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All computational models that predict something have certain underlying assumptions that constitute the physical basis for the model. In protein structure prediction, there are two physical/biological processes that can be modeled: the process of evolution, or the process of folding.We name these two paradigms, Darwin and Boltzmann, after the scientists who defined the fundamental principles of evolutionary biology and statistical thermodynamics, respectively. Most of the work in protein structure prediction is Darwin-based, using the well-known premise that sequences that have a common ancestor have similar folds, and they strive to extrapolate this principle to increasingly distant sequence relationships. Methods that use multiple sequence alignment, structural alignment, or "threading potentials" are implicitly searching for a common ancestor. Despite the often-used "energy-like" scoring functions, these methods do not address the physical process of folding. Evolution happens on the time scale of millions of years, folding on the time scale of fractions of a second. [ABSTRACT FROM AUTHOR]

Almost every biochemist and molecular biologist with an interest in protein research confronts the question of the role played by individual amino acid residues in a specific polypeptide. The wide variety of experimental techniques available to address this question can be categorized into two general approaches: functional and structural. In the former case, the residue in question is chemically modified or mutated; in the latter, the relationships with neighboring residues are defined and biological function is inferred. Each approach has its advantages and limitations and the most accurate information is provided when both are used together. [ABSTRACT FROM AUTHOR]

The increasing information in databases of protein sequences and structures together with the development of bioinformatic tools has helped the biochemists to identify and validate the function of many different proteins. In this chapter, we will show the successful application of two methods, proteinfold recognition (FR) and evolutionary-trace (ET) analysis to learn about the function of a group of proteins which belong to the class restriction endonucleases, namely the type II restriction endonuclease (REase) Sau3AI and the mismatch repair (MMR) protein MutH. [ABSTRACT FROM AUTHOR]

An assembly of functioning ribosomes starts with the biosynthesis of ribosomal RNAs and proteins. In all living species, polycistronic pre-ribosomal RNA (pre-rRNA) is processed to mature rRNAs and is covalently modified at multiple positions, with the aid of specific protein enzymes and small nucleolar guide RNAs (snoRNAs). There are more than 40 known types of covalent rRNA modifications, the two most common ones being pseudouridylation and methylation (Crain et al. 2003). Ribosomal proteins, some of which are also covalently modified, are then assembled into mature ribosome subunits with rRNA. In eukaryotic cells, cytoplasmically synthesized ribosomal proteins have to be imported into the nucleus, and the assembled ribosomes are exported from the nucleus back into the cytoplasm. [ABSTRACT FROM AUTHOR]

As the adapters between mRNAs and the elongating peptide chain, transfer RNAs (tRNA) are at the nexus of the genetic code and of the translation apparatus. Prior to their participation in translation, tRNAs must undergo extensive processing of the nascent transcript. The post-transcriptional processing of tRNAs involves a number of functionally distinct events essential for tRNA maturation (Altman et al. 1995; Björk 1995; Deutscher 1995; Westaway and Abelson 1995). The phenomenon of nucleoside modification is perhaps the most remarkable of these events, and results in a wealth of structural changes to the canonical nucleosides (Björk 1995). Although other RNA species also exhibit varying degrees of nucleoside modification, it is only in the tRNA that a rich structural diversity is realized. [ABSTRACT FROM AUTHOR]

Naturally occurring RNAs contain numerous chemically altered nucleosides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process. To date, a total of 96 structurally distinguishable modified nucleosides originating from different types of RNAs from many diverse organisms of the three major phylogenetic domains of life have been reported (Rozenski et al. 1999); http://medstat.med.utah.edu/RNAmods; and references therein). The pattern of modifications (type and location) depends on the RNA molecule considered, as well as, on the organism or the organelle they originate from.However, the largest number of modified nucleosides with the greatest structural diversity (a total of 81) is found in transfer RNAs, especially in tRNAs from higher organisms (Sprinzl et al. 1998; http://www.uni-bayreuth.de/departments/biochemie/trna). Other types of RNA (snRNA, snoRNA, rRNA,mRNA) also contain modified nucleosides (see http://rna.wustl.edu/snoRNAdb), however, their occurrence and particularly their diversity are lower than in tRNAs (see, for example,Limbach et al. 1995;Motorin and Grosjean 1998). [ABSTRACT FROM AUTHOR]

It has become common ground to start a bioinformatics article by mentioning the flood of data overwhelming the research community. Indeed, the large amount of data has led to the insight that even the average wet lab needs several computers, e.g. to manage micro array results or to run BLAST searches via the internet.However, it is more than that: bioinformatics has matured to a research discipline in its own right. The main reason is that bioinformatics not only allows for the solving of problems that are tedious in a traditional approach (e.g. protein function can often be inferred from homologous proteins in other species), but contributes to a new way of looking at biological systems: from a reductionist approach, to a systemic view of biology (Noble 2002).With the large amounts of data on whole systems, the focus in biomedical research is increasing steadily: from a single protein to complexes, from an enzyme-catalyzed reaction to metabolic networks.Even virtual cells or tissues are no longer science fiction. Recently, the term bioinformatics has been used more and more to describe research related to databases (integration of resources, web access, etc.) and sequence analysis (homology searches,multiple sequence alignments, phylogenetic trees).Computational biology refers to the simulation of complex networks, e.g. metabolic or signalling pathways, cell and tissue simulations. In recent years, the field has seen an -omics explosion (e.g. genomics, proteomics, transcriptomics, metabolomics), five of which have created several research communities eager to integrate their data. [ABSTRACT FROM AUTHOR]

