Download Construction of recombinant plasmid carrying the λ DNA fragment

Volume 5 Number 6 June 1978
NliCleJC A c i d s Research
Construction of recombinant plasmid carrying the X DNA fragment responsible for prophage integration
N. Strizhov and L. Tikhomirova
Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences,
Pushchino, 142292, USSR
Received 4 April 1978
ABSTRACT
The recombinant DNA molecules were constructed from plasmid
RSF2124 and the EcoRI fragment of A DNA containing the genes responsible for prophage integration. The presence of
these genes in recombinant plasmids was detected genetically.
A int-gene was shown to be expressed in either orientation
of insertion in the plasmid. We found that recombinant plasmid was able to integrate into chromosome of A lysogens.
The integration of plasmid into host chromosome was demonstrated by contransduction of chromosome and plasmid markers
using generalized transducer P1 and by specialized transduction with A phages.
INTRODUCTION
The stabilization of foreign DNA fragment in the recipient
cells is one of the major problems of recombinant DNA research. The stabilization of foreign DNA sequences in the cell
can be achieved by integration of these sequences into host
chromosome. It may be carried out by means of A vectors
containing the genes necessary for stable lysogenization.
However, the maximal length of the fragment which can be
cloned by the use of such phages as cloning vehicles is
limited. Plasmid Col E1 and its derivatives are particularly
convenient as cloning vehicles for the construction of recombinant DNA molecules. However, these plasmids can not
integrate into host chromosome and are maintained in cells
as independent replicons. In some cases it is important for
succesful cloning that foreign DNA sequences are present in
only one copy per cell otherwise the superproduction of the
foreign gene product may be fatal for recipient cell. Taking into account the widespread application of plasmids as
cloning vehicles for recombinant DNA research we investigat© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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ed the possibility of integration of plasmid DNA into the
chromosome of E.coli. It was carried out using the in vitro
constructed recombinant plasmid RSF2124 with A DNA fragment
containing att-site and int-xis genes integrated into the
EcoRI site.
MATERIALS AND METHODS
Containment classification; These experiments do not give
rise to any recombinants which are thought to create novel
biohazards. They were handled using normal sterile microbiological procedures.
Bacteria and bacteriophage strains. The Escherichia coli K12
strains used and their origin were: C600 (from A.Kaiser),
QD5OO3 (from L.Siminovitch), C600/RSF2124 from H.Boyers
laboratory. C6OO( A. cI857) and C600 (A 1434) were-lysogenized in our laboratory. JC5088 rec A,-g (from A.Clark via
K.Zlotnikov) 953 gal E~galK~trp~lac~su" (from Gellert)
RW842 - the indicator strain for red plaque test
from
L.Enquist.
The phages and their sources were: Agt- AC
from G.Zavilgelsky, P1KC from K.Zlotnikov, A red 3 1 4 C I 8 5 7 and
Aint4gam210CI857 from B.Malone via K.Zlotnikov, Ab2c, A + ,
ACI857, Ai434 from A.Kaiser and Aint am29 CI857 and Axisam6
from L.Enquist.
Media. The standard rich media used were NB broth and NB
broth agar. The NB broth medium contained (per liter of water): 5 g of Nutrient broth (Difco), 10 g of peptone (Spofa,
CSR), 5 g of NaCl. It was solidified by the addition of
Difco agar to a concentration of 1.3# for plates and 0.796
for top agar. EMBO-agar was prepared as described '. The minimal medium used was M9. For identification of gal + transductants 50 mg/1 2,3,5-triphenyltetrazolium chloride (TTC)
was added.
DNAs and enzymes. RSF2124 plasmid DNA was purified as described by Tanaka and Weisblum . The DNA of recombinant plasmids were purified according to Meyers et al. . Preparation
of A g t - A C phage lysate was made by infection of exponentially growing culture of E.coli C600 in NB broth containing
10"**' M MgS0A« The phages were recovered by precipitation
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with polyethyleneglycol and purified by centrifugation
through a CsCl gradient . The purified phage was dialysed
against 10 mM tris-HCl pH 7,5, 50 mM NaCl, 1 mM MgSO^ and
the DNA was extracted by gentle rolling with freshly distilled phenol 7 followed by dialysis against 10 mM tris-HCl pH
7,5, 50 mM NaCl.
