SMART, a simple modular architecture research tool: Identification of signaling domains

Accurate multiple alignments of 86 domains that occur in signaling proteins have been constructed and used to provide a Web-based tool (SMART: simple modular architecture research tool) that allows rapid identification and annotation of signaling domain sequences. The majority of signaling proteins are multidomain in character with a considerable variety of domain combinations known. Comparison with established databases showed that 25% of our domain set could not be deduced from SwissProt and 41% could not be annotated by Pfam. SMART is able to determine the modular architectures of single sequences or genomes; application to the entire yeast genome revealed that at least 6.7% of its genes contain one or more signaling domains, approximately 350 greater than previously annotated. The process of constructing SMART predicted (i) novel domain homologues in unexpected locations such as band 4.1-homologous domains in focal adhesion kinases; (ii) previously unknown domain families, including a citron-homology domain; (iii) putative functions of domain families after identification of additional family members, for example, a ubiquitin-binding role for ubiquitin-associated domains (UBA); (iv) cellular roles for proteins, such predicted DEATH domains in netrin receptors further implicating these molecules in axonal guidance; (v) signaling domains in known disease genes such as SPRY domains in both marenostrin/pyrin and Midline 1; (vi) domains in unexpected phylogenetic contexts such as diacylglycerol kinase homologues in yeast and bacteria; and (vii) likely protein misclassifications exemplified by a predicted pleckstrin homology domain in a Candida albicans protein, previously described as an integrin.

The TIGR Gene Indices: analysis of gene transcript sequences in highly sampled eukaryotic species

While genome sequencing projects are advancing rapidly, EST sequencing and analysis remains a primary research tool for the identification and categorization of gene sequences in a wide variety of species and an important resource for annotation of genomic sequence. The TIGR Gene Indices (http://www.tigr.org/tdb/tgi.shtml) are a collection of species-specific databases that use a highly refined protocol to analyze EST sequences in an attempt to identify the genes represented by that data and to provide additional information regarding those genes. Gene Indices are constructed by first clustering, then assembling EST and annotated gene sequences from GenBank for the targeted species. This process produces a set of unique, high-fidelity virtual transcripts, or Tentative Consensus (TC) sequences. The TC sequences can be used to provide putative genes with functional annotation, to link the transcripts to mapping and genomic sequence data, to provide links between orthologous and paralogous genes and as a resource for comparative sequence analysis.

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety.

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

Piperonylic Acid, a Selective, Mechanism-Based Inactivator of the trans-Cinnamate 4-Hydroxylase: A New Tool to Control the Flux of Metabolites in the Phenylpropanoid Pathway1

Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy function that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway. We show that when incubated in vitro with yeast-expressed CYP73A1, PA behaves as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase. Inactivation requires NADPH, is time dependent and saturable (KI = 17 μm, kinact = 0.064 min−1), and results from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex is reversible with substrate or other strong ligands of the enzyme. In plant microsomes PA seems to selectively inactivate the CYP73A P450 subpopulation. It does not form detectable complexes with other recombinant plant P450 enzymes. In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicited tobacco (Nicotiana tabacum) cell suspension. It also strongly decreases the formation of scopoletin in tobacco leaves infected with tobacco mosaic virus.

Photodissociation of a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)-cobalt(III)] complex: a tool to study fast biological reactions involving O2.

Upon photolysis at 355 nm, dioxygen is released from a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)cobalt-(III)] complex in aqueous solutions and at physiological pH with a quantum yield of 0.04. The [Co(bpy)2(H2O)2]2+ (bpy = bipyridyl) photoproduct was generated on a nanosecond or faster time scale as determined by time-resolved optical absorption spectroscopy. A linear correspondence between the spectral changes and the oxygen production indicates that O2 is released on the same time scale. Oxyhemoglobin was formed from deoxyhemoglobin upon photodissociation of the (mu-peroxo) (mu-hydroxo)bis[bis(bipyridyl)cobalt(III)] complex, verifying that dioxygen is a primary photoproduct. This complex and other related compounds provide a method to study fast biological reactions involving O2, such as the reduction of dioxygen to water by cytochrome oxidase.