G4 motifs affect origin positioning and efficiency in two vertebrate replicators

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome‐wide studies in vertebrates have recently identified a consensus G‐rich motif potentially able to form G‐quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200‐bp cis‐regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.     

Effects of biological DNA precursor pool asymmetry upon accuracy of DNA replication in vitro

Deoxyguanosine triphosphate is underrepresented among the four common deoxyribonucleoside triphosphates (dNTPs), typically accounting for just 5-10% of the total dNTP pool. We have asked whether this pool asymmetry affects the fidelity of DNA replication, by use of an in vitro assay in which an M13 phagemid containing the Escherichia coli lacZalpha gene and an SV40 replication origin is replicated by extracts of human cells. By monitoring reversion of either a TGA or TAA codon within the lacZalpha gene, we found that replication in "biologically biased" dNTPs, representing our estimate of the concentrations in HeLa cell nuclei, is not significantly more accurate than when measured in reaction mixtures containing the four dNTPs at equimolar concentrations. However, sequence analysis of revertants revealed significantly different patterns of mispairing events leading to mutation. During replication at biased dNTP levels, mutations at the site 5' to C in the template strand for the TGA triplet were less frequent than seen in equimolar reaction mixtures, suggesting that extension from mismatches at this site is relatively slow, and proofreading efficiency high, when dGTP is the next nucleotide to be incorporated. Mismatches opposite template C, which might have been favored by the low physiological concentrations of dGTP, were not favored in our in vitro system, although one particular substitution at this site, TGA-->TTA, was strongly favored at low [dGTP]. An excess of one dNTP was found in our system to be more mutagenic than a corresponding deficiency. We also estimated dNTP concentrations in non-transformed human fibroblasts and found that in vitro replication at these levels caused significantly fewer mutations than we observed under equimolar conditions (100 microM each dNTP). This increased replication fidelity may result from increased proofreading efficiency at the lower dNTP levels; however, replication rates were decreased only slightly at these non-transformed fibroblast concentrations.     

Identification of Yeast Factors Involved in the Replication of Mungbean Yellow Mosaic India Virus Using Yeast Temperature-Sensitive Mutants

正Dear Editor,The geminiviruses are small single-stranded plant DNA viruses belonging to the family Geminiviridae, which cause serious diseases in many economically important     

Incorporating functional annotation information in prioritizing disease associated SNPs from genome wide association studies

With recent advances in genotyping and sequencing technologies,many disease susceptibility loci have been identified.However,much of the genetic heritability remains unexplained and the replication rate between independent studies is still low.Meanwhile,there have been increasing efforts on functional annotations of the entire human genome,such as the Encyclopedia of DNA Elements(ENCODE)project and other similar projects.It has been shown that incorporating these functional annotations to prioritize genome wide association signals may help identify true association signals.However,to our knowledge,the extent of the improvement when functional annotation data are considered has not been studied in the literature.In this article,we propose a statistical framework to estimate the improvement in replication rate with annotation data,and apply it to Crohn’s disease and DNase I hypersensitive sites.The results show that with cell line specific functional annotations,the expected replication rate is improved,but only at modest level.     

Surviving chromosome replication: the many roles of the S-phase checkpoint pathway

Checkpoints were originally identified as signalling pathways that delay mitosis in response to DNA damage or defects in chromosome replication, allowing time for DNA repair to occur. The ATR (ataxia- and rad-related) and ATM (ataxia-mutated) protein kinases are recruited to defective replication forks or to sites of DNA damage, and are thought to initiate the DNA damage response in all eukaryotes. In addition to delaying cell cycle progression, however, the S-phase checkpoint pathway also controls chromosome replication and DNA repair pathways in a highly complex fashion, in order to preserve genome integrity. Much of our understanding of this regulation has come from studies of yeasts, in which the best-characterized targets are the stimulation of ribonucleotide reductase activity by multiple mechanisms, and the inhibition of new initiation events at later origins of DNA replication. In addition, however, the S-phase checkpoint also plays a more enigmatic and apparently critical role in preserving the functional integrity of defective replication forks, by mechanisms that are still understood poorly. This review considers some of the key experiments that have led to our current understanding of this highly complex pathway.     

