Cloning of NruI and Sbo13I restriction and modification sstems in E. coli and amino acid sequence comparison of M.NruI and M.Sbo13I with other amino-methyltransferasesReportar como inadecuado




Cloning of NruI and Sbo13I restriction and modification sstems in E. coli and amino acid sequence comparison of M.NruI and M.Sbo13I with other amino-methyltransferases - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Research Notes

, 3:139

First Online: 24 May 2010Received: 23 April 2010Accepted: 24 May 2010DOI: 10.1186-1756-0500-3-139

Cite this article as: Zhu, Z., Pedamallu, C.S., Fomenkov, A. et al. BMC Res Notes 2010 3: 139. doi:10.1186-1756-0500-3-139

Abstract

BackgroundNruI and Sbo13I are restriction enzyme isoschizomers with the same recognition sequence 5- TCG↓CGA 3- cleavage as indicated↓. Here we report the cloning of NruI and Sbo13I restriction-modification R-M systems in E. coli. The NruI restriction endonuclease gene nruIR was cloned by PCR and inverse PCR using primers designed from the N-terminal amino acid sequence. The NruI methylase gene nruIM was derived by inverse PCR walking.

ResultsThe amino acid sequences of NruI endonuclease and methylase are very similar to the Sbo13I R-M system which has been cloned and expressed in E. coli by phage selection of a plasmid DNA library. Dot blot analysis using rabbit polyclonal antibodies to N6mA- or N4mC-modified DNA indicated that M.NruI is possibly a N6mA-type amino-methyltransferase that most likely modifies the external A in the 5- TCGCGA 3- sequence. M.Sbo13I, however, is implicated as a probable N4mC-type methylase since plasmid carrying sbo13IM gene is not restricted by Mrr endonuclease and Sbo13I digestion is not blocked by Dam methylation of the overlapping site. The amino acid sequence of M.NruI and M.Sbo13I did not show significant sequence similarity to many known amino-methyltransferases in the α, β, and γ groups, except to a few putative methylases in sequenced microbial genomes.

ConclusionsThe order of the conserved amino acid motifs blocks in M.NruI-M.Sbo13I is similar to the γ. group amino-methyltranferases, but with two distinct features: In motif IV, the sequence is DPPY instead of NPPY; there are two additional conserved motifs, IVa and Xa as extension of motifs IV and X, in this family of enzymes. We propose that M.NruI and M.Sbo13I form a subgroup in the γ group of amino-methyltransferases.

Electronic supplementary materialThe online version of this article doi:10.1186-1756-0500-3-139 contains supplementary material, which is available to authorized users.

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Autor: Zhenyu Zhu - Chandra Sekhar Pedamallu - Alexey Fomenkov - Jack Benner - Shuang-yong Xu

Fuente: https://link.springer.com/







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