Construction of a modular dihydrofolate reductase cDNA gene: Analysis of signals utilized for efficient expression

Journal Article · Mon Nov 01 04:00:00 EST 1982 · Mol. Cell. Biol.; (United States)

DOI:https://doi.org/10.1128/MCB.2.11.1304· OSTI ID:5998638

Kaufman, J; Sharp, P A

Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome, DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR/sup -/ cells to the DHFR/sup +/ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFr mRNA produced in amplified lines indicated the following: (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA generated a recombinant capable of transforming cells to the DHFR/sup +/ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer 72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.

• Research Organization: Center for Cancer Research and Dept. of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
• OSTI ID: 5998638
• Journal Information: Mol. Cell. Biol.; (United States), Journal Name: Mol. Cell. Biol.; (United States) Vol. 2:11; ISSN MCEBD
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
550400 -- Genetics
59 BASIC BIOLOGICAL SCIENCES
ADENOVIRUS
AMINO ACIDS
ANIMAL CELLS
ANTIMETABOLITES
AROMATICS
AZAARENES
BIOSYNTHESIS
CARBOXYLIC ACIDS
CELL PROLIFERATION
CELL TRANSFORMATIONS
CHO CELLS
CLONING
CULTURE MEDIA
DNA
DNA-CLONING
DRUGS
ENZYMES
FOLIC ACID
GENE REPRESSORS
GENES
GENETIC ENGINEERING
GENOME MUTATIONS
GLOBULINS
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYBRIDIZATION
HYDROXY COMPOUNDS
IMMUNOGLOBULINS
METHOTREXATE
MICROORGANISMS
MODULATION
MOLECULAR BIOLOGY
MUTANTS
MUTATIONS
NUCLEIC ACIDS
NUCLEOPROTEINS
ONCOGENIC TRANSFORMATIONS
ONCOGENIC VIRUSES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
PARASITES
PHENOTYPE
PROTEINS
PTERIDINES
RECOMBINANT DNA
REVERTANTS
SIMIAN VIRUS
SYNTHESIS
TOXICITY
VIRUSES
VITAMIN B GROUP
VITAMINS