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Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cellcell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.

Background Changes in DNA methylation have been associated with traffic-related air pollution in observational studies, but the specific mechanisms and temporal dynamics therein have not been explored in a controlled study of asthmatics. In this study, we investigate short-term effects of diesel exhaust inhalation on DNA methylation levels at CpG sites across the genome in circulating blood in asthmatics. Methods A double-blind crossover study of filtered air and diesel exhaust exposures was performed on sixteen non-smoking asthmatic subjects. Blood samples were collected pre-exposure, and then 6 and 30 hours post-exposure. Peripheral blood mononuclear cell DNA methylation was interrogated using the Illumina Infinium HumanMethylation450 Array. Exposure-related changes in DNA methylation were identified. In addition, CpG sites overlapping with Alu or LINE1 repetitive elements and candidate microRNA loci were also analyzed. Results DNA methylation at 2827 CpG sites were affected by exposure to diesel exhaust but not filtered air; these sites enriched for genes involved in protein kinase and NFkB pathways. CpG sites with significant changes in response to diesel exhaust exposure primarily became less methylated, with a site residing within GSTP1 being among the significant hits. Diesel exhaust-associated change was also found for CpG sites overlapping with Alu and LINE1 elements as well as for a site within miR-21 . Conclusion Short-term exposure to diesel exhaust resulted in DNA methylation changes at CpG sites residing in genes involved in inflammation and oxidative stress response, repetitive elements, and microRNA. This provides plausibility for the role of DNA methylation in pathways by which airborne particulate matter impacts gene expression and offers support for including DNA methylation analysis in future efforts to understand the interactions between environmental exposures and biological systems.

Transitions between sex determination systems have occurred in many lineages of squamates and it follows that novel sex chromosomes will also have arisen multiple times. The formation of sex chromosomes may be reinforced by inhibition of recombination and the accumulation of repetitive DNA sequences. The karyotypes of monitor lizards are known to be highly conserved yet the sex chromosomes in this family have not been fully investigated. Here, we compare male and female karyotypes of three Australian monitor lizards, Varanus acanthurus, V. gouldii and V. rosenbergi, from two different clades. V. acanthurus belongs to the acanthurus clade and the other two belong to the gouldii clade. We applied C-banding and comparative genomic hybridization to reveal that these species have ZZ/ZW sex micro-chromosomes in which the W chromosome is highly differentiated from the Z chromosome. In combination with previous reports, all six Varanus species in which sex chromosomes have been identified have ZZ/ZW sex chromosomes, spanning several clades on the varanid phylogeny, making it likely that the ZZ/ZW sex chromosome is ancestral for this family. However, repetitive sequences of these ZW chromosome pairs differed among species. In particular, an (AAT)n microsatellite repeat motif mapped by fluorescence in situ hybridization on part of W chromosome in V. acanthurus only, whereas a (CGG)n motif mapped onto the W chromosomes of V. gouldii and V. rosenbergi. Furthermore, the W chromosome probe for V. acanthurus produced hybridization signals only on the centromeric regions of W chromosomes of the other two species. These results suggest that the W chromosome sequences were not conserved between gouldii and acanthurus clades and that these repetitive sequences have been amplified rapidly and independently on the W chromosome of the two clades after their divergence.

Barley (Hordeum vulgare L.) is among the world's earliest domesticated and most important crop plants. It is diploid with a large haploid genome of 5.1 gigabases (Gb). Here we present an integrated and ordered physical, genetic and functional sequence resource that describes the barley gene-space in a structured whole-genome context. We developed a physical map of 4.98 Gb, with more than 3.90 Gb anchored to a high-resolution genetic map. Projecting a deep whole-genome shotgun assembly, complementary DNA and deep RNA sequence data onto this framework supports 79,379 transcript clusters, including 26,159 'high-confidence' genes with homology support from other plant genomes. Abundant alternative splicing, premature termination codons and novel transcriptionally active regions suggest that post-transcriptional processing forms an important regulatory layer. Survey sequences from diverse accessions reveal a landscape of extensive single-nucleotide variation. Our data provide a platform for both genome-assisted research and enabling contemporary crop improvement.

