Method for producing the Eag I restriction endonuclease and methylase

The present invention is directed to a method for cloning and producing the Eag I restriction endonuclease by (1) introducing the restriction endonuclease gene from E. agglomerans ATCC into a host whereby the restriction gene is expressed; (2) fermenting the host which contains the vector encoding and expressing the Eag I restriction endonuclease, and (3) purifying the Eag I restriction endonuclease from the fermented host which contains the vector encoding and expressing the Eag I restriction endonuclease activity.

BACKGROUND OF THE INVENTION 
The present invention relates to clones for the Eag I restriction 
endonuclease and modification methylase, and to the production of these 
enzymes from the clones. 
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. Over one hundred different restriction 
endonucleases have been identified among many hundreds of bacterial 
species that have been examined to date. 
Bacteria usually possess only a small number restriction endonucleases per 
species. The 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 (AT)GGCC(AT), 
PuGCGCPy and GGCC respectively. Escherichia coli RY13, on the other hand, 
synthesizes only one enzyme, EcoRI, which recognizes the sequence GAATTC. 
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 
molecules and cleaving them each time 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 bacterial protective systems are the modification 
methylases. These enzymes are complementary to restriction endonucleases 
and they provide the means by which bacteria are able to protect their own 
DNA and distinguish it from foreign, infecting DNA. Modification 
methylases recognize and bind to the same 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 fully modified, by 
virtue of the activity of its modification methylase and it is therefore 
completely 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. 
With the advent of genetic engineering technology, it is now possible to 
clone genes and to produce the proteins and enzymes that they encode in 
greater quantities than are obtainable by conventional purification 
techniques. The key to isolating clones of restriction endonuclease genes 
is to develop a simple and reliable method to identify such clones within 
complex `libraries` i.e. populations of clones derived by `shotgun` 
procedures, when they occur at frequencies as low as 10.sup.-3 to 
10.sup.-4. Preferably, the method should be selective, such that the 
unwanted, majority, of clones are destroyed while the desirable, rare, 
clones survive. 
Type II restriction-modification systems are being cloned with increasing 
frequency. The first cloned systems used bacteriophage infection as a 
means of identifying or selecting restriction endonuclease clones (HhaII: 
Mann et al., Gene 3: 97-112, (1978); EcoRII: Kosykh et al., Molec. gen. 
Genet 178: 717-719, (1980); PstI: Walder et al., Proc. Nat. Acad. Sci. USA 
78 1503-1507, (1981)). Since the presence of restriction-modification 
systems in bacteria enables them to resist infection by bacteriophages, 
cells that carry cloned restriction-modification genes can, in principle, 
be selectively isolated as survivors from libraries that have been exposed 
to phage. This method has been found, however, to have only limited value. 
Specifically, it has been found that cloned restriction-modification genes 
do not always manifest sufficient phage resistance to confer selective 
survival. 
Another cloning approach involves transferring systems initially 
characterized as plasmid-borne into E.coli cloning plasmids (EcoRV: 
Bougueleret et al., Nucleic Acids Res. 12:3659-3676, (1984); PaeR7: 
Gingeras and Brooks, Proc. Natl. Acad. Sci. SA 80:402-406, (1983); 
Theriault and Roy, Gene 19:355-359, (1982); PvuII: Blumenthal et al., 
J.Bacteriol. 164:501-509, (1985)). 
A third approach, and one that is being used to clone a growing number of 
systems, involves selecting for an active methylase gene referring to our 
patent application No. 707079 and (BsuRI: Kiss et al., Nucleic Acids Res. 
13:6403-6421, (1985)). Since restriction and modification genes tend to be 
closely linked, clones containing both genes can often be isolated by 
selecting for just the one gene. Selection for methylation activity does 
not always yield a complete restriction-modification system however, but 
instead sometimes yields only the methylase gene (BspRI: Szomolanyi et 
al., Gene 10:219-225, (1980); BcnI: Janulaitis et al, Gene 20: 197-204 
(1982); BsuRI: Kiss and Baldauf, Gene 21: 111-119, (1983); and MspI: 
Walder et al., J. Biol. Chem. 258:1235-1241, (1983)). 
