Cloning and sequencing of two functional rabbit germ-line immunoglobulin V λ genes

Gene, 80 (1989) 185-191 Elsevier

185

GENE 03043

Cloning and sequencing of two functional rabbit germ-line immunogiobulin VI genes (V-regions; promoters; diversity; cDNA; alignments; subcloning)

David J. Hayzer and Jean-Claude Jaton Department of Medical Biochemistry, Faculty of h4edicine, University Medical Center, CH-I21 I Geneva 4 (Switzerland) Received by H.G. Zachau: 16 December 1988 Accepted after revision: 27 February 1989

SUMMARY

A recombin~t-phage library of rabbit genomic DNA was screened for ~unogiobul~ V1 genes. Two functionat genes, \I,42 and VL3, which are separated by 1.6 kb were isolated and sequenced. Both are accompanied by signals required for transcription-translation and the recombination with J1 genes. The two genes, which are 95 % similar in their framework regions, may be the parents of several cDNAs encoding V1 regions. Rabbit VIZ-region diversity is likely to be generated by somatic mutation and V-J junction flexibility. Two cDNA clones encoding I-light chains may have arisen by gene conversion, exchanging the complement~ity-detesting regions, CDR3s, of the V12 and V13 genes.

INTRODUCTION

In common with other Ig hght chains, rabbit I chains are comprised of V, J and C regions, each encoded by a distinct genetic element. The rabbit genome contains at least eight CL genes (Duvoisin

Co~es~on~enee too:Dr. D.J. Hayzer, Department of Medical Bi~hemis~, Faculty of Medicine, University Medical Center, CH-1211 Geneva 4 (Switzerland) Tel. 022122-91-40. Abbreviations: aa, amino acid(s); bp, base pair(s); cap, 5’ protected end of mRNA; CDR, complementarity-determining region; C, constant; dc box, decanucleotide box; FR, framework region; Ig, immunoglobulin; J, joining; kb, kilobase or 1000 bp; MCS, multiple cloning site; ORF, open reading frame; pd box, pentadec~ucleotide box; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/O.O15M Na, . citrate pH 7.5; V, variable.

et al., 1986; 1988). Six of these have been sequenced. The first four may be pseudogenes since they are not linked with JI segments (Duvoisin et al., 1986; 1988). The other two, CL5 and CX, are transcribed since they have been found in cDNA clones (Hayzer and Jaton, 1987) and are associated with their respective JI gene segments. Sequence information from cDNAs (Hayzer et al., 1987) showed that rabbit VI regions have a number of features not found in the L or fc chains of other species. There is an extra residue at position -10 of the leader region, an insertion of four extra residues close to the junction of FR2 and CDR2, a significant homology between the rabbit V1 CDR2 and the human D2 mini-gene, and length variation of CDR3, analogous to the length variation of the CDR3 of rabbit V1 chains (Heidmann and Rougeon,

0378-l 119/89/$03 50 Q 1989 Elsewer Science Publishers B V. (Biomedical Diwsion)

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1984). Southern-blot analysis of genomic DNA with a VIZ-specificprobe disclosed at least five hybridizing bands, suggesting a restricted number of V1 germline genes (Hayzer et al., 1987). It should be emphasized, however, that the probe used may detect only a subset of the total VI gene pool. The rabbit 1 locus is more complex than those of other species. In chicken, there are single functional VI, JI and CI gene elements and a large number of VA pseudogenes (Reynaud et al., 1985). Rat has a single VIZgene and two J,?-C1 gene pairs, one of which is unlikely to be expressed due to a defective JIZgene (Steen et al., 1987). Two gene clusters have been identified in the laboratory mouse (Selsing et al., 1982), each with one VA gene and two J1-Gil gene pairs. Recently, a third mouse cDNA-derived V,? region, VAX, in association with the J12-CA2 combination, has been identified (Sanchez and Cazenave, 1987; Dildrop et al., 1987; Sanchez et al., 1987). Southern analysis of wild-mice genomic DNA indicates several hundred potential germ-line VL genes (Scott and Potter, 1984). Currently, seven human VI-chain subgroups have been recognized (Kabat et al., 1987; Anderson et al., 1984); a human V,? probe revealed about ten hybridizing genomic DNA fragments (Anderson et al., 1984) and six J/Z-CA gene pairs are known (Hieter et al., 1981; Udey and Blomberg, 1987), a degree of complexity surpassing that of the rabbit. A competent immune system requires the generation of extensive Ig diversity. A number of mechanisms operate individually or in concert to this purpose. With a restricted repertoire of germ-line V and J genes limiting combinato~~ pairing and junctional flexibility, the principal effector would be somatic mutation (Yancopoulos and Alt, 1986). To determine the relative contributions of the various mechanisms to the formation of rabbit a chains, germ-line VAgenes have been cloned, sequenced and compared with VZ regions encoded by cDNAs.

