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Prions are transmissible, propagating alternative states of proteins. Prions in budding yeast propagate heritable phenotypes and can function in large-scale gene regulation, or in some cases occur as diseases of yeast. Other 'prionogenic' proteins are likely prions that have been determined experimentally to form amyloid in vivo, and to have prion-like domains that are able to propagate heritable states. Furthermore, there are over 300 additional 'prion-like' yeast proteins that have similar amino-acid composition to prions (primarily a bias for asparagines and glutamines). Here, we examine the protein functional and interaction networks that involve prion, prionogenic and prion-like proteins. Set against a marked overall preference for N/Q-rich prion-like proteins not to interact with each other, we observe a significant tendency of prion/prionogenic proteins to interact with other, N/Q-rich prion-like proteins. This tendency is mostly due to a small number of networks involving the proteins NUP100p, LSM4p and PUB1p. In general, different data analyses of functional and interaction networks converge to indicate a strong linkage of prionogenic and prion-like proteins, to stress-granule assembly and related biological processes. These results further elucidate how prions may impact gene regulation, and reveal a broader horizon for the functional relevance of N/Q-rich prion-like domains.

Pseudogenes are non-functioning copies of genes in genomic DNA, which may either result from reverse transcription from an mRNA transcript (processed pseudogenes) or from gene duplication and subsequent disablement (non-processed pseudogenes). As pseudogenes are apparently ‘dead’, they usually have a variety of obvious disablements (e.g., insertions, deletions, frameshifts and truncations) relative to their functioning homologs. We have derived an initial estimate of the size, distribution and characteristics of the pseudogene population in the Caenorhabditis elegans genome, performing a survey in ‘molecular archaeology’. Corresponding to the 18 576 annotated proteins in the worm (i.e., in Wormpep18), we have found an estimated total of 2168 pseudogenes, about one for every eight genes. Few of these appear to be processed. Details of our pseudogene assignments are available from http://bioinfo.mbb.yale.edu/genome/worm/pseudogene. The population of pseudogenes differs significantly from that of genes in a number of respects: (i) pseudogenes are distributed unevenly across the genome relative to genes, with a disproportionate number on chromosome IV; (ii) the density of pseudogenes is higher on the arms of the chromosomes; (iii) the amino acid composition of pseudogenes is midway between that of genes and (translations of) random intergenic DNA, with enrichment of Phe, Ile, Leu and Lys, and depletion of Asp, Ala, Glu and Gly relative to the worm proteome; and (iv) the most common protein folds and families differ somewhat between genes and pseudogenes—whereas the most common fold found in the worm proteome is the immunoglobulin fold and the most common ‘pseudofold’ is the C-type lectin. In addition, the size of a gene family bears little overall relationship to the size of its corresponding pseudogene complement, indicating a highly dynamic genome. There are in fact a number of families associated with large populations of pseudogenes. For example, one family of seven-transmembrane receptors (represented by gene B0334.7) has one pseudogene for every four genes, and another uncharacterized family (represented by gene B0403.1) is approximately two-thirds pseudogenic. Furthermore, over a hundred apparent pseudogenic fragments do not have any obvious homologs in the worm.

Prion disease is caused by the propagation of a particle containing PrPSc, a misfolded form of the normal cellular prion protein (PrPC). PrPC can re-fold to form PrPSc with loss of alpha-helical structure and formation of extensive beta-sheet structure. Here, we model this prion folding problem with a simple, low-resolution lattice model of protein folding. If model proteins are allowed to re-fold upon dimerization, a minor proportion of them (up to approximately 17%) encrypts an alternative native state as a homodimer. The structures in this homodimeric native state re-arrange so that they are very different in conformation from the monomeric native state. We find that model proteins that are relatively less stable as monomers are more susceptible to the formation of alternative native states as homodimers. These results suggest that less-stable proteins have a greater need for a well-designed energy landscape for protein folding to overcome an increased chance of encrypting substantially different native conformations stabilized by multimeric interactions. This conceptual framework for aberrant folding should be relevant in Alzheimer's disease and other disorders associated with protein aggregation.

Cripto, also known as human teratocarcinoma-derived growth factor 1 (TDGF-1), contains a 40 amino acid region with some similarity to the epidermal growth factor (EGF) domain. However, sequence homology is largely restricted to the classical cysteine/glycine motif with only limited similarities in other regions. Significant differences to human EGF include the absence of all seven residues between the two N-terminal half-cystines and a five-residue shorter loop between the third and fourth half-cystines. We examine the hypothesis that, in spite of these differences, cripto can adopt the characteristic EGF-like 1-3, 2-4, 5-6 disulfide bond pattern. A comparative structural model of the growth factor cripto was constructed on the basis of its similarity to EGF, transforming growth factor alpha (TGF-alpha), and the EGF-like domain of human clotting factor IX. The predicted disulfide bridges and disulfide-bridged loops were analyzed and appear viable in the modeled structure. Moreover, to ascertain the importance of disulfide arrangement for cripto bioactivity, two 47-residue peptides were synthesized and then refolded using either a simple oxidative or a controlled sequential refolding protocol. The cripto peptides were tested for their ability to stimulate MAP-kinase activity, for inhibition of beta-casein induction, and for Shc phosphorylation in MDA-MB 453 human mammary carcinoma cells and HC-11 mouse mammary epithelial cells. Data suggest that cripto does adopt the 1-3, 2-4, 5-6 disulfide pattern and thus forms the classical EGF-like fold in spite of the significant deletions within the folding domain. The predicted structure of cripto shows some of the characteristics of both the ErbB1- and ErbB3/ErbB4-binding growth factors.

Prion diseases are neurodegenerative disorders in which dramatic conformational change in the structure of the prion protein is the fundamental event. This structural transition involves the loss of substantial α-helical content and the acquisition of β-sheet structure. A convergence of recent biological and structural studies argues that the mechanism underlying the prion diseases is truly unprecedented.

Despite the central importance of noncoding DNA to gene regulation and evolution, understanding of the extent of selection on plant noncoding DNA remains limited compared to that of other organisms. Here we report sequencing of genomes from three Brassicaceae species (Leavenworthia alabamica, Sisymbrium irio and Aethionema arabicum) and their joint analysis with six previously sequenced crucifer genomes. Conservation across orthologous bases suggests that at least 17% of the Arabidopsis thaliana genome is under selection, with nearly one-quarter of the sequence under selection lying outside of coding regions. Much of this sequence can be localized to approximately 90,000 conserved noncoding sequences (CNSs) that show evidence of transcriptional and post-transcriptional regulation. Population genomics analyses of two crucifer species, A. thaliana and Capsella grandiflora, confirm that most of the identified CNSs are evolving under medium to strong purifying selection. Overall, these CNSs highlight both similarities and several key differences between the regulatory DNA of plants and other species.

