scholarly journals Direct demonstration of genetic alterations at the dihydrofolate reductase locus after gamma irradiation.

1982 ◽  
Vol 2 (1) ◽  
pp. 93-96 ◽  
Author(s):  
L H Graf ◽  
L A Chasin
Keyword(s):  
Dihydrofolate Reductase ◽  
Gamma Ray ◽  
Reductase Activity ◽  
Chinese Hamster Ovary Cells ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Genetic Alterations ◽  
Ovary Cells ◽  
Direct Demonstration ◽  
Dna Deletions

Gamma ray-induced mutants of Chinese hamster ovary cells lacking dihydrofolate reductase activity were screened for DNA sequence changes at the locus specifying this activity by using a cloned cDNA probe. Two of nine mutants screened displayed an altered restriction fragment pattern suggesting the occurrence of DNA deletions or rearrangements.

Direct demonstration of genetic alterations at the dihydrofolate reductase locus after gamma irradiation

1982 ◽  
Vol 2 (1) ◽  
pp. 93-96
Author(s):  
L H Graf ◽  
L A Chasin
Keyword(s):  
Dihydrofolate Reductase ◽  
Gamma Ray ◽  
Reductase Activity ◽  
Chinese Hamster Ovary Cells ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Genetic Alterations ◽  
Ovary Cells ◽  
Direct Demonstration ◽  
Dna Deletions

Gamma ray-induced mutants of Chinese hamster ovary cells lacking dihydrofolate reductase activity were screened for DNA sequence changes at the locus specifying this activity by using a cloned cDNA probe. Two of nine mutants screened displayed an altered restriction fragment pattern suggesting the occurrence of DNA deletions or rearrangements.

scholarly journals Overproduction of dihydrofolate reductase and gene amplification in methotrexate-resistant Chinese hamster ovary cells.

1982 ◽  
Vol 2 (3) ◽  
pp. 275-285 ◽  
Author(s):  
W F Flintoff ◽  
M K Weber ◽  
C R Nagainis ◽  
A K Essani ◽  
D Robertson ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Selection Process ◽  
Chinese Hamster Ovary Cells ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Wild Type Enzyme ◽  
Ovary Cells ◽  
Reductase Gene ◽  
Elevated Rate

Stable isolates of Chinese hamster ovary cells that are highly resistant to methotrexate have been selected in a multistep selection process. Quantitative immunoprecipitations have indicated that these isolates synthesize dihydrofolate reductase at an elevated rate over its synthesis in sensitive cells. Restriction enzyme and Southern blot analyses with a murine reductase cDNA probe indicate that the highly resistant isolates contain amplifications of the dihydrofolate reductase gene number. Depending upon the parenteral line used to select these resistant cells, they overproduce either a wild-type enzyme or a structurally altered enzyme. Karyotype analysis shows that some of these isolates contain chromosomes with homogeneously staining regions whereas others do not contain such chromosomes.

Overproduction of dihydrofolate reductase and gene amplification in methotrexate-resistant Chinese hamster ovary cells

1982 ◽  
Vol 2 (3) ◽  
pp. 275-285
Author(s):  
W F Flintoff ◽  
M K Weber ◽  
C R Nagainis ◽  
A K Essani ◽  
D Robertson ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Selection Process ◽  
Chinese Hamster Ovary Cells ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Wild Type Enzyme ◽  
Ovary Cells ◽  
Reductase Gene ◽  
Elevated Rate

Stable isolates of Chinese hamster ovary cells that are highly resistant to methotrexate have been selected in a multistep selection process. Quantitative immunoprecipitations have indicated that these isolates synthesize dihydrofolate reductase at an elevated rate over its synthesis in sensitive cells. Restriction enzyme and Southern blot analyses with a murine reductase cDNA probe indicate that the highly resistant isolates contain amplifications of the dihydrofolate reductase gene number. Depending upon the parenteral line used to select these resistant cells, they overproduce either a wild-type enzyme or a structurally altered enzyme. Karyotype analysis shows that some of these isolates contain chromosomes with homogeneously staining regions whereas others do not contain such chromosomes.

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1926-1935
Author(s):  
P J Mitchell ◽  
G Urlaub ◽  
L Chasin
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Splice Sites ◽  
Ovary Cells ◽  
Splicing Mutations ◽  
Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes

1983 ◽  
Vol 3 (9) ◽  
pp. 1598-1608
Author(s):  
R J Kaufman ◽  
P A Sharp
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Ovary Cells ◽  
Dividing Cells ◽  
Major Late Promoter ◽  
The Stability

Dihydrofolate reductase (DHFR) synthesis is regulated in a growth-dependent fashion. Dividing cells synthesize DHFR at a 10-fold-higher rate than do stationary cells. To study this growth-dependent synthesis. DHFR genes have been constructed from a DHFR cDNA segment, the adenovirus major late promoter, and fragments of simian virus 40 (SV40) which provide signals for polyadenylation. These genes have been introduced into Chinese hamster ovary cells. The DHFR mRNAs produced in different transformants are identical at their 5' ends, but differ in sequences in their 3' ends as different sites are utilized for polyadenylation. Three transformants that utilize either DHFR polyadenylation signals or the SV40 late polyadenylation signal exhibit growth-dependent DHFR synthesis. The level of DHFR mRNA in growing cells is approximately 10 times that in stationary cells for these transformants. This growth-dependent DHFR mRNA production probably results from posttranscriptional events. In contrast, three transformants that utilize the SV40 early polyadenylation signal and another transformant that utilizes a cellular polyadenylation signal do not exhibit growth-dependent DHFR synthesis. In these three cell lines, the fraction of mRNAs polyadenylated at different sites in a tandem array shifts between growing and stationary cells. These results suggest that the metabolic state of the cell is important in determining either the efficiency of polyadenylation at various sites or the stability of mRNA polyadenylated at various sites.

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.

Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells

1977 ◽  
Vol 55 (4) ◽  
pp. 445-452 ◽  
Author(s):  
Radhey S. Gupta ◽  
Wayne F. Flintoff ◽  
Louis Siminovitch
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Class I ◽  
Class Iii ◽  
Wild Type ◽  
Ovary Cells ◽  
I Cells ◽  
Resistant Cells

We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levels of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W. F., Davidson, S. V., and Siminovitch, L. (1976) Somatic Cell Genet. 2, 245–261). By selecting for increased resistance from these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In this report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and a class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.

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