Andrea Lautner-Rieske, Christian Huber, Alfons Meindl., Walter Pargent., Karlheinz F. Schable, Rainer Thiebe, Ines Zocher and Hans G. Zachau lnstitut fiir Physiologische Chemie der Universitat Munchen, Munchen

1023

The A regions of the human immunoglobulin x locus

Eur. J. Immunol. 1992. 22: 1023-1029

The human immunoglobulin x locus. Characterization of the duplicated A regions” The central regions of the x locus, the so-called A regions, have been fully characterized on cosmid and phage h clones. The regions, which are parts of the C,-proximal and -distal copies of the locus and are, therefore, called Ap and Ad regions, comprise about 140 kb each and contain together 30V, genes and pseudogenes. The A regions have been linked on their 5’ sides to the 0 regions and on their 3‘ sides to the L regions. Chromosomal walking has eliminated a previous gap in the A p region. Detailed restriction maps of the Ap and Ad regions and the sequences of 9 V, genes are reported. Four events, which have occurred in evolution probably after the duplication of the A region, were identified: the insertion of an Alu element in Ad; the insertion of part of a LINE element in Ap; the deletion of a 17.5-kb fragment including oneV, gene from Ap; the sequence divergence of duplicated V, gene regions which ranges among the five pairs studied here from 0 to 14 bp per kb and converted two genes to pseudogenes while their duplicates stayed functional. An analysis of the A regions of the lymphoid cell lines RPMI 6140 and GM607 confirmed the previous finding that the V,-J, rearrangement in these cell lines had occurred by deletion and inversion mechanisms, respectively. Thus, the structural data contribute to the understanding of the evolution and the functioning of the A regions of the x locus.

1 Introduction The structure and the functioning of the human immunoglobulin x locus have been studied for the past dozen years. After early work by Bentley and Rabbitts [1,21 we cloned systematically V, genes and their surroundings. Contigs were constructed from overlapping cosmid and phage clones and were linked by chromosomal walking. A comprehensive map of the locus was eventually established. Large parts of the locus turned out to be duplicated, giving rise to a C,-proximal (p) copy and a similar, but not identical, distal (d) copy. In the course of the work three fully or partly duplicated “regions” and one unique region emerged from the respective contigs: Op/Od [3], Ap/Ad [4], Lp/Ld [5], and the B-J,-C, region [6,7]. Several structural and functional aspects of the x locus have been reviewed [S, 91 and a general overview was given in a recent study on polymorphisms and haplotypes in the locus [lo]. In the ongoing effort to elucidate the structure of the 3t locus, structurally or functionally important aspects are being published at intervals. However, for reasons of

clarity, it is necessary to present also the structures of the various regions in context.This has been done for all regions at the early stages of the investigation but it has to be done again once the gaps within and between the regions are closed by chromosomal walking, by detailed restriction maps being established and by all V, genes, which we have detected, being cloned and sequenced. Reports which fully describe parts of the x locus have been published for the Op/Od regions [3] and the B-J,-C, region [6, 71. The corresponding data on the Ap/Ad regions are presented here and the ones on the Lp/Ld regions will follow soon (C. Huber et al., in preparation). In these reports all available data are compiled; they will, therefore, serve as sources of reference on the regions of the x locus. Certain aspects concerning all regions of the x locus will be described separately, as for instance the structures of some pseudogenes, the cloning of the locus in yeast artificial chromosomes, and the search for transcribed regions between theV, genes, etc. It is probable that one day somebody will sequence the whole 3t locus as part of the Human Genome Project.

2 Materials and methods [I 101681

*

This work was supported by the Bundesministerium fur Forschung und Technologie, Center Grant 0316200A, and the Fonds der Chemischen Industrie. Present address: Abteilung fur padiatrische Genetik und pranatale Diagnostik der Kinderpoliklinik der Universitat Munchen, Miinchen Present address: Dorner KG, Mullheim/Baden

Correspondence: Hans G . Zachau, Institut fur Physiologische Chemie, Schillerstr. 44, W-8000 Miinchen, FRG Abbreviations: OpIOd, ApIAd, LpILd, B regions: V, gene containing contigs of the human x locus, proximal (p) and distal (d) copies, rcspectively

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992

2.1 Enzymes, reagents, blot hybridization, sequencing The reagents and techniques were essentially the same as in [3,4]. All sequences have been submitted to the EMBL Data Library. The accession numbers are as follows: A l , X63402; A7, X63401; A13, X63395; A17, X63403, A18, X63396; A19 X63397; A20 X63398; A26, X63399; A29, X63400; ~256-1, X63392; ~656-11, X63393; ~659-12, X63394.