Small disulphide-rich protein folds (SDFs) tend to have less, regular secondary structure than larger protein folds and are thus problematic in protein structure taxonomy and prediction. We report regularities for disulphide-bridged beta-sheet and for cystine clustering that are particularly relevant to such proteins. The repertoire of cystine conformations results in preferences in disulphide distribution between/within beta-sheets. For example, disulphides seldom bridge between beta-sheets with antiparallel orientation for the flanking polypeptide segments, as the separations between packed sheets are such that the only rotamers that straddle them easily are those that generally require parallel orientation. A left-handed chirality preference is described for the intervening connection for disulphides bridging between berta-strands in different sheets in such a parallel orientation. Geometrical analysis of clusters of two cystine residues has shown that closely clustered cystine residues tend to have approximately orthogonal relative orientation. A positive orientation of this type is most often accommodated by a recurrent motif of disulphide-bridged beta-sheet that we call the disulphide beta-cross. The consensus features of this motif are described. It occurs in non-homologous proteins with a variety of folds, subsuming partial similarities previously noted by several other workers. Further examples are discussed, such as a two-cystine/two-beta-hairpin assembly common to hirudin and the three-fingered toxin folds. We suggest that the consensus features enable it to act as a good folding nucleus. We classify similar three-cystine arrangements that may be described as a ladder or stack, that tend to contain a disulphide beta-cross and that recur in folds that can otherwise be quite different. The preferences for disulphide-beta-sheet distribution and for cystine clusters contribute to an array of partial similarities for SDFs, many of which incorporate the disulphide beta-cross. It is suggested that SDF taxonomy cannot properly be considered without using both the relationship between clustered cystine residues and that between cystine residues and the regular secondary structures that they connect (here, we study beta in particular). The implications for SDF classification are demonstrated.

Profile cutting of thin sheet bright metals can be achieved in a cost effective manner through the use of a pulsed fiber laser in conjunction with a galvanometric scanhead without co-axial assist gas. This is attractive where many batches of distinct small parts are manufactured in volume, for instance within the jewellery and the micro-electronics industries.In this paper the capabilities of a pulsed fiber laser are considered in detail when processing bright metals such as brass, copper and silver. The effect of key laser parameters such as peak power, temporal pulse shape and laser pulse frequency will be considered along with several process parameters such as pulse overlap and beam wobbling techniques. This is with the aim of producing parts at high speed and/or at high quality and to determine the maximum thickness that can be cut at effective speeds. The results will be compared and contrasted with other metals such as stainless steel and aluminium to determine the range of processing strategies needed to cut these materials.Profile cutting of thin sheet bright metals can be achieved in a cost effective manner through the use of a pulsed fiber laser in conjunction with a galvanometric scanhead without co-axial assist gas. This is attractive where many batches of distinct small parts are manufactured in volume, for instance within the jewellery and the micro-electronics industries.In this paper the capabilities of a pulsed fiber laser are considered in detail when processing bright metals such as brass, copper and silver. The effect of key laser parameters such as peak power, temporal pulse shape and laser pulse frequency will be considered along with several process parameters such as pulse overlap and beam wobbling techniques. This is with the aim of producing parts at high speed and/or at high quality and to determine the maximum thickness that can be cut at effective speeds. The results will be compared and contrasted with other metals such as stainless steel and aluminium to determine the range of processing strategies ne...

Prion Proteins (PrP) are among a small number of proteins for which large numbers of NMR ensembles have been resolved for sequence mutants and diverse species. Here, we perform a comprehensive principle components analysis (PCA) on the tertiary structures of PrP globular proteins to discern PrP subdomains that exhibit conformational change in response to point mutations and clade-specific evolutionary sequence mutation trends. This is to our knowledge the first such large-scale analysis of multiple NMR ensembles of protein structures, and the first study of its kind for PrPs. We conducted PCA on human (n = 11), mouse (n = 14), and wildtype (n = 21) sets of PrP globular structures, from which we identified five conformationally variable subdomains within PrP. PCA shows that different non-local patterns and rankings of variable subdomains arise for different pathogenic mutants. These subdomains may thus be key areas for initiating PrP conversion during disease. Furthermore, we have observed the conformational clustering of divergent TSE-non-susceptible species pairs; these non-phylogenetic clusterings indicate structural solutions towards TSE resistance that do not necessarily coincide with evolutionary divergence. We discuss the novelty of our approach and the importance of PrP subdomains in structural conversion during disease., Author Summary Prion Proteins (PrP) cause a variety of incurable TSE diseases, and are among a small number of proteins for which large numbers of NMR ensembles have been resolved for sequence mutants and diverse species. Here, we perform a comprehensive PCA study to assess conformational variation and discern the landscape of the PrP structural response to sequence mutation. This is to our knowledge the first large-scale analysis of multiple NMR ensembles for a specific protein, and the first study to perform a multivariate PCA on the native globular structures of PrP. We conducted exhaustive PCA on three PrP subsets: human and mouse subsets that include structures of sequence mutants, and the set of wild-type PrP (16 PrP species). PCA shows that different non-local patterns of variable subdomains arise for different pathogenic mutants. These subdomains may thus be key areas for initiating PrP conversion during disease. Furthermore, we observed that some evolutionarily divergent species that are non-susceptible to TSEs have surprising structural similarities in their PrPs. We discuss the novelty of our approach with respect to prions, and the advantage of this analysis as a fast, reliable starting point to identify interesting domains that may warrant further experimental and computational analysis.

Prions are units of propagation of an altered state of a protein or proteins. Prions can propagate from cell to cell, and from organism to organism, through cooption of other protein copies. Prions contain no necessary nucleic acids, and are important both as both pathogenic agents, and as a potential force in epigenetic phenomena. The original prions were derived from a misfolded form of the mammalian Prion Protein PrP. Infection by these prions causes neurodegenerative diseases. Other prions cause non-Mendelian inheritance in budding yeast, and sometimes act as diseases of yeast. We have compiled a database of >2000 prion-related sequences, called the PrionOme. The database comprises seven PrionOme classification categories: prionogenic sequences (i.e., sequences that can make prions), ‘prionoids’ (i.e., phenomena that have some prion characteristics), orthologs, paralogs, pseudogenes, prion interactors, and prion-like molecules. Database entries list: supporting information for PrionOme classifications, prion-determinant areas (where relevant), and disordered and compositionally-biased regions. Also included are original references for the PrionOme classifications, transcripts and genomic coordinates, and structural data (including comparative models). We provide database usage examples for both vertebrate and fungal prion contexts. As development of this resource is on-going, we will be very happy to receive and act on any constructive comments from peer scientists in the areas of prion biology and protein misfolding, either by email or using the feedback form provided on the PrionOme website. We hope that this database will be a valuable experimental aid and reference resource. It is freely available at: http://libaio.biol.mcgill.ca/prion.

The HET-s prion-forming domain from the filamentous fungus Podospora anserina is gaining considerable interest since it yielded the first well-defined atomic structure of a functional amyloid fibril. This structure has been identified as a left-handed beta solenoid with a triangular hydrophobic core. To delineate the origins of the HET-s prion-forming protein and to discover other amyloid-forming proteins, we searched for all homologs of the HET-s protein in a database of protein domains and fungal genomes, using a combined application of HMM, psi-blast and pGenThreader techniques, and performed a comparative evolutionary analysis of the N-terminal alpha-helical domain and the C-terminal prion-forming domain of HET-s. By assessing the tandem evolution of both domains, we observed that the prion-forming domain is restricted to Sordariomycetes, with a marginal additional sequence homolog in Arthroderma otae as a likely case of horizontal transfer. This suggests innovation and rapid evolution of the solenoid fold in the Sordariomycetes clade. In contrast, the N-terminal domain evolves at a slower rate (in Sordariomycetes) and spans many diverse clades of fungi. We performed a full three-dimensional protein threading analysis on all identified HET-s homologs against the HET-s solenoid fold, and present detailed structural annotations for identified structural homologs to the prion-forming domain. An analysis of the physicochemical characteristics in our set of structural models indicates that the HET-s solenoid shape can be readily adopted in these homologs, but that they are all less optimized for fibril formation than the P. anserina HET-s sequence itself, due chiefly to the presence of fewer asparagine ladders and salt bridges. Our combined structural and evolutionary analysis suggests that the HET-s shape has “limited scope” for amyloidosis across the wider protein universe, compared to the ‘generic’ left-handed beta helix. We discuss the implications of our findings on future identification of amyloid-forming proteins sharing the solenoid fold.

