Proc. Nati. Acad. Sci. USAVol. 81, pp. 7544-7548, December 1984Immunology

Role of a disulfide bridge in the immune function of majorhistocompatibility class I antigen as studied byin vitro mutagenesis

(synthetic oligonucleotides/H-2 antigen)

ToSHIHIKO SHIROISHI*, GLEN A. EVANSt, ETTORE APPELLAt, AND KEIKO OZATO**Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda,MD 20205; tCancer Biology Laboratory, Salk Institute, San Diego, CA 92138; and tLaboratory of Cell Biology, National Cancer Institute,National Institutes of Health, Bethesda, MD 20205

Communicated by Igor B. Dawid, July 26, 1984

ABSTRACT Polymorphic major histocompatibility class Iantigens have highly conserved disulfide bridges in the secondand third external domains. To study the role of a disulfidebridge, we have introduced a mutation into the mouse H-2Ldgene by oligonucleotide-directed site-specific mutagenesis, dis-rupting the disulfide bridge in the second domain of the pro-tein by changing cysteine at amino acid position 101 into ser-ine. Upon introduction of the mutant gene into L cells, themutant transplantation antigens were synthesized, insertedinto the membrane, and displayed on the cell surface, indicat-ing that the disulfide bridge is not essential for surface expres-sion of the H-2 antigen. Binding studies carried out with 16monoclonal antibodies specific for the H-2Ld antigen showedthat most of the allodeterminants are lost or greatly altered inthe mutant antigen. Further, almost complete loss of the rec-ognition by H-2Ldd-specific alloreactive cytotoxic T cells wasobserved. These results indicate that polymorphic determi-nants are dependent on a protein folding pattern dictated bythe disulfide bridge. However, two antibodies previouslyfound to react with antigenic sites present in the first and thirddomains were reactive with the mutant, implying an elementof domain independence with respect to the determinants rec-ognized by these antibodies.

The class I antigens of the major histocompatibility complex(MHC) are integral cell membrane proteins composed of a45-kilodalton polymorphic heavy chain encoded by genes onchromosome 17 in the mouse and expressed on the cell sur-face in association with a 12-kilodalton nonpolymorphic lightchain, f2-microglobulin. The heavy chain consists of threeextracellular domains, N, C1, and C2, which are attached toa transmembrane domain, M, and a cytoplasmic domain, I.This domain structure correlates with the exon-intron orga-nization of class I genes (1-6). The class I antigens are highlypolymorphic; multiple amino acid substitutions are foundthroughout the sequences of various alleles (7, 8). The poly-morphic amino acid substitutions are regarded as the struc-tural basis for allorecognition and for self-restriction.

In spite of extensive polymorphism, certain structural fea-tures of the class I heavy chains are highly conserved. Forexample, all of the functional class I antigens contain disul-fide bridges in the second (Cl) and the third (C2) externaldomains of the molecule (1-6, 9, 10). Two cysteines involvedin forming the disulfide bridges are located in homologouspositions: Cys-101 Cys-164 in the second domain, and

Cys-203 Cys-259 in the third domain. Glycosylation sites are

also highly conserved. Because of this high degree of conser-

vation, the disulfide bridges and the glycosylation sites maybe of crucial importance in the expression and normal func-tion of class I major histocompatibility antigen. In this paperwe utilized oligonucleotide-directed site-specific mutagene-sis (11, 12) to produce a mutant H-2Ld gene lacking the disul-fide bridge in the C1 domain and to investigate the immuno-logical properties of the altered gene product. Similar ap-proaches have generated a variety of specific mutationsintroduced into cloned genes at precise locations (11-15). Inthe present study, the cysteine at amino acid 101 of the H-2Ld antigen has been changed to serine by substituting a thy-midine for adenosine in the corresponding codon.We present here the construction of the mutant H-2Ld

gene, cell surface expression of the gene in mouse L cells,and characterization of the mutant antigen by a series ofmonoclonal antibodies and by alloreactive T cells.

