The human low affinity immunoglobulin G Fc receptor IIC gene is a result of an unequal crossover event.

We have isolated and characterized three genes coding for hFc gamma RIIA, IIB, and IIC. Each gene spans approximately 15-19 kilobases of DNA and consists of eight exons. Two exons encode the 5'-untranslated region and signal peptides, two exons code for homologous Ig-like extracellular domains, a single exon encodes the transmembrane spanning region, and three exons encode the cytoplasmic domains and 3'-untranslated regions. Analysis of gene structures support the concept that the hFc gamma RIIA and hFc gamma RIIB genes originated via gene duplication and divergence processes. The hFc gamma RIIC gene, however, showed a remarkable homology at its 5' end with the hFc gamma RIIB gene, whereas its 3' region was highly homologous with the hFc gamma RIIA gene, suggesting that the hFc gamma RIIC gene results from an unequal crossover event between the hFc gamma RIIA and IIB genes. This hypothesis was supported by nucleotide sequence analyses of the putative break-point region. The proposed site of recombination was located approximately 300 nucleotides downstream from the sixth (C1) exon. These data provide a unique model for the evolutionary generation of a receptor family with multiple biological functions.

Human IgG Fc receptors comprise a family of glycoproteins that can bind the Fc moiety of immunoglobulin type G. Interaction of antigen-antibody complexes with FcyR triggers multiple cell type-specific functions (reviewed in Refs. 1, 2). On human leukocytes three distinct h'FcyR classes are currently recognized, hFcyRI (CD64), hFcyRII (CD32), and hFcyRIII (CD16). The second class consists of different, albeit structurally related, 40-kDa molecules with low affinity for IgG (3-6). These molecules were found to be encoded by three genes, denoted hFcyRIIA, IIB, and IIC (7), all localized on chromosome 1q23-24 (7, 8). The transcripts derived from genes IIA (hFcyRIIa) and IIB (hFcyRIIb) differ both in their signal peptides and cytoplasmic domain encoding regions, whereas extracellular and transmembrane encoding regions are ~9 2 % homologous. The transcript derived from gene IIC (hFcyRIIc) was found identical in its signal peptide, extracel-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Apart from distinct genes, allelic variations have been identified (hFcyRIIA gene consists of HR and/or LR alleles, Ref.6), and their transcripts can be alternatively spliced (hFcyRIIb1,IIb2,and IIb3,Ref. 5 ) . The structural heterogeneity within the hFcyRII class of molecules results in functionally different receptors. For example, heterogeneity within the extracellular regions (hFcyRIIb, or IIc uersus hFcyRIIaLR, and IIaHR) results in profound differences in ligand binding capacity between the receptor isoforms (9, IO), while cytoplasmic tail variation (hFcyRIIa or IIc, uersus hFcyRIIbl, or IIb2) results in different functional properties between hFcyRII isoforms (11-13).' The molecular basis for diversity within this receptor family was studied by analysis of the genomic structure of human FcyRII. We observed several important differences compared to the hFcyRII genomic organization reported recently (7). Our data support the hypothesis that hFcyRIIA and IIB genes originated via gene duplication and divergence processes. More interestingly, restriction mapping and sequence analyses data suggest that the hFcyRIIC gene originated from an unequal crossover event between hFcrRIIA and IIB genes.

MATERIALS AND METHODS
Screening and Isolation of Genomic DNA Encoding Human FcyRZZ-A genomic DNA library, constructed in EMBL3 (14) using Sau3AI partial digests of human leukocyte DNA derived from a CML patient, was a generous gift of Dr. J. Hoeijmakers (Erasmus University, Rotterdam, The Netherlands). A second genomic DNA library, constructed in X FIX, using Sau3AI partial digests of human placental DNA, was obtained from Stratagene (La Jolla, CA). Approximately 2 X IO6 plaques of each library were screened with: 1) an EcoRI probe of cDNA clone pPW3 (nt: 1-1440, Ref. The probes were labeled with [a-32P]dCTP using a random primer labeling kit (Gibco, Paisley, Scotland). Hybridizing phage clones were isolated by repeated plaque purification and rescreening. Genomic DNA inserts from phage clones were isolated according to standard procedures (15).
