JOURNAL
OF BACTERIOLOGY, Aug. 1991, p. 5207-5219 0021-9193/91/165207-13$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 173, No. 16
Characterization and Expression of the Escherichia coli Mrr Restriction System PHYLLIS A. WAITE-REES,t CAROLE J. KEATING, LAURIE S. MORAN, BARTON E. SLATKO, LINDA J. HORNSTRA, AND JACK S. BENNER*
New England Biolabs Inc., 32 Tozer Road, Beverly, Massachusetts 01915 Received 15 March 1991/Accepted 24 May 1991
The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CvilRI, Hinc, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.
Restriction and modification systems appear to be widespread among bacteria and are generally believed to function in the destruction of foreign DNA (68, 70) and the reduction of the DNA to recombinationally usable pieces (33). Of the restriction-modification systems characterized to date, two types have been closely examined: type II restriction systems, which are generally composed of an endonuclease and a methylase, and type I systems, such as EcoK, consisting of three subunits, one of which confers sequence specificity (70). Escherichia coli has been shown to contain at least two restriction systems, McrA and McrBC, which restrict N4- or C5-cytosine-methylated DNA (39, 41, 44, 45). These two systems were initially called rglA and rglB because they restricted glucoseless, 5-hydroxylmethyl cytosine-containing DNA, present in many T-even phage (42). One system (rglB) was found to produce an endonuclease activity (16). These restriction systems differ from the classically recognized ones since they require modified DNA as the substrate for their action rather than using modification for selfprotection. An additional methylation-specific restriction system of E. coli, Mrr, was described by Heitman and Model (20) and was shown to interfere with the maintenance of certain N6adenine methylases. The HhaII and PstI N6-adenine methylase genes, when maintained in several E. coli K-12 strains, produced DNA damage as evidenced by induction of the SOS DNA repair response (20). They also demonstrated that several cytosine methylases induced the SOS response. Transposon insertion mapping and Southern blotting were used to position mrr on the E. coli chromosome at 98.5 min.
In addition, the plasmid pBg3 (Fig. 1), isolated from the E. coli K-12 genome (strain CR63), was identified as sufficient to complement mrr (40) (reference 48 gives a detailed description of the preparation of this clone). This locus was originally named mrr, the acronym for methylated adenine recognition and restriction. The use of mrr rather than mar was fortuitous since we have found that the product of the gene restricts both adenine- and cytosine-methylated DNA; to date, this is the only well-characterized function which restricts both. Restriction of transformation of methylated DNA by bacteria other than E. coli has been observed with Streptococcus pneumoniae (26), multiple Streptomyces strains (29), and Acholeplasma laidlawii (52). In Streptomyces avermitilis, transformation frequencies of E. coli dam and M. TaqI N6-adenine-methylated DNAs and AluI, E. coli dcm, HhaI, and HphI C5-cytosine-modified DNA are reduced by >103. Only one endonuclease, DpnI (and its isoschizomers), is known to be specific for adenine-methylated DNA. DpnI resembles a type II endonuclease since it requires a divalent cation to cleave in the sequence G' ATC (26). These strains of bacteria and mycoplasma have been proposed to contain Mcr- and Mrr-like systems which might serve to control entry and expression of foreign DNA by using methylation patterns as a recognition criterion. In this report, we extend the characterization of the specificity of mrr restriction to the presently available cloned adenine and many cytosine methylases, report the DNA sequence of the gene, and examine the levels of expression of Mrr under various conditions. MATERIALS AND METHODS
Bacterial strains and media. The bacterial strains used in this study and their sources are listed in Table 1. E. coli strains were grown in Luria broth (4). Media were supple-
Corresponding author. t Present address: Department of Chemistry, Florida State University, Tallahassee, FL 32306. *
5207
5208
J. BACTERIOL.
WAITE-REES ET AL.
/"..uc II
PAvsNi 4
T7 p
p(pET3b)mrr5.3-A3' 5300 p Sat! l
'AlIll-
+1kam-
E-olRZ
ECOR~~~~~W(
ill I
Nsi
.5aci-1 Bldai
*fr:
1\
/ ,A/! JU[IN
A.
