Molecular Determination of T-Cell Receptor and/3 Chain Genotypes in Human Families

M. A. Robinson and T. J. Kindt

ABSTRACT: Polymorphism in genes encoding the ce and/3 chain of the human T cell receptor has been detected by Southern blot analysis. Genomic DNA samples were isolated from B /ymphoblastoid cell lines derived from members of families, each family including at least one individual with a recombinant HLA haplotype. T cell receptor ce and/3 chain hap/otypes could be assigned/n the families on the basis of observed restriction fragment length polymorphism (RFLP). Polymorphism in the ce chain gene was detected in BgllI digests using an ce chain probe that included the V. J, C. and _3' untranslated sequences, A probe consisting of only the constant region (C,d revealed no polymorphism indicating that the po]ymorphic fragment hybridized to V.J. or 3' untranslated sequences of the ce chain. Polymorphism in/3 chain genes was observed in Bglll digested DNA samples using a probe that corresponds to the constant region (C~). Polymorphic C~ restriction fragments of 10.0 and 9.2 kilobase segregated in six of the eight famik~s studied. Recent structural data for the C~ region suggest that the polymorphie BgllI site lies in the region 5' to the C~2 gene. These polymorphisms should serve as markers for ce and/3 chain complexes alkm,ing g:enetic studies of these immunologically important gene families.

A B B R E V I A T I O N S BLCL B lymphoblastoid cell line(s) D diversity bp base pair(s) J joining C constant kb kilobase(s) Cot constant region of the T cell re- RFLP restriction fragment length

ceptor a chain gene polymorphism C/3 constant region of the T cell re- V variable

ceptor/3 chain gene

I N T R O D U C T I O N

Genetic analysis of human immune responses has relied upon polymorphism of genes and gene products involved in immune processes. Studies of the HLA complex have revealed associations between particular antigens and immune responses as well as associations with particular disease states [ 1,2]. HLA class I, class II, and class III products are highly polymorphic [3]. The contribution of immunoglobin genes has been studied using all allotypic markers of immu- noglobulin heavy and light chains. Associations between specific responses and disease states have been reported for G m and Km alleles [4-6] . Similar genetic analysis of T cell function has not yet been possible because there are no sera that define allotypic markers on human T cell receptors.

From the Laboratory of lmmunogenetics. National Institute of Allergy and Infectious Disease.*. National Institutes of Health. Bethesda. MD.

Address reprint requests to Mart Ann Robinson, LIG, NIAID. NIH, Building 5 Room BI-04. Bethesda. MD, 20205.

Human Immunology 14, 195-205 (1985) (© Elsevier Science Publishing Co., Inc., 1985 52 Vanderbilt Ave., New York, NY 10017

195 0 L 98~8859/85/$ ~. 2,0

196 M.A. Robinson and T. J. Kindt

However , monoclonal antibodies that recognize idiotypic determinants on T cells have been used to characterize the protein structure of the T cell antigen receptor. The T cell antigen receptor is a heterodimeric cell surface glycoprotein consisting of disulfide linked ~x and /3 polypeptide chains. The ce chain has an approximate molecular weight of 46,000 and is acidic. The /3 chain is neutral or slightly basic and has an approximate molecular weight of 38,000 [7,8]. Genes corresponding to the characterized protein have been cloned [9-12] . In addition, a third gene designated 7, has been cloned. This gene is similar to ex and/3 chain genes and is transcribed in murine cytolytic T lymphocytes [13,14].

The c~ and/3 chain gene complexes have striking similarities to immunoglobulin genes in sequence and in organization into clusters containing variable (V), joining (J), and constant (C) region gene segments [11,12,15,16]. The /3 chain has, in addition, a diversity (D) segment [15,16]. The/3 chain V, D, andJ gene segments rearrange during T cell differentiation [17,18]. cDNA sequences of the ce chain indicate that the ce chain gene segments also undergo rearrangement [ 11,12].

The present report describes the use of ce and/3 chain cDNA probes to define restriction fragment length polymorphisms (RFLP) for human T cell receptor genes to follow the segregation of these genes in human families. These RFLPs should provide useful markers for genetic studies of these immunologically im- portant gene complexes.

