scholarly journals Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases.

1983 ◽  
Vol 3 (5) ◽  
pp. 892-902 ◽  
Author(s):  
R E Cirullo ◽  
S Dana ◽  
J J Wasmuth
Keyword(s):  
Human Genome ◽  
Cell Lines ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Trna Synthetase ◽  
Temperature Sensitive ◽  
Efficient Procedure ◽  
Human Dna ◽  
Human Genes ◽  
Genes Encoding

We have developed a simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, we have constructed Chinese hamster cell lines that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure. Analysis of these cell lines with Southern blots confirmed the presence of a small number of restriction endonuclease fragments containing human DNA specifically. These cell lines represent a convenient and simple means to clone the human genomic sequences of interest.

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases

1983 ◽  
Vol 3 (5) ◽  
pp. 892-902
Author(s):  
R E Cirullo ◽  
S Dana ◽  
J J Wasmuth
Keyword(s):  
Human Genome ◽  
Cell Lines ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Trna Synthetase ◽  
Temperature Sensitive ◽  
Efficient Procedure ◽  
Human Dna ◽  
Human Genes ◽  
Genes Encoding

We have developed a simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, we have constructed Chinese hamster cell lines that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure. Analysis of these cell lines with Southern blots confirmed the presence of a small number of restriction endonuclease fragments containing human DNA specifically. These cell lines represent a convenient and simple means to clone the human genomic sequences of interest.

Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification

1984 ◽  
Vol 4 (12) ◽  
pp. 2932-2936
Author(s):  
R L Stallings ◽  
A C Munk ◽  
J L Longmire ◽  
C E Hildebrand ◽  
B D Crawford
Keyword(s):  
Cell Lines ◽  
Chromosome Rearrangement ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Chromosome 3 ◽  
Chromosome 3P ◽  
Genes Encoding ◽  
Cytogenetic Analyses ◽  
Chinese Hamster Chromosome

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.

scholarly journals Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.

1984 ◽  
Vol 4 (12) ◽  
pp. 2932-2936 ◽  
Author(s):  
R L Stallings ◽  
A C Munk ◽  
J L Longmire ◽  
C E Hildebrand ◽  
B D Crawford
Keyword(s):  
Cell Lines ◽  
Chromosome Rearrangement ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Chromosome 3 ◽  
Chromosome 3P ◽  
Genes Encoding ◽  
Cytogenetic Analyses ◽  
Chinese Hamster Chromosome

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.

Concanavalin A as a selective agent in tissue culture for temperature-sensitive hamster cell lines

1975 ◽  
Vol 21 (10) ◽  
pp. 1650-1654 ◽  
Author(s):  
Jim A. Wright
Keyword(s):  
Tissue Culture ◽  
Cell Lines ◽  
Concanavalin A ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Resistant Cell ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Selective Agent ◽  
Growth Properties

A novel method for isolating Chinese hamster cell cultures with temperature-sensitive growth properties is described. Concanavalin A can be used as a selective agent in tissue culture to isolate lectin-resistant cell lines which exhibit colony-forming abilities at the nonpermissive temperature relative to the permissive temperature as low as 10−4 to 10−6. A general correlation exists between resistance to the lectin and temperature sensitivity.

Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression

1985 ◽  
Vol 5 (2) ◽  
pp. 320-329
Author(s):  
B D Crawford ◽  
M D Enger ◽  
B B Griffith ◽  
J K Griffith ◽  
J L Hanners ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Chinese Hamster Cells ◽  
Future Studies ◽  
Noncoding Regions ◽  
Genes Encoding ◽  
Chinese Hamster Cell Line

We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.

Cloning of a human S-phase cell cycle gene: use of transient expression for screening

1987 ◽  
Vol 7 (2) ◽  
pp. 775-779
Author(s):  
A Fainsod ◽  
G Diamond ◽  
M Marcus ◽  
F H Ruddle
Keyword(s):  
Cell Cycle ◽  
Genomic Library ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
S Phase ◽  
Phase Cell ◽  
Cell Cycle Gene ◽  
Temperature Sensitive ◽  
Specific Cell ◽  
Restrictive Temperature

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.

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.

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