Proc. Nail. Acad. Sci. USA Vol. 87, pp. 2856-2860, April 1990 Immunology Genomic organization of mouse Fcy receptor genes (Fc receptors/exon-ntron junctions/protein domains/immunoglobulin gene superfamily) ANTHONY KULCZYCKI, JR.*t, JENNIFER WEBBER*, HAUNANI A. SOARES*, MICHAEL D. ONKEN*, JAMES A. THOMPSON*, DAVID D. CHAPLIN*t, DENNIS Y. LOH*t, AND JEFFREY P. TILLINGHAST* *Department of Medicine and tHoward Hughes Medical Institute, Washington University School of Medicine, Saint Louis, MO 63110 Communicated by David M. Kipnis, January 16, 1990 ABSTRACT We have isolated and characterized the gene responsible for intrathymic deletion of a large fraction of coding for the mouse Fc receptor that is termed Fc,,RHa. The potentially self-reactive T-cell clones (11-13). gene contains five exons and spans approximately 9 kilobases. An understanding of the relationship of the various Fc Unlike most members of the immunoglobulin gene superfam- receptor genes to each other, the factors influencing their ily, this gene utilizes multiple exons to encode its leader peptide. evolution, and molecular mechanisms regulating their The first exon encodes the hydrophobic region of the signal expression requires a detailed analysis of the structures of sequence; the second exon, which contains only 21 base pairs, their genes. Here we report the characterization of two encodes a segment of the signal peptidase recognition site; and members of this gene family. the beginning of the third exon encodes the predicted site of peptidase cleavage. The third and fourth exons each code for immunoglobulin-like extracellular domains. The fifth exon MATERIALS AND METHODS encodes the hydrophobic transmembrane domain and the Screening of Mouse cDNA and Genomic Libraries. A NZW cytoplasmic tail. Partial characterization of the FcyRIIb gene mouse thymus cDNA library in phage AgtlO was screened by indicates that it also contains multiple leader exons, including using the 32P-end-labeled oligonucleotide probes 5'-GGA- a 21-base-pair exon and two exons coding for homologous CCTGGCTCCGGATGGACCTCCCATTGTGGAAC- immunoglobulin-like extracellular domains. However, the CACTG-3' and 5'-GAAATAAAGGCCCGTGTCCACTG- Fc,,Rlb gene uses four exons to encode its intracytoplasmic CAAACAGGAGGCACATC-3', which correspond to region. Analysis using contour-clamped homogeneous electric FcrRIIb1 cDNA and FcRIIa cDNA (nucleotides 544-583 field (CHEF) gels indicates that the FcRlIa and FcRIlb genes and 723-762, respectively) (3). Two NZW cDNA clones were are linked within 160 kilobases on mouse chromosome 1. isolated. One clone, "a," contained an insert of 0.8 kilobase (kb) specific for FcyRIIa starting at "FcyRa" nucleotide 706 Fc receptors are a family of cell surface proteins that bind to (3). A second clone, "b," contained a 1.5-kb insert that the Fc regions of immunoglobulins and thereby enable anti- corresponds to "FcrRI31," starting at nucleotide 273 and gen-antibody interactions to influence cellular responses (for including 510 nucleotides with 96.7% identity between reviews, see refs. 1 and 2). Interestingly, not only can FcrRIIb and FcrRIIa (3). distinctive Fc receptor genes be expressed on different cell The 1.5-kb cDNA insert of clone b was labeled with 32P by types, but also different spliced messages from the same Fc the random priming method (14) and was used to screen two receptor gene can be expressed in a tissue-specific manner genomic libraries: a mouse BALB/c library in EMBL-3 (1-6). The result of antigen binding to antibody molecules (Clontech), and a mouse BALB/c library in the cosmid that are bound to different Fc receptors is also dependent vector pTCF (15). Screening, hybridization, and washing upon cell type (1, 2)-e.g., ingestion and processing of procedures were carried out as described (16-18). Isolated antigen in macrophages (6), regulation of antibody synthesis colonies were rescreened with an Fc,,RIIa-specific probe (a in B cells, and triggering ofhistamine release from mast cells. 0.42-kb Bgl I fragment of the cDNA insert of clone a). Mouse macrophages express three distinct types (1) of Fc Initial screening of 500,000 plaques of the BALB/c ge- receptors specific for IgG (FcR): one (FczRI) that specifi- nomic library in EMBL-3 yielded three independent clones cally binds monomeric IgG2a; a second (FcrRII) that binds containing exons 4 and 5 of the FcrRIIa gene (see Fig. 1) and aggregated IgG1, IgG2a, and IgG2b (7, 8); and a third three independent clones containing portions of the FcYRIIb FcRIII) that binds only the minor subclass, IgG3. Two gene (see Fig. 3). From a BALB/c genomic cosmid library of different but homologous cDNAs (FcyRIIa and FcyRIIb) 300,000 colonies, four independent overlapping cosmid each