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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)

A sensitive and versatile assay is described for the nuclear transport of 35S-labeled proteins obtained by the in vitro translation of SP6 plasmid-generated mRNAs. A specific nuclear accumulation of greater than 20-fold is observed for the transformation-related nuclear proteins, p53 and E1b, and the nuclear enzyme, thymidine kinase, whereas transport of the nonnuclear proteins, dihydrofolate reductase and simian virus 40 small t antigen, is negligible within 30 min.
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PMID:Nuclear transport of proteins translated in vitro from SP6 plasmid-generated mRNAs. 213 54

In a previous report it was shown that mammalian ribosomes were capable of initiating translation at a non-AUG triplet when the initiation codon of mouse dihydrofolate reductase (dhfr) was mutated to ACG (Peabody, D.S. (1987) J. Biol. Chem. 262, 11847-11851). In order to assess the capacity of the mammalian translation apparatus to initiate at other non-AUG triplets, the initiator AUG of dihydrofolate reductase was converted to GUG, UUG, CUG, AGG, AAG, AUA, AUC, and AUU. These represent (with ACG) all the possible triplets that differ from AUG by only one nucleotide. The ability of each mutant to produce dihydrofolate reductase was assessed by in vitro transcription/translation of the mutant dhfr sequences under control of the bacteriophage SP6 promoter. Each of the triplets (with the exceptions of AGG and AAG) was able to direct the synthesis of apparently normal dihydrofolate reductase. Incorporation of [35S]tRNAifMet into the products of in vitro translation indicates that in each case the non-AUG triplet is able to direct initiation of the polypeptide chain with methionine. The mutant dhfr sequences were also inserted into the mammalian expression vector SVGT5 for expression in cultured monkey cells. The hierarchy of relative translation efficiencies was similar in vivo and in vitro.
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PMID:Translation initiation at non-AUG triplets in mammalian cells. 253 69

The human transferrin receptor (TR) is a protein comprising 760 amino acid residues that spans the membrane once with its N terminus towards the cytoplasm. It is synthesized without a cleavable signal peptide. We have tested whether the signal responsible for its membrane insertion is present within its transmembrane peptide using a combined recombinant DNA/in vitro translation approach. The complete TR coding region was first reconstructed from overlapping TR cDNA clones and then engineered into an SP6-based transcription vector. In vitro transcription and subsequent translation in the presence of rough microsomes yielded TR molecules that were glycosylated and correctly inserted into the membrane. Two kinds of experiments demonstrated that the spanning region of the TR polypeptide contained the signal for translocation across the membrane of the rough endoplasmic reticulum. First, we deleted the spanning region of TR and showed that this deletion mutant could not be inserted. Second, we showed that two cytoplasmic proteins (the mouse dihydrofolate reductase and the chimpanzee alpha-globin) could be inserted into the microsomal membrane in the expected orientation when the TR transmembrane segment was added to their N termini. Thus, the spanning peptide was shown to be both necessary and sufficient for chain translocation. Further analyses demonstrated that the translocation event was dependent on the signal recognition particle.
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PMID:The transmembrane segment of the human transferrin receptor functions as a signal peptide. 301 1

We have designed cosmid vectors for rapid genomic "walking" and restriction mapping. These vectors contain the transcription promoters from either bacteriophage SP6, T7, or T3 flanking a unique BamHI cloning site. Mammalian expression modules encoding the dominant marker neomycin phosphotransferase or the amplifiable dihydrofolate reductase gene expressed from SV40 promoters were inserted for use in gene transfer studies. Restriction sites for the enzymes Not I and Sfi I, which cut mammalian DNA very infrequently, have been engineered near the transcriptional promoters to enable the excision of most inserts as single, full-length fragments. Genomic libraries representative of mouse, human, and hamster genomes were constructed by inserting 33- to 44-kilobase-pair (kbp) DNA fragments, generated by partial cleavage of genomic DNA with Mbo I or Sau3A, into the unique BamHI site. Digestion of recombinant cosmids with restriction enzymes that cleave frequently but do not disrupt the transcriptional promoters generates two small DNA templates for the synthesis of end-specific RNA probes to facilitate directional "walking." Cosmid restriction maps can be determined rapidly by one of several methods. The cosmids and methods we describe should have wide utility in determining the functional and structural organization of complex eukaryotic genomes and for physically linking distant genetic loci.
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PMID:Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. 303 68

The initiator AUC of the mouse dihydrofolate reductase gene (dhfr) was converted to ACG by site-directed mutagenesis and assayed for expression in cultured monkey cells using an SV40 recombinant called SVGT5dhfr26m2. Synthesis of apparently full-length dihydrofolate reductase (DHFR) protein was significantly reduced compared to wild-type, but not entirely abolished, suggesting that the ACG triplet was being utilized for translation initiation. In addition, a truncated form of DHFR was produced, apparently by initiation at the next in-frame AUG downstream. This result was confirmed in vitro. Transcripts of the dhfr sequence were produced by SP6 RNA polymerase in the presence of m7GpppG and translated in vitro using reticulocyte lysates and wheat germ extracts. The results paralleled those observed in vivo. Synthesis of full-length DHFR was reduced, but not eliminated, and a new species was produced by initiation at an internal site. Amino acid sequence analysis of the products of in vitro translation demonstrated that translation does indeed initiate at the ACG triplet and that it initiates with methionine. Additional mutations were introduced which altered the sequence context of the ACG triplet. Mutation of the translation initiation consensus sequence by substitution of the A residue at position -3, or of the G at +4 resulted in a significant decrease in initiation at the ACG and an increase in the level of the internal initiation product. Thus, translation initiation at a non-AUG triplet depends on a favorable sequence context.
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PMID:Translation initiation at an ACG triplet in mammalian cells. 304 Jul 20

A coupled transcription/translation system from Escherichia coli has been developed that is very active for protein synthesis but deficient in chaperone proteins. The chaperones GroEL and DnaK distribute during the first ultracentrifugation of the E. coli extract partially with the ribosomes and partially in a liquid, viscous fraction above the ribosomes. Gel filtration chromatography of this latter fraction separates GroEL and DnaK as high-molecular-weight components from the peak of activity of the factors required for protein synthesis. Thus, a chaperone-deficient transcription/translation system can be reconstituted with salt-washed ribosomes. This chaperone-deficient system was used to study synthesis and folding of bacterial dihydrofolate reductase and of rhodanese, a eukaryotic mitochondrial enzyme. Both enzymes were synthesized from nonlinearized plasmids that had the respective coding sequence under the SP6 promoter. Both enzymes were synthesized in active form and with high specific activity in the chaperone-deficient system. A high proportion, about 20% of newly synthesized dihydrofolate reductase and about 50% of rhodanese, stayed with the ribosomes after coupled transcription/translation. No enzymatic activity was detected in this fraction. Addition of the chaperones GroEL/ES and DnaK resulted in a shift of rhodanese molecules from the ribosomes into the supernatant fraction. Nearly all molecules in the supernatant were enzymatically active.
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PMID:Development of a chaperone-deficient system by fractionation of a prokaryotic coupled transcription/translation system. 791 Dec 83