Key Points DNA methylation is an epigenetic mark that can be mitotically inherited and is involved in adding stability to the repression of transcription when it is located at the start sites of mammalian genes. Our ability to obtain complete methylomes has transformed our appreciation of the role of DNA methylation in epigenetic processes. DNA methylation in the bodies of genes has long been ignored but might be involved in differential promoter usage and also in transcription elongation and alternative splicing. Repetitive DNA from intragenomic parasites is heavily methylated, which allows transcription of the host gene at the same time as preventing transcription initiation of the repetitive DNA. Methylation of control regions outside of the transcription start sites — such as enhancers and insulators — is increasingly being recognized as being functionally important. Demethylation of DNA is now accepted as being essential for embryonic development and seems to occur mainly in regions of DNA that are not CpG islands; thus, methylation patterns are increasingly being realized as being far more dynamic than previously recognized. Our understanding of the function of DNA methylation is developing now that we are able to look beyond CpG-rich regions at transcriptional start sites. The emerging picture is of a complex relationship between DNA methylation and transcription and of possible additional roles of methylation. DNA methylation is frequently described as a 'silencing' epigenetic mark, and indeed this function of 5-methylcytosine was originally proposed in the 1970s. Now, thanks to improved genome-scale mapping of methylation, we can evaluate DNA methylation in different genomic contexts: transcriptional start sites with or without CpG islands, in gene bodies, at regulatory elements and at repeat sequences. The emerging picture is that the function of DNA methylation seems to vary with context, and the relationship between DNA methylation and transcription is more nuanced than we realized at first. Improving our understanding of the functions of DNA methylation is necessary for interpreting changes in this mark that are observed in diseases such as cancer.

Affinity maturation in B cells generates antibodies with increasingly enhanced antigen-binding properties. Imkeller et al. investigated the maturation of human B cells that express protective antibodies against the circumsporozoite protein of the malaria-causing parasite Plasmodium falciparum (PfCSP). The repetitive structure of PfCSP induces mutations in B cells, facilitating direct interactions between two repeat-bound antibodies against PfCSP, which enhance antigen affinity and B cell activation. Such interactions may optimize binding and promote clustering of surface antibodies in general. Science , this issue p. 1358 A repetitive malaria antigen induces clonal selection and affinity maturation of human B cells expressing protective antibodies. Affinity maturation selects B cells expressing somatically mutated antibody variants with improved antigen-binding properties to protect from invading pathogens. We determined the molecular mechanism underlying the clonal selection and affinity maturation of human B cells expressing protective antibodies against the circumsporozoite protein of the malaria parasite Plasmodium falciparum (PfCSP). We show in molecular detail that the repetitive nature of PfCSP facilitates direct homotypic interactions between two PfCSP repeat-bound monoclonal antibodies, thereby improving antigen affinity and B cell activation. These data provide a mechanistic explanation for the strong selection of somatic mutations that mediate homotypic antibody interactions after repeated parasite exposure in humans. Our findings demonstrate a different mode of antigen-mediated affinity maturation to improve antibody responses to PfCSP and presumably other repetitive antigens.

Clustered regularly interspaced short palindromic repeats (CRISPR)‐encoded immunity in Type I systems relies on the Cascade (CRISPR‐associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4‐Cas (CRISPR‐associated) system (St‐CRISPR4‐Cas), we isolated an effector complex (St‐Cascade) containing 61‐nucleotide CRISPR RNA (crRNA). We show that St‐Cascade, guided by crRNA, binds in vitro to a matching proto‐spacer if a proto‐spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (−1 position) of the proto‐spacer. In the presence of a correct PAM, St‐Cascade binding to the target DNA generates an R‐loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R‐loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St‐CRISPR4‐Cas and other Type I systems. Bacterial CRISPR systems confer immunity by degrading invading DNA species. Reconstituting the Type I CRISPR Cascade complex from S. thermophilus demonstrates the molecular basis for recognition and cleavage of DNA by the nuclease Cas3.

