Arrayed transposase-binding sequences on the ends of transposon Tn5090/Tn402

The transposon Tn5090/Tn402 encodes a 559 amino acid transposase, TniA, with a DDE motif. Gel mobility shifting and cleavage protection analysis with DNase I and hydroxyl radical probes revealed that TniA binds to multiple repeat sequences on either terminus of Tn5090/Tn402. Four of these TniA-binding 19mers occurred on the left-hand (t) end and two on the right-hand (i) end. Hydroxyl radical cleavage protection demonstrated the presence of 3–6 bp contact sequences on one face of the DNA helix. The binding pattern and organisation of repeats suggested parallels between Tn5090/Tn402 and Mu, which controls its transpositional activity in the assembly step of a higher order transpososome complex. The complex terminal structure and genes of transposase and nucleotide-binding proteins in tandem are hallmarks of the handful of Mu-like elements that are known to date.

Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues

p13suc1 has two native states, a monomer and a domain-swapped dimer. We show that their folding pathways are connected by the denatured state, which introduces a kinetic barrier between monomer and dimer under native conditions. The barrier is lowered under conditions that speed up unfolding, thereby allowing, to our knowledge for the first time, a quantitative dissection of the energetics of domain swapping. The monomer–dimer equilibrium is controlled by two conserved prolines in the hinge loop that connects the exchanging domains. These two residues exploit backbone strain to specifically direct dimer formation while preventing higher-order oligomerization. Thus, the loop acts as a loaded molecular spring that releases tension in the monomer by adopting its alternative conformation in the dimer. There is an excellent correlation between domain swapping and aggregation, suggesting they share a common mechanism. These insights have allowed us to redesign the domain-swapping propensity of suc1 from a fully monomeric to a fully dimeric protein.

Spatially independent activity patterns in functional MRI data during the Stroop color-naming task

A method is given for determining the time course and spatial extent of consistently and transiently task-related activations from other physiological and artifactual components that contribute to functional MRI (fMRI) recordings. Independent component analysis (ICA) was used to analyze two fMRI data sets from a subject performing 6-min trials composed of alternating 40-sec Stroop color-naming and control task blocks. Each component consisted of a fixed three-dimensional spatial distribution of brain voxel values (a “map”) and an associated time course of activation. For each trial, the algorithm detected, without a priori knowledge of their spatial or temporal structure, one consistently task-related component activated during each Stroop task block, plus several transiently task-related components activated at the onset of one or two of the Stroop task blocks only. Activation patterns occurring during only part of the fMRI trial are not observed with other techniques, because their time courses cannot easily be known in advance. Other ICA components were related to physiological pulsations, head movements, or machine noise. By using higher-order statistics to specify stricter criteria for spatial independence between component maps, ICA produced improved estimates of the temporal and spatial extent of task-related activation in our data compared with principal component analysis (PCA). ICA appears to be a promising tool for exploratory analysis of fMRI data, particularly when the time courses of activation are not known in advance.

Functional neuroimaging studies of encoding, priming, and explicit memory retrieval

Human functional neuroimaging techniques provide a powerful means of linking neural level descriptions of brain function and cognition. The exploration of the functional anatomy underlying human memory comprises a prime example. Three highly reliable findings linking memory-related cognitive processes to brain activity are discussed. First, priming is accompanied by reductions in the amount of neural activation relative to naive or unprimed task performance. These reductions can be shown to be both anatomically and functionally specific and are found for both perceptual and conceptual task components. Second, verbal encoding, allowing subsequent conscious retrieval, is associated with activation of higher order brain regions including areas within the left inferior and dorsal prefrontal cortex. These areas also are activated by working memory and effortful word generation tasks, suggesting that these tasks, often discussed as separable, might rely on interdependent processes. Finally, explicit (intentional) retrieval shares much of the same functional anatomy as the encoding and word generation tasks but is associated with the recruitment of additional brain areas, including the anterior prefrontal cortex (right > left). These findings illustrate how neuroimaging techniques can be used to study memory processes and can both complement and extend data derived through other means. More recently developed methods, such as event-related functional MRI, will continue this progress and may provide additional new directions for research.

