Recombinant DNA Part F

593 Pages·1988·12.657 MB·English

by  Kaplan N.P. (ed.), Colowick N.P. (ed.), Wu R. (ed.)

Preface
Recombinant DNA methods are powerful, revolutionary techniques
for at least two reasons. First, they allow the isolation of single genes in
large amounts from a pool of thousands or millions of genes. Second, the
isolated genes or their regulatory regions can be modified at will and re-
introduced into cells for expression at the RNA or protein levels. These
attributes allow us to solve complex biological problems and to produce
new and better products in the areas of health, agriculture, and industry.
Volumes 153, 154, and 551 supplement Volumes 68, 100, and 101 of
Methods in Enzymology. During the past few years, many new or im-
proved recombinant DNA methods have appeared, and a number of them
are included in these three new volumes. Volume 351 covers methods
related to new vectors for cloning DNA and for expression of cloned
genes. Volume 154 includes methods for cloning cDNA, identification of
cloned genes and mapping of genes, chemical synthesis and analysis of
oligodeoxynucleotides, site-specific mutagenesis, and protein engineer-
ing. Volume 551 includes the description of several useful new restriction
enzymes, detail of rapid methods for DNA sequence analysis, and a num-
ber of other useful methods.
RAY WU
xiii
Contributors to Volume 155
srebmun elcitrA are ni parentheses gniwollof the seman of .srotubirtnoc
snoitailiffA listed era .tnerruc
ASAD AHMED (14), Department of Genetics, tory, Meyerhofstrasse Heidelberg, 6900 ,1
University of Alberta, Edmonton, Al- Federal Republic of Germany
berta, Canada 2E9 G6T PAMELA F. CRAIN (23), Department of Me-
AMY ARROW (15), Biotix Inc., Commerce dicinal Chemistry, College of Pharmacy,
Park, Danbury, Connecticut 01860 ehT University of Utah, Salt Lake City,
A. T. BANKIER (7), MedicalResearch Coun- Utah 21148
cil Laboratory of Molecular Biology, RODERIC M. K. DALE (15), Biotix Inc.,
Cambridge 2BC 2QH, England Commerce Park, Danbury, Connecticut
B. G. BARKELL (7), Medical Research 01860
Council Laboratory of Molecular Biol- LUCIANA DENTE (9), Universitd di Napoli,
ogy, Cambridge 2BC 2QH, England Istituto di Scienze Biochimiche, H Fa-
KIRK BAUMEISTER (11), E. I. du Pont de coltd di Medicina, 13108 Napoli, Italy
Nemours & Company, Inc., Central Re- BERNHARD DOBBERSTEIN (26), European
search and Development Department, Molecular Biology Laboratory, D-6900
Experimental Station, Wilmington, Dela- Heidelberg, Federal Republic of Ger-
ware 89891 many
STEPHEN BECK (18), Medical Research BETH A. DOMBROSKI (33), Department of
Council Laboratory of Molecular Biol- Chemistry, ehT Johns Hopkins Univer-
ogy, Cambridge 2BC 2QH, England sity, Baltimore, Maryland 81212
JUDITH BERMAN (32), Department of Bot- SHLOMO EISENBERG (32), Department of
any, University of Minnesota, Twin Cit- Microbiology, University of Connecticut
ies, St. Paul, Minnesota 80155 Health Center, Farmington, Connecticut
HERMANr~ BUJARD (26), Zentrum far Mole- 23060
kularbiologie, Universitiit Heidelberg, FRED A. FAEOONA (21), Molecular Biology
D-6900 Heidelberg, Federal Republic of Department, Xytronyx, Inc., 6555 Nancy
Germany Ridge Drive, San Diego, California 12129
CHARLES R. CANTOR (28), Departments of REINER GENTZ (26), Central Research
Genetics and Development, College of Units, Hoffman-La Roche and Company