encode receptors for aggregated IgG (2-5). The gene clones were isolated, and three of these clones contained all encoding FcYRIIa is expressed only in macrophages (3, 4), five exons of the FcrRIIa gene. and its expression is selectively induced by y interferon (6). Characterization ofGenomic Clones. DNAs from phage and The gene encoding FcRIIb is expressed in both macro- cosmid clones and specific subcloned fragments were char- phages and lymphocytes, with alternative splicing producing acterized by standard restriction endonuclease mapping and two transcripts, b, and b2, which differ only in' their cyto- Southern blot analyses. DNA sequencing of specific restric- plasmic regions (3-5). The genes for FcYR that have been tion fragments subcloned into pBluescript SK vectors (Strat- cloned (FcRIIa and Fc7RIIb genes) are genetically insepa- agene) was performed by using the chain-termination se- rable from the Mls-i locus on mouse chromosome 1 (9-11). quencing method. Coding regions and exon-intron junctions The Mls-i locus controls B-cell products that stimulate mixed were sequenced in both directions by using T3 and T7 lymphocyte reactions in H-2-compatible strains and, with the promoter primers of pBluescript SK and synthetic oligonu- class II major histocompatibility complex molecule I-E, are cleotides (20-mers) corresponding to known exon or intron The publication costs of this article were defrayed in part by page charge Abbreviations: FcR, Fc receptor(s) specific for IgG; CHEF, con- payment. This article must therefore be hereby marked "advertisement" tour-clamped homogeneous electric field. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2856 Downloaded by guest on September 30, 2021 Immunology: Kulczycki et al. Proc. Natl. Acad. Sci. USA 87 (1990) 2857 1kb Fc1R]b A|T C T T G C T G C T GGGA C T C A Fc1Rla c TET T GC GACAGGCAGAGTG FceR oc chain C T G T GT CA TAACAW RB R H B B H H R )I - D Leu Ala Ala Gly Thr His (Asp) exons 1 2 3 4 5 FcRllb (Asn) FcRla (Ala) Phe Ala Asp Arg Gln Ser (Ala) [AIa ; Leu] FcER os chain (Ser) Leu Gly Val Met Leu Thr (Ala) B 3.7 . B 3.9 P. R 2.5 I- R 10.1 . l FIG. 2. Comparison of the 21-bp exons of the FcYRIIb, FcYRIIa, and FcRI a-chain genes and the segments of signal sequence that FIG. 1. Structure of the mouse gene. Exons are num- FcYRIIa they encode. Nucleotide homologies are indicated in boxes. Amino bered and shown as boxes. All restriction sites forBamHl (B), EcoRI acids in parentheses are encoded the exons, (R), and HindIll (H) are indicated. The four subclones used for partially by 21-bp whereas bracketed amino acids are encoded by the adjacent exon. are labeled their restriction site bound- mapping and sequencing by Arrows indicate preferred predicted sites of peptidase cleavage for aries and size. Exon 1 is represented from the start of translation. Fc,,RIIb (3-5), FczRIIa (see text), and FcRI a-chain (25) gene products. sequences. Nucleotide sequences were analyzed by using the MicroGenie program (Beckman). lular segment, the transmembrane and cytoplasmic regions, Contour-Clamped Homogeneous Electric Field (CHEF) and the 3' noncoding region. The exons range in size from 21 Gels. Mouse J774 DNA was prepared in low-gelling- bp (exon 2) to 648 bp (exon 5). All nucleotides in the coding temperature agarose as described (19), and aliquots were region for mouse Fc,,RIIa match the published cDNA clone digested with restriction enzymes. Samples were electropho- (3). resed in 1% agarose gels with a CHEF hexagonal array Introns range in size from 0.66 kb (intron A) and 0.85 kb apparatus (20) for 28 hr. Field direction was reoriented by (intron B) to 3.5 kb (intron C). The shortest introns flank the using a switch ramp from 0.3 to 15 sec with the field strength smallest exon. The poly(A) signal AATAAA (27) is found in at 6 V/cm. Gels were hybridized at 65°C with 32P-labeled exon 5 (nucleotides 1307-1312). Fc,,RIIb-specific (Apa I/Bgl I) or FcYRIIa-specific cDNA Partial Map ofthe Gene Encoding FcRIIb. In screening the probes and were washed with 0.5x SSC at 650C (lx = 0.15 BALB/c genomic library in EMBL-3, we isolated three M NaCI/0.015 M sodium citrate, pH 7). clones that contained portions ofthe FcYRIIb gene. A partial map of the FcYRIIb gene was constructed from these clones RESULTS (Fig. 3) and includes six of the exons. The FcYRIIb gene, like the Fc,,RIIa gene, contains a 21-bp exon that encodes a Organization and Sequence of the Mouse Gene Encoding portion of the leader peptide required for the peptidase FclRla. A restriction map derived from cosmid clones of cleavage site [Fig. 2, top (Fc,,RIIb) sequences; also denoted genomic DNA containing the FczRIIa gene is shown in Fig. L* in Fig. 3]. The FcYRIIb gene also contains two exons that 1.