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A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments
Gene, 19 (1982) 269-276 Elsevier Biomedical Press
269
A new pair of Ml3 vectors for selecting either DNA strand of double-digest (Single-stranded strategy)
DNA phage; inserts with controlled
orientation;
res~iction
fragments
multiple cloning sites; sequencing
Joachim Messing and Jeffrey Vieira Depurtment of Biochemistry, University of Minnesota, St. Paul, MN 55108 (U.S.A.) (Received April 5th. 1982) (Accepted July 12th, 1982)
SUMMARY
The strategy of shotgun cloning with Ml3 is based on obtaining random fragments used for the rapid accumulation of sequence data. A strategy, however, is sometimes needed for obtaining subcloned sequences preferentially out of a mixture of fragments. Shotgun sequencing experiments have shown that not all DNA fragments are obtained with the same frequency and that the redundant information increases during the last third of a sequencing project, In addition, experiments have shown that particular fragments are obtained more frequently in one orientation, allowing the use of only one of the two DNA strands as a template for Ml3 shotgun sequencing. Two new Ml3 vectors, Ml3mp8 and M13mp9, have been constructed that permit the cloning of the same restriction fragment in both possible orientations. Consequently, each of the two strands becomes a (t) strand in a pair of vectors. The fragments to be cloned are cleaved with two restriction enzymes to produce a fragment with two different ends. The insertion of such a fragment into the vector can occur only in one orientation. Since M 13mp8 and M 13mp9 have their array of cloning sites in an antiparallel order, either orientation for inserting a double-digest fragment can be selected by the choice of the vector.
INTRODUCTION
A number of techniques have been used for the preparation of single-stranded DNA. These include poly(UG)-CsCl gradients (see review by Szybalski et al., 1971), alkaline CsCl gradients (Vinograd et al., 1963), polyacrylamide gels Abbreviations: kb, kifobase pairs; PEG, polyethylene glycol: RF, double-stranded replicative form; SS, single-stranded viral form. 0378- 1I 19/82/~0-~0/$02.75
0 1982 Elsevier Biomedical Press
(Maxam and Gilbert, 1977; Szalay et al., 1977), and exonuclease treatment (Smith, 1979). An alternative biological approach has been developed involving the bacteriophage M13. The RF of the phage DNA is a circular double-stranded molecule; it can be isolated from infected cells, used to clone DNA fragments, and reintroduced into Esch~richi~ co/i cells by transfection. The infected cells extrude the Ml3 phage particles, each of which contains a circular SS DNA molecule. Large amounts of SS DNA containing a cloned
270
insert
(5-10
pg phage
SS DNA/ml
ture) can be easily and rapidly et al., 1977; Barnes, Ohsumi
bacterial
obtained
1978; Ray and Kook,
et al., 1978; Hermann
cul-
{Messing 1978;
et al., 1978; Nomura
et al., 1978). The cloning
of DNA
the M 13mp7 cloning
gene)
and was
addition,
vehicle
1978; Messing,
et al., 1981). A fragment (the promoter In
into the RF of
by a series of improve-
and Messing,
galactosidase genome.
fragments
facilitated
ments which produced (Gronenborn Messing
1979;
of the E. eoli Iac
N-terminus inserted
a small
into DNA
of the @the
ified
The synthesized
DNA
(Langley fragment
et
al.,
contains
1975; two
sites each for the EcoRI, BarnHI, SalI, AccI, and IJinciI restriction enzymes arranged symmetrically to a centrally
located
and
Ml3mp9
restriction
orientations by
guarantees
forced
fragment
the clones
the (-t-) strand and
arranged
in
to the M 13 genome.
cloning.
that each strand
will become
have this mod-
site region
relative
a given restriction
orientated
that has
can be directly This
procedure
of the cloned
fragment
in one or the other of
thus be extruded
as SS DNA
in
phage particles.
Ml3
ment. In spite of these insertions the Ml3mp7 DNA is still infective and the modified luc DNA is able to encode the synthesis of a functional /3galactosidase cu-peptide Messing et al., 198 1).
Thus,
Ml3mp8
multiple
vehicles
with the same pair of restriction
fragment
synthesized in vitro and containing an array of restriction cleavage sites was inserted into the structural region of the P-galactosidase gene frag-
with respect
enzymes. opposite
Ml 3 has been
operon
to one of these new Ml3 cloning also been “cut”
Pst I site. Thus,
MATERIALS
AND
METHODS
(a) Strains Two new plasmids, pUC8 and pUC9, have been constructed from the pBR322 plasmid and the Ml3mp7 vector (Bolivar et al., 1977; Sutcliffe, 1979; Messing et al., 1977, 1981; Ruther, 1980; Vieira and Messing, 1982). The 2297-bp EcoRI/ PuuII fragment of pBR322 has had its PstI and HincII sites removed by single base pair changes without loss of /?-lactamase activity. The AccI site in this fragment has been removed by cleavage
by chance either strand of a cloned restriction fragment can become part of the viral (-I-) strand. This depends on the fragment’s orientation relative to the Ml3 genome after they have been joined by ligase. The insertion of a DNA fragment into one of these restriction sites is readily monitored because the insertion results in a nonfunctional a-peptide and the loss of P-galactosidase
and treatment with BAL31 (Legerski et al., 1978). The 433-bp iac Hue11 fragment of Ml3mp7 RF containing the multiple restriction sites was then inserted into this modified pBR322 fragment to yield the plasmid pUC7. The nudeotide sequence containing the multiple restriction sites was then
activity.
modified
Under
appropriate
plating
conditions,
the
to contain
the following
restriction
sites
functional a-peptide results in blue plaques; a nonfunctional a-peptide results in colorless plaques
ordered in the same polarity as lac transcription: EcoRI, XmaI, SmaI, BarnHI, WI, AccI, HincII,
(Messing and Gronenborn, 1978). Ml3mp7 has found wide application in the dideoxy nucleotide
PstI, and HindHI. A second modified
sequencing procedure (Sanger et al., 1977). This paper reports the construction of two new SS DNA bacteriophage vectors, Ml3mp8 and Ml3mp9, and their applications to DNA sequencing and strand-sp~ifi~ hyb~dization. The nucleotide sequence of Ml3mp7, containing the multiple restriction sites, has been modified to have only one copy of each restriction site and in addition, single HindIII, SmaI, and XmaI sites. Thus, DNA fragments whose ends correspond to two of these restriction sites can be “forced cloned” by ligation
site was used to produce the plasmid pUC9. This plasmid contains the multiple restriction sites of pUC8 in a reverse orientation relative to the lat promoter. A detailed description of these plasmids, their construction, and their uses are presented in Vieira and Messing (1982). (b) Construction
the
Ml3mp7 Hue11
This plasmid is termed pUC8. form of the multiple restriction
of M13mp8 and M13mp9
RF DNA was partially cleaved with enzyme (all enzymes were obtained
271
from the Bethesda England
Research
Biolabs)
fied by gel electrophoresis (Heidecker
Laboratory
and the linear
et al.,
and eluted
1980).