Physicians and Surgeons of Columbia AG, Basel, CH-4002 Switzerland
University, New York, New York 23001 R. S. GOODY (13), Department of Biophys-
GEORGES F. CARLE (29), Department of -eG ics, Max-Planck Institut fiir medizinische
netics, Washington University School of Forschung, Heidelberg, 6900 Federal Re-
Medicine, St. Louis, Missouri 01136 public of Germany
MAIR E. A. CHURCHILL (33), Department of MARIE-THERESE HAEUPTLE (26), European
Chemistry, The Johns Hopkins Univer- Molecular Biology Laboratory, D-6900
sity, Baltimore, Maryland 81212 Heidelberg, Federal Republic of Ger-
RICCARDO CORTESE (9), Uniuersitd di Na- many
poli, lstituto di Scienze Biochimiche, H NAOHIRO HANYU (24), National Cancer
Facoltd di Medicina, 13108 Napoli, Italy, Center Research Institute, Tsukiji ,1-1-5
and European Molecular Biology Labora- Chuo-ku, Tokyo 104, Japan
ix
X SROTUBIRTNOC OT EMULOV 551
STEVEN HENIKOFF (12), Fred Hutchinson P. A. KRIEG (25), Department of Zoology,
Cancer Research Center, Seattle, Wash- University of Texas at Austin, Austin,
ington 98104 Texas 21787
PHiLiP HIETER (22), Department of Molecu- KR6GER MANFRED (1), Institute of Microbi-
lar Biology and Genetics, The John ology and Molecular Biology, Justus-
Hopkins University School of Medicine, Liebig University, D-6300 Giessen, Fed-
Baltimore, Maryland 21205 eral Republic of Germany
DAVID E. HILL (34), Genetics Institute Inc., IKUYIHSOY KUCHINO (24), Biology Divi-
Cambridge, Massachusetts 02140 sion, National Cancer Center Research
GERD HOBOM (1), Institute of Microbiology Institute, TsukUi 5-1-1, Chuo-ku, Tokyo
and Molecular Biology, Justus-Liebig 104, Japan
University, D-6300 Giessen, Federal Re- S. LABEIT (13), National Cancer Research
public of Germany Institute, Department of Cell Biology, Im
Guo-FAN HUNG (8), Shanghai Institute of Neuerheimerfeld, 6900 Heidelberg, Fed-
Biochemistry, Academia Sinica, Shang- eral Republic of Germany
hai 200031, China MICHAEL LANZER (26), Zentrum fiir Mole-
LEROY E. HOOD (19), Division of Biology, kularbiologie, Universitdt Heidelberg,
California Institute of Technology, Pasa- D-6900 Heidelberg, Federal Republic of
dena, California 91125 Germany
HANS-DIETER HUNGER (20), Abteilung Mo- H. LEHRACH (13), Imperial Cancer Re-
search Fund, Lincoln's Inn Fields, Lon-
lekulare Humangenetik, Zentralinstitut
don WC2A 3PX, England
fiir Molekularbiologie, Akademie der Wis-
senschaften der DDR, 1115 Berlin-Buch, DRANOEL S. LERMAN (30, 31), Department
German Democratic Republic of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts
MIHARBI IBRAHIMI (26), European Molecu-
83120
lar Biology Laboratory, D-6900 Heidel-
berg, Federal Republic of Germany F. I. LEWITTER (36), Life Sciences Re-
search Systems, BBN Laboratories Incor-
GABOR L. IGLOI (27), Institut fiir Biologie
porated, 01 Moulton Street, Cambridge,
III der Universitdt Freiburg, D-7800 Frei-
Massachusetts 02238
burg, Federal Republic of Germany
MOT MANIATIS (31), Department of Bio-
RUDOLF JUNG (20), Zentralinstitut far
chemistry and Molecular Biology, Har-
Genetik und Kulturpflanzenforschung,
vard University, Cambridge, Massachu-
Akademie der Wissenschaften der DDR,
setts 02138
4325 Gatersleben, German Democratic
Republic MICHAEL MCCLELLAND (4, 5), Department
of Biochemistry and Molecular Biology,
TREBOR J. KAISER (19), Division of Biology, University of Chicago, Chicago, Illinois
California Institute of Technology, Pasa-
73606
dena, California 52119
