Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Expression of human interleukin 2 (IL-2) at high levels has been achieved in Chinese hamster ovary (CHO) cells by amplification of transfected sequences. Plasmids containing the human IL-2 cDNA or genomic DNA and mouse dihydrofolate reductase (DHFR) cDNA were transfected into DHFR-negative CHO cells. Transformants expressing DHFR were selected in media lacking nucleosides, and cells which amplified both DHFR and IL-2 genes were obtained by exposure to increasing methotrexate (MTX) concentrations. These cell lines constitutively expressed elevated levels of IL-2 at a concentration of 2 mg/liter. These cell lines continued to produce IL-2 stably through at least 1 month, even in the absence of MTX.
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PMID:Expression of amplified cDNA and genomic sequences encoding human interleukin 2 in Chinese hamster ovary cells. 282 76

An expression cassette of mouse dihydrofolate reductase (Mdhfr) cDNA under control of the yeast cytochrome c promoter was inserted in a yeast plasmid containing the ARS1 sequence. The ARS replicating function was destroyed by BglII treatment prior to yeast transformation. Using this linearized plasmid, genomic transformants could be obtained from either laboratory or industrial strains of bakers' yeast based on direct methotrexate (MTX)-resistance selection. The entire sequence of the linearized plasmid was integrated by homologous recombination at the ARS region of the host chromosome. The results indicate that repetitive and homologous recombination occurs readily in such transformations. The stability of the constructed integrants was more than 99.95% per generation in non-selective medium, and tandem repeats of up to six copies (i.e., about 44 kb) were not changed even after 30 generations in rich medium. Expression in rich medium of cointegrated, human interleukin 2 cDNA under control of the triose phosphate isomerase promoter was shown by Western blot experiments in both laboratory and industrial yeast strains. Furthermore, a comparison of the transcription efficiency of the Mdhfr gene in the chromosome with that in the plasmid revealed that the efficiency was almost proportional to the number of gene copies, irrespective of the location of the transcription unit. These results show that by using the MTX/Mdhfr dominant selection-amplification system one can construct stable recombinant yeast strains suitable for heterologous gene expression in laboratory as well as in industrial fermentation conditions.
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PMID:Construction of stable laboratory and industrial yeast strains expressing a foreign gene by integrative transformation using a dominant selection system. 355 23