Stephan C. Schuster

Archaea predominate among ammonia-oxidizing prokaryotes in soils.

Ammonia oxidation is the first step in nitrification, a key process in the global nitrogen cycle that results in the formation of nitrate through microbial activity. The increase in nitrate availability in soils is important for plant nutrition, but it also has considerable impact on groundwater pollution owing to leaching. Here we show that archaeal ammonia oxidizers are more abundant in soils than their well-known bacterial counterparts. We investigated the abundance of the gene encoding a subunit of the key enzyme ammonia monooxygenase (amoA) in 12 pristine and agricultural soils of three climatic zones. amoA gene copies of Crenarchaeota (Archaea) were up to 3,000-fold more abundant than bacterial amoA genes. High amounts of crenarchaeota-specific lipids, including crenarchaeol, correlated with the abundance of archaeal amoA gene copies. Furthermore, reverse transcription quantitative PCR studies and complementary DNA analysis using novel cloning-independent pyrosequencing technology demonstrated the activity of the archaea in situ and supported the numerical dominance of archaeal over bacterial ammonia oxidizers. Our results indicate that crenarchaeota may be the most abundant ammonia-oxidizing organisms in soil ecosystems on Earth.

Next-generation sequencing transforms today's biology

A new generation of non-Sanger-based sequencing technologies has delivered on its promise of sequencing DNA at unprecedented speed, thereby enabling impressive scientific achievements and novel biological applications. However, before stepping into the limelight, next-generation sequencing had to overcome the inertia of a field that relied on Sanger-sequencing for 30 years.

Ancestral polyploidy in seed plants and angiosperms

Whole-genome duplication (WGD), or polyploidy, followed by gene loss and diploidization has long been recognized as an important evolutionary force in animals, fungi and other organisms, especially plants. The success of angiosperms has been attributed, in part, to innovations associated with gene or whole-genome duplications, but evidence for proposed ancient genome duplications pre-dating the divergence of monocots and eudicots remains equivocal in analyses of conserved gene order. Here we use comprehensive phylogenomic analyses of sequenced plant genomes and more than 12.6 million new expressed-sequence-tag sequences from phylogenetically pivotal lineages to elucidate two groups of ancient gene duplications—one in the common ancestor of extant seed plants and the other in the common ancestor of extant angiosperms. Gene duplication events were intensely concentrated around 319 and 192 million years ago, implicating two WGDs in ancestral lineages shortly before the diversification of extant seed plants and extant angiosperms, respectively. Significantly, these ancestral WGDs resulted in the diversification of regulatory genes important to seed and flower development, suggesting that they were involved in major innovations that ultimately contributed to the rise and eventual dominance of seed plants and angiosperms.

Diverse somatic mutation patterns and pathway alterations in human cancers.

The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.

Microbial community gene expression in ocean surface waters

Metagenomics is expanding our knowledge of the gene content, functional significance, and genetic variability in natural microbial communities. Still, there exists limited information concerning the regulation and dynamics of genes in the environment. We report here global analysis of expressed genes in a naturally occurring microbial community. We first adapted RNA amplification technologies to produce large amounts of cDNA from small quantities of total microbial community RNA. The fidelity of the RNA amplification procedure was validated with Prochlorococcus cultures and then applied to a microbial assemblage collected in the oligotrophic Pacific Ocean. Microbial community cDNAs were analyzed by pyrosequencing and compared with microbial community genomic DNA sequences determined from the same sample. Pyrosequencing-based estimates of microbial community gene expression compared favorably to independent assessments of individual gene expression using quantitative PCR. Genes associated with key metabolic pathways in open ocean microbial species—including genes involved in photosynthesis, carbon fixation, and nitrogen acquisition—and a number of genes encoding hypothetical proteins were highly represented in the cDNA pool. Genes present in the variable regions of Prochlorococcus genomes were among the most highly expressed, suggesting these encode proteins central to cellular processes in specific genotypes. Although many transcripts detected were highly similar to genes previously detected in ocean metagenomic surveys, a significant fraction (≈50%) were unique. Thus, microbial community transcriptomic analyses revealed not only indigenous gene- and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.

Nucleosome organization in the Drosophila genome

Comparative genomics of nucleosome positions provides a powerful means for understanding how the organization of chromatin and the transcription machinery co-evolve. Here we produce a high-resolution reference map of H2A.Z and bulk nucleosome locations across the genome of the fly Drosophila melanogaster and compare it to that from the yeast Saccharomyces cerevisiae. Like Saccharomyces, Drosophila nucleosomes are organized around active transcription start sites in a canonical -1, nucleosome-free region, +1 arrangement. However, Drosophila does not incorporate H2A.Z into the -1 nucleosome and does not bury its transcriptional start site in the +1 nucleosome. At thousands of genes, RNA polymerase II engages the +1 nucleosome and pauses. How the transcription initiation machinery contends with the +1 nucleosome seems to be fundamentally different across major eukaryotic lines.

