Cooperative interaction of branch signals in the actin intron of Saccharomyces cerevisiae.

In pre-mRNA splicing, specific spliceosomal components recognize key intron sequences, but the mechanisms by which splice sites are selected arenot completely understood. In the Saccharomyces cerevisiae actin intron a silent branch point-like sequence (UACUAAG) is located 7 nt upstream of the canonical sequence. Mutation of the canonicalUACUAAC sequence to UAAUAAC reduces utilization of this signal and activates the cryptic UACUAAG. Splicing-dependent beta-galactosidase assays have shown that these two splice signals cooperate to enhance splicing. Analyses of several variants of this double branch point intron demonstrate that the upstream UACUAAG sequence significantly increases usage of the UAAUAAC as a site of lariat formation. This activation is sequence-specific and unidirectional. However the ability of the UACUAAG signal to activate the downstream branch point is dependent on the presence of a short non-conserved sequence located a few nucleotides upstream of the UACUAAG. Mutation of this sequence leads to the disappearance of the cooperative interactions between the two branch signals. Our results show that this non-conserved sequence and the UACUAAG signal must both be present to achieve activation of the downstream branch point and suggest that a specific structure may be necessary to allow efficient recognition of the UAAUAAC.     

Two models of the sound-signal frequency dependence on the animal body size as exemplified by the ground squirrels of Eurasia (mammalia,rodentia)

Dependence of the sound-signal frequency on the animal body length was studied in 14 ground squirrel species (genus Spermophilus) of Eurasia. Regression analysis of the total sample yielded a low determination coefficient (R² = 26%), because the total sample proved to be heterogeneous in terms of signal frequency within the dimension classes of animals. When the total sample was divided into two groups according to signal frequency, two statistically significant models (regression equations) were obtained in which signal frequency depended on the body size at high determination coefficients (R² = 73 and 94% versus 26% for the total sample). Thus, the problem of correlation between animal body size and the frequency of their vocal signals does not have a unique solution.     

Genome level analysis of rice mRNA 3'-end processing signals and alternative polyadenylation

The position of a poly(A) site of eukaryotic mRNA is determined by sequence signals in pre-mRNA and a group of polyadenylation factors. To reveal rice poly(A) signals at a genome level, we constructed a dataset of 55 742 authenticated poly(A) sites and characterized the poly(A) signals. This resulted in identifying the typical tripartite cis-elements, including FUE, NUE and CE, as previously observed in Arabidopsis. The average size of the 3′-UTR was 289 nucleotides. When mapped to the genome, however, 15% of these poly(A) sites were found to be located in the currently annotated intergenic regions. Moreover, an extensive alternative polyadenylation profile was evident where 50% of the genes analyzed had more than one unique poly(A) site (excluding microheterogeneity sites), and 13% had four or more poly(A) sites. About 4% of the analyzed genes possessed alternative poly(A) sites at their introns, 5′-UTRs, or protein coding regions. The authenticity of these alternative poly(A) sites was partially confirmed using MPSS data. Analysis of nucleotide profile and signal patterns indicated that there may be a different set of poly(A) signals for those poly(A) sites found in the coding regions. Based on the features of rice poly(A) signals, an updated algorithm termed PASS-Rice was designed to predict poly(A) sites.     

Slit scanning of Saccharomyces cerevisiae cells: quantification of asymmetric cell division and cell cycle progression in asynchronous culture.

Slit scanning flow cytometry has been applied to the analysis of the cell cycle and cell-cycle-dependent events in Saccharomyces cerevisiae, yielding information on the low-resolution spatial distribution of cellular components in single cells of unperturbed cell populations. Because this process is rapid, large numbers of cells can be analyzed to give distributions of parameters in a given population. To study asymmetric cell division and cell cycle progression, forward-angle light scattering (FALS) signals together with fluorescence signals from acriflavine-stained nuclei have been measured in cells from exponentially growing yeast populations. An algorithm has been developed that assigns the position of the bud neck in the FALS signals so that both FALS and DNA signals can be analyzed in terms of the contributions from the mother cell and the cell bud. The data indicate that mother cell FALS, on average, remains constant while FALS due to the cell bud increases as a cell progresses through the cell cycle. By identifying mitotic cells and measuring their properties, we have found that the coefficient of variation for the distribution of FALS is smallest within the dividing cell population and largest within the newborn cell population, in accordance with the critical size control mechanism of yeast cell growth. The use of this experimental approach to provide data for statistical population models is discussed.     

