The murine CR genes Crry (previously termed mCRY) and Crry-ps (previously termed mCRX) are two distinct, but related, sequences which are the evolutionary homologs to sequences contained within the human CR1 gene. Screening a BALB/c genomic DNA library with the Crry/Crry-ps specific cDNA resulted in the isolation of two clusters of genomic sequences: those specific for Crry and those specific for Crry-ps. The coding sequences of the Crry gene encompass over 25 kb of DNA, whereas the Crry-ps sequences are included within a single 5.6-kb Eco-R1 fragment. The Crry gene consists of 10 separate exons. The first of these contains both the signal sequence and an alternatively spliced 129 bp present in approximately 10% of the Crry transcripts. Of the remaining exons, two encode a single sixty amino acid repeat domain each (A and E), two encode a split sixty amino acid repeat (B), and another encodes two 60 amino acid domains (C and D) fused as one exon. The transmembrane and cytoplasmic regions are both split into two exons each. RNA protection analysis indicates that although there is alternative splicing in the 5' region of the gene, the 3' exons encoding the terminal 60 amino acid repeat, the transmembrane region and cytoplasmic exons are used in the same order in all Crry transcripts. This suggests that the Crry gene product is not found as a secreted protein, but only as a cell surface bound protein. DNA sequence analysis of the Crry-ps gene indicates that this sequence most likely represents a pseudogene resulting from a processed mRNA transcript from the Crry gene. This conclusion is based on the lack of intervening sequences in the Crry-ps gene and the observation that the Crry-ps gene sequence contains both an 11-bp deletion within the "coding" region and a degenerate poly A tail at the 3' end of the homologous sequence. Additionally, RNA protection analysis indicates that mRNA cannot be detected which matches the Crry-ps sequence.
The mouse genome contains two sets of gene sequences which are highly homologous to the gene encoding the human C3b/C4b receptor (CR1). These genes, termed murine CRY (mCRY) and murine CRX (mCRX) reside on murine chromosomes 1 and 8, respectively. Analysis of cDNA isolated by using these sequences as probes indicates that there are two related but distinct mRNA which are expressed in a wide variety of murine tissues including spleen, liver, lung, and brain. Both of these transcripts encode proteins which should contain a signal sequence for membrane insertion, a transmembrane/cytoplasmic tail region for membrane anchoring, and five extracellular domains made up of 60 amino acid consensus repeat sequences. The difference between the two is the presence of an additional exon of 129 bp immediately 3' of the signal sequence. This additional exon does not encode a 60 amino acid repeat. The sizes of the mature proteins predicted from the cDNA sequences are 43,998 Mr and 48,680 Mr; however, antisera raised against carboxy-terminal sequences detects a 70,000 Mr protein from murine fibroblasts suggesting a high degree of post-translational modification of the mature protein. A comparison of these murine gene sequences with a partial human CR1 sequence suggests that the human CR1 gene evolved by direct duplication of the ancestral coding sequences contained within these murine genes including those sequences important for membrane anchoring and cytoplasmic protein attachment.
CR2, a 145,000 to 150,000 Mr protein which binds specific breakdown products of C3, has been identified on the surface of both human and murine B cells. In order to understand the evolutionary relatedness of the human and murine proteins, we have used the coding sequences from the human CR2 gene to investigate those homologous sequences of murine Cr2. Human CR2 cDNA sequences were used as probes on a cDNA library derived from BALB/c spleen mRNA to identify cross-reacting cDNA sequences. A number of putative cDNA clones encoding murine Cr2 have been isolated and examined. DNA sequence analysis of these Cr2 cDNA clones indicates that they represent the murine homolog to human CR2. mRNA analysis with these Cr2 cDNA clones has revealed a transcription pattern similar to, but distinct from that seen for CR2. Whereas human CR2 coding sequences identify a single mRNA species of approximately 5 kb from human tonsillar mRNA, the murine counterpart identifies four transcripts from murine spleen of approximately 3, 5, 9 and 11 kb in size. The Cr2 cDNA clones which detect the four forms of spleen mRNA overlap in coding sequences and contain exons mapping to three colinear fragments as defined by EcoRI digestion. This suggests that the 3- 5-, 9-, and 11-kb mRNA forms arise by alternative splicing from a single gene. Use of these murine Cr2-specific cDNA clones to isolate their respective genomic sequences has allowed for the linkage of the 3' end of the Cr2 gene to the 5' end of the Crry gene, the evolutionary homolog to human CR1.
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