Vol. 8, No. 10MOLECULAR AND CELLULAR BIOLOGY, OCt. 1988, p. 4342-43520270-7306/88/104342-11$02.00/0Copyright © 1988, American Society for Microbiology

Microrecombinations Generate Sequence Diversity in the MurineMajor Histocompatibility Complex: Analysis of the K , Kb

Kbm and KbmJJ MutantsJAN GELIEBTERt AND STANLEY G. NATHENSON*

Departments of Cell Biology and Microbiology and Immunology, Albert Einstein College of Medicine,Bronx, New York 10461

Received 28 March 1988/Accepted 24 June 1988

The mechanism that generates spontaneous mutants of the Kb histocompatibility gene was analyzed.Nucleotide sequence analysis of four mutant genes (KbP3, Kb^4, Kbmlo, and Kb^'l) revealed that each mutant

K gene contains clustered, multiple nucleotide substitutions. Hybridization analyses of parental B6 genomicDNA and cloned class I genes with mutant-specific oligonucleotide probes, followed by sequence analyses, haveidentified major histocompatibility complex class I genes in the K, D, and Tla regions (K), Db, and TS,respectively) that contain the exact sequences as substituted into mutant Kb genes. These data provide evidencefor the hypothesis that the mutant Kb genes are generated by a microrecombination (gene conversion)mechanism that results in the transfer of small DNA segments from class I genes of all four regions of the majorhistocompatibility complex (K, D, Qa, and Tla) to Kb. Many of the nucleotides substituted into the mutant Kbgenes were identical to those found in other naturally occurring K alleles such as Kd. Thus, we propose that theaccumulation of microrecombination products within the K genes of a mouse population is responsible for thehigh sequence diversity among H-2 alleles.

Class I genes of the murine major histocompatibilitycomplex (MHC) are tandemly arrayed in four regions (K, D,Qa, and Tla) along chromosome 17. Members of this multi-gene family share sequence homology and similar intron-exon organization, and their products share similar biochem-ical characteristics (8, 12, 26, 35). The K and D regionscontain the genes encoding the classical H-2 transplantationantigens (K, D, L) which are found on all somatic cells andwhich function as restricting elements in the presentation offoreign, cell-associated antigens to T cells. Alleles of eachH-2 locus exhibit approximately 10 to 15% nucleotide se-quence diversity (diversity refers to differences in primarysequence) and are very polymorphic at the population level(12, 14, 15, 35). The diversity and polymorphism of H-2 geneproducts are thought to be important in their function asantigen-presenting molecules. Products of the Qa and Tlaregion genes have a more restricted tissue distribution andan, as yet, unknown function. Alleles of Qa and Tla locidisplay limited sequence diversity (8, 23, 30). The particularassortment of alleles of the various class I genes defines thehaplotype of the mouse strain (e.g., C57BL/6, H-2b; BALB/c, H-2d).H-2 molecules are integral membrane glycoproteins whose

three extracellular domains (axl, oa2, and a3) are noncova-lently associated with P2 microglobulin. This multimericcomplex interacts with the T-cell receptor. Sequence analy-sis of H-2 genes and their products indicates that sequencediversity among H-2 alleles is not randomly distributedthroughout the molecule but is rather concentrated in vari-able regions which are primarily in the otl and at2 domains(exons 2 and 3) (2, 12). Interestingly, there is greaterhomology between class I variable-region sequences ofnonallelic genes than between those of alleles of the same

* Corresponding author.t Present address: The Rockefeller University, New York, NY

10021.

locus. The sharing of variable regions among nonallelicgenes results in H-2 alleles that resemble combinatorialassortments of various class I gene segments. These findingssuggested that the sequence diversity of H-2 genes is theresult of recombination (e.g., crossing over or gene conver-sion) among the various class I genes (6, 11, 18, 28, 39).However, the level of sequence variability among H-2 allelesis so high that it is very difficult to determine evolutionaryrelationships between H-2 genes and to provide evidencethat H-2 diversity is generated by a recombination mecha-nism.

Spontaneous, in vivo mutants of the H-2Kb gene havebeen detected in the C57BL/6 mouse strain by skin graftanalysis at a frequency of 2 x 1i4 per gamete (13). (Mutantmice are denoted bml, bm2, etc.; mutant Kb genes aredenoted Kbml, Kbm2, etc.) In contrast to the analysis ofnaturally occurring, diverse H-2 alleles, the analysis ofmutant Kb genes provides a model system for the study ofthe individual steps that lead to the diversification of H-2genes. Partial amino acid sequence analysis of several Kbmutant molecules indicate that they differ from the parentalKb molecule by clustered, multiple amino acid substitutions(25). It has also been observed that the amino acids substi-tuted into the mutant Kb glycoproteins are also present, athomologous positions, in other class I molecules of variousH-2 haplotypes (6, 29). These findings suggest that a recom-bination mechanism between class I genes, such as geneconversion, may be responsible for the generation of the Kbmutants (6, 29). Nucleic acid analysis of the KbmI, Kb", andKbm9 mutants indicates that they each contain a singlecluster of altered nucleotides (Kbmb and Kbm9 arose indepen-dently but contain the same mutations) (9, 10, 34, 40). TwoQa region genes of the H-2b haplotype, QJO and Q4, containsequences identical to those substituted into Kbml and Kb-6/Kbm9, respectively (9, 10, 21). These data support a recom-bination hypothesis which suggests that these two donor

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ANALYSIS OF Kb MUTANTS 4343

genes recombined with the Kb gene to generate their respec-tive mutant Kb genes.

Since only two different Kb mutants have been analyzed,it was important to study more mutant genes to expand ourunderstanding of the mechanism of mutant generation. Thisanalysis would determine whether recombination is a majorfactor in the generation of Kb mutants and, by implication, inthe diversification of H-2 genes. Also, the identification of allpossible donor genes from all regions of the MHC wouldexpand our understanding of the concerted evolution of theclass I multigene family.