M E T H O D S

Preparation of DNA samples. B lymphoblastoid cell lines (BLCL) were initiated on the members of all families by culturing 5 x 10 ~' peripheral blood lymphocytes in the presence of cyclosporin A (2 /.tg/ml) and supernatant from the Epstein- Barr virus producing cell lines B95-8. RPMI-1640 culture medium supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 ~g/ml streptomycin (Grand Island Biological Inc., Grand Island, NY), and 10% heat inactivated fetal calf serum (Dutchland Laboratories, Inc., Denver, PA) was used to maintain the cell lines. The identity of all BLCL was confirmed by typing for HLA-A,B,C and, - DR. Genomic D N A was extracted from BLCL using a previously described procedure [19].

Digestion of genomic DNA with restriction endonucleases. High molecular weight D N A (24-48 ~g) was digested in the appropriate buffer with 2 units of en- zyme/b~g D N A for 4 hr at 37°C followed by an additional aliquot of enzyme (2 units//xg DNA) and incubation for 4 hr at 37°C. Digests using two enzymes were performed simultaneously. Digestion was monitored by adding an aliquot of the D N A mixture containing 2 ~g of genomic D N A to 1 htg of bacteriophage A D N A after the second addition of enzyme. The a test samples were incubated at 37°C for 4 hr and complete digestion of a D N A in each sample was monitored by agarose gel electrophoresis. Digested D N A samples were extracted once with phenol, once with chloroform, and then precipitated with ethanol. Precipitated D N A was resuspended in 10 mM Tris pH 7.6, 1 mM EDTA. BgllI was purchased from Bethesda Research Laboratories, Gaithersburg, MD. EcoRI, BamHl, PvulI, and PstI purchased from New England Biolabs, Beverly, MA.

~DNA probes. The T cell receptor/3 chain probe was prepared from the murine c DNA clone 86T1 provided by Dr. Mark Davis [20]. A 615 bp BamHI-EcoRl fragment that corresponds to 40 bp of the V region, the entire D region, J region, and C region was used. This probe cross-reacts with human Cr3 but not human V region genes.

T Cell Receptor c~ and 13 Chain Genotypes 197

Two different T cell receptor ~ chain probes were obtained from the human cDNA clone pY14 provided by Dr. Tak Mak [21]. pY14 contains an EcoRI insert of 1101 bp that corresponds to the 5' untranslated region, leader sequence, V region, J region, C region, and 3' untranslated sequences. The entire 1101 bp EcoRI insert and a PvulI fragment of 389 bp corresponding to the C region, designated C,, were used in these studies.

Probes were prepared by electroblotting onto DEAE paper [22]. Probes (200 ng DNA) were nick translated using the Nick Translation Kit and 100 /.tCi deoxycytidine 5'-[c~32P] triphosphate (3000 Ci/mM) from Amersham Corpora- tion (Arlington Heights, IL) and had a specific activity of approximately l0 s cpm//.tg DNA.

Southern blot analysis. Restriction endonuclease digested DNA (10/*g/lane) was electrophoresed in 0.6% agarose gels for 36-48 hr in 40 mM Tris/10 mM sodium acetate/1 mM EDTA brought to pH 7.2 with acetic acid. Lambda DNA digested with HindlIl was used for size markers. Blots were performed according to the method of Southern [23] using nitrocellulose filters (Millipore Corp., Bedford, MA).

Following transfer, filters were prehybridized overnight at 42°C in the presence of 10% dextran sulfate and 40% formamide [24], then hybridized for 18 hr at 42°C using probe at 3.5-5.0 x 106 cpm/ml. After three low stringency washes at room temperature, the blots were washed twice in 15 mM sodium chloride/1.5 mM sodium citrate, pH 7.0/0.5% NaDodSO~ at 50°C for 30 rain for blots using the 13 chain probe and at 60°C for 30 min for blots using the ce chain probes. Blots were exposed for 24-72 hr to Kodak Xomat AR film at -70°C using Dupont Lightning Plus Screens.

RESULTS

Polymorphism in C a Genes

Southern blot analysis of genomic DNA digested with the restriction enzyme BgllI and hybridized with a probe corresponding to the C region of the/3 chain of the T cell receptor (C~) reveals restriction fragments of approximately 10.0, 9.2, and 0.8 kb [25]. The 0.8 kb fragment is present in all DNA samples; however, the presence of the 10.0 and 9.2 kb fragments is variable.

