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

We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers--SV2-gpt, SV2-DHFR, and SV2-neo--in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human alpha-globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of alpha-globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult alpha-globin genes were more actively expressed than the embryonic zeta-globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.
 ...
PMID:Versatile cosmid vectors for the isolation, expression, and rescue of gene sequences: studies with the human alpha-globin gene cluster. 631 May 66

We studied the effects of gene amplification on human globin gene expression in Chinese hamster ovary cells. The normal human alpha-globin gene (N alpha 2) and a hybrid gene (M alpha G) containing the 5' promoter-regulator region of the mouse metallothionein gene and the human structural alpha 2-globin gene were linked to a modular SV2-cDNA dihydrofolate reductase (DHFR) gene. The recombinant DNA molecules were introduced into Chinese hamster ovary cells by calcium phosphate precipitation. After initial selection to retain the DHFR and linked sequences, the cells were cultured in increasing concentrations of methotrexate up to 0.2 mM. Southern blot analysis of total cellular DNA showed an approximately 500- to 1,000-fold increase in the number of copies of DHFR and human alpha-globin genes. The transcription of the alpha-globin and DHFR genes increased as their copy number within the cells increased. The transcription of the amplified hybrid M alpha G gene was also inducible with cadmium treatments. Both mature mRNA and "read-through" transcripts were observed. DHFR constituted approximately 10% of pulse-labeled cellular proteins in these cells, but no human alpha-globin was detected. In vitro translation of polyadenylated RNA from these cells showed that alpha-globin mRNA transcribed from the amplified alpha-globin genes was functional and directed alpha-globin chain synthesis. In situ hybridization of 3H-labeled alpha-globin and DHFR DNA probes in chromosome preparations from the two cell lines indicated that both genes were coamplified in the same chromosomal locations in each cell type. These results indicate that gene amplification enhances human globin gene expression in cultured Chinese hamster ovary cells.
 ...
PMID:Amplification and expression of human alpha-globin genes in Chinese hamster ovary cells. 649 27

We compared (i) the enhancer/promoter (mCMV promoter) from the murine cytomegalovirus (CMV) major immediate early gene,(ii) the enhancer/promoter from human CMV major immediate early gene, containing a short promoter (h1CMV) or a long stretch of 5' untranslated region (UTR) from the gene promoter (h2CMV) and (iii) the simian virus 40 (SV40) enhancer/early region promoter (SV2) for their ability to direct foreign gene expression in transiently transfected mammalian cell lines. Two series of recombinant plasmids containing the different viral promoters fused to the cat reporter gene and 3'-UTR for processing of transcripts from either the SV40 early region or the rabbit Beta 1-globin-encoding gene (Glb) were also analyzed for their effect on transient gene expression. The mCMV was the most active in dihydrofolate reductase-deficient Chinese hamster ovary (CHOdhfr-) cells and BALB/3T3 clone A31 mouse embryo cells. The h2CMV was more active than the other promoters in Bowes human melanoma cells and in Vero African green monkey kidney cells. In human hepatoma (Hep G2) cells, similar levels of CAT synthesis were observed with the h2CMV- and the mCMV-based vectors. In Hep G2 and Bowes cells, 3'-UTR from the SV40 early region resulted in consistently higher levels of cat expression, as compared to the rabbit beta 1-Glb gene, while the converse was true in BALB/3T3 clone A31 and Vero cells. SV40 early region and rabbit beta1-Glb gene 3'-UTR resulted in similar cat expression in CHOdhfr- cells.
 ...
PMID:Efficiency of different viral promoters in directing gene expression in mammalian cells: effect of 3'-untranslated sequences. 865 43

When maintained under continuous selection with the folate inhibitor, methotrexate, cultured Aedes albopicfus mosquito cells amplify an 200 kb region of DNA containing the dihydrofolate reductase gene. To determine whether the amplicon contained additional coding regions, Southern blots of cosmid clones containing amplicon DNA were probed separately with reverse-transcribed mRNA from methotrexate-sensitive and methotrexate-resistant cells. Cosmid pWED118 contained five EcoRI fragments (A, B, C, F, G) ranging in size from 2 to 5 kb that hybridized with cDNA from resistant cells. Of these, fragments B and F also hybridized to probe representing mRNA from sensitive cells, and all but fragment G hybridized to repetitive DNA from wild-type cells. Fragment G, which appeared to encode a low copy number gene in wild-type cells that subsequently became part of the dihydrofolate reductase amplicon in methotrexate-resistant cells, hybridized strongly to a 7 kb band and more weakly to bands measuring 9 and 3 kb on Northern blots containing RNA from resistant cells. Fragment G contained a 1203 bp open reading frame, encoding 401 amino acids homologous to synaptic vesicle protein SV2, a member of a transmembrane transporter family expressed in neural and endocrine cells. The region of homology included the six N-terminal transmembrane domains, an internal cytoplasmic loop, a seventh transmembrane domain, and most of an intravesicular loop. This partial sequence, which appears to correspond to a truncated gene generated during formation of the dihydrofolate reductase amplicon, provides a useful basis for more extensive characterization of an important gene family that may be the target of novel insecticides.
 ...
PMID:The mosquito dihydrofolate reductase amplicon contains a truncated synaptic vesicle protein gene. 972 69