The function of a protein is defined by its interactions with other molecules in its environment. The interactions can be either transient, such as protein-protein interactions involved in intracellular signaling, or relatively stable, such as the protein-protein and protein-RNA interactions in ribosomes. A structural description of these interactions is an important step toward understanding the mechanisms of biochemical, cellular, and higher order biological processes. There is a need to integrate structural information gathered at multiple levels of the biological hierarchy-from atoms to cells-into a common framework. Recent developments in several experimental and computational techniques allow structural biology to shift its focus from the structures of the individual proteins to larger assemblies (Sali et al. 2003; Baumeister 2002). [ABSTRACT FROM AUTHOR]

Today, the pace of genome sequencing rapidly increases the number of protein sequences. This may lead to a description of living organisms at an unprecedented level of both detail and completeness. It will require the characterization of the biophysical properties and of the biological role of each macromolecular assembly. The growing number of known protein sequences largely exceeds the number of protein structures determined experimentally by NMR and X-ray crystallography (Baker and Sali 2001). However, at the same time, new folds are now rarely discovered despite significant efforts to determine structures of unrelated proteins (see CASP5 results).Meanwhile, a huge number of small molecules can now be easily synthesized and tested experimentally thanks to robotics. Libraries of chemical compounds are rapidly growing while the structural, thermodynamic and dynamic characterization of ligand-macromolecule complexes is still tedious and difficult. These observations suggest that new in silico methods (taking advantage of the increasing power of computers) need to be developed in the field of pharmacogenomics. [ABSTRACT FROM AUTHOR]

The computational assignment of three-dimensional structures to newly determined protein sequences is becoming an increasingly important element in experimental structure determination and in structural genomics (Fischer et al. 2001a). In particular, fold-recognition methods aim to predict approximate three-dimensional (3D) models for proteins bearing no evident sequence similarity to any protein of known structure (see the review by Cymerman et al., this Vol.). The assignment is carried out by searching a library of known structures (usually obtained from the Protein Data Bank) for a compatible fold. [ABSTRACT FROM AUTHOR]

Amino acid sequence analysis provides important insight into the structure of proteins,which in turn greatly facilitates the understanding of its biochemical and cellular function. Efforts to use computational methods in predicting protein structure based only on sequence information started 30 years ago (Nagano 1973; Chou and Fasman 1974).However, only during the last decade, has the introduction of new computational techniques such as protein fold recognition and the growth of sequence and structure databases due to modern high-throughput technologies led to an increase in the success rate of prediction methods, so that they can be used by the molecular biologist or biochemist as an aid in the experimental investigations. [ABSTRACT FROM AUTHOR]

RNA editing is the sequence alteration of RNA molecules by nucleotide insertion/deletion or conversion mechanisms. In this chapter, I describe how the different forms of RNA editing may have evolved from pre-existing activities. It appears that repeated and widespread independent evolution of RNA editing occurred. The diversity in origins seems to be mirrored in the range of possible functions of editing: (1) Multiple proteins could be encoded by one gene. Different editing patterns would generate several proteins from one gene. Conversion editing in vertebrate mRNAs seems to be an instance of such an adaptive function. (2) RNA editing could provide organisms with an extra level of regulation of gene expression, and indications for this function are seen in most RNA editing forms. (3) Editing could serve as a defence against viruses and transposons. This could be another role of editing of vertebrate mRNAs. (4) Editing might counteract mutations which have occurred in the genome. These could occur particularly in organellar genomes, when selective pressures are absent. This role may be the raison d'être of mitochondrial tRNA editing. (5) RNA editing could offer the possibility to retain ‘difficult' coding sequences, and such a function might be performed by mitochondrial RNA editing in myxomycetes. (6) Last but not least, RNA editing could speed up evolution by creating higher amounts of genetic variation over a shorter period of time. For its function, this model relies heavily on an analogy with splicing, where the possibility of domain shuffling has been invoked as a functional advantage. All these explanations seem not to suffice for kinetoplastid panediting, the most complex and extensive form of RNA editing known. In this case, I propose that the original advantage was found in the gene fragmentation it entails, protecting against loss of temporarily non-expressed mt genes during periods of intense intraspecific competition. Present-day kinetoplastid editing, however, reflects the effects of a long history of opposing selective forces obscuring its evolutionary origin. [ABSTRACT FROM AUTHOR]