Funding begets biodiversity

Aim Effective conservation of biodiversity relies on an unbiased knowledge of its distribution. Conservation priority assessments are typically based on the levels of species richness, endemism and threat. Areas identified as important receive the majority of conservation investments, often facilitating further research that results in more species discoveries. Here, we test whether there is circularity between funding and perceived biodiversity, which may reinforce the conservation status of areas already perceived to be important while other areas with less initial funding may remain overlooked. Location Eastern Arc Mountains, Tanzania. Methods We analysed time series data (1980–2007) of funding (n = 134 projects) and plant species records (n = 75,631) from a newly compiled database. Perceived plant diversity, over three decades, is regressed against funding and environmental factors, and variances decomposed in partial regressions. Cross‐correlations are used to assess whether perceived biodiversity drives funding or vice versa. Results Funding explained 65% of variation in perceived biodiversity patterns – six times more variation than accounted for by 34 candidate environmental factors. Cross‐correlation analysis showed that funding is likely to be driving conservation priorities and not vice versa. It was also apparent that investment itself may trigger further investments as a result of reduced start‐up costs for new projects in areas where infrastructure already exists. It is therefore difficult to establish whether funding, perceived biodiversity, or both drive further funding. However, in all cases, the results suggest that regional assessments of biodiversity conservation importance may be biased by investment. Funding effects might also confound studies on mechanisms of species richness patterns. Main conclusions Continued biodiversity loss commands urgent conservation action even if our knowledge of its whereabouts is incomplete; however, by concentrating inventory funds in areas already perceived as important in terms of biodiversity and/or where start‐up costs are lower, we risk losing other areas of underestimated or unknown value.     

Deletion analysis of ors12, a centromeric,early activated,mammalian origin of DNA replication

We have generated a panel of deletion mutants of ors12 (812-bp), a mammalian origin of DNA replication previously isolated by nascent strand extrusion from early replicating African Green monkey (CV-1) DNA. The deletion mutants were tested for their replication activity in vivo by the bromodeoxyuridine substitution assay, after transfection into HeLa cells, and in vitro by the DpnI resistance assay, using extracts from HeLa cells. We identified a 215-bp internal fragment as essential for the autonomous replication activity of ors12. When subcloned into the vector pML2 and similarly tested, this subfragment was capable of autonomous replication in vivo and in vitro. Several repeated sequence motifs are present in this 215-bp fragment, such as TGGG(A) and G(A)AG (repeated four times each); TTTC, AGG, and CTTA (repeated 3 times each); the motifs CACACA and CTCTCT, and two imperfect inverted repeats, 22 and 16 bp long, respectively. The overall sequence of the 215-bp fragment is G/C-rich (50.2%), by comparison to the 186-bp (33.5% G/C-rich) minimal sequence required for the autonomous replication activity of ors8, another functional ors that was similarly isolated and characterized. J. Cell. Biochem. 66:87–97, 1997. © 1997 Wiley-Liss, Inc.     

Prereplicative complexes assembled in vitro support origin‐dependent and independent DNA replication

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2‐7 helicase is first loaded into prereplicative complexes (pre‐RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre‐RCs assembled with purified proteins support complete and semi‐conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK). DDK phosphorylation of Mcm2‐7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin‐dependent in this system. These experiments indicate that Mcm2‐7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins.     

