Mapping an origin of DNA replication at a single-copy locus in exponentially proliferating mammalian cells.

A general method for determining the physical location of an origin of bidirectional DNA replication has been developed recently and shown to be capable of correctly identifying the simian virus 40 origin of replication (L. Vassilev and E. M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989). The advantage of this method over others previously reported is that it avoids the use of metabolic inhibitors, the requirement for cell synchronization, and the need for multiple copies of the origin sequence. Application of this method to exponentially growing Chinese hamster ovary cells containing the nonamplified, single-copy dihydrofolate reductase gene locus revealed that DNA replication begins bidirectionally in an initiation zone approximately 2.5 kilobases long centered about 17 kilobases downstream of the DHFR gene, coinciding with previously described early replicating sequences. These results demonstrate the utility of this mapping protocol for identifying cellular origins of replication and suggest that the same cellular origin is used in both the normal and the amplified DHFR locus.     

Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation.

In the presence of emetine, an inhibitor of protein synthesis, nascent DNA on forward arms of replication forks in hamster cell lines containing either single or amplified copies of the DHFR gene region was enriched 5- to 7-fold over nascent DNA on retrograde arms. This forward arm bias was observed on both sides of the specific origin of bidirectional DNA replication located 17 kb downstream of the hamster DHFR gene (OBR-1), consistent with at least 85% of replication forks within this region emanating from OBR-1. However, the replication fork asymmetry induced by emetine does not result from conservative nucleosome segregation, as previously believed, but from preferentially inhibiting Okazaki fragment synthesis on retrograde arms of forks to produce 'imbalanced DNA synthesis'. Three lines of evidence support this conclusion. First, the bias existed in long nascent DNA strands prior to nuclease digestion of non-nucleosomal DNA. Second, the fraction of RNA-primed Okazaki fragments was rapidly diminished. Third, electron microscopic analysis of SV40 DNA replicating in the presence of emetine revealed forks with single-stranded DNA on one arm, and nucleosomes randomly distributed to both arms. Thus, as with cycloheximide, nucleosome segregation in the presence of emetine was distributive.     

Nucleosome assembly in mammalian cell extracts before and after DNA replication.

Protein-free DNA in a cytosolic extract supplemented with SV40 large T-antigen (T-Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori- and ori+) were assembled into chromatin with similar efficiencies whether T-Ag was present or not. Approximately 50-80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre-replicated and post-replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome.     

Gap junction structures: Analysis of the x-ray diffraction data

Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W.C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.     

Apoptosis in yeasts

Although yeasts lack some elements of the complex apoptotic machinery of metazoan cells, recent studies show that many features of apoptosis, including a caspase-like activity, can be induced in these organisms by DNA damage and other apoptotic triggers. These remarkable findings provide a compelling argument for increased efforts to bring the powerful genetic approaches available to yeast researchers more directly to bear on questions related to apoptosis and its induction or inhibition by drugs. Yeasts may provide a particularly useful model for understanding connections between DNA damage, cell cycle regulation and apoptosis. Here we summarize these recent findings and explore their implications, particularly for the development of more effective therapeutic strategies for treating cancer.     

DNA replication stress, genome instability and aging

Genome instability is a fundamentally important component of aging in all eukaryotes. How age-related genome instability occurs remains unclear. The free radical theory of aging posits oxidative damage to DNA and other cellular constituents as a primary determinant of aging. More recent versions of this theory predict that mitochondria are a major source of reactive oxygen species (ROS) that cause oxidative damage. Although substantial support for the free radical theory exists, the results of some tests of this theory have been contradictory or inconclusive. Enhanced growth signaling also has been implicated in aging. Many efforts to understand the effects of growth signaling on aging have focused on inhibition of oxidative stress responses that impact oxidative damage. However, recent experiments in the model organism Saccharomyces cerevisiae (budding yeast) and in higher eukaryotes suggest that growth signaling also impacts aging and/or age-related diseases—including cancer and neurodegeneration—by inducing DNA replication stress, which causes DNA damage. Replication stress, which has not been broadly considered as a factor in aging, may be enhanced by ROS that signal growth. In this article, we review evidence that points to DNA replication stress and replication stress-induced genome instability as important factors in aging.     

The role of nitric oxide in cancer

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including va-sodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic pro     

Caloric Restriction Engages Hepatic RNA Processing Mechanisms in Rhesus Monkeys

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