Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2002-05-17
2004-09-21
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S320100, C435S252300, C536S023200
Reexamination Certificate
active
06794172
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to recombinant DNA that encodes the PpuMI restriction endonuclease (PpuMI endonuclease or PpuMI) as well as PpuMI methyltransferase (PpuMI methylase or M.PpuMI), and expression of PpuMI endonuclease and methylase in
E. coli
cells containing the recombinant DNA.
PpuMI endonuclease is found in the bacterium
Pseudomonas putida
(NEB#372, New England Biolabs, Beverly, Mass.). It recognizes the double-stranded DNA sequence 5′RG/GWCCY3′ (W=A or T, R=A or G, Y═C or T, / indicates the cleavage position) and cleaves between the two guanines to generate 3-base cohesive ends. Due to degeneracy at the central position of the recognition sequence, the cohesive ends derived from two different PpuMI sites may or may not be complementary. PpuMI methylase (M.PpuMI) is also found in the same strain and it recognizes the same DNA sequence as PpuMI endonuclease. M.PpuMI displays homology to the C5-cytosine DNA methyltransferase family. Therefore, M.PpuMI presumably methylates the C5 position of one of the cytosines present within the recognition sequence to protect DNA from PpuMI endonuclease cleavage. The substrate for M.PpuMI may be non-methylated or hemi-methylated DNA.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases recognize and bind particular sequences of nucleotides (the ‘recognition sequence’) along DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and twenty-eight restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis,
Nucl. Acids Res.
29:268-269 (2001)).
Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species
Deinococcus radiophilus
for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5′TTT/AAA3′, 5′RG/GNCCR3′ and 5′CACNNN/GTG3′ respectively.
Escherichia coli
RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5′G/AATTC3′.
It is thought that in nature, restriction endonucleases play a protective role in the welfare of the bacterial cells. The enzymes cleave invading foreign DNA molecules such as plasmids or viral DNA that would otherwise destroy or parasitize the bacteria while the host bacterial DNA remains intact. The cleavage that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific nucleases.
A second component of the bacterial protective systems are the modification methylases that protect host DNA from cleavage with restriction endonuclease with which they coexist. The restriction endonuclease and modification methylase form the restriction-modification (R-M) system. The methylase provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign 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 particular nucleotide within the sequence by the addition of a methyl group to produce C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine. 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 the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiable foreign DNA, is susceptible to restriction endonuclease recognition and cleavage. During and after DNA replication, usually hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction endonuclease.
With the advancement of recombinant DNA technology, it is now possible to clone restriction-modification genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction-modification genes is to develop an efficient method to identify such clones within genomic DNA 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 clones with non-methylase inserts are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used 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 expression of restriction-modification systems in bacteria enables them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into
E. coli
cloning vectors (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); Tsp45I: Wayne et al.
Gene
202:83-88 (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al.,
Nucl. Acids. Res.
13:6403-6421 (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but 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 PstI: Walder et al.,
J. Biol. Chem.
258:1235-1241 (1983)).
A more recent method, the “endo-blue method”, has been described for direct cloning of thermostable restriction endonuclease genes into
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, (1996); Fomenkov et al.,
Nucl. Acids Res.
22:2399-2403 (1994)). This method utilizes the
E. coli
SOS response signal following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that some positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.
There are three major groups of methyltransferases identified as C5-cytosine methylases, and the amino-transferases—N4-cytosine methylases and N6-adenine methylases. (Malone et al.
J. Mol. Biol.
253:618-632 (1995)). These groups of methylases derive their names from the position and the ba
Samuelson James
Xu Shuang-yong
New England Biolabs Inc.
Patterson Jr. Charles L.
Strimpel Harriet M.
Williams Gregory D.
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