EXPERIMENTAL AND DISCUSSION

(a) Screening of a rabbit genomic DNA library and cloning of VL germ-line genes A library of rabbit genomic DNA, fragmented by partial MbaI digestion and cloned into the phage

vector aEMBL4 (Knight et al., 1985), was used. Approximately 2 x lo5 recombinant phage were grown on Escherichia coli 4359 and screened with a 419-bp probe, derived from cDNA clone pDH7 (Hayzer and Jaton, 1987), and encoding an entire L-chain leader sequence, V;1region and the first eight aa residues of a J®ion. The probe was labeled with [K-32P]dCTP by nick-translation. Filters were hybridized at 42” C with 50% formamide, 5 x SSC and 10% dextran sulfate and washed twice at room temperature with 2 x SSC, 0.1 y0 SDS and twice at 50°C with 0.1 x SSC, 0.1% SDS. Positively hybridizing plaques were purified and rescreened. Phage DNA was prepared by the method of Kaslow (1986). EcoRI fragments of phage DNA, hybridizing to the cDNA-derived probe, were subcloned into vector pUC9 and restriction-mapped by standard methods (Maniatis et al., 1982). Four recombinant phages hybridizing to the V~-r~on-sp~~c probe were isolated. Restriction analysis of the cloned DNA inserts grouped the genomic regions into two pairs, non-overlapping one another. The inserts of phages lVL1 and I/VL2 carried a single VAgene, VA1 (data not shown). The second phage pair, 1VL25 and IVL40, embracing 16.5 kb of the genome, each carried three VAgenes, V;12,V13 and V14 (Fig. 1). Only genes VA2 and VA3 will be described below. VLl and V14 are pseudogenes due to some unusual features, to be reported in detail elsewhere. Intergenic distances are 1.6 kb between VA2 and VA3 and 4.6 kb between VA3 and VA4. Southern analysis, with a CA regionspecific probe described by Duvoisin et al. (1986), disclosed no cross-hyb~~~able C1 genes within the region of the genome incorporated into 1VL25 and IVL40. (b) Characterization of Vi12 and I7123 V1-encoding germ-line genes were subcloned into the vectors M 13mp 18 and M 13mp 19 and sequenced by the dideox~ucleotide method of Sanger et al. (1977), using the protocol supplied by Gibco BRL Ltd. A modified, alternative procedure [Focus (BRL Ltd), 9 (3), pp. 12 and 131 was also used. Genes V12 and V13 have the same orientation in the genome and alignment of the nucleotide sequences is possible from 325 bp upstream from the ATG start codon (bp position 361, Fig. 2). Both

