Quantitation and subcellular localization of proliferating cell nuclear antigen (PCNA/cyclin) in oocytes and eggs of Xenopus laevis

Proliferating cell nuclear antigen (PCNA/cyclin) is a 36-kDa polypeptide present in the nuclei of mitotically active cells. It is known to be involved in DNA replication through an association with DNA polymerase δ. We examined the total content as well as the subcellular distribution of PCNA in the oocyte and the egg of Xenopus laevis by employing immunocytological staining and immunoblot analysis. While oocytes are not capable of replicating chromosomes, PCNA is abundant in the nucleus (about 65 ng per nucleus). The oocyte cytoplasm, on the other hand, does not contain a significant quantity of this protein. The amount of total PCNA does not change appreciably during oocyte maturation and the subsequent stages of egg cleavage. Thus, PCNA belongs to a class of proteins which are stockpiled during oogenesis in order to be utilized later for early embryogenesis.     

Egg maturation and parthenogenetic recovery of diploidy in the scorpion Liocheles australasiae (Fabricius) (Scorpions, ischnuridae)

In the scorpion Liocheles australasiae, egg maturation and parthenogenetic recoveries of chromosome number and nuclear DNA content were examined by histological, karyological observations and quantitative measurements of DNA. The primary oocyte becomes mature through two successive maturation divisions. The first maturation division takes place in the primary oocyte to produce a secondary oocyte and a first polar body. The second maturation division soon occurs in the secondary oocyte, in which the nucleus is divided into a mature egg nucleus and a second polar body nucleus, not followed by cytoplasmic fission. The first polar body, in one case, was successively divided into two second polar bodies; in the other case it was not divided. In either case, these polar bodies remained attached to the early embryo. The fate of these polar bodies during further embryogenesis were studied. In the karyological analysis, the chromosome number was divided into two groups, one from 27-32, the other was 54-64. The former was presumably the metaphase chromosome number at the meiotic division; the latter was presumably the metaphase chromosome number at the mitotic division. DNA content in the diploid nucleus of the primary oocyte, doubled before the maturation divisions, was reduced through the maturation divisions by one-half in the nuclei of the secondary oocyte and the first polar body and by one-fourth in the nuclei of the egg and the second polar bodies. The first reduction of DNA content corresponded to halving the number of the chromosomes in the first maturation division and the second to the nuclear division in the secondary oocyte. These reductions represent a common process of egg maturation, except the final production of the mature egg with two haploid nuclei, an egg nucleus, and a second polar body nucleus. These two nuclei, which were formed apart in the mature egg, drew near to fuse into a zygote nucleus. The chromosome number and nuclear DNA content were doubled in the zygote and each blastomere in embryos, supporting the hypothesis that the egg nucleus fuses with the second polar body nucleus and this conjugation initiates subsequent embryonic development.     

Maturation-promoting factor induces nuclear envelope breakdown in cycloheximide-arrested embryos of Xenopus laevis

We have studied the effect of maturation-promoting factor (MPF) on embryonic nuclei during the early cleavage stage of Xenopus laevis development. When protein synthesis is inhibited by cycloheximide during this stage, the embryonic cell cycle arrests in an artificially produced G2 phase-like state, after completion of one additional round of DNA synthesis. Approximately 100 nuclei can be arrested in a common cytoplasm if cytokinesis is first inhibited by cytochalasin B. Within 5 min after injection of MPF into such embryos, the nuclear envelope surrounding each nucleus disperses, as determined histologically or by immunofluorescent staining of the nuclear lamina with antilamin antiserum. The breakdown of the nuclear envelope occurs at levels of MPF comparable to or slightly lower than those required for oocyte maturation. Amplification of MPF activity, however, does not occur in the arrested egg as it does in the oocyte. These results suggest that MPF can act to advance interphase nuclei into the first events of mitosis and show that the nuclear lamina responds rapidly to MPF.     

