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)

A large cassette, 4.6 x 10(3) bases (4.6 kb) in length, containing an inducible expression system (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene) and a bacterial neomycin-resistance gene (neo) has been cloned into the noncoding region of a GAL1-regulated Ty1 retrotransposon. Galactose was used to induce retrotransposition in Saccharomyces cerevisiae, and cells containing integrations were selected by resistance to the aminoglycoside G418. Integrations of neo and CUP1p-lacZ were verified, and beta-galactosidase activity was confirmed. Analysis via Southern blots demonstrated integrations at various chromosomal locations, and the number of insertions obtained ranged from one to five after three rounds of induction. Therefore, the packaging limit of Ty1 virus-like particles for RNA is at least 10.3 kb and Ty1 can transpose foreign genes as large as 4.6 kb, demonstrating the practical application of Ty1 for the insertion of large regulated expression cassettes.
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PMID:Application of Ty1 for cloned gene insertion: amplification of a large regulated expression cassette in Saccharomyces cerevisiae. 870 32

An intron module was developed for Saccharomyces cerevisiae that imparts conditional gene regulation. The kanMX marker, flanked by loxP sites for the Cre recombinase, was embedded within the ACT1 intron and the resulting module was targeted to specific genes by PCR-mediated gene disruption. Initially, recipient genes were inactivated because the loxP-kanMX-loxP cassette prevented formation of mature transcripts. However, expression was restored by Cre-mediated site-specific recombination, which excised the loxP-kanMX-loxP cassette to generate a functional intron that contained a single loxP site. Cre recombinase activity was controlled at the transcriptional level by a GAL1::CRE expression vector or at the enzymatic level by fusing the protein to the hormone-dependent regulatory domain of the estrogen receptor. Negative selection against leaky pre-excision events was achieved by growing cells in modified minimal media that contained geneticin (G418). Advantages of this gene regulation technique, which we term the conditional knock-out approach, are that (i) modified genes are completely inactivated prior to induction, (ii) modified genes are induced rapidly to expression levels that compare to their unmodified counterparts, and (iii) it is easy to use and generally applicable.
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PMID:Controlling gene expression in yeast by inducible site-specific recombination. 1112 95

The budding yeast species Saccharomyces castellii has provided important new insights into molecular evolution when incorporated in comparative genomics studies and studies of mitochondrial inheritage. Although it shows some diversity in the specific molecular details, several analyses have shown that it contains many genetic pathways similar to those of S. cerevisiae. Here we have investigated the possibility of performing genetic analyses in S. castellii. We optimized the LiAc transformation protocol to achieve 200-300 transformants/microg plasmid DNA. We found that the commonly used plasmids for S. cerevisiae are stably maintained in S. castellii under selective conditions. Surprisingly, both 2micro and CEN/ARS plasmids are kept at a high copy number. Moreover, the kanMX cassette can be used as a resistance marker against the selective drug geneticin (G418). Finally, we determined that the S. cerevisiae GAL1 promoter can be used for the activation of transcription in S. castellii, thus enabling the controlled overexpression of genes when galactose is present in the medium. The availability of these tools provides the possibility of performing genetic analyses in S. castellii, and makes it a promising new model system in which hypotheses derived from bioinformatics studies can be experimentally tested.
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PMID:Tools and methods for genetic analysis of Saccharomyces castellii. 1743 26