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Fig. 1. Cloning and strategies for the determination of the nucleotide sequences of rabbit germ-line genes, V12 and VL3. (A) Cloning and sequencing strategy for V12. The extents of the genomic DNA stretches cloned in the recombinant lEMBL4 phages rZVL25and lVL40 are indicated (-). Open boxes, EcoRI fragments hybridizing with the cDNA-derived VA-region-specific probe. Blackened boxes, VL ORFs. EcoRI sites E* and E** are not present in genomic DNA but derive from the MCSs of the phage vectors of lVL40 and IVL25, respectively. The direction of transcription-translation is indicated by the large, open arrow. A 2.1-kb EcoRI fragment from 1VL25 was subcloned to give plasmid pVL25E2.1 for subsequent sequencing. Arrows below the indicated subcloned regions (ti), give the extents and directions of the sequencing runs. (B) Cloning and sequencing strategy for V13. A 2.3-kb EcoRI fragment from 1VL25 was subcloned to give plasmid pVL25E2.3 for subsequent sequencing. See A for other designations. Restriction sites: B, BumHI; E, EcoRI; K, KPnI; P, &I; Pv, PVUII.

mdc230 240 250 260 270 280 ggtccc~gabgtgcatgggCcctcagacc~~g~gg~~c~~~ tttgc.tga.ggacgt6tcaggctccEctggggc _---g_--t__gatg___t_t__a-___g________g____~t~____________t___~___t____________~_______~------_____

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genes are interrupted by introns; that in V12 is 109 bp and that in VA3,105 bp, with 89% similarity. Conserved RNA-splice sites at the intron/exon boundaries are present. The nt analysis shows that VA2 and V13 encode the 20-aa-long leader peptide and the N-terminal, 100 aaresidues, of IgVIZ-regions. Located at the 3’ ends of the VA-coding segments are heptamer and nonamer recombination signal sequences with 100 % and 90 % similarity, respectively, to the consensus sequences CACAGTG and ACAAAAACT (Sakano et al., 1979). An expected 23-bp spacer separates the two signals (Honjo, 1983). The 3’ flanking regions are much less conserved, if at all, with the exception of the first 14 bp, than are the regions 5’ upstream from both genes. The V12 and VA3 genes share 95% similarity in the FRs, have two base differences in the leader and are identical in the FR2, CDR2 and half of the FR3. Both contain the 12 extra bp at the FR2-CDR2 junction, previously observed in all rabbit VAcDNA clones studied (Hayzer et al., 1987). Nucleotide sequence alignment (not shown) shows 70 % similarity between the FRs of the rabbit V,?genes and those of the mouse cDNA-derived Vlx sequence (Sanchez et al., 1987) and only 40% with the FRS of either mouse VA1 or V12 gene. Interestingly, the Vlx sequence also includes four extra residues, homologous to those of the rabbit residue positions 48a-48d (Fig. 2). (c) 5’ regulatory transcription signal sequences Transcription-associated sequences have been identified 5’ upstream from IgV genes. Consensus TATA and cap-site sequences are TATAtAS and N~A~~~~~ (Wall and Kuehl, 1983). The only possible TATA site of V122 and VA3 is bp position 308-314, GATAAGA, with a potential cap site, TGAGCCCT at 339-346. A potential CAAT site is

at bp position 249-252. A normally conserved dc box, TNATTTGCAT, which may play a role in enhancing VA gene expression (Faulkner and Zachau, 1984) is identifiable at bp position 259-270. The effect of the insertion of the CT doublet into the dc box of VA3 upon transcription of this gene, is not known. Faulkner and Zachau (1984) also described a less well-conserved pd box, TGCACCTGTGNCCAG. A related sequence, CAGCTGTGATCAC, with only two differences (the first two bp positions are absent from the VA2 sequence) is at bp position 1-13. If this is, indeed, equivalent to the pd box it lies considerably farther upstream (-360 bp) from the translation start point, than reported for other VIZgenes. Steen et al. (1987) describe a sequence of unknown function, GCCCCTgAc, where the lowercase g and c represent less well conserved nt, located at varying distances upstream from the V1 genes of mouse, rat and human. A similar stretch of bp, at position 161-169, is upstream from VA2 and V13, albeit without a succeeding pd element. Unique to the 5’ flanking regions of V12 and V13 is a 13-bp motif, CCNCTGGGGNCAG, repeated five times at positions 86-98, 149-161, 203-215, 342-354 and 459-471. The central CTGGGG is especially well conserved. The longest intervening distance, between blocks 203-215 and 340-352, embraces the CAAT, dc and TATA sites, while preceding blocks are periodic, spaced 50 bp apart. The V12 and VA3 genes satisfy all criteria necessary to be considered as genes capable of being expressed. ORFs of 120 aa align with reported VAencoding cDNA sequences (Hayzer et al., 1987), with no in-frame stop codons or introduced gaps (Fig. 2). Their respective 5’ and 3’ flanking regions include all structures necessary for the transcription and translation of the two genes and adequate 3’ recombinational sequences.

