Homology-based annotation yields 1,042 new candidate genes in the Drosophila melanogaster genome.

The approach to annotating a genome critically affects the number and accuracy of genes identified in the genome sequence. Genome annotation based on stringent gene identification is prone to underestimate the complement of genes encoded in a genome. In contrast, over-prediction of putative genes followed by exhaustive computational sequence, motif and structural homology search will find rarely expressed, possibly unique, new genes at the risk of including non-functional genes. We developed a two-stage approach that combines the merits of stringent genome annotation with the benefits of over-prediction. First we identify plausible genes regardless of matches with EST, cDNA or protein sequences from the organism (stage 1). In the second stage, proteins predicted from the plausible genes are compared at the protein level with EST, cDNA and protein sequences, and protein structures from other organisms (stage 2). Remote but biologically meaningful protein sequence or structure homologies provide supporting evidence for genuine genes. The method, applied to the Drosophila melanogaster genome, validated 1,042 novel candidate genes after filtering 19,410 plausible genes, of which 12,124 matched the original 13,601 annotated genes. This annotation strategy is applicable to genomes of all organisms, including human.

Structural genomics: beyond the human genome project.

With access to whole genome sequences for various organisms and imminent completion of the Human Genome Project, the entire process of discovery in molecular and cellular biology is poised to change. Massively parallel measurement strategies promise to revolutionize how we study and ultimately understand the complex biochemical circuitry responsible for controlling normal development, physiologic homeostasis and disease processes. This information explosion is also providing the foundation for an important new initiative in structural biology. We are about to embark on a program of high-throughput X-ray crystallography aimed at developing a comprehensive mechanistic understanding of normal and abnormal human and microbial physiology at the molecular level. We present the rationale for creation of a structural genomics initiative, recount the efforts of ongoing structural genomics pilot studies, and detail the lofty goals, technical challenges and pitfalls facing structural biologists.

Minimum information about a microarray experiment (MIAME)-toward standards for microarray data.

Microarray analysis has become a widely used tool for the generation of gene expression data on a genomic scale. Although many significant results have been derived from microarray studies, one limitation has been the lack of standards for presenting and exchanging such data. Here we present a proposal, the Minimum Information About a Microarray Experiment (MIAME), that describes the minimum information required to ensure that microarray data can be easily interpreted and that results derived from its analysis can be independently verified. The ultimate goal of this work is to establish a standard for recording and reporting microarray-based gene expression data, which will in turn facilitate the establishment of databases and public repositories and enable the development of data analysis tools. With respect to MIAME, we concentrate on defining the content and structure of the necessary information rather than the technical format for capturing it.

Reprogramming of the macrophage transcriptome in response to interferon-gamma and Mycobacterium tuberculosis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase.

Macrophage activation determines the outcome of infection by Mycobacterium tuberculosis (Mtb). Interferon-gamma (IFN-gamma) activates macrophages by driving Janus tyrosine kinase (JAK)/signal transducer and activator of transcription-dependent induction of transcription and PKR-dependent suppression of translation. Microarray-based experiments reported here enlarge this picture. Exposure to IFN-gamma and/or Mtb led to altered expression of 25% of the monitored genome in macrophages. The number of genes suppressed by IFN-gamma exceeded the number of genes induced, and much of the suppression was transcriptional. Five times as many genes related to immunity and inflammation were induced than suppressed. Mtb mimicked or synergized with IFN-gamma more than antagonized its actions. Phagocytosis of nonviable Mtb or polystyrene beads affected many genes, but the transcriptional signature of macrophages infected with viable Mtb was distinct. Studies involving macrophages deficient in inducible nitric oxide synthase and/or phagocyte oxidase revealed that these two antimicrobial enzymes help orchestrate the profound transcriptional remodeling that underlies macrophage activation.

Whole-genome analysis: annotations and updates.

The most important advances in the field of genome annotation over the past two years involve the use of cDNA sequences, protein structures and gene expression data to predict genes. These types of information not only improve gene identification, but they also give insights into variation in gene structure and function.

Archaeal genomics.

