The theoretical limits of DNA sequence discrimination by linked polyamides

Linked polyamides bind in the minor groove of double-stranded DNA in a partially sequence-specific manner. This report analyzes the theoretical limits of DNA sequence discrimination by linked polyamides composed of two to four different types of heterocyclic rings, determining (i) the optimal choice of base-binding specificity for each ring and (ii) the optimal design for a polyamide composed of these rings to target a given DNA sequence and designed to maximize the fraction of the total polyamide binding to the specified target sequence relative to all other sequences. The results show that, fortuitously, polyamides composed of pyrrole, a naturally occurring G-excluding element, and imidazole, a rationally designed G-favoring element, have features similar to the theoretical optimum design for polyamides composed of two different rings. The results also show that, in polyamides composed of two or three types of heterocyclic rings, choosing a nonspecific “placeholder” ring, which binds equally strongly to each of the four bases, along with one or two base-specific rings will often enhance sequence specificity over a polyamide composed entirely of base-specific rings.

Discrimination of the closely related A and D genomes of the hexaploid oat Avena sativa L.

A satellite DNA sequence, As120a, specific to the A-genome chromosomes in the hexaploid oat, Avena sativa L., was isolated by subcloning a fragment with internal tandem repeats from a plasmid, pAs120, that had been obtained from an Avena strigosa (As genome) genomic library. Southern and in situ hybridization showed that sequences with homology to sequences within pAs120 were dispersed throughout the genome of diploid (A and C genomes), tetraploid (AC genomes), and hexaploid (ACD genomes) Avena species. In contrast, sequences homologous to As120a were found in two A-genome species (A. strigosa and Avena longiglumis) and in the hexaploid A. sativa whereas this sequence was little amplified in the tetraploid Avena murphyi and was absent in the remaining A- and C-genome diploid species. In situ hybridization of pAs120a to hexaploid oat species revealed the distribution of elements of the As120a repeated family over both arms of 14 of 42 chromosomes of this species. By using double in situ hybridization with pAs120a and a C genome-specific probe, three sets of 14 chromosomes were revealed corresponding to the A, C, and D genomes of the hexaploid species. Simultaneous in situ hybridizations with pAs120a and ribosomal probes were used to assign the SAT chromosomes of hexaploid species to their correct genomes. This work reports a sequence able to distinguish between the closely related A and D genomes of hexaploid oats. This sequence offers new opportunities to analyze the relationships of Avena species and to explore the possible evolution of various polyploid oat species.

Subtle atomic group discrimination in the RNA minor groove

As a problem in molecular recognition and for drug discovery, great interest has developed around the possibility that RNA structures could be discriminated by peptides and other small molecules. Although small peptides have been shown to have the capacity to discriminate specific bulges and loops in RNA molecules, discrimination of double helical regions by a peptide binder has not been reported. Indeed, the most accessible part of an RNA helix is the minor groove, and fundamental stereochemical considerations have suggested that discrimination of at least some base pairs would be difficult in the minor groove. Here we report the design and isolation of a peptide binder that manifests the most subtle kind of discrimination of base pair differences in the RNA minor groove. Functional discrimination of a single atomic group is demonstrated as well as the difference between two different angular orientations of the same group. This report of RNA helix discrimination by a peptide binder suggests a richer potential for RNA minor groove recognition than previously thought.

Perceptual learning in motion discrimination that generalizes across motion directions

When human subjects discriminate motion directions of two visual stimuli, their discrimination improves with practice. This improved performance has been found to be specific to the practiced directions and does not transfer to new motion directions. Indeed, such stimulus-specific learning has become a trademark finding in almost all perceptual learning studies and has been used to infer the loci of learning in the brain. For example, learning in motion discrimination has been inferred to occur in the visual area MT (medial temporal cortex) of primates, where neurons are selectively tuned to motion directions. However, such motion discrimination task is extremely difficult, as is typical of most perceptual learning tasks. When the difficulty is moderately reduced, learning transfers to new motion directions. This result challenges the idea of using simple visual stimuli to infer the locus of learning in low-level visual processes and suggests that higher-level processing is essential even in “simple” perceptual learning tasks.

The α-helical FXXΦΦ motif in p53: TAF interaction and discrimination by MDM2

Transcriptional activation domains share little sequence homology and generally lack folded structures in the absence of their targets, aspects that have rendered activation domains difficult to characterize. Here, a combination of biochemical and nuclear magnetic resonance experiments demonstrates that the activation domain of the tumor suppressor p53 has an FXXΦΦ motif (F, Phe; X, any amino acids; Φ, hydrophobic residues) that folds into an α-helix upon binding to one of its targets, hTAFII31 (a human TFIID TATA box-binding protein-associated factor). MDM2, the cellular attenuator of p53, discriminates the FXXΦΦ motif of p53 from those of NF-κB p65 and VP16 and specifically inhibits p53 activity. Our studies support the notion that the FXXΦΦ sequence is a general α-helical recognition motif for hTAFII31 and provide insights into the mechanistic basis for regulation of p53 function.

