Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (E mu Myc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from E mu Myc Pim1(-/-) Pim2(-/-) mice and 27 tumors from E mu Myc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of E mu Myc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.

SummaryLow environmental pH strongly affected the organization of the Saccharomyces cerevisiae cell wall, resulting in rapidly induced resistance to b1,3-glucanase. At a molecular level, we found that a considerable amount of Cwp1p became anchored through a novel type of linkage for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins, namely an alkali-labile linkage to b1,3-glucan. This novel type of modification for Cwp1p did not require the presence of a GPI-derived structure connecting the protein with b1,6-glucan. In addition, we found high levels of Cwp1p, which was double-anchored through both the novel alkali-sensitive bond to b1,3-glucan and the alkali-resistant GPI-derived linkage to b1,6-glucan. Further cell wall analyses demonstrated that Pir2p/Hsp150 and possibly other Pir cell wall proteins, which were already known to be linked to the b1,3-glucan framework by an alkali-sensitive linkage, were also more efficiently retained in the cell wall at pH 3.5 than at pH 5.5. Consequently, the alkali-sensitive type of linkage of cell wall proteins to b1,3-glucan was induced by low pH. The low pHinduced alterations in yeast cell wall architecture were demonstrated to be dependent on a functional HOG1 gene, but not on the Slt2p-mediated MAP kinase pathway. Consistent with this observation, DNA microarray studies revealed transcriptional induction of many known high-osmolarity glycerol (HOG) pathway-dependent genes, including four cell wall-related genes, namely CWP1, HOR7, SPI1 and YGP1.

Yeast cell wall proteins, including Cwp1p and alpha-agglutinin, could be released by treating the cell wall with either beta-1,3-or beta-1,6-glucanases, indicating that both polymers are involved in anchoring cell wall proteins. It was shown immunologically that both beta-1,3- and beta-1,6-glucan were linked to yeast cell wall proteins, including Cwp1p and alpha-agglutinin. It was further shown that beta-1,3-glucan was linked to the wall protein through a beta-1,6-glucan moiety. The beta-1,6-glucan moiety could be removed from Cwp1p and other cell wall proteins by cleaving phosphodiester bridges either enzymatically using phosphodiesterases or chemically using ice-cold aqueous hydrofluoric acid. These observations are consistent with the notion that cell wall proteins in Saccharomyces cerevisiae are linked to a beta-1,3-/beta-1,6-glucan heteropolymer through a phosphodiester linkage and that this polymer is responsible for anchoring cell wall proteins. It is proposed that this polymer is identical to the alkali-soluble beta-1,3-/beta-1,6-glucan heteropolymer characterized by Fleet and Manners (1976, 1977).

Background: Mutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5/sterolin-1 and ABCG8/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8. These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion.

Abstract. The analysis of complex distributed systems requires dedicated software tools. The mCRL2 language and toolset have been developed to support such analysis. We highlight changes and improvements made to the toolset in recent years. On the one hand, these affect the scope of application, which has been broadened with extended support for data structures like infinite sets and functions. On the other hand, considerable progress has been made regarding the performance of our tools for state space generation and model checking, due to improvements in symbolic reduction techniques and due to a shift towards parity gamebased solving. We also discuss the software architecture of the toolset, which was well suited to accommodate the above changes, and we address a number of case studies to illustrate the approach.

We arrange various classes of probabilistic systems studied in the literature in an expressiveness hierarchy. Our expressiveness criterion is the existence of a system translation, from the less expressive type into the more expressive type, that preserves and reflects probabilistic bisimilarity. We model the different system types as coalgebras of suitable behaviour functors and argue that the corresponding coalgebraic bisimilarity coincides with probabilistic bisimilarity for the classes for which the latter notion has been proposed in the literature. The theory of coalgebras provides a unified framework for the presentation of the different classes and the system translations we needed to establish the hierarchy. All these translations arise in a standard way from natural transformations between the two behaviour functors involved. Such a translation generally preserves coalgebraic bisimilarity. We exploit a new result that, under mild assumptions on the behaviour functors, a system translation induced by a natural transformation with injective components also reflects bisimilarity.

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Abstract. The paradigm of service-oriented computing revolutionized the field of software engineering. According to this paradigm, new systems are composed of existing stand-alone services to support complex cross-organizational business processes. Correct communication of these services is not possible without a proper coordination mechanism. The Reo coordination language is a channel-based modeling language that introduces various types of channels and their composition rules. By composing Reo channels, one can specify Reo connectors that realize arbitrary complex behavioral protocols. Several formalisms have been introduced to give semantics to Reo. In their most basic form, they reflect service synchronization and dataflow constraints imposed by connectors. To ensure that the composed system behaves as intended, we need a wide range of automated verification tools to assist service composition designers. In this paper, we present our framework for the verification of Reo using the mCRL2 toolset. We unify our previous work on mapping various semantic models for Reo, namely, constraint automata, timed constraint automata, coloring semantics and the newly developed action constraint automata, to the process algebraic specification language of mCRL2, address the correctness of this mapping, discuss tool support, and present a detailed example that illustrates the use of Reo empowered with mCRL2 for the analysis of dataflow in service-based process models.