J. clin. Path., 32, Suppl. (Roy. Coll. Path.), 13, 76-84

Roy Cameron Lecture

Control of antibody formation: certainuncertaintiesALAN R. WILLIAMSON

From the Department of Biochemistry, University of Glasgow, Glasgow

The formation of antibodies has been the subject ofhypothesis and experimentation for the whole of thiscentury. Our knowledge of antibody formation hasmade dramatic advances on several occasions. Manyof these advances have become crystallised ascertainties in the text books. The fascination of thesubject, however, has always been its uncertainties,which have continued to undermine the text bookpicture of antibody formation. As ever in research,the questions are more important than the answers.For an understanding of antibody formation, or

of any other biological system, one must ultimatelyturn to the molecular level. This necessity was plainlyseen by Roy Cameron who, in recreating his researchdepartment in 1946, 'determined to explore the newtechniques for examination of cell fractions, andlethal syntheses, and biochemical lesions in the cell'(Oakley, 1968). Progress in the study of antibodyformation during the past three decades has beenachieved by exploring the paths pointed out by RoyCameron. Those paths have led us well over anyhorizon that could have been foreseen in 1946 andeven beyond horizons visible in 1966. At our presentstage in the exploration I will attempt to drawtogether the apparent certainties and indicate someof the uncertainties. I will begin with the beguilingquestion of antibody diversity, a subject for whichthe cellular, genetic, and molecular basis continuesto hold many uncertainties.

Antibody repertoire

The antibody repertoire is defined here as the totalpopulation of antibodies that a single animal iscapable of elaborating. This repertoire can be sub-divided into collections of antibodies each specificfor particular determinants (epitopes). Antibodiesselected to be specific for a particular epitope arealmost invariably diverse. This diversity is based onamino-acid sequence variation. Reviewing the

amino-acid sequences of murine immunoglobulins(of myeloma tumour origin) there is a clearly non-random distribution of sequence variation (Kabat etal., 1976). V region sequences show a clustering ofvariability into regions which have been called hyper-variable regions with the remainder of the V regionsequences being termed framework regions. Thissequence data led to the hypothesis that hyper-variable regions constituted complementarity-deter-mining regions of the antibody molecule (Cohn etal., 1974).

Determination of the three-dimensional structureof the antibody-combining site confirmed the hypo-thesis that hypervariable regions contribute comple-mentarity-determining residues (Amzel et al., 1974;Segal et al., 1974) but shows that not all hyper-variable regions are involved in a particular antibody-combining site, and some hypervariable regions maybe too remote ever to contribute to an antibody-combining site. Hypervariability remote from theantibody-combining site and the sequence varianceof the framework regions must be put in a categoryof non-specificity-related variation. Thus we candefine a molecular basis for the redundancy in theantibody system. The redundancy within the reper-toire of antibodies leads one to question how muchof the total repertoire is significant in terms of pro-viding a complete immune response. Biologically theredundancy appears to explain the lack of gaps in theantibody repertoire.

Recent advances in molecular genetics have shedlight on the organisation of antibody genes. Thehypothesis (Dreyer and Bennett, 1965) that eachimmunoglobulin chain is coded for by two genes,one V gene and one C gene, with rearrangement ofthe genes at the DNA level being necessary for anti-body production, has in essence been verified. Theimmunoglobulin chains, however, need no longer beconsidered to be genetic oddities since othereukaryotic proteins have now been shown to be

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Control ofantibody formation: certain uncertainties

encoded in multiple distinct gene segments-forexample, ovalbumin (Breathnach et al., 1977; Doelet al., 1977). It has now been shown that the antibodylight chain is encoded in four not two gene segments(Brack et al., 1978). The arrangement of these seg-ments differs between the embryonic genome and theexpressed genome of the antibody-producing cell(Fig. 1). The evidence indicates that there are veryfew (maybe only one) C gene segments (Rabbitts,1977), of the order of 10 J segments (Weigert et al.,1978), and a much larger number of V segments. Theexact number of V gene segments remains uncertain.

P vE mbryo c

v I

J C

DNA

, 12 , "

Conimitted Cell DNA

in,RNA

Fig. 1 Immunoglobulin L chain gene segments,commitment and expression. Gene segments coding formurine immunoglobulin A light chain (based on datafrom Brack et al., 1978).

