Specific amplification of cDNA ends (SPACE): A new tool for the analysis of rare transcripts and its application for the promoter analysis of killer cell receptor genes

Abstract

The expression control of activating and inhibitory killer cell Ig-like receptors (KIR) on natural killer (NK) cells is highly relevant for the initiation of NK cell mediated cytolysis and cytokine secretion. Transcription start points of nine human KIR genes from two Caucasian donors and the NK cell line NK3.3 were investigated. To overcome sensitivity problems due to the low abundance of the respective transcripts, a novel protocol, specific amplification of cDNA ends (SPACE) with superior specificity and sensitivity was applied. A total of 235 individual SPACE clones resulting from different KIR genes were analysed and revealed a series of transcription start sites tightly clustered between 10 and 60 bp upstream of the start codon. The comparison of the adjacent putative promoter region of the human, chimpanzee and macaque KIR genes revealed a very high conservation for almost all of the KIR family members. An inter-gene and inter-species comparative approach revealed transcription factor binding sites at regions of maximal homology for all primate KIR genes analysed.