Waseem, Naushin Halim; (1991) A functional and immunological analysis of the proliferating cell nuclear antigen. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Proliferating Cell Nuclear Antigen (PCNA) is a cell cycle regulated protein involved in DNA replication. The cDNA of rat PCNA was subcloned in various expression vectors. A fusion protein of PCNA with p-galactosidase was produced by subcloning rat PCNA cDNA in plasmid pUR288. The PCNA cDNA was also expressed as protein A fusion protein. PCNA in an unfused form was produced by subcloning rat PCNA cDNA in a T7 polymerase based expression system. These bacterially expressed fusion proteins were used to immunise Balb/c mice which were then used to raise monoclonal antibodies against PCNA. Eleven new monoclonal anti-PCNA antibodies were raised and designated PC1 to PC11. All the eleven antibodies recognised denatured antigen on a western blot. Ten antibodies recognised native antigen in an immunoprecipitation reaction. Antibodies PC4, PC7 and PC9 do not stearically compete with the rest of the anti-PCNA antibodies. Immunofluorescence studies performed with these antibodies showed three distinct patterns. A large group comprising of PC1, PC2, PCS, PC4, PCS, PC6, PCS, PC10 and PC11 showed nuclear staining which showed cell cycle variation. PC7 failed to give any positive reaction on immunofluorescence while PC9 showed only nucleolar staining. Monoclonal antibodies PC2, PC3, PCS, PCS and PC10 recognised PCNA from Schizosaccharomyces pombe and Spodoptera frugiperda. One of the antibodies showing strongest reactivity with PCNA in S. pombe, PC10, was used to screen a S. pombe lamda gt11 library. A cDNA of PCNA homologue in S. pombe was isolated. Sequence comparison with other known PCNA cDNA showed that this cDNA was incomplete at its 5'end. This was then used to screen a S. pombe genomic library. A PCNA gene was isolated, sequenced and analysed. This 2.1 kb gene contains a single intron. Gene disruption experiments were carried out in S. pombe. The PCNA gene was found to be essential for the viability of S. pombe.

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