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)

Screening tests for antibodies to the human immunodeficiency virus (HIV), based on the indirect ELISA principle using viral preparations as antigen, yield a substantial number of false-positive and false-negative results. These failures are due to the lack of certain viral polypeptides or contaminating cellular polypeptides in viral preparations. Therefore, the accuracy of the screening tests should be improved by using highly purified, synthetic viral antigens. With establishment of such an ELISA antigen in mind, we examined a bacterially synthesized polypeptide [ENV(80)] that corresponds to 80 conserved amino acids of the HIV gp41 transmembrane glycoprotein. ENV(80) was expressed as a DHFR fusion protein in Escherichia coli. Results obtained by HIV ELISA and immunoprecipitation with 497 serum samples from various groups at risk of AIDS were compared with those obtained with the ENV(80) ELISA. The ENV(80) ELISA was found to be superior to the H9/HTLV-III ELISA with respect to sensitivity and specificity and is almost equivalent in accuracy to immunoprecipitation.
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PMID:A new ELISA test for HIV antibodies using a bacterially produced viral env gene product. 354 57

The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.
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PMID:Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant. 383 74

The structure and activity of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from the protozoan parasite Leishmania tropica were examined by limited proteolysis with five different endopeptidases. Each reaction resulted in a rapid, time-dependent loss of TS activity and no effect on DHFR activity. The proteolytic products were examined by NaDodSO4/PAGE; each digestion produced a fragment of apparent Mr approximately 35,000, and three of the five digestions generated a fragment of Mr approximately 20,000. Attempts to separate the fragments under nondenaturing conditions failed, suggesting that the proteolyzed protein remains a dimer with the gross structure of the subunits more or less undisturbed. In contrast, kinetic data indicate that some aspects of higher-order structure in the native protein are affected by proteolysis. The fragments (Mr 36,600 and 20,000) generated by Staphylococcus aureus V8 protease were subjected to sequence analysis. Whereas neither the native protein nor the Mr 36,600 fragment yielded an NH2-terminal amino acid, we obtained the sequence of the first 28 amino acids of the Mr 20,000 fragment. This sequence bore strong homology with sequences situated within TS of human, Lactobacillus casei, Escherichia coli, and bacteriophage T4. These and other data indicate that the TS-DHFR polypeptide consists of a DHFR sequence at the blocked NH2-terminal and a TS sequence at the COOH-terminal end of the protein. The region that is the target of the five proteases corresponds to a highly variable region within the sequences of the other four TSs. We suggest that an insertion occurs within the TS-DHFR sequence, positioned on the surface of the protein and quite vulnerable to the action of endopeptidases.
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PMID:Limited proteolysis of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica. 390 47

The cleavable prepiece of the precursor to yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) was attached to the amino-terminus of mouse dihydrofolate reductase (a cytosolic protein) by gene fusion. The resulting fusion protein was imported into the matrix of isolated, energized yeast mitochondria and cleaved to a polypeptide whose size was similar to that of authentic dihydrofolate reductase.
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PMID:The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix. 609 70

A recombinant plasmid (pSVS dhfr) encoding the pre-S region and the S gene of human hepatitis B virus (HBV) and murine dihydrofolate reductase (DHFR) cDNA has been used for the transfection of Chinese hamster ovary (CHO) DHFR- cells. Selection of clones resistant to methotrexate has permitted amplification of HBV sequences and an increase in production of hepatitis B surface antigen (HBsAg). HBV-specific transcripts have been characterized. The HBsAg 22-nm particles contain a receptor for polymerized human serum albumin (pHSA) and elicit in animals the synthesis of antireceptor antibodies. This property is ascribed to a 34,000-dalton polypeptide in the particles, which is most likely encoded by the S gene and part of the pre-S region. Especially because the pHSA receptor is most abundantly present on the virion and because, in hepatitis B infection, the appearance of anti-pHSA receptor antibodies seems to be a highly reliable criterion for viral clearance, the HBsAg particles obtained may constitute a particularly efficient vaccine.
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PMID:Synthesis in animal cells of hepatitis B surface antigen particles carrying a receptor for polymerized human serum albumin. 609 51