Model calculations of casein micelle size distributions.

A previously proposed model for the formation and structure of casein micelles from subunits of variable composition is used to calculate theoretical micelle size distributions. Using the fractional content of k-casein as the only variable but with a value near that observed in a sample of milk serum, the model successfully reproduces experimentally determined distributions. Predicted size distributions are quite sensitive to the value of the variable and shift toward smaller average size as the assumed fractional content k-casein gets larger. Also, there is a discontinuity in the distributions which predicts that there will be essentially no micelles with radii smaller than 25-30 nm. These predictions are all in accord with experimental observations. The good agreement between theory and experimenet supports the micelle structure suggested by the model.     

Adaptation and selective information transmission in the cricket auditory neuron AN2

Sensory systems adapt their neural code to changes in the sensory environment, often on multiple time scales. Here, we report a new form of adaptation in a first-order auditory interneuron (AN2) of crickets. We characterize the response of the AN2 neuron to amplitude-modulated sound stimuli and find that adaptation shifts the stimulus-response curves toward higher stimulus intensities, with a time constant of 1.5 s for adaptation and recovery. The spike responses were thus reduced for low-intensity sounds. We then address the question whether adaptation leads to an improvement of the signal's representation and compare the experimental results with the predictions of two competing hypotheses: infomax, which predicts that information conveyed about the entire signal range should be maximized, and selective coding, which predicts that "foreground" signals should be enhanced while "background" signals should be selectively suppressed. We test how adaptation changes the input-response curve when presenting signals with two or three peaks in their amplitude distributions, for which selective coding and infomax predict conflicting changes. By means of Bayesian data analysis, we quantify the shifts of the measured response curves and also find a slight reduction of their slopes. These decreases in slopes are smaller, and the absolute response thresholds are higher than those predicted by infomax. Most remarkably, and in contrast to the infomax principle, adaptation actually reduces the amount of encoded information when considering the whole range of input signals. The response curve changes are also not consistent with the selective coding hypothesis, because the amount of information conveyed about the loudest part of the signal does not increase as predicted but remains nearly constant. Less information is transmitted about signals with lower intensity.     

Quantum nature of photon signal emitted by Xanthoria parietina and its implications to biology

The properties of living systems are usually described in the semi-classical framework that makes phenomenological division of properties into four classes--matter, psyche, soft consciousness and hard consciousness. Quantum framework provides a scientific basis of this classification of properties. The scientific basis requires the existence of macroscopic quantum entity entangled with quantum photon field of a living system. Every living system emits a photon signal with features indicating its quantum nature. Quantum nature of the signal emitted by a sample of X. parietina is confirmed by analysing photo count distributions obtained in 20000 measurements of photon number in contiguous bins of sizes of 50, 100, 200, 300 and 500 ms. The measurements use a broadband detector sensitive in 300-800 nm range (Photo count distributions of background noise and observed signal are measured similarly. These measurements background noise corrected squeezed state parameters of the signal. The parameters are signal strength expressed in counts per bin, r = 0.06, theta = 2.76 and phi = 0.64. The parameters correctly reproduce photo count distribution of any bin size in 50 ms-6 s range. The reproduction of photo count distributions is a credible evidence of spontaneous emission of photon signal in a quantum squeezed state for macroscopic time by the sample. The evidence is extrapolated to other living systems emitting similar photon signals. It is suggested that every living system is associated with a photon field in squeezed state. The suggestion has far reaching implications to biology and provides two ways of observing and manipulating a living system--either through matter or field or a combination of the two. Some implications and possible scenarios are elaborated.     

Fission yeast gene structure and recognition.

