GENOMICS 4,445-448 (1989)

SHORT COMMUNICATION

Isolation and Restriction Map of the V-J Interval of the Human T Cell Receptor y Chain Locus

VICTOR L. Fox,*,’ w. M. STRAUSS,t AND J. G. SEIDMANt

*Combined Program in Pediatric Gastroenterology and Nutrition, The Children’s Hospital, and Wepartment of Genetics, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115

ReceivedAugust31, 1988;revisedDecemberl. 1988

The human T cell receptor y chain locus encodes the immunoglobulin-like y chain polypeptide and spans a distance of approximately 150 kb. Previous studies have not precisely characterized the interval sepa- rating variable regions from joining-constant regions which is excised during 7 gene rearrangement. We re- port a series of overlapping cosmids which includes the portion of the y chain locus beginning with V2 and extends to the second exon of C2. Sixteen kilobases separate the most 3’ variable region gene, V4, from the most 5’ joining segment, Jl. 1. o 1~8s Academic

Press, Inc.

The human T cell receptor y chain (TCR y) locus encodes one polypeptide of the heterodimer yS T cell receptor (Brenner et aZ., 1986). This locus and its mu- rine counterpart have been characterized extensively. The human y locus on chromosome 7 (Fig. 1) comprises six variable region families (Vl, +A, V2, V3, +B, and V4) and two constant regions (Cl and C2) preceded by a total of five joining segments (Jl.1, 51.2, 51.3, 52.1, and 52.3) (Strauss et al., 1987; Lefranc et aL, 1986, Forster et al., 1987; Huck et al., 1988; Quertermous et al., 1986; Pelicci et al., 1987). No diversity segments have been identified. The compact organization and complete characterization of the TCR y locus make it an ideal model for studying the mechanisms involved in site-specific gene rearrangement. We have directed our attention to detailed structural analysis of this locus since structure has direct bearing on the mechanism of site-specific recombination. The size of this locus is approximately 150 kb in length as determined by a combination of deletion mapping and field inversion gel electrophoresis (FIGE) of high-molecular-weight fragments of germline DNA (Strauss et al., 1987). This

1 To whom correspondence should be addressed.

locus is much smaller than other T cell receptor and immunoglobulin gene loci. For example, the murine TCR /3 locus reportedly spans between 450 and 800 kb (Chou et al., 1987; Lai et aZ., 1987; Lindsten et al., 1987) and the human Ig heavy chain locus extends 2500- 3000 kb in length (Berman et aZ., 1988; Matsuda et aZ., 1988).

Little is known about the interval of DNA separating variable and constant regions of immunoglobulin-like loci. Only recently has a first approximation of physical distance separating these regions been reported for the human TCR y locus (Strauss et al., 1987), the murine TCR y locus (Woolf et al., 1988), the murine TCR /3 locus (Chou et al, 1987; Lai et al., 1987; Lindsten et aZ., 1987), and the human immunoglobulin heavy chain locus (Berman et al., 1988, Buluwela et aZ., 1988). Nothing is known about the presence of related tran- scription products or regulatory elements contained within the V-J interval. This report presents a detailed structural characterization of this portion of the human genome.

FIGE analysis of the human TCR y locus permitted an estimation of 50 kb separating the most 3’ variable region from the most 5’ joining region (Strauss et al., 1987). Cosmid clones were isolated because of their larger insert size, limiting the number of anticipated chromosome steps. Figure 1 shows three cosmid clones isolated from two genomic libraries and their relative positions along the TCR y locus. Each library repre- sented a base of 3 X lo6 transformants. Cosmid 2.1 was initially identified by a probe for 52.3. A 5’ end fragment from cos 2.1, designated 5’5, was used to select cos 2.2 and COB 27. Cosmid 2.2 begins 7 kb 5’ of Jl.1 and in- cludes all five joining regions, Cl, and the first two exons of C2 (Fig. 2 and data not shown). The 5’ and 3’ ends of cos 2.1 and cos 2.2 are identical. However, the 5-kb EcoRl fragment found in the JlCl region is pres-

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446 SHORT COMMUNICATION

16 Kb 1 KB

H

1v 2 3 4 5 5W 6Y 7’4’ 6 AV BY 12 3 I II Ill 1 3 I Ila Ilbllc Ill

I I I I I I III I,,, II I [II II I IIII mom- 7 1 I B B B BB B B B B (BNB)(B)

I -27

II cos 2.1

I cc6 2.2

I I I I I I I I

Probes: v2 v3 v4 5’J (52.1) (J2.3) J2.1 J2.3

wmQw=4

Kb 0 50 100 150 1 1 I 1 I I 1 I I I 1 I

FIG. 1. Genomic organization of the human TCR y locus and restriction enzyme map of the V-J interval. Enzymes include BamHl (B), KpnI (K), and HindHI (H). Pseudogene are indicated by #. Polymorphic BamHl sites due to exon II triplication in the C2 region are enclosed by parentheses. Cos 2.1 and cos 2.2 were isolated from a genomic library generated from Raji cell line (Burkitt’s B-cell lymphoma) DNA ligated to cloning vector pJBS (Ref. (9)). The hatched area in co8 2.1 indicates an internal deletion resulting in a 25-kb aberrant clone. Cos 27 was isolated from a genomic library generated from fetal liver DNA ligated to cloning vector CPRB (Ref. (1)). Probe 5’5 repreaenta a 2.1-kb 5’ end fragment subcloned from cos 2.1. The remaining probes for V2, V3, and V4 (W. M. Strauss, unpublished data) and 52.1 and 52.3 (Ref. (15) and unpublished data) were previously isolated from germline or rearranged clones. Jl.1 and 51.3 can be detected by cross-hybridization with probes for 52.1 and 52.3, respectively, indicated in parentheses.

ent in cos 2.2 but not in cos 2.1. Cosmid 2.1, therefore, represents an aberrant clone in which genomic DNA beginning near the EcoRl site 3’ of 51.3 and ending near the EcoRl site 3’ of 52.3 has been deleted (Fig. 1). Probes for V2, V3, V4, Jl.1, 51.2, and the 5’ end fragment of cos 2.1 all hybridize to cos 27 (Figs. 2C and D and data not shown). A genomic clone directly linking V2 to V3 has not been previously reported.

