Construction and use of a dominant, selectable marker: a Harvey sarcoma virus-dihydrofolate reductase chimera.

Murray, M J; Kaufman, Randal J.; Latt, S A; Weinberg, R A

doi:10.1128/mcb.3.1.32

Cited by 32 publications

(9 citation statements)

References 28 publications

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“…Plasmid pMUD1 was constructed by inserting the gene encoding the murine DHFR, amplified by PCR from plasmid pLTRdhfr26 (Murray et al, 1983), into plasmid pJMB100A. The PCR primers introduced a unique NcoI site at the 5′ end of the gene and a unique XbaI site at the 3′ end of the gene.…”

Section: Methodsmentioning

confidence: 99%

Determination of Regions in the Dihydrofolate Reductase Structure That Interact with the Molecular Chaperonin GroEL

Hugo

Frieden

1996

Biochemistry

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Dihydrofolate reductase (DHFR) from Escherichia coli does not interact with the molecular chaperonin GroEL regardless of whether the interaction is initiated from the native or the unfolded state. In contrast, murine DHFR shows a strong interaction with GroEL. Using the structure of human DHFR as a model for the murine protein, a superimposition of the two structures shows that there are three distinct external loops in the eukaryotic DHFR that are not present in the E. coli protein. Removal of one loop (residues 99-108) from the eukaryotic murine DHFR has no effect on the interaction with GroEL. On the basis of the differences in structures, we inserted either of two surface loops of murine DHFR into the corresponding regions of E. coli DHFR. In the first mutant (EcDHFR-i(9)36), residues 36 and 37 (L-N) of E. coli DHFR were replaced with the nine amino acid sequence T-T-S-S-V-E-G-K-Q. In the second mutant (EcDHFR-i(7)136), residues 136-139 (V-F-S-E) of E. coli DHFR were replaced with the seven amino acid sequence L-P-E-Y-P-G-V. Both E. coli DHFR mutants formed a complex with GroEL starting from either the native or the unfolded states of DHFR. The binding was specific since the presence of MgATP caused the release of the proteins from GroEL. As with murine DHFR, nonnative conformations of EcDHFR-i(9)36 and EcDHFR-i(7)136 are bound to GroEL. Fluorescence titration techniques were used to quantitate the interaction between GroEL and these proteins. A simple chromatographic procedure was developed to remove contaminating tryptophan containing peptides from GroEL samples. The mutant EcDHFR-i(7)136 binds to GroEL with a stoichiometry of 4-5 mol of DHFR per mol of GroEL tetradecamer, while murine DHFR binds to GroEL with a stoichiometry of 2 mol of DHFR per mol of GroEL tetradecamer. Both murine DHFR and EcDHFR-i(7)136 bind to GroEL very tightly, with equilibrium dissociation constants of less than 85 nM.

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Section: Methodsmentioning

confidence: 99%

Determination of Regions in the Dihydrofolate Reductase Structure That Interact with the Molecular Chaperonin GroEL

Hugo

Frieden

1996

Biochemistry

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“…D-dot hybridization analysis (Fig. SB) indicated that there were an average of 20 and 1,150 copies of pdhOD1 per cell in the initial and final DFMO-resistant populations, respectively. Southern blot analysis (Fig.…”

Section: Expression Of Heterologousmentioning

confidence: 99%

“…Amplification vectors based on dihydrofolate reductase (dhfr) can be used to obtain high levels of expression of a variety of heterologous genes, since genes linked to it can be coamplified and thus overexpressed (14,15,18,34). However, amplification of dhfr-based vectors is generally limited to DHFR-deficient Chinese hamster ovary (CHO) cells, and dominant vectors based on dhfr have reduced ability to amplify (20,27). An amplifiable vector based on adenosine deaminase (ADA) (13) is more easily used in wild-type cells.…”

mentioning

confidence: 99%

Amplification and expression of heterologous ornithine decarboxylase in Chinese hamster cells.

Chiang¹,

McConlogue²

1988

Mol. Cell. Biol.