Identification of ambiguous encoding in protein secondary structure is paramount to develop an understanding of key protein segments underlying amyloid diseases. We investigate two types of structurally ambivalent peptides, which were hypothesized in the literature as indicators of amyloidogenic proteins: discordant α-helices and chameleon sequences. Chameleon sequences are peptides discovered experimentally in different secondary-structure types. Discordant α-helices are α-helical stretches with strong β-strand propensity or prediction. To assess the distribution of these features in known protein structures, and their potential role in amyloidogenesis, we analyzed the occurrence of discordant α-helices and chameleon sequences in nonredundant sets of protein domains (n = 4263) and amyloidogenic proteins extracted from the literature (n = 77). Discordant α-helices were identified if discordance was observed between known secondary structures and secondary-structure predictions from the GOR-IV and PSIPRED algorithms. Chameleon sequences were extracted by searching for identical sequence words in α-helices and β-strands. We defined frustrated chameleons and very frustrated chameleons based on varying degrees of total β propensity ≥α propensity. To our knowledge, this is the first study to discern statistical relationships between discordance, chameleons, and amyloidogenicity. We observed varying enrichment levels for some categories of discordant and chameleon sequences in amyloidogenic sequences. Chameleon sequences are also significantly enriched in proteins that have discordant helices, indicating a clear link between both phenomena. We identified the first set of discordant-chameleonic protein segments we predict may be involved in amyloidosis. We present a detailed analysis of discordant and chameleons segments in the family of one of the amyloidogenic proteins, the Prion Protein.

An almost unique place within protein databases, twenty-five years of study has underscored the enigmatically subtle role of PrP(C) in normal cell biology. It seems that PrP has evolved (and survived) to perform a function that does not have a precedent amongst transmembrane cell-surface proteins, perhaps representing a new type of plasma membrane ecosystem. In a context where we await a clarifying insight to unify a panoply of PrP(C) data into logical molecular framework, the GPI-anchored N-glycosylated Doppel and Sho proteins are tantalizing in that they correspond roughly to the front and back halves of PrP(C) itself. These molecules may be simpler - and more "understandable" - entities that can be pursued in parallel to PrP(C), and could open up the biology of mammalian prion proteins from fresh directions. Dpl has a profound role in successful gametogenesis that warrants close scrutiny and a case for deeper study can be made for Sho, a recently discovered CNS-expressed protein with many parallels to established facets of PrP biochemistry. In an aerial view of biomedical research, Sho and Dpl can be considered as adjacent islands in a prion protein archipelago. As such, the coming years of molecular exploration should be extremely interesting.

Background The role of long non-coding RNAs (lncRNAs) in controlling gene expression has garnered increased interest in recent years. Sequencing projects, such as Fantom3 for mouse and H-InvDB for human, have generated abundant data on transcribed components of mammalian cells, the majority of which appear not to be protein-coding. However, much of the non-protein-coding transcriptome could merely be a consequence of ‘transcription noise’. It is therefore essential to use bioinformatic approaches to identify the likely functional candidates in a high throughput manner. Principal Findings We derived a scheme for classifying and annotating likely functional lncRNAs in mammals. Using the available experimental full-length cDNA data sets for human and mouse, we identified 78 lncRNAs that are either syntenically conserved between human and mouse, or that originate from the same protein-coding genes. Of these, 11 have significant sequence homology. We found that these lncRNAs exhibit: (i) patterns of codon substitution typical of non-coding transcripts; (ii) preservation of sequences in distant mammals such as dog and cow, (iii) significant sequence conservation relative to their corresponding flanking regions (in 50% cases, flanking regions do not have homology at all; and in the remaining, the degree of conservation is significantly less); (iv) existence mostly as single-exon forms (8/11); and, (v) presence of conserved and stable secondary structure motifs within them. We further identified orthologous protein-coding genes that are contributing to the pool of lncRNAs; of which, genes implicated in carcinogenesis are significantly over-represented. Conclusion Our comparative mammalian genomics approach coupled with evolutionary analysis identified a small population of conserved long non-protein-coding RNAs (lncRNAs) that are potentially functional across Mammalia. Additionally, our analysis indicates that amongst the orthologous protein-coding genes that produce lncRNAs, those implicated in cancer pathogenesis are significantly over-represented, suggesting that these lncRNAs could play an important role in cancer pathomechanisms.

Pseudogenes arise from the decay of gene copies following either RNA-mediated duplication (processed pseudogenes) or DNA-mediated duplication (nonprocessed pseudogenes). Here, we show that long protein-coding genes tend to produce more nonprocessed pseudogenes than short genes, whereas the opposite is true for processed pseudogenes. Protein-coding genes longer than 3000 bp are 6 times more likely to produce nonprocessed pseudogenes than processed ones. Reviewers This article was reviewed by Dr. Dan Graur and Dr. Craig Nelson (nominated by Dr. J Peter Gogarten).

Background Transcribed pseudogenes are copies of protein-coding genes that have accumulated indicators of coding-sequence decay (such as frameshifts and premature stop codons), but nonetheless remain transcribed. Recent experimental evidence indicates that transcribed pseudogenes may regulate the expression of homologous genes, through antisense interference, or generation of small interfering RNAs (siRNAs). Here, we assessed the genomic evidence for such transcribed pseudogenes of potential functional importance, in the human genome. The most obvious indicators of such functional importance are significant evidence of conservation and selection pressure. Results A variety of pseudogene annotations from multiple sources were pooled and filtered to obtain a subset of sequences that have significant mid-sequence disablements (frameshifts and premature stop codons), and that have clear evidence of full-length mRNA transcription. We found 1750 such transcribed pseudogene annotations (TPAs) in the human genome (corresponding to ~11.5% of human pseudogene annotations). We checked for syntenic conservation of TPAs in other mammals (rhesus monkey, mouse, rat, dog and cow). About half of the human TPAs are conserved in rhesus monkey, but strikingly, very few in mouse (~3%). The TPAs conserved in rhesus monkey show evidence of selection pressure (relative to surrounding intergenic DNA) on: (i) their GC content, and (ii) their rate of nucleotide substitution. This is in spite of distributions of Ka/Ks (ratios of non-synonymous to synonymous substitution rates), congruent with a lack of protein-coding ability. Furthermore, we have identified 68 human TPAs that are syntenically conserved in at least two other mammals. Interestingly, we observe three TPA sequences conserved in dog that have intermediate character (i.e., evidence of both protein-coding ability and pseudogenicity), and discuss the implications of this. Conclusion Through evolutionary analysis, we have identified candidate sequences for functional human transcribed pseudogenes, and have pinpointed 68 strong candidates for further investigation as potentially functional transcribed pseudogenes across multiple mammal species.