A potential obstacle to cloning restriction-modification genes lies in 
trying to introduce the endonuclease gene into a host not already 
protected by modification. If the methylase gene and endonuclease gene are 
introduced together as a single clone, the methylase must protectively 
modify the host DNA before the endonuclease has the opportunity to cleave 
it. On occasion, therefore, it might only be possible to clone the genes 
sequentially, methylase first then endonuclease. Another obstacle to 
cloning restriction-modification systems lies in the discovery that some 
strains of E.coli react adversely to cytosine modification; they possess 
systems that destroy DNA containing methylated cytosine (Raleigh and 
Wilson, Proc. Natl. Acad. Sci., USA 83:9070-9074, (1986)). 
Cytosine-specific methylase genes cannot be cloned easily into these 
strains, either on their own, or together with their corresponding 
endonuclease genes. To avoid this problem it is necessary to use mutant 
strains of E.coli (McrA.sup.- and McrB.sup.-) in which these systems are 
defective. 
Because purified restriction endonucleases, and to a lesser extent, 
modification methylases, are useful tools for characterizing and 
rearranging DNA in the laboratory, there is a commercial incentive to 
obtain strains of bacteria through recombinant DNA techniques that 
synthesize these enzymes in abundance. Such strains would be useful 
because they would simplify the task of purification as well as providing 
the means for production in commercially useful amounts. 
SUMMARY OF THE INVENTION 
In accordance with the present invention there is provided a clone 
containing the genes for the Eag I restriction endonuclease and 
modification methylase derived from Enterobacter agglomerans (ATCC 53769), 
as well as related methods for the production of the enzymes. More 
specifically, this invention relates to clones which express the 
restriction endonuclease Eag I, an enzyme which recognizes the DNA 
sequence CGGCCG and cleaves after the first C. Eag I restriction 
endonuclease produced in accordance with the present invention is 
substantially pure and free of the contaminants normally found in Eag I 
preparations made by conventional techniques from E.agglomerans. 
The preferred method for cloning this enzyme comprises forming a library 
containing the DNA from E.agglomerans (ATCC,53769), isolating those clones 
which contain DNA coding for the Eag I modification methylase by 
resistance to Not I endonuclease cleavage (GC GGCCGC) and screening among 
these to identify those that also contain the Eag I restriction 
endonuclease gene.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to clones of the Eag I restriction and 
modification genes, as well to the restriction endonuclease Eag I produced 
from such clones. The Eag I genes are cloned by a method which takes 
advantage of the fact that certain clones which are selected on the basis 
of containing and expressing the Eag I modification methylase gene also 
contain the Eag I restriction gene. The DNA of such clones is resistant to 
digestion, in vitro, by the Not I and Eag I restriction endonucleases. The 
resistance to digestion by Not I, which recognizes a subset of Eag I sites 
(three out of four Eag I sites on pLSN3), allowed the use of a more active 
endonuclease; resulting in a greater probability of selectively isolating 
clones encoding the Eag I methylase and restriction endonuclease. 
The method described herein by which the Eag I restriction gene and 
methylase gene are preferably cloned and expressed are illustrated in FIG. 
1, and they include the following steps: 
1. The DNA of Enterobacter agglomerans is purified. Samples of this 
bacterium are available from: ATCC 53769. 
2. The DNA is digested with a restriction endonuclease such as Pst I. 
3. A vector containing four Eag I sites (three Not I sites) spread 
throughout the plasmid was constructed from pBR322 (ATCC 37017) (See FIG. 
2). 
4. The digested DNA is ligated to a cloning vector such as pLSN3, that 
contains one or more Eag I sites. The ligated DNA is transformed into an 
appropriate host such as Escherichia coli strain RR1 (ATCC 31343). 
5. The transformed mixture is plated onto media selective for transformed 
cells, such as the antibiotic tetracycline. After incubation, the 
transformed colonies are collected together into a single culture, the 
cell library. 
6. The recombinant plasmids are purified in toto from the cell library to 
make the plasmid library. 
7. The plasmid library is digested to completion with the Not I restriction 
endonuclease, prepared from Nocardia otitidis-caviarum (ATCC 14630). Not I 
digestion differentially destroys unmodified, non-methylase-containing, 
clones, increasing the relative frequency of Eag I methylase clones. 
8. The digested plasmid library is transformed back into an appropriate 
host such as E.coli RR1, and transformants are recovered by plating onto 
selective media. The colonies are picked and their DNA is analyzed for the 
presence of the Eag I modification gene: the plasmids that they carry are 
purified and incubated with the Eag I restriction endonuclease to 
determine whether they are resistant to digestion. Total cellular DNA 
(chromosomal and plasmid) is also purified and incubated with the Eag I 
restriction endonuclease. The DNA of clones that carry the Eag I 
modification gene should be fully modified, and both plasmid DNA and total 
DNA should be substantially resistant to digestion. 
