Nucleic Acids Research, Vol 25, Issue 20 3991-3994, Copyright © 1997 by Oxford University Press
S Xu, J Xiao, J Posfai, R Maunus and J Benner 2nd
BssHII restriction endonuclease cleaves 5'-GCGCGC-3' on double-stranded DNA
between the first and second bases to generate a four base 5'overhang.
BssHII restriction endonuclease was purified from the native Bacillus
stearothermophilus H3 cells and its N-terminal amino acid sequence was
determined. Degenerate PCR primers were used to amplify the first 20 codons
of the BssHII restriction endonuclease gene. The BssHII restriction
endonuclease gene (bssHIIR) and the cognate BssHII methyltransferase gene
(bssHIIM) were cloned in Escherichia coli by amplification of Bacillus
stearothermophilus genomic DNA using PCR and inverse PCR. BssHII
methyltransferase (M.BssHII) contains all 10 conserved cytosine-5
methyltransferase motifs, but motifs IX and X precede motifs I-VIII. Thus,
the conserved motifs of M. BssHII are circularly permuted relative to the
motif organizations of other cytosine-5 methyltransferases. M.BssHII and
the non-cognate multi-specific phiBssHII methyltransferase, M.phiBss HII
[Schumann,J. et al . (1995) Gene, 157, 103-104] share 34% identity in amino
acid sequences from motifs I-VIII, and 40% identity in motifs IX- X. A
conserved arginine is located upstream of a TV dipeptide in the N- terminus
of M.BssHII that may be responsible for the recognition of the guanine 5'
of the target cytosine. The BssHII restriction endonuclease gene was
expressed in E.coli via a T7 expression vector.
ARTICLES
Cloning of the BssHII restriction-modification system in Escherichia coli : BssHII methyltransferase contains circularly permuted cytosine-5 methyltransferase motifs
New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA. xus@neb.com
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