In this paper, we present the nucleotide sequences of theKbm3 bm4 Kbmno , and Kbm"I mutants. Each mutant genecontains multiple nucleotide substitutions localized to asingle cluster of 11, 35, 37, or 38 nucleotides. The shortsegments containing the mutant sequences are also presentin other class I donor genes from the K, D, and Tla regions.These data provide overwhelming evidence that mutant Kbgenes are generated by a microrecombination process thatresults in the transfer of short segments ofDNA from donorgenes to Kb. The data also indicate that genes from allregions of the MHC can engage in microrecombinationevents with the Kb gene. The finding that the positions of thealtered nucleotides in the mutant Kb genes coincide withvariable regions of H-2 alleles suggests that microrecombi-nations among class I genes can provide the raw materialsfor the diversification of H-2 genes. The accumulation ofmany microrecombination products would result in themosaic structure that is characteristic of these genes. Thus,these data provide evidence that the concerted evolution ofmultigene families can include recombination events whichdiversify, as well as homogenize, the member genes.

MATERIALS AND METHODS

Mice. C57BL/6 (B6), B6H-22bm3, B6.C-H-2bm4, B6.C-H-2bmlO, and B6.C-H-2bmll mice were obtained from the breed-ing and screening laboratories of R. Melvold (NorthwesternUniversity Medical School, Chicago, Ill.), C. David (MayoClinic, Rochester, Minn.), and The Jackson Laboratory.C57B/6Y (B6/Y) mice were obtained from the Academy ofMedical Sciences of the USSR, Moscow.

Preparation of DNA, RNA, and synthetic oligonucleotides.The preparation of genomic DNA, polyadenylated liverRNA, and synthetic oligonucleotides and procedures forhybridization analysis of DNA and RNA with 32P-labeledoligonucleotides have been described elsewhere (9, 10).RNA and uncloned-cDNA sequence analysis. The RNA and

uncloned-cDNA dideoxynucleotide-sequencing techniquesutilizing Kb-specific, 32P-labeled oligonucleotide primershave been described elsewhere (9). A total of 849 nucleotidesof each of the transcripts for the Kb-3, Kb-4, Kbi'lo, andK)m] genes were sequenced. The 849 nucleotides corre-spond to the codons for the amino-terminal 283 amino acidsof the mutant Kb molecules and include the three externaldomains, al, a2, and at3 (exons 2, 3, and 4), and nine aminoacids encoded for by the transmembrane exon.

Molecular genetic mapping of donor genes. Cosmid clonescontaining class I genes of the H-2b haplotype were obtainedfrom Richard A. Flavell (38). DNA from 15 clones weredigested with BamHI, size fractionated on agarose gels, andtransferred to GeneScreen. The filter was prehybridized for2 h (5x SSPE, 0.3% sodium dodecyl sulfate, 200 ,ug ofdenatured salmon sperm DNA per ml), hybridized for 2 to 16h in fresh prehybridization solution containing 100 ng of32P-labeled, mutant-specific oligonucleotide probe. (See Fig.

3 for hybridization temperatures.) Following hybridization,the filter was twice washed in 5 x SSPE (1 x SSPE is 180 mMNaCl, 10 mM NaH2PO4 H20, 1 mM disodium EDTA [pH7.4]) at room temperature for 30 min each and once at thehybridization temperature for 10 min. After being washed,filters were exposed to Kodak XAR film. Bound oligonucle-otide probes were removed from the filter by washing in 5 xSSPE at 80°C for 5 min.DNA sequencing of donor genes. Following the identifica-

tion of a potential donor gene in a cosmid clone, the exoncontaining the donor sequence was subcloned into M13mplOand M13mpll vectors and sequenced with the Pharmacia(Piscataway, N.J.) dideoxynucleotide-sequencing kit. Uni-versal M13-sequencing primers and mutant-specific primerswere used, and [a35S]dATP (Amersham Corp., ArlingtonHeights, Ill.) was incorporated as label.

RESULTS

Kbi4. Sequence analysis of the Kbm4 transcript indicatedthat it differs from the parental Kb transcript by six nucleo-tides in the codons for amino acids 162, 163, 165, 173, and174 (Fig. la). Kb and Kbm4 were otherwise identical over the849 nucleotides sequenced, which included the exons for thethree external domains (domains al, a2, and a3; exons 2, 3,and 4) and part of the transmembrane exon. All of thenucleotide substitutions occurred in base positions 1 and 2 oftheir respective codons and resulted in amino acid replace-ments.To confirm the sequence data generated by RNA and

uncloned-cDNA sequencing, mutant nucleotides were ana-lyzed to determine whether they generated or destroyedrestriction enzyme sites. The mutant sequence at positions173 and 174 (GAGCTC) generated a Sacl restriction enzymesite in Kb'4 that is absent in Kb (Fig. la). This sequence wasconfirmed by restriction enzyme digestion and hybridizationanalysis. Hybridization analysis of SacI-digested B6 andbm4 DNA with an H-2K-specific oligonucleotide to thetransmembrane region demonstrated that the Kbm4 genecontained the predicted additional SacI site (Fig. 2a). Thisfinding is confirmed by the fact that the oligonucleotideprobe hybridized to the smaller, 2.6-kilobase Kbm4 genefragment in bm4 DNA, compared with the larger, 6.3-kilobase Kb fragment in parental B6 DNA (Fig. 2a). Weknow that the new 2.6-kilobase Kb"4 SacI fragment has thepredicted size because both the Kb and Kbm4 genes con-tained a Sacl site in intron 6, which was approximately 2.6kilobases downstream from positions 173 and 174.The nucleotide alterations in positions 162 and 163 of Kbm?

generated an Avall restriction enzyme site (GGACC) notpresent in the Kb gene (Fig. la). The parental Kb genecontains AvaIl sites at codon 104 and 363 nucleotides intothe large intron between exons 3 and 4 (domains 2 and 3).This 600-nucleotide restriction fragment was detectable ingenomic DNA by an oligonucleotide probe to codons 151through 157 (Fig. 2b). The presence of a new Avall restric-tion site in Kb,4 was confirmed by the reduction of theparental 600-nucleotide Avall fragment to approximately 172nucleotides, the linear distance between codons 104 and 162(Fig. 2b). The G -- C alteration in codon 165 of the Kbm4gene generated a BstNI restriction enzyme site. However,this sequence is particularly difficult to demonstrate byrestriction enzyme digestion and hybridization analysis be-cause the alteration in codon 173 generated another BstNIsite and would require the resolution and detection of a21-nucleotide genomic DNA fragment.

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4344 GELIEBTER AND NATHENSON MOL. CELL. BIOL.