and New
forms were puri-
This
DNA
from the gel was
then
cleaved with the E’coRI enzyme to produce phage DNA molecules with HaeII and EcoRI ends. The lac HaeII
modified pUC8
DNA
EcoRI
and PuuI.
lac HaeII
EcoRI
with HaeII
and
Cleavage
fragment
and HaeII
fragments
fragment
was excised
from
also digested
with
at the EcoRI site of the
produces ends. PuuI
two fragments
with
cleaves one of these
and blocks its later incorporation
into a
vector with EcoRI and HaeII ends. The linearized Ml3mp7 DNA was mixed with the triple-enzyme-digested DNA from pUC8 and joined with ligase. This leads to the “forced” insertion of the EcoRI-HaeII subfragment of the modified fat HaeII fragment. The DNA products of the ligation reaction were used to transform competent E. coli JM103 cells (Messing et al., 1981). Candidates for properly constructed vectors were identified as blue plaques. These isolates were saved for further characterization and designated according
to their plasmid
A similar procedure M 13mp9. In this case prepared
as before.
origin as M 13mp8. was used to construct the M13mp7 DNA was
The modified
lac HaeII
frag-
ment was excised from pUC9 DNA with HaeII and digested with EcoRI and HinfI. HinfI cleavage blocks the later incorporation of one of the EcoRI-HaeII subfragments. The restriction-enzyme-digested phage and the pUC9 DNAs were joined by ligase and used to transform competent JM103 cells. Blue plaques characterization.
were picked
for further
after about cleared
30 min at room temperature
by centrifugation
B. The supernatant pipet avoiding
was removed
any damage
ual fluid was removed was resuspended mM NaCl,
in a Beckman
with a Kimwipe.
After another
again by adding
by a short centrifugation
Microfuge
B. The pellet
again in low Tris buffer
of 300 ~1 and extracted
was then
in a volume
twice with an equal volume
of phenol-chloroform.
was precipitated
100
and 100 ~1 of 5 M
30 min at room temperature
the phage was collected
of a 1: 1 mixture
(10
10 mM Tris . HCl pH
7.5). Phage were precipitated
resuspended
The pellet
in 800 ~1 of low Tris buffer
1 mM EDTA,
in a Beckman
with a Pasteur
to the soft pellet; resid-
~1 of a 20% PEG 6000 solution NaCl.
and was Microfuge
with ethanol
from
The DNA the aqueous
phase, washed once with ethanol, taken up in 10 ~1 of low Tris buffer, and used directly to provide template for the DNA sequencing reaction with chain terminators (Sanger et al., 1977). (d) Primer and DNA sequencing An aliquot from the template solution (1 ~1) was annealed with the synthetic master primer and subjected to the chain termination reaction (Sanger et al., 1977) as described previously (Messing et al., 1981; Gardner
RESULTS
AND
et al., 1981).
DISCUSSION
(a) Scheme of forced cloning Restriction
endonuclease
cleavage
fragments
(c) Preparation of templates
with non-complementing joined during a ligation
Templates for DNA sequencing reactions with chain terminating inhibitors (Sanger et al., 1977)
with two different restriction endonucleases and a fragment with two non-complementing ends is
were prepared as follows. Infected cells from a blue plaque were diluted into 1.5 ml of 2 YT(Miller, 1972), mixed with about 10’ non-infected log phase JM103 cells (Messing et al., 1981), and grown for 8 h at 37’C. The infected cells were sedimented by a short centrifugation and the phage in the supernatant were concentrated by the addition to 1.3 ml of the supernatant of 200 ~1 of 27% PEG 6000 (Sigma) in 3.3 M NaCl. The mixture became turbid
produced, the resulting fragment can be neither circularized nor joined to another fragment in both orientations during a ligation reaction. Consequently, two “double-digest” fragments can be forced to form a chimeric molecule under the appropriate ligation conditions. In addition, a recombinant molecule is formed during the ligation
ends are rarely, if at all, reaction. If DNA is cleaved
reaction with the defined order of the two fragments. Since the orientation of a cloned DNA
272
9
x
“PI
Lo
BamKI
1
Hind Ill
Fig. 1. Scheme of forced cloning. Restriction cleavage sites used as cloning sites are unique and located in a region of the circular vector molecule convenient for the insertion of foreign DNA. The DNA sequencewhich has been selected for cloning is cut out of its original DNA with the use of two restriction endonucleases. In the described example the enzymes used are BumHI and HindHI. The DNA fragment produced has two different cohes;ve ends which cannot join in a DNA ligation experiment. The vector molecule is treated with the same enzymes and the same type of cohesive ends are produced. Both DNA fragments, cannot
form a circular
the formation
the vector molecule
of a circular
and the DNA to be cloned
themselves
chimeric
and therefore
molecule,
which
favor
cloning into M13mp7, the symm~t~ around the PstI site of M13mp7 has been replaced by an asymmetric array of sites. The two resulting orders of sites have been produced on two different phage molecules so that both strands of a DNA fragment can serve as the viral strand. In addition, IlindIII. XmaI and SmaI have been added as new cloning sites. The latter serves as a second blunt end cloning site in the case that the I-fincII site cannot be used. A detailed cloning guide which illustrates the combinatorial variety of tobesive and blunt ends produced by a large number of different restriction endonucleases has been described elsewhere (Messing and Seeburg, 198 1). The engineering of this segment of lac DNA has been conducted with pUC7 which contains the luc region of M13mp7 on a plasmid vector as outlined in MATERIALS AND METHODS. The modified cloning sites from the two plasmid vectors pUC8 and pUC9, which contain the cleavage sites in tbe two possible orientations, were transferred to M 13mp7 as described in ~AT~RIA~~ AND METI-IODS. (c)
DNA sequence
determination
for both DNA
strands by the same primer
can be
for by the transformation of competent cells. The location oF the two ctoning sites determines bow the cloned DNA is oriented within the vector molecule.
selected
fragment in the RF of Ml3 vectors determines which of the two DNA strands is going to be the viral strand, this procedure allows the direct preparation of one of the two DNA strands by cloning. Fig. 1 illustrates this scheme when the DNA to be cloned is cut out with Hind111 and BumHI. In addition to the strand separation, a “double-digest” restriction fragment with two non-cornpIementing ends is easiry seIected from DNA fragments in the pool that have been generated by cleavage with only one of the two enzymes. (b) Modification of the Ia& gene
To avoid the random selection of either strand of an insert fragment arising from the use of
A blue plaque from both transfer experiments has been retained and single-stranded DNA prepared as described in MATERIALS AND METHODS. Using the synthetic master primer (Messing et al., 1981 f. sequencing reactions with chain terminators @anger et al., 1977) have been performed and analyzed by polyac~lamide gel electrophoresis. The pertinent sequence containing the cloning sites can be read from the autoradiogram in both orientations (Fig. 2). The primary structure of the Z gene of Ml3mp7 is compared with the structures from the two new isolates which are named M13mp8 and Ml 3mp9 (Fig. 3). Both strands of the insert have been sequenced with the same primer because of the reverse polarity of the synthetic ~c~~I/~i~dIII insert in Ml?mp8 and Ml 3mp9. Therefore, every *‘double-digest” restriction fragment cloned in both vectors will provide both strands of the restriction fragment directly as template which can be sequenced with the same primer.
