Amounts of nuclear DNA in anurans of the USSR

Summary The amounts of nuclear DNA in blood erythrocytes of 18 species of Anura from the USSR have been determined to be in the range of 4.0–20.6 pg (10^–12 g). Brown frogs of genusRana have lower mean genome sizes than green frogs. Palaearctic Anura, as a whole, have a greater content of nuclear DNA than the species of the same families from regions further south.     

DNA sequence organisation in relation to genome size in birds

Summary DNA sequence interspersion was investigated in pheasant and pelican nuclear DNA. As is typical for birds, these genomes are organized in a long period pattern. Altogether, 5 bird species with genome sizes between 1.6 and 1.9 pg DNA are compared with regard to the extent of repetitive and single copy sequence interspersion. The result indicates that the average length of interspersed repetitive sequences increases with genome size.Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft. Mrs H. Tuczynski and Mr J. S. Jackson are thanked for editorial assistance.     

Stable transmission of reversible modifications: maintenance of epigenetic information through the cell cycle

Even though every cell in a multicellular organism contains the same genes, the differing spatiotemporal expression of these genes determines the eventual phenotype of a cell. This means that each cell type contains a specific epigenetic program that needs to be replicated through cell divisions, along with the genome, in order to maintain cell identity. The stable inheritance of these programs throughout the cell cycle relies on several epigenetic mechanisms. In this review, DNA methylation and histone methylation by specific histone lysine methyltransferases (KMT) and the Polycomb/Trithorax proteins are considered as the primary mediators of epigenetic inheritance. In addition, non-coding RNAs and nuclear organization are implicated in the stable transfer of epigenetic information. Although most epigenetic modifications are reversible in nature, they can be stably maintained by self-recruitment of modifying protein complexes or maintenance of these complexes or structures through the cell cycle.     

Regulation of the cell cycle following DNA damage in normal and Ataxia telangiectasia cells

A proportion of the population is exposed to acute doses of ionizing radiation through medical treatment or occupational accidents, with little knowledge of the immedate effects. At the cellular level, ionizing radiation leads to the activation of a genetic program which enables the cell to increase its chances of survival and to minimize detrimental manifestations of radiation damage. Cytotoxic stress due to ionizing radiation causes genetic instability, alterations in the cell cycle, apoptosis, or necrosis. Alterations in the G1, S and G2 phases of the cell cycle coincide with improved survival and genome stability. The main cellular factors which are activated by DNA damage and interfere with the cell cycle controls are: p53, delaying the transition through the G1-S boundary; p21^WAF1/CIPI, preventing the entrance into S-phase; proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), blocking DNA replication; and the p53 variant protein p53as together with the retinoblastoma protein (Rb), with less defined functions during the G2 phase of the cell cycle. By comparing a variety of radioresistant cell lines derived from radiosensitive ataxia talangiectasia cells with the parental cells, some essential mechanisms that allow cells to gain radioresistance have been identified. The results so far emphasise the importance of an adequate delay in the transition from G2 to M and the inhibition of DNA replication in the regulation of the cell cycle after exposure to ionizing radiation.     

Integrase, LEDGF/p75 and HIV replication

HIV integrates a DNA copy of its genome into a host cell chromosome in each replication cycle. The essential DNA cleaving and joining chemistry of integration is known, but there is less understanding of the process as it occurs in a cell, where two complex and dynamic macromolecular entities are joined: the viral pre-integration complex and chromatin. Among implicated cellular factors, much recent attention has coalesced around LEDGF/p75, a nuclear protein that may act as a chromatin docking factor or receptor for lentiviral pre-integration complexes. LEDGF/p75 tethers HIV integrase to chromatin, protects it from degradation, and strongly influences the genome-wide pattern of HIV integration. Depleting the protein from cells and/or over-expressing its integrase-binding domain blocks viral replication. Current goals are to establish the underlying mechanisms and to determine whether this knowledge can be exploited for antiviral therapy or for targeting lentiviral vector integration in human gene therapy. Received 25 November 2007; received after revision 7 January 2008; accepted 10 January 2008     

Compared expression of murine plasmacytoma associated virus in tumor cells and in mouse embryo cells]

A DNA complementary to the viral genome of C-type particles produced by a Mouse myeloma derived cell line (MF2 cell line) was synthesized. This cDNA was used as a probe to study the viral genome expression among the total RNA and the poly (A)-rich RNA extracted from the MF2 and Balb/c embryonic cells. As evidenced by molecular hybridization experiments, the presence of at least one endogenous Balb/c virus in the MF2 virus stocks is suggested. In the productive cells, the viral RNA sequences are expressed in the poly (A)-rich RNA fraction.     