Four euryarchaeal genomes have been completely sequenced and are publicly available: Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Pyrococcus horikoshii and Archaeoglobus fulgidus. Four more genome sequences, two crenarchaeal and two pyrococci, will soon be released. In addition, seven more archaeal genome sequencing projects are under way, including two halophiles, two Thermoplasma, and a methanogen. These projects cover all branches of the archaeal domain and will lead to new insights into archaeal metabolism, DNA processing, and evolutionary relationships with the Bacteria and Eukarya.

The Sulfolobus solfataricus P2 genome project.

Over 800 kbp of the 3-Mbp genome of Sulfolobus solfataricus have been sequenced to date. Our approach is to sequence subclones of mapped cosmids, followed by sequencing directly on cosmid templates with custom primers. Using a prototype automated system for genome-scale analysis, known as MAGPIE, along with other tools, we have discovered one open reading frame of at least 100 amino acids per kbp of sequence, and have been able to associate 50% of these with known genes through database searches. An examination of completely sequenced cosmids suggests a clustering of genes by function in the S. solfataricus genome.

Fully automated genome analysis that reflects user needs and preferences. A detailed introduction to the MAGPIE system architecture.

A system called MAGPIE (Multipurpose Automated Genome Project Investigation Environment) has been designed and implemented to meet the challenges of automated whole genome analysis. The system initiates large numbers of remote and local transactions, each depending on evolving criteria and on changing remote and local conditions. Transactions are requested from different types of remote and local resources. The remote request load is fairly balanced with other community demands on server resources. Local decision modules monitor and obey user preferences and combine evidence from multiple sources to formulate credible hypotheses about sequence function. Consistency checks from multiple types of data are integrated into the ongoing local analysis. The system performs reliably on local UNIX workstations and communicates with remote resources through standard networking protocols.

Characterizing the relationship between protein-fusion and gene co-expression.

A pair of distinct proteins in one organism may most closely match different parts of the same protein in another organism. A comparison of all proteins from the genome of Saccharomyces cerevisiae and all proteins from 24 prokaryotic genomes yields 1010 pairs of yeast proteins whose homologs are parts of one protein from a prokaryotic genome. Marcotte et al. (Science 285:751-3) showed that proteins related in this manner are more likely to interact than proteins chosen at random. In this paper, we investigated whether genes coding for such proteins are also likely to be concurrently transcribed. We identified 1010 fused pairs of proteins encoded in the yeast genome and analyzed expression of the corresponding genes at the transcriptional level. We found that the transcriptional profiles of fused gene pairs are significantly closer than those of randomly selected pairs. This finding is reproducible and established by multiple distance metrics. Moreover, such pairs frequently share additional biologically relevant properties. Thus, while protein fusion patterns are not predictive of co-expression, they are an important element in explaining co-expression. This justifies the use of curated protein fusion events to help characterize gene co-expression clusters.

Vascular tone pathway polymorphisms in relation to primary open-angle glaucoma.

AIMS: Vascular perfusion may be impaired in primary open-angle glaucoma (POAG); thus, we evaluated a panel of markers in vascular tone-regulating genes in relation to POAG. METHODS: We used Illumina 660W-Quad array genotype data and pooled P-values from 3108 POAG cases and 3430 controls from the combined National Eye Institute Glaucoma Human Genetics Collaboration consortium and Glaucoma Genes and Environment studies. Using information from previous literature and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we compiled single-nucleotide polymorphisms (SNPs) in 186 vascular tone-regulating genes. We used the 'Pathway Analysis by Randomization Incorporating Structure' analysis software, which performed 1000 permutations to compare the overall pathway and selected genes with comparable randomly generated pathways and genes in their association with POAG. RESULTS: The vascular tone pathway was not associated with POAG overall or POAG subtypes, defined by the type of visual field loss (early paracentral loss (n=224 cases) or only peripheral loss (n=993 cases)) (permuted P≥0.20). In gene-based analyses, eight were associated with POAG overall at permuted P<0.001: PRKAA1, CAV1, ITPR3, EDNRB, GNB2, DNM2, HFE, and MYL9. Notably, six of these eight (the first six listed) code for factors involved in the endothelial nitric oxide synthase activity, and three of these six (CAV1, ITPR3, and EDNRB) were also associated with early paracentral loss at P<0.001, whereas none of the six genes reached P<0.001 for peripheral loss only. DISCUSSION: Although the assembled vascular tone SNP set was not associated with POAG, genes that code for local factors involved in setting vascular tone were associated with POAG.