Characterization of Genomic DNA Clones-Genomic DNA inserts were isolated from a total of 39 hybridizing phage clones, digested with SalI (in polylinker of EMBL3 and X FIX) and BarnHI, separated on 0.8% agarose gels, and analyzed with different probes (see below). Several independent phage clones were selected for further analysis.
using BarnHI, EcoRI, HindIII, and SalI, followed by Southern blotting The DNA inserts were analyzed by restriction endonuclease mapping of Three hFcyRII Genes analyses (16). Eight phage clones were selected, based on their restriction maps and hybridization patterns. Nucleotide sequences of the DNA inserts or subcloned DNA fragments were determined by the dideoxy chain termination method (17) using a T7 polymerase sequencing kit (Pharmacia) and synthetic oligonucleotides (16-18mers). The data obtained from Southern blotting and sequence analyses showed that the inserts of three phage clones contained parts of the hFcyRIIA gene, three phage clones parts of hFcyRIIC, and two phage clones hFcyRIIB gene fragments.
Southern Blot Analyses-High molecular weight genomic DNA was isolated from human leukocytes of 19 healthy donors by standard procedures (15). The genomic DNA was digested with BamHI, EcoRI, or HindIII, separated on 0.8% agarose gels, and blotted to Zeta-probe nylon membranes (Bio-Rad). Southern blots were hybridized in 0.25 M NaCl, 7% SDS, 1 mM EDTA, 50% formamide, supplemented with 50 pg/ml denatured salmon sperm DNA at 42 'C with either of the following cDNA probes: SIG A, EcoRI-PstI fragment of pPW3 After overnight hybridization, blots were washed at stringency in 0.3 X SSC, 1% SDS at 65 "C, and exposed for 20-72 h to Kodak XAR5 x-ray film.

RESULTS AND DISCUSSION
Organization of Three Distinct hFcyRII Genes-Several overlapping phage clones were isolated upon screening of two human genomic libraries with three hFcyRII-specific cDNA probes (see "Materials and Methods"). Restriction endonuclease mapping, Southern blotting analysis, and nucleotide sequencing of eight independent clones resulted in the construction of a complex genomic pattern consistent with three distinct genes encoding hFcyRI1: A, B, and C ( Fig. 1). All three genes were composed of eight exons located on =15-19 kb of DNA. The first two exons (S1 and S2) encoded the signal peptides, with the predicted signal peptidase cleavage sites located in the second, 21-nucleotide, mini-exon (Sa). A corresponding mini-exon has been found in all members of the FcyR gene families both in man and mouse, as well as in the gene encoding the a-chain of FctRI (7,(18)(19)(20)(21). In all three hFcyRII genes, two exons (EC1 and EC2) were found to encode a single C2 set Ig-like domain (22), and a single exon (TM) for a hydrophobic transmembrane segment. The sixth exon (Cl), present in all three genes, was previously found to be alternatively spliced in hFcyRIIB transcripts (3, 5, 10). Interestingly, this exon as well as its splice borders displayed extensive homology in all three genes, but until now no hFcyRIIa or IIc cDNA clones have been identified which include information encoded by this C1 exon. Moreover, insertion of the C1 exon in hFcyRIIa or IIc transcripts does not change their reading frames. Furthermore, two exons ( c 2 and C3) were found t o encode the main parts of the cytoplasmic regions. The deduced amino acid sequence of both of these exons of hFcyRIIa and IIc is remarkably similar (98% homology), in contrast to that of IIb (8% homology) (4-7). The 3"untranslated region of gene IIA can be =1 kb longer than that of IIC, due to the presence of one versus two polyadenylation signals in IIC, and IIA, respectively.
In conclusion, the physical maps of several overlapping independent genomic clones clearly show the presence of the three distinct hFcyRII genes. The organization of the hFcyRII genes show a remarkable resemblance between genes IIB and IIC upstream from the C1 exon, whereas downstream from C1 the organization of gene IIC was virtually identical to that of gene IIA (Fig. 1).
