Recombination activating genes-1 and -2 of the rabbit: Cloning and characterization of germline and expressed genes

hfolecular Immunology, Vol. 30, NO. 11, pp. 1021-1032, 1993 Printed in Great Britain.

0161-5890/93$6.00 + 0.00 Pergamon Press Ltd

RECOMBINATION ACTIVATING GENES-l AND -2 OF THE RABBIT: CLONING AND CHARACTERIZATION OF GERMLINE AND EXPRESSED GENES PATRIZIA FUSCHIOTTI,*NAGARADONAHARINDRANATH,~~ROSEG. MAGE,*§ WAYNE T. MCCORMACK,(IPUSHPARANI DHANARAJAN~and KENNETHH. Roux~**

*Laboratory of Immunology, NIAID and TLaboratory of Oral Medicine, NIDR, NIH, Bethesda, MD 20892, U.S.A.; IIDepartment of Pathology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A.; IDepartment of Biological Science, Florida State University, Tallahassee, FL 32306, U.S.A. (First received 9 November 1992; accepted in revised form 19 January 1993)

Abstract-The recombination activating genes RAG-l and RAG-2 appear to be necessary components of the machinery needed for the Ig or TCR gene rearrangements that occur in developing B and T lymphocytes. In addition RAG-2 has been implicated in the process of V-gene diversification by somatic gene conversion in the chicken. Because gene conversion may be an important mechanism for V-gene diversification in the rabbit, we cloned the rabbit RAG locus and characterized the coding regions of the genomic RAG-l and RAG-2. In addition, we sequenced cDNAs encompassing the RAG-2 coding region, part of the RAG-2 5’ untranslated region and a 967 bp fragment of cDNA from the RAG-l coding region. Northern analysis revealed a RAG-l mRNA of 6.6 kb which is similar in size to the RAG-l mRNA reported previously for other species, and a major species of RAG-2 mRNA of 4.4 kb, which is larger than that from the mouse (2.2 kb). Analysis of the genomic clones showed that, as in other species, the RAG-l and RAG-2 genes are oriented so as to be convergently transcribed. The DNA sequence analysis showed that the rabbit RAG-l coding region is 91,85 and 72% identical to human, mouse and chicken, respectively. The deduced RAG-l protein sequence for rabbit is 93, 90 and 78% identical to human, mouse and chicken. Comparison of the rabbit RAG-2 coding region revealed 90, 87 and 71% identity to human, mouse and chicken, respectively, at the nucleotide level, and 91, 90 and 72% at the protein level. Although there is considerable conservation of sequence between species, we obtained evidence for allelic forms of the rabbit RAG locus both by Southern analyses and by sequencing. A remarkable degree of polymorphism was found in our rabbit colonies, particularly in the region 3’ of the rabbit RAG-2 coding region. A 5’ cDNA probe hybridized with one or more additional fragments that are not detected with the coding region probes, suggesting that the 5’ cDNA sequence results from splicing of one or more upstream exons.

INTRODUCTION Complete, active Ig and TCR genes are generated by somatic recombination that occurs during the differen-

tiation of lymphoid precursor cells. In developing lymphocytes, distinct gene segments, variable (V), joining (J), and sometimes diversity (D), are joined at the §Author to whom correspondence should be addressed. SCurrent address: Department of Pathology, Ohio State University, 139 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210-1238, U.S.A. **K.H.R. was supported by grants from NIH, NIAID, AI16596 and the American Cancer Society, Florida Division. Abbreviations: PCR, polymerase chain reaction; RAG, recombination activating gene; RSS, recombination signal sequences; SSPE, saline sodium phosphate EDTA (20 x SSPE is 3 M NaCl, 200 mM NaH,PO,. H,O, 20 mM EDTA). GenBank accession numbers: genomic RAG-l M77666; genomic RAG-2 M77667; SUT cDNA clone 5F M99310; RAG-2 cDNA clone 1 M99311; RAG-2 cDNA clone 2 M99312.

appropriate loci to form the coding sequences for Igs and TCR variable regions of diverse binding specificity. Joining is directed by recombination signal sequences (RSS) in the noncoding DNA adjacent to each of these coding segments. Each RSS consists of a highly conserved palindromic heptamer and an A/T-rich nonamer separated from each other by a spacer region whose length in most cases is either 12 or 23 base pairs (bp). These signals are conserved among the different Ig and TCR loci and among species that carry out V(D)J recombination and are functionally interchangeable, suggesting that the joining reaction is catalyzed by the same, evolutionarily conserved, V(D)J recombinase complex (Tonegawa, 1983; Alt et al., 1986; Hunkapiller and Hood, 1989; Blackwell and Alt, 1988; Yancopoulos et al., 1986; Hesse et al., 1989; Litman et al., 1985; Schatz et al., 1992). The complexity of the V(D)J recombination process suggests that multiple enzymatic activities are involved including: sequence-specific DNA recognition, endonucleolytic cleavage between the RSS and gene segment, base trimming (deletion) and addition (N and P seg-

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the RAG-l and the RAG-2 deficient mice failed to generate mature T or B lymphocytes owing to complete lack of ability to undergo V(D)J recombination. The finding that RAG-2 but not RAG-l is selectively expressed in the chicken B cells that undergo Ig gene conversion during development in the bursa of Fabricius (Carlson et al., 1991) suggested that RAG-2 contributes to the process of Ig gene conversion in the chicken (Carlson et al., 1991). However, gene knockout experiments in a bursal lymphoma cell line. that constitutively undergoes gene conversion in vitro suggest that RAG-2 expression is not essential for gene conversion (Takeda et al., 1992). Although RAG-2 expression may only contribute to one of several stages in the gene conversion process (Takeda et al., 1992), the finding is of particular interest to laboratories studying the immunoglobulins of the rabbit because it has been suggested that gene conversion may play a major role in generating antibody diversity in the rabbit (Becker and Knight, 1990; Allegrucci et al., 1991; Roux et al., 1991; Fitts and Metzger, 1990). In this paper we describe the cloning and sequencing of the coding regions of the rabbit genomic RAG-l and RAG-2 genes at the RAG locus, the cloning and characterization of rabbit RAG-l and RAG-2 cDNA probes, and cDNA encoding the entire RAG-2 coding region.

ments), and ligation of the cleaved ends. Some of these activities may be carried out by proteins found in many cell types (Lieber et al., 1987; Aguilera et al., 1987; Halligan and Desiderio, 1987; Hope et al., 1986; Schatz and Baltimore, 1988), but others are likely to be specific to recombinationally active lymphocytes. Recently two genes that appear to encode components involved in V(D)J recombinase activity, recombination activating genes 1 and 2 (RAG- 1 and RAG-2), have been cloned and characterized (Schatz et al., 1989; Oettinger et al., 1990; Ichihara et al., 1992). These two genes are evolutionarily conserved in vertebrates and closely linked on the chromosomes of mouse, human (Schatz et al., 1989; Oettinger et al., 1990, 1992) and chicken (Carlson et al., 1991). Coexpression of RAG-l and RAG-2 is both necessary and sufficient to induce V(D)J recombination of introduced recombination substrates in fibroblasts, and their expression in lymphoid cells correlates precisely with the presence of V(D)J recombinase activity, suggesting a direct participation of the RAG-l and RAG-2 gene products in the recombination reaction. Important in viuo data further support this hypothesis; using the targeted gene disruption technique, transgenic mice were produced that were deficient for production of RAG-l (Mombaerts et al., 1992) or RAG-2 (Shinkai et al., 1992). Both

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Fig. I. Northern blot analyses of poly(A) + rabbit thymic mRNA and total mouse thymus RNA with: (A) human RAG-l, (B) mouse RAG-2, and (C) rabbit RAG-2 probes.

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Fig. 2. Schematic diagram of the rabbit RAG locus. A partial restriction map of the 16.7 kb phage insert is shown. Open boxes depict the open reading frames of RAG-l and RAG-2 with the arrow heads indicating direction of transcription. The restriction sites shown are L, MI; S, SacI; B, BumHI; and H, HindlII. The SalI sites are derived from the phage polylinker. MATERIALS AND METHODS

mRNA was extracted from thymus using the FastTrack” kit (Invitrogen, San Diego, CA) according to procedures described by the manufacturer. The mRNA samples [3 pg poly (A+ ) and 1.5pg total RNA] were electrophoresed on 1% agarose-1 .l M formaldehyde gels and transferred to Gene Screen Plus (NEN Du Pont, Boston. MA) membranes. Filters were hybridized with appropriate probes that were labeled with [R-~~P] dCTP using the random hexamer labeling method (Feinberg and Volgelstein, 1983). Prehybridization and hybridization (20 hr) were performed at 42°C in a solution containing 50% formamide, 5 x Denhardt’s solution, 50mM sodium phosphate buffer (pH 6.8) 0.5% (w/v) SDS, and 100 pug/ml sheared and denatured salmon sperm DNA. The filters were washed at room temp for 30 min in 2 x SSC, 0.1% SDS, followed by washing at 60°C for 15 min in 0. I x SSC, 0.1% SDS. Autoradiography for periods ranging from 24 hr to 7 days was done using Kodak X-AR film with intensifying screens at -70°C.

human (Schatz et al., 1989) and chicken RAG-l (Carlson et al., 1991) (human positions 2093-3060). The rabbit RAG-2 probes were a PCR-amplified fragment of 667 bp (rabbit thymus mRNA donor EE21 l-3) which again utilized oligonucleotides from regions of interspecies sequence conservation, as well as a PCRamplified 1.6 kb fragment corresponding to the entire coding region of the gene (the details of which are described below; rabbit thymus mRNA donor lHH3101). A rabbit RAG-2 cDNA probe corresponding to sequences 5’ of the known coding region was obtained from a cDNA library constructed using rabbit thymus mRNA (5 pg) and the TimeSaver’m cDNA synthesis kit (Pharmacia, Piscataway, NJ), according to the manufacturer’s directions. A specific 3’ primer complementary to positions starting 64 bases 3’ of the RAG-2 start codon (PF 55_5’GGGATCGATATCAGTGAGAAGCCTGGTTGA) was used for the cDNA synthesis. We used adaptors containing EcoRI and Not I restriction sites to clone the cDNA synthesized with this kit in the EcoRI site of pUC18. The resultant colonies were screened by hybridization with an end-labeled oligonucleotide that was the 5’ oligonucleotide (PF 27 described below), used to synthesize the full-length RAG-2 coding region. A 291 bp long positive clone (SF) was isolated, sequenced and used as a probe. The sequence of clone 5F includes 65 bases at the 3’ end that overlap with the RAG-2 coding region, and 226 bp of unique sequence at the 5’ end. A 279 bp fragment was amplified using rabbit cDNA clone 2 (see below) in order to produce a probe for Southern analyses that originated 3’ of an internal BamHI site. The primers used produced a fragment corresponding to positions 1141-1420 in the rabbit RAG-2 cDNA sequence.