Murine VK gene segments appear to be organisedin sets comprising about 10 per set, V genes within aset being more similar in sequence to one anotherthan to V genes in other sets (Seidman et al., 1978;Valbuena et al., 1978). Analyses by molecular cloningand DNA sequencing, however, shows that there isboth framework and hypervariable region diversityamong V genes in the same set (Seidman et al., 1978).Interpretation of VK amino-acid sequences in termsof V gene-segment sets indicates a minimum of 50and possibly as many as 100 V gene sets, giving a

total complement of up to 1000 V gene segments.Each V region expressed requires the joining of a Vgene and a J segment. With 10 different J segmentsand combinatorial joining of V and J we arrive atan estimate of 104 VE region sequences coded forentirely by germ line genes. If by similar logic we

accept 104 VH region sequences then, by com-

binatorial association, a repertoire of 108 antibodiescan be coded for by germ line genes.There have been many calculations of the size of

the antibody repertoire, based on extrapolation frommeasurements of the size of individual repertoires ofantibodies of a given specificity (Williamson, 1976).Most estimates place the total repertoire at between107 and 108. Therefore quite conceivably germ linegenes could account for all the diversity in straightnumerical terms. Nevertheless, three lines of argu-ment suggest further uncertainties. (1) It is reason-able to question whether the repertoire is really

limited to 108 antibodies. The measurements ofindividual specificity repertoires lack precision andthere are many assumptions in scaling up to a totalrepertoire. (2) The large number of inherited genesegments coding for antibody variable regionsprovides a large target size for mutation, and thismust increase the pool of both germ line and somaticvariability. (3) There is already evidence that amino-acid sequence diversity within certain V regiongroups may exceed the estimated number of V genesegments within the corresponding set (Tonegawa etal., 1977; Brack et al., 1978; Weigert et al., 1978).

All estimates of the size of specific antibodyrepertoires and of the extent of diversity in themyeloma protein repertoire have presupposed thatall mice of the same inbred strain have an identicalcomplement of germ line genes. The repertoire ofthe inbred strain is equated with the repertoire ofeach mouse of that strain. Indications that the pheno-typic diversity of antibodies might exceed the geno-typic diversity have therefore been accepted asevidence that somatic diversification of V regionsequences must take place. But uncertainty mustremain about the extent to which germ line V regionsequences accumulate diversity within the animalpopulation as opposed to within the somatic cellpopulation. There is no evidence for the generationby mutation of new hypervariable regions occurringin committed clones of B lymphocytes (Scharff et al.,1975; Milstein et al., 1977). Antibody variationduring expansion of a committed clone has beenreported (Cunningham, 1974) but evidence that suchvariation is due to mutation is lacking. Germ linevariation among inbred mice has not been examinedas a factor in estimates of the antibody repertoire.There is evidence for such genetic variation within astrain of mice (Bailey, 1978) and the multiple V genefamily could readily accumulate such variation.

Selection is the complement to mutation inshaping the antibody phenotype. Somatic diversifica-tion hypotheses have emphasised the need for posi-tive selection of variants (Cohn et al., 1974), butevidence for positive selection of particular hyper-variable regions, generated either by somatic or germline mutation, is lacking. The effect of negativeselection, eliminating non-viable V region pheno-types, is evident. Negative selective pressure allowsexpression of all mutations, whether specificitydetermining or non-specificity determining, that donot interfere with the essential process of assembly,surface expression, and secretion of antibody mole-cules (Hood et al., 1974; Williamson, 1976). Negativeselective pressure operates on mutations arisingeither somatically or in the germ line. The allowedphenotype, showing only conservative variation inlarge regions of the molecule but more extensive

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Stem Cell Pre-B Lymphocyte Plasma Cell

Fig. 2 Stages in thedifferentiation of cells of the Blymphocyte lineage.

No Ab receptor Ab secreted Ab(104 105/cell) (2000/cell/ se)

variation in those regions for which a wide range ofstructures can be accommodated in the basictertiary and quaternary structure, is consistent withthe known pattern of framework and hypervariableregions.The expression of the extensive antibody repertoire

must be controlled during development and must beresponsive to the antigenic environment. In thefollowing sections I shall discuss some aspects of thegenetic and molecular controls on the expression ofthe antibody phenotype.

