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)

DNA sequences corresponding to specific genes may be prepared by chemical synthesis, isolation of naturally occurring DNA, or reverse transcription. Such DNA may then be inserted into vectors such as plasmids or bacteriophages which carry the DNA into bacterial cells. Although significant differences exist in the basic molecular biology of eucaryotic and procaryotic organisms, these differences do not constitute absolute barriers to the expression of eucaryotic genes in bacteria. Several eucaryotic proteins, including insulin, growth hormone, ovalbumin, dihydrofolate reductase and somatostatin have been produced in bacteria. The use of chimeric microorganisms harboring recombinant DNA offers a completely new approach to the production of biologically useful polypeptides.
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PMID:Use of recombinant DNA technology for the production of polypeptides. 9 11

Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C. Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity. Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein. The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated. However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer. The fusion protein was highly purified by means of a methotrexate affinity chromatography.
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PMID:Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle. 133 Oct 37

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from approximately 1-5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr approximately 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence and pH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P6-P'3 sequence of human angiotensinogen.
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PMID:Recombinant human prorenin from CHO cells: expression and purification. 196 33

A hybrid cell line was constructed by fusion of mouse L-cells with an NIH3T3 cell line derivative containing a hybrid gene consisting of the mouse immunoglobulin kappa (IgK) variable gene promoter linked to the Escherichia coli gpt gene. Such hybrids grew to a much higher density compared to either of the parental cell lines. The utility of this cell line as a host to express foreign genes was tested by the expression of TGF-beta cDNA using the cytomegalovirus promoter. The vector also contained the human dihydrofolate reductase (DHFR) gene driven by SV40 early promoter, to allow for the amplification of the transfected gene. Initial transformants, selected at 100 nM methotrexate (MTX), were subsequently selected for resistance to a higher concentration of MTX (2 microM). Such clones expressed an increased level of TGF-beta when compared to the initial transformants. Both the initial transformants and the clones with the amplified DHFR gene produced TGF-beta in an acid-activatable precursor form. This mouse hybrid host cell line also allowed the expression of foreign genes cloned in an eukaryotic expression vector with the mouse IgK variable region promoter and human growth hormone as the reporter gene, whereas such vectors did not function in CHO cells. The mouse hybrid cell line was also found to be capable of being used with a broad range of promoters.
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PMID:A mouse hybrid cell line that supports gene expression from a variety of promoters in amplifiable vectors. 251 81

A cDNA library of Ob1771 preadipocytes was constructed, and a cDNA clone designated pOb24 was isolated by differential screening. The pOb24 mRNA, 6 kilobases in length, rose sharply in early differentiating Ob1771 and 3T3-F442A cells and decreased thereafter. In mouse adipose tissue, it was present at a high level in stromal-vascular cells (containing adipose precursor cells) and at a low level in mature adipocytes. Thus, pOb24 mRNA appears to be both in vitro and in vivo an unique marker of the preadipose state, i.e. of cell commitment during adipose cell differentiation. In contrast to glycerol-3-phosphate dehydrogenase mRNA, the emergence of pOb24 mRNA in Ob1771 cells required neither growth hormone or triiodothyronine as obligatory hormones nor insulin as a modulating hormone. Comparative studies of the expression of pOb24 and dihydrofolate reductase genes during the cell cycle suggest that arrest at the G1/S boundary was critical for the entry into the preadipose state. Tumor necrosis factor and transforming growth factor-beta were able to induce a large decrease of pOb24 mRNA level in growth-arrested Ob1771 cells. This decrease was shown to be only confined to early differentiating, glycerol-3-phosphate dehydrogenase negative cells as no decrease of pOb24 mRNA level was observed in glycerol-3-phosphate dehydrogenase positive cells. This result suggests that signals generated by tumor necrosis factor and transforming growth factor-beta have no effect on a commitment-related gene in late differentiated cells.
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PMID:Cloning and regulation of a mRNA specifically expressed in the preadipose state. 272 62

We have constructed two new recombinant cosmid vectors that can be used for direct expression and amplification of genomic DNA in mammalian cells. The vectors allow cloning of DNA fragments up to 40 kb in size. Each carries two dominant selectable markers: the bacterial neo gene and the mouse DHFR gene. In the first vector, pCV001, the neo and DHFR genes are regulated by the SV40 early promoter, and in the second, pAVCV007, by the avian sarcoma virus LTR promoter. The neo gene served as a dominant marker for the selection of transformants in all mammalian cell types, and we demonstrate here that the LTR promoter significantly improved the efficiency of DNA-mediated transformation of a human cell line. We isolated the human growth hormone genes from genomic libraries prepared in these cosmid vectors and used these recombinant cosmids for direct transfections of cultured cells. Selection of transformants in increasing concentrations of methotrexate led to the outgrowth of resistant cell populations carrying amplified copies of the DHFR marker. A 40-1000-fold coamplification of the hGH genes was observed in the different transfected cell lines, along with a corresponding increase in transcription and translation activity of the hGH gene. Gene amplification could be achieved in both DHFR deficient or normal cell lines. High level expression of a cloned gene mediated by gene amplification should facilitate characterization of DNA sequences, as well as isolation of specific gene products for biochemical, functional, and pharmacological studies.
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PMID:Cosmid vectors for high efficiency DNA-mediated transformation and gene amplification in mammalian cells: studies with the human growth hormone gene. 346 45

A mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. This vector was constructed to facilitate X-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. Proteins expressed with this vector possess an N-terminal human growth hormone domain and an octahistidine tag separated from the desired polypeptide sequences by a tobacco etch virus protease recognition site. Advantages of this vector are high levels of expression, simple detection and purification of expressed proteins, and reliable cleavage of the fusion protein. Cotransfection of this vector with a dihydrofolate reductase gene allows amplification of expression levels with methotrexate. Over one dozen cysteine-rich secreted proteins have been expressed in sufficient quantity for structural studies using this vector; the structure of at least one of these proteins has been determined.
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PMID:A mammalian expression vector for expression and purification of secreted proteins for structural studies. 1108 90