(a)155 160 165

Kb AGA CTC AGG GCC TAC CTG GAG GGC ACG TGC GTG GAG TGG CTC170 175

CGC AGA TAC CtG AAG AAC GGG AAC GCG

Kbm4 ___ --- --- --- --- --- --- -A- C-- --- C-- ------ ---- --- --- --- G-- cTr- --- ---

T5 -CG --- --- --- --- --- --- -A- C-- --- C-- --- --- --- --- --- --- --- G-- CT- --- --G -A-

(b)156 160 165 170 175 180CTC AGG GCC TAC CTG GAG GGC ACG TGC GTG GAG TGG CTC CGC AGA TAC CTG AAG AAC G,GG AAC GCG ACG CTG CTG CGC ACA GKb

Kbm10o --- --- --- --- --- --- --- G-- --- A-- --- -C- --- --- --- --- --- G-- CT- --- --- --- ___ -_- -__ __- ___

KI GAG --- --- --- --- --- --- G-- --- A-- --- -C- --- --- --- --- --- G-- CT- --- --- --- ----------

(c)85

TAC TAC MC CAG90

AGC AAG GGCKb

.m IKDMII --- --- --- --- AG- -- --- -- --- --- --- --- --- .-- --- --- --- ---

Db t-G --- --- --- AG- -----A-G-- - - - - - - C- ---

(d)75 80 85 90

b **** *** *** *** *** ***********K AGT TtC CGA GTG GAC CTG AGG ACC CTG CTC GGC TAC TAC AAC CAG AGC AAG GGC Ggtgag

KN ... ... ... ... AG- --- --- --- --- --- --- --- --- --- --- --- GC----

Q8 --- --- --- --- AG- --- --- --- GCA

Db T-G --- --- --- AG- -- --- -- --- --- --- --- --- --- --- --- GC----

FIG. 1. Nucleotide sequence comparisons of K', mutant Kb, and donor genes. The nucleotide sequence of Kb is given in comparison withthose of mutants Kbnm4 (a), Kbm]0 (b), Kbml]l (c), and Kbm3 (d) and the respective' donor genes. The numbers on top of the Kb sequence indicatethe amino acid position coded for by the codon. Dashes in mutant K" and donor gene sequences indicate identity to Kb. Asterisks indicatethe positions where mutant-specific oligonucleotides would hybridize to mutant K" and donor gene DNA and RNA. Lower-case lettersindicate intron sequences. The Kb and Kl (gene A) sequences are according to Weiss et al. (39); the Db sequence is taken from Hemmi et al.(S. Hemmi, J. Geliebter, R. W. Melvold, and S. G. Nathenson, J. Exp. Med., in press). The Q8 sequence is from Devlin et al. (5). The Kbsequence in (d) is from both the B6/J and B6NY strains.

If the six nucleotide substitutions in Kbm4 are the result ofa recombination event, an identical sequence should bepresent elsewhere in the B6 genome. An oligonucleotideprobe specific for the Kbm4 mutations should hybridize to theKbm4 gene and the donor sequence. To determine whetherthe six nucleotide substitutions in Kbm4 are the result ofrecombination between the K" gene and a donor sequence, a24-base oligonucleotide complementary to the mutant se-quence for codons 160 through 167 (encompassing the mu-tations at 162, 163, and 165) was synthesized (Fig. la). Thisoligonucleotide (the bm4-mer) was radiolabeled and hybrid-ized to BamHI-digested B6, bm4, and BALB/c mouse ge-nomic DNA (Fig. 3a). The hybridization of the bm4-mer tothe 4.8-kilobase BamHl fragment in bm4 DNA that containsexons 1, 2, and 3 of the Kbm4 gene confirmed the mutantsequence. The specificity of the bm4-mer was demonstratedby the fact that it did not hybridize to the 4.8-kilobaseBamHl fragment of Kb in B6 DNA. The bm4-mer alsohybridized to a 9-kilobase donor sequence in both B6 andbm4 DNA. A potential donor sequence was also present inBALB/c mouse genomic DNA (the ofiginal bm4 mouse wasa B6 x BALB/c Fj; see Discussion). To determine whetherthe donor sequence is present in a class I gene, radiolabeledbm4-mer was hybridized to a filter containing BamHl-di-gested DNA from 15 class I cosmid clones, corresponding toall of the class I genes of the H-2b haplotype. The bm4-mer

hybridized to a single class I gene, TS, located in the Tlaregion of the murine MHC (data not shown).To determine whether the TS gene contains the same

sequence as that substituted into K"'4, a restriction enzymemap of the TS gene was constructed, and an approximately525-nucleotide BamHI-Pstl fragment containing exon 3 (cor-responding to the a2 domain) was subcloned into M13mplland M13mplO and sequenced. The TS gene sequence wasidentical to that of Kbm4 at the altered positions 162, 163,165, 173, and 174 (Fig. la). This result provides very strongevidence that TS is the donor gene for Kbm4 mutations. The37 nucleotides between the base substitutions at positions162 and 174 represent the minimum size of the recombina-tion event between Kb and TS that is necessary to generatethe Kbm4 gene. The sequences of Kbm4 and TS are identicalfrom the codon for amino acid 156 until position 3 of codon176. Thus, 62 nucleotides represent the maximum recombi-nation between Kb and TS. Otherwise, Kbm4 would havecontained additional nucleotide substitutions at positions 155and 176. Comparative sequence analysis of exons 3 from Kband TS indicate that they are 86.6% homologous (Fig. 4).Kbm'. Dideoxynucleotide sequencing of Kb-10 mRNA

revealed that it contains six nucleotide alterations in thecodons for amino acids 163, 165, 167, 173, and 174 (Fig. lb).All six nucleotide substitutions were in codon positions 1and 2 and resulted in amino acid replacements. At positions

75 80AGT TTC CGA GTG GAC CTG AGG ACC CTG CTC GGC

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ANALYSIS OF Kb MUTANTS 4345

bp m -o1631 -506 o3967344f,

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FIG. 2. Confirmation of mutant sequence by restriction enzyme digestion and hybridization analysis. A 50-p.g sample of genomic DNAfrom parental B6 and various mutant mice was digested with various restriction enzymes, size fractionated by agarose gel electrophoresis,transferred to GeneScreen, and hybridized with 32P-labeled, oligonucleotide probes for 16 h as previously described (10). The enzymes usedfor digestion, percent agarose gel used for fractionation, and hybridization and washing temperatures were as follows: (a) Sacl, 0.8%, 59°C;(b) AvaIl, 2.25%, 55°C; (c) ThaI, 3%, 55°C; (d) NlaIV, 3%, 55°C; (e) AvaIl, 3%, 60°C; and (f) RsaI-AvaIl, 3%, 60°C (lanes 1 and 2) and ThaI,3%, 60°C (lanes 3 and 4). After hybridization, filters were washed and exposed to Kodak XAR film, as previously described (10). Numbersin kilobases (kb) are either HindIll-digested A DNA, or Hinfi-digested pBR322 DNA molecular size markers. Restriction enzyme sites in Kbare from Weiss et al. (39). bp, Base pair.

