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Unformatted text preview: P roc. Natl. Acad. Sci. USA
Vol. 93, pp. 14008–14013, November 1996
Immunology In vitro V(D)J recombination: Signal joint formation
(recombination activating protein RAG1recombination activating protein RAG2) PATRICIA CORTES*, FRANCES WEIS-GARCIA*, ZIVA MISULOVIN*, A NDRE NUSSENZWEIG†, JIANN-SHIUN L AI‡,
GLORIA LI†, MICHEL C. NUSSENZWEIG*, AND DAVID BALTIMORE‡
*Laboratory of Molecular Immunology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021; †Department of Medical Physics
and Radiation Oncology, Memorial Sloan–Kettering Cancer Center, 1275 York Avenue, New York, NY 10021; and ‡Department of Biology, Massachusetts
Institute of Technology, Cambridge, MA 02139 Contributed by David Baltimore, August 26, 1996 catalyze signal joint formation. This system depends on Ku
proteins and may provide the means for identification of the
range of factors involved in generation of the signal joint. ABSTRACT
The first step of V(D)J recombination, specific cleavage at the recombination signal sequence (RSS), can
be carried out by the recombination activating proteins RAG1
and RAG2. In vivo, the cleaved coding and signal ends must
be rejoined to generate functional antigen receptors and
maintain chromosomal integrity. We have investigated signal
joint formation using deletion and inversion substrates in a
cell free system. RAG1 and RAG2 alone or in combination
were unable to generate signal joints. However, RAG1 and
RAG2 complemented with nuclear extracts were able to
recombine an extrachromosomal substrate and form precise
signal joints. The in vitro reaction resembled authentic V(D)J
recombination in being Ku-antigen-dependent. METHODS
PCR Analysis. Recombined DNA was amplified and labeled
with -32P by using 30 cycles of 94C for 0.5 min, 65C for 1 min,
and 75C for 1 min, followed by incubation at 72C for 10 min.
The PCR amplification mixture contained 10 mM TrisHCl
(pH 9.0), 50 mM KCl, 5.5 mM MgCl2, BSA (0.1 mgml), 100
ng of each appropriate primer (R5, CCAGTCTGTAGCACTGTGCAC; R14, TCCAGCTGAACGGTCTGGT), all four
dNTPs (each at 20 M), 1 mCi of [-32P]dCTP (3000 Ci
mmol; 1 Ci 37 GBq; Amersham), and 1 unit of Taq DNA
polymerase (Boehringer Mannheim). When primer R14 was
used in combination with primer R3 (TGTTCCAGTCTGTAGCACTG), PCR was for 30 cycles of 95C for 10 sec, 55C for 0.5
min, 72C for 1 min, followed by incubation at 72C for 10 min.
Purification of Truncated Glutathione S-Transferase Fusion RAG1 and RAG2 Proteins. Truncated versions of RAG1
(amino acids 330-1040) and RAG2 (amino acids 1–383) were
expressed as glutathione S-transferase fusion proteins under
the transcriptional control of the elongation factor 1 promoter (21). 293T cells [293 cells expressing the simian virus 40
large tumor (T) antigen] were transiently transfected with the
RAG1 and RAG2 constructs by calcium phosphate precipitation (22). Two days after transfection, the cells were harvested and lysed for 5 min in RSB buffer (10 mM TrisHCl, pH
7.4 10 mM NaCl5 mM MgCl20.5% Nonidet P-40 protease
inhibitors). Lysates were brought to 0.6 M NaCl with buffer
LSB (20 mM TrisHCl, pH 7.4 1.0 M NaCl0.2 mM MgCl2
0.1% Nonidet P-40 protease inhibitors) and incubated for 30
min on ice. Extracts were centrifuged and the supernatant was
incubated with glutathione-agarose beads. RAG proteins were
eluted (50 mM TrisHCl, pH 8.3 20 mM glutathione1 M
NaCl10% glycerolprotease inhibitors) and dialyzed against
buffer D (20 mM HepesNaOH, pH 7.5 1 mM DTT10%
glycerol0.3 M NaCl0.1 mM EDTAprotease inhibitors).
Both proteins were expressed at levels corresponding to approximately 1 to 2 g of each protein from one 100-mm tissue
culture dish. Although RAG1 and RAG2 were highly purified
(as determined by Coomassie blue staining, data not shown),
we cannot rule out the possibility that our preparations
contained additional factors that contributed to the biochemical activities described. All of the experiments described were
done at least three times with two different preparations of
extracts and proteins.
