Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.4.17 (adenosine deaminase)
 5,206 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fluorescent in situ hybridization was used to localize the adenylate deaminase 2 (AMPD2) genes and flanking sequences on the chromosomes of the Chinese hamster line GMA32 and to study the distribution of additional copies of these genetic sequences in amplified mutants selected at several early stages of the amplification process. The synteny of AMPD2 genes and MDR1 genes, located on chromosomes 1, was demonstrated; in GMA32 the existence of a rearrangement positioning the two AMPD2 genes at different distances from the telomeres was disclosed. Using this structural marker, we showed that the amplified copies distribute along only one of the chromosomes 1. Their organization in different cells of clonal mutant populations at a very early stage of amplification was extremely heterogeneous; classes of organization could be recognized however. Their quantitative distribution at this stage and in cells which went through 10 more division cycles suggests an evolution pathway common to the mutant clones under study: as a rule, tandems of few units of identical and very large size (47 Mb) appear to be the first detected product of amplification; this organization is progressively overtaken by structures with more units of reduced and irregular size, while, in a growing number of cells, clusters of much shorter units can be observed. The nature of segregative amplification mechanisms operating in these processes and the possible involvement of replicative ones are discussed.
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PMID:The evolution of the amplified adenylate deaminase 2 domains in Chinese hamster cells suggests the sequential operation of different mechanisms of DNA amplification. 137 18

A fusion between a selectable multidrug resistance (MDR1) cDNA and an adenosine deaminase (ADA) cDNA concomitantly confers multidrug resistance and ADA activity on transfected cells. We have produced a Harvey murine sarcoma virus-derived, replication-defective, recombinant retrovirus to transduce this chimeric MDR-ADA gene efficiently into a great variety of cells. Infection with the MDR-ADA retrovirus conferred the multidrug resistance phenotype on drug-sensitive cells, therefore allowing selection in the presence of colchicine. Colchicine-resistant cells synthesized large amounts of a membrane-associated 210-kDa MDR-ADA fusion protein that preserved both MDR and ADA functional activities. To monitor expression of the chimeric gene in vivo, Kirsten virus-transformed NIH cells were infected with the MDR-ADA retrovirus, and after drug-selection, injected into athymic nude mice. Tumors developed that contained the bifunctionally active MDR-ADA fusion protein. When these mouse tumor cells were placed in tissue culture without the selecting drug, they did not lose the bifunctionally active MDR-ADA fusion protein. The replication-defective, recombinant MDR-ADA retrovirus should be useful to stably introduce the chimeric MDR-ADA gene into a variety of cell types for biological experiments in vitro and in vivo.
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PMID:Retroviral transfer of a chimeric multidrug resistance-adenosine deaminase gene. 196 8

Current gene therapy protocols designed to treat adenosine deaminase (ADA) deficiency and other metabolic disorders suffer from low-efficiency delivery to target cells and a lack of long-term stability in expression of the therapeutic proteins. These problems may be resolved by use of an in vivo dominant selection. The multidrug transporter (MDR1) has been suggested as a useful selective marker for gene therapy. In this work, we co-expressed ADA and MDR1 cDNA in a retroviral vector using an internal ribosome entry site (IRES) from encephalomyocarditis virus. This system produced a bicistronic mRNA containing both ADA and MDR1, which enables co-expression of ADA and MDR1, and also allows the two proteins to be translated separately. After in vitro selection using a cytotoxic MDR1 substrate, vincristine, we demonstrated that functional ADA was co-expressed with MDR1 in proportion to the expression level of MDR1, whereas MDR1 expression was proportional to the stringency of the vincristine selection. Because the efficiency of IRES-dependent translation was much lower than that of cap-dependent translation in this system, we observed lower expression of the genes positioned after the IRES. This asymmetric expression caused a lower viral titer when MDR1 was placed downstream from the IRES, but it also provided a way of modulating the relative expression of ADA and MDR1. The retroviral system described in this work may serve as a useful tool to evaluate the strategies involving in vivo dominant selection for gene therapy of ADA-deficient patients.
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PMID:Co-expression of human adenosine deaminase and multidrug resistance using a bicistronic retroviral vector. 950 46