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EcoK restriction during in vitro packaging of coliphage lambda DNA
313
Gene, 39 (1985) 313-315 Elsevier GENE
1397A
EcoK restriction during in vitro packaging of coliphage lambda DNA
(Recombinant DNA; bacteriophage I vectors; cosmids; genomic library; E. coli C host bacteria)
Susan M. Rosenberg Institute of Molecular Biology, University of Oregon, Eugene, OR 97403 (U.S.A.) Tel. (503)686-5146
SUMMARY
The K restriction system of Escherichiu coli works in vitro [Meselson and Yuan, Nature 217 (1968) 11 lo-11141. E. coli C lacks the K restriction system. I show that in vitro packaging in standard E. coli K- 1Zderived systems effects a loss of plaque-former output from K-unmodified I DNA relative to K-modified I DNA when compared with packaging in the E. coli C-derived system of Rosenberg et al. [Gene 38 (1985) 165-1751. I conclude that the EcoK restriction system is active in standard in vitro packaging systems. EcoK restriction during in vitro packaging could specifically depress recovery of some I and cosmid clones of eukaryotic DNA or any other DNA not modified for EcoK restriction.
INTRODUCTION
Bertani and Weigle (1953) observed a phenomenon that has since become known as restriction and modification of DNA by E. coli K-12’s EcoK system (see Bickle, 1982, for review). EcoK is a Type I restriction enzyme. Subunits encoded by genes hsdS (sequence specificity), hsdR, (restriction) and hsdM, (modification) sequence-specifically bind, then nonspecifically cut DNA not modified by adenine-methylation in the recognition sequence. The enzyme complex works in vitro (Meselson and Yuan, 1968). E. coli strain C (and all strains other than K-12) lack the EcoK system. Except for a one-strain in vitro packaging system for J DNA (Rosenberg et al., 1985), all published and commercially available packaging systems are extracts of E. coli K-12 cells. In vitro packaging is extensively used to recover clones of non-E. coli DNA ligated into either I or cosmids. Since such clones originate from unmodified DNA, cloners should wonder whether EcoK restriction happens during in vitro packaging, thereby biasing ,? and 0378-I119/85/$03.30
0
1985 Elsevier
Science
Publishers
cosmid libraries against DNA inserts which carry the heptameric EcoK recognition sequence. Because packaging efficiency of any system is usually measured with EcoK-modified 2 DNA, the extent of loss of unmodified DNA during packaging would be a hidden variable. Phage 1 contains five EcoK sites. I show here that unmodified 2 DNA suffers a loss of plaque-former output relative to modified L DNA in K-12-derived packaging systems when compared with the C-derived system of Rosenberg et al. (1985). EXPERIMENTAL
AND DISCUSSION
(a) Strains and packaging systems
Bacteria (Table I) not denoted as E. coli C are derivatives of E. coli K-12. Preparation of ADNA from strains SMR47, BHB2365 and FS168, and preparation of SMRlO in vitro packaging extract are as in Rosenberg et al. (1985). Phage ;1mutations are also described therein. Gigapack is a commercially distributed in vitro packaging extract sold by Vector Cloning Systems (San Diego).
314
TABLE
I
Bacterial
strains
Name
Relevant
genotypea
Source
C600 hsdr, - mK -
SuII + , does not EcoK restrict
E. coli C-1192
E. coli C SuIII +
FS186
SuII + (A cIts857
BHB2365
Su-
Sam7)
(2 imm434cIts
C600 hsdr,-m,E. coli C-1792 (2)
SMRSO
E. coli C-1192 (A imm434)
SMRlO
E. coli C Su-(1
BHB2688
recA Su-(l
BHB2689
recA Su-(i
BHB2690
recA Su-
(b)
of fragment
of
a
B. Hohn,
(1 imm434cIts Sam7)
This work This work
cos2 AB x&l red3 gam210
nin5 Sam7)/1
cIts857
Rosenberg
Earn4 Ab2 red3 imm434cIts Sam7)/1
B. Hohn,
et al. (1985) Base1
Aamll
Ab2 red3 imm434cIts Sam’T)/I
B. Hohn,
Base1
(Darn15
&2 red3 imm434cIts Samll)/l,
B. Hohn,
Base1
et al. (1985) for properties
OR
Base1
This work
of i, genetic elements.
( ), indicates
prophage;
/,a, resistant
to phage I; A, deletion;
EcoRI-B.
Packaging efficiencies Aliquots
Cambridge
et al. (1971)
F.W. Stahl, Eugene,
Sam7)
SMR49
dB, deletion
M. Meselson, Sunshine
SMR47
“See Table II in Rosenberg
or modify DNA
mixture
Aimml DNA and unmodified
even between batches of the same examined a limited sample. of
type.