Within the vast range of laser materials processing applications, every type of successful commercial laser has been driven by a major industrial process. For high average power, high peak power, nanosecond pulse duration Nd:YAG DPSS lasers, the enabling process is high speed surface engineering. This includes applications such as thin film patterning and selective coating removal in markets such as the flat panel displays (FPD), solar and automotive industries. Applications such as these tend to require working spots that have uniform intensity distribution using specific shapes and dimensions, so a range of innovative beam delivery systems have been developed that convert the gaussian beam shape produced by the laser into a range of rectangular and/or shaped spots, as required by demands of each project. In this paper the authors will discuss the key parameters of this type of laser and examine why they are important for high speed surface engineering projects, and how they affect the underlying laser-material interaction and the removal mechanism. Several case studies will be considered in the FPD and solar markets, exploring the close link between the application, the key laser characteristics and the beam delivery system that link these together.

This article offers a case study of the evaluation of a redesigned and redeveloped laboratory-based cell biology course. The course was a compulsory element of the biology program, but the laboratory had become outdated and was inadequately equipped. With the support of a faculty-based teaching improvement project, the teaching team redesigned the course and re-equipped the laboratory, using a more learner-centered, constructivist approach. The focus of the article is on the project-supported evaluation of the redesign rather than the redesign per se. The evaluation involved aspects well beyond standard course assessments, including the gathering of self-reported data from the students concerning both the laboratory component and the technical skills associated with the course. The comparison of pre- and postdata gave valuable information to the teaching team on course design issues and skill acquisition. It is argued that the evaluation process was an effective use of the scarce resources of the teaching improvement project.

Background Protein kinases and phosphatases regulate protein phosphorylation, a critical means of modulating protein function, stability and localization. The identification of functional networks for protein phosphatases has been slow due to their redundant nature and the lack of large-scale analyses. We hypothesized that a genome-scale analysis of genetic interactions using the Synthetic Genetic Array could reveal protein phosphatase functional networks. We apply this approach to the conserved type 1 protein phosphatase Glc7, which regulates numerous cellular processes in budding yeast. Results We created a novel glc7 catalytic mutant (glc7-E101Q). Phenotypic analysis indicates that this novel allele exhibits slow growth and defects in glucose metabolism but normal cell cycle progression and chromosome segregation. This suggests that glc7-E101Q is a hypomorphic glc7 mutant. Synthetic Genetic Array analysis of glc7-E101Q revealed a broad network of 245 synthetic sick/lethal interactions reflecting that many processes are required when Glc7 function is compromised such as histone modification, chromosome segregation and cytokinesis, nutrient sensing and DNA damage. In addition, mitochondrial activity and inheritance and lipid metabolism were identified as new processes involved in buffering Glc7 function. An interaction network among 95 genes genetically interacting with GLC7 was constructed by integration of genetic and physical interaction data. The obtained network has a modular architecture, and the interconnection among the modules reflects the cooperation of the processes buffering Glc7 function. Conclusion We found 245 genes required for the normal growth of the glc7-E101Q mutant. Functional grouping of these genes and analysis of their physical and genetic interaction patterns bring new information on Glc7-regulated processes.

The process of rapid laser patterning (RLP) of ITO thin films on glass has been adopted into mass production by the flat panel displays industry. This process involves high speed selective removal of thin films for the manufacture of transparent electrode structures and uses high average power, high peak power, nanosecond pulse duration Nd:YAG DPSS lasers. In this paper the laser patterning process and removal mechanism are described in detail with consideration given to how laser pulse characteristics influence features of the process. Subsequent issues such as the effect of the laser ablation plume, debris, substrate heating and redeposition of ITO residue are also considered.The process of rapid laser patterning (RLP) of ITO thin films on glass has been adopted into mass production by the flat panel displays industry. This process involves high speed selective removal of thin films for the manufacture of transparent electrode structures and uses high average power, high peak power, nanosecond pulse duration Nd:YAG DPSS lasers. In this paper the laser patterning process and removal mechanism are described in detail with consideration given to how laser pulse characteristics influence features of the process. Subsequent issues such as the effect of the laser ablation plume, debris, substrate heating and redeposition of ITO residue are also considered.

Background Efforts to gather genomic evidence for the processes of gene evolution are ongoing, and are closely coupled to improved gene annotation methods. Such annotation is complicated by the occurrence of disrupted mRNAs (dmRNAs), harbouring frameshifts and premature stop codons, which can be considered indicators of decay into pseudogenes. Results We have derived a procedure to annotate dmRNAs, and have applied it to human data. Subsequences are generated from parsing at key frame-disruption positions and are required to align significantly within any original protein homology. We find 419 high-quality human dmRNAs (3% of total). Significant dmRNA subpopulations include: zinc-finger-containing transcription factors with long disrupted exons, and antisense homologies to distal genes. We analysed the distribution of initial frame disruptions in dmRNAs with respect to positions of: (i) protein domains, (ii) alternatively-spliced exons, and (iii) regions susceptible to nonsense-mediated decay (NMD). We find significant avoidance of protein-domain disruption (indicating a selection pressure for this), and highly significant overrepresentation of disruptions in alternatively-spliced exons, and 'non-NMD' regions. We do not find any evidence for evolution of novelty in protein structures through frameshifting. Conclusion Our results indicate largely negative selection pressures related to frame disruption during gene evolution.

A combined bioinformatic and genetic approach was used to conduct a systematic analysis of the relationship between ribosomal protein genes and Minute loci in Drosophila melanogaster, allowing the identification of 64 Minute loci corresponding to ribosomal genes., Background Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes. Results We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci. Conclusion This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome.

Rapid Laser Patterning (RLP) of ITO thin films on glass offers a credible industrial alternative to wet-etch lithography for the manufacture of Flat Panel Displays – particularly for Plasma Displays. For any disruptive technology it is necessary to compare and contrast it with the existing manufacturing technique so that end users can make an informed decision as to its relative merits – both technical and commercial.Both processes are compared empirically and it is demonstrated that RLP offers benefits in terms of edge resolution, taper and is less prone to residue. Furthermore a commercial cost model is generated based on real capital and operational cost data. This model suggests that RLP can offer cost savings in the order of tens of millions of dollars for a single production line over a three year depreciation period.Rapid Laser Patterning (RLP) of ITO thin films on glass offers a credible industrial alternative to wet-etch lithography for the manufacture of Flat Panel Displays – particularly for Plasma Displays. For any disruptive technology it is necessary to compare and contrast it with the existing manufacturing technique so that end users can make an informed decision as to its relative merits – both technical and commercial.Both processes are compared empirically and it is demonstrated that RLP offers benefits in terms of edge resolution, taper and is less prone to residue. Furthermore a commercial cost model is generated based on real capital and operational cost data. This model suggests that RLP can offer cost savings in the order of tens of millions of dollars for a single production line over a three year depreciation period.

Liquid Crystal Displays (LCDs) represent the most widely manufactured form of Flat Panel Display (FPD) in the world today. This market is extremely price sensitive and so competition is fierce. In an attempt to reduce costs, manufacturers are always interested in new technology to gain a lead on their competition. In recent years laser use has been increasing in the manufacture of FPDs – most notably as a replacement for wet-etch lithography to pattern ITO thin films on the screens of Plasma Display Panels.In this paper the authors describe a new laser-based technique which allows the direct patterning of the Black Matrix resin used as a contrast enhancer between the RGB pixels in an LCD. This technique solves the problems experienced in previous attempts at laser processing of this material: edge taper, feature variation, residue and debris. This technique has the potential to displace wet-etch lithography for this process by offering both cost and quality advantages over the incumbent technology.Liquid Crystal Displays (LCDs) represent the most widely manufactured form of Flat Panel Display (FPD) in the world today. This market is extremely price sensitive and so competition is fierce. In an attempt to reduce costs, manufacturers are always interested in new technology to gain a lead on their competition. In recent years laser use has been increasing in the manufacture of FPDs – most notably as a replacement for wet-etch lithography to pattern ITO thin films on the screens of Plasma Display Panels.In this paper the authors describe a new laser-based technique which allows the direct patterning of the Black Matrix resin used as a contrast enhancer between the RGB pixels in an LCD. This technique solves the problems experienced in previous attempts at laser processing of this material: edge taper, feature variation, residue and debris. This technique has the potential to displace wet-etch lithography for this process by offering both cost and quality advantages over the incumbent technology.

