Eur. J. Immunol. 1990. 20: 545-550

Walter SchulerAo, Amelie SchulerO and Melvin J. BosmaO Fox Chase Cancer Centero, Institute for Cancer Research, Philadelphia and Basel Institute for ImmunologyA+,Basel

Defective V-to-J recombination in scid mice

545

Defective V-to-J recombination of T cell receptor y chain genes in scid mice* The status of T cell receptor y chain genes (TcR y) in 11 spontaneous T cell lymphomas from mice with severe combined immune deficiency (scid) was analyzed. We found that as a result of large abnormal deletions accompanying attempted site-specific V-to-J recombinations, 36 of 47 rearranged TcR y genes lacked the variable (V) andor joining (J) region gene segment involved in this attempted recombination. No such deletions were found in T cell lymphomas from normal mice. We interpret our data as indicating a defective V-to-J recombination in scid lymphocytes consistent with our earlier observation of faulty D-to-J recombination in transformed scid lymphocytes (Schuler et al., Cell 1986. 46: 963). The present results further support the hypothesis that the scid mutation affects a component of the VDJ-recombinase system used in common by B and Tcells to assemble antigen receptor genes.

1 Introduction Lymphocyte antigen receptor genes, i.e. Ig and TcR genes, are assembled from separate gene segments (V, D and J) during early lymphoid differentiation. V(D)J recombination is mediated by highly conserved heptamer and nonamer DNA sequences which flank the V, D and J segments of both Ig and TcR genes. These sequences appear to serve as recognition sites for a site-specific recombination system (here referred to as VDJ-recombinase system) which is thought to catalyzeV(D)J joining in bothTand B cells (for reviews see [ 1 4 . The mechanism of antigen receptor gene rearrangement appears to be adversely affected by the mouse mutation scid [5], which occurred spontaneously in the C.B-17Icr inbred mouse strain [6]. Mice homozygous for this mutation (C.B-17Icr scidlscid), here referred to as scid mice, lack functional Tand B cells [6-81, but show evidence of earlyT and B lineage committed cells [9].We previously reported [5] a high frequency of abnormal Igh and TcR p gene rearrangements in transformed immature scid lymphoid cells. Such abnormalities typically involved large J segmentassociated deletions resulting from attempted D-to-J recombination.These findings were recently confirmed and extended in several laboratories [lo-131. Our results prompted us to propose that a component of the VDJrecombinase system, common to bothTand B lineage cells, might be altered or missing in scid mice [5]. As a result,

developing scid lymphocytes are generally unable to make functional antigen receptor gene rearrangements and therefore fail to express an antigen receptor. If this hypothesis is correct, one would expect to find that the impairment of antigen receptor gene rearrangement is not restricted to D-to-J recombination but that rearrangement of Vgene segments would also be affected in the same manner. Previously, we could not detect rearrangements of VH genes in A-MuLV-transformed scid BM cells [lo], or of Vg genes in spontaneous scid T cell lymphomas (W. S., unpublished results). A likely explanation for this is the lack of an acceptor site for a V gene segment, since these sites were generally deleted as a result of a preceding attempt to join D and J [lo]. Igl-x, Igl-h and TcR a genes, which lack D segments, were in germ-line configuration in the transformed scid cell lines ([5]; W. S., unpublished results), but TcR y genes, which also lack D segments, were rearranged in the scid T cell lymphomas and thus allowed the analysis of V-to-J recombination.

2 Materials and methods 2.1 Mice C.B-17Icr (C.B-17) normal mice and C.B-17Icr scidlscid mice (scid mice) were bred and maintained as previously described [8] under specified pathogen-free conditions in the barrier facility of the Laboratory Animal Facility of the Fox Chase Cancer Center, Philadelphia, PA.

[I 80211

* This work was supported by grants AI-13323, CA-04946 and

+

CA-06927from the National Institutes of Health, an appropriation form the Commonwealth of Pennsylvania, and a research fellowship from the Deutsche Forschungsgemeinschaft to W.S. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland.

