structural gene. Thus, we examined four independently derived RIII-type mutants and found that each had a moderate amplification of the dhfr gene sequences, and karyotype analysis revealed that each carried a translocation involving the 2p arm at or near band 2p25. That this chromosomal rearrangement involves a site near the dhfr locus was demonstrated by mapping the altered but unamplified structural gene coding for the RI phenotype to the short arm of an unaltered chromosome 2. This suggests that a highly specific rearrangement involving an exchange at or near the site of the unamplified gene is a necessary prerequisite for the amplification process. A model for gene amplification involving chromosomal rearrangements and sister chromatid exchange is described.

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