The complete sequences of Takifugu Toll-like receptor (TLR) loci and gene predictions from many draft genomes enable comprehensive molecular phylogenetic analysis. Strong selective pressure for recognition of and response to pathogen-associated molecular patterns has maintained a largely unchanging TLR recognition in all vertebrates. There are six major families of vertebrate TLRs. This repertoire is distinct from that of invertebrates. TLRs within a family recognize a general class of pathogen-associated molecular patterns. Most vertebrates have exactly one gene ortholog for each TLR family. The family including TLR1 has more speciesspecific adaptations than other families. A major family including TLR11 is represented in humans only by a pseudogene. Coincidental evolution plays a minor role in TLR evolution. The sequencing phase of this study produced finished genomic sequences for the 12 Takifugu rubripes TLRs. In addition, we have produced >70 gene models, including sequences from the opossum, chicken, frog, dog, sea urchin, and sea squirt. coincidental evolution ͉ multigene family ͉ concerted evolution T he Toll-like receptor (TLR) multigene family encodes important recognition receptors of the innate immune system that have been conserved in both the invertebrate and vertebrate lineages (1, 2). TLRs recognize a variety of endogenous and exogenous ligands; many of the latter are conserved molecules essential for pathogen survival. TLR genes have been recognized in a number of vertebrate genomes, and many partial and full-length sequences are available. Recent additions include draft predictions from the Japanese pufferfish Takifugu rubripes (3), the zebrafish Danio rerio (4-6), and the chicken Gallus gallus (7), and partially or fully sequenced mRNAs, including one from the goldfish Carassius auratus (8), several from the Japanese flounder Paralichthys olivaceus (9), and several from the rainbow trout Oncorhynchus mykiss (10). These papers provide incremental molecular phylogenetic analyses, and several reviews are available (11-13). Additionally, the draft genomes of the frog Xenopus tropicalis, chicken G. gallus, and opossum Monodelphis domesticus are now available. We present a complete molecular phylogenetic analysis of the known vertebrate TLR genes in the context of the complete genomic sequences of the T. rubripes TLRs. MethodsSequencing and Assembly. A draft genome sequence of T. rubripes was obtained by pairwise shotgun sequencing (14) through the efforts of an international collaboration (15). Sequence finishing was performed in part as described (16), with additional details provided in Supporting Text, which is published as supporting information on the PNAS web site.Bioinformatics. TLRs were identified as genes coding for both an N-terminal leucine-rich repeat (LRR) domain and a C-terminal Toll-IL-resistance (TIR) domain. To form the basis of our study, vertebrate sequences from the nonredundant DDBJ͞EMBL͞ NCBI database (GenBank) were identified by similarity to known TLRs (Data Set 1, which is...

The environment significantly influences the dynamic expression and assembly of all components encoded in the genome of an organism into functional biological networks. We have constructed a model for this process in Halobacterium salinarum NRC-1 through the data-driven discovery of regulatory and functional interrelationships among approximately 80% of its genes and key abiotic factors in its hypersaline environment. Using relative changes in 72 transcription factors and 9 environmental factors (EFs) this model accurately predicts dynamic transcriptional responses of all these genes in 147 newly collected experiments representing completely novel genetic backgrounds and environments-suggesting a remarkable degree of network completeness. Using this model we have constructed and tested hypotheses critical to this organism's interaction with its changing hypersaline environment. This study supports the claim that the high degree of connectivity within biological and EF networks will enable the construction of similar models for any organism from relatively modest numbers of experiments.

Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals, we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sublethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II), and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large-scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up-regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down-regulation of Mn(II) uptake and up-regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all of these discoveries into a unified systems-level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals, we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms.[Supplemental material is available online at www.genome.org. The microarray data from this study have been submitted to GEO under accession nos. GSM109343-GSM109461 and GSM109514-GSM109522.]Transition metals such as manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni) copper (Cu), and zinc (Zn) are essential cofactors in the physiology of all organisms. In fact, recent estimates suggest that over half of all proteins in every organism are metalloproteins (Degtyarenko 2000). Although essential in trace amounts, at higher levels these metals can be toxic to cells because they directly or indirectly compromise DNA, protein, and membrane integrity and function. For example, cycling in redox states of metals such as Fe and Cu and antioxidant scavenging by redoxinactive metals such as Zn can both cause oxidative damage to cell components through increased production of reactive oxygen species (ROS) (Nelson 1999). Organisms usually avoid metal toxicity through selective uptake, trafficking, and efflux of metal ions, enzymatic conversion of metals into non-or less-toxic redox states, or sequestering toxic metal ions with ferritins and metallothioneins (Silver 1992;Blindauer et al. 2002;Reindel et al. 2002;Zeth et al. 2004). These mechanisms are believed to be often regulated by free metal-ion concentration (Raab and Feldman 2003). In this regard, other factors such as salinity,...