Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-04-27
2001-06-12
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S193000, C435S252300, C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06245545
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the recombinant DNA which encodes the SwaI restriction endonuclease and modification methylase, and the production of SwaI restriction endonuclease from the recombinant DNA. SwaI restriction endonuclease (see U.S. Pat. No. 5,158,878) is originally isolated from
Staphlococcus warneri.
It recognizes the DNA sequence 5′ ATTTAAAT 3′ and cleaves the phosphodiester bond 5′ to the second A of the recognition sequence to produce a blunt end.
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 precise fragments for molecular cloning and gene characterization.
Type II restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the ‘recognition sequence’) along the DNA molecule. Once bound, they cleave the DNA molecule at specific positions. Different restriction endonucleases have affinity for different recognition sequences. More than 3000 restriction endonucleases have been characterized so far, and they recognize 212 different recognition sequences (Roberts, R. J., Macelis, D. Nucleic Acids Res. 26:338-350 (1998)).
It is thought that in nature, restriction endonucleases play a protective role in the welfare of the bacterial cell. They enable bacteria to resist infection by foreign DNA molecules like bacteriophages and plasmids that would otherwise destroy or parasitize them. They impart resistance by cleaving invading foreign DNA molecules each time that the recognition sequence occurs. 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 bacterial protective systems are the modification methylases. These enzymes are complementary to restriction endonucleases 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 the target nucleotide within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer cleaved by the restriction endonuclease. The DNA of a bacterial cell is always modified by virtue of the activity of its modification methylase. It is therefore insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiable foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
With the advent of genetic engineering technology, it is now possible to clone genes and to produce the proteins that they encode in greater quantities than are obtainable by conventional purification techniques. 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 bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Molec. 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 phage. 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. Acid. 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 which is being used to clone a growing number of systems, involves selection for an active methylase gene (see, e.g., U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acid. Res. 13: 6403-6421, (1985)). Since restriction and modification genes are often closely linked, 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 MspI: Walder et al., J. Biol. Chem. 258: 1235-1241, (1983)).
Another method for cloning methylase and endonuclease genes is based on a colorimetric assay for DNA damage (see, U.S. Pat. No. 5,492,823). When screening for a methylase, the plasmid library is transformed into the host
E. coli
strain such as AP1-200. The expression of a methylase will induce the SOS response in an
E. coli
strain which is McrA+, McrBC+, or Mrr+. The AP1-200 strain is temperature sensitive for the Mcr and Mrr systems and includes a lac-Z gene fused to the damage inducible dinD locus of
E. coli
. The detection of recombinant plasmids encoding a methylase or endonuclease gene is based on induction at the restrictive temperature of the lacZ gene. Transformants encoding methylase genes are detected on LB agar plates containing X-gal as blue colonies. (Piekarowicz, et. al., Nucleic Acids Res. 19:1831-1835, (1991) and Piekarowicz, et. al. J. Bacteriology 173:150-155 (1991),). Likewise, the
E. coli
strain ER1992 contains a dinD1-Lac Z fusion but is lacking the methylation dependent restriction systems McrA, McrBC and Mrr. In this system (called the “endo-blue” method), the endonuclease gene can be detected in the absence of it's cognate methylase when the endonuclease damages the host cell DNA, inducing the SOS response. The SOS-induced cells form deep blue colonies on LB agar plates supplemented with X-gal. (Xu et. al. Nucleic Acids Res. 22:2399-2403 (1994)).
Sometimes the straight-forward methylase selection method fails to yield a methylase (and/or endonuclease) clone due to various obstacles. See, e.g., Lunnen, et al., Gene, 74(1):25-32 (1988). One potential obstacle to cloning restriction-modification genes lies in trying to introduce the endonuclease gene into a host not already protected by modification. If the methylase gene and endonuclease gene are introduced together as a single clone, the methylase must protectively modify the host DNA before the endonuclease has the opportunity to cleave it. On occasion, therefore, it might only be possible to clone the genes sequentially, methylase first then endonuclease (see, U.S. Pat. No. 5,320,957).
Another obstacle to cloning restriction-modification systems lies in the discovery that some strains of
E. coli
react adversely to cytosine or adenine modification; they possess systems that destroy DNA containing methylated cytosine (Raleigh and Wilson, Proc. Natl. Acad. Sci., USA 83:9070-9074, (1986)) or methylated adenine (Heitman and Model, J. Bact. 196:3243-3250, (1987); Raleigh, Trimarchi, and Revel, Genetics, 122:279-296, (1989) Waite-Rees, et al., J. Bacteriology, 173:5207-5219 (1991)). Cytosine-specific or adenine-specific methylase genes cannot be cloned easily into these strains, either on their
Dalton Michael A.
Higgins Lauren S.
Kong Huimin
Cullem James Gregory
New England Biolabs Inc.
Slobodyansky Elizabeth
Williams Gregory D.
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