Cellular basis of antibody diversity

'One cell, one antibody' is a statement that con-veniently summarises the evidence relating to thecellular basis of antibody diversity. Our presentpicture of the development of the antibody-formingcell is summarised in Fig. 2. A stem cell undergoes aninitial commitment to the synthesis of a single lightchain and a single heavy chain variable region. Thiscommitment step involves a rearrangement of DNAsegments in the genome (Fig. 1). For murine A andK chains the V gene segment undergoes rearrange-ment so that it becomes precisely joined to the smallJ segment (Brack et al., 1978). In the case of K chainsone of about 10 J segments, located at variousdistances from the C, gene segment, can be chosen.Although the rearrangement of H chain gene seg-ments has not yet been demonstrated we mayreasonably assume that the mechanism of V-Jjoining evolved before the evolution of separateheavy and light chain gene families.Commitment of a cell to the expression of a single

antibody means that only two out of the six chromo-somes carrying antibody genes contribute to thephenotype. This is observed and recorded as twophenomena-(1) allelic exclusion, and (2) K/Aexclusion. We therefore need to know how manyrearrangement events occur in a single cell. Doescommitment involve only two chromosomes withrearrangement of a single VL gene segment on oneand a single VH gene segment on the other? Or doesrearrangement take place on several chromosomes

leading to multiple VL and VH expression withphenotypic selection?There is good evidence that allelic exclusion occurs

at the level of DNA arrangement. Physical mappingof V and C gene segments in the DNA of immuno-globulin-producing cells shows two patterns, onecorresponding to the expressed V-J-C gene arrange-ment and the other to the non-expressed embryonicarrangement of these segments (Brack et al., 1978;Seidman and Leder, 1978). Similar evidence pointsto the choice between K and A expression being madeat the level of DNA arrangement. Evidence that theK/A exclusion is a random process comes from theobservation that in newborn mice about equalnumbers of K- and A-bearing B lymphocytes aregenerated (Haughton et al., 1978). This contrastswith the predominant (90-95 %) K chain productionin the phenotype of the mature mouse. Apparentlythe much greater diversity of the K chain populationrelative to the A chain population allows K chainsto be involved in more distinct antibody specificities,so that antigenic selection eventually leads to themature preponderance of K synthesising cells.

Two forms of antibody expression

Antibodies were first observed as serum proteins andwere subsequently shown to be secreted by plasmacells. Plasma cells arise by proliferation and differ-entiation of B lymphocytes. The B lymphocytesynthesises and displays on its surface a smallamount (104-105 molecules) of antibody of a singlespecificity (Fig. 2). A basic tenet of the clonal selec-tion hypothesis is that the cell surface antibodymolecules act as receptors for antigens. Under thecorrect conditions interaction of antigen withreceptor antibody initiates proliferation and differ-entiation of the B lymphocyte. The involvement ofreceptor antibody seems certain-the nature of thatinvolvement is most uncertain.The existence of both secreted serum antibody and

membrane-bound antibody raises many biochemicalquestions. How does the immunoglobulin moleculeadapt itself to serve two such different functions?

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What controls the flow of immunoglobulin either tothe membrane or to secretion from the cell? How isthe switch from membrane deposition to activesecretion of antibody effected during proliferationand differentiation in a clone of B lymphocytes? Toseek answers to these questions we must first look atthe characteristics which generally designate aprotein for secretion or for surface membranedeposition.

Proteins destined for secretion are generallysynthesised on membrane-bound polyribosomes(Palade, 1975). The nascent polypeptide chain issynthesised and released vectorially across themembrane into the lumen of the endoplasmicreticulum (Sabatini and Kreibich, 1976). Location ofribosomes on the membrane and transfer of thenascent chain across the membrane is thought to beeffected by the amino terminal peptide of the nascentchain (Blobel and Dobberstein, 1975a). For mostsecreted polypeptide chains this amino terminalpeptide is a predominantly hydrophobic sequence ofabout 20 amino-acids in length, which is cleavedeither shortly before or shortly after completion ofpolypeptide chain synthesis. Precursor peptides havebeen identified for both heavy and light chains ofimmunoglobulin (Milstein et al., 1972; Schechter andBurstein, 1976; Jilka and Pestka, 1977). For themurine A chain it has been shown by sequencing ofcloned DNA (Tonegawa et al., 1978) that the pre-cursor peptide is encoded by a separate genesegment, P (Fig. 1). Probably separate P genesegments exist proximal to V,K and VH gene segments.Many secreted proteins are glycosylated. For the