RNA editing of mitochondrial mRNAs in trypanosomes is characterized by the post-transcriptional insertion or deletion of uridine residues. This process is directed by a diverse family of small, guide RNAs (gRNAs). The trypanosome mitochondrial genome contains nine genes for mRNAs that are extensively edited, three genes for mRNAs that are edited at a limited number of sites, and six genes that encode mRNAs, which are not edited. Editing is essential in both insect and bloodstream developmental stages of the parasite, and results in the formation of initiation codons and extended open reading frames in mRNA, necessary for maintaining mitochondrial electron transport and oxidative phosphorylation. In essence, RNA editing is responsible in producing functional mRNAs so that conventional mitochondrial proteins can be made. Another potential raison d'être for RNA editing in trypanosomes has recently been demonstrated. Trypanosome mitochondrial mRNAs can be differentially edited, producing mRNAs with extended open reading frames. This suggests that multiple proteins can be produced from a single mitochondrial gene. A product of alternative editing of the cytochrome oxidase subunit III (COIII) mRNA encodes a mitochondrial membrane protein with a unique, arginine-rich N-terminal sequence and the C-terminal sequence of COIII. Alternative mRNA editing thus expands the repertoire of mitochondrial proteins in trypanosomes. This review addresses the functions of RNA editing in both RNA repair and generation of protein diversity in these organisms. [ABSTRACT FROM AUTHOR]

The majority of mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of a U nucleotide-specific, insertion/deletion-type RNA editing reaction. The process converts nonfunctional pre-mRNAs into translatable molecules, and can generate protein diversity by alternative editing. A high molecular mass enzyme complex, the editosome, catalyzes the reaction. Editosomes provide a molecular platform for the individual catalytic steps of the reaction cycle. While the molecular composition of the editosome has been studied in detail, dynamic aspects of the reaction have by and large been ignored. Here, we focus on accessory proteins that bind to the editosome only at defined steps of the reaction cycle, thereby modulating the structure and function of the catalytic machinery. As an example, we concentrate on the mitochondrial DExH/D protein mHel61p, a putative RNA helicase and/or RNPase. We summarize the current structural, genetic and biochemical knowledge on mHel61p, and provide an outlook onto dynamic processes of the editing reaction. [ABSTRACT FROM AUTHOR]

The proteins in the ∼20S editosome function in a coordinated fashion to catalyze the precise insertion and deletion of uridine nucleotides (Us) in mitochondrial pre-mRNAs, thereby generating functional mRNAs. Many catalytic components of the editosome have been identified, but the abundance of apparently non-catalytic components is a conspicuous reminder that the editosome is much more than the sum of its catalytic activities. As the functions of the various editosome proteins are elucidated, the importance of how they interact in vivo is becoming more evident. Indeed, ∼20S editosomes are compositionally heterogeneous in vivo, and higher-order interactions with proteins of other complexes affect editing. Here, we review the general process of editing, and discuss how the known editosome proteins interact and function to produce translatable mRNAs in vivo. [ABSTRACT FROM AUTHOR]

RNA editing in plant mitochondria alters more than 400 cytidines to uridines in flowering plants. In other plants such as ferns and mosses, the reverse reaction is observed at almost equal frequency. In the last few years, the development of transfection systems with isolated mitochondria and of in vitro systems with mitochondrial extracts has considerably improved our understanding of the parameters of site recognition. However, the biochemistry and the enzymes involved are still open questions. We here summarize our present knowledge of RNA editing as an essential part of RNA maturation in flowering plant mitochondria. [ABSTRACT FROM AUTHOR]

RNA editing has been observed in chloroplast transcripts from most land plants, but not from algae. In higher plant chloroplasts, C-to-U conversion occurs at around 30 specific sites in mRNAs, many of which result in amino acid alteration. Occasionally, RNA editing creates an AUG initiation codon from ACG, and a UAA termination codon from CAA. No obvious consensus element or secondary structure was found in the sequences surrounding these editing sites. Chloroplast transformation approaches have revealed that cis-elements for editing are commonly located in the upstream and proximal regions of editing sites. Development of an in vitro chloroplast RNA editing system has enabled us to investigate biochemical processes of RNA editing, e.g., detection of trans-acting factors. [ABSTRACT FROM AUTHOR]

Mitochondrial gene expression in the acellular slime mold Physarum polycephalum requires a diverse array of RNA editing events. Virtually all transcripts encoded in the organelle are subject to changes at the RNA level, including both mRNAs and stable RNAs. Roughly 500 editing events involving nucleotide insertion or deletion have been confirmed thus far; these occur co-transcriptionally. Base changes also occur in Physarum mitochondria, but these are much rarer and occur post-transcriptionally. This chapter focuses on the experimental approaches used to dissect the unique mechanism of insertion/deletion editing in Physarum mitochondria. [ABSTRACT FROM AUTHOR]

