Decoding the dynamic representation of musical pitch from human brain activity.

In music, the perception of pitch is governed largely by its tonal function given the preceding harmonic structure of the music. While behavioral research has advanced our understanding of the perceptual representation of musical pitch, relatively little is known about its representational structure in the brain. Using Magnetoencephalography (MEG), we recorded evoked neural responses to different tones presented within a tonal context. Multivariate Pattern Analysis (MVPA) was applied to "decode" the stimulus that listeners heard based on the underlying neural activity. We then characterized the structure of the brain's representation using decoding accuracy as a proxy for representational distance, and compared this structure to several well established perceptual and acoustic models. The observed neural representation was best accounted for by a model based on the Standard Tonal Hierarchy, whereby differences in the neural encoding of musical pitches correspond to their differences in perceived stability. By confirming that perceptual differences honor those in the underlying neuronal population coding, our results provide a crucial link in understanding the cognitive foundations of musical pitch across psychological and neural domains.

A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide.

We describe a modification of the DNA extraction method, in which cetyltrimethylammonium bromide (CTAB) is used to extract nucleic acids from plant tissues. In contrast to the original method, the modified CTAB procedure is faster, omits the selective precipitation and CsCl gradient steps, uses less expensive and toxic reagents, requires only inexpensive laboratory equipment and is more readily adapted to high-throughput DNA extraction. This protocol yields approximately 5-30 microg of total DNA from 200 mg of tissue fresh weight, depending on plant species and tissue source. It can be completed in as little as 5-6 h.

The role of cell differentiation state and HMG-I/Y in the expression of transgenes flanked by matrix attachment regions.

The tobacco nuclear matrix attachment region (MAR), RB7, has been shown to have a much greater effect on transgene expression in cultured cells than in transgenic plants. This is comparable to work in mouse systems showing that MARs have a positive effect on transgene expression in embryonic tissues but not adult tissues. There are several possible explanations for these observations. One is that cell differentiation state and proliferation rate can affect MAR function. We tested this possibility by initiating suspension cell cultures from well-characterized transgenic plants transformed with 35S::GUS with and without flanking MARs and then comparing GUS specific activity in the cell lines to those of the transgenic plants from which the cell lines were derived. If cell differentiation state and proliferation rate do affect MAR function, we would expect the ratio of transgene expression (cell suspensions : plants) to be greater in MAR lines than in control lines. This turned out not to be the case. Thus, it appears that MAR function is not enhanced simply because cells in culture divide rapidly and are not differentiated. Because in animal systems the chromosomal protein HMG-I/Y has been shown to be upregulated in proliferating cells and may have a role in MAR function, we have also examined the levels of the tobacco HMG-I/Y homolog by immunoblotting. The level of this protein does not differ between primary transformant cultured cells (NT-1) and Nicotiana tabacum plants (SR-1). However, a higher molecular weight cross-reacting polypeptide was found in nuclei from the NT-1 cell suspensions but was not detected in SR-1 leaf nuclei or cell suspensions derived from the SR-1 plants.

Arousal, mood, and the Mozart effect.

The "Mozart effect" refers to claims that people perform better on tests of spatial abilities after listening to music composed by Mozart. We examined whether the Mozart effect is a consequence of between-condition differences in arousal and mood. Participants completed a test of spatial abilities after listening to music or sitting in silence. The music was a Mozart sonata (a pleasant and energetic piece) for some participants and an Albinoni adagio (a slow, sad piece) for others. We also measured enjoyment, arousal, and mood. Performance on tbe spatial task was better following the music than the silence condition but only for participants who heard Mozart. The two music selections also induced differential responding on the enjoyment, arousal and mood measures. Moreover, when such differences were held constant by statistical means, the Mozart effect disappeared. These findings provide compelling evidence that the Mozart effect is an artifact of arousal and mood.

The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants.

To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.

The speed of pitch resolution in a musical context.