ATP bound to the origin recognition complex is important for preRC formation

The origin recognition complex (ORC) binds origins of replication and directs the assembly of a higher order protein complex at these sites. ORC binds and hydrolyzes ATP in vitro. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA. These mutants inhibit formation of the prereplicative complex when overexpressed. The dominant lethal phenotype of these mutant ORC complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the origin. Based on these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly.

Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30°C but not 20°C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G1, consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.

Sir3-dependent assembly of supramolecular chromatin structures in vitro

Baculovirus-expressed recombinant Sir3p (rSir3p) has been purified to near homogeneity, and its binding to naked DNA, mononucleosomes, and nucleosomal arrays has been characterized in vitro. At stoichiometric levels rSir3p interacts with intact nucleosomal arrays, mononucleosomes, and naked DNA, as evidenced by formation of supershifted species on native agarose gels. Proteolytic removal of the core histone tail domains inhibits but does not completely abolish rSir3p binding to nucleosomal arrays. The linker DNA in the supershifted complexes remains freely accessible to restriction endonuclease digestion, suggesting that both the tail domains and nucleosomal DNA contribute to rSir3p–chromatin interactions. Together these data indicate that rSir3p cross-links individual nucleosomal arrays into supramolecular assemblies whose physical properties transcend those of typical 10-nm and 30-nm fibers. Based on these data we hypothesize that Sir3p functions, at least in part, by mediating reorganization of the canonical chromatin fiber into functionally specialized higher order chromosomal domains.

Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome.

Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome.

Genetic and molecular analysis of the gypsy chromatin insulator of Drosophila.

Boundary or insulator elements set up independent territories of gene activity by establishing higher order domains of chromatin structure. The gypsy retrotransposon of Drosophila contains an insulator element that represses enhancer-promoter interactions and is responsible for the mutant phenotypes caused by insertion of this element. The gypsy insulator inhibits the interaction of promoter-distal enhancers with the transcription complex without affecting the functionality of promoter-proximal enhancers; in addition, these sequences can buffer a transgene from chromosomal position effects. Two proteins have been identified that bind gypsy insulator sequences and are responsible for their effects on transcription. The suppressor of Hairy-wing [su(Hw)] protein affects enhancer function both upstream and downstream of its binding site by causing a silencing effect similar to that of heterochromatin. The modifier of mdg4 [mod(mdg4)] protein interacts with su(Hw) to transform this bi-directional repression into the polar effect characteristic of insulators. These effects seem to be modulated by changes in chromatin structure.

The temporal lobe is a target of output from the basal ganglia.

The basal ganglia are known to receive inputs from widespread regions of the cerebral cortex, such as the frontal, parietal, and temporal lobes. Of these cortical areas, only the frontal lobe is thought to be the target of basal ganglia output. One of the cortical regions that is a source of input to the basal ganglia is area TE, in inferotemporal cortex. This cortical area is thought to be critically involved in the recognition and discrimination of visual objects. Using retrograde transneuronal transport of herpes simplex virus type 1, we have found that one of the output nuclei of the basal ganglia, the substantia nigra pars reticulata, projects via the thalamus to TE. Thus, TE is not only a source of input to the basal ganglia, but also is a target of basal ganglia output. This result implies that the output of the basal ganglia influences higher order aspects of visual processing. In addition, we propose that dysfunction of the basal ganglia loop with TE leads to alterations in visual perception, including visual hallucinations.

Structural analysis of p185c-neu and epidermal growth factor receptor tyrosine kinases: oligomerization of kinase domains.

The epidermal growth factor receptor (EGFR) and p185c-neu proteins associate as dimers to create an efficient signaling assembly. Overexpression of these receptors together enhances their intrinsic kinase activity and concomitantly results in oncogenic cellular transformation. The ectodomain is able to stabilize the dimer, whereas the kinase domain mediates biological activity. Here we analyze potential interactions of the cytoplasmic kinase domains of the EGFR and p185c-neu tyrosine kinases by homology molecular modeling. This analysis indicates that kinase domains can associate as dimers and, based on intermolecular interaction calculations, that heterodimer formation is favored over homodimers. The study also predicts that the self-autophosphorylation sites located within the kinase domains are not likely to interfere with tyrosine kinase activity, but may regulate the selection of substrates, thereby modulating signal transduction. In addition, the models suggest that the kinase domains of EGFR and p185c-neu can undergo higher order aggregation such as the formation of tetramers. Formation of tetrameric complexes may explain some of the experimentally observed features of their ligand affinity and hetero-receptor internalization.