Preparation of Nuclear Extracts. 293 and CHO cells stably
transfected with full-length RAG1 and RAG2 are indicated as
293-S and CHO-S, respectively. BASC6C2 is a pro-B-cell line
and 22D6 is an Abelson virus-transformed pre-B-cell line. Lymphocyte antigen receptors are encoded by multiple copies of
gene segments in germ-line DNA. During B- and T-lymphocyte
development, these gene segments are assembled into functional
transcription units by the mechanism of V(D)J recombination.
The DNA sequence requirements for V(D)J recombination
consist of highly conserved heptamer and nonamer DNA motifs
separated by a spacer of 12 or 23 base pairs (12 RSS and 23 RSS,
where RSS is recombination signal sequence). During the V(D)J
recombination reaction, two types of DNA joints are formed:
signal joints, generally involving precise head-to-head ligation of
two heptamers, and coding joints, usually containing deletions or
additions of a few nucleotides (1).
Several lymphoid-specific factors are known to be involved
in V(D)J recombination. These include terminal deoxynucleotidyltransferase (2–4) and the recombination activating proteins RAG1 and RAG2 (5–7). RAG1 and RAG2 are sufficient
for the formation of specific double-strand DNA breaks at
RSSs (8, 9). Efficient recombination occurs almost exclusively
between RSSs with different spacers. This restriction is known
as the 12 23 rule and is evident at the initial cleavage event
(10–12). The cleavage reaction involves the formation of hairpin
loops at the coding ends and precise double-strand breaks at the
signal ends (8, 9, 13–16). RAG1 and RAG2 can be coimmunoprecipitated from cells, suggesting that they function as part of a
complex during V(D)J recombination (17, 18).
Other proteins that are not restricted in their pattern of
expression to lymphoid cells are also required for V(D)J
recombination. These include two components of the DNAdependent protein kinase, the p86 subunit of the p70 p86 Ku
antigen (product of XRCC5), and the large catalytic subunit
(product of SCID), as well as the XRCC4 protein. These
proteins are involved in DNA repair as well as in V(D)J
recombination (19, 20). In addition other as yet unidentified
factors may be required for antigen receptor assembly.
Herein we report an in vitro system in which RAG1 and
RAG2 complemented with a nuclear extract are able to
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: RAG, recombination activating protein; RSS, recombination signal sequence. 14008 Immunology: Cortes et al. Proc. Natl. Acad. Sci. USA 93 (1996) 14009 FIG. 1. PCR assay for signal joint formation. ( A ) Schematic representation of the pJH200 substrate (28). The unrecombined plasmid and the
recombined product are shown on the left and right, respectively. The relative positions of the primers (R5 and R14) used to amplify the 252-bp
region of the recombined product are indicated by arrows (not to scale). (B ) Nucleotide sequence to which primer R5 hybridizes in the unrecombined
(pJH200) and recombined (pJH200R) plasmids. Note that in the unrecombined substrate there is a 1-bp mismatch at the 3 end of R5 (arrowhead).
( C ) Evaluation of primer combination R5 R14 on recombined and unrecombined sequences. The PCR assay was performed using the indicated
amounts of recombined plasmid as template in the presence (lanes 1–5) or absence (lanes 6–10) of 2.5 ng of unrecombined substrate. The 456and 252-bp fragments are the amplified products from pJH200 and pJH200R, respectively. Nuclear extracts were prepared using a modification of the
Dignam protocol (23). Briefly, cells were harvested and resuspended in 5 vol of buffer A (10 mM HepesKOH, pH
7.9 1.5 mM MgCl210 mM KCl0.1% Nonidet P-40 protease
inhibitors). After 10 min of incubation on ice, samples were
centrifuged at 1000 g for 10 min. The pellet was resuspended
in 1.5 vol of buffer C (20 mM HepesKOH, pH 7.9 20%
glycerol0.6 M NaCl1.5 mM MgCl20.2 mM EDTA0.5 mM
DTT0.1% Nonidet P-40 protease inhibitors) and incubated
on ice for 30 min. Cellular debris was removed by high-speed
centrifugation, and the supernatant was dialyzed against low
salt buffer C (100 mM NaCl) as described by Dignam et al. (23).
