SHRiMP: Accurate Mapping of Short Color-space Reads

The development of Next Generation Sequencing technologies, capable of sequencing hundreds of millions of short reads (25–70 bp each) in a single run, is opening the door to population genomic studies of non-model species. In this paper we present SHRiMP - the SHort Read Mapping Package: a set of algorithms and methods to map short reads to a genome, even in the presence of a large amount of polymorphism. Our method is based upon a fast read mapping technique, separate thorough alignment methods for regular letter-space as well as AB SOLiD (color-space) reads, and a statistical model for false positive hits. We use SHRiMP to map reads from a newly sequenced Ciona savignyi individual to the reference genome. We demonstrate that SHRiMP can accurately map reads to this highly polymorphic genome, while confirming high heterozygosity of C. savignyi in this second individual. SHRiMP is freely available at http://compbio.cs.toronto.edu/shrimp

First Neutrino Observations from the Sudbury Neutrino Observatory

The first neutrino observations from the Sudbury Neutrino Observatory are presented from preliminary analyses. Based on energy, direction and location, the data in the region of interest appear to be dominated by 8B solar neutrinos, detected by the charged current reaction on deuterium and elastic scattering from electrons, with very little background. Measurements of radioactive backgrounds indicate that the measurement of all active neutrino types via the neutral current reaction on deuterium will be possible with small systematic uncertainties. Quantitative results for the fluxes observed with these reactions will be provided when further calibrations have been completed.Comment: Latex, 7 pages, 10 figures, Invited paper at Neutrino 2000 Conference, Sudbury, Canada, June 16-21, 2000 to be published in the Proceeding

Drosophila Argonaute-1 is critical for transcriptional cosuppression and heterochromatin formation

Argonaute-1 (Ago-1) plays a crucial role in gene regulation and genome stability via biogenesis of small non-coding RNAs. Two “Argonaute” family genes, piwi and Ago-2 in Drosophila are involved in multiple silencing mechanisms in the nucleus, transgene cosuppression, long-distant chromosome interaction, nuclear organization and heterochromatin formation. To investigate whether Ago-1 also plays a similar role, we have generated a series of Ago-1 mutations by excising P element, inserted in the Ago-1 promoter (Ago-1k08121). AGO-1 protein is distributed uniformly in the nucleus and cytosol in early embryos but accumulated predominantly in the cytoplasm during the gastrulation stage. Repeat induced silencing produced by the mini-white (mw) array and transcriptional cosuppression of non-homologous transgenes Adh-w/w-Adh was disrupted by Ago-1 mutation. These effects of Ago-1 are distict from its role in microRNA processing because Dicer-1, a critical enzyme for miRNA biogenesis, has no role on the above silencing. Reduction of AGO-1 protein dislodged the POLYCOMB, EZ (enhancer of zeste) and H3me3K27 binding at the cosuppressed Adh-w transgene insertion sites suggesting its role in Polycomb dependent cosuppression. An overall reduction of methylated histone H3me2K9 and H3me3K27 from the polytene nuclei precisely from the mw promoters was also found that leads to concomitant changes in the chromatin structure. These results suggest a prominent role of Ago-1 in chromatin organization and transgene silencing and demonstrate a critical link between transcriptional transgene cosuppression, heterochromatin formation and chromatin organization. We propose Drosophila Ago-1 as a multifunctional RNAi component that interconnects at least two unrelated events, chromatin organization in the nucleus and microRNA processing in the cytoplasm, which may be extended to the other systems

Interaction of RNA polymerase II and the small RNA machinery affects heterochromatic silencing in Drosophila

<p>Abstract</p> <p>Background</p> <p>Heterochromatin is the tightly packaged dynamic region of the eukaryotic chromosome that plays a vital role in cellular processes such as mitosis and meiotic recombination. Recent experiments in <it>Schizosaccharomyces pombe </it>have revealed the structure of centromeric heterochromatin is affected in RNAi pathway mutants. It has also been shown in fission yeast that the heterochromatin barrier is traversed by RNA Pol II and that the passage of RNA Pol II through heterochromatin is important for heterochromatin structure. Thus, an intricate interaction between the RNAi machinery and RNA Pol II affects heterochromatin structure. However, the role of the RNAi machinery and RNA Pol II on the metazoan heterochromatin landscape is not known. This study analyses the interaction of the small RNA machinery and RNA Pol II on <it>Drosophila </it>heterochromatin structure.</p> <p>Results</p> <p>The results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit) and small RNA silencing machinery components (<it>dcr-2, ago1, ago2, piwi, Lip [D], aub </it>and <it>hls</it>). Immunofluorescence analysis of polytene chromosomes from trans-heterozygotes of RNA Pol II and different mutations of the small RNA pathways show decreased H3K9me2 and mislocalization of Heterochromatin protein-1. A genetic analysis performed on these mutants showed a strong suppression of <it>white-mottled4h </it>position effect variegation. This was further corroborated by a western blot analysis and chromatin immunoprecipitation, which showed decreased H3K9me2 in trans-heterozygote mutants compared to wild type or single heterozygotes. Co-immunoprecipitation performed using <it>Drosophila </it>embryo extracts showed the RNA Pol II largest subunit interacting with Dcr-2 and dAGO1. Co-localization performed on polytene chromosomes showed RNA Pol II and dAGO1 overlapping at some sites.</p> <p>Conclusion</p> <p>Our experiments show a genetic and biochemical interaction between RNA Pol II (largest and second largest subunits) and the small RNA silencing machinery in <it>Drosophila</it>. The interaction has functional aspects in terms of determining H3K9me2 and HP-1 deposition at the chromocentric heterochromatin. Thus, RNA Pol II has an important role in establishing heterochromatin structure in <it>Drosophila</it>.</p

Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish

© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Genomics 11 (2010): 643, doi:10.1186/1471-2164-11-643.Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development. Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters. Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.This work was supported by NIH grants R01ES015912 and P42ES007381 (Superfund Basic Research Program at Boston University) (to JJS). MEJ was a Guest Investigator at the Woods Hole Oceanographic Institution (WHOI) and was supported by grants from the Swedish research council Formas and Carl Trygger's foundation. AK was a Post-doctoral Fellow at WHOI, and was supported by a fellowship from the Japanese Society for Promotion of Science (JSPS). JZ and TP were Guest Students at the WHOI and were supported by a CAPES Ph.D. Fellowship and CNPq Ph.D. Sandwich Fellowship (JZ), and by a CNPq Ph.D. Fellowship (TP), from Brazil

Insertion of Horizontally Transferred Genes within Conserved Syntenic Regions of Yeast Genomes

Horizontal gene transfer has been occasionally mentioned in eukaryotic genomes, but such events appear much less numerous than in prokaryotes, where they play important functional and evolutionary roles. In yeasts, few independent cases have been described, some of which corresponding to major metabolic functions, but no systematic screening of horizontally transferred genes has been attempted so far. Taking advantage of the synteny conservation among five newly sequenced and annotated genomes of Saccharomycetaceae, we carried out a systematic search for HGT candidates amidst genes present in only one species within conserved synteny blocks. Out of 255 species-specific genes, we discovered 11 candidates for HGT, based on their similarity with bacterial proteins and on reconstructed phylogenies. This corresponds to a minimum of six transfer events because some horizontally acquired genes appear to rapidly duplicate in yeast genomes (e.g. YwqG genes in Kluyveromyces thermotolerans and serine recombinase genes of the IS607 family in Saccharomyces kluyveri). We show that the resulting copies are submitted to a strong functional selective pressure. The mechanisms of DNA transfer and integration are discussed, in relation with the generally small size of HGT candidates. Our results on a limited set of species expand by 50% the number of previously published HGT cases in hemiascomycetous yeasts, suggesting that this type of event is more frequent than usually thought. Our restrictive method does not exclude the possibility that additional HGT events exist. Actually, ancestral events common to several yeast species must have been overlooked, and the absence of homologs in present databases leaves open the question of the origin of the 244 remaining species-specific genes inserted within conserved synteny blocks

The Baker's Yeast Diploid Genome Is Remarkably Stable in Vegetative Growth and Meiosis

Accurate estimates of mutation rates provide critical information to analyze genome evolution and organism fitness. We used whole-genome DNA sequencing, pulse-field gel electrophoresis, and comparative genome hybridization to determine mutation rates in diploid vegetative and meiotic mutation accumulation lines of Saccharomyces cerevisiae. The vegetative lines underwent only mitotic divisions while the meiotic lines underwent a meiotic cycle every ∼20 vegetative divisions. Similar base substitution rates were estimated for both lines. Given our experimental design, these measures indicated that the meiotic mutation rate is within the range of being equal to zero to being 55-fold higher than the vegetative rate. Mutations detected in vegetative lines were all heterozygous while those in meiotic lines were homozygous. A quantitative analysis of intra-tetrad mating events in the meiotic lines showed that inter-spore mating is primarily responsible for rapidly fixing mutations to homozygosity as well as for removing mutations. We did not observe 1–2 nt insertion/deletion (in-del) mutations in any of the sequenced lines and only one structural variant in a non-telomeric location was found. However, a large number of structural variations in subtelomeric sequences were seen in both vegetative and meiotic lines that did not affect viability. Our results indicate that the diploid yeast nuclear genome is remarkably stable during the vegetative and meiotic cell cycles and support the hypothesis that peripheral regions of chromosomes are more dynamic than gene-rich central sections where structural rearrangements could be deleterious. This work also provides an improved estimate for the mutational load carried by diploid organisms

Redundancy and the Evolution of Cis-Regulatory Element Multiplicity

The promoter regions of many genes contain multiple binding sites for the same transcription factor (TF). One possibility is that this multiplicity evolved through transitional forms showing redundant cis-regulation. To evaluate this hypothesis, we must disentangle the relative contributions of different evolutionary mechanisms to the evolution of binding site multiplicity. Here, we attempt to do this using a model of binding site evolution. Our model considers binding sequences and their interactions with TFs explicitly, and allows us to cast the evolution of gene networks into a neutral network framework. We then test some of the model's predictions using data from yeast. Analysis of the model suggested three candidate nonadaptive processes favoring the evolution of cis-regulatory element redundancy and multiplicity: neutral evolution in long promoters, recombination and TF promiscuity. We find that recombination rate is positively associated with binding site multiplicity in yeast. Our model also indicated that weak direct selection for multiplicity (partial redundancy) can play a major role in organisms with large populations. Our data suggest that selection for changes in gene expression level may have contributed to the evolution of multiple binding sites in yeast. We conclude that the evolution of cis-regulatory element redundancy and multiplicity is impacted by many aspects of the biology of an organism: both adaptive and nonadaptive processes, both changes in cis to binding sites and in trans to the TFs that interact with them, both the functional setting of the promoter and the population genetic context of the individuals carrying them