9. Clones carrying the Eag I restriction endonuclease are identified by 
preparing cell extracts of the Eag I methylase clones, identified in step 
8, and assaying the extracts for Eag I restriction endonuclease activity. 
10. The quantity of Eag I restriction endonuclease produced by the clones 
may be increased by elevating the gene dosage, through the use of high 
copy number vectors, and by elevating the transcription rate, through the 
use of highly active, exogenous promotors. 
11. The Eag I restriction endonuclease may be produced from clones carrying 
the Eag I restriction and modification genes by propagation in a fermenter 
in a rich medium containing tetracycline. The cells are collected by 
centrifugation and disrupted by sonication to produce a crude cell extract 
containing the Eag I restriction endonuclease activity. 
12. The crude cell extract containing the Eag I restriction endonuclease 
activity is purified by standard protein purification techniques such as 
affinity-chromatography and ion-exchange chromatography. 
Although the above-outlined steps represent 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. 
The following example is given to illustrate embodiments of the present 
invention as it is presently preferred to practice. It will be understood 
that this example is illustrative, and that the invention is not to be 
considered as restricted thereto except as indicated in the appended 
claims. 
EXAMPLE 
Cloning of Eag I Restriction Endonuclease Gene 
1. DNA purification: Three grams of frozen Enterobacter agglomerans (ATCC 
53769) cells were thawed on ice and resuspended in 25 ml of 0.1 M Tris-HCl 
pH 7.1; 0.1M EDTA. 60mg of lysozyme in 35ml of the above buffer was added. 
The suspension was incubated at 37.degree. C. for 15 minutes. SDS was 
added from a 10% stock solution to adjust the final concentration of SDS 
to 1%. Proteinase K was added to a final concentration of 50.mu.g/ml and 
the solution was incubated for one hour at 37.degree. C. SDS and sarcosyl 
were added to the final concentrations of 3% each and incubated for 2 
hours at 55.degree. C. The DNA was dialyzed overnight into TE (10mM 
Tris-HCl pH.7.1; 1mM EDTA). The DNA was then diluted with an equal volume 
of TE. One gram/ml of CsCl and 100.mu.g/ml of EtBr were added and the DNA 
was spun in a Ti70 rotor for 48 hours at 44,000rpm. The bands were 
removed and extracted with isopropanol saturated with water and CsCl. The 
DNA was then dialyzed overnight into TE and then phenol and chloroform 
extracted. This solution was dialyzed once again against TE overnight. 
2. Digestion of DNA: 8 and 4 .mu.g of E.agglomerans DNA were diluted into 
200 and 100 .mu.l of Pst I restriction endonuclease digestion buffer (10mM 
Tris pH 7.5, 10mM MgCl.sub.2, 10mM mercaptoethanol, 100mM NaCl), 
respectively. 10 units of Pst I restriction endonuclease was added to the 
first tube containing 200.mu.l. 100.mu.l was then transfered from this 
tube to the second tube and both were incubated at 37.degree. C. for 1 hr, 
then digestion was terminated by phenol extraction. 
3. Ligation and transformation: 2 .mu.g (40.mu.l) of the combined Pst I 
digestions of E.agglomerans DNA was mixed with 0.5 .mu.g (10 .mu.l) of 
PstI-cleaved and dephosphorylated pLSN3 (FIG. 2). 75 .mu.l of 4X ligation 
buffer (200mM Tris pH 7.5, 40mM MgCl.sub.2, 80mM DTT, 4mM ATP), and 170 
.mu.l of sterile distilled water were added to bring the volume to 300 
.mu.l. 5 .mu.l of T4 DNA ligase was added and the solution was incubated 
at 16.degree. C. overnight. In each of five tubes, 24 .mu.l of the 
ligation solution was mixed with 0.2 ml of ice-cold, competent E.coli RR1 
(ATCC 31343) cells. The solution was incubated on ice for 25 minutes and 
then at 42.degree. C. for 2 mins. Then 1.0 ml of Luria-broth (L-broth) was 
added and incubation was continued at 37.degree. C. for 1 hr. 
4. Cell Library: The transformed culture was gently centrifuged, 1.0 ml of 
the supernatant was discarded and the cells were resuspended in the 
remaining L-broth and spread onto Luria-agar (L-agar) plates containing 20 
.mu.g/ml tetracyline. The plates were incubated overnight at 37.degree. C. 