D. A. MELTON (25), Department of Bio-
ECNARUAL KAM (33), Department of Chem- chemistry and Molecular Biology, Har-
istry, The Johns Hopkins University, Bal- vard University, Cambridge, Massachu-
timore, Maryland 21218 setts 02138
SALGUOD KOSHLAND (22), Department of TAPAN K. MISR(A1 0), Department of Mi-
Embryology, Carnegie Institution of crobiology and Immunology, University
WashMgton, Baltimore, Maryland 21210 of lllinois College of Medicine, Chicago,
HANS KOSSEL (27), Institut far Biologie III Illinois 60612
der Universitdt Freiburg, D-7800 Frei- LEAHCIM MUELLER (26), Zentrumfiir Mole-
burg, Federal Republic of Germany kularbiologie, Universitdit Heidelberg,
SROTUBIRTNOC OT EMULOV 551 xi
D-6900 Heidelberg, Federal Republic of crobiology, University of Liverpool, Liv-
Germany erpool L69 3BX, England
KARY B. MULLIS (21), Molecular Biology ARI (3, SCHILDKRAUT 6), New England
Department, Xytronyx, Inc., 6555 Nancy Biolabs, Inc., 32 Tozer Road, Beverly,
Ridge Drive, San Diego, California 12129 Massachusetts 51910
RICHARD M. MYERS (31), Department of OKUBON ADAKO-ODNIHS (23), Biology Divi-
Physiology, School of Medicine, Univer- sion, National Cancer Center Research
sity of California at San Francisco, San Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo
Francisco, California 34149 104, Japan
MICHAEL NELSON (5, 6), New England KAREN (30), SILVERSTEIN Department of
Biolabs, Inc., 32 Tozer Road, Beverly, Mathematical Sciences, Memphis State
Massachusetts 01915 University, Memphis, Tennessee 25183
SUSUMU NISHIMUaA (23, 24), National ARDNASSAC L..SMITH (28), Departments of
Cancer Center Research Institute, Tsuk(]i Microbiology and Psychiatry, College of
5-1-1, Chuo-ku, Tokyo 104, Japan Physicians and Surgeons of Columbia
University, New York, New York 23001
C. DAVID O'CONNOR (2), Department of
Biochemistry, University of Southamp- LLOYD M. SMITH (19), Division of Biology,
ton, Southampton S09 3TU, England California Institute of Technology, Pasa-
dena, California 52119
DLONRA R. (34, 35), OLIPHANT Department
of Biological Chemistry, Harvard Medi- (34, 35), STRUHL KEVIN Department of Bio-
cal School, Boston, Massachusetts 51120 logical Chemistry, Harvard Medical
School, Boston, Massachusetts 51120
DRANYAM V. OLSON (29), Department of
Genetics, Washington University School (26), STUEBER DIETRICH Central Research
of Medicine, St. Louis, Missouri 01136 Units, Hoffman-La Roche and Company
AG, CH-4002 Basel, Switzerland
ZE-GUO PENG (16), National Cancer Insti-
SAMOHT D. TULLIUS (33), Department of
tute, Frederick Cancer Research Facility ~
Chemistry, The Johns Hopkins Univer-
Frederick, Maryland 10712
sity, Baltimore, Maryland 21218
FRITZ M. POHL (18), Fakultiitfiir Biologie,
TYE (32), BIK-KWOON Section of Biochem-
Universitiit Konstanz, D-7750 Konstanz,
istry, Molecular and Cell Biology, Cornell
Federal Republic of Germany
University, Ithaca, New York 35841
Bo-QIN (3), QIANG Department of Biochem-
GUIDO (17), VOLCKAERT Rega Institute,
istry and Molecular Biology, Institute of
Faculty of Medicine, University of
Basic Medical Sciences, Chinese Acad-
Leuven, B-3000 Leuven, Belgium
emy of Medical Sciences, Beijing, China
YMEREJ N. B. (2), WALKER Amersham In-
W. P. RINDONE (36), Life Sciences Re- ternational plc, Little Chalfont, Bucking-
search Systems, BBN Laboratories Incor- hamshire HP7 9LL, England
porated, 01 Moulton Street, Cambridge,
K. M. WESTON (7), Medical Research