Eucaryotic DNA Replication Complex: Study of Structure and Function Using the Affinity Modification Technique

Eucaryotic DNA replication complex is now one of the most intensively studied subjects of molecular biology and biochemistry. In addition to detailed studies on the structures and functions of individual DNA polymerases involved in this process, other enzymes and protein factors are also given much attention. The structures and functions of proteins in the replication complexes are studied by various approaches, including X-ray diffraction analysis. At present, this approach provides sufficient information about the structures and functions of individual biopolymers and their complexes with ligands. However, this approach is unsuitable for studies on proteins, which cannot be cloned and isolated in amounts sufficient for X-ray diffraction analysis. Moreover, this approach is inapplicable for studies on multicomponent systems, such as DNA replication and repair complexes. Furthermore, data of X-ray diffraction analysis virtually never characterize the variety of dynamic interactions in enzymatic systems. Affinity modification is an alternative and rather successful approach for studies on structure-functional organization of supramolecular structures. This approach can be used for studies on individual enzymes and their complexes with substrates and also on systems consisting of numerous interacting proteins and nucleic acids. The purpose of this review is to analyze the available data obtained by affinity modification studies on the eucaryotic replication complex.     

Reproducible Research Practices and Transparency across the Biomedical Literature

There is a growing movement to encourage reproducibility and transparency practices in the scientific community, including public access to raw data and protocols, the conduct of replication studies, systematic integration of evidence in systematic reviews, and the documentation of funding and potential conflicts of interest. In this survey, we assessed the current status of reproducibility and transparency addressing these indicators in a random sample of 441 biomedical journal articles published in 2000–2014. Only one study provided a full protocol and none made all raw data directly available. Replication studies were rare (n = 4), and only 16 studies had their data included in a subsequent systematic review or meta-analysis. The majority of studies did not mention anything about funding or conflicts of interest. The percentage of articles with no statement of conflict decreased substantially between 2000 and 2014 (94.4% in 2000 to 34.6% in 2014); the percentage of articles reporting statements of conflicts (0% in 2000, 15.4% in 2014) or no conflicts (5.6% in 2000, 50.0% in 2014) increased. Articles published in journals in the clinical medicine category versus other fields were almost twice as likely to not include any information on funding and to have private funding. This study provides baseline data to compare future progress in improving these indicators in the scientific literature.     

The case for increasing the statistical power of eddy covariance ecosystem studies: why,where and how?

Eddy covariance (EC) continues to provide invaluable insights into the dynamics of Earth's surface processes. However, despite its many strengths, spatial replication of EC at the ecosystem scale is rare. High equipment costs are likely to be partially responsible. This contributes to the low sampling, and even lower replication, of ecoregions in Africa, Oceania (excluding Australia) and South America. The level of replication matters as it directly affects statistical power. While the ergodicity of turbulence and temporal replication allow an EC tower to provide statistically robust flux estimates for its footprint, these principles do not extend to larger ecosystem scales. Despite the challenge of spatially replicating EC, it is clearly of interest to be able to use EC to provide statistically robust flux estimates for larger areas. We ask: How much spatial replication of EC is required for statistical confidence in our flux estimates of an ecosystem? We provide the reader with tools to estimate the number of EC towers needed to achieve a given statistical power. We show that for a typical ecosystem, around four EC towers are needed to have 95% statistical confidence that the annual flux of an ecosystem is nonzero. Furthermore, if the true flux is small relative to instrument noise and spatial variability, the number of towers needed can rise dramatically. We discuss approaches for improving statistical power and describe one solution: an inexpensive EC system that could help by making spatial replication more affordable. However, we note that diverting limited resources from other key measurements in order to allow spatial replication may not be optimal, and a balance needs to be struck. While individual EC towers are well suited to providing fluxes from the flux footprint, we emphasize that spatial replication is essential for statistically robust fluxes if a wider ecosystem is being studied.     