In five experiments, we investigated the speed of pitch resolution in a musical context. In experiments 1-3, listeners were presented an incomplete scale (doh, re, mi, fa, sol, la, ti) and then a probe tone. Listeners were instructed to make a rapid key-press response to probe tones that were relatively proximal in pitch to the last note of the scale (valid trials), and to ignore other probe tones (invalid trials). Reaction times were slower if the pitch of the probe tone was dissonant with the expected pitch (i.e., the completion of the scale, or doh) or if the probe tone was nondiatonic to the key implied by the scale. In experiments 4 and 5, listeners were presented a two-octave incomplete arpeggio, and then a probe tone. In this case, listeners were asked to make a rapid key-press response to probe tones that were relatively distant in pitch from the last note of the arpeggio. Under these conditions, registral direction and pitch proximity were the dominant influences on reaction time. Results are discussed in view of research on auditory attention and models of musical pitch.

Illusory conjunctions of pitch and duration in unfamiliar tone sequences.

In 3 experiments, the authors examined short-term memory for pitch and duration in unfamiliar tone sequences. Participants were presented a target sequence consisting of 2 tones (Experiment 1) or 7 tones (Experiments 2 and 3) and then a probe tone. Participants indicated whether the probe tone matched 1 of the target tones in both pitch and duration. Error rates were relatively low if the probe tone matched 1 of the target tones or if it differed from target tones in pitch, duration, or both. Error rates were remarkably high, however, if the probe tone combined the pitch of 1 target tone with the duration of a different target tone. The results suggest that illusory conjunctions of these dimensions frequently occur. A mathematical model is presented that accounts for the relative contribution of pitch errors, duration errors, and illusory conjunctions of pitch and duration.

The 5' end of the pea ferredoxin-1 mRNA mediates rapid and reversible light-directed changes in translation in tobacco.

Ferredoxin-1 (Fed-1) mRNA contains an internal light response element (iLRE) that destabilizes mRNA when light-grown plants are placed in darkness. mRNAs containing this element dissociate from polyribosomes in the leaves of transgenic tobacco (Nicotiana tabacum) plants transferred to the dark for 2 d. Here, we report in vivo labeling experiments with a chloramphenicol acetyl transferase mRNA fused to the Fed-1 iLRE. Our data indicate that the Fed-1 iLRE mediates a rapid decline in translational efficiency and that iLRE-containing mRNAs dissociate from polyribosomes within 20 min after plants are transferred to darkness. Both events occur before the decline in mRNA abundance, and polyribosome association is rapidly reversible if plants are re-illuminated. These observations support a model in which Fed-1 mRNA in illuminated leaves is stabilized by its association with polyribosomes, and/or by translation. In darkness a large portion of the mRNA dissociates from polyribosomes and is subsequently degraded. We also show that a significant portion of total tobacco leaf mRNA is shifted from polyribosomal to non-polyribosomal fractions after 20 min in the dark, indicating that translation of other mRNAs is also rapidly down-regulated in response to darkness. This class includes some, but not all, cytoplasmic mRNAs encoding proteins involved in photosynthesis.

Use of matrix attachment regions (MARs) to minimize transgene silencing.

Matrix attachment regions (MARs) are operationally defined as DNA elements that bind specifically to the nuclear matrix in vitro. It is possible, although unproven, that they also mediate binding of chromatin to the nuclear matrix in vivo and alter the topology of the genome in interphase nuclei. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable expression in transgenic plants or cell lines, most likely by minimizing gene silencing. Our review explores current data and presents several plausible models to explain MAR effects on transgene expression.

Expectancies generated by recent exposure to melodic sequences.

In four experiments, we examined the effects of exposure to unfamiliar tone sequences on melodic expectancy and memory. In Experiment 1, 30 unfamiliar tone sequences (target sequences) were presented to listeners three times each in random order (exposure phase), and listeners recorded the number of notes in each sequence. Listeners were then presented target and novel sequences and rated how well the final note continued the pattern of notes that preceded it. Novel sequences were identical to target sequences, except for the final note. Ratings were significantly higher for target sequences than for novel sequences, illustrating the influence of exposure on melodic expectancy. Experiment 2 confirmed that without exposure to target sequences, ratings were equivalent for target and novel sequences. In Experiment 3, new listeners were assessed for explicit memory for target sequences following the exposure phase. Recognition of target sequences was above chance, but unrelated to expectancy judgments in Experiment 1. Experiment 4 replicated the exposure effect, using a modified experiment design, and confirmed that the effect is not dependent on explicit memory for sequences. We discuss the idea that melodic expectancies are influenced by implicit memory for recently heard melodic patterns.

Heat shock protein HSP101 binds to the Fed-1 internal light regulator y element and mediates its high translational activity.

