U.S. patent application number 11/249274 was filed with the patent office on 2006-02-16 for recombinational cloning using engineered recombination sites.
This patent application is currently assigned to Invitrogen Corporation. Invention is credited to Michael A. Brasch, James L. Hartley.
Application Number | 20060035269 11/249274 |
Document ID | / |
Family ID | 27048589 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060035269 |
Kind Code |
A1 |
Hartley; James L. ; et
al. |
February 16, 2006 |
Recombinational cloning using engineered recombination sites
Abstract
Recombinational cloning is provided by the use of nucleic acids,
vectors and methods, in vitro and in vivo, for moving or exchanging
segments of DNA molecules using engineered recombination sites and
recombination proteins to provide chimeric DNA molecules that have
the desired characteristic(s) and/or DNA segment(s).
Inventors: |
Hartley; James L.;
(Frederick, MD) ; Brasch; Michael A.;
(Gaithersburg, MD) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Invitrogen Corporation
Carlsbad
CA
|
Family ID: |
27048589 |
Appl. No.: |
11/249274 |
Filed: |
October 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10162879 |
Jun 6, 2002 |
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11249274 |
Oct 14, 2005 |
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09432085 |
Nov 2, 1999 |
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10162879 |
Jun 6, 2002 |
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09233493 |
Jan 20, 1999 |
6143557 |
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09432085 |
Nov 2, 1999 |
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08663002 |
Jun 7, 1996 |
5888732 |
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09233493 |
Jan 20, 1999 |
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08486139 |
Jun 7, 1995 |
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08663002 |
Jun 7, 1996 |
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Current U.S.
Class: |
435/6.18 ;
435/91.2 |
Current CPC
Class: |
C12N 15/66 20130101;
C12N 15/10 20130101; C12N 9/00 20130101; C12N 15/64 20130101; C12P
19/34 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Claims
1. A method for synthesizing a double stranded nucleic acid
molecule comprising: (a) mixing one or more nucleic acid templates
with a polypeptide having polymerase activity and one or more
primers comprising at least a first recombination site or portions
thereof; (b) incubating said mixture under conditions sufficient to
synthesize a first nucleic acid molecule which is complementary to
all or a portion of said one or more templates and which comprises
at least said first recombination site or portions thereof; and (c)
incubating said first nucleic acid molecule in the presence of one
or more primers comprising at least a second recombination site or
portions thereof under conditions sufficient to synthesize a second
nucleic acid molecule complementary to all or a portion of said
first nucleic acid molecule, thereby producing a double stranded
nucleic acid molecule comprising at least said first and second
recombination sites or portions thereof, wherein at least one of
said first and second recombination sites comprises one or more
mutations that remove one or more stop codons from said
recombination sites.
2. A method for synthesizing a double stranded nucleic acid
molecule comprising: (a) mixing one or more nucleic acid templates
with a polypeptide having polymerase activity and one or more
primers comprising at least a first recombination site or portions
thereof; (b) incubating said mixture under conditions sufficient to
synthesize a first nucleic acid molecule which is complementary to
all or a portion of said one or more templates and which comprises
at least said first recombination site or portions thereof; and (c)
incubating said first nucleic acid molecule in the presence of one
or more primers comprising at least a second recombination site or
portions thereof under conditions sufficient to synthesize a second
nucleic acid molecule complementary to all or a portion of said
first nucleic acid molecule, thereby producing a double stranded
nucleic acid molecule comprising at least said first and second
recombination sites or portions thereof, wherein at least one of
said first and second recombination sites comprises one or more
mutations that avoids hairpin formation in said recombination
sites.
3. A method for synthesizing a double stranded nucleic acid
molecule comprising: (a) mixing one or more nucleic acid templates
with a polypeptide having polymerase activity and one or more
primers comprising at least a first recombination site or portions
thereof; (b) incubating said mixture under conditions sufficient to
synthesize a first nucleic acid molecule which is complementary to
all or a portion of said one or more templates and which comprises
at least said first recombination site or portions thereof; and (c)
incubating said first nucleic acid molecule in the presence of one
or more primers comprising at least a second recombination site or
portions thereof under conditions sufficient to synthesize a second
nucleic acid molecule complementary to all or a portion of said
first nucleic acid molecule, thereby producing a double stranded
nucleic acid molecule comprising at least said first and second
recombination sites or portions thereof, wherein at least one of
said first and second recombination sites comprises at least one
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 1-16 or a DNA sequence complementary thereto.
4. The method of claim 1, wherein said recombination sites or
portions thereof are located at or near one terminus of said double
stranded nucleic acid molecule.
5. The method of claim 2, wherein said recombination sites or
portions thereof are located at or near one terminus of said double
stranded nucleic acid molecule.
6. The method of claim 3, wherein said recombination sites or
portions thereof are located at or near one terminus of said double
stranded nucleic acid molecule.
7. The method of claim 1, wherein said first or second
recombination sites are selected from the group consisting of attB
sites, attP sites, attL sites, attR sites, lox sites, and portions
thereof.
8. The method of claim 2, wherein said first or second
recombination sites are selected from the group consisting of attB
sites, attP sites, attL sites, attR sites, lox sites, and portions
thereof.
9. The method of claim 3, wherein said first or second
recombination sites are selected from the group consisting of attB
sites, attP sites, attL sites, attR sites, lox sites, and portions
thereof.
10. The method of claim 1, further comprising amplifying said first
and second nucleic acid molecules.
11. The method of claim 2, further comprising amplifying said first
and second nucleic acid molecules.
12. The method of claim 3, further comprising amplifying said first
and second nucleic acid molecules.
13. The method of claim 1, wherein said recombination sites or
portions thereof are located at or near one or both termini of said
double stranded nucleic acid molecule.
14. The method of claim 2, wherein said recombination sites or
portions thereof are located at or near one or both termini of said
double stranded nucleic acid molecule.
15. The method of claim 3, wherein said recombination sites or
portions thereof are located at or near one or both termini of said
double stranded nucleic acid molecule.
16. The method of claim 1, wherein said first and second
recombination sites do not recombine with each other.
17. The method of claim 2, wherein said first and second
recombination sites do not recombine with each other.
18. The method of claim 3, wherein said first and second
recombination sites do not recombine with each other.
19. A method for synthesizing a double stranded nucleic acid
molecule comprising: (a) mixing one or more nucleic acid templates
with a polypeptide having polymerase activity and one or more
primers comprising at least a first recombination site or portions
thereof; (b) incubating said mixture under conditions sufficient to
synthesize a first nucleic acid molecule which is complementary to
all or a portion of said one or more templates and which comprises
at least said first recombination site or portions thereof; and (c)
incubating said first nucleic acid molecule in the presence of one
or more primers comprising at least a second recombination site or
portions thereof under conditions sufficient to synthesize a second
nucleic acid molecule complementary to all or a portion of said
first nucleic acid molecule, thereby producing a double stranded
nucleic acid molecule comprising at least said first and second
recombination sites or portions thereof, wherein at least one of
said first and second recombination sites comprises at least one
nucleotide sequence that has at least 80-99% homology to a
nucleotide sequence selected from the group of sequences consisting
of SEQ ID NOs: 1-16, and a corresponding or complementary DNA or
RNA sequence.
20. The method of claim 19, wherein said recombination sites or
portions thereof are located at or near one terminus of said double
stranded nucleic acid molecule.
21. The method of claim 19, further comprising amplifying said
first and second nucleic acid molecules.
22. The method of claim 19, wherein said recombination sites or
portions thereof are located at or near one or both termini of said
double stranded nucleic acid molecule.
23. The method of claim 19, wherein said first and second
recombination sites do not recombine with each other.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of, and claims the
benefit under 35 U.S.C. .sctn. 120 of, U.S. application Ser. No.
10/162,879, filed Jun. 6, 2002, which is a continuation of, and
claims the benefit under 35 U.S.C. .sctn. 120 of, U.S. application
Ser. No. 09/432,085, filed Nov. 2, 1999, which is a divisional of,
and claims the benefit under 35 U.S.C. .sctn. 120 of, U.S.
application Ser. No. 09/233,493, filed Jan. 20, 1999 (now U.S. Pat.
No. 6,143,557), which is a continuation of, and claims the benefit
under 35 U.S.C. .sctn. 120 of, U.S. application Ser. No.
08/663,002, filed Jun. 7, 1996 (now U.S. Pat. No. 5,888,732), which
is a continuation-in-part of, and claims the benefit under 35
U.S.C. .sctn. 120 of, U.S. application Ser. No. 08/486,139, filed
Jun. 7, 1995 (now abandoned), which applications are entirely
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to recombinant DNA technology.
DNA and vectors having engineered recombination sites are provided
for use in a recombinational cloning method that enables efficient
and specific recombination of DNA segments using recombination
proteins. The DNAs, vectors and methods are useful for a variety of
DNA exchanges, such as subcloning of DNA, in vitro or in vivo.
[0004] 2. Related Art
[0005] Site specific recombinases. Site specific recombinases are
enzymes that are present in some viruses and bacteria and have been
characterized to have both endonuclease and ligase properties.
These recombinases (along with associated proteins in some cases)
recognize specific sequences of bases in DNA and exchange the DNA
segments flanking those segments. The recombinases and associated
proteins are collectively referred to as "recombination proteins"
(see, e.g., Landy, A., Current Opinion in Biotechnology 3:699-707
(1993)).
[0006] Numerous recombination systems from various organisms have
been described. See, e.g., Hoess et al., Nucleic Acids Research
14(6):2287 (1986); Abremski et al., J Biol. Chem. 261(1):391
(1986); Campbell, J. Bacteriol. 174(23):7495 (1992); Qian et al., J
Biol. Chem. 267(11):7794 (1992); Araki et al., J. Mol. Biol.
225(l):25 (1992); Maeser and Kahnmann (1991) Mol. Gen. Genet.
230:170-176).
[0007] Many of these belong to the integrase family of recombinases
(Argos et al. EMBO J. 5:433-440 (1986)). Perhaps the best studied
of these are the Integrase/att system from bacteriophage .lamda.
(Landy, A. Current Opinions in Genetics and Devel. 3:699-707
(1993)), the Cre/loxP system from bacteriophage P1 (Hoess and
Abremski (1990) In Nucleic Acids and Molecular Biology, vol. 4.
Eds.: Eckstein and Lilley, Berlin-Heidelberg: Springer-Verlag; pp.
90-109), and the FLP/FRT system from the Saccharomyces cerevisiae
2.mu. circle plasmid (Broach et al. Cell 29:227-234 (1982)).
[0008] Backman (U.S. Pat. No. 4,673,640) discloses the in vivo use
of .lamda. recombinase to recombine a protein producing DNA segment
by enzymatic site-specific recombination using wild-type
recombination sites attB and attP.
[0009] Hasan and Szybalski (Gene 56:145-151 (1987)) discloses the
use of .lamda. Int recombinase in vivo for intramolecular
recombination between wild type attP and attB sites which flank a
promoter. Because the orientations of these sites are inverted
relative to each other, this causes an irreversible flipping of the
promoter region relative to the gene of interest.
[0010] Palazzolo et al. Gene 88:25-36 (1990), discloses phage
lambda vectors having bacteriophage .lamda. arms that contain
restriction sites positioned outside a cloned DNA sequence and
between wild-type loxP sites. Infection of E. coli cells that
express the Cre recombinase with these phage vectors results in
recombination between the loxP sites and the in vivo excision of
the plasmid replicon, including the cloned cDNA.
[0011] Posfai et al. (Nucl. Acids Res. 22:2392-2398 (1994))
discloses a method for inserting into genomic DNA partial
expression vectors having a selectable marker, flanked by two
wild-type FRT recognition sequences. FLP site-specific recombinase
as present in the cells is used to integrate the vectors into the
genome at predetermined sites. Under conditions where the replicon
is functional, this cloned genomic DNA can be amplified.
[0012] Bebee et al. (U.S. Pat. No. 5,434,066) discloses the use of
site-specific recombinases such as Cre for DNA containing two loxP
sites is used for in vivo recombination between the sites.
[0013] Boyd (Nucl. Acids Res. 21:817-821 (1993)) discloses a method
to facilitate the cloning of blunt-ended DNA using conditions that
encourage intermolecular ligation to a dephosphorylated vector that
contains a wild-type loxP site acted upon by a Cre site-specific
recombinase present in E. coli host cells.
[0014] Waterhouse et al. (PCT No. 93/19172 and Nucleic Acids Res.
21 (9):2265 (1993)) disclose an in vivo method where light and
heavy chains of a particular antibody were cloned in different
phage vectors between loxP and loxP 511 sites and used to transfect
new E. coli cells. Cre, acting in the host cells on the two
parental molecules (one plasmid, one phage), produced four products
in equilibrium: two different cointegrates (produced by
recombination at either loxP or loxP 511 sites), and two daughter
molecules, one of which was the desired product.
[0015] In contrast to the other related art, Schlake & Bode
(Biochemistry 33:12746-12751 (1994)) discloses an in vivo method to
exchange expression cassettes at defined chromosomal locations,
each flanked by a wild type and a spacer-mutated FRT recombination
site. A double-reciprocal crossover was mediated in cultured
mammalian cells by using this FLP/FRT system for site-specific
recombination.
[0016] Transposases. The family of enzymes, the transposases, has
also been used to transfer genetic information between replicons.