Probes

Genomic

cDNAs corresponding to the entire human RAG-l (Schatz et al., 1989) (6.6 kb) and the mouse RAG-2 (2.2 kb) (Oettinger et al., 1990) were kindly provided by Dr Marjorie Oettinger, Whitehead Institute, Cambridge, MA. The human RAG-l probe used was a fragment of 908 bp corresponding to the 3’ end of the coding region (positions 2273-3 181) produced by Xho I/Hind111 digestion. Mouse RAG-2 probes were either a 851 bp PstI fragment from the coding region of the gene (positions 162-1013) or the entire 2.1 kb insert obtained by digestion of the pBluescript KS + vector with Not I. The rabbit RAG-l probe was a reverse transcriptase-PCR (RTPCR)-amplified fragment of 1 kb (rabbit thymus mRNA donor EE21 l-3). PCR oligo primers were chosen from regions with conserved coding sequences in the cloned

A partial Mb01 library of rabbit genomic liver DNA was cloned into 1. FIX (Stratagene) according to the manufacturer’s directions. The genomic library was plated and plaques were lifted in duplicate onto nitrocellulose filters. RAG-l and RAG-2 genes were identified by hybridization to nick translated RAG-l or RAG-2 probe in 5 x SSPE at 65°C overnight, followed by two or three washes at room temp for 10 min each in 2 x SSC. Clones that were positive for one or both probes were plaque-purified. A single clone that was positive for both probes was isolated and used for further analysis. Aliquots of DNA from the selected phage clone were digested with SalI, SacI, BamHI or HindIII. Digested DNA was electrophoresed in a 1% agarose gel, stained

Rabbits The donor of liver DNA for cloning of the genomic RAG-l and RAG-2 was rabbit E220- 1 VHa2/a2, Clc b4/b4 from the allotype-defined colony at Florida State University. Donors of tissues for mRNA and the genomic DNAs used for Southern analyses were from allotype-defined pedigreed rabbit lines at the National Institutes of Health, Bethesda, MD. The thymus donors for messengers RNA were rabbits EE21 l-3 VHa2/a2 CK b9/b9 and lHH3 10-l VHa*/a*, CK b5/bb”” Or5. Northern

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PATRIZIA FUSCHIOTTI

with ethidium bromide, and photographed. The gels was subsequently dried and hybridized with RAG-l or RAG-2 probes according to the method of Kidd (1984). The RAG-l and RAG-2 genes were localized to 7.4 kb and 2.3 kb %rI/HindIII fragments, respectively, and subcloned into pBluescript

et al.

II SK+. The Sal1 sites were derived from the phage polylinker sites. Bacterial colonies containing recombinant clones were identified by hybridization to the RAG-l and RAG-2 probes according to a commercially available protocol (Stratagene, La Jolla, CA).

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70 40 80 90 100 50 60 20 30 I I I I I I I I I MetAlaValSerCysThrProTh~LeuG1yLeuSerSerAlaProAspGluIleGlnHisProHisIleLysPheSerGluT CCAGGTMTTCAGCCATCATGGCTGTGTCTTGCACACCAAT I I I I I I I I I I I

AMGCGAGAGACAAAGCMTCCACCAAGC~~~~CTGTGG~MTTCTTT~CTGATGMCACMCAGGA~TACCCTG

400 I I I I I alHisGlyProValAspSerL sThfGlnG1 LeuLeuArgLysL sGluL sLysAlaThrSerTrpPmAspLeuIleAlaLysValPheArgIleAs GAGA3; GAAAGCCACTTCCTGGCCAGACCTCATTGCCAAGGTTTTCCGGATTGA 500 TCCATGGGCCTGTGGACAGT &A CCCAAGG&TTTTACd I pVslLysTh~AspVslAsp~erIle"~sP~~ThrG1"Ph!CysH1sAsndysTrpA~gIleMet"lsA~~LysPhel AlsPr&Gl"VslTy~ ! GCTCCATGTGAGGTTTAC 600 TGTGMGAC:GATGTTGACTCGATCCACCACC~ACTGAGTT~GCCATMCTGCTGGCGTA~GMGTTTAGCGG

PheProArgAsnAlaThrHetGluTrpHisProHisAlsP~~Se~CysA~pIleCySTyrThrAlsArg~rgGlyLsuL:fArgArgAshHlsGl"Pro~ TTCCCMGGMTGCGACCATGGAGTGGCACCCCCATGCACCATCCTGTGACATCTGTTACACTGCCCGTCGGGGTCTC~GAG~GGMTCATCAGCC~ I I I I I I I I I snValGlnLeuSerLysLysLeuLysThrValLeuAspGl~laArgGlnAlaArgGlnArgLysArgArgVslGlnAlaArgIleThrSerLysG1uVa ATGTGCAGCTTAGCAAAAAA CTCAAAACTGTGCTTGACCAGCGAGACAAGCCCGTCAGCGCMGAGGAGGGTTCAGGCMGGATCACCAGCMG~GGT

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700 I BOO

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lnetLysMe:IleAlaAs"~ysSerLysIleHisLe"Se=Th=Lys~"~e"AlsVslA~pPheP=~A1~"lsPheVsl~ sSe=Ile&sGlnIl: ~ATCTATCTC~XTGCCAGATT CATGAAGATGATTGCCMCTGCAGTRAGATACATCTTAGTACCMGCTCCTTGCAGTGGACTTCCCGGCGCACTTTGTG I I I I I I I CysGluHisIleLeuAlaAspProValGluThrSerCysLysHisValPheCysArgIleCysIleLeuArgCySLeuLySVslHetGlySerTyrCySP TGTGAGCACATTCTGGCTGATCCTGTGGAr;ACCAGCTGT~GCATGTGTTTTGTAGGATCTGCATTCTGAGGTGCCT~~GCTATTGCC I I I I I I roSerCysGlnTyrProCysPheProThrAspLeuGluSerProValLysSerPheLeuCysIleLeuAsnSerLeuI1eVslLysC sSerAlaProG1 zTFCACCAGA CTTCTTGTCMTATCCATGCTTCCCTACT~CCTG~~~CCAGTG~TCCTTTCTGTGTATCTT~CTCCTTGAT~TG~T I I I I I I uCysAsnGluGluValSerLeuGluLysTyrAsnHisHisValSerSerHisLysGluSerArgAspThrPheVslHisIleAs~LySG1yGlyA~gP~o GTGCMTGAr;GAGGTCAGCTTGGAAAAAT:CAATCACCA~TTCMGT~C~GMT~MGAGATAC~TTTGTGCAT~TTMT~G~GGCcGGCCC

I

ArgGlnHisLeuLeuSerLeuThrArgArgAlaGlnLysHisArgLeuArgGluLeuLysLeuGlnVslLysAlaPheAlsAspLysGluGluGlYGlYA CGTCMCATCTCCTATCGCTGACCCGGAG:GCTCAGAAGGTGGAG I I

