Activation of gene transcription in mammalian cells requires several classes of coactivators that participate in different steps of the activation cascade. Using conventional and affinity chromatography, we have isolated a human coactivator complex that interacts directly with the C-terminal domain (CTD) of RNA polymerase II (Pol II). The CTD-binding complex is structurally and functionally indistinguishable from our previously isolated CRSP coactivator complex. The closely related, but transcriptionally inactive, ARC-L complex failed to interact with the CTD, indicating a significant biochemical difference between CRSP and ARC-L that may, in part, explain their functional divergence. Electron microscopy and three-dimensional single-particle reconstruction reveals a conformation for CTD-CRSP that is structurally distinct from unliganded CRSP or CRSP bound to SREBP-1a, but highly similar to CRSP bound to the VP16 activator. Together, our findings suggest that the human CRSP coactivator functions, at least in part, by mediating activator-dependent recruitment of RNA Pol II via the CTD.

The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a K(m) of 0.32 mM and a V(max) of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li(+) (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

The understanding of the molecular basis of the endocrine control of insect metamorphosis has been hampered by the profound differences in responses of the Lepidoptera and the Diptera to juvenile hormone (JH). In both Manduca and Drosophila, the broad (br) gene is expressed in the epidermis during the formation of the pupa, but not during adult differentiation. Misexpression of BR-Z1 during either a larval or an adult molt of Drosophila suppressed stage-specific cuticle genes and activated pupal cuticle genes, showing that br is a major specifier of the pupal stage. Treatment with a JH mimic at the onset of the adult molt causes br re-expression and the formation of a second pupal cuticle in Manduca, but only in the abdomen of Drosophila. Expression of the BR isoforms during adult development of Drosophila suppressed bristle and hair formation when induced early or redirected cuticle production toward the pupal program when induced late. Expression of BR-Z1 at both of these times mimicked the effect of JH application but, unlike JH, it caused production of a new pupal cuticle on the head and thorax as well as on the abdomen. Consequently, the ’status quo’ action of JH on the pupal-adult transformation is mediated by the JH-induced re-expression of BR.

Although cellular mitochondrial DNA (mtDNA) copy number varies widely among cell lines and tissues, little is known about the mechanism of mtDNA copy number control. Most nascent replication strands from the leading, heavy-strand origin (O(H)) are prematurely terminated, defining the 3’ boundary of the displacement loop (D-loop). We have depleted mouse LA9 cell mtDNA to approximately 20% of normal levels by treating with 2’,3’-dideoxycytidine (ddC) and subsequently allowed recovery to normal levels of mtDNA. A quantitative ligation-mediated PCR assay was used to determine the levels of both terminated and extended nascent O(H) strands during mtDNA depletion and repopulation. Depleting mtDNA leads to a release of replication termination until mtDNA copy number approaches a normal level. Detectable total nascent strands per mtDNA genome remain below normal. Therefore, it is likely that the level of replication termination plays a significant role in copy number regulation in this system. However, termination of D-loop strand synthesis is persistent, indicating formation of the D-loop structure has a purpose that is required under conditions of rapid recovery of depleted mtDNA.

Wiring a brain presents a formidable problem because neural circuits require an enormous number of fast and durable connections. We propose that evolution was likely to have optimized neural circuits to minimize conduction delays in axons, passive cable attenuation in dendrites, and the length of "wire" used to construct circuits, and to have maximized the density of synapses. Here we ask the question: "What fraction of the volume should be taken up by axons and dendrites (i.e., wire) when these variables are at their optimal values?" The biophysical properties of axons and dendrites dictate that wire should occupy 3/5 of the volume in an optimally wired gray matter. We have measured the fraction of the volume occupied by each cellular component and find that the volume of wire is close to the predicted optimal value.

The evolution of body form is believed to involve changes in expression of developmental genes, largely through changes in cis-regulatory elements. Recent studies suggest that changes in the sequences of key developmental regulators, such as the Hox proteins, may also play an important role.

The Roundabout (Robo) receptors have been intensively studied for their role in regulating axon guidance in the embryonic nervous system, whereas a role in dendritic guidance has not been explored. In the adult giant fiber system of Drosophila, we have revealed that ectopic Robo expression can regulate the growth and guidance of specific motor neuron dendrites, whereas Robo2 and Robo3 have no effect. We also show that the effect of Robo on dendritic guidance can be suppressed by Commissureless coexpression. Although we confirmed a role for all three Robo receptors in giant fiber axon guidance, the strong axon guidance alterations caused by overexpression of Robo2 or Robo3 have no effect on synaptic connectivity. In contrast, Robo overexpression in the giant fiber seems to directly interfere with synaptic function. We conclude that axon guidance, dendritic guidance, and synaptogenesis are separable processes and that the different Robo family members affect them distinctly.

Small molecules that alter protein function provide a means to modulate biological networks with temporal resolution. Here we demonstrate a potentially general and scalable method of identifying such molecules by application to a particular protein, Ure2p, which represses the transcription factors Gln3p and Nil1p. By probing a high-density microarray of small molecules generated by diversity-oriented synthesis with fluorescently labelled Ure2p, we performed 3,780 protein-binding assays in parallel and identified several compounds that bind Ure2p. One compound, which we call uretupamine, specifically activates a glucose-sensitive transcriptional pathway downstream of Ure2p. Whole-genome transcription profiling and chemical epistasis demonstrate the remarkable Ure2p specificity of uretupamine and its ability to modulate the glucose-sensitive subset of genes downstream of Ure2p. These results demonstrate that diversity-oriented synthesis and small-molecule microarrays can be used to identify small molecules that bind to a protein of interest, and that these small molecules can regulate specific functions of the protein.

Changes in synaptic connectivity patterns through the formation and elimination of dendritic spines may contribute to structural plasticity in the brain. We characterize this contribution quantitatively by estimating the number of different synaptic connectivity patterns attainable without major arbor remodeling. This number depends on the ratio of the synapses on a dendrite to the axons that pass within a spine length of that dendrite. We call this ratio the filling fraction and calculate it from geometrical analysis and anatomical data. The filling fraction is 0.26 in mouse neocortex, 0.22-0.34 in rat hippocampus. In the macaque visual cortex, the filling fraction increases by a factor of 1.6-1.8 from area V1 to areas V2, V4, and 7a. Since the filling fraction is much smaller than 1, spine remodeling can make a large contribution to structural plasticity.

The sense of taste provides animals with valuable information about the nature and quality of food. Mammals can recognize and respond to a diverse repertoire of chemical entities, including sugars, salts, acids and a wide range of toxic substances. Several amino acids taste sweet or delicious (umami) to humans, and are attractive to rodents and other animals. This is noteworthy because L-amino acids function as the building blocks of proteins, as biosynthetic precursors of many biologically relevant small molecules, and as metabolic fuel. Thus, having a taste pathway dedicated to their detection probably had significant evolutionary implications. Here we identify and characterize a mammalian amino-acid taste receptor. This receptor, T1R1+3, is a heteromer of the taste-specific T1R1 and T1R3 G-protein-coupled receptors. We demonstrate that T1R1 and T1R3 combine to function as a broadly tuned L-amino-acid sensor responding to most of the 20 standard amino acids, but not to their D-enantiomers or other compounds. We also show that sequence differences in T1R receptors within and between species (human and mouse) can significantly influence the selectivity and specificity of taste responses.