Distinct mechanistic responses to replication fork stalling induced by either nucleotide or protein deprivation

Incidents that slow or stall replication fork progression, collectively known as replication stress, represent a major source of spontaneous genomic instability. Here, we determine the requirement for global protein biosynthesis on DNA replication and associated downstream signaling. We study this response side by side with dNTP deprivation; one of the most commonly used means to investigate replication arrest and replicative stress. Our in vitro interrogations reveal that inhibition of translation by cycloheximide (CHX) rapidly impairs replication fork progression without decoupling helicase and polymerase activities or inducing DNA damage. In line with this, protein deprivation stress does not activate checkpoint signaling. In contrast to the direct link between insufficient dNTP pools and genome instability, our findings suggest that replication forks remain stable during short-term protein deficiency. We find that replication forks initially endure fluctuations in protein supply in order to efficiently resume DNA synthesis upon reversal of the induced protein deprivation stress. These results reveal distinct cellular responses to replication arrest induced by deprivation of either nucleotides or proteins.     

Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET)

Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB-VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB-3D, VPg-3D, and 3AB-VPg were 3.9 ± 0.4% (n = 36), 4.5 ± 0.4% (n = 39), and 8.3 ± 0.6% (n = 44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg-3D and 3AB-3D, with FRET efficiencies in the virus-infected cells of 10.7 ± 1.1% (n = 39) and 9.0 ± 0.9% (n = 37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.     

Proteasome-dependent degradation of replisome components regulates faithful DNA replication

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF^Pof3 (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.     

Replication study of 34 common SNPs associated with prostate cancer in the Romanian population

Prostate cancer is the third‐most common form of cancer in men in Romania. The Romanian unscreened population represents a good sample to study common genetic risk variants. However, a comprehensive analysis has not been conducted yet. Here, we report our replication efforts in a Romanian population of 979 cases and 1027 controls, for potential association of 34 literature‐reported single nucleotide polymorphisms (SNPs) with prostate cancer. We also examined whether any SNP was differentially associated with tumour grade or stage at diagnosis, with disease aggressiveness, and with the levels of PSA (prostate specific antigen). In the allelic analysis, we replicated the previously reported risk for 19 loci on 4q24, 6q25.3, 7p15.2, 8q24.21, 10q11.23, 10q26.13, 11p15.5, 11q13.2, 11q13.3. Statistically significant associations were replicated for other six SNPs only with a particular disease phenotype: low‐grade tumour and low PSA levels (rs1512268), high PSA levels (rs401681 and rs11649743), less aggressive cancers (rs1465618, rs721048, rs17021918). The strongest association of our tested SNP's with PSA in controls was for rs2735839, with 29% increase for each copy of the major allele G, consistent with previous results. Our results suggest that rs4962416, previously associated only with prostate cancer, is also associated with PSA levels, with 12% increase for each copy of the minor allele C. The study enabled the replication of the effect for the majority of previously reported genetic variants in a set of clinically relevant prostate cancers. This is the first replication study on these loci, known to associate with prostate cancer, in a Romanian population.     

Nurse Practitioner and Physician's Assistant Clinics in Rural California: Part I: Issues

The primary health care needs of at least 26 rural California communities are being served by nurse practitioners (NP''s) or physician''s assistants (PA''s). All of these have physician supervision and support. NP''s and PA''s have proved to be acceptable and effective. With 230 rural areas in California identified as having unmet health care needs, this type of service is likely to increase and should be supported.NP/PA clinics serve total populations or concentrate on Indians, Chicanos or the poor. Many barriers have been overcome, especially over the past four years, to allow these clinics to flourish and increase in number. The availability of nurse practitioners and physician''s assistants has increased due to support to schools and to school policies. Clinic funding has greatly improved; federal funds for general rural clinics, Indians, migrants, family planning and maternalchild health have been greatly supplemented by California state funds. Beginning in 1978, rural NP and PA services can be reimbursed by Medicare and Medi-Cal (California''s Medicaid program).Since 1975 state laws have defined PA and NP roles broadly, and these roles are more precisely defined at the local level. Although nurse practitioners and physician''s assistants generally cannot prescribe or dispense drugs (a major problem in many clinics), demonstration legislation allows special pilot projects to do both. As remaining funding and legal problems are corrected, NP''s and PA''s will serve an even greater role in rural areas.     