2.2 Recombinant DNA libraries The cos 607 clones are derived from a cosmid library of lymphoid cell line GM607 DNA [7]. Cos 780 was isolated

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Figure 1. Restriction maps of Ad and Ap regions of the human x 1ocus.Thescale in kb is a continuation of the one of the 0 regions [3].The last 0 genes and the first L gene are included into the map of Ad.The two maps are aligned for maximum homology. Parentheses in Ad (position 183; 0.7 kb) and Ap (position 152; 0.3 kb and positions 251-268.5) indicate parts that have no equivalent on the other copy. Positions of restriction sites that are identical in both regions are indicated by small vertical bars. Arrowheads point to sites that are present in only one of the regions; some of the sites have been characterized as duplication-differentiating polymorphisms [lo]. No cleavage sites were found for the restriction nucleases Nru I, Not I, Sal I, and Pvu I. The genes and their leaders are indicated by dark boxes; the roman numerals refer to their subgroup specificities. In addition to the cosmid clones described in [4] the following ones are shown: cos 256 [14]; cos 146, cos 149, and cos 607/7 [3]; cos 60711-3were isolated and mapped by H. G. Klobeck and G. m Combriato and kindly donated; they contain the recombination points between the J,-C, region (dashed lines) and the Ad region (solid lines). cos 60711 comprises about 4 kb of Ad, the A3-JX1 C7 joint, and reaches beyond the kde element [24]; cos 607/2 and 3 contain 12-13 kb of 5‘ J, region, the signal joint of the A3 flank and the J,1 flank [35] and parts of the Ad region as indicated; 4 cos 607/5 was isolated from the same library [7] by screening with the probe m656-1 [11]; the isolation of cos 780 and the h phage clones h5, h313, and h314 are described in Sect. 2.3. Subclones are shown as small horizontal bars and designated p for plasmids (pBR322, pUC, or Bluescript SK-) or m for M13 clones.The subclones in gene regions, which have been used for sequencing are described in Fig. 4 and in [4, 14, 16, 17, 18-21l.The duplication-differentiating probes have also been published: m142-2in [4], p516-16 in [lo], m146-5,6in [lo], m237-1 in [4], p313-1 in [lo], m659-2 in [lo]; m656-6 is a 0.9-kb Bam HI / Sp HI clone in M13mp 18 (G. Weichhold, unpublished); p780-1 is a 1.1-kb Xba I fragment in Bluescript SK-.

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Eur. J. Immunol. 1992. 22: 1023-1029

with the help of the probe p313-1 from a cosmid library of placenta N DNA partially digested by Bgl I1 and cloned in the vector pHC 79 (library Vb; [ll]). The h clone h5 stems from the libraryof Lawn et al. [12, 13].The clones 1313und A314 were isolated from a library of partially Eco RIdigested placenta N DNA in the vector Charon 40 [14] by screening with the probe m237-1.

3 Results and discussion 3.1 The restriction maps of the Ap and Ad regions

When the data on the A regions were compiled 4 years ago [4] there was still a gap in the Ap region and only one of the 0 regions had been linked to Ap by chromosomal walking. The linkage of both 0 and A regions has been described in the meantime [3] and the closing of the gap in Ap is reported here. It was achieved by two sequential steps of chromosomal walking: screening of a phage h library with m237-1 yielded the clone h313 and screening of a cosmid library with the subclone p313-1 eventually yielded the clone cos 780 (Fig. 1). The linking of the A and the L regions has been achieved recently and will be described (C. Huber et al., in preparation). We show in Fig. 1all cosmid and h clones covering the A regions and include also some subclones, which have been published previously (Fig. 1in [4]) but still play a role in current work and in the present report. More detailed restriction maps of individual cosmid clones, of subclones and of V, gene regions are presented in a number of dissertations [ l l , 13-18] and other publications [19-211. 3.2 The V, genes of the A regions

The 30 V, genes and pseudogenes of the A regions have been sequenced. Some of them have already been published [4, 19-21] and some pseudogenes will be compiled together with pseudogenes from other regions of the x locus (K. F. Schable et al., in preparation). In the present report the sequences of eight potentially functional V, genes (or genes with only minor defects) of the A regions are described and reference to the systematic survey of the A region genes in [4] is given. Since all nucleic acid sequences are being transmitted to the EMBL Data Library and for most genes the sequencing strategies are available in dissertations, we restrict ourselves here in most cases to reporting the formal translation products of the genes. It should be mentioned, however, that all gene sequences were determined on both strands. Sequence differences