I have
EcoK-modified
Aimm434 DNA were
packaged in various in vitro packaging mixtures. Titers of il immA and A imm434 phage produced were measured on E. coli C lysogens SMRSO and SMR49. The ratios of 1 imm434: AimmA particles produced by each packaging system are given in Table II. Since that ratio is 1.3 for the E. coli C-derived system, ratios less than 1.3 reflect restriction of unmodified DNA in vitro. The K-12-derived Gigapack system manifests a seven-fold loss of Aimm434 particles. Losses of kmm434 particles produced from tubes of a crude E. coli K- 1Zderived packaging system (Kobayashi and Ikeda, 1978) ranged from two- to seven-fold. When a mixture of modified DNAs of both immunities was packaged in the SMRlO and Gigapack systems, the ratios of &mm434 to AimmA plaques produced were 1.9 k 0.21 and 1.5 f 0.10, respectively; no loss occurred. Thus, the loss of plaque formers produced from unmodified limm434 DNA was due to EcoK restriction in vitro. When plated on E. coli K-12, I grown on nonmodifying host C600 hsdr,- mK forms plaques with an efficiency approx. lOOO-fold lower than when plated on E. coli K- 12 hsdr, - . The EcoK system appears active but less efficient in these packaging mixtures than in vivo. However, the efficiency of EcoK restriction may vary between different types of K- 1Zderived packaging extract or
TABLE
II
Restriction packaging
of unmodified
I chromosomes
in various
in vitro
systems
In vitro packaging
Ratio of:
Lysogens
system
(titer of 1 produced from unmodified DNA) to (titer of I produced from modified
Gigapack
BHB2689
DNA)
0.19 5 0.015”
BHB2690 Kobayashi
and
BHB2688
Ikeda (1977); (modified Hohn
0.44 + 0.30b
BHB2690
from
and Hohn,
1974) Rosenberg
et al.
SMRlO
1.3 & 0.26’
(1985) a Two tubes of extract b Three
tubes
produced
significant
formed
plaques
of extract SMR49.
were examined.
of extract
were
examined.
Since
levels of limm434 endogenous
on SMR49, endogenous
phage from eight tubes
(to which no DNA had been added) The average
endogenous
from the titers on SMR49 ’ Six tubes of extract
phage
were assayed
on
titer was subtracted
in the experiment.
were examined.
this system phage which
315
When packaging ligations of nonbacterial fragments plus il or cosmid DNA in vitro, EcoK restriction may depress recovery of hybrid molecules and specifically bias libraries against large inserts and genes containing many EcoK sites. The SMRlO in vitro packaging system should relieve such bias and give a higher true efficiency of packaging from ligations with unmodified insert DNA.
REFERENCES Bertani, G. and Weigle, J.J.: Host controlled viruses.
J. Bacterial.
Bickle, T.A.: The ATP-dependent Linn, S.M. and Roberts, Harbor Hohn,
Laboratory,
restriction
endonucleases,
R.J. (Eds.), Nucleases.
Cold
Spring
B. and Hohn, T.: Activity
packaging Acad.
Harbor,
in
Cold Spring
NY,
1982, pp.
particles
for
1 in vitro. Proc. Natl.
Sci. USA 71 (1974) 2372-2376. I. and Ikeda, H.: Formation
without ration.
of empty headlike
of DNA of bacteriophage
bacteriophage
M. Cieb suggested the importance of a nonrestricting packaging system. Frank Stahl and David Thaler offered helpful suggestions and read the manuscript. I thank John Adelman and Mark Grimes for gifts of packaging extract. I am supported by N.I.H. predoctoral fellowship No. 5-T32GM07413. This work was supported by N.I.H. grant No. GM 33677 and N.S.F. grant No. PCM 8409843 to F.W. Stahl.
in bacterial
85-108.
Kobayashi,
ACKNOWLEDGEMENTS
variation
65 (1953) 113-121.
lambda
duplication,
transcription,
Mol. Gen. Genet.
Meselson,
M. and Yuan,
E. coli. Nature Rosenberg,
of recombinant
by recA function
translation
coli
matu-
restriction
enzyme
from
217 (1968) 1110-l 114.
in vitro packaging
one-strain
and
153 (1977) 237-245. R.: DNA
S.M., Stahl, M.M., Kobayashi,
Improved
DNA of
of Escherichia
system
free from
I. and Stahl, F.W.:
of coliphage
lambda
endogenous
phage.
DNA: Gene
a 38
(1985) 165-175. Sunshine,
M.G., Thorn,
B.: P2 phage polarity
amber
suppressor.