Massachusetts 02238
Council Laboratory of Molecular Biol-
~IRDNA LAHTNESOR (20), Abteilung Moleku- ogy, Cambridge CB2 2QH, England
lare Humangenetik, Zentralinstitut far
YAR WU (16), Section of Biochemistry, Mo-
Molekularbiologie, Akademie der Wis-
lecular and Cell Biology, Cornell Univer-
senschaften der DDR, 1115 Berlin-Buch,
sity, Ithaca, New York 35841
German Democratic Republic
TREBOR YKSRUGAZ (11), E. I. du Pont de
JANE Z. SREDNAS (19), Division of Biology, Nemours & Company, Inc., Central Re-
California Institute of Technology, Pasa- search and Development Department,
dena, California 52119 Experimental Station, Wilmington, Dela-
JON R. (2), SAUNDERS Department of Mi- ware 89891
]1[ NOITCIRTSER EMYZNE ICigH 3
[1] Restriction Enzyme HgiCI Characterization of the
6-Nucleotide Staggered Cut Sequence and Its Application in
Mismatch Cloning
By
MANFRED KROGER and GERD HOBOM
Thanks to the availability of the rich collections of Drs. Reichenbach 1
and Brown, 2 the gliding bacterium giganteus Herpetosiphon became one
of the most intensively screened groups of organisms in the search for
new restriction enzymes. Among the 01 strains tested, 71 enzymes could
be found with seven different but related recognition sequences. This led
to a hypothesis regarding the evolutionary relationship among these en-
zymes and could be a basis for a better understanding of the biochemical
mechanism of restriction enzyme-DNA target interaction. 3
Among these enzymes HgiCI is remarkably different from all other
previously described endonucleases, since it produces 5'-hexanucleotide
protruding ends. Combined with the fact that HgiCI recognizes a degen-
erated sequence, specific applications of this enzymatic activity in gene
technology are possible. Usually, for specific base pairing within 5'- or 3'-
protruding ends, a match of 2 bp is fair, while four matching base pairs
lead to highly efficient ligase reactions. Since a perfect match of 6 bp may
not be required, we used HgiCI-restricted DNA fragments in order to test
whether DNA ligase reactions among hexanucleotide protruding ends
could proceed in spite of some mismatch positions. Our results presented
here allow the conclusion that it is possible to obtain mismatched ligase
reaction products in considerable fractions. A wider application of this
observation seems possible, since an isoschizomer of HgiCI BanI, is
available commercially and is obtained from an unrelated strain sullicaB
sucityloniruena (IAM 1077). In contrast to the data given in the litera-
ture, 4 we have determined via cross-ligation that BanI also produces 5'-
hexanucleotide protruding DNA fragments.
In this article we intend to focus on the methodology used to charac-
terize recognition sequences and on the application of HgiCI )InaB( frag-
ment ends in mismatch cloning rather than on enzyme purification proce-
dures.
H. Mayer and H. Reichenbach, J. Bacteriol. 136, 708 (1978).
2 N. L. Brown, M. McClelland, and P. R. Whitehead, Gene 9, 49 (1980).
3 M. Kr6ger, G. Hobom, S. Schiatte, and H. Mayer, Nucleic Acids Res. 12, 3127 ~.)4891(
4 I. Schildkraut, cited in R. J. Roberts, Nucleic Acids Res. 12, r167 (1984).
thgirypoC © 7891 yb Inc. Press, Academic
SDOHTEM IN ,YGOLOMYZNE VOL. 551 llA rights of noitcudorper reserved. form any in
4 RESTRICTION ENZYMES [1]
Purification of HgiCI
Herpetosiphon giganteus strain Hpg9 has been obtained from Dr. H.