900 1000 1100 1200 1300

GlyArgGlnIlePheGlnProLeuHisAlaLeuArgAsnAlaGluLysValLeuLeuProGlyTyrHisProPheGlUTrpGlnP~oPKOLeULysAs~V GGGAGGCAGATTTTTCAGCCCTTGCATGCCCTTCGGAATGGTCCTTCTGCCAGGCTACCACCCCTTTGAGTGGCAGCCGCCTCTGMG~TG 1600 I I I I I I I I I alSerSerSerThrAspValGlyIleIleAspGlyLeuSerGlyLeuSerSerSerValAspAspTyrProValAspThrIleAlsLysArgPheAKgTy TGTCTTCCAGCACTGATGTTGGCATTATCGATGGACTGTCAGGACTCTCCTCCTCTGTGGACGACTACCCGGTGGACACTATTGC~GAGGTTCCGCTA 1700 I I I I I I I I I I rAspSerAlaLeuValSerAlaLeuMetAspMetGluGluAspIleLeuGluGlyMetArgSerG1nAspLeuGluAspTyrLeuAsnGlyProPheThr TGATTCAGCTTTGGTGTCA~TTTGATGG:CATGGAAGAAGACATCTTGGMGGCATGAGGTCCC~GACCTTGAGGACTACTTGMTGGCCCATTCACC 1800 I I I I I I I ValValValLysGluSerCysAspGlyMetGlyAspValSerGluLysHisGlySerGlyProAlaValProGluLysAlaValArgPheSeKPheTh~V GTGGTGGTGRRAGAGTCTTGTGATGGAATGGGGAGACGTGAGTGAGMGCATGGGAGTGGTCCAGCAGTTCCAGAGMGGCAGTTCGTTTTTCATTCACAG 1900 I I I I I I I I I I alMetLysIleThrIleAlaHisGlySerGlnAsnValLysValPheGluGluAlaLysProAsnSerGluLeuCysCysLysP~OLeUCysLeUMetLe TCATGAAAATCACCATAGCl;ICACGGCTCA~MTGTG~GTGTTTGAGGMGCC~CCT~CTCTGMCTGTGCTGC~GCCGTTGTGCCTTATGTT 2000 I I I I I I I uAlaAspGluSerAspHisGluThrLeuThrAlaIleLeuSerProLeuIleAlaGluArgGluAlaMetLysSerSerGluLeuMetLeuGluMetGly GGCAGATGAGTCTGACCATGAGACCCTGACTGCCATCCTGAGCCCTCTGATTGCTGAGCGGGAGGCCATG~GAGCAGTG~TTGATGCTTGAGATGGGA 2100 I I I I I I I I I I GlyIleLeuArgThrPheLysPheIlePheArgGlyThrGlyTyrAspGluLysLeuValArgGluValGluGlyLeuGluAlaSerGlySe~VslTy~I GGCATTCTCAGGACTTTCAAGTTCATCTTTAGGGGCACTGGATATGATGAACTTGTCCGGGAAGTGGRAGGCCTTGAGGCTTCTGGCTCAGTCTATA 2200 I I I I I I I I I I leCysThrLeuCysAspAlaThrArgLeuGluAlaSerGlnAsnLeuValPheHisSerIleThrArgSerHisAlaG1uRsnLeuGluArgTyrGluVa TTTGTACTCTTTGTGATGCfACCCGCTTG~~GCCTCTCTTGTCTTCCACTCCATAACCAGAAGCCATGCTGAGMTCTGG~~GCTATGAGGT 2300 I I I I I I I lTrpArgSerAsnProTyrHisGluSerValGluGluLeuArgAspArgValLysGlyValSerAlaLysP~OPheIleGlUThrVslP~OSe~IleAsp CTGGCGTTCCAACCCCTAC~ACGAGTCTGTGGAAGAACTGAGGGATCGGGTG~GGGGTCTCAGCC~GCCTTTCATTGAGACAGTCC~TTCCATCGAT 2400 I I I I I I I I AlaLeuHisCysAspIleGlyAsnAlaAlaGluPheTyrLysIlePheGlnLeuGluIleGlyGluValTyrLysAsnProSerAlaSerLysGlUGluA GCACTCCACTGTGACATTGGCAATGCAGCTGAGTTCTACRAGATTTTCCAGCTAGAGATAGGGGAGGTGTATAAGAATCCCCAGTGCTTCCAAGGAGGAAA 2500 I I I I I I I I I I rgLysArgTrpGlnAlaThrLeuAspLysHisLeuArgLysLysMetAsnLeuLysP~OIleMetArgMetAsnGlyAsnPheAlaArgLysLeuMetTh GGRRAAGATGGCAGGCCACACTGGACAAGCATCTTAGGAAGAAGATGAACCTGAAACCAATCATGAGGATGAATGGCARCTTTGCCAGGAAGCTCATGAC 2600 I I I I I I I I I I rIleGluThrValGluAlaValCysGluLeuIleProSerLysGluArgHisGluAlLLeuArgGluLeuMetGluLeuTyrLeuLysMetLysP~OVal CATCGAGACFGTGGAGGCAFTTTGTGAGTTAATTCCTTCCAAGGAGAGGCACGAAGCTCTCCGGGAGCTGATGGAACTATACCTGMGATGRRACCCGTA 2700 I I I I I I I TrpArgSerSerCysProAlaLysGluCysProGluSerLeuCysGlnTyrSerPheAsnSerGlnArgPheAlaGluLeuLeuSerThrLysPheLysT TGGCGCTCATCATGTCCAGCTAAAGAGTGCCCAGTGCCCAGMTCCCTCTGCCAGTACAGTTTC~TTCACAGCGTTTCGCTGAGCTTCTCTCTACC~GTTCAAGT2800 I I I I I yrArgTyrGl"GlyLysIleThrAsnTyrPheHlsLysT~~Le"Als~l~VslP~~Gl"~leIleGl"A~gAspGlySe~IleGlyAlsT~pAlsSe=Gl ATAGATATGAGGGCAAAATCACCAATTATTTTCACAAARCCCTGGCGCATGTGCCTGAAATTATTGAGCGGGATGGCTCTATTGGGGCCTGGGCAAGTGA 2900 I I I I I I I I I I uGlyAsnGluSerGlyAsnLysLeuPheArgArgPheArgLysMetAsnAlsArgGlnSerLysCysTyrGluMetGlUAspValLeULysHiSHisT~p GGGAAATGAGTCAGGCAACARACTGTTCCGGCGCTTCCGGARAATGMTGCCAGGCAGTCCAAGTGCTATGAAATGGAAGACGTACTGAAACACCATTGG 3000 I I I I I I I I I I LeuTyrThrSerLysTyrLeuGlnLysPheMetAsnAlaHisAsnAlsVa1LysAsnSerGlyPheThrMetAsnSerGlnVa1SerLeuGlUAspP~oL CTGTACACCTCCAAATACCTCCAGAAGTTTATGAATGCTCACAATGCAGTTAAAAACTCTGGGTTTACCATGAACTCACAAGTGAGCTTAGAAGACCCAT 3100 I I I I I I I I I I euGlyIleGl"AspSerLeuGluSerGlnTyrSerMetGluPhe*** TGGGCATAGrPGGACTCTCTGGAGAGCCAATATTCAATGG~TTTAAGGAGGGTTCAACCACTTATGAGTTGGTTTTTA:AATTTTCTTTCGGGTTACAT 3200 I I I I I I TGAGACCTTCTCCTAGCACCCTTCACTACTGTGTGAGGGGCTTTGCACCCRAGAGGTGGTAGGTTGGAGAGAGAGGTCAAGATGCTGAAACCATGCCAGG 3300 I I I I I I I I I 3398 AATGGTTAATGATGAGCTGGTTGCTTGGGCTGTTTAGTGAGTTCAGAAAAGCAAAAGGAGATATCAGTTATTTGRARGCTCACTACTCAGAGCAGGAG

Fig. 3(A).

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200 300 isGlyGlyL:sThrPr~A~~s~G~"L~"~~rAspLysIi~Tyr"~lH~:S~r"~1"~l~ysLysAsnA~~LysLys"~:ThrPh~Arg~ysThrG~uA~ AAAAMGTTACTTTTCGCTGCACAGAMG ACGGAGG~CACCAAAC~TGAGCTTTCTGATMGATTTATGTCATGTCTGTTGTTTGC~CMC I gAspLau"a:GlyAsp"albroG~"A~~A~gTyrG~y~~~S~r~~sp~~~"~~TyrS~rArgG~yLy~S~r"~tG~y~~~L~"Ph~G~yG~yArgS~r GAAAAAGTATGGGA~TTCTCTTTG~GGTC~ AGACTTGGTAGGAGATGTTCCAGMGCCATCGAG I I I I I TyrMetProSerAsnGlnhrgThrThrGluLysTrpAsnSerValAlaAspCysLe"ProHlsValPhe~"ValAspPheGl"PheGlyCySAlaThrS TACATGCCTTCTMTCAAAGCCACAG~TGGMT~GCGTCGCTGATTGCCTGCCCCATGTTTTCTTGGTG~TT~~TTTGG~~~~

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erTyrIleLe"ProGl"Le"GlnAspGlyLeuSerPheHisValSe~~ArgAs"AspThrV=1T~rIleLe"GlyGlyHisS~r~~~~A*~* CCTACATTCTTCCAGAGCTTCAGW\TGGG~GTCATTTCATGTCTCTATTGCCAG~T~TACCGTTTATATTTTA~~~CATTCT~MT~ I I I I I nIlaArgProAlaAsnLe"TyrArgIleArgValAspLeu"GlySerProAlaIleAsnCysThrValLe"ProGlyGlyIleSarValSerS~r TATCCGCCCTGCCMTCTGTACAGMTCAGGGTTGATCTTCCCTTGGGTAGCCCAGCCAT~TTGCACAGTTTTGCCAGGAG~TTTCTGTCTCCAGT I I I I I I I AlaIleLe"ThrGlnThrAsnAsnAspGluPheValIle?alGlyGlyTyrGl"Le"GluAsnGlnLys~rg~~tValCysAs~IleValSerLe"Gl"A GAATGGTCT~AACATTGT~CTTTAGI; GCMTCCTGACTCAGACTMCMTGATTTGTTATCG I I I I I I I spAonLysIleGl"IleGlnGl"MetGl"ThrProAspThrProAspIleLysHisSerLysIleTrpPheGlySeCAsnnetGlyAsnGlySerVa ATMCMGATTGAAATTCAP;rGAGATGGAG:CCCCCAGATTGGACTCCAGATATTMGCACAGC~TATGGTTTG~GCMCAT~~ATCCG~ 1PheLe"G1:IleProGlyAspAsnLysG~~I~=V~~S~~G~"A~~Ph~~yrPh~Tyr"~t~"LysCy~ThrG~~sp~spV=~H~sG~"As~~~Arg TTTCCTTGGCATCCCAGGAGACMT~CAGATTGTTTCAGMGCATTTTATTTCTATATGTTG~TGTACTGMGATGATGTGCATGM~TCAGAGA I I I I I ThrPheThrAsnSerGlnThrSerThrGluAspP=~G~yAspS~=ThrP~"Ph~G~"As~S~=G~"G~"~~~CysPh~S~~A~=G~"A~~As~SerPh~A ACTTTCACAMTAGTCAGACATCMCAGAAGATCCAGGGGACTCCACTCCCTTTGAG~CTCGG~G~TTTTGTTTCAGTGCAGMGC~TAGTTTTG I I I I I I I I spGlyAspAspGl"PheAspThrTyrAsnGluAspAs~~"AspAspG~"S~~G~"Th~G~yTy~TrpI~~Th~CysCy~P~oThrCysAspV~~Asp~~ ATGGCGATGATCAATTTGACACTTATAATGAAGATGATGAGATGATGAGTCTGAGACAGGCTACTGW\TTACCTGCTGCCCTACTTGTGATGTG~~T I I I I I I I I I eArnThrTrpValPrOPheTyrSerThrGl"LeuAsnLysProAlaMetIleTyrCysSerHisGlyAspGlyHisTrpValHlsAlaLysCysMetAsp TMCACTTGGGTGCCATTCTATTCMCGGAGCTCAACAAGT I L~"Al=Gl"ArgThrLe"I~eHiSLe"Se~Gl"GlySer~s"LysTy=T~rCy~A~~G~~H~sV~~G~"~~~A~~ArgA~~~"G~~Th~ProLysArgT CTGGCAGMCGTACCCTCATCCATCTGTCAGAAGGAAGCA I I I I I I I I I hrIlePrOLe"ArgLysProProMetLYsSeKLe"HisLysLysGlySerGlyLysIleLe"ThrProAlaLysLysSerPheLeuArgArgLe"PheAs CCATACCCTTMG~GCCCCCMTCCCTCCACAAGCGGTTGTTTGA I I I I I I I I I p.*t CTMTTTTGCAAAlUCTTTTCAGATCCATGCACATTMGCTCTT~CCTATCTTTCMRACCTTGACAATGATCAACATTATATTTGTATTTTTGTTTA TTGGAMTGiCC