Microsatellite spreading in the human genome: evolutionary mechanisms and structural implications.

Microsatellites are tandem repeat sequences abundant in the genomes of higher eukaryotes and hitherto considered as "junk DNA." Analysis of a human genome representative data base (2.84 Mb) reveals a distinct juxtaposition of A-rich microsatellites and retroposons and suggests their coevolution. The analysis implies that most microsatellites were generated by a 3'-extension of retrotranscripts, similar to mRNA polyadenylylation, and that they serve in turn as "retroposition navigators," directing the retroposons via homology-driven integration into defined sites. Thus, they became instrumental in the preservation and extension of primordial genomic patterns. A role is assigned to these reiterating A-rich loci in the higher-order organization of the chromatin. The disease-associated triplet repeats are mostly found in coding regions and do not show an association with retroposons, constituting a unique set within the family of microsatellite sequences.

In vitro motility from recombinant dynein heavy chain.

The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain.

Glutamate receptor blockade at cortical synapses disrupts development of thalamocortical and columnar organization in somatosensory cortex.

The segregation of thalamocortical inputs into eye-specific stripes in the developing cat or monkey visual cortex is prevented by manipulations that perturb or abolish neural activity in the visual pathway. Such findings show that proper development of the functional organization of visual cortex is dependent on normal patterns of neural activity. The generalisation of this conclusion to other sensory cortices has been questioned by findings that the segregation of thalamocortical afferents into a somatotopic barrel pattern in developing rodent primary somatosensory cortex (S1) is not prevented by activity blockade. We show that a temporary block of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat S1 during the critical period for barrel development disrupts the topographic refinement of thalamocortical connectivity and columnar organization. These effects are evident well after the blockade is ineffective and thus may be permanent. Our findings show that neural activity and specifically the activation of postsynaptic cortical neurons has a prominent role in establishing the primary sensory map in S1, as well as the topographic organization of higher order synaptic connections.

Transcriptional activation by protein-induced DNA bending: evidence for a DNA structural transmission model.

Integration host factor (IHF) is a DNA-bending protein that binds to an upstream activating sequence (UAS1) and, on a negatively supercoiled DNA template, activates transcription from the ilvPG promoter of the ilvG-MEDA operon of Escherichia coli. The transcriptional initiation site of the ilvGMEDA operon is located 92 bp downstream of UAS1. Activation is still observed when the orientation of the upstream IHF binding site is reversed. This manipulation places the IHF binding site on the opposite face of the DNA helix, directs the IHF-induced DNA bend in the opposite direction, and presents the opposite face of the nonsymmetrical, heterodimeric, IHF molecule to the downstream RNA polymerase. Lymphoid enhancer-binding factor, LEF-1, is a DNA-bending, lymphoid-specific, mammalian transcription factor that shares no amino acid sequence similarity with IHF. When the IHF site in UAS1 is replaced with a LEF-1 site, LEF-1 activates transcription from the downstream ilvPG promoter in E. coli as well as it is activated by its natural activator, IHF. These results suggest that specific interactions between IHF and RNA polymerase are not required for activation. The results of DNA structural studies show that IHF forms a protein-DNA complex in the UAS1 region that, in the absence of RNA polymerase, alters the structure of the DNA helix in the -10 hexanucleotide region of the downstream ilvPG promoter. The results of in vitro abortive transcription assays show that IIIF also increases the apparent rate of RNA polymerase isomerization from a closed to an open complex. We suggest, therefore, that IHF activates transcription by forming a higher-order protein-DNA complex in the UAS1 region that structurally alters the DNA helix in a way that facilitates open complex formation at the downstream ilvPG promoter site.