273
(d) Phenotypic
compa~son
of the different
IacZ
sequences The addition region
of the synthetic
of M13mp2
1978) leads to the synthesis with
the insertion
cloning
to the lac
and
Messing,
of a modified
a-peptide
of 11 amino
M 13mp8 and M 13mp9 compared (Messinget
DNA
(Gronenborn
acid residues
to 14 in Ml 3mp7
al., 1981) and 19 in M13mp5,
phage
polypeptides
(Messing,
a IIindIII
1979). In all cases
were functional
in
the
in a-complementa-
tion and gave rise to blue colored
plaques
under
the appropriate plating conditions (Messing et al., 1977). We have noted that the insertion of these small tion
synthetic test
DNAs
to different
affects degrees,
the complementaand
the
various
M13mp phage molecules can be differentiated by the intensity of the blue color of their plaques. Indeed, the new phage vectors produce a more intense color than Ml3mp7. Moreover, the sequencing analysis of this different 1acZ sequence causes a different degree of secondary structure affecting compression in the gel electrophoresis (not shown). As expected, M13mp7, with a perfect symmetry, shows the highest degree of compression followed by M 13mp5. M 13mp8 and M 13mp9, however,
do not give an indication
for compres-
sion as shown in Fig. 2. (e) “Double-digest” quencing
cloning in shotgun DNA se-
Since all the constructions are based on the use of a master primer (Heidecker et al., 1980) any insert can be conveniently copied by DNA polymerase. Using chain terminators 1977) the sequence of the DNA
(Sanger et al., can be deduced
from selected templates. The shotgun sequencing method, for instance, leads to a less efficient effort Fig. 2. DNA
sequence
analysis
of M13mp8
and MI3mp9.
M13mp8 and M13mp9 have been constructed MATERIALS
AND METHODS.
as described
in
Starting from a single plaque
template from both phage vectors were prepared and a primer extension
reaction in presence of chain terminators was carried
out as described in MATERIALS
AND METHODS.
products were loaded on a 8% polyacrylamide
Reaction
gel in the GATC
order and the sequence was read from the Hue111 site (bottom) in the position of the 8th amino acid residue of the Z gene to the ATG
start codon.
with M13mp7 in Fig. 3.
The resulting sequences
are compared
in determining the last 10-20X of the nucleotides of a contiguous sequence (Gardner et al., 1981). A direct way of obtaining missing sequences of both strands is the use of selective cleavage and forced cloning into the RF of M 13mp8 and M 13mp9. If these resulting clones, and therefore the gaps between two blocks of sequences, are too large to be sequenced with one primer extension reaction these clones in turn can be used as master sequences to identify smaller templates from both strands within
274
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Fig. 3. A genetic in respect detail
by the nucleotide
underlined inserted
and labeled.
sequence
M13mpS
charge
marker,
pUC7,
of the pertinent
from Bethesda
Research
TP
ALA
GFT
GCA Pst
region
of the wild-type
is set up and numbered. pUC8
AL4
III
and Ml3mp9.
and pUC9 Laboratory
10
Ii
LEU
ALA
AGC
TT;G GCA
7
8
LEU CTG
k-?
ALA GCC
k?
nindm
I
GLY
ARG ARG
9
10
11
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FRO
GLY
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y
ATC
7
8
ASN
SER
LEU
ALA
C,CC GG,G AAT
The HueII-cleavage
TCA
CTG
GCC
acid sequence
The same fat cloning
as described
6
sites of the three different
of the IucZ gene. The recognition amino
5
in Ml3mp2
between
AND
vectors
sites for restriction (Gronenborn
METHODS.
have been mapped
the three molecules
sites exist also on the pBR322
in MATERIALS
Absm
ECORI ‘
LS;i--iBW
et al., 1977). Map units are given in kb. The difference
The interruption
amino acid sequences
as a selection without
map of M13mp7,
to the genetic map (Messing
CC?
9 SER
AccI HincIt
LEU
AGC
Hind
PRO
CM
12345676 12 THR
kzl?
Jpstl
and Messing, derivative
All strains
is given in
endonucleases
are
1978) by the
with ampicillin
are made
available
upon request.
pool of the different shotgun clones obtained in the first cloning experiment. Using a new procedure of making Ml3 probes and a dot hybridization procedure, a library of Ml3 shotgun clones can be rapidly screened for statistically underrepresented templates (Hu and Messing, 1982). the
(f) Comparative sequencing, transcriptional ping, and in vitro mutagenesis
map-
The same template selection can be used to compare mutant sequences. Once the primary
structure of a DNA sequence is known its genetic structure has to be determined by comparison of the primary structure with mutant sequences. Since this requires the comparison of a small part of the entire sequence, the appropriate doubly digested fragment of interest can be used to readily obtain mutant sequences in template form in M13mp8 and M13mp9. Defining the orientation of a cloned fragment in M13 is very useful in at least two other experimental procedures; first, for making single-strand specific hyb~dization probes (Hu and Messing,
275
1982), second for in vitro mutagenesis. Singlestrand specific hybridization probes are helpful in determining the strand polarity of inserts in other Ml3 viral DNAs or for physically mapping transcripts. If site-specific changes in a particular nucleotide sequence are planned by using a synthetic primer as a mutagen (Smith and Gillam, 198 1). it may be useful to have the extension of the mutagen primer carried out in the absence of the complementary strand. A discussion of an integrative strategy of shotgun DNA sequencing and exploration of gene structure has been described in more detail elsewhere (Messing, 198 1; 1982).
Hu, N. and Messing, probes.
Gene
Langley,
J.: The making
of strand
specific
Ml3
17 (1982) 271-277.
K.E., Villarejo,
M.R.,
A.V., Zamenhof,
Fowler,
I.: Molecular
mentation.
Proc. Nat]. Acad. Sci. USA 72 (197.5) 1254- 1257.
Legerski.
R.H..
Hodnett,
basis of fl-galactosidase-a
P.J.
and Zabin,
J.L. and Gray,
nucleases
of Pseudomonas
stranded
deoxyriboexonuclease
convenient
method
produced
BAL31,
for
by cleavage
H.B.:
comple-
Extracellular
III. Use of the double-
activity
as the basis
mapping
of fragments
with restriction
enzymes.
of a
of DNA Nucl. Acids
Res. (1978) 144% 1463.. Maxam.
A. and Gilbert,
W.: A new method
for sequencing
DNA. Proc. Nati. Acad. Sci. USA 74 (1977) 560-564. Messing,
J.: A multi-purpose
single-stranded DNA
DNA
Technical
cloning
system
bacteriophage
Bulletin,
NIH
based
M13.
on the
Recombinant
Publication
No. 79-99,
2,
No. 2 (1979) 43-48. Messing,
J.: M13mp2
system ACKNOWLEDGEMENTS
for DNA
and
and in vitro mutagenesis, ings of the Third
This work was supported by the Minnesota Experiment Station, grant No. MN 15-030, and a grant No. DE-AC02-81 ER 10901 from the Department of Energy. We thank Gisela Heidecker for her stimulating discussions and Jim Fuchs, Perry Hackett, Irwin Rubenstein, Kris Kohn and Bonnie Allen for their aid in preparing this manuscript.