In Vitro Recombination. Fifty nanograms of pJH200 or 10 ng
of pJH288 was incubated with RAG proteins and nuclear
extracts in 20 l in the presence of 12.5 mM HepesKOH, pH
7.5 100 mM KCl1 mM MnCl20.05 mM EDTA5% glycerol
0.5 mM ATPall four dNTPs (each at 50 mM). RAG1 (200
ng), RAG2 (200 ng), or nuclear extracts from 293-S (8 mgml),
BASC6C2 (11 mgml), HeLa (10 mgml), CHO-S (9 mgml),
or 22D6 (3 mgml) were added to the reaction mixtures. After
a 5-hr incubation at 30C, the samples were treated with
proteinase K for 2 hr. The samples were then extracted once with phenolchloroform and twice with chloroform. The DNA
was recovered by ethanol precipitation, using 3 g of
poly(dIdC) as carrier, and resuspended in 20 l of 1 mM
TrisHCl, pH 7.5 0.1 mM EDTA. Five percent of the recovered DNA was used as template in the PCR detection assay.
In Vivo Recombination. 293T cells (293 cells expressing the
simian virus 40 large tumor antigen) were transiently transfected with the recombination substrate alone or together with
the RAG1 and RAG2 constructs by a calcium phosphate
precipitation (22). Plasmid DNA was recovered from the cells
2 days later by performing a standard alkaline lysis protocol.
Southern Blot Analysis. PCR products were resolved by
electrophoresis through a 2% agarose gel, denatured, renatured within the gel, and then transferred onto Biotrans
membrane (ICN). The DNA was UV-crosslinked to the membrane with a Stratalinker (Stratagene) and probed with an
oligonucleotide which encompasses a correct signal joint (CTGTGCACAGTGGTA) according to the protocol described by
Oettinger et al. (7). RESULTS AND DISCUSSION
As a further step toward defining the biochemical requirements for the V(D)J recombination reaction, we have devel- 14010 Immunology: Cortes et al. Proc. Natl. Acad. Sci. USA 93 (1996) FIG. 2. In vitro signal joint formation by deletion. (A ) In vitro signal joint formation by deletion requires RAG1, RAG2, and nuclear extracts.
pJH200 was incubated at 30C with the indicated combinations of purified RAG1 (R1), RAG2 (R2), and nuclear extracts (NE) from 293-S cells
under the conditions describe. The DNA was then purified and analyzed by PCR assay as described in Fig. 1. The autoradiograph from one such
experiment is presented on the left (lanes 1–9), while the right (lanes 10 and 11) shows the 32P-labeled PCR products amplified from in
vivo-recombined DNA purified from 293T cells transfected with pJH200 alone () or cotransfected with RAG1 and RAG2 (). Lane 9 correspond
to a control reaction, RAG1, RAG2, and NE but not template DNA. (B ) Time course of in vitro signal joint formation. The reaction conditions
were identical to those described in A, lane 7 (with RAGs and nuclear extract) and the products were analyzed as explained in Fig. 1. Lane 11 is
identical to lane 10 except that template DNA was not included in the reaction. ( C ) Digestion pattern of the in vivo- and in vitro-recombined products.
Purified 252-bp 32P-labeled PCR products were incubated with no enzyme (), RsaI (RI), or HindIII (HIII) and separated on an 8%
polyacrylamide1 TBE gel. The 252-bp fragment contains a unique HindIII site 151 bp from the R14 oligonucleotide. This fragment also contains
two RsaI sites, one in the 23 RSS and the other 25 bp from oligonucleotide R14. ( D ) Different nuclear extracts can complement RAG proteins
in signal joint formation. RAG1 (R1) and RAG2 (R2) were supplemented with extracts prepared from BASC6C2 (BASC), 22D6, HeLa, CHO-S,
or 293-S cells, as indicated. Each sample was processed as in A and the bands were visualized by autoradiography. oped an in vitro system to study signal joint formation. The
system uses the active core regions of RAG1 (amino acid
330-1040) and RAG2 (amino acid 1–383), which were purified
as fusion proteins with glutathione S-transferase (24–27).
Truncated RAG1 and RAG2 fusion proteins mediated V(D)J
recombination in vivo and specific cleavage of the RSSs in vitro
(data not shown). The truncated fusion proteins were used for
all of the experiments shown herein and will be referred to as
RAG1 and RAG2.