173 and 174, Kbm10 contained substitutions identical to thoseof Kbm4. The nucleotide changes at positions 163 and 165were different from the alterations in the same codons ofKbm4KA .

As described above, the mutant sequence at positions 173and 174 (GAGCTC) created a new SacI restriction enzymesite in KbmJo (and Kbm4) that is not present in the Kb gene(Fig. lb). Hybridization analysis of SacI-digested B6 andbmlO genomic DNA with an H-2K-specific probe confirmedthe presence of an additional SacI site in the Kbm'o gene(Fig. 2a). The smaller, 2.6-kilobase Kbmb0 fragment gener-ated by Sacl had the predicted size of a Kb gene with anadditional SacI recognition site at positions 173 and 174.The A -- C substitution in codon 163 of Kbmn0 generated

the new ThaI restriction enzyme site (CGCG) (Fig. lb). The

parental Kb gene contains ThaI sites at codons 111 and 176,which yielded a 197-nucleotide fragment detectable in ge-nomic B6 DNA with an oligonucleotide probe to codons 151through 157 (Fig. 2c). The presence of a new ThaI site incodon 163 of KbmIo was demonstrated by the hybridizationof the probe to a smaller, 155-nucleotide fragment in bmlODNA (Fig. 2c). A 155-nucleotide fragment corresponded tothe size of a restriction fragment from codons 111 through163. The G -* C alteration at position 167 of Kbmlo destroyed

an NlaIV restriction enzyme site (GGNNCC) present in Kb(Fig. lb). The Kb gene contains NlaIV sites at the codons foramino acid positions 112 and 167 and six nucleotides into theintron between exons 3 and 4 (domains 2 and 3). Hybridiza-tion analysis with an oligonucleotide complementary topositions 151 through 157 detected the 165-nucleotide NlaIV

a b C0

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4346 GELIEBTER AND NATHENSON

aC-.

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FIG. 3. Hybridization of genomic DNA with mutant-specific oligonucleotide probes. DNA from parental B6, BALB/c, and mutant micewere digested with BamHI, size fractionated by 1% agarose gel electrophoresis, transferred to GeneScreen, and hybridized with 32P-labeledmutant-specific oligonucleotides for 16 h as previously described (10). Oligonucleotide probes are described in the text and shown in Fig. 1.Hybridization and washing temperatures were as follows: (a) 65°C, (b) 64°C, (c) 61°C, and (d) 61°C (lane 1 [bm3-merl]) and 65°C (lane 2[bm3-mer-2]). Both lanes in panel d contain bm3 DNA. The arrow in panel d indicates the Kb??3 band. After hybridization, filters were washedand exposed to Kodak XAR film, as previously described (10). Numbers in kilobases (kb) are HindIlI-digested A DNA molecular sizemarkers.

fragment in B6 DNA, a fragment corresponding to codons112 through 167 of Kb (Fig. 2d). The same oligonucleotidehybridized to the 220-nucleotide NlaIV fragment of Kb,?I(position 112 to six nucleotides into intron 3), a resultdemonstrating the loss of the NlaIV site at position 167 ofKbm°o (Fig. 2d). The G -* A substitution at position 165 in

Kbmo' has already been confirmed by amino acid sequencingtechniques which have identified methionine at position 165of the mutant glycoprotein (25). Since ATG is the only codoncoding for methionine, the G -- A substitution can be

inferred by the genetic code.To ascertain whether recombination was responsible for

generating the six nucleotide substitutions in Kbm'o, a mu-tant-specific oligonucleotide was synthesized to identify a

donor sequence. The mutant-specific oligonucleotide (thebmlO-mer) was composed of 22 nucleotides and was com-

plementary to the KbmIo sequence from codons 162 to 169(Fig. lb). Radiolabeled bmlO-mer was hybridized to BamHI-digested B6, bmlO, and BALB/c mouse genomic DNA (Fig.3b). The mutant sequence of Kbmo' was confirmed by thehybridization of the bmlO-mer to the 4.8-kilobase BamHIfragment in bmlO DNA corresponding to the first threeexons (leader and domains al and (2) of Kb?'o. ThebmlO-mer did not hybridize to the corresponding Kb frag-ment in parental B6 genomic DNA. A 2.3-kilobase BamHIfragment in both B6 and bmlO DNA hybridized to thebmlO-mer, representing the donor sequence. A potentialdonor sequence was not present in BALB/c mouse DNA(the original bmlO mouse was a B6 x BALB/c F1; seeDiscussion). When hybridized to a filter containing class Icosmid clones, the bmlO-mer hybridized to a single gene, KJ(data not shown). The KJ gene is approximately 15 kilobases

91 100 110T5 GCTCTCACACATTTCAGAGGATGTACGGCTGTGACCTGGGGTCGGACGGGCGCCTCCTCCGCGGGTACTKb - TA-----GT---C-CT--------AG-------C--------A---------------C

120 130T5 GGCAGTTCGCTTACGACGGCAGCGATTACATCGCCCTGAACCAAGACCTGAAAACGTGGACGGCGGCGGKb A---A----C---------T--------------------G---------------------------

140 150 1T5 ACTTGGCGGCGCAGATCACTCGACGCAGGTGGGAGCAGGGTGGTGTTGCAGAGACGCTCAGGGCCTACCKb -_-A---------T------CAA--A--A -----------C------AA------- GA-------------

60 170 180T5 TGGAGGACCCGTGCCTGGAGTGGCTCCGCAGATACCTGGAGCTCGGGAAGGAGACGCTGCTGCGCACAGKb ------G-A-----G-----------------------A--AA------C-C-----------------

FIG. 4. Sequence comparison of exons 3 (ox2 domains) of T5 and Kb. Numbers indicate the amino acid positions coded for by thecorresponding codons. Dashes in Kb indicate identity to T5. The Kb sequence is from Weiss et al. (39).

