Host expressing NgoAIII restriction endonuclease and modification methylase from neisseria

The present invention is directed to recombinant hosts which contain and express various Type II restriction endonuclease and/or modification methylase genes. In particular, the present invention is concerned with the cloned restriction endonucleases, NgoAIII and NgoAI, which recognize and cleave within or near the double-stranded DNA sequence, 5' CCGCGG 3' and 5' PuGCGCPy 3', respectively. Also provided in this invention are cloned modification methylase genes corresponding to said restriction endonucleases. This invention is further concerned with a cloned modification methylase, M.NgoAII. One source of these enzymes is Neisseria gonorrhoeae, although other microorganisms may be used to isolate the restriction endonuclease isoschizomers and modification methylase isoschizomers of this invention.

FIELD OF THE INVENTION 
This invention is directed to recombinant hosts expressing restriction 
endonuclease and modification methylase genes from the genus Neisseria. 
This invention is specifically directed to the recombinant hosts and their 
cloning vectors which contain the genes coding for the restriction 
endonuclease NgoAI and its corresponding methylase M.NgoAI, the 
restriction endonuclease NgoAIII and its corresponding methylase 
M.NgoAIII, or the modification methylase M.NgoAII. This invention is also 
directed to cloned restriction endonuclease and modification methylase 
isoschizomers of these enzymes. 
BACKGROUND OF THE INVENTION 
Restriction endonucleases are a class of enzymes that occur naturally in 
prokaryotic and eukaryotic organisms. When they are purified away from 
other contaminating cellular components, restriction endonucleases can be 
used in the laboratory to cleave 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 
are 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, 
this sequence in both strands. Different restriction endonucleases have 
affinity for different recognition sequences. About 100 kinds of different 
endonucleases have so far been isolated from many microorganisms, each 
being identified by the specific base sequence it recognizes and by the 
cleavage pattern it exhibits. In addition, a number of restriction 
endonucleases, called restriction endonuclease isoschizomers, have been 
isolated from different microorganisms which in fact recognize the same 
recognition sequence as those restriction endonucleases that have 
previously been identified. These isoschizomers, however, may or may not 
cleave the same phosphodiester bond as the previously identified 
endonuclease. 
In nature, restriction endonucleases play a protective role in the welfare 
of the microbial cell. They enable the microorganism to resist infection 
by foreign DNA molecules like viruses and plasmids that would otherwise 
destroy or parasitize them. They achieve this resistance by scanning the 
lengths of the infecting DNA molecule and cleaving them each time that the 
recognition sequence occurs. The DNA cleavage that takes place disables 
many of the infecting genes and renders the DNA susceptible to further 
degradation by nonspecific exonucleases. 
A second component of microbial protective systems are the modification 
methylases. Modification methylases are complementary to their 
corresponding restriction endonucleases in that they recognize and bind to 
the same recognition sequence. Modification methylases, in contrast to 
restriction endonucleases, chemically modify certain nucleotides within 
the recognition sequence by addition of a methyl group. Following this 
methylation, the recognition sequence is no longer bound or cleaved by the 
restriction endonuclease. The microbial cell modifies its DNA by virtue of 
its modification methylases and therefore is completely insensitive to the 
presence of its endogenous restriction endonucleases. Thus, endogenous 
restriction endonuclease and modification methylase provide the means by 
which a microorganism is able to identify and protect its own DNA, while 
destroying unmodified foreign DNA. 
The combined activities of the restriction endonuclease and the 
modification methylase are referred to as the restriction-modification 
system. Three types of restriction-modification systems have been 
identified that differ according to their subunit structure, substrate 
requirements and DNA cleavage. Specifically, Type-I and Type-III 
restriction systems carry both modification and ATP-requiring restriction 
(cleavage) activity in the same protein. Type-II restriction-modification 
systems, on the other hand, consist of a separate restriction endonuclease 
and modification methylase, i.e., the two activities are associated with 
independent proteins. 
Type II restriction endonucleases are endodeoxyribonucleases which are 
commonly used in modern genetic research. These enzymes recognize and bind 
to particular DNA sequences and once bound, cleave within or near this 
recognition sequence. Phosphodiester bonds are thereby hydrolyzed in the 
double stranded DNA target sequence, i.e., one in each polynucleotide 
strand. Type-II restriction endonucleases can generate staggered breaks 
within or near the DNA recognition sequence to produce fragments of DNA 
with 5' protruding termini, or DNA fragments with 3' protruding termini. 
Other Type-II restriction endonucleases which cleave at the axis of 
symmetry, produce blunt ended DNA fragments. Therefore, Type-II 
restriction endonucleases can differ according to their recognition 
sequence and/or the location of cleavage within that recognition sequence. 
Type-II restriction endonucleases are frequently used by the genetic 
engineers to manipulate DNA in order to create novel recombinant 
molecules. Specific Type-II restriction endonucleases are known for 
numerous DNA sequences, but there is still a need to provide new Type-II 
restriction endonucleases. These new enzymes will add to the list of 
indispensable tools needed for modern genetic research. 
SUMMARY OF THE INVENTION 
The present invention is directed to recombinant hosts which contain and 
express various Type II restriction endonuclease and/or modification 
methylase genes. This invention is further directed to a process for 
obtaining these enzymes and the use thereof. 
In particular, the present invention is concerned with the cloned 
restriction endonuclease, NgoAIII, which recognizes the palindromic 
sequence: 
EQU 5' CCGC .dwnarw.GG 3' 
EQU GG.uparw.CGCC 5' 
and cleaves the sequence between the second C and G residues from the 5' 
end producing a two-base 3' extension. The cloned modification methylase 
corresponding to this endonuclease is called M.NgoAIII. 
In addition, this invention relates to the cloned restriction endonuclease, 
NgoAI, and its corresponding modification methylase (M.NgoAI) which 
recognize the palindromic sequence 
EQU 5' PuGCGCPy 3' 
EQU 3'PyCGCGPu 5'. 
Furthermore, the present invention is directed to a recombinant host 
expressing the modification methylase, M.NgoAII which recognizes and 
chemically modifies the palindromic sequence 
EQU 5' GGCC 3' 
EQU 3' CCGG 5'.

DEFINITIONS 
In the description that follows, a number of terms used in recombinant DNA 
technology are extensively utilized. In order to provide a clear and 
consistent understanding of the specification and claims, including the 
scope to be given such terms, the following definitions are provided. 