Prebiotic Chemistry and the Origin of the RNA World

The demonstration that ribosomal peptide synthesis is a ribozyme-catalyzed reaction makes it almost certain that there was once an RNA World. The central problem for origin-of-life studies, therefore, is to understand how a protein-free RNA World became established on the primitive Earth. We first review the literature on the prebiotic synthesis of the nucleotides, the nonenzymatic synthesis and copying of polynucleotides, and the selection of ribozyme catalysts of a kind that might have facilitated polynucleotide replication. This leads to a brief outline of the Molecular Biologists' Dream, an optimistic scenario for the origin of the RNA World. In the second part of the review we point out the many unresolved problems presented by the Molecular Biologists' Dream. This in turn leads to a discussion of genetic systems simpler than RNA that might have “invented” RNA. Finally, we review studies of prebiotic membrane formation.     

Facing the credit crunch. Politics sends mixed messages for science in the wake of the global financial crisis

The global response to the credit crunch has varied from belt tightening to spending sprees. Philip Hunter investigates how various countries react to the financial crisis in terms of supporting scientific research.The overall state of biomedical research in the wake of the global financial crisis remains unclear amid growing concern that competition for science funding is compromising the pursuit of research. Such concerns pre-date the credit crunch, but there is a feeling that an increasing amount of time and energy is being wasted in the ongoing scramble for grants, in the face of mounting pressure from funding agencies demanding value for money. Another problem is balancing funding between different fields; while the biomedical sciences have generally fared well, they are increasingly dependent on basic research in physics and chemistry that are in greater jeopardy. This has led to calls for rebalancing funding, in order to ensure the long-term viability of all fields in an increasingly multidisciplinary and collaborative research world.For countries that are cutting funding—such as Spain, Italy and the UK—the immediate priority is to preserve the fundamental research base and avoid a significant drain of expertise, either to rival countries or away from science altogether. This has highlighted the plight of postdoctoral researchers who have traditionally been the first to suffer from funding cuts, partly because they have little immediate impact on on a country''s scientific competitiveness. Postdocs have been the first to go whenever budgets have been cut, according to Richard Frankel, a physicist at California Polytechnic State University in Saint Luis Obispo, who investigates magnetotaxis in bacteria. “In the short term there will be little effect but the long-term effects can be devastating,” he said.…there is a feeling that an increasing amount of time and energy is being wasted in the ongoing scramble for grants, in the face of mounting pressure from funding agencies…According to Peter Stadler, head of a bioinformatics group at the University of Leipzig in Germany, such cuts tend to cause the long-term erosion of a country''s science skills base. “Short-term cuts in science funding translate totally into a brain drain, since they predominantly affect young researchers who are paid from the soft money that is drying up first,” said Stadler. “They either leave science, an irreversible step, or move abroad but do not come back later, because the medium-term effect of cuts is a reduction in career opportunities and fiercer competition giving those already in the system a big advantage.”Even when young researchers are not directly affected, the prevailing culture of short-term funding—which requires ongoing grant applications—can be disruptive, according to Xavier Salvatella, principal investigator in the Laboratory of Molecular Biophysics at the Institute for Research in Biomedicine in Barcelona, Spain. “I do not think the situation is dramatic but too much time is indeed spent writing proposals,” he commented. “Because success rates are decreasing, the time devoted to raise funds to run the lab necessarily needs to increase.”At the University of Adelaide in Australia, Andrew Somogyi, professor of pharmacology, thinks that the situation is serious: “[M]y postdocs would spend about half their time applying for grants.” Somogyi pointed out that the success rate has been declining in Australia, as it has in some other countries. “For ARC [Australian Research Council] the success rate is now close to 20%, which means many excellent projects don''t get funding because the assessment is now so fine cut,” he said.Similar developments have taken place in the USA at both the National Institutes of Health (NIH)—which provides US$16 billion funding per year and the American Cancer Society (ACS), the country''s largest private non-profit funder of cancer