Copy DNA (cDNA) was prepared from induced leucocyte poly(A) RNA and cloned in Escherichia coli. IFN-alpha cDNA clones were isolated by subculture cloning with the use of a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, for IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids. A human chromosomal library was screened with IFN cDNA and 17 distinct IFN-alpha-related sequences were isolated and identified, of which 7 proved to be nonallelic authentic genes and 4 pseudogenes; 6 sequences remain to be elucidated. Taking into account the work of Goeddel and his colleagues, 13 non-allelic authentic genes and 6 pseudogenes can be distinguished. In addition, 9 genes believed to be allelic to the 13 authentic genes have been sequenced. The IFN-alpha genes may be classified into two major subfamilies, which diverged at least 33 Ma ago, but perhaps much earlier, if sequence rectification occurred. At least one IFN-alpha gene appears to have resulted by a recombinational event between members of the subfamily I and II. IFN-beta is distantly related to IFN-alpha's and may have diverged from a common ancestor at least 500 Ma ago. Both IFN-alpha and IFN-beta genes differ from most other genes of higher organisms by being devoid of introns. The mouse was found to possess an IFN-alpha gene family of a size similar to that of man; the murine genes also do not have introns. IFN-alpha genes devoid of their signal sequence were joined to prokaryotic promoters to produce the mature interferons in E. coli in high yield. IFN-alpha 2, purified to homogeneity, has been crystallized by T. Unge and B. Strandberg (Uppsala). Hybrid genes consisting of IFN-alpha 1 and IFN-alpha 2 segments were constructed and expressed in E. coli; the target cell specificities of such hybrids were dependent on the arrangement of the segments and were different from those of either parent. The chromosomal gene for HuIFN-alpha 1 was introduced into mouse L cells to study the mechanism of its expression. Correct transcription was only detected after induction (with Newcastle disease virus); expression was transient, with the same kinetics as those of the endogenous mouse IFN mRNA. Natural murine IFNs and human IFN-beta and IFN-gamma are glycosylated. Because E. coli cells transformed with the genes of eukaryotic glycoproteins are not expected to yield correctly glycosylated polypeptides, we prepared lines of hamster cells permanently transformed with hybrid plasmids, which contained an IFN gene linked to the SV40 early promoter, as well as dihydrofolate reductase as a selective marker. After intracellular amplification of the introduced genes, cell lines were obtained which constitutively produced IFN at about 40 000 units ml-1 and could be propagated for at least several months.
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PMID:Structure and expression of human IFN-alpha genes. 612 51

Bisbrusatolyl malonate, which was shown previously to be active against P-388 lymphocytic leukemia cell growth, was investigated for inhibitory effects on nucleic acid and protein synthesis. DNA and RNA synthesis as well as protein synthesis were markedly inhibited at 10,25, and 50 mu mole final concentrations in vitro. The major sites of inhibition of nucleic acid synthesis appeared to be DNA polymerase, messenger and transfer RNA polymerases, orotidine-5'-monophosphate decarboxylase, phosphoribosyl pyrophosphate amino transferase, and dihydrofolate reductase. Moderate inhibition of nucleotide kinase activities and oxidative phosphorylation processes occurred after drug treatment. Cyclic adenosine monophosphate levels were reduced. Protein synthesis was inhibited during the elongation step of peptide synthesis. The data suggested that bisbrusatolyl malonate interfered with the peptide bond formation. However, the ongoing polypeptide synthesis must be completed before the drug can bind to the ribosome effectively.
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PMID:Antitumor agents XLVII: The effects of bisbrusatolyl malonate on P-388 lymphocytic leukemia cell metabolism. 627 24