Messing, @is.),
beyond,
Genetic
4. Plenum, Messing,
Engineering,
Insertion
of a Hind11
Harbor, W.M.:
DNA
sequence
from the histidine
Seven histidine
codons
in a row.
operon
con-
Proc.
Nat].
F.,
Rodriguez,
Heynecker,
and characterization
II. A multi-purpose R.C.,
Shepherd,
cloning
Howarth, R.J. and
sequence
Greene.
P.J.,
H.L., Boyer, H.W., Crosa,
Construction Gardner,
R.L.,
A.J., Messing,
of an infectious
by M 13mp7 shotgun
Betlach,
S.:
‘vehicles,
Gene 2 (1977) 95-113.
Hahn,
P., Brown-Luedi,
J.: The complete
clone of cauliflower
sequencing.
M.C.,
J.H. and Falkow,
of new cloning
system.
M.,
nucleotide mosaic
virus
Nucl. Acids Res. 9 (1981)
DNA
B. and Messing, J.: Methylation in vitro
cleavage
sites. Nature
Heidecker, primer
G.. Messing, for DNA
tem. Gene Hermann,
introduces
of single-stranded
restriction
endonuclease
J. and
sequencing
Gronenbom,
B.: A versatile
in the M13mp2
cloning
sys-
B.: The filamentous
Transposition
K., Zentgraf,
of DNA
sequence
into the single-stranded
fd. Mol. Gen. Genet.
H. and Schaller,
determining genome
I53 (1978) 171-178.
H.:
kanamycin
of bacteriophage
vehicle: region
J., Crea, R. and Seeburg,
Cold Spring
and Cellular
P.H.: A system for shotgun
P.H.: A strategy
in Brown,
using Purified
Genes,
D. (Ed.), ICN-UCLA
Biology,
for high speed DNA
Developmental Symposia
Biology
on Molecular
Vol. XXIII.
Academic
in Molecular
Genetics.
Press,
New
1981, 659-663.
Miller, J.H.: Harbor
Experiments Laboratory,
Cold Spring Harbor,
N., Yamagichi,
acterization Ohsumi,
Laboratory,
Nucl. Acids Res. 9 (1981) 309-321.
J. and Seeburg,
sequencing,
Nomura,
Ml3
D. and Ray, D.S. (Eds.), The Single-stranded
DNA sequencing.
York,
phage
fusions in vitro, in Denhardt.
coliphage
Virology
Id carrying
N.D.:
fl
and
a kana-
The isolation
of an in vivo recombinant bacteriophage
and char-
I.
gene. Gene 3 (1978) 39-5
M., Vovis, G.F. and Zinder,
characterization
Cold Spring
NY, 1972.
H. and Oka. A.: Isolation
of transducing
between
the plasmid
and the
pSCIO1.
89 (1978) 438-449.
Ray, D.S. and Kook, Y.: Insertion the genome of the single-stranded
10 (1980) 69-73.
as a cloning
NY, 1978, pp. 449-453.
filamentous
272 (1978) 375-377.
R., Neugebauer,
resistance
new
Vol.
form in vitro. Proc. Natl. Acad. Sci. USA
DNA for operon
mycin resistance
287 I-2888. Gronenbom,
Messing, Messing,
Acad. Sci. USA 75 (1978) 4261-4285.
and Methods.
of the iac regulatory
DNA Phages. Cold Spring Harbor
trol region:
J.K.
B. and Hofschneider,
Ml3
fragment
J. and Gronenborn,
D.T.. Dressier,
Bolivar,
Principles
and
Setlow,
74 (1977) 36423646. as a carrier
REFERENCES
sequencing
A. and
1982, pp. 19-36.
coliphage
in Ml3 replicative
of DNA
B., Muiler-Hill,
Filamentous
on Macromole-
1981. pp. 143- 153. strategy
New York,
cloning
hybridization,
A.G. (Ed.), Proceed-
Symposium
in Hollaender,
.I., Gronenborn,
P.H.:
specific
in Walton,
Cleveland
J.: An integrative
experiments
A molecular
strand
cules. Elsevier, Amsterdam,
Messing,
Barnes,
derivatives:
sequencing,
of the Tn3 transposon
into
DNA phage M13. Gene 4
(1978) 103-I 19. Ruther,
U.:
Construction
vehicle, allowing Mol. Gen. Genet.
and
properties
direct screening
of a new cloning
for recombinant
1978 (1980) 475-477.
plasmids.
276
Sanger,
F., Nickien,
S. and Cot&en.
with chain-terminating
A.R.:
inhibitors.
Proc.
DNA sequencing
derived
Natl.
with synthetic
Acad.
Sci.
Vinograd,
USA 74 (1977) 5463-5467. Smith, A.J.H.:
The use of exonuclease
stranded
DNA for use as a template
tion sequencing
methods.
Nucl.
HI for preparing
single-
in the chain
Acids
termina-
system
J., Morris.
buoyant
co/i plasmid
pBR322.
nucleotide
sequence
Cold Spring
J., Davidson,
Gene
and
sequencing
19 (1982) 259-268.
N. and Dove, W.F.:
of viral and bacterial
DNA
The
in alkaline
CsCl. Proc. Nat]. Acad. Sci. USA 49 (1963) 12-17. Communicated
J.G.: Complete
behavior
mutagenesis
primers.
Res. 6 (1979) 831-
848. Sutcliffe.
for insertion universal
by Z. HradeEna.
of the &cberichia
Harbor
Symposium
43
Note added in proof
( 1979) 77-90. Szalay,
A.A., Grohmann,
of the complementary acrylamide Szybalski, tary
DNA
New York, J. and
of DNA
H., HradeEnl,
and preparative strands,
(Eds.). Methods Vieira,
strands
R.L.: Separation fragments
on poly-
gels. Nucl. Acids Res. (1977) 1563-1578.
W., Kubinski,
Analytical
K. and Sinsheimer
2. and Summers,
separation
in Grossman,
in Enzymology,
W.C.:
of the complemenL. and
Moldave,
Vol. 21D. Academic
K.
Press,
1971, pp. 383-413. Messing,
J.: The pUC
plasmids,
a M13mp7
In the meantime, a new Ml3 vector pair has been constructed. They have two additional cloning sites for SstI and X&z1 in their polylinker. ~13mplO is derived from M13mp8, and M13mpll from Ml3mp9, except that Ml3mp 11 does not contain the extra piece of pBR322 sequence as one of the isolates of M13mp9 does.