Because BLCL were used as the source of genomic DNA it is expected that the observed differences in C~ genes were due to polymorphism in Bglll restric- tion sites and not due to gene rearrangements. However, to verify that DNA samples contained T cell receptor/3 chain genes in germline configuration, diges- tions with BamHI were performed. A fragment of approximately 24 kb that was anticipated for unrearranged/3 chain genes was observed [ 17 ]. There was a single exception. As reported previously, the BLCL from one individual had a rear- ranged 13 chain gene [25]. Therefore the observed BgllI 10.0 and 9.2 fragments are interpreted to represent polymorphic configurations of germline T cell re- ceptor 13 chain genes. DNA samples from a total of 61 individual members of eight families have been tested and no additional BgllI restriction fragment poly- morphisms have been identified.

Segregation of C o Genes in Family 2

The results of analysis of DNA samples from the members of Family 2 digested with BgllI are shown in Figure 1. Fragments of 10.0, 9.2, and 0.8 kb are present

198

FAMILY 2

Bgl II

FA MO $1 $ 2 $ 3 $ 4 $ 5 $ 6 $ 7

_10.0 ~ ~ ~ ~ ~ - 9.2

J~

- 0 . 8

F I G U R E 1 Southern blot analysis of family 2 with the C o probe and D N A samples digested with BgllI. Lanes are identified by the D N A donor: FA, father; MO, mother; and S1-$7, siblings 1-7. Approximate sizes of hybridizing fragments in kb are indicated to the right of the blot. Arrowheads mark the position of Hindlll fragments of phage A D N A of 23.5, 9.7, 6.6, 4.3, 2.2, and 2.1 kb.

T Cell Receptor ~ and/3 Chain Genotypes 199

in DNA samples from the parents. The 0.8 kb fragment is present in DNA from all family members whereas the polymorphic 10.0 and 9.2 kb fragments segregate differentially in the offspring. Like the parents, offspring $2, $4, and $7 are heterozygous for the 10.0 and 9.2 kb fragments, whereas S1 and $3 are homo- zygous for the 9.2 kb fragment and $5 and $6 are homozygous for the 10.0 kb fragment. Southern blot analysis of DNA samples from the members of Family 2 digested with BamHI revealed a fragment of approximately 24 kb which in- dicates that all DNA samples had C~ genes in germline configuration (data not shown).

A schematic representation of the polymorphic BglI1 fragments segregating in Family 2 is shown in Figure 2. T cell receptor/3 chain genotyping is possible by making the following conventional haplotype designations: a to the paternal 10.0 kb fragment, b to the paternal 9.2 kb fragment, c to the maternal 10.0 kb fragment, and d to the maternal 9.2 kb fragment. Thus, offspring S1 and $3 who carry only the 9.2 kb polymorphic fragment inherited b and d haplotypes. Likewise offspring $5 and $6 carrying only the 10.0 kb polymorphic fragment inherited a and c haplotypes. Definitive haplotype assignments cannot be made fi3r the three offspring heterozygous for the 10.0 and 9.2 kb fragments. Offspring $2, $4, and $7 inherited either a and d haplotypes or b and c haplotypes. An additional polymorphic marker would make haplotype assignments possible in these het- erozygous offspring. Studies are currently underway using probes that correspond to V region gene segments. Restriction enzymes that produce polymorphic frag- ments hybridizing with a V region probe derived from the murine cDNA clone C5 [26] have been identified and complete testing of the family is being performed.

Conservation of Restriction Sites in the C o Gene Region

BglII was the only restriction enzyme that produced polymorphic C~ restriction fragment in the DNA samples examined. Identical restriction fragments were observed in DNA samples from randomly selected individuals when digested with enzymes XbaI, SstI, PvuII, PstI, HpaI, EcoRV, HindlII, EcoRI, BamHl, MspI, or TaqI ([25], Figure 3, and data not shown).

In order to further examine/3 chain polymorphism, DNA samples from two

FIGURE 2 T cell receptor/3 chain haplotypes in family 2. Pedigree chart of family 2 with schematic representation of 10.0 and 9.2 kb polymorphic BX/I1 C,j fragments and haplotype assignments. Data taken from Figure 1.