Monoubiquitination of proliferating cell nuclear antigen induced by stalled replication requires uncoupling of DNA polymerase and mini-chromosome maintenance helicase activities

Proliferating cell nuclear antigen (PCNA) is a homotrimeric, ring-shaped protein complex that functions as a processivity factor for DNA polymerases. Following genotoxic stress, PCNA is modified at a conserved site by either a single ubiquitin moiety or a polyubiquitin chain. These modifications are required to coordinate DNA damage tolerance processes with ongoing replication. The molecular mechanisms responsible for inducing PCNA ubiquitination are not well understood. Using Xenopus egg extracts, we show that ultraviolet radiation and aphidicolin treatment induce the mono- and diubiquitination of PCNA. PCNA ubiquitination is replication-dependent and coincides with activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent DNA damage checkpoint pathway. However, loss of ATR signaling by depletion of the ATR-interacting protein (ATRIP) or Rad1, a component of the 911 checkpoint clamp, does not impair PCNA ubiquitination. Primed single-stranded DNA generated by uncoupling of mini-chromosome maintenance helicase and DNA polymerase activities has been shown previously to be necessary for ATR activation. Here we show that PCNA ubiquitination also requires uncoupling of helicase and polymerase activities. We further demonstrate that replicating single-stranded DNA, which mimics the structure produced upon uncoupling, is sufficient to induce PCNA monoubiquitination. Our results suggest that PCNA ubiquitination and ATR activation are two independent events that occur in response to a common single-stranded DNA intermediate generated by functional uncoupling of mini-chromosome maintenance (MCM) helicase and DNA polymerase activities.     

Immunoelectron microscopy of PCNA as an efficient marker for studying replication times and sites during pollen development

Here we report for the first time the ultrastructural localization of DNA replication sites in the nucleus of plant cells and the timing of replication through the pollen developmental programme by proliferating cell nuclear antigen (PCNA) immunogold labelling. Replication sites were identified by labelling with anti-PCNA antibodies in fibrils of the interchromatin region close to the condensed chromatin, defining a perichromatin subdomain in the interchromatin space where DNA replication takes place. The same nuclear structures are decorated by anti-BrdU (5-bromo-2'-deoxyuridine) immunogold after short pulses of BrdU labelling. Double immunogold labelling for PCNA and DNA show colocalization on these perichromatin structures. PCNA immunoelectron microscopy also allows correlation of replicative activity with the dynamics of chromatin condensation. DNA replication was also monitored at different phases during pollen development by PCNA immunoelectron microscopy, revealing two peaks of DNA synthesis, at the beginning (early tetrad), and the end (late vacuolate), of microspore interphase. High-resolution autoradiography after [3H]thymidine incorporation also showed high replicative activity at the same two periods of microspore interphase. In the bicellular pollen grain, PCNA immunogold labelling revealed that DNA replication in the generative cell starts at an intermediate stage of pollen maturation, whereas the vegetative nucleus does not replicate and is arrested in G1. The use of anti-PCNA antibodies at the ultrastructural level is an easier, faster and more feasible method than the detection of in vivo-incorporated nucleotides, especially in plant systems with long cell cycles. PCNA immunogold labelling is, therefore, proposed as an efficient marker for mapping the sites and timing of replication at the electron microscopy level.     

Nuclear-cytoplasmic interactions during ovine oocyte maturation

The present studies have been undertaken to investigate the interactions that occur between the nucleus and cytoplasm of ovine oocytes at various stages during meiotic maturation. We report that the nucleus of ovine fully grown dictyate stage oocytes can be efficiently removed by a microsurgical enucleation procedure. It is demonstrated that between the initiation of maturation and germinal vesicle breakdown certain newly synthesized polypeptides are selectively sequestered in the oocyte nucleus and the major sequestered polypeptide has a relative molecular mass of 28,000, which represent at least 9% of the total labelled polypeptides transferred to the oocyte nucleus during the first 4 h of maturation. The experiments provide evidence that the removal of the oocyte nucleus at various times before germinal vesicle breakdown (GVBD) does not prevent the major series of changes in protein synthesis that occurs after entry into a metaphase. We conclude therefore that the mixing of the nucleoplasm and cytoplasm is not essential for the initiation or progression of the protein reprogramming process during maturation. In addition, the experiments show that the development of the ability to condense chromatin during ovine oocyte maturation is independent of the oocyte nucleus. The combined results strongly support the hypothesis that the extensive series of translational changes that occur in oocytes during maturation are controlled by cytoplasmic rather than nuclear factors.     

