Cell, Vol.

54, 453-460,

August 12, 1988, Copyright

0 1988 by Cell Press

The scid Defect Affects the Final Step of the

lmmunoglobulin VDJ Recombinase Mechanism
Barbara A. Malynn: T. Keith Blackwell:
Gabrielle M. Fulop,f Gary A. Rathbun:
Andrew J. W. Furley, Pierre Ferrier: L. Bruce Heinke:
Robert A. Phillips,f George D. YanCOpOUlOS,
and Frederick W. Alt
*Howard Hughes Medical Institute
and Departments of Biochemistry and Microbiology
Columbia University
College of Physicians and Surgeons
New York, New York 10032
t Department of Medical Genetics
University of Toronto
and Division of HaematologyIOncology
The Hospital for Sick Children
Toronto, Canada

Summary
Abelson murine leukemia virus-transformed
precursor B lymphocytes from scid (severe combined immunodeficient)
mice, like A-MuLV transformants from
normal mice, actively rearrange segments of their lg
heavy chain variable region gene locus during growth
in culture. Targeting of recombination to appropriate
segments appears normal in these lines as evidenced
by initial rearrangement of sequences from within the
D and JH locus to form aberrant WI+ rearrangements
and secondary rearrangement
of sequences from
within the VH locus to the aberrant DJ intermediates. A detailed analysis of the joints in these rearrangements indicates that the VW recombinase in
scid pre-B cells can correctly recognize heptamernonamer signal sequences and perform precise endonucleolytic
scissions at these sequences. We propose that the scid defect involves the inability of scid
precursor lymphocytes to join correctly the cleaved
ends of the coding strands of variable region gene
segments.
ntroduction
The variable regions of immunoglobulin (lg) and T cell
receptor (TCR) lymphocyte antigen receptors are encoded
by DNA segments (V, D, and J) that are separate in the
germ line but are assembled into complete variable region
genes by rearrangement events during precursor (pre)lymphocyte differentiation (reviewed in Tonegawa, 1983;
Alt et al., 1986; Marrack and Kappler, 1987). These somatic rearrangement events are mediated by conserved
recognition sequences which flank each of the germ-line
elements and consist of a palindromic heptamer and a
conserved nonamer separated by a nonconserved spacer
sequence of 12 or 23 bp (reviewed in Tonegawa, 1983);
only segments flanked by recognition sequences with
spacers of different length are joined (the 12/23 rule; Early
et al., 1980; Sakano et al., 1980). The recognition se-

quences are thought to be targets of a common sitespecific recombination system (Yancopoulos et al., 1986),
subsequently referred to as VDJ recombinase. Assembly
of variable region gene segments has been postulated to
involve a two-step nonreciprocal recombination mechanism (Alt and Baltimore, 1982; Figure 1A). The initial event
is a precise endonucleolytic scission between the involved segments and their flanking heptamers. Subsequently, the heptamers are precisely joined to each other,
but the two coding strands are often imprecisely joined.
Imprecision in coding strand joining may involve loss of
nucleotides from one or both strands and addition of novel
nucleotides at the point of joining (N regions) (reviewed by
Alt et al., 1987).
scid (severe combined immune deficiency) is an autosomal, recessive mutation that arose spontaneously in
CB-17 mice (Bosma et al., 1983). Mice that are homozygous for this mutation fail to develop mature T or B lymphocytes (Bosma et al., 1983; Dorshkind et al., 1984).
Although scid mice lack detectable numbers of mature B
lineage cells, they have normal numbers of targets for
A-MuLV transformation, suggesting that they generate
normal numbers of B cell precursors (Fulop et al., 1988).
Southern blot analyses of DNA rearrangements in A-MuLV
transformants or spontaneous thymomas in scid mice
suggested that both B and T lineage cells of scid mice
made highly aberrant rearrangements, often large deletions, at their respective lg or TCR loci (Schuler et al.,
1986). This finding strongly implied that the scid mutation
affects the activity of some component of the common
VDJ recombinase. To elucidate the nature of the scid defect, we generated A-MuLV transformed cell lines from
scidmice and their normal C.B-17 counterparts. Comparative analyses of these lines demonstrated that scid transformants are identical to those from normal mice with respect to the expression of a large variety of tested pre-B
cell markers and activities except for production of mRNA
capable of encoding complete lg heavy chain molecules
(Blackwell et al., submitted). In this paper, we define in
molecular terms the nature of rearrangement events
within the IgH variable region gene locus in scidpre-B cell
lines and propose a mechanistic explanation for their
structure.
Results
Ongoing Rearrangement of the JH Locus in scid
A-MuLV Transformants
Transformation of murine bone marrow or fetal liver with
A-MuLV generates pre-B cell lines of which many actively
assemble lg heavy chain variable region genes during
growth in culture (reviewed in Alt et al., 1986). Studies of
these transformants demonstrated that heavy chain variable region genes are assembled in an ordered process:
first a D segment is joined to a J, segment, ancl subsequently a VH segment is appended to the preexisting DJn
complex (Alt et al., 1981; Sugiyama et al., 1983; Alt et al.,