The EcoRI endonuclease was prepared by D.Vorozheikina from
E.coli strain 1100/RI as described by Yoshimori 8 . The diQ
gestion of DNAs was carried out as described . 1 pi of purified enzyme was enough for complete digestion of 1 ug of
ADNA after 30 min at 37°C. The DNA ligase of phage T4 was
kindly given us by A.Solonin. The hydroxylapatite fraction
of the enzyme was used with the activity as high as
520 units/ml. Bam H1 restriction endonuclease was a generous
gift from N.Kuzmin.
The construction of recombinant plasmids. The central DNA
fragment of phage Agt- AC was separated in a glycerol gradient 1 0 at 32.000 rev/min, 14 h, 15°C, ultracentrifuge
L5-5O. The gradient solution was passed from the bottom of
the tube through the Analabs u.v.monitor. The fractions of
central fragment were collected and used in the ligase reaction. There was no noticeable contamination of the central fragment by the end fragments, as was shown by electrophoresis through an agarose gel. RSF2124 plasmid DNA was
digested with EcoRI treated with phenol and after dialysis
against 10 mM Tris-HCl pH 7,5, 50 mM NaCl was mixed with
central fragment of Agt- Ac DNA (1,4 y.g and 1.7 ^g correspondingly) . Ligation and transformation were carried out as
q
described .
Electrophoresis of DNAs was carried out on 1% agarose gels
(Bio Rad) in 0.04 M Tris-acetate, pH 8.0, 0.02 M sodium acetate, 0.002 M EDTA at 2 v/cm for 20 h.
Transduction experiments with P1 were carried out as described by Rhotman
.
Lft and Hft lysates of A phages were obtained by heat induction of A CI857 from bacteria containing parental or recombinant plasmids. All transducing phage lysates used were
sterile.
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The assay of the Lft and Hft lysates was carried out in the
preformed gradient of CsCl in SW 50.1 at 30.000 rev/min for
5 h. The fractions were collected in 2 ml of Nutrient broth.
Colicine E1 was isolated from E.coli JC411/Col E1 (from
D.Helinski) by the method of Schwartz and Helinski
.
Electron microscopic observation of plasmid DNA was carried
out by micro-method of Lang and Mitani
.
RESULTS AND DISCUSSION
The construction of hybrid plasmid. The mixture of central
Agt- Ac DNA fragment and EcoRI endonuclease digested DNA of
plasmid RSF2124 was treated with ligase and used to transform CaCl2-treated QD5OO3 cells. The results of transformation experiment are presented in table 1.
Table 1. The appearance of ampicillin resistant (Apr) clones
in the transformation experiments.
DNA
Transformants/ }ig RSF2124 DNA
RSF2124,native
RSF2124, E.coRI
RSF2124, EcoRI+ligase
RSF2124,EcoRI+ ADNA fragment+ligase
3«10 5
2,3«1O2
6,2«103
8,6«103
The efficiency of transformation increased 27 times when
mixture of EcoRI-digested DNA of plasmid RSF2124 and central
fragment of Agt- Ac DNA was treated with T4 ligase. 100 Apresistant transformants were assayed for the ability to produce colicine E1. It was found that 9 of 100 assayed clones
were immune to colicine E1, but not able to synthesize it.
Plasmid DNA was purified from these clones. The electrophoresis in agarose gels of EcoRI digestion products of purified plasmids shows two fragments - one corresponding to
linear form of RSF2124 DNA and other corresponding to central EcoRI fragment of A g t - A C (fig.1).
The insertion of DNA fragment into plasmid RSF2124 was confirmed by electron microscopy of parental and recombinant
plasmid DNAs. The measuring of the contour lengths of plas1770
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a b e d
Fig.1. Agarose gel electrophoresis
of DNA digested with EcoRI endonuclease:
a - RSF2124
b - RSF2124 - AC
<?c - Agt - AC
d - RSF2124 - AC + Agt - AC
mids showed that recombinant plasraid differs from parental
RSF2124 by length corresponding to Ac fragment (fig.2).
Expression of A genes integrated into plasmid RSF2124. Expression of the int-gene of A inserted into RSF2124 DNA molecule by an in vitro recombination was studied by assaying
the ability of recombinant plasmids to complement lnt~ mutation of A phage . The int-mutants of A are not able to produce stable lysogens. Abortive lysogens picked from the turbid plaques of integration-negative mutants may be distinguished from stable lysogens picked from the plaques of wild
type phage by their response on a test plate to a continuous
challenge to their immunity. On a EMBO-test plate spread with
Ab2c large pink colonies appear after incubation at 33 C
for 20 h if the A lysogens transferred to the plate are
14
stable
. Abortive lysogens appear as a small dark colonies.
