Type II Restriction endonuclease, HpyCH4V, obtainable from helicobacter pylori CH4 and a process for producing the same

In accordance with the present invention, there is provided a novel restriction endonuclease and its DNA obtainable from Helicobacter pylori CH4 (NEB#1236), hereinafter referred to as "HpyCH4V", which endonuclease: PA1 (1) recognizes the nucleotide sequence 5'-TGCA-3' in a double-stranded DNA molecule as shown below, EQU 5'-TG.dwnarw.CA-3' EQU 3'-AC.Arrow-up bold.GT-5' (wherein G represents guanine, C represents cytosine, A represents adenine, T represents thymine and N represents either G, C, A, or T); PA1 (2) cleaves said sequence in the phosphodiester bonds between the G and C as indicated with the arrows; and PA1 (3) cleaves double-stranded pBR322 DNA to produce 21 fragments, including fragments of 576, 498, 441, 335, 315, 312, 296, 244 and 205 base pairs, and 12 fragments smaller than 200 base pairs.

BACKGROUND OF THE INVENTION 
The present invention relates to a new Type II restriction endonuclease, 
HpyCH4V, obtainable from Helicobacter pylori CH4, 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 restriction 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 asymmetric sequences tend to cleave at a distance of from 1 to 
18 nucleotides away from the recognition site. More than two hundred 
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 5'-(W)GGCC(W)-3' (SEQ ID NO:1), 
5'-(Pu)GCGC(Py)-3' and 5'-GGCC-3' respectively. Escherichia coli RY13, on 
the other hand, synthesizes only one enzyme, EcoRI, which recognizes the 
sequence 5'-GAATTC-3' (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 3000 
restriction endonucleases have been isolated from various bacterial 
strains. Of these, more than 200 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 CH4 
(NEB#1236), hereinafter referred to as "HpyCH4V", which endonuclease: 
(1) recognizes the nucleotide sequence 5'-TGCA-3' in a double-stranded DNA 
molecule as shown below, 
EQU 5'-TG.dwnarw.CA-3' 
EQU 3'-AC.Arrow-up bold.GT-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 G and C 
as indicated with the arrows to create blunt ends; and 
(3) cleaves double-stranded pBR322 DNA to produce 21 fragments, including 
fragments of 576, 498, 441, 335, 315, 312, 296, 244 and 205 base pairs, 
and 12 fragments smaller than 200 base pairs. 
The present invention further relates to a process for the production of 
the novel restriction endonuclease HpyCH4V. This process comprises either 
culturing Helicobacter pylori Ch4 under conditions suitable for expressing 
HpyCH4V, collecting the cultured cells, obtaining a cell-free extract 
therefrom and separating and collecting the restriction endonuclease 
HpyCH4V from the cell-free extract, or culturing a transformed host, such 
as E. coli, containing the genes for the HpyCH4V methylase and 
endonuclease, collecting the cultured cells, obtaining a cell-free extract 
therefrom and aseparating and collecting the restriction endonuclease 
HpyCH4V from the cell-free extract.

DETAILED DESCRIPTION OF THE INVENTION 
The recognition sequence of the endonuclease of the present invention may 
be determined by mapping the locations of several HpyCH4V cleavage sites 
in various DNAs and comparing the DNA sequences of these regions for 
homology, then comparing the predicted cleavage fragments of the putative 
recognition sequence with the observed restriction fragments produced by 
HpyCH4V cleavage of various DNAS. The endonuclease HpyCH4V was found to 
cleave pBR322 DNA more than ten times, producing fragments of 
approximately 575, 500, 450, 340, 320, 300, 250 and 200 bp, along with a 
number of smaller fragments. The location of several cut sites were mapped 
to approximate positions of 250 and 4040 (the 575 bp fragment) by 
simultaneously digesting PhiX174 DNA with HpyCH4V and with endonucleases 
which cleave at known positions, such as ClaI, NruI, NdeI, and PstI (FIG. 