Transposons are structurally variable, being described as simple or
compound, but typically encode the recombinase gene flanked by DNA
sequences organized in inverted orientations. Integration of
transposons can be random or highly specific. Representatives such
as Tn7, which are highly site-specific, have been applied to the in
vivo movement of DNA segments between replicons (Lucklow et al., J.
Virol. 67:4566-4579 (1993)).
[0017] Devine and Boeke Nucl. Acids Res. 22:3765-3772 (1994),
discloses the construction of artificial transposons for the
insertion of DNA segments, in vitro, into recipient DNA molecules.
The system makes use of the integrase of yeast TY1 virus-like
particles. The DNA segment of interest is cloned, using standard
methods, between the ends of the transposon-like element TY1. In
the presence of the TY1 integrase, the resulting element integrates
randomly into a second target DNA molecule.
[0018] DNA cloning. The cloning of DNA segments currently occurs as
a daily routine in many research labs and as a prerequisite step in
many genetic analyses. The purpose of these clonings is various,
however, two general purposes can be considered: (1) the initial
cloning of DNA from large DNA or RNA segments (chromosomes, YACs,
PCR fragments, mRNA, etc.), done in a relative handful of known
vectors such as pUC, pGem, pBlueScript, and (2) the subcloning of
these DNA segments into specialized vectors for functional
analysis. A great deal of time and effort is expended both in the
initial cloning of DNA segments and in the transfer of DNA segments
from the initial cloning vectors to the more specialized vectors.
This transfer is called subcloning.
[0019] The basic methods for cloning have been known for many years
and have changed little during that time. A typical cloning
protocol is as follows: [0020] (1) digest the DNA of interest with
one or two restriction enzymes; [0021] (2) gel purify the DNA
segment of interest when known; [0022] (3) prepare the vector by
cutting with appropriate restriction enzymes, treating with
alkaline phosphatase, gel purify etc., as appropriate; [0023] (4)
ligate the DNA segment to vector, with appropriate controls to
estimate background of uncut and self-ligated vector; [0024] (5)
introduce the resulting vector into an E. coli host cell; [0025]
(6) pick selected colonies and grow small cultures overnight;
[0026] (7) make DNA minipreps; and [0027] (8) analyze the isolated
plasmid on agarose gels (often after diagnostic restriction enzyme
digestions) or by PCR.
[0028] The specialized vectors used for subcloning DNA segments are
functionally diverse. These include but are not limited to: vectors
for expressing genes in various organisms; for regulating gene
expression; for providing tags to aid in protein purification or to
allow tracking of proteins in cells; for modifying the cloned DNA
segment (e.g., generating deletions); for the synthesis of probes
(e.g., riboprobes); for the preparation of templates for DNA
sequencing; for the identification of protein coding regions; for
the fusion of various protein-coding regions; to provide large
amounts of the DNA of interest, etc. It is common that a particular
investigation will involve subcloning the DNA segment of interest
into several different specialized vectors.
[0029] As known in the art, simple subclonings can be done in one
day (e.g., the DNA segment is not large and the restriction sites
are compatible with those of the subcloning vector). However, many
other subclonings can take several weeks, especially those
involving unknown sequences, long fragments, toxic genes,
unsuitable placement of restriction sites, high backgrounds, impure
enzymes, etc. Subcloning DNA fragments is thus often viewed as a
chore to be done as few times as possible.
[0030] Several methods for facilitating the cloning of DNA segments
have been described, e.g., as in the following references.
[0031] Ferguson, J., et al. Gene 16:191 (1981), discloses a family
of vectors for subcloning fragments of yeast DNA. The vectors
encode kanamycin resistance. Clones of longer yeast DNA segments
can be partially digested and ligated into the subcloning vectors.
If the original cloning vector conveys resistance to ampicillin, no
purification is necessary prior to transformation, since the
selection will be for kanamycin.
[0032] Hashimoto-Gotoh, T., et al. Gene 41:125 (1986), discloses a
subcloning vector with unique cloning sites within a streptomycin
sensitivity gene; in a streptomycin-resistant host, only plasmids
with inserts or deletions in the dominant sensitivity gene will
survive streptomycin selection.
[0033] Accordingly, traditional subcloning methods, using
restriction enzymes and ligase, are time consuming and relatively
unreliable. Considerable labor is expended, and if two or more days
later the desired subclone can not be found among the candidate
plasmids, the entire process must then be repeated with alternative
conditions attempted. Although site specific recombinases have been
used to recombine DNA in vivo, the successful use of such enzymes
in vitro was expected to suffer from several problems. For example,
the site specificities and efficiencies were expected to differ in
vitro; topologically-linked products were expected; and the
topology of the DNA substrates and recombination proteins was
expected to differ significantly in vitro (see, e.g., Adams et al,
J. Mol. Biol. 226:661-73 (1992)). Reactions that could go on for
many hours in vivo were expected to occur in significantly less
time in vitro before the enzymes became inactive. Multiple DNA
recombination products were expected in the biological host used,
resulting in unsatisfactory reliability, specificity or efficiency
of subcloning. In vitro recombination reactions were not expected
to be sufficiently efficient to yield the desired levels of
product.
[0034] Accordingly, there is a long felt need to provide an
alternative subcloning system that provides advantages over the
known use of restriction enzymes and ligases.
SUMMARY OF THE INVENTION
[0035] The present invention provides nucleic acid, vectors and
methods for obtaining chimeric nucleic acid using recombination
proteins and engineered recombination sites, in vitro or in vivo.
These methods are highly specific, rapid, and less labor intensive
than what is disclosed or suggested in the related background art.
The improved specificity, speed and yields of the present invention
facilitates DNA or RNA subcloning, regulation or exchange useful
for any related purpose. Such purposes include in vitro
recombination of DNA segments and in vitro or in vivo insertion or
modification of transcribed, replicated, isolated or genomic DNA or
RNA.
[0036] The present invention relates to nucleic acids, vectors and
methods for moving or exchanging segments of DNA using at least one
engineered recombination site and at least one recombination
protein to provide chimeric DNA molecules which have the desired
characteristic(s) and/or DNA segment(s). Generally, one or more
parent DNA molecules are recombined to give one or more daughter
molecules, at least one of which is the desired Product DNA segment
or vector. The invention thus relates to DNA, RNA, vectors and
methods to effect the exchange and/or to select for one or more
desired products.
[0037] One embodiment of the present invention relates to a method
of making chimeric DNA, which comprises
[0038] (a) combining in vitro or in vivo [0039] (i) an Insert Donor
DNA molecule, comprising a desired DNA segment flanked by a first
recombination site and a second recombination site, wherein the
first and second recombination sites do not recombine with each
other; [0040] (ii) a Vector Donor DNA molecule containing a third
recombination site and a fourth recombination site, wherein the
third and fourth recombination sites do not recombine with each
other; and [0041] (iii) one or more site specific recombination
proteins capable of recombining the first and third recombinational
sites and/or the second and fourth recombinational sites;
[0042] thereby allowing recombination to occur, so as to produce at
least one Cointegrate DNA molecule, at least one desired Product
DNA molecule which comprises said desired DNA segment, and
optionally a Byproduct DNA molecule; and then, optionally,
[0043] (b) selecting for the Product or Byproduct DNA molecule.
[0044] Another embodiment of the present invention relates to a kit
comprising a carrier or receptacle being compartmentalized to
receive and hold therein at least one container, wherein a first
container contains a DNA molecule comprising a vector having at
least two recombination sites flanking a cloning site or a
Selectable marker, as described herein. The kit optionally further
comprises: [0045] (i) a second container containing a Vector Donor
plasmid comprising a subcloning vector and/or a Selectable marker
of which one or both are flanked by one or more engineered
recombination sites; and/or [0046] (ii) a third container
containing at least one recombination protein which recognizes and
is capable of recombining at least one of said recombination
sites.
[0047] Other embodiments include DNA and vectors useful in the
methods of the present invention. In particular, Vector Donor
molecules are provided in one embodiment, wherein DNA segments
within the Vector Donor are separated either by, (i) in a circular
Vector Donor, at least two recombination sites, or (ii) in a linear
Vector Donor, at least one recombination site, where the
recombination sites are preferably engineered to enhance
specificity or efficiency of recombination.
[0048] One Vector Donor embodiment comprises a first DNA segment
and a second DNA segment, the first or second segment comprising a
Selectable marker. A second Vector Donor embodiment comprises a
first DNA segment and a second DNA segment, the first or second DNA
segment comprising a toxic gene. A third Vector Donor embodiment
comprises a first DNA segment and a second DNA segment, the first
or second DNA segment comprising an inactive fragment of at least
one Selectable marker, wherein the inactive fragment of the
Selectable marker is capable of reconstituting a functional
Selectable marker when recombined across the first or second
recombination site with another inactive fragment of at least one
Selectable marker.
[0049] The present recombinational cloning method possesses several
advantages over previous in vivo methods. Since single molecules of
recombination products can be introduced into a biological host,
propagation of the desired Product DNA in the absence of other DNA
molecules (e.g., starting molecules, intermediates, and
by-products) is more readily realized. Reaction conditions can be
freely adjusted in vitro to optimize enzyme activities. DNA
molecules can be incompatible with the desired biological host
(e.g., YACs, genomic DNA, etc.), can be used. Recombination
proteins from diverse sources can be employed, together or
sequentially.
[0050] Other embodiments will be evident to those of ordinary skill
in the art from the teachings contained herein in combination with
what is known to the art.
BRIEF DESCRIPTION OF THE FIGURES
[0051] FIG. 1 depicts one general method of the present invention,
wherein the starting (parent) DNA molecules can be circular or
linear. The goal is to exchange the new subcloning vector D for the
original cloning vector B. It is desirable in one embodiment to
select for AD and against all the other molecules, including the
Cointegrate. The square and circle are sites of recombination:
e.g., loxP sites, att sites, etc. For example, segment D can
contain expression signals, new drug markers, new origins of
replication, or specialized functions for mapping or sequencing
DNA.
[0052] FIG. 2A depicts an in vitro method of recombining an Insert
Donor plasmid (here, pEZC705) with a Vector Donor plasmid (here,
pEZC726), and obtaining Product DNA and Byproduct daughter
molecules. The two recombination sites are attP and loxP on the
Vector Donor. On one segment defined by these sites is a kanamycin
resistance gene whose promoter has been replaced by the tetOP
operator/promoter from transposon Tn10. See Sizemore et al., Nucl.
Acids Res. 18(10):2875 (1990). In the absence of tet repressor
protein, E. coli RNA polymerase transcribes the kanamycin
resistance gene from the tetOP. If tet repressor is present, it
binds to tetOP and blocks transcription of the kanamycin resistance
gene. The other segment of pEZC726 has the tet repressor gene
expressed by a constitutive promoter. Thus cells transformed by
pEZC726 are resistant to chloramphenicol, because of the
chloramphenicol acetyl transferase gene on the same segment as
tetR, but are sensitive to kanamycin. The recombinase-mediated
reactions result in separation of the tetR gene from the regulated
kanamycin resistance gene. This separation results in kanamycin
resistance in cells receiving only the desired recombination
products. The first recombination reaction is driven by the
addition of the recombinase called Integrase. The second
recombination reaction is driven by adding the recombinase Cre to
the Cointegrate (here, pEZC7 Cointegrate).
[0053] FIG. 2B depicts a restriction map of pEZC705.
[0054] FIG. 2C depicts a restriction map of pEZC726.
[0055] FIG. 2D depicts a restriction map of pEZC7 Cointegrate.
[0056] FIG. 2E depicts a restriction map of Intprod.
[0057] FIG. 2F depicts a restriction map of Intbypro.
[0058] FIG. 3A depicts an in vitro method of recombining an Insert
Donor plasmid (here, pEZC602) with a Vector Donor plasmid (here,
pEZC629), and obtaining Product (here, EZC6prod) and Byproduct
(here, EZC6Bypr) daughter molecules. The two recombination sites
are loxP and loxP 511. One segment of pEZC629 defined by these
sites is a kanamycin resistance gene whose promoter has been
replaced by the tetOP operator/promoter from transposon Tn10. In
the absence of tet repressor protein, E. coli RNA polymerase
transcribes the kanamycin resistance gene from the tetOP. If tet
repressor is present, it binds to tetOP and blocks transcription of
the kanamycin resistance gene. The other segment of pEZC629 has the
tet repressor gene expressed by a constitutive promoter. Thus cells
transformed by pEZC629 are resistant to chloramphenicol, because of
the chloramphenicol acetyl transferase gene on the same segment as
tetR, but are sensitive to kanamycin. The reactions result in
separation of the tetR gene from the regulated kanamycin resistance
gene. This separation results in kanamycin resistance in cells
receiving the desired recombination product. The first and the
second recombination events are driven by the addition of the same
recombinase, Cre.
[0059] FIG. 3B depicts a restriction map of EZC6Bypr.
[0060] FIG. 3C depicts a restriction map of EZC6prod.
[0061] FIG. 3D depicts a restriction map of pEZC602.
[0062] FIG. 3E depicts a restriction map of pEZC629.
[0063] FIG. 3F depicts a restriction map of EZC6coint.
[0064] FIG. 4A depicts an application of the in vitro method of
recombinational cloning to subclone the chloramphenicol acetyl
transferase gene into a vector for expression in eukaryotic cells.