MATERIALS AND METHODSSynthetic Oligonucleotides. The mutagenic oligonucleotide

5'-C-A-C-G-T-C-A-C-T-G-C-C-G-T-A-C-A-T-3' and primeroligomers 5'-G-C-G-C-T-C-T-G-C-T-T-G-T-A-G-T-A-G-3',5'-T-A-A-T-C-A-C-A-G-C-C-G-T-C-G-3', and 5'-G-C-C-C-G-C-G-G-C-C-C-C-T-G-C-A-C-3' were synthesized by thesolid-phase phosphotriester method (16), using an automatedsynthesizer (Vega Biochemicals, 280), and were purified byhigh-pressure liquid chromatography. Oligonucleotides werefurther purified through 20% denaturing polyacrylamide gelsto remove contaminating smaller oligonucleotides.

Strategy for in Vitro Mutagenesis. Preparation of therecombinant M13 phage m9.33.6 will be described else-where. Briefly, two restriction fragments containing the 5'and 3' portions of the H-2Ld gene and flanking DNA wereintroduced into M13.mp9. This construction results in thedeletion of a portion of the intervening sequence separatingthe C1 and C2 exons. The mutagenic oligonucleotide wasphosphorylated with T4 polynucleotide kinase (New En-gland Biolabs) and used for primer extension. Two addition-al oligonucleotides, which hybridize to M13 sequences oneither side of the insert, were added to primer extension re-actions (Bethesda Research Laboratories, Collaborative Re-search). Twenty picomoles of each of the three primers wasadded to 1 ug of m9.33.6 single- (+) strand DNA template in20 ,ul of 20 mM Tris HCl, pH 7.6/200 mM NaCl/9 mM mag-nesium acetate/20 mM 2-mercaptoethanol and heated to65°C for 5 min. The reaction mixtures were allowed to coolto room temperature over 30 min. After cooling further to12°C in ice water, 1-,u portions of 10 mM dCTP, dATP,dGTP, and dTTP and 100 mM ATP (Bethesda Research Lab-oratories) were added and the volume was adjusted to 30 ,ul

Abbreviations: CTL, cytotoxic T lymphocytes; TK, thymidine ki-nase.

7544

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Proc. NatL Acad Sci. USA 81 (1984) 7545

in 20 mM Tris HCl, pH 7.6/200 mM NaCl/9 mM magnesiumacetate/20 mM 2-mercaptoethanol. Primer extension wasinitiated by adding 5 units of DNA polymerase I (large frag-ment) (Boehringer Mannheim) and 40 units of T4 polynucle-otide ligase (New England Biolabs), followed by incubationat 12'C for 12-18 hr. This reaction mixture was used directlyto transform CaCl2-treated competent Escherichia coli hoststrain JM103. Phage plaques were isolated and single-strand-ed DNA was prepared as described (17).Dot Hybridization Screening. Procedures for dot hybridiza-

tion using 32P-labeled oligonucleotides have been describedelsewhere (11, 12, 17, 18). Briefly, 1 gg of single-strandedDNA purified from independent phage plaques was blottedonto nitrocellulose filters. The filters were incubated in 0.9M NaCl/0.09 M sodium citrate/lOx Denhardt's solu-tion/0.2% NaDodSO4 at 650C for 1 hr, then incubated withthe 32P-labeled mutagenic primer (2 x 106 cpm) in 0.9 MNaCl/0.09 M sodium citrate/lOx Denhardt's solution atroom temperature for 1 hr for hybridization. The filters werewashed at increasing temperatures-i.e., 58TC, 60TC, and62°C-each for 1 min. After each wash the filters were ex-posed to the XAR5 x-ray film (Kodak) for 5-18 hr.DNA Sequence Analysis. DNA sequence was analyzed by

