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Abstract
The human C3d/Epstein-Barr virus receptor (CR2/CD21) is a 145-kDa protein primarily expressed on mature B lymphocytes. CR2 is a member of the regulators of complement activation (RCA) gene family found on band q32 of chromosome 1. The RCA proteins are characterized by the presence of 60-70 amino acid short consensus repeats (SCR). A full length CR2 cDNA was cloned and used to identify overlapping cosmid genomic clones. Analysis of CR2 exon-intron junctions revealed the presence of three types of exons in the short consensus repeat region of CR2. First, four exons each of which encodes two SCR are present. Five exons encode a single SCR. Six exons encode SCRs which are split in identical positions. The order of these types of exons is in a repeated array of four SCRs, indicating that the contemporary CR2 gene likely evolved from a more primitive gene containing four SCRs. The CR2 full length cDNA clone was used to find restriction fragment length polymorphisms (RFLPs). Restriction enzyme TaqI generated 2.55- and 2.10-kilobase (kb) polymorphic bands. This RFLP was mapped near the exon containing the first two SCRs. HaeIII digestion generated polymorphic bands of 1.45, 1.55, and 1.75 kb. The HaeIII 1.45-kb RFLP band maps near the exon containing the 15th SCR. The TaqI and HaeIII RFLPs will provide tools for the genetic analysis of CR2. The organization of the CR2 gene provides insights into the evolution of human CR2 and the RCA gene family.
Highlights
CD21) is a 145-kDa protein primarily expressed on cleavage of C3b
A large surface glycoprotein lators of complement activation (RCA) genefamily found on band q32 of chromosome 1.The RCA proteins are characterized by the presenocfe60-70 amino acid short consensus repeats (SCR).A full length CcRD2NA was cloned and used to identify overlapping cosmid of the Epstein-Barr virus, gp350/220, is the viral ligand for CR2 [5, 6]
We describe here the isolation of cDNA and genomic clones forCR2 as well asthe exon-intronstructure of the SCR containing and more 3‘ exons of the CR2 gene.The genomic structure demonstrates anovel type of exon in the regulators
Summary
Isolation, Subcloning, and Sequencing of CR2 cDNAs-Total RNA was isolated from the lymphoblastoid cell line Raji using the guanidine isothiocyanate method [26]. 400,000 independent cosmid clones were screened with the same conditions as cDNA screening, except hybridization was performed at 42 "C in 50% formamide and the wash at 56 "C. Overlapping cosmids were initially mapped using at least six restriction enzymes (EcoRI, BamHI, PstI, HincII, HindIII, and KpnI) and specific cDNA subclones (see RFLP analysis below). GenomicDNA Purification, Electrophoresis, and Transfer-Genomic DNA was purified from peripheral leukocytes of 31 healthy. Caucasians and their family members and from Epstein-Barr virustransformed human B cell lines by proteinase K digestion and phenol extraction.
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