The Insert Donor plasmid, pEZC843, is comprised of the
chloramphenicol acetyl transferase gene of E. coli, cloned between
loxP and attB sites such that the loxP site is positioned at the
5'-end of the gene. The Vector Donor plasmid, pEZC1003, contains
the cytomegalovirus eukaryotic promoter apposed to a loxP site. The
supercoiled plasmids were combined with lambda Integrase and Cre
recombinase in vitro. After incubation, competent E. coli cells
were transformed with the recombinational reaction solution.
Aliquots of transformations were spread on agar plates containing
kanamycin to select for the Product molecule (here CMVProd).
[0065] FIG. 4B depicts a restriction map of pEZC843.
[0066] FIG. 4C depicts a restriction map of pEZC1003.
[0067] FIG. 4D depicts a restriction map of CMVBypro.
[0068] FIG. 4E depicts a restriction map of CMVProd.
[0069] FIG. 4F depicts a restriction map of CMVcoint.
[0070] FIG. 5A depicts a vector diagram of pEZC1301.
[0071] FIG. 5B depicts a vector diagram of pEZC1305.
[0072] FIG. 5C depicts a vector diagram of pEZC1309.
[0073] FIG. 5D depicts a vector diagram of pEZC1313.
[0074] FIG. 5E depicts a vector diagram of pEZC1317.
[0075] FIG. 5F depicts a vector diagram of pEZC1321.
[0076] FIG. 5G depicts a vector diagram of pEZC1405.
[0077] FIG. 5H depicts a vector diagram of pEZC1502.
[0078] FIG. 6A depicts a vector diagram of pEZC1603.
[0079] FIG. 6B depicts a vector diagram of pEZC1706.
[0080] FIG. 7A depicts a vector diagram of pEZC2901.
[0081] FIG. 7B depicts a vector diagram of pEZC2913
[0082] FIG. 7C depicts a vector diagram of pEZC3101.
[0083] FIG. 7D depicts a vector diagram of pEZC1802.
[0084] FIG. 8A depicts a vector diagram of pGEX-2TK.
[0085] FIG. 8B depicts a vector diagram of pEZC3501.
[0086] FIG. 8C depicts a vector diagram of pEZC3601.
[0087] FIG. 8D depicts a vector diagram of pEZC3609.
[0088] FIG. 8E depicts a vector diagram of pEZC3617.
[0089] FIG. 8F depicts a vector diagram of pEZC3606.
[0090] FIG. 8G depicts a vector diagram of pEZC3613.
[0091] FIG. 8H depicts a vector diagram of pEZC3621.
[0092] FIG. 8I depicts a vector diagram of GST-CAT.
[0093] FIG. 8J depicts a vector diagram of GST-phoA.
[0094] FIG. 8K depicts a vector diagram of pEZC3201.
DETAILED DESCRIPTION OF THE INVENTION
[0095] It is unexpectedly discovered in the present invention that
subcloning reactions can be provided using recombinational cloning.
Recombination cloning according to the present invention uses DNAs,
vectors and methods, in vitro and in vivo, for moving or exchanging
segments of DNA molecules using engineered recombination sites and
recombination proteins. These methods provide chimeric DNA
molecules that have the desired characteristic(s) and/or DNA
segment(s).
[0096] The present invention thus provides nucleic acid, vectors
and methods for obtaining chimeric nucleic acid using recombination
proteins and engineered recombination sites, in vitro or in vivo.
These methods are highly specific, rapid, and less labor intensive
than what is disclosed or suggested in the related background art.
The improved specificity, speed and yields of the present invention
facilitates DNA or RNA subcloning, regulation or exchange useful
for any related purpose. Such purposes include in vitro
recombination of DNA segments and in vitro or in vivo insertion or
modification of transcribed, replicated, isolated or genomic DNA or
RNA.
Definitions
[0097] In the description that follows, a number of terms used in
recombinant DNA technology are utilized extensively. In order to
provide a clear and consistent understanding of the specification
and claims, including the scope to be given such terms, the
following definitions are provided.
[0098] Byproduct: is a daughter molecule (a new clone produced
after the second recombination event during the recombinational
cloning process) lacking the DNA which is desired to be
subcloned.
[0099] Cointegrate: is at least one recombination intermediate DNA
molecule of the present invention that contains both parental
(starting) DNA molecules. It will usually be circular. In some
embodiments it can be linear.
[0100] Host: is any prokaryotic or eukaryotic organism that can be
a recipient of the recombinational cloning Product. A "host," as
the term is used herein, includes prokaryotic or eukaryotic
organisms that can be genetically engineered. For examples of such
hosts, see Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).
[0101] Insert: is the desired DNA segment (segment A of FIG. 1)
which one wishes to manipulate by the method of the present
invention. The insert can have one or more genes.
[0102] Insert Donor: is one of the two parental DNA molecules of
the present invention which carries the Insert. The Insert Donor
DNA molecule comprises the Insert flanked on both sides with
recombination signals. The Insert Donor can be linear or circular.
In one embodiment of the invention, the Insert Donor is a circular
DNA molecule and further comprises a cloning vector sequence
outside of the recombination signals (see FIG. 1).
[0103] Product: is one or both the desired daughter molecules
comprising the A and D or B and C sequences which are produced
after the second recombination event during the recombinational
cloning process (see FIG. 1). The Product contains the DNA which
was to be cloned or subcloned.
[0104] Promoter: is a DNA sequence generally described as the
5'-region of a gene, located proximal to the start codon. The
transcription of an adjacent DNA segment is initiated at the
promoter region. A repressible promoter's rate of transcription
decreases in response to a repressing agent. An inducible
promoter's rate of transcription increases in response to an
inducing agent. A constitutive promoter's rate of transcription is
not specifically regulated, though it can vary under the influence
of general metabolic conditions.
[0105] Recognition sequence: Recognition sequences are particular
DNA sequences which a protein, DNA, or RNA molecule (e.g.,
restriction endonuclease, a modification methylase, or a
recombinase) recognizes and binds. For example, the recognition
sequence for Cre recombinase is loxP which is a 34 base pair
sequence comprised of two 13 base pair inverted repeats (serving as
the recombinase binding sites) flanking an 8 base pair core
sequence. See FIG. 1 of Sauer, B., Current Opinion in Biotechnology
5:521-527 (1994). Other examples of recognition sequences are the
attB, attP, attL, and attR sequences which are recognized by the
recombinase enzyme .lamda. Integrase. attB is an approximately 25
base pair sequence containing two 9 base pair core-type Int binding
sites and a 7 base pair overlap region. attP is an approximately
240 base pair sequence containing core-type Int binding sites and
arm-type Int binding sites as well as sites for auxiliary proteins
IHF, FIS, and Xis. See Landy, Current Opinion in Biotechnology
3:699-707 (1993). Such sites are also engineered according to the
present invention to enhance methods and products.
[0106] Recombinase: is an enzyme which catalyzes the exchange of
DNA segments at specific recombination sites.
[0107] Recombinational Cloning: is a method described herein,
whereby segments of DNA molecules are exchanged, inserted,
replaced, substituted or modified, in vitro or in vivo.
[0108] Recombination proteins: include excisive or integrative
proteins, enzymes, co-factors or associated proteins that are
involved in recombination reactions involving one or more
recombination sites. See, Landy (1994), infra.
[0109] Repression cassette: is a DNA segment that contains a
repressor of a Selectable marker present in the subcloning
vector.
[0110] Selectable marker: is a DNA segment that allows one to
select for or against a molecule or a cell that contains it, often
under particular conditions. These markers can encode an activity,
such as, but not limited to, production of RNA, peptide, or
protein, or can provide a binding site for RNA, peptides, proteins,
inorganic and organic compounds or compositions and the like.
Examples of Selectable markers include but are not limited to: (1)
DNA segments that encode products which provide resistance against
otherwise toxic compounds (e.g., antibiotics); (2) DNA segments
that encode products which are otherwise lacking in the recipient
cell (e.g., tRNA genes, auxotrophic markers); (3) DNA segments that
encode products which suppress the activity of a gene product; (4)
DNA segments that encode products which can be readily identified
(e.g., phenotypic markers such as .beta.-galactosidase, green
fluorescent protein (GFP), and cell surface proteins); (5) DNA
segments that bind products which are otherwise detrimental to cell
survival and/or function; (6) DNA segments that otherwise inhibit
the activity of any of the DNA segments described in Nos. 1-5 above
(e.g., antisense oligonucleotides); (7) DNA segments that bind
products that modify a substrate (e.g. restriction endonucleases);
(8) DNA segments that can be used to isolate a desired molecule
(e.g. specific protein binding sites); (9) DNA segments that encode
a specific nucleotide sequence which can be otherwise
non-functional (e.g., for PCR amplification of subpopulations of
molecules); and/or (10) DNA segments, which when absent, directly
or indirectly confer sensitivity to particular compounds.
[0111] Selection scheme: is any method which allows selection,
enrichment, or identification of a desired Product or Product(s)
from a mixture containing the Insert Donor, Vector Donor, and/or
any intermediates, (e.g. a Cointegrate) Byproducts. The selection
schemes of one preferred embodiment have at least two components
that are either linked or unlinked during recombinational cloning.
One component is a Selectable marker. The other component controls
the expression in vitro or in vivo of the Selectable marker, or
survival of the cell harboring the plasmid carrying the Selectable
marker. Generally, this controlling element will be a repressor or
inducer of the Selectable marker, but other means for controlling
expression of the Selectable marker can be used. Whether a
repressor or activator is used will depend on whether the marker is
for a positive or negative selection, and the exact arrangement of
the various DNA segments, as will be readily apparent to those
skilled in the art. A preferred requirement is that the selection
scheme results in selection of or enrichment for only one or more
desired Products. As defined herein, to select for a DNA molecule
includes (a) selecting or enriching for the presence of the desired
DNA molecule, and (b) selecting or enriching against the presence
of DNA molecules that are not the desired DNA molecule.
[0112] In one embodiment, the selection schemes (which can be
carried out reversed) will take one of three forms, which will be
discussed in terms of FIG. 1. The first, exemplified herein with a
Selectable marker and a repressor therefor, selects for molecules
having segment D and lacking segment C. The second selects against
molecules having segment C and for molecules having segment D.
Possible embodiments of the second form would have a DNA segment
carrying a gene toxic to cells into which the in vitro reaction
products are to be introduced. A toxic gene can be a DNA that is
expressed as a toxic gene product (a toxic protein or RNA), or can
be toxic in and of itself. (In the latter case, the toxic gene is
understood to carry its classical definition of "heritable
trait".)
[0113] Examples of such toxic gene products are well known in the
art, and include, but are not limited to, restriction endonucleases
(e.g., DpnI) and genes that kill hosts in the absence of a
suppressing function, e.g., kicB. A toxic gene can alternatively be
selectable in vitro, e.g., a restriction site.
[0114] In the second form, segment D carries a Selectable marker.
The toxic gene would eliminate transformants harboring the Vector
Donor, Cointegrate, and Byproduct molecules, while the Selectable
marker can be used to select for cells containing the Product and
against cells harboring only the Insert Donor.
[0115] The third form selects for cells that have both segments A
and D in cis on the same molecule, but not for cells that have both
segments in trans on different molecules. This could be embodied by
a Selectable marker that is split into two inactive fragments, one
each on segments A and D.
[0116] The fragments are so arranged relative to the recombination
sites that when the segments are brought together by the
recombination event, they reconstitute a functional Selectable
marker. For example, the recombinational event can link a promoter
with a structural gene, can link two fragments of a structural
gene, or can link genes that encode a heterodimeric gene product
needed for survival, or can link portions of a replicon.
[0117] Site-specific recombinase: is a type of recombinase which
typically has at least the following four activities: (1)
recognition of one or two specific DNA sequences; (2) cleavage of
said DNA sequence or sequences; (3) DNA topoisomerase activity
involved in strand exchange; and (4) DNA ligase activity to reseal
the cleaved strands of DNA. See Sauer, B., Current Opinions in
Biotechnology 5:521-527 (1994). Conservative site-specific
recombination is distinguished from homologous recombination and
transposition by a high degree of specificity for both partners.
The strand exchange mechanism involves the cleavage and rejoining
of specific DNA sequences in the absence of DNA synthesis (Landy,
A. (1989) Ann. Rev. Biochem. 58:913-949).
[0118] Subcloning vector: is a cloning vector comprising a circular
or linear DNA molecule which includes an appropriate replicon. In
the present invention, the subcloning vector (segment D in FIG. 1)
can also contain functional and/or regulatory elements that are
desired to be incorporated into the final product to act upon or
with the cloned DNA Insert (segment A in FIG. 1). The subcloning
vector can also contain a Selectable marker (contained in segment C
in FIG. 1).
[0119] Vector: is a DNA that provides a useful biological or
biochemical property to an Insert. Examples include plasmids,
phages, and other DNA sequences which are able to replicate or be
replicated in vitro or in a host cell, or to convey a desired DNA
segment to a desired location within a host cell. A Vector can have
one or more restriction endonuclease recognition sites at which the
DNA sequences can be cut in a determinable fashion without loss of
an essential biological function of the vector, and into which a
DNA fragment can be spliced in order to bring about its replication
and cloning. Vectors can further provide primer sites, e.g., for
PCR, transcriptional and/or translational initiation and/or
regulation sites, recombinational signals, replicons, Selectable
markers, etc. Clearly, methods of inserting a desired DNA fragment
which do not require the use of homologous recombination or
restriction enzymes (such as, but not limited to, UDG cloning of
PCR fragments (U.S. Pat. No. 5,334,575, entirely incorporated
herein by reference), T:A cloning, and the like) can also be
applied to clone a fragment of DNA into a cloning vector to be used
according to the present invention. The cloning vector can further
contain a Selectable marker suitable for use in the identification
of cells transformed with the cloning vector.