/
.t p(pACYCI84)mrr6.3-4 Nsi S.aI(. 1}
I flfft
+Nsi I
6:300 bp
T7 p
/
14
'colEllorniE'COR I
p(pPET3b-pACYC)2 1 1.6-1
+
1100 14()O }3p15a orw
Fink.y 1-iX
.14. .C
'
Sal1cS Nsi I+
INd. I
SSp) .&:fv
I
M
VaoR
I\
\ I
_
P~i 4
Nsi I
p(pUC19)mrr4.7-20 4700 bp
T7
PCR 2) + pET' r3b
Nde I
-
p(pET3b)mrr5.3-A3 5300 bp
1)
+
44
+ I3arI 11I1
Banrd 1 1
b ' .W11 1
lac lacZ
-]3(i I I'l I I
i
I-flad
A'!
I I
..21 f
III
FVc
IA
iN
lr--j -r
.
.-.
-,
........, ,_
pBg3 p
Bg
~~~L-i
L.-... I kO)
FIG. 1. Plasmid constructions of mrr. Representative restriction sites are indicated on each construct. The positions of genes and fragments of interest are shown with stippled boxes or arcs. The stippled boxes and arcs for mrr indicated the entire fragment which contains mrr and not just the coding region. The hsd region of E. coli K-12 is shown at the bottom.
VOL. 173,
1991
E. COLI Mrr
TABLE 1. E. coli strains used in this study Phenotype
Strain
RR1 HB101 GM2931 NM554 K802 MM294 ER1398 ER1563 ER1562 ER1821
ER1378 ER1381 ER1564 ER1565 ER1648 ER1797 BL21(DE3)
Refer-
RecA
McrA
McrBC
Hsd
Mrr
Dam
ence
+
+
-
-
+ +
-
-
+ +
39 39
+
+ + +
-
-
+ +
-
+
+
+ -
+ -
-
-
-
_ -
-
+ +
+ + +
+ + + + +
+
+ + -
-
_
+
+
+ + + + +
+ +
_ + + + +
+
-
+
-
-
_ +
_
-
-
+
-
+ + + + + + + + + + +
a
39 39 39 39 39 39
b 40 40 39 39 40 b c
a GM2931 is a derivative of RR1, prepared and kindly provided by M. Marinus. b ER1797 is a derivative of ER1370, and ER1821 is a derivative of MM294, both prepared and kindly provided by E. Raleigh (38). c The phenotype of BL21(DE3) was determined by using M.HpaII, M.PvuII, and RM.HincII [p(pBIIHI.2)HincIIRM8.0-A1] as testers for McrA, McrBC, and Mrr phenotypes.
mented with antibiotics (ampicillin [100 ,ug/ml], tetracycline [25 p.g/ml], and kanamycin [35 p.g/ml]) as required. Plasmids. The plasmids pACYC184 (9), pUC19 (69), pET3b (58), and pBg3 (48) were used for cloning and as sources of DNA fragments. Plasmid DNA was prepared by the alkaline lysis method (30), followed by CsCl-ethidium bromide ultracentrifugation. The sources of the plasmid constructs used in the transformation studies are listed in Table 2. Plasmid constructs containing mrr were named as follows: the vector used in the construction is enclosed in parentheses, followed by the designation mrr, the total size in kilobases, and an isolate number [i.e., p(pUC19)mrr4.720, a clone in the plasmid pUC19 containing some part of mrr with a vector plus insert size of 4.7 kb and an isolate number of 20]. Deletion clones contain in addition the restriction endonuclease site(s) used to make the deletion. Cloning enzymes and techniques. All restriction enzymes were produced at New England Biolabs, Inc., and used according to recommended conditions. Dephosphorylated vectors were prepared from linearized vectors treated with calf intestine alkaline phosphatase (Pharmacia) by using conditions previously described (30). All ligations were done with 1,000 to 2,000 U of T4 DNA ligase (New England Biolabs, Inc.) in a 100-,ul reaction volume under prescribed conditions, unless noted. Transformations were performed by using the CaCl2 heat shock method (11). For nick translations of DNA fragments, E. coli DNA polymerase I (New England Biolabs), DNase I (Boehringer Mannheim), and [oa-32P]ATP (800 Ci/mM; New England Nuclear) were used. Bidirectional blots (55) and Southern hybridizations (56) were performed as described previously. Nitrocellulose blots were washed at 65°C (43, 60, 66). DNA preparation. E. coli DNA from strains RR1, HB101, NM554, ER1648, and K802 was purified in a fashion similar to that used for Bacillus amyloliquefaciens DNA (5) with the modification that 2 g of frozen E. coli cells was used in the procedure rather than 0.5 g. Approximately 1 mg of DNA