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ANALYSIS OF Kb MUTANTS 4347

centromeric to the Kb gene on chromosome 17 (38). Exon 3(domain a2) of the K) gene is present on a 2.3-kilobaseBamHI fragment identifying K) as the gene identified ingenomic DNA (data not shown). A restriction enzymeprofile of the K) gene was constructed, and a 600-nucleotideXmaI-PstI fragment that contained exon 3 of K) was sub-cloned into M13mplO and M13mpll and sequenced. Thesequence of the KJ gene at codons 163, 165, 167, 173, and174 is identical to the sequence substituted into the mutantKbmlo gene (Fig. lb), an observation confirming that K) canbe the donor gene for the Kbmlo mutations. The 35 nucleo-tides between codons 163 and 174 represent the minimumrecombination between Kb and K) to generate the Kbmlogene. The Kbmlo and K) genes are identical in at least 79nucleotides, beginning with codon 157 and extending intointron 3. Because the KbmlO sequence was determined bymRNA sequencing, the intron sequence was not obtainable,and the maximum recombination between Kb and K) is notknown.Kbml. The nucleotide sequence of KbmIl differs from that

of Kb by three nucleotides at codons 77 and 80 (Fig. lc). Thedouble nucleotide substitution at position 77 and the singlealteration at position 80, were changes in base position 1 or2 and resulted in amino acid replacements.The nucleotide substitutions in codons 77 and 80 de-

stroyed two AvaIl restriction enzyme sites (GGACC) (Fig.lc). This fact was confirmed by restriction enzyme digestionand hybridization analysis. In addition to positions 77 and80, the parental Kb gene contains Avall sites at 30 nucleo-tides 5' and 60 nucleotides 3' to exon 2. An oligonucleotideprobe to positions 53 to 59 hybridized to a 256-nucleotideAvall restriction fragment of Kb (Fig. 2e). The loss of twoAvall sites in Kbmll was demonstrated by the hybridizationof the probe to the approximately 364-nucleotide AvaIIrestriction fragment of Kbml) (Fig. 2e). Thus, restrictionenzyme and hybridization analysis confirmed the loss of theparental sequence at positions 77 and 80 of Kbm"I.A Kbm"-specific oligonucleotide (bmll-mer) complemen-

tary to the mutant sequences at positions 77 and 80 wassynthesized (Fig. lc). The bmll-mer was radiolabeled andhybridized to digested genomic DNA to detect the presenceof a donor sequence (Fig. 3c). The specificity of the bmll-mer was demonstrated by the fact that it hybridized to the4.8-kilobase BamHI gene fragment of Kbml' DNA (thusconfirming the mutant sequence) but not to the correspond-ing Kb fragment in B6 DNA. A 2.1-kilobase potential donorsequence was present in both the parental B6 and mutantbmll genomic DNA, as well as in BALB/c mouse DNA (thestrain of the father of the original bmll mutant; see Discus-sion). Hybridization analysis of radiolabeled bmll-mer oncloned class I genes revealed that the Db gene is the onlyclass I gene that hybridizes to the bmll-mer (data notshown). The Db gene fragment that hybridized to the bmll-mer was 2.1 kilobases long, a result confirming the genomicDNA data. An inspection of the nucleotide sequence of Db(Hemmi et al., in press) indicates that the DI and KbmIIsequences are identical at positions 77 and 80 (Fig. lc).Thus, the Db gene is the donor gene for the Kbmln mutations.The 11 nucleotides that encompass the three nucleotidesubstitutions define the minimum recombination length be-tween Kb and Db to generate the Kbm)l mutant. The 45nucleotides that are identical in Db and Kbml) delineate themaximum recombination between Kb and Db to produce theKbml' mutations. A longer recombination event would haveresulted in additional nucleotide alterations at positions 73and 89.

Kbm3. Nucleotide sequence analysis of Kb` transcriptsindicated that Kb?? differs from the parental Kb sequence ofB6/J and B6/Y mice by four nucleotides, two each in thecodons for amino acids 77 and 89 (Fig. ld). The nucleotidesubstitutions in codon 77 were identical to those substitutedinto the Kbm-l gene. All four nucleotide alterations werechanges in codon position 1 or 2 and resulted in amino acidreplacements.

All the nucleotide replacements in Kb?? were confirmedby restriction enzyme and hybridization analysis. As withthe KbmII gene, the alteration at position 77 of Kbm3 dis-rupted an AvaIl site (GGACC) (Fig. ld). However, becauseboth Kb and Kb3 contain AvaII restriction sites at codon 80,digestion with AvaII will yield a mutant Kbn3 fragment thatis only eight nucleotides longer than the parental Kb frag-ment. To enhance the detection of this eight-nucleotidedifference, B6 and bm3 genomic DNA were restricted withboth RsaI and Avall. Since RsaI digests both the parentaland mutant Kb genes at codon 22, the double digestionshould yield a parental fragment of 164 nucleotides (codons22 through 77) and a mutant fragment of 172 nucleotides(codons 22 through 80). Hybridization analysis with a radio-labeled oligonucleotide probe to codons 53 to 59 confirmedthe loss of an Avall site in Kb?? by showing that hybridiza-tion was to a slightly larger Kbl fragment (Fig. 2f, lanes 1and 2).The AA -- GC substitution at position 89 in Kbl3 created

a new ThaI restriction site (Fig. ld). The Kb gene containsThaI restriction sites at codon 49 and 133 nucleotides intointron 2 (the intron between exons 2 and 3). Hybridizationanalysis with an oligonucleotide probe to positions 53 to 59indicated that the 258-nucleotide parental Kb fragment wasreduced to 118 nucleotides in Kb? (Fig. 2f, lanes 3 and 4).This reduction corresponded to a ThaI restriction fragmentextending from codons 49 to 89. Thus, both altered codons inKbm3 were confirmed by restriction enzyme digestion andhybridization analysis.Two Kb?-specific oligonucleotide probes were con-

structed, one probe spanning the Kb?? alteration at position77 (bm3-mer-1) and the other spanning the alteration atposition 89 (bm3-mer-2) (Fig. ld). Hybridization analysis ofbm3 genomic DNA with these oligonucleotides indicatedthat each probe hybridized to the Kb?: gene (see arrow inFig. 3d), as well as to several potential donor sequences (Fig.3d). However, the two Kb?3 probes did not bind to the samepotential donor fragment, a result indicating that whereas theB6 genome contains several sequences with the AGC codonat position 77 (e.g., Q8 and Db) and several sequences withthe GCG codon at position 89 (e.g., K) and Db), no singledonor gene was identical to Kb?? at positions 77 through 89(Fig. ld). The absence of a single donor gene identical toKb?? at positions 77 through 89 was also indicated whenthese two oligonucleotides (and other, smaller Kb7-specificprobes) were hybridized to H-2b class I cosmid clones (datanot shown). Interestingly, the Db gene was identical to Kb??3at position 77 and 89 but contained a mismatch at position 80(Fig. ld).

