We have developed a novel cloning technique called recursive antisense cloning that allows, for the first time, the cloning of multiple genes of phenotypically-defined biochemical pathways. This is accomplished by reproducing any biochemical pathway (whose endpoint can be measured) in Xenopus oocytes, by injecting the oocytes with the appropriate poly A+RNA that is first prehybridized with groups of degenerate oligodeoxynucleotides that, in the aggregate, inhibit 96% of vertebrate mRNA species. Each group that demonstrates inhibition of the measured phenotype is then subdivided and extended in an iterative process. Seven iterations result in the generation of an oligonucleotide probe that recognizes, on average, a unique cDNA. We propose to apply this technique to the isolation of the genes encoding the proteolytic processing enzymes of Beta-amyloid peptide precursor protein. These enzymes are present in cells of the cerebral microvasculature, and their discovery has important implications for the understanding and treatment of Alzheimer's disease. For three reasons, this novel approach is highly appropriate for these studies: firstly, Xenopus oocytes injected with poly A+RNA have proven remarkably accurate in their reproduction of post-translational modifications and biochemical pathways, even reproducing the pathway of neurotransmitter precursor uptake, synthesis, vesicular packaging, and calcium-mediated release. Secondly, standard techniques have repeatedly failed to isolate these enzymes. Thirdly, we have shown that the study is feasible by injecting Xenopus oocytes with Beta-amyloid precursor protein messenger RNA and demonstrating both Beta-amyloid precursor protein and Beta-amyloid peptide synthesis.
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