Interspecific comparisons of protein sequences can reveal regions of evolutionary conservation that are under purifying selection because of functional constraints. Interpreting these constraints requires combining evolutionary information with structural, biochemical, and physiological data to understand the biological function of conserved regions. We take this integrative approach to investigate the evolution and function of the nuclear-encoded subunits of cytochrome c oxidase (COX). We find that the nuclear-encoded subunits evolved subsequent to the origin of mitochondria and the subunit composition of the holoenzyme varies across diverse taxa that include animals, yeasts, and plants. By mapping conserved amino acids onto the crystal structure of bovine COX, we show that conserved residues are structurally organized into functional domains. These domains correspond to some known functional sites as well as to other uncharacterized regions. We find that amino acids that are important for structural stability are conserved at frequencies higher than expected within each taxon, and groups of conserved residues cluster together at distances of less than 5 A more frequently than do randomly selected residues. We, therefore, suggest that selection is acting to maintain the structural foundation of COX across taxa, whereas active sites vary or coevolve within lineages.

Current thinking about long-term memory in the cortex is focused on changes in the strengths of connections between neurons. But ongoing structural plasticity in the adult brain, including synapse formation/elimination and remodelling of axons and dendrites, suggests that memory could also depend on learning-induced changes in the cortical 'wiring diagram'. Given that the cortex is sparsely connected, wiring plasticity could provide a substantial boost in storage capacity, although at a cost of more elaborate biological machinery and slower learning.

The evolution of the ancestral eukaryotic flagellum is an example of a cellular organelle that became dispensable in some modern eukaryotes while remaining an essential motile and sensory apparatus in others. To help define the repertoire of specialized proteins needed for the formation and function of cilia, we used comparative genomics to analyze the genomes of organisms with prototypical cilia, modified cilia, or no cilia and identified approximately 200 genes that are absent in the genomes of nonciliated eukaryotes but are conserved in ciliated organisms. Importantly, over 80% of the known ancestral proteins involved in cilia function are included in this small collection. Using Drosophila as a model system, we then characterized a novel family of proteins (OSEGs: outer segment) essential for ciliogenesis. We show that osegs encode components of a specialized transport pathway unique to the cilia compartment and are related to prototypical intracellular transport proteins.

In Drosophila most thoracic neuroblasts have two neurogenic periods: an initial brief period during embryogenesis and a second prolonged phase during larval growth. This study focuses on the adult-specific neurons that are born primarily during the second phase of neurogenesis. The fasciculated neurites arising from each cluster of adult-specific neurons express the cell-adhesion protein Neurotactin and they make a complex scaffold of neurite bundles within the thoracic neuropils. Using MARCM clones, we identified the 24 lineages that make up the scaffold of a thoracic hemineuromere. Unlike the early-born neurons that are strikingly diverse in both form and function, the adult specific cells in a given lineage are remarkably similar and typically project to only one or two initial targets, which appear to be the bundled neurites from other lineages. Correlated changes in the contacts between the lineages in different segments suggest that these initial contacts have functional significance in terms of future synaptic partners. This paper provides an overall view of the initial connections that eventually lead to the complex connectivity of the bulk of the thoracic neurons.

MHR3 is an ecdysone-inducible transcription factor whose expression in both Manduca sexta epidermis and the Manduca GV1 cell line is induced by 20-hydroxyecdysone (20E) in vitro. There are four putative ecdysone response elements (EcRE) in the 2.6-kb flanking region of the MHR3 promoter. The most proximal, EcRE1, is necessary for activation of the promoter by 20E in the GV1 cells because the mutation of EcRE1 caused the loss of responsiveness to 20E. Previous studies showed that EcR-B1/USP-1 bound only to EcRE1 and high levels of this complex increased the 20E-induced activation, whereas the presence of high USP-2 prevented this increased activation. When we expressed EcR-A alone or in combination with USP-1 under the control of Autographa californica baculovirus promoter (pIE1hr), the activation of the 2.6-kb promoter by 20E was reduced by about 50%. Moreover, when EcR-A was expressed together with both EcR-B1 and USP-1, it reduced the normal activation caused by EcR-B1 and USP-1 by 50%. Gel mobility shift assays showed no binding of EcR-A/USP-1 to EcRE1. The presence of EcR-A, however, reduced the binding of EcR-B1/USP-1 by about 50%. These findings suggest that EcR-A competes with EcR-B1 for binding of USP-1, leading to a decline in activity of the promoter. In addition, E75A, another ecdysone-induced transcription factor, and MHR3 itself suppressed MHR3 promoter activity by binding to the monomeric response element (MRE2). Therefore, MHR3 can be down-regulated both by itself and by E75A.