1). The approximate size of several of the DNA fragments produced by 
HpyCH4V digestion of pBR322 DNA was entered into the program SITES 
(Gingeras, et al., Nucl. Acids Res. 5:4105 (1978)), which generates 
potential recognition sequences for the input data by comparing the 
fragment sizes which would result from cleavage of the DNA at any given 
recognition pattern with the input fragment sizes. One such potential 
pattern generated was 5'-TGCA-3', which occurs in pBR322 DNA at positions 
consistent with the mapping data obtained, i.e. at positions 250 and 4037. 
The size of fragments predicted from cleavage at the sequence 5'-TGCA-3' 
in pBR322, pUC19 and PhiX174 DNAs matched the observed size of fragments 
from cleavage of these DNAs with HpyCH4V, from which we conclude that 
HpyCH4V recognizes the sequence 5'-TGCA-3'. 
The point of cleavage within the HpyCH4V recognition sequence may be 
determined through dideoxy sequencing analysis of the terminal base 
sequence obtained from HpyCH4V cleavage of a suitable DNA substrate 
(Sanger, et al., PNAS 74:5463-5467 (1977) Brown, et al., J. Mol. Biol. 
140:143-148 (1980)). By the above referenced method (FIG. 2, exemplified 
in Example II) it was found that HpyCH4V cleaves the phosphodiester bond 
between the G and the C in the recognition sequence 5'-TGCA-3' to produce 
a blunt end extension, as indicated by the arrows: 
EQU 5'-TG.dwnarw.CA-3' 
EQU 3'-AC.Arrow-up bold.GT-5' 
In accordance with the present invention, HpyCH4V is obtained by culturing 
Helicobacter pylori CH4 and recovering the endonuclease from the cells. A 
sample of Helicobacter pylori CH4 (NEB#1236) has been deposited under the 
terms and conditions of the Budapest Treaty with the American Type Culture 
Collection (ATCC) on Sep. 23, 1999 and bears the Patent Accession No. 
PTA-781. 
For recovering the enzyme of the present invention Helicobacter pylori CH4 
may be grown using any suitable technique. For example, Helicobacter 
pylori CH4 may be grown in Brucella broth media (BBL Microbiology Systems, 
Cockeysville, Md.) incubated at 37.degree. C. Cells in the late 
logarithmic stage of growth are collected by centrifugation and either 
disrupted immediately or stored frozen at -70.degree. C. 
The HpyCH4V enzyme can be isolated from Helicobacter pylori CH4 cells by 
conventional protein purification techniques. For example, cell paste is 
suspended in a buffer solution and treated by sonication, high pressure 
dispersion or enzymatic digestion to allow extraction of the endonuclease 
by the buffer solution. Intact cells and cellular debris are then removed 
by centrifugation to produce a cell-free extract containing HpyCH4V. The 
HpyCH4V endonuclease is then purified from the cell-free extract by 
ion-exchange chromatography, affinity chromatography, molecular sieve 
chromatography, or a combination of these methods to produce the 
endonuclease of the present invention. 
The endonuclease of the present invention along with its corresponding 
methylase may also be obtained using recombinant DNA techniques, such as 
the methylation selection technique disclosed by Wilson, et al., U.S. Pat. 
No. 5,200,333. As an example, DNA from a bacterial strain which contains 
an R-M system, such as Helicobacter pylori, is purified, partially 
digested with suitable type II endonucleases, and ligated to an 
appropriate cleaved, dephosphorylated cloning vector. The ligated DNA is 
transformed into an appropriate host, such as E. coli, the transformants 
are pooled and the population of cloning vectors are purified to form 
libraries. The library of clones is then challenged by digesting with an 
endonuclease which will selectively destroy vectors which do not contain 
and express the methylase of the R-M system being cloned. Vectors which 
contain and express the methylase gene of interest will be modified at the 
endonuclease recognition sites of the challenging endonuclease and thus be 
immune from cleavage. The challenged clone pools are then transformed back 
into the appropriate host to recover the undigested, presumably methylase 
expressing clones. The transformants may be screened for endonuclease 
activity or cycled through further rounds of purification and selection. 
Finally, individual transformants are selected and their DNA purified. 