269
A new pair of Ml3 vectors for selecting either DNA strand of double-digest (Single-stranded strategy)
DNA phage; inserts with controlled
orientation;
res~iction
fragments
multiple cloning sites; sequencing
Joachim Messing and Jeffrey Vieira Depurtment of Biochemistry, University of Minnesota, St. Paul, MN 55108 (U.S.A.) (Received April 5th. 1982) (Accepted July 12th, 1982)
SUMMARY
The strategy of shotgun cloning with Ml3 is based on obtaining random fragments used for the rapid accumulation of sequence data. A strategy, however, is sometimes needed for obtaining subcloned sequences preferentially out of a mixture of fragments. Shotgun sequencing experiments have shown that not all DNA fragments are obtained with the same frequency and that the redundant information increases during the last third of a sequencing project, In addition, experiments have shown that particular fragments are obtained more frequently in one orientation, allowing the use of only one of the two DNA strands as a template for Ml3 shotgun sequencing. Two new Ml3 vectors, Ml3mp8 and M13mp9, have been constructed that permit the cloning of the same restriction fragment in both possible orientations. Consequently, each of the two strands becomes a (t) strand in a pair of vectors. The fragments to be cloned are cleaved with two restriction enzymes to produce a fragment with two different ends. The insertion of such a fragment into the vector can occur only in one orientation. Since M 13mp8 and M 13mp9 have their array of cloning sites in an antiparallel order, either orientation for inserting a double-digest fragment can be selected by the choice of the vector.
INTRODUCTION
A number of techniques have been used for the preparation of single-stranded DNA. These include poly(UG)-CsCl gradients (see review by Szybalski et al., 1971), alkaline CsCl gradients (Vinograd et al., 1963), polyacrylamide gels Abbreviations: kb, kifobase pairs; PEG, polyethylene glycol: RF, double-stranded replicative form; SS, single-stranded viral form. 0378- 1I 19/82/~0-~0/$02.75
0 1982 Elsevier Biomedical Press
(Maxam and Gilbert, 1977; Szalay et al., 1977), and exonuclease treatment (Smith, 1979). An alternative biological approach has been developed involving the bacteriophage M13. The RF of the phage DNA is a circular double-stranded molecule; it can be isolated from infected cells, used to clone DNA fragments, and reintroduced into Esch~richi~ co/i cells by transfection. The infected cells extrude the Ml3 phage particles, each of which contains a circular SS DNA molecule. Large amounts of SS DNA containing a cloned
270
insert
(5-10
pg phage
SS DNA/ml
ture) can be easily and rapidly et al., 1977; Barnes, Ohsumi
bacterial
obtained
1978; Ray and Kook,
et al., 1978; Hermann
cul-
{Messing 1978;
et al., 1978; Nomura
et al., 1978). The cloning
of DNA
the M 13mp7 cloning
gene)
and was
addition,
vehicle
1978; Messing,
et al., 1981). A fragment (the promoter In
into the RF of
by a series of improve-
and Messing,
galactosidase genome.
fragments
facilitated
ments which produced (Gronenborn Messing
1979;
of the E. eoli Iac
N-terminus inserted
a small
into DNA
of the @the
ified
The synthesized
DNA
(Langley fragment
et
al.,
contains
1975; two
sites each for the EcoRI, BarnHI, SalI, AccI, and IJinciI restriction enzymes arranged symmetrically to a centrally
located
and
Ml3mp9
restriction
orientations by
guarantees
forced
fragment
the clones
the (-t-) strand and
arranged
in
to the M 13 genome.
cloning.
that each strand
will become
have this mod-
site region
relative
a given restriction
orientated
that has
can be directly This
procedure
of the cloned
fragment
in one or the other of
thus be extruded
as SS DNA
in
phage particles.
Ml3
ment. In spite of these insertions the Ml3mp7 DNA is still infective and the modified luc DNA is able to encode the synthesis of a functional /3galactosidase cu-peptide Messing et al., 198 1).
Thus,
Ml3mp8
multiple
vehicles
with the same pair of restriction
fragment
synthesized in vitro and containing an array of restriction cleavage sites was inserted into the structural region of the P-galactosidase gene frag-
with respect
enzymes. opposite
Ml 3 has been
operon
to one of these new Ml3 cloning also been “cut”
Pst I site. Thus,
MATERIALS
AND
METHODS
(a) Strains Two new plasmids, pUC8 and pUC9, have been constructed from the pBR322 plasmid and the Ml3mp7 vector (Bolivar et al., 1977; Sutcliffe, 1979; Messing et al., 1977, 1981; Ruther, 1980; Vieira and Messing, 1982). The 2297-bp EcoRI/ PuuII fragment of pBR322 has had its PstI and HincII sites removed by single base pair changes without loss of /?-lactamase activity. The AccI site in this fragment has been removed by cleavage
by chance either strand of a cloned restriction fragment can become part of the viral (-I-) strand. This depends on the fragment’s orientation relative to the Ml3 genome after they have been joined by ligase. The insertion of a DNA fragment into one of these restriction sites is readily monitored because the insertion results in a nonfunctional a-peptide and the loss of P-galactosidase
and treatment with BAL31 (Legerski et al., 1978). The 433-bp iac Hue11 fragment of Ml3mp7 RF containing the multiple restriction sites was then inserted into this modified pBR322 fragment to yield the plasmid pUC7. The nudeotide sequence containing the multiple restriction sites was then
activity.
modified
Under
appropriate
plating
conditions,
the
to contain
the following
restriction
sites
functional a-peptide results in blue plaques; a nonfunctional a-peptide results in colorless plaques
ordered in the same polarity as lac transcription: EcoRI, XmaI, SmaI, BarnHI, WI, AccI, HincII,
(Messing and Gronenborn, 1978). Ml3mp7 has found wide application in the dideoxy nucleotide
PstI, and HindHI. A second modified
sequencing procedure (Sanger et al., 1977). This paper reports the construction of two new SS DNA bacteriophage vectors, Ml3mp8 and Ml3mp9, and their applications to DNA sequencing and strand-sp~ifi~ hyb~dization. The nucleotide sequence of Ml3mp7, containing the multiple restriction sites, has been modified to have only one copy of each restriction site and in addition, single HindIII, SmaI, and XmaI sites. Thus, DNA fragments whose ends correspond to two of these restriction sites can be “forced cloned” by ligation
site was used to produce the plasmid pUC9. This plasmid contains the multiple restriction sites of pUC8 in a reverse orientation relative to the lat promoter. A detailed description of these plasmids, their construction, and their uses are presented in Vieira and Messing (1982). (b) Construction
the
Ml3mp7 Hue11
This plasmid is termed pUC8. form of the multiple restriction
of M13mp8 and M13mp9
RF DNA was partially cleaved with enzyme (all enzymes were obtained
271
from the Bethesda England
Research
Biolabs)
fied by gel electrophoresis (Heidecker
Laboratory
and the linear
et al.,
and eluted
1980).
and New
forms were puri-
This
DNA
from the gel was
then
cleaved with the E’coRI enzyme to produce phage DNA molecules with HaeII and EcoRI ends. The lac HaeII
modified pUC8
DNA
EcoRI
and PuuI.
lac HaeII
EcoRI
with HaeII
and
Cleavage
fragment
and HaeII
fragments
fragment
was excised
from
also digested
with
at the EcoRI site of the
produces ends. PuuI
two fragments
with
cleaves one of these
and blocks its later incorporation
into a
vector with EcoRI and HaeII ends. The linearized Ml3mp7 DNA was mixed with the triple-enzyme-digested DNA from pUC8 and joined with ligase. This leads to the “forced” insertion of the EcoRI-HaeII subfragment of the modified fat HaeII fragment. The DNA products of the ligation reaction were used to transform competent E. coli JM103 cells (Messing et al., 1981). Candidates for properly constructed vectors were identified as blue plaques. These isolates were saved for further characterization and designated according
to their plasmid
A similar procedure M 13mp9. In this case prepared
as before.
origin as M 13mp8. was used to construct the M13mp7 DNA was
The modified
lac HaeII
frag-
ment was excised from pUC9 DNA with HaeII and digested with EcoRI and HinfI. HinfI cleavage blocks the later incorporation of one of the EcoRI-HaeII subfragments. The restriction-enzyme-digested phage and the pUC9 DNAs were joined by ligase and used to transform competent JM103 cells. Blue plaques characterization.
were picked
for further
after about cleared
30 min at room temperature
by centrifugation
B. The supernatant pipet avoiding
was removed
any damage
ual fluid was removed was resuspended mM NaCl,
in a Beckman
with a Kimwipe.