The transformed cells that grew up on the surfaces of the plates were 
collected together by flooding each of the plates with 5 ml of L-broth, 
scraping the colonies together, and pooling the suspensions into a single 
tube. 
5. Plasmid Library The cell library was centrifuged at 10K rpm for 5 
minutes. The supernatant was discarded and the cell pellet was resuspended 
in 10 ml of ice-cold 10% sucrose, 50mM Tris-HCl (pH 8.0). 2 ml of lysozyme 
(10mg/ml in 0.25M Tris-HCl, pH8.0) and 8 ml of 0.25M EDTA were added. The 
solution was gently mixed and set on ice for 10 min. Then 4 ml of 10% SDS 
was added and the suspension was gently stirred; immediately following, 6 
ml of 5M NaCl was added and mixed gently. The solution was placed on ice 
for 1 hour to induce cell lysis. 
After cell lysis, the mixture was transferred to a 50 ml tube and 
centrifuged for 45 min. at 17K rpm, 4.degree. C. The supernatant was 
extracted twice with phenol/CHCl.sub.3 and once with CHCl.sub.3. The DNA 
was then precipitated with two volumes of ethanol at -70.degree. C. for 15 
min. The DNA was spun at 15K rpm for 20 min at 4.degree. C. The pellet was 
washed with 70% ethanol and then resuspended in 8 ml of TE (pH 8.0). 
One g/ml of CsCl and 100.mu.g/ml EtBr was added. The solution was 
transferred to a 5/8 in..times.3 in. centrifuge tubes and spun in a 
Beckman Ti70 rotor for 42 hours at 44K rpm, 17.degree. C. 
To collect the plasmids, the tubes were opened, illuminated with 
ultraviolet light, and the lower of the two fluorescent bands was 
collected by syringe. The DNA was diluted with two volumes of water and 
then precipitated with two volumes of ethanol in a dry ice-ethanol bath. 
The solution was spun for 20 minutes at 12K rpm, 0.degree. C. The DNA was 
resuspended in 500 .mu.l of TE and extracted with phenol and then 
CHCl.sub.3. The DNA was reprecipitated with two volumes of ethanol and 
resuspended in 500 .mu.l of TE. The plasmid DNA concentration was found to 
be approximately 100 .mu.g/ml. 
6. Digestion of the Plasmid Library: 2 .mu.g (20 .mu.l) of the plasmid 
library was diluted into 120 .mu.l of Not I restriction endonuclease 
digestion buffer (10mM Tris pH 7.9, 10mM MgCl.sub.2, 100.mu.g/ml bovine 
serum albumin, 150mM NaCl, 0.01% Triton X-100). 60 units (6 .mu.l) of Not 
I restriction endonuclease were added and the tube was incubated at 
37.degree. C. for 1 hr. Then another 60 units of Not I and 1 .mu.l of Calf 
intestinal phosphotase was added and the reaction was incubated an 
additional 30 minutes at 37.degree. C. The reaction was terminated by 
CHCl.sub.3 extraction. 
7. Transformation: 3, 6 and 12 .mu.l (0.05, 0.1 and 0.2 .mu.g) each of the 
digested library was mixed with 0.2 ml ice-cold competent E.coli RR1 (ATCC 
31343) cells (section 3) and incubated on ice for 25 min. The mixture was 
warmed to 42.degree. C. for 2 min. and then 1 ml of L-broth was added and 
incubated for 1 hour at 37.degree. C. The suspension was briefly 
microfuged and 1 ml was removed. The cell pellet was resuspended in the 
remaining L-broth and plated onto an L-agar containing 20 .mu.g/ml 
tetracycline. The plate was incubated overnight at 37.degree. C. Not I 
digestion reduced the number of transformants 10.sup.3 -fold compared with 
transformation by undigested plasmids. A total of thirteen colonies 
survived on the three plates. Five ml of L-broth containing tetracycline 
was inoculated with each of the survivors to prepare a miniculture, and 
streaked onto an L-agar plate containing tetracycline, to prepare a master 
stock. 
8. Analysis of surviving individuals: The thirteen 5 ml cultures of the 
surviving colonies obtained from section 7 were minipreped to isolate the 
plasmids that they carried by the purification procedure adapted from the 
method of Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982) Molecular 
Cloning. A Laboratory Manual. Cold Spring Harbor Laboratories, New York; 
pages 368-369. 
