Cell, Vol. 54, 453-460, August 12, 1988, Copyright 0 1988 by Cell Press

The scid Defect Affects the F inal 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 precur- sor B lymphocytes from scid (severe combined im- munodeficient) 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 intermedi- ates. A detailed analysis of the joints in these rear- rangements indicates that the VW recombinase in scid pre-B cells can correctly recognize heptamer- nonamer signal sequences and perform precise en- donucleolytic scissions at these sequences. We pro- pose 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 so- matic 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 site- specific 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 mecha- nism (Alt and Baltimore, 1982; Figure 1A). The initial event is a precise endonucleolytic scission between the in- volved segments and their flanking heptamers. Subse- quently, 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 au- tosomal, recessive mutation that arose spontaneously in CB-17 mice (Bosma et al., 1983). Mice that are homozy- gous for this mutation fail to develop mature T or B lym- phocytes (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 dele- tions, 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 de- fect, we generated A-MuLV transformed cell lines from scidmice and their normal C.B-17 counterparts. Compara- tive analyses of these lines demonstrated that scid trans- formants are identical to those from normal mice with re- spect 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 vari- able region genes are assembled in an ordered process: first a D segment is joined to a J, segment, ancl subse- quently a VH segment is appended to the preexisting DJn complex (Alt et al., 1981; Sugiyama et al., 1983; Alt et al.,

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1984; Desiderio et ai., 1984). To characterize the molecu- lar nature of the scid defect, we isolated A-MuLV transfor- mants from normal CB-17 and CB-17scid mice (subse- quently 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 hy- bridization 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 indi- cate 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.

Figure 1. Model for VDJ Recombinase-Medi- ated 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 pro- posed normal joining mechanism is shown in panel A (adapted from Alt and Baltimore, 1982). Illegitimate recombination events pro- posed 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 fur- ther details.

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 seg- ments 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 cul- ture it also underwent rearrangements that fused se- quences from the D locus to sequences within the Jn lo- cus (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 Defect 455

SCID C.B-17

-23-

-23-

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J,, probe 6.4 kb c

Figure 2. JHAssociated Rearrangements in Pre-B Cell Lines from scid and Normal CB-17 Mice

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-la- beled 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 transfor- mants, did not express normal lg heavy chain mRNA (Blackwell et al., submitted), which suggests that the on- going 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 se- quences are compared to germ-line sequences in Figure 3A and are schematically represented in Figure 38. We re- fer 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 rear- rangements analyzed involved normal ligation at both ends of two joined segments. Three rearrangements (SC3, SC7A, and SC7B) involved joining of sequences lo- cated 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 rearrange- ments (Figure 3A, #l and #3) were identified by sequence homology to be upstream of DSPP and DQ52 coding se- quences, 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) demon- strated that this portion of the SC-/B rearrangement de- rived 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 nor- mal 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 cod- ing 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 rear- ranged 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 in- verted orientation relative to the D (Figure 3A, #8); down- stream of this joint, the inverted JH4 coding region was joined to a site 90 bp upstream of the EcoRl site that is immediately 3 of the Jn coding segments (IFigure 3A, #9). A short sequence (ATTTTA) of unknown origin was in- serted 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 repre- senting N regions (Blackwell, et al., submitted). However, it is also possible that the novel bases may have been de- rived by other mechanisms (see below and legend to Fig- ure 3A).

In summary, even rearrangements that appeared nor- mal 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: replace- ment of the initial DJn rearrangement bsy joining an up- stream D to a downstream Jr, and/or appendage of a Vu segment to the DJH intermediate (Reth et al., 1986). The

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B t Germline v Dcm

J,, JH2 JH3 Em

Ii4

Figure 3. Nucleotide Sequences of scid JH-As- sociated Rearrangements

(A) JH-associated rearrangements were cloned from the indicated cell lines as previously de- scribed (Alt et al., 1984) and the nucleotide se- quences 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 in- dicated 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 5se- quences 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 re- striction 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 hybrid- ized to a single, 6.4 kb EcoRl fragment in genomic DNA; 4) the DNA sequence deter- mined further 5from the site of rearrangement in clone SC7A corresponds to sequence re- cently 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 se- quences 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 rear- rangements shown in Figure 3A. Coding regions are represented by boxes and hep- tamer-nonamer signal sequences by trian- gles. 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.