Prions are transmissible self-replicating alternative states of proteins. Four prions ([PSI+], [URE3], [RNQ+] and [NU+]) can be inherited cytoplasmically in Saccharomyces cerevisiae laboratory strains. In the case of [PSI+], there is increasing evidence that prion formation may engender mechanisms to uncover hidden genetic variation. Here, we have analysed the evolution of the prion-determinant (PD) domains across 21 fungi, focusing on compositional biases, repeats and substitution rates. We find evidence for constraint on all four PD domains, but each domain has its own evolutionary dynamics. For [PSI+], the Q/N bias is maintained in fungal clades that diverged one billion years ago, with purifying selection observed within the Saccharomyces species. The degree of Q/N bias is correlated with the degree of local homology to prion-associated repeats, which occur rarely in other proteins (

Motivation: Mammalian genomes contain many ‘genomic fossils’ i.e. pseudogenes. These are disabled copies of functional genes that have been retained in the genome by gene duplication or retrotransposition events. Pseudogenes are important resources in understanding the evolutionary history of genes and genomes. Results: We have developed a homology-based computational pipeline (‘PseudoPipe’) that can search a mammalian genome and identify pseudogene sequences in a comprehensive and consistent manner. The key steps in the pipeline involve using BLAST to rapidly cross-reference potential “parent” proteins against the intergenic regions of the genome and then processing the resulting “raw hits” -- i.e. eliminating redundant ones, clustering together neighbors, and associating and aligning clusters with a unique parent. Finally, pseudogenes are classified based on a combination of criteria including homology, intron-exon structure, and existence of stop codons and frameshifts. Availability: The PseudoPipe program is implemented in Python and can be downloaded at Contact: Mark.Gerstein@yale.edu or zhaolei.zhang@utoronto.ca

Flat panel display manufacturing has recently been revolutionized by the industrial uptake of laser direct write techniques to replace lithography for patterning active thin films on glass. The use of diode pumped, high average power, nanosecond pulsed, kilohertz repetition rate infrared lasers to directly pattern electrically active thin films on glass substrates has eliminated as many as five production stages in the manufacture of flat panel displays. Such lasers are now available in average powers nearing one kilowatt, which allows sufficient productivity to take this process from the lab into industry.One key issue of concern is quantifying the removal of the thin films and how that relates to beam homogeneity and energy density on the workpiece. In this paper the authors attempt to quantify the removal of the thin films from glass by comparing advanced analytical techniques such as Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) with optical microscopy for a range of laser parameters. This analysis is put into context of the wider laser direct write technique of thin films on glass; particularly that of Indium Tin Oxide (ITO) for the manufacture of Plasma Display Panels (PDPs).Flat panel display manufacturing has recently been revolutionized by the industrial uptake of laser direct write techniques to replace lithography for patterning active thin films on glass. The use of diode pumped, high average power, nanosecond pulsed, kilohertz repetition rate infrared lasers to directly pattern electrically active thin films on glass substrates has eliminated as many as five production stages in the manufacture of flat panel displays. Such lasers are now available in average powers nearing one kilowatt, which allows sufficient productivity to take this process from the lab into industry.One key issue of concern is quantifying the removal of the thin films and how that relates to beam homogeneity and energy density on the workpiece. In this paper the authors attempt to quantify the removal of the thin films from glass by comparing advanced analytical techniques such as Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) with optical microscopy for a range of laser param...

Laser marking of metals and plastics is one of the most widespread of all laser materials processing techniques used throughout the industrial sectors. One remaining challenge is the creation of scanner readable marks on highly reflective materials such as aluminium without the aid of a surface coating. In previous work the authors have presented a novel technique employing a high average power diode pumped solid-state Q-switched laser to locally change the surface roughness, creating high contrast regions between highly scattering laser roughened marks and the unmarked reflective substrate. In this paper the effect of surface modification is quantified and analysed for aluminium, and the techniques are transferred and adapted to other widespread industrial materials such as stainless and galvanized steel. In this way the authors demonstrate the applicability of this laser technique for a range of reflective metals. Applications such as traceability and security are discussed for high volume industrial sectors such as automotive manufacture.Laser marking of metals and plastics is one of the most widespread of all laser materials processing techniques used throughout the industrial sectors. One remaining challenge is the creation of scanner readable marks on highly reflective materials such as aluminium without the aid of a surface coating. In previous work the authors have presented a novel technique employing a high average power diode pumped solid-state Q-switched laser to locally change the surface roughness, creating high contrast regions between highly scattering laser roughened marks and the unmarked reflective substrate. In this paper the effect of surface modification is quantified and analysed for aluminium, and the techniques are transferred and adapted to other widespread industrial materials such as stainless and galvanized steel. In this way the authors demonstrate the applicability of this laser technique for a range of reflective metals. Applications such as traceability and security are discussed for high volume industrial se...

DNA transposons are known to frequently capture duplicated fragments of host genes. The evolutionary impact of this phenomenon depends on how frequently the fragments retain protein-coding function as opposed to becoming pseudogenes. Gene fragment duplication by Mutator-like elements (MULEs) has previously been documented in maize, Arabidopsis, and rice. Here we present a rigorous genome-wide analysis of MULEs in the model plant Oryza sativa (domesticated rice). We identify 8274 MULEs with intact termini and target-site duplications (TSDs) and show that 1337 of them contain duplicated host gene fragments. Through a detailed examination of the 5% of duplicated gene fragments that are transcribed, we demonstrate that virtually all cases contain pseudogenic features such as fragmented conserved protein domains, frameshifts, and premature stop codons. In addition, we show that the distribution of the ratio of nonsynonymous to synonymous amino acid substitution rates for the duplications agrees with the expected distribution for pseudogenes. We conclude that MULE-mediated host gene duplication results in the formation of pseudogenes, not novel functional protein-coding genes; however, the transcribed duplications possess characteristics consistent with a potential role in the regulation of host gene expression.

The authors investigate the high-speed laser cutting of flexible printed circuit boards (PCBs) using a low M2 532nm laser to create features 120mm/s are reported with a high quality kerf.The authors investigate the high-speed laser cutting of flexible printed circuit boards (PCBs) using a low M2 532nm laser to create features 120mm/s are reported with a high quality kerf.