After another
again by adding
by a short centrifugation
Microfuge
B. The pellet
again in low Tris buffer
of 300 ~1 and extracted
was then
in a volume
twice with an equal volume
of phenol-chloroform.
was precipitated
100
and 100 ~1 of 5 M
30 min at room temperature
the phage was collected
of a 1: 1 mixture
(10
10 mM Tris . HCl pH
7.5). Phage were precipitated
resuspended
The pellet
in 800 ~1 of low Tris buffer
1 mM EDTA,
in a Beckman
with a Pasteur
to the soft pellet; resid-
~1 of a 20% PEG 6000 solution NaCl.
and was Microfuge
with ethanol
from
The DNA the aqueous
phase, washed once with ethanol, taken up in 10 ~1 of low Tris buffer, and used directly to provide template for the DNA sequencing reaction with chain terminators (Sanger et al., 1977). (d) Primer and DNA sequencing An aliquot from the template solution (1 ~1) was annealed with the synthetic master primer and subjected to the chain termination reaction (Sanger et al., 1977) as described previously (Messing et al., 1981; Gardner
RESULTS
AND
et al., 1981).
DISCUSSION
(a) Scheme of forced cloning Restriction
endonuclease
cleavage
fragments
(c) Preparation of templates
with non-complementing joined during a ligation
Templates for DNA sequencing reactions with chain terminating inhibitors (Sanger et al., 1977)
with two different restriction endonucleases and a fragment with two non-complementing ends is
were prepared as follows. Infected cells from a blue plaque were diluted into 1.5 ml of 2 YT(Miller, 1972), mixed with about 10’ non-infected log phase JM103 cells (Messing et al., 1981), and grown for 8 h at 37’C. The infected cells were sedimented by a short centrifugation and the phage in the supernatant were concentrated by the addition to 1.3 ml of the supernatant of 200 ~1 of 27% PEG 6000 (Sigma) in 3.3 M NaCl. The mixture became turbid
produced, the resulting fragment can be neither circularized nor joined to another fragment in both orientations during a ligation reaction. Consequently, two “double-digest” fragments can be forced to form a chimeric molecule under the appropriate ligation conditions. In addition, a recombinant molecule is formed during the ligation
ends are rarely, if at all, reaction. If DNA is cleaved
reaction with the defined order of the two fragments. Since the orientation of a cloned DNA
272
9
x
“PI
Lo
BamKI
1
Hind Ill
Fig. 1. Scheme of forced cloning. Restriction cleavage sites used as cloning sites are unique and located in a region of the circular vector molecule convenient for the insertion of foreign DNA. The DNA sequencewhich has been selected for cloning is cut out of its original DNA with the use of two restriction endonucleases. In the described example the enzymes used are BumHI and HindHI. The DNA fragment produced has two different cohes;ve ends which cannot join in a DNA ligation experiment. The vector molecule is treated with the same enzymes and the same type of cohesive ends are produced. Both DNA fragments, cannot
form a circular
the formation
the vector molecule
of a circular
and the DNA to be cloned
themselves
chimeric
and therefore
molecule,
which
favor
cloning into M13mp7, the symm~t~ around the PstI site of M13mp7 has been replaced by an asymmetric array of sites. The two resulting orders of sites have been produced on two different phage molecules so that both strands of a DNA fragment can serve as the viral strand. In addition, IlindIII. XmaI and SmaI have been added as new cloning sites. The latter serves as a second blunt end cloning site in the case that the I-fincII site cannot be used. A detailed cloning guide which illustrates the combinatorial variety of tobesive and blunt ends produced by a large number of different restriction endonucleases has been described elsewhere (Messing and Seeburg, 198 1). The engineering of this segment of lac DNA has been conducted with pUC7 which contains the luc region of M13mp7 on a plasmid vector as outlined in MATERIALS AND METHODS. The modified cloning sites from the two plasmid vectors pUC8 and pUC9, which contain the cleavage sites in tbe two possible orientations, were transferred to M 13mp7 as described in ~AT~RIA~~ AND METI-IODS. (c)
DNA sequence
determination
for both DNA
strands by the same primer
can be
for by the transformation of competent cells. The location oF the two ctoning sites determines bow the cloned DNA is oriented within the vector molecule.
selected
fragment in the RF of Ml3 vectors determines which of the two DNA strands is going to be the viral strand, this procedure allows the direct preparation of one of the two DNA strands by cloning. Fig. 1 illustrates this scheme when the DNA to be cloned is cut out with Hind111 and BumHI. In addition to the strand separation, a “double-digest” restriction fragment with two non-cornpIementing ends is easiry seIected from DNA fragments in the pool that have been generated by cleavage with only one of the two enzymes. (b) Modification of the Ia& gene
To avoid the random selection of either strand of an insert fragment arising from the use of
A blue plaque from both transfer experiments has been retained and single-stranded DNA prepared as described in MATERIALS AND METHODS. Using the synthetic master primer (Messing et al., 1981 f. sequencing reactions with chain terminators @anger et al., 1977) have been performed and analyzed by polyac~lamide gel electrophoresis. The pertinent sequence containing the cloning sites can be read from the autoradiogram in both orientations (Fig. 2). The primary structure of the Z gene of Ml3mp7 is compared with the structures from the two new isolates which are named M13mp8 and Ml 3mp9 (Fig. 3). Both strands of the insert have been sequenced with the same primer because of the reverse polarity of the synthetic ~c~~I/~i~dIII insert in Ml?mp8 and Ml 3mp9. Therefore, every *‘double-digest” restriction fragment cloned in both vectors will provide both strands of the restriction fragment directly as template which can be sequenced with the same primer.