Cloning vector. A plasmid or phage DNA or other DNA sequence which is able 
to replicate autonomously in a host cell, and which is characterized by 
one or a small number of endonuclease recognition sites at which such DNA 
sequences may be cut in a determinable fashion without loss of an 
essential biological function of the vector, and into which DNA may be 
spliced in order to bring about its replication and cloning. The cloning 
vector may further contain a marker suitable for use in the identification 
of cells transformed with the cloning vector. Markers, for example, are 
tetracycline resistance or ampicillin resistance. 
Expression vector. A vector similar to a cloning vector but which is 
capable of enhancing the expression of a gene which has been cloned into 
it, after transformation into a host. The cloned gene is usually placed 
under the control of (i.e., operably linked to) certain control sequences 
such as promoter sequences. 
Restriction endonuclease isoschizomer. A restriction endonuclease 
isoschizomer is a term used to designate a group of restriction 
endonucleases that recognize and bind to the same recognition sequence but 
are isolated from different microbial sources. Restriction endonucleases 
isoschizomers may or may not cleave in the exact location as the 
restriction endonuclease with which it is being compared. 
Modification methylase isoschizomer. A modification methylase isoschizomer 
is a term used to designate a group of modification methylase that 
recognize the same recognition sequence but are isolated from different 
microbial sources. Modification methylase isoschizomers may or may not 
chemically modify the same nucleotides within the recognition sequence as 
the modification methylase with which it is being compared. 
Recognition sequence. Recognition sequences are particular sequences which 
restriction endonucleases and modification methylase recognize and bind 
along the DNA molecule. Recognition sequences are typically four to six 
(and in some cases eight) nucleotides in length with a two fold axis of 
symmetry. 
Recombinant host. Any prokaryotic or eukaryotic microorganism which contain 
the desired cloned genes on an expression vector or cloning vector. 
Host. Any prokaryotic or eukaryotic microorganism that is the recipient of 
a replicable expression vector or cloning vector. 
Promoter. A DNA sequence generally described as the 5' region of a gene, 
located proximal to the start codon. At the promoter region, transcription 
of an adjacent gene(s) is initiated. 
Gene. A DNA sequence that contains information encoding for a polypeptide 
or protein, and as used herein, includes the 5' and 3' ends. 
Structural gene. A DNA sequence that is transcribed into messenger RNA that 
is then translated into a sequence of amino acids characteristic of a 
specific polypeptide. Typically the first nucleotide of the first 
translated codon is numbered +1, and the nucleotides are numbered 
consecutively with positive integers through the translated region of the 
structural gene and into the 3' untranslated region. The numbering of 
nucleotides in the promoter and regulatory region 5' to the translated 
region proceeds consecutively with negative integers within the 5' 
nucleotide next to the first translated nucleotide being numbered -1. 
Operably linked. As used herein means that the promoter controls the 
initiation of the expression of the polypeptide encoded by the structural 
gene. 
Expression. Expression is the process by which a structural gene produces a 
polypeptide. It involves transcription of the gene into messenger RNA 
(mRNA) and the translation of such mRNA into polypeptide(s) 
DETAILED DESCRIPTION OF THE INVENTION 
This invention is directed to recombinant hosts which express genes 
encoding for Type II restriction endonucleases including NgoAIII or NgoAI. 
NgoAIII recognizes the palindromic sequence 5' CCGCGG 3', and cleaves 
between the second C and G residues from the 5' end, producing a two-base 
3' extension. The double-stranded recognition site of NgoAIII is thus 
characterized as follows: 
EQU 5' CCGC .dwnarw.GG 3' 
EQU 3' GG.uparw.CGCC 5' 
(wherein G represents deoxyguanosine and C represents deoxycytidine. NgoAI 
recognizes and cleaves within or near the double-stranded palindromic 
sequence: 
EQU 5' PuGCGCPy 3' 
EQU 3' PyCGCGPu 5' 
(wherein Py represents deoxythymidine (T) and deoxycytidine (C), and Pu 
represents deoxyguanosine (G) and deoxyadenosine (A)). 
This invention is also directed to recombinant hosts which express the 
corresponding modification methylase genes of NgoAIII and NgoAI. M.NgoAIII 
recognizes and binds to the same recognition sequence as NgoAIII while 
M.NgoAI recognizes and binds to the sequence recognized by NgoAI. Rather 
than cleaving the DNA, these methylase chemically modify certain 
nucleotides within the recognition sequence by addition of a methyl group, 
thus making the modified sequence resistant to cleavage with its 
corresponding restriction endonuclease. 
This invention is further concerned with a recombinant host which express a 
gene encoding for the modification methylase, M.NgoAII. M.NgoAII 
recognizes and chemically modifies, by addition of a methyl group, the 
double-stranded palindromic sequence 
EQU 5' GGCC 3' 
EQU 3' CCGG 5'. 
Also provided in this invention are recombinant hosts which express genes 
encoding for isoschizomers of the restriction endonuclease and 
modification methylase of the present invention (NgoAII, M.NgoAIII, NgoAI, 
M.NgoAI and M.NgoAII). 
I. Isolation of the Genes Encoding Restriction Endonucleases and 
Modification Methylases of the Present Invention or Isoschizomers thereof 
The restriction endonucleases and their corresponding modification 
methylases of the present invention (NgoAIII, M.NgoAIII, NgoAI, M.NgoAI, 
and M.NgoAII) may be obtained from any species of Neisseria gonorrhoeae. 
Genes encoding isoschizomers of these enzymes can be obtained from any 
genus including, but not limited to, Arthrobacter, Bacillus, Citrobacter, 
Enterobacter, Escherichia, Flavobacterium, Haemophilus, Klebsiella, 
Micrococcus, Neisseria, Nocardia, Pseudomonas, Salmonella, and 
Streptomyces. The preferred genus to isolate isoschizomers of the 
modification methylases and restriction endonucleases of the present 
invention is Neisseria. The genus Neisseria are gram negative cocci 
occurring in pairs or in masses and are aerobic or facultatively 
anaerobic. These organisms may be found in the oropharynx or nasopharynx 
and the genitourinary tract of humans and animals. 
Nomenclature for naming the restriction endonucleases are in accord with 
the proposal of Smith et al., J. Mol. Biol. 81: 419-423 (1973). Briefly, 
the first letter "N" of NgoAIII designates the genus Neisseria while the 
lower case letters "go" designates the species, gonorrhoeae. 