These clones are analyzed for resistance to cleavage by the endonuclease 
of interest and for common insert DNA. Cell extracts prepared from 
transformants which demonstrate endonuclease resistance are assayed in 
vitro for methyltransferase and endonuclease activities. 
The present invention is further illustrated by the following Examples. 
These Examples are provided to aid in the understanding of the invention 
and are not construed as a limitation thereof. 
The references cited above and below are herein incorporated by reference. 
EXAMPLE I 
Production of HpyCH4V Endonuclease 
Helicobacter pylori CH4 strain NEB#1236 was grown in Brucella broth media. 
The cells were incubated anaerobically under 5% CO.sub.2 at 37.degree. C. 
until late logarithmic stage. The cells were then harvested by 
centrifugation and stored frozen at -70.degree. C. 
8 grams of the cells obtained above were suspended in 40 mls buffer A (20 
mM Tris-HCl, 0.1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, pH 7.6 at 
25.degree. C.) adjusted to 50 mM NaCl. The cell suspension was sonicated 
until approximately 50 mg protein per gram of cells was released. The 
lysate was centrifuged at 15,000 rpm for 20 minutes at 4.degree. C. in a 
Beckman JA17 rotor. 44 ml of supernatant was obtained containing 
approximately 400 mg of soluble protein. 
The supernatant solution was applied to a 20 ml Heparin Hyper-D column 
(Biosepra, Marlborough, Mass.) equilibrated in buffer A adjusted to 50 mM 
NaCl. A 40 ml wash of buffer A adjusted to 50 mM NaCl was applied, then a 
200 ml linear gradient of NaCl from 50 mM to 1M in buffer A was applied 
and fractions of 4 ml were collected. Fractions were assayed for HpyCH4V 
endonuclease activity by incubation with 1 .mu.g Lambda DNA (NEB) in 50 
.mu.l NEBuffer 4 for one hour at 37.degree. C. HpyCH4V activity eluted at 
0.31M to 0.49M NaCl. 
The Heparin Hyper-D column fractions containing the HpyCH4V activity were 
pooled, diluted to 100 mM NaCl in buffer A and applied to a 3 ml 
Heparin-TSK column (Toso-Haas, Philadelphia, Pa.), and a 50 ml linear 
gradient from 0.1M to 0.6M NaCl in buffer A was applied to the Heparin-TSK 
column. The HpyCH4V activity eluted between 0.2M to 0.4M NaCl. The 
Heparin-TSK fractions containing HpyCH4V activity were pooled, diluted to 
50 mM NaCl in buffer A and applied to a 1 ml Mono-Q column (Pharmacia, 
Piscataway, N.J.), and a 50 ml linear gradient from 0.1 M to 0.6 M NaCl in 
buffer A was applied to the column. The HpyCH4V activity eluted between 
0.12M to 0.15M NaCl and contained approximately 1000 units of HpyCH4V 
endonuclease activity. The HpyCH4V obtained was substantially pure and 
free of contaminating endonuclease and exonuclease activities. Bovine 
serum albumin was added as a stabilizer to a final concentration of 200 
.mu.g/ml and the HpyCH4V enzyme was dialyzed against storage buffer (50% 
glycerol, 50 mM NaCl, 20 mM Tris-HCl, 0.1 mM dithiothreitol, pH 7.5). 
Activity determination 
HpyCH4V activity: Samples of from 1 to 10 .mu.l were added to 50 .mu.l of 
substrate solution consisting of 1X NEBuffer 4 containing 1 .mu.g Lambda 
phage DNA. The reaction was incubated at 37.degree. C. for 5 to 60 mins. 
The reaction was terminated by adding 15 .mu.ls of a stop solution (50% 
glycerol, 50 mM EDTA pH 8.0, and 0.02% Bromophenol Blue). The reaction 
mixture was applied to a 1.2% agarose gel and electrophoresed. The bands 
obtained were identified in comparison with DNA size standards. 
Unit Definition: One unit of HpyCH4V is defined as the amount of HpyCH4V 
required to completely cleave 1.0 .mu.g of Lambda DNA in a total reaction 
volume of 50 .mu.l NEBuffer 4, supplemented with 100 .mu.g/ml bovine serum 
albumin, within one hour at 37.degree. C. 