Cell
454

Figure 1. Model for VDJ Recombinase-Mediated Rearrangements

The germ-line lg locus is represented by a D
coding segment (open box) flanked by a 12 bp
spacer signal element (shaded triangles) and
positioned at an undefined distance upstream
of a JH coding segment (black box) flanked by
a23 bp signal element (open triangles). Arrows
indicate the transcriptional orientation. A proposed normal joining mechanism is shown in
panel A (adapted from Alt and Baltimore,
1982). Illegitimate recombination events proposed to resolve the defective joining process
in scidpre-B cells may result in deletion of one
(6) or both (D) coding segments or inversion of
a chromosomal fragment (C). See text for further details.

- -

- -

- - - -

1984; Desiderio et ai., 1984). To characterize the molecular nature of the scid defect, we isolated A-MuLV transformants from normal CB-17 and CB-17scid mice (subsequently referred to as scid mice) and assayed the lines for
rearrangements of their lg JH locus. For this assay,
genomic DNA from the various lines was digested with
EcoRl and assayed by Southern blot procedures for hybridization to a Ju-specific probe (Figure 2).
Genomic DNA from the normal C.B-17 lines contained
multiple Jn-hybridizing EcoRl fragments (Figure 2, right
panel), a phenomenon previously demonstrated to indicate ongoing heavy chain gene rearrangement (Alt et al.,
1981,1984). EcoRI-digested genomic DNA from most scid
transformants also contained multiple Jn-hybridizing
fragments; many, like those from initial isolates of normal
C.B-17 transformants, hybridized to 5.flanking D probes.

This result indicates that these fragments represented

rearrangements that fused sequences from the D locus to
sequences from the JH locus (Figure 2, left panel and
data not shown). Subcloning analyses confirmed that the
various scid cell lines continued to rearrange DNA segments within this region during propagation in culture
(Blackwell et al., submitted; see below). Although two scid
A-MuLV transformants (SC24 and SC44) apparently
differed from A-MuLV transformants of normal mice in that
they retained a germ-line Jn allele (Figure 2), subcloning
analyses of the SC44 line demonstrated that during culture it also underwent rearrangements that fused sequences from the D locus to sequences within the Jn locus (data not shown). Therefore, based on these and other
data (Blackwell et al., submitted), we conclude that A-M&V
transformed pre-6 lines from both the C.B-17 and scid

The scid VDJ Recombinase

455

Defect

C.B-17

SCID

-23-

-23-2.0-

J,, probe
6.4kb
Figure 2. JHAssociated
Rearrangements
scid and Normal CB-17 Mice

c
in Pre-B Cell Lines from

Genomic DNA (10 Kg) from the scid (left panel) and normal C.B-17
(right panel) A-MuLV transformed cell lines was digested with EcoRl
and analyzed by Southern blot analysis for hybridization to the 32P-labeled JH probe indicated in the figure. DNA from CB-17 liver served
as the germ-line control. Fragment sizes of Hindlll-digested
lambda
phage DNA are indicated in kb.

