Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Angiotensinogen mRNA is found in many extrahepatic tissues, where it may participate in local angiotensin-generating systems. In this study we explore the feasibility of using anti-sense RNA to decrease angiotensinogen production in rat H4IIEC3 hepatoma cells. An amplifiable shuttle vector was modified to allow the production of high levels of stable anti-sense RNA from two regions of the mouse angiotensinogen gene under the control of the inducible sheep metallothionein promoter. Stably transformed, clonal cell lines expressing anti-sense RNA for angiotensinogen were isolated after selection with the aminoglycoside G418. Subsequently, the number of chromosomally integrated copies of the angiotensinogen anti-sense constructs was coamplified by methotrexate selection for dihydrofolate reductase activity carried on the shuttle vector. With a 20- to 30-fold induction of the anti-sense RNAs, the target angiotensinogen mRNA level was reduced to 25-30% of control values. The specificity of this effect was confirmed by showing no decrease in either beta-tubulin or neomycin phosphotransferase mRNA levels. Using tissue-specific promoters, it should be possible to direct these effects to specific organs in transgenic mice. However, in agreement with results from other groups, our findings suggest that it will not be possible to eradicate completely the target gene product using the anti-sense RNA strategy.
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PMID:Inducible anti-sense RNA for angiotensinogen in stably transformed hepatoma cell lines. 169 79

We have further analyzed parameters affecting stable transformation of Trypanosoma brucei. Linear DNA was much more efficient than circular DNA and in the vast majority of transformants analyzed the plasmid DNA had inserted into the chromosomes by homologous recombination. The presence of non-homologous (vector) DNA at one or both ends of linear constructs inhibited transformation efficiency. Less than 1 kb of homologous flanking sequence was sufficient for efficient targeting of a marker gene into the tubulin gene array. When transformants with a single neomycin phosphotransferase (neo(r)) gene replacing a beta-tubulin gene were selected for higher levels of G418 resistance, the neo(r) gene was amplified and spread through the tubulin gene cluster. The additional neo(r) gene copies were adjacent in the tubulin gene array and were added to the array rather than replacing beta-tubulin genes. These results are compatible with asymmetric post-replication recombination (unequal sister chromatid exchange) as the mechanism for neo(r) gene amplification. Starting with a circular construct containing the neo(r) gene between tubulin intergenic regions, we obtained a single transformant that maintained the neo(r) genes as an extrachromosomal plasmid. We show this plasmid to consist of a circular pentamer of the input construct. All other attempts to derive a shuttle vector that replicates extrachromosomally in T. brucei were unsuccessful. Our experiments extend previous observations suggesting that T. brucei has a strong preference for chromosomal insertion of exogenous DNA by homologous recombination.
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PMID:Stable transformation of Trypanosoma brucei. 851 75

Human brain and testis specific betaIII-tubulin was amplified from a cDNA library, modified to encode a C-terminal hemagglutinin antigen epitope tag, and cloned into a vector that allows tetracycline regulated expression in mammalian cells. Immunofluorescence analysis of transfected Chinese hamster ovary cells demonstrated that expressed HA-tagged betaIII-tubulin is able to assemble with endogenous tubulin into microtubules even though betaIII-tubulin is not a normal constituent of these cells. A stable G418-resistant clone with moderate HAbetaIII-tubulin expression displayed weak (1.5-2-fold) resistance to paclitaxel. A second clone with higher HAbetaIII-tubulin expression could not grow unless tetracycline was present to repress transcription of the transfected cDNA. Analysis of cellular microtubules in each of these clones indicated that incorporation of HAbetaIII-tubulin led to a significant expression-dependent decrease in assembled tubulin. Paclitaxel resistant cells were also directly selected from the transfected cell population using a paclitaxel concentration 4 times higher than the minimum toxic dose. Few cells were able to survive the selection and they grew very slowly. Western blot analysis of these resistant cells revealed very high HAbetaIII-tubulin expression that led to almost complete replacement of endogenous beta-tubulin at steady state. Transfected betaIII-tubulin with no epitope tag behaved in a very similar fashion indicating that presence of the HA tag had no discernible functional effect. The results demonstrate that betaIII-tubulin diminishes microtubule assembly, is toxic when present at high levels, but is able to confer weak resistance to paclitaxel when expressed at moderate levels in mammalian cells.
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PMID:Expression of class III beta-tubulin reduces microtubule assembly and confers resistance to paclitaxel. 1290 30

Mycosphaerella graminicola is a major pathogen of wheat worldwide, causing Septoria leaf blotch disease. Targeted gene disruption in M. graminicola, by Agrobacterium tumefaciens-mediated transformation, has become an established functional genomics tool for M. graminicola research in recent years. However, in order to advance research into this economically important pathogen, further functional genomics tools need to be developed. Here, we report three new capabilities for M. graminicola research: (i) two selectable markers have been shown to work robustly in M. graminicola, namely G418 and the fungicide carboxin; (ii) the generation of a strain of M. graminicola in which the KU70 (MUS-51) homologue has been disrupted; in this strain, homologous recombination efficiencies increased to more than 95%, whilst maintaining wild-type growth in vitro and full pathogenicity on wheat leaves; (iii) the ability to efficiently target and generate precise mutations of specific genes in the genomic context in M. graminicola. In addition, the insertion of the E198A mutation into the beta-tubulin gene (MgTUB1), conferring resistance to the fungicide benomyl, suggests that this mutant allele may provide an additional selectable marker. The collective use of these tools will permit further advancements in our knowledge of the biology and pathogenicity of this important plant pathogen.
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PMID:New capabilities for Mycosphaerella graminicola research. 2069 6