Reconstruction of metabolic networks using incomplete information.

This paper describes an approach that uses methods for automated sequence analysis (Gaasterland et al. August 1994) and multiple databases accessed through an object+attribute view of the data (Baehr et al. 1992), together with metabolic pathways, reaction equations, and compounds parsed into a logical representation from the Enzyme and Metabolic Pathway Database (Selkov, Yunus, & et.al. 1994), as the sources of data for automatically reconstructing a weighted partial metabolic network for a prokaryotic organism. Additional information can be provided interactively by the expert user to guide reconstruction.

Constructing multigenome views of whole microbial genomes.

We have designed and implemented a system to carry out cross-genome comparisons of open reading frames (ORFs) from multiple genomes. This implementation includes a genome profiling system that allows us to explore pairwise comparisons at different levels of match similarity and ask biologically motivated queries involving number and identity of ORFs, their function, functional category, distribution in genomes or in biological domains, and statistics on their matches and match families. This analysis required precise definition of new classification terms and concepts. We define the terms genomic signature, summary signature, biologic domain signature, domain class, match level, match family, and extended match family, then use these terms to define concepts, including genomically universal proteins and proteins characteristics of sets of genomes. We initiate an analysis based on automated FASTA (Pearson, 1996) comparison of 22,419 conceptually translated protein sequences from nine microbial genomes.

Microbial genescapes: phyletic and functional patterns of ORF distribution among prokaryotes.

We have implemented a statistically based approach to comparative genomics that allows us to define and characterize distributional patterns of conceptually translated open reading frames (ORFs) at different confidence levels based on pairwise FASTA matches. In this report, we apply this methodology to nine microbial genomes, focusing particularly on phyletic and functional patterns of ORF distribution within and between the two prokaryotic domains of life, Bacteria and Archaea. We examine patterns of presence and absence of matches, determine the universal ORF set, analyze features of genome specialization between closely related organisms, and present genomic evidence for the monophyly of Archaea. These analyses illustrate how a quantitative approach to comparative genomics can illuminate questions of fundamental biological significance.

Microbial genescapes: a prokaryotic view of the yeast genome.

We examine the translated open reading frames (ORFs) of the yeast Saccharomyces cerevisiae, focusing on those that have FASTA matches in phyletically defined sets of completely sequenced genomes. On this basis, we identify archaeal yeast, bacterial yeast, universal yeast, and yeast ORFs that do not have a match in any of nine prokaryote genomes. Similarly, we examine the yeast mitochondrial genome and the subset of the yeast nuclear ORFs identified as being involved in mitochondrial biogenesis. For the yeast ORFs that match one or more ORFs in these prokaryote genomes, we examine the phyletic and functional distributions of these matches as a function of match strength. These results provide genome level insights into the origin of the eukaryotic cell and the origin of mitochondria. More generally, they exemplify how the growing database of prokaryote genome sequences can help us understand eukaryote genomes.

Assigning function to CDS through qualified query answering: beyond alignment and motifs.

In this paper, we show how to use qualitative query answering to annotate CDS-to-function relationships with confidence in the score, confidence in the tool, and confidence in the decision about the function. The system, implemented in Prolog, provides users with a powerful tool to analyze large quantities of data that have been produce by multiple sequence analysis programs. Using qualified query answering techniques, users can easily change the criteria for how tools reinforce each other and for how numbers of occurrences of particular functions reinforce each other. They can also alter how different scores for different tools are categorized.

Presence of prokaryotic and eukaryotic species in all subgroups of the PP(i)-dependent group II phosphofructokinase protein family.

Inorganic pyrophosphate-dependent phosphofructokinase (PP(i)-PFK) of the amitochondriate eukaryote Mastigamoeba balamuthi was sequenced and showed about 60% identity to PP(i)-PFKs from two eubacteria, Propionibacterium freudenreichii and Sinorhizobium meliloti. These gene products represent a newly recognized lineage of PFKs. All four lineages of group II PFKs, as defined by phylogenetic analysis, contained both prokaryotic and eukaryotic species, underlining the complex evolutionary history of this enzyme.