mice have an active VDJ recombinase system. However,

the scid pre-B transformants, unlike the C.B-17 transformants, did not express normal lg heavy chain mRNA
(Blackwell et al., submitted), which suggests that the ongoing rearrangements generated in the scid pre-B lines
are always aberrant.
The Nature of JH-Associated Rearrangements
in scid A-MuLV Transformants
To define the defective rearrangements, JH-associated
EcoRl fragments from SC3, SC7, SC24, and SC44 scid
A-MuLV transformants were isolated and the nucleotide
sequences of junctional regions determined. These sequences are compared to germ-line sequences in Figure
3A and are schematically represented in Figure 38. We refer to normal D to JH joining as recombinations that fuse
a D coding region to a J, coding region with the possible
loss of a few base pairs from one or both coding ends and
addition of N regions (Figure 1A). None of the scid rearrangements analyzed involved normal ligation at both
ends of two joined segments. Three rearrangements
(SC3, SC7A, and SC7B) involved joining of sequences located 5 to a D coding region into sequences 90 to 1100
bp 3to the JH coding region, thereby deleting both D and

J coding regions. Origins of the regions irnmediately 5to

the recombination points of the SC3 and SC7A rearrangements (Figure 3A, #l and #3) were identified by sequence
homology to be upstream of DSPP and DQ52 coding sequences, respectively. Sequences 5of the recombination
point in the SC7B rearrangement (Figure 3A, #4) could not
be similarly identified; however, restriction mapping and
hybridization analyses (see legend to Figure 3A) demonstrated that this portion of the SC-/B rearrangement derived from a region within l-2 kb upstream1 of DQ52. Even
larger deletions at the JH locus have been observed
(Schuleret al., 1986; Blackwell et al., submitted); however,
such rearrangements would not have been detected with
the probe utilized.
Although none of the scid rearrangements involved normal recombination at the borders of both joined segments,
six of nine joins had recombination joints that could be
considered normal (i.e., rearranged precisely at the coding regionlheptamer junction with the possible loss and/or
addition of several base pairs) with respect to one of the
participating segments but not the other (Figure 38). For
example, the 4.4 kb EcoRl fragment in SC44 DNA (which
was cloned from SC44-40, a subclone of X44, Figure 3A,
#2) contained a normal join of the JH1 coding segment to
flanking sequences upstream of a DFL16 coding region.
Two rearrangements (SC7C and SC24A; Figure 3A, #5
and #7, respectively) contained joins that were apparently
normal with respect to D coding regions but wsere rearranged into sequences 300 to 400 bp 3 to Jr, coding
regions. Rearrangement SC24B contained normal joins
with respect to both the involved D and Jr. segments;
however, these segments were not joined to each other
but rather to noncoding sequences within the JH region.
Thus, a D coding sequence was joined to a sequence just
downstream of the JH4 coding region, with the latter in inverted orientation relative to the D (Figure 3A, #8); downstream of this joint, the inverted JH4 coding region was
joined to a site 90 bp upstream of the EcoRl site that is
immediately 3of the Jn coding segments (IFigure 3A, #9).
A short sequence (ATTTTA) of unknown origin was inserted at this junction (Figure 3A, #9). Nucleotides that do
not appear to be derived from germ-line sequences were
observed in several other rearrangements isolated from
scid lines (SC3 and SC24A; Figure 3A), perhaps representing N regions (Blackwell, et al., submitted). However,
it is also possible that the novel bases may have been derived by other mechanisms (see below and legend to Figure 3A).
In summary, even rearrangements that appeared normal by Southern blot analyses (i.e., that hybridized to both
5 D and Ju probes) were grossly aberrant but often had
some characteristics in common with normal joins.
Secondary Rearrangements
in scid A-MuLV
Transformants
A-MuLV transformants from normal milce undergo two
types of secondary rearrangements of DJn joins: replacement of the initial DJn rearrangement bsy joining an upstream D to a downstream Jr, and/or appendage of a Vu
segment to the DJH intermediate (Reth et al., 1986). The