T~3 HAPLOTYPES

FAMILY 2

[ - ~ l a c l ~ ~ I ' ~ [ - ~ ~ 10.Okb / o r / /

200

Barn HI Pst I Eco RI

Bgl II Bgl II Pst I 'A B A B A B A B A B A B

M. A. Robinson and T. J. Kindt

Bgl II

Eco RI A B A B

_ 1 0 . 0 - 9 .2

~ - 0 . 8

FIGURE 3 Southern blot analysis of DNA samples from two individuals with Ct~ probe. Lanes are identified by the DNA donor; A, homozygous for the 10.0 kb Bglll C,~ fragment; and B, homozygous for the 9.2 kb Bglll C o fragment. Approximate sizes of BgllI fragments in kb are indicated to the right of the blot. Arrowheads mark the position of Hindlll fragments of phage A DNA of 23.5, 9.7, 6.6, 4.3, 2.2, and 2,1 kb.

characterized individuals (one homozygous for the 10.0 kb BgllI C~ fragment and the other homozygous for the 9.2 kb BgllI C~ fragment) were digested with BamHI, PstI, EcoRI, and BgllI individually as well as with mixtures of two enzymes (Figure 3). The BgIII pattern of restriction fragments (0.8 and 10.0 or 9.2 kb) was observed in samples digested with BgllI alone or with a mixture of Bglll and BamHI. Therefore , the BamHI recognition sites must lie outside the BgllI sites at both the 5' and 3' ends. Samples digested with mixtures of BglI1 and Pstl or BgllI and EcoRI contain a 0.8 kb fragment that is found in digestions using BgllI alone. These double digestions contain, in addition, a fragment with a size dif- ferent from D N A digested with any of the enzymes alone. The polymorphism observed with BgllI alone is not maintained in the double digests. Thus, conserved PstI and EcoRI sites must lie within the BglIl sites that flank the polymorphic fragments.

T Cell Receptor c~ and/3 Chain Genotypes 201

BglII Restriction Map of the C o Region

Available sequence data for the human C~ region and the present results allow a tentative localization of the polymorphic BgllI site that gives rise to the RFLP. The human T cell receptor /3 chain gene complex contains two nearly identical C region genes. All reported C¢3 sequences contain a BglII site approximately 60 bp from the 5' end of the C region. In addition, two of the sequences, JUR/732 [27] and HPB-ALL [28], contain an additional BgllI site that is located near the 3' end of the coding region. The JUR/32 and HPB-ALL sequences differ signif- icantly from the other three reported sequences at the 3' end of the coding region and the 3' untranslated region, and are therefore considered to represent a second distinct (isotypic) C~ gene [27,28]. TheJUR/32 sequence is thought to correspond to the 3' member (C¢32) of the Cf3 pair [27] which are about 10 kb apart [29].

A schematic representation of the C~ loci and the hypothetical placement of the polymorphic BgllI site is depicted in Figure 4. This map is based on the cDNA information, on genomic sequence data, restriction maps of genomic clones ([29] and Tunnacliffe and Rabbitts, manuscript in preparation) as well as the present results. The fragment of approximately 0.8 kb observed in all D N A samples tested, would correspond to the sequence between BglII sites in C~2 (Figure 4). The polymorphic BgllI site is most likely located in the region between the two C~3 genes and we speculate that it would lie approximately 0.8 kb 5' to Cf32. The 9.2 kb fragment would therefore span the region from the 5' Bg/lI site in C/31 to the polymorphic BgllI site, whereas the 10 kb fragment would terminate at the 5' Bglll site in C/32 (Figure 4).