Translational and rotational settings of H2A.Z nucleosomes across the Saccharomyces cerevisiae genome

The nucleosome is the fundamental building block of eukaryotic chromosomes. Access to genetic information encoded in chromosomes is dependent on the position of nucleosomes along the DNA. Alternative locations just a few nucleotides apart can have profound effects on gene expression. Yet the nucleosomal context in which chromosomal and gene regulatory elements reside remains ill-defined on a genomic scale. Here we sequence the DNA of 322,000 individual Saccharomyces cerevisiae nucleosomes, containing the histone variant H2A.Z, to provide a comprehensive map of H2A.Z nucleosomes in functionally important regions. With a median 4-base-pair resolution, we identify new and established signatures of nucleosome positioning. A single predominant rotational setting and multiple translational settings are evident. Chromosomal elements, ranging from telomeres to centromeres and transcriptional units, are found to possess characteristic nucleosomal architecture that may be important for their function. Promoter regulatory elements, including transcription factor binding sites and transcriptional start sites, show topological relationships with nucleosomes, such that transcription factor binding sites tend to be rotationally exposed on the nucleosome surface near its border. Transcriptional start sites tended to reside about one helical turn inside the nucleosome border. These findings reveal an intimate relationship between chromatin architecture and the underlying DNA sequence it regulates.

Simultaneous Assessment of Soil Microbial Community Structure and Function through Analysis of the Meta-Transcriptome

Background Soil ecosystems harbor the most complex prokaryotic and eukaryotic microbial communities on Earth. Experimental approaches studying these systems usually focus on either the soil communitys taxonomic structure or its functional characteristics. Many methods target DNA as marker molecule and use PCR for amplification. Methodology/Principal Findings Here we apply an RNA-centered meta-transcriptomic approach to simultaneously obtain information on both structure and function of a soil community. Total community RNA is random reversely transcribed into cDNA without any PCR or cloning step. Direct pyrosequencing produces large numbers of cDNA rRNA-tags; these are taxonomically profiled in a binning approach using the MEGAN software and two specifically compiled rRNA reference databases containing small and large subunit rRNA sequences. The pyrosequencing also produces mRNA-tags; these provide a sequence-based transcriptome of the community. One soil dataset of 258,411 RNA-tags of ∼98 bp length contained 193,219 rRNA-tags with valid taxonomic information, together with 21,133 mRNA-tags. Quantitative information about the relative abundance of organisms from all three domains of life and from different trophic levels was obtained in a single experiment. Less frequent taxa, such as soil Crenarchaeota, were well represented in the data set. These were identified by more than 2,000 rRNA-tags; furthermore, their activity in situ was revealed through the presence of mRNA-tags specific for enzymes involved in ammonia oxidation and CO2 fixation. Conclusions/Significance This approach could be widely applied in microbial ecology by efficiently linking community structure and function in a single experiment while avoiding biases inherent in other methods.

The genome of Theobroma cacao

We sequenced and assembled the draft genome of Theobroma cacao, an economically important tropical-fruit tree crop that is the source of chocolate. This assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of these genes anchored on the 10 T. cacao chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example, flavonoid-related genes. It also provides a major source of candidate genes for T. cacao improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten T. cacao chromosomes were shaped from an ancestor through eleven chromosome fusions.

Genome 10K: A Proposal to Obtain Whole-Genome Sequence for 10 000 Vertebrate Species

The human genome project has been recently complemented by whole-genome assessment sequence of 32 mammals and 24 nonmammalian vertebrate species suitable for comparative genomic analyses. Here we anticipate a precipitous drop in costs and increase in sequencing efficiency, with concomitant development of improved annotation technology and, therefore, propose to create a collection of tissue and DNA specimens for 10,000 vertebrate species specifically designated for whole-genome sequencing in the very near future. For this purpose, we, the Genome 10K Community of Scientists (G10KCOS), will assemble and allocate a biospecimen collection of some 16,203 representative vertebrate species spanning evolutionary diversity across living mammals, birds, nonavian reptiles, amphibians, and fishes (ca. 60,000 living species). In this proposal, we present precise counts for these 16,203 individual species with specimens presently tagged and stipulated for DNA sequencing by the G10KCOS. DNA sequencing has ushered in a new era of investigation in the biological sciences, allowing us to embark for the first time on a truly comprehensive study of vertebrate evolution, the results of which will touch nearly every aspect of vertebrate biological enquiry.