5209
was obtained from each preparation. DNA fragments from genomic and plasmid DNA restriction digests were gel purified as described previously (64). Deletion subclones requiring only recircularization after digestion [p(pUC19)mrr4.1-AEcoRV, p(pUC19)mrr3.4-A BamHI, p(pUC19)mrr4.4-ANsiI/PstI, and p(pUC19)mrr3.0AAccI; Fig. 2) were prepared as described previously (65). One deletion plasmid, p(pUC19)mrr4.7-ABssHII, was made by sequential digestion of p(pUC19)mrr4.7-20 with BssHII and S1 nuclease, followed by ligation (Fig. 2). Coupled in vitro transcription-translation. A prokaryotic in vitro transcription-translation kit (New England Nuclear) was used according to recommended conditions, except reactions were performed at one-half scale (10 ,ul, total volume). All reactions utilized [35S]methionine (900 Ci/ mmol; New England Nuclear) as the label. Samples containing the [35S]methionine-labeled proteins were electrophoresed on 10 to 20% sodium dodecyl sulfate (SDS)polyacrylamide minigels (ISS-Enprotech), fixed in methanolacetic acid, rinsed briefly in water, and soaked for 30 min in 1 M sodium salicylate before drying and autoradiography. DNA sequencing and oligonucleotide synthesis. Sanger dideoxy sequencing (49, 50) was performed by a modification of the "collapsed plasmid" protocol (10, 17, 19). All reaction mixtures utilized [a-355]dATP (600 to 1,200 Ci/mM; New England Nuclear) or [oa-32P]dATP (3,000 Ci/mM; New England Nuclear) and were subjected to electrophoresis on 6 to 8% standard, "wedge," or buffer gradient sequencing gels (1, 67). The following five plasmid constructs were used as templates for sequencing: p(pUC19)mrr4.7-20, p(pUC19) mrr4.1 - AEcoRV, p ( pUC19) mrr3.4 - ABamHI, p (pUC19) mrr4.4-ANsiI/PstI, and p(pUC19)mrr3.0-AAccI (see Fig. 2) by using primers or probes synthesized on a Biosearch 8600 automatic synthesizer. One template, p(pET3b)mrr5.3-A3 (Fig. 1), was used to verify the DNA sequence between the NdeI and NsiI sites in this plasmid construct. DNA sequence data were computer acquired by using a Grafbar digitizer (SAC Corporation) and analyzed by computer programs written by us for the Macintosh (Apple Computer) and computer programs from Cold Spring Harbor Laboratory (24), the National Biomedical Research Foundation (12), and the Genetics Computer Group (14) by using a VAX (Digital Equipment Corp.). Overexpression of the Mrr protein. Two oligonucleotide primers were used with p(pUC19)mrr4.7-20 for polymerase chain reaction (PCR) (47) to introduce convenient cloning sites into the E. coli mrr sequence. A forward primer (5' CAAGGATGTACATATGACGGTTCCT 3') introduced a single base mutation and created an NdeI site, and a reverse primer (5' TACCCGGTAGATCTCAGCGAAGTTT 3') changed a single base and created a BglII site. The reaction mixture contained 100 ng of p(pUC19)mrr4.7-20, 3 ,ug of each primer, and 200 ,uM of the four deoxynucleoside triphosphates in a total volume of 100 ,ul of the supplied buffer. The plasmid DNA was denatured for 2 min at 95°C and cooled to 45°C before the addition of 10 U of Vent DNA polymerase (New England Biolabs) and bovine serum albumin (BSA) to a final concentration of 100 ,ug/ml. Twenty-five cycles of 93°C (30 s), 45°C (60 s), and 65°C (180 s) were carried out in a Techne thermal cycler. The product from the reactions, approximately 20 ,ug, was purified by agarose gel electrophoresis as previously described (65). The purified DNA fragment was subjected to microdialysis on a VSWP (0.045 Rm; Millipore) filter for 1 h versus 10 mM Tris-HCl-1 mM EDTA, pH 8.0. The DNA was digested with BamHI (this site was internal to the PCR primer) and NdeI, ex-
5210
WAITE-REES ET AL.