Polymorphism in T-Cel l R e c e p t o r ~ Chain Genes

Genomic D N A samples from the members of family 7 were digested with BgllI and subjected to Southern blot analysis using a probe that corresponds to the human T cell receptor ~ chain [2 l]. As shown in Figure 5, numerous fragments hyridized with the ~ chain probe. Seven fragments ranging in size from approx- imately 8.5 to 2.15 kb were present in D N A samples from all family members. These seven ~ chain fragments have also been observed in D N A from other individuals digested with BgllI (data not shown). In addition to the seven common fragments, the father of family 7 has a fragment of approximately 2.9 kb (indicated by an arrow to the right of the blot in Figure 5) that is not present in D N A from the mother. This polymorphic BgllI ~ chain fragment segregates in the children;

FIGURE 4 A tentative map of the human C~ region. Known BglIl restriction sites (Bg) and the postulated polymorphic BglII site (Bg) are indicated. Approximate sizes of BglII restriction fragments are given in kb. The map was adapted from Ref. 20 and data of T. Rabbitts (personal communication).

C/31 C32

Bg SgBg I i i , 1 o.o ,o.~

Bg

9.2 I 01~

202

FA

FAMILY 7

MO $1 $2 $3 $4

i I i

C-"*

C'--*

FIGURE 5 Southern blot analysis of family 7 with T cell receptor 6e chain probe and DNA samples digested with BgllI. Lanes are identified by the DNA donor: FA, father; MO, mother; S1-$4, siblings 1-4. Star (*) designates the polymorphic fragment of --2.9 kb; C designates fragments hybridizing with C,~ probe. Arrowheads mark the position of HindlII fragments of phage a DNA of 23.5, 9.7, 6.6, 4.3, 2.2, and 2.1 kb.

T Cell Receptor ~ and/3 Chain Genotypes 203

it is present in D N A from S1 and $4 but is not present in D N A from $2 and $3. On the basis of presence or absence of the polymorphic restriction fragment, paternal T cell receptor haplotypes can be assigned. Thus, S1 and $4 inherited paternal T cell receptor haplotype designated a and $2 and $3 inherited the b haplotype. D N A from the mother lacked the polymorphic fragments; therefore, maternal T cell receptor ce chain haplotypes can not be differentiated on the basis of BgllI digests.

The T cell receptor ce chain probe, pY14 [21], used for this analysis contained approximately 125 bp of V region and complete J, C, and 3' untranslated se- quences. To further characterize the hybridizing region a 390 bp Pvull fragment that corresponds to the C region (Ca) was derived from pY14. When the C,~ insert was used as a probe in Southern blot analysis of BgllI digested D N A from family 7, two nonpolymorphic fragments of approximately 3.8 and 2.15 bp were observed in the autoradiogram (indicated by arrows to the left of the blot in Figure 5). Thus, the polymorphic fragment of paternal origin corresponds not to constant region sequences but to V region, J region, or 3' untranslated sequences; the nature of this fragment is being investigated with other probes derived from clone pY14.

No RFLP hybridizing with the C,, probe were identified in D N A samples from the parents of family 7 when digested with any of a total of 12 restriction en- donucleases. Identical fragments were observed in both D N A samples when digested with PstI, SstI, Xbal, XhoI, NdeI, BstEII, EcoRl, BamHI, HindlII, BgllI, EcoRV, and HpaI. Polymorphism in the C~ gene may be detectable with D N A from other individuals and/or with other restriction enzymes.

D I S C U S S I O N

This report demonstrates that genes encoding both the ce chain and the/3 chain of the human T cell receptor can be distinguished by RFLPs that will serve as markers for T cell receptor ex chain and /3 chain haplotypes in families. T cell receptor 13 chain gene polymorphism was detectable in genomic D N A samples digested with Bg/II [25] using a probe corresponding to the constant region of the/3 chain (Cl3). A total of 61 individual members of eight families were analyzed and polymorphic Bglll restriction fragments were observed to segregate in six of the eight families. In many cases maternal and/or paternal C¢3 haplotypes could be assigned.

The Ct3 region itself is not anticipated to be a determinant o f T cell specificity. Structural studies have linked both J and D gene segments to the C/3 region and V region gene segments are likewise expected to be closely linked [15,16]. Therefore , the C¢3 polymorphisms may serve as markers for complete T cell receptor /3 chain haplotypes. Additional studies will be probes that correspond to V region genes. By identifying V region RFLP and following their segregation in the families characterized for C~ haplotypes, linkage can be evaluated and more complete haplotype assignments should be possible.