273
(d) Phenotypic
compa~son
of the different
IacZ
sequences The addition region
of the synthetic
of M13mp2
1978) leads to the synthesis with
the insertion
cloning
to the lac
and
Messing,
of a modified
a-peptide
of 11 amino
M 13mp8 and M 13mp9 compared (Messinget
DNA
(Gronenborn
acid residues
to 14 in Ml 3mp7
al., 1981) and 19 in M13mp5,
phage
polypeptides
(Messing,
a IIindIII
1979). In all cases
were functional
in
the
in a-complementa-
tion and gave rise to blue colored
plaques
under
the appropriate plating conditions (Messing et al., 1977). We have noted that the insertion of these small tion
synthetic test
DNAs
to different
affects degrees,
the complementaand
the
various
M13mp phage molecules can be differentiated by the intensity of the blue color of their plaques. Indeed, the new phage vectors produce a more intense color than Ml3mp7. Moreover, the sequencing analysis of this different 1acZ sequence causes a different degree of secondary structure affecting compression in the gel electrophoresis (not shown). As expected, M13mp7, with a perfect symmetry, shows the highest degree of compression followed by M 13mp5. M 13mp8 and M 13mp9, however,
do not give an indication
for compres-
sion as shown in Fig. 2. (e) “Double-digest” quencing
cloning in shotgun DNA se-
Since all the constructions are based on the use of a master primer (Heidecker et al., 1980) any insert can be conveniently copied by DNA polymerase. Using chain terminators 1977) the sequence of the DNA
(Sanger et al., can be deduced
from selected templates. The shotgun sequencing method, for instance, leads to a less efficient effort Fig. 2. DNA
sequence
analysis
of M13mp8
and MI3mp9.
M13mp8 and M13mp9 have been constructed MATERIALS
AND METHODS.
as described
in
Starting from a single plaque
template from both phage vectors were prepared and a primer extension
reaction in presence of chain terminators was carried
out as described in MATERIALS
AND METHODS.
products were loaded on a 8% polyacrylamide
Reaction
gel in the GATC
order and the sequence was read from the Hue111 site (bottom) in the position of the 8th amino acid residue of the Z gene to the ATG
start codon.
with M13mp7 in Fig. 3.
The resulting sequences
are compared
in determining the last 10-20X of the nucleotides of a contiguous sequence (Gardner et al., 1981). A direct way of obtaining missing sequences of both strands is the use of selective cleavage and forced cloning into the RF of M 13mp8 and M 13mp9. If these resulting clones, and therefore the gaps between two blocks of sequences, are too large to be sequenced with one primer extension reaction these clones in turn can be used as master sequences to identify smaller templates from both strands within
274
SER
10 THR
11 ASP
12 PRO
13 GLN
14 ASN
6
?
6
AG,G TCG
AC?
CAT
Ccl;
GGG AAT
%:
%:
?k:
123456789 1234
Ml3mp7
THR ATG ACC
IpUC7
MET ATG
ILE ATT
THR XG
5
SER
PRO
ASP
PRO
SER
THR
ASN AAT
TCC
CCG
GAT
CCG
TCG
AC,C TCC
km-
-%ii;il
-Sal
CYS
AM
EcoRI
I
ACCI
Ace1 Hint II
HinclI
12345678 12
M13mp8/pUC8
ATG
34
5
THA
MET
ILE
THR
ASN
ACC
ATG
ATT
AC,O AAT Eco
6 SER
ARG
TF
CGG GGA
RI
GLY
SER TCC
L__&!F=
VAL GTC
ASP GAC
l_;&
XmaI
LEU CTG
GLN
MltmpQ/pUCQ
ATG
ACC
MET
3 ILE
4 THR
PRO
ATG
ATT
ACG CCF
SER
Fig. 3. A genetic in respect detail
by the nucleotide
underlined inserted
and labeled.
sequence
M13mpS
charge
marker,
pUC7,
of the pertinent
from Bethesda
Research
TP
ALA
GFT
GCA Pst
region
of the wild-type
is set up and numbered. pUC8
AL4
III
and Ml3mp9.
and pUC9 Laboratory
10
Ii
LEU
ALA
AGC
TT;G GCA
7
8
LEU CTG
k-?
ALA GCC
k?
nindm
I
GLY
ARG ARG
9
10
11
ILE
FRO
GLY
G,GT CGA
y
ATC
7
8
ASN
SER
LEU
ALA
C,CC GG,G AAT
The HueII-cleavage
TCA
CTG
GCC
acid sequence
The same fat cloning
as described
6
sites of the three different
of the IucZ gene. The recognition amino
5
in Ml3mp2
between
AND
vectors
sites for restriction (Gronenborn
METHODS.
have been mapped
the three molecules
sites exist also on the pBR322
in MATERIALS
Absm
ECORI ‘
LS;i--iBW
et al., 1977). Map units are given in kb. The difference
The interruption
amino acid sequences
as a selection without
map of M13mp7,
to the genetic map (Messing
CC?
9 SER
AccI HincIt
LEU
AGC
Hind
PRO
CM
12345676 12 THR
kzl?
Jpstl
and Messing, derivative
All strains
is given in
endonucleases
are
1978) by the
with ampicillin
are made
available
upon request.
pool of the different shotgun clones obtained in the first cloning experiment. Using a new procedure of making Ml3 probes and a dot hybridization procedure, a library of Ml3 shotgun clones can be rapidly screened for statistically underrepresented templates (Hu and Messing, 1982). the
(f) Comparative sequencing, transcriptional ping, and in vitro mutagenesis
map-
The same template selection can be used to compare mutant sequences. Once the primary
structure of a DNA sequence is known its genetic structure has to be determined by comparison of the primary structure with mutant sequences. Since this requires the comparison of a small part of the entire sequence, the appropriate doubly digested fragment of interest can be used to readily obtain mutant sequences in template form in M13mp8 and M13mp9. Defining the orientation of a cloned fragment in M13 is very useful in at least two other experimental procedures; first, for making single-strand specific hyb~dization probes (Hu and Messing,
275
1982), second for in vitro mutagenesis. Singlestrand specific hybridization probes are helpful in determining the strand polarity of inserts in other Ml3 viral DNAs or for physically mapping transcripts. If site-specific changes in a particular nucleotide sequence are planned by using a synthetic primer as a mutagen (Smith and Gillam, 198 1). it may be useful to have the extension of the mutagen primer carried out in the absence of the complementary strand. A discussion of an integrative strategy of shotgun DNA sequencing and exploration of gene structure has been described in more detail elsewhere (Messing, 198 1; 1982).
Hu, N. and Messing, probes.
Gene
Langley,
J.: The making
of strand
specific
Ml3
17 (1982) 271-277.
K.E., Villarejo,
M.R.,
A.V., Zamenhof,
Fowler,
I.: Molecular
mentation.
Proc. Nat]. Acad. Sci. USA 72 (197.5) 1254- 1257.
Legerski.
R.H..
Hodnett,
basis of fl-galactosidase-a
P.J.
and Zabin,
J.L. and Gray,
nucleases
of Pseudomonas
stranded
deoxyriboexonuclease
convenient
method
produced
BAL31,
for
by cleavage
H.B.:
comple-
Extracellular
III. Use of the double-
activity
as the basis
mapping
of fragments
with restriction
enzymes.
of a
of DNA Nucl. Acids
Res. (1978) 144% 1463.. Maxam.