using the dideoxy chain termination method (19, 20). Onemicrogram samples of single- (+) stranded DNA purifiedfrom the mutant and the wild-type phage were used as tem-plates. The mutagenic primer and four additional syntheticoligonucleotides were used for priming: 5'-G-C-G-C-T-C-T-G-C-T-T-G-T-A-G-T-A-G-3, complementary to the se-quence at the end of the N exon, 5'-T-A-A-T-C-A-C-A-G-C-C-G-T-C-G-3', complementary to the sequence about 60base pairs downstream of the mutation site, and 5'-G-C-C-C-G-C-G-G-C-C-C-C-T-G-C-A-C-3', complementary to the be-ginning of the intron between the C1 and the C2 domains.Availability of these primers allowed us to sequence the por-tion of DNA covering the N and the C1 domains withoutsubcloning the genes. [a-32P]dATP (specific activity 400Ci/mmol; Amersham; 1 Ci = 37 GBq), deoxy- and dideoxy-nucleoside triphosphates (Bethesda Research Laboratories)were used for chain extension. Sequence determination ofeach gene was carried out at least twice for the entire N-Clregion and several times more for the region of the expectedmutation. A 6% polyacrylamide gel with a gradient of 0.5-2.5 times XM Tris/YM borate/ZM EDTA (TBE) was usedfor electrophoresis.DNA-Mediated Gene Transfer. Double-stranded DNAs

were prepared from the mutant and the wild-type phage (17)and introduced into the thymidine kinase-deficient (TK-) Lcells, DAP-3, by a calcium phosphate precipitation methodas described elsewhere (3, 21-23). Twenty micrograms ofM13 double-stranded DNA and 500 ng ofpBR TK DNA con-taining the herpesvirus TK gene were precipitated with calci-um phosphate and then added to 1 x 106 DAP-3 cells. Selec-tion with hypoxanthine/aminopterin/thymidine mediumyielded TK+ transformed colonies from which cells express-ing mutant genes were cloned by a limiting dilution method.Antibody Binding Assay. Details of antibody binding as-

says carried out on monolayers of transformed L cells with1251I-labeled sheep anti-mouse Ig (Amersham) have been de-scribed elsewhere (23, 24). Monoclonal antibodies specificfor the H-2Ld antigen contained in culture supernatants (23-25) have been tested. Antibodies 1634 and 174.1 were gener-ous gifts from T. H. Hansen (University of Washington) andJ. A. Frelinger (University of North Carolina), respectively.Antibodies 66.2.4, 66.3.5, 64.3.7, and 66.13.5 were kindlydonated by S. Chatterjee-Das and D. H. Sachs (NationalCancer Institute). Antibodies T.O.101, T.O.102, T.O.105,and T.O.106 produced by N. Tada and K. Kimura will bedescribed in detail elsewhere. For flow cytofluorographyanalysis, fluorescein isothiocyanate-conjugated goat F(ab')2

anti-mouse F(ab')2 (Cappel Laboratories, Cochranville, PA)was used as a second reagent (23, 26).Generation of Alloreactive Cytotoxic T Lymphocytes

(CTL). Primary CTL were generated in vitro as describedelsewhere (26, 27). Briefly, CTL specific for the H-2Ld anti-gen were generated by stimulation of 5 x 106 BALB/c.H-2dm2 spleen cells with 2 x 106 irradiated BALB/c spleen cellsin RPMI medium (GIBCO) supplemented with 10% fetal bo-vine serum and other ingredients (26) for 5 days at 5% CO2 inhumidified air at 370C. BALB/c anti-C3H/HeJ CTL weregenerated by the identical procedure. Cytolytic activity wasmeasured by 51CrO4 release from the transformed targetcells after 6-hr incubation as described (26, 27).