The synthesis and storage of histones during the oogenesis of Xenopus laevis

Further data, including two-dimensional gel electrophoresis and peptide mapping of newly synthesized proteins, confirms the view that oocytes make several types of histone. The newly synthesized histone is present in both nucleus and cytoplasm, but at a higher concentration in the oocyte nucleus and in great excess over the DNA binding sites. The unfertilized egg seems to contain a pool of histones detectable on two-dimensional electrophoretograms. The peptide maps of these proteins are consistent with their identification as histones. The egg contains enough histone to support nuclear replication through most of cleavage.     

Rad18 guides poleta to replication stalling sites through physical interaction and PCNA monoubiquitination

The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase eta (poleta) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that poleta does not form nuclear foci in RAD18(-/-) cells after UV irradiation. Both Rad18 and Rad6 are required for poleta focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with poleta constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, poleta interacts preferentially with monoubiquitinated PCNA, but poldelta does not. These results suggest that Rad18 is crucial for recruitment of poleta to the damaged site through protein-protein interaction and PCNA monoubiquitination.     

Dynamic changes in subnuclear NP95 location during the cell cycle and its spatial relationship with DNA replication foci

We determined the expression and subcellular localization of nuclear protein NP95 during the cell cycle in mouse 3T3 cells. The levels of NP95 mRNA and protein were extremely low in quiescent cells; however, stimulation with 10% serum increased their expressions in a time course similar to that of the late growth-regulated gene proliferating cell nuclear antigen (PCNA). Subnuclear location of NP95 dynamically changed during the cell cycle. Double immunostaining for NP95 and chromatin-bound PCNA, a marker of DNA replication sites, revealed that NP95 was almost exclusively colocalized with chromatin-bound PCNA throughout the nucleus in early S phase and partly in mid-S phase. Distinct localization of the two proteins, however, became evident in mid-S phase, and thereafter, many chromatin-bound PCNA foci not carrying NP95 foci could be detected. In G2 phase, nodular NP95 foci were still identified without any chromatin-bound PCNA foci. Chromatin-bound PCNA was observed as a pre-DNA replication complex at the G1/S boundary synchronized by hydroxyurea treatment, while NP95 was detected in nucleolar regions as unique large foci. There was no significant redistribution of NP95 foci shortly after DNA damage by gamma-irradiation. Nodular NP95 foci characteristically seen in G2 phase were also detected in G2-arrested cells following gamma-irradiation. Taken together, our results indicate that NP95 is assigned to a late growth-regulated gene and suggest that NP95 does not take a direct part in DNA replication as part of the DNA synthesizing machinery, like PCNA, but is presumably involved in other DNA replication-linked nuclear events.     

Regulation of proliferating cell nuclear antigen during the cell cycle

The proliferating cell nuclear antigen (PCNA), also known as cyclin and DNA polymerase delta auxiliary factor, is present in reduced amounts in nongrowing cells and is synthesized at a greater rate in the S phase of growing cells. The recently discovered involvement of PCNA in DNA replication suggested that this pattern of expression functions to regulate DNA synthesis. We have investigated this possibility further by examining the synthesis, stability, and accumulation of PCNA in HeLa cells fractionated by centrifugal elutriation into nearly synchronous populations of cells at various positions in the cell cycle. In these fractionated cells we found that there is an increase in the rate of PCNA synthesis with a peak in early S phase of the cell cycle, but the magnitude of the increase is only 2-3-fold. This change reflects similar changes in the amount of PCNA mRNA. The fluctuating synthesis of PCNA maintains this protein at a roughly constant proportion of the total cell protein, although the amount doubles/cell in the cell cycle. Consistent with this observation, the stability of PCNA does not differ significantly from that of total cellular protein in synchronized HeLa cells. We also observed that a maximum of one-third of the total PCNA is tightly associated with the nucleus, presumably in replication complexes, at the peak of S phase. We conclude that the cyclic synthesis of PCNA in cycling HeLa cells maintains PCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.     

Proliferating cell nuclear antigen recruits cyclin-dependent kinase inhibitor Xic1 to DNA and couples its proteolysis to DNA polymerase switching

The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.     