Miniprep Procedure 
Each 5 ml culture was centrifuged at 5K rpm for 5 minutes; the supernatant 
was discarded and the cell pellet was resuspended in 0.1 ml of 25mM 
Tris-HCl,pH 8.0, 10mM EDTA, 50mM glucose, containing 4 mg/ml lysozyme. The 
solution was transfered to a 1.5 ml Eppendorf tube. After 5 minutes at 
room temperature, 0.2 ml of 0.2N NaOH, 1% SDS was added to each tube and 
the tubes were immediately inverted gently to lyse the cells, then placed 
on ice for 5 minutes. Then 0.15 ml of 3M/5M potassium acetate, pH 4.8, was 
added to each, vortexed for 10 seconds and incubated on ice for 5 minutes. 
The precipitates that formed were spun down in an Eppendorf centrifuge at 
4.degree. C. for 5 minutes. Each supernatant was transfered to a new 
Eppendorf tube containing 1 ml of ethanol and mixed. After 2 minutes at 
room temperature, the tubes were spun for 2 minutes at room temp. to 
pellet the precipitated nucleic acids. The supernatants were discarded and 
the pellets were rinsed with 1 ml of 70% ethanol and then air-dried at 
room temperature for 5 minutes. The pellets were resuspended in 50 .mu.l 
of 10mM Tris, 1mM EDTA, pH 8.0 containing DNase-free pancreatic RNase (20 
.mu.g/ml). The plasmid minipreps were subsequently analyzed by digestion 
with Eag I, Not I and Pst I. 
9. Eag I Methylase Gene Clones: Twelve of the 13 plasmids that were 
analyzed were found to be resistent to Eag I and Not I digestion and to 
carry a 2.9 Kb Pst I fragment of E.agglomerans DNA.. One of the 12 
resistant plasmids contained an additional 0.9 Kb Pst I fragment of 
E.agglomerans DNA. The presence of the additional fragment did not affect 
the properties of the plasmid, so for simplicity only plasmids carrying 
the 2.9 Kb fragment were analyzed in detail (FIG. 3). These plasmids, 
typical of which is pEagRM2.9, were shown to encode not only the Eag I 
modification methylase but also the Eag I restriction endonuclease. A 
sample of plasmid pEagRM2.9 has been deposited at the American Type 
Culture Collection under ATCC Accession No. 40853. 
10. Eag I Restriction Gene Clone: pEggRM2.9, and similar plasmids, were 
found to encode and express the Eag I restriction endonuclease by assaying 
extracts of E.coli RR1 that carried the plasmids. 
Endonuclease Assay 
A 1 liter culture of the cells to be assayed was grown overnight at 
37.degree. C. in L-broth containing 20 .mu.g/ml tetracycline. The culture 
was centrifuged at 10K rpm for 5 min and the cell pellet (.about.7 grams) 
was resuspended in 20 ml of 10mM KPO.sub.4 pH 7.5, 0.1M NaCl, 0.1mM EDTA. 
1 ml of 10 mg/ml lysozyme and 2 ml of 0.1M EDTA was added and the 
suspension was left on ice for 15 minutes. The suspension was sonicated 
gently for four 20-second bursts to disrupt the cells. 0.2 ml of 5M NaCl 
was added to the sonicated extract and the cell debris was then removed by 
centrifugation for 10 minutes at 12K rpm. The supernatant was assayed for 
endonuclease activity in the following way: 
14 .mu.g (28 .mu.l) of Bam HI-linearized pLSN3 was diluted into 700 .mu.l 
of Eag I restriction endonuclease digestion buffer (150mM NaCl, 10mM 
Tris-HCl,pH 8.0, 10mM MgCl2, 10mM 2-mercaptoethanol, 100.mu.g/ml BSA). The 
solution was dispensed into 6 tubes, 200 .mu.l into the first tube and 100 
.mu.l into each of the remaining 5 tubes. 1 .mu.l of the extract was added 
to the first tube to achieve 0.25 .mu.l extract/.mu.g DNA. 100 .mu.l was 
then removed from the first tube and transferred to the second tube to 
achieve 0.125 .mu.l/.mu.g. 100.mu.l serial transfers were continued into 
tubes 3 (0.06 .mu.l/.mu.g), 4 (0.03 .mu.l/.mu.g) and 5 (0.015 
.mu.l/.mu.g). The sixth tube received no extract and served as a negative 
control. The tubes were incubated at 37.degree. C. for 30 minutes, then 
20 .mu.l from each was analyzed by gel electrophoresis. The extract was 
found to contain approximately 3.times.10.sup.4 units of Eag I restriction 
endonuclease per ml, which corresponds to about 1.times.10.sup.5 units per 
gram of cells. 