Importance of newly generated neurons in the adult olfactory bulb for odor discrimination

In adult rodents, neurons are continually generated in the subventricular zone of the forebrain, from where they migrate tangentially toward the olfactory bulb, the only known target for these neuronal precursors. Within the main olfactory bulb, they ascend radially into the granule and periglomerular cell layers, where they differentiate mainly into local interneurons. The functional consequences of this permanent generation and integration of new neurons into existing circuits are unknown. To address this question, we used neural cell adhesion molecule-deficient mice that have documented deficits in the migration of olfactory-bulb neuron precursors, leading to about 40% size reduction of this structure. Our anatomical study reveals that this reduction is restricted to the granule cell layer, a structure that contains exclusively γ-aminobutyric acid (GABA)ergic interneurons. Furthermore, mutant mice were subjected to experiments designed to examine the behavioral consequences of such anatomical alteration. We found that the specific reduction in the newly generated interneuron population resulted in an impairment of discrimination between odors. In contrast, both the detection thresholds for odors and short-term olfactory memory were unaltered, demonstrating that a critical number of bulbar granule cells is crucial only for odor discrimination but not for general olfactory functions.

Increased discrimination of “false memories” in autism spectrum disorder

Individuals with autism spectrum disorder (ASD) have impaired ability to use context, which may manifest as alterations of relatedness within the semantic network. However, impairment in context use may be more difficult to detect in high-functioning adults with ASD. To test context use in this population, we examined the influence of context on memory by using the “false memory” test. In the false memory task, lists of words were presented to high-functioning subjects with ASD and matched controls. Each list consists of words highly related to an index word not on the list. Subjects are then given a recognition test. Positive responses to the index words represent false memories. We found that individuals with ASD are able to discriminate false memory items from true items significantly better than are control subjects. Memory in patients with ASD may be more accurate than in normal individuals under certain conditions. These results also suggest that semantic representations comprise a less distributed network in high-functioning adults with ASD. Furthermore, these results may be related to the unusually high memory capacities found in some individuals with ASD. Research directed at defining the range of tasks performed superiorly by high-functioning individuals with ASD will be important for optimal vocational rehabilitation.

Assessment and discrimination of odor stimuli in rat olfactory bulb by dynamic functional MRI

Dynamic blood oxygenation level-dependent functional MRI was applied at 7 T in the rat olfactory bulb (OB) with pulsed delivery of iso-amyl acetate (IAA) and limonene. Acquisition times for single-slice and whole OB data were 8 and 32 s, respectively, with spatial resolution of 220 × 220 × 250 μm. On an intrasubject basis, short IAA exposures of 0.6 min separated by 3.5-min intervals induced reproducible spatial activity patterns (SAPs) in the olfactory nerve layer, glomerular layer, and external plexiform layer. During long exposures (≈10 min), the initially dominant dorsal SAPs declined in intensity and area, whereas in some OB regions, the initially weak ventral/lateral SAPs increased first and then decreased. The SAPs of different concentrations were topologically similar, which implies that whereas an odor at various concentrations activates the same subsets of receptor cells, different concentrations are assessed and discriminated by variable magnitudes of laminarspecific activations. IAA and limonene reproducibly activated different subsets of receptor cells with some overlaps. Whereas qualitative topographical agreement was observed with results from other methods, the current dynamic blood oxygenation level-dependent functional MRI results can provide quantitative SAPs of the entire OB.

Surgically created neural pathways mediate visual pattern discrimination

Combined lesions of retinal targets and ascending auditory pathways can induce, in developing animals, permanent retinal projections to auditory thalamic nuclei and to visual thalamic nuclei that normally receive little direct retinal input. Neurons in the auditory cortex of such animals have visual response properties that resemble those of neurons in the primary visual cortex of normal animals. Therefore, we investigated the behavioral function of the surgically induced retino-thalamo-cortical pathways. We showed that both surgically induced pathways can mediate visually guided behaviors whose normal substrate, the pathway from the retina to the primary visual cortex via the primary thalamic visual nucleus, is missing.

Electrostatic surface plasmon resonance: Direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches

We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surface-immobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum.

