CHARACTERIZATION OF HORSE (EQUUS-CABALLUS) IMMUNOGLOBULIN-MU-CHAIN-ENCODING GENES

Horse (Equus caballus) immunoglobulin mu chain-encoding (IgM) variable , joining, and constant gene segments were cloned and characterized. N ucleotide sequence analyses of 15 cDNA clones from a mesenteric lymph node library identified 7 unique variable gene segments, 5 separate jo ining segments, and a single constant region. Based on comparison with human sequences, horse variable segments could be grouped into either family 1 of immunoglobulin (Ig) clan I or family 4 of Ig clan II subc lan IV. All horse sequences had a relatively conserved 16 base pair (b p) segment in framework 3 which was recognized with high specificity i n polymerase chain reaction by a degenerate oligonucleotide primer. Ho rse complementarity determining regions (CDR) had considerable variabi lity in predicted amino acid content and length but also included the presence of relatively conserved residues and several canonical sequen ces that may be necessary in formation of the beta chain main structur e and conformation of antigen-binding sites through interaction with l ight chain CDR, Sequence analysis of joining regions revealed the pres ence of nearly invariant 3' regions similar to those found in human an d mouse genes. A single horse IgM constant region comprising 1472 bp a nd encoding 451 residues was also identified. Direct comparison of the horse constant region predicted amino acid sequence with those from e leven other species revealed the presence of 53 invariant residues wit h particularly conserved sequences within the third and fourth exons. Phylogenetic analysis using a neighbor-joining algorithm showed closes t similarity of the horse mu chain-encoding constant region gene to hu man and dog sequences. Together, these findings provide insights into the comparative biology of IgM as well as data for additional detailed studies of the horse immune system and investigation of immune-relate d diseases.