Two discrete modes of histone gene expression during oogenesis in Drosophila melanogaster

We have used in situ hybridization to ovarian tissue sections to study the pattern of histone gene expression during oogenesis in Drosophila melanogaster. Our studies suggest that there are two distinct phases of histone gene expression during oogenesis. In the first phase, which occurs during early to middle oogenesis (stages 5-10A), we observe a mosaic pattern of histone mRNA in the 15 nurse cells of the egg chamber: some cells have very high levels of mRNA, while others have little or no mRNA. Our analysis suggests that there is a cyclic accumulation and subsequent degradation of histone mRNA in the egg chamber and that very little histone mRNA is transported into the growing oocyte. Moreover, since the endomitotic replication cycles of the nurse cells are asynchronous during this period, the mosaic distribution of histone message would suggest that the expression of the histone genes in each nurse cell nucleus is probably coupled to DNA replication as in most somatic cells. The second phase begins at stage 10B. During this period, histone gene expression appears to be "induced" in all 15 nurse cells of the egg chamber, and instead of a mosaic pattern, high levels of histone mRNA are found in all cells. Unlike the earlier phase, this expression is apparently uncoupled from the endomitotic replication of the nurse cells (which are completed by the end of stage 10A). Moreover, much of the newly synthesized histone mRNA is transported from the nurse cells into the oocyte where it accumulates and is stored for use during early embryogenesis. Finally, we have also observed tightly clustered grains within nurse cell nuclei in non-denatured tissue sections. As was the case with cytoplasmic histone mRNA, there is a mosaic distribution of nuclear grains from stages 5 to 10A, while at stage 10B, virtually all nurse cell nuclei have grain clusters. These grain clusters appear to be due to the hybridization of nurse cell histone gene DNA to our probe, and are localized in specific regions of the nucleus.     

A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells.

Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free SV40 origin-dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF-Cp145 we have mapped the DNA binding domain of RF-Cp145 to amino acid residues 369-480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent in other subunits of RF-C. The PCNA binding domain maps to amino acid residues 481-728 and is conserved in all five subunits of RF-C. The PCNA binding domain of RF-Cp145 inhibits several functions of RF-C, such as: (i) in vitro DNA replication of SV40 origin-containing DNA; (ii) RF-C-dependent loading of PCNA onto DNA; and (iii) RF-C-dependent DNA elongation. The PCNA binding domain of RF-Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481-728 of human RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in DNA replication in vivo.     

Effects of starvation on reproduction of the predacious mite <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> (Acari: Phytoseiidae)

Effects of starvation on gravid females of Neoseiulus californicus were investigated at 20°C and 85% RH. When females that had been reared with abundant prey were swapped, just after laying their first egg, to conditions without any prey and water, they laid 1.8 eggs and survived for 4.3 days. In the body of well-fed females, an egg with eggshell and/or two oocytes were observed in the ventral and dorsal regions, respectively. The larger oocyte had two roundish nuclei and abundant yolk granules, and was enveloped with a vitelline membrane. These two nuclei were not fused but were just close to each other. The smaller oocyte had a nucleus, but had not yet formed yolk granules and vitelline membrane. Females after 12 h starvation had an egg in the ventral region and an oocyte in the dorsal region of the body. After more than 24 h starvation females maintained an oocyte in the dorsal region of the body, but had no egg in the ventral region. The oocyte was filled with abundant yolk granules and contained two irregular nuclei when females were starved for 24 h, but when starved for more than 36 h it contained one irregular nucleus. These findings suggest that (1) gravid females maintained an oocyte in the dorsal region after laying two eggs during starvation, (2) the oocyte was not absorbed during starvation, (3) the oocyte advanced vitellogenesis and the fusion of two nuclei, and (4) the vitellogenic oocyte was not enveloped with an eggshell and had not started embryogenesis.     

Human CDC6/Cdc18 Associates with Orc1 and Cyclin-cdk and Is Selectively Eliminated from the Nucleus at the Onset of S Phase

In a two-hybrid screen for proteins that interact with human PCNA, we identified and cloned a human protein (hCdc18) homologous to yeast CDC6/Cdc18 and human Orc1. Unlike yeast, in which the rapid and total destruction of CDC6/Cdc18 protein in S phase is a central feature of DNA replication, the total level of the human protein is unchanged throughout the cell cycle. Epitope-tagged protein is nuclear in G₁ and cytoplasmic in S-phase cells, suggesting that DNA replication may be regulated by either the translocation of this protein between the nucleus and the cytoplasm or the selective degradation of the protein in the nucleus. Mutation of the only nuclear localization signal of this protein does not alter its nuclear localization, implying that the protein is translocated to the nucleus through its association with other nuclear proteins. Rapid elimination of the nuclear pool of this protein after the onset of DNA replication and its association with human Orc1 protein and cyclin-cdks supports its identification as human CDC6/Cdc18 protein.     