Abstract The degeneracy of the genetic code is implied in the need for 61 sense codons to specify 20 different amino acids; with the exception of methionine and tryptophan, each amino acid is encoded by more than one codon. This discrepancy between codon and amino acid numbers was first explained by Crick's wobble hypothesis, which invoked the need for base-pairing flexibility between the first anticodon and third codon positions during decoding. Since the inception of the wobble rules, over 100 posttranscriptional modifications have been described, with the largest number affecting the anticodon of tRNA. As anticodon modifications accrue, new findings lead to a constant reinterpretation of the wobble rules to include novel effects on tRNA function. In general, anticodon modifications play key roles in translational fidelity and efficiency. However, anticodon-sequence alterations to a particular tRNA that permit decoding of multiple codons are part of a growing number of posttranscriptional changes collectively known as tRNA editing. In fact, the decoding changes imparted by tRNA editing provide a mechanism to effectively accommodate genetic code degeneracy. Although a number of editing events have direct effects in expanding a tRNA's decoding capacity, some editing events indirectly affect tRNA function by repairing otherwise non-functional tRNAs. This chapter will attempt to summarize what is currently known about both types of tRNA editing in various organisms, with the proviso that due to the serendipitous nature of editing discoveries, the work presented here will undoubtedly not be conclusive. This chapter will rather compile the few existing examples of tRNA editing, and whenever possible will try to illustrate current efforts to characterize the different tRNA editing enzymes and the various mechanisms. [ABSTRACT FROM AUTHOR]

Adenosine deaminases that act on RNA (ADARs) give rise to the most abundant form of RNA editing found in Metazoa. ADAR proteins convert adenosines to inosines within structured and double-stranded RNAs. Since inosines are interpreted as guanosines by several cellular machineries, the consequences of editing can be widespread. In messenger RNA, alterations of codons, changes in splice patterns, and influences on RNA stability have been observed as a result of RNA editing. Moreover, A to I editing has been shown to interconnect with the RNA interference machinery. In this chapter, an overview on ADAR enzymes, their molecular architecture, occurrence, and substrate specificity is given. Consequences of editing, studies in model organisms, and implications for other double-stranded RNA-dependent processes are discussed. [ABSTRACT FROM AUTHOR]

Abstract RNA editing belongs to the large group of processing reactions that are required to convert primary RNA transcripts into mature and functional transcripts. The main determinants of specificity rest in the three-dimensional structures of RNA and protein molecules that act in concert to coordinate and regulate the posttranscriptional steps in gene expression. Many high-resolution structures of RNA-protein complexes, including the ribosome, have become available during the last decade and have offered detailed views of the intracellular RNA world. The focus of this review is to highlight the contributions of RNA structure to the specificity and efficiency of RNA editing. Editing occurs by a variety of mechanisms, but the fidelity of the reactions critically depends on the specific sequences and structures of the RNA molecules involved and on their recognition by trans-acting factors, including proteins and RNA. Hence, the editing machineries, also termed "editosomes", make use of RNA-RNA, RNA-protein and protein-protein interactions to achieve specificity and efficiency. High-resolution structures of protein components of various editosomes exist, but reports of RNA structures and RNA-protein complexes are still limited. Progress can be expected in the near future from more efficient purification and crystallization techniques developed in other fields of RNA processing, like RNA interference, splicing and catalysis. Although each structure reveals only a static view of a multistep reaction, they will eventually lead to a better understanding of the dynamic molecular machines involved in RNA editing. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are evolutionary conserved, germ-line encoded molecules that express an ectodomain with leucine-rich regions, a single transmembrane domain, and a cytoplasmic region that contains the Toll-IL-1R resistance (TIR) signaling domain. TLRs recognize bacterial and viral pathogen-associated molecular patterns (PAMPs), as well as certain endogenous mammalian "danger signals," i.e., proteins, oligosaacharides, and nucleic acids released from damaged cells as a consequence of stress, inflammation, and wounding. TLR2, TLR4, TLR5, TLR9, and TLR11 preferentially respond to bacterial, yeast, and protozoan PAMPs, while TLR3, TLR7, and TLR8 sense viral nucleic acids. TLRs exhibit different cellular localization: TLR2, TLR4, and TLR5 are expressed on the cell surface, while TLR3, TLR7, TLR8, and TLR9 are localized intracellularly in endosomal compartments. Recognition of PAMPs by TLRs triggers TLR oligomerization and conformational changes within TIR domains, leading to recruitment of various adapter proteins and kinases. This, in turn, activates MAP kinases, transcription factors, and, ultimately, production of nitric oxide (NO) and reactive oxygen intermediates, up-regulation of expression of accessory and co-stimulatory molecules, and secretion of cytokines and chemokines. TLR-mediated up-regulation of co-stimulatory and MHC molecules on dendritic cells (DC), coupled with cytokine production and DC maturation, facilitates the adaptive immune response, providing a link between the innate and adaptive immunity. This review focuses on mechanisms of TLR signaling, known mutations/polymorphisms in genes encoding TLRs and IRAK-4 and their implication for susceptibility to infectious and autoimmune diseases and asthma. [ABSTRACT FROM AUTHOR]