Correspondence: Walter Schuler, Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland Abbreviation: scid: Severe combined immune deficiency 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

2.2 T cell lymphomas Scid mice are highly disposed t o thymic lymphomas. About 15% of the mice spontaneously develop malignant lymphomas which appear to originate in the thymus. They show T cell surface markers [8] as well as rearrangement (mostly aberrant) of theTcR p locus [ 5 ] .These lymphomas are thus classified asTcell lymphomas. No such spontaneous tumors have been observed in C.B-17 normal mice.Therefore,Tcell lymphomas induced by X-irradiation of C.Bl7 normal mice were used as controls. 0014-2980/90/0303-0545$02.50/0

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W. Schuler, A. Schuler and M. J. Bosma

2.3 Hybridization probes The TcR y-specific full-length cDNA clones p8/10-2yl.1 and p5/10-13y1.2 [14] were a kind gift of A. Iwamoto. The genomic DNA clone p10.5 [15] which contains J2-C2 was kindly provided by A. Hayday. Probe pV1 specific for theV1 family, and probe pC2, hybridizing to the C1, C2 and C3 C region genes were subcloned from p8/10-2yl.l. pV1 is a 500 bp Eco RI-Ava I fragment subcloned into the transcription vector pSP64; pC2 is an 850-bp Eco RI-Ava I fragment subcloned into pSP65. Probe pC4, containing 54 and the first C, exon, is a 392-bp Sau 3A-Hind 111fragment from p5/10-13y1.2 subcloned into the transcription vector pGEM-3. Probe pJ2 is a 1.2-kb Ava II-Hind I11 fragment from p10.5 containing the J2 segment and about 1.1kb of its 3’-flanking region subcloned into pGEM-3. pJ2 crosshybridizes to J1 and 53 and to their respective 3’-flanking regions, but does not hybridize to the J4 segment. Probes pV2, pV3 and pV4 (17y, 13y and l l y [16]), specific for theV genes V2,V3 and V4, respectively, were kindly provided by D. Raulet.TheV5-specific probe pV5 [16] was a kind gift of J. Pelkonen. 32P-labeled hybridization probes were prepared by either nick-translation [18] using 32P-dCTPor by transcription of linearized plasmids employing SP6 RNA polymerase and using 32P-GTP [191. 2.4 Southern blot analyses Preparation of DNA from tumor tissue of primary T cell lymphomas and Southern blot analyses were performed as described previously [5] with the modification that hybridization with 32P-labeled RNA hybridization probes was carried out for 21 h at 48 “C in the presence of 50% formamide. 2.5 Genomic cloning, subcloning and DNA sequencing

Eco FU-digested and size-selected DNA was ligated with EMBLA bacteriophage vector arms, packaged in vitro and phage libraries screened with TcR y-specific probes. Phage inserts containing rearranged TcR y genes were subcloned into pGEM-3 and characterized by restriction enzyme mapping and comparison to the known germ-line TcR y restriction maps [ 15, 171. Restriction fragments spanning the respective recombination sites were subcloned into pGEM-3 and subsequently sequenced. To determine the breakpoint junction in DNA clone g343-4.6, a series of plasmids containing overlapping, nested deletions were generated following the Erase-a-Base protocol (Promega, Madison, WI) and sequenced. DNA sequencing was performed according to Chen and Seeburg by the dideoxy chain termination method [20] using SP6 and T7 promotorspecific oligonucleotide primers. Sequenase (US Biochemical Corporation, Cleveland, OH) was used following the manufacturer’s protocol.

3 Results and discussion 3.1 Introductory remarks: organization of the TcR y locus Fig. 1 schematically outlines the genomic organization of the mouse TcR y genes based on previously published

--- V5

V2

V4

V3

Eur. J. Immunol. 1990. 20: 545-550 J1

c1

r

I

E Figure 1. Organization of the TcR y locus. Nomenclature adopted from [16], see Sect. 3.1. Arrows indicate transcriptional orientation. (Map not drawn to scale.)

studies [14-16,21,22].The TcR y locus contains the four C region genes C1, C2, C3 and C4 (the nomenclature used in this study follows that of Garman et al. [16],Vy4.4 [17] is here referred to asV5 to be consistent with this nomenclature). C1,C2 and C3 are highly homologous and hybridize to the cDNA probe pC2 derived from C2. C4 shows little homology to the other C region genes and does not hybridize to pC2 under the conditions used here. Each C region gene is associated with one J segment and with one to four Vgenes. SevenVgenes are known;V1.1,Vl.2and V1.3 which are homologous, hybridizing to the cDNA probe pV1 derived fromV1.2.The remainingvgenes, i.e.V,,V,,V, and V5, show little homology to each other and to theV1 family. D elements have not been found at the TcR y locus. The TcR y genes are clustered, and V-to-J recombination appears to follow a restricted pattern, with rearrangements occurring predominantly within a given cluster [14-16, 21,221.Vl.l rearranges to J4 and V1.2 rearranges to J2.Vi.3 rearrangement, which seems to occur only infrequently, links V1.3 to J3. V2, V3, V4 and V5 each rearrange to J1. Deviations from this strict pattern have been observed in T cells of mouse strains other than BALB/c, where rearrangements of V2 [23] and V5 [17] to J4-C4 were found. 3.2 Abnormal size of Eco RI fragments of scid T cell lymphoma DNA containing rearranged TcR y genes