J. BACTERIOL.
TABLE 2. Plasmids and phage used in this study Plasmid or phage
pSCaccIRM121-2 ............................................ pNWhhaIIRM2-13 ...........................................
pGWpstIRM10-3 ........................................... p(pUC19)HincIIM5.7-WL-5 ........................................... pUCHinf2.3 ........................................... p(pBIIHI.2)HpaIM ...........................................
Reference or source
28 G. Wilson and D. Nwankwo G. Wilson and D. Nwankwo 64 8 63
p(ACYC)NlaIIIM-4, p(pUC)NlaIIIM-H64, pBspHIRM-4 and pAseIRMEcolO.1-1 ............................................
R. Morgan 53 I. Hall and P. Riggs 57 7 31 G. Wilson T. Barshevsky and J. Benner 65 61 T. Jager-Quinton, G. Wilson, L. M. Ar Sznyter, naud, and J. Brooks D. Nwankwo and 28 pDNHindIII-G18 ........................................... 27 pAEL201 and pAFJ1 ........................................... 28 and C. Vacarro, M. Amaud, L. Sznyter, and pSphM1.4 ........................................... V. Bernan, and J. Brooks 6 and 28 pGW1 ........................................... 36 pNW106RM2-63 ........................................... 51 pIL-3A.22 ........................................... D. Heiter and J. Benner pPvuIIRM8.7-M15 ........................................... 22 pBcnl ............................................ pBamM1.8 and pBamM2.2 ............................................4 P. Levin, T. Lee, and J. Benner pBLSboC13RM9.7-A8 ........................................... J. Benner pDraIIM14.0-A1 ............................................ J. Benner pDraIRM12.5-A5 ........................................... P. Waite-Rees, and J. Benner A. Eldridge, L. Hornstra, pSspIM14.4-B6 ............................................ pDNAatIIRM-H7, pLNXmnIRM-7 and pDNSpeIM-9 ..................................... J. Lynch and D. Nwankwo 62 pEVCncoIM612-1 ........................................... 62 pEVCxbaIRM-101 ............................................ 54 pHaeIIRM4-5 ........................................... M. Meda, F. Perler, and G. Wilson pMMfspIM-35 ........................................... 28 pKLaflIIRM19-2 ........................................... 21 pDdeM1.6 ........................................... Hodges, andW. Jack R. J. Forney, pCAL7 ............................................ Mu momA cts62.K12 and Mu cts62.K12 ........................................... S. Hattman
pSW149RM-3B ............................................ pIH913 ............................................ pXZ-6E5.9 ........................................... pHhaI2-1 ........................................... pTP166 ........................................... pEco3-10 ............................................ pEcoRVRM8.7-2 ............................................ pNdeIM6.7-H5 ........................................... pPaeM2.7 and pPaeRM3.8 ............................................ pSalRM3242 ...........................................