Cell
456

Figure 3. Nucleotide Sequences

sociated Rearrangements

B
Germline

Dcm

J,,

JH2

JH3

JH-associated rearrangements SCX, SC24A, and SC24B,

although aberrant, have intact upstream heptamer-nonamer signal sequences on the involved D segment and
thus theoretically have the potential to undergo VDJ
recombinase-mediated secondary rearrangements to upstream elements. Subclones of the primary scid A-MuLV

Ii4

Em

of scid JH-As-

(A) JH-associated rearrangements were cloned

from the indicated cell lines as previously described (Alt et al., 1984) and the nucleotide sequences of junctional regions determined. The
scid sequences are compared to homologous
germ-line sequences (JHI sequence, Early et
al., 1980; J$-JH~, Gough and Bernard, 1981;
Cl region sequences in #I and #5, Kurosawa et
al., 1981 and in #7 and #8, Kurosawa and
Tonegawa, 1982). Identical nucleotides are indicated by dashes; nucleotide differences are
noted. The germ-line sequences were derived
form a different strain of mice; thus, some of
the observed nucleotide differences may be
due to polymorphisms. The origins of the 5sequences in SC44-40 (#2), SC7A (#3), and SC78
(#4) were identified by the following criteria: 1)
phage clones containing the rearrangements
from SC44-40 and SC7A hybridized to 5DFLlG
and to 5 DQ52 probes, respectively; 2) the restriction maps of the 5 portions of phage
clones containing rearrangements from SC7A
and SC76 were similar to each other and to the
germ-line EcoRl fragment which contains the
JH locus and extends 5 to DQ52 (Figure 2); 3)
the EcoRI-Ncpl fragment isolated from the 5
end of clone SC7A hybridized to both SC78 and
to the cloned genomic 6.4 kb EcoRl fragment
which contains the JH segments and hybridized to a single, 6.4 kb EcoRl fragment in
genomic DNA; 4) the DNA sequence determined further 5from the site of rearrangement
in clone SC7A corresponds to sequence recently reported by Nottenberg et al. (1987) and
identified
as 5-flanking
DQ52 (data not
shown). Lines are drawn to indicate probable
sites of rearrangement.
Coding regions are
boxed and heptamer-nonamer
recognition sequences are marked. The rearrangement
in
clone SC24B was the result of an inversion
event; the sequences of the 5 end joint (#8)
and the 3 end joint (#9) are shown. To simplify
the comparison of the inverted sequence, part
of the germ-line sequences are given in a 3to
5 orientation as indicated by arrows. Short
repeats are indicated by arrows which span the
length of the repeated sequence.
(B) A schematic representation
of the rearrangements
shown in Figure 3A. Coding
regions are represented by boxes and heptamer-nonamer
signal sequences
by triangles. The arrows above the germ-line map of
the immunoglobulin heavy chain locus indicate
the approximate sites of the rearrangement
events. A solid arrow denotes a rearrangement
which resulted in a normal join at the D or J
coding regions. Shaded arrows represent joins
that resulted in deletion of the coding regions
and some flanking regions. The arrows under
SC24B represent the 5 to 3 orientation of
those regions.

transformants often contained novel JH-hybridizing bands

relative to the parent line; genomic blotting analyses suggested that some of these represented rearrangements of
sequences from more upstream regions of the D locus or
from the VH locus into the region of the remaining portion
of the JH locus (Blackwell et al., submitted). To define