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We have developed an amplifiable mammalian expression vector based on the enzyme ornithine decarboxylase (ODC). We show greater than 700-fold amplification of this vector in ODC-deficient Chinese hamster ovary cells. A passive coamplified marker, dihydrofolate reductase (dhfr), was amplified and overexpressed 1,000-fold. This ODC vector was a dominant marker in a variety of cell types and displayed at least 300-fold amplification in wild-type Chinese hamster ovary cells.Selectable genetic markers have allowed the introduction of cloned DNA into animal cells, allowing study of eucaryotic gene regulation and function. Amplification vectors based on dihydrofolate reductase (dhfr) can be used to obtain high levels of expression of a variety of heterologous genes, since genes linked to it can be coamplified and thus overexpressed (14,15,18,34). However, amplification of dhfr-based vectors is generally limited to DHFR-deficient Chinese hamster ovary (CHO) cells, and dominant vectors based on dhfr have reduced ability to amplify (20,27). An amplifiable vector based on adenosine deaminase (ADA) (13) is more easily used in wild-type cells. However, the selections required for the ADA markers are complicated, and some cell types do not grow well under selective conditions (13).To independently amplify more than one polypeptide in the same cell requires the use of more than one amplifiable marker. There are currently a limited number of amplifiable markers. We have developed a simple dominant amplifiable vector system, based on ornithine decarboxylase (ODC; EC 4.1.1.17), the initial enzyme in the synthesis of polyamines by animal cells and an essential enzyme for cellular growth (32). MATERIALS AND METHODSOerivgtion of the ODC expression vector. pdhOD1 ( Fig. 1) contains the HindIII-PvuI fragment of pSVMdhfr (16) that includes the dhfr expression unit and the 626-base-pair (bp) EcoRI-PvuI fragment of pBR322. The HindIII-XhoI fragment containing the simian virus 40 (SV40) early promoter and the XhoI-PvuI fragment containing the SV40 late polyadenylation sequences and flanking pBR322 sequences were obtained from pCD-X (21). The TaqI-PvuII fragment of pOD20.7 (11,17), containing the ODC cDNA adapted with XhoI linkers is between the SV40 early promoter and SV40 polyadenylation signals of the pCD-X-derived sequences so that ODC is expressed from the SV40 early promoter.Cell culture. ODC-deficient (ODC-) CHO cells (clone C55.7) were grown as described (31). Other cells were maintained in Dulbecco modified Eagle medium containing 10% fetal calf serum.Cells were transfected with calcium phosphate-precipitated DNA as described (9, 10) with the following modifications. CHO cells in 100-nmm dishes were exposed to 20 ,g of precipitated DNA for 3 h and then shocked for 3 min in * Corresponding author. 15% glycerol. African green monkey kidney (American Type Culture Collection) CV1 cells were treated with the precipitated DNA for 16 h and were not treated with glycerol. Mouse fibroblast NIH 3T3 cells were exposed to the precipitated DNA...

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“…Amplification ofthis MTX-resistantDHFR is limited due to the higher concentrations of MTX required as a result of the increased Ki of the mutant DHFR. An alternative approach has been the use of a very efficient DHFR expression vector (17). In this case it has been difficult to obtain the optimal MTX concentration necessary for the selection ofthe foreign DHFR gene.…”

mentioning

confidence: 99%

Selection and amplification of heterologous genes encoding adenosine deaminase in mammalian cells.

Kaufman¹,

Murtha²,

Ingolia³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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Construction and use of a dominant, selectable marker: a Harvey sarcoma virus-dihydrofolate reductase chimera.

Cited by 32 publications

References 28 publications

Determination of Regions in the Dihydrofolate Reductase Structure That Interact with the Molecular Chaperonin GroEL

Determination of Regions in the Dihydrofolate Reductase Structure That Interact with the Molecular Chaperonin GroEL

Amplification and expression of heterologous ornithine decarboxylase in Chinese hamster cells.

Selection and amplification of heterologous genes encoding adenosine deaminase in mammalian cells.

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