In this paper a progressive empirical study is conducted refining the laser cutting of Polycrystalline Diamond (PCD) as ongoing work.PCD is one of the hardest substances known and is used extensively in cutting tools for non-ferrous materials. It is extremely difficult to cut by conventional means and the current incumbent technology, Electro-Discharge Machining (EDM), is relatively slow and has some serious drawbacks in terms of tool wear, unidirectional cutting and post processing. The PCD is extremely hard but brittle and so is typically mounted on Tungsten Carbide (WC) in the form of a laminar disk. Cutting this laminar structure creates additional challenges because the materials have dissimilar properties.In previous work the authors have successfully demonstrated and modelled rapid laser cutting and drilling of PCD using high average power Q-switched diode pumped solid-state lasers. In this paper the cutting process is refined empirically to improve cut edge quality, reduce surface dross and create complex shapes whilst still achieving a comparatively high cutting velocity.In this paper a progressive empirical study is conducted refining the laser cutting of Polycrystalline Diamond (PCD) as ongoing work.PCD is one of the hardest substances known and is used extensively in cutting tools for non-ferrous materials. It is extremely difficult to cut by conventional means and the current incumbent technology, Electro-Discharge Machining (EDM), is relatively slow and has some serious drawbacks in terms of tool wear, unidirectional cutting and post processing. The PCD is extremely hard but brittle and so is typically mounted on Tungsten Carbide (WC) in the form of a laminar disk. Cutting this laminar structure creates additional challenges because the materials have dissimilar properties.In previous work the authors have successfully demonstrated and modelled rapid laser cutting and drilling of PCD using high average power Q-switched diode pumped solid-state lasers. In this paper the cutting process is refined empirically to improve cut edge quality, reduce surface dross and create...

This paper investigates deep engraving of steel and aluminium by laser. By examination of laser and scanner parameters an optimal balance between feature quality and processing speed is achieved. Material removal rates of up to 20 mm3/min for steel and 40 mm3/min for aluminium are demonstrated up to a maximum engraved depth of 1mm. The effect of feature shape and feature size on the process is also investigated.Deep engraving of metals using latest generation Q-switched diode pumped solid-state lasers is a developing application across a range of market sectors. One key use is in creating indelible laser milled marks on high value components in the automotive industry for security reasons. These marks must be resistant to tampering, visible after subsequent painting, difficult to reproduce and not affecting the working properties of the component.

Pseudogenes are inheritable genetic elements formally defined by two properties: their similarity to functioning genes and their presumed lack of activity. However, their precise characterization, particularly with respect to the latter quality, has proven elusive. An opportunity to explore this issue arises from the recent emergence of tiling-microarray data showing that intergenic regions (containing pseudogenes) are transcribed to a great degree. Here we focus on the transcriptional activity of pseudogenes on human chromosome 22. First, we integrated several sets of annotation to define a unified list of 525 pseudogenes on the chromosome. To characterize these further, we developed a comprehensive list of genomic features based on conservation in related organisms, expression evidence, and the presence of upstream regulatory sites. Of the 525 unified pseudogenes we could confidently classify 154 as processed and 49 as duplicated. Using data from tiling microarrays, especially from recent high-resolution oligonucleotide arrays, we found some evidence that up to a fifth of the 525 pseudogenes are potentially transcribed. Expressed sequence tags (EST) comparison further validated a number of these, and overall we found 17 pseudogenes with strong support for transcription. In particular, one of the pseudogenes with both EST and microarray evidence for transcription turned out to be a duplicated pseudogene in the cat eye syndrome critical region. Although we could not identify a meaningful number of transcription factor-binding sites (based on chromatin immunoprecipitation-chip data) near pseudogenes, we did find that approximately 12% of the pseudogenes had upstream CpG islands. Finally, analysis of corresponding syntenic regions in the mouse, rat and chimp genomes indicates, as previously suggested, that pseudogenes are less conserved than genes, but more preserved than the intergenic background (all notation is available from http://www.pseudogene.org).

Laser milling of diverse materials has been demonstrated with short pulse lasers ranging from microsecond to femtosecond pulse durations, and with wavelengths from the far infrared to vacuum ultra-violet. In all cases a balance between quality, throughput and cost of ownership must be struck in order to determine commercial relevance. Latest generation Q-switched Diode Pumped Solid State Lasers offer the potential to enable the industrial uptake of laser milling for a wide variety of materials including aerospace alloys, thermal barrier coatings, tool steels, diamond and diamond substitutes. This paper will investigate these practical applications of laser milling with reference to comparative laser and non-laser processes.

Laser milling of a variety of substrates is investigated with the intention of achieving high quality material removal to create three-dimensional shapes in the material. A high power Q-switched Diode Pumped Solid State Nd:YAG Laser at 1064nm is used in all cases. Materials investigated include Nickel Superalloys, Thermal Barrier Coatings, Steels, Tungsten Carbide and Polycrystalline Diamond. Multi-layer substrates are also considered. The effects of laser intensity, plasma formation, pulse duration, material properties, and resulting removal rate, recast and surface finish are explored for this process. This paper defines the findings of this study within the context of commercial imperatives.

Processed pseudogenes were created by reverse-transcription of mRNAs; they provide snapshots of ancient genes existing millions of years ago in the genome. To find them in the present-day human, we developed a pipeline using features such as intron-absence, frame-disruption, polyadenylation, and truncation. This has enabled us to identify in recent genome drafts approximately 8000 processed pseudogenes (distributed from http://pseudogene.org). Overall, processed pseudogenes are very similar to their closest corresponding human gene, being 94% complete in coding regions, with sequence similarity of 75% for amino acids and 86% for nucleotides. Their chromosomal distribution appears random and dispersed, with the numbers on chromosomes proportional to length, suggesting sustained "bombardment" over evolution. However, it does vary with GC-content: Processed pseudogenes occur mostly in intermediate GC-content regions. This is similar to Alus but contrasts with functional genes and L1-repeats. Pseudogenes, moreover, have age profiles similar to Alus. The number of pseudogenes associated with a given gene follows a power-law relationship, with a few genes giving rise to many pseudogenes and most giving rise to few. The prevalence of processed pseudogenes agrees well with germ-line gene expression. Highly expressed ribosomal proteins account for approximately 20% of the total. Other notables include cyclophilin-A, keratin, GAPDH, and cytochrome c.

Prokaryote gene annotation is complicated by large numbers of short open reading frames (ORFs) that arise naturally from genetic code design. Historically, many hypothetical ORFs have been annotated as genes in microbes, usually with an arbitrary length threshold (e.g. greater than 100 codons). Given the use of such thresholds, what is the extent of genuine undiscovered short genes in the current sampling of prokaryote genomes? To assess rigorously the potential under-annotation of short ORFs with homology, we exhaustively compared the polyORFome--all possible ORFs in 64 prokaryotes (53 bacteria and 11 archaea) plus budding yeast--to itself and to all known proteins. The novelty of our analysis is that, firstly, sequence comparisons to/between both annotated and un-annotated ORFs are considered, and secondly a two-step disabled-homology filter is applied to set aside putative pseudogenes and spurious ORFs. We find that un-annotated homologous short ORFs (uhORFs) correspond to a small but non-negligible fraction of the annotated prokaryote proteomes (0.5-3.8%, depending on selection criteria). Moreover, the disabled-homology filter indicates that about a third of uhORFs correspond to putative pseudogenes or spurious ORFs. Our analysis shows that the use of annotation length thresholds is unnecessary, as there are manageable numbers of short ORF homologies conserved (without disablements) across microbial genomes. Data on uhORFs are available from http://pseudogene.org/polyo

A DNA microarray representing nearly all of the unique sequences of human Chromosome 22 was constructed and used to measure global-transcriptional activity in placental poly(A)+RNA. We found that many of the known, related and predicted genes are expressed. More importantly, our study reveals twice as many transcribed bases as have been reported previously. Many of the newly discovered expressed fragments were verified by RNA blot analysis and a novel technique called differential hybridization mapping (DHM). Interestingly, a significant fraction of these novel fragments are expressed antisense to previously annotated introns. The coding potential of these novel expressed regions is supported by their sequence conservation in the mouse genome. This study has greatly increased our understanding of the biological information encoded on a human chromosome. To facilitate the dissemination of these results to the scientific community, we have developed a comprehensive Web resource to present the findings of this study and other features of human Chromosome 22 athttp://array.mbb.yale.edu/chr22.