Transcription analysis of donor genes. The Q4 and Q1Odonor genes are transcribed in various tissues (4, 10). Thisfact is consistent with the possibility of microrecombinationvia an mRNA or cDNA intermediate (21, 39), as well as theobservation of a linkage between transcription and recombi-nation (37). To determine the transcriptional activity of thedonor genes identified in this study, 10 p.g of liver poly(A)+RNA from parental B6 and mutant bm4, bmlO, and bmllmice were size fractionated on agarose-formaldehyde gels,

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(2, 24), a finding that supports the hypothesis that microre-C. combinations are an ongoing process in nature.

That the microrecombination process plays a major role in- the diversification process of H-2 genes is also supported bySE the finding that the most variable codons (here termed

hypervariable codons) in exons 2 and 3 among the K allelesare also those that are altered in the spontaneous Kb mu-tants. Figure 6b illustrates that of the 12 codons that containthree or more different triplets in the four K alleles se-quenced, 7 positions were also altered in the Kb mutants(positions 24, 77, 116, 152, 156, 163, and 173). Further, asdiscussed above, in many instances, the triplet of the natu-rally occurring K allele was identical to that of the mutantKbl gene. These data suggest that the hypervariability ofcertain codons of various K alleles may be the result of themicrorecombination process that operates in the differentmouse strains and leads to sequence diversity among H-2alleles.

bm4-mer bmIO-mer bmII-merFIG. 5. Hybridization analysis of B6 and mutant mouse RNA.

Whole-cell liver RNA was prepared from parental B6 mice andvarious mutant mice. A 10->g sample of poly(A)+ RNA was

fractionated by formaldehyde-agarose gel electrophoresis, trans-ferred to GeneScreen, and hybridized with 32P-labeled mutant-specific oligonucleotides for 16 h as previously described (10).Oligonucleotide probes are described in the text and shown in Fig.la to c. Hybridization and washing temperatures were as follows: (a)65°C, (b) 64°C, and (c) 61°C. After hybridization, filters were washedand exposed to Kodak XAR film, as previously described (10). The28S and 18S rRNAs are 5,000- and 1,950-base molecular sizemarkers present in the poly(A)- fraction.

transferred to GeneScreen, and hybridized with mutant-specific oligonucleotide probes. (The Db donor gene isexpressed in all tissues, as are all classical transplantationmolecules, and the analysis of its transcriptional activity isincluded for completeness.) Hybridization analysis indicatedthat the KJ, T5, and Db donor genes were all transcribed inthe parental B6 liver (Fig. 5). (The faint darkenings below17S in B6 RNA (Fig. 5b) were not confined to the lane. Theapproximately 12S band in bmll RNA (Fig. Sc) was artifac-tual.)

Sequence comparison of spontaneous Kb mutants and natu-rally occurring K alleles. The identification of donor se-quences for all the mutations of the Kb mutants sequenced todate establishes the microrecombination process as themechanism generating spontaneous H-2 mutants. If micro-recombinations are a continuously occurring, natural phe-nomenon, natural K alleles would be expected to containsome of the same donor gene sequences as those found in themutant Kb genes. Figure 6a indicates that the Kd allele of theDBA/2J mouse contains many nucleotides that are identicalto those substituted into Kb mutant genes. This observationimplicates the microrecombination process in the sequencediversification of H-2 genes and reassortment of class Isequences in nature. It is possible that the progenitor of theKd allele may have undergone several microrecombinationswith donor genes to attain its present mosaic structure ofDb-, Q4-, Q10-, T5-, and KJ-like sequences. Conversely, themutant Kb microrecombinations can be considered as indi-vidual events that, if allowed to accumulate, would diversifythe Kb gene such that it would resemble Kd. It should also benoted that the Kk and K'28 alleles also contain codons thatare identical to those substituted into the various Kb mutants

DISCUSSION

H-2 genes, members of the class I MHC multigene family,exhibit extensive allelic sequence diversity and are verypolymorphic at the population level (12, 14, 15, 35). Incontrast, the Qa and Tla class I MHC genes display littlesequence diversity among the few alleles detected (8, 14).The mosaic structure of H-2 genes hint that a recombinationmechanism such as gene conversion between class I genescould promote the diversification of H-2 genes (6, 11, 18, 28,39). The analysis of spontaneous H-2 mutants that representsingle-generation events (13, 22) has provided a system ofstudying the role of recombination in the concerted evolu-tion of class I genes (9, 10, 21, 25, 34, 40).The Kbm3, Kbm4, Kbmlo, and KbmlI mutants contained

four, six, six, and three nucleotide substitutions, respec-tively (Fig. la to d) and were otherwise identical to Kb overthe 849 nucleotides sequenced. Within each mutant gene, thenucleotide substitutions were clustered into segments of 38or fewer nucleotides. The clustering of multiple alterationsand the finding that identical nucleotide substitutions occurin independently arising mutants (positions 77, 173, and 174)are consistent with the mutants being generated by a non-random, complex mechanism such as recombination. Criti-cal to a recombination hypothesis is a donor sequence in theparental genome that would contain the exact sequencesubstituted into the mutant Kb gene. All nucleotide substi-tutions in Kbm3 Kbm4, Kbmlo, and KbmlI had donor counter-parts in the B6 genome (Fig. la to d). The Kbm4 gene resultedfrom the interaction of the T5 gene with Kb. The KJ gene wasthe donor gene for the Kbm)o mutations. Recombinationbetween the Kb and Db genes generated the Kbmll mutant.Although a single donor gene for the Kbm3 mutant has notbeen definitively established, potential donor sequences foreach of its substitutions have been identified.

Nucleotide sequence comparisons of the Kb gene, mutantKb genes, and donor genes defined the minimum and maxi-mum extents of recombination between Kb and the donorgene to generate a particular mutant. For example, the Kb,4mutant resulted from the transfer of at least 37 but fewerthan 62 nucleotides from T5 to Kb (Fig. la). The analysis ofother mutant Kb genes in this and other studies indicates thatthe extent of recombination between Kb and donor genes togenerate each of the Kb mutants has also been less than 100nucleotides (9, 21). Such recombinations can be calledmicrorecombinations, a term reflecting the transfer of verysmall segments of DNA (tens of nucleotides). A similar

b.a.