[0120] Vector Donor: is one of the two parental DNA molecules of
the present invention which carries the DNA segments encoding the
DNA vector which is to become part of the desired Product. The
Vector Donor comprises a subcloning vector D (or it can be called
the cloning vector if the Insert Donor does not already contain a
cloning vector) and a segment C flanked by recombination sites (see
FIG. 1). Segments C and/or D can contain elements that contribute
to selection for the desired Product daughter molecule, as
described above for selection schemes. The recombination signals
can be the same or different, and can be acted upon by the same or
different recombinases. In addition, the Vector Donor can be linear
or circular.
Description
[0121] One general scheme for an in vitro or in vivo method of the
invention is shown in FIG. 1, where the Insert Donor and the Vector
Donor can be either circular or linear DNA, but is shown as
circular. Vector D is exchanged for the original cloning vector A.
It is desirable to select for the daughter vector containing
elements A and D and against other molecules, including one or more
Cointegrate(s). The square and circle are different sets of
recombination sites (e.g., lox sites or att sites). Segment A or D
can contain at least one Selection Marker, expression signals,
origins of replication, or specialized functions for detecting,
selecting, expressing, mapping or sequencing DNA, where D is used
in this example.
[0122] Examples of desired DNA segments that can be part of Element
A or D include, but are not limited to, PCR products, large DNA
segments, genomic clones or fragments, cDNA clones, functional
elements, etc., and genes or partial genes, which encode useful
nucleic acids or proteins. Moreover, the recombinational cloning of
the present invention can be used to make ex vivo and in vivo gene
transfer vehicles for protein expression and/or gene therapy.
[0123] In FIG. 1, the scheme provides the desired Product as
containing vectors D and A, as follows. The Insert Donor
(containing A and B) is first recombined at the square
recombination sites by recombination proteins, with the Vector
Donor (containing C and D), to form a Co-integrate having each of
A-D-C-B. Next, recombination occurs at the circle recombination
sites to form Product DNA (A and D) and Byproduct DNA (C and B).
However, if desired, two or more different Co-integrates can be
formed to generate two or more Products.
[0124] In one embodiment of the present in vitro or in vivo
recombinational cloning method, a method for selecting at least one
desired Product DNA is provided. This can be understood by
consideration of the map of plasmid pEZC726 depicted in FIG. 2. The
two exemplary recombination sites are attP and loxP. On one segment
defined by these sites is a kanamycin resistance gene whose
promoter has been replaced by the tetOP operator/promoter from
transposon Tn10. In the absence of tet repressor protein, E. coli
RNA polymerase transcribes the kanamycin resistance gene from the
tetOP. If tet repressor is present, it binds to tetOP and blocks
transcription of the kanamycin resistance gene. The other segment
of pEZC726 has the tet repressor gene expressed by a constitutive
promoter. Thus cells transformed by pEZC726 are resistant to
chloramphenicol, because of the chloramphenicol acetyl transferase
gene on the same segment as tetR, but are sensitive to kanamycin.
The recombination reactions result in separation of the tetR gene
from the regulated kanamycin resistance gene. This separation
results in kanamycin resistance in cells receiving the desired
recombination Product.
[0125] Two different sets of plasmids were constructed to
demonstrate the in vitro method. One set, for use with Cre
recombinase only (cloning vector 602 and subcloning vector 629
(FIG. 3)) contained loxP and loxP 511 sites. A second set, for use
with Cre and integrase (cloning vector 705 and subcloning vector
726 (FIG. 2)) contained loxP and att sites. The efficiency of
production of the desired daughter plasmid was about 60 fold higher
using both enzymes than using Cre alone. Nineteen of twenty four
colonies from the Cre-only reaction contained the desired product,
while thirty eight of thirty eight colonies from the integrase plus
Cre reaction contained the desired product plasmid.
[0126] Other Selection Schemes A variety of selection schemes can
be used that are known in the art as they can suit a particular
purpose for which the recombinational cloning is carried out.
Depending upon individual preferences and needs, a number of
different types of selection schemes can be used in the
recombinational cloning method of the present invention. The
skilled artisan can take advantage of the availability of the many
DNA segments or methods for making them and the different methods
of selection that are routinely used in the art. Such DNA segments
include but are not limited to those which encodes an activity such
as, but not limited to, production of RNA, peptide, or protein, or
providing a binding site for such RNA, peptide, or protein.
Examples of DNA molecules used in devising a selection scheme are
given above, under the definition of "selection scheme"
[0127] Additional examples include but are not limited to: [0128]
(i) Generation of new primer sites for PCR (e.g., juxtaposition of
two DNA sequences that were not previously juxtaposed); [0129] (ii)
Inclusion of a DNA sequence acted upon by a restriction
endonuclease or other DNA modifying enzyme, chemical, ribozyme,
etc.; [0130] (iii) Inclusion of a DNA sequence recognized by a DNA
binding protein, RNA, DNA, chemical, etc.) (e.g., for use as an
affinity tag for selecting for or excluding from a population)
(Davis, Nucl. Acids Res. 24:702-706 (1996); J. Virol. 69: 8027-8034
(1995)); [0131] (iv) In vitro selection of RNA ligands for the
ribosomal L22 protein associated with Epstein-Barr virus-expressed
RNA by using randomized and cDNA-derived RNA libraries; [0132] (vi)
The positioning of functional elements whose activity requires a
specific orientation or juxtaposition (e.g., (a) a recombination
site which reacts poorly in trans, but when placed in cis, in the
presence of the appropriate proteins, results in recombination that
destroys certain populations of molecules; (e.g., reconstitution of
a promoter sequence that allows in vitro RNA synthesis). The RNA
can be used directly, or can be reverse transcribed to obtain the
desired DNA construct; [0133] (vii) Selection of the desired
product by size (e.g., fractionation) or other physical property of
the molecule(s); and [0134] (viii) Inclusion of a DNA sequence
required for a specific modification (e.g., methylation) that
allows its identification.
[0135] After formation of the Product and Byproduct in the method
of the present invention, the selection step can be carried out
either in vitro or in vivo depending upon the particular selection
scheme which has been optionally devised in the particular
recombinational cloning procedure.
[0136] For example, an in vitro method of selection can be devised
for the Insert Donor and Vector Donor DNA molecules. Such scheme
can involve engineering a rare restriction site in the starting
circular vectors in such a way that after the recombination events
the rare cutting sites end up in the Byproduct. Hence, when the
restriction enzyme which binds and cuts at the rare restriction
site is added to the reaction mixture in vitro, all of the DNA
molecules carrying the rare cutting site, i.e., the starting DNA
molecules, the Cointegrate, and the Byproduct, will be cut and
rendered nonreplicable in the intended host cell. For example,
cutting sites in segments B and C (see FIG. 1) can be used to
select against all molecules except the Product. Alternatively,
only a cutting site in C is needed if one is able to select for
segment D, e.g., by a drug resistance gene not found on B.
[0137] Similarly, an in vitro selection method can be devised when
dealing with linear DNA molecules. DNA sequences complementary to a
PCR primer sequence can be so engineered that they are transferred,
through the recombinational cloning method, only to the Product
molecule. After the reactions are completed, the appropriate
primers are added to the reaction solution and the sample is
subjected to PCR. Hence, all or part of the Product molecule is
amplified.
[0138] Other in vivo selection schemes can be used with a variety
of E. coli cell lines. One is to put a repressor gene on one
segment of the subcloning plasmid, and a drug marker controlled by
that repressor on the other segment of the same plasmid. Another is
to put a killer gene on segment C of the subcloning plasmid (FIG.
1). Of course a way must exist for growing such a plasmid, i.e.,
there must exist circumstances under which the killer gene will not
kill. There are a number of these genes known which require
particular strains of E. coli. One such scheme is to use the
restriction enzyme DpnI, which will not cleave unless its
recognition sequence GATC is methylated. Many popular common E.
coli strains methylate GATC sequences, but there are mutants in
which cloned DpnI can be expressed without harm.
[0139] Of course analogous selection schemes can be devised for
other host organisms. For example, the tet repressor/operator of
Tn10 has been adapted to control gene expression in eukaryotes
(Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA
89:5547-5551 (1992)). Thus the same control of drug resistance by
the tet repressor exemplified herein can be applied to select for
Product in eukaryotic cells.
Recombination Proteins
[0140] In the present invention, the exchange of DNA segments is
achieved by the use of recombination proteins, including
recombinases and associated co-factors and proteins. Various
recombination proteins are described in the art. Examples of such
recombinases include:
[0141] Cre: A protein from bacteriophage PI (Abremski and Hoess, J.
Biol. Chem. 259(3):1509-1514 (1984)) catalyzes the exchange (i.e.,
causes recombination) between 34 bp DNA sequences called loxP
(locus of crossover) sites (See Hoess et al., Nucl. Acids Res.
14(5):2287 (1986)). Cre is available commercially (Novagen, Catalog
No. 69247-1). Recombination mediated by Cre is freely reversible.
From thermodynamic considerations it is not surprising that
Cre-mediated integration (recombination between two molecules to
form one molecule) is much less efficient than Cre-mediated
excision (recombination between two loxP sites in the same molecule
to form two daughter molecules). Cre works in simple buffers with
either magnesium or spermidine as a cofactor, as is well known in
the art. The DNA substrates can be either linear or supercoiled. A
number of mutant loxP sites have been described (Hoess et al.,
supra). One of these, loxP 511, recombines with another loxP 511
site, but will not recombine with a loxP site.
[0142] Integrase: A protein from bacteriophage lambda that mediates
the integration of the lambda genome into the E. coli chromosome.
The bacteriophage .lamda. Int recombinational proteins promote
irreversible recombination between its substrate att sites as part
of the formation or induction of a lysogenic state. Reversibility
of the recombination reactions results from two independent
pathways for integrative and excisive recombination. Each pathway
uses a unique, but overlapping, set of the 15 protein binding sites
that comprise att site DNAs. Cooperative and competitive
interactions involving four proteins (Int, Xis, IHF and FIS)
determine the direction of recombination.
[0143] Integrative recombination involves the Int and IHF proteins
and sites attP (240 bp) and attB (25 bp). Recombination results in
the formation of two new sites: attL and attR. Excisive
recombination requires Int, IHF, and Xis, and sites attL and attR
to generate attP and attB. Under certain conditions, FIS stimulates
excisive recombination. In addition to these normal reactions, it
should be appreciated that attP and attB, when placed on the same
molecule, can promote excisive recombination to generate two
excision products, one with attL and one with attR. Similarly,
intermolecular recombination between molecules containing attL and
attR, in the presence of Int, IHF and Xis, can result in
integrative recombination and the generation attP and attB. Hence,
by flanking DNA segments with appropriate combinations of
engineered att sites, in the presence of the appropriate
recombination proteins, one can direct excisive or integrative
recombination, as reverse reactions of each other.
[0144] Each of the att sites contains a 15 bp core sequence;
individual sequence elements of functional significance lie within,
outside, and across the boundaries of this common core (Landy, A.,
Ann. Rev. Biochem. 58:913 (1989)). Efficient recombination between
the various att sites requires that the sequence of the central
common region be identical between the recombining partners,
however, the exact sequence is now found to be modifiable.
Consequently, derivatives of the att site with changes within the
core are now discovered to recombine as least as efficiently as the
native core sequences.
[0145] Integrase acts to recombine the attP site on bacteriophage
lambda (about 240 bp) with the attB site on the E. coli genome
(about 25 bp) (Weisberg, R. A. and Landy, A. in Lambda II, p. 211
(1983), Cold Spring Harbor Laboratory)), to produce the integrated
lambda genome flanked by attL (about 100 bp) and attR (about 160
bp) sites. In the absence of Xis (see below), this reaction is
essentially irreversible. The integration reaction mediated by
integrase and IHF works in vitro, with simple buffer containing
spermidine. Integrase can be obtained as described by Nash, H. A.,
Methods of Enzymology 100:210-216 (1983). IHF can be obtained as
described by Filutowicz, M., et al., Gene 147:149-150 (1994).
[0146] In the presence of the .lamda. protein Xis (excise)
integrase catalyzes the reaction of attR and attL to form attP and
attB, i.e., it promotes the reverse of the reaction described
above. This reaction can also be applied in the present
invention.
[0147] Other Recombination Systems. Numerous recombination systems
from various organisms can also be used, based on the teaching and
guidance provided herein. See, e.g., Hoess et al., Nucleic Acids
Research 14(6):2287 (1986); Abremski et al., J. Biol. Chem.
261(1):391 (1986); Campbell, J. Bacteriol 174(23):7495 (1992); Qian
et al., J. Biol. Chem. 267(11):7794 (1992); Araki et al., J. Mol.
Biol. 225(1):25 (1992)). Many of these belong to the integrase
family of recombinases (Argos et al. EMBO J. 5:433-440 (1986)).
