CLONING OF THE GENE FOR BOVINE MONOCYTE CHEMOATTRACTANT PROTEIN-2

Bovine monocyte chemoattractant protein-1 (bovine MCP-1) cDNA has rece ntly been characterized and shown to be highly expressed in bovine sem inal vesicles secretory epithelium as well as in phytohemagglutinin (P HA)-stimulated peripheral blood mononuclear leukocytes (PMNLs). In an attempt to isolate the MCP-1 gene, we screened a bovine genomic cosmid library with a MCP-1-specific probe pH42. A positive clone, c11/1, wa s subjected to restriction analysis and fragments probed with pH42 by southern blotting. pH42-positive fragments were subcloned and sequence d. The sequence revealed three exon-like regions that coded for a prot ein displaying an identity of 51% with bovine MCP-1. Employing this se quence information from c11/1, the c11/1-specific cDNA was generated f rom poly(A)CRNA of bovine PMNLs by reverse transcription sad a combina tion of polymerase chain reaction (PCR) methods. The assembled c11/1 c DNA comprised a 5' UTR coding region as well as 3' UTR for the gene pr oduct c11/1. Amino acid sequence comparison of the bovine c11/1 gene p roduct with human monocyte chemotactic proteins yielded the highest se quence identity with human MCP-2, and it is assumed that the c11/1 gen e product represents the bovine MCP-2. The exon/intron structure of th e bovine MCP-2 gene was found to be similar to the human MCP-1 gene. T he bovine MCP-2 gene consists of three exons separated by two introns. In the 5'-flanking region of the 3.3-kb gene, a TATA box as well as a n AP-1 sequence motif were identified. The bovine MCP-2 is specified b y a single-copy gene. Contrary to MCP-1, MCP-2 is not expressed in bov ine seminal vesicle tissue. In bovine PMNLs, expression of MCP-1 and M CP-2 is stimulated by PHA.