A. and Gilbert,
W.: A new method
for sequencing
DNA. Proc. Nati. Acad. Sci. USA 74 (1977) 560-564. Messing,
J.: A multi-purpose
single-stranded DNA
DNA
Technical
cloning
system
bacteriophage
Bulletin,
NIH
based
M13.
on the
Recombinant
Publication
No. 79-99,
2,
No. 2 (1979) 43-48. Messing,
J.: M13mp2
system ACKNOWLEDGEMENTS
for DNA
and
and in vitro mutagenesis, ings of the Third
This work was supported by the Minnesota Experiment Station, grant No. MN 15-030, and a grant No. DE-AC02-81 ER 10901 from the Department of Energy. We thank Gisela Heidecker for her stimulating discussions and Jim Fuchs, Perry Hackett, Irwin Rubenstein, Kris Kohn and Bonnie Allen for their aid in preparing this manuscript.
Messing, @is.),
beyond,
Genetic
4. Plenum, Messing,
Engineering,
Insertion
of a Hind11
Harbor, W.M.:
DNA
sequence
from the histidine
Seven histidine
codons
in a row.
operon
con-
Proc.
Nat].
F.,
Rodriguez,
Heynecker,
and characterization
II. A multi-purpose R.C.,
Shepherd,
cloning
Howarth, R.J. and
sequence
Greene.
P.J.,
H.L., Boyer, H.W., Crosa,
Construction Gardner,
R.L.,
A.J., Messing,
of an infectious
by M 13mp7 shotgun
Betlach,
S.:
‘vehicles,
Gene 2 (1977) 95-113.
Hahn,
P., Brown-Luedi,
J.: The complete
clone of cauliflower
sequencing.
M.C.,
J.H. and Falkow,
of new cloning
system.
M.,
nucleotide mosaic
virus
Nucl. Acids Res. 9 (1981)
DNA
B. and Messing, J.: Methylation in vitro
cleavage
sites. Nature
Heidecker, primer
G.. Messing, for DNA
tem. Gene Hermann,
introduces
of single-stranded
restriction
endonuclease
J. and
sequencing
Gronenbom,
B.: A versatile
in the M13mp2
cloning
sys-
B.: The filamentous
Transposition
K., Zentgraf,
of DNA
sequence
into the single-stranded
fd. Mol. Gen. Genet.
H. and Schaller,
determining genome
I53 (1978) 171-178.
H.:
kanamycin
of bacteriophage
vehicle: region
J., Crea, R. and Seeburg,
Cold Spring
and Cellular
P.H.: A system for shotgun
P.H.: A strategy
in Brown,
using Purified
Genes,
D. (Ed.), ICN-UCLA
Biology,
for high speed DNA
Developmental Symposia
Biology
on Molecular
Vol. XXIII.
Academic
in Molecular
Genetics.
Press,
New
1981, 659-663.
Miller, J.H.: Harbor
Experiments Laboratory,
Cold Spring Harbor,
N., Yamagichi,
acterization Ohsumi,
Laboratory,
Nucl. Acids Res. 9 (1981) 309-321.
J. and Seeburg,
sequencing,
Nomura,
Ml3
D. and Ray, D.S. (Eds.), The Single-stranded
DNA sequencing.
York,
phage
fusions in vitro, in Denhardt.
coliphage
Virology
Id carrying
N.D.:
fl
and
a kana-
The isolation
of an in vivo recombinant bacteriophage
and char-
I.
gene. Gene 3 (1978) 39-5
M., Vovis, G.F. and Zinder,
characterization
Cold Spring
NY, 1972.
H. and Oka. A.: Isolation
of transducing
between
the plasmid
and the
pSCIO1.
89 (1978) 438-449.
Ray, D.S. and Kook, Y.: Insertion the genome of the single-stranded
10 (1980) 69-73.
as a cloning
NY, 1978, pp. 449-453.
filamentous
272 (1978) 375-377.
R., Neugebauer,
resistance
new
Vol.
form in vitro. Proc. Natl. Acad. Sci. USA
DNA for operon
mycin resistance
287 I-2888. Gronenbom,
Messing, Messing,
Acad. Sci. USA 75 (1978) 4261-4285.
and Methods.
of the iac regulatory
DNA Phages. Cold Spring Harbor
trol region:
J.K.
B. and Hofschneider,
Ml3
fragment
J. and Gronenborn,
D.T.. Dressier,
Bolivar,
Principles
and
Setlow,
74 (1977) 36423646. as a carrier
REFERENCES
sequencing
A. and
1982, pp. 19-36.
coliphage
in Ml3 replicative
of DNA
B., Muiler-Hill,
Filamentous
on Macromole-
1981. pp. 143- 153. strategy
New York,
cloning
hybridization,
A.G. (Ed.), Proceed-
Symposium
in Hollaender,
.I., Gronenborn,
P.H.:
specific
in Walton,
Cleveland
J.: An integrative
experiments
A molecular
strand
cules. Elsevier, Amsterdam,
Messing,
Barnes,
derivatives:
sequencing,
of the Tn3 transposon
into
DNA phage M13. Gene 4
(1978) 103-I 19. Ruther,
U.:
Construction
vehicle, allowing Mol. Gen. Genet.
and
properties
direct screening
of a new cloning
for recombinant
1978 (1980) 475-477.
plasmids.
276
Sanger,
F., Nickien,
S. and Cot&en.
with chain-terminating
A.R.:
inhibitors.
Proc.
DNA sequencing
derived
Natl.
with synthetic
Acad.
Sci.
Vinograd,
USA 74 (1977) 5463-5467. Smith, A.J.H.:
The use of exonuclease
stranded
DNA for use as a template
tion sequencing
methods.
Nucl.
HI for preparing
single-
in the chain
Acids
termina-
system
J., Morris.
buoyant
co/i plasmid
pBR322.
nucleotide
sequence
Cold Spring
J., Davidson,
Gene
and
sequencing
19 (1982) 259-268.
N. and Dove, W.F.:
of viral and bacterial
DNA
The
in alkaline
CsCl. Proc. Nat]. Acad. Sci. USA 49 (1963) 12-17. Communicated
J.G.: Complete
behavior
mutagenesis
primers.
Res. 6 (1979) 831-
848. Sutcliffe.
for insertion universal
by Z. HradeEna.
of the &cberichia
Harbor
Symposium
43
Note added in proof
( 1979) 77-90. Szalay,
A.A., Grohmann,
of the complementary acrylamide Szybalski, tary
DNA
New York, J. and
of DNA
H., HradeEnl,
and preparative strands,
(Eds.). Methods Vieira,
strands
R.L.: Separation fragments
on poly-
gels. Nucl. Acids Res. (1977) 1563-1578.
W., Kubinski,
Analytical
K. and Sinsheimer
2. and Summers,
separation
in Grossman,
in Enzymology,
W.C.:
of the complemenL. and
Moldave,
Vol. 21D. Academic
K.
Press,
1971, pp. 383-413. Messing,
J.: The pUC
plasmids,
a M13mp7
In the meantime, a new Ml3 vector pair has been constructed. They have two additional cloning sites for SstI and X&z1 in their polylinker. ~13mplO is derived from M13mp8, and M13mpll from Ml3mp9, except that Ml3mp 11 does not contain the extra piece of pBR322 sequence as one of the isolates of M13mp9 does.