The A regions of the human immunoglobulin

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1025

between duplicate gene pairs were verified by comparing the autoradiograms; the mostly minor corrections in, and the extensions of, published sequences (A2, A10, A l l , A23, A27, A28) have been transmitted to the EMBL Data Library. In the following the potentially functional V, genes of the A regions are surveyed together with some genes bearing only minor defects. The gene A1 was sequenced in the subclone p211-1 (Fig. 1) and the 3'adjacent p211-2 [16] and the duplicate gene A17 in the m237 subclones (Fig. 1) [17].The two genes differ in the coding regions at one position (TGG/Trp as codon 54 in A1 vs. a CGG/Arg codon in A17; Fig. 2a). There are seven differences in the intron and numerous others in the upstream and downstream sequences. The rearranged V, gene of the lymphoblastoid cell line RPMI6410 ([22]; correction of three non-coding positions in [17]) is clearly derived from A17: the two genes differ only in codon 95 (TCC/Ser vs. CCTPro) and in six positions in the noncoding region while there are altogether 23 differences to the A1 gene. The RPMI6410 and the A17 genes share also the above-mentioned Arg codon, which distinguishes them from the A1 gene. While the gene A2 has been published already [21], its duplicate A18 as sequenced in m237 subclones [17] is reported here.The two genes differ in seven positions in the coding regions and in five positions in the intron. The translation products are shown in Fig. 2b. A18 contains a stop codon at the end of FR3 and is, therefore, a pseudogene. A2 codes for the light chain of a major anti-Haemophilus influenzae antibody [21]. An individual who is lacking the distal copy of the x locus [3, 101including A2 cannot use A18 instead because of its pseudogene character but on vaccination with the Haemophilus polysaccharide resorts to other genes of the proximal copy which are adapted to the antigen by somatic mutations [23]. The gene A19 was sequenced in the p313 subclones by a strategy analogous to that published for its duplicate A3 [4]. Since the coding parts of the two genes are identical, no sequences are shown. There are three differences in the introns, however, and several others in the upstream and downstream region. The intron and upstream differences also identify the rearranged V, gene of the cell line GM607 as a product of the A3 and not the A19 gene.The aberrantly rearranged V, gene of the cell line GM2132 is probably derived from A19, at least according to the one difference in the intron which was spanned by the (incompletely determined) sequence of the rearranged gene [24]. Consequently, the GM607 gene was formed by an inversion of the

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Figure 2. Formal translation products of threeV,II gene pairs of the A regions. Full sequences are given for A l , A N , and A 7 and the differences in the duplicates A17, A2, and A23 are indicated. Invariant amino acids ([29]; in the leader region own sequence comparisons) are underlined. A line above amino acid symbols indicates that an invariant amino acid has been replaced by another amino acid.

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Eur. J. Immunol. 1992. 22: 1023-1029

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found in this cell line (H. G. Klobeck, personal communication). The gene A4 was found to be a pseudogene in sequencing two independent isolates ([4]; V52 in [25]) while the duplicate A20, which was recently sequenced in p780-1[18] is potentially functional. As can be seen in Fig. 3, A4 becomes a pseudogene by a fifth G compared to four G in the A20 sequence (arrow in Fig. 3). Since the sequencing of G clusters is error prone the A4 situation was rechecked and confirmed. For the A20 situation there is independent proof in the sequences of cDNA clones derived from this gene ([26]; R. Klein, unpublished). After the duplication of the x. locus, probably both the A4 and A20 genes were functional and later the A4 gene became a pseudogene upon insertion of a G, possibly by an error in replication. The heptanucleotide signal sequence CACTGTG, present in both A4 and A20, instead of the normal CACAGTG

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Eur. J. Imrnunol. 1992. 22: 1023-1029