tracted with phenol-chloroform, and precipitated with two volumes of ethanol. The resuspended fragment was ligated into the T7 overexpression vector pET3b (58), which had been cleaved with BamHI and NdeI and treated with phosphatase as described above. The construct obtained by the above PCR procedure, p(pET3b)mrr5.3-A3, was subjected to a replacement of the 3' portion of the gene (approximately 90%) from the NsiI site within the mrr gene to the external BamHI site (see Fig. 1, 2, and 3). This was done to ensure that no mutation(s) generated in the PCR would be carried into the final construct used to determine the biochemical function of the Mrr protein. This was accomplished by first linearizing both p(pET3b)mrr5.3-A3 and p(pACYC184)mrr6.3-4 with NsiI, treating p(pET3b)mrr5.3-A3 with phosphatase, ligating the two, and transforming into RR1 cells (Fig. 1). The intermediate construct p(pET3b-pACYC)mrr211.6-1 containing the desired structure was isolated (see Fig. 1). This plasmid DNA was then cut with BamHI endonuclease, diluted 10fold in ligation buffer, ligated, and transformed into RR1 cells. From these colonies, a representative isolate with the desired plasmid structure was selected and designated p(pET3b)mrr5.3-A3'.
Purification of the Mrr protein. One gram of frozen cells [E. coli BL21(DE3)/pLysS/p(pET3b)mrr5.3-A3] was resuspended in 10 ml of ice-cold buffer A (20 mM K2HPO4, 5 mM dithiothreitol, 0.1 mM EDTA, 0.05 M NaCl, pH 7.0) and subjected to several 30-s periods of sonication. Cellular debris was removed by centrifugation at 12,000 x g for 20 min at 4°C. The supernatant was applied to a heparinSepharose (Pharmacia) column (10.0 by 2.5 cm) which had been equilibrated in buffer B (0.02 mM Tris-HCl, 50 mM KCI, 1 mM dithiothreitol [pH 7.4], 10% glycerol) and washed with two volumes of the same buffer. The column was developed with a linear gradient of 0.05 to 1.0 M KCl in buffer B (pH 7.4) at a flow rate of 1.3 ml/min, with 7.5-ml fractions being collected. Fractions were assayed for Mrr protein by SDS-polyacrylamide gel electrophoresis. A portion of each of the above fractions was diluted with an equal volume of glycerol and stored at -20°C. Protein sequencing. The Mrr protein (crude and purified), prepared from p(pET3b)mrr5.3-A3, was subjected to electrophoresis on a precast 10 to 20o SDS-polyacrylamide gradient gel (ISS-Enprotech) with a 4% polyacrylamide stacking gel and electrophoresed at 100 V for 4 h. After electrophoresis, the gel was electroblotted (Hoeffer TE-52)
VOL. 173, 1991
ILi-"licKi "CII el1
E.
=
A
.V,I
i
) I -.h I (
I
I
mrr~PU
E~~~I
Ie 'v'r11
M~I
I
I
COLI Mrr
5211
p(pUC19)
nurr4.7
p(pUC)l9
mrr4. I-AEcoR V
-20
mrr+
unknown ORF
M-=W
'..OM II.I.-M, --p M4MI '.
lin IL-
.............................
p(pUC19) mnur3.4-ABamH
...............
p(pUC19) nrT4.4-A&Nsi
k2222222Z:.22222
I
rmrr-
I
I/Pst
I
ffwr-
p(pUC 19) mrr3. I1-AAcx I
I
p(pACYC 184) mrr4.7-ANsI I
mzt--
p(pUC19) mirr4.7-,&BssH II
mrrf+
nIL-
IkbI
FIG.
2. EcoRI
fragment restriction
directions of the three ORF
map and deletion clones.
indicated. The
are
mrr
Regions present in deletion phenotype of each clone is also noted.
ProBlott polyvinylidene difluoride transfer membrane (Applied Biosystems) according to the procedure of Mat-
to
lanes of 2
(200
m-A).
