Random symmetry breaking and freezing in chaotic networks.

Parameter space of a driven damped oscillator in a double well potential presents either a chaotic trajectory with sign oscillating amplitude or a nonchaotic trajectory with a fixed sign amplitude. A network of such delay coupled damped oscillators is shown to present chaotic dynamics while the sign amplitude of each damped oscillator is randomly frozen. This phenomenon of random broken global symmetry of the network simultaneous with random freezing of each degree of freedom is accompanied by the existence of exponentially many randomly frozen chaotic attractors with the size of the network. Results are exemplified by a network of modified Duffing oscillators with infinite range pseudoinverse delayed interactions.

Coexistence of exponentially many chaotic spin-glass attractors.

A chaotic network of size N with delayed interactions which resembles a pseudoinverse associative memory neural network is investigated. For a load α = P/N < 1, where P stands for the number of stored patterns, the chaotic network functions as an associative memory of 2P attractors with macroscopic basin of attractions which decrease with α. At finite α, a chaotic spin-glass phase exists, where the number of distinct chaotic attractors scales exponentially with N. Each attractor is characterized by a coexistence of chaotic behavior and freezing of each one of the N chaotic units or freezing with respect to the P patterns. Results are supported by large scale simulations of networks composed of Bernoulli map units and Mackey-Glass time delay differential equations.

Multiplicative noise induces zero critical frequency.

Stochastic Bloch equations which model the fluorescence of two-level molecules and atoms, NMR experiments, and Josephson junctions are investigated to illustrate the profound effect of multiplicative noise on the critical frequency of a dynamical system. Using exact solutions and the cumulant expansion we find two main effects: (i) even very weak noise may double or triple the number of critical frequencies, which is related to an instability of the system, and (ii) strong multiplicative noise may induce a nontrivial zero critical frequency thus wiping out the overdamped phase.

Eukaryotic expression: developments for structural proteomics.

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens.

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.

SPINE bioinformatics and data-management aspects of high-throughput structural biology.

SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.

Recombinant protein expression and solubility screening in Escherichia coli: a comparative study.

Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His(6)-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore ;expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization.

Asm-1+, a Neurospora crassa gene related to transcriptional regulators of fungal development.

This report describes the identification, cloning, and molecular analysis of Asm-1+ (Ascospore maturation 1), the Neurospora crassa homologue of the Aspergillus nidulans stuA (stunted A) gene. The Asm-1+ gene is constitutively transcribed and encodes an abundant, nucleus-localized 68.5-kD protein. The protein product of Asm-1+ (ASM-1), contains a potential DNA-binding motif present in related proteins from A. nidulans (StuA), Candida albicans (EFGTF-1), and Saccharomyces cerevisiae (Phd1 and Sok2). This motif is related to the DNA binding motif of the Swi4/Mbp1/Res family of transcription factors that control the cell cycle. Deletion of Asm-1+ destroys the ability to make protoperithecia (female organs), but does not affect male-specific functions. We propose that the APSES domain (ASM-1, Phd1, StuA, EFGTF-1, and Sok2) defines a group of proteins that constitute a family of related transcription factors involved in the control of fungal development.

Translocation of Neurospora crassa transcription factor NUC-1 into the nucleus is induced by phosphorus limitation.

NUC-1, a basic helix-loop-helix zipper protein, activates the expression of several genes involved in phosphorus acquisition in Neurospora crassa. In the present study we investigated whether posttranscriptional mechanisms control the activity of NUC-1. The NUC-1 level was higher (up to fivefold) in wild-type cells grown at low external phosphate concentration and in mutant strains expressing the phosphorus acquisition genes constitutively than in a wild-type strain grown at high external phosphate concentration. Using indirect immunofluorescence we demonstrated that NUC-1 is localized at least predominantly in the cytosol when wild-type N. crassa is grown with an adequate supply of phosphate, whereas NUC-1 is largely concentrated in the nucleus upon limitation of external phosphate. In mutant strains expressing the phosphorus acquisition genes constitutively, NUC-1 localization was also primarily in the nucleus. Thus, subcellular compartmentation of regulatory proteins is an important mechanism in regulating gene expression in filamentous fungi.

