The cloning and sequencing of the plant nuclear poly ADP-ribose polymerase gene
The project was to clone the gene encoding a nuclear enzyme poly-ADP-ribose polymerase (PARP) involved in the posttranslational modification of nuclear proteins. This modification is important in the regulation of various cellular processes such as cell differentiation, proliferation and in the molecular events involved in the recovery of cells from DNA damage. At the start of this project this enzyme had been well characterized in animal systems but had not as yet been explored in plants. Firstly, I showed the enzyme to be present in higher plant nuclei, by a series of experiments including enzyme assays and western blotting (probed with a polyclonal antibody specific to the protein). Molecular biology approaches were used to isolate the gene encoding the PARP enzyme. These techniques included screening of cDNA libraries, constructed in λZAPII, with both the polyclonal antibody and a gene probe (a restriction digest of a human PARP gene was performed and a 1.4Kb fragment containing the C-terminal region of the gene used as a probe). This identified a number of bacteriophages from which sequence information was obtained (by making and extracting phagemids). Subsequent translation of these nucleotide sequences revealed that one of the sequences (245941) obtained in this manner showed homology to the PARP protein. Degenerate oligonucleotide primers, designed to the conserved C-terminal region of the gene, were also used in an attempt to amplify a 375bp region of the gene thought to contain the PARP signature. PCR products of the expected size (375bp) were obtained from an SST cDNA library. These PCR products hybridized to the gene probe (mentioned earlier). Subsequent subcloning revealed multiple products of 375bp comigrating in the agarose gels. Two different sequences were obtained which were, upon translation, shown not be PARP-like sequence.