Any strain of Neisseria capable of producing restriction endonuclease 
isoschizomers of NgoAIII or NgoAI--or modification methylase isoschizomers 
of M.NgoAIII, M.NgoAI or M.NgoAII--can be used for the purpose of this 
invention. For example, Neisseria meningitidis, Neisseria sicca, Neisseria 
mucosa, Neisseria lactamica, Neisseria ovis, Neisseria subflava, and 
Neisseria flavescens may be used to obtain the genes expressing the 
restriction endonuclease isoschizomers of NgoAIII or NgoAI. Any species of 
Neisseria may also be used to isolate the genes coding for the 
modification methylase isoschizomers of M.NgoAIII, M.NgoAIV or M.NgoAII. 
The preferred species in the present invention for obtaining the genes 
encoding enzymes of the present invention (NgoAIII, M.NgoAIII, NgoAI, 
M.NgoAI, and M.NgoAII) is Neisseria gonorrhoeae as described in the 
examples. 
II. Cloning and Expressing the Genes Encoding for the Restriction 
Endonucleases and Modification Methylases of the Present Invention or 
Isoschizomers thereof 
NgoAIII, M.NgoAIII, NgoAI, M.NgoAI and M.NgoAII are preferably obtained by 
isolating the genes encoding for the enzymes from Neisseria gonorrhoeae 
and then cloning and expressing them. It is understood in this invention 
that genes coding for isoschizomers of the restriction endonucleases and 
modification methylases of the present invention may be obtained from any 
microorganism including the genus Neisseria by using the recombinant 
techniques described herein. 
DNA molecules which code for NgoAIII, M.NgoAIII, NgoAI, M.NgoAI, and 
M.NgoAII, or isoschizomers thereof, can be recombined into a cloning 
vector and introduced into a host cell to enable the expression of the 
restriction endonuclease or modification methylase by that cell. DNA 
molecules may be recombined with vector DNA in accordance with 
conventional techniques, including blunt-ended or stagger-ended termini 
for ligation, restriction receptor molecule digestion to provide 
appropriate termini, filling in of cohesive ends as appropriate, alkaline 
phosphatase treatment to avoid undesirable joining, and ligation with 
appropriate ligases. 
a. Hosts for Cloning and Expressing 
The present invention encompasses the expression of the desired restriction 
endonuclease or modification methylase in prokaryotic cells. Preferred 
prokaryotic hosts include bacteria such as Escherichia coli, Bacillus, 
Streptomyces, Pseudomonas, Salmonella, Serratia, Neisseria etc. The most 
preferred prokaryotic host is E. coli. 
It has been found that E. coli has several mechanisms (restriction systems) 
for identifying foreign DNA and destroying it. This can be a significant 
problem in cloning experiments, resulting in reduced recovery of the 
desired sequences. In particular, it has been found that E. coli contains 
restriction systems that degrade DNA when it is methylated, either 
cytosine residues or adenine residues. Specifically, the well known 
methylcytosine-specific systems include mcrA (rglA), and mcrB (rglB). The 
methyladenine-specific restriction system has been designated mrr. Thus, 
the preferred host for cloning and expressing the genes encoding for the 
enzymes of the present invention is an E. coli host in which these 
restriction systems have been inactivated through mutation or loss. 
Bacterial hosts of particular interest in the present invention include E. 
coli K12 strain K802 (mcrA, mcrB, r.sub.k.sup.- and m.sub.k.sup.-), E. 
coli K12 DH5.alpha.MCR (F.sup.- endo Al, hsdR17[r.sub.k.sup.-, 
m.sub.k.sup.+ ], supE44, thi-1, .lambda..sup.-, recAl, gyrA96, relAl, 
.phi.80dlacZ, .DELTA.MI5, .DELTA.mcrB, mcrA, mrr) and DH10B (F.sup.-, 
araD139 .DELTA. (ara, leu) 7697, .DELTA.lacX74, galU, galK, mcrA, 
.DELTA.(mrr hsd RMS mcrB), rpsL dor. .phi.80 dlac Z .DELTA.M15, endAl, 
nupG, recAl). The prokaryotic host must be compatible with the replicon 
and control sequences in the cloning vector. 
b. Methods for Cloning and Expression 
NgoAIII, M.NgoAIII, NgoAI, M.NgoAI, and M.NgoAII or isoschizomers thereof 
are preferably obtained by isolating the genes coding for the enzymes and 
then cloning and expressing them. Wilson, "Cloned restriction-modification 
system--a review," Gene 74:281-289 (1988), describes four techniques for 
isolating and cloning restriction endonuclease and modification methylase. 
The four methods reviewed include (1) subcloning of natural plasmids; (2) 
selection based on phage restriction; (3) selection based on vector 
modification involving methylation protection; and (4) multi-step 
isolation. Any one of these four methods can be used for isolating and 
cloning the genes encoding for the enzymes of the present invention or 
isoschizomers thereof. 
The preferred method according to this invention is vector modification 
technique, i.e., methylation protection. Methylation protection involves 
digestion of a plasmid library with the restriction enzyme to be cloned so 
that only plasmids whose sequences are modified, because of the presence 
of the methylase, will produce transformants in a suitable host. This 
selection has worked well to clone endonuclease and methylase genes 
together as well as methylase genes alone (Szomolanyi et al., 1980; 
Janulaitis et al., 1982; Walder et al., 1983; Kiss and Baldanf, 1983; and 
Wilson, 1988). 
Specifically, selection based on modification requires that the vector used 
to construct the plasmid library contain at least one recognition site 
(recognition sequence) corresponding to the modification methylase to be 
cloned. Clones that contain the modification gene on the plasmid vector 
will methylate their own plasmid DNA, provided that the modification 
methylase is expressed in the host used. Thus, plasmid DNA isolated from 
such clones will therefore be resistant to digestion in vitro by the 
corresponding restriction endonuclease. 
It is known that linear plasmid DNA will transform competent cells at a 
much lower frequency than uncleaved circular DNA. It therefore follows 
that restriction endonuclease digestion of plasmid library followed by 
transformation into a suitable host will result in the selection survival 
of methylase-encoding clones. Moreover, if the methylase-encoding clone 
also contains the corresponding restriction gene, then such clones will 
also provide the means for expressing and harvesting the restriction 
enzyme itself. 
Restriction genes and their corresponding modification genes are usually 
closely linked in the DNA of many bacteria. This being the case, selection 
for methylase-containing cells can be used as a simple and reliable method 
for selectively co-isolating methylase and endonuclease clones. In brief, 
selection of methylase-carrying clones from plasmid libraries which also 
contain DNA fragments coding for the corresponding restriction genes 
frequently results in the isolation of clones that carry both the 
modification methylase gene and the corresponding restriction endonuclease 
gene. Methylase-selection is therefore an indirect way of selecting a 
restriction endonuclease clone. 