The scici VDJ Recombinase

Defect

457

B
1

123

56

23-

23-

9.4 -

9,4-

68-

6b-

4A-

2.3-

21)-

4.4-

23ZD-

I
ECORI

HindIU

BamHI

EcoRI

Figure 4. Attempted VH to DJH Joining in scid A-MuLV-;Transformants

(A) Genomic DNA (10 ug) from scid liver was digested with the indicated restriction endonucleases and Southern blots were hybridized
to 32P-labeled probes derived from either the coding region of a
VnQ52 gene (Yancopoulos et al., 1988) (lanes 1, 3, and 5) or from sequence 5 to the join in the subcloned plasmid from the phage clone
SC7D (probe 32, OxaNl fragment) (lanes 2, 4, and 6).
(B) Deletion mapping of probe 32. Southern blots of EcoRCdigested
DNA (10 pg) from BALB/c liver (lane l), 22D6-5 (lane 2) and BCLI (lane
3) were hybridized to probe 32. The 22D6-5 cell line has DJn rearrangements on both alleles (Alt et al., 1984) and the BCLl cell line has
apparently duplicated copies of chromosome 12 that contain identical
VHJ558DJe rearrangements (Chen et al., 1986); thus, this line deleted
the downstream V&52 and V,7183 families (Rathbun et al., 1987).

such a potential secondary rearrangement, we isolated,

from the genomic DNA of the SC7 line, JH-hybridizing
EcoRl fragments that failed to hybridize to 5 D probes.
The nucleotide sequence of the junctional region of one
such rearrangement (SC7D) demonstrates that it derived
from a secondary rearrangement of an aberrant DJH join
represented by clone SC7C (Figure 3A, compare sequences 5 and 6). The single recombination joint of SC7C consists of a DSP2 coding region joined to intervening sequence between JH3 and JH4; this joint is identical to the
3 recombination joint in clone SC7D. Thus, SC7D represents a secondary rearrangement in which novel sequences have joined into the 5 border of an aberrant
DSP2 to JH rearrangement; in this case, the recombination was normal with respect to the 5 border of the involved D.
The sequence appended to the 5 flank of the D sequence in SC-/D had no homology to any known nucleotide sequence. To determine its source, a probe derived
from the 5 portion of SC7D (probe 32) was hybridized to
a Southern blot of scici liver DNA digested with variousrestriction enzymes. Hybridization patterns obtained were
almost identical to those of a probe specific for V&l52
codingregions (Figure 4A). V&52 is one of the most JHproximal Vn families in tested mouse strains (Brodeur et
al., 1984; Reth et al., 1986; Rathbun et al., 1987; Blanken-