A sensitive PCR assay was used to test for signal joint
formation in a cell-free system (Fig. 1). The substrate used for
the joining reaction was pJH200, a plasmid that undergoes
deletional V(D)J recombination (28). Recombined signal
joints were detected by amplification with primers R5 and R14
to yield a 252-bp PCR fragment (Fig. 1 A). Hybridization of the
R5 primer to recombined DNA is precise, whereas hybridization to the unrecombined substrate results in a mismatch at the
3 end of R5 (Fig. 1B). Despite the mismatch, some amplification of a 456-bp PCR fragment was observed with R5 and
R14 on the unrecombined substrate (Fig. 1C, lane 5). However, there was a preference for the recombined product even
when the unrecombined plasmid was present in vast excess
(Fig. 1C). Furthermore, the presence of the unrecombined
substrate did not significantly inhibit detection of the recombined product (Fig. 1C). As a further control for the in vitro signal joining reaction, pJH200 was cotransfected with or
without RAG1 and RAG2 into 293T cells. The 252-bp product
was only amplified from DNA recovered from cells transfected
with the combination of pJH200, RAG1, and RAG2 (Fig. 2A).
Using this PCR assay, we found that RAG1 and RAG2 or
a combination of both were not sufficient to carry out signal
joint formation in vitro. However, when RAG1 and RAG2
proteins were complemented with nuclear extracts, signal
joints were generated as evidenced by the presence of the
252-bp PCR-amplified fragment (Fig. 2 A, B, and D). Signal
joints were detected after approximately 2 hr and the amount
of product increased for up to 12 hr (Fig. 2 B). In contrast,
cleavage was observed after a few minutes under the same
reaction conditions (data not shown). The time lag between
cleavage and joining may reflect the time required for the two
signals ends to find one another and be joined. A significant
time lag between cleavage and signal joint formation was
observed in vivo (29).
To estimate the number of signal joints formed in vitro, we
constructed a standard curve with different amounts of prerecombined molecules diluted in 2.5 ng of the unrecombined
substrate (Fig. 1C). We found that a typical in vitro reaction
resulted in formation of approximately 107 recombined molecules, corresponding to an average of 0.2% of the pJH200
substrate being transformed into recombined product. Immunology: Cortes et al. FIG. 3. Signal joints generated in vitro and detected by PCR are
precise. ( A ) Southern blot analysis of pJH200 recombined in vitro and
PCR-amplified with flanking oligonucleotides R3 and R14. Because
primers R3 and R14 hybridize equally well to the recombined and
unrecombined pJH200, the DNA recovered from in vitro recombination reactions was first digested with HincII before PCR amplification,
to enrich for the recombined DNA. The in vitro recombination assay
was performed with the indicated combinations of RAGs, 293-S
nuclear extracts, and 50 ng of pJH200 (lanes 1–5). The HincII-digested
DNA from these recombination reactions, as well as a titration (0–100
pg) of pJH200R without (lanes 6–10) or with (lanes 11–15) 2.5 ng of
pJH200, was then subjected to PCR with the following modifications:
no [32P]dCTP was added to the mixture and the primer pair used was
R3 R14 (which generates a 256-bp fragment). Unlike R5, which
overlaps the signal joint, the 3 end of R3 stops 3 bp before the signal
joint and is, therefore, considered a flanking primer. The PCR
products were separated on an agarose gel, transferred to nylon
membrane, subjected to Southern blot analysis using a 32P-labeled
oligonucleotide that overlaps the signal joint and visualized by autoradiography. ( B ) pJH200 recombined in vitro and PCR-amplified with
flanking oligonucleotides R3 and R14, in the presence of [32P]dCTP.
