Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids

Nucleolar dominance is an epigenetic phenomenon in which one parental set of ribosomal RNA (rRNA) genes is silenced in an interspecific hybrid. In natural Arabidopsis suecica, an allotetraploid (amphidiploid) hybrid of Arabidopsis thaliana and Cardaminopsis arenosa, the A. thaliana rRNA genes are repressed. Interestingly, A. thaliana rRNA gene silencing is variable in synthetic Arabidopsis suecica F1 hybrids. Two generations are needed for A. thaliana rRNA genes to be silenced in all lines, revealing a species-biased direction but stochastic onset to nucleolar dominance. Backcrossing synthetic A. suecica to tetraploid A. thaliana yielded progeny with active A. thaliana rRNA genes and, in some cases, silenced C. arenosa rRNA genes, showing that the direction of dominance can be switched. The hypothesis that naturally dominant rRNA genes have a superior binding affinity for a limiting transcription factor is inconsistent with dominance switching. Inactivation of a species-specific transcription factor is argued against by showing that A. thaliana and C. arenosa rRNA genes can be expressed transiently in the other species. Transfected A. thaliana genes are also active in A. suecica protoplasts in which chromosomal A. thaliana genes are repressed. Collectively, these data suggest that nucleolar dominance is a chromosomal phenomenon that results in coordinate or cooperative silencing of rRNA genes.

Spatial organization of retinal information about the direction of image motion.

The visual stimuli that elicit neural activity differ for different retinal ganglion cells and these cells have been categorized by the visual information that they transmit. If specific visual information is conveyed exclusively or primarily by a particular set of ganglion cells, one might expect the cells to be organized spatially so that their sampling of information from the visual field is complete but not redundant. In other words, the laterally spreading dendrites of the ganglion cells should completely cover the retinal plane without gaps or significant overlap. The first evidence for this sort of arrangement, which has been called a tiling or tessellation, was for the two types of "alpha" ganglion cells in cat retina. Other reports of tiling by ganglion cells have been made subsequently. We have found evidence of a particularly rigorous tiling for the four types of ganglion cells in rabbit retina that convey information about the direction of retinal image motion (the ON-OFF direction-selective cells). Although individual cells in the four groups are morphologically indistinguishable, they are organized as four overlaid tilings, each tiling consisting of like-type cells that respond preferentially to a particular direction of retinal image motion. These observations lend support to the hypothesis that tiling is a general feature of the organization of information outflow from the retina and clearly implicate mechanisms for recognition of like-type cells and establishment of mutually acceptable territories during retinal development.

Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species

The genome sequence of the extremely thermophilic archaeon Methanococcus jannaschii provides a wealth of data on proteins from a thermophile. In this paper, sequences of 115 proteins from M. jannaschii are compared with their homologs from mesophilic Methanococcus species. Although the growth temperatures of the mesophiles are about 50°C below that of M. jannaschii, their genomic G+C contents are nearly identical. The properties most correlated with the proteins of the thermophile include higher residue volume, higher residue hydrophobicity, more charged amino acids (especially Glu, Arg, and Lys), and fewer uncharged polar residues (Ser, Thr, Asn, and Gln). These are recurring themes, with all trends applying to 83–92% of the proteins for which complete sequences were available. Nearly all of the amino acid replacements most significantly correlated with the temperature change are the same relatively conservative changes observed in all proteins, but in the case of the mesophile/thermophile comparison there is a directional bias. We identify 26 specific pairs of amino acids with a statistically significant (P < 0.01) preferred direction of replacement.

PRIM: Proximity imaging of green fluorescent protein-tagged polypeptides

We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins in vivo. The method, which we term proximity imaging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces.

Neural systems underlying learning and representation of global motion

We demonstrate performance-related changes in cortical and cerebellar activity. The largest learning-dependent changes were observed in the anterior lateral cerebellum, where the extent and intensity of activation correlated inversely with psychophysical performance. After learning had occurred (a few minutes), the cerebellar activation almost disappeared; however, it was restored when the subjects were presented with a novel, untrained direction of motion for which psychophysical performance also reverted to chance level. Similar reductions in the extent and intensity of brain activations in relation to learning occurred in the superior colliculus, anterior cingulate, and parts of the extrastriate cortex. The motion direction-sensitive middle temporal visual complex was a notable exception, where there was an expansion of the cortical territory activated by the trained stimulus. Together, these results indicate that the learning and representation of visual motion discrimination are mediated by different, but probably interacting, neuronal subsystems.

Invariance between subjects of brain wave representations of language

In three experiments, electric brain waves of 19 subjects were recorded under several different experimental conditions for two purposes. One was to test how well we could recognize which sentence, from a set of 24 or 48 sentences, was being processed in the cortex. The other was to study the invariance of brain waves between subjects. As in our earlier work, the analysis consisted of averaging over trials to create prototypes and test samples, to both of which Fourier transforms were applied, followed by filtering and an inverse transformation to the time domain. A least-squares criterion of fit between prototypes and test samples was used for classification. In all three experiments, averaging over subjects improved the recognition rates. The most significant finding was the following. When brain waves were averaged separately for two nonoverlapping groups of subjects, one for prototypes and the other for test samples, we were able to recognize correctly 90% of the brain waves generated by 48 different sentences about European geography.