stein and Krawinkel, 1987). To further define its origin,

probe 32 was hybridized to EcoRI-digested DNA from
B-lineage cell lines that either had DJH [rearrangements
and retained all VH gene segments (22D6-5) or that had
deleted all members of the V&52 family but retained
members of upstream VH families (BCLl) (see legend to
Figure 4). Sequences hybridizing to probe 32 were deleted from BCLl DNA (Figure 4B, lane 3) but not from
22D6-5 DNA (Figure 48, compare lanes 1 and 2) confirming that this sequence originated from within the proximal
portion of the VH locus. We conclude that the rearrangement represented by SC7D arose as an attempt to append
a VH segment to the aberrant, primary DJH rearrangement, but resulted in deletion of the V coding region.
Thus, in this strikingly novel rearrangement, an essentially complete DSP2 coding segment was joined at its 3
border to sequences downstream of a &3 segment and
at its 5 border to sequences upstream of a VH segment.
Discussion
scid Pre-B Lines Have an Active, but Defective,
VW Recombinase
scid A-MuLV transformants, like normal pre-5 transformants, have an active VDJ recombinase system, as
shown by their ability to rearrange portions of the Ig heavy
chain variable region locus during growth in culture. Initial
rearrangements often generate grossly aberrant joins of
sequences in the D locus to sequences in the Jn locus;
secondary rearrangements can involve joining of sequences from the VH locus to the aberrant DJH joins. In
contrast to our current findings, previous analyses found
that most scid A-MuLV transformants had deleted the region of the JH locus shown in Figure 2 (Schuler et al.,
1986). One explanation for the apparent discrepancy between the results of these two studies is our finding that
rearrangements of the JH locus continue during culture of
scid A-MuLV transformants, resulting not only in accumulation of aberrant rearrangements but also the accumulation of further deletions at the locus. Thus, after long-term
propagation, we observed deletion of the JH locus in several scid lines (SC7, SC24, and SC44) that initially had
JH-hybridizing sequences (Blackwell et al., submitted);
these deletions appeared similar in extent to those previously described (Schuler et al., 1986). Such deletions
might derive from VDJ recombinase-mediated events or
from other types of rearrangements. For example, scid
pre-B lines appear to have active heavy chain class-switch
recombinase activity (Lutzker et al., 1988; Blackwell et al.,
submitted); in normal A-MuLV transformants, this activity
is believed to generate large deletions in the JH-C, intron, with some extending far into the JH region (e.g., Alt
et al., 1982b). We intentionally analyzed lines soon after
establishment in culture to define ongoing rearrangements and to avoid possible outgrowth of cells that completely deleted the JH locus.
Studies of A-MuLV transformants from scid mice indicate that the scid defect is manifested as i:he inability of
immature precursor B cells to produce mRNA capable of
encoding complete lg heavy chain molecules (Blackwell

Cell
458

et al., submitted; Fulop et al., 1988; Witte et al., 1987;

Hirayoshi et al., 1987). Our current studies demonstrate
that this phenomenon does not result from complete absence of the VDJ recombinase activity nor from its inappropriate targeting with respect to lg loci. Targeting of
recombinase to appropriate loci appears normal as evidenced by sequential attempts to make first D to JH and
subsequently Vu to DJH rearrangements, all generally in
the absence of rearrangement at light chain loci (Figures
3 and 4; Blackwell et al., submitted). The finding that scid
lines attempt VH rearrangements to the grossly aberrant
DJH rearrangements is consistent with previous indications that once a DJu is formed, subsequent recombination events are targeted to more 5 regions of the locus
(JH-distal D segments or Vu segments) (Reth et al., 1986).
Many aspects of the VDJ recombination mechanism
also appear to function normally in scid pre-B cells. Comparison of the sequences of JH-associated rearrangements in scidpre-B lines to known germ-line counterparts
did not reveal pseudo recognition sequences adjacent to
abnormally joined partners, suggesting that the scid defect does not affect the fine specificity of VDJ recombinase cleavage. Furthermore, many primary and secondary
joins were normal with respect to one or the other involved
coding segment; this finding indicates that scid recombinase can recognize, and precisely cut at, heptamernonamer signal sequences. Although none of the rearrangements had normal joins with respect to both partners
involved, all derived from abortive joining attempts involving two appropriate segments (i.e., D to JH or Vn to DJH)
flanked by complementary recognition sequences in accord with the 12/23 rule. Therefore, it is unlikely that the
scid mutation results in an abnormal recombinase that
generally recognizes only a single gene segment. Finally,
a large percentage (6 of 18) of involved D or J segments
in the joins analyzed did not have excessive deletions.
This finding suggests that the scid defect does not result
simply from a hyperactive exonucleolytic activity; this percentage of normal ends should result in the generation of
detectable numbers of mature 6 and T cells in scid mice
if all other joining steps are normal.
Our findings suggest that the most likely explanation for
the scid defect is the impaired joining of coding segment
ends that have been generated by a normal site-specific
endonucleolytic activity. Several different mechanisms
could lead to an inability to join free coding segment ends
correctly. One explanation may be an abnormality in a Iigase activity responsible for fusing the ends; alternatively,
a complex that normally holds the free ends or brings
them together after endonucleolytic cleavage may be
defective such that normal ligation of the coding segment
ends cannot occur. Whatever the defect, the absence of
detectable B and T cells in the majority of scid mice alS0
suggests that such free ends are not fused end-to-end by
any nonspecific mechanism to create normal joins at Significant frequency.
Model for the Molecular Basis of Defective
Rearrangements
in scid Mice
In yeast (Malone and Esposito, 1980; Weiffenbach and
Haber, 1981; Klar et al., 1984) and in other eukaryotic sys-