The internal light-regulatory element (iLRE) of ferredoxin (Fed-1) mRNA, comprising the 5' leader and at least the first 13 codons of the open reading frame, controls transcript abundance after illumination of the plant in a translation-dependent manner. We have characterized the RNA binding activities associated with the Fed-1 iLRE and have identified one activity as the heat shock protein HSP101, a protein shown to bind the 5' leader of tobacco mosaic virus. HSP101 was sufficient and necessary to mediate a high level of translational activity from a Fed-1 iLRE-containing mRNA in yeast. Moreover, the Fed-1 iLRE substantially enhanced translation of reporter mRNAs in plant protoplasts expressing HSP101. Expression of HSP101 was subject to developmental regulation in leaves in that expression was highest in young leaves. These data suggest that Fed-1 mRNA may use the HSP101 regulatory mechanism as a means of ensuring a high level of translation required for the light-mediated regulation of Fed-1 mRNA stability.

Premature termination codons destabilize ferredoxin-1 mRNA when ferredoxin-1 is translated.

Ferredoxin-1 (Fed-1) mRNA is poorly translated in dark-treated tobacco (Nicotiana tabacum) leaves, resulting in destabilization of Fed-1 mRNA and a differential light/dark accumulation of the mRNA. Insertion of nonsense codons within the Fed-1 coding sequence disrupts the light regulation of Fed-1 mRNA abundance. Here we show that the nonsense codon effect results primarily from lowering the Fed-1 mRNA stability in light-treated leaf tissue and in rapidly growing tobacco cell cultures, but not in dark-treated leaf tissue. These results suggest that nonsense codons trigger a decay pathway distinct from that seen for Fed-1 mRNA in the dark. We propose that nonsense-mediated decay of nonsense-containing Fed-1 mRNA occurs in light-treated leaves and in non-photosynthetic tobacco culture cells where Fed-1 mRNA is being actively translated.

Technical advance: stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system.

We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10:mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.

Post-transcriptional light regulation of nuclear-encoded genes.

A significant number of studies have detected a post-transcriptional component in the light responses of nuclear genes. As yet there are few in-depth studies of the mechanism(s) involved, and it seems likely some additional examples have been missed. For instance, transcriptional responses have sometimes been inferred on the basis of experiments with translational fusions containing both the promoter and 5' UTR of the test gene, but we now know that elements within the 5' UTR can mediate post-transcriptional light responses. Similarly, because of possible changes in translation rates and protein turnover, the common assumption that mRNA levels directly dictate protein levels is tenuous at best. It is no longer permissible to assume that the biological effect of a gene is a simple function of its transcription. Thus it is likely that with careful experimental design, reports of nuclear-encoded post-transcriptional gene regulation will become increasingly prevalent.

Transposon tagging of the sulfur gene of tobacco using engineered maize Ac/Ds elements.

The Sulfur gene of tobacco is nuclearly encoded. A Su allele at this locus acts as a dominant semilethal mutation and causes reduced accumulation of chlorophyll, resulting in a yellow color in the plant. An engineered transposon tagging system, based upon the maize element Ac/Ds, was used to mutate the gene. High frequency of transposon excision from the Su locus produced variegated sectors. Plants regenerated from the variegated sector exhibited a similar variegated phenotype. Genetic analyses showed that the variegation was always associated with the transposase construct and the transposon was linked to the Su locus. Sequences surrounding the transposon were isolated, and five revertant sectors possessed typical direct repeats following Ds excisions. These genetic and molecular data are consistent with the tagging of the Su allele by the transposon.

Characterization of randomly-obtained matrix attachment regions (MARs) from higher plants.

Matrix attachment regions (MARs) can be operationally defined as DNA fragments that bind to the nuclear matrix. We have created a library of randomly obtained MARs from tobacco (Nicotiana tobacum) by cloning DNA fragments that co-isolate with nuclear matrixes prepared by a method involving lithium diiodosalicylate. The interactions of several of the cloned MARs with nuclear matrixes were tested by an in vitro binding assay in which genomic DNA was used as competitor. Based on this assay, the MARs were classified as strong, medium, and weak binders. Examples of each of the binding classes were further studied by in vitro binding using self- and cross-competition. Estimates of dissociation constants for several MARs ranged from 6 to 11 nM and correlated inversely with binding strength. The number of binding sites per matrix for several MARs ranged from 4 x 10(5) to 9 x 10(5) and correlated directly with binding strength. We conclude that binding strength, as we have measured it, is a function of both numbers of binding sites and affinity for the sites. The tobacco MARs were sequenced and analyzed for overall AT content, for distribution of AT-rich regions, and for the abundance of several MAR-related motifs. Previously identified MAR motifs correlate to various degrees with binding strength. Notably, the Drosophila topoisomerase II motif does not correlate with binding strength of the tobacco MARs. A newly identified motif, the "90%AT Box," correlates better with binding strength than any of the previously identified motifs we investigated.