The preferred methods to clone and express the genes of the present 
invention (NgoAIII, M.NgoAIII, NgoAI, M.NgoAI, and M.NgoAII) or 
isoschizomers thereof, are described in the following steps: 
1. The DNA of the bacterial species to be cloned is purified. 
2. The DNA is digested partially with a convenient restriction 
endonuclease. 
3. The resulting fragments are ligated into a cloning vector, such as pCP13 
(Darzins, A. et al., J. Bacteriol. 159:9-18 (1984)), and the mixture is 
used to transform an appropriate host cell, such as E. coli. 
4. The DNA/cell mixture is plated on antibiotic media selective for 
transformed cells. After incubation, the transformed cell colonies are 
pooled and an aliquot of this cell suspension is grown to create the cell 
library. 
5. The recombinant plasmids are purified in toto from the cell library to 
make a plasmid library. 
6. The plasmid library is then digested to completion in vitro with the 
restriction enzyme whose corresponding methylase gene is sought. 
Exonuclease and/or phosphatase may also be added to the digestion to 
enhance the destruction of non-methylase plasmids. 
7. The digested plasmid DNA is transformed into E. coli and transformed 
colonies are again obtained by plating on antibiotic plates. Individual 
colonies are picked and analyzed for the presence of the modification 
methylase. 
8. If clones are found to express modification methylase, they are further 
analyzed for the simultaneous expression of the restriction endonuclease. 
9. Methylase screening may be performed by: (a) The recombinant plasmid DNA 
molecule of the clone may be purified and exposed in vitro to the 
selecting restriction endonuclease to establish that it is resistant to 
digestion. Provided that the plasmid vector carries several sites for that 
endonuclease, resistance indicates modification rather than mutational 
site loss. 
(b) The total chromosomal DNA of the clone may be purified and exposed to 
the selective restriction endonuclease. If the clone carries the methylase 
gene, the bacterial chromosome should be fully methylated and, like the 
plasmid, should be found to be resistant to digestion. 
(c) The cell extract from the clone may be prepared and assayed in vitro 
for methylase activity (methylase protection and radioactive labelling). 
10. Restriction endonuclease screening may be carried out as follows: 
(a) The cell extract from the clone may be prepared and assayed in vitro 
for its ability to digest substrate DNA, such as Ad-2. Cleavage of Ad-2 
DNA indicates the presence of cloned restriction endonuclease. 
(b) The cells themselves may be tested in vivo for their ability to resist 
phage infection. Resistance to phage infection indicates the presence of 
the restriction endonuclease. 
The restriction endonuclease used to selectively digest the plasmid library 
in step 6 is usually the same as that encoded by the 
restriction-modification system to be cloned. Occasionally, the selective 
restriction endonuclease can be used to clone identical 
restriction-modification systems from other microorganisms, i.e., to clone 
exact isoschizomers of the prototype enzyme (the first example isolated). 
It has been shown, for example, that HaeIII was used to select the 
isoschizomeric restriction-modification system of BsuRI (Kiss et al., 
Nucleic Acid Res. 13:6403-6421 (1983)). Both HaeIII and BsuRI cleave at 
the same location within the recognition sequence, 5'GG.dwnarw.CC3'. 
Selection for isoschizomeric restriction-modification systems can be 
accomplished, provided that the modification methylase isoschizomer to be 
cloned can cross-protect against cleavage with the selective endonuclease. 
Applicants have used commercially available prototype restriction 
endonucleases to select isoschizomeric restriction-modification systems 
that recognize and cleave the same recognition sequence as the 
endonuclease used to select these clones. Applicants have shown, for 
example, that SstII can be used to select recombinant hosts expressing 
NgoAIII and M.NgoAIII; HaeII can be used to isolate recombinant hosts 
expressing NgoAI and M.NgoAI; and HaeIII can be used to select recombinant 
hosts expressing M.NgoAII. 
Although the steps outlined above are the preferred mode for practicing the 
present invention, it will be apparent to those skilled in the art that 
the above-described approach can vary in accordance with techniques known 
in the art. 
c. Methods for Enhancing Expression 
Once the desired restriction endonuclease and modification methylase genes 
have been isolated, a number of recombinant DNA strategies exist for 
enhanced production of the desired proteins in these hosts. These 
strategies which will be appreciated by those skilled in the art, utilize 
high copy number cloning vectors or expression vectors. 
Furthermore, those skilled in the art will recognize that both the 
restriction endonuclease and modification methylase genes need not be 
maintained on the same cloning or expression vector within the same 
recombinant host. The endonuclease gene, for example, may be located on 
one vector, while its corresponding methylase gene may be located on a 
separate vector or located on the host genome. Various combinations of 
maintaining both the modification and restriction genes within the same 
recombinant host can be constructed. The only requirement, when cloning 
restriction endonuclease genes, is that the recombinant host contain and 
express the methylase gene corresponding to the endonuclease gene being 
cloned. 
In order to enhance the production of the desired restriction endonuclease 
in a prokaryotic cell, it is important to maintain expression of the 
corresponding modification methylase gene sufficient to protect the DNA of 
the recombinant host against cleavage with the cloned restriction 
endonuclease. Therefore, it may be necessary to enhance the level of 
methylase expression in conjunction with increased endonuclease activity. 
Enhanced production of these enzymes can be accomplished, for example, by 
operably linking the desired gene(s) to a strong prokaryotic promoter. 
Such promoters may be either constitutive or, more preferably, regulatable 
(i.e., inducible or derepressible). Examples of constitutive promoters 
include the int promoter of bacteriophage .lambda., and the bla promoter 
of the .beta.-lactamase gene of pBR322, etc. Examples of inducible 
prokaryotic promoters include the major left and right promoters of 
bacteriophage .lambda. (P.sub.L and P.sub.R), the trp. recA. lacZ. gal. 
and tac promoters of E. coli. the .alpha.-amylase (Ulmanen, I., et al., J. 
Bacteriol. 162:176-182 (1985)), the .alpha.-28-specific promoters of B. 
subtilis (Gilman, M. Z., et al., Gene 32:11-20 (1984)), the promoters of 
the bacteriophages of Bacillus (Gryczan, T. J., In: The Molecular Biology 
of the Bacilli, Academic Press, Inc., N.Y. (1982)), and Streptomyces 
promoters (Ward, J. M., et al., Mol. Gen. Genet. 203:468-478 (1986)). 
Prokaryotic promoters are reviewed by Glick, B. R., (J. Ind. Microbiol. 