A novel method has been derived to assess compositional biases in biological sequences. It is based on finding the lowest-probability subsequences for a given residue-type set., We have derived a novel method to assess compositional biases in biological sequences, which is based on finding the lowest-probability subsequences for a given residue-type set. As a case study, the distribution of prion-like glutamine/asparagine-rich ((Q+N)-rich) domains (which are linked to amyloidogenesis) was assessed for budding and fission yeasts and four other eukaryotes. We find more than 170 prion-like (Q+N)-rich regions in budding yeast, and, strikingly, many fewer in fission yeast. Also, some residues, such as tryptophan or isoleucine, are unlikely to form biased regions in any eukaryotic proteome.

Mammals have 79 ribosomal proteins (RP). Using a systematic procedure based on sequence-homology, we have comprehensively identified pseudogenes of these proteins in the human genome. Our assignments are available at http://www.pseudogene.org orhttp://bioinfo.mbb.yale.edu/genome/pseudogene. In total, we found 2090 processed pseudogenes and 16 duplications of RP genes. In relation to the matching parent protein, each of the processed pseudogenes has an average relative sequence length of 97% and an average sequence identity of 76%. A small number (258) of them do not contain obvious disablements (stop codons or frameshifts) and, therefore, could be mistaken as functional genes, and 178 are disrupted by one or more repetitive elements. On average, processed pseudogenes have a longer truncation at the 5′ end than the 3′ end, consistent with the target-primed-reverse-transcription (TPRT) mechanism. Interestingly, on chromosome 16, an RPL26 processed pseudogene was found in the intron region of a functional RPS2 gene. The large-scale distribution of RP pseudogenes throughout the genome appears to result, chiefly, from random insertions with the numbers on each chromosome, consequently, proportional to its size. In contrast to RP genes, the RP pseudogenes have the highest density in GC-intermediate regions (41%–46%) of the genome, with the density pattern being between that of LINEs and Alus. This can be explained by a negative selection theory as we observed that GC-rich RP pseudogenes decay faster in GC-poor regions. Also, we observed a correlation between the number of processed pseudogenes and the GC content of the associated functional gene, i.e., relatively GC-poor RPs have more processed pseudogenes. This ranges from 145 pseudogenes for RPL21 down to 3 pseudogenes for RPL14. We were able to date the RP pseudogenes based on their sequence divergence from present-day RP genes, finding an age distribution similar to that for Alus. The distribution is consistent with a decline in retrotransposition activity in the hominid lineage during the last 40 Myr. We discuss the implications for retrotransposon stability and genome dynamics based on these new findings.

Protein families can be used to understand many aspects of genomes, both their "live" and their "dead" parts (i.e. genes and pseudogenes). Surveys of genomes have revealed that, in every organism, there are always a few large families and many small ones, with the overall distribution following a power-law. This commonality is equally true for both genes and pseudogenes, and exists despite the fact that the specific families that are enlarged differ greatly between organisms. Furthermore, because of family structure there is great redundancy in proteomes, a fact linked to the large number of dispensable genes for each organism and the small size of the minimal, indispensable sub-proteome. Pseudogenes in prokaryotes represent families that are in the process of being dispensed with. In particular, the genome sequences of certain pathogenic bacteria (Mycobacterium leprae, Yersinia pestis and Rickettsia prowazekii) show how an organism can undergo reductive evolution on a large scale (i.e. the dying out of families) as a result of niche change. There appears to be less pressure to delete pseudogenes in eukaryotes. These can be divided into two varieties, duplicated and processed, where the latter involves reverse transcription from an mRNA intermediate. We discuss these collectively in yeast, worm, fly, and human. The fly has few pseudogenes apparently because of its high rate of genomic DNA deletion. In the other three organisms, the distribution of pseudogenes on the chromosome and amongst different families is highly non-uniform. Pseudogenes tend not to occur in the middle of chromosome arms, and tend to be associated with lineage-specific (as opposed to highly conserved) families that have environmental-response functions. This may be because, rather than being dead, they may form a reservoir of diverse "extra parts" that can be resurrected to help an organism adapt to its surroundings. In yeast, there may be a novel mechanism involving the [PSI+] prion that potentially enables this resurrection. In worm, the pseudogenes tend to arise out of families (e.g. chemoreceptors) that are greatly expanded in it compared to the fly. The human genome stands out in having many processed pseudogenes. These have a character very different from those of the duplicated variety, to a large extent just representing random insertions. Thus, their occurrence tends to be roughly in proportion to the amount of mRNA for a particular protein and to reflect the extent of the intergenic sequences. Further information about pseudogenes is available at http://genecensus.org/pseudogene

SNPs are useful for genome-wide mapping and the study of disease genes. Previous studies have focused on SNPs in specific genes or SNPs pooled from a variety of different sources. Here, a systematic approach to the analysis of SNPs in relation to various features on a genome-wide scale, with emphasis on protein features and pseudogenes, is presented. We have performed a comprehensive analysis of 39,408 SNPs on human chromosomes 21 and 22 from the SNP consortium (TSC) database, where SNPs are obtained by random sequencing using consistent and uniform methods. Our study indicates that the occurrence of SNPs is lowest in exons and higher in repeats, introns and pseudogenes. Moreover, in comparing genes and pseudogenes, we find that the SNP density is higher in pseudogenes and the ratio of nonsynonymous to synonymous changes is also much higher. These observations may be explained by the increased rate of SNP accumulation in pseudogenes, which presumably are not under selective pressure. We have also performed secondary structure prediction on all coding regions and found that there is no preferential distribution of SNPs in α-helices, β-sheets or coils. This could imply that protein structures, in general, can tolerate a wide degree of substitutions. Tables relating to our results are available from http://genecensus.org/pseudogene.

We report here the discovery of 137 previously unappreciated genes in yeast through a widely applicable and highly scalable approach integrating methods of gene-trapping, microarray-based expression analysis, and genome-wide homology searching. Our approach is a multistep process in which expressed sequences are first trapped using a modified transposon that produces protein fusions to beta-galactosidase (beta-gal); non-annotated open reading frames (ORFs) translated as beta-gal chimeras are selected as a candidate pool of potential genes. To verify expression of these sequences, labeled RNA is hybridized against a microarray of oligonucleotides designed to detect gene transcripts in a strand-specific manner. In complement to this experimental method, novel genes are also identified in silico by homology to previously annotated proteins. As these methods are capable of identifying both short ORFs and antisense ORFs, our approach provides an effective supplement to current gene-finding schemes. In total, the genes discovered using this approach constitute 2% of the yeast genome and represent a wealth of overlooked biology.