RESULTS

Isolation and Identification of the Mutant H-2Ld Clones. Anoligonucleotide primer, complementary to the sequence ofthe H-2Ld gene at amino acid positions 99-104 (Phe-Gly-Cys-His-Asp-Gly) was synthesized. The single nucleotidesubstitution, from A to T at position 9 of the oligomer, re-places cysteine at 101 by serine. The mutant primer was an-nealed with the wild-type DNA m9.33.6 and primer exten-sion was carried out with DNA polymerase and T4 DNAligase. Twenty-four independent plaques were isolated, andDNA was prepared from each and screened by dot hybrid-ization using the 32P-labeled mutagenic primer as the hybrid-ization probe. The probe hybridized with all 24 DNAs atroom temperature (Fig. 1 Left). A stepwise increase in thewashing temperature removed the labeled probe from mostof the clones. The final wash at 620C removed the probecompletely from 21 clones, leaving 3 strongly hybridizingclones (Fig. 1 Right). These clones were isolated as putativemutants and characterized by restriction endonucleasecleavage. Double-stranded (replicative form) DNA was pre-pared and digested with Xba I, BamHI, Bgl III, and the com-bination of EcoRI and HindIII; identical patterns were foundfor both mutant and wild-type DNA, indicating that the dif-ferential hybridization was not due to gross structuralchanges in the gene.To confirm the mutation, two positive clones, designated

SH6-10 and SH6-17, were subjected to sequence analysisencompassing the N domain exon, the intron between the Nand the C1 coding sequences, and the C1 exon (except for 10base pairs at the 3' end) (19, 20). Four primers were used fordideoxy sequencing directly from the single-stranded phageDNA, eliminating the necessity of subcloning the genes. Fig.2 shows the comparison of one mutant and one wild-typegene sequence through the region of the mutation. The ex-pected alteration of ACA to ACT was found in both SH6-10and SH6-17. This particular region was sequenced severaltimes, using a primer that hybridized 80 base pairs down-stream from the beginning of the C1 coding sequence. Noother alteration was detected in the remaining portion of theDNA sequence in either mutant clone.

.00000.00* @000000000

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FIG. 1. Screening for mutant genes by dot hybridization. Sam-ples (1 A.g) of different cloned DNAs were placed onto a nitrocellu-lose filter and hybridized with the 32P-labeled mutagenic primer (2 x106 cpm per filter). The filter was washed at room temperature(Left), followed by stepwise increasing temperature reaching 620C(Right). The filter was exposed for 5 hr (Left) or 18 hr (Right).

Immunology: Shiroishi et aL

7546 Immunology: Shiroishi et al.

cv,A C GT H

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FIG. 2. Comparison of the mutant and the wild-type DNA se-quence. An autoradiograph of a 6% polyacrylamide gel with a gradi-ent of 0.5-2.5x TBE shows the sequence of the mutant SH6-17(Right) and wild-type (Left) DNA around the region of the mutation.The synthetic oligonucleotide 5'-T-A-A-T-C-A-C-A-G-C-C-G-T-C-G-3' was used for priming. The mutagenic primer sequence is under-lined; arrows indicate the site of mutation.