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cDNA

cloning

cDNA corresponding to the coding region of RAG-2 was amplified by RT-PCR using an RNA amplification kit (Perkin Elmer Cetus). PF 27 and PF 28 used in these experiments were from sequences immediately 5’ and 3’ of the coding region of the rabbit genomic RAG-2 sequence. In addition, a Clul restriction site (underlined bases) was incorporated into each primer 5’ of the homologous regions. The sequences were: PF 27: 5’GGGATCGATCCGAAAACATGTCACTGCAG-3’; PF 28: 5’-GGGATCGATGCTTAATGTGCATGGATCTGA-3’. mRNA (0.3 pg) was reverse transcribed using approximately 200 ng of the specific 3’ oligonucleotide primer, PF 28. This cDNA was used in PCR reactions in a final volume of 100 ~1 containing 300 ng each of 3’ (PF 28) and 5’ (PF 27) oligonucleotide primers. Amplification reactions were performed initially for 5 cycles of 1 min at 94°C 2 min at 52°C and 3 min at 72°C and then for 33 cycles of 94°C for 1 min, 64°C for 2 min and 3 min at 72°C. After the final cycle the samples were incubated at 72°C for an additional 7 min. The PCR product was filled in with Klenow fragment and phosphorylated using T4 polynucleotide kinase prior to cloning into SmaI digested dephosphorylated pUC 13 vector (Pharmacia, Piscataway, NJ). DNA

sequencing

The coding regions of genomic DNA subclones were sequenced by the dideoxy chain termination

method either manually (Sanger et al., 1977) or on an automated sequencer (Applied Biosystems Model 373A). The manual method consisted of a modification of the dideoxy chain termination method (Sanger et al., 1977) described by Brow (1990). Briefly, dsDNA was annealed to end-labeled primer and repeatedly (eight cycles) extended in the presence of Taq DNA polymerase, dNTPs and ddNTP using a DNA Thermal Cycler (Perkin Elmer Cetus). Double stranded cDNA from plasmid clones were sequenced by the dideoxynucleotide chain termination method using [cc-35S]dATP and Taq polymerase (TaqTrak’“, Promega, Madison WI) according to the manufacturer’s protocols. The complete sequences of two different cDNA clones as well as the 5’ region in cDNA clone 5F were confirmed on both strands using commercial oligonucleotide primers and rabbit RAG-specific oligonucleotides (18 bases).

Southern

blotting

Genomic DNA (15 ,ug) was digested with various restriction enzymes and analyzed by Southern blotting (Maniatis et al., 1982). DNA probes were labeled with [c~-~~P] dCTP by the random hexamer method (Feinberg and Volgelstein, 1983). Blots were pre-hybridized and hybridized at 60°C in 6 x SSC, 0.5% SDS, 5 x Denhardt’s solution, and lOOpgg/ml of denatured salmon sperm DNA. Blots were washed

PATRIZIA FUSCHIOTTI et al.

1026

first at room temp in 2 x SSC and 0.1 x SDS for 30 min, and then at 60°C in 0.1 x SSC and 0.1% SDS for 20-30 min. Autoradiography was done using Kodak X-AR film with an intensifying screen at - 70°C.

ii%%

Mouse Chickn E::? Mouse Chicken

RESULTS N,rthern

blot analysis

In our initial experiments we tested whether mouse RAG-2 cDNA and human RAG-l cDNA probes

t+~~S~;PTL&LSSAPDEIQ~PHIKFSEWK~KLFRVRSF&~TTEENQK//E .................................. ..A.LPS..SF..........Q................v.

48

..P..A..//.

AP..A..//. .//.I.SRMD.///.E.....Y A ........ ..K..P.Q.EPSDKSQCI N KdSS/EGKPSLEbSPAVL///DKAS~QKPEVAPPA~KVHPKFSKK~HDDG .KD.F.............///...D....VPTQ.LL.A..........N .D../ .. ..Y ... ..V.P///E.PG..NSILTQR.L.L.........A .DQEQ.V/A.TDKNITLHKDEEVPR.E.LILTQKD.MGNTQALE.DV.M

94 E .. 144

Chicken

KARDKAIHQANLRHLARICGNSLKT d EHNRRYPVHAPVDSKTQGLLRKKE ....... . ..G..................FRA...............G..L .SS...V...R...F......RF.S.G.S..........A...S.F .... .IQ.NTA..N..KQ......V.F...CYK.TH.......DE.LW ...... KKATS?PDLIAKVFR!DVKTDVDSI~PTEFCHNCWf(IM"RKFSGA 6 CEVY .R.................A...............S.......S ...... .RV........RI......A............D..S.......SSHSQ .. ..... ............ ..K...RG...T....R......S.I.....NT

194

ii%tna blouse Chicken

FPRNAJMEWHPHAPS c DICYTARRGLKRRNHQPNVbLSKKLKTVLdQARQ ............... .. ..V.......T......N........K.....L ../ . ..KV.V.....T......F..H.....KR...............NH ..V .. ..S....Q..SAN.EV.H.PS..V..KSQP....HG.RV.IIAER

244

ii%!;' Mouse Chicken

ARQRKARVQARITSK A VMKMIANCSl&HLSTKLLA?DFPAHFVKS 4SCQI .......... ..... ..A....S..D...K...................E /.D.RK.T...VS....L.K.s ............................ N.GI.l;lQ.///.KN.~...E.T..K~R........i.Y..D.I..i ....

294

CEHILADPVETSCKHVFCRICILRCLKVMGSYCPSC#YPCFPTDLESPVK ......... ..N.......V ............................. ............. ..L .................... ..... ;::::::::ir+::: ......... ..T.R.L...T...S.IR...C

344

SFLCI~NSLIVKCSA~ECNEEVSL~~YNHHVSSHK~sRDT,,FVHIN~GG ........ . ..SV....M...P.K..............I......KEI// ....... . ..N ... ..M . ..P.QD....................KE.//L ... N..D..SIR.PV...D..ILHG..GQ.F.N...MK.KELYNP .....

392

RPRQHLL~LTRRAQKHR~RELKLQVKA~ADKEEGGDV~SVCLTLFLL~LR ..M ........ ....................................... .................... ..I...E...........A ........... ..R......E......I.A ..M ........ ....................

442

!%% Mouse Chicken

ARNEHRQ~ELEAIMQGI!GSGLQPAVC!,AIRRVNTFLS~SQYHKMYRT?KA ............... ..K ................................ .................................................. .K...K................H........I ..................

492

~%E' Mouse Chicken

542

E%' Mouse Chicken

ITGRQIFbPLHALRNAE~VLLPGYHPF~WQPPLKNVSBSSG ..H ........................ ....................... ..R ........... .................................... V ........... ..T...A..........K.......TN .E ......... I LsssVDD~PVDTIAKRFdYDSALVSALMDMEEDILEG~RsQDLEDYL~GP ......................................... ..D ...... D .A....E ..i::::6:::::l%I%:D .PL.I..i.I.......i..T

Rabbit Human MOUSIZ Chicken

642 FTVVVKESCDGMGDVsEKHGsGP;~PE~A"RFsFTVM~ITIAHGsQN~KV ..........I.......S .................. . .............................................. ................ ..L .................. R...E ........ N.A.D.ENERIRI ..... ..c .............................

Rabbit Human Mouse Chicken

692 FEEAKPN&LCCKPLCLt!fLADESDHETtTAILsPLIAkREAMKSSELhLE .................................................. T .... ......... :::~:::::::::.........:::::::::::::::::::::i:::L

Rabbit Human MOUSe Chicken

~""~~RT~KFIFRGTGYbEKLVREVEG~EAsGsVYIC~LCDATRL~A~QN ............................................. T ........ .. ..P. ................................... T ................ I ................................