The A regions of the human immunoglobulin x locus

1027

seems to permit V,-J, rearrangements as would be expected from studies in model systems [27, 281. The pseudogene pairs A5lA21 and A6lA22 have been published [4, 201 or will be elsewhere (K. F. Schable et al., in preparation). The gene A7 which is the duplicate of the published A23 gene [4] was sequenced in the p212 subclones [16].The two genes differ in four positions of the coding regions and in several positions in the sequenced upstream and downstream parts. The protein sequences are shown in Fig. 2c. It is noteworthy that in A7 the invariant cysteine in framework 1 [29] is replaced by a phenylalanine residue. The pseudogene pairs ASIA24 and A9lA25 will be published elsewhere (K. F. Schable et al., in preparation). The gene A26 as sequenced in the subclone p663-5 [15] is remarkable since it is fully identical to its duplicate A10 [19] from 262 bp upstream of the leader to 115bp downstream of the gene. The formal translation products of the genes A10 and A26 do not belong to one of the classical V, subgroups I-IV. We previously classified the gene A10 together with A14 as members of a sequence family N [19]. Since cDNA derived from A10 or A26 and from B2=EV15 [30] have recently been found and, therefore, also the corresponding proteins probably exist, one may as well talk of subgroupV for the B2 gene and subgroup VI for A10lA26 and A14 (Fig. 1). TheV,gene pair AlllA27 has been published [4,31,32], as was the pseudogene A28 [4], while the duplicate of the latter, the A12 gene, will be included into a later report. For the duplicate genes A13 and A29, which are not documented in a dissertation, the sequencing strategies together with the nucleic acid and protein sequences are shown (Fig. 4).The two genes, which differ from each other in several positions, are potentially functional except for a change in the initiation codon: ATA is found instead of ATG. Apparently, a mutation has occured prior to the duplication of the n locus. Since a gene with only one defect may be completely intact in another individual, as it was observed e.g. for an Lregion gene [5], we hesitate to classifyA13 and A29 as generally non-functional but rather call them genes with minor defects. The gene A30 [4, 171 turned out to be the duplicate of L14 [14] and will be dealt with in the context of the L region genes (C. Huber et al., in preparation). The pseudogene pairs 09lA15 and 010/A16 at the transition between the 0 and the A regions will be described elsewhere. 3.3 Insertions and deletions in the A regions

The maps of the Ap and Ad regions were aligned for maximal homology. At three positions, gaps had to be introduced in order to achieve this homology. At position 152 of the map (Fig. 1)an Alu element seems to have been inserted into the d copy after the duplication of the n locus. Formally, an insertion of the element before the duplication and subsequent deletion from the p copy

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Figure5 An Alu element in the Ad region helps to identify a rearranged V, gene in a lymphoid cell line. The Alu probe BLURS (see text) was kindly donated by P. L. Deininger. The restriction maps around the Alu element are shown for the germline situation of Ad (Ap for comparison) and for the rearranged allele of GM 607 as represented in cos 60712 and 3. The Alu element is indicated by an open box and regions that hybridize to the duplicationdifferentiating clone p313-1 are shown as bars. The rhomboid designates the signal joint between the 3' flank of the A3 gene and the 5' flank of a J,1 element (see text). This 5' flank is shown as a dotted line (as in Fig. 1).

cannot be excluded, but this is unlikely. The genomic situation is depicted in Fig. 5. An Eco RI fragment was found to be 0.3 kb larger in Ad than in A p and the pertinent fragments of the cosmid clones from the Ad region hybridize with the authentic Alu clone BLUR8 [33]. The subclone p313-1, which spans in Ap the site of Alu insertion into Ad, is unique and can serve as a duplicationdifferentiating probe [lo]. A 0.7-kb size difference was detected at position 183 between homologous fragments of the duplicated regions [4]. The fragment from A p was now shown to hybridize with a LINE probe (pcD 11, a,b of [34], kindly donated by M. F. Singer) while the corresponding Ad fragment does not. This indicates that part of a LINE element had been inserted into Ap; but the situation has not been further investigated. It was previously found that the gene A14 of the d copy had no duplicate on the p copy, and that the homology between Ap and Ad ends at position 251. It was not clear, however, whether this is due to a local deletion or whether we had reached the end of the duplication of the x locus [4]. The reason is now shown to be large deletion in Ap between positions 251 and 268.5. A detailed analysis of the genomic situation at the 3' end of the A regions and in the continuing L regions [14] showed that the homology is resumed at the 5' side of the A30 gene which is the duplicate of the L14 gene (Fig. 1 in [14]). We consider it likely that a 17.5-kb fragment including the duplicate of the A14 gene was deleted from Ap after the duplication of the n locus. The presumptive deletion breakpoints were sequenced in the subclones p659-12 of A p and compared to the sequences of the clones p656-11 and p256-1 of Ad (Fig. 1). As can be seen from Fig. 6 the 5' break in Ap had occurred within a LINE element [34] and the 3' break within the upstream region of a V,I gene. Although recombinations and deletions at repetitive sequences are known to occur no specific mechanistic scheme can be drawn for the present case.