Expression of the Mutant H-2Ld Gene in Mouse L Cells.Double-stranded DNA purified from the mutants SH6-17,SH6-10, and the wild-type gene m9.33.6 were introducedinto TK- mouse L cells as described (3, 21, 22). Ten TK'colonies were isolated from the cells transformed with thewild-type gene, all of which expressed the H-2Ld antigen asdetected by several representative monoclonal antibodies,including antibody 28.14.8, which reacts with a determinantin the C2 domain (23). Of the TK' colonies obtained with themutant gene SH6-17, 8 out of 18 colonies were positive with28.14.8. The cells were cloned to ensure homogeneity of thepopulation. Results of a binding assay carried out with 16monoclonal antibodies reactive with the H-2Ld antigen test-ed on mutant clone SH6-17B and wild-type clones W-12 andW-13 are shown in Table 1. All of the anti-H-2Ld antibodiesexhibited the expected reactivity toward the wild-type H-2Ldproducts: clone W-12 expressed the H-2Ld antigens at signif-icantly higher level than W-13. No reactivity was found onthe untransformed DAP-3 cells with these antibodies. Since125I-labeled antibody used as a second reagent reacted withIgG2 more strongly than with IgM or IgG3, variability of thebinding among antibodies of different isotypes was noted.Only two antibodies, 28.14.8 and 64.3.7, showed appreciablebinding to the mutant clone. All the rest of the antibodiesshowed either no or very low reactivity to the mutant. Theantibodies 64.3.7 and 28.14.8 have been previously assignedto react with determinants in the N and C2 domains, respec-tively, from studies of hybrid H-2 genes having chimeric do-mains (refs. 23 and 24; unpublished observations). Antibod-ies 23.10.1, 30.5.7, 1634, 174.1, 66.2.4, and 66.3.5, which didnot bind the mutant antigen, have been assigned to reactwith sites located in the C1 domain (ref. 24; S. Chatterjee-Das, personal communication). Other antibodies, whosesites have not been localized to a specific domain due tocross-reactions between Ld and Dd, also lost all reactivity.Thus, the mutant Ld, while lacking many determinants in theC1 domain, retained at least one determinant each in the Nand C2 domains. The antibody binding was further analyzedat the single-cell level by cytofluorography. The transformedclone SH6-17B gave a single peak in the fluorescence stain-

Table 1. Binding of anti-H-2Ld monoclonal antibodies to themutant cells

Reactivity,1251 cpm bound per well

Monoclonalantibody

16.1.230.5.728.14.823.10.11634174.127.11.1328.11.534.4.20T.O.101T.O.102T.O.105T.O.10666.2.466.3.564.3.766.13.514.4.4None

SpecificityKkDkLd, ClLd, C2Ld, ClLd, ClLd, ClLd and DdLd and DdLd and DdLd and DdLd and DdLd and DdLd and DdLd, ClLd, ClLd, NLd and DdI-Ek

Isotype

IgG2aIgG2aIgG2aIgMIgMIgMIgG2aIgMIgG2aIgG2aIgG2aIgG2aIgG2aIgG3IgG2aIgG2bIgG2bIgG2a

W-12cells

424165726146522265314

3288621

38465295562159949463775439592030911490

W-13cells

787050294545331196179

1973399

25663790445861

2717138

374817001989587

DAP-3cells

10,5011038986109901217914389109919081110971027586

SH6-17Bcells

7855524109713912411617311915612774928082

3181728

938392

Cells (4 x 103) were placed in wells of 96-well microtiter platesand incubated overnight. Monoclonal antibodies in 50 ,ul were addedto each well, and binding was assayed by subsequent incubationwith "15I-labeled sheep anti-mouse whole 1g. Each value representsthe mean of triplicate wells. Specificity of monoclonal antibodies forthe H-2 products and for the domains have been assigned previously(22, 23). W-12 and W-13 are cells transformed with the wild-type H-2Ld gene; DAP-3 cells are untransformed cells; SH6-17B cells aretransformed with the mutant gene.

ing profile with both 28.14.8 and 64.3.7 antibodies, indicatingthat all the cells in the cloned population displayed the mu-tant H-2Ld antigen. Additional transformed cells other thanSH6-17B (Table 1) were also studied. All the cells obtainedby transformation with the SH6-17 gene showed serologicalproperties identical to those of SH6-17B cells.

