Preventing DNA over-replication: a Cdk perspective

The cell cycle is tightly controlled to ensure that replication origins fire only once per cycle and that consecutive S-phases are separated by mitosis. When controls fail, DNA over-replication ensues: individual origins fire more than once per S-phase (re-replication) or consecutive S-phases occur without intervening mitoses (endoreduplication). In yeast the cell cycle is controlled by a single cyclin dependent kinase (Cdk) that prevents origin licensing at times when it promotes origin firing, and that is inactivated, via proteolysis of its partner cyclin, as cells undergo mitosis. A quantitative model describes three levels of Cdk activity: low activity allows licensing, intermediate activity allows firing but prevents licensing, and high activity promotes mitosis. In higher eukaryotes the situation is complicated by the existence of additional proteins (geminin, Cul4-Ddb1Cdt2, and Emi1) that control licensing. A current challenge is to understand how these various control mechanisms are co-ordinated and why the degree of redundancy between them is so variable. Here the experimental induction of DNA over-replication is reviewed in the context of the quantitative model of Cdk action. Endoreduplication is viewed as a consequence of procedures that cause Cdk activity to fall below the threshold required to prevent licensing, and re-replication as the result of procedures that increase that threshold value. This may help to explain why over-replication does not necessarily require reduced Cdk activity and how different mechanisms conspire to prevent over-replication. Further work is nevertheless required to determine exactly how losing just one licensing control mechanism often causes over-replication, and why this varies between cell systems

Identification of a novel gene family that includes the interferon-inducible human genes 6–16and ISG12

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background The human 6–16 and ISG12 genes are transcriptionally upregulated in a variety of cell types in response to type I interferon (IFN). The predicted products of these genes are small (12.9 and 11.5 kDa respectively), hydrophobic proteins that share 36% overall amino acid identity. Gene disruption and over-expression studies have so far failed to reveal any biochemical or cellular roles for these proteins. Results We have used in silico analyses to identify a novel family of genes (the ISG12 gene family) related to both the human 6–16 and ISG12 genes. Each ISG12 family member codes for a small hydrophobic protein containing a conserved ~80 amino-acid motif (the ISG12 motif). So far we have detected 46 family members in 25 organisms, ranging from unicellular eukaryotes to humans. Humans have four ISG12 genes: the 6–16 gene at chromosome 1p35 and three genes (ISG12(a), ISG12(b) and ISG12(c)) clustered at chromosome 14q32. Mice have three family members (ISG12(a), ISG12(b1) and ISG12(b2)) clustered at chromosome 12F1 (syntenic with human chromosome 14q32). There does not appear to be a murine 6–16 gene. On the basis of phylogenetic analyses, genomic organisation and intron-alignments we suggest that this family has arisen through divergent inter- and intra-chromosomal gene duplication events. The transcripts from human and mouse genes are detectable, all but two (human ISG12(b) and ISG12(c)) being upregulated in response to type I IFN in the cell lines tested. Conclusions Members of the eukaryotic ISG12 gene family encode a small hydrophobic protein with at least one copy of a newly defined motif of ~80 amino-acids (the ISG12 motif). In higher eukaryotes, many of the genes have acquired a responsiveness to type I IFN during evolution suggesting that a role in resisting cellular or environmental stress may be a unifying property of all family members. Analysis of gene-function in higher eukaryotes is complicated by the possibility of functional redundancy between family-members. Genetic studies in organisms (e.g. Dictyostelium discoideum) with just one family member so far identified may be particularly helpful in this respect.Peer Reviewe

Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics

<p>Abstract</p> <p>Background</p> <p>The ability to regulate transgene expression has many applications, mostly concerning the analysis of gene function. Desirable induction characteristics, such as low un-induced expression, high induced expression and limited cellular heterogeneity, can be seriously impaired by chromosomal position effects at the site of transgene integration. Many clones may therefore need to be screened before one with optimal induction characteristics is identified. Furthermore, such screens must be repeated for each new transgene investigated, and comparisons between clones with different transgenes is complicated by their different integration sites.</p> <p>Results</p> <p>To circumvent these problems we have developed a "screen and insert" strategy in which clones carrying a transgene for a fluorescent reporter are first screened for those with optimal induction characteristics. Site-specific recombination (SSR) is then be used repeatedly to insert any new transgene at the reporter transgene locus of such clones so that optimal induction characteristics are conferred upon it. Here we have tested in a human fibrosarcoma cell line (HT1080) two of many possible implementations of this approach. Clones (e.g. Rht14-10) in which a GFP reporter gene is very stringently regulated by the tetracycline (tet) transactivator (tTA) protein were first identified flow-cytometrically. Transgenes encoding luciferase, I-<it>Sce</it>I endonuclease or Rad52 were then inserted by SSR at a <it>LoxP </it>site adjacent to the GFP gene resulting stringent tet-regulated transgene expression. In clone Rht14-10, increases in expression from essentially background levels (+tet) to more than 10⁴-fold above background (-tet) were reproducibly detected after Cre-mediated insertion of either the luciferase or the I-<it>Sce</it>I transgenes.</p> <p>Conclusion</p> <p>Although previous methods have made use of SSR to integrate transgenes at defined sites, none has effectively combined this with a pre-selection step to identify integration sites that support optimal regulatory characteristics. Rht14-10 and similar HT1080-derived clones can now be used in conjunction with a convenient delivery vector (pIN2-neoMCS), in a simple 3-step protocol leading to stringent and reproducible transgene regulation. This approach will be particularly useful for transgenes whose products are very active at low concentrations and/or for comparisons of multiple related transgenes.</p

The Impact of the C-Terminal Region on the Interaction of Topoisomerase II Alpha with Mitotic Chromatin.

