A novel restriction endonuclease designated XcyI recognizes and cleaves the sequence 5'-C.dwnarw.CCGGG-3', where the arrow indicates the cleavage site. The enzyme may be obtained from Xanthamonas cyanopsidis. Xanthamonas cyanopsidis strain 13D5 was deposited at the American Type Culture Collection on Jan. 20, 1984, and granted accession No. 39587.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The ability to specifically cleave a duplex DNA molecule into discrete 
fragments is essential for the manipulation and modification of DNA in 
vitro. Such specific cleavage is accomplished using restriction 
endonucleases which are enzymes capable of cutting a DNA molecule at or 
about a specific recognition site. Type II restriction endonucleases 
recognize and cleave DNA at particular sequences of from four to seven 
nucleotides which usually have an axis of rotational symmetry. The site of 
cleavage may be within the recognition sequence or may lie a fixed number 
of base pairs away from the sequence. Moreover, the cleavage may be cut 
straight across the duplex producing flush or blunt-ended fragments, or 
may be staggered to produce either 5'- or 3'- cohesive ends. 
Isoschizomers are different restriction enzymes which recognize the same 
target sequence and which may share a common cleavage pattern. Often, 
however, the isoschizomers will be affected differently by methylation of 
the substrate DNA. For example, while one member of an isoschizomer pair 
may be inhibited by methylation of a particular cytosine residue in the 
recognition sequence, the other member of the pair may be unaffected. 
Thus, two isoschizomers may produce different restriction patterns, 
depending on methylation of the DNA being cleaved. 
To perform genetic manipulation in vitro, it is desirable to have a very 
large number of restriction endonucleases available to perform cleavage at 
preselected locations. Under certain circumstances, the use of one 
isoschizomer may be favored over another because of different cleavage 
patterns brought about by methylation of the substrate DNA. Moreover, one 
isoschizomer may be preferred over another because of greater inherent 
stability, greater purity from other contaminating enzymes, and the like. 
2. Description of the Prior Art 
Many restriction enzymes are reported in the literature, and a number of 
the reported enzymes are commercially available. Restriction enzyme XmaI 
recognizes the sequence 5'-CCCGGG-3' and cleaves between the first and 
second residues to produce a 5'-CCGG cohesive end. The isolation and use 
of XmaI are described by Endow and Roberts (1977) J. Mol. Biol. 
112:521-529 and Kunkel et al. (1979) J. Mol. Biol. 132:133-139. The 
purification of XmaI is complicated by the presence of two other 
restriction enzymes, XmaII and XmaIII, and preparations of XmaI are often 
contaminated by these other enzyme activities. Moreover, XmaI is 
insufficiently stable to allow long-term storage and routine use. 
SUMMARY OF THE INVENTION 
A novel restriction enzyme obtained from the bacterial strain Xanthamonas 
cyanopsidis is provided. The endonuclease, designated XcyI, cleaves the 
sequence 5'-C.dwnarw.CCGGG-3', where the arrow indicates the cleavage 
site. XcyI can be isolated at a high degree of purity, being substantially 
free from contaminating exonuclease and endonuclease activities. The XcyI 
enzyme preparations appear to be stable and may be stored for long periods 
prior to use. XcyI is an isoschizomer of both XmaI and SmaI and shares a 
common cleavage pattern with XmaI. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Restriction endonuclease XcyI is a Type II restriction enzyme which is an 
isoschizomer of both XmaI and SmaI. XcyI is isolated from bacterial strain 
Xanthamonas cyanopsidis and has been named in accordance with the 
nomenclature of Smith and Nathans (1973) J. Mol. Biol. 81:419-423. Optimal 
enzyme activity is obtained at about 37.degree. C. 
XcyI may be isolated from Xanthamonas cyanopsidis strain 13D5, deposited 
with the American Type Culture Collection for patent purposes on Jan. 20, 
1984, and granted accession No. 39587. Isolation may be performed using 
the rapid purification procedure of Greene et al. (1978) Nucl. Acids Res. 
5:2373-2380. Cells are grown in a suitable culture medium, e.g., Luria 
broth, and suspended in an extraction buffer. The cell walls are lysed 
using lysozyme followed by sonication, the resulting lysate cleared of 
cell debris by centrifugation, and the supernatant decanted. The enzyme 
may be isolated from the supernatant by phosphocellulose chromatography 
followed by hydroxylapatite chromatography. Peak fractions are pooled and 
dialyzed, and the concentrated enzyme may be stored at -20.degree. C. The 
enzyme preparation obtained by this procedure is substantially free from 
contaminating exonuclease and endonuclease activity. 
Alternatively, XcyI may be produced by first isolating the gene which 
encodes XcyI and introducing the gene into an appropriate expression host. 