742

LVFHsIT~SHAENLERY&RSNPYHdVEELRDRVdVSAKPFIET~S .................................................. .............. Q...i:::::: ..... .................... .................... ..D ....................

792

Rabbit Human MOUSe Chicken

IDALHCD~GNAAEFYKI~QLEIGEVYK~PSASKEERdwQATLDKHL~K ........................... ..N .................... ................. ..;::::: ..H.N ................. ..R ..i.D.T.....i..L......i .. ..T...R .............

842

Rabbit Human MOUSe Chicken

.NL,P,,~NGNFAR,,iT~,~"~~~C~~IP~~~RH~ALR~~~~~YL~~ .................. ................... Q..:D..:.:::.E::::::::..D.::::: .K...M...S........SK..........KCE......K...D ......

892

Rabbit Human Mouse Chicken

942 p"wRssc~AKECPESLCbYSFNSQRFA~LLSTKFKYR?EGKITNYFH~T~ .................................................. .............i~~ .............. ..Y ............................................................. ...

i%iz' MOUS0

i%%' Mouse chicken

K%' Mouse Chicken

%%' Mouse Chicken Rabbit Human Mouse Chicken

592

992 AH"PEIIkRDGSIGAWA~EGNESGNKL~RRFRKMNARbSXCYEMED"~KH .................................................. ..........F::::: .... ................................... ................... . . .... ....... . .. 'i" HWLYTSK~LQKFMNAHNA~ISGFTMN~QVSLEDPLG~EDSLESQYS~EF* 1042 ..P.A..G............D ..... ....................... .................. L.S ..... ..KET.G............D ..... ................ KTLRSQ ..AIDPDDG.G.S.PP.I....ND.V.L.

Fig. 3(C).

Rabbit RAG-l 10

fk%c’ HOUSe Chicken

1027

and RAG-2

20

50

40

30

t4SLQ"ITV&TALIQpGF~LMNFDGQIF~FGQKGWPKR~CpTGvF"FD~ . . . ..~..~H.................V...................L.V . . . . . . . . . . . . . . . .V . . . . . . . . . . . . . . . . . .. ... . . . . . ..VSAVS.SS.L...S..L....HV..................FL..

50

.....

I I I I I KaNH~KLKPAVFSKDSCYLPPLRYPATCT~~GS~~SEKaIIHGGKTPN . ..TI.................. :::::: . . . . . . . . . .I.................SYK::ID:ti.H:.: . ..E..M...A..R............I..LR.NG..D.H....:......

100

Chicken

I I I I I NE$SD;IYVMSWCKNNKKVTFRCTEfLVGDVPEARYGv%;WYSRGK .... ..I ............. I..:::::: I .K::::::::+::: .A .... .... :rj::::::I::M.N.TT::+::b:i.K..G.........:+IN..H

Rabbit Human

SMGVLFGGR&MpSNQRTTkKWNSVADCL$FLVDFEF&CATSYILPE :. ..:~.::: .......... . . ........... ..TH .............

Chicken

::i:~::::::i:i~::::::::::ir::::s::..:...::~::::::::

Rabbit HUmall Mouse Chicken

QDGLSFHVS~ARNDTVYIL~GHSLANNIR~ANLYRIRVD~PLGSPAINC& ........... K...I..............................V S.................T..V ......................... ......... V..D..I........Q..T..PS..KLK........CVT

Rabbit HUmaIl Mouse Chicken

VLPGGISVS~AILTQTNND~FVIVGGYQL~NQKRMVCNI?SLEDNKISI~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..I...I.........R .SL...G..T...S

300

Rabbit Human Mouse Chicken

EMETPDWTPLjIKHSKIWFG~NMGNGSVFL~IPGDNKQIV~EAFYFYMLK~ . . . . . . . . . . . . . . . . . . . . . . . ..T...........V...G........ ..s.. AM.......T.R. TI...... :~~~~::.....::~RM:::CD::K:..L.....~:::LI.D.N...I.R.

350

Rabbit Human Mouse Chicken

//TSDDVHE~QRTFTNSQTBTEDPGDSTP~EDSEEFCFSAEANSFDGDD~ //A...TN.E.T........... //S.E.LS...KIVS........:::::::::::::::::::T::::::: NKA.E.EE.ELTAQ.C..A....Q............S.....S...V../

398

Rabbit Human Mouse Chicken

FDTYNEDDE~DESETGYWI&CpTCDVDI/JTWVPFYSTE~NKPAMIYCS~ . . . . . . . ..E........................................ .v.. I::::::: :i::....:::. I::A~:NI:::::::::::::::::::i::S

440

Rabbit Human Mouse Chicken

GDGHWVHA;$lDLAERTLI~LSEGSNKYY~NEHVETARA$ . ... . .A.................H..Q.VL..K ::i::::::: ..S.SM.LQ.::ti+::f.:::::%ljI%:::::6%:#

498

Rabbit Human Mouse Chicken

KPPMKSL"Kl!GSGKILTPAiKSFLRRLFD! . . . . . . .R..................... ..V.............. :Q:::~::::I\+N.LT..V.......,.E

527

~z%'

Mouse

MOUSe

150

200

250 ... ... .S

Fig. 3(D). Fig. 3. (A) Nucleotide and conceptually translated protein sequence of genomic rabbit RAG-I gene (GenBank Accession Number M77666). Asterisks (*** ) represent the termination codon. (B) Nucleotide and conceptually translated protein sequence of genomic rabbit RAG-2 gene (GenBank Accession Number M77667). Asterisks (*** ) represent the termination codon. (C) Comparison of deduced amino acid sequences of rabbit, human, mouse and chicken RAG-l protein. Slashes (/) represent gaps inserted to maximize identity and dots (. . .) indicate identity to the rabbit sequence. The asterisk (*) represents the termination codon. (D) Comparison of deduced amino acid sequences of rabbit, human, mouse and chicken RAG-2 protein. Slashes (/) represent gaps inserted to maximize identity and dots (. .) indicate identity to the rabbit sequence. The asterisk (*) represents the termination codon. detected rabbit RAG-2 and RAG-l in mRNA from rabbit thymus. When poly (A)+ RNA isolated from rabbit thymus and total RNA from mouse thymus were probed with the human RAG-l probe, bands of 6.6 kb were detected [Fig. l(A)], which is the same size as previously reported for mouse and human RAG-l

(Schatz et al., 1989). This result was confirmed using a rabbit RAG-l cDNA probe (data not shown). However, the major species of mRNA detected with the mouse

RAG-2 probe were of different sizes in the rabbit (~4.4 kb) and mouse (2.2 kb) [Fig. l(B)] Hybridization patterns using a rabbit RAG-2 probe confirmed these results [Fig. l(C)]. Genomic DNA cloning and sequencing

Screening of a rabbit genomic library yielded plaques which hybridized with the RAG-l probe (2), the RAG-2

Table 1. Percent identity to rabbit RAG coding sequences RAG-l Human Mouse Chicken

91 85 72

DNA RAG-l

protein RAG-2 DNA RAG-2 protein

93 (99) 90 (99) 78 (94)

90 87 71

“Values in parentheses include conservative substitutions.

91 (97) 90 (98) 72 (94)

PATRIZIAFUSCHIOTTI et al.

1028

probe (3), and with both probes (1). The double-positive plaque was selected for purification and subcloning. The 16.7 kb insert was double digested with Sal1 and Hind111 to generate a 7.4 kb RAG-l-positive &/I/Hind111 fragment, and a 2.3 kb RAG-Zpositive SulI/Hir?dIII fragment, each of which was subcloned into pBIuescript II SK +, The orientations and locations of the fragments, determined in part by sequence analysis, showed that, as with other species, both genes are oriented so as to be convergently transcribed (Fig. 2). Sequence data from the 3’ end of the subcloned RAG-l-positive fragment revealed that the Hind111 cloning site was at a position corresponding to bp 6538 of the 3’ untranslated region of the human RAG-l gene (sequence not shown). This suggested that the cloned RAG-I fragment was likely to contain a sequence homologous to the entire known sequence of RAG-l and that there was a general conservation in size of the coding and noncoding regions between these species. Sequencing of the two ends of the RAG-Zpositive 2.3 kb clone showed that the Sal1 end shared identity with the 5’ end of the previously described RAG-2 genes. We did not detect identity with the mouse 3’ untranslated region (extending to 1.9 kb from the mouse start codon) upon sequencing 300 bp in from the HindI (3’) end of the 2.3 kb RAG-Z fragment. The entire coding regions and a portion of the 3’ untranslated regions of both rabbit RAG genes were sequenced [Fig. 3 (A) and (B)]. DNA sequence analysis showed that the rabbit RAG-l gene is most similar to human (91% identity) and less so to mouse and chicken (85 and 72% identity, respectively) (Table 1). These differences are similarly reflected at the protein level [Fig. 3(C)] (93% human, 90% mouse, and 78% chicken). When conservative substitutions are included in the comparisons, the values increase to 99, 99 and 94%, respectively. Most of the differences were located in the N-terminal 40% of the protein (codons l-395) with the first 100 codons showing the least identity (82% human, 68% mouse, 29% chicken). The C-terminal 33 codons are also poorly conserved. In contrast, certain regions (i.e. codons 647-813 and 887-1008) appear highly conserved among all species studied thus far. Comparison of the rabbit RAG-2 gene and predicted translation product to those of human, mouse and chicken revealed 90, 87 and 71% identity, respectively, at the nucleotide level, and 91,90 and 72%, respectively,

at the protein level (Table 1). Inclusion of conservative substitutions yielded values of 97, 98 and 94%, respectively. Unlike the RAG-l comparisons, RAG-2 substitutions were more uniformly distributed throughout the sequence with a few clusters of differences evident [Fig. 3(D)]. RAG-I

cDNA

cloning and sequencing

A portion of the RAG-l coding region corresponding to positions 19962963 of the genomic clone [Fig. 3(A)] was amplified by RT-PCR from thymus mRNA and cloned in pBluescript SK + . The sequence of this 967 bp fragment was almost identical to the genomic clone with the exception of one silent change and one replacement (position 2931, TTT-Phe to TCT-Ser). This difference might reflect a genetic polymorphism or a PCR artifact. This fragment was only used as a probe and was not investigated further.