NMR studies of intracellular sodium ions in amphibian oocytes, ovulated eggs, and early embryos

23Na NMR, in combination with an anionic paramagnetic shift reagent dysprosium bis(tripolyphosphate), has been used to study intracellular Na+ in Rana oocytes, ovulated eggs, and early cleavage embryos. The technique allows accurate and simultaneous determination of both extracellular space and intracellular Na+ concentration. In prophase-arrested, follicle-enclosed oocytes, only about 17% of the total oocyte Na+ (approximately 40 mmol/kg of cells) was NMR-visible. Homogenizing oocytes in 0.24 M sucrose did not significantly affect the 23Na resonance. About 30% of the total oocyte Na+ was associated with the yolk platelets isolated at room temperature by differential centrifugation. NMR analysis, however, did not yield a detectable 23Na signal from these intact platelets. Thus, while yolk platelets are rich in Na+, this Na+ does not contribute to the oocyte 23Na NMR signal. Denuded oocytes, obtained by removing the follicular epithelium, gained about 10 mmol of total Na+/kg of cells and exhibited a comparable increase in NMR-visible Na+, suggesting the existence of compartments with varying degree of NMR visibility within the oocyte. Partially relaxed 23Na Fourier transform NMR spectra revealed the existence of at least two major intracellular compartments of NMR-visible Na+ with different magnetic environments and relaxation behavior in denuded oocytes. Since platelet Na+ appears to be NMR-invisible, one of the two observed compartments may be the nucleus. Progesterone action on the amphibian oocyte caused measurable changes in NMR-visible Na+. By ovulation (second metaphase), there is a gain in total egg Na+, and the NMR-visible Na+ is also increased. Following fertilization, however, there is some loss of total cell Na+ but, by the 2-4 cell stage, about 70% of the total Na+ becomes NMR-visible. These results indicate that a sizable fraction of the Na+ in follicle-enclosed, prophase oocyte is sequestered and located in NMR-invisible compartments and that changes in NMR-visible intracellular Na+ occur following hormonal and developmental stimuli.     

The modification of stored histones H3 and H4 during the oogenesis and early development of Xenopus laevis

In amphibian development large amounts of histone are accumulated at early stages and used in assembling nuclei at later stages, when cell proliferation is rapid. The acetate groups on stored H3 and H4 molecules turn over. In the oocyte nucleus H4 exists primarily in a diacetylated state, whereas it probably exists in a phosphorylated form in the cytoplasm. This represents, at steady state, what is normally a transitory stage in the transport of newly synthesized H4 into chromatin. Stored H3 of the oocyte has acetylated, and probably phosphorylated forms, in the same proportions as is normally seen in the chromatin of somatic cells. The various forms of H3 occur in similar proportions in the nucleus and cytoplasm of the oocyte. H3 does not therefore need to associate with DNA to be acetylated, even though the appearance of modified forms of H3 in cultured cells is seen only after the incorporation of H3 into chromatin.     

Arabidopsis thaliana: Proliferating cell nuclear antigen 1 and 2 possibly form homo- and hetero-trimeric complexes in the plant cell

The proliferating cell nuclear antigen (PCNA) is a key component of the eukaryotic DNA replication machinery. It also plays an important role in DNA repair mechanisms. Despite the intense scientific research on yeast and human PCNA, information describing the function of this protein in plants is still very limited. In the previous study Arabidopsis PCNA2 but not PCNA1 was proposed to be functionally important in DNA polymerase η-dependent postreplication repair. In addition to the above study, PCNA2 but not PCNA1 was also shown to be necessary for Arabidopsis DNA polymerase λ-dependent oxidative DNA damage bypass. Taking into account the reported differences between PCNA1 and PCNA2, we tested the idea of a possible cooperation between PCNA1 and PCNA2 in the plant cell. In a bimolecular fluorescence complementation assay an interaction between PCNA1 and PCNA2 was observed in the nucleus, as well as in the cytoplasm. This finding, together with our previous results, indicates that PCNA1 and PCNA2 may cooperate in planta by forming homo- and heterotrimeric rings. The observed interaction might be relevant when distinct functions for PCNA1 and PCNA2 are considered.