The C~, region appears to be at least as highly conserved as C~3 where BgllI as the only restriction enzyme of 12 tested that produced RFLP hydridizing with the Ct3 probe. The identification of an ae chain gene polymorphism relied upon the use of a human cDNA probe that contained sequences corresponding to V, J, C, and 3' untranslated regions. One polymorphic BgllI fragment of approxi- mately 2.9 kb was present in D N A from the father and two of the children in family 7. Paternal T cell receptor ee chain haplotypes could be assigned on the basis of presence or absence of the RFLP. In addition, seven nonpolymorphic

204 M.A. Robinson and T. J. Kindt

BgllI fragments ranging in size from 8.5 to 2.15 kb were observed, including C,, fragments of 3.8 and 2.15 kb. No C~ RFLP were observed in D N A samples from two individuals when digested with 12 different restriction endonucleases.

The families studied with T cell receptor ce and/3 chain gene probes have also been studied extensively using HLA class II and class III gene probes [19,30]. Each family includes at least one individual who inherited a recombinant HLA haplotype, and the region where each cross-over event occurred has been iden- tified. Thus, these families provide the means of analyzing the role of discrete regions of the HLA complex as well as T cell receptor ce and ~ chain haplotypes in all allogeneic and HLA restricted immune responses. The ability to differentiate allelic forms ofce and/3 chain genes should make it possible to investigate whether ce and ~ chain haplotypes are correlated with heritable variation in T cell function or with disease susceptibility.

A C K N O W L E D G M E N T S

We thank Dr. Terry Rabbitts for access to unpublished information, Dr. Mark Davis for providing the T cell receptor/3 chain eDNA clones, and Dr. Tak Mak for providing the T cell receptor ~ chain cDNA clone. We appreciate the detailed criticism of Dr. Edward Max. The expert technical assistance of Mary Mitchell and the secretarial help provided by Ginny Shaw are gratefully acknowledged.

REFERENCES

1. Amos DB, Kostyu DD: HLA--A central immunological agency of man. Adv Hum Genet 10:137, 1979.

2. Moiler G: HLA and disease susceptibility, Immunol Rev 70:218, 1983.

3. Bodmer WF, Albert ED, Bodmer JG, Dausset J, Kissmeyer-Nielsen F, Mayr W, Payne R, van Rood JJ, Trnka Z, Walford RL: Nomenclature for the HLA system 1984. Tissue Antigens 24:73, 1984.

4. Pandey JP, Johnson AH, Fudenberg HH, Amos DB, Gutterman JU, Hersh EM: HLA antigens and immunoglobulin allotypes in patients with malignant melanoma. Hum Immunol 2:185, 1981.

5. Whittingham S, Mathews JD, Schanfield MS, Matthews JV, Tait BD, Morris P J, Mackay IR: interactive effect of Gm allotypes and HLA-B locus antigens on the, human antibody response to a bacterial antigen. Clin Exp hnmunol 40:8, 1980.

6. Whittingham S, Propert DN, McKay IR: A strong association between the antinuclear antibody anti-La (SS-B) and the kappa chain allotype Kin(1). lmmunogenetics I q):295, 1984.

7. Acuto O, Fabbi M, Smart J, Poole CB, Protentis J, Royuer HD, Schlossman SF, Reinherz EL: Purification and NH2-terminal amino acid sequencing of the/3 subunit of a human T-cell antigen receptor. Proc Natl Acad Sci USA 81:3851, 1984.

8. Hannum CH, Kappler JW, Trowbridge IS, Marrack PH, FreedJH: lmmunoglobulin- like nature of the cr-chain of a human T-cell antigen/MHC receptor. Nature 312:63, 1984.

9. Yanagi Y, Yoshikai Y, Leggett K, Clark SP, Aleksander I, Mak TW: A human T cell- specific cDNA encodes a protein having extensive homology to immunoglobulin chains. Nature 308:145, 1984.

10. Hedrick SM, Cohen D1, Nielsen EA, Davis MM: Isolation of eDNA chines encoding T cell-specific membrane-associated protein. Nature 308:149, 1984.

1 l. Sire GK, Yagiie J, Nelson J, Marrack P, Palmer E, Augustin A, Kappler J: Primary structure of human T-cell receptor c~-chain. Nature 312:771, 1984.

