Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-06-02
2001-04-03
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06210945
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to recombinant DNA that encodes the RsaI restriction endonuclease, as well as the RsaI methylase, and to the production of the RsaI restriction endonuclease from the recombinant DNA.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other bacterial components, restriction endonucleases can be used in the laboratory to cleave DNA molecules into fragments for molecular cloning and gene characterization.
Restriction endonucleases act by binding to particular sequences of nucleotides (the ‘recognition sequence’) along the DNA molecule. Once bound, they cleave the DNA molecule within, to one side of, or to both sides of the recognition sequence. Different restriction endonucleases recognize and cleave different nucleotide sequences. Over two hundred restriction endonucleases with unique specificities have been identified among thousands of bacterial species that have been examined (Roberts and Macelis,
Nucl. Acids Res.
24:223-235, (1996)).
Restriction endonucleases are named according to the bacteria from which they derive. Thus, the bacterium
Deinococcus radiophilus
for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5′-TTTAAA-3′, 5′-PuGGNCCPy-3′ and 5′-CACNNNGTG-3′ respectively.
Escherichia coli
RY13, on the other hand, produces only one restriction enzyme, EcoRI, which recognizes the sequence 5′ GAATTC 3′.
Restriction endonucleases usually occur together with one or more companion enzymes termed methyltransferase, the whole forming a restriction-modification (R-M) system. Methyltransferases are complementary to the restriction endonuclease they accompany and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one of the nucleotides within the sequence by the addition of a methyl group to form 5-methylcytosine, N4-methylcytosine, or N6-methyladenine. Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by virtue of the activity of its modification methylase(s), and therefore it is completely insensitive to the presence of the restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
With the advent of recombinant DNA technology, it is possible to clone genes and overproduce the enzymes they encode in large quantities. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex ‘libraries’, i.e. populations of clones derived by ‘shotgun’ procedures, when they occur at frequencies as low as 10
−3
to 10
−4
. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.
Type II restriction-modification systems are being cloned with increasing frequency. The first cloned systems used resistance to bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al.,
Mol. Gen. Genet.
178:717-719, (1980); HhaII: Mann et al.,
Gene
3:97-112, (1978); PstI: Walder et al.,
Proc. Nat. Acad. Sci.
78:1503-1507, (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phages. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into
E. coli
cloning plasmids (EcoRV: Bougueleret et al.,
Nucl. Acids. Res.
12:3659-3676, (1984); PaeR7: Gingeras and Brooks,
Proc. Natl. Acad. Sci. USA
80:402-406, (1983); Theriault and Roy,
Gene
19:355-359 (1982); PvuII: Blumenthal et al.,
J. Bacteriol.
164:501-509, (1985)).
A third approach to clone R-M systems is by selection for an active methylase gene (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al.,
Nucl. Acids. Res.
13:6403-6421, (1985)). Since R and M genes are usually closely linked, both genes can often be cloned simultaneously by selecting for only one. Selection for the M gene does not always yield a complete restriction system however, but often instead yields only the methylase gene (BspRI: Szomolanyi et al.,
Gene
10:219-225, (1980); BcnI: Janulaitis et al.,
Gene
20:197-204 (1982); BsuRI: Kiss and Baldauf,
Gene
21:111-119, (1983); and MspI: Walder et al.,
J. Biol. Chem.
258:1235-1241, (1983)).
Another approach is to clone R-M Systems in
E.coli
by making use of the fact that certain modification genes, when cloned into a new host and adequately expressed, enable the host to tolerate the presence of a different restriction gene (Wilson et al; U.S. Pat. No. 5,246,845).
A more recent method, the “endo-blue method”, has been described for direct cloning of restriction endonuclease genes in E. coli based on the indicator strain of
E. coli
containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535; Fomenkov et al.,
Nucl. Acids Res.
22:2399-2403, (1994)). This method utilizes the
E. coli
SOS response following DNA damages caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535).
Because purified restriction endonucleases, and to a lesser extent modification methylases, are useful tools for manipulating DNA molecules in the laboratory, there is a commercial incentive to create bacterial strains through recombinant DNA techniques that produce these enzymes in large quantities. Such overexpression strains also simplify the task of enzyme purification.
SUMMARY OF THE INVENTION
The methylase selection method was used to clone the RsaI methylase gene (rsaIM) from
Rhodopseudomonas sphaeroides
(NEB (New England Biolabs, Beverly, Mass.) Culture Collection #233, (Lynn, et al.,
J. Bacteriol.
142:380-383 (1980)) into the
E.coli
plasmid vector pBR322. Subcloning, deletion mapping, and DNA sequencing verified the location of the inserted RsaI methylase gene (ORF1) and revealed the presence of a second incomplete converging open reading frame (ORF2).
Because methylase endonuclease genes usually occur next to each other in bacterial DNA, ORF2 was assumed to be the rsaIR gene and efforts were made to clone the missing portion of ORF2. Southern blots revealed that Bc/I-, BstYI-, and PstI-fragments could potentially contain rsaIM as well as enough adjacent DNA to include the whole ORF2. Methylase selection on de novo libraries made with Bc/I and BstYI, as well as with size-fractionated, gel-purified, PstI-digested, chromosomal DNA failed to yield any RsaI methylase clones whatsoever, suggesting that these fragments were perhaps toxic in
E.coli.
Native RsaI restriction endonuclease was purified to near homogeneity from a
Rhodopseudomonas sphaeroides
cell extract. Two proteins of approximately 18 kDa and 22 kDa were found to be present in the prep by SDS-PAGE gel analysis. The N-terminal amino acid sequences of both of these proteins were determined, and they were used to synthesize primers for PCR of a fragment containing rsaIM and the converging ORF2. These PCR attempts also failed to yield the desired clone.
Finally, inverse PCR, was used to isolate the adjacent c
Lunnen Keith D.
Meixsell Timothy
Morgan Richard D.
Wilson Geoffrey G.
New England Biolabs Inc.
Patterson Jr. Charles L.
Williams Gregory D.
LandOfFree
Method for cloning and producing the RsaI restriction... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for cloning and producing the RsaI restriction..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for cloning and producing the RsaI restriction... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2445113