When Eco RI is used as restriction enzyme, three fragments are found on Southern blots of liver (germ-line) DNA that hybridize to the pC2 probe (Fig. 2A). These are a 13.4-kb fragment containing Jl-Cl, a 10.5-kb fragment containing J 2 - c ~and a 7.5-kb fragment which contains the C3 gene [15]. A restricted set of non-germ-line fragments between 14 and 18 kb hybridizing to the pC2 probe is normally seen in Southern blot analyses of Eco RI-digested DNA from T cell lines, reflecting the restricted types of rearrangements described above. This is seen in Fig. 2A for DNA from transformed cell lines NL151, NL33 and NL37, which all originate from normal C.B-17 mice. These non-germ-line fragments contain the rearranged C1 and C2 genes. The 7.5-kb Eco RI fragment containing C3 does not change upon rearrangement since the Eco RI restriction site is located downstream from 53 (Fig. 2A). In contrast, in DNA derived from spontaneous scid T cell lymphomas (prefix “SL” in Fig. 2), a diverse spectrum of novel non-germ-line fragments hybridizing to pC2 was found, the majority being

Eur. J. Immunol. 1990. 20: 545-550

547

ments of 14 a n d o r 16 kb are normally observed in Southern blots of Tcell DNA.This is seen, for example, in the C.B-17 lines NL151, NL33 and NL37 (Fig. 2B). Again, as seen with probe pC2, different results were obtained for scid Tcell lymphoma DNA using the pC4 probe (Fig. 2B). Although fragments of the expected size were found in two lines (SL2847 and SL195), the majority of C4 containing fragments were considerably smaller and ranged from 3.1 kb (SL2847) to 9.5 kb (SL275, SL350).

kb

9.4 6.6-

23.1

4.4

Defective V-to-J recombination in scid mice

3.3 Lack of V and J segments in rearranged TcR y genes from scid T cell lymphomas To ascertain whether the abnormally small TcR y-containing Eco RI fragments described above might reflect deletions accompanying attempted site-specific V-to-J recombinations, we consecutively probed Southern blots of Eco RI-digested T cell lymphoma DNA with probes specific for the four known C genes, for J1, J2 and J3 and for the seven known V genes. Within a given cluster, the C genes and their respectivevgenes are located on separate Eco RI fragments (Figs. 1and 3A). The exception is V1.1, which is located on the same Eco RI fragment as C4. Upon V gene rearrangement, the Eco RI site upstream of the respective J element is lost, generating a non-germ-line fragment that contains bothVand J-C genes.Therefore, the same Eco RI fragment is usually revealed with pJ2, pC2 and the appropriatevgene probe. (The exception is C3, where the respective J element is located on a separate Eco RI fragment, see Fig. 1.) This is seen, for instance, in Tcell lymphoma line NL37, derived from a normal C.B-17 mouse, which serves as an example for the normal TcR y rearrangement pattern (Fig. 3B). In NL37 rearrangement of V1.1 and V1.2 generated a non-germ-line 14-kb Eco RI fragment containing the rearranged Vl.l-J4-C4 and V1.1J2-C2 genes, as demonstrated by the appearance of comigrating fragments that hybridized to the respectivev-, Jand C-specific probes (Fig. 3B, arrowhead 1, lanes a-d). Co-migrating 17-kb fragments hybridizing to probes pC2, pJ2 and pV2, and loss of the V2 germ-line fragment indicated rearrangement of V2 to J1-C1 on both alleles (Fig. 3, arrowhead 2, lanes c-e). V3, which is located downstream from V2, was deleted as a result of the V2 rearrangement as can be seen from the lack of the V3 germ-line fragment (Fig. 3B, arrowhead 3, lane f).