11. Phage-resistance of the Eag I RM clones: Some restriction-modification 
systems express the restriction phenotype when they are cloned into 
E.coli, and some do not. The restriction phenotype is the ability of cells 
to survive infection by phages and the inability of phages to reproduce. 
E.coli RR1 carrying pEagRM2.9, or other plasmids containing the 2.9 Kb Pst 
I fragment, displays a restriction phenotype. Lambdoid phages plaque with 
an efficiency of approximately 10.sup.-2 on these clones compared to an 
efficiency of 1 on the same strain carrying pLSN3. 
12. Transfer of the 2.9 Kb fragment to pUC19: 32 .mu.g (100 .mu.l) of 
purified pEagRM2.9 DNA was prepared in 800 .mu.l of Pst I restriction 
endonuclease digestion buffer (section 2). 300 units of Pst I restriction 
endonucleases was added and the solution was incubated at 37.degree. C. 
for 90 minutes. The digestion mix was run out on a 0.7% Tris-Acetate 
agarose gel (140 .mu.l/well). The desired 2.9 Kb Pst I E.agglomerans 
fragment ran sufficiently below the 4.3 Kb band of the vector and was 
easily cut out of the gel. 
To extract the 2.9 Kb fragment from the agarose, it was first frozen at 
-70.degree. C. and then thawed. An equal volume of running buffer was 
added and then the mixture was extruded through an 18-guage needle and 
refrozen. The thawed mixture was extruded again, diluted to 11 ml total 
volume and spun in a Ti70 rotor at 55K rpm for 1 hour. 
The supernatant was brought to 0.1M NaCl and precipitated with two volumes 
of ethanol. The DNA pellet was resuspended in TE (10mM Tris-HCl, pH8.0, 
10mM EDTA) and then extracted with phenol, phenol/CHCl.sub.3 and finally 
CHCl.sub.3. The DNA was reprecipitated; rinsed with 70% ethanol; and 
resuspended in 60 .mu.l TE. 
0.5 .mu.g (5 .mu.l) of the gel purified 2.9 Kb Pst I fragment was mixed 
with 0.1 .mu.g of Pst I-cleaved and dephosphorylated pUC19. 15 .mu.l of 4X 
ligation buffer (section 3) and 38 .mu.l of sterile distilled water were 
added to bring the volume to 60 .mu.l. 1 .mu.l of T4 DNA ligase was added 
and the solution was incubated at 16.degree. C. for 2 hr. The ligation was 
sterilized by extraction with 60 .mu.l of chloroform, then clarified by 
brief microcentrifugation. 1, 5 and 10 .mu.l of the sterile ligation was 
mixed with 200 .mu.l of competent, ice-cold, E.coli RR1 and incubated on 
ice for 25 minutes and then heat shocked at 42.degree. C. for 2 min. 1 ml 
of L-broth was then added to each mixture and incubated at 37.degree. C. 
for 1 hour. The cells were pelleted and 1 ml of supernatant was removed. 
The cells were then resuspended in the remaining L-broth and then plated 
onto L-agar plates containing 100 .mu.g/ml ampicillin and transformants 
were recovered after incubation at 37.degree. C., overnight. 
8 transformant colonies were picked and screened by the miniprep procedure 
(section 8) to identify plasmids composed of pUC19 with the 2.9 Kb 
fragment inserted at the Pst I site. All plasmids were found to contain 
the 2.9 Kb fragment; 2 contain the 2.9 Kb fragment in one orientation 
(pEagRM2.9-19a) and the other 6 were in the opposite orientations (pEagRM2 
9-19b). All exhibit complete resistance to Eag I digestion. A crude cell 
extract of E.coli RR1 carrying each of these plasmids was assayed for Eag 
I restriction endonuclease activity (FIG. 4). 
The extracts from pEagRM2.9-19b were found to contain approximately 5-fold 
more Eag I restriction endonuclease than the parental clone and extracts 
from pEagRM2.9-19a contain .about.10-fold more endonuclease, 
.about.1.times.106 units/gm cells. 
E.coli RR1 carrying pEagRM2.9-19a is the preferred host from which the Eag 
I restriction endonuclease can be purified. The strain should be grown to 
stationary phase at 37.degree. C. in a fermenter, in L-broth containing 
ampicillin. The cells should then be collected by centrifugation and 
either broken immediately for extract preparation, or stored frozen at 
-70.degree. C. until it is convenient to do so.