Perhaps the best studied of these are the lntegrase/att system from
bacteriophage .lamda. (Landy, A. (1993) Current Opinions in
Genetics and Devel. 3:699-707), the Cre/loxP system from
bacteriophage P1 (Hoess and Abremski (1990) In Nucleic Acids and
Molecular Biology, vol. 4. Eds.: Eckstein and Lilley,
Berlin-Heidelberg: Springer-Verlag; pp. 90-109), and the FLP/FRT
system from the Saccharomyces cerevisiae 2.mu. circle plasmid
(Broach et al. Cell 29:227-234 (1982)).
[0148] Members of a second family of site-specific recombinases,
the resolvase family (e.g., .gamma..delta., Tn3 resolvase, Hin,
Gin, and Cin) are also known. Members of this highly related family
of recombinases are typically constrained to intramolecular
reactions (e.g., inversions and excisions) and can require
host-encoded factors. Mutants have been isolated that relieve some
of the requirements for host factors (Maeser and Kahnmann (1991)
Mol. Gen. Genet. 230:170-176), as well as some of the constraints
of intramolecular recombination.
[0149] Other site-specific recombinases similar to .lamda. Int and
similar to P1 Cre can be substituted for Int and Cre. Such
recombinases are known. In many cases the purification of such
other recombinases has been described in the art. In cases when
they are not known, cell extracts can be used or the enzymes can be
partially purified using procedures described for Cre and Int.
[0150] While Cre and Int are described in detail for reasons of
example, many related recombinase systems exist and their
application to the described invention is also provided according
to the present invention. The integrase family of site-specific
recombinases can be used to provide alternative recombination
proteins and recombination sites for the present invention, as
site-specific recombination proteins encoded by bacteriophage
lambda, phi 80, P22, P2, 186, P4 and P1. This group of proteins
exhibits an unexpectedly large diversity of sequences. Despite this
diversity, all of the recombinases can be aligned in their
C-terminal halves.
[0151] A 40-residue region near the C terminus is particularly well
conserved in all the proteins and is homologous to a region near
the C terminus of the yeast 2 mu plasmid Flp protein. Three
positions are perfectly conserved within this family: histidine,
arginine and tyrosine are found at respective alignment positions
396, 399 and 433 within the well-conserved C-terminal region. These
residues contribute to the active site of this family of
recombinases, and suggest that tyrosine-433 forms a transient
covalent linkage to DNA during strand cleavage and rejoining. See,
e.g., Argos, P. et al., EMBO J. 5:433-40 (1986).
[0152] Alternatively, IS231 and other Bacillus thuringiensis
transposable elements could be used as recombination proteins and
recombination sites. Bacillus thuringiensis is an entomopathogenic
bacterium whose toxicity is due to the presence in the sporangia of
delta-endotoxin crystals active against agricultural pests and
vectors of human and animal diseases. Most of the genes coding for
these toxin proteins are plasmid-borne and are generally
structurally associated with insertion sequences (IS231, IS232,
IS240, ISBT1 and ISBT2) and transposons (Tn4430 and Tn5401).
Several of these mobile elements have been shown to be active and
participate in the crystal gene mobility, thereby contributing to
the variation of bacterial toxicity.
[0153] Structural analysis of the iso-IS231 elements indicates that
they are related to IS1151 from Clostridium perfringens and
distantly related to IS4 and IS186 from Escherichia coli. Like the
other IS4 family members, they contain a conserved
transposase-integrase motif found in other IS families and
retroviruses.
[0154] Moreover, functional data gathered from IS231A in
Escherichia coli indicate a non-replicative mode of transposition,
with a preference for specific targets. Similar results were also
obtained in Bacillus subtilis and B. thuringiensis. See, e.g.,
Mahillon, J. et al., Genetica 93:13-26 (1994); Campbell, J.
Bacteriol. 7495-7499 (1992).
[0155] The amount of recombinase which is added to drive the
recombination reaction can be determined by using known assays.
Specifically, titration assay is used to determine the appropriate
amount of a purified recombinase enzyme, or the appropriate amount
of an extract.
[0156] Engineered Recombination Sites. The above recombinases and
corresponding recombinase sites are suitable for use in
recombination cloning according to the present invention. However,
wild-type recombination sites contain sequences that reduce the
efficiency or specificity of recombination reactions as applied in
methods of the present invention. For example, multiple stop codons
in attB, attR, attP, attL and loxp recombination sites occur in
multiple reading frames on both strands, so recombination
efficiencies are reducted, e.g., where the coding sequence must
cross the recombination sites, (only one reading frame is available
on each strand of loxP and attB sites) or impossible (in attP, attR
or attL).
[0157] Accordingly, the present invention also provides engineered
recombination sites that overcome these problems. For example, att
sites can be engineered to have one or multiple mutations to
enhance specificity or efficiency of the recombination reaction and
the properties of Product DNAs (e.g., att1, att2, and att3 sites);
to decrease reverse reaction (e.g., removing P1 and H1 from attB).
The testing of these mutants determines which mutants yield
sufficient recombinational activity to be suitable for
recombination subcloning according to the present invention.
[0158] Mutations can therefore be introduced into recombination
sites for enhancing site specific recombination. Such mutations
include, but are not limited to: recombination sites without
translation stop codons that allow fusion proteins to be encoded;
recombination sites recognized by the same proteins but differing
in base sequence such that they react largely or exclusively with
their homologous partners allow multiple reactions to be
contemplated. Which particular reactions take place can be
specified by which particular partners are present in the reaction
mixture. For example, a tripartite protein fusion could be
accomplished with parental plasmids containing recombination sites
attR1 and attR2; attL1 and attL3; and/or attR3 and attL2.
[0159] There are well known procedures for introducing specific
mutations into nucleic acid sequences. A number of these are
described in Ausubel, F. M. et al., Current Protocols in Molecular
Biology, Wiley Interscience, New York (1989-1996). Mutations can be
designed into oligonucleotides, which can be used to modify
existing cloned sequences, or in amplification reactions. Random
mutagenesis can also be employed if appropriate selection methods
are available to isolate the desired mutant DNA or RNA. The
presence of the desired mutations can be confirmed by sequencing
the nucleic acid by well known methods.
[0160] The following non-limiting methods can be used to engineer a
core region of a given recombination site to provide mutated sites
suitable for use in the present invention: [0161] 1. By
recombination of two parental DNA sequences by site-specific (e.g.
attL and attR to give attB) or other (e.g. homologous)
recombination mechanisms. The DNA parental DNA segments containing
one or more base alterations resulting in the final core sequence;
[0162] 2. By mutation or mutagenesis (site-specific, PCR, random,
spontaneous, etc) directly of the desired core sequence; [0163] 3.
By mutagenesis (site-specific, PCR, random, spontanteous, etc) of
parental DNA sequences, which are recombined to generate a desired
core sequence; and [0164] 4. By reverse transcription of an RNA
encoding the desired core sequence.
[0165] The functionality of the mutant recombination sites can be
demonstrated in ways that depend on the particular characteristic
that is desired. For example, the lack of translation stop codons
in a recombination site can be demonstrated by expressing the
appropriate fusion proteins. Specificity of recombination between
homologous partners can be demonstrated by introducing the
appropriate molecules into in vitro reactions, and assaying for
recombination products as described herein or known in the art.
Other desired mutations in recombination sites might include the
presence or absence of restriction sites, translation or
transcription start signals, protein binding sites, and other known
functionalities of nucleic acid base sequences. Genetic selection
schemes for particular functional attributes in the recombination
sites can be used according to known method steps. For example, the
modification of sites to provide (from a pair of sites that do not
interact) partners that do interact could be achieved by requiring
deletion, via recombination between the sites, of a DNA sequence
encoding a toxic substance. Similarly, selection for sites that
remove translation stop sequences, the presence or absence of
protein binding sites, etc., can be easily devised by those skilled
in the art.
[0166] Accordingly, the present invention provides a nucleic acid
molecule, comprising at least one DNA segment having at least two
engineered recombination sites flanking a Selectable marker and/or
a desired DNA segment, wherein at least one of said recombination
sites comprises a core region having at least one engineered
mutation that enhances recombination in vitro in the formation of a
Cointegrate DNA or a Product DNA.
[0167] The nucleic acid molecule can have at least one mutation
that confers at least one enhancement of said recombination, said
enhancement selected from the group consisting of substantially (i)
favoring excisive integration; (ii) favoring excisive
recombination; (ii) relieving the requirement for host factors;
(iii) increasing the efficiency of said Cointegrate DNA or Product
DNA formation; and (iv) increasing the specificity of said
Cointegrate DNA or Product DNA formation.
[0168] The nucleic acid molecule preferably comprises at least one
recombination site derived from attB, attP, attL or attR. More
preferably the att site is selected from att1, att2, or att3, as
described herein.
[0169] In a preferred embodiment, the core region comprises a DNA
sequence selected from the group consisting of: TABLE-US-00001 (a)
RKYCWGCTTTYKTRTACNAASTSGB (SEQ ID NO: 1) (m-att); (b)
AGCCWGCTTTYKTRTACNAACTSGB (SEQ ID NO: 2) (m-attB); (c)
GTTCAGCTTTCKTRTACNAACTSGB (SEQ ID NO: 3) (m-attR); (d)
AGCCWGCTTTCKTRTACNAAGTSGB (SEQ ID NO: 4) (m-attL); (e)
GTTCAGCTTTYKTRTACNAAGTSGB (SEQ ID NO: 5) (m-attP1);
or a corresponding or complementary DNA or RNA sequence, wherein
R=A or G; K=G or T/U; Y=C or T/U; W=A or T/U; N=A or C or G or T/U;
S=Cor G; and B=C or G or T/U, as presented in 37 C.F.R.
.sctn.1.822, which is entirely incorporated herein by reference,
wherein the core region does not contain a stop codon in one or
more reading frames.
[0170] The core region also preferably comprises a DNA sequence
selected from the group consisting of: TABLE-US-00002 (a)
AGCCTGCTTTTTTGTACAAACTTGT (SEQ ID NO: 6) (attB1); (b)
AGCCTGCTTTCTTGTACAAACTTGT (SEQ ID NO: 7) (attB2); (c)
ACCCAGCTTTCTTGTACAAACTTGT (SEQ ID NO: 8) (attB3); (d)
GTTCAGCTTTTTTGTACAAACTTGT (SEQ ID NO: 9) (attR1); (e)
GTTCAGCTTTCTTGTACAAACTTGT (SEQ ID NO: 10) (attR2); (f)
GTTCAGCTTTCTTGTACAAAGTTGG (SEQ ID NO: 11) (attR3); (g)
AGCCTGCTTTTTTGTACAAAGTTGG (SEQ ID NO: 12) (attL1); (h)
AGCCTGCTTTCTTGTACAAAGTTGG (SEQ ID NO: 13) (attL2); (i)
ACCCAGCTTTCTTGTACAAAGTTGG (SEQ ID NO: 14) (attL3); (j)
GTTCAGCTTTTTTGTACAAAGTTGG (SEQ ID NO: 15) (attP1); (k)
GTTCAGCTTTCTTGTACAAAGTTGG (SEQ ID NO: 16) (attP2, P3);
or a corresponding or complementary DNA or RNA sequence.
[0171] The present invention thus also provides a method for making
a nucleic acid molecule, comprising providing a nucleic acid
molecule having at least one engineered recombination site
comprising at least one DNA sequence having at least 80-99%
homology (or any range or value therein) to at least one of SEQ ID
NOS:1-6, or any suitable recombination site, or which hybridizes
under stringent conditions thereto, as known in the art.
[0172] Clearly, there are various types and permutations of such
well-known in vitro and in vivo selection methods, each of which
are not described herein for the sake of brevity. However, such
variations and permutations are contemplated and considered to be
the different embodiments of the present invention.
[0173] It is important to note that as a result of the preferred
embodiment being in vitro recombination reactions, non-biological
molecules such as PCR products can be manipulated via the present
recombinational cloning method. In one example, it is possible to
clone linear molecules into circular vectors.
[0174] There are a number of applications for the present
invention. These uses include, but are not limited to, changing
vectors, apposing promoters with genes, constructing genes for
fusion proteins, changing copy number, changing replicons, cloning
into phages, and cloning, e.g., PCR products (with an attB site at
one end and a loxP site at the other end), genomic DNAs, and
cDNAs.
[0175] The following examples are intended to further illustrate
certain preferred embodiments of the invention and are not intended
to be limiting in nature.
EXAMPLES
[0176] The present recombinational cloning method accomplishes the
exchange of nucleic acid segments to render something useful to the
user, such as a change of cloning vectors. These segments must be
flanked on both sides by recombination signals that are in the
proper orientation with respect to one another. In the examples
below the two parental nucleic acid molecules (e.g., plasmids) are
called the Insert Donor and the Vector Donor. The Insert Donor
contains a segment that will become joined to a new vector
contributed by the Vector Donor. The recombination intermediate(s)
that contain(s) both starting molecules is called the
Cointegrate(s). The second recombination event produces two
daughter molecules, called the Product (the desired new clone) and
the Byproduct.
Buffers
[0177] Various known buffers can be used in the reactions of the
present invention. For restriction enzymes, it is advisable to use
the buffers recommended by the manufacturer. Alternative buffers
can be readily found in the literature or can be devised by those
of ordinary skill in the art.