Protein refolding/misfolding to an alternative form plays an aetiologic role in many diseases in humans, including Alzheimer's disease, the systemic amyloidoses, and the prion diseases. Here we have discovered that such refolding can occur readily for a simple lattice model of proteins in a propagatable manner without designing for any particular alternative native state. The model uses a simple contact energy function for interactions between residues and does not consider the peculiarities of polypeptide geometry. In this model, under conditions where the normal (N) native state is marginally stable or unstable, two chains refold from the N native state to an alternative multimeric energetic minimum comprising a single refolded conformation that can then propagate itself to other protein chains. The only requirement for efficient propagation is that a two-faced mode of packing must be in the ground state as a dimer (a higher-energy state for this packing leads to less efficient propagation). For random sequences, these ground-state dimeric configurations tend to have more beta-sheet-like extended structure than almost any other sort of dimeric ground-state assembly. This implies that propagating states (such as for prions) are beta-sheet rich because the only likely propagating forms are beta-sheet rich. We examine the details of our simulations to see to what extent the observed properties of prion propagation can be predicted by a simple protein folding model. The formation of the alternative state in the present model shows several distinct features of amyloidogenesis and of prion propagation. For example, an analog of the phenomenon of conformationally distinct strains in prions is observed. We find a parallel between 'glassy' behavior in liquids and the formation of a propagatable state in proteins. This is the first report of simulation of conformational propagation using any heteropolymer model. The results imply that some (but not most) small protein sequences must maintain a sequence signal that resists refolding to propagatable alternative native states and that the ability to form such states is not limited to polypeptides (or reliant on regular hydrogen bonding per se) but can occur for other protein-like heteropolymers.

The evolutionary origin of prion genes, only known to exist in the vertebrate lineage, had remained elusive until recently. Following a lead from interactome investigations of the murine prion protein, our previous bioinformatic analyses revealed the evolutionary descent of prion genes from an ancestral ZIP metal ion transporter. However, the molecular mechanism of evolution remained unexplored. Here we present a computational investigation of this question based on sequence, intron-exon, synteny and pseudogene analyses. Our data suggest that during the emergence of metazoa, a cysteine-flanked core domain was modularly inserted, or arose de novo, in a preexisting ZIP ancestor gene to generate a prion-like ectodomain in a subbranch of ZIP genes. Approximately a half-billion years later, a genomic insertion of a spliced transcript coding for such a prion-like ZIP ectodomain may have created the prion founder gene. We document that similar genomic insertions involving ZIP transcripts, and probably relying on retropositional elements, have indeed occurred more than once throughout evolution.

BACKGROUND: Alternatively-spliced (AS) forms can vary protein function, intracellular localization and post-translational modifications. AS coupled with mRNA nonsense-mediated decay (NMD) can also control the transcript abundance. Here, we have investigated the genome-scale conservation of alternatively-spliced NMD candidates (AS-NMD candidates), in mammals. METHODOLOGY/PRINCIPAL FINDINGS: We mapped>12 million cDNA/EST library transcripts, comprising pooled data from both older and next-generation sequencing techniques, against genomic sequences to annotate AS-NMD candidates generated by in-frame premature termination codons (PTCs), in the human, mouse, rat and cow genomes. In these genomes, we found populations of genes that harbour AS-NMD candidates, varying in number from approximately 149 to 2,051 genes. We discovered that a highly-significant proportion (27%-35%) of AS-NMD candidate genes in mouse, rat and cow, also have human orthologs targeted for NMD. Intron retention was the most abundant type of AS-NMD, ranging from 43% to 67% of genes harbouring an AS-NMD candidate. Groupings of AS-NMD candidate genes either with or without intron retentions also have highly significant AS-NMD conservation, indicating that the trend is not due primarily to conservation of intron retentions. As a subset, the AS-NMD intron retentions are distinguished from non-retained introns by higher GC content, and codon usage similar to the usage in protein-coding sequences. This indicates that most of these alternatively spliced sequences have coded for proteins in the recent evolutionary past. In general, the AS-NMD candidate genes showed a similar pattern of Gene Ontology functional category enrichments in all four species. Genes linked to nucleic-acid interaction and apoptosis, and involved in pathways linked with cancer, were the most common. Finally, we mapped the AS-NMD candidates to mass spectrometry-derived proteomics data, and gathered evidence of truncated polypeptides for at least 10% of all human AS-NMD candidate transcripts. CONCLUSIONS/SIGNIFICANCE: In summary, our analysis provides strong statistical evidence for conservation of functional AS-NMD candidature across Mammalia for a large subset of genes. However, because codon usage of AS-NMD intron retentions is similar to the usage in exons, it is difficult to de-couple conservation of AS-NMD-based regulation from conservation for protein-coding ability, for intron retentions.

Background Functional diversification of genes in mammalian genomes is engendered by a number of processes, e.g., gene duplication and alternative splicing. Gene duplication is classically discussed as leading to neofunctionalization (generation of new functions), subfunctionalization (generation of a varied function), or pseudogenization (loss of the gene and its function). Results Here, we focus on the process of pseudogenization, but specifically for individual exons from genes. It is at present unclear to what extent pseudogenization of individual exon duplications affects gene evolution, i.e., is it a random phenomenon, or is it associated with specific types of genes and encoded proteins, and positions in gene structures? We gathered genomic evidence for pseudogenic exons (ΨEs, i.e., exons disabled by frameshifts and premature stop codons), to examine for significant trends in their distribution across four mammalian genomes (specifically human, cow, mouse and rat). Across these four genomes, we observed a consistent population of ΨEs, associated with 0.4–1.0% of genes. These ΨE populations exhibit codon substitution patterns that are typical of an endemic population of decaying sequences. In human, ΨEs have significant over-representation for functional categories related to 'ion binding' and 'nucleic-acid binding', compared to duplicated exons in general. Also, ΨEs tend to be associated with some protein domains that are abundant generally, e.g., Zinc-finger and immunoglobulin protein domains, but not others, e.g., EGF-like domains. Positionally, ΨEs are also significantly associated with the 5' end of genes, but despite this, individual stop codons are positioned so that there is significant avoidance of potential targeting to nonsense-mediated decay. In human, ΨEs are often associated with alternative splicing (in 22 out of 284 genes with ΨEs in their milieu), and can have different parts of their sequence differentially spliced in alternative transcripts. Some unusual cases of ΨEs embedded within 5' and 3' non-coding exons are observed. Conclusion Our results indicate the types of genes that harbour ΨEs, and demonstrate that ΨEs have non-random distribution within gene structures. These ΨEs may function in gene regulation through generation of transcribed pseudogenes, or regulatory alternate transcripts.

Patterning the Black Matrix (BM) layer in an LCD typically employs wet-etch lithography. This paper describes an alternative laser-based patterning technique called Assist Liquid™. This technique meets rigorous customer targets and may offer a more cost effective solution for LCD manufacture.

Background: Pseudogenes often manifest themselves as disabled copies of known genes. In prokaryotes, it was generally believed (with a few well-known exceptions) that they were rare. Results: We have carried out a comprehensive analysis of the occurrence of pseudogenes in a diverse selection of 64 prokaryote genomes. Overall, we find a total of around 7,000 candidate pseudogenes. Moreover, in all the genomes surveyed, pseudogenes occur in at least 1 to 5% of all gene-like sequences, with some genomes having considerably higher occurrence. Although many large populations of pseudogenes arise from large, diverse protein families (for example, the ABC transporters), notable numbers of pseudogenes are associated with specific families that do not occur that widely. These include the cytochrome P450 and PPE families (PF00067 and PF00823) and others that have a direct role in DNA transposition. Conclusions: We find suggestive evidence that a large fraction of prokaryote pseudogenes arose from failed horizontal transfer events. In particular, we find that pseudogenes are more than twice as likely as genes to have anomalous codon usage associated with horizontal transfer. Moreover, we found a significant difference in the number of horizontally transferred pseudogenes in pathogenic and non-pathogenic strains of Escherichia coli.