immunoglobulins the heavy chains are glycosylatedbut not the light chains. The human g chain, forexample, has five oligosaccharide units, each onelinked to an asparagine residue via a N-glycosidicbond (Shimizu et a!., 1971; Putnam et al., 1973).The process of glycosylation at asparagine residuesbegins with the attachment of core oligosaccharidesto the nascent polypeptide chain (Behrens et al.,1973; Hsu et al., 1974; Lennarz, 1975). This stepmay well be important in the correct folding of thepolypeptide chain, but glycosylation itself does notseem to be a necessary event for protein secretion(Schachter, 1974; Weitzman and Scharff, 1976).Correct secondary, tertiary, and possibly quaternarystructure of the protein does, however, seem to benecessary for secretion. The presence of the proteinin the lumen seems to be necessary, but not sufficient,for secretion. Immunoglobulin heavy chains aregenerally not found to be secreted in the absence oflight chains. On the other hand, light chain secretionin the absence of heavy chains is not unusual. It is,however, possible to select structural mutants oflight chains that have lost the ability to be secreted

(Mosmann et al., 1979). These mutants are syn-thesised with apparently normal precursor peptidesbut are defective in some structural feature requiredfor the transport from the lumen to the outside ofthe cell (Mosmann and Williamson, 1979). Thedefect is thought to involve a structural featureneeded for selective passage of the protein from thelumen to the Golgi apparatus.

Proteins designated for the surface membrane areassumed to traverse a similar route to that followedby proteins heading for secretion. Transfer across themembrane is effected by the precursor peptide withsubsequent cleavage, folding, glycosylation, andtransport through the cell to the surface (Fig. 3). In

I Vectorial'0aythl

iam&popt$. I e., jv ts

? esie or,ma,tpn

Fig. 3 Biosynthetic route for surface membraneproteins.

this regard the two roles for immunoglobulin areconsistent with one another. However, in order tobe retained at the surface of the cell the membraneprotein must be attached to the plasma membraneor to another protein which is itself an integralmembrane protein. Attachment of the integralmembrane proteins appears to involve a hydrophobicpeptide (Marchesi et al., 1976), generally found at ornear the carboxy-terminal end of the molecule(although an example of amino-terminal attachmentof a protein to a membrane has been reported(Kenny et al., 1978)). This part of the model poses aproblem for immunoglobulin. The only knownhydrophobic sequences associated with immuno-globulin molecules are the precursor peptides whichinitiate synthesis of the heavy and light chains. Theseprecursor peptides are rapidly removed fromimmunoglobulins destined for secretion (Schmeck-peper et al., 1975). The possibility that the precursorpeptide might act as an anchor in the membrane has

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been ruled out; the absence of the precursor peptideon membrane immunoglobulin was shown byN-terminal sequence analysis (Bergman and Haimo-vich, 1978; Singer et al., unpublished observation).A number of differences between membrane-

bound and secreted immunoglobulin have beenreported without any of these differences giving aclear indication of the mode of attachment ofimmunoglobulin to the membrane. A more promisingfinding that we have made recently is that the non-glycosylated heavy chain of membrane immuno-globulin is longer than the non-glycosylated heavychain of secretory immunoglobulin (Singer, 1979).This finding was made by allowing the heavy chainsto be synthesised in the presence of the drug tunica-mycin, which blocks glycosylation (Tkacz andLampen, 1975; Bettinger and Young, 1975; Takat-suki et al., 1976; Lehle and Tanner, 1976). The sizedifference corresponds to an extra peptide of about20 amino-acids long on the membrane-bound heavychain. Comparative analysis of membrane-boundsecretory heavy chain using carboxypeptidase pointsto a carboxy-terminal location of the extra peptideand suggests a hydrophobic nature for that peptide(Williams et al., 1978; Singer, 1979). The postulatedrole for the carboxy-terminal hydrophobic peptidein anchoring a , heavy chain to membrane firstly atthe point of synthesis, then during assembly of four-chain IgM, through the intracellular transportsystem, and finally for display of surface IgM isshown in Fig. 4.The hydrophobic anchor peptide provides us with

a static structural answer to the problem of howimmunoglobulin fulfils its two different roles, but westill require answers to the dynamic questions con-cerning the biosynthesis of surface and secretedimmunoglobulins. Some answers may be suggestedin the form of provocative hypotheses discussed herein the context of recent evidence concerning Blymphocyte maturation.

B lymphocyte differentiation

The development of B lymphocytes may be dividedinto two phases, (1) pre-antigenic stimulation, and(2) antigen-driven clonal expansion.