pJH200 was subjected to the in vitro recombination assay in the
absence (lane 1) or presence of RAGs and 293-S nuclear extract (lane
2). As a control, pJH200 was transiently transfected into 293T cells
alone (lane 3) or along with RAGs (lane 4) to generate in vivorecombined plasmid. DNA recovered from recombination reactions was
digested with HincII and PCR-amplified using flanking oligonucleotides
R3 and R14. [32P]dCTP was used during PCR amplification. The
32P-labeled PCR products were fractionated on an 8% native polyacrylamide1 TBE gel and detected by autoradiography. (C) Restriction
enzyme digestion of signal joints formed in vivo and in vitro. The 256-bp
product from in vivo and in vitro reactions was extracted from a gel and
submitted to restriction enzyme digestion with ApaL1 (A1), RsaI (RI),
and HindIII (HIII). Undigested and digested aliquots were separated on
an 12% native polyacrylamide gel and the 32P-labeled PCR products were
detected by autoradiography. ApaL1 cuts at the correct signal joint,
located 20 bp from the end of the PCR product. Proc. Natl. Acad. Sci. USA 93 (1996) 14011 Nuclear extracts from BASC6C2, 22D6, HeLa, CHO-S, or
293-S cells complemented the RAG proteins in signal joint
formation (Fig. 2D). However, nuclear extracts prepared from
293 cells stably expressing RAG1 and RAG2 (293-S) were at
least 5 times more active than all other extracts tested for signal
joint formation. Low levels of recombination were seen in
unsupplemented extracts both from BASC6C2 pro-B cells,
which constituitively express RAG1 and RAG2 and actively
recombine their immunoglobulin genes, and CHO-S cells,
which stably express low levels of RAG1 and RAG2. This
recombination activity was enhanced by the addition of recombinant RAG1 and RAG2 (Fig. 2D).
To determine whether the amplified products from in vivo
and in vitro reactions were molecularly identical, we purified
the 252-bp fragment from both reaction mixtures and performed restriction digests with RsaI and HindIII. We found
that the digestion pattern of the PCR products of the in vitro
V(D)J joining reaction was identical to that displayed by
authentic in vivo recombination reactions (Fig. 2C).
Because the R5 primer crosses the recombination border
and could theoretically give an artifactual result, we used an
alternative pair of flanking primers, R14 and R3 (Fig. 1 A), to
confirm in vitro signal joint formation. The recombined product was detected by Southern blot analysis with a probe that
covered the recombination border. The results obtained with
the flanking primers were identical to those obtained with the
R5 and R14 primers. Using these primers, we found in vitro
signal joint formation only when the RAG proteins and
nuclear extract were present (Fig. 3A). Since the heptamer–
heptamer junction generates an ApaL1 site, digestion with this
enzyme was used as to define the precision of the signals joints
amplified with the flanking primers. PCR was performed with
R3 and R14 flanking primers, on in vitro and in vivo recombination reactions, in the presence of [32P]dCTP (Fig. 3B) and
the 256-bp recombined product was analyzed by restriction
digestion (Fig. 3C). Almost 100% of the amplified products
from the in vitro and in vivo reaction were ApaL1-sensitive (Fig.
3C), indicating that the majority of the signal joints produced
in vitro were precise.
Inversion V(D)J reactions are characteristic of antigen
receptor gene rearrangement in lymphoid cells. To examine
signal joint formation in an inversion substrate, we used
pJH288 (30) and the PCR assay with oligonucleotides R5 and
R14. In this assay, the recombined plasmid should generate a
PCR product of 231 bp. Neither RAG1 or RAG2 alone nor the
combination was sufficient to generate an inversion product.
However, extracts from 293-S cells were able to complement
the purified RAG proteins and produce signal joints by
inversion (Fig. 4A). To verify the accuracy of the inversionmediated signal joints, the products of in vivo and in vitro
reactions were compared by restriction enzyme digestion (Fig.
4B). Just as with deletional joining, the joints mediated by
inversion in vitro were indistinguishable from their counterparts produced in vivo. Signal joints in this inversion substrate
were detected after approximately 2 hr (Fig. 4C), whereas
cleavage could be observed after a few minutes (data not shown).
During in vivo V(D)J recombination, a DNA sequence with
a 12 RSS recombines with a sequence containing a 23 RSS.
This restriction is known as the 12 23 rule and has been shown
in vitro to regulate cleavage when Mg2 is included in the
reaction (10, 12). Under our reaction conditions, we were
unable to observe the 12 23 rule in cleavage and, therefore,
signal joint formation could not obey the 12 23 rule. We are
pursuing further studies to attempt reconstitution of signal
joint formation under conditions that allow observation of the
12 23 rule. Reproduction of this restriction is key to producing
an in vitro system that completely mimics the in vivo reaction.