Communicated
M., Gibbs, W., Calendar, mutants:
R. and Kelly,
Characterization
Virology 46 (1971) 691-702.
by A.J. Podhajska.
by use of
Gene, 39 (1985) 313-315 Elsevier GENE
1397A
EcoK restriction during in vitro packaging of coliphage lambda DNA
(Recombinant DNA; bacteriophage I vectors; cosmids; genomic library; E. coli C host bacteria)
Susan M. Rosenberg Institute of Molecular Biology, University of Oregon, Eugene, OR 97403 (U.S.A.) Tel. (503)686-5146
SUMMARY
The K restriction system of Escherichiu coli works in vitro [Meselson and Yuan, Nature 217 (1968) 11 lo-11141. E. coli C lacks the K restriction system. I show that in vitro packaging in standard E. coli K- 1Zderived systems effects a loss of plaque-former output from K-unmodified I DNA relative to K-modified I DNA when compared with packaging in the E. coli C-derived system of Rosenberg et al. [Gene 38 (1985) 165-1751. I conclude that the EcoK restriction system is active in standard in vitro packaging systems. EcoK restriction during in vitro packaging could specifically depress recovery of some I and cosmid clones of eukaryotic DNA or any other DNA not modified for EcoK restriction.
INTRODUCTION
Bertani and Weigle (1953) observed a phenomenon that has since become known as restriction and modification of DNA by E. coli K-12’s EcoK system (see Bickle, 1982, for review). EcoK is a Type I restriction enzyme. Subunits encoded by genes hsdS (sequence specificity), hsdR, (restriction) and hsdM, (modification) sequence-specifically bind, then nonspecifically cut DNA not modified by adenine-methylation in the recognition sequence. The enzyme complex works in vitro (Meselson and Yuan, 1968). E. coli strain C (and all strains other than K-12) lack the EcoK system. Except for a one-strain in vitro packaging system for J DNA (Rosenberg et al., 1985), all published and commercially available packaging systems are extracts of E. coli K-12 cells. In vitro packaging is extensively used to recover clones of non-E. coli DNA ligated into either I or cosmids. Since such clones originate from unmodified DNA, cloners should wonder whether EcoK restriction happens during in vitro packaging, thereby biasing ,? and 0378-I119/85/$03.30
0
1985 Elsevier
Science
Publishers
cosmid libraries against DNA inserts which carry the heptameric EcoK recognition sequence. Because packaging efficiency of any system is usually measured with EcoK-modified 2 DNA, the extent of loss of unmodified DNA during packaging would be a hidden variable. Phage 1 contains five EcoK sites. I show here that unmodified 2 DNA suffers a loss of plaque-former output relative to modified L DNA in K-12-derived packaging systems when compared with the C-derived system of Rosenberg et al. (1985). EXPERIMENTAL
AND DISCUSSION
(a) Strains and packaging systems
Bacteria (Table I) not denoted as E. coli C are derivatives of E. coli K-12. Preparation of ADNA from strains SMR47, BHB2365 and FS168, and preparation of SMRlO in vitro packaging extract are as in Rosenberg et al. (1985). Phage ;1mutations are also described therein. Gigapack is a commercially distributed in vitro packaging extract sold by Vector Cloning Systems (San Diego).
314
TABLE
I
Bacterial
strains
Name
Relevant
genotypea
Source
C600 hsdr, - mK -
SuII + , does not EcoK restrict
E. coli C-1192
E. coli C SuIII +
FS186
SuII + (A cIts857
BHB2365
Su-
Sam7)
(2 imm434cIts
C600 hsdr,-m,E. coli C-1792 (2)
SMRSO
E. coli C-1192 (A imm434)
SMRlO
E. coli C Su-(1
BHB2688
recA Su-(l
BHB2689
recA Su-(i
BHB2690
recA Su-
(b)
of fragment
of
a
B. Hohn,
(1 imm434cIts Sam7)
This work This work
cos2 AB x&l red3 gam210
nin5 Sam7)/1
cIts857
Rosenberg
Earn4 Ab2 red3 imm434cIts Sam7)/1
B. Hohn,
et al. (1985) Base1
Aamll
Ab2 red3 imm434cIts Sam’T)/I
B. Hohn,
Base1
(Darn15
&2 red3 imm434cIts Samll)/l,
B. Hohn,
Base1
et al. (1985) for properties
OR
Base1
This work
of i, genetic elements.
( ), indicates
prophage;
/,a, resistant
to phage I; A, deletion;
EcoRI-B.
Packaging efficiencies Aliquots
Cambridge
et al. (1971)
F.W. Stahl, Eugene,
Sam7)
SMR49
dB, deletion
M. Meselson, Sunshine
SMR47
“See Table II in Rosenberg
or modify DNA
mixture
Aimml DNA and unmodified
even between batches of the same examined a limited sample. of
type.
