Method for screening restriction endonucleases

a technology of restriction endonucleases and endonucleases, which is applied in the field of screening and identifying restriction endonucleases, can solve the problems of complex type i systems, failure to select, and dna susceptible to further degradation by non-specific endonucleases, and achieve the effect of facilitating characterization, cloning and production

Inactive Publication Date: 2005-10-06
MORGAN RICHARD
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  • Abstract
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Benefits of technology

[0030] In accordance with one embodiment of the present invention, a novel method for screening for restriction endonucleases is provided. This method has been successfully employed and may be used to identify heretofore unknown restriction endonucleases as well as isoschizomers of known restriction endonucleases, such isoschizomers possessing a desired physical property, such as thermostability. This novel method will also facilitate the characterization, cloning and production of newly identified restriction endonucleases and isoschizomers.

Problems solved by technology

The cleavage that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific endonucleases.
The Type I systems are complex.
Only when expression of the methylase gene is poor or coexpression of flanking sequences is lethal does the selection fail.
In general, genes encoding cytotoxic products are extremely difficult to clone, even when care has been taken to remove sequences that might enable their expression in the plasmid host.
Attempting to clone a toxic gene into a plasmid designed to facilitate high expression is, in many cases, futile.
Thus, some expression of a cloned gene is unavoidable, creating a powerful selective pressure against cells that faithfully replicate the lethal gene.
This approach to cloning a cytotoxic gene, however, suffers from several shortcomings: 1 ) a high copy replicon significantly raises the dosage of the toxic allele, increasing the likelihood for undesired expression; 2) placement of operator sequences on a high copy replicon, while the genes encoding the repressor proteins are present at substantially lower copy number, does not provide optimal repression; 3) strong repression of gene expression and elective induction of gene expression are mutually exclusive.
In this case, pattern matching algorithms do fairly well, but cannot provide conclusive evidence whether a newly sequenced gene encodes an N6A or an N4C methyltransferase.
However, for many of the other patterns we are a long way away from the theoretically possible number.

Method used

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  • Method for screening restriction endonucleases
  • Method for screening restriction endonucleases

Examples

Experimental program
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Effect test

example i

MjaI Restriction Endonuclease

[0077] The restriction endonuclease MjaI, from Methanococcus jannaschii, has previously been characterized biochemically and shown to recognize the sequence CTAG (Zerler, B., Myers, P. A., Escalante, H. and Roberts, R. J. cited in REBASE—see Roberts, R. J. and Macelis, D. Nucl. Acids Res. 26: 338-350 (1998)), but the gene had not been cloned. With the recent determination of the complete sequence of the M. jannaschii genome (Bult et al. Science 273: 1058-1073 (1996)) the sequence was searched using the BLAST program (Altschul, et al. J. Mol. Biol. 215: 403-410 (1990)) to identify candidate restriction enzyme and methylase genes. In brief, all open reading frames in the sequence were compared with the RM sequence database that contained the published sequences of all DNA methylases and restriction endonucleases that had been compiled from entries in GenBank. Each match against an entry in this database was recorded and the corresponding region of the M. ...

example ii

Hhal Restriction Endonuclease

[0087] The genes encoding the restriction endonuclease and methylase of the Hhal system have previously been cloned and sequenced (U.S. Pat. No. 4,999,293). Examination of the sequence showed a characteristic 5-methyl cytosine gene followed by an open reading frame on the complementary strand that was known to be the Hhal restriction endonuclease. This system was used as a test to show that it would be possible to make a sufficient quantity of the restriction enzyme in vitro to allow its detection using standard procedures.

[0088] First, plasmid DNA encoding the Hhal restriction system was prepared from E. coli NEB691 (New England Biolabs). The E. coli cells containing the recombinant plasmid were incubated in 10 ml LB in a roller at 37° C. overnight. Cells were pelleted at 4,000 rpm for 30 sec at 4° C. and the supernatant was discarded. The pellet was resuspended in 1 ml 1×GTE (50 mM glucose, 25 mM Tris.HCl, 10 mM EDTA, pH 8.0) and lysed by adding 0.2 ...

example iii

A 2nd Putative New Restriction Endonuclease from M. jannaschii (ORF 1328—GTNNAC, MjaIV)

[0093] Another of the open reading frames that showed a good match to a known methylase gene was MJ1328. This gene is similar to the gene for M.HincII, which recognizes the sequence GTYRAC. The open reading frame immediately preceding MJ1 328 shows some low similarity to the gene for the HincII restriction enzyme and so is a good candidate for a new restriction enzyme of the same or related specificity. This open reading frame, MJ1327, comprises residues 1748-2485 of GenBank entry U67573. However, because M. jannaschii is a thermophile that normally grows at high temperatures, this new putative restriction enzyme encoded by the open reading frame MJ1327 may be anticipated to work at much higher temperatures than

[0094] HincII, isolated from the mesophile Haemophilus influenzae serotype c (Landy et al. Biochemistry 13: 449-456, 1974).

[0095] The ORF designated MJ1328 by TIGR (The Institute for Gen...

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Abstract

A method is provided for identifying a restriction endonuclease, which includes the steps of (a) screening a target DNA sequence for the presence of known methylase sequence motifs, (b) identifying any open reading frames which lie close to the methylase sequence motifs screened in step (a), and (c) assaying the protein products of these open reading frames for restriction endonuclease activity. Methods for identifying isoschizomers of known restriction endonucleases, which isoschizomers possess a desired physical property, such as thermostability, are also provided by the present invention, as are several novel restriction endonucleases isolated from M. jannaschii, MjaIII and MjaIV. Additionally, a gene was identified that encoded a previously observed endonuclease activity, designated MjaII. Also provided by the present invention are vectors suitable for cloning a DNA sequence encoding a cytotoxic protein, via independent transcription promoters which may be selectively controlled by several conditions. A method for producing these cytotoxic proteins using such vectors is also provided, as are stable clones of Pacl and NlaIII.

Description

RELATED APPLICATIONS [0001] This Application is a Divisional Application of U.S. application Ser. No. 10 / 208,557 filed Jul. 30, 2002, which is a Continuation-In-Part Application of U.S. application Ser. No. 09 / 486,356 filed Feb. 25, 2000, now U.S. Pat. No. 6,383,770, which Application is a 371 of PCT / US98 / 18124 filed Sep. 1, 1998 which claims the benefit of U.S. Provisional Application No. 60 / 057,873 filed Sep. 2, 1997.BACKGROUND OF THE INVENTION [0002] The present invention relates to a novel method for screening and identifying restriction endonucleases based on the proximity of their genes to the genes of their cognate methylases. A similar method for identifying isoschizomers of known endonucleases, which isoschizomers possess a desired physical property is also provided. Related methods for producing and cloning such endonucleases or other cytotoxic proteins are provided, as are several novel M. jannaschii restriction endonucleases. [0003] Nucleases are a class of enzymes which...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): B26D1/00B26D1/08B42C5/00C07H21/04C12N9/22C12N15/74
CPCB26D1/0006B26D1/08B26D2001/006B42C5/00C12Q1/44G01N2333/922C12P21/02G06F19/00C12N9/22
Inventor MORGAN, RICHARD
Owner MORGAN RICHARD
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