The stable lysogenization of hybrid plasmid carrying cells
by A int mutants is possible if the plasmids provides the
int product defficient in A int mutants. Other possibility
for stable lysogenization by int" phages may occur from the
recombination of int" phages with plasmid DNA in homologous
regions producing phages of int+ genotype. The EMBO-test
allow us to differenciate stable lysogens from abortive
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B
0,2?
Fig.2. Electron micrographs of RSF2124 - Ac (A) and RSF2124 (B)
plasmid DNA.
ones. When bacteria containing RSF2124-AC plasmids were infected with int-mutants of A stable lysogens appeared with
high efficiency. They did not appear at all when int-mutants
infected the strain containing parental plasmid RSF2124. High
frequency of stable lysogenization in the first case indicates that rather the complementation than the recombination
may be a reason for lysogenization by int~ phages.
It was supported by the fact that phages obtained from
lysogenic bacteria containing recombinant plasmids were of
int~ genotype.
Expression of int gene carried out by the RSF2124-AC plasmid
was confirmed by red plaque test developed by L.Enquist and
R.Weisberg . According to this test the expression of int
and xis genes of A can be detected by their ability to promote the int and xis dependent excision of a cryptic prophage
inserted within gal T gene of E.coli. Excision of a cryptic
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prophage results in gal + reversion of the cells that survived infection. For this purpose parental and recombinant plasmid DNAs were transformed to the strain RW842 containing the
cryptic prophage. When int~ xis + phages were plated on this
strain red plaques similar to ones of wild type A were observed if the strain carried the plasmid RSF2124- AC. If the
strain RW842/RSF2124 was infected with Aint~xis + phages there appeared plaques with turbid but completely colourless
centers. Excision of prophage requires both int and xis gene
products
. The int gene is expressed from its own promoter
AC. A*"?
*
therefore its expression does not depend on orientation of A DNA fragment in plasmid. Expression of xis gene
within the plasmid probably must be regulated by transcription starting from a promoter for colicin E1 protein synthesis. The red plaque test can give a semiquantitative estimate of int and xis functions . The expression of xis gene
inserted into the plasmid in both orientation (it was shown
by endonuclease EcoRI and Bam H1 restriction analysis of recombinant plasmids; data not shown) was very low. When gal~
strain RW842 was transformed with parental plasmid RSF2124
the red gal + clones were not observed. RW842 does not revert spontaneously to gal + . In the case of transformation by
RSF2124- AC plasmid the red gal + clones appeared but with
low frequency. In contrast to the plating of int~xis+ phages
the effect of complementation was not observed when int+xis~
phages were plated on the RW842 carrying recombinant plasmid.
About 2/3 of the int+xis~ phage plaques were completely colourless and 1/3 contained one to four red cured clones. We
cannot exclude that cured clones may be the int + xis + recombinant phages, formed as a result of recombination of infecting int+xis~ phages with RSF2124-AC plasmid.
Insertion of recombinant plasmid into chromosome. Having
convinced that inserted DNA fragment contained functional
int gene of A the possibility of integration of plasmid
RSF2124-AC in the chromosome of bacteria was assayed. The
P1 mediated cotransduction of chromosome gal marker and
Ap r Col10111 plasmid markers must be observed if the recombinant plasmid is able to integrate in the attachment site for
A. However, we could not observe cotransduction of these
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Table 2. P1 mediated transduction.
Donor s t r a i n
gal +
gal +
An1*
C6OO/RSF2124
C6OO/RSF2124- AC
C6OO(AcI857)/RSF2124
C600(AcI857)/RSF2124- Ac
JC5088(Ared 314)/RSF2124
JC5088(Ared 314)/RSF2124- AC
225
735
411
258
467
596
Ap
Col i m m
0
0
0
% of cotransduction
0
0
0
17
6,6
0
0
0,8
5
markers if the transduction was carried out by P1 phage obtained from the gal + strain C600/RSF2124- AC. The results of
transduction experiments are shown in the table 2. It is
known that besides the int gene expression the function of
18 19
other genes are necessary for stable lysogenization by A ' .
Cotransduction of gal + Ap r Col
markers to gal" strain 953
was observed when P1 transducing phages were obtained from
A-lysogenic E.coli. Frequency of cotransduction for gal +
Ap r Colm m markers in these experiments was 6£>% (table 2 ) .