Reichenbach (Gesellschaft fiir Biotechnologische Forschung, Braun-
schweig-St6ckheim, Federal Republic of Germany). The conditionally
anaerobic strain was grown at ° 30 as described by Mayer and Reichen-
bach. ~ After centrifugation at 24,000 g for 51 min at °, 5 01 g of cells was
used in a standard purification procedure, 5 which involved breaking the
cells through a Branson Sonifier followed by a single centrifugation step
(30 min, 45,000 g). The supernatant was used for column chromatography
on DEAE-cellulose DE-52 (2.6 x 51 cm) without any further treatment.
The appropriate restriction enzyme-containing fraction was obtained
through gradient elution from 0 to 0.3 M NaC1 in l0 mM potassium phos-
phate (pH 7.5), 1 mM EDTA, 0.1% (v/v) 2-mercaptoethanol, and 10%
glycerol. Restriction endonucleolytic activity was assayed for every frac-
tion by incubation with h DNA or some other substrate. Fractions with
identical activities were pooled and dialyzed against the buffer given
above. The dialyzed enzyme solution was rechromatographed on a phos-
phocellulose P11 column (2.6 x 51 cm). NaCI-Dependent elution yields
three different restriction enzymes named according to the order of elu-
tion: HgiCI, HgiCII, and HgiCIII. A more detailed description of the
purification procedure is given by Kr6ger et al. 3
Recognition Sequence Determination
The purified enzymes were used to generate a series of fragmentation
patterns from completely sequenced plasmid DNAs. Incubation was gen-
erally for 2 hr at ° 37 in 01 mM MgCI2 and 01 mM Tris. HC1, pH 7.5. The
patterns obtained after agarose gel electrophoresis usually provided
enough information to distinguish between cleavage reactions already
known and new digestion specificities. Within the H. giganteus strain
Hpg9 (C) we could identify HgiCII as an isoschizomer of AvaII(GGT/
ACC) and HgiCIII as an isoschizomer of SatI (G/TCGAC). However,
HgiCI digestion resulted in an unknown pattern that could be resolvedb y
double digestions with other enzymes as described in detail by Kr6ger et
al. 3 The HgiCI recognition sequence was finally identified as the degener-
ated GGPyPuCC sequence.
Determination of the Cleavage Site for HgiCI and BanI
In principle, two strategies were used to identify the endonucleolytic
cleavage sites relative to the respective recognition sequences: (1) chemi-
v. Pirrotta dna .T .A ,¢lkciB series, this .loV ,56 .p .98
[1] RESTRICTION ENZYME HgiCI 5
cal characterization by determination of the 5' nucleotide(s) plus size
determination of denatured DNA fragments resulting from enzymatic di-
gestion in comparison to a sequencing ladder for that DNA segment, and
(2) mixed ligase reaction between restriction fragments obtained after
cleavage with two different enzymes. The latter procedure is applicable
for any (suspected) isoschizomers or for enzymes producing fragments
with identical cohesive ends. In the case of isoschizomers, additional
confirmation can be provided by recutting the interligation products with
either of the two enzymes.
The chemical characterization of the endonucleolytic cleavage posi-
tion has been applied for HgiCI as the first enzyme discovered with the
recognition sequence GGPyPuCC. An initial determination of the nature
of the 5'-terminal nucleotide for several different HgiCI fragments com-
prising a full representation of the pyrimidine and purine degeneracies at
the two central positions resulted in a G residue as the 5'-terminal nucleo-
tide (93 to 95% pG).