The complete RAG-2 coding region (1.64 kb) was amplified by RT-PCR using oligonucleotides chosen from the sequence of the genomic rabbit RAG-2 clone. Two of six individual recombinant RAG-2 positive clones were selected for sequencing (clones 1 and 2) [Fig. 4(B)]. The sequence of a cDNA clone (SF) containing some of the region 5’ of the coding segment was also determined {Fig. 4(A)]. In Fig. 4(A), the 5’ region is compared with available rabbit genomic and mouse cDNA sequences; in Fig. 4(B), the two cDNA clones are compared with each other and with the genomic clone. Clones 1 and 2 differ from each other at nine positions, which include four silent changes and five replacement changes. Such differences could reflect different allelic forms of the rabbit RAG-2 genes and/or errors introduced during the PCR amplification protocol. Both of the cDNA sequences agree with each other and differ from the genomic sequence at seven positions (an eighth difference at the 5’ end is due to the design of the oligonucleotide primer); two differences result in amino acid replacements (ATA Ile-ACA Thr and AGT SerACT Thr). These differences are likely to reflect genetic differences between the rabbits. RAG-2

RFLP

In order to further investigate genetic differences in the rabbit RAG-2 region, genomic DNA from genetically

A cDNAclone

SF

Rabbit muse

50 40 60 10 30 ~ACAGTGGGCATTCAGTG~TCTTCCCC~GTGC~GATA*~~TACCTG~T*AGTGCTGATTCAG -------------------------G..G...--------.....T.....C..G..C.A..C.......

Rabbit Hoabe

120 60 90 100 110 AGAGGGGCGAtCAGTCCCTCTGGCCTTCA~TCCTA1GGACC G..A.AAT.AG.AAGAG.A.. . . . . ..ATA.....C................

130

I40

___--- . ..c..c.----

180 190 160 150 110 CCTTATGMTATTTGGATAGGCCACAMCAARCATCTTCCCAGAGTATTTCA~TCA~T~GA -;;o-------;;o--.C.A.~~G..GT.Cii~..AT..CT~C..----;;;;--..A..jjd.. Rabbit nouse GMlOlldC (RabbltJ

70

200

CCCTGCAGATW\TAACAGTCAGGAATMCATAGCCCTTAAcaaCCagqCttCtCaCtqat TCTAChCAC~TCCG~~ P.C . . . . . . . ..G.......GG.TC................. C...TT.......---..a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..c..........

Fig. 4(A).

Rabbit

RAG-l

1029

and RAG-2

90 100 10 80 60 40 50 20 30 10 Genomlc CL1 tccgaaaa~ CL2 . . . . . . . . . . . . . WetSerLeuGlnMetIleThrVslArgAsnAs "c"L: . . .. . . . .. . ... . .. . . . .. .. . . . .. . .. . GenOmiC I I I I "ck: TTGGCCIUAAACGATGGCC:AAGAGATCC:GCCCCACTG~AGTTTTTCA~TTTGATAT~ACAG~CCATCTC~CT~AAGcc~Gc~GTTTTcTcT~ 200 . . . .. . . . .. . .. .. .. .. . . . . .. .. .. . .. . . .. . . . .. .. . . . ... .. .. .. .. ... . .. .. .. .. .. .. .. .. .. .. .. .. .. . . . .. . . . .. . .. CL1 heGlyGlnLysGlyTrpProLysA~gSerCysPr~ThrGlyValPheHisPheAspIleLysGl~AsnHiS~"LysLe"LYsP~oAlsVslPheSe~LY CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..".....".."................i.........i.........i.........i.........i I I I Genomic CTCCTCTCCGTTACCCAGCCACTTGCACATTCC~GGCAGCTCAGAGTCTG~~C~CAATATATCATCCACGGA~G~ 300 CL1 GGATTCCTGCTATC . . . . .. .. . . . .. . ... . .. . . .. . . .. .. .. . . . .. . . ... .. . . .. .. .. .. .. . . .. . . . .. ... . .. .. .. . . .. .. .. . CL2 . . . . . . . . . . . . . . CL1 sAspSerCysTyrLeu ' 4' roProLeuArgTyrProAlaThrCy.ThrPheGlnGlySerSeKGl"SerGl"LysG1nGlnTY~IleIle~~sGlYG~YLYs CL2 . . . . . . . . .."...................................................................i'......"i....."..i Genomic CL1 ACACCAAACAATGAGCTTT CL2 CL1 CL2 Genomic Genomic CL1 CL2 CL1 CL2

TGCTCTACAGTCGAGGAAAAGTATGGGAGTTCTCTTTGGAGGACGGTCATACATGCCTTC 500 GAGATGTTCCAGAAGCCAGATATGGTCATTCCCTTG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .# . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lyAspValProGluAlaArgTyrGlyHisSecLe"AspVslValTyrSerA~gGlyLysSeKMetGlYVslLe"PheGlyG1YA~9Se~TY~~etP~oSe .i.'.......i .."..".i..'......i..".....i.........i..."....i..'......i.........i.........i........

Genomic CL1 T CAAAGAACCAC AAAATGGAATAGCGTCGCTGATTGCCTGCCCC TTTTCTTGGTGGATTTTGAATTTGGATGTGCCACATCCTACATTCTT 600 CL2 .. .. .. .. .. .. .. . . .. .. . . .. .. .. .. .. . . . .. . . .. . . .. . . . e 1nArgThrTh LysTrpAsnSerValAlaAspCysLeuProHisValPheLe"ValAspPheGluPheGlyCysAlaThrSerTyrIleLeu CL1 . . . . . . . . . ..".................................................................... CL2 As . . . . . . . . . . . G(i L Genomic l!r As bl , I I I Genomic CL1 CCAGAGCTTCAGGATGGGC CATTTCATGTCT TTGCCAGI\AATGATACCGTTTATATTTTAGGAGGACATTCTCTTGCC~T~TATCCGCCCTG 700 CL2 . . . . . . . . . . . . . . . . . . . 6 .. . . .. . . .. .. .. .. .. . . .. .. .. .. .. . . .. .. .. .. .. .. . . .. . . . .. . . .. . . . .. .. CL1 ProGluLeuGlnAspGlyLeuSerPheHisVslSe~IleAlaArgAsnAspThrValTyrIleLeuGlyClyGlyHisSeK~"AlsAsnASnIleArgP~OA CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .._....................................................... I I I I I I I I GenOmiC CL1 CCAATCTGTACAGAATCAGGGTTGATC CCTTGGGTAGCCCAGCCATAAATTGCAGTTTTGCCAGGCCTGAC 800 CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . .# . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CL1 laAsnLe"TyrArgIleArgValAspLe"P~oLe"GlySe~Pr~AlsIleAsnCysTh?ValLeuProGlyGlyIleSerValSerSe~AlaIleLe"Th CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .._........................................................

. ... ........ . ..... .... ........ .... ............ I

I

I

I

I

.I

CL1 TCAGACTI\PI:AATGATGM:TTGTTATCG:TGGTGGGTA~CAGCTTG~~TC-~~AATGGTCTG~~CATTGTC~CTTTAGAGG~T~C~GAT~ 900 CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CL1 rGlnThrAsnAsnAspGluPheVslIleVslGlyGlyTy~GlnLe"Gl"AsnGlnLysA~gUetValCysAsnIleValSerLe"GluAspAsnLysIle CL2 . . . . . . . ..i.......""........"...................................."....'......."................. I BanHI I I I I I I I I Genomic GTTTTCCTTGGCA 1000 CL1 GA~TTCI\AGAGATGGAGRCCCCAGATTGGA~T~~AGATATT~G~A~AGC~~TATGGTTTGG~G~~~ATGGG~ CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CL1 Gl"IleGlnGl"UetGl"ThrP~oAspTrpThrProAspIleLysHisSerLysIleTrpPheGlySerAs~etGlyAs CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genomic I I GenOmIC 1100

I

I

I

I

I

l"AspAspValHisGl"AspClnRrgThrPheThrAs . . .. .. .. . . . . .. . .. . . . .. .. .. .. . . . . .. .. . . . .. .. .. .. .. .. .. .. .. . Genomic CL1 TAGTCAGACATC~~AGAAGATC~AGGGGA~T~CA~T~C~TTTGAGGACT~GGAAGAATTTTGTTT~AGTG~AGAAG~A~TAGTTTTGATGG~GATGAT 1200 CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EL.; nSerGlnThrSerThrGluAspP?oGlyAspSerThrProPheGluAspSerGl"GluPheCysPheSerAlaGl"AlsAsnSe~PheAs~lyAspAsp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .._.................................................. CL1 GAATTTGA~~CTTAT~TG!AGATGATG~~GATG~TGAG~~TGAG~~~G~~TA~TGGAT~A~~TG~TG~~~TACTTGTG~TGTGG~~~T~~~~~TTGG~ 1300 CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .._...._............................._.............._...._...... CL1 Gl"PheAspThrTy~AsnG1uAspAspCluAspAspGl"SerGl"ThrGlyTyrTrpIleThrCysCysProTh~CysAspVs1AspIleAsnThrTrpV CL2 ..""...i.........i..".............'.......‘................".."....".......................... I I I I I Genomic CL1 TGCCATTCTATTCAA CCTGCCATGATCTACTGTTCTCATGGAGATGGGCACTGGGTCCATGCCAAGTGCATGGATCTGGCAGAACG 1400 CL2 . . . . . . . . . . . . . . . . . .. .. .. .. . . . .. . . .. . . .. . . . .. . . .. ... . . . .. . .. . . .. . .. . . . .. . .. .. .. . . .. . . . . CL1 alProPheTyrSerThrGluLe"As CoAlaMetIleTyrCysSerHisGlyAspGlyHisT~pValHisAlaLysCysMetAspLe"AlaGl"Ar CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .. .. .. .. . . . .. . . ... .. . . . .. . . .. . . .. . . . .. .. .. .. .. .. . . . .. . . . . . .. . . .. Genomic