EXAMPLE II 
Determination of the HpyCH4V Cleavage Site 
The location of HpyCH4V cleavage relative to the recognition sequence was 
determined by cleavage of a primer extension product, which was then 
electrophoresed alongside a set of standard dideoxy sequencing reactions 
produced from the same primer and template. M13mp18 DNA was employed as 
the template utilizing two HpyCH4V recognition sites at positions 6272 and 
6280, which sites were conveniently located 30 and 38 bp 3' of a priming 
site for a standard sequencing primer: Sequenase -40 primer 
(5'-dGTTTTCCCAGTCACGAC-3' (SEQ ID NO:5) 
Sequencing Reactions 
The sequencing reactions were performed using the Sequenase version 2.0 DNA 
sequencing kit (Amersham Life Science) with modifications for the cleavage 
site determination. The template and primer were assembled in a 0.5 mL 
eppendorf tube by combining 2.5 .mu.l dH2O, 3 .mu.l 5X sequencing buffer 
(200 mM Tris pH 7.5, 250 mM NaCl, 100 mM MgCl2), 8 .mu.l M13mp18 
single-stranded DNA (1.6 .mu.g) and 1.5 .mu.l of primer (Sequenase -40 
primer at 0.5 .mu.M concentration). The primer-template solutions were 
incubated at 65.degree. C. for 2 minutes, then cooled to 37.degree. C. 
over 20 minutes in a beaker of 65.degree. C. water on the benchtop to 
anneal the primer. The labeling mix (diluted 1:20) and sequenase were 
diluted according to manufacturer's instructions. The annealed primer and 
template tube was placed on ice. To this tube were added 1.5 .mu.l 100 mM 
DTT, 3 .mu.l diluted dGTP labeling mix, 1 .mu.l [.alpha.-.sup.33 P] DATP 
(2000 Ci/mmole, 10 mCi/ml) and 3 .mu.l diluted T7 Sequenase polymerase. 
The reaction was mixed and incubated at room temperature for 4 minutes. 
3.5 .mu.l of this reaction was then transferred into each of four tubes 
containing 2.5 .mu.l termination mix for the A, C, G and T sequencing 
termination reactions. To the remaining reaction was added to 10 .mu.l of 
Sequence Extending Mix, which is a mixture of dNTPs (no ddNTPs) to allow 
extension of the primer through and well beyond the HpyCH4V site with no 
terminations to create a labeled strand of DNA extending through the 
HpyCH4V recognition site for subsequent cleavage. The reactions were 
incubated 5 minutes at 37.degree. C. To the A, C, G and T reactions were 
added 4 .mu.l of stop solution and the samples were stored on ice. The 
extension reaction was then incubated at 70.degree. C. for 20 minutes to 
inactivate the DNA polymerase (Sequenase), then cooled on ice. 10 .mu.l of 
the extension reaction was then placed in one 0.5 ml eppendorf tube while 
7 .mu.l was placed in a second tube. To the first tube was added 1 .mu.l 
(approximately 0.5 unit) HpyCH4V endonuclease, the reaction was mixed, and 
then 2 .mu.l was transferred to the second tube. These enzyme digest 
reactions were mixed and then incubated at 37.degree. C. for 1 hour, 
following which the reactions were divided in half. To one half 4 .mu.l of 
stop solution was added and mixed (the minus polymerase reaction). To the 
second half was added 0.4 ul Klenow DNA polymerase (NEB#210) containing 80 
.mu.M dNTPs and the reactions were incubated at room temperature for 15 
minutes, following which 4 .mu.l of stop solution was added. The 
sequencing reaction products were electrophoresed on an 6% Bis-Acrylamide 
sequencing gel (Novex QuickPoint system), with the HpyCH4V digestions of 
the extension reaction next to the set of sequencing reactions produced 
from the same primer and template combination. 
