Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-09-23
2001-05-29
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S199000, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06238901
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a new Type II restriction endonuclease, Hpy188III, obtainable from
Helicobacter pylori
J188, and to the process for producing the same.
Restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other contaminating bacterial components, restriction endonucleases can be used in the laboratory to break DNA molecules into precise fragments. This property enables DNA molecules to be uniquely identified and to be fractionated into their constituent genes. Restriction endonucleases have proved to be indispensable tools in modern genetic research. They are the biochemical ‘scissors’ by means of which genetic engineering and analysis is performed.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the ‘recognition sequence’) along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, the sequence. Different restriction endonucleases have affinity for different recognition sequences. The majority of restriction endonucleases recognize sequences of 4 to 6 nucleotides in length, although recently a small number of restriction endonucleases which recognize 7 or 8 uniquely specified nucleotides have been isolated. Most recognition sequences contain a dyad axis of symmetry and in most cases all the nucleotides are uniquely specified. However, some restriciton endonucleases have degenerate or relaxed specificities in that they recognize multiple bases at one or more positions in their recognition sequence, and some restriction endonucleases recognize asymmetric sequences. HaeIII, which recognizes the sequence 5′ GGCC 3′, is an example of a restriction endonuclease having a symmetrical, non-degenerate recognition sequence, while HaeII, which recognizes 5′ (Pu)GCGC(Py) 3′ typifies restriction endonucleases having a degenerate or relaxed recognition sequence. Endonucleases with symmetrical recognition sequences generally cleave symmetrically within or adjacent to the recognition site, while those that recognize assymmetric sequences tend to cleave at a distance of from 1 to 18 nucleotides away from the recognition site. Over one hundred twenty-five unique restriction endonucleases have been identified among several thousands of bacterial species that have been examined to date.
Endonucleases are named according to the bacteria from which they are derived. Thus, the species
Haemophilus aegyptius
, for example synthesizes 3 different restriction endonucleases, named HaeI, HaeII and HaeIII. These enzymes recognize and cleave the sequences (W)GGCC(W) (SEQ ID NO:1), (Pu)GCGC(Py) and GGCC respectively.
Escherichia coli
RY13, on the other hand, synthesizes only one enzyme, EcoRI, which recognizes the sequence GAATTC (SEQ ID NO:2).
While not wishing to be bound by theory, 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 viruses and plasmids that would otherwise destroy or parasitize them. They impart resistance by binding to infecting DNA molecule and cleaving them in each place that the recognition sequence occurs. The disintegration that results inactivates many of the infecting genes and renders the DNA susceptible to further degradation by exonucleases.
A second component of restriction 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 nucleotide recognition sequence as the corresponding restriction endonuclease, but instead of breaking the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease. The DNA of a bacterial cell is always modified, by virtue of the activity of its modification methylase and it is therefore insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign, DNA that is sensitive to restriction endonuclease recognition and attack.
More than 2,500 restriction endonucleases have been isolated from bacterial strains. Of these, more than 1xx recognize unique sequences, while the rest share common recognition specificities. Restriction endonucleases which recognize the same nucleotide sequence are termed “isoschizomers.” Although the recognition sequences of isoschizomers are the same, they may vary with respect to site of cleavage (e.g., XmaI v. SmaI, Endow, et al.,
J. Mol. Biol
. 112:521 (1977); Waalwijk, et al.,
Nucleic Acids Res
. 5:3231 (1978)) and in cleavage rate at various sites (XhoI v. PaeR7I, Gingeras, et al.,
Proc. Natl. Acad. Sci
. U.S.A. 80:402 (1983)).
There is a continuing need for novel Type II restriction endonucleases. Although Type II restriction endonucleases which recognize a number of specific nucleotide sequences are currently available, new restriction endonucleases which recognize novel sequences provide greater opportunities and ability for genetic manipulation. Each new unique endonuclease enables scientists to precisely cleave DNA at new positions within the DNA molecule, with all the opportunities this offers.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a novel restriction endonuclease obtainable from
Helicobacter pylori
NEB#1174, hereinafter referred to as “Hpy188III”, which endonuclease:
(1) recognizes the nucleotide sequence TCNNGA (SEQ ID NO:3) in a double-stranded DNA molecule as shown below,
5′-TC↓NNGA-3′
3′-AGNN↑CT-5′
(wherein G represents guanine, C represents cytosine, A represents adenine, T represents thymine and N represents either G, C, A, or T);
(2) cleaves said sequence in the phosphodiester bonds between the C and first N as indicated with the arrows; and
(3) cleaves double-stranded PhiX174 DNA to produce 26 fragments, including fragments of 704, 560, 497, 477, 423, 309, 308, 304, 261, 225 basepairs, and 16 fragments smaller than 200 basepairs.
The present invention further relates to a process for the production of the novel restriction endonuclease Hpy188III. This process comprises either culturing
Helicobacter pylori
J188 under conditions suitable for expressing Hpy188III, collecting the cultured cells, obtaining a cell-free extract therefrom and separating and collecting the restriction endonuclease Hpy188III from the cell-free extract, or culturing a transformed host, such as
E. coli
, containing the genes for the Hpy188III methylase and endonuclease, collecting the cultured cells, obtaining a cell-free extract therefrom and separating and collecting the restriction endonuclease Hpy188III from the cell-free extract.
In accordance with the present invention, isoschizomers of Hpy188III have been identified, which are substantially similar to Hpy188III (having greater than about 90% homology at the amino acid level), in other Helicobacter strains, such as
Helicobacter pylori
J178 and
Helicobacter pylori
CH4. Of nine different Helicobacter strains studied, for two of which the DNA sequence is known, it was observed that the genes for various given restriction endonucleases and methylases are present and functional in a number, though not all, of these strains. The presence of Hpy188III or isoschizomers thereof, can be readily assertained in various Helicobacter strains by any of several methods. The genomic DNA of the strain may be prepared (as outlined in the Example below) and digested with the enzyme of the current application. If Hpy188III cleaves the genomic DNA, that strain does not contain an active Hpy188III methyltransferase, whereas if the DNA is not cleaved, it is likely the Hpy188III methyltransferase is present in tha
Morgan Richard D.
Xu Qing
New England Biolabs Inc.
Patterson Jr. Charles L.
Williams Gregory D.
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