Molecular cloning and expression of a cDNA encoding the proliferating cell nuclear antigen from Brassica napus (oilseed rape)

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Abstract
A cDNA clone encoding the proliferating cell nuclear antigen (PCNA) has been isolated from a Brassica napus apical meristem cDNA library. The putative full-length cDNA contains an open reading frame of 1004 nucleotides, which predicts a protein of 263 amino acids (Mr = 29 231). Sequence analysis has revealed that the plant PCNA exhibits 81.6% amino acid similarity with the human PCNA. Genomic Southern blot analysis indicates the presence of at least two copies of PCNA per genome. The B. napus PCNA mRNA (1.0 kb) was expressed in rapidly dividing tissues such as flower buds, apical meristems, and young leaves, while mature stem and fully expanded leaves showed significantly lower levels of PCNA transcript. The B. napus PCNA cDNA was expressed in Escherichia coli as a fusion protein with glutathione-5-transferase (GST) in the bacterial expression vector pGEX-2T. A broad specificity monoclonal antibody raised against rabbit PCNA cross-reacted with the GST–PCNA fusion peptide but not with the GST moiety alone. This antibody also recognized the human PCNA (36 kDa) polypeptide, confirming the structural similarities between the human and plant PCNA. The high degree of structural conservation of PCNA from such diverse organisms as humans and higher plants suggests that the plant PCNA may function in a manner analogous to that found in mammals with respect to plant cell DNA replication. Such conservation suggests that PCNA is also a critical component of the plant cell DNA replication complex.Key words: proliferating cell nuclear antigen, DNA replication, DNA polymerase-δ, cell cycle regulation, Brassica napus (oilseed rape).

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