Results: 
Digestion of the extension reaction product from the -40 primer with 
HpyCH4V endonuclease produced a band which co-migrated with the G 
nucleotide of the HpyCH4V recognition sequence 5'-TGCA-3', indicating 
cleavage between the G and the C of the recognition sequence. Treatment of 
the cleaved extension reaction product with Klenow DNA polymerase produced 
a band which also co-migrated with the G nucleotide of the HpyCH4V 
recognition sequence 5'-TGCA-3', indicating cleavage between the G and the 
C of the recognition sequence on the opposite strand of DNA as well (FIG. 
2). These results indicate HpyCH4V cleaves DNA between the G and C in its 
recognition sequence on both DNA strands 5'-TG.dwnarw.CA-3', to produce 
blunt-ended fragments. 
EXAMPLE III 
Cloning the HpyCH4V Endonuclease and Methylase 
1. DNA purification: To prepare the genomic DNA of Helicobacter pylori CH4, 
1 gram of cell paste was resuspended in 10 ml of 25% sucrose, 0.05 M 
Tris-HCl pH 8.0, to which was added 5 ml of 0.25 M EDTA, pH 8.0. Then 3 ml 
of lysozyme solution (10 mg/ml lysozyme in 0.25 M Tris-HCl, pH 8.0) was 
added and the cell suspension was incubated at 4.degree. C. for 16 hours. 
12 ml of Lytic mix (1% Triton-X100, 0.05 M Tris, 62 mM EDTA, pH 8.0) and 
2.5 ml of 10% SDS was then added and the solution was incubated at 
37.degree. C. for 5 minutes. The solution was extracted with one volume of 
equilibrated phenol:chloroform:isoamyl alcohol (50:48:2, v/v/v) and the 
aqueous phase was recovered and extracted with one volume of 
chloroform:isoamyl alcohol (24:1, v/v) two times. The aqueous solution was 
then dialysed against four changes of 2 L of 10 mM Tris, 1 mM EDTA, pH 
8.0. The dialysed DNA solution was digested with RNase (100 .mu.g/ml) at 
37.degree. C. for 1 hour. The DNA was precipitated by the addition of 
1/10th volume 5 M NaCl and 0.55 volumes of 2-propanol and spooled on a 
glass rod. The DNA was briefly rinsed in 70% ethanol, air dried and 
dissolved in 3 ml TE (10 mM Tris, 1 mM EDTA, pH 8.0) to a concentration of 
approximately 300 mg/ml and stored at 4.degree. C. 
2. Construction of libraries of genomic 
Helicobacter pylori CH4 DNA in a selectable vector: 
Helicobacter pylori CH4 genomic DNA was partially digested with either of 
two frequent cutting enzymes, Sau3AI or AciI. The partial digestion was 
carried out by serial dilution of the Sau3AI or the AciI restriction 
endonuclease from 0.5 units/.mu.g DNA to 0.016 units/.mu.g in the 
manufacturer's reaction buffer and digesting at 37.degree. C. for 1 hour. 
The reactions were subsequently terminated by phenol:chloroform 
extraction. Reactions which produced an average size range of fragments 
from 2 to 6 kb were used for library construction. 3 .mu.g of this 
partially digested HpyCH4V genomic DNA was ligated to 1 .mu.g of the 
vector pUC19 (previously cleaved by BamHI (Sau3AI) or AccI (AciI) and 
dephosphorylated with calf intestinal alkaline phosphatase) in a final 
volume of 50 .mu.l in 1X NEB ligase buffer with 1000 units (NEB) of T4 DNA 
ligase. The ligation reactions were incubated at 16.degree. C. for 16 
hours. 10 ul of each ligation reaction mixture was then transformed by 
electroporation into E. coli ER2683 cells and grown out in 10 ml L-Broth 
for 1 hour. 10 .mu.l was then plated onto L-Broth agar plates supplemented 
with 100 .mu.g/ml ampicillin to count the number transformants and the 
plates were incubated at 37.degree. C. overnight. The remaining outgrowth 
was grown overnight in 250 ml L-Broth supplemented with 100 .mu.g/ml 
ampicillin with shaking at 37.degree. C. A total of 1.times.10.sup.6 
individual transformants were obtained for the Sau3Ai library, and 
6.times.10.sup.5 transformants for the AciI library. The cells of the 250 
ml liquid culture were harvested by centrifugation at 5 K rpm for 5 
minutes. The plasmids from these cells were purified by a standard 
alkaline lysis procedure, followed by four rounds of desalting in an 
Amicon Centricon-50 microconcentration device, washing with TE buffer each 
round, and then the plasmids were precipitated by PEG precipitation 
(combined 672 .mu.l centricon purified plasmid, 128 .mu.l 5 M NaCl and 800 
.mu.l 13% PEG-8000, incubated at 4.degree. C. for 30 min, microfuged at 
4.degree. C. at maximum speed for 10 minutes, washed 2X with 70% cold 
ethanol) and resuspended in TE buffer at a concentration of 250 .mu.g/ml. 