12. Croce CM, Isobe M, Palumbo A, Puck J, Ming J, Tweardy D, Erikson J, Davis M, Rovera G: Gene for or-chain of human T-cell receptor: Location on chromosome 14 region involved in T-cell neoplasms. Science 227:1044, 1985.

T Cell Receptor c~ and/3 Chain Genotypes 205

13. Hayday DC, Saito H, Gillies SD, Kranz DM, Tanigawa G, Eisen HN, Tonegawa S: Structure, organization, and somatic rearrangement of T cell gamma genes. Cell 4(}:259, 1985.

14. Kranz DM, Saito H, Heller M, Takagaki Y, Haas W, Eisen HN, Tonegawa S: Limited diversity of the rearranged T-cell A gene. Nature 313 752, 1985.

13. Siu G, Clark SP, Yoshikai Y, Malissen M, Yanagi Y, Strauss E, Mak TW, Hood L: The human T cell antigen receptor is encoded by variable, diversity and joining gene segments that rearrange to generate a complete V gene. Cell 37 ~,9~, 1984.

16. Clark SP, Yoshikai Y, Taylor S, Siu G, Hood L, Mak TW: Identification of a diw:rsity segment of human T-cell receptor/3-chain, and comparison with the analogous m urine element. Nature 311:388, 1984.

17. Toyonaga B, Uanagi Y, Suciu-Foca N, Minden M, Mak TW: Rearrangements of T- cell receptor gene YT35 in human DNA from thymic leukaemia T-cell lines anti functional T-cell clones. Nature 3 l 1:383, 1984.

18. Minden MD, Toyonaga B, Ha K, Yanagi Y, Chin B, Gelfand E, Mak T: Somatic rearrangement of T-cell antigen receptor gene in human T-cell malignancies. Proc Natl Acad Sci USA 82:1224, 1983.

19. Robinson MA, Long EO, Johnson AH, Hartzman RJ, Mach B, Kindt TJ: Recom- bination within the HLA-D region. Correlation of molecular genotyping with func- tional data. J Exp Med 160:222, 1984.

20. Hedrick SM, Nielsen EA, Kawder J, Cohen DI, Davis MM: Sequence relationships between putative T-cell receptor polypeptides and immunoglobulins. Nature 308:153, 1984.

21. Yanagi U, Chan A, Chin B, Minden M, Mak TW: Analysis cDNA clones specific for human T cells and the c~ and /3 chains of the T-cell receptor heterodimer from a human T-cel lines. Proc Natl Acad Sci USA 82 3430, 1983.

22. Winberg G, Hammarskjold M.-L.: Isolation of DNA from agarose gels using DEAE paper. Application to restriction site mapping of adenovirus type 16 DNA. Nucleic Acids Res 8:487, 198/).

23. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:5{)3, 1975.

24. Wahl GM, Stern M, Stark GR: Efficient transfer of large DNA fragments from agarose gels to diazobenzylomethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683, 1979.

23. Robinson MA, Kin& TJ: Segregation of polymorphic T-cell receptor genes in human families. Proc Natl Acad Sci USA 82:3804, 1985.

26. Patten P, Yokota T, Rothbard J, Chief Y, Arai K1, Davis MM: Structure, expression and divergence of T-cell receptor/3-chain variable regions. Nature 312:40, l{)84.

27. Yoshikai Y, Anatoniou D, Clark SP, Yanagi Y, Sangster R, Van den Elsen P, Terhorst C, Mak TW: Sequence and expression of transcripts of the human T-cell receptor/3- chain genes. Nature 312:~21, 1984.

28. Jones N, Leiden J, Dialynas D, Fraser J, Clabby M, Kishimoto T, Strominger JL, Andrews D, Lane W, Woody J: Partial primary structure of the alpha and beta chains of human tumor T-cell receptors. Science 227:311, 1985.

29. Sims JE, Tunnacliffe A, Smith WJ, Rabbitts TH: Complexity of human T-cell antigen receptor/3-chain constant- and variable-regk)n genes. Nature 312:541, 1984.

30. Robinson MA, Carroll MC, Johnson AH, Hartzman RJ, Belt KT, Kin& TJ: Local- ization of C4 genes within the HLA complex by molecular genotyping. Immunoge- netics, 21:143, 1985.