Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mutational potency of apurinic/apyrimidinic (AP) sites induced by heat-treatment under acidic conditions has been studied in mammalian cells. Abasic sites were induced on a single-stranded DNA shuttle vector carrying the supF tRNA gene, eliminating, therefore, any ambiguity concerning the damaged strand. This vector was able to replicate both in mammalian cells and in bacteria where the mutations induced in animal cells on the supF tRNA gene were screened by the white/blue beta-galactosidase assay in the presence of isopropyl-1-thio-beta-D-galactopyranoside and 5-bromo-4-chloro-3-indoyl-beta-D-galactoside. All white colonies contained plasmid with a mutation on the target gene which was directly sequenced. Our results show that one AP site was induced/22 min of heating as measured by sensitivity of DNA to alkali denaturation or treatment with the AP-endonuclease activity of the FPG protein (Fapy-DNA glycosylase). Putative AP sites decrease survival of the plasmid with a lethal hit of one AP site/single-stranded molecule. Mutation frequency was increased by a factor of approximately six after 2 h at 70 degrees C. Most of the induced mutations were point mutations not distributed at random and clustered in the gene region which will give rise to the mature tRNA. Mutations were abolished by treatments that eliminated AP sites such as alkali treatment or incubation with the Fapy-DNA glycosylase protein. Under our experimental conditions, when only single mutations were taken into account, the order of base insertion opposite AP sites was G greater than A greater than T greater than C.
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PMID:Mutation spectrum of heat-induced abasic sites on a single-stranded shuttle vector replicated in mammalian cells. 152 92

The aims of this research were to construct prokaryotic expression vector containing fusion gene of Cholecystokinin 39 (CCK39) of pig and Urease subunit B (UreB) of coliform bacteria, and then to express the fusion protein in recombinant Escherichia coli BL21(DE3). The CCK39 gene was amplified by RT-PCR from the extracted total RNA of pig's duodenum, and the UreB gene was then amplified by PCR from the extracted plasmid DNA of bacillus of coliform bacteria from pig's intestinal content. Then the CCK39 and the UreB were inserted into the prokaryotic expression vector pET43a(+) to construct a recombinant fusion expression vector pET43a(+)/CCK39/UreB and then, the recombinant vector was identified by PCR, endonuclease digestion and sequence analysis. It was identified that the gene fragment of CCK39 at length of 117 bp and UreB at length of 324 bp were amplified and cloned into the vector pET43a(+) successfully. The recombinant vector was transformed into Escherichia coli BL21(DE3) and induced the expression of CCK39/UreB fusion protein with a molecular mass of approximately 80 kD by using isopropylthio-beta-D-galactoside (IPTG) as inducer. The fusion protein was mostly located in the cytoplasm and it was soluble. The soluble protein was collected and purified by Ni2+-NTA column chromatograph and then reached a purity of more than 95%. It was proved by western blotting that the fusion protein could react with rabbit anti-CCK8 antiserum and rabbit anti-UreB antiserum. Therefore, the expressed fusion protein has good antigenicity. This work established a good foundation for further study on the production of anti-CCK/Urease vaccines.
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PMID:[Construction and expression of the fusion gene CCK39/UreB in recombinant Escherichia coli BL-21(DE3)]. 1944 Dec 24