tems (reviewed in Hickson and Harris, 1988) unrepaired

double-strand DNA breaks are lethal. The scid defect that
we have suggested would result in a high frequency of
double-strand breaks in scid pre-B transformants. In this
regard, the accumulation of scid A-MuLV transformants
that have deleted the entire JH locus (Schuler et al., 1986;
Blackwell et al., submitted) may reflect a selective advantage of cells that no longer have target sequences for VDJ
recombination-removing
targets for potentially lethal
double-strand breaks. In this context, the putative inability
to normally link the free ends would require alternative
recombination mechanisms to restore chromosomal integrity in scid pre-B transformants that attempted rearrangements of the JH locus.
Illegitimate recombination events (i.e., not mediated by
homologous or site-specific recombination) have been implicated in restoring chromosomal integrity in other eukaryotic systems that generate unrepaired double-strand
breaks (Hawley and Tartof, 1983; Hawley et al., 1988). An
illegitimate recombination mechanism is consistent with
the abnormal structures of the joins in the scid lines.
There were no long stretches of homology between the involved partners, eliminating homologous recombination
as a mechanism. Furthermore, several rearrangements
had direct repeats of 3-8 bp precisely at the junction
(SC7B, SC-/C, and SC24B3; Figure 3A); such short direct
repeats have been observed at junctions of illegitimate
recombination sites and may result from the mechanism
of ligation and repair (Roth et al., 1985; Roth and Wilson,
1986). We propose that rearrangements in scid pre-B lines
that are normal for one partner involve illegitimate recombination between one of the free ends and the chromosomal fragment containing the other free end (Figure
1B). Illegitimate recombination events involving free ends
generated by VDJ recombinase may also account for the
rearrangement containing the inversion that was isolated
from SC24 (Figure 3A, #8 and #9 and Figure 1C). In this
rearrangement, site-specific cleavage is proposed to occur adjacent to the involved D and JH coding segments at
the positions used for conventional deletional joining
(compare Figure 1A and 1C); the inverted sequence would
result if the free ends containing the D and JH segments
underwent illegitimate recombination at the positions illustrated (Figure 1C). Finally, rearrangements that are not
normal with respect to either partner also can be explained in the context of this model (Figure 1D); for example, loss of varying numbers of base pairs often occurs at
the ends of linear DNA sequences that have been introduced into eukaryotic cells (Folger et al., 1982; Wake et al.,
1984; Kopchick and Stacey, 1984). With respect to the
substantial loss of sequence from one or both partners in
scid joins, it is possible that failure to ligate immediately
or to protect properly and/or position free ends may make
them subject to additional double-stranded breaks or allow exonucleolytic digestion to proceed further than
normal.
Aberrant rearrangements similar to those that predominate in scid transformants are occasionally observed in
normal A-MuLV transformants (Reth and Alt, unpublished
data), within recombination substrates introduced into
normal A-MuLV transformants (Yancopoulos et al., 1986)