It should be mentioned here that one of the two mutantgenes, SH6-10, did not express any detectable H-2Ld antigenafter transfer into L cells, although we tested more than 50independent colonies. The nonexpressing mutant gene pro-duced H-2Ld-specific mRNA in the transformed cells as de-tected by a synthetic probe specific for the H-2Ld gene (28)(data not shown). This is reminiscent of an HLA class I"pseudogene" reported by Jordan and co-workers (29, 30),in which cysteine at amino acid 164 was replaced by phenyl-alanine and failed to be expressed on the surface of L cells.The DNA sequence analysis of the nonexpressor SH6-10 in-dicated no alteration in the N and C1 coding regions and theintron except for the expected mutation. We reasoned that adefect must exist in SH6-10 upstream of the N-domain-en-coding sequence. To test this possibility, the 5' end of theSH6-10 gene, containing the leader and N- and Cl-encodingsequence was recombined with the 3' end of the wild-type Ldgene (m9.33.6) encoding the C2, membrane, and cytoplasmicdomains (23). A mosaic gene with the reverse combinationwas prepared as well. Expression was observed only withthe mosaic gene having the 5' end of the wild-type gene andthe 3' end of the nonexpressor SH6-10. The mosaic gene ofthe reverse combination was totally negative for antigenexpression (data not shown). These findings suggest that thedefect of the nonexpressor SH6-10 gene is located upstreamof the N domain, representing an unrelated secondary muta-tion.

Proc. NatL Acad Sci. USA 81 (1984)

OIO

Proc. Natl. Acad Sci. USA 81 (1984) 7547

LL30

0-

10

Anti-H-2Ld

* W-12o W-13* SH6-17Bo DAP

60r

50

40 -

30-

20-

10

50 25 12.5 6.5 3.2 50 25

EFFECTOR TO TARGET RATIO

FIG. 3. Specific lysis of transformed cells ex

H-2Ld antigen by alloreactive CTL. EffectorBALB/c.H-2dm2 anti-BALB/cKh (Left) andBALB/cKh (Right) were incubated with 5"Cr-lab6 hr. The values represent means ± SD of triplicSpontaneous 51Cr release from the transform(25%. DAP, untransformed L cells; W-12 andformed with the wild-type H-2Ld gene; SH6-17Ewith the mutant gene.

Reactivity of Anti-H-2Ld Allospecific CTLGene. Previous studies performed with hybr(27, 31) indicated that antigenic determinanalloreactive or H-2-restricted CTL are local]C1 domains but not in the C2 domain. We e

of the disulfide bridge in the formation oftected by alloreactive T cells by testing thethe mutant cells to in vitro generated H-21Fig. 3 shows the cytotoxicity of BALBBALB/c specific for H-2Ld antigen and tanti-C3H specific for H-2Kk and H-2Dkagainst W-12, W-13, SH6-17B, and DAP-3ed, all the cells tested were killed by the CT]2Kk and H-2Dk antigens, corresponding to Ithe host L cells (Fig. 3 Right). Also, the willysed effectively by the H-2Ld-specific CT]detectable lysis by H-2Ld-specific CTL wa

the SH6-17B mutant cells at any effector-tcamined. These results indicate a critical rolebridge for the formation of alloantigenicity icells. The fact that W-13 wild-type transfcpressing less H-2Ld antigens were still lysedgests that the absence of the lysis is not duedifference in the amount of antigens on thesupport of this contention we found that C

which lacked a glycosylation site and expmuch lower degree than the wild-type, w

highly susceptible to the CTL (unpublished

DISCUSSIONThis paper uses oligonucleotide-directed mu

dress the role of the disulfide bridge in the Cexpression and function of a MHC class I genbridges present in the C1 and C2 domainsserved throughout mammalian class I antigmutant animals are available to study their r

addition, no mutant cell lines established irhave been shown to have a defect in the d(34). Site-specific mutagenesis, which allowsvirtually any amino acid at any position, w,this work, since it is most suitable to studyrole of conserved amino acids.