JH-associated rearrangements SCX, SC24A, and SC24B, transformants often contained novel JH-hybridizing bands although aberrant, have intact upstream heptamer-non- relative to the parent line; genomic blotting analyses sug- amer signal sequences on the involved D segment and gested that some of these represented rearrangements of thus theoretically have the potential to undergo VDJ sequences from more upstream regions of the D locus or recombinase-mediated secondary rearrangements to up- from the VH locus into the region of the remaining portion stream elements. Subclones of the primary scid A-MuLV of the JH locus (Blackwell et al., submitted). To define

The scici VDJ Recombinase Defect 457

B 1 2 3 4 56 123

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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 indi- cated 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 se- quence 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 rear- rangements 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 sequen- ces 5 and 6). The single recombination joint of SC7C con- sists of a DSP2 coding region joined to intervening se- quence between JH3 and JH4; this joint is identical to the 3 recombination joint in clone SC7D. Thus, SC7D repre- sents a secondary rearrangement in which novel se- quences have joined into the 5 border of an aberrant DSP2 to JH rearrangement; in this case, the recombina- tion was normal with respect to the 5 border of the in- volved D.

The sequence appended to the 5 flank of the D se- quence in SC-/D had no homology to any known nucleo- tide 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 variousre- striction 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 JH- proximal 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 de- leted from BCLl DNA (Figure 4B, lane 3) but not from 22D6-5 DNA (Figure 48, compare lanes 1 and 2) confirm- ing that this sequence originated from within the proximal portion of the VH locus. We conclude that the rearrange- ment represented by SC7D arose as an attempt to append a VH segment to the aberrant, primary DJH rearrange- ment, but resulted in deletion of the V coding region. Thus, in this strikingly novel rearrangement, an essen- tially 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 transfor- mants, 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 se- quences 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 re- gion of the JH locus shown in Figure 2 (Schuler et al., 1986). One explanation for the apparent discrepancy be- tween 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 accumu- lation of aberrant rearrangements but also the accumula- tion of further deletions at the locus. Thus, after long-term propagation, we observed deletion of the JH locus in sev- eral scid lines (SC7, SC24, and SC44) that initially had JH-hybridizing sequences (Blackwell et al., submitted); these deletions appeared similar in extent to those previ- ously 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, in- tron, 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 rearrange- ments and to avoid possible outgrowth of cells that com- pletely deleted the JH locus.

Studies of A-MuLV transformants from scid mice indi- cate 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 ab- sence of the VDJ recombinase activity nor from its inap- propriate targeting with respect to lg loci. Targeting of recombinase to appropriate loci appears normal as evi- denced 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 indica- tions that once a DJu is formed, subsequent recombina- tion 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. Com- parison of the sequences of JH-associated rearrange- ments in scidpre-B lines to known germ-line counterparts did not reveal pseudo recognition sequences adjacent to abnormally joined partners, suggesting that the scid de- fect does not affect the fine specificity of VDJ recombi- nase cleavage. Furthermore, many primary and secondary joins were normal with respect to one or the other involved coding segment; this finding indicates that scid recom- binase can recognize, and precisely cut at, heptamer- nonamer signal sequences. Although none of the rear- rangements had normal joins with respect to both partners involved, all derived from abortive joining attempts involv- ing two appropriate segments (i.e., D to JH or Vn to DJH) flanked by complementary recognition sequences in ac- cord 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 per- centage 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 Ii- gase 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 Sig- nificant 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 advan- tage 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 in- tegrity in scid pre-B transformants that attempted rear- rangements of the JH locus.

Illegitimate recombination events (i.e., not mediated by homologous or site-specific recombination) have been im- plicated in restoring chromosomal integrity in other eu- karyotic 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 in- volved 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 recom- bination between one of the free ends and the chro- mosomal 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 oc- cur 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 il- lustrated (Figure 1C). Finally, rearrangements that are not normal with respect to either partner also can be ex- plained in the context of this model (Figure 1D); for exam- ple, loss of varying numbers of base pairs often occurs at the ends of linear DNA sequences that have been intro- duced 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 al- low exonucleolytic digestion to proceed further than normal.

Aberrant rearrangements similar to those that predomi- nate 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 Defect 459

and in normal B cells (Nottenberg et al., 1987). For exam- ple, 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 re- peat at the site of the join (Nottenburg et al., 1987). In addi- tion, a VDJ recombinase-specif ic cutting and subsequent illegitimate recombination event has been invoked to ex- plain joins between lg variable region gene segments and unrelated sequences (not containing recombination rec- ognition sequences) that result in chromosomal translo- cations characteristic of certain human lymphomas (Bakh- shi et al., 1987). These findings suggest that even in cells with a normal VDJ recombinase, attempted VDJ recombi- nations 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 recom- binations 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 character- ized 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 rear- rangements in lymphomas (Bakhshi et al., 1987) probably results from a selective growth advantage (transformation) conferred by the translocation.

Several human genetic disorders (xeroderma pigmen- tosum, 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 lympho- cyte 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 unique- ly 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 previ- ously described (Alt et al., 1984). 3*P-labeling of DNA fragments was performed by nick translation as previously described (Alt et al., 1982a).

preparation of DNA and Southern Blot Analysis Preparation 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 molecu- larly 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 (Beck- man 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 Mallinck- rodt 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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