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme's residence time on mitotic chromatin, as well as its subcellular localisation

Probable detection of starlight reflected from the giant exoplanet orbiting tau Bootis

Giant planets orbiting stars other than the Sun are clearly detectable through precise radial-velocity measurements of the orbital reflex motion of the parent star. In the four years since the discovery of the companion to the star 51 Peg, similar low-amplitude ``Doppler star wobbles'' have revealed the presence of some 20 planets orbiting nearby solar-type stars. Several of these newly-discovered planets are very close to their parent stars, in orbits with periods of only a few days. Being an indirect technique, however, the reflex-velocity method has little to say about the sizes or compositions of the planets, and can only place lower limits on their masses. Here we report the use of high-resolution optical spectroscopy to achieve a probable detection of the Doppler-shifted signature of starlight reflected from one of these objects, the giant exoplanet orbiting the star tau Bootis. Our data give the planet's orbital inclination i=29 degrees, indicating that its mass is some 8 times that of Jupiter, and suggest strongly that the planet has the size and reflectivity expected for a gas-giant planet.Comment: 15 pages, 4 figures. (Fig 1 and equation for epsilon on p1 para 2 revised; changed from double to single spacing

Effect of spirometry on intra-thoracic pressures

Due to the high intra-thoracic pressures associated with forced vital capacity manoeuvres, spirometry is contraindicated for vulnerable patients. However, the typical pressure response to spirometry has not been reported. Eight healthy, recreationally-active men performed spirometry while oesophageal pressure was recorded using a latex balloon-tipped catheter. Peak oesophageal pressure during inspiration was - 47 ± 9 cmH O (37 ± 10% of maximal inspiratory pressure), while peak oesophageal pressure during forced expiration was 102 ± 34 cmH O (75 ± 17% of maximal expiratory pressure). The deleterious consequences of spirometry might be associated with intra-thoracic pressures that approach maximal values during forced expiration

Scalar soliton quantization with generic moduli

This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credArticle funded by SCOAP3. CP is a Royal Society Research Fellow and partly supported by the U.S. Department of Energy under grants DOE-SC0010008, DOE-ARRA-SC0003883 and DOE-DE-SC0007897. ABR is supported by the Mitchell Family Foundation. We would like to thank the Mitchell Institute at Texas A&M and the NHETC at Rutgers University respectively for hospitality during the course of this work. We would also like to acknowledge the Aspen Center for Physics and NSF grant 1066293 for a stimulating research environment which led to questions addressed in this paper

Neighbourhood, Route and Workplace-Related Environmental Characteristics Predict Adults' Mode of Travel to Work

Commuting provides opportunities for regular physical activity which can reduce the risk of chronic disease. Commuters' mode of travel may be shaped by their environment, but understanding of which specific environmental characteristics are most important and might form targets for intervention is limited. This study investigated associations between mode choice and a range of objectively assessed environmental characteristics.Participants in the Commuting and Health in Cambridge study reported where they lived and worked, their usual mode of travel to work and a variety of socio-demographic characteristics. Using geographic information system (GIS) software, 30 exposure variables were produced capturing characteristics of areas around participants' homes and workplaces and their shortest modelled routes to work. Associations between usual mode of travel to work and personal and environmental characteristics were investigated using multinomial logistic regression.Of the 1124 respondents, 50% reported cycling or walking as their usual mode of travel to work. In adjusted analyses, home-work distance was strongly associated with mode choice, particularly for walking. Lower odds of walking or cycling rather than driving were associated with a less frequent bus service (highest versus lowest tertile: walking OR 0.61 [95% CI 0.20–1.85]; cycling OR 0.43 [95% CI 0.23–0.83]), low street connectivity (OR 0.22, [0.07–0.67]; OR 0.48 [0.26–0.90]) and free car parking at work (OR 0.24 [0.10–0.59]; OR 0.55 [0.32–0.95]). Participants were less likely to cycle if they had access to fewer destinations (leisure facilities, shops and schools) close to work (OR 0.36 [0.21–0.62]) and a railway station further from home (OR 0.53 [0.30–0.93]). Covariates strongly predicted travel mode (pseudo r-squared 0.74).Potentially modifiable environmental characteristics, including workplace car parking, street connectivity and access to public transport, are associated with travel mode choice, and could be addressed as part of transport policy and infrastructural interventions to promote active commuting