There has been a revolution in immunology in recent years that has transformed the paradigmatic underpinnings of vertebrate immunology to include the innate immune response. The utilization of basally diverging model systems, like the zebrafish, provides particular insight into the origins and evolution of vertebrate immunity. Investigations aimed at exposing the breadth and complexity of innate immunity using the zebrafish model system have uncovered a broad spectrum of mechanisms, both novel and conserved, that add depth to our understanding of how the immune system functions. Of particular significance is the fact that, during the first 4-6 weeks of development, the zebrafish relies upon innate immunity as its sole mechanism of defense. This unique characteristic, combined with the zebrafish model's inherent advantages including high fecundity, external development, and optical transparency during early development, make the zebrafish a particularly attractive model of study. The establishment of bacterial and viral infectious disease models such as Edwardsiella tarda and snakehead rhabdovirus, respectively, as well as the addition of a wide range of reagents and techniques, including robust forward and reverse genetics approaches, have facilitated the zebrafish model's usage to study of a variety of innate immunity questions. Close examination of the zebrafish's innate immune system reveals a strong degree of sequence conservation in many of areas of study, including but not limited to pattern recognition receptors like the Toll-like receptors, their pathway components, and a variety of cytokines. Studies are currently underway to determine whether such sequence homology equates to functional homology. In addition, a variety of zebrafish genes encoding proteins of unique function are currently under study, including assorted lectins and novel immune type receptors. Close examination of these genes may provide needed insight into the evolutionary history of immunity in vertebrates. [ABSTRACT FROM AUTHOR]

Currently existing urochordates (sea squirts) and vertebrates diverged in evolution around 570 million years (myr) ago. Phylogenetic analyses based on molecular data provide compelling evidence that these animals are the closest living relatives of vertebrates. Urochordares, therefore, are of critical importance for understanding the origin of vertebrate immune system. For a number of species a large body of molecular data is now available. An extensive EST project and the draft genome sequences of Ciona intestinalis and C. savignyi allow rapid "in silico" searches for immunorelevant molecules. Recent data convincingly demonstrate that urochordates possess nearly full repertoire of vertebrate innate immune system, but totally lack most of the components of an adaptive immunity such as MHC, TCRs and antibodies. In this review we show that knowledge of immunity in lower vertebrate and invertebrate species is now rapidly increasing. Elucidating the details of the origin of the immune systems from a comparative point of view in vertebrate's closest relatives, may finally lead to a better understanding of our own immune system. [ABSTRACT FROM AUTHOR]

Complement is an important component of the innate immune defence of animals against infectious agents. The complement system in mammals is well characterised and consists of about 35-40 proteins, present in blood plasma and other body fluids, and also on cell surfaces. The function of complement is to rec-ognise and opsonise particulate materials including invading micro-organisms and "altered-self" cells (dying, infected or damaged host cells). Recognition of a target by large polymeric complement proteins including C1q, MBL and the ficolins results in activation of proteases which cleave complement protein C3, a thiolester-containing protein (TEP) which binds covalently to the target. Target-bound complement proteins opsonise the target by promoting interaction with phagocytic cells which express complement receptors. The complement system appears to be highly conserved in vertebrates, although research on reptiles and amphibians is limited. Only a few invertebrate animals have been studied, but likely ortho-logues of complement target-recognition proteins, proteases and TEPs have been demonstrated in cephalochordates, urochordates, echinoderms, arthropods and coe-lenterates. This suggests that complement-like activity has been important in host defence since an early stage in the evolution of multicellular animals. [ABSTRACT FROM AUTHOR]

Findings of the past two decades clearly document that epithelial cells have the capacity to mount a "chemical barrier" apart from the physical defense shield against invading microorganisms. This "chemical barrier" includes preformed antimicrobial proteins present at the uppermost layers of the epithelium as well as newly synthesized compounds that are produced upon stimulation after contact with pathogenic bacteria or bacterial products, endogenous proinflammatory cytokines and/or the disruption of the physical barrier by wounding with subsequently released growth factors. This chapter introduces the reader into the field by giving an overview of the most important human epithelial and phagocyte derived anti-microbial peptides. Furthermore, strategies for the putative action of antimicrobial peptides in the healthy human are presented. The third part of the review gives an overview of several diseases which are in connection with a decreased or impaired antimicrobial peptide expression: skin diseases and wound healing, diseases of the airway epithelia and the gastrointestinal tract as well as diseases associated with phagocyte dysfunction. Exogenous application of antimicrobial peptides could be a promising therapeutic option in the near future for the treatment of patients with epithelial infections and chronic wounds but a much more promising option would be the promotion of the endogenous expression of antimicrobial peptides. [ABSTRACT FROM AUTHOR]

In mammals, a tissue-specific set of Nod-like receptors (NLRs) enables collectively a swift and differential cytosolic detection of evolutionary distant microbial- and/or danger-associated molecular patterns from the extracellular and intracellular microenvironment. Repressing and de-repressing this surveil-lance machinery contribute to vital immune homeostasis and protective responses within specific tissues. Conversely, defective biology of NLR signaling pathways drives the development of recurrent infectious and/or inflammatory diseases by failing to mount barrier functions, to instruct the adaptive immune response and/or to ignore self and non-self antigens. Better decoding of microbial shedding and immune escape strategies will provide clues for the development of rational therapies striving to cure and prevent common and emerging immunopathologies in humans. [ABSTRACT FROM AUTHOR]