Southern Blotting Analyses-Leukocyte DNA from 19 Caucasian individuals was digested with BamHI, EcoRI, and HindIII and subjected to Southern blotting (Fig. 2 shows data from six individuals). Upon digestion with HindIII, three hybridizing bands sized 5.9, 9.5, and =19 kb were observed using an EC2-TM probe. The three hybridizing bands were found present in all 19 individuals and were consistent with the maps for genes IIA, IIC, and IIB, respectively. Southern blots probed with the SIG A probe revealed a single hybridizing fragment for each digest (Z20-kb BamHI, 3.8-kb EcoRI, and 1-kb HindIII fragments), as predicted from the restriction map of hFcyRIIA genomic clones. Southern analyses with the SIG B probe revealed single hybridizing fragments of 6.4 kb (BamHI) and 4.2 kb (HindIII) in all 19 individuals. This pattern was again consistent with the maps from the 5' region of genes IIB and IIC. One hybridizing band of 5.8 kb was noted with EcoRI-digested DNA in most individuals tested. However, in two out of 19 unrelated donors (one of which is shown in Fig. 2, lane 4, SIG B probe), we found two hybridizing bands, 5.8 and -8 kb. This extra band may reflect an EcoRI restriction fragment length polymorphism (RFLP) in the 5' region of either the hFcyRIIB or IIC gene. Moreover, the relative intensities of the two bands (EcoRI-digested DNA, probed with SIG B) in donor 4 versus the single ones in the other individuals, supported the hypothesis that the single hybridizing band represented identical fragments derived from two genes. Southern analyses of the 3' parts of the three hFcyRII genes further supported a strong homology between the hFcyRIIA and IIC genes. The 4.1-kb BamHI, 4.3-kb EcoRI, and 7.5-kb HindIII fragments observed to hybridize with the CYT A probe were compatible with sizes predicted from genomic hFcyRIIA and IIC clones. Southern blots with the CYT B probe revealed 3.5-kb BamHI, 4.6-kb EcoRI, and ~2 0 -k b HindIII fragments, all corresponding with the hFcyRIIB structural map (Fig. 1). Notably, a weakly hybridizing 4.2-kb BamHI band was visible upon longer exposure in genomic DNA probed with CYT B. This fragment corresponds to a BamHI fragment containing the C2 exon of hFcyRIIB, and the inefficient hybridization is likely attributable to the exon size (38 bp).
Taken together, the Southern analyses of genomic DNA from 19 unrelated Caucasian donors proved to be consistent  designated 1-6) was digested with BarnHI, EcoRI, or HindIII, separated on agarose gels, and transferred to nylon membranes. The Southern blots were analyzed using five different probes, SIG A , SIG B, EC2-TM, CYT A , and CYT B, as indicated.
Hybridizing fragments from the three genes are indicated by A, B, or C. Hind111 fragments of phage X-DNA served as size markers, indicated by horizontal bars, and are given in kb.

SIG A -
with the genomic organization of the three hFcyRII genes shown in Fig. 1.
The identification of three distinct genes encoding hFcyRII is consistent with the number of genes described by Qiu et al.  (7). However, we found several differences in the genomic organization of these genes, which lead to a significantly different concept of the evolution of this gene family. We identified two HindIII sites between the C2 and C3 exons in hFcyRIIA, whereas a single HindIII site was found by Qiu et al. (7). The presence or absence of this restriction site could reflect a HindIII RFLP. However, such a polymorphism may then be rare in Caucasians because we did not observe a HindIII RFLP in genomic DNA of 19 individuals. Next, we localized the EC1 exon within the hFcyRIIA gene at a different position than Qiu et al. (7). To confirm our data, we determined the location of EC1 in four independent genomic clones (two of which are shown in Fig. 1). In all four clones, EC1 was found located in a 0.9-kb HindIII-EcoRI fragment and not in the adjacent 1.8-kb EcoRI fragment (7).