It is not clear how prophage can promote the site specific
recombination between plasmid. and chromosome. It is not
excluded that recombinant plasmid may be inserted into the
chromosome of bacteria by means of general recombination in
the homologic region of plasmid and prophage.
In order to eliminate the possibility of recombination between prophage and plasmid DNA the recombinant and parental
plasmids were transferred into strain defective for general
recombination system and lysogenic for recombination defective A prophage JC5088 recAcg ( A red 314). In this strain
insertion of plasmid RSF2124— AC into chromosome may occur
only by means of site specific recombination responsible
for prophage integration. The frequency of cotransduction of
gal + A p r Colm m markers reduced when P1 lysate was obtained
from rec A~ (A red) strain, however, it was sufficiently
high to allow us to suppose that integration should be carried out by site-specific recombination. It is known that
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Table 3. Specialized transduction by A .
Donor strain
Lft C600(AcI857)/RSF2124
_n_
_ti_
_n —
Hft
C600(AcI857)/RSF2124- Ac
C600/RSF2124
C600/RSF2124 - AC
C600(AcI857)AprColimm
Preparation of
phage
lysate
Recipient
strain
Ap r Col inun
transducing phage
frequency
heat induction
C600
1«10~ 9
C6OO
C600
C6OO
C6OO
5*10~6
5«10~ 9
4'10~7
1.10"4
1.10"5
— n_
infection
_n_
heat induction
c6oo(/V)
rec~ mutations have no apparent effect on the ability of the
cells to maintain the Col E1 factor but can prevent the
spontaneous and induced production of colicine. These mutations do not affect the expression of immunity of Col fac20
tor
. If the expression of some function from colicine gene promoter is necessary the frequency of plasmid insertion
in rec A~ strain may be reduced.
Other confirmation for recombinant plasmid insertion into
chromosome of bacteria was obtained from specialized transduction experiments. A transducing phages were prepared from
appropriate lysogens by heat induction. Lft lysates were
used to infect E.coli C600 and Ap r C 6 1 i m m transductants were
counted on the plates with ampicillin and colicin E1. The
frequency of transducing phages in Lft lysates was 5*10
(table 3). When Lft lysate was obtained from the strain containing parental plasmid RSF2124 Ap r Col i m m transductants
were not observed. Hft lysates transduced corresponding
markers to recipient strain with frequency 1.10 . The recombination in homology regions is not excluded when propagation of phages occur in recombinant plasmid carrying
strains. In order to estimate this the bacteria carrying
parental or recombinant plasmids were infected with phage
AcI857 at multiplicity of infection equal to 0.1 and lysates obtained were assayed for the presence of transducing
phages. It was shown that recombinant Ap r Col m m transducing
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TO*
A
I
A
o
1
6
i
I
I.1B0
Air
/o2
i;
i
f.375
1.370
5
10
•"— bottom
\5
25
30
3S
40
10
(5
20
2*
30
FRACTIONS
Fig.3. Density gradient analysis of Lft (A) and. Hft (B) lysates. Gradient fractions were titrated for total phage
(closed circles) and for transducing phage (open circles).
phages arise with 10 times lower frequency as compeared with
prophage induction (table 3 ) .
Additional information was obtained about structure of
transducing phages by assaying Lft and Hft lysates in the
CsCl gradient. The fractions of the gradients were plated
on C600 (A ) and C600 ( A i434) and the peak fractions of
marker phage A i43^, phages from Lft and Hft lysates were
identified. The presence of transducing phages in the fractions was determined by incubation of fraction aliquots with
starved E.coli C600 cells and subsequent plating on the medium containing ampicillin and colicin E1. The results of
these experiments are presented in figure 3. The sedimentogram of Lft lysate shows wide distribution of Ap r transducing phages in the gradient fractions. Transducing phages from
Hft lysate were localized as a sharp peak . The frequency of
transducing phages increased 10 times if the presence of
transducing phages in the gradient fractions was assayed on
the A lysogenic strain. Experiments, are in progress to elucidate the structure of transducing phages and the mechanism
of recomblnant plasmid integration.
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ACKNOWLEDGEMENTS
We are indebted to Dr. N.Matvienko for helpful advice and
discussions. We thank Dr. I.Fodor for his critical reading
of the manuscript. We are also grateful to Dr. L.Enquist for
making the red plaque test available for us and L.Matrosova for
technical help.
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