1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and 
Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)). In is important to 
note that the restriction endonuclease gene may be cloned in a host which 
is not protected with the methylase gene, provided that the endonuclease 
gene is operably linked to a controllable promoter. 
Proper expression in a prokaryotic cell also requires the presence of a 
ribosome binding site upstream from the gene-encoding sequence. Such 
ribosome binding sites are disclosed, for example, by Gold, L., et al. 
(Ann. Rev. Microbiol. 35:365-404 (1981)). 
III. Isolation and Purification of the Restriction Endonucleases and 
Modification Methylase Enzymes from Recombinant Hosts 
The enzymes of this invention (NgoAIII, M.NgoAIII, NgoAI, M.NgoAI, and 
M.NgoAII) or isoschizomers thereof are preferably produced by fermentation 
of the recombinant host containing and expressing the cloned restriction 
endonuclease and/or modification methylase genes. The recombinant host, 
such as E. coli producing the cloned proteins, can be grown and harvested 
according to techniques well known in the art. 
Any nutrients that can be assimilated by the host containing the cloned 
restriction endonuclease and modification methylase genes may be added to 
the culture medium. Glucose, sucrose, maltose, lactose, glycerol, ethanol, 
lactates, various fats and oils, and others may be used as carbon source, 
while yeast extract, peptone, defatted soybeans, corn steep liquor, 
bouillon and others are suitable as nitrogen source. Minerals and metal 
salts, e.g., phosphates, potassium salts and magnesium salts, iron, as 
well as vitamins and growth-promoting substances, may also be added as 
required. 
Optimal culture conditions should be selected case by case according to the 
strain used and the composition of the culture medium. Restriction 
endonucleases and modification methylases produced by the recombinant 
hosts of this invention are accumulated inside the microbial cells. 
The recombinant host cells producing the restriction endonuclease and/or 
modification methylase of this invention can be separated from the culture 
liquid, for example, by centrifugation. Both of these enzymes can be 
extracted and purified by using known protein purification techniques 
commonly employed for these types of enzymes. 
In general, the collected microbial cells are dispersed in a suitable 
buffer, and then broken down by ultrasonic treatment to allow extraction 
of the enzyme by the buffer solution. After removal of the residue by 
ultracentrifugation, ammonium sulfate can be added to the supernatant of 
the crude lysate for salting out, and the precipitate which separates out 
is dissolved in a Tris-HCl buffer (pH: 7.6) and dialyzed against a buffer 
of the same composition. The dialyzed sample can be purified by 
ion-exchange chromatography, molecular-sieve chromatography and affinity 
chromatography, giving the restriction endonuclease or modification 
methylase of this invention. 
In an example to purify NgoAIII from a recombinant host expressing the 
genes encoding the restriction-modification system of NgoAIII, the crude 
lysate is absorbed directly onto a heparin-agarose (BRL) column, followed 
by elution with 0.05 to 0.6M NaCl solutions. The active fractions are then 
pooled and dialyzed to reduce the NaCl concentration to 0.02 M. The 
dialyzed material containing the active enzyme is then bound to a MONO-Q 
column (Pharmacia), followed by elution with 0 to 0.4 M NaCl solutions. 
The active fractions collected are then dilated 1:1 with buffer lacking 
NaCl and absorbed to a MONO-S column (Parmacia) which is eluted with 0.05 
to 0.6 M NaCl solutions. The active peak fractions are made 50% (V/V) in 
glycerol affording a standard sample of NgoAIII. 
According to the present invention, assays to detect the presence of the 
restriction endonucleases and modification methylases can be used during 
the conventional biochemical purification methods to determine the 
presence of these enzymes. 
Restriction endonuclease can be identified on the basis of the cleavage of 
its recognition sequence. As substrate, there can be used, for example, 
Adenovirus-2 (Ad-2) DNA. The DNA fragments obtained are separated 
electrophoretically in agarose gels in the buffer systems conventional for 
the fragment separation in the presence of ethidium bromide. 
Demonstration of modification methylase activity can be, but is not limited 
to, a two-step identification process. First, DNA substrate (Ad-2 DNA) 
that contains the recognition sequence is incubated with column fractions 
to be tested for methylase activity. Secondly, this DNA is then challenged 
with the corresponding restriction activity to identify those fractions 
which contain methylase activity. For example, while assaying for 
M.NgoAIII, the DNA samples will be challenged with NgoAIII. Thus, DNA 
samples which do not exhibit cleavage with NgoAIII contain M.NgoAIII 
activity. 
The recombinant host containing the genes encoding for NgoAIII and 
M.NgoAIII (designated DHIOB/pRMNgoAIII) was put on deposit with the Patent 
Culture Collection, Northern Regional Research Center, USDA, 1815 N. 
University Street, Peoria, Ill. 61604 USA (NRRL) as deposit no. NRRL 
B-18657. 
The recombinant host containing the genes encoding for NgoAI and M.NgoAI 
(designated DH5.alpha.MCR/pRMNgoAI) was put on deposit with the NRRL as 
deposit no. NRRL B-18656. 
EXAMPLE 1 
Bacterial Strains and Growth Conditions 
Neisseria qonorrhoeae FA1090 (provided by Dr. M. S. Cohen, University of 
North Carolina, Chapel Hill) was grown at 37.degree. C. in the presence of 
5% (v/v) CO.sub.2 in CTA medium (1.125% [w/v] Protease Peptone No. 3; 1% 
[w/v] nutrient agar; 0.1% [w/v] KH.sub.2 PO.sub.4 ; 0.4% [w/v] K.sub.2 
HPO.sub.4 ; 0.5% w/v] NaCl; 0.1% [w/v] soluble starch; and 1% [v/v] of a 
solution containing 40% [w/v] glucose; 0.5% [w/v] L-glutamine; 0.05% [w/v] 
Fe(NO.sub.3).sub.3 ; 0.002% [w/v] thiamine pyrophosphate). The cells were 
centrifuged and stored at -70.degree. C. as a cell pellet prior to total 
genomic DNA isolation. 
E. coli strains were grown at 37.degree. C. in YET broth (10 g/1 Bacto 
trypton, 5 g/l yeast extract and 5 g/l NaCL) with antibiotic supplements 
of ampicillin (Ap), 100 .mu.g/ml; or tetracycline (Tc), 20 .mu.g/ml as 
required. E. coli strains, K802 (Maniatis et al.: Molecular cloning. A 
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y., 1982), DH5.alpha.MCR, or DH10B were used interchangeably for cloning 
the restriction modification genes from Neisseria gonorrhoeae. 