Cleavage of the RSS at the heptamer results in blunt-ended
5-phosphorylated linear DNA molecules (8, 9, 13–16). These
ends could potentially be joined to one another nonspecifically 14012 Immunology: Cortes et al. Proc. Natl. Acad. Sci. USA 93 (1996) FIG. 4. In vitro signal joint formation by inversion. ( A ) Autoradiogram of the 32P-labeled PCR products amplified from the recombination
reaction using oligonucleotides R5 and R14 and separated in an 8% polyacrylamide1 TBE gel. pJH288 inversion substrate was incubated with
RAG1 (R1), RAG2 (R2), and nuclear extract (NE) as indicated. The in vivo results, to the right, show the 32P-labeled PCR products from 293T
cells transfected with pJH288 alone () or cotransfected with RAG1 and RAG2 (). The 231-bp 32P-labeled PCR product is indicated. ( B )
Digestion pattern of the in vivo and in vitro products, recombined by inversion. The 231-bp 32P-labeled PCR products were incubated with no enzyme
(), ApaLI (A1), BamHI (BI), and SmaI (SI) as indicated and separated in an 8% polyacrylamide1 TBE gel. The sizes of the fragments are
indicated. ( C ) Time course of in vitro signal joint formation by invertion. The reaction conditions were identical to those described in A, lane 7
(with RAGs and nuclear extract). by DNA ligases to complete the signal joining reaction. In fact,
complementation of RAG1 and RAG2 with purified T4 ligase
will cause signal joint formation (data not shown). However,
genetic experiments indicate that several factors other than
RAG1 and RAG2 are required for efficient V(D)J recombination in vivo including the p86 subunit of Ku (19). To
determine whether in vitro signal joint formation has similar
requirements, we performed immunodepletion experiments
with antibodies against the Ku antigen. We found that polyclonal antibodies to p70, one of the Ku subunits, and monoclonal antibodies that recognize the p70 p86 Ku heterodimer
(31) specifically inhibited signal joint formation (Fig. 5).
Preimmune serum and monoclonal isotype controls had no
effect on the reaction. Thus, the in vitro reaction resembles
authentic V(D)J recombination in that it involves genetically
defined factors other than RAG1, RAG2, and ligase.
In summary, we have developed a cell-free system that
mediates signal joint formation. This in vitro reaction recapitulates key aspects of signal joint formation as observed during in vivo V(D)J recombination. The joints are precise head to
head heptamer fusions and their formation involves factors
beyond RAG1 and RAG2. Because RAG1 and RAG2 are
known to be sufficient for cleavage at RSSs (ref. 8 and data not
shown), the other cellular proteins must be needed for the
joining reaction. Our antibodies identify Ku-70 as one component that we assume acts in complex with Ku-86 and the
DNA protein kinase. Other factors could well be necessary.
The availability of this system to study signal joint formation
should allow for rapid progress in identifying all of the factors
required to mediate this reaction.
We thank Dr. David Schatz for critical review of this manuscript and
also for providing the DNA substrate to test the 12 23 rule, Bruce
Meyer for providing the pEBG plasmid, and Juan Carcamo and
Dennis Sawchuk for comments on the manuscript and very helpful
discussions. P.C. was supported by a fellowship from the Irvington
Institute and is now supported by the Lymphoma Research Foundation of America. J.-C.L. is supported by a fellowship from the Irvington
Institute. This work was supported by grants from the National
Institutes of Health to D.B. and M.C.N. M.C.N. is an associate
investigator in the Howard Hughes Medical Institute and D.B. is an
American Cancer Society Research Professor.
8. FIG. 5. Anti- () Ku70 antibodies specifically inhibit in vitro signal
joint formation. On the left (lanes 1–5), the in vitro recombination
assay was performed with the indicated combinations of RAGs, 293-S
nuclear extracts, and 50 ng of pJH200. Lane 4 corresponds to a control
reaction: RAG1, RAG2, and NE but no template DNA. Lanes 6–11
show complete reactions in which the RAGs and nuclear extracts were
preincubated for 15 min at 4C with a preimmune sera (PI), a
polyclonal Ku70 (I-Ku70) raised against rat Ku-70, Ku monoclonal
(m-Ku70 80) raised to the human p70 p86 heterodimer (ref. 31,
monoclonal 162), or an isotype-matched monoclonal antibody (HB95;
American Type Culture Collection). The 1 and 2 refer to the
relative amounts of sera used. Each in vitro recombination reaction was
then analyzed by PCR as described in Fig. 1. 9.
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