The complete nucleotide sequence of the type I dihydrofolate reductase gene from Tn7 was determined. The structural gene coded for a polypeptide of 157 amino acid residues. The polypeptide deduced from the DNA sequence had a molecular weight of 17,577 which was in good agreement with that estimated by mobility in SDS-polyacrylamide gels. Sequences were identified proximal to the coding region which were similar to those found in the consensus E. coli promoter region and for the initiation of protein synthesis. Features consistent with the termination of RNA transcription were present distal to the structural gene. No homology was apparent when the DNA sequence of the type I gene was compared to the sequence of the type II plasmid DHFR genes, but sequence homology was evident when the type I and E. coli chromosomal enzymes were compared. Homology was greatest in the regions coding for amino acids which in the E. coli chromosomal enzyme are associated with substrate, cofactor and inhibitor binding.
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PMID:The nucleotide sequence of the trimethoprim-resistant dihydrofolate reductase gene harbored by Tn7. 630 74

We have transformed a dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary cell line to the DHFR+ phenotype with a recombinant cosmid (cH1) containing a functional Chinese hamster DHFR gene (J.D. Milbrandt et al., Mol. Cell. Biol. 3:1266-1273, 1983). After exposure of cells to successive increases in methotrexate, we have isolated a resistant cell line (JSH-1) that grows in 1 microM methotrexate. We show here that JSH-1 contains 300 to 500 copies of the integrated cosmid and that these copies are located predominantly at one position on a chromosome identified as Z5a. Hybridization analysis of restriction digests of genomic DNA indicates that the cosmid has been integrated intact into the genome and that upon amplification, the original cosmid/genomic junction fragments are also amplified in JSH-1. Furthermore, the pattern of amplified bands observed in ethidium bromide-stained gels indicates that the unit amplified sequence (amplicon) may be as large as 120 to 135 kilobases and therefore includes considerable amounts of flanking DNA in addition to the 45 kilobases of integrated cosmid. We also show that the protein overproduced by the amplified cosmid in JSH-1 comigrates with the 21,000-dalton polypeptide characteristic of the methotrexate-resistant cell line (CHOC 400) from which cH1 was cloned. However, the DHFR mRNA species overproduced in JSH-1 appear to be larger than those detected in CHOC 400, indicating that not all of the normal transcription and processing signals are preserved in the integrated recombinant cosmid.
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PMID:Amplification of a cloned Chinese hamster dihydrofolate reductase gene after transfer into a dihydrofolate reductase-deficient cell line. 631 Mar 71

We have determined the nucleotide sequence of a 1075-base-pair HindIII fragment of the T4 phage genome. This fragment contains the structural gene (frd) for dihydrofolate reductase and part of the gene (td) encoding thymidylate synthase. The fragment contains a 579-base-pair open reading frame, encoding a 193-residue polypeptide with a calculated mass of 21,603 Da, in agreement with our reported subunit molecular mass of 23,000. The deduced amino acid sequence shows partial homology with other dihydrofolate reductases, with most of the identities lying in regions known to be involved in substrate binding and catalysis. The 3' end of the coding strand overlaps the coding region for thymidylate synthase; the sequence - ATGA -includes an opal terminator for the frd gene and an initiating triplet for the td gene. The deduced amino acid sequence from this initiating ATG is identical, for the first 20 residues, with the NH2-terminal 20 residues reported for the td protein (M. Belfort , A. Moelleken , G. F. Maley , and F. Maley (1983) J. Biol. Chem. 258, 2045-2051). The sequenced HindIII fragment was transferred into a high expression plasmid vector for large scale production of homogeneous T4 dihydrofolate reductase. The experimentally determined sequence of 20 residues at the NH2-terminus of this protein is identical with that deduced from the nucleotide sequence for T4 dihydrofolate reductase.
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PMID:Nucleotide sequence reveals overlap between T4 phage genes encoding dihydrofolate reductase and thymidylate synthase. 632 73


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