Modulation of a human dihydrofolate reductase minigene following release from amino acid deprivation involves both 5' and 3' nucleotide sequences

M. E. Goldsmith, K. H. Cowan

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3 Citations (Scopus)

Abstract

A dihydrofolate reductase (DHFR) expression system composed of a DHFR minigene constructed from human DHFR genomic and cDNA sequences stably transfected into DHFR- Chinese hamster ovary cells used to study the modulation of DHFR levels in response to release from amino acid deprivation. The addition of complete medium to cells grown for 48 hr in medium lacking isoleucine and glutamine caused the transfected cells to undergo a synchronous cycle of DNA replication. When DHFR protein levels assayed at the time of maximum DNA synthesis were compared to that present in the deprived state, levels rose 3.2- to 4.9-fold. By contrast, DHFR levels in cells transfected with a DHFR expression construct made from mouse DHFR cDNA fused to viral promoter, intervening, and polyadenylation sequences were not inducible under the identical conditions. Human DHFR minigene deletion or substitution constructs were used to determine which nucleotide sequences were responsible for amino acid-modulated expression. Although deletion of sequences upstream from 322 base pair 5' to the start of transcription did not affect DHFR expression, removal of sequences between 322 and 113 base pairs reduced DHFR induction by approximately 50%. Deletion of nucleotide sequences within the 3' nontranslated region of the gene also reduced the level of induction by approximately 50%. Reduction in the levels of DHFR RNA relative to total cellular RNA was also found. Thus, both 5' and 3' nucleotide sequences are involved in the modulation of DHFR levels following release from amino acid deprivation.

Original languageEnglish (US)
Pages (from-to)378-383
Number of pages6
JournalMolecular pharmacology
Volume33
Issue number4
StatePublished - Jan 1 1988

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Tetrahydrofolate Dehydrogenase
Amino Acids
Base Pairing
Complementary DNA
RNA
Polyadenylation
Isoleucine
Sequence Deletion
Cricetulus
Glutamine
DNA Replication
Introns

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

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title = "Modulation of a human dihydrofolate reductase minigene following release from amino acid deprivation involves both 5' and 3' nucleotide sequences",
abstract = "A dihydrofolate reductase (DHFR) expression system composed of a DHFR minigene constructed from human DHFR genomic and cDNA sequences stably transfected into DHFR- Chinese hamster ovary cells used to study the modulation of DHFR levels in response to release from amino acid deprivation. The addition of complete medium to cells grown for 48 hr in medium lacking isoleucine and glutamine caused the transfected cells to undergo a synchronous cycle of DNA replication. When DHFR protein levels assayed at the time of maximum DNA synthesis were compared to that present in the deprived state, levels rose 3.2- to 4.9-fold. By contrast, DHFR levels in cells transfected with a DHFR expression construct made from mouse DHFR cDNA fused to viral promoter, intervening, and polyadenylation sequences were not inducible under the identical conditions. Human DHFR minigene deletion or substitution constructs were used to determine which nucleotide sequences were responsible for amino acid-modulated expression. Although deletion of sequences upstream from 322 base pair 5' to the start of transcription did not affect DHFR expression, removal of sequences between 322 and 113 base pairs reduced DHFR induction by approximately 50{\%}. Deletion of nucleotide sequences within the 3' nontranslated region of the gene also reduced the level of induction by approximately 50{\%}. Reduction in the levels of DHFR RNA relative to total cellular RNA was also found. Thus, both 5' and 3' nucleotide sequences are involved in the modulation of DHFR levels following release from amino acid deprivation.",
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