Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots

Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a two-phase integration mechanism. In the “preintegration” phase, transforming plasmid molecules (either intact or partial) are spliced together. This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences. Subsequently, integration of transgenic DNA into the host genome is initiated. Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus. The resulting transgenic locus may have plant DNA separating the transgenic sequences. Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism. Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically. Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through Agrobacterium, the qualitative and/or quantitative efficiency of transformation events is not compromised. Our results cast doubt on the role of Agrobacterium vir genes in the integration process.

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.

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.

On representations of finite type

Representations of the (infinite) canonical anticommutation relations and the associated operator algebra, the fermion algebra, are studied. A “coupling constant” (in (0,1]) is defined for primary states of “finite type” of that algebra. Primary, faithful states of finite type with arbitrary coupling are constructed and classified. Their physical significance for quantum thermodynamical systems at high temperatures is discussed. The scope of this study is broadened to include a large class of operator algebras sharing some of the structural properties of the fermion algebra.

Adeno-associated virus (AAV) site-specific integration: Formation of AAV–AAVS1 junctions in an in vitro system

An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo.

T-strand integration in maize protoplasts after codelivery of a T-DNA substrate and virulence genes

We describe a plant protoplast transformation method that provides transformants with a simple pattern of integration of a foreign gene. The approach is to deliver into plant protoplasts by direct gene transfer the Agrobacterium virulence genes virD1 and virD2 with or without virE2, together with a target plasmid containing a gene of interest flanked by Agrobacterium T-DNA border repeat sequences of 25 bp. We present evidence of T-DNA formation in maize protoplasts and its integration into the maize genome. The frequency of VirD1-VirD2-mediated integration events was about 20–35% of the total number of transformants. The addition of virE2 doubled the transformation efficiency. The method described here is of sufficient efficiency and simplicity to be useful for the production of transgenic plants with single-copy well-defined transgenic inserts.

Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries.

Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression

Little is known about plant circadian oscillators, in spite of how important they are to sessile plants, which require accurate timekeepers that enable the plants to respond to their environment. Previously, we identified a circadian clock-associated (CCA1) gene that encodes an Myb-related protein that is associated with phytochrome control and circadian regulation in plants. To understand the role CCA1 plays in phytochrome and circadian regulation, we have isolated an Arabidopsis line with a T DNA insertion that results in the loss of CCA1 RNA, of CCA1 protein, and of an Lhcb-promoter binding activity. This mutation affects the circadian expression of all four clock-controlled genes that we examined. The results show that, despite their similarity, CCA1 and LHY are only partially redundant. The lack of CCA1 also affects the phytochrome regulation of gene expression, suggesting that CCA1 has an additional role in a signal transduction pathway from light, possibly acting at the point of integration between phytochrome and the clock. Our results indicate that CCA1 is an important clock-associated protein involved in circadian regulation of gene expression.

Genetic analysis using genomic representations

Analysis of the genetic changes in human tumors is often problematical because of the presence of normal stroma and the limited availability of pure tumor DNA. However, large amounts of highly reproducible “representations” of tumor and normal genomes can be made by PCR from nanogram amounts of restriction endonuclease cleaved DNA that has been ligated to oligonucleotide adaptors. We show here that representations are useful for many types of genetic analyses, including measuring relative gene copy number, loss of heterozygosity, and comparative genomic hybridization. Representations may be prepared even from sorted nuclei from fixed and archived tumor biopsies.

Maintenance of a somatotopic cortical map in the face of diminishing thalamocortical inputs

This study addresses the extent of divergence in the ascending somatosensory pathways of primates. Divergence of inputs from a particular body part at each successive synaptic step in these pathways results in a potential magnification of the representation of that body part in the somatosensory cortex, so that the representation can be expanded when peripheral input from other parts is lost, as in nerve lesions or amputations. Lesions of increasing size were placed in the representation of a finger in the ventral posterior thalamic nucleus (VPL) of macaque monkeys. After a survival period of 1–5 weeks, area 3b of the somatosensory cortex ipsilateral to the lesion was mapped physiologically, and the extent of the representation of the affected and adjacent fingers was determined. Lesions affecting less than 30% of the thalamic VPL nucleus were without effect upon the cortical representation of the finger whose thalamic representation was at the center of the lesion. Lesions affecting about 35% of the VPL nucleus resulted in a shrinkage of the cortical representation of the finger whose thalamic representation was lesioned, with concomitant expansion of the representations of adjacent fingers. Beyond 35–40%, the whole cortical representation of the hand became silent. These results suggest that divergence of brainstem and thalamocortical projections, although normally not expressed, are sufficiently great to maintain a representation after a major loss of inputs from the periphery. This is likely to be one mechanism of representational plasticity in the cerebral cortex.

Computation, prediction, and experimental tests of fitness for bacteriophage T7 mutants with permuted genomes

We created a simulation based on experimental data from bacteriophage T7 that computes the developmental cycle of the wild-type phage and also of mutants that have an altered genome order. We used the simulation to compute the fitness of more than 105 mutants. We tested these computations by constructing and experimentally characterizing T7 mutants in which we repositioned gene 1, coding for T7 RNA polymerase. Computed protein synthesis rates for ectopic gene 1 strains were in moderate agreement with observed rates. Computed phage-doubling rates were close to observations for two of four strains, but significantly overestimated those of the other two. Computations indicate that the genome organization of wild-type T7 is nearly optimal for growth: only 2.8% of random genome permutations were computed to grow faster, the highest 31% faster, than wild type. Specific discrepancies between computations and observations suggest that a better understanding of the translation efficiency of individual mRNAs and the functions of qualitatively “nonessential” genes will be needed to improve the T7 simulation. In silico representations of biological systems can serve to assess and advance our understanding of the underlying biology. Iteration between computation, prediction, and observation should increase the rate at which biological hypotheses are formulated and tested.