Dq1E

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28S -18S -

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(a)77 116 121 155 156 173 174

Kb GAC TA TGC AMA CTC AM AAC

Kbl3e 11 AG-

Kbm6, 9 --- -T- C --- --- --- ---

Kbm --- --- --- TAT TA- --- ---

Kbn4,l0 --- --- --- ---. -- G-- CT-

K' AG- -T- C-- TAT TA- G-- CT-

(b)24 63 66 69 73 77 99 116 152 156

Kb GM GAG MA GGC AMT GAC TCT TAC GM CTC

Kd

Kk

KW28

163 173ACG AMG

-CT C-- -G- A-- T-- AG- -TC -T- --T TA- GA- G--

TCT A-C -TC --- -t- A-- -AC --- --T GA- --- C--

TCT A-C -TC -A- --- --- -AC GT- -CT 4T G-- ---

FIG. 6. Sequence comparisons of codons from K alleles and mutant Kb genes. (a) The nucleotide sequence of Kb is given in comparisonwith mutant Kb and K" sequences. The numbers above the Kb sequence indicate the amino acid position coded for by the codon. Dashes inmutant Kb and Kd sequences indicate identity to Kb. The Kb sequence is from Weiss et al. (39); the K" sequence is from Kvist et al. (17). Kbm6and Kbm9 sequences are from Geliebter et al. (9), and the KbmI sequedce is from Weiss et al. (40) and Schulze et al. (34). (b) The nucleotidesequences of hypervariable codons of Kb, Kd, Kk, and KJ28 are given. The numbers above the Kb sequence indicate the amino acid positioncoded by the hypervariable codon. Dashes in the Kd, Kk, and K%Y28 sequences indicate identity to Kb. Asterisks above numbers indicatehypervariable codons altered in the various Kb mutants. The Kk sequence is from Arnold et al. (2); the KY28 sequence is from Morita et al.(24).

microrecombination has been described in the generation ofthe class II I-Abml2 mutant, in which fewer than 44 nucleo-tides were recombined (20, 41). Microrecombinations maynot be a phenomenon restricted to MHC genes or even tomammals, since the somatic diversification of the chickenimmunoglobulin light chains may proceed by a similar mech-anism (31).The identification of KJ, Db, and TS as donor genes for the

Kb mutants firmly establishes and broadens our understand-ing of the microrecombination process in the concertedevolution of class I genes. Previously, only the Qa regiongenes Q4 and QJO were identified as donors, capable ofinteraction with the Kb gene (9, 10, 21). The lack of a knownfunction for the Qa genes fueled speculation that the approx-imately 25 non-H-2 class I genes (Qa and Tla) function toprovide a reservoir of alternative sequences to diversify H-2genes. The finding that the Db gene, whose product is afunctional antigen-presenting molecule, is also a donor gene,suggests that donation of genetic information to H-2 genes isnot the sole function of non-H-2 class I genes. That Q4 andQJO reside in the telomeric portion of the MHC also sug-gested that microrecombination processes were directional,proceeding from telomeric donor genes to the centromericKb gene. The fact that KJ is approximately 15 kilobasescentromeric to Kb (38) and is the donor gene for the KbmIomutant indicates that microrecombination can occur in atelomeric direction. Thus, there does not appear to be adirectionality factor in the recombination among class Igenes along chromosome 17.The finding that the T5 gene is a donor gene is significant

because comparative hybridization and sequence analyseshave indicated that those Tla region genes analyzed to dateare very divergent from H-2 and Qa genes (7, 27, 30, 32). Inparticular, exons 3 of Tla region genes are approximately25% divergent from exons 3 of H-2 and Qa genes (7, 27, 30,32). These findings suggested that recombination between

H-2 and Tla genes occur infrequently, if at all. The T5 geneis the donor gene for the Kbm4 mutant, a finding thatdemonstrates that microrecombination between Kb and a Tlagene can occur. Further, the extent of homology betweenexons 3 of T5 and Kb (86.6%) is comparable with the level ofhomology among H-2 and Qa region genes (Fig. 4) (2, 30).This comparability suggests that some Tla region genes maybe more closely related to H-2 genes than previouslythought.The Kbm3 allele was segregating in the C57BL/6Y (Yur-

lovo, USSR) mouse colony when it was isolated duringroutine skin graft testing for homozygosity (16). The B6/Ymouse subline had been separated from its parent line B6/Jfor less than 5 years when the bm3 mutant was detected. TheKbm3 gene contains double nucleotide substitutions in eachof codons 77 and 89. Although there are several potentialdonor genes for the alterations at position 77 (i.e., Db andQ8) or 89 (i.e., Db and KJ) there is no one potential donorgene that is identical to Kbm3 from positions 77 through 89(Fig. ld). Several explanations may account for this finding.One possibility is that the Kb? gene may have been gener-ated in a single generation by recombination with the Dbgene (Fig. ld). This recombination could involve heterodu-plex formation between Kb and Db with mismatch repair atposition 80 to the Kb sequence, followed by resolution of theheteroduplex in favor of the Db sequence at positions 77 and89. The patchwork resolution of artificially created hetero-duplexes has been described (1). A second possibility is thatthe Kbm3 gene was generated by the recombination of the Kbgene with two donor genes. Whatever the nature of therecombination events that generated the Kbm3 mutant, theidentification of potential donor genes for each of its doublenucleotide substitutions in the B6 genome is consistent withthe recombination hypothesis.The microrecombinations described for the Kb mutants

occur in the germ line (mitotically or meiotically) or in the

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zygote. This latter possibility is of particular importancebecause the bm4, bmlO, and bmll mutants were detected inthe F1 generation of B6 x BALB/c mouse matings (16), anddonor genes could conceivably come from the genome ofeither parent during zygotic recombination. Hybridizationanalysis with mutant-specific oligonucleotides for Kbm4 andKbm'l (but not Kbmlo) detected potential donor sequences inboth the B6 (TS and Db) and BALB/c mouse genomes.However, because donor sequences within the H-2b MHChave been identified for all of the Kb mutants analyzed andbecause several microrecombinations must have occurredduring the mitotic amplification of the germ line (bm6, bm9,and bm23; 9), it is probable that microrecombinations occurin the germ line and zygotic events are of little or noconsequence.The microrecombination process may be regulated by