(1) Initial immunoglobulin gene expression by thecell that has become committed at the level of DNArearrangement appears to result in the production ofa single sort of g chain and a single sort of lightchain. This stage in development at which the com-mitted genome is first expressed is termed a pre-Blymphocyte (Melchers et al., 1975; Osmond et al.,1976; and see J. J. T. Owen at page 1). Althoughthis pre-B cell is producing small amounts of u chainand light chain it does not appear to be capable of

Fig. 4 Postulated biosynthetic route for surface-membrane immunoglobulin (based on data from Singer,1979). The heavy and light chains grow vectorially acrossthe membrane of the endoplasmic reticulum andassembly could begin, as shown, before completion of thenascent heavy chain. The assembledfour-chain moleculeremains integrated into the membrane via a hydrophobictail peptide through intracellular transport to the plasmamembrane (see Fig. 3).

inserting an IgM receptor antibody into the plasmamembrane in a stable and functional manner(Melchers et al., 1975; Raff et al., 1976). The nextstage in development is the maturation of the pre-Bcell into the virgin B lymphocyte which does exhibitfunctional IgM receptor antibody. The differencebetween the pre-B lymphocyte and the B lymphocytethat accounts for the latter expressing a surfacereceptor antibody requires an explanation. The pre-Bcell synthesises IgM. There is good evidence, how-ever, that receptor antibody on B lymphocytes canbe of any class or subclass of immunoglobulin(Abney et al., 1978) and indeed that two classes ofimmunoglobulin, apparently associated with thesame idiotype or antibody specificity, can be foundon the surface of a single cell (Pernis et al., 1974;Stern and McConnell, 1976). It therefore appearsnecessary to explain how the B lymphocyte acquiresthe ability to produce surface receptor antibodies ofany class or subclass of immunoglobulin, and theexplanation must allow simultaneous production of

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any two classes of antibody of the same specificity.At the pre-B cell or B lymphocyte stage only verysmall amounts of antibody are produced (of theorder of 104-105 molecules per day).

(2) Once the B lymphocyte encounters antigenunder circumstances suitable for the initiation ofclonal expansion a dramatic increase in the rate ofexpression of antibody takes place. From the pro-duction of small amounts of surface receptor anti-body the cell switches to high rate secretion of anti-body. Production of light and heavy chains increasesby four to five orders of magnitude. This increase inimmunoglobulin synthesis and the switch fromsurface to secreted antibody accompanies cell pro-liferation. The extent of differentiation occurringduring proliferative clonal expansion is regulated sothat the clone gives rise not only to high rateantibody secreting cells but also to memory cellswhich are B lymphocytes closely resembling theprogenitor B lymphocyte of the clone. Duringantigen-driven clonal expansion leading to eitherantibody secreting cells or to memory cells it appearsthat an ordered sequential production of differentclasses of immunoglobulin, each exhibiting theoriginal antibody specificity of the clone, can occur(Gearhart et al., 1975). Although antibody-secretingcells devoted to the production of different classes ofimmunoglobulin arise during clonal expansion thespectrum of class and subclass switches permitted isnot yet understood.

Discussion of B lymphocyte development at amolecular level yields few certainties but we canperhaps limit the uncertainties. The switch fromproduction of surface receptor antibody to high-ratesecretion of antibody could be due either to changesat the DNA level, with rearrangement of gene seg-ments, or changes at the RNA level involving eitherregulation of transcription or processing of thetranscript, or changes in the posttranslationalmodification of the antibody polypeptide chains. Inmolecular terms we have to account for an alterationin the polypeptide structure of the heavy chain,proceeding from production of the heavy chainhaving a carboxy-terminal hydrophobic tail peptideto a heavy chain lacking the tail peptide. This changeis not accompanied by any change in class or, on thebasis of allotype expression evidence (Goding et al.,1977), by any more subtle change in the expressionof constant region gene segments. The simultaneousproduction of surface and secreted forms of the sameimmunoglobulin molecule by monoclonal lympho-blast cells in culture (Singer and Williamson, 1979)adds to the evidence against rearrangement of DNAsegments being required for the switch from surfaceto secreted immunoglobulin. Recently we haveobtained evidence that the surface and secreted

NUCLEUS CYTOPLASM PMASMAMEMBRANE

mRNAti'- 5t"--plus-L---- L - L L

DNA-4-nRNA plus carbohydrate -

plus L +JmRNAji L2i ( 2j2CISTERNAE

GOLGI SECRETINFig. 5 Postulated separate pathways in biosynthesis ofsurface membrane and secreted immunoglobulin (basedon data from Singer, 1979).