I

I

I

CL1 TA~~CTCAT~CAT~TGT~~GAAGGAAG~A~~~GTATTA~TGT~TGAG~ATGTGGAGA~~G~~G~G~~~TA~~~T~~~~~G~~~~TA~~~TT~ 1500 CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CL1 9ThrLeuIleRisLeuSerGluGlySerA.nLysTyrTyrCysAsnGluHisValGl"IleAlaA~gAlaLeuGlnTh~ProLysA~gThrIlePro~u CL2 "....".i.........i........"...."........""......"........"............."........"......... I I I I I Genomic TGTTTGACTAATTTTGCA 1600 CL1 AGRPIAGC~~~~AATG~T~~~T~~A~~G~GGTTCTGGG~~TCTTG~CTCCTGCCAAGAAAT~CTTTCTTAGAC CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .._............._............................... CL1 A~gLYSP~oPro~etLysSerLe"~isLysLysGlySerGlyLysIleLe"ThrP~oAlaLysLysSerPheLeuA~gArg~uPheAsp~*~ CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .._...._..................................................

I

CL1 AAAACTTTtcagstccatgcscsttssgc CL2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I

I

1629

Fig. 4(B). Fig. 4. (A) Nucleotide sequence of RAG-2 cDNA clone 5F (GenBank Accession Number M99310) compared with available sequences of the mouse RAG-2 cDNA (Oettinger et al., 1990) and rabbit genomic DNA. Dashes (---) represent gaps inserted to maximize identity and dots (. .) indicate identity to the clone 5F sequence. A box is drawn around the ATG start codon. (B) Sequences of RAG-2 PCR derived cDNA clones 1 (GenBank Accession Number M99311) and 2 (GenBank Accession Number M99312) compared with genomic DNA and with each other. Lower case letters are used to indicate the locations of oligonucleotide primers used to generate clones 1 and 2 and a box is drawn around the atg start codon and around the sequence the complement of which was used to prime for the 5F cDNA. The genomic sequence is only shown where it differs from one or both of the cDNA sequences. Sequence differences are enclosed in boxes.

1030

PATRIZIAFUSCHIOTTIet al. Table 2. Rabbit RAG-2 RFLP

(A) Restriction fragments probes.” Enzyme Hind111 Probe kb

detected

Eco RI kb

Pst I kb

3P Clone 1 5F

7.4, 8.1 7.4, 8.1 2.5, (7.4),’ (8.1)

3.0, 7.6 3.0, 7.6 1.9

3.3, 4.1 3.3, 4.1 3.0,b 3.3, 4.1

with

RAG-2

ible association. In Fig. 5, the deduced map of these polymorphic fragments is shown. Additional fragments that are not detected with the coding region probes were found with the 5F probe (Table 2) and these may map to the genomic area indicated with a dashed line. There are no Hind111 sites in any of the probes yet, in Hind111 digests, clone 1 (and 3P) hybridized to a single band of 3.3 kb (or two bands of 3.3 and 4.1 kb in putative heterozygotes); the 5’ probe 5F hybridized with the same fragment(s) plus an additional one of 3.0 kb. Similarly the 5’ probe reveals a new band of - 1.9 kb in PstI digests. This suggests that the polymorphic 7.6 or 3 kb PstI fragments extend 3’ from the known PstI site near the 5’ end of the RAG-2 coding region (see Fig. 4). EcoRI digestion gave fragments of 8.1 and 7.4 kb when hybridized with clone 1 or the 3P probe; the 5F probe showed faint 8.1 and 7.4 kb fragments and a new strong 2.5 kb band in all digests tested. We conclude from these results that the 5’ cDNA sequence resulted from splicing of one or more upstream exons; for this reason the 5F probe hybridizes to some fragments containing the coding region (identified with clone 1 and the 3P probes) and to additional genomic DNA.

cDNA

(B) Results of backcrosses involving Hind111 and PstI RFLP Hin dII1 PstI __-.

Sire kb 3.313.3

Dam kb

Sire kb

Dam kb

3.314.1

313

311.6

Progeny 3.313.3 4

Progeny 3.314.1 3

317.6 3

313 4

“No Hind111 or EcoRI sites are present in the probes used. A PstI site is present near the 5’ end of the RAG-2 coding region [positions 16-21 in Fig. 4(B)]. ‘Underlined fragments were only detected with the 5’UT region probe 5F [see Fig. 4(A)]. ‘Parentheses indicate fragments that were faintly visible with the probe.

DISCUSSION The diversification mechanism contributing to the antibody repertoire in birds is somatic gene conversion (McCormack et al., 1991). The molecular events that occur during rabbit V, gene rearrangement and diversification may be, in several respects, more like those of the chicken than those so far described in mammals. For example, although the rabbit V, cluster contains more than 100 V, genes they appear to be members of one large family of rather closely spaced gene elements many of which are pseudogenes (Becker and Knight, 1990; Roux et al., 1991; Fitts and Metzger, 1990; Gallarda et al., 1985; Currier et al., 1988; Mage et al., 1984; Kabat et al., 1991). As in the chicken, the most 3’ V, gene V, 1 is found preferentially (but in the rabbit not exclusively) rearranged in B cells and appears to undergo somatic diversification by gene conversion with upstream elements serving as sequence donors (Becker and Knight, 1990; Allegrucci et al., 1991; Rouxetal., 1991; FittsandMetzger, 1990). Asinmiceand

defined pedigreed animals was subjected to Southern blot analysis using different restriction enzymes and rabbit RAG-2 cDNA probes. The probes included clone 1, clone 5F, which includes 65 bp at the 5’ end of the coding region and an additional 226 bp 5’ of untranslated region [Fig. 4(A)], and a PCR-amplified fragment (3P) corresponding to the region 3’ of the BamHI site in the sequences of clone 2 and the genomic DNA. In Table 2, we list the sizes of restriction endonuclease fragments found with these probes. Although the numbers of progeny examined are small, backcross analyses are consistent with allelic segregation of the RFLP detected with clone 1 and 3P in Hind111 and Pst I digests. In the families studied, three progeny inherited the larger (4.1 kb) Hind111 fragment trait. The same three also inherited the larger PstI (7.6 kb) fragment trait suggesting their poss-

Cq)p (HI

E(E)

PE

H

.SqeG-25

P

Rs(llo” withh which . . ..~.....~.........

. . . . . ..-

2 I

4 I

6

8

10

I

I

I

12 I

14 I

5’ eronr

16 I

my . exist . . . . . . . . . . . . ..-

18 I

20 I

kb

Fig. 5. Genomic map indicating some of the polymorphic sites observed in the rabbit RAG-2 locus. The filled box depicts the RAG-2 open reading frame and the arrow beneath it the direction of transcription. The region where our studies indicate the existence of additional 5’ exon(s) is indicated with a dashed line.

Rabbit RAG-l humans, RAG-l and RAG-2 are coordinately expressed in chicken thymus, the site of T-cell maturation. However, in the chicken bursa of Fabricius, the site of B-cell maturation and gene conversion, RAG-2 is expressed in the absence of RAG-l (Carlson et al., 1991). Moreover RAG-2 is expressed in the absence of RAG-l in a chicken B cell line (DT40) that undergoes constitutive somatic gene conversion in vitro (Carlson et al., 1991). Gene conversion continues in DT40 subclones in which the RAG-2 coding regions have been deleted by homologous recombination, indicating that expression of RAG2 in the cell lines is not essential for gene conversion to occur (Takeda et al., 1992). However, the possibility remains that RAG-2 is required for a developmental step in bursal lymphocytes, such as the activation of other components of the gene conversion enzymatic machinery. We sequenced the rabbit RAG-l and RAG-2 genes for comparison to their human, mouse and chicken counterparts in an attempt to correlate any structural differences with the suspected species-specific functional differences. In addition we are currently using the probes characterized in the present study to investigate tissuespecific expression of RAG-l and RAG-2. Our results show that the organization of rabbit RAG-l and RAG-2 genes is similar to that reported for mouse, human and chicken. All RAG genes thus far described are in close proximity to each other and are convergently transcribed. Comparison of the known RAG-l amino acid sequences [Fig. 3(C)] reveals that the great majority of substitutions are clustered in the N-terminal 40%. However, within this region (codon positions 291-330), all four RAG-l sequences have the eight conserved Cys/His and one Pro recently described for a family of sequences including the putative transcription factor PML (Kakizuka et al., 1991; de The’ et al., 1991). Unlike the RAG-l comparisons, RAG-2 substitutions were more uniformly distributed throughout the sequence with a few clusters of differences evident, suggesting a generally conserved structure. Although the putative nuclear localization signal RKKEKR in RAG-l of mouse and man (Schatz et al., 1989) was found to be RKKEKK in rabbit and chicken (codon positions 14&146), the data did not reveal any striking regions in which the two species that express somatic gene conversion activity (rabbit and chicken) showed greater identity to each other than to the mouse. It is possible that the as yet uncharacterized 5’ ends of RAG-2 could reveal such relationships. A striking finding is that the predominant rabbit RAG-2 mRNA transcript is larger than the major species in the mouse (4.4 kb compared to 2.2 kb). Our data suggest that there are one or more upstream exons present in the region 5’ of the known RAG-2 coding region. There is nucleotide sequence conservation of 5’ cDNA sequences between mouse and rabbit in the region that was available for comparison [Fig. 4(A)] We find that this rabbit cDNA is derived from splicing of additional exons; and this is probably true in the human and mouse as well (Ichihara et al., 1992; Schatz et al., 1992). A potential splice site was noted in the human

