CLONING AND CHARACTERIZATION OF A NOVEL NK CELL-SPECIFIC SERINE-PROTEASE GENE AND ITS FUNCTIONAL 5'-FLANKING SEQUENCES

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30000 M(r) serine p rotease (granzyme) found in the cytolytic granules of CD3- large granu lar lymphocytes (LGL) with natural killer (NK) activity. To characteri ze the genomic sequences responsible for the CD3- LGL-restricted expre ssion of this gene, we screened a rat genomic library with RNK-Met-1 c DNA, and obtained bacteriophage clones that contained the RNK-Met-1 ge ne. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 k ilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5'-flanking region of the RNK-M et-1 gene contains a number of putative promoter and enhancer regulato ry elements and shares several regions of homology with the 5'-flankin g region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5'-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransfe rase (CAT) reporter gene. These 5'-flanking RNK-Met-1-CAT constructs w ere transiently transfected into rat LGL leukemia, T-lymphoma, and bas ophilic leukemia cell lines. The transcriptional activity of the RNK-M et-1 5'-flanking region was strong, restricted to the RNK-16 LGL leuke mia and controlled by several positive cis-acting regions spread over at least 3.3 kb. The longest and most active 5'-flanking region (-3341 to -33) was also used to drive specific expression of beta-galactosid ase in RNK-16. These data are consistent with the NK cell-specific exp ression of RNK-Met-1 and suggest the potential utility of this gene pr omoter in the development of transgene models of NK cell biology in vi vo.