Intrachain disulfide bridges play a pivotaling of biologically active proteins. For exarmatic activity of ribonuclease A is lost upon i

Anti-H-2K Dk disulfide bridges but is recoverable upon renaturation by ox-idation (35). Antigen binding capacity of F(ab')2 fragments ofan antibody is dependent on inter- as well as intrachain disul-fide bridges of heavy and light chains (36). These approach-es, which rely on external modification of molecules, do notallow us to study the role of disulfide bridge in the biosynthe-sis and transport of proteins inserted in the cell membrane.In this paper we showed that the removal of the disulfidelinkage does not prevent biosynthesis, insertion into themembrane, and expression on the cell surface of the mutantH-2 antigen. This observation indicates that selective pres-sure to conserve the disulfide bridge through evolution, if it

12.5 6.5 3.2 existed, is not directly related to the ability of the antigen tobe expressed on the cell surface. Whether or not there is aquantitative disadvantage in the synthetic processes or in the

pressing the mutant stability of the antigens, however, has not been studied incells generated by detail in this paper. In this regard Jordan and co-workers (29,by C3H/HeJ anti- 30) have described the absence of detectable HLA antigen,eled target cells for upon L cell transformation by an HLA pseudogene that con--ate determinations. t azd cells was about tained an apparent amino acid substitution of a cysteine byW-13, cells trans- phenylalanine at amino acid 164. These authors suggested3,cells transformed that the disruption of a disulfide bridge may be responsible

for the failure of cell surface expression. Although these re-sults are not directly comparable with ours, since the posi-tion and the amino acid substituted were not identical, our

with the Mutant results indicate that the disruption of a disulfide bridge alonerid gene products is not responsible for the lack of surface expression.ts recognized by The examination of binding of 16 monoclonal antibodiesized in the N and revealed that many of the determinants were either com-xamined the role pletely lost or had greatly reduced activity in this mutantdeterminants de- gene product. All the antibodies previously mapped to reactsusceptibility of with the determinants in the C1 domain were affected. In

_ specific CTL. addition, all the antibodies whose determinants have not

/hat anti been localized to a specific domain showed almost complete:hat of BALB/c loss of the reactivity. Furthermore, no appreciable cytolysisantigens tested of the mutant cells was found by alloreactive CTL. Thesecells. As expect- results indicate that the tertiary structure created by the di-L specific for H- sulfide bridge is essential for the formation of numerous allo-the haplotype of geneic determinants. The contribution of the serine residueI-type cells were that replaced the cysteine in the mutant is not clear at pres-L. However, no ent. Studies on substitutions with other amino acids willLs observed with eventually clarify this point. The two antibodies previously)-target ratio ex- found to react with determinants in the N or C2 domain werefor the disulfide reactive with the mutant, suggesting that disulfide bridgesrecognized by T control an overall tertiary structure largely within each do-)rmant cells ex- main. However, conformational interactions between do-Ieffectively sug- mains (37) are not negated by these results, since most anti-to a quantitative genic sites were affected in the mutant.cell surface. In In vitro mutagenesis is an effective means to study struc-another mutant, ture-function relationships of class I antigens. Using this ap-

,ressed Ld to a proach, additional site-specific mutations in the H-2Ld genetas nevertheless have been generated. These mutations result in an altereddata). glycosylation site or a single amino acid substitution in a

polymorphic region (unpublished data). By this method itshould be possible to identify the precise amino acids that

itagenesis to ad- correspond to polymorphic determinants in H-2 antigens re-1 domain in the acting with antibodies and T cells.e. The disulfideare highly con- We are grateful to Dr. Gilbert Jay for providing an H-2Ld-specificyens; no class I probe and thoughtful advice on the RNA blot experiment and to Dr.oles (32, 33). In K. Okayama and Dr. K. Tanaka for valuable suggestions and stimu-n tissue culture lating discussions. We thank Drs. D. H. Sachs, S. Chatterjee-Das,isulfide bridges J. A. Frelinger, and T. H. Hansen for their gifts of monoclonal anti-bodies. We also thank Ms. B. Orrison and Mr. M. Gibson for pro-substitution of viding expert technical assistance and Ms. K. Miceli for superb sec-as employed in retarial help.ythe functional

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