Dataset Information

Characterization of a heat resistant beta-glucosidase as a new reporter in cells and mice.

ABSTRACT: Reporter genes are widely used in biology and only a limited number are available. We present a new reporter gene for the localization of mammalian cells and transgenic tissues based on detection of the bglA (SYNbglA) gene of Caldocellum saccharolyticum that encodes a thermophilic beta-glucosidase.SYNbglA was generated by introducing codon substitutions to remove CpG motifs as these are associated with gene silencing in mammalian cells. SYNbglA expression can be localized in situ or detected quantitatively in colorimetric assays and can be co-localized with E. coli beta-galactosidase. Further, we have generated a Cre-reporter mouse in which SYNbglA is expressed following recombination to demonstrate the general utility of SYNbglA for in vivo analyses. SYNbglA can be detected in tissue wholemounts and in frozen and wax embedded sections.SYNbglA will have general applicability to developmental and molecular studies in vitro and in vivo.

SUBMITTER: McCutcheon SC

PROVIDER: S-EPMC2909947 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Characterization of a heat resistant beta-glucosidase as a new reporter in cells and mice.

McCutcheon Susan C SC Jones Ken K Cumming Sarah A SA Kemp Richard R Ireland-Zecchini Heather H Saunders John C JC Houghton Carol A CA Howard Louise A LA Winton Douglas J DJ

BMC biology 20100622

<h4>Background</h4>Reporter genes are widely used in biology and only a limited number are available. We present a new reporter gene for the localization of mammalian cells and transgenic tissues based on detection of the bglA (SYNbglA) gene of Caldocellum saccharolyticum that encodes a thermophilic beta-glucosidase.<h4>Results</h4>SYNbglA was generated by introducing codon substitutions to remove CpG motifs as these are associated with gene silencing in mammalian cells. SYNbglA expression can b ...[more]

PMID: 20569471

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Project description:Ipecac alkaloids produced in the medicinal plant Psychotria ipecacuanha such as emetine and cephaeline possess a monoterpenoid-tetrahydroisoquinoline skeleton, which is formed by condensation of dopamine and secologanin. Deglucosylation of one of the condensed products N-deacetylisoipecoside (1 alpha(S)-epimer) is considered to be a part of the reactions for emetine biosynthesis, whereas its 1 beta(R)-epimer N-deacetylipecoside is converted to ipecoside in P. ipecacuanha. Here, we isolated a cDNA clone Ipeglu1 encoding Ipecac alkaloid beta-D-glucosidase from P. ipecacuanha. The deduced protein showed 54 and 48% identities to raucaffricine beta-glucosidase and strictosidine beta-glucosidase, respectively. Recombinant IpeGlu1 enzyme preferentially hydrolyzed glucosidic Ipecac alkaloids except for their lactams, but showed poor or no activity toward other substrates, including terpenoid-indole alkaloid glucosides. Liquid chromatography-tandem mass spectrometry analysis of deglucosylated products of N-deacetylisoipecoside revealed spontaneous transitions of the highly reactive aglycons, one of which was supposed to be the intermediate for emetine biosynthesis. IpeGlu1 activity was extremely poor toward 7-O-methyl and 6,7-O,O-dimethyl derivatives. However, 6-O-methyl derivatives were hydrolyzed as efficiently as non-methylated substrates, suggesting the possibility of 6-O-methylation prior to deglucosylation by IpeGlu1. In contrast to the strictosidine beta-glucosidase that stereospecifically hydrolyzes 3 alpha(S)-epimer in terpenoid-indole alkaloid biosynthesis, IpeGlu1 lacked stereospecificity for its substrates where 1 beta(R)-epimers were preferred to 1 alpha(S)-epimers, although ipecoside (1 beta(R)) is a major alkaloidal glucoside in P. ipecacuanha, suggesting the compartmentalization of IpeGlu1 from ipecoside. These facts have significant implications for distinct physiological roles of 1 alpha(S)- and 1 beta(R)-epimers and for the involvement of IpeGlu1 in the metabolic fate of both of them.

Dataset Information

Characterization of a heat resistant beta-glucosidase as a new reporter in cells and mice.

Publications

Characterization of a heat resistant beta-glucosidase as a new reporter in cells and mice.

Similar Datasets

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