A database of 210 Schizosaccharomyces pombe DNA sequences (524,794 bp) was extracted from GenBank (release number 81.0) and examined by a number of methods in order to characterize statistical features of these sequences that might serve as signals or constraints for messenger RNA splicing. The statistical information compiled includes splicing signal (donor, acceptor and branch site) profiles, translational initiation start profile, exon/intron length distributions, ORF distribution, CDS size distribution, codon usage table, and 6-tuple distribution. The information content of the various signals are also presented. A rule-based interactive computer program for finding introns called INTRON.PLOT has been developed and was used to successfully analyze 7 newly sequenced genes.     

Simulation of <Superscript>125</Superscript>I decay in a synthetic oligodeoxynucleotide with normal and distorted geometry and the role of radiation and non-radiation actions

Within the track structure code PARTRAC, DNA strand break induction by direct and indirect radiation action was calculated for the E. coli catabolite gene activator protein (CAP) DNA complex with ¹²⁵I located at the position of the H₅ atom of the cytosine near the center. The shape of the resulting DNA fragment size distributions was found to be in reasonable agreement with corresponding experimental results. However, the calculated yield was considerably lower than the measured one. To study possible reasons for this, recently published experimental data on DNA strand breaks in a 41-mer synthetic oligodeoxynucleotide (oligoDNA) with incorporated ¹²⁵I were analyzed aiming at an evaluation of the non-radiation-related component due to the neutralization of the initially highly charged ^125mTe daughter ion. This was done by assuming that the differences between simulated radiation-induced distribution and the measured total fragment size distributions were due to the neutralization process. The neutralization effect defined in this way was found to dominate the strand breakage frequency within a range of 5–7 base pairs around the ¹²⁵I decay site on both strands. After implementing this neutralization effect derived from the oligoDNA analysis into the PARTRAC simulation for the CAP-DNA complex, the agreement of the calculated DNA fragment distributions with the corresponding experimental data was considerably improved. The results indicate that DNA conformation may be explored by incorporation of ¹²⁵I into the DNA, measurement of fragment size distributions, and comparison with simulation calculation for various hypothetical DNA models.     

Strength of translation initiation signal sequence of mRNA as studied by quantification method: effect of nucleotide substitutions upon translation efficiency in rat preproinsulin mRNA.

Concerning the translation initiation signals in vertebrate mRNAs, both the ATG initiation codon and the sequences flanking the initiation codon are required to direct the position of initiation. A consensus sequence for the signal, (GCC)GCC(A or G)CCATGG, has been proposed, but actual initiation sequences differ from it to a greater or lesser degree. In the present report, the translation initiation signal sequences of rat preproinsulin and its mutant mRNAs were analyzed using a quantification method proposed previously. In this method, each 16 nt sequence in the mRNA was characterized by its sample score, which shows strength of the signal. So far, Kozak has constructed a number of preproinsulin mutant mRNAs in which nucleotides flanking the ATG codon are systematically varied, and measured the translation initiation efficiency in terms of the proinsulin product. Her experimental results were well understood on the basis of the strength of the translation initiation signal sequence.     

Characterization of elementary Ca2+ release signals in NGF-differentiated PC12 cells and hippocampal neurons

Elementary Ca2+ release signals in nerve growth factor- (NGF-) differentiated PC12 cells and hippocampal neurons, functionally analogous to the "Ca2+ sparks" and "Ca2+ puffs" identified in other cell types, were characterized by confocal microscopy. They either occurred spontaneously or could be activated by caffeine and metabotropic agonists. The release events were dissimilar to the sparks and puffs described so far, as many arose from clusters of both ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Increasing either the stimulus strength or loading of the intracellular stores enhanced the frequency of and coupling between elementary release sites and evoked global Ca2+ signals. In the PC12 cells, the elementary Ca2+ release preferentially occurred around the branch points. Spatio-temporal recruitment of such elementary release events may regulate neuronal activities.     

