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Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called 'microbial dark matter'. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet., Microorganisms are the most diverse and abundant cellular life forms on Earth, occupying every possible metabolic niche. The large majority of these organisms have not been obtained in pure culture [...]

Permafrost contains an estimated 1672 Pg carbon (C), an amount roughly equivalent to the total currently contained within land plants and the atmosphere (1-3). This reservoir of C is vulnerable to decomposition as rising global temperatures cause the permafrost to thaw (2). During thaw, trapped organic matter may become more accessible for microbial degradation and result in greenhouse gas emissions (4, 5). Despite recent advances in the use of molecular tools to study permafrost microbial communities (6-9), their response to thaw remains unclear. Here we use deep metagenomic sequencing to determine the impact of thaw on microbial phylogenetic and functional genes, and relate these data to measurements of methane emissions. Metagenomics, the direct sequencing of DNA from the environment, allows the examination of whole biochemical pathways and associated processes, as opposed to individual pieces of the metabolic puzzle. Our metagenome analyses reveal that during transition from a frozen to a thawed state there are rapid shifts in many microbial, phylogenetic and functional gene abundances and pathways. After one week of incubation at 5°C, permafrost metagenomes converge to be more similar to each other than while they are frozen. We find that multiple genes involved in cycling of C and nitrogen shift rapidly during thaw. We also construct the first draft genome from a complex soil metagenome, which corresponds to a novel methanogen. Methane previously accumulated in permafrost is released during thaw and subsequently consumed by methanotrophic bacteria. Together these data point towards the importance of rapid cycling of methane and nitrogen in thawing permafrost., We collected three intact frozen permafrost soil cores with their overlying seasonally thawed active layers from Hess Creek, Alaska. This is a black-spruce forest site containing many metres of frozen [...]

Permafrost microbes await the thaw Permafrost soil in the Arctic contains a huge reservoir of carbon. If the climate warms and the permafrost thaws, this carbon would become accessible to microbial degradation, releasing greenhouse gases in the process. The microbes responsible for this process are largely unknown. Metagenomic analysis of DNA isolated from two permafrost soils collected in Alaska reveals a rapid microbial response to thawing, with many functional gene abundances increasing. A draft genome of a novel methanogen was constructed from the sequence data. This study highlights the importance of rapid cycling of methane and nitrogen in thawing permafrost. Permafrost contains an estimated 1672 Pg carbon (C), an amount roughly equivalent to the total currently contained within land plants and the atmosphere.sup.1,2,3. This reservoir of C is vulnerable to decomposition as rising global temperatures cause the permafrost to thaw.sup.2. During thaw, trapped organic matter may become more accessible for microbial degradation and result in greenhouse gas emissions.sup.4,5. Despite recent advances in the use of molecular tools to study permafrost microbial communities.sup.6,7,8,9, their response to thaw remains unclear. Here we use deep metagenomic sequencing to determine the impact of thaw on microbial phylogenetic and functional genes, and relate these data to measurements of methane emissions. Metagenomics, the direct sequencing of DNA from the environment, allows the examination of whole biochemical pathways and associated processes, as opposed to individual pieces of the metabolic puzzle. Our metagenome analyses reveal that during transition from a frozen to a thawed state there are rapid shifts in many microbial, phylogenetic and functional gene abundances and pathways. After one week of incubation at 5 °C, permafrost metagenomes converge to be more similar to each other than while they are frozen. We find that multiple genes involved in cycling of C and nitrogen shift rapidly during thaw. We also construct the first draft genome from a complex soil metagenome, which corresponds to a novel methanogen. Methane previously accumulated in permafrost is released during thaw and subsequently consumed by methanotrophic bacteria. Together these data point towards the importance of rapid cycling of methane and nitrogen in thawing permafrost., Author(s): Rachel Mackelprang [sup.1] [sup.2] , Mark P. Waldrop [sup.3] , Kristen M. DeAngelis [sup.4] , Maude M. David [sup.4] , Krystle L. Chavarria [sup.4] , Steven J. Blazewicz [sup.5] [...]