3. HpyCH4V methylase selection: 1 .mu.g of the plasmid library was digested 
for 2 hours at 37.degree. C. in 50 .mu.l 1X NEB#4 buffer with 4 units of 
the HpyCH4V prepared as above from Helicobacter pylori CH4 cells. 10 .mu.l 
of the HpyCH4V digestion reaction was then transformed into 100 .mu.l E. 
coli ER2683 competent cells and plated on L-broth plates containing 100 
ug/ml ampicillin and the plates incubated at 37.degree. C. overnight. In 
the initial attempt, a total of 3 transformants were obtained from the 
AciI library digested with 4 units from HpyCH4V and none from the Sau3AI 
library. The 3 AciI library clones were analyzed as follows: Plasmid from 
each colony was isolated by miniprep procedures and digested with HpyCH4V 
endonuclease. 
Analysis of plasmid clones: Individual transformants were inoculated into 
10 ml cultures of L-broth containing 100 .mu.g/ml ampicillin and the 
plasmids that they carried were prepared by Qiagen QIAprep.RTM. Spin 
Miniprep columns according to the manufacturers instructions. Plasmids 
were assayed for the presence of the HpyCH4V methylase by digestion with 
HpyCH4V endonuclease. 
All three of the clones analyzed from the AciI library were found to be 
fully protected from HpyCH4V digestion. All three clones were tested for 
the expression of HpyCH4V restriction activity when grown in L-Broth 
containing 100 .mu.g/ml ampicillin. 1 of the 3 clones analyzed was found 
to express HpyCH4V restriction activity. This clone was designated 
pHpyCH4VA3 (strain NEB#1240) and may be used to produce HpyCH4V 
endonuclease by propagation to mid-log phase in a fermenter containing 
L-broth medium with ampicillin (100 .mu.g/ml). The cells are harvested by 
centrifugation and may be stored at -20.degree. C. or used immediately. 
4. Purification of the HpyCH4V restriction endonuclease from NEB #1240 can 
be accomplished by a combination of standard protein purification 
techniques, such as affinity-chromatography or ion-exchange 
chromatography, as outlined above. The HpyCH4V restriction endonuclease 
obtained from this purification is substantially pure and free of 
non-specific endonuclease and exonuclease contamination. 
EXAMPLE IV 
Sequencing the HpyCH4V Endonuclease and Methylase 
1. DNA Sequencing: DNA sequencing was performed on double-stranded 
templates on an ABI 373 automated sequencer. Individual clones were 
sequenced from primers located in the vector on both sides of the inserts. 
The DNA sequencing of the clones was incomplete at time of filing. 
Computer analyses of the DNA sequences obtained were performed with the 
Genetics Computer Group programs (Deverenx, et al., Nucleic Acids Res. 
12:387-395 (1984)) and database similarity searches were performed via the 
internet at the National Center for Biotechnology Information site 
(http://www.ncbi.nlm.nih.gov/BLAST/) using the BLASTX algorithm (Altschul, 
et al., J. Mol. Biol 215:403-410 (1990) and Gish, et al., Nature Genet. 