1031

and RAG-2

genomic RAG-2 sequence 27 bp 5’ of the start codon (Ichihara et al., 1992). Unfortunately, we have had difficulty in obtaining further sequence 5’ possibly due to sequences which have unusual secondary structure or instability during cloning. Prior to using the PCR approach for synthesis of RAG-2 cDNA clones, we had constructed a cDNA library from rabbit thymic mRNA and screened with the mouse RAG-2 probe. Although we obtained full-length clones, we were unable to grow and subclone them from the Agtl 1 vector, even when we used bacterial strains designed to limit rearrangement events resulting from the propagation of cDNA libraries. PCR analyses of preparations from attempts to grow these clones indicated that deletions were occurring in the recombinants during growth. There are many examples of single genes with alternatively spliced exons (Foulkes and Sassone-Corsie, 1992). The exons define functional, structural or regulatory domains, and the splicing pattern of the transcripts can modulate the expression or function of the resultant product. The possibility that there are alternative functions for alternately spliced forms of the RAG-2 gene is raised by the finding of a longer mRNA transcript and additional 5’ exon(s). For example, alternative splicing of the RAG-2 gene might affect its tissue distribution or role in activating other factors in the recombination or gene conversion machinery. Further cloning of the region 5’ of the rabbit RAG-2 gene is in progress to characterize the portion upstream which appears to generate a longer message in the rabbit. The complex organization of the RAG-2 locus’ allelic forms, a long mRNA transcript with the possibility of additional spliced exon(s), suggests that the rabbit provides a good model for further exploration of functional activities of the RAG-2 gene. REFERENCES Aguilera R. J., Akira S., Okazaki K. and Sakano H. (1987) A pre-B cell nuclear protein which specifically interacts with the immunoglobulin V-J recombination sequences. Cell 51, 909-9 17. Allegrucci M., Young-Cooper G. O., Alexander C. B., Newman B. A. and Mage R. G. (1991) Preferential rearrangement in normal rabbit of the 3’ Vna allotype gene that is Alicia deleted in mutants; somatic hypermutation/conversion may play a major role in generating the heterogeneity of rabbit heavy chain variable region sequence. Eur. J. Immun. 21, 411-417. Alt F. W., Blackwell T. K., DePinho R. A. and Yancopoulos G. D. (1986) Regulation of genome rearrangement events during lymphocyte differentiation. Immun. Rev. 89, 5-30. Becker R. S. and Knight K. L. (1990) Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits. Cell 63, 987-997. Blackwell T. K. and Ah F. W. (1988) In Molecular Immunology (Edited by Hames B. D. and Glover D. M.), pp. l-60. IRL Press, Washington, DC. Brow M. A. D. (1990) Sequencing with Taq DNA polymerase. In PCR Protocols (Edited by Innis M. A., Gelfand D. H., Sninsky J. J. and White T. J.), pp. 189-196. Academic Press, New York.

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Carlson L. M., Oettinger M. A., Schatz D. G., Masteller E. L., Hurley E. A., McCormack W. T., Baltimore D. and Thompson C. B. (1991) Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion. Cell 64, 201-208. Currier S. L., Gallarda J. L. and Knight K. L. (1988) Partial molecular genetic map of the rabbit V, chromosomal region. J. Immun. 140, 1651-1657. de The’ H., Lavau C., Marchio A., Chomlenne C., Degos L. and Dejean A. (1991) The PML-RARc( fusion mRNA generated by the t( 15; 17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell 66, 6755686. Feinberg A. P. and Volgelstein B. (1983) A technique; for radiolabeling DNA restriction fragments to high specific activity. Analyt. Biochem. 132, 613. Fitts M. G. and Metzger D. W. (1990) Identification of rabbit genomic Ig-V, pseudogenes that could serve as donor sequences for latent allotype expression. J. Immun. 145, 2713-2717. Foulkes N. S. and Sassone-Corsi P. (1992) More is better: activators and repressors from the same gene. Cell 68, 411414. Gallarda T. L., Gleason K. S. and Knight K. L. (1985) Organization of rabbit immunoglobulin genes. I Structure and multiplicity of germline V, genes. J. Immun. 135, 42224227. Halligan B. D. and Desiderio S. V. (1987) Identification of a DNA binding protein that recognizes the nonamer recombinational signal sequences of immunoglobulin genes. Proc. natn. Acad. Sci. U.S.A. 84, 7019-7023. Hesse J. E., Lieber M. R., Mizuuchi K. and Gellert M. (1989) V(D)J recombination: a functional definition of the joining signals. Genes Dev. 3, 1053-1061. Hope T. J., Aquilera R. J., Mime M. E. and Sakano H. (1986) Endonucleolitic activity that cleaves immunoglobulin recombination sequences. Science 231, 1141-I 145. Hunkapiller T. and Hood L. E. (1989) Diversity of the immunoglobulin gene superfamily. Adv. Immun. 44, 1-63. Ichihara Y., Hirai M. and Kurosawa Y. (1992) Sequence and chromosome assignment to llpl3-pl2 of human RAG genes. Immun. Lett. 33, 277-284. Kabat E. A., Wu T. T., Perry H. M., Gottesman K. S. and Foeller C. (1991) Sequences of Proteins of Immunological Interest, 5th Edition. NIH Publication No. 91-3242, US Department of Health and Human Services, PHS, NIH, Bethesda, MD. Kakizuka A., Miller W. H. Jr, Umesono K., Warrell R. P. Jr, Frankel S. R., Murty V. V. V. S., Dmitrovsky E. and Evans R. M. (1991) Chromosomal translocation t( 15; 17) in human acute promyelocytic leukemia fuses RARc( with a novel putative transcription factor, PML. Cell 66, 663-672. Kidd V. J. (1984) In-situ hybridization to agarose gels. Focus 6,3-4. Lieber M. R., Hesse J. E., Mizuuchi K. and Gellert M. (1987) Developmental stage specificity of the V(D)J recombination activity. Genes Dev. 1, 751-761.

Litman G. W., Berger L., Murphy K., Litman R., Hind K. and Erickson B. W. (1985) Immunoglobulin V, gene structure and diversity in Heterodontus, a phylogenetically primitive shark. Proc. natn. Acad. Sci. U.S.A. 82, 2082-2086. Mage R. G., Bernstein K. E., McCartney-Francis N., Alexander C. B., Young-Cooper G. O., Padlan E. A. and Cohen G. H. (1984) The structure and genetic basis for expression of normal and latent Vna allotypes of the rabbit. Molec. Immun. 21, 1067-1081. Maniatis T., Fritsch E. F. and Sambrook J. (1982) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. McCormack W. T., Tjoelker L. W. and Thompson C. B. (1991) Avian B-cell development: generation of an immunoglobulin repertoire by gene conversion. A. Rev. Immun. 9, 219-241. Mombaerts P., Iacomini J., Johnson R. S., Herrup K., Tonegawa S. and Papaioannou V. E. (1992) RAG-l deficient mice have no mature B and T lymphocytes. Cell 68, 869-877. Oettinger M. A., Schatz D. G., Gorka C. and Baltimore D. (1990) RAG-l and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248, 1517-1523. Oettinger M. A., Stanger B., Schatz D. G., Glaser T., Call K., Housman D. and Baltimore D. (1992) The recombination activating genes, RAG-l and RAG-2, are on chromosome 1 lp in humans and chromosome 2p in mice. Immunogenetics 35,97-101. Roux K. H., Dhanarajan P., Gottschalk V., McCormack W. T. and Renshaw R. W. (1991) Latent al V, germline genes in an a*a* rabbit. Evidence for gene conversion at both the germline and somatic levels. J. Immun. 146, 2027-2036. Sanger F., Nicklen S. and Coulson A. R. (1977) DNA sequencing with chain terminating inhibitors. Proc. natn. Acad. Sci. U.S.A. 14, 5463-5467. Schatz D. G. and Baltimore D. (1988) Stable expression of immunoglobulin gene V(D)J recombinase activity by gene transfer into 3T3 fibroblast. Cell 53, 107-l 15. Schatz D. G., Oettinger M. A. and Baltimore D. (1989) The V(D)J recombination activating gene, RAG-l. Cell 59, 1035-1048. Schatz D. G., Oettinger M. A. and Schlissel M. S. (1992) V(D)J recombination: molecular biology and regulation. A. Rev. Immun. 10, 359-383. Shinkai Y., Rathbun G., Lam K.-P., Oetz E. M., Stewart V., Mendelson M., Charron J., Datta M., Young F., Stall A. M. and Alt F. W. (1992) RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855-867. Takeda S., Masteller E. L., Thompson C. B. and Buerstedde J. M. (1992) RAG-2 expression is not essential for chicken immunoglobulin gene conversion. Proc. natn. Acad. Sci. U.S.A. 89, 40234027. Tonegawa S. (1983) Somatic generation of antibody diversity. Nature 302, 575-58 1. Yancopoulos G., Blackwell T. K., Suh H., Hood L. and Alt F. W. (1986) Introduced T cell receptor variable region gene segments recombine in pre-B cells: evidence that B and T cells use a common recombinase. CeN 44, 251-259.