Sequencing of bacterial and archaeal genomes has revolutionized our understanding of the many roles played by microorganisms (1). There are now nearly 1,000 completed bacterial and archaeal genomes available (2), most of which were chosen for sequencing on the basis of their physiology. As a result, the perspective provided by the currently available genomes is limited by a highly biased phylogenetic distribution (3-5). To explore the value added by choosing microbial genomes for sequencing on the basis of their evolutionary relationships, we have sequenced and analysed the genomes of 56 culturable species of Bacteria and Archaea selected to maximize phylogenetic coverage. Analysis of these genomes demonstrated pronounced benefits (compared to an equivalent set of genomes randomly selected from the existing database) in diverse areas including the reconstruction of phylogenetic history, the discovery of new protein families and biological properties, and the prediction of functions for known genes from other organisms. Our results strongly support the need for systematic 'phylogenomic' efforts to compile a phylogeny-driven 'Genomic Encyclopedia of Bacteria and Archaea' in order to derive maximum knowledge from existing microbial genome data as well as from genome sequences to come., Since the publication of the first complete bacterial genome, sequencing of the microbial world has accelerated beyond expectations. The inventory of bacterial and archaeal isolates with complete or draft sequences [...]

In contrast to protein-coding sequences, the significance of variation in non-coding DNA in human disease has been minimally explored. A great number of recent genome-wide association studies suggest that non-coding variation is a significant risk factor for common disorders, but the mechanisms by which this variation contributes to disease remain largely obscure. Distant-acting transcriptional enhancers--a major category of functional non-coding DNA--are involved in many developmental and disease-relevant processes. Genomewide approaches to their discovery and functional characterization are now available and provide a growing knowledge base for the systematic exploration of their role in human biology and disease susceptibility., Multiple lines of evidence indicate that important functional properties are embedded in the non-coding portion of the human genome, but identifying and defining these features remains a major challenge. An [...]

A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover because they are scattered among the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here we present the results of chromatin immunoprecipitation with the enhancer-associated protein p300 followed by massively parallel sequencing, and map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain and limb tissue. We tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases demonstrated reproducible enhancer activity in the tissues that were predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities, and suggest that such data sets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale., The initial sequencing of the human genome (1,2), complemented by effective computational and experimental strategies for mammalian gene discovery (3,4), has resulted in a virtually complete list of protein-coding sequences. [...]

The development of alternatives to fossil fuels as an energy source is an urgent global priority. Cellulosic biomass has the potential to contribute to meeting the demand for liquid fuel, [...]

Author(s): Eric D. Green (corresponding author) [1]; Edward M. Rubin [2]; Maynard V. Olson [3] Forty years ago, two papers [1, 2] described the first tractable methods for determining the [...]

Author(s): Falk Warnecke [1, 8]; Peter Luginbühl [2, 8]; Natalia Ivanova [1]; Majid Ghassemian [2]; Toby H. Richardson [2, 9]; Justin T. Stege [2]; Michelle Cayouette [2]; Alice C. McHardy [...]

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during ligno-cellulose degradation. Globally, termites [...]

Despite the greater information content of genomic DNA, ancient DNA studies have largely been limited to the amplification of mitochondrial sequences. Here we describe metagenomic libraries constructed with unamplified DNA extracted from skeletal remains of two 40,000-year-old extinct cave bears. Analysis of ~1 megabase of sequence from each library showed that despite significant microbial contamination, 5.8 and 1.1% of clones contained cave bear inserts, yielding 26,861 base pairs of cave bear genome sequence. Comparison of cave bear and modern bear sequences revealed the evolutionary relationship of these lineages. The metagenomic approach used here establishes the feasibility of ancient DNA genome sequencing programs., Genomic DNA sequences from extinct species can help reveal the process of molecular evolution that produced modern genomes. However, the recovery of ancient DNA is technologically challenging, because the molecules [...]

