PHYSICAL MAPPING OF THE RAT-TISSUE KALLIKREIN FAMILY IN 2 GENE CLUSTERS BY ANALYSIS OF P1-BACTERIOPHAGE CLONES

In several mammalian species the tissue kallikreins constitute gene fa milies whose members encode a highly related subgroup of the simple se rine proteases. Previous characterization of kallikrein genes in the r at identified 13 of the potential 15-20 members present in this specie s. To characterize comprehensively all members of the rat gene family and to define the linkage of family members, we have isolated clones b earing kallikrein genes from a rat genomic library constructed with th e P1 bacteriophage vector pAd10-SacBII. Library pools containing kalli krein genes were identified by polymerase chain reaction with primers complementary to regions highly conserved among members of the rat gen e family. Individual members of the family within the library pools we re identified with gene-specific PCR assays that rely upon short diver gent regions among the family members. Detection of multiple kallikrei n family members within single library pools suggested tight linkage o f the individual genes. Isolation and analysis of 12 kallikrein P1 clo nes confirmed the linkage of gene family members and established a phy sical map for two clusters of genes at the kallikrein locus. The linka ge relationships of the known gene family members within the two gene contigs are rKLK1-rRLK3-rKLK7rKLRS and rKLK8-rRLK2-rKLK6-rKLK4-rKLK10- rKLK12. Pulsed-field electrophoretic analysis of rat genomic DNA demon strated linkage between the two gene clusters, which form an extended locus that is most narrowly defined by a 440-kb BssHII fragment, and i dentified an unmethylated CpG island that is tightly linked to this lo cus. (C) 1994 Academic Press, Inc.