Ferredoxin-1 mRNA is destabilized by changes in photosynthetic electron transport.

In transgenic tobacco, pea Ferredoxin-1 (Fed-1) mRNA accumulates rapidly in response to photosynthesis even when the transgene is driven by a constitutive promoter. To investigate the role of photosynthesis on Fed-1 mRNA stability, we used the tetracycline repressible Top10 promoter system to specifically shut off transcription of the Fed-1 transgene. The Fed-1 mRNA has a half-life of approximately 2.4 hr in the light and a half-life of only 1.2 hr in the dark or in the presence of the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These data indicate that cessation of photosynthesis, either by darkness or DCMU results in a destabilization of the Fed-1 mRNA. Furthermore, the Fed-1 mRNA half-life is reduced immediately upon transfer to darkness, suggesting that Fed-1 mRNA destabilization is a primary response to photosynthesis rather than a secondary response to long-term dark adaptation. Finally, the two different methods for efficient tetracycline delivery reported here generally should be useful for half-life measurements of other mRNAs in whole plants.

Light regulation of Fed-1 mRNA requires an element in the 5' untranslated region and correlates with differential polyribosome association.

Light regulation of Fed-1 mRNA abundance in the leaves of green plants is primarily a post-transcriptional process. Previously, we have shown that the Fed-1 mRNA light response requires an open reading frame, indicating that the light regulation of the mRNA depends on its concurrent translation. We now show that light-induced increases in Fed-1 mRNA abundance are associated with increases in polyribosome association that require both a functional AUG and a normal Fed-1 translational start context. We also present evidence that light regulation of Fed-1 mRNA levels requires more than efficient translation per se. Substitution of the efficiently translated tobacco mosaic virus Omega 5' untranslated region resulted in a loss of Fed-1 light regulation. In addition, we identified a CAT T repeat element located near the 5' terminus of the Fed-1 5' untranslated region that is essential for light regulation. We introduced two different mutations in the CAT T repeat element, but only one of these substitutions blocked the normal light effect on polyribosome association, whereas both altered dark-induced Fed-1 mRNA disappearance. The element may thus be important for Fed-1 mRNA stability rather than polyribosome loading. We propose a model in which Fed-1 mRNA is relatively stable when it is associated with polyribosomes in illuminated plants but in darkness is not polyribosome associated and is thus rapidly degraded by a process involving the CAT T repeat element.

Gene silencing from plant DNA carried by a Geminivirus.

The geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to asses silencing: (1) the sulfur allele (su) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene (luc). Various portions of both marker genes were inserted into TGMV in place of the coat protein open-reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild type Nicotiana benthamiana, circular, yellow spots with an area of several hundred cells formed after 3-5 days. Systemic movement of TGMV::su subsequently produced varigated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5' fragment of the 1392 bp su cDNA in sense and anti-sense orientation, and a 403 bp 3' fragment. TGMV::su-induced silencing was propogated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expresse luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti-sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes.

Expectancies generated by melodic intervals: evaluation of principles of melodic implication in a melody-completion task.

Bottom-up principles of melodic implication (Narmour, 1990) were evaluated in a melody-completion task. One hundred subjects (50 low training; 50 high training in music) were presented each of eight melodic intervals. For each interval, the subjects were asked to compose a short melody on a piano keyboard, treating the interval provided as the first two notes of the melody. For each melody, the first response--the note immediately following the initial interval--was analyzed. Multinomial log linear analyses were conducted to assess the extent to which responses could be predicted by Narmour's (1990, 1992) bottom-up principles. Support was found for all of Narmour's principles, and two additional predictors based on implied tonal structure. Responses of low- and high-training groups were similar.