Author(s): Kirsten K. Youngren [1]; Douglas Coveney [3]; Xiaoning Peng [4]; Chitralekha Bhattacharya [4]; Laura S. Schmidt [5]; Michael L. Nickerson [6]; Bruce T. Lamb [1]; Jian Min Deng [4]; [...]

The species complexity of microbial communities and challenges in culturing representative isolates make it difficult to obtain assembled genomes. Here we characterize and compare the metabolic capabilities of terrestrial and marine microbial communities using largely unassembled sequence data obtained by shotgun sequencing DNA isolated from the various environments. Quantitative gene content analysis reveals habitat-specific fingerprints that reflect known characteristics of the sampled environments. The identification of environment-specific genes through a gene-centric comparative analysis presents new opportunities for interpreting and diagnosing environments., Despite their ubiquity, relatively little is known about the majority of environmental microorganisms, largely because of their resistance to culture under standard laboratory conditions. A variety of environmental sequencing projects [...]

Author(s): Joel Martin [1]; Cliff Han [2]; Laurie A. Gordon [1]; Astrid Terry [1]; Shyam Prabhakar [4]; Xinwei She [5]; Gary Xie [1, 2]; Uffe Hellsten [1]; Yee Man Chan [...]

Author(s): Marcelo A. Nóbrega [2]; Yiwen Zhu [2]; Ingrid Plajzer-Frick; Veena Afzal; Edward M. Rubin (corresponding author) The functional importance of the roughly 98% of mammalian genomes not corresponding to [...]

Author(s): Jeremy Schmutz (corresponding author) [1]; Joel Martin [2]; Astrid Terry [2]; Olivier Couronne [3]; Jane Grimwood [1]; Steve Lowry [2]; Laurie A. Gordon [2, 4]; Duncan Scott [2]; Gary [...]

Author(s): Jane Grimwood (corresponding author) [1]; Laurie A. Gordon [2, 3]; Anne Olsen [2, 3]; Astrid Terry [2]; Jeremy Schmutz [1]; Jane Lamerdin [2, 3]; Uffe Hellsten [2]; David Goodstein [...]

Author(s): Gene W. Tyson [1]; Jarrod Chapman [3, 4]; Philip Hugenholtz [1]; Eric E. Allen [1]; Rachna J. Ram [1]; Paul M. Richardson [4]; Victor V. Solovyev [4]; Edward M. [...]

Nonhuman primates represent the most relevant model organisms to understand the biology of Homo sapiens. The recent divergence and associated overall sequence conservation between individual members of this taxon have [...]

Author(s): Mouse Genome Sequencing Consortium ; Genome Sequencing Center: ; Asif T. Chinwalla [1, 47]; Lisa L. Cook [1]; Kimberly D. Delehaunty [1]; Ginger A. Fewell [1]; Lucinda A. Fulton [...]

Author(s): Edward M. Rubin (corresponding author); Alan Tall [1] Although many of the genes responsible for rare single-gene vascular disorders have been described, the vast majority of genes that contribute [...]

A mouse model for Down syndrome, Ts1Cje, has been developed. This model has made possible a step in the genetic dissection of the learning, behavioral, and neurological abnormalities associated with segmental trisomy for the region of mouse chromosome 16 homologous with the so-called 'Down syndrome region' of human chromosome segment 21q22. Tests of learning in the Morris water maze and assessment of spontaneous locomotor activity reveal distinct learning and behavioral abnormalities, some of which are indicative of hippocampal dysfunction. The triplicated region in Ts1Cje, from Sod1 to Mx1, is smaller than that in Ts65Dn, another segmental trisomy 16 mouse, and the learning deficits in Ts1Cje are less severe than those in Ts65Dn. In addition, degeneration of basal forebrain cholinergic neurons, which was observed in Ts65Dn, was absent in Ts1Cje.

