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.

Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: Induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal

We have recently shown that VEGF functions as a survival factor for newly formed vessels during developmental neovascularization, but is not required for maintenance of mature vessels. Reasoning that expanding tumors contain a significant fraction of newly formed and remodeling vessels, we examined whether abrupt withdrawal of VEGF will result in regression of preformed tumor vessels. Using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, we showed that shutting off VEGF production leads to detachment of endothelial cells from the walls of preformed vessels and their subsequent death by apoptosis. Vascular collapse then leads to hemorrhages and extensive tumor necrosis. These results suggest that enforced withdrawal of vascular survival factors can be applied to target preformed tumor vasculature in established tumors. The system was also used to examine phenotypes resulting from over-expression of VEGF. When expression of the transfected VEGF cDNA was continuously “on,” tumors became hyper-vascularized with abnormally large vessels, presumably arising from excessive fusions. Tumors were significantly less necrotic, suggesting that necrosis in these tumors is the result of insufficient angiogenesis.

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.

An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives

We have developed a universally applicable system for conditional gene expression in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expression vectors that allow transgene expression from endogenous cellular promoters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the β-galactosidase/neoR fusion protein and the Cre-ERT2 (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bicistronic gene-trap vector encoding the hygroR protein and the human alkaline phosphatase (hAP), the expression of which is prevented by tandemly repeated stop-of-transcription sequences flanked by loxP sites. In selected clones, hAP expression was shown to be regulated accurately by 4′hydroxy-tamoxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitro in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtually all the tissues of the 10-day-old chimeric fetus (after injection of 4′hydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can be applied to drive conditional expression of virtually any transgene in a large variety of cell types, both in vitro and in vivo.

A method for the generation of conditional gene repair mutations in mice

Conditional gene repair mutations in the mouse can assist in cell lineage analyses and provide a valuable complement to conditional gene inactivation strategies. We present a method for the generation of conditional gene repair mutations that employs a loxP-flanked (floxed) selectable marker and transcriptional/translational stop cassette (neostop) located within the first intron of a target gene. In the absence of Cre recombinase, expression of the targeted allele is suppressed generating a null allele, while in the presence of Cre, excision of neostop restores expression to wild-type levels. To test this strategy, we have generated a conditional gene repair allele of the mouse Huntington’s disease gene homolog (Hdh). Insertion of neostop within the Hdh intron 1 generated a null allele and mice homozygous for this allele resembled nullizygous Hdh mutants and died after embryonic day 8.5. In the presence of a cre transgene expressed ubiquitously early in development, excision of neostop restored Hdh expression and rescued the early embryonic lethality. A simple modification of this strategy that permits the generation of conventional gene knockout, conditional gene knockout and conditional gene repair alleles using one targeting construct is discussed.

A conditional probability analysis of cystic fibrosis transmembrane conductance regulator gating indicates that ATP has multiple effects during the gating cycle

ATP-binding cassette (ABC) transporters bind and hydrolyze ATP. In the cystic fibrosis transmembrane conductance regulator Cl− channel, this interaction with ATP generates a gating cycle between a closed (C) and two open (O1 and O2) conformations. To understand better how ATP controls channel activity, we examined gating transitions from the C to the O1 and O2 states and from these open states to the C conformation. We made three main observations. First, we found that the channel can open into either the O1 or O2 state, that the frequency of transitions to both states was increased by ATP concentration, and that ATP increased the relative proportion of openings into O1 vs. O2. These results indicate that ATP can interact with the closed state to open the channel in at least two ways, which may involve binding to nucleotide-binding domains (NBDs) NBD1 and NBD2. Second, ATP prolonged the burst duration and altered the way in which the channel closed. These data suggest that ATP also interacts with the open channel. Third, the channel showed runs of specific types of open–closed transitions. This finding suggests a mechanism with more than one cycle of gating transitions. These data suggest models to explain how ATP influences conformational transitions in cystic fibrosis transmembrane conductance regulator and perhaps other ABC transporters.

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.