Paper-like electronic displays: Large-area rubber-stamped plastic sheets of electronics and microencapsulated electrophoretic inks

Electronic systems that use rugged lightweight plastics potentially offer attractive characteristics (low-cost processing, mechanical flexibility, large area coverage, etc.) that are not easily achieved with established silicon technologies. This paper summarizes work that demonstrates many of these characteristics in a realistic system: organic active matrix backplane circuits (256 transistors) for large (≈5 × 5-inch) mechanically flexible sheets of electronic paper, an emerging type of display. The success of this effort relies on new or improved processing techniques and materials for plastic electronics, including methods for (i) rubber stamping (microcontact printing) high-resolution (≈1 μm) circuits with low levels of defects and good registration over large areas, (ii) achieving low leakage with thin dielectrics deposited onto surfaces with relief, (iii) constructing high-performance organic transistors with bottom contact geometries, (iv) encapsulating these transistors, (v) depositing, in a repeatable way, organic semiconductors with uniform electrical characteristics over large areas, and (vi) low-temperature (≈100°C) annealing to increase the on/off ratios of the transistors and to improve the uniformity of their characteristics. The sophistication and flexibility of the patterning procedures, high level of integration on plastic substrates, large area coverage, and good performance of the transistors are all important features of this work. We successfully integrate these circuits with microencapsulated electrophoretic “inks” to form sheets of electronic paper.

Functional analysis of primary visual cortex (V1) in humans

Human area V1 offers an excellent opportunity to study, using functional MRI, a range of properties in a specific cortical visual area, whose borders are defined objectively and convergently by retinotopic criteria. The retinotopy in V1 (also known as primary visual cortex, striate cortex, or Brodmann’s area 17) was defined in each subject by using both stationary and phase-encoded polar coordinate stimuli. Data from V1 and neighboring retinotopic areas were displayed on flattened cortical maps. In additional tests we revealed the paired cortical representations of the monocular “blind spot.” We also activated area V1 preferentially (relative to other extrastriate areas) by presenting radial gratings alternating between 6% and 100% contrast. Finally, we showed evidence for orientation selectivity in V1 by measuring transient functional MRI increases produced at the change in response to gratings of differing orientations. By systematically varying the orientations presented, we were able to measure the bandwidth of the orientation “transients” (45°).

Linear and nonlinear pathways of spectral information transmission in the cochlear nucleus

At the level of the cochlear nucleus (CN), the auditory pathway divides into several parallel circuits, each of which provides a different representation of the acoustic signal. Here, the representation of the power spectrum of an acoustic signal is analyzed for two CN principal cells—chopper neurons of the ventral CN and type IV neurons of the dorsal CN. The analysis is based on a weighting function model that relates the discharge rate of a neuron to first- and second-order transformations of the power spectrum. In chopper neurons, the transformation of spectral level into rate is a linear (i.e., first-order) or nearly linear function. This transformation is a predominantly excitatory process involving multiple frequency components, centered in a narrow frequency range about best frequency, that usually are processed independently of each other. In contrast, type IV neurons encode spectral information linearly only near threshold. At higher stimulus levels, these neurons are strongly inhibited by spectral notches, a behavior that cannot be explained by level transformations of first- or second-order. Type IV weighting functions reveal complex excitatory and inhibitory interactions that involve frequency components spanning a wider range than that seen in choppers. These findings suggest that chopper and type IV neurons form parallel pathways of spectral information transmission that are governed by two different mechanisms. Although choppers use a predominantly linear mechanism to transmit tonotopic representations of spectra, type IV neurons use highly nonlinear processes to signal the presence of wide-band spectral features.

Plasticity in the neural coding of auditory space in the mammalian brain

Sound localization relies on the neural processing of monaural and binaural spatial cues that arise from the way sounds interact with the head and external ears. Neurophysiological studies of animals raised with abnormal sensory inputs show that the map of auditory space in the superior colliculus is shaped during development by both auditory and visual experience. An example of this plasticity is provided by monaural occlusion during infancy, which leads to compensatory changes in auditory spatial tuning that tend to preserve the alignment between the neural representations of visual and auditory space. Adaptive changes also take place in sound localization behavior, as demonstrated by the fact that ferrets raised and tested with one ear plugged learn to localize as accurately as control animals. In both cases, these adjustments may involve greater use of monaural spectral cues provided by the other ear. Although plasticity in the auditory space map seems to be restricted to development, adult ferrets show some recovery of sound localization behavior after long-term monaural occlusion. The capacity for behavioral adaptation is, however, task dependent, because auditory spatial acuity and binaural unmasking (a measure of the spatial contribution to the “cocktail party effect”) are permanently impaired by chronically plugging one ear, both in infancy but especially in adulthood. Experience-induced plasticity allows the neural circuitry underlying sound localization to be customized to individual characteristics, such as the size and shape of the head and ears, and to compensate for natural conductive hearing losses, including those associated with middle ear disease in infancy.