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 Streptococcus mutans LM7 gene gtfA was cloned in Escherichia coli along with flanking regions of the chromosome as a fragment representing 10.3 kilobases (kb) of streptococcal DNA. Restriction endonuclease mapping revealed that the cloned DNA consisted of four EcoRI fragments with gtfA sucrase activity localized to one fragment, EcoRI-B (2.4 kb). Subsequent analysis with E. coli minicells indicated that three polypeptides were encoded on the 10.3-kb insert (55 [GtfA], 45, and 35 kilodaltons). Neither the 45- nor 35-kilodalton polypeptide exhibited any detectable sucrase activity. The approximate positions and directions of transcription of the two larger proteins were determined from minicell protein profiles displaying truncated versions of these polypeptides. The restriction endonuclease data for the cloned gtfA gene were used to develop a strategy for insertional inactivation of this locus in vivo. An internal HincII fragment of the gtfA gene was removed and replaced with a DNA fragment containing a tetracycline resistance determinant. This new recombinant plasmid was linearized and then transformed into S. mutans GS5 and S. mutans V403 where it was incapable of replication. It was predicted that Tcr colonies would result from double-crossover recombinational events involving homologous regions flanking the gtfA gene. This was verified by Southern DNA hybridization analyses. The inactivation of the gtfA gene in both S. mutans GS5 and S. mutans V403 resulted in a decrease of water-soluble exopolysaccharide but no detectable changes in the amounts of water-insoluble polymers.
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PMID:Molecular organization and expression of the gtfA gene of Streptococcus mutans LM7. 301 93

Streptococcus mutans LM7 (Bratthall serotype e) chromosomal DNA was partially digested with EcoRI and ligated into the positive-selection plasmid vector pOP203(A2+). The ligation mixture was transformed into Escherichia coli, and transformants were selected for tetracycline resistance. Recombinant-bearing clones were screened for their ability to ferment raffinose, using the procedure of Robeson et al. (J. Bacteriol. 153:211-221, 1983). One raffinose-fermenting clone was isolated and found to contain a plasmid with an insert consisting of four EcoRI fragments totalling approximately 10.3 kilobases (kb). This strain was capable of growth on defined medium plus raffinose or sucrose and generated reducing sugars from a sucrose substrate. Southern hybridization analysis of the four EcoRI fragments revealed homology not only to S. mutans LM7 chromosomal DNA but also to S. mutans serotypes b, c, and f. Subcloning of this fragment array into a streptococcal E. coli shuttle vector indicated that a 2.4-kb EcoRI fragment was essential for sucrase activity. E. coli minicell experiments revealed a gene product of 55 kilodaltons. These data along with restriction endonuclease analysis and Southern hybridizations suggested that the cloned S. mutans LM7 gene was closely related to the gtfA gene cloned by Robeson et al. from S. mutans PS13 (Bratthall serotype c). The shuttle plasmid containing the 2.4-kb fragment was transformed into Streptococcus sanguis, which subsequently displayed increased sucrase activity in both intracellular and extracellular fractions. Elevated levels of synthesis of alcohol-insoluble and water-insoluble glucans were observed with crude extracellular fractions of the S. sanguis strain bearing the 2.4-kb fragment. An isolate cured of the shuttle plasmid plus the 2.4-kb fragment displayed wild-type S. sanguis glucan synthesis. In S. sanguis, this gtfA allele may play a role in glucan synthesis by interacting with extant high-molecular-weight glucosyltransferases.
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PMID:Cloned gtfA gene of Streptococcus mutans LM7 alters glucan synthesis in Streptococcus sanguis. 399 43