Male infertility, affecting as many as 10% of the adult population, is an extremely prevalent disorder. In most cases, the cause of the condition is unknown, and genetic factors that might affect male fertility, other than some sequences on the Y chromosome, have not been identified. We report here that male mice heterozygous for a targeted mutation of the apolipoprotein B (apo B) gene exhibit severely compromised fertility. Sperm from these mice failed to fertilize eggs both in vivo and in vitro. However, these sperm were able to fertilize eggs once the zona pellucida was removed but displayed persistent abnormal binding to the egg after fertilization. In vitro fertilization-related and other experiments revealed reduced sperm motility, survival time, and sperm count also contributed to the infertility phenotype. Recognition of the infertility phenotype led to the identification of apo B mRNA in the testes and epididymides of normal mice, and these transcripts were substantially reduced in the affected animals. Moreover, when the genomic sequence encoding human apo B was introduced into these animals, normal fertility was restored. These findings suggest that this genetic locus may have an important impact on male fertility and identify a previously unrecognized function for apo B.

RNA editing in the nucleus of higher eukaryotes results in subtle changes to the RNA sequence, with the ability to effect dramatic changes in biological function. The first example to be described and among the best characterized, is the cytidine-to-uridine editing of apolipoprotein B (apo-B) RNA. The editing of apo-B RNA is mediated by a novel cytidine deaminase, apobec-1, which has acquired the ability to bind RNA. The stop translation codon generated by the editing of apo-B RNA truncates the full-length apo-B100 to form apo-B48. The recent observations of tumor formation in Apobec-1 transgenic animals, together with the fact that Apobec-1 is expressed in numerous tissues lacking apo-B, raises the issue of whether this enzyme is essential for a variety of posttranscriptional editing events. To directly test this, mice were created with a null mutation in Apobec-1 using homologous recombination in embryonic stem cells. Mice, homozygous for this mutation, were viable and made apo-B100 but not apo-B48. The null animals were fertile, and a variety of histological, behavioral, and morphological analyses revealed no phenotype other than abnormalities in lipoprotein metabolism, which included an increased low density lipoprotein fraction and a reduction in high density lipoprotein cholesterol. These studies demonstrate that neither apobec-1 nor apo-B48 is essential for viability and suggest that the major role of apobec-1 may be confined to the modulation of lipid transport.

The human apolipoprotein B (apoB) was successfully expressed in mice due to the efficient accumulation of the atherogenic macromolecule lipoprotein(a). A 90-kilobase P1 phagemid having the human apoB gene was isolated to generate B lines of transgenic mice DNA, on which 11 lines expressed the human apoB.

Fetal hematopoietic stem cell (HSC) transplantation has the potential to play a vital role in the treatment of erythroid chimerism. HSCs from non thalassemic syngeneic murine fetal donors were transplanted into recipients with nonirradiated neonatal beta-thalassemic properties. Erythrocyte chimerism was observed in several recipients within a month.

To create mice expressing exclusively human sickle hemoglobin (HbS), transgenic mice expressing human [Alpha]-, [Gamma]-, and [Β.sup.S]-globin were generated and bred with knockout mice that had deletions of the murine [Alpha]- and Β-globin genes. These sickle cell mice have the major features (irreversibly sickled red cells, anemia, multiorgan pathology) found in humans with sickle cell disease and, as such, represent a useful in vivo system to accelerate the development of improved therapies for this common genetic disease., A single base pair change in codon 6 of the Β-globin gene causes sickle cell anemia in individuals who are homozygous for the mutation (1). Sickle hemoglobin [HbS ([[Alpha].sub.2][Β.sub.2])] undergoes [...]

Quantification of the area of lipid-strained lesions in aorta of transgenic mice suggested that the presence of apolipoprotein AII, which is absent in the mice, reduces the anti-atherogenic influence of Al-HDL. Human plasma contains AII, and so is more anti-atherogenic than transgenic mice. It is hypothesized that HDL transports cholestrol from peripheral tissues to the liver, and this may attribute to its anti-atherogenic functions. AI-HDL is more efficient in this than AI/AII-HDL.