DNAP V I C I T I

I

nRNA - 1

mRNAsurface

orsecretory

polyA

_==>- poly A

Fig. 6 Hypothetical arrangement and expression ofagene segment (T) coding for tail peptide to provide ahydrophobic site for integration of immunoglobulin heavychain into the plasma membrane. Only interveningsequences (I) between V and C andflanking the Tsegment are shown but other I sequences may be presentbetween P and V and dividing C into smaller segments.

forms of ,u chain are produced by translation ofdifferent messenger RNA molecules. The evidenceis consistent with the switch from surface to secretedimmunoglobulin occurring at the level of RNAprocessing (Fig. 5). It is postulated that the carboxy-terminal hydrophobic tail peptide is encoded by aseparate DNA segment, T (Fig. 6). Each CH genesegment could have its own T segment or a single Tsegment could be shared by some or all of the CHgene segments. The major selective pressure wouldbe for T segments to code for very hydrophobicpeptides; almost certainly a more consistent hydro-phobic nature would be required for the T peptidethan for the P peptide, but as with the P peptidesequence diversity of T segment could occur. In thismodel the T segment functions as a part of the heavychain transcription unit. Production of the two typesof messenger RNA, one coding for receptor-typeheavy chain and one coding for secreted-type heavychain, can then be regulated by changes in processingof the RNA transcript. This model may also beapplied to the problem of the earlier switch inimmunoglobulin production at the pre-B cell to Blymphocyte differentiation step when stable surface

g

mRNA

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receptor antibody first appears. The immunoglobulinof the pre-B cell may also lack the heavy chain Tpeptide, thus resembling secreted immunoglobulin.The pre-B cell heavy chain, however, might arisefrom yet another type of messenger RNA, one inwhich the intervening sequence between the C and Tsegments has not been removed so that the T codingsequence forms part of the 3'-untranslated region ofthe messenger RNA.The switch from one immunoglobulin class to

another during clonal expansion can also beexplained in a variety of ways. Some explanationsinvolve special arrangements or progressive re-arrangements of the gene segment with appropriatechanges in transcription (Williamson and Fitz-maurice, 1977) and some involve changes in theprocessing of the RNA transcripts (Tonegawa et al.,1978). Studies on hybrid cells have so far failed toshow evidence for an enzymic mechanism whichmight be involved in differential RNA processingrequired for switching the class of immunoglobulinproduced by the cell (Shulman and Kbhler, 1978).Nevertheless, some variation on the RNA processingmodel still remains an attractive possibility forexplaining switches in immunoglobulin class eitherbefore or after exposure to antigen. Simultaneous orsequential expression of the same V regions coupledto different CH regions could be explained byduplication of the VH gene segment with multipleDNA joining events. However, the postulate of aVH-JH joining event complicates switch models atthe DNA level. Amino-acid sequence data point toa multiplicity of putative JH segments. Thereforethe rearrange:nent of a VH segment might imply thata choice be made among a number of different JHsegments for each CH gene. It is therefore necessaryto ask questions about the exact sequence of the JHregion when switches of immunoglobulin class occurwithin an antibody forming clone. Sequentialexpression of a given VH gene with different CHgenes could occur either with or without a change ofthe J region sequence according to the mechanism ofthe switch at a molecular level.One thing is certain, there are sufficient uncer-

tainties to make antibody formation a fascinatingarea of study for some time to come. I hope that thisaccount has illustrated the way in which the studyof a complex biological system such as that involvedin antibody formation has progressed from beingdescriptive at the cellular level to more precisedescription at the genetic and the molecular level,eventually seeking for molecular mechanisms. RoyCameron was far-sighted in his determination toprobe the biochemical mechanisms of the cell. Thetechniques now available to us, in particular those ofgenetic manipulation, appear to offer us more certain

answers than ever before. Nevertheless, it is when weare most certain that we understand something thatwe should look for inherent uncertainties.

References

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Amzel, L. M., Poljak, R. J., Saul, F., Varga, J. M., andRichards, F. F. (1974). The three dimensional structureof a combining region-ligand complex of immuno-globulin NEW at 3.5-A resolution. Proceedings of theNational Academy of Sciences, USA, 71, 1427-1430.

Bailey, D. W. (1978). In Origins ofInbred Mice (Proceed-ings of a Workshop), edited by H. C. Morse. AcademicPress, New York.

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