The scid VDJ Recombinase

459

Defect

and in normal B cells (Nottenberg et al., 1987). For example, a rearrangement isolated from normal splenic B cells
consisted of sequences 5 of the DQ52 coding region
joined to sequences 3 of JM4, and contained a 4 bp repeat at the site of the join (Nottenburg et al., 1987). In addition, a VDJ recombinase-specific cutting and subsequent
illegitimate recombination event has been invoked to explain joins between lg variable region gene segments and
unrelated sequences (not containing recombination recognition sequences) that result in chromosomal translocations characteristic of certain human lymphomas (Bakhshi et al., 1987). These findings suggest that even in cells
with a normal VDJ recombinase, attempted VDJ recombinations may be resolved by illegitimate recombination
mechanisms. Although the observed frequency of such
aberrant rearrangements appears much higher in scid
pre-B cells, it is possible that the actual rate of these
events may be similar in normal and scid pre-B cells, but
in normal cells the aberrant events may be masked by a
much higher rate of normal VDJ recombinase activity. The
apparent frequency of occurrence of the aberrant recombinations in scid cells may also be amplified by selection
mechanisms as discussed above. It is notable that both
partners of the recombination events that we characterized in scid pre-B cells derived from within the IgH locus.
Recombinations into other chromosomes may occur but
at a lower frequency; the frequent observance of such rearrangements in lymphomas (Bakhshi et al., 1987) probably
results from a selective growth advantage (transformation)
conferred by the translocation.
Several human genetic disorders (xeroderma pigmentosum, Fanconis anaemia, ataxia-telangiectasia,
and
Blooms syndrome) that are associated with chromosomal
breakage, hypersensitivity to environmental carcinogenic
agents, and neoplasia may reflect defects in general DNA
repair and/or replication mechanisms (reviewed in Ray
and German, 1983). Thus far, such pleiotropic effects
have not been reported for scid mice (Bosma et al., 1983;
Dorshkind et al., 1984; Custer et al., 1985). The restricted
association of the scid mutation with a defect in lymphocyte development suggests that the scid defect is limited
to a specific VDJ recombinase component rather than a
more general recombination disorder.
Experimental

Procedures

Cell Lines
A-MuLV transformants were derived by transformation of bone marrow
from 3-6 week old C.B.-17 (prefix CB) or CB-17scid(prefix
SC) mice as
previously described (Fulop et al., 1988). The one exception is that
SC44 was the result of transformation of spleen cells from C.B-17scid.
The clonality of cell isolates was confirmed by the criterion of a uniquely integrated Abelson provirus. BCLI has been described (Chen et al.,
1986; Rathbun et al., 1987). 22D6-5, a subclone of 22D6 (Alt et al.,
1984), has the same rearrangements as the parent line (unpublished
data).
Probes
DNA fragments used as probes were isolated and prepared as previously described (Alt et al., 1984). 3*P-labeling of DNA fragments was
performed by nick translation as previously described (Alt et al.,
1982a).
preparation
Preparation

of DNA and Southern Blot Analysis

of genomic DNA, restriction enzyme digestions,

agarose

gel electrophoresis, and DNA blotting procedures were performed as

previously described (Yancopoulos et al., 1984; Yancopotulos and Alt,
1985).
DNA Cloning, Sequencing,
and Analysis
Genomic DNA cloning procedures into Charon 16A and subcloning
into plasmid vectors have previously been described (Yancopoulos et
al., 1984). The nucleotide sequence of relevant portions of the molecularly cloned rearrangements were determined by the method of Maxam
and Gilbert (1980). DNA sequence homologies were determined by
using the Beckman Microgenie Sequence Analysis Program (Beckman Instruments, Inc., Palo Alto, CA).
Acknowledgments
The authors thank Hamish Young, Rodney Rothstein, and R. Scott
Hawley for helpful discussions, and P Tucker for the BCLI cell line.
B. A. M. and G. A. R. are Leukemia Society of America fellows;
T K. B. and G. D. Y. are fellows of the Howard t-lughes Medical Institute;
A. J. W. F. is a Jane Coffin Childs fellow; i? F. is an EMBCl fellow. This
work was supported by the Howard Hughes Medical Institute, grants
from the NIH (Al-20047 and CAa27),
and an award from the Mallinckrodt Foundation to F. W. A.; and grants from the Medical Research
Council and the National Cancer Institute of Canada to R. A. P
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby
marked advertisement in accordance with 18 1J.S.C. Section 1734
solely to indicate this fact.
Received

March 25, 1988; revised June 2, 1988.

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