-

6.6,

4.4 -

c4 Figure2. Status of TcR y genes as revealed by Southern blot analysis of Eco RI-digested DNA from scid liver (SC-L), T cell lymphoma lines from normal C.B-17 mice (NL) and from scid mice (SL). DNA was hybridized either (A) to probe pC2, specific for TcR y C region genes C1,C2 and C3or (B) to probe pC4, specific for the C region gene C4.

shorter than 14 kb.The size of these fragments ranged from 4.4 kb (SL179) to about 12 kb (SL12), although at least one fragment of the expected size was found in six of the eleven scid T cell lymphomas studied (i.e. SL12, SL273, SL350, SL357, SL2847 and SL4172 in Fig. 2A). Probing Eco RI-digested liver DNA with probe pC4 reveals a 10.8-kb germ-line fragment (Fig. 2B), containing the C region gene C, [ 141. Non-germ-line C4 hybridizing frag-

An example of the typical situation found in scid T cell lymphoma lines is seen in line SL179. As already demonstrated in Fig. 2 the Eco RI fragments of SL179 containing the rearranged TcR y genes were of abnormal size. As shown in Fig. 3C,V1.3 and V5 were in germ-line configuration in SL179 (arrowheads 1, 2 and 3; lanes b, d and g, respectively), while loss of the germ-line fragments containing V1.1, V1.2,Vz and V3 indicated rearrangement of these gene segments (lanes b, e and f). However, non-germ-line fragments could not be detected with any of the V-specific probes; also, unlike the normal situation, neither of the two J-C-containing fragments hybridized with a V gene-specific probe (Fig. 3C, arrowheads 4 and 5, c.p. lanes c and d to lanes b, e , and f). These V genes were obviously deleted from the TcR y locus. Normally, a deletion of V2 and Vfld is seen upon rearrangement of the upstream V5 gene [17], but V5 is still in germ-line configuration in SL179 (Fig. 3C, arrowhead 3, lane g).

Eur. J. Immunol. 1990.20: 545-550

W.Schuler, A. Schuler and M. J. Bosma

548

Recombination of both V1.1 and V1.2 normally results in a 14-kb Eco RI fragment that hybridizes to pC2 and pC4 as well as to pV1 and pJ2 (see NL37, arrowhead 1in Fig. 3B). In contrast, in the scid thymoma line SL179, an Eco FU fragment of only 4.3 kb hybridized to both pC2 and pC4, but did not hybridize to any Vgene-specific probes or to pJ2 (Fig. 3C, arrowhead 6, lanes a and c vs. lanes b, d, e and f). The presence of both the C2 and C4 gene on one, short Eco RI fragment can be best explained by deletions accompanying attempted v ~ . ~ - t oand - J ~V1.1-to-J4recombination; upon this rearrangement bothvgenes, as well as the J2 segment, were lost. Similarly, in scid Tcell lymphoma line SL343 a 7-kb Eco RI fragment was found that still hybridized to pC2 and pC4 as well as to pV1, but that did not hybridize to pJ2 (Fig. 3D, arrowhead 1, lanes a, b and c vs. lane d). Again, the interpretation is that the J2 element and at least 1.2 kb of 3'-flanking DNA (the size of the probe pJ2) was deleted upon an attempted V1.2-to-J2 recombination.

CYl

cy4-vy1.1 -vy1.2

cy2*

CY3, vy3-vy4Vy 1.3-

vy2vy5-

JY3,

2 b-

Figure 3. Status of the TcR y genes as revealed by Southern blot analysis of Eco RI-digested DNA employing variousTcR y-specific DNA probes. Southern blot filters were consecutively probed (after eluting the preceding probe) with probes specific for the TcR y C region genes C4 (a), C1, C2 and C3 (c), for the J region segments J1, Jz and 53 (d) and for theVgenesV1.1,Vl.z and V1.3 (b), Vz (e),V3 (9and V5(g). (Probing with a probe for the V4 gene, which is located on the same Eco RI fragment asV3, revealed the same fragments as the Vrspecific probe did. These results are therefore omitted here.)