Self/non-self recognition Bacteria and archaea that take up exogenous DNA are equipped with host immunity systems that can recognize and eliminate the foreign DNA. One such system is mediated by the CRISPR genes, which encode small RNAs (crRNAs). CRISPR loci consist of repeats and spacer sequences. When the crRNA spacer sequence pairs with complementary invading DNA, it is marked for elimination. Luciano Marraffini and Erik Sontheimer resolve how the spacer DNA within the CRISPR loci themselves is not recognized as foreign; sequences outside the spacer show perfect pairing to the crRNAs while invading DNA will have mismatches. Differential complementarity of this type may underlie many types of self/non-self recognition — a key function in all immune systems. Distinguishing self from non-self is a vital function for immune systems to repel invaders without inducing autoimmunity. One system, which protects bacteria and archaea from invasion by phage and plasmid DNA, involves clustered, regularly interspaced, short palindromic repeat (CRISPR) loci. Here, in Staphylococcus epidermidis , the mechanism of CRISPR self/non-self discrimination is defined. All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . CRISPR loci are present in ∼40% and ∼90% of sequenced bacterial and archaeal genomes, respectively 10 , and evolve rapidly, acquiring new spacer sequences to adapt to highly dynamic viral populations 1 , 11 , 12 , 13 . Immunity requires a sequence match between the invasive DNA and the spacers that lie between CRISPR repeats 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . Each cluster is genetically linked to a subset of the cas (CRISPR-associated) genes 14 , 15 , 16 that collectively encode >40 families of proteins involved in adaptation and interference. CRISPR loci encode small CRISPR RNAs (crRNAs) that contain a full spacer flanked by partial repeat sequences 2 , 17 , 18 , 19 . CrRNA spacers are thought to identify targets by direct Watson–Crick pairing with invasive ‘protospacer’ DNA 2 , 3 , but how they avoid targeting the spacer DNA within the encoding CRISPR locus itself is unknown. Here we have defined the mechanism of CRISPR self/non-self discrimination. In Staphylococcus epidermidis , target/crRNA mismatches at specific positions outside of the spacer sequence license foreign DNA for interference, whereas extended pairing between crRNA and CRISPR DNA repeats prevents autoimmunity. Hence, this CRISPR system uses the base-pairing potential of crRNAs not only to specify a target, but also to spare the bacterial chromosome from interference. Differential complementarity outside of the spacer sequence is a built-in feature of all CRISPR systems, indicating that this mechanism is a broadly applicable solution to the self/non-self dilemma that confronts all immune pathways.

We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co- opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non- protein- coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation.

The Tol2 element is a naturally occurring active transposable element found in vertebrate genomes. The Tol2 transposon system has been shown to be active from fish to mammals and considered to be a useful gene transfer vector in vertebrates. However, cis-sequences essential for transposition have not been characterized. Here we report the characterization of the minimal cis-sequence of the Tol2 element. We constructed Tol2 vectors containing various lengths of DNA from both the left (5′) and the right (3′) ends and tested their transpositional activities both by the transient excision assay using zebrafish embryos and by analyzing chromosomal transposition in the zebrafish germ lineage. We demonstrated that Tol2 vectors with 200 bp from the left end and 150 bp from the right end were capable of transposition without reducing the transpositional efficiency and found that these sequences, including the terminal inverted repeats (TIRs) and the subterminal regions, are sufficient and required for transposition. The left and right ends were not interchangeable. The Tol2 vector carrying an insert of >11 kb could transpose, but a certain length of spacer, <276 but >18 bp, between the left and right ends was necessary for excision. Furthermore, we found that a 5-bp sequence, 5′-(A/G)AGTA-3′, is repeated 33 times in the essential subterminal region. Mutations in the repeat sequence at 13 different sites in the subterminal region, as well as mutations in TIRs, severely reduced the excision activity, indicating that they play important roles in transposition. The identification of the minimal cis-sequence of the Tol2 element and the construction of mini-Tol2 vectors will facilitate development of useful transposon tools in vertebrates. Also, our study established a basis for further biochemical and molecular biological studies for understanding roles of the repetitive sequence in the subterminal region in transposition.