several factors including sex, mouse strain, and the donorand recipient genes involved. For example, most microre-combinations can be traced to the maternal chromosome. Ofthe 11 independently arising spontaneous class I H-2 mu-tants detected in the F1 generation of B6 x BALB/c mousematings, all have occurred in the maternal H-2b chromo-some, 10 occurred in the Kb gene, and 1 occurred in the Dbgene (25). It has been suggested that the preponderance ofmutants in the maternal H-2b haplotype is caused by thepreferential occurrence of recombination (gene conversion)in the female (B6 in these matings) (19), perhaps because ofthe longer duration of meiosis in females compared withmales (42). This hypothesis is not totally correct because atleast some, if not all, of the Kb mutants were generatedduring the mitotic amplification of the germ line and notduring meiosis, and thus, the duration of meiosis would notbe a factor (9). Alternatively, because the mother of all of theabove-described F1 mutants was B6, the preponderance ofH-2b mutants may be due to strain differences, which areeither MHC linked or background. A possible example ofstrain-dependent recombination is that whereas H-2b mu-tants are of the microrecombination type, H1-2d mutantsinvolve single crossovers (33, 36). Additional parametersmust also control microrecombinations, because the differ-ential in the number of Kb versus Db mutants (10:1) cannotbe explained by strain or sex differences. Structural ormechanistic factors, such as the location or orientation of theKb and Db genes relative to each other and to other class Igenes on chromosome 17 and the presence or absence ofrecombinogenic sequences, may be important in the micro-recombination process.The product of Kbm microrecombination, a Kb gene con-

taining a small segment of nonallelic class I DNA, may bethe result of a double, unequal crossover or a gene conver-sion-like event. Because we cannot recover all of the prod-ucts of a mammalian germ cell recombination, we cannotdistinguish between these two processes. We also cannotdetermine whether microrecombination events involve non-allelic genes on the same chromatid, sister chromatids, orhomologous chromosomes. We can, however, conclude thatsingle crossovers are not involved in the generation of the Kbmutants. Such events would yield hybrid products, genesthat are one-half Kb and one-half donor gene, similar to thatdescribed for the DdmJ mutant (36). This possibility can beruled out because all Kb mutants analyzed were entirely Kbwith a very small substitution of donor gene sequences.Single crossovers between Kb and other class I genes mayoccur, but their products may be eliminated by selection.Depending on the relative 5' -* 3' orientation of the two

genes, single crossovers may result in large inversions oraccentric and dicentric chromosomes.The observation that intron sequences of allelic H-2 genes

are more conserved than exon sequences suggested thatdiversification by microrecombination events may be medi-ated by an RNA or cDNA intermediate (21, 39). Thishypothesis is consistent with the finding that all donor genesanalyzed to date are transcriptionally active in varioustissues, a fact indicating their potential to also be active ingerm cells where microrecombinations must occur. Thetranscriptional activity of the TS, KJ, and Db donor genes(Fig. 5) is also consistent with the proposed linkage betweentranscription and recombination (37).A comparison of H-2K locus alleles indicates that they are

composed of regions of conserved and variable primarysequences (2, 12). When the K alleles, Kb, Kd, Kk, and K1'28were compared, several hypervariable codons were appar-ent within the variable regions (Fig. 6b). At these positions,the hypervariable codons code for at least three differentamino acid residues among the four K alleles. Most strikingis the fact that of the 12 hypervariable codons 7 were incodons altered in the Kb mutant series. This finding supportsthe hypothesis that K gene sequence variability (diversity)and hypervariability are a direct result of the microrecombi-nation process; i.e., the regions of the K gene that arediverse are those that have been involved in microrecombi-nations. Therefore, on an evolutionary level, microrecombi-nation between class I genes would provide the raw materi-als for the diversification of the H-2 genes. The accumulationof various mutant sequences within a single Kb gene viaseveral, sequential microrecombinations with different do-nor genes will result in a chimeric K gene that is diverse fromKb. This result is indeed the observation with naturallyoccurring K alleles; they appear as mosaics containingblocks of sequences also present in other class I genes (39).Further support for the contention that H-2 allelic diversityis a result of the microrecombination process is the fact thatmany of the nucleotides substituted into the various Kbmutants are identical to those at the homologous positions ofthe naturally occurring Kd allele (Fig. 6a). Thus, it appearsthat the mutant Kb genes are at a transient first step tobecoming divergent, naturally occurring K alleles.The microrecombination-diversification scheme for the

dynamic evolution of H-2 genes has significant implicationson the rate at which H-2 alleles will diverge. H-2 genes, andparticularly the Kb gene, mutate at a relatively high fre-quency (13). The ultimate source of almost all de novogenomic sequence variation is point mutation. However,H-2 genes are part of a much larger multigene family ofapproximately 30 genes, which would collectively accumu-late point mutations at a rate approximately 30 times fasterthan a single gene does. When the point mutations of theentire multigene family are coupled with a microrecombina-tion mechanism that can reassort sequence segments be-tween H-2 genes and other class I genes, H-2 evolution anddiversification can proceed at a rate many times faster thanthat of a single gene. The finding that several mutant Kbmolecules cannot present certain viral antigens to the T-cellreceptor (3) suggests that the maintenance of microrecombi-nation products in the population probably depends onnatural selection. Thus, the demand for antigen-presentingmolecules of many different specificities may be the drivingforce that utilizes the microrecombination process for H-2sequence diversification.

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ACKNOWLEDGMENTS

We thank R. A. Flavell, E. H. Weiss. and A. L. Mellor for makingavailable their H-2b class I cosmid clones to help identify donorgenes. We thank I. K. Egorov and R. W. Melvold for valuablediscussions on the genealogy of the bm mutant mice and R. Zeff andEdith Palmieri-Lehner for the preparation and purification of syn-thetic oligonucleotides. We also thank Catherine Whelan for secre-tarial assistance. We thank A. M. Malashenko and Z. K. Blandovaof the Academy of Medical Sciences of the USSR for providingC57BL/6Y mice.The studies were supported in part by grants to S.G.N. from the

National Institutes of Health (Al-10702, AI-07289, and NCI P30CA-13330), the American Cancer Society (IM-236), and The Irving-ton House Institute for Medical Research. J.G. gratefully acknowl-edges the support of National Institutes of Health Training GrantCA-09173.

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