The risk factors for atherosclerosis are well known, but with one exception its causes remain unclear. A study using transgenic mice that express human apolipoprotein(a) may help clarify matters. Experimental mice exposed to a high fat diet develop more lipid-staining lesions in the aorta than non-transgenic mice on a similar diet or transgenic mice on a normal diet. Apolipoprotein(a) was found to localize with lipids in lesion areas suggesting that the observed differences in atherogenesis results from the protein.

The health implications of cholesterol in the blood are determined by the types and quantities of cholesterol in the blood plasma. High levels of low-density lipoprotein (LDL) cholesterol are correlated with increased risk for atherosclerosis, and high levels of high-density lipoprotein (HDL) cholesterol are associated with decreased risk for atherosclerosis, and its attendant heart disease and strokes. Since the protein apolipoprotein AI (ApoA-I) is the major protein component of HDL, it is not surprising that higher amounts of ApoA-I are also associated with lower risk for disease. Since HDL cholesterol is carried back to the liver from the body tissues, it is plausible to think that higher amounts might carry back more cholesterol and provide benefit directly. However, increases in HDL cholesterol may result from some unknown factor that directly reduces the risk for atherosclerosis; if this is the case, manipulating HDL directly would have no effect on the risks for atherosclerosis. To examine the role of HDL in preventing disease, the direct physiological effects of ApoA-I have now been examined in laboratory mice. Some inbred strains of mice are predisposed to develop fatty streak lesions when fed diets rich in fats. These lesions represent the early stages of what will become atherosclerosis. Researchers examined the effect of ApoA-I on these lesions by using the techniques of molecular biology. With genetic engineering, the gene for human ApoA-I was inserted into the mouse genes. The resulting animals make all the normal mouse proteins, but they also make the human ApoA-I as well. The large amounts of ApoA-I in these mice resulted in a correspondingly large amount of HDL in the blood. It was found that the high levels of HDL actually did result in some protection, and the development of the fatty lesions in these mice was significantly inhibited. It is possible that ApoA-I may inhibit the development of mature atherosclerotic plaques as well. (Consumer Summary produced by Reliance Medical Information, Inc.)

Author(s): Len A. Pennacchio (corresponding author) [1, 2]; Nadav Ahituv [2]; Alan M. Moses [2]; Shyam Prabhakar [2]; Marcelo A. Nobrega [2, 5]; Malak Shoukry [2]; Simon Minovitsky [2]; Inna [...]

Author(s): Tanja Woyke [1, 2]; Hanno Teeling [3]; Natalia N. Ivanova [1]; Marcel Huntemann [3]; Michael Richter [3]; Frank Oliver Gloeckner [3, 4]; Dario Boffelli [1, 2]; Iain J. Anderson [...]

Author(s): Gill Bejerano (corresponding author) [1]; Craig B. Lowe [1]; Nadav Ahituv [2, 3]; Bryan King [1, 4]; Adam Siepel [1, 5]; Sofie R. Salama [1, 4]; Edward M. Rubin [...]

Comparison of genomic DNA sequences from human and mouse revealed a new apolipoprotein (APO) gene (APOAV) located proximal to the well-characterized APOAI/CIII/AIV gene cluster on human 11q23. Mice expressing a human APOAV transgene showed a decrease in plasma triglyceride concentrations to one-third of those in control mice; conversely, knockout mice lacking Apoav had four times as much plasma triglycerides as controls. In humans, single nucleotide polymorphisms (SNPs) across the APOAV locus were found to be significantly associated with plasma triglyceride levels in two independent studies. These findings indicate that APOAV is an important determinant of plasma triglyceride levels, a major risk factor for coronary artery disease., Plasma lipid levels are a major determinant of cardiovascular disease susceptibility (1). Members of the apolipoprotein gene family have been shown to play a significant role in determining an organism's [...]