I have
EcoK-modified
Aimm434 DNA were
packaged in various in vitro packaging mixtures. Titers of il immA and A imm434 phage produced were measured on E. coli C lysogens SMRSO and SMR49. The ratios of 1 imm434: AimmA particles produced by each packaging system are given in Table II. Since that ratio is 1.3 for the E. coli C-derived system, ratios less than 1.3 reflect restriction of unmodified DNA in vitro. The K-12-derived Gigapack system manifests a seven-fold loss of Aimm434 particles. Losses of kmm434 particles produced from tubes of a crude E. coli K- 1Zderived packaging system (Kobayashi and Ikeda, 1978) ranged from two- to seven-fold. When a mixture of modified DNAs of both immunities was packaged in the SMRlO and Gigapack systems, the ratios of &mm434 to AimmA plaques produced were 1.9 k 0.21 and 1.5 f 0.10, respectively; no loss occurred. Thus, the loss of plaque formers produced from unmodified limm434 DNA was due to EcoK restriction in vitro. When plated on E. coli K-12, I grown on nonmodifying host C600 hsdr,- mK forms plaques with an efficiency approx. lOOO-fold lower than when plated on E. coli K- 12 hsdr, - . The EcoK system appears active but less efficient in these packaging mixtures than in vivo. However, the efficiency of EcoK restriction may vary between different types of K- 1Zderived packaging extract or
TABLE
II
Restriction packaging
of unmodified
I chromosomes
in various
in vitro
systems
In vitro packaging
Ratio of:
Lysogens
system
(titer of 1 produced from unmodified DNA) to (titer of I produced from modified
Gigapack
BHB2689
DNA)
0.19 5 0.015”
BHB2690 Kobayashi
and
BHB2688
Ikeda (1977); (modified Hohn
0.44 + 0.30b
BHB2690
from
and Hohn,
1974) Rosenberg
et al.
SMRlO
1.3 & 0.26’
(1985) a Two tubes of extract b Three
tubes
produced
significant
formed
plaques
of extract SMR49.
were examined.
of extract
were
examined.
Since
levels of limm434 endogenous
on SMR49, endogenous
phage from eight tubes
(to which no DNA had been added) The average
endogenous
from the titers on SMR49 ’ Six tubes of extract
phage
were assayed
on
titer was subtracted
in the experiment.
were examined.
this system phage which
315
When packaging ligations of nonbacterial fragments plus il or cosmid DNA in vitro, EcoK restriction may depress recovery of hybrid molecules and specifically bias libraries against large inserts and genes containing many EcoK sites. The SMRlO in vitro packaging system should relieve such bias and give a higher true efficiency of packaging from ligations with unmodified insert DNA.
REFERENCES Bertani, G. and Weigle, J.J.: Host controlled viruses.
J. Bacterial.
Bickle, T.A.: The ATP-dependent Linn, S.M. and Roberts, Harbor Hohn,
Laboratory,
restriction
endonucleases,
R.J. (Eds.), Nucleases.
Cold
Spring
B. and Hohn, T.: Activity
packaging Acad.
Harbor,
in
Cold Spring
NY,
1982, pp.
particles
for
1 in vitro. Proc. Natl.
Sci. USA 71 (1974) 2372-2376. I. and Ikeda, H.: Formation
without ration.
of empty headlike
of DNA of bacteriophage
bacteriophage
M. Cieb suggested the importance of a nonrestricting packaging system. Frank Stahl and David Thaler offered helpful suggestions and read the manuscript. I thank John Adelman and Mark Grimes for gifts of packaging extract. I am supported by N.I.H. predoctoral fellowship No. 5-T32GM07413. This work was supported by N.I.H. grant No. GM 33677 and N.S.F. grant No. PCM 8409843 to F.W. Stahl.
in bacterial
85-108.
Kobayashi,
ACKNOWLEDGEMENTS
variation
65 (1953) 113-121.
lambda
duplication,
transcription,
Mol. Gen. Genet.
Meselson,
M. and Yuan,
E. coli. Nature Rosenberg,
of recombinant
by recA function
translation
coli
matu-
restriction
enzyme
from
217 (1968) 1110-l 114.
in vitro packaging
one-strain
and
153 (1977) 237-245. R.: DNA
S.M., Stahl, M.M., Kobayashi,
Improved
DNA of
of Escherichia
system
free from
I. and Stahl, F.W.:
of coliphage
lambda
endogenous
phage.
DNA: Gene
a 38
(1985) 165-175. Sunshine,
M.G., Thorn,
B.: P2 phage polarity
amber
suppressor.
Communicated
M., Gibbs, W., Calendar, mutants:
R. and Kelly,
Characterization
Virology 46 (1971) 691-702.
by A.J. Podhajska.
by use of