The
membrane
Coomassie blue R-250, and two
stained
was
protein
101
201
boxed and
stippled. The positions
and
excised and treated as previously sequential degradation of proteins was performed with an Applied Biosystems model 470A gasphase sequenator using a Blottl program. The first 35 amino acid residues, with the exceptions of residues 15 and 22,
with
bands from identical
el
are
each
were
described (27). The
sudaira (32), with modification of the transfer time (12 h) and current
~Lg
clones
sl
as
CCTTTCCGGG1CAATGACCACGGTCACAGCAACTGACTCATTTCTAACGTGEZITATTTTTGTAGTGCIEAaGCCGAAAAACATCTACCTGATTC TGCEEFGTACTATGACGGTTCCTACCTATGACAAATTTATTGAACCTGTTCTGCGTTATCTGGCAACAAAACCGGAAGGTGCAGCCGCGCGTGATGT M TV PT Y DK F I E P VL RY L ATK P E GA A AR DV bl
s2
n
3 01
H
501
601 701 801 901
EA A AD AL G
L
D
D
S Q
R AK V
I TS
G OL
V
Y
K N
RAG W A
H D RL KR A GL SO0S L S R G K WCL TP AG FD WV AS H PQ P a2 CAGCGGAGGCACACGCTCCTTTATTAATAGCCGCGTCGCATAACGAGCAT M TE QE THNHL A FA F V NVK L KS RP D AV D LDP K AD S bl p TCGTAGAACTCAGGACCGCACGTGTAGGTAAACTGGTCGGCGTAGTTGAA P D H E E L A KS S P D D R L D OA L K E L R D A VA D E V LE N
TATCGTTTCTGGTTAGCTGTTGTTTGACCTGGAGCGCCGGTATGACTTGC L L QV SP S R FEV I VL D VL H RLG YGG HR D DL Q RVG G c e5 GATGGTGGCTGTGGGTTGTGTACTGCGAAATTTTCGCAAGTGAATCGAGA T G D G G I D GV I S L D K L GL E K V Y V QA K R W QN T VG R
GCGATCGCTTAGCCCGCGGAAACAAGGGTTTTACCTTGTTCTTAGGGGCT P E LQ A FYG AL AGQ0K A KR G VF I T TS G FT SOA R D F 1
1001
A Q
V
S
E G MVL VD
G E
R
N
L V
L M
E N
I
E V GV
S S
R
L
L
K V
P
1101 K
L
D
MD
Y
F
E b
1201
CCATTTCTCGACGACCAAAACTGGTTCGTTCTGCCCGCGCCGCGGTTGGC h2
1301
CGAGCTGAACGCGTGTCCGCACAACTCTTTAGACGTACGCATCGACGTTC
1401
ACCCGAATCCGTTTCGGATCGTCCAATGCAAACTTGTACGGTAGGAAGGT
1501
TGGTAGCTTCTCGAAACGTGGTACGAGCAACAGCCGGGGCGGAACGGGGG
e5
c
b2 1601
h2
al
a
GTAGGGCGCTCTCGTTCGTCCTCAGCGTTGCGAATCGCGGACACTTATGC
1701
AGTACGTCTTCCTGCTTCGACACGGATAGCTCGAAACCGCTAATCTGAAA sl
1801
AGCGAATGGCGACGCTCGTCTCAGCAAGTGATATTGCTGCAATTCGAGCA
1901
TGGGCTCGTGTGGGGAACGTCGCTGTACTTGTTCTGGGTGCACGTACTAC
2001
GAGACTTCGCCGAATTC
el
FIG. the
3.
(a),
P,
areas
upstream of
and y. The following restriction sites are indicated: Aatll (a), Accl (al), Aflhl (a2), Aflhll protein are marked a, (h), AlwNI (w), ApaLI (1), Asel (as), BamHl (b), BspHI (bl), BssHII (b2), Clal (c), Dral (d), EcoRI (el), EcoRV (eS), Nsil (n),
coding region for
Pvul
2017
Nucleotide sequence. The protein sequence verified from the amino terminus is underlined. The three boxed
Ahall (p), Sacll
the Mrr
(s2), and
Sspl (sl).
5212
J. BACTERIOL.
WAITE-REES ET AL. TABLE 3. Efficiency of transformationa of several restriction-modification system in various E. coli strains EOT of host
Plasmidb
HinclI RM(pBIIHI.2) HpaI RM TaqI RM HincII M
HincII RM(pBIIHI.2) HincII RM(pUC19-4) HincII RM(pUC19-10) HincII M
RR1 1.0 1.0 1.0 1.0 NM554