Centrosome positioning and directionality of cell movements

In several cell types, an intriguing correlation exists between the position of the centrosome and the direction of cell movement: the centrosome is located behind the leading edge, suggesting that it serves as a steering device for directional movement. A logical extension of this suggestion is that a change in the direction of cell movement is preceded by a reorientation, or shift, of the centrosome in the intended direction of movement. We have used a fusion protein of green fluorescent protein (GFP) and γ-tubulin to label the centrosome in migrating amoebae of Dictyostelium discoideum, allowing us to determine the relationship of centrosome positioning and the direction of cell movement with high spatial and temporal resolution in living cells. We find that the extension of a new pseudopod in a migrating cell precedes centrosome repositioning. An average of 12 sec elapses between the initiation of pseudopod extension and reorientation of the centrosome. If no reorientation occurs within approximately 30 sec, the pseudopod is retracted. Thus the centrosome does not direct a cell’s migration. However, its repositioning stabilizes a chosen direction of movement, most probably by means of the microtubule system.

An extracellular signaling component in propagation of astrocytic calcium waves

Focally evoked calcium waves in astrocyte cultures have been thought to propagate by gap-junction-mediated intercellular passage of chemical signal(s). In contrast to this mechanism we observed isolated astrocytes, which had no physical contact with other astrocytes in the culture, participating in a calcium wave. This observation requires an extracellular route of astrocyte signaling. To directly test for extracellular signaling we made cell-free lanes 10–300 μm wide in confluent cultures by deleting astrocytes with a glass pipette. After 4–8 hr of recovery, regions of confluent astrocytes separated by lanes devoid of cells were easily located. Electrical stimulation was used to initiate calcium waves. Waves crossed narrow (<120 μm) cell-free lanes in 15 of 36 cases, but failed to cross lanes wider than 120 μm in eight of eight cases. The probability of crossing narrow lanes was not correlated with the distance from the stimulation site, suggesting that cells along the path of the calcium wave release the extracellular messenger(s). Calculated velocity across the acellular lanes was not significantly different from velocity through regions of confluent astrocytes. Focal superfusion altered both the extent and the direction of calcium waves in confluent regions. These data indicate that extracellular signals may play a role in astrocyte–astrocyte communication in situ.

Orientation of the transcription preinitiation complex in Archaea

The basal transcription machinery of Archaea corresponds to the minimal subset of factors required for RNA polymerase II transcription in eukaryotes. Using just two factors, Archaea recruit the RNA polymerase to promoters and define the direction of transcription. Notably, the principal determinant for the orientation of transcription is not the recognition of the TATA box by the TATA-box-binding protein. Instead, transcriptional polarity is governed by the interaction of the archaeal TFIIB homologue with a conserved motif immediately upstream of the TATA box. This interaction yields an archaeal preinitiation complex with the same orientation as the analogous eukaryal complex.

The structural basis for the oriented assembly of a TBP/TFB/promoter complex

Recently the definition of the metazoan RNA polymerase II and archaeal core promoters has been expanded to include a region immediately upstream of the TATA box called the B recognition element (BRE), so named because eukaryal transcription factor TFIIB and its archaeal orthologue TFB interact with the element in a sequence-specific manner. Here we present the 2.4-Å crystal structure of archaeal TBP and the C-terminal core of TFB (TFBc) in a complex with an extended TATA-box-containing promoter that provides a detailed picture of the stereospecific interactions between the BRE and a helix–turn–helix motif in the C-terminal cyclin repeat of TFBc. This interaction is important in determining the level of basal transcription and explicitly defines the direction of transcription.

Role of intrinsic synaptic circuitry in collicular sensorimotor integration

The superficial gray layer of the superior colliculus contains a map that represents the visual field, whereas the underlying intermediate gray layer contains a vector map of the saccades that shift the direction of gaze. These two maps are aligned so that a particular region of the visual field is represented directly above the neurons that orient the highest acuity area of the retina toward that region. Although it has been proposed that the transmission of information from the visuosensory to the motor map plays an important role in the generation of visually guided saccades, experiments have failed to demonstrate any functional linkage between the two layers. We examined synaptic transmission between these layers in vitro by stimulating the superficial layer while using whole-cell patch-clamp methods to measure the responses of intermediate layer neurons. Stimulation of superficial layer neurons evoked excitatory postsynaptic currents in premotor cells. This synaptic input was columnar in organization, indicating that the connections between the layers link corresponding regions of the visuosensory and motor maps. Excitatory postsynaptic currents were large enough to evoke action potentials and often occurred in clusters similar in duration to the bursts of action potentials that premotor cells use to command saccades. Our results indicate the presence of functional connections between the superficial and intermediate layers and show that such connections could play a significant role in the generation of visually guided saccades.

Invariance of brain-wave representations of simple visual images and their names

In two experiments, electric brain waves of 14 subjects were recorded under several different conditions to study the invariance of brain-wave representations of simple patches of colors and simple visual shapes and their names, the words blue, circle, etc. As in our earlier work, the analysis consisted of averaging over trials to create prototypes and test samples, to both of which Fourier transforms were applied, followed by filtering and an inverse transformation to the time domain. A least-squares criterion of fit between prototypes and test samples was used for classification. The most significant results were these. By averaging over different subjects, as well as trials, we created prototypes from brain waves evoked by simple visual images and test samples from brain waves evoked by auditory or visual words naming the visual images. We correctly recognized from 60% to 75% of the test-sample brain waves. The general conclusion is that simple shapes such as circles and single-color displays generate brain waves surprisingly similar to those generated by their verbal names. These results, taken together with extensive psychological studies of auditory and visual memory, strongly support the solution proposed for visual shapes, by Bishop Berkeley and David Hume in the 18th century, to the long-standing problem of how the mind represents simple abstract ideas.