3:266-722 (1993).). An open reading frame (ORF) of 462 bp which contained 
motifs 1 and 4 characteristic of gamma type N6-methyl adenine DNA 
methyltransferases was identified in DNA sequence reading from the vector 
into one end of the insert (SEQ ID NO:3 and SEQ ID NO:4, FIG. 3). This 
partial open reading frame was identified as the N-terminal portion of the 
HpyCH4V methyltransferase, designated HpyCH4VM. DNA 5' to the HpyCH4V 
methyltransferase matches with genes JHP1440 and JHP1441 in the sequenced 
genome of strain J99, with the ATG start of the HpyCH4V methyltransferase 
gene located 13 bp 3' to the stop codon of the JHP1441 gene. The sequence 
of the HpyCH4V methyltransferase clone diverges from the J99 strain 
immediately following the stop codon of the JHP1441 gene, and the sequence 
of the HpyCH4V methyltransferase gene is not present in strain J99, nor in 
the other sequenced Helicobacter pylori strain, 26695. This is consistent 
with the observation that the genomic DNA of both strains J99 and 26695 is 
cleaved with the HpyCH4V endonuclease of the present invention. DNA 
sequence from the other end of the HpyCH4V methyl-transferase clones 
matches sequence in ORFs JHP1444 or JHP1445 of strain J99, and thus is 
presumed to be located beyond the HpyCH4V methylase and endonuclease 
genes. 
A sample of an E. coli containing pHpyCH4VA3 (NEB#1240) has been deposited 
under the terms and conditions of the Budapest Treaty with the American 
Type Culture Collection on Sep. 23, 1999, and received ATCC Patent 
Accession No. PTA-782. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - &lt;160&gt; NUMBER OF SEQ ID NOS: 5 
- - &lt;210&gt; SEQ ID NO 1 
&lt;211&gt; LENGTH: 6 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Haemophilus aegyptius 
- - &lt;400&gt; SEQUENCE: 1 
- - wggccw - # - # - 
# 6 
- - - - &lt;210&gt; SEQ ID NO 2 
&lt;211&gt; LENGTH: 6 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Escherichia coli 
- - &lt;400&gt; SEQUENCE: 2 
- - gaattc - # - # - 
# 6 
- - - - &lt;210&gt; SEQ ID NO 3 
&lt;211&gt; LENGTH: 900 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Helicobacter pylori 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: CDS 
&lt;222&gt; LOCATION: (439)..(900) 
- - &lt;400&gt; SEQUENCE: 3 
- - cggaatgttt tcctacccgc aaaagaaatt gcgcaagctt tttgaagtct tc - 
#cctttagc 60 
- - cttgatggtt gaaaaagcta aaggggaagc gttttatttt gataaggggg tg - 
#aaaaagcg 120 
- - tttgctagag caaagcgtag aaaattacca tgaaaaaagc gaatgctatt ta - 
#gctagcca 180 
- - gcatgaagct caaattttag aaaaatattt aaagggaaaa tgatgcaaaa ta - 
#gtgctaaa 240 
- - aaattagaat atgaagagcg ttttaatgac gctcttttga aattaaaagc at - 
#gccaagaa 300 
- - gaaaaacaag tagcaagttg tttgaaatgc gagaaggttt taaaatgcga ga - 
#ttcgcaac 360 
- - aactatgtgg atgcggctta tgaaagcatg agtttaggcg aagcgggcgg gt - 
#ttgatttc 420 
- - aactaaaatg ggcttaaa atg gtt agt aac act acc ttg - #caa aag aat tta 