[0178] Examples 1-3. One exemplary buffer for lambda integrase is
comprised of 50 mM Tris-HCl, at pH 7.5-7.8, 70 mM KCl, 5 mM
spermidine, 0.5 mM EDTA, and 0.25 mg/ml bovine serum albumin, and
optionally, 10% glycerol.
[0179] One preferred buffer for P1 Cre recombinase is comprised of
50 mM Tris-HCl at pH 7.5, 33 mM NaCl, 5 mM spermidine, and 0.5
mg/ml bovine serum albumin.
[0180] The buffer for other site-specific recombinases which are
similar to lambda Int and P1 Cre are either known in the art or can
be determined empirically by the skilled artisans, particularly in
light of the above-described buffers.
Example 1
Recombinational Cloning Using Cre and Cre & Int
[0181] Two pairs of plasmids were constructed to do the in vitro
recombinational cloning method in two different ways. One pair,
pEZC705 and pEZC726 (FIG. 2A), was constructed with loxP and att
sites, to be used with Cre and .lamda. integrase. The other pair,
pEZC602 and pEZC629 (FIG. 3A), contained the loxP (wild type) site
for Cre, and a second mutant lox site, loxP 511, which differs from
loxP in one base (out of 34 total). The minimum requirement for
recombinational cloning of the present invention is two
recombination sites in each plasmid, in general X and Y, and X' and
Y'. Recombinational cloning takes place if either or both types of
site can recombine to form a Cointegrate (e.g. X and X'), and if
either or both (but necessarily a site different from the type
forming the Cointegrate) can recombine to excise the Product and
Byproduct plasmids from the Cointegrate (e.g. Y and Y'). It is
important that the recombination sites on the same plasmid do not
recombine. It was found that the present recombinational cloning
could be done with Cre alone.
Cre-Only
[0182] Two plasmids were constructed to demonstrate this conception
(see FIG. 3A). pEZC629 was the Vector Donor plasmid. It contained a
constitutive drug marker (chloramphenicol resistance), an origin of
replication, loxP and loxP 511 sites, a conditional drug marker
(kanamycin resistance whose expression is controlled by the
operator/promoter of the tetracycline resistance operon of
transposon Tn10), and a constitutively expressed gene for the tet
repressor protein, tetR. E. coli cells containing pEZC629 were
resistant to chloramphenicol at 30 .mu.g/ml, but sensitive to
kanamycin at 100 .mu.g/ml. pEZC602 was the Insert Donor plasmid,
which contained a different drug marker (ampicillin resistance), an
origin, and loxP and loxP 511 sites flanking a multiple cloning
site.
[0183] This experiment was comprised of two parts as follows:
[0184] Part I: About 75 ng each of pEZC602 and pEZC629 were mixed
in a total volume of 30 .mu.l of Cre buffer (50 mM Tris-HCl pH 7.5,
33 mM NaCl, 5 mM spermidine-HCl, 500 .mu.g/ml bovine serum
albumin). Two 10 .mu.l aliquots were transferred to new tubes. One
tube received 0.5 .mu.l of Cre protein (approx. 4 units per .mu.l;
partially purified according to Abremski and Hoess, J. Biol. Chem.
259:1509 (1984)). Both tubes were incubated at 37.degree. C. for 30
minutes, then 70.degree. C. for 10 minutes. Aliquots of each
reaction were diluted and transformed into DH5.alpha.. Following
expression, aliquots were plated on 30 .mu.g/ml chloramphenicol;
100 .mu.g/ml ampicillin plus 200 .mu.g/ml methicillin; or 100
.mu.g/ml kanamycin. Results: See Table 1. The reaction without Cre
gave 1.11.times.10.sup.6 ampicillin resistant colonies (from the
Insert Donor plasmid pEZC602); 7.8.times.10.sup.5 chloramphenicol
resistant colonies (from the Vector Donor plasmid pEZC629); and 140
kanamycin resistant colonies (background). The reaction with added
Cre gave 7.5.times.10.sup.5 ampicillin resistant colonies (from the
Insert Donor plasmid pEZC602); 6.1.times.10.sup.5 chloramphenicol
resistant colonies (from the Vector Donor plasmid pEZC629); and 760
kanamycin resistant colonies (mixture of background colonies and
colonies from the recombinational cloning Product plasmid).
Analysis: Because the number of colonies on the kanamycin plates
was much higher in the presence of Cre, many or most of them were
predicted to contain the desired Product plasmid. TABLE-US-00003
TABLE 1 Chlo- Enzyme Ampicillin ramphenicol Kanamycin Efficiency
None 1.1 .times. 10.sup.6 7.8 .times. 10.sup.5 140 140/7.8 .times.
10.sup.5 = 0.02% Cre 7.5 .times. 10.sup.5 6.1 .times. 10.sup.5 760
760/6.1 .times. 10.sup.5 = 0.12%
[0185] Part II: Twenty four colonies from the "+Cre" kanamycin
plates were picked and inoculated into medium containing 100
.mu.g/ml kanamycin. Minipreps were done, and the miniprep DNAs,
uncut or cut with SmaI or HindIII, were electrophoresed. Results:
19 of the 24 minipreps showed supercoiled plasmid of the size
predicted for the Product plasmid. All 19 showed the predicted SmaI
and HindIII restriction fragments. Analysis: The Cre only scheme
was demonstrated. Specifically, it was determined to have yielded
about 70% (19 of 24) Product clones. The efficiency was about 0.1%
(760 kanamycin resistant clones resulted from 6.1.times.10.sup.5
chloramphenicol resistant colonies).
Cre Plus Integrase
[0186] The plasmids used to demonstrate this method are exactly
analogous to those used above, except that pEZC726, the Vector
Donor plasmid, contained an attP site in place of loxP 511, and
pEZC705, the Insert Donor plasmid, contained an attB site in place
of loxP 511 (FIG. 2A).
[0187] This experiment was comprised of three parts as follows:
[0188] Part I: About 500 ng of pEZC705 (the Insert Donor plasmid)
was cut with ScaI, which linearized the plasmid within the
ampicillin resistance gene. (This was done because the .lamda.
integrase reaction has been historically done with the attB plasmid
in a linear state (H. Nash, personal communication). However, it
was found later that the integrase reaction proceeds well with both
plasmids supercoiled.) Then, the linear plasmid was ethanol
precipitated and dissolved in 20 .mu.l of .lamda. integrase buffer
(50 mM Tris-HCl, about pH 7.8, 70 mM KCl, 5 mM spermidine-HCl, 0.5
mM EDTA, 250 .mu.g/ml bovine serum albumin). Also, about 500 ng of
the Vector Donor plasmid pEZC726 was ethanol precipitated and
dissolved in 20 .mu.l .lamda. integrase buffer. Just before use,
.lamda. integrase (2 .mu.l, 393 .mu.g/ml) was thawed and diluted by
adding 18 .mu.l cold .lamda. integrase buffer. One .mu.l IHF
(integration host factor, 2.4 mg/ml, an accessory protein) was
diluted into 150 .mu.l cold .lamda. integrase buffer. Aliquots (2
.mu.l) of each DNA were mixed with .lamda. integrase buffer, with
or without 1 .mu.l each .lamda. integrase and IHF, in a total of 10
.mu.l. The mixture was incubated at 25.degree. C. for 45 minutes,
then at 70.degree. C. for 10 minutes. Half of each reaction was
applied to an agarose gel. Results: In the presence of integrase
and IHF, about 5% of the total DNA was converted to a linear
Cointegrate form. Analysis: Activity of integrase and IHF was
confirmed.
[0189] Part II: Three microliters of each reaction (i.e., with or
without integrase and IHF) were diluted into 27 .mu.l of Cre buffer
(above), then each reaction was split into two 10 .mu.l aliquots
(four altogether). To two of these reactions, 0.5 .mu.l of Cre
protein (above) were added, and all reactions were incubated at
37.degree. C. for 30 minutes, then at 70.degree. C. for 10 minutes.
TE buffer (90 .mu.l; TE: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) was
added to each reaction, and 1 .mu.l each was transformed into E.
coli DH5.alpha.. The transformation mixtures were plated on 100
.mu.g/ml ampicillin plus 200 .mu.g/ml methicillin; 30 .mu.g/ml
chloramphenicol; or 100 .mu.g/ml kanamycin. Results: See Table 2.
TABLE-US-00004 TABLE 2 Chloram- Kana- Enzyme Ampicillin phenicol
mycin Efficiency None 990 20000 4 4/2 .times. 10.sup.4 = 0.02% Cre
only 280 3640 0 0 Integrase* 1040 27000 9 9/2.7 .times. 10.sup.4 =
0.03% only Integrase* + Cre 110 1110 76 76/1.1 .times. 10.sup.3 =
6.9% *Integrase reactions also contained IHF.
[0190] Analysis: The Cre protein impaired transformation. When
adjusted for this effect, the number of kanamycin resistant
colonies, compared to the control reactions, increased more than
100 fold when both Cre and Integrase were used. This suggests a
specificity of greater than 99%.
[0191] Part III: 38 colonies were picked from the Integrase plus
Cre plates, miniprep DNAs were made and cut with HindIII to give
diagnostic mapping information. Result: All 38 had precisely the
expected fragment sizes. Analysis: The Cre plus .lamda. integrase
method was observed to have much higher specificity than Cre-alone.
Conclusion: The Cre plus .lamda. integrase method was demonstrated.
Efficiency and specificity were much higher than for Cre only.
Example 2
Using in vitro Recombinational Cloning to Subclone the
Chloramphenicol Acetyl Transferase Gene into a Vector for
Expression in Eukaryotic Cells (FIG. 4A)
[0192] An Insert Donor plasmid, pEZC843, was constructed,
comprising the chloramphenicol acetyl transferase gene of E. coli,
cloned between loxP and attB sites such that the loxP site was
positioned at the 5'-end of the gene (FIG. 4B). A Vector Donor
plasmid, pEZC1003, was constructed, which contained the
cytomegalovirus eukaryotic promoter apposed to a loxP site (FIG.
4C). One microliter aliquots of each supercoiled plasmid (about 50
ng crude miniprep DNA) were combined in a ten microliter reaction
containing equal parts of lambda integrase buffer (50 mM Tris-HCl,
pH 7.8, 70 mM KCl, 5 mM spermidine, 0.5 mM EDTA, 0.25 mg/ml bovine
serum albumin) and Cre recombinase buffer (50 mM Tris-HCl, pH 7.5,
33 mM NaCl, 5 mM spermidine, 0.5 mg/ml bovine serum albumin), two
units of Cre recombinase, 16 ng integration host factor, and 32 ng
lambda integrase. After incubation at 30.degree. C. for 30 minutes
and 75.degree. C. for 10 minutes, one microliter was transformed
into competent E. coli strain DH5.alpha. (Life Technologies, Inc.).
Aliquots of transformations were spread on agar plates containing
200 .mu.g/ml kanamycin and incubated at 37.degree. C. overnight. An
otherwise identical control reaction contained the Vector Donor
plasmid only. The plate receiving 10% of the control reaction
transformation gave one colony; the plate receiving 10% of the
recombinational cloning reaction gave 144 colonies. These numbers
suggested that greater than 99% of the recombinational cloning
colonies contained the desired product plasmid. Miniprep DNA made
from six recombinational cloning colonies gave the predicted size
plasmid (5026 base pairs), CMVProd. Restriction digestion with NcoI
gave the fragments predicted for the chloramphenicol acetyl
transferase cloned downstream of the CMV promoter for all six
plasmids.
Example 3
Subcloned DNA Segments Flanked by attB Sites Without Stop
Codons
Part I: Background
[0193] The above examples are suitable for transcriptional fusions,
in which transcription crosses recombination sites. However, both
attR and loxP sites contain multiple stop codons on both strands,
so translational fusions can be difficult, where the coding
sequence must cross the recombination sites, (only one reading
frame is available on each strand of loxP sites) or impossible (in
attR or attL).
[0194] A principal reason for subcloning is to fuse protein
domains. For example, fusion of the glutathione S-transferase (GST)
domain to a protein of interest allows the fusion protein to be
purified by affinity chromatography on glutathione agarose
(Pharmacia, Inc., 1995 catalog). If the protein of interest is
fused to runs of consecutive histidines (for example His6), the
fusion protein can be purified by affinity chromatography on
chelating resins containing metal ions (Qiagen, Inc.). It is often
desirable to compare amino terminal and carboxy terminal fusions
for activity, solubility, stability, and the like.
[0195] The attB sites of the bacteriophage X integration system
were examined as an alternative to loxP sites, because they are
small (25 bp) and have some sequence flexibility (Nash, H. A. et
al., Proc. Natl. Acad. Sci. USA 84:4049-4053 (1987). It was not
previously suggested that multiple mutations to remove all stop
codes would result in useful recombination sites for
recombinational subcloning.
[0196] Using standard nomenclature for site specific recombination
in lambda bacteriophage (Weisber, in Lambda III, Hendrix, et al.,
eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1989)), the nucleotide regions that participate in the
recombination reaction in an E. coli host cell are represented as
follows: ##STR1## where: O represents the 15 bp core DNA sequence
found in both the phage and E. coli genomes; B and B' represent
approximately 5 bases adjacent to the core in the E. coli genome;
and P1, H1, P2, X, H2, C, C', H', P'1, P'2, and P'3 represent known
DNA sequences encoding protein binding domains in the bacteriophage
.lamda. genome.
[0197] The reaction is reversible in the presence of the protein
Xis (excisionase); recombination between attL and attR precisely
excise the .lamda. genome from its integrated state, regenerating
the circular .lamda. genome containing attP and the linear E. coli
genome containing attB.
Part II: Construction and Testing of Plasmids Containing Mutant att
Sites
[0198] Mutant attL and attR sites were constructed. Importantly,
Landy et al. (Ann. Rev. Biochem. 58:913 (1989)) observed that
deletion of the P1 and H1 domains of attP facilitated the excision
reaction and eliminated the integration reaction, thereby making
the excision reaction irreversible. Therefore, as mutations were
introduced in attR, the P1 and H1 domains were also deleted. attR
sites in the present example lack the P1 and H1 regions and have
the NdeI site removed (base 27630 changed from C to G), and contain
sequences corresponding to bacteriophage .lamda. coordinates
27619-27738 (GenBank release 92.0, bg:LAMCG, "Complete Sequence of
Bacteriophage Lambda").
[0199] The sequence of attB produced by recombination of wild type
attL and attR sites is: TABLE-US-00005 (SEQ. ID NO 31) B O B'
attBwt: 5' AGCCT GCTTTTTTATACTAA CTTGA 3' (SEQ. ID NO: 32) 3' TCGGA
CGAAAAAATATGATT GAACT 5'
The stop codons are italicized and underlined. Note that sequences
of attL, attR, and attP can be derived from the attB sequence and
the boundaries of bacteriophage .lamda. contained within attL and
attR (coordinates 27619 to 27818).
[0200] When mutant attR1 and attL1 sites were recombined the
sequence attB1 was produced (mutations in bold, large font):
TABLE-US-00006 (SEQ ID NO: 6) B O B' attB1: 5' AGCCT GCTTTTTTTACAA
CTTG 3' (SEQ. ID NO: 33) 3' TCGGA CGAAAAAAATGTT GAAC 5'
Note that the four stop codons are gone.
[0201] When an additional mutation was introduced in the attR1 and
attL1 sequences (bold), attR2 and attL2 sites resulted.
Recombination of attR2 and attL2 produced the attB2 site:
TABLE-US-00007 (SEQ. ID NO: 7) B O B' attB2: 5' AGCCT
GCTTTTTGTACAAA CTTGT 3' 3' TCGGA CGAAAAACATGTTT GAACA 5' (SEQ. ID
NO: 34)
[0202] The recombination activities of the above attL and attR
sites were assayed as follows. The attB site of plasmid pEZC705
(FIG. 2B) was replaced with attLwt, attL1, or attL2. The attP site
of plasmid pEZC726 (FIG. 2C) was replaced with attRwt (lacking
regions P1 and H1), attR1, or attR2. Thus, the resulting plasmids
could recombine via their loxP sites, mediated by Cre, and via
their attR and attL sites, mediated by Int, Xis, and IHF. Pairs of
plasmids were mixed and reacted with Cre, Int, Xis, and IHF,
transformed into E. coli competent cells, and plated on agar
containing kanamycin. The results are presented in Table 3:
TABLE-US-00008 TABLE 3 # of kanamycin resistant Vector donor att
site Gene donor att site colonies* attRwt (pEZC1301) None 1
(background) '' attLwt (pEZC1313) 147 '' attL1 (pEZC1317) 47 ''
attL2 (pEZC1321) 0 attR1 (pEZC1305) None 1 (background) '' attLwt
(pEZC1313) 4 '' attL1 (pEZC1317) 128 '' attL2 (pEZC1321) 0 attR2
(pEZC1309) None 9 (background) '' attLwt (pEZC1313) 0 '' attL2
(pEZC1317) 0 '' attL2 (pEZC1321) 209 (*1% of each transformation
was spread on a kanamycin plate.)
[0203] The above data show that whereas the wild type att and att1
sites recombine to a small extent, the att1 and att2 sites do not
recombine detectably with each other.
[0204] Part III. Recombination was demonstrated when the core
region of both attB sites flanking the DNA segment of interest did
not contain stop codons. The physical state of the participating
plasmids was discovered to influence recombination efficiency.
[0205] The appropriate att sites were moved into pEZC705 and
pEZC726 to make the plasmids pEZC1405 (FIG. 5G) (attR1 and attR2)
and pEZC1502 (FIG. 5H) (attL1 and attL2). The desired DNA segment
in this experiment was a copy of the chloramphenicol resistance
gene cloned between the two attL sites of pEZC1502. Pairs of
plasmids were recombined in vitro using Int, Xis, and IHF (no Cre
because no loxP sites were present). The yield of desired kanamycin
resistant colonies was determined when both parental plasmids were
circular, or when one plasmid was circular and the other linear as
presented in Table 4: TABLE-US-00009 TABLE 4 Kanamycin resistant
Vector donor.sup.1 Gene donor.sup.1 colonies.sup.2 Circular
pEZC1405 None 30 Circular pEZC1405 Circular pEZC1502 2680 Linear
pEZC1405 None 90 Linear pEZC1405 Circular pEZC1502 172000 Circular
pEZC1405 Linear pEZC1502 73000 .sup.1DNAs were purified with Qiagen
columns, concentrations determined by A260, and linearized with Xba
I (pEZC1405) or AlwN I (pEZC1502). Each reaction contained 100 ng
of the indicated DNA. All reactions (10 .mu.l total) contained 3
.mu.l of enzyme # mix (Xis, Int, and IHF). After incubation (45
minutes at 25.degree., 10 minutes at 65.degree.), one .mu.l was
used to transform E. coli DH5.alpha. cells. .sup.2Number of
colonies expected if the entire transformation reaction (1 ml) had
been plated. Either 100 .mu.l or 1 .mu.l of the transformations
were actually plated.
[0206] Analysis: Recombinational cloning using mutant attR and attL
sites was confirmed. The desired DNA segment is subcloned between
attB sites that do not contain any stop codons in either strand.
The enhanced yield of Product DNA (when one parent was linear) was
unexpected because of earlier observations that the excision
reaction was more efficient when both participating molecules were
supercoiled and proteins were limiting (Nunes-Duby et al., Cell
50:779-788 (1987).
Example 4
Demonstration of Recombinational Cloning Without Inverted
Repeats
Part I: Rationale
[0207] The above Example 3 showed that plasmids containing inverted
repeats of the appropriate recombination sites (for example, attL1
and attL2 in plasmid pEZC1502) (FIG. 5H) could recombine to give
the desired DNA segment flanked by attB sites without stop codons,
also in inverted orientation. A concern was the in vivo and in
vitro influence of the inverted repeats. For example, transcription
of a desired DNA segment flanked by attB sites in inverted
orientation could yield a single stranded RNA molecule that might
form a hairpin structure, thereby inhibiting translation.
[0208] Inverted orientation of similar recombination sites can be
avoided by placing the sites in direct repeat arrangement att
sites. If parental plasmids each have a wild type attL and wild
type attR site, in direct repeat the Int, Xis, and IHF proteins
will simply remove the DNA segment flanked by those sites in an
intramolecular reaction. However, the mutant sites described in the
above Example 3 suggested that it might be possible to inhibit the
intramolecular reaction while allowing the intermolecular
recombination to proceed as desired.
Part II: Structure of Plasmids Without Inverted Repeats for
Recombinational Cloning
[0209] The attR2 sequence in plasmid pEZC1405 (FIG. 5G) was
replaced with attL2, in the opposite orientation, to make pEZC1603
(FIG. 6A). The attL2 sequence of pEZC1502 (FIG. 5H) was replaced
with attR2, in the opposite orientation, to make pEZC1706 (FIG.
6B). Each of these plasmids contained mutations in the core region
that make intramolecular reactions between att1 and att2 cores very
inefficient (see Example 3, above).
[0210] Plasmids pEZC1405, pEZC1502, pEZC1603 and pEZC1706 were
purified on Qiagen columns (Qiagen, Inc.). Aliquots of plasmids
pEZC1405 and pEZC1603 were linearized with Xba I. Aliquots of
plasmids pEZC1502 and pEZC1706 were linearized with AlwN I. One
hundred ng of plasmids were mixed in buffer (equal volumes of 50 mM
Tris HCl pH 7.5, 25 mM Tris HCl pH 8.0, 70 mM KCl, 5 mM spermidine,
0.5 mM EDTA, 250 .mu.g/ml BSA, 10% glycerol) containing Int (43.5
ng), Xis (4.3 ng) and IHF (8.1 ng) in a final volume of 10 .mu.l.
Reactions were incubated for 45 minutes at 25.degree. C., 10
minutes at 65.degree. C., and 1 .mu.l was transformed into E. coli
DH5.alpha.. After expression, aliquots were spread on agar plates
containing 200 .mu.g/ml kanamycin and incubated at 37.degree.
C.
[0211] Results, expressed as the number of colonies per 1 .mu.l of
recombination reaction are presented in Table 5: TABLE-US-00010
TABLE 5 Vector Donor Gene Donor Colonies Predicted % product
Circular 1405 -- 100 -- Circular 1405 Circular 1502 3740 3640/3740
= 97% Linear 1405 -- 90 -- Linear 1405 Circular 1502 172,000
171,910/172,000 = 99.9% Circular 1405 Linear 1502 73,000
72,900/73,000 = 99.9% Circular 1603 -- 80 -- Circular 1603 Circular
1706 410 330/410 = 80% Linear 1603 -- 270 -- Linear 1603 Circular
1706 7000 6730/7000 = 96% Circular 1603 Linear 1706 10,800
10,530/10,800 = 97%
[0212] Analysis. In all configurations, i.e., circular or linear,
the pEZC1405.times.pEZC1502 pair (with att sites in inverted repeat
configuration) was more efficient than pEZC1603.times.pEZC1706 pair
(with att sites mutated to avoid hairpin formation). The
pEZC1603.times.pEZC1706 pair gave higher backgrounds and lower
efficiencies than the pEZC1405.times.pEZC1502 pair. While less
efficient, 80% or more of the colonies from the
pEZC1603.times.pEZC1706 reactions were expected to contain the
desired plasmid product. Making one partner linear stimulated the
reactions in all cases.
Part III: Confirmation of Product Plasmids's Structure
[0213] Six colonies each from the linear pEZC1405 (FIG.
5G).times.circular pEZC1502 (FIG. 5H), circular
pEZC1405.times.linear pEZC1502, linear pEZC1603 (FIG.
6A).times.circular pEZC1706 (FIG. 6B), and circular
pEZC1603.times.linear pEZC1706 reactions were picked into rich
medium and miniprep DNAs were prepared. Diagnostic cuts with Ssp I
gave the predicted restriction fragments for all 24 colonies.
[0214] Analysis. Recombination reactions between plasmids with
mutant attL and attR sites on the same molecules gave the desired
plasmid products with a high degree of specificity.
Example 5
Recombinational Cloning with a Toxic Gene
Part I: Background
[0215] Restriction enzyme Dpn I recognizes the sequence GATC and
cuts that sequence only if the A is methylated by the dam
methylase. Most commonly used E. coli strains are dam.sup.+.
Expression of Dpn I in dam.sup.+ strains of E. coli is lethal
because the chromosome of the cell is chopped into many pieces.
However, in dam.sup.- cells expression of Dpn I is innocuous
because the chromosome is immune to Dpn I cutting.
[0216] In the general recombinational cloning scheme, in which the
vector donor contains two segments C and D separated by
recombination sites, selection for the desired product depends upon
selection for the presence of segment D, and the absence of segment
C. In the original Example segment D contained a drug resistance
gene (Km) that was negatively controlled by a repressor gene found
on segment C. When C was present, cells containing D were not
resistant to kanamycin because the resistance gene was turned
off.
[0217] The Dpn I gene is an example of a toxic gene that can
replace the repressor gene of the above embodiment. If segment C
expresses the Dpn I gene product, transforming plasmid CD into a
dam+ host kills the cell. If segment D is transferred to a new
plasmid, for example by recombinational cloning, then selecting for
the drug marker will be successful because the toxic gene is no
longer present.
Part II: Construction of a Vector Donor Using Dpn I as a Toxic
Gene
[0218] The gene encoding Dpn I endonuclease was amplified by PCR
using primers 5'CCA CCA CAA ACG CGT CCA TGG AAT TAC ACT TTA ATT
TAG3' (SEQ. ID NO: 17) and 5' CCA CCA CAA GTC GAC GCA TGC CGA CAG
CCT TCC AAA TGT3' (SEQ. ID NO: 18) and a plasmid containing the Dpn
I gene (derived from plasmids obtained from Sanford A. Lacks,
Brookhaven National Laboratory, Upton, N.Y.; also available from
American Type Culture Collection as ATCC 67494) as the
template.
[0219] Additional mutations were introduced into the B and B'
regions of attL and attR, respectively, by amplifying existing attL
and attR domains with primers containing the desired base changes.
Recombination of the mutant attL3 (made with oligo Xis115) and
attR3 (made with oligo Xis112) yielded attB3 with the following
sequence (differences from attB 1 in bold): TABLE-US-00011 B O B'
ACCC GCTTTTTGTACAAA TGT (SEQ. ID NO: 8) TGGT CGAAAAACATGTTT CACA
(SEQ. ID NO: 35)
The attL3 sequence was cloned in place of attL2 of an existing Gene
Donor plasmid to give the plasmid pEZC2901 (FIG. 7A). The attR3
sequence was cloned in place of attR2 in an existing Vector Donor
plasmid to give plasmid pEZC2913 (FIG. 7B) Dpn I gene was cloned
into plasmid pEZC2913 to replace the tet repressor gene. The
resulting Vector Donor plasmid was named pEZC3101 (FIG. 7C). When
pEZC3101 was transformed into the dam.sup.- strain SCS110
(Stratagene), hundreds of colonies resulted. When the same plasmid
was transformed into the dam+ strain DH5.alpha., only one colony
was produced, even though the DH5.alpha. cells were about 20 fold
more competent than the SCS110 cells. When a related plasmid that
did not contain the Dpn I gene was transformed into the same two
cell lines, 28 colonies were produced from the SCS110 cells, while
448 colonies resulted from the DH5.alpha. cells. This is evidence
that the Dpn I gene is being expressed on plasmid pEZC3101 (FIG.
7C), and that it is killing the dam.sup.+ DH5.alpha. cells but not
the dam.sup.- SCS110 cells. III: Demonstration of Recombinational
Cloning Using Dpn I Selection
[0220] A pair of plasmids was used to demonstrate recombinational
cloning with selection for product dependent upon the toxic gene
Dpn I. Plasmid pEZC3101 (FIG. 7C) was linearized with Mlu I and
reacted with circular plasmid pEZC2901 (FIG. 7A). A second pair of
plasmids using selection based on control of drug resistance by a
repressor gene was used as a control: plasmid pEZC1802 (FIG. 7D)
was linearized with Xba I and reacted with circular plasmid
pEZC1502 (FIG. 5H). Eight microliter reactions containing the same
buffer and proteins Xis, Int, and IHF as in previous examples were
incubated for 45 minutes at 25.degree. C., then 10 minutes at
75.degree. C., and 1 .mu.l aliquots were transformed into
DH5.alpha. (i.e., dam+) competent cells, as presented in Table 6.
TABLE-US-00012 TABLE 6 Reac- Basis of tion # Vector donor selection
Gene donor Colonies 1 pEZC3101/Mlu Dpn I toxicity -- 3 2
pEZC3101/Mlu Dpn I toxicity Circular pEZC2901 4000 3 pEZC1802/Xba
Tet repressor -- 0 4 pEZC1802/Xba Tet repressor Circular pEZC1502
12100
[0221] Miniprep DNAs were prepared from four colonies from reaction
#2, and cut with restriction enzyme Ssp I. All gave the predicted
fragments.
[0222] Analysis: Subcloning using selection with a toxic gene was
demonstrated. Plasmids of the predicted structure were
produced.
Example 6
Cloning of Genes with Uracil DNA Glycosylase and Subcloning of the
Genes with Recombinational Cloning to Make Fusion Proteins
Part I: Converting an Existing Expression Vector to a Vector Donor
for Recombinational Cloning
[0223] A cassette useful for converting existing vectors into
functional Vector Donors was made as follows. Plasmid pEZC3101
(FIG. 7C) was digested with Apa I and Kpn I, treated with T4 DNA
polymerase and dNTPs to render the ends blunt, further digested
with Sma I, Hpa I, and AlwN I to render the undesirable DNA
fragments small, and the 2.6 kb cassette containing the attR1
--Cm.sup.R--Dpn I--attR-3 domains was gel purified. The
concentration of the purified cassette was estimated to be about 75
ng DNA/.mu.l.
[0224] Plasmid pGEX-2TK (FIG. 8A) (Pharmacia) allows fusions
between the protein glutathione S transferase and any second coding
sequence that can be inserted in its multiple cloning site.
pGEX-2TK DNA was digested with Sma I and treated with alkaline
phosphatase. About 75 ng of the above purified DNA cassette was
ligated with about 100 ng of the pGEX-2TK vector for 2.5 hours in a
5 .mu.l ligation, then 1 .mu.l was transformed into competent BRL
3056 cells (a dam.sup.- derivative of DH10B; dam.sup.- strains
commercially available include DM1 from Life Technologies, Inc.,
and SCS 110 from Stratagene). Aliquots of the transformation
mixture were plated on LB agar containing 100 .mu.g/ml ampicillin
(resistance gene present on pGEX-2TK) and 30 .mu.g/ml
chloramphenicol (resistance gene present on the DNA cassette).
Colonies were picked and miniprep DNAs were made. The orientation
of the cassette in pGEX-2TK was determined by diagnostic cuts with
EcoR I. A plasmid with the desired orientation was named pEZC3501
(FIG. 8B).
Part II: Cloning Reporter Genes Into an Recombinational Cloning
Gene Donor Plasmid in Three Reading Frames
[0225] Uracil DNA glycosylase (UDG) cloning is a method for cloning
PCR amplification products into cloning vectors (U.S. Pat. No.
5,334,515, entirely incorporated herein by reference). Briefly, PCR
amplification of the desired DNA segment is performed with primers
that contain uracil bases in place of thymidine bases in their 5'
ends. When such PCR products are incubated with the enzyme UDG, the
uracil bases are specifically removed. The loss of these bases
weakens base pairing in the ends of the PCR product DNA, and when
incubated at a suitable temperature (e.g., 37.degree. C.), the ends
of such products are largely single stranded. If such incubations
are done in the presence of linear cloning vectors containing
protruding 3' tails that are complementary to the 3' ends of the
PCR products, base pairing efficiently anneals the PCR products to
the cloning vector. When the annealed product is introduced into E.
coli cells by transformation, in vivo processes efficiently convert
it into a recombinant plasmid.
[0226] UDG cloning vectors that enable cloning of any PCR product
in all three reading frames were prepared from pEZC3201 (FIG. 8K)
as follows. Eight oligonucleotides were obtained from Life
Technologies, Inc. (all written 5'.fwdarw.3': rf1 top (GGCC GAT TAC
GAT ATC CCA ACG ACC GAA AAC CTG TAT TTT CAG GGT) (SEQ. ID NO:19),
rf1 bottom (CAG GTT TTC GGT CGT TGG GAT ATC GTA ATC)(SEQ. ID
NO:20), rf2 top (GGCCA GAT TAC GAT ATC CCA ACG ACC GAA AAC CTG TAT
TTT CAG GGT)(SEQ. ID NO:21), rf2 bottom (CAG GTT TTC GGT CGT TGG
GAT ATC GTA ATC T)(SEQ. ID NO:22), rf3 top (GGCCAA GAT TAC GAT ATC
CCA ACG ACC GAA AAC CTG TAT TTT CAG GGT)(SEQ. ID NO:23), rf3 bottom
(CAG GTT TTC GGT CGT TGG GAT ATC GTA ATC TT)(SEQ. ID NO:24),
carboxy top (ACC GTT TAC GTG GAC)(SEQ. ID NO:25) and carboxy bottom
(TCGA GTC CAC GTA AAC GGT TCC CAC TTA TTA)(SEQ. ID NO:26). The rf1,
2, and 3 top strands and the carboxy bottom strand were
phosphorylated on their 5' ends with T4 polynucleotide kinase, and
then the complementary strands of each pair were hybridized.
Plasmid pEZC3201 (FIG. 8K) was cut with Not I and Sal I, and
aliquots of cut plasmid were mixed with the carboxy-oligo duplex
(Sal I end) and either the rf1, rf2, or rf3 duplexes (Not I ends)
(10 .mu.g cut plasmid (about 5 pmol) mixed with 250 pmol carboxy
oligo duplex, split into three 20 .mu.l volumes, added 5 .mu.l (250
pmol) of rf1, rf2, or rf3 duplex and 2 .mu.l=2 units T4 DNA ligase
to each reaction). After 90 minutes of ligation at room
temperature, each reaction was applied to a preparative agarose gel
and the 2.1 kb vector bands were eluted and dissolved in 50 .mu.l
of TE.
Part III: PCR of CAT and phoA Genes
[0227] Primers were obtained from Life Technologies, Inc., to
amplify the chloramphenicol acetyl transferase (CAT) gene from
plasmid pACYC184, and phoA, the alkaline phosphatase gene from E.
coli. The primers had 12-base 5' extensions containing uracil
bases, so that treatment of PCR products with uracil DNA
glycosylase (UDG) would weaken base pairing at each end of the DNAs
and allow the 3' strands to anneal with the protruding 3' ends of
the rf1, 2, and 3 vectors described above. The sequences of the
primers (all written 5'.fwdarw.3') were: CAT left, UAU UUU CAG GGU
ATG GAG AAA AAA ATC ACT GGA TAT ACC (SEQ. ID NO:27); CAT right, UCC
CAC UUA UUA CGC CCC GCC CTG CCA CTC ATC (SEQ. ID NO:28); phoA left,
UAU UUU CAG GGU ATG CCT GTT CTG GAA AAC CGG (SEQ. ID NO:29); and
phoA right, UCC CAC UUA UUA TTT CAG CCC CAG GGC GGC TTT C (SEQ. ID
NO:30). The primers were then used for PCR reactions using known
method steps (see, e.g., U.S. Pat. No. 5,334,515, entirely
incorporated herein by reference), and the polymerase chain
reaction amplification products obtained with these primers
comprised the CAT or phoA genes with the initiating ATGs but
without any transcriptional signals. In addition, the
uracil-containing sequences on the amino termini encoded the
cleavage site for TEV protease (Life Technologies, Inc.), and those
on the carboxy terminal encoded consecutive TAA nonsense
codons.
[0228] Unpurified PCR products (about 30 ng) were mixed with the
gel purified, linear rf1, rf2, or rf3 cloning vectors (about 50 ng)
in a 10 .mu.l reaction containing 1.times.REact 4 buffer (LTI) and
1 unit UDG (LTI). After 30 minutes at 37.degree. C., 1 .mu.l
aliquots of each reaction were transformed into competent E. coli
DH5.alpha. cells (LTI) and plated on agar containing 50 .mu.g/ml
kanamycin. Colonies were picked and analysis of miniprep DNA showed
that the CAT gene had been cloned in reading frame 1
(pEZC3601)(FIG. 8C), reading frame 2 (pEZC3609)(FIG. 8D) and
reading frame 3 (pEZC3617)(FIG. 8E), and that the phoA gene had
been cloned in reading frame 1 (pEZC3606)(FIG. 8F), reading frame 2
(pEZC3613)(FIG. 8G) and reading frame 3 (pEZC3621)(FIG. 8H).
Part IV: Subcloning of CAT or phoA from UDG Cloning Vectors into a
GST Fusion Vector
[0229] Plasmids encoding fusions between GST and either CAT or phoA
in all three reading frames were constructed by recombinational
cloning as follows. Miniprep DNA of GST vector donor pEZC3501(FIG.
8B) (derived from Pharmacia plasmid pGEX-2TK as described above)
was linearized with Cla I. About 5 ng of vector donor were mixed
with about 10 ng each of the appropriate circular gene donor
vectors containing CAT or phoA in 8 .mu.l reactions containing
buffer and recombination proteins Int, Xis, and IHF (above). After
incubation, 1 .mu.l of each reaction was transformed into E. coli
strain DH5.alpha. and plated on ampicillin, as presented in Table
7. TABLE-US-00013 TABLE 7 Colonies (10% of each DNA transformation)
Linear vector donor (pEZC3501/Cla) 0 Vector donor + CAT rf1 110
Vector donor + CAT rf2 71 Vector donor + CAT rf3 148 Vector donor +
phoA rf1 121 Vector donor + phoA rf2 128 Vector donor + phoA rf3
31
Part V: Expression of Fusion Proteins
[0230] Two colonies from each transformation were picked into 2 ml
of rich medium (CIRCLEGROW.RTM. brand culture medium, Bio101 Inc.)
in 17.times.100 mm plastic tubes (FALCON.RTM. brand plasticware,
Cat. No. 2059, Becton Dickinson) containing 100 .mu.g/ml ampicillin
and shaken vigorously for about 4 hours at 37.degree. C., at which
time the cultures were visibly turbid. One ml of each culture was
transferred to a new tube containing 10 .mu.l of 10% (w/v) IPTG to
induce expression of GST. After 2 hours additional incubation, all
cultures had about the same turbidity; the A600 of one culture was
1.5. Cells from 0.35 ml each culture were harvested and treated
with sample buffer (containing SDS and .beta.-mercaptoethanol) and
aliquots equivalent to about 0.15 A600 units of cells were applied
to a Novex 4-20% gradient polyacrylamide gel. Following
electrophoresis the gel was stained with Coomassie blue.
[0231] Results: Enhanced expression of single protein bands was
seen for all 12 cultures. The observed sizes of these proteins
correlated well with the sizes predicted for GST being fused
(through attB recombination sites without stop codons) to CAT or
phoA in three reading frames: CAT rf1=269 amino acids; CAT rf2=303
amino acids; CAT rf3=478 amino acids; phoA rf1=282 amino acids;
phoA rf2=280 amino acids; and phoA rf3=705 amino acids.
[0232] Analysis: Both CAT and phoA genes were subcloned into a GST
fusion vector in all three reading frames, and expression of the
six fusion proteins was demonstrated.
[0233] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art from a reading of this
disclosure that various changes in form and detail can be made
without departing from the true scope of the invention and appended
claims. All patents and publications cited herein are entirely
incorporated herein by reference.
Sequence CWU 1
1
* * * * *