The context-dependence of mutations: a linkage of formalisms

Defining the extent of epistasis - the non-independence of the effects of mutations - is essential for understanding the relationship of genotype, phenotype, and fitness in biological systems. The applications cover many areas of biological research, including biochemistry, genomics, protein and systems engineering, medicine, and evolutionary biology. However, the quantitative definitions of epistasis vary among fields, and its analysis beyond just pairwise effects remains obscure in general. Here, we show that different definitions of epistasis are versions of a single mathematical formalism - the weighted Walsh-Hadamard transform. We discuss that one of the definitions, the backgound-averaged epistasis, is the most informative when the goal is to uncover the general epistatic structure of a biological system, a description that can be rather different from the local epistatic structure of specific model systems. Key issues are the choice of effective ensembles for averaging and to practically contend with the vast combinatorial complexity of mutations. In this regard, we discuss possible approaches for optimally learning the epistatic structure of biological systems.Comment: 6 pages, 3 figures, supplementary informatio

The Wanassa and the Damokoro: a new interpretation of a Linear B text from Pylos

This paper aims to highlight a number of problems involved with current interpretations and identifications of persons in Ta 711, a Linear B tablet from the Mycenaean palace of Pylos, which records a number of objects that were presented on the occasion of the appointment of a da- mo-ko-ro. Contrary to current thinking, we argue that Pu2-ke-qi- ri, the person who appears to have recorded or received the gifts listed in this tablet, was not necessarily a male, and that an argument can be made that this person was attached to the household of the Queen. In addition, we propose that the da- mo-ko-ro, the official whose appointment is mentioned in the same text, may be compared to the Hittite LÚ.MEŠ AGRIG, the keeper of royal storerooms

On the expression strategy of the tospoviral genome

The work described in this thesis was aimed at the unravelling of the molecular biology of tospoviruses, with special emphasis on the process of replication of the tripartite RNA genome.At the onset of the research the complete genome sequence of tomato spotted wilt virus (TSWV), type species of the genus Tospovirus, became available. These sequence data indicated that the tospoviruses represent plant-infecting members of the large family of the arthropod-born Bunyaviridae. Genome sequence comparisons indicated however that the L RNA segment of TSWV would encode a much larger viral polymerase (331.5 kDa) than, as far as known, its animal-infecting counterparts (reported sizes of 241 to 259 kDa). To verify whether a large polymerase represents a characteristic i.e. genus-specific property of tospoviruses the complete sequence of the L RNA segment of a second tospovirus, impatiens necrotis spot virus (INSV), was elucidated (Chapter 2). These sequence data revealed that the L RNA of INSV appeared to be comparable in size to that of TSWV (8675 nucleotides versus 8897 for TSWV), containing an open reading frame with a predicted size of 330.3 kDa of the INSV polymerase. Therefore the next question to be answered was whether the large primary translation product of the tospoviral L RNA acts as an unprocessed polymerase or whether this protein would undergo some cleavages to obtain smaller, functional replication proteins. Answering this question was even more necessary since the theoretical size of the TSWV L RNA ORF greatly exceeded previously determined sizes (110 to 220 kDa) for a large protein reported to copurify with TSWV particles. To this end both the 5'-terminal and 3'-terminal parts of the ORF in the TSWV L RNA were expressed in Escherichia coli and antibodies raised against these regions. Using these tools it could be established that the polymerase (L protein) of TSWV, though significantly larger than that of other bunyaviruses, is present in virus particles (10 to 20 copies per virion) in an unprocessed, full length form (Chapter 3). To allow further analyses of the TSWV polymerase, attempts were made to clone and express the complete L RNA ORF in the baculovirus/insect cell system. In spite of all efforts, only a shorter translation product of 67 kDa was obtained from a baculovirus recombinant containing a complete DNA copy of the TSWV L RNA (Chapter 3). Sequence analysis of the cloned copy revealed a 80 basepairs deletion, resulting in two premature stop codons, which most likely have led to the resulting truncated L protein.To gain more insight in the "cap-snatching" event which takes place during initiation of tospovirus transcription, nucleoprotein (N) mRNAs were partially purified from TSWV-infected N.rustica leaves and cloned (Chapter 4). Sequence analysis of the cloned, 5'-proximate regions of 20 cloned mRNAs showed the presence of extra, non-templated sequences, ranging in length from 12 to 21 nucleotides, confirming our earlier primer extension studies. As these sequences were of non-viral origin a cap-snatching mechanism for tospoviral transcription initiation could thus be definitively identified. None of the hostderived leader sequences analyzed were identical and only limited sequence specificity at the endonucleolytic site was observed (some preference for cleavage at a U residue). During the course of this Ph.D. research, Adkins et al. (1995) reported that in vitro transcriptase activity was associated with freshly isolated TSWV particles. It was investigated (Chapter 5) whether the reported levels of in vitro activity could be further improved and whether this system would lend itself for analysis of the viral proteins involved by e.g. inhibition studies using specific antibodies. Trichloroacetic acid-precipitable products could consistently be obtained after incubation of detergent-disrupted TSWV virions under the assay conditions reported by Adkins et al. (1995) and using (α- 32P)CTP. No significant improvement in CMP incorporation levels could be achieved by testing variable conditions and varying concentrations of assay components. The reaction products obtained hybridized with clones from all three genomic RNA segments. No discrimination between transcription and replication could be made however, and since none of the available specific antibodies directed against any viral protein had an inhibitory effect, it was concluded that the current in vitro system will be of limited value for unravelling the RNA synthesizing process and the role of the individual viral proteins therein.As a first step towards a manipulatable transcription/replication system, a hybrid baculovirus/bacteriophage T7 vector system was developed for transient expression in insect cells of all factors involved in TSWV genome transcription and replication. The results obtained (Chapter 6) illustrate the potential of the system. Although various foreign genes could successfully be expressed to measurable amounts, the reconstitution of a TSWV transcription/replication complex was hampered due to the apparent impossibility (Chapter 3) to clone the complete polymerase gene. Finally, in Chapter 7 (General discussion and concluding remarks), the results obtained are compared with the data reported for animalinfecting bunyaviruses, leading to a discussion of some evolutionary aspects. Furthermore, suggestions are made to circumvent some of the problems encountered during the course of the studies presented in this thesis