471 
- # Met Val Ser Asn Thr Thr Leu Gln Lys - #Asn Leu 
- # 1 - # 5 - # 10 
- - gac gct ttt tac acc cac ccc aaa atc gca cg - #a ttt tgt ttg gat tta 
519 
Asp Ala Phe Tyr Thr His Pro Lys Ile Ala Ar - #g Phe Cys Leu Asp Leu 
15 - # 20 - # 25 
- - tta aaa gat ctc atc cat caa aat cta ggg ct - #a gac ttg aac gcg ttc 
567 
Leu Lys Asp Leu Ile His Gln Asn Leu Gly Le - #u Asp Leu Asn Ala Phe 
30 - # 35 - # 40 
- - cat ttt tta gag cca agt gca ggg agt ggg ag - #c ttt gtt ggc gcg tta 
615 
His Phe Leu Glu Pro Ser Ala Gly Ser Gly Se - #r Phe Val Gly Ala Leu 
45 - # 50 - # 55 
- - aaa gga tta ggg att gct gat tgt ctc gcc ct - #t gat att gcc cct aaa 
663 
Lys Gly Leu Gly Ile Ala Asp Cys Leu Ala Le - #u Asp Ile Ala Pro Lys 
60 - # 65 - # 70 - # 75 
- - gct caa ggc att caa caa aaa gat tat ttg tt - #g gaa ttg att gag ttt 
711 
Ala Gln Gly Ile Gln Gln Lys Asp Tyr Leu Le - #u Glu Leu Ile Glu Phe 
80 - # 85 - # 90 
- - aac aaa aag cgc atc att att ggc aac cct cc - #t ttt gga cat agg ggg 
759 
Asn Lys Lys Arg Ile Ile Ile Gly Asn Pro Pr - #o Phe Gly His Arg Gly 
95 - # 100 - # 105 
- - aaa ctg gct cta aat ttc tta aac aaa tct tt - #g aat gaa gcg cct att 
807 
Lys Leu Ala Leu Asn Phe Leu Asn Lys Ser Le - #u Asn Glu Ala Pro Ile 
110 - # 115 - # 120 
- - gta gcg ttt att ttg ccc aat tta ttc aaa cg - #c tat tct att caa aaa 
855 
Val Ala Phe Ile Leu Pro Asn Leu Phe Lys Ar - #g Tyr Ser Ile Gln Lys 
125 - # 130 - # 135 
- - cac att gat aag cgt gca aaa ttg gtt tta aa - #c gct gat tta gaa 
90 - #0 
His Ile Asp Lys Arg Ala Lys Leu Val Leu As - #n Ala Asp Leu Glu 
140 1 - #45 1 - #50 
- - - - &lt;210&gt; SEQ ID NO 4 
&lt;211&gt; LENGTH: 154 
&lt;212&gt; TYPE: PRT 
&lt;213&gt; ORGANISM: Helicobacter pylori 
- - &lt;400&gt; SEQUENCE: 4 
- - Met Val Ser Asn Thr Thr Leu Gln Lys Asn Le - #u Asp Ala Phe Tyr Thr 
1 5 - # 10 - # 15 
- - His Pro Lys Ile Ala Arg Phe Cys Leu Asp Le - #u Leu Lys Asp Leu Ile 
20 - # 25 - # 30 
- - His Gln Asn Leu Gly Leu Asp Leu Asn Ala Ph - #e His Phe Leu Glu Pro 
35 - # 40 - # 45 
- - Ser Ala Gly Ser Gly Ser Phe Val Gly Ala Le - #u Lys Gly Leu Gly Ile 
50 - # 55 - # 60 
- - Ala Asp Cys Leu Ala Leu Asp Ile Ala Pro Ly - #s Ala Gln Gly Ile Gln 
65 - # 70 - # 75 - # 80 
- - Gln Lys Asp Tyr Leu Leu Glu Leu Ile Glu Ph - #e Asn Lys Lys Arg Ile 
85 - # 90 - # 95 
- - Ile Ile Gly Asn Pro Pro Phe Gly His Arg Gl - #y Lys Leu Ala Leu Asn 
100 - # 105 - # 110 
- - Phe Leu Asn Lys Ser Leu Asn Glu Ala Pro Il - #e Val Ala Phe Ile Leu 
115 - # 120 - # 125 
- - Pro Asn Leu Phe Lys Arg Tyr Ser Ile Gln Ly - #s His Ile Asp Lys Arg 
130 - # 135 - # 140 
- - Ala Lys Leu Val Leu Asn Ala Asp Leu Glu 
145 1 - #50 
- - - - &lt;210&gt; SEQ ID NO 5 
&lt;211&gt; LENGTH: 17 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Helicobacter pylori 
- - &lt;400&gt; SEQUENCE: 5 
- - gttttcccag tcacgac - # - # 
- # 17 
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