The ABCA1 gene, a member of the ATP-binding cassette A (ABCA1) transporter superfamily, encodes a membrane protein that facilitates the cellular efflux of cholesterol and phospholipids. Mutations in ABCA1 lead to familial high density lipoprotein deficiency and Tangier disease. We report the complete human ABCA1 gene sequence, including 1,453 bp of the promoter, 146,581 bp of introns and exons, and 1 kb of the 3' flanking region. The ABCA1 gene spans 149 kb and comprises 50 exons. Sixty-two repetitive Alu sequences were identified in introns 1-49. The transcription start site is 315 bp upstream of a newly identified initiation methionine codon and encodes an ORF of 6,783 bp. Thus, the ABCA1 protein is comprised of 2,261 aa. Analysis of the 1,453 bp 5' upstream of the transcriptional start site reveals multiple binding sites for transcription factors with roles in lipid metabolism. Comparative analysis of the mouse and human ABCA1 promoter sequences identified specific regulatory elements, which are evolutionarily conserved. The human ABCA1 promoter fragment -200 to -80 bp that contains binding motifs for SP1, SP3, E-box, and AP1 modulates cellular cholesterol and cAMP regulation of ABCA1 gene expression. These combined findings provide insights into ABCA1-mediated regulation of cellular cholesterol metabolism and will facilitate the identification of new pharmacologic agents for the treatment of atherosclerosis in humans.

In their Report 'Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons' (7 Apr., p. 136), G. G. Loots and colleagues identify conserved noncoding [...]

Although cardiac hypertrophy has been the subject of intensive investigation, regression of hypertrophy has been significantly less studied, precluding large-scale analysis of the relationship between these processes. In the present study, using pharmacological models of cardiac hypertrophy in mice, expression profiling was performed with fragments of more than 4,000 genes to characterize and contrast expression changes during induction and regression of hypertrophy. Administration of angiotensin II and isoproterenol by osmotic minipump produced increases in heart weight (15 and 45%, respectively) that returned to preinduction size after drug withdrawal. From multiple expression analyses of left ventricular RNA isolated at daily time-points during cardiac hypertrophy and regression, we identified sets of genes whose expression was altered at specific stages of this process. While confirming the participation of 25 genes or pathways previously shown to be altered by hypertrophy, a larger set of 30 genes was identified whose expression had not previously been associated with cardiac hypertrophy or regression. Of the 55 genes that showed reproducible changes during the time course of induction and regression, 32 genes were altered only during induction, and 8 were altered only during regression. This study identified both known and novel genes whose expression is affected at different stages of cardiac hypertrophy and regression and demonstrates that cardiac remodeling during regression utilizes a set of genes that are distinct from those used during induction of hypertrophy.

To accelerate the biological annotation of novel genes discovered in sequenced regions of mammalian genomes, we are creating large deletions in the mouse genome targeted to include clusters of such genes. Here we describe the targeted deletion of a 450-kb region on mouse chromosome 11, which, based on computational analysis of the deleted murine sequences and human 5q orthologous sequences, codes for nine putative genes. Mice homozygous for the deletion had a variety of abnormalities, including severe hypertriglyceridemia, hepatic and cardiac enlargement, growth retardation, and premature mortality. Analysis of triglyceride metabolism in these animals demonstrated a several-fold increase in hepatic very-low density lipoprotein triglyceride secretion, the most prevalent mechanism responsible for hypertriglyceridemia in humans. A series of mouse BAC and human YAC transgenes covering different intervals of the 450-kb deleted region were assessed for their ability to complement the deletion induced abnormalities. These studies revealed that OCTN2, a gene recently shown to play a role in carnitine transport, was able to correct the triglyceride abnormalities. The discovery of this previously unappreciated relationship between OCTN2, carnitine, and hepatic triglyceride production is of particular importance because of the clinical consequence of hypertriglyceridemia and the paucity of genes known to modulate triglyceride secretion.

Approximately 25% of the genome consists of gene-poor regions greater than 500 kb, termed gene deserts (1). These segments have been minimally explored, and their functional significance remains elusive. One [...]