The human CRSP-Med coactivator complex is targeted by a diverse array of sequence-specific regulatory proteins. Using EM and single-particle reconstruction techniques, we recently completed a structural analysis of CRSP-Med bound to VP16 and SREBP-1a. Notably, these activators induced distinct conformational states upon binding the coactivator. Ostensibly, these different conformational states result from VP16 and SREBP-1a targeting distinct subunits in the CRSP-Med complex. To test this, we conducted a structural analysis of CRSP-Med bound to either thyroid hormone receptor (TR) or vitamin D receptor (VDR), both of which interact with the same subunit (Med220) of CRSP-Med. Structural comparison of TR- and VDR-bound complexes (at a resolution of 29 A) indeed reveals a shared conformational feature that is distinct from other known CRSP- Med structures. Importantly, this nuclear receptor-induced structural shift seems largely dependent on the movement of Med220 within the complex.

The goal of our study was to examine whether the in vivo force-length behavior, work and elastic energy savings of distal muscle-tendon units in the legs of tammar wallabies (Macropus eugenii) change during level versus incline hopping. To address this question, we obtained measurements of muscle activation (via electromyography), fascicle strain (via sonomicrometry) and muscle-tendon force (via tendon buckles) from the lateral gastrocnemius (LG) and plantaris (PL) muscles of tammar wallabies trained to hop on a level and an inclined (10 degrees, 17.4% grade) treadmill at two speeds (3.3 m s(-1) and 4.2 m s(-1)). Similar patterns of muscle activation, force and fascicle strain were observed under both level and incline conditions. This also corresponded to similar patterns of limb timing and movement (duty factor, limb contact time and hopping frequency). During both level and incline hopping, the LG and PL exhibited patterns of fascicle stretch and shortening that yielded low levels of net fascicle strain [LG: level, -1.0+/-4.6% (mean +/- S.E.M.) vs incline, 0.6+/-4.5%; PL: level, 0.1+/-1.0% vs incline, 0.4+/-1.6%] and muscle work (LG: level, -8.4+/-8.4 J kg(-1) muscle vs incline, -6.8+/-7.5 J kg(-1) muscle; PL: level, -2.0+/-0.6 J kg(-1) muscle vs incline, -1.4+/-0.7 J kg(-1) muscle). Consequently, neither muscle significantly altered its contractile dynamics to do more work during incline hopping. Whereas electromyographic (EMG) phase, duration and intensity did not differ for the LG, the PL exhibited shorter but more intense periods of activation, together with reduced EMG phase (P<0.01), during incline versus level hopping. Our results indicate that design for spring-like tendon energy savings and economical muscle force generation is key for these two distal muscle-tendon units of the tammar wallaby, and the need to accommodate changes in work associated with level versus incline locomotion is achieved by more proximal muscles of the limb.

The efficient silylation of alcohols with di- and trialkynylsilanes was achieved under base-catalyzed conditions to afford alkynyl silyl ethers and symmetrical alkynyl silaketals in good yield. A selective alcoholysis of dialkynyl silyl ethers to mixed silaketals was also demonstrated. These products served as substrates for enyne ring-closing metathesis and, consequently, as precursors to stereochemically defined 1,3-dienes.

Genetic transformation in insects holds great promise as a tool for genetic manipulation in species of particular scientific, economic, or medical interest. A number of transposable elements have been tested recently as potential vectors for transformation in a range of insects. Minos is one of the most promising elements because it appears to be active in diverse species and has the capacity to carry large inserts. We report here the use of the Minos element as a transformation vector in the red flour beetle Tribolium castaneum (Coleoptera), an important species for comparative developmental and pest management studies. Transgenic G(1) beetles were recovered from 32.4% of fertile G(0)’s injected with a plasmid carrying a 3xP3-EGFP-marked transposon and in vitro synthesized mRNA encoding the Minos transposase. This transformation efficiency is 2.8-fold higher than that observed when using a plasmid helper. Molecular and genetic analyses show that several independent insertions can be recovered from a single injected parent, but that the majority of transformed individuals carry single Minos insertions. These results establish Minos as one of the most efficient vectors for genetic transformation in insects. In combination with piggyBac-based transgenesis, our work allows the introduction of sophisticated multicomponent genetic tools in Tribolium.

Evolution perfected brain design by maximizing its functionality while minimizing costs associated with building and maintaining it. Assumption that brain functionality is specified by neuronal connectivity, implemented by costly biological wiring, leads to the following optimal design problem. For a given neuronal connectivity, find a spatial layout of neurons that minimizes the wiring cost. Unfortunately, this problem is difficult to solve because the number of possible layouts is often astronomically large. We argue that the wiring cost may scale as wire length squared, reducing the optimal layout problem to a constrained minimization of a quadratic form. For biologically plausible constraints, this problem has exact analytical solutions, which give reasonable approximations to actual layouts in the brain. These solutions make the inverse problem of inferring neuronal connectivity from neuronal layout more tractable.