In six of the eleven scid lines (SL12, SL179, SL343, SL357, SL4172, SL6188), the V/J-associated deletions extended into the C4 coding regions as seen, for example, in line SL343. In SL343, both C4-containing alleles underwent rearrangements as indicated by the loss of the germ-line C4 fragment (Fig. 3D, lane a), but only one novel Eco RI fragment was found, the VJC; 7-kb fragment, that hybridized t o pC4 (Fig. 3D, arrowhead 1, lane a). A second Eco RI fragment, hybridizing to pV1, pJ2 and pC2 (Fig. 3D, arrowhead 2), did not hybridize to pC4 (Fig. 3D, lane a). The strong intensity of the hybridization signal of this 8.6-kb Eco RI fragment after probing with pC2 and pJ2 (Fig. 3D, lanes c and d) suggested that this fragment contained J 2 - c ~ .(Since pJ2 and pC2 were both derived from the J 2 - c ~gene, these probes most strongly hybridize to J2/C2 and only to a lesser degree to J1/C1; compare the 10.5-kb J2/C2 germ-line bands to the respective 13.4 kb J1/C1 bands in liver DNA, Fig. 3A, lanes c and d.) As already mentioned, J2-C2 is normally found together with J4-C4 on one, 14-kb Eco RI fragment.Therefore, the most likely interpretation is that this 8.6-kb fragment, and the respective Eco RI fragments of line SL12, SL179, SL357, SL4172and SL6188, resulted from deletions accompanying an attempted V1.1-to-J4 recombination which affected the J4 segment and extended beyond the first exon of the C region gene C4. (Probe pC4, which was derived from a cDNA clone covers predominantly the first exon of C4.)

Table 1. Status of TcR y genes in scid T cell lymphomasa)

Line

SL 12 SL 179 SL 1% SL 273

v+c:,

J+C&

V+ J+CL

1/4

2/4

on

2n 112

w4 w3 lL?

113

w3

113 ll2

w3 ll2

ll2 ll3

sz.275

on

SL343 SL 350 SL 357 SL 2847 SL 4172

2J2 ll4 213

rn m

2/3

SL 6188

w4

lJ4

34

v4

113 lL?

w3 u2 113 w4

v+c,+ W1 (A)b) 011 (A) ll2 GL ll2 111 (A) ll2 011 (A) ll2 1/1 (A) 011 (A)

a) Summary of Southern blot analysis results. Given are the numbers of non-germ-line Eco RI fragments containing TcR y C region genes C1 and C2 or C4 and hybridizing to either V o r J probes or to both V and J probes compared to the total number of C:, or C: non-germ-line fragments. b) Deletion of C4 on one allele.

Eur. J. Immunol. 1990. 20: 545-550

Defective V-to-J recombination in scid mice

We have analyzed in the way described in this section 11T cell lymphomas from scid mice and 3 from normal C.B-17 mice. The results are summarized in Table 1. We found V/J-associated deletions for each of the 11 scid T cell lymphomas tested: of 47 non-germ-line Eco RI fragments containing C1, C2 or C4, 36 fragments lacked V andor J gene segments. No such absence of Vandor J segments was found in the T cell lymphomas from normal C.B-17 mice.

9313-7.0

549

TTTTflTTGRGTGflCflflTTGflRGCCCflGCflflTTTTCRflfl

I uy1.2

TTAGAGCCTC

9313-4.6A BU3.8.1

9313-1.66

3.4 Molecular characterization of rearranged scid TcR y genes

Our interpretation of the Southern blot data was confirmed by genomic cloning and nucleotide sequence analyses of some of theTcR y rearrangements of scid Tcell lymphomas. The analysis of two cloned Eco RI fragments of line SL343, the 7.0-kb V1-C2-C: fragment (g343-7.0, Fig. 3D, arrowhead 1) and the 4.6-kbVl fragment (g343-4.6, Fig. 3D, arrowhead 3) serves as an example. Comparison of a partial restriction map of g343-7.0 to that of the C2-containing germ-line fragment (Fig. 4A) revealed a loss of the Ava I site and of the Hind I11 site 3-prime of J2. This loss indicated that recombination had occurred to a DNA sequence about

(A) GERNL I NE Uyl /Cy4

GERMLINE CY2

JY3

Figure 5. Partial DNA sequences of g343-7.0 aligned with germline sequence of Vl.2 (from [15]), of g343-4.6A (see Fig. 4B) aligned with sequence of BW3.8.1, a rearranged V5J4-containing DNA clone (from [17]) and of g343-4.6B (see Fig. 4B) aligned with germ-line sequence of 53 (from [22] and W. S., unpublished). Coding region sequences are indicated by shaded boxes. Heptamerhonamer signal sequences are underlined. Arrows show the recombination breakpoints.

