A homologue of the ubiquitous eukaryotic cell cycle regulatory gene,
cdc2, has been cloned from
Pisum sativum, the garden pea. A novel immunological strategy was devised and implemented for screening PCR products generated by degenerate oligonucleotide primers. We used PCR to construct a deletion derivative of an
Escherichia coli expression plasmid carrying the
Schizosaccharomyces pombe cdc2 gene. The deleted segment encoded the domain recognized by monoclonal antibody MAb-J4, a reagent which also detects a single protein in extracts of all plant species we have examined. PCR products, generated by appropriate
cdc2 primers, were ligated into new restriction sites flanking the deletion, reconstituting the deleted epitope. This strategy, first validated on a cloned yeast
cdc2 template as control, was applied to the highly efficient cloning of a cDNA segment comprising 60% of the pea
cdc2 homologue. DNA sequencing revealed strong amino acid sequence conservation among the
cdc2 gene products from pea, yeast and animal cells. Genomic Southern analysis indicated that the
cdc2 gene occurs as a single copy in pea. An additional
cdc2-like clone was recovered which displays amino acid sequence similarity with that of pea
cdc2. The reported cloning and screening strategy, though limited by the availability of appropriate immunological reagents, provides not only an efficient means of screening heterogeneous PCR products generated by degenerate probes and/or low stringency PCR, but also product verification by immunological criteria.
Key words cell cycle - cell division - mitosis - phosphorylation - protein kinase