Methods for manipulating and identifying genetic material in vitro are now 
well known and need not be described in detail. Briefly, however, the XcyI 
gene may be isolated by first cleaving the chromosomal DNA, typically by 
endonuclease digestion or mechanical shearing, followed by cloning in an 
appropriate usually bacterial cloning vector. Clones carrying the XcyI 
gene may be selected and expanded. The XcyI gene may then be introduced 
into an expression host, either directly or by means of an expression 
vector. The XcyI gene product may then be isolated from the expression 
host by conventional means. The gene product will have an amino acid 
sequence which is substantially identical to the naturally-occurring 
product, usually differing by fewer than 5% of the amino acids. 
Substrate DNA may be restricted in a suitable buffer, e.g., Tris-HCl at pH 
7.5, at 37.degree. C. for a suitable time, e.g., 1 hour.

The following examples are offered by way of illustration, and not by way 
of limitation. 
EXPERIMENTAL 
MATERIALS AND METHODS 
1. Growth of Cells 
Xanthamonas cyanopsidis 13D5 was grown in Luria broth (LB) medium (Miller 
(1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 
Cold Spring Harbor, N.Y.) at 30.degree. C. in a rotary shaking incubator. 
A one liter culture grown for 20 hours yielded 5 gm of cells (wet weight). 
2. Preparation of Cell Extract 
The purification procedure of Greene et al. (1978) Nucl. Acids Res. 
5:2373-2380 was followed with minor modifications. All steps were 
performed at 4.degree. C. Extraction buffer (EB) was 10 mM K.sub.2 
HPO.sub.4 -KH.sub.2 PO.sub.4, pH 7.0, 7 mM 2-mercaptoethanol, 1 mM EDTA, 
25 .mu.g/ml phenylmethylsulfonyl fluoride, 1 mM NaN.sub.3. Frozen cells 
(10 gm) were resuspended in 100 ml of EB +0.4 M NaCl. Lysozyme (10 mg) was 
added to the solution and dissolved by stirring. After 30 minutes, cells 
were lysed by sonication with a Heat System model W140D sonifier with a 
1/2 inch probe. Sonication was performed at maximum power in three 60 
second pulses. The lysate was cleared of cell debris by centrifugation in 
the Beckman Type 35 rotor (35,000 rpm, 4.degree. C., 1 hour). The 
supernatant was decanted, diluted with EB to a conductivity equal to that 
of 0.2 M NaCl, and applied to a phosphocellulose column. 
3. Phosphocellulose Chromatography 
The crude extract was loaded onto a 2 cm.times.8.5 cm P-11 phosphocellulose 
column equilibrated in EB +0.2 M NaCl. The column was washed with EB +0.2 
M NaCl until the A.sub.280 was less then 0.05. A 300 ml gradient of 0.2 M 
NaCl-1 M NaCl in EB was applied to the column and 5 ml fractions were 
collected and assayed. XcyI activity eluted between fractions 12 and 30. 
4. Hydroxylapatite Chromatography 
Peak fractions from the phosphocellulose column were pooled and applied to 
a 1.5 cm.times.6 cm hydroxylapatite column equilibrated in EB. Activity 
was eluted with a 180 ml gradient of 0.01 M-0.50 potassium phosphate, pH 
7.0, containing 1 mM EDTA, 7 mM 2-mercaptoethanol, 1 mM NaN.sub.3, and 0.2 
M NaCl. Fractions of 3 ml were collected and assayed for activity. XcyI 
eluted between fractions 12 and 30. Peak fractions were pooled and 
dialysed against storage buffer (50% glycerol, 20 mM potassium phosphate, 
pH 7.0, 0.2 M NaCl, 1 mM EDTA, 7 mM 2-mercaptoethanol, 1 mM NaN.sub.3). 
The concentrated enzyme may be stored at -20.degree. C. 
5. XcyI Assay Conditions 
Restriction reactions were performed in 6 mM Tris-HCl, pH 7.5, 6 mM 
2-mercaptoethanol, 6 mM MgCl.sub.2. Reactions of 18 .mu.l volume 
containing 0.2 .mu.g DNA were initiated by the addition of 2 .mu.l of the 
fractions to be assayed. Reactions were incubated at 37.degree. C. for 1 
hour, then examined by agarose gel electrophoresis. Gels were run using 
the Tris-borate EDTA system (Bolivar et al. (1977) Gene 2:75-93) with 1% 
agarose submarine minigels. 
6. Ligation Conditions 
T4 DNA ligase was purified and used according to Tait et al. (1980) J. Mol 
Biol. 255:813-815. 
7. DNA Sequencing 
DNA sequencing was carried out according to the method of Sanger et al. 
(1977) Proc. Natl. Acad. Sci. USA 74:5463-5467. Plasmid DNA was denatured 
and primed for sequencing reactions as follows. Plasmid DNA (2 .mu.g) was 
added to 18 .mu.l ddH.sub.2 O (twice distilled water). Following the 
addition of 2 .mu.l of 2 N NaOH, 2 mM EDTA, and 5 minutes incubation at 
room temperature, the DNA was precipitated by the addition of 45 .mu.l of 
95% EtOH. The precipitate was collected by centrifugation in an Eppendorf 
microcentrifuge for 15 minutes at 4.degree. C. The supernatant was poured 
off and the pellet dried 15 minutes in a vacuum over. The DNA pellet was 
resuspended in 3 .mu.l of 2 M ammonium acetate, pH 4.8+ .mu.l ddH.sub.2 
O+3 .mu.l 3 M sodium acetate, pH 4.8. DNA was precipitated by the addition 
of 80 .mu.l of 95% EtOH, incubated 10 minutes at 70.degree. C., then 
centrifuged 15 minutes at 4.degree. C. in a microcentrifuge. The 
supernatant was removed and the pellet washed once in 1 ml of cold 70% 
EtOH. The pellet was dried 15 minutes in a vacuum oven, resuspended in 15 
.mu.l ddH.sub.2 O, then used in the standard dideoxy-sequencing reactions. 