Probes for both 5’5 and V4 hybridize to the same 19- kb Hind111 and KpnI fragments in cos 27 (Fig. 2B). The genomic correlates of these restriction fragments were confirmed on Southern blots of germline genomic DNA digests (Fig. 2A), thereby excluding the possibility of an error in mapping due to deletion. A 52.1 probe (homologous to Jl.1) identifies the same 1.2-kb HindI fragment in cosmids 2.1, 2.2, and 27 (Fig. 2D). Figure 1 shows a limited restriction enzyme map of the V-J interval. Restriction sites were confirmed by analyzing subcloned fragments. The KpnI site located approxi- mately 4 kb 5’ to V4 corresponds to the recently re- ported site of V$B (Huck et al., 1933). The distance

separating V2 and V3 is no greater than 4 kb. The distance separating V4 and Jl.1 measures 16 kb.

The resolving power of FIGE (Carle et aZ., 1936) is considerably limited in the simultaneous separation of DNA fragments ranging from >600 to <20 kb. Band- width thickness alone may account for significant error in measurement. Given these limitations we were sur- prised to find that the direct V-J interval measurement corresponded with relative accuracy to our first ap- proximation. The utility and relative accuracy of FIGE are confirmed for the purposes of mapping expansive loci within the human genome.

A V-J interval of 16 kb fits with the compact ar- chitecture of the TCR y locus. Recent reports permit comparisons of V-J interval estimations in the human Ig heavy chain locus with those of the murine TCR @ and TCR y loci. Matsuda et al. (1933) showed that the human Ig Vn to Du regions were approximately 240 kb apart, while the Dn region is separated from the Jn region by approximately 22 kb. Berman et al. (1988) reported direct Vn to Jn linkage within 30 kb. Buluwela

SHORT COMMUNICATION 447

A. Germline

r12 34’

Probe: V4 5’5 v4 S’J

c. ;;z D. ;ss

Kb Kb

2.5 - J2.3 2.5 - J2.1

2.1 - Jl.2

1.5- J1.3 1.2 - J1.l

Probe: J2.3 J2.1

FIG. 2. (A) Germline DNA analyzed with probes for V4 and 5’5 demonstrating shared size of restriction fragment. Genomic DNA was extracted from EBV lymphoblastoid cell line CHRF, digested with KpnI (lanes 1 and 3) or Hind111 (lanes 2 and 4), and size fractionated on 1% agarose gels. Southern blots were hybridized to probes for V4 and 5’5. (B) Southern blot of HindIII-digested cos 2.1, cos 2.2, and cos 27 hybridized to probes for V4 and 5’J. (C) Southern blot of EcoRl-digested cos 2.1 and COB 2.2 hybridized to a probe for 52.3. (D) Southern blot of HindHI-digested cos 2.1, cos 2.2, and cos 27 hybridized to a probe for 52.1. Moderate stringency wash with 0.2X SSC at 50°C permits hybridization to partially homologous J regions.

et al. (1988) have similarly identified a 98-kb interval separating a pseudo-Vn from Jn. Chou et al. (1987) reported a distance of 320 kb linking the most proximal variable region gene, V/37, to the D-J-Cfl cluster in the murine T cell receptor fi chain locus. Lindsten et al. (1987) similarly found 280-360 kb separating V/37 from DJC within the murine TCR fl locus which measured 700-800 kb. In contrast, Lai et al. (1987) found only 100 kb separating the same V to DJC interval within this locus measuring approximately 450 kb. This dis- crepancy may reflect a deletion occurring in the L-cell line used by these investigators as a source of germline DNA. Liver DNA was used by both Chou et al. and Lindsten et al. as a source of germline DNA. Woolf et al. (1988) resolved the murine TCR y locus to 195 kb

of DNA and estimated the smallest V-J interval to be 60-70 kb. Genomic organization of the human TCR y locus V, J, and C regions differs from that of the murine TCR y locus in that all of the human V region genes lie 5’ of the J and C regions. The murine TCR 7 locus comprises separate VJC clusters. However, the overall size of both loci and the distances separating V from J regions are remarkably similar. Yancopoulos et al. (1986), Chou et al. (1987), and others have proposed that large distances separating V and DJ regions within the Ig gene and TCR gene loci may be important in limiting accessibility to recombinase activity and pre- serving the appropriate order of DJ followed by V to DJ rearrangement. By analogy, the short V-J interval of the TCR y locus may promote early rearrangement, in particular, relative to the TCR a! and fl loci.

ACKNOWLEDGMENTS

We thank Dr. John H. Weis, Harvard Medical School (Boston, MA), for the use of the Raji cell cosmid library and Dr. Randall F. Holcombe, Division of Hematology, Brigham and Women’s Hospital (Boston, MA), for the use of the lymphoblastoid cell line CHRF. This work was supported by NIH Training Grant T32-DK07477 to V.L.F. and NIH Grants AI19148 and AI18436 to J.G.S.

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