Regionale Energiestrategie: juridisch instrumentarium en bestuurlijke praktijk

In een Regionale Energiestrategie (RES) beschrijft een energieregio waar en hoe binnen de betreffende regio het best duurzame elektriciteit op land (wind en zon) opgewekt kan worden. Dat gebeurt ter uitvoering van het Klimaatakkoord. De uitkomsten van de RES zullen juridisch moeten worden verankerd in bindende besluiten. De RES kan worden gezien als een groot experiment. De RES geeft vorm aan een belangrijke transitie in de fysieke leefomgeving, waarvan de toekomst zal moeten uitwijzen of die aan de verwachting kan voldoen. De tijd zal leren of de RES daadwerkelijk bijdraagt aan effectievere, zorgvuldigere en voldoende legitieme besluiten over het realiseren van zonneparken en windmolens

Effects of Epistasis and Pleiotropy on Fitness Landscapes

The factors that influence genetic architecture shape the structure of the fitness landscape, and therefore play a large role in the evolutionary dynamics. Here the NK model is used to investigate how epistasis and pleiotropy -- key components of genetic architecture -- affect the structure of the fitness landscape, and how they affect the ability of evolving populations to adapt despite the difficulty of crossing valleys present in rugged landscapes. Populations are seen to make use of epistatic interactions and pleiotropy to attain higher fitness, and are not inhibited by the fact that valleys have to be crossed to reach peaks of higher fitness.Comment: 10 pages, 6 figures. To appear in "Origin of Life and Evolutionary Mechanisms" (P. Pontarotti, ed.). Evolutionary Biology: 16th Meeting 2012, Springer-Verla

Optimality and evolution of transcriptionally regulated gene expression

<p>Abstract</p> <p>Background</p> <p>How transcriptionally regulated gene expression evolves under natural selection is an open question. The cost and benefit of gene expression are the driving factors. While the former can be determined by gratuitous induction, the latter is difficult to measure directly.</p> <p>Results</p> <p>We addressed this problem by decoupling the regulatory and metabolic function of the <it>Escherichia coli lac </it>system, using an inducer that cannot be metabolized and a carbon source that does not induce. Growth rate measurements directly identified the induced expression level that maximizes the metabolism benefits minus the protein production costs, without relying on models. Using these results, we established a controlled mismatch between sensing and metabolism, resulting in sub-optimal transcriptional regulation with the potential to improve by evolution. Next, we tested the evolutionary response by serial transfer. Constant environments showed cells evolving to the predicted expression optimum. Phenotypes with decreased expression emerged several hundred generations later than phenotypes with increased expression, indicating a higher genetic accessibility of the latter. Environments alternating between low and high expression demands resulted in overall rather than differential changes in expression, which is explained by the concave shape of the cross-environmental tradeoff curve that limits the selective advantage of altering the regulatory response.</p> <p>Conclusions</p> <p>This work indicates that the decoupling of regulatory and metabolic functions allows one to directly measure the costs and benefits that underlie the natural selection of gene regulation. Regulated gene expression is shown to evolve within several hundreds of generations to optima that are predicted by these costs and benefits. The results provide a step towards a quantitative understanding of the adaptive origins of regulatory systems.</p

Predicting evolution using regulatory architecture

The limits of evolution have long fascinated biologists. However, the causes of evolutionary constraint have remained elusive due to a poor mechanistic understanding of studied phenotypes. Recently, a range of innovative approaches have leveraged mechanistic information on regulatory networks and cellular biology. These methods combine systems biology models with population and single-cell quantification and with new genetic tools, and they have been applied to a range of complex cellular functions and engineered networks. In this article, we review these developments, which are revealing the mechanistic causes of epistasis at different levels of biological organization¤mdash¤in molecular recognition, within a single regulatory network, and between different networks¤mdash¤providing first indications of predictable features of evolutionary constraint