DH5.alpha.MCR and DH10B competent cells were obtain commercially from Life 
Technologies Inc. (LTI), Gaithersburg, Md. Competent cells of K802 were 
made by a protocol previously described (Hanahan, D., J. Mol. Biol. 
51:557-580 (1983)). 
EXAMPLE 2 
DNA Isolation 
Small scale plasmid DNA isolations were performed by an alkaline lysis 
method (Maniatis et al., 1982). For large scale preparations, alkaline 
lysis was followed by standard CsCl-EtdBr gradient centrifugation 
(Maniatis et al., 1982). 
Neisseria qonorrhoeae total genomic DNA was isolated by resuspending 2 
grams of frozen cells in 8 mls of TNE buffer (50 mM Tris-HCl pH 8.0, 50 mM 
NaCl and 10 mM EDTA). A 10 mg/ml lysozyme solution in TNE buffer was added 
to the cell suspension to a final concentration of 1 mg/ml. After a one 
hour incubation at 37.degree. C., 10% SDS was added to a 2% final 
concentration and the suspension was shaken gently until lysis was 
complete. After cell lysis, the lysate was extracted once with phenol and 
twice with phenol:chloroform:isoamylalcohol (1:24:1). DNA was spooled with 
a glass rod under two volumes of cold ethanol (-20.degree. C.), dissolved 
in TE (10 mM Tris-HCl pH 8.0, and mM EDTA) and purified by CsCl-EtdBr 
gradient centrifugation. 
EXAMPLE 3 
Construction of Genomic Libraries 
Genomic DNA of Neisseria gonorrhoeae was digested partially with HpaII as 
follows: Purified genomic DNA was digested with HpaII in a 10 .mu.l volume 
with 0.42, 0.21, 0.105, 0.0525, 0.026 or 0.013 u/.mu.g. Each digestion 
reaction contained 0.3 .mu.g of DNA in 20 mM Tris-HCl (pH 7.4), and 10 mM 
MgCl.sub.2. After the samples were incubated one hour at 37.degree. C., 
the DNA was analyzed by agarose gel electrophoresis. 
Conditions required to achieve minimal digestion (90% of the DNA greater 
than 15 Kb in length) were chosen as determined by gel electrophoresis. 
Enzyme concentrations of 0.105 and 0.0525 u/.mu.g provided the conditions 
necessary for minimal digestion of the genomic DNA. These reactions were 
subsequently scaled up 10 fold. 
Two tubes, each containing 3 .mu.g of genomic DNA, were partially digested 
in a 100 .mu.l reaction volume as described above with 0.105 or 0.0525 
u/.mu.g of HpaII. After incubation, a 20 .mu.l sample of each was analyzed 
by agarose gel electrophoresis to confirm minimal digestion of the scaled 
up reactions. The fragmented DNA from these reactions were combined, 
extracted with phenylchloroform, ethanol precipitated, and dissolved in 10 
.mu.l of TE buffer. 
One .mu.g of ClaI cleaved and dephosphorylated pCP13 vector was ligated 
with 2 .mu.g of the partially digested genomic DNA using two units of T4 
DNA ligase in 1.times.ligase buffer (0.05 M Tris-HCl (pH 7.6), 10 mM 
MgCl.sub.2, 1 mM ATP, 1 mM DTT, and 5% (w/v) polyethylene glycol-8000). 
The 20 .mu.l ligation reaction was incubated at room temperature 
(25.degree. C.) overnight. 
Approximately 0.75 .mu.g of ligated DNA (5 .mu.l of the ligation reaction 
mixture) was packaged using Stratagene's Gigapack Gold Lambda Packaging 
System according to the manufactures recommended procedure. After the 
packaging reaction was complete, E. coli cells were infected with the 
packaging mix as follows: DH5.alpha.MCR cells were prepared by growing an 
overnight culture in YET media containing 0.2% maltose. The next day, 500 
.mu.ls of these cells were inoculated into 10 mls of YET containing 0.2% 
maltose and grown to mid-log phase. These cells were then centrifuged and 
resuspended in 4.0 mls of sterile 10 mM MgSO.sub.4 buffer. 200 .mu.ls of 
the cell suspension was mixed with 100 .mu.ls of packaging mix. After a 30 
minute incubation at 37.degree. C. without shaking, a 700 .mu.l volume of 
SOC media (2% Bacto tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 
10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM glucose) was added. The cells 
were allowed to grow at 37.degree. C. in an air shaker-incubator for 30 
minutes. The cells were then plated onto 9 YET agar plates containing 
tetracycline and incubated overnight. 
Approximately 5.times.10.sup.4 tetracycline resistant colonies were pooled 
together by scraping the cells from the agar surface. This was 
accomplished by flooding each plate with 2.5 mls of filter sterilized PEB 
I (50 mM glucose, 25 mM Tris-HCl pH 8.0 and 10 mM EDTA). After carefully 
resuspending the cells in buffer with a sterilized glass rod, the cell 
suspension was transferred to a sterile tube and stored at -70.degree. C. 
Before freezing these cells, a 5 ml aliquot was removed and immediately 
inoculated into I liter of YET media containing tetracycline. 
After a five hour growth, the cells were harvested, resuspended in 100 mls 
of PEB I and then combined with the pooled cells which were previously 
stored at -70.degree. C. Plasmid DNA was isolated from this cell 
suspension according to Example 2. The isolated cosmid library was 
designated Neisseria gonorrhoeae plasmid library. 
EXAMPLE 4 
Selection of Clones Expressing Methylase and Restriction Enzymes 
Clones expressing M.NgoIII, M.NgoAI or M.NgoAII methylase were selected by 
digesting the cosmid library with an excess amount of SstII, HaeII or 
HaeIII, respectively. To select M.NgoAIII, Neisseria gonorrhoeae plasmid 
library (5 .mu.g) was digested in a reaction volume of a 100 .mu.l 
containing 1 X REact 2 buffer (50 mM Tris-HCl pH 8.0, and 10 mM 
MgCl.sub.2, 50mM NaCl) with 70 units of SstII at 37.degree. C. for 4.5 
hours. One half of the digested DNA was dephosphorylated by adding 2 units 
of calf intestinal alkaline phosphatase (supplied by Boehringer Mannheim) 
to 50 .mu.ls of the reaction mix. After a 1 hour incubation at 37.degree. 
C., the DNA was extracted with an equal volume of phenol:chloroform (1:1), 
ethanol precipitated, and resuspended in 10 .mu.1 of TE buffer. 