The fumR gene encoding fumarase in the filamentous fungus Rhizopus oryzae: cloning, structure and expression.

The filamentous fungus Rhizopus oryzae (Ro) is known for its ability to overproduce and accumulate high levels of fumaric acid (FA) under stress conditions. In order to study the molecular mechanisms involved in the increased biosynthesis of FA, the gene (designated fumR) encoding Ro fumarase was cloned and analysed for its structure and expression. Nucleotide (nt) sequence and comparison of the fumR product with fumarases from various sources established that fumR contains nine introns and encodes a deduced product of 494 amino acids (aa), related to class-II fumarases. A fumarase protein of 50 kDa was immuno-detected in crude Ro extracts. Primer extension experiments mapped the 5' end of the fumR RNA 159 nt upstream from the putative translation start codon. Both primer extension and Northern analysis showed the existence of one transcript of fumR. The level of fumR RNA increased in cells producing FA under stress conditions (high carbon and low nitrogen levels in the medium), suggesting that transcriptional regulation of fumR might be involved in the overproduction and accumulation of FA by Ro cells under stress conditions. The possibility that additional mechanisms are responsible for this phenomenon is discussed.

Analysis of the DNA-binding and dimerization activities of Neurospora crassa transcription factor NUC-1.

NUC-1, a positive regulatory protein of Neurospora crassa, controls the expression of several unlinked target genes involved in phosphorus acquisition. The carboxy-terminal end of the NUC-1 protein has sequence similarity to the helix-loop-helix family of transcription factors. Bacterially expressed and in vitro-synthesized proteins, which consist of the carboxy-terminal portion of NUC-1, bind specifically to upstream sequences of two of its target genes, pho2+ and pho-4+. These upstream sequences contain the core sequence, CACGTG, a target for many helix-loop-helix proteins. A large loop region (47 amino acids) separates the helix I and helix II domains. Mutations and deletion within the loop region did not interfere with the in vitro or in vivo functions of the protein. Immediately carboxy-proximal to the helix II domain, the NUC-1 protein contains an atypical zipper domain which is essential for function. This domain consists of a heptad repeat of alanine and methionine rather than leucine residues. Analysis of mutant NUC-1 proteins suggests that the helix II and the zipper domains are essential for the protein dimerization, whereas the basic and the helix I domains are involved in DNA binding. The helix I domain, even though likely to participate in dimer formation while NUC-1 is bound to DNA, is not essential for in vitro dimerization.

The single translation product of the FUM1 gene (fumarase) is processed in mitochondria before being distributed between the cytosol and mitochondria in Saccharomyces cerevisiae.

The yeast mitochondrial and cytosolic isoenzymes of fumarase, which are encoded by a single nuclear gene (FUM1), follow a unique mechanism of protein subcellular localization and distribution. Translation of all FUM1 messages initiates only from the 5'-proximal AUG codon and results in a single translation product that contains the targeting sequence located within the first 32 amino acids of the precursor. All fumarase molecules synthesized in the cell are processed by the mitochondrial matrix signal peptidase; nevertheless, most of the enzyme (80 to 90%) ends up in the cytosol. The translocation and processing of fumarase are cotranslational. We suggest that in Saccharomyces cerevisiae, the single type of initial translation product of the FUM1 gene is first partially translocated, and then a subset of these molecules continues to be fully translocated into the organelle, whereas the rest are folded into an import-incompetent state and are released by the retrograde movement of fumarase into the cytosol.

Cloning and characterization of the pho-2+ gene encoding a repressible alkaline phosphatase in Neurospora crassa.

The Neurospora crassa phosphate-repressible alkaline phosphatase-encoding gene pho-2+ was cloned and its nucleotide sequence was determined. An open reading frame was found that contains four introns and encodes a putative protein of 555 amino acids. 'Activator-independent expression' of ectopically integrated pho-2+ was observed, as noted before for ectopically integrated pho-4+.