Type II restriction endonuclease MamI

Details Type II restriction endonuclease MamI Type II restriction endonuclease MamI

1990-07-25

1992-10-06

Weimar, Elizabeth C.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435199, 435822, C12P 1934, C12N 922

Patent

active

051531224

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The invention concerns the new type II restriction endonuclease MamI, a process for its isolation and its use.


BACKGROUND

Type II restriction endonucleases are endodeoxyribonucleases which are able to recognize and cleave particular DNA sequence. In this process, one phosphodiester bridge in each polynucleotide strand of the target sequence is hydrolyzed. Type II restriction endonucleases are thus of value for the analysis of DNA molecules. Although type II restriction endonucleases are known which are specific for numerous DNA sequences, there is still a need for further type II restriction endonucleases which are specific for DNA sequences which up to now have not been recognized by any of the known restriction endonucleases. The object of the invention is therefore to provide a new restriction endonuclease which is able to recognize and cleave a sequence which has not been recognized up to now by any such enzyme.


SUMMARY OF THE INVENTION

This object is achieved according to the present invention by a type II restriction endonuclease having the recognition sequence ##STR2## and the cleavage site indicated by the arrows


DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The new restriction endonuclease according to the present invention, which is denoted MamI hereafter, has a temperature optimum at ca. 37.degree. C. The enzyme has good activity between pH 8.5 and pH 9.0 in 33 mmol/l Tris/HCl buffer with 0.5 mmol/l DTE (dithioerythritol), 10 mmol Mg acetate and 66 mmol K acetate. The pH optimum is at ca. pH 8.8. An isoschizomer for MamI is not known.
The recognition sequence can be confirmed by the complete digestion of the DNA's of the SV40 and adeno 2, viruses of the lambda phage, phix-174 viruses, of the phage derivatives M13mp8 and of the p-asmids pBR322 and pBR328. These DNA molecules are treated with MamI.
Table 1 shows a comparison of the cleavage site specificity observed experimentally with a cleavage site specificity determined by a computer for an enzyme which recognizes the sequence GATNNNNATC.


TABLE 1 __________________________________________________________________________ Number of Number of Fragment lengths Fragment lengths Cleavage positions cleavage sites cleavage sites (base pairs) (base pairs) determined determined determined by determined determined by by computer DNA experimentally computer analysis experimentally computer analysis analysis __________________________________________________________________________ SV40 3* 3 2400, 2400 1083, 1278, at bp 1682, 2765, 2882 4043 phiX174 2 2 2400, 3000 2375, 3011 at bp 341, 2716 M13mp9 2 2 2900, 4800 2825, 4774 at bp 1149, 3974 pBR322 1* 1 undigested 4363 at bp 1668 pBR328 0 0 0 0 0 __________________________________________________________________________ *has a cleavage site which is not cleaved whtn the adenine is methylated.
The cleavage position within the recognition sequence of the enzyme can be determined on a M13 derivative having this recognition sequence at an interval of ca. 30-200 bases from the binding site of the universal sequencing primer (Messing, J. et al., (1981) Nucl. Acids Res. 9, 309-321). At first sequence reactions according to the dideoxy chain-termination method (Sanger, F. et al., (1977) Proc. Natl. Acad. Sci. USA 74, 560-564, Messing, J. et al., (1981) Nucl. Acids Res. 9, 309-321) are carried out on the single-stranded DNA of the M13 derivative with the universal sequencing primer.
Parallel to this, the sequencing primer is radioactively labelled at the 5' end with T4-polynucleotide kinase and [.gamma.-.sup.32 P]ATP. After hybridization of this 5' end-labelled sequencing primer to the single-stranded M13 DNA, a partially double-stranded DNA is prepared in a "filling up" reaction with DNA-polymerase I, Klenow fragment and a deoxynucleotide triphosphate mixture of dATP, dCTP, dGTP and dTTP. This DNA, of which the newly synthesized strand is radioactively labelled at the 5' end, is now cleaved with the restr

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