M.NgoAI and M NgoAII methylase clones were selected by digesting Neisseria 
gonorrhoeae plasmid library by following the general procedure used to 
select M.NgoAIII except that 70 units of HaeII was used to select 
recombinant hosts expressing M.NgoAI and 35 units of HaeIII was used to 
select clones expressing M.NgoAII. 
E. coli DH5.alpha.MCR competent cells were transformed with the digested 
DNA library according to the manufacturers suggested protocol. Briefly, 
100 .mu.ls of cold competent cells were mixed with the 10 .mu.l sample of 
the SstII, HaeII or HaeIII digested DNA. The cells were incubated without 
shaking for 30 minutes on ice. After a 45 second heat shock at 42.degree. 
C., the cells were diluted with 900 .mu.1 of SOC and grown for 30 minutes 
at 37.degree. C. Approximately 100-300 tetracycline resistant colonies 
were isolated after plating the transformed cells on YET agar plates 
containing tetracycline. 
Colonies that survived the methylase selection scheme were analyzed for the 
presence of methylase activity. Thirty clones, ten clones each that 
survived SstII, HaeII or HaeIII selection, were individually inoculated 
and grown overnight in 2 mls of YET media containing tetracycline. Small 
scale plasmid isolations were preformed as previously described. DNA 
preparations isolated from clones which survived SstII, HaeII or HaeIII 
digestion were then tested for their ability to resist cleavage with 
SstII, HaeII or HaeIII, respectively, as follows: 
A 0.5 to 1.0 .mu.g amount of isolated DNA was digested in 1 X REact 2 
buffer with 10 units of the appropriate restriction endonuclease at 
37.degree. C. for hour in a 20 .mu.l reaction. Protection of the resident 
plasmid and the host chromosomal DNA from digestion indicated the presence 
of methylase activity. Analysis of all clones isolated by agarose gel 
electrophoresis demonstrated that neither the host chromosomal DNA nor the 
resident plasmid DNA of these clones were cleaved by their respective 
enzymes. Two clones from each group were saved and later assayed for 
restriction enzyme activity according to Example 5. These clones were 
designated numerically: 61 and 62-recombinant hosts which express 
M.NgoAIII, selected with SstII; 25 and 26-recombinant hosts which express 
M.NgoAI, selected with HaeII; and 35 and 37-recombinant hosts which 
express M.NgoAII, selected with HaeIII. 
Recombinant hosts (61, 62, 25, 26, 35, and 37) were tested for restriction 
endonuclease activity according to Example 5. All recombinant hosts except 
26, 35 and 37 exhibited expression of restriction endonuclease activity. 
Thus, clones 61 and 62 expressed the genes encoding for M.NgoAIII and 
NgoAIII while clone 25 expressed the genes encoding for M.NgoAI and NgoAI. 
Recombinant hosts 35 and 37 appeared to express M.NgoAII activity without 
any detectable restriction endonuclease activity. 
EXAMPLE 5 
Assay for Restriction Enzyme Activity 
A 20 ml overnight culture was harvested and resuspended in 1 ml buffer 
containing 10 mM Tris-HCl (pH 7.5), 10 mM beta-mercaptoethanol and 1 mM 
EDTA. Cells were sonicated on ice by 3 to 4, 10 second blast with a 
microtip probe. After sonication, the cell extract was centrifuged at 
4.degree. C. for 30 sec. using a microfuge (1.5 ml tubes) in order to 
separate the cell debris. 
Adenovirus-2 (Ad-2) DNA substrate (0.75 .mu.g) was digested in 
1.times.REact 2 buffer with serial dilutions of extract as follows: Ad-2 
DNA was diluted to a concentration of 0.038 .mu.g/ul in 1.times.REact 2 
buffer. A 30 .mu.l aliquot of the sample DNA was then added to the first 
tube and 20 .mu.l aliquots were dispensed into the second, third and 
fourth tubes. A 3 .mu.l volume of crude extract was mixed into the first 
tube. A 10 .mu.l sample was then removed and serially diluted into the 
remaining tubes, with the final tube having the highest dilution of 
extract. The samples were incubated at 37.degree. C. for 1 hour and a 20 
.mu.l aliquot was analyzed by agarose gel electrophoresis. 
EXAMPLE 6 
Over Expression of the M.NgoAII Modification Methylase 
To enhance expression M.NgoAII methylase, the gene encoding for M.NgoAII 
was cloned into a high copy number vector, pUC19 (Yanisch-Perron, C. et 
al., Gene 33:103-119(1985)). Plasmid DNA was isolated from clone 35 using 
the small scale isolation procedure. Approximately 1 .mu.g of this DNA was 
digested for 1 hour with 100 units of EcoRI in 100 .mu.l of 1.times.REact 
3 buffer (50 mM Tris-HCL pH 8.0, 10 mM MgCl.sub.2 and 0.1 M NaCl) at 
37.degree. C. After incubation, the reaction mixture was extracted with 
phenylchloroform, ethanol precipitated and dissolved in TE buffer. 
Approximately 0.5 .mu.g of EcoRI cleaved and dephosphorylated pBR322 vector 
(Bolivar F. et al Gene 2:95-113 (1977)) was ligated with 0.5 .mu.g of 
EcoRI digested plasmid DNA from clone 35. The ligation mixture was 
incubated at room temperature overnight in a 20 .mu.l reaction containing 
x ligase buffer and 2 units of T4 DNA ligase enzyme. 
Competent K802 cells were transformed with 3 .mu.l of the ligation mixture 
according to the protocol described in Example 4. After the 30 minutes 
expression step, the cells were plated on YET plates containing ampicillin 
and incubated overnight. 
The next day, approximately 1.times.10.sup.3 Ap resistant cells were pooled 
together by scraping the cells from the outer surface as described in 
Example 3. A 100 .mu.l volume of the cell suspension was removed and a 
standard small scale plasmid purification was performed. The isolated DNA 
was then digested with 80 u/.mu.g of HaeIII in 100 .mu.l of 1.times.REact 
2 buffer. The sample was dephosphorylated with 2 units of calf intestinal 
alkaline phosphatase at 37.degree. C. for 1 hour, extracted, ethanol 
precipitated and taken up in 10 .mu.l of TE. 100 .mu.ls of cold competent 
E. coli K802 cells were transformed with 5 .mu.l of this DNA sample. The 
cells were then diluted and plated on YET agar plates containing 
ampicillin. 