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Eur. J. Immunol. 1992. 22: 1023-1029

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G C c G T C CA TC T C TAGC TGC C T T T A A C A T T T T T T T T C T T T CA TTTTA A C.TG A A G A TTA

LINE

I I I I I I I I I I I I I1 I l l I I I I I I I I TCTCTAGCTGCATATACTTACAAATATATATGCATCATCATGTC~TATATTTTATTTTTAA I I I I IlIIIIIIIIIIIIIlIIIIIIIIIIIIIllllIlIIIIIlIIlIII

659-12

ACACCATTACCTCATACTTACAAATATATATGCATG

656-11

256-1

Figure 6. Sequences defining the deletion break points at the 3' side of the A regions. The sequence of p659-12 of the A p region is compared to the sequences p656-11 and 256-1 of Ad. Sequences were determined in one strand only.The parts near the breakpoints are shown, but the full sequences [14] have been sent to the EMBL Data Library. The homologous part of a LINE sequence ([34]; here in reverse orientation) which is 78 % identical in the compared regions is shown at the deviating positions. Dots are introduced for missing nucleotides in order to maximize homology.The 256-1and 659-12sequences contain 5' flanks of the L14 and A30 genes, respectively.They are highly homologous to the flanks of other V,I genes of the L regions [5].

3.4 The A regions in two lymphoid cell lines with rearranged V, genes

explained by defining the borders of a deletion between which most probably the missing V, gene was located originally. Another event that had occurred after the In the two cell lines RPMI6410 and GM607 one of the two duplication of the 1c locus is the insertion of an Alu element alleles is in the germ-line configuration while in the other at position 152 of Ad. This insertion is an easily recognizalleles the genes A17 and A3, respectively, are rearranged able difference between the two copies, which, together to J,1 (Fig. 1 and [22,35]). In RPMI6410 the A17-Jn1 with other duplication-differentiating polymorphisms [lo], rearrangement had occurred by a deletion mechanism and, allows to study the prevalence of the duplication in human consequently, in blot hybridizations with digested cell line populations. At the level of DNA sequences, which were DNA duplication-differentiating probes, as m237-1, determined mostly in the V, gene regions, the differences detected an about twofold higher amount of the d copy- between the two copies vary in different parts of the derived fragments than of the p copy-derived fragments. A regions.There are no differences at all within the 1043bp This is true for all probes on the 3' side of Al/A17 but not of the A10 and A26 gene regions but a 21-bp difference for the ones on the 5' side of the genes (see Fig. 1 and between the 1482 bp of the A1 and A17 gene regions. A Table 2 of [lo]). In GM607 the A3-J,1 rearrangement had more detailed analysis of sequence differences between the taken place by an inversion mechanism and hence no DNA duplicated V, genes will be presented in a later report was lost. The amounts of Ap- and Ad-derived fragments (K. F. Schable et al., in preparation), but for a number of were about equal except for the Bam HI-generated frag- reasons it will be difficult to give, on the basis of such ments detected by the probe p313-I; here the dcopy- sequence differences, a reliable estimate of how old the derived band is weaker than the p copy-derived one, but an duplication of the x locus is. For instance, there may have additional fragment of 7.5 kb is seen which stems from the been, in addition to the primary mutations, some gene conversion-like events and recombinations between the signal joint-carrying region (Fig. 5; Table 2 in [lo]). two copies of the locus which render the sequences more similar again.

4 Concluding remarks In the present report new data on the Ap and Ad regions are presented. The A regions, which constitute the central parts of the x. locus, comprise 140 kb each and contain together 30 V, genes and pseudogenes.The data allow us to draw the following conclusions as to the evolution of this part of the human genome. The duplication of the ?c locus including the A regions must have been a relatively late event .This is concluded from the fact that the gross organization of the Ap and Ad copies is practically identical: most V, genes and also most restriction sites occur in both copies in the same positions; the transcriptional polarities of the V, genes are the same within the copies and several alterations which convert genes into pseudogenes are found in both duplicate copies. The one exception of the V, gene duplication, i.e. the absence of a duplicate of the A14 gene, has now been

'

The Ap and Ad regions are the central parts of the p and d copies of the x. locus, respectively, which continues with the 0 regions on one side and the L regions on the other side. The structural information presented here contributes to the understanding of the functioning and the evolution of this part of the locus. We thank H . Schek and R. Larnrn for expert technical assistance.

Received December 2, 1991.

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