The "run-off" DNA synthesis reaction was essentially the same as the 
dideoxy A sequencing reaction except that XcyI cleaved denatured linear 
DNA was used as the template and the dideoxy ATP was omitted. 
RESULTS AND DISCUSSION 
Xanthamonas strains were initially screened on agarose minigels using phage 
lambda DNA as a substrate. On the basis of those results, the activity 
present in X. cyanopsidis 13D5 appeared to be an isoschizomer of SmaI. 
SmaI recognizes the sequence 5'-CCC.dwnarw.GGG, and cleaves where 
indicated by the arrow. However, the DNA fragments generated by digestion 
of lambda DNA with SmaI are difficult to resolve on agarose minigels. A 
recombinant pSa727 plasmid containing a 25 kb KpnI-generated fragment of 
the Agrobacterium tumefaciens plasmid pTiC58 (Tait et al. (1983) 
Biotechnology 1:269-275) was also used as a substrate for screening. To 
verify that the digestion specificity of XcyI was the same as that of 
SmaI, this recombinant plasmid (pSa727:Kpn3) was digested with KpnI to 
generate a 15 kb vector DNA fragment that contains no SmaI cleavage sites 
and a 25 kb DNA fragment that contains 4 SmaI cleavage sites. Digestion of 
pSa727:Kpn3 with KpnI and SmaI or XcyI produced an identical restriction 
pattern, confirming that SmaI and XcyI are isoschizomers. 
XcyI digested DNA was ligated with T4 DNA ligase at room temperature for 10 
minutes, then examined by agarose gel electrophoresis. Ligation was 
efficient, and digestion of the ligation reaction with XcyI regenerated 
the original DNA fragments. Prolonged digestion of DNA with XcyI (72 
hours) did not result in the appearance of additional DNA fragments or in 
the apparent loss or smearing of existing DNA bands. It has been 
previously demonstrated that the ability of T4 DNA ligase to ligate blunt 
ends can be inhibited by the presence of high concentrations of ATP 
(Ferretti and Sgaramella (1981) Nuc. Acids Res. 9:3695-3705). No 
difference was detected when XcyI digested DNA was ligated in the presence 
of 0.6 or 6.0 mM ATP, suggesting that the termini produced by XcyI are 
cohesive in nature. 
In order to verify the cohesive nature of the termini and determine whether 
a 5' or 3' terminal extension was generated, the plasmid pUC8 (Vieira and 
Messing (1982) Gene 19:269-276) was digested with XcyI, then denatured. A 
17 base pair primer and DNA polymerase I Klenow fragment were used to 
prime "run-off" DNA synthesis on the denatured linear plasmid DNA. The 
Klenow fragment will only elongate the primer to the point where the 
template DNA has been cleaved by the XcyI enzyme. When the products of 
this reaction are electrophoresed adjacent to a standard dideoxy-sequence 
of pUC8, the migration position of the run-off DNA synthesis reaction will 
correspond to the site cleaved by XcyI. The results indicated that XcyI 
cleaves the SmaI site of pUC8 between the first and second residues to 
generate a protruding 5'-CCGG. These results demonstrate that XcyI and 
XmaI recognize the same sequence and display an identical cleavage 
pattern. 
It was observed that certain recognition sites are somewhat resistant to 
cleavage by XcyI. By increasing the amount of enzyme in the reaction, 
however, even recalcitrant sequences could be digested. It is possible 
that although XcyI recognizes and cleaves the sequence 5'-CCCGGG-3', the 
methylation of residues within or adjacent to this sequence may affect the 
function of the enzyme. Although purification of DNA from the dcm.sup.- 
strain E. coli GM48 increased the relative rate of cleavage of these 
sequences, the deficiency of cytosine methylation did not completely 
alleviate the inhibition of cleavage. The XcyI cleavage site in pUC88 is a 
slowly digested site, and it is located adjacent to a BamHI site. BamHI 
cleavage sites are believed to contain a recognition site for adenine 
methylase (Vander Ploeg and Flavell (1980) Cell 19:947-958). 
According to the subject invention, a novel restriction endonuclease is 
provided which is capable of cleaving the sequence 5'-C.dwnarw.CCGGG-3'. 
The enzyme is found to be particularly stable and may be isolated from X. 
cyanopsidis substantially free from contaminating enzymes. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be understood that certain changes and modifications may be practiced 
within the scope of the appended claims.