A locus for female discrimination behavior causing sexual isolation in Drosophila

The genetic basis of sexual isolation that contributes to speciation is one of the unsolved questions in evolutionary biology. Drosophila ananassae and Drosophila pallidosa are closely related, and postmating isolation has not developed between them. However, females of both species discriminate their mating partners, and this discrimination contributes to strong sexual isolation between them. By using surgical treatments, we demonstrate that male courtship songs play a dominant role in female mate discrimination. The absence of the song of D. pallidosa dramatically increased interspecies mating with D. ananassae females but reduced intraspecies mating with D. pallidosa females. Furthermore, genetic analysis and chromosomal introgression by repeated backcrosses to D. pallidosa males identified possible loci that control female discrimination in each species. These loci were mapped on distinct positions near the Delta locus on the middle of the left arm of the second chromosome. Because the mate discrimination we studied is well developed and is the only known mechanism that prevents gene flow between them, these loci may have played crucial roles in the evolution of reproductive isolation, and therefore, in the speciation process between these two species.

Operant conditioning of H-reflex changes synaptic terminals on primate motoneurons.

Operant conditioning of the primate triceps surae H-reflex, the electrical analog of the spinal stretch reflex, creates a memory trace that includes changes in the spinal cord. To define the morphological correlates of this plasticity, we analyzed the synaptic terminal coverage of triceps surae motoneurons from animals in which the triceps surae H-reflex in one leg had been increased (HRup mode) or decreased (HRdown mode) by conditioning and compared them to each other and to motoneurons from unconditioned animals. Motoneurons were labeled by intramuscular injection of cholera toxin-horseradish peroxidase. A total of 5055 terminals on the cell bodies and proximal dendrites of 114 motoneurons from 14 animals were studied by electron microscopy. Significant differences were found between HRup and HRdown animals and between HRup and naive (i.e., unconditioned) animals. F terminals (i.e., putative inhibitory terminals) were smaller and their active zone coverage on the cell body was lower on motoneurons from the conditioned side of HRup animals than on motoneurons from the conditioned side of HRdown animals. C terminals (i.e., terminals associated with postsynaptic cisterns and rough endoplasmic reticulum) were smaller and the number of C terminals in each C complex (i.e., a group of contiguous C terminals) was larger on motoneurons from the conditioned side of HRup animals than on motoneurons either from the conditioned side of HRdown animals or from naive animals. Because the treatment of HRup and HRdown animals differed only in the reward contingency, the results imply that the two contingencies had different effects on motoneuron synaptic terminals. In combination with other recent data, they show that H-reflex conditioning produces a complex pattern of spinal cord plasticity that includes changes in motoneuron physiological properties as well as in synaptic terminals. Further delineation of this pattern should reveal the contribution of the structural changes described here to the learned change in behavior.

Binding of a hairpin polyamide in the minor groove of DNA: sequence-specific enthalpic discrimination.

Hairpin polyamides are synthetic ligands for sequence-specific recognition in the minor groove of double-helical DNA. A thermodynamic characterization of the DNA-binding properties exhibited by a six-ring hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (where Im = imidazole, Py = pyrrole, gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide), reveals an approximately 1-2 kcal/mol greater affinity for the designated match site, 5'-TGTTA-3', relative to the single base pair mismatch sites, 5'-TGGTA-3' and 5'-TATTA-3'. The enthalpy and entropy data at 20 degrees C reveal this sequence specificity to be entirely enthalpic in origin. Correlations between the thermodynamic driving forces underlying the sequence specificity exhibited by ImPyPy-gamma-PyPyPy-beta-Dp and the structural properties of the heterodimeric complex of PyPyPy and ImPyPy bound to the minor groove of DNA provide insight into the molecular forces that govern the affinity and specificity of pyrrole-imidazole polyamides.

Discrimination among pheromone component blends by interneurons in male antennal lobes of two populations of the turnip moth, Agrotis segetum.

A difference in female pheromone production and male behavioral response has previously been found in two populations of the turnip moth, Agrotis segetum, originating from Sweden and Zimbabwe, respectively. In this study, we investigated the pheromone response of antennal lobe interneurons of males of the two populations by intracellular recordings, stimulating with single pheromone components and various inter- and intra-populational pheromone blends. Three major physiological types of antennal lobe neurons were established in the two populations according to their responses to different stimuli. One type responded broadly to almost all the stimuli tested. The second type responded selectively to some of the single components and blends. The third type did not respond to any single components but did respond to certain blends. Furthermore, some neurons of the second and third type recognized strain specific differences in ratios between pheromone components. Both projection neurons and local interneurons were found among these three types. Two pheromone responding bilateral projection neurons are reported for the first time in this paper.