Geminivirus DNA replication

Geminiviruses are DNA viruses which infect plants. They have a small genome and encode only a few proteins. Therefore, their DNA replication cycle relies largely on the use of cellular DNA replication proteins. The strategy used by geminiviruses to replicate their single-stranded DNA (ssDNA) genome consists of a first stage of conversion of ssDNA into double-stranded DNA (dsDNA) intermediates and, then, the use of dsDNA as a template to amplify viral dsDNA and to produce mature ssDNA genomes by a rolling-circle replication mechanism. In addition, the accumulating evidence indicates that viral DNA replication is somehow coupled to the cell cycle regulatory network of the infected cell. For these reasons, geminiviruses are excellent model systems to understand the regulation of DNA replication and cell cycle in plant cells. Recent years have witnessed significant progress in the identification of cis-acting signals and their interaction with trans-acting factors that contribute to geminivirus origin function. These and other aspects of the geminivirus DNA replication cycle will be reviewed.     

S-phase-coupled apoptosis in tumor suppression

DNA replication is essential for accurate transmission of genomic information from parental to daughter cells. DNA replication is licensed once per cell division cycle. This process is highly regulated by both positive and negative regulators. Over-replication, under-replication, as well as DNA damage in a cell all induce the activation of checkpoint control pathways such as ATM/ATR, CHK kinases, and the tumor suppressor protein p53, which provide “damage controls” via either DNA repairs or apoptosis. This review focuses on accumulating evidence, with the emphasis on recently discovered Killin, that S-phase checkpoint control is crucial for a mammalian cell to make a life and death decision in order to safeguard genome integrity.     

Chromosomal territory segmentation in apoptotic cells

The nuclear architecture of selected chromosomes in apoptotic nuclei of human leukemic cells K-562 and HL-60 was investigated. Etoposide and prolonged confluence were used for the induction of apoptosis. DAPI as well as TUNEL labeling of apoptotic nuclear bodies was combined with visualization of chromosomal territories by the FISH technique. Simultaneous vital staining by annexin V, propidium iodide, and Hoechst 33342 was applied to distinguish apoptotic, necrotic, and intact cell fraction of tested populations. Our FISH analyses revealed that the three-dimensional (3D) structure of apoptotic nuclei as well as the 3D structure of apoptotic bodies is preserved in formaldehyde-fixed cells. High-molecular-weight DNA fragmentation was determined in apoptotic K-562 cells in contrast to oligonucleosomal cleavage observed in apoptotic HL-60 cells. In K-562 populations, chromosomal territories were located separately either in one apoptotic body or underwent disassembly into chromosomal segments dispersed into single and/or several apoptotic bodies. The apoptotic disorganization of chromosomal territories was irregular, leading mainly to chromosomal segments of different sizes and, consequently, chromosomal disassembly was not observed at specific sites. In comparison with the control, an increased number of centromeric FISH signals were observed in prolonged confluence-treated K-562 cells induced to apoptosis. This finding can be explained either as a consequence of apoptosis or by polyploidization. Sequential staining of the same apoptotic nuclei by the FISH and TUNEL techniques revealed that chromosomal territory segmentation precedes the formation of nuclear apoptotic bodies.     

Induction of vanadium accumulation and nuclear sequestration causing cell suicide in human Chang liver cells

Very little is known about the modulation of vanadium accumulation in cells, although this ultratrace element has long been seen as an essential nutrient in lower life forms, but not necessarily in humans where factors modulating cellular uptake of vanadium seem unclear. Using nuclear microscopy, which is capable of the direct evaluation of free and bound (total) elemental concentrations of single cells we show here that an NH₄Cl acidification prepulse causes distinctive accumulation of vanadium (free and bound) in human Chang liver cells, concentrating particularly in the nucleus. Vanadium loaded with acidification but leaked away with realkalinization, suggests proton-dependent loading. Vanadyl(4), the oxidative state of intracellular vanadium ions, is known to be a potent source of hydroxyl free radicals (OH^.). The high oxidative state of nuclei after induction of vanadyl(4) loading was shown by the redox indicator methylene blue, suggesting direct oxidative damage to nuclear DNA. Flow cytometric evaluation of cell cycle phase-specific DNA composition showed degradation of both 2N and 4N DNA phases in G₁, S and G₂/M cell cycle profiles to a solitary 1N DNA peak, in a dose-dependent manner, effective from micromolar vanadyl(4) levels. This trend was reproduced with microccocal nuclease digestion in a time response, supporting the notion of DNA fragmentation effects. Several other approaches confirmed fragmentation occurring in virtually all cells after 4 mM V(4) loading. Ultrastructural profiles showed various stages of autophagic autodigestion and well defined plasma membrane outlines, consistent with programmed cell death but not with necrotic cell death. Direct intranuclear oxidative damage seemed associated with the induction of mass suicide in these human Chang liver cells following vanadium loading and nuclear sequestration.     

