Molecular cloning of the gene for plant proliferating‐cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells

Abstract

A cDNA library was screened for plant proliferating-cell nuclear antigen (PCNA) from Catharanthus roseus (periwinkle). A 1 gtll cDNA library was constructed using poly(A)-rich RNA isolated from the cells in the S phase. A cDNA clone for PCNA was isolated by using a rice genomic clone, pCJ-1, which contains PCNA-related gene sequences. The cDNA contains an open reading frame of 804 nucleotides, encoding a protein of 268 amino acids with a molecular mass of 29765 Da. When conservative substitutions were included, a high degree of similarity (about 85%) was observed between the predicted amino acid sequence of periwinkle PCNA and that of human PCNA. Expression of mRNA for periwinkle PCNA was undetectable or very weak in quiescent cells, such as phosphate-starved cells, auxin-starved cells and cells in the stationary phase. In the synchronous progression of the cell cycle induced by the addition of phosphate or auxin, the active accumulation of periwinkle PCNA mRNA was observed preferentially in the S phase. When an inhibitor of DNA synthesis, aphidicolin, was added to the cells at the G I phase, an increase in the level of PCNA mRNA was observed. The partial inhibition of protein synthesis at the G1 phase by a protein inhibitor, anisomycin, caused the arrest of cells in the G1 phase. No increase of the level of periwinkle PCNA mRNA was observed in cells arrested at the GI phase by the inhibition of protein synthesis. These results indicate that the induction of mRNA for periwinkle PCNA occurred independently of the initiation of DNA replication, but that synthesis of certain proteins at the G1 phase was required for the induction of periwinkle PCNA mRNA at the S phase.Proliferating-cell nuclear antigen (PCNA) was initially recognized as a nuclear autoantigen which reacts with autoimmune sera from a certain group of patients with systemic lupus erythematosus (Miyachi et al., 1978). This antigen is an acidic, nuclear, non-histone protein and is present preferentially in the nuclei of proliferating mammalian cells and not in those of quiescent cells (Takasaki et al., 1984). Bravo et al. (1981) identified another nuclear protein, 'cyclin', with a molecular mass of about 36 kDa, whose synthesis is also correlated with the proliferative state of mammalian cells. Mathews et al. (1984) showed that PCNA and cyclin are identical. Immunofluorescence studies of the distribution of PCNA during the cell cycle have revealed that the expression of PCNA increases during the late G1 and early S phase, and also that the nuclear localization of PCNA changes dramatically in cells in the S phase Celis and Celis, 1985;Bravo, 1986;Kurki et al., 1986;Sadaie and Mathews, 1986;Moore et al., 1987). When the site Science, Tohoku University, Sendai, Japan 980 proliferating-cell nuclear antigen ; SV40, simian virus 40 of DNA synthesis was identified by labeling with bromodeoxyuridine or [3H]thymidine, a remarkable correlation was found between the distribution of PCNA and patterns of DNA synthesis . These observations suggest a role for PCNA ...

Isolation and characterization of the E2F‐like gene in plants

“…After removal of propyzamide, cells immediately exited the M phase and the mitotic index dropped rapidly and remained at low levels until 12 h. A second peak of the mitotic index was observed from 12 to 16 h. In contrast, a peak in the DNA synthesis was observed at 9 h, after which the rate of DNA synthesis gradually decreased. We have chosen the PCNA gene as a control, since expression of the Catharanthus roseus PCNA gene was preferentially observed in the S phase [17]. Consistent with this observation, the tobacco PCNA gene was also accumulated predominantly during the late G1 and S phases (Fig.…”

Fatty Acid Desaturation during Chilling Acclimation Is One of the Factors Involved in Conferring Low-Temperature Tolerance to Young Tobacco Leaves

“…Eight micrograms of total RNA were denatured and fractionated as described previously (Kodama et al, 1991). RNAs were blotted onto nylon membranes.…”

Molecular cloning of the cDNA for the catalytic subunit of plant DNA polymerase α and its cell‐cycle dependent expression

“…In higher plants, little is known about the cell cycle regulated genes and the mechanisms that regulate their transcription. We have previously isolated plant PCNA cDNA and demonstrated that the expression of the PCNA gene fluctuates during the cell cycle, with a peak during S phase, in synchronous cultures of Catharanthus roseus cells (Kodama et al 1991). Recently, S-phase specific expression of the ribonucleotide reductase gene has also been described (Philipps et al 1995).…”