2 kb downstream from the 52 element. Sequencing demonstrated V1.2 gene sequences upstream of this recombination site, while J2 sequences were not found (Fig. 5). Normally, when V and J segments are joined during rearrangement modifications at the junctional borders occur, i.e. loss and/or addition of several base pairs are found at the junctions of the coding segments. In g343-7.0, however, the recombination breakpoint was located 22 base pairs upstream from the 3-prime end of Vl.2, i.e. considerably out of range of deletions normally seen at the junctional borders, while J2 was entirely deleted. Comparison of the 5-prime end of clone g343-7.0 with the V1.1-C4-containing germ-line fragment (Fig. 4A) showed that the Hind 111, Bam HI and Bgl I1 restriction sites between the first exon of C4 and the 54 segment were lost. Furthermore, by sequencing we could assign the single Kpn I site found in g343-7.0 to theV1.2 gene.This indicated that bothV1.1and 54 had been deleted, including about 2 kb of DNA 3-prime of J4 as well as at least 2 kb of DNA 5-prime of V1.l.

9343-7.0

fl

fl

P PU

KKHp

P Pu K K H p

9343-4.6

t

n

+ B

I kb

Figure 4. (A) Partial restriction map of DNA clone g343-7.0, cloned from scid T cell lymphoma line SL343, compared to the germ-linemapsV1/C4(V10.8, [15] and Cz (J-C10.5, [15]. A, Ava I; B, Barn HI; G, Bgl 11; H, Hind III; K, Kpn I; R, Eco RI; X, Xba I. (B) Partial restriction map of DNA clone g343-4.6 compared to the V5J4 containing clone BW3.8.1 [17]. Hc, Hind 11; Hp, Hpa 11; K, Kpn I; P, Pst I; Pu, Pvu II; R, Eco RI. Arrows A and B refer to the localization of the DNA sequences g343-4.6A and g343-4.6B in Fig. 5.

Another example of deletion of both V and J coding segments is clone g343-4.6. This non-germ-line fragment hybrized to the pV5, and apparently to the pJ2 probe, but not to probes for the C region genes (Fig. 3D, arrowhead 3). Comparison to clone BW3.8.1, derived from an AKR mouse and containing a V~-to-J4rearrangement [17] confirmed the presence of Vs in clone g343-4.6; however,Vs turned out to be in germ-line configuration (Figs. 4B and 5). At the 3-prime end of g343-4.6, 2.5 kb downstream from V,, a DNA sequence identical to that of the 3-prime flanking region of J3 was found; sequence homology started 36 base pairs downstream from the 3-prime end of the J3 coding segment (g343-4.6B in Fig. 5). The sequence 5'-prime of this recombination breakpoint lacked any homology toTcR y V region genes. Our explanation for this result is an attempted recombination of V2, or of another unknownV region gene downstream fromV5, to 53 resulting in the deletion of this particular V gene and the 53 coding segment.

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W. Schuler, A. Schuler and M. J. Bosma

4 Concluding remarks The results reported here demonstrate that V-to-J rearrangement at theTcR y locus is defective inTcell lines from scid mice. We have analyzed the status of TcR y genes in eleven scid T cell lymphoma lines. Analogous to the situation found for D-to-J recombinations at theTcR @ and the Igh locus in transformed cell lines from scid mice [5, 10-131, we found that V and/or J segments had been deleted in 36 of 47 rearrangements containing TcR y C region genes. These deletions have been shown to result from attempted V-to-J recombination. In line with these results, Blackwell et al. [24] recently reported, in two transformed pre-B cell lines from scid mice, two rearrangements of Igh-x genes, one lacking V, the other lacking V, and J,. Although not directly shown, these deletions probably resulted from attempted V,-to-J, recombination. The above observations are consistent with, and give further support to, the hypothesis that the scid mutation affects a component of theVDJ recombinase system used in common by Tand B cells to assemble their antigen receptor genes [ 5 ] . Although most TcR y rearrangements were defective in the scid T cell lymphomas as reflected by the irregular size of TcR y-containing Eco RI fragments, we consistently found regular size fragments suggesting that normal rearrangement might still be possible in scid lymphocytes. In fact, we were able to clone normal TcR y rearrangements from scid T lymphoma lines which predominantly harbor defective TcR and TcR y rearrangements (W. Schuler, M. Amsler, M. Bosma, manuscript in preparation). We are indebted to Dawn E. Kelley for advice and assistence in the techniques of genomic cloning. We thank Drs. A . Hayday, A. Iwamoto, J. Pelkonen and D. Raulet for their gifts of TcR y-specific DNA probes, Drs. Steven Bauer and Klaus Karjalainen for critical reading of our manuscript, and Janette Millar and Nicole Schoepflin for excellent secretarial assistance.

Received October 10, 1989.

5 References 1 Tonegawa, S., Nature 1983. 302: 575. 2 Kronenberg, M., Siu, G., Hood, L. and Shastri, N., Annu. Rev. lmmunol. 1986. 4: 529.

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