The ability of blood cells, known as hemocytes, to detect and eliminate pathogens is vital to the Drosophila immune response. Various pathogens that can subvert the cellular immune response are often lethal to the fly. For example, parasitoid wasps deposit their eggs with chemicals targeting Drosophila hemocytes. These chemicals increase parasitoid success. Similarly, when hemocyte counts are drastically lowered through mutations like domino, mutant larvae are vulnerable to large—scale colonization by live bacteria. Further, the inhibition of phagocytic ability in hemocytes leads to a dramatic increase in susceptibility to Escherichia coli infection in flies lacking a humoral response. This chapter discusses our current understanding of encapsulation and phagocytosis, two cellular immune responses important for defense against parasites and bacteria. Both responses initiate with recognition, followed by activation of the blood cells, and finish with either encapsulation or uptake of the microbe. Recent works from many laboratories have used whole-genome RNAi screens, forward genetic screens, and fluorescent visualization of cellular processes to identify old and new players in these cellular immune responses. [ABSTRACT FROM AUTHOR]

Insects mount a robust innate immune response against a wide array of microbial pathogens. For example, the fruit fly Drosphila melanogaster uses both cellular and humoral innate immune responses to combat pathogens. The hallmark of the Drosophila humoral immune response is the rapid induction of antimicrobial peptide genes in the fat body, the homolog of the mammalian liver. Expression of these antimicrobial peptide genes is rapidly induced by two immune signaling pathways, which respond to distinct microorganisms. The Toll pathway is activated by fungal and Gram-positive bacterial infections, whereas the IMD pathway responds to Gram-negative bacteria. In this chapter, we discuss recent advances in understanding the mechanisms involved in microbial recogni-tion, signal transduction, and immune protection mediated by these pathways, highlighting similarities and differences between Drosophila immune responses and mammalian innate immunity. [ABSTRACT FROM AUTHOR]

The phylum Cnidaria is one of the earliest branches in the animal tree of life. Cnidarians possess most of the gene families found in bilaterians and have retained many ancestral genes that have been lost in Drosophila and Caenorhabditis elegans. Characterization of the innate immune repertoire of extant cnidarians is, therefore, of both fundamental and applied interest — it not only provides insights into the basic immunological "tool kit" of the common ancestor of all animals, but is also likely to be important in understanding human barrier disorders by describing ancient mechanisms of host/microbial interactions and the resulting evolutionary selection processes. The chapter summarizes four aspects of immunity which can be studied particularly well within cnidarians — and which may be of interest from a comparative point of view to all immunologists: intraspecies competition in sea anemones, allorecognition and cell lineage competition in the marine hydrozoan Hydractinia, antimicrobial defense reactions in Hydra and jellyfish, and symbiotic relationships in both corals and Hydra. Studies in cnidarians reveal that there is no problem in innate immunity these basal metazoans did not attempt to solve. Thus, whatever we experience with our own innate immune system, whatever we hope to learn, we will see that the cnidarians have been there before us. [ABSTRACT FROM AUTHOR]

Potential pathogens deliver effector proteins into plant cells to suppress microbe-associated molecular pattern (MAMP)-triggered immunity in plants, resulting in host—pathogen coevolution. To counter pathogen suppression, plants evolved disease resistance (R) proteins to detect the presence of the pathogen effectors and trigger R-dependent defenses. Most isolated R genes encode proteins possessing a leucine-rich-repeat (LRR) domain, of which the majority also contain a nucleotidebinding site (NBS) domain. There is structural similarity and/or domain homology between plant R proteins and animal immunity proteins, suggesting a common origin or convergent evolution of the defense proteins. Two basic strategies have evolved for an R protein to recognize a pathogen effector (then called avirulence factor; Avr): direct physical interaction and indirect interaction via association with other host proteins targeted by the Avr factor. Direct R-Avr recognition leads to high genetic diversity at paired R and Avr loci due to diversifying selection, whereas indirect recognition leads to simple and stable polymorphism at the R and Avr loci due to balancing selection. Based on these two patterns of R-Avr coevolution, investigation of the sequence features at paired R and Avr may help infer the R-Avr interaction mechanisms, assess the role and strength of natural selection at the molecular level in host—pathogen interactions and predict the durability of R gene-triggered resistance. [ABSTRACT FROM AUTHOR]

Fragments of the non-coding portion of mtDNA of various lengths have been successfully isolated from a total of eight Neanderthal specimens. This has provided an insight into the mtDNA gene pool and has enabled some aspects of the diversity and age of the Neanderthal lineage to be assessed. No admixture between modern humans and Neanderthals has been detected, but the limited number of samples available for molecular analysis limit the conclusions that can be made with respect to potential admixture. Other explanations for the lack of Neanderthal lineages in the modern mtDNA gene pool, in particular genetic drift, can also explain the results, especially as the conclusions are based on the analysis of one haploid locus, the mtDNA. Further analysis will provide a better view of the Neanderthal gene pool, but the number of potential samples is limited: in total 70 sites have yielded Neanderthal bones (Klein 2003). Many of the sites, particularly those from southern Europe, do not show good molecular preservation (Cooper et al. 1997; Smith et al. 2003). [ABSTRACT FROM AUTHOR]