Eukaryotic DNA damage checkpoint activation in response to double-strand breaks

Double-strand breaks (DSBs) are the most detrimental form of DNA damage. Failure to repair these cytotoxic lesions can result in genome rearrangements conducive to the development of many diseases, including cancer. The DNA damage response (DDR) ensures the rapid detection and repair of DSBs in order to maintain genome integrity. Central to the DDR are the DNA damage checkpoints. When activated by DNA damage, these sophisticated surveillance mechanisms induce transient cell cycle arrests, allowing sufficient time for DNA repair. Since the term “checkpoint” was coined over 20 years ago, our understanding of the molecular mechanisms governing the DNA damage checkpoint has advanced significantly. These pathways are highly conserved from yeast to humans. Thus, significant findings in yeast may be extrapolated to vertebrates, greatly facilitating the molecular dissection of these complex regulatory networks. This review focuses on the cellular response to DSBs in Saccharomyces cerevisiae, providing a comprehensive overview of how these signalling pathways function to orchestrate the cellular response to DNA damage and preserve genome stability in eukaryotic cells.     

Absence of low molecular weight DNA polymerase activity from the nuclei ofAmoeba discoides

Summary Amoeba discoides nuclear protein partially purified by passage through Sephadex G-200 showed 3 high-mol.-wt DNA polymerase activities which eluted in and just following the void volume. No low-mol.-wt (45,000 daltons) DNA polymerase activity was detected. Nuclear protein layered on 5–20% sucrose gradients also showed an absence of lowmol.-wt DNA polymerase . The void volume enzyme showed deoxyribonuclease activity, but no low-mol.-wt nuclease activity was detected.     

Localization of integrated adenovirus DNA in the hamster genome

Adenovirus DNA integrated into the genomes of adenovirus-transformed hamster cells or of adenovirus type 12 (Ad12)-induced hamster tumor cells can be located at many different chromosomal sites. This raises the question as to whether distinct isochores of the hamster cell genome might be more accessible to recombination with adenovirus DNA. In Ad12- or Ad2-transformed hamster cell lines, and in Ad12 revertants, the investigated integrated viral DNA sequences were assigned to isochore families by analyzing DNA fractions from preparative CsCl density gradients for their buoyant densities (and, therefore, GC levels) and for the presence of viral DNA. Adenovirus DNA sequences were found in different isochores, which did not generally match the base composition of viral sequences. This is in apparent contrast to what was previously observed with retroviral integration. However, in cell lines carried in culture for many years, the viral DNA sequences might have been transposed to different isochore positions, since the host sequences flanking the viral DNA appear to have been conserved.Received 6 July 2004; received after revision 23 August 2004; accepted 6 October 2004     

Diacylglycerol kinases in nuclear lipid-dependent signal transduction pathways

Several independent groups have shown that lipid-dependent signal transduction systems operate in the nucleus and that they are regulated independently from their membrane and cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation and differentiation. Diacylglycerol is a fundamental lipid second messenger which is produced in the nucleus. The levels of nuclear diacylglycerol fluctuate during the cell cycle progression, suggesting that such a molecule has important regulatory roles. Most likely, nuclear diacylglycerol serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases, i.e. the enzymes that, by converting diacylglycerol into phosphatidic acid, terminate diacylglycerol-dependent events. A number of diacylglycerol kinases encoded by separate genes are present in the mammalian genome. This review aims at highlighting the different isotypes of diacylglycerol kinases identified at the nuclear level as well as at discussing their potential function and regulation. Received 4 December 2001; received after revision 28 January 2002; accepted 31 January 2002