Identification of carrot cDNA clones encoding a second putative proliferating cell‐nuclear antigen, DNA polymerase δ auxiliary protein

Abstract

The proliferating cell-nuclear antigen (PCNA) plays a key role in the control of eukaryotic DNA replication. We have isolated two cross-hybridizing groups of cDNA encoding carrot homologs of PCNA. Sequence analysis and Southern-blot experiments showed that the cDNA were derived from two distinct genes. One corresponded to the typical PCNA, which is known to be highly conserved in eukaryotes from yeast to man; its mRNA is 1.2 kb in size and the calculated molecular mass of the protein is 29 kDa. The other encoded a larger PCNA homolog which has not previously been reported; the mRNA is 1.5 kb in size, the N-terminal three quarters (calculated molecular mass, 29 kDa) of the protein product is 88% identical at the amino acid level to the typical PCNA, but the protein has an extra C-terminal domain of 11 kDa. Both PCNA homologs were apparently coexpressed concomitant with somatic embryogenesis. The mRNA level of the novel homolog is 10 -20% that of the typical PCNA in the embryos. The presence of the second putative PCNA may provide new insight into studies on the mechanism of DNA replication in eukaryotes.The proliferating cell-nuclear antigen (PCNA) was originally discovered as a nuclear antigen in human proliferative cells [I]. Subsequently, it was identified as an auxiliary protein for DNA polymerase 6 [2, 31. PCNA plays an important role in eukaryotic DNA replication by increasing the processibility of the polymerase 6 during elongation of the leading strand [4-61. In addition, PCNA is a highly conserved protein in widely divergent species [7]. Previously, we showed that PCNA is also present in higher plants [8, 91 and that its expression is localized to the proliferating organs [lo]. So far, it has been believed that each organism contains only one molecular species of PCNA.Somatic embryogenesis in cultured carrot cells is an attractive system for investigating the mechanism of plant cell differentiation [Ill. A number of studies on the gene-expression program of somatic embryogenesis have been carried out and several genes were reported to be induced in the process [12-141. In those studies, however, the structures and functions of the genes remained unclear. We have recently Correspondence to S. Hata, Faculty of Science, Himeji Institute of Ahhrc4ution. PCNA, proliferating cell-nuclear antigen. Enzymes. DNA polymerase 6, modified T7 DNA polymerase (EC 2.7.7.7); restriction enzymes BcoRI, EcoT141, HindlII, PstI, XbuI (EC 3.1.21.4).Nore. The novel nucleotide sequence data published here have been deposited in the EMBL sequence data bank and are available under the accession numbers X62976 and X62977.Technology, Kamigori, Ako-gun, Hyogo, demonstrated that a mitotic cyclin gene is expressed concomitant with the embryogenesis [15].We undertook the present investigation to determine whether or not somatic embryogenesis occurs concomitant with the induction of PCNA expression. As a first step, we isolated cDNA for carrot PCNA using PCNA clones of other plants as probes. Unexpectedly, however, the cDNA formed two c...

A proposed conserved role for an avocado fw2.2-like gene as a negative regulator of fruit cell division

“…An analogous phenomenon has been reported in carrot, where two PCNA genes, a short and a long one, were found. SpeciWcally, the long atypical carrot PCNA contains the short typical PCNA sequence with an extra C-terminal extension of unknown function (Hata et al 1992). Although the justiWcation of the existence of two related versions of PCNA transcripts is unclear, expression studies, performed in carrot somatic embryos, suggested that the long PCNA protein might act as a 'helper' of the short typical protein (Hata et al 1992).…”

“…SpeciWcally, the long atypical carrot PCNA contains the short typical PCNA sequence with an extra C-terminal extension of unknown function (Hata et al 1992). Although the justiWcation of the existence of two related versions of PCNA transcripts is unclear, expression studies, performed in carrot somatic embryos, suggested that the long PCNA protein might act as a 'helper' of the short typical protein (Hata et al 1992). It is also worth noting, that in various plants species, two PCNA genes encoding distinct PCNA family members were isolated (Lopez et al 1997;Touneille et al 2002;Strzalka et al 2009Strzalka et al , 2010.…”

PCNA: structure, functions and interactions

“…PCNA, however, may also exist as a dimeric ring. During somatic embryogenesis of Daucus carota, the carrot, two distinct PCNA genes are expressed (Hata et al, 1992). One encodes a typical size PCNA (264 amino acids) while the other encodes a longer form (365 amino acids).…”

β*, a UV-inducible Shorter Form of the β Subunit of DNA Polymerase III of Escherichia coli

“…PCNA has usually a size of 29 kDa, and it forms a trimeric hexagonal ring-shaped structure very similar to that of the ␤ subunit (37). Interestingly, it was recently reported that two PCNA genes were found in carrot, encoding putative PCNA molecules of 29 and 40 kDa (38). Based on its size, its trimeric structure and its activity as a processivity factor, ␤* could thus be viewed as an analog of the T4 gp45 and the eukaryotic PCNAs, with a possible evolutionary link.…”