Modeling Sequence-Space Exploration and Emergence of Epistatic Signals in Protein Evolution

During their evolution, proteins explore sequence space via an interplay between random mutations and phenotypic selection. Here, we build upon recent progress in reconstructing data-driven fitness landscapes for families of homologous proteins, to propose stochastic models of experimental protein evolution. These models predict quantitatively important features of experimentally evolved sequence libraries, like fitness distributions and position-specific mutational spectra. They also allow us to efficiently simulate sequence libraries for a vast array of combinations of experimental parameters like sequence divergence, selection strength, and library size. We showcase the potential of the approach in reanalyzing two recent experiments to determine protein structure from signals of epistasis emerging in experimental sequence libraries. To be detectable, these signals require sufficiently large and sufficiently diverged libraries. Our modeling framework offers a quantitative explanation for different outcomes of recently published experiments. Furthermore, we can forecast the outcome of time- and resource-intensive evolution experiments, opening thereby a way to computationally optimize experimental protocols.     

Small sequence insertions within the branch point region dictate alternative sites of lariat formation in a yeast intron.

The problem of intron recognition in S. cerevisiae appears to be in part solved by the strong conservation of intron encoded splicing signals, in particular the 5' GUAUGU and the branch point UACUAAC which interact via base pairing with the RNA components of U1 and U2 snRNPs respectively. Nevertheless, the mere presence of such signals is insufficient for splicing to occur. In the S. cerevisiae ACT1 intron, a silent UACUAAC-like sequence (UACUAAG) is located 7 nucleotides upstream of the canonical branch point signal. In order to investigate whether other factors, in addition to the U2-UACUAAC base-pair interactions, affect branch point selection in yeast, we created a cis-competition assay by converting the UACUAAG to a strong branch point signal (UACUAAC). If simply having a canonical UACUAAC sequence were sufficient for lariat formation, a 1:1 ratio in usage of the two signals should have been observed. In this double branch point intron, however, the downstream UACUAAC is utilized preferentially (4:1). Results obtained from the analyses of numerous sequence variants flanking the two UACUAAC sequences, demonstrate that non-conserved sequences in the branch point region are able to define lariat formation. Consequently, we conclude that U2 base-pairing is not the only requirement determining branch point selection in yeast, and local structure in the vicinity of the branch point could play a critical role in its recognition.     

Context matters: sexual signaling loss in digital organisms

Sexual signals are important in attracting and choosing mates; however, these signals and their associated preferences are often costly and frequently lost. Despite the prevalence of signaling system loss in many taxa, the factors leading to signal loss remain poorly understood. Here, we test the hypothesis that complexity in signal loss scenarios is due to the context‐dependent nature of the many factors affecting signal loss itself. Using the Avida digital life platform, we evolved 50 replicates of ~250 lineages, each with a unique combination of parameters, including whether signaling is obligate or facultative; genetic linkage between signaling and receiving genes; population size; and strength of preference for signals. Each of these factors ostensibly plays a crucial role in signal loss, but was found to do so only under specific conditions. Under obligate signaling, genetic linkage, but not population size, influenced signal loss; under facultative signaling, genetic linkage does not have significant influence. Somewhat surprisingly, only a total loss of preference in the obligate signaling populations led to total signal loss, indicating that even a modest amount of preference is enough to maintain signaling systems. Strength of preference proved to be the strongest single force preventing signal loss, as it consistently overcame the potential effects of drift within our study. Our findings suggest that signaling loss is often dependent on not just preference for signals, population size, and genetic linkage, but also whether signals are required to initiate mating. These data provide an understanding of the factors (and their interactions) that may facilitate the maintenance of sexual signals.     

Application of Photoshop and Scion Image analysis to quantification of signals in histochemistry, immunocytochemistry and hybridocytochemistry

OBJECTIVE: To describe a simple method to achieve the differential selection and subsequent quantification of the strength signal using only one section. STUDY DESIGN: Several methods for performing quantitative histochemistry, immunocytochemistry or hybridocytochemistry, without use of specific commercial image analysis systems, rely on pixel-counting algorithms, which do not provide information on the amount of chromogen present in the section. Other techniques use complex algorithms to calculate the cumulative signal strength using two consecutive sections. To separate the chromogen signal we used the "Color range" option of the Adobe Photoshop program, which provides a specific file for a particular chromogen selection that could be applied on similar sections. The measurement of the chromogen signal strength of the specific staining is achieved with the Scion Image software program. CONCLUSION: The method described in this paper can also be applied to simultaneous detection of different signals on the same section or different parameters (area of particles, number of particles, etc.) when the "Analyze particles" tool of the Scion program is used.     

Inferring local competition intensity from patch size distributions: a test using biological soil crusts

Dryland vegetation is inherently patchy. This patchiness goes on to impact ecology, hydrology, and biogeochemistry. Recently, researchers have proposed that dryland vegetation patch sizes follow a power law which is due to local plant facilitation. It is unknown what patch size distribution prevails when competition predominates over facilitation, or if such a pattern could be used to detect competition. We investigated this question in an alternative vegetation type, mosses and lichens of biological soil crusts, which exhibit a smaller scale patch‐interpatch configuration. This micro‐vegetation is characterized by competition for space. We proposed that multiplicative effects of genetics, environment and competition should result in a log‐normal patch size distribution. When testing the prevalence of log‐normal versus power law patch size distributions, we found that the log‐normal was the better distribution in 53% of cases and a reasonable fit in 83%. In contrast, the power law was better in 39% of cases, and in 8% of instances both distributions fit equally well. We further hypothesized that the log‐normal distribution parameters would be predictably influenced by competition strength. There was qualitative agreement between one of the distribution's parameters (μ) and a novel intransitive (lacking a ‘best’ competitor) competition index, suggesting that as intransitivity increases, patch sizes decrease. The correlation of μ with other competition indicators based on spatial segregation of species (the C‐score) depended on aridity. In less arid sites, μ was negatively correlated with the C‐score (suggesting smaller patches under stronger competition), while positive correlations (suggesting larger patches under stronger competition) were observed at more arid sites. We propose that this is due to an increasing prevalence of competition transitivity as aridity increases. These findings broaden the emerging theory surrounding dryland patch size distributions and, with refinement, may help us infer cryptic ecological processes from easily observed spatial patterns in the field.     

Nucleotide sequence analysis of human beta-globin gene by the quantification method: mutations in 3'-splice junction sequence and beta-thalassemia

The nucleotide sequence at the intron-exon junction in the human beta-globin gene was analyzed by the quantification method (categorical discriminant analysis) proposed previously. Using the sample score of a 16-nucleotide sequence at a 3'-splice junction, we studied to what extent such a sequence contains the 3'-splice signal. To examine the applicability of our method, we further studied several mutants of beta-thalassemia, where nucleotide changes exist at 3'-splice junction sequences of the first and second introns. Other mutants involve point mutations which generate new 3'-splice signals within the first intron. Experimental results on the abnormal splicing in those mutants could be explained in terms of the sample scores of 16-nucleotide sequences and their locations relative to the branch point.     

Transport of proteins into chloroplasts. Delineation of envelope "transit" and thylakoid "transfer" signals within the pre-sequences of three imported thylakoid lumen proteins.

The targeting of cytosolically synthesized proteins into the thylakoid lumen is mediated by an aminoterminal pre-sequence consisting of an "envelope transit" and a "thylakoid transfer" signal in tandem. We have investigated the structural characteristics of several thylakoid transfer signals by determining the intermediate sites at which the stromal processing peptidase cleaves to remove the transit sequences. Using this approach we have found that the thylakoid transfer signals of Silene pratensis plastocyanin, 23-kDa oxygen-evolving complex protein from wheat, and 33-kDa oxygen-evolving complex protein from wheat, are 25, 39, and 48 residues in length, respectively. All of the transfer signals contain hydrophobic core sequences and a "-3,-1" motif reminiscent of those found in signal sequences, but the amino-terminal regions of the transfer signals of the 23- and 33-kDa proteins are both longer and more highly charged. The net charge of each amino-terminal region of the transfer sequences is +1, including the amino-terminal amino group. In each case, the stromal processing peptidase cleaves immediately after a positively charged residue, but otherwise the cleavage sites exhibit no common elements of either primary or secondary structure.