For this determination of the 5'-terminal nucleotide we used paper
electrophoresis, after the unlabeled 5'-phosphate group was changed en-
zymatically into a 32p-labeled 5'-phosphate group using alkaline phospha-
tase and T4 polynucleotide kinase, following the Maxam-Gilbert proto-
col. 6 Usually the 32p-labeling procedure was performed using a mixture of
DNA fragments produced from the same plasmid DNA. In order to obtain
fragments with only a single labeled end, the primary digests were con-
verted into a mixture of subfragments by secondary restriction enzyme
digestion prior to isolation. Only those fragments known to contain a
single 32p-labeled HgiCI end were isolated and treated further to identify
the labeled nucleotide. Each fragment was digested completely into
mononucleotides within a volume of 30/zl containing 01 mM Tris (pH 8.5)
and 01 mM MgCI2 plus 1/zg DNase I and 1 ~g snake venom phosphodies-
terase for 1 hr at 37 .° Then 40/zl of a mixture of unlabeled mononucleo-
tides (about 20 mg/ml each) was added as cartier to achieve optical visibil-
ity. As described by Kr6ger and Singer 7 the reaction mixture was applied
onto Whatman 3MM paper and the mononucleotides were separated in a
Savant paper electrophoresis system using 0.12 M ammonium formate
buffer, pH 3.5 (2.1 g ammonium formate and 3.3 ml formic acid/liter). The
four mononucleotide spots observable on the dry paper sheet were cut out
under UV light and were used directly for measuring their p23 activities.
The ordero f separation at pH 3.5 was pC, pA, pG, and pT. Application of
a high-performance liquid chromatography (HPLC) separation technique
may be recommended as a more modern alternative, especially since the
A. M. Maxam and W. Gilbert, this series, Vol. 65, p. 499.
7 M. Krrger and B. Singer, yrtsimehcoiB 18, 3493 (1979).
6 NOITCIRTSER SEMYZNE [1]
Savant paper electrophoresis requires huge amounts of inflammable pe-
troleum.
Since this result does not lead to an unambiguous interpretation for the
cleavage position, due to the two G residues within the GGPyPuCC
HgiCI recognition sequence, a second identification procedure had to be
applied.
For molecular-weight determination, use has been made of an HgiCI
cleavage product gel electrophoretic sizing against a DNA sequencing
ladder of a DNA fragment which contains within its known sequence a
single cleavage site for HgiCI. For this purpose endonucleolytic cleavage
by HgiCI prior to isolation of the desired subfragment was used on part of
the terminally labeled material, while the main fraction was converted
into a DNA sequencing ladder following the conventional Maxam-Gilbert
procedure. The HgiCI cleavage product(s) of the same fragment were
loaded in a fifth lane of the sequencing gel. To ensure identical ionic
conditions when all five samples are applied onto the acrylamide gel, the
HgiCI endonucleolytic digest had been subsequently treated with phenol
to remove all of the protein, and precipitated with alcohol in the presence
of tRNA.
For the correct assignment of the resulting electrophoresis pattern, it
is necessary to take into consideration the presence of a phosphate group
at the 3' end of each of the chemical fragmentation products. Endonu-
cleolytically derived fragments, however, contain free 3'-OH groups.
This results in a shift in electrophoretic mobility as shown in Fig. .1 The
given assignment indicates that restriction enzyme-generated fragments
migrate a shorter distance than the 3'-phosphorylated counterparts of
identical chain length. As a control experiment a similar fragmentation
reaction has been performed for the well-established Sau3A endonu-
cleolytic cleavage site, as is shown in the lower part of Fig. .1 Here we
used a DNA fragment with both ends labeled, a smaller part of which was
digested with Sau3A, while the main part was used for strand separation
on a denaturating acrylamide gel 6 and for DNA sequencing. Thus sizing
was possible using two Maxam-Gilbert sequencing ladders for both com-
plementary cleavage products, although only an unseparated fragment
mixture was used after Sau3A digestion. In light of the two results we
were able to show by this procedure that HgiCI produces a hexanu-
cleotide staggered cut with an extended 5' terminus, the first enzyme to be
observed with this fragmentation characteristic.