4 Fig. 2. Nucleotide sequences of rabbit VI genes, VI.2 and V13 and alignments with cDNA-derived V1 sequences. The nt of noncoding regions are in lower-case letters and of V&encoding regions in upper-case letters. Leader, intron, FR, CDRs, the dc and TATA boxes, and the 3’ heptamer and nonamer recombination signals are indicated above the nucleotide sequences. Numbering below the amino acid sequences, -19, -i, i, etc, are aa residue positions in accordance with the scheme of Kabat et al. (1987). Additional aa residues are also given; a, b, C, d. Sequences were aligned visually, with gaps introduced to minimize differences. The nt and aa residues identical to those of the rVI sequence are indicated by dashes and aa residues absent t?om the CDR3s encoded by the cDNAs pDHlO1 and pDH31 are indicated by parentheses. rV12 and rVI.3 are the rabbit V12 and VI3 germ-line genes, respectively. cDNA sequences from clones pDH2, 7, 8, 9, 31 and 101 are taken from Hayzer et al. (1987).

190

(d) Generation of rabbit VA-region diversity

Neither VA2 nor VA3 is the parental gene for the VA-regions of cDNAs pDH2 and pDH9 (Fig. 2). The FR3 of each differs markedly from those in the germ-line genes and the CDR3 of pDH2 is longer than that of VR2 or V13. VJ.3, rather than Vd2, is the potential parent of the V-regions of cDNAs pDH7, pDH8 and pDH 101; there is commonality of bp at positions 530, 559-60,581,632 and 768 not found with V122(Fig. 2). However, only the V®ion of cDNA pDH101 probably originated in toto from V13. The CDR3 of pDH 101 is 7 1% similar to the CDR3 of Vi23 and 29% to the CDR3 of V12; pDH101 and VA3 have additional commonality at bp positions 624-626,794,801-803,811,813-814, 816-817,821-822 and 826-827, which is not found with V122,at these positions (Fig. 2). Interestingly, the V1 regions encoded by two other cDNAs, pDH7 and pDH8 appear to be composite sequences derived from both VJ.2 and V123genes. The CDR3s of pDH7 and pDH8 are 86% and 81 y0 similar, respectively, to the V12 CDR3 but only 48% and 52% similar to the CDR3 of VA3. The V-regions of these two cDNAs may, therefore, have arisen either by gene conversion exchange of CDR3s, or from, as yet, uncloned Vd genes.

ACKNOWLEDGEMENTS

We express our thanks to Dr. K. Knight, University of Illinois College of Medicine, Chicago, IL, for her generous gift of the AEMBL4-phage rabbit DNA library. We thank Ms. Ruth Bisig for her excellent technical assistance throughout this work and MS Thi Nha Huynh for her secrete&l assistance. This work was supported by grant No. 3.342-0.86 from the Swiss National Science Foundation.

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globulin lambda locus in rat consists of two C 1 genes and a single V I gene. Gene 55 (1987) 75-84. Udey, J.A. and Blomberg, B.: Human lambda light chain locus: organization and DNA sequences of three genomic J-regions. Immunogenetics 25 (1987) 63-70. Wall, R. and Kuehl, M.: Biosynthesis and regulation of immunoglobulins. Annu. Rev. Immunol. 1 (1983) 393-422. Yancopoulos, G.D. and Alt, F.W.: Regulation of the assembly and expression ofvariable region genes. Annu. Rev. Immunol. 4 (1986) 339-368.