research, with a much smaller pot of US$120 million per year. The NIH funded 21% of research proposals submitted to it in 2009, compared with 32% a decade earlier, while the ACS approves only 15% of grant applications, down several percentage points over the past few years.While the NIH is prevented by federal law from allowing observers in to its grant review meetings, the ACS did allow a reporter from Nature to attend one of its sessions on the condition that the names of referees and the applications themselves were not revealed (Powell, 2010). The general finding was that while the review process works well when around 30% of proposals are successful, it tends to break down as the success rate drops, as more arbitrary decisions are made and the risk of strong pitches being rejected increases. This can also discourage the best people from being reviewers because the process becomes more tiring and time-consuming.Even when young researchers are not directly affected, the prevailing culture of short-term funding—which requires ongoing grant applications—can be disruptive…In some countries, funding shortfalls are also leading to the loss of permanent jobs, for example in the UK where finance minister George Osborne announced on October 20 that the science budget would be frozen at £4.6 billion, rather than cut as had been expected. Even so, combined with the cut in funding for universities that was announced on the same day, this raises the prospect of reductions in academic staff numbers, which could affect research projects. This follows several years of increasing funding for UK science. Such uncertainty is damaging, according to Cornelius Gross, deputy head of the mouse biology unit, European Molecular Biology Laboratory in Monterotondo, Italy. “Large fluctuations in funding have been shown to cause damage beyond their direct magnitude as can be seen in the US where the Clinton boom was inevitably followed by a slowdown that led to rapid and extreme tightening of budgets,” he said.Some countries are aware of these dangers and have acted to protect budgets and, in some cases, even increase spending. A report by the OECD argued that countries and companies that boosted research and development spending during the ‘creative destruction'' of an economic downturn tended to gain ground on their competitors and emerge from the crisis in a relatively stronger position (OECD, 2009). This was part of the rationale of the US stimulus package, which was intended to provide an immediate lift to the economy and has been followed by a slight increase in funding. The NIH''s budget is set to increase by $1 billion, or 3% from 2010 to 2011, reaching just over $32 billion. This looks like a real-term increase, since inflation in the USA is now between 1 and 2%. However, there are fears that budgets will soon be cut; even now the small increase at the Federal level is being offset by cuts in state support, according to Mike Seibert, research fellow at the US Department of Energy''s National Renewable Energy Laboratory. “The stimulus funds are disappearing in the US, and the overall budget for science may be facing a correction at the national level as economic, budget, and national debt issues are addressed,” he said. “The states in most cases are suffering their own budget crises and will be cutting back on anything that is not nailed down.”…countries and companies that boosted research and development spending during the ‘creative destruction'' of an economic downturn tended to gain ground on their competitors…In Germany, the overall funding situation is also confused by a split between the Federal and 16 state governments, each of which has its own budget for science. In contrast to many other countries though, both federal and state governments have responded boldly to the credit crisis by increasing the total budget for the DFG (Deutsche Forschungsgemeinschaft)—Germany''s largest research funding agency—to €2.3 billion in 2011. Moreover, total funding for research and education from the BMBF (Federal Ministry for Education and Research) is expected to increase by another 7% from €10.9 billion in 2010 to €11.64 billion, although the overall federal budget is set to shrink by 3.8% under Germany''s austerity measures (Anon, 2010). There have also been increases in funding from non-government sources, such as the Fraunhofer Society, Europe''s largest application-oriented research organization, which has an annual budget of €1.6 billion.The German line has been strongly applauded by the European Union, which since 2007 has channelled its funding for cutting-edge research through the European Research Council (ERC). The ERC''s current budget of €7.5 billion, which runs until 2013, was set in 2007 and negotiations for the next period have not yet begun, but the ERC''s executive agency director Jack Metthey has indicated that it will be increased: “The Commission will firmly sustain in the negotiations the view that research and innovation, central to the