Mixed ligase reactions are a quick and easy method to characterize an
endonucleolytic cleavage reaction, if a suspected isoschizomer is avail-
able. We have applied this technique for the AvalI/HgiCII cleavage site
and used gel electrophoretic separation to identify the multiple ligation
[1] NOITCIRTSER EMYZNE ICigH 7
HgiCI
CT.CA G E
,CACTACG-
- ~CACTACG HoCCACTACG-
~CACTACG-
- pACTACG -- ---~
-GCATC- / ,i.,
-GCATp ~
pACG- ~ i
-GCp ~ ~
-Gp ~ -- im
Sau3A
G AT.CC E C ToCA G E
- GTGGATTG ~ ~" ~ I
-GTGGATTp ,..~ ,..~
- GTGGATp. ,.~
GTGGAp - GTGGATToH - I
pGTG- ___~__.~-- ~ ~ I j j I
-% ------------~" I I .. -CTGo"
J
FIG. 1. Determination of cleavage sites for endonucleases ItgiCI and Sau3A through gel
electrophoretic sizing of cleavage products. Three Maxam-Gilbert DNA sequencing ladders
are produced, each serving as a molecular-weight marker series for the respective fragment
cleaved either by HgiCI (upper panel) or by Sau3A (lower panels). The terminal fraction of
the DNA sequences corresponding to the autoradiographically visible bands is notified at the
left margin. The main structural difference between the chemically and the endonucleolyti-
caily cleaved products is the absence of the 3'-phosphate group from the fragment in the E
lane (E = enzyme treated). In all three cases this leads to an electrophoretic mobility
decreased by almost one unit for a pair of the otherwise identical fragments.
products in an agarose gel system (for details, see Ref. 3). An alternative
to tracing the (multiple) reaction products on gels is provided by determi-
nation of cloning frequencies for the respective (co)ligation products. This
technique was applied in a coligation analysis for BanI/HgiCI cleavage
sites. Both enzymes have been reported to recognize the same degener-
ated sequence GGPyPuCC, but BanI cleavage reaction has been de-
scribed to result in a four-nucleotide rather than a six-nucleotide stag-
gered cut as observed for ngiCI. 8'4 We wanted to redetermine the
8 H. Sugisaki, Y. Maekawa, S. Kanazawa, and M. Takanami, Nucleic Acids Res. 10, 5747
(1982).
8 RESTRICTION ENZYMES [1]
cleavage position for the endonuclease BanI relative to HgiCI and have
used a mixed ligation procedure as outlined in Fig. 2.
In order to provide an easier characterization of the resulting clones
we chose two different but closely related plasmids with two HgiCI/BanI
sites within two regions of identical sequence. One BanI/HgiCI recogni-
tion site was located within the ampicillin resistance gene, thus clones
were expected only after successful ligation. The replication function was
supplied by one HgiCI fragment only, and the complementing BanI frag-
ment contained two extra landmark restriction sites (SphI and SnaBI).
(For experimental details, see Fig. 2.) The cloning yield was excellenta nd
all 21 clones analyzed showed the correct restriction pattern for the calcu-
lated coligation product, using SphI and BanI for characterization. Thus
BanI and HgiCI cleave at identical positions and are true isoschizomers.
Eco., .<-,...2 .,n0,,,
®".n
CCGTGGI~GGA*CCGCGG~
FIG. 2. Cloning strategy used for a mixed ligase reaction between fragments derived from
HgiCI-restricted plasmid pHK255 and the closelyr elated BanI-restricted plasmid pHK402.
Plasmid pHK255 is digested first with EcoRI and HindlII and subsequently treated with
alkaline phosphatase to remove 5'-phosphate groups from these termini, and the mixture is
finally cleaved with HgiCI. The resulting four fragments are without isolation, mixed, and
coligated with the isolated BanI fragment, which contained no origin of replication. Using
ampiciUin selectiont he coligation product plasmid pHK422 was obtained exclusively (12 out
of 21 clones analyzed) and in high yield. Both analysis by SphI dna BanI digestion led to two
fragments each (the third BanI/HgiCI site is methylated in Escherichia coli and thus is not
cleaved), as expected for the given map of pHK422.