The structural organization of hFcyRIIC was not entirely consistent with the IIC gene (referred to as hFcyRIIa') characterized in Ref. 7. Briefly, this study points out differences in the 5' regions between gene IIC and gene IIB and in the 3' region between IIC and IIA. Furthermore, the structural map of gene IIC concerning the region in which C1 and C2 exons were located in Ref. 7 was observed to be different from the corresponding region of the hFcyRIIC gene presented in this paper. Unfortunately, however, no Southern data were shown by Qiu et al. (7) precluding the verification of both the 5' and 3' regions of hFcyRIIC. Our data, however, argue against restriction length differences in the 5' regions of genes IIB and IIC (Fig. 2,probe SZCB). The differences in the 3' regions of genes IIA and IIC (7) could not be confirmed, neither in our genomic clones, nor by Southern analyses (Fig. 2, probe CYTA). The most outstanding differences, located in the region containing the C1 and C2 exons, were further analyzed in two genomic hFcyRIIC clones isolated from different libraries. Both clones contained a 1.4-kb EcoRI fragment (including the C1 exon), identical to such a fragment in gene IIA. Apart from the differences described above, the Southern blotting data presented by Qiu et al. (7) showed two BamHI and three HindIII hybridizing bands with a probe recognizing EC1. The hybridizing fragments and the corresponding genes (HindIII: =20 kb (B), = 10 kb (a'/C), and 3.3 kb (A); and for BamHI: =18 kb (A) and 6 kb (B and a'/C) Ref. 7) are in perfect agreement with the hFcyRII genomic organization shown in Fig. 1.
Evidence for an Unequal Crossover Event-The striking homology between genes IIB and IIC upstream from the C1 exon, and downstream between genes IIA and IIC, prompted us to evaluate whether an unequal crossover event between ancestral gene by duplication. The high percent of homology between IIA and IIB genes may have allowed these genes to align unequally. This resulted in an unequal crossover (indicated by X), resulting in the presence of three homologous hFcyRII genes (hFc-yRIIA, IIB, and IIB/A=IIC) encoding receptor molecules with multiple biological functions.
IIA and IIB genes resulted in the generation of IIC. Therefore, we sequenced the region surrounding the C1 exon from single phage clones containing each of the three genes. The EC2, TM, and C2 exons were also sequenced to identify the genetic origin of the genomic clones. Fig. 3 contains a schematic representation of the sequencing data of clones encoding genes IIA (upper row), IIC (center row), and IIB (bottom row). Each vertical line represents a single nucleotide difference between the genes IIA/IIB and gene IIC. The 5' region was found to be remarkably similar (99.8% homology) between genes IIB and IIC. In contrast, homology between genes IIA and IIC in this region is approximately 80%, including several insertions and deletions relative to the corresponding regions of genes IIC and IIB. The 3' portion, the sequences of genes IIA and IIC displayed 98.7% sequence homology, with less homology between genes IIB and IIC (-78%). Remarkably, the region indicated between the arrows (Fig. 3) exhibited the highest homology between all three hFcyRII genes (99.8% homology between IIA and IIC, 98.9% between IIB and IIC, and 97.9% between IIA and IIB). This strong homology, as well as the absence of insertions or deletions render this region a likely candidate for a homologous recombination event. We, therefore, postulate that the hFcyRIIC gene resulted from an unequal crossover event between the IIA and IIB genes with a break-point located -300 bp downstream from the sixth (Cl) exon. It is important to note that the pattern of gene evolution supported by our data is different from that derived from work of Qiu et al. (7). These authors suggested that the hFcyRIIA gene was generated via recombination of the hFcyRIIC and hFcyRIIIA genes.
It is noteworthy that we found an at instead of a regular gt (23) splice border of the C2 exon in gene IIC. This unusual splice border was also identified by Qiu et al. (7) in -75% of the donors and was found to be accompanied by a stop codon in exon EC1. Upon sequencing the genomic hFcyRIIC clone, however, we did not observe such a stop codon in exon EC1 and can, therefore, exclude that the genomic IIC clone (shown in Fig. 3) contains a pseudogene.
In conclusion, we have characterized three genes encoding the class I1 Fc receptor for IgG. Our data support the concept that hFcyRIIA and IIB genes are derived from a common ancestral gene. An unequal crossover event between the hFcyRIIA and IIB genes most likely lead to the generation of a third gene, hFcyRIIC (as depicted in Fig. 4). This recombination event may have occurred relatively recent in evolution since intron sequences displayed a high degree of homology. This notion is further supported by the observation that in the murine system only a single gene has been identified for FcyRII.