The SCR of the pioneer phase of the peopling of the vast territories of Asia has gained increasingly strong experimental support, thanks to recently acquired deeper phylogenetic and phylogeographic knowledge about the spread of mtDNA (and Y-chromosomal) variation in this continent. Much, if not all, of the early settlement process can be seen as a ‘fast train to Southeast Asia and Australia along the SCR'—indeed, so fast that the founder haplotypes at the base of haplogroups M, N, and R reached all major destinations alongside the route, as far down as Australia. It appears that Central Asia and southern Siberia were not involved in the initial peopling of the continent. It is also evident that the initial fast train phase was followed by a long-lasting freezing of the major geographic pools of maternal lineages in the south and further gene flows northwards from Southeast Asia and subsequently back westwards along the Steppe Belt extending from Manchuria to Europe. At present, western Siberia, the Urals, and Central Asia form a huge continuous admixture zone encompassing East and West Eurasian maternal lineages—a process that has so far had only a minimal influence on the essentially distinct autochthonous patterns of mtDNA variation in most of South Asia, East Asia, Southeast Asia, and Australasia. [ABSTRACT FROM AUTHOR]

If present at all, endogenous DNA within an ancient sample will have experienced two general damage processes, analogous to those seen in vivo. The quantity of potential PCR template and the size of the amplifiable fragment will be limited by cross-linking, radiation-induced double-strand breaks, hydrolytic and oxidative depurination, and the oxidative formation of hydantoins. In addition to this, even when amplification is successful, the DNA sequence is likely to contain miscoding lesions as a result of further hydrolytic and oxidative damage. While these processes are a hindrance to the aDNA researcher, they have also provided us with new insights into the significance of secondary structure on in vivo mitochondrial mutation, and hence mitochondrial evolution. As further studies into mitochondrial postmortem degradation are undertaken, it can be hoped that they will help resolve both the hotly contested debate on mitochondrial heteroplasmy, and the presence and significance of mitochondrial mutational hotspots. [ABSTRACT FROM AUTHOR]

Following the publication of the first complete sequence of human mtDNA, and the widespread use of semiautomated mtDNA sequencing, there has been a major growth of interest in mtDNA and its role in human evolution and disease. Although superficially these two topics may appear to be unrelated, there is increasing evidence that human mtDNA evolution has an important role in disease expression, both for monogenic disorders (such as LHON) and possibly for complex traits (such as Parkinson's disease). Conversely, these and other diseases, coupled with the environment, may have shaped the evolution of mtDNA in concert with the nuclear genome. [ABSTRACT FROM AUTHOR]

The recent advent of genomic tools has provided us with very powerful ways for the identification of functional genes, thus enhancing our understanding of the molecular basis of both normal and disease phenotypes. Now that sequence information is available for many genomes, a simpler and more definitive technology for the rapid identification of functional genes is a current focus of interest. A simple screening system based on the catalytic activity of ribozymes, whose target specificities are coupled with loss-of-function mutants, has been developed to isolate key genes involved in a defined phenotype. The system was validated for functional gene screens including apoptosis, transformation, metastasis, and muscle and normal differentiation. This system should be applicable to the identification of functional genes involved in many cellular processes and diseases. [ABSTRACT FROM AUTHOR]

In animals noncoding RNAs are involved in a large variety of gene silencing mechanisms. These include post-transcriptional RNA interference (RNAi) that is mediated by small double-stranded RNAs and results in degradation of messenger RNAs as well as epigenetic silencing of genes. RNAi as a naturally occurring silencing mechanism has been well investigated in various eukaryotic organisms. Sequencing of the human and mouse genomes and careful analyses of the related transcriptomes led to the identification of some hundred microRNAs that might regulate endogenous gene expression by RNAi-like mechanisms or repression of translation. In mammals the major protein components of the RNAi machinery have been identified, and RNAi has become a tool for artificial gene silencing in mammalian systems. There is also evidence that in mammalian cells genes can be regulated by noncoding antisense transcripts that are transcribed from the opposite DNA strand. Besides their potential roles in RNAi and repression of translation, short double-stranded RNAs and also long noncoding RNAs are involved in epigenetic gene silencing. In this chapter we give an overview of prominent features of naturally occurring RNAi and also of the potential role of RNAs in epigenetic gene silencing mechanisms in animals, especially in mammals. [ABSTRACT FROM AUTHOR]

Transposons are mobile genetic elements that live parasitically within the genome of cellular organisms. They can affect the fitness of their hosts by influencing gene function, gene activity, genome structure, and overall DNA content. Since excessive transposon activity can result in a high mutagenic rate and genomic instability, eukaryotes have evolved epigenetic mechanisms to reduce transposition to manageable levels. The alga Chlamydomonas reinhardtii appears to have several, at least partly independent, transposon repression pathways that operate at either the transcriptional or the post-transcriptional level. Two genes have been implicated in the transcriptional silencing of transposons and single-copy transgenes: Mut9, which encodes a novel serine/ threonine protein kinase capable of phosphorylating histones H3 and H2A, and Mut11, which encodes a WD40-repeat containing protein. The Mut11 protein functions as a subunit of a histone methyltransferase complex(es) that is required for monomethylation of histone H3 lysine 4 and the maintenance of repressed euchromatic domains. These mechanisms of transcriptional gene silencing operate independently from the RNA interference (RNAi) machinery. [ABSTRACT FROM AUTHOR]