The next day, single colony isolates were screened for methylase activity 
as in example 4. A recombinant clone was isolated which contained and 
expressed the M.NgoAII gene on a greater than 15 kb EcoRI fragment 
inserted into pBR322. This intermediate clone was subsequently used to 
isolate an approximately 4.0 kb AvaI DNA fragment from the 15 Kb EcoRI 
fragment. This 4.0 kb AvaI DNA fragment was ligated into the AvaI site in 
pUC19 and then transformed into DH5.alpha.MCR. The recombinant host 
containing pUC19 with a 4.0 kb AvaI insert exhibited high levels of 
M.NgoAII methylase activity. This strain was designated 
DH5.alpha.MCR/pMNgoAII. 
EXAMPLE 7 
Over Expression of the M.NgoAIII and NgoAIII Restriction Endonuclease 
To enhance the expression of NgoAIII methylase and restriction enzymes, the 
genes encoding for M.NgoAIII and NgoAII were cloned into a high copy 
number plasmid pUC19 (see Example 6). Plasmid DNA from clone 61 (Example 
4) was used for the overexpression of NgoAIII restriction-modification 
system. Approximately 1 .mu.g of this plasmid was digested with XhoI by a 
standard protocol suggested by the manufacture, extracted with 
phenol:chloroform/isoamyl alcohol (1:1) and ethanol precipitated. The XhoI 
digested DNA was then self-ligated and the ligated DNA transformed into 
DHIOB. The rationale of XhoI digestion and self-ligation was to eliminate 
extra portion of the unwanted DNA but to leave the genes coding for the 
NgoAIII restriction-modification enzymes. Indeed, the XhoI fragment 
deletion resulted in clones containing much smaller (approximately 4.4 kb 
DNA compared to about 30 kb portion of N. gonorrhoeae DNA in the original 
clone 61). The resulting clone designating 61X3 produced both NgoAIII 
methylase and restriction enzyme. A linear approximate map of plasmid DNA 
in 61X3 is shown in FIG. 1. 
To subclone the remaining portion of N. gonorrhoeae DNA containing genes 
for M.NgoAIII and NgoAIII, the 61X3 plasmid DNA was digested with BamHI 
and EcoRI and a 2.8 kb fragment was purified and ligated into pUC19 at 
BamHI-EcoRI sites. Note that the BamHI site was derived from the vector 
pCP13. The ligated DNA was introduced into a protected host, DHIOB 
containing plasmid DNA from 61X3. The transformants were selected with 
both ampicillin and tetracycline antibiotics in the presence of XGal. All 
white clones contained the desired 2.8 kb fragment. Finally, a 1.6 kb 
EcoRI fragment was reconstructed at the EcoRI site of pUC19-2.8 kb 
plasmid. Two types of NgoAIII activities were noticed; clones with about 5 
times more activity than others. Presumably, this difference was due to 
the different orientation of the EcoRI fragment in the final construct. 
However, no attempts were made to confirm the speculation. The clones with 
the highest activities were saved. One of the clones was designated as 
DHIOB/pRMNgoAIII. 
A simplified restriction map of plasmid DNA, pRMNgoAIII, is shown in FIG. 
2. The map was established by a standard mapping protocol. 
EXAMPLE 8 
Purification of NgoAIII Restriction Endonuclease 
NgoAIII restriction enzyme was purified from the overproducing recombinant 
host, DHIOB/pRMNgoAIII. Approximately 100,000 units of purified enzyme can 
be obtained from 3.3 grams of cells by following the procedure described 
below. 
In an example to purify NgoAIII from a recombinant host expressing the 
genes encoding the restriction-modification system of NgoAIII, the crude 
lysate is absorbed directly onto a heparin-agarose (BRL) column, followed 
by elution with 0.05 to 0.6M NaCl solutions. The active fractions are then 
pooled and dialyzed to reduce the NaCl concentration to 0.02 M. The 
dialyzed material containing the active enzyme is then bound to a MONO-Q 
column (Pharmacia), followed by elution with 0 to 0.4 M NaCl solutions. 
The active fractions collected are then dialyzed 1:1 with buffer lacking 
NaCl and absorbed to a MONO-S column (Parmacia) which is eluted with 0.05 
to 0.6 M NaCl solutions. The active peak fractions are made 50% (V/V) in 
glycerol, affording a standard sample of NgoAIII. 
EXAMPLE 9 
Characterization of the NgoAIII Restriction Endonuclease 
The NgoAIII restriction enzyme purified in example 8 was characterized to 
determine its nucleotide recognition sequence as well as the location of 
cleavage within this recognition site. As detailed below, NgoAIII was 
determined to be a type II restriction endonuclease, which recognizes the 
sequence 5'CCGC.dwnarw.GG3' producing a 2-base 3'-extension. 
The fragments generated after the cleavage of Ad-2 DNA by NgoAIII were 
identical to the fragment profile obtained when Ad-2 DNA is cleaved with 
SstII, which recognizes the same sequence. Therefore, the cleavage site of 
NgoAIII was determined and compared with that of SstII. 
The position of phosphodiester bond cleavage within the recognition site 
was determined by the method of Brown and Smith (Brown, N.L. et al., 
Methods Enzymol. 65:391-404 (1980)). Sequencing reactions were performed 
as described by Sanger, et al. (J. Mol. Biol. 143:161-178 (1980)). A DNA 
fragment containing an SstII site was introduced into M13mp19 for 
cleavage-site determination. Single-stranded DNA template containing the 
SStII site was used to synthesize double-stranded DNA through the SstII 
site using BRL universal primer. The extended DNA was cleaved with NgoAIII 
and SstII separately. Aliquots of the digested products were run on a 
sequencing gel next to DNA sequencing reaction products produced with the 
same template and primer. The results showed that both NgoAIII and SstII 
produced a fragment which comigrated with the 3'C within the recognition 
sequence 5'CCGCGG3'. Treatment with Klenow fragment or T4 DNA polymerase 
in the presence of all four deoxyribonucleotides, subsequent to NgoAIII or 
SstII digestion, shifted the migration of the fragments to a position 
corresponding to the second 5'C. These results demonstrate that both 
NgoAIII and SstII cleave at the same site within the sequence. In 
addition, Ad-2 DNA fragments generated by NgoAIII digestion can be cloned 
into a SstII-cleaved vector and all cloned fragments are recovered by 
digestion with either SstII or NgoAIII. These results demonstrate that 
NgoAIII cleaves between the second C and G from the 5' end to produce a 2 
-base 3' extension: 
EQU 5'CCGC.dwnarw.GG3' 
EQU 3'GG.uparw.CGCC5'