Europe 2020 Strategy agreed by the Member States, should be a top budgetary priority.” Metthey also implied that governments cutting funding, as the UK had been planning to do, were making a false economy that would gain only in the short term. “Situations vary at the national level but the European Commission believes that governments should maintain and even increase research and innovation investments during difficult times, because these are pro-growth, anti-crisis investments,” he said.Many other countries have to cope with flat or declining science budgets; some are therefore exploring ways in which to do more with less. In Japan, for instance, money has been concentrated on larger projects and fewer scientists, with the effect of intensifying the grant application process. Since 2002, the total Japanese government budget for science and technology has remained flat at around ¥3,500 billion—or €27 billion at current exchange rates—with a 1% annual decline in university support but increased funding for projects considered to be of high value to the economy. This culminated in March 2010 with the launch of the ¥100 billion (€880 million) programme for World Leading Innovative Research and Development on Science and Technology.But such attempts to make funding more competitive or focus it on specific areas could have unintended side effects on innovation and risk taking. One side effect can be favouring scientists who may be less creative but good at attracting grants, according to Roger Butlin, evolutionary biologist at the University of Sheffield in the UK. “Some productive staff are being targeted because they do not bring in grants, so money is taking precedence over output,” said Butlin. “This is very dangerous if it results in loss of good theoreticians or data specialists, especially as the latter will be a critical group in the coming years.”“Scientists are usually very energetic when they can pursue their own ideas and less so when the research target is too narrowly prescribed”There have been attempts to provide funding for young scientists based entirely on merit, such as the ERC ‘Starting Grant'' for top young researchers, whose budget was increased by 25% to €661 million for 2011. Although they are welcome, such schemes could also backfire unless they are supported by measures to continue supporting the scientists after these early career grants expire, according to Gross. “There are moves to introduce significant funding for young investigators to encourage independence, so called anti-brain-drain grants,” he said. “These are dangerous if provided without later independent positions for these people and a national merit-based funding agency to support their future work.”Such schemes might work better if they are incorporated into longer-term funding programmes that provide some security as well as freedom to expand a project and explore promising side avenues. Butlin cited the Canadian ‘Discovery Grant'' scheme as an example worth adopting elsewhere; it supports ongoing programmes with long-term goals, giving researchers freedom to pursue new lines of investigation, provided that they fit within the overall objective of the project.To some extent the system of ‘open calls''—supported by some European funding agencies—has the same objective, although it might not provide long-term funding. The idea is to allow scientists to manoeuvre within a broad objective, rather than confining them to specific lines of research or ‘thematic calls'', which tend to be highly focused. “The majority of funding should be distributed through open calls, rather than thematic calls,” said Thomas Höfer from the Modeling Research Group at the German Cancer Research Center & BioQuant Center in Heidelberg. “Scientists are usually very energetic when they can pursue their own ideas and less so when the research target is too narrowly prescribed. In my experience as a reviewer at both the national and EU level, open calls are also better at funding high-quality research whereas too narrow thematic calls often result in less coherent proposals.”“Cutting science, and education, is the national equivalent of a farmer eating his ‘seed corn'', and will lead to developing nation status within a generation”Common threads seems to be emerging from the different themes and opinions about funding: budgets should be consistent over time and spread fairly among all disciplines, rather than focused on targeted objectives. They should also be spread across the working lifetime of a scientist rather than being shot in a scatter-gun approach at young researchers. Finally, policies should put a greater emphasis on long-term support for the best scientists and projects, chosen for their merit. Above all, funding policy should reflect the fundamental importance of science to economies, as Seibert concluded: “Cutting science, and education, is the national equivalent of a farmer eating his ‘seed corn'', and will lead to developing nation status within a generation.”