Gene silencing by RNA interference (RNAi) and by antisense RNA are powerful tools to interfere with the expression of eukryotic genes. Since the first description of RNAi in 1998, antisense-mediated gene silencing has been considered to have essentially the same mechanism as gene silencing by RNAi. However, while substantial effort has been made to dissect the RNAi pathway, the cellular machinery that is responsible for posttranscriptional regulation by antisense RNA is rather poorly defined and direct comparisons between the RNAi and antisense experiments are rare. Even though similarities are very likely, recent data suggest that in addition to the expected overlaps in the pathways, there are also mechanistic differences and different requirements for specific gene products. We will summarize the current state of knowledge of the antisense RNA and RNAi mechanisms and address some of the open questions in the field. We will further provide some evidence suggesting that gene silencing by antisense RNA and by RNAi represent related but not identical mechanisms. A model to explain the partially overlapping pathways will be presented and may contribute to the further understanding of posttranscriptional gene regulation. [ABSTRACT FROM AUTHOR]

RNA silencing is a conserved regulatory mechanism that plays an important role in genome integrity and defense in eukaryotic organisms. A key molecule in this sequence-specific RNA degradation mechanism is double-stranded RNA, which is processed by an RNase-III like enzyme (Dicer) into small interfering RNAs (siRNAs). The initial pool of siRNAs can be amplified through the action of RNA-dependent RNA polymerases, which could account for the observed spreading of RNA silencing along the target gene (transitive silencing) and throughout the organism (systemic silencing). In this chapter we discuss the mechanism of RNA amplification and its possible involvement in transitive and systemic RNA silencing in different organisms. [ABSTRACT FROM AUTHOR]

Double-stranded RNA has long been known to be a trigger for cellular responses to viral infections, leading to dramatic changes in cellular processes. Since the advent of RNA interference, it has become clear that double-stranded RNA also causes specific effects, regulating gene expression on the transcriptional, post-transcriptional and translational levels. An essential prerequisite for double-stranded RNA effects is proteins that specifically recognise these molecules in order to elicit the cellular response. This chapter focuses on the function and molecular architecture of those proteins that interact with double-stranded RNA and that are key players in the RNA interference, editing and the PKR response. After summarising the origin of double-stranded RNA molecules and structural features of A-type helices, the way proteins can interact with this secondary structure is discussed. The variability of domain structures of proteins that are functional homologues in processes triggered by double-stranded RNA is reviewed and consequences resulting from the different design of proteins from various organisms are discussed. Finally, differences and similarities of pathways with respect to their subcellular localisation and the length of the double-stranded RNA trigger are summarised. [ABSTRACT FROM AUTHOR]

Micro-RNAs (miRNAs) are single-stranded RNA molecules of about 20 nucleotides and represent a class of small non-coding regulatory RNAs found in higher eukaryotes across kingdoms. miRNAs originate from endogenous chromosomal genes that encode a transcript, called pre-miRNA, of about 70 nucleotides in length. Several premiRNAs can be combined in a polycistronic pri-miRNA. Both precursors are processed by specific ribonucleases: Drosha cleaves a pri-miRNA to several pre-miRNAs, which are processed by a Dicer nuclease into the mature miRNA. The miRNAs themselves interfere with gene expression by base-pairing with a messenger RNA (mRNA) target, but one miRNA can bind in a specific manner to several mRNAs; therefore, miRNAs form a regulatory network that controls gene expression. In this way they are believed to determine tissue-specific gene expression and to act as checkpoints for developmentally important processes. Only a few plant miRNAs show a perfect match to their mRNA target, but the majority of miRNAs, including mammalian miRNAs, regulate translation by imperfect base-pairing. Each species is expected to have a couple of hundred miRNAs and only a few validated targets are known so far. Several attempts have been made to identify miRNA targets by bioinformatics. [ABSTRACT FROM AUTHOR]

Over the last few years several specialized software tools have been developed, each allowing a certain class of RNAs insequencedatatobe found.Herewedescribeageneral tool that allows us to specify many different non-coding RNAs and structural RNA elements by a simple pattern description language.To take into account that RNA is normally conserved in structure as well as in sequence, the pattern description language combines methods to describe sequence and structural similarities as well as further characteristics, e.g., thermodynamic constraints. Structure- and sequence-based patterns describing certain classes of RNAs are collected in a web-based pattern library. These include simple patterns, e.g., describing extrastable tetraloops and small regulatory stem-loop structures, as well as more complex patterns, for example describing pseudoknots, ribozymes, SRP RNAs, 5S RNA and selenocysteine insertion sequences.Aweb-based service allows a user to search the patterns fromthe library in sequences given by the user. Alternatively, the user can specify a pattern that is searched for in public genomic sequence data. Here we give a comprehensive introduction of the pattern language, describe how to systematically derive pattern descriptions, and show some results on purine riboswitches obtained using this computational approach. [ABSTRACT FROM AUTHOR]