Heat shock-induced relaxation of restriction enzyme specificity inEscherichia coli |
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Authors: | M C Raja K Dharmalingam |
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Institution: | 1. Genetic Engineering Research Unit, Department of Biotechnology, Madurai Kamaraj University, 625 021, Madurai, India
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Abstract: | Methylated and hydroxymethylated cytosine containing DNA was restricted by proteins encoded by themcrBC (rglB) loci ofE. coli. In vivo, RglB proteins recognize and cleave hmCT2 and hmCT4 DNAs at 30°C and 42°C but hmCT6 DNA was unaffected at both temperatures,
However, cells carrying therglB genes cloned on pBR322 (pDSS17) did not restrict hmCT6 at 30°C, but hmCT6 DNA was cleaved efficiently at 42°C. Heat shock
treatment for five minutes was enough to induce this promiscuity in recognition specificity. We call this activity RglB star.
A single copy ofrglB located on the chromosome or cloned on a low copy vector pMU575 failed to show RglB star activity.De novo protein synthesis was not required for the manifestation of RglB star activity. |
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Keywords: | RglB star heat shock McrBC McrA |
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