s'Nucleotide sequences influence serum-modulated expression of a human dihydrofolate reductase minigene

M. E. Goldsmith, C. A. Beckman, K. H. Cowan

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Human dihydrofolate reductase (DHFR) gene sequences were isolated from DHFR gene-amplified breast cancer cell line MCF-7. These genomic sequences plus human DHFR cDNA sequences were used to construct a DHFR minigene. Calcium phosphate-mediated transfer of minigene DNA into DHFR gene-deleted Chinese hamster ovary cells converted these cells to a DHFR+ phenotype at a frequency of 0.12%. Minigene-transfected cels contained 20 to 30 minigene copies per cell and had DHFR enzyme levels similar to those of wild-type MCF-7 human cells (1.4 pmol/mg of protein). In contrast to gene-amplified MCF-7 cells, which contained multiple DHFR mRNA species (1.1, 1.6, 3.8, and 5.3 kilobases), only a single 3.8-kilobase DHFR mRNA was found in minigene-transfected cells. Previous studies on normal cells demonstrated modulation of DHFR levels by a variety of conditions which altered cell growth. When cell growth was induced in minigene-transfected cells by release from serum deprivation and DHFR levels were assayed at the time of maximum DNA synthesis, these levels were increased 2.4 to 3.7-fold. In contrast, the DHFR levels in cells transfected with a construct made from DHFR cDNA and viral promoter, intron, and termination sequences were unchanged. Minigene deletions were made and analyzed to determine the DHFRgene sequences reponsible for regulation. Deletion of sequences upstream from 322 base pairs 5' to the start of transcription or 90 base pairs downstream from the termination of translation (which removed most of the 3' nontranslated region of the gene) did not alter the responsiveness of minigene-transfected cells to serum deprivation. However, when sequences between 322 and 113 base pairs 5' to the start of transcription were deleted, serum-dependent expression in minigene-transfected cells was affected.

Original languageEnglish (US)
Pages (from-to)878-886
Number of pages9
JournalMolecular and cellular biology
Volume6
Issue number3
StatePublished - Jan 1 1986

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Tetrahydrofolate Dehydrogenase
Serum
Base Pairing
Genes
MCF-7 Cells
Complementary DNA
Messenger RNA
Sequence Deletion
DNA
Growth
Cricetulus
Introns
Ovary

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

s'Nucleotide sequences influence serum-modulated expression of a human dihydrofolate reductase minigene. / Goldsmith, M. E.; Beckman, C. A.; Cowan, K. H.

In: Molecular and cellular biology, Vol. 6, No. 3, 01.01.1986, p. 878-886.

Research output: Contribution to journalArticle

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abstract = "Human dihydrofolate reductase (DHFR) gene sequences were isolated from DHFR gene-amplified breast cancer cell line MCF-7. These genomic sequences plus human DHFR cDNA sequences were used to construct a DHFR minigene. Calcium phosphate-mediated transfer of minigene DNA into DHFR gene-deleted Chinese hamster ovary cells converted these cells to a DHFR+ phenotype at a frequency of 0.12{\%}. Minigene-transfected cels contained 20 to 30 minigene copies per cell and had DHFR enzyme levels similar to those of wild-type MCF-7 human cells (1.4 pmol/mg of protein). In contrast to gene-amplified MCF-7 cells, which contained multiple DHFR mRNA species (1.1, 1.6, 3.8, and 5.3 kilobases), only a single 3.8-kilobase DHFR mRNA was found in minigene-transfected cells. Previous studies on normal cells demonstrated modulation of DHFR levels by a variety of conditions which altered cell growth. When cell growth was induced in minigene-transfected cells by release from serum deprivation and DHFR levels were assayed at the time of maximum DNA synthesis, these levels were increased 2.4 to 3.7-fold. In contrast, the DHFR levels in cells transfected with a construct made from DHFR cDNA and viral promoter, intron, and termination sequences were unchanged. Minigene deletions were made and analyzed to determine the DHFRgene sequences reponsible for regulation. Deletion of sequences upstream from 322 base pairs 5' to the start of transcription or 90 base pairs downstream from the termination of translation (which removed most of the 3' nontranslated region of the gene) did not alter the responsiveness of minigene-transfected cells to